150 results on '"Seyer, J M"'
Search Results
2. Suppression of murine collagen-induced arthritis by nasal administration of collagen
- Author
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MYERS, L. K., SEYER, J. M., STUART, J. M., and KANG, A. H.
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- 1997
3. Evidence for the presence of numerous protein components in immature bovine dental enamel
- Author
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Seyer, J. M. and Glimcher, M. J.
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- 1977
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4. The isolation of two types of collagen from embryonic bovine epiphyseal cartilage
- Author
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Seyer, J. M., Brickley, D. M., and Glimcher, M. J.
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- 1974
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5. The identification of two types of collagen in the articular cartilage of postnatal chickens
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Seyer, J. M., Brickley, D. M., and Glimcher, M. J.
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- 1974
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6. Effect of peptides of bovine myelin basic protein on dermal fibroblasts.
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Chiang, T. M., Whitaker, J. N., Seyer, J. M., and Kang, A. H.
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- 1980
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7. Identification and characterization of a major tolerogenic T-cell epitope of type II collagen that suppresses arthritis in B10.RIII mice.
- Author
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Miyahara, H., Myers, L. K., Rosloniec, E. F., Brand, D. D., Seyer, J. M., Stuart, J. M., and Kang, A. H.
- Subjects
T cells ,EPITOPES ,COLLAGEN ,ARTHRITIS ,DISEASES ,JOINT diseases ,IMMUNOLOGY ,MEDICAL sciences - Abstract
Tolerization of B10.RIII mice (H-2
r ) with intravenously injected type II collagen (CII) renders the animals resistant to induction of collagen-induced arthritis (CIA). In order to clarify 11-2r - restricted T-cell responses that modulate CIA, we have analysed the T-cell proliferative response of B10.RIII mice against cyanogen bromide (CB) peptides of CII, and detected the strongest response to α(II)-CB10 (CII 552–897). A panel of chemically synthesized overlapping peptide homologues was used to deduce the minimum structure of this determinant which was found to be CII 610–618. A 15-residue synthetic peptide flanking this region. CII 607–621, was found to effectively suppress arthritis when administered as a tolerogen. Collectively, these data identify the structural component within α1(II)-CB10 which is capable of inducing tolerance in BIO.RIII mice. A similar approach to the treatment of autoimmune arthritis, involving the institution of self-tolerance, has potential applicability to human rheumatoid arthritis. [ABSTRACT FROM AUTHOR]- Published
- 1995
8. HLA ASSOCIATIONS WITH T CELL REACTIVITY TO TYPE II COLLAGEN EPITOPES IN PATIENTS WITH RHEUMATOID ARTHRITIS.
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Postlethwaite, A. E., Seyer, J. M., Endres, R., Ward, F., Kang, A. H., Myers, L., McKown, K., Barrow, K., and Carbone, L.
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- 2005
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9. Induction of immune tolerance to human type I collagen in patients with systemic sclerosis by oral administration of bovine type I collagen.
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McKown KM, Carbone LD, Bustillo J, Seyer JM, Kang AH, and Postlethwaite AE
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- Administration, Oral, Animals, Cattle, Collagen administration & dosage, Collagen immunology, Down-Regulation, Female, Humans, Immune Tolerance physiology, Interferon-gamma metabolism, Leukocytes, Mononuclear metabolism, Male, Middle Aged, Patient Compliance, Scleroderma, Systemic immunology
- Abstract
Objective: To determine whether oral tolerance to type I collagen (CI) could be induced in patients with systemic sclerosis (SSc)., Methods: Twenty adult patients with limited or diffuse SSc were enrolled in a study to receive 0.1 mg of solubilized native bovine CI daily for 1 month, followed by 0.5 mg daily for 11 months. Peripheral blood mononuclear cells (PBMC) were obtained from the patients and cultured with human alpha1(I) and alpha2(I) chains, before and after CI treatment. Culture supernatants were analyzed for levels of interferon-gamma (IFNgamma) and interleukin-10 (IL-10). Sera obtained before and after treatment were analyzed for levels of soluble IL-2 receptor (sIL-2R). Although this study was not intended to assess the clinical efficacy of oral CI administration in SSc, selected measures of disease severity and organ involvement were evaluated., Results: Oral administration of CI to SSc patients induced significant reductions in levels of IFNgamma and IL-10 in alpha1(I)- and alpha2(I)-stimulated PBMC culture supernatants, indicating that T cell immunity to CI was decreased by this treatment. Serum levels of sIL-2R also decreased significantly after oral CI treatment, suggesting a reduction in T cell activation. Significant improvements occurred in the modified Rodnan skin thickness score and the modified Health Assessment Questionnaire after 12 months of oral CI in this open trial. The lung carbon monoxide diffusing capacity improved statistically and showed a trend toward clinically significant improvement., Conclusion: Oral administration of bovine CI to patients with diffuse or limited SSc induces a reduction in T cell reactivity to human CI, appears to be well tolerated, and does not worsen the disease. Further evaluation of oral tolerance to CI in patients with SSc is justified to determine whether it has therapeutic efficacy.
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- 2000
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10. Lack of efficacy of oral bovine type II collagen added to existing therapy in rheumatoid arthritis.
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McKown KM, Carbone LD, Kaplan SB, Aelion JA, Lohr KM, Cremer MA, Bustillo J, Gonzalez M, Kaeley G, Steere EL, Somes GW, Myers LK, Seyer JM, Kang AH, and Postlethwaite AE
- Subjects
- Administration, Oral, Adolescent, Adult, Aged, Animals, Arthritis, Rheumatoid pathology, Cattle, Collagen administration & dosage, Double-Blind Method, Drug Therapy, Combination, Female, Humans, Joints drug effects, Joints pathology, Male, Middle Aged, Pain Measurement, Severity of Illness Index, Surveys and Questionnaires, Treatment Outcome, Antirheumatic Agents therapeutic use, Arthritis, Rheumatoid drug therapy, Collagen therapeutic use
- Abstract
Objective: To investigate the efficacy of oral type II collagen (CII) in the treatment of rheumatoid arthritis (RA), when added to existing therapy., Methods: Patients with active RA (n = 190) were randomized into a 6-month, double-blind, placebo-controlled trial. Patients continued to take their current arthritis medications. Patients received either placebo or bovine CII, 0.1 mg/day for 1 month, then 0.5 mg/day for 5 months., Results: There were no significant differences between the baseline characteristics of either group. The primary response parameter was the American College of Rheumatology (ACR) preliminary definition of improvement in RA (ACR 20). There was no statistically significant difference in the ACR 20 after 6 months (20.0% of placebo patients; 16.84% of bovine CII patients). There were significant differences in several clinical variables after treatment, all favoring the placebo group., Conclusion: Oral solubilized bovine CII, added to existing therapy, did not improve disease activity in patients with RA.
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- 1999
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11. Identification of specific sites in the TNF-alpha molecule promoting insulin resistance in H-411E cells.
- Author
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Solomon SS, Mishra SK, Palazzolo MR, Postlethwaite AE, and Seyer JM
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- Amino Acid Sequence, Animals, Antibodies immunology, Blotting, Northern, Calmodulin biosynthesis, Calmodulin genetics, Carcinoma, Hepatocellular, Gene Expression Regulation drug effects, Glucose metabolism, Humans, Insulin pharmacology, Lipid Metabolism, Molecular Sequence Data, Peptide Fragments chemical synthesis, Peptide Fragments chemistry, Peptide Fragments pharmacology, RNA, Messenger genetics, RNA, Messenger metabolism, Rats, Receptors, Tumor Necrosis Factor antagonists & inhibitors, Receptors, Tumor Necrosis Factor immunology, Receptors, Tumor Necrosis Factor metabolism, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha antagonists & inhibitors, Insulin Antagonists pharmacology, Insulin Resistance physiology, Tumor Necrosis Factor-alpha chemistry, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Data from a number of laboratories support a potential role for tumor necrosis factor-alpha (TNF-alpha) in the loss of insulin sensitivity and the pathogenesis of insulin resistance (IR) in diabetic animal models and human patients. We designed experiments to establish a dose-response relationship for TNF-alpha and IR in H-411E cells in culture. IR was measured by inhibition of the ability of graded amounts of insulin to stimulate expression of calmodulin (CaM) mRNA in these cells. This was assessed by autoradiographs of Northern blot(s) of CaM mRNA probed with labeled oligonucleotide cDNA for rat CaM. We found that TNF-alpha at 0.1, 1.0, and 10.0 ng/ml opposed 10,000 microU/ml insulin (i.e., %IR = 20%, 67%, and 88%, respectively). At 1.0 ng/ml TNF-alpha, insulin at the concentration of 1000 microU/ml (0.006 micromol/L) stimulated CaM mRNA at a 41% level and at 10,000 microU/ml (0.06 micromol/L) at a 63% level. Furthermore, oligopeptide TNF-alpha homologs (at 1000 x the molar concentration of TNF-alpha) TNF-alpha 69-100 and TNF-alpha 133-157 conferred 66% and 101% IR, respectively, while all other peptide fragments of TNF-alpha were essentially without effect. Studies done with both monoclonal and polyclonal antibodies to the TNF-alpha receptor demonstrated blocking activity by polyclonal but not by monoclonal anti TNF-alpha receptor antibody. This supports the concept that the activity of the peptide fragments occurs through the TNF-alpha receptor and not through nonspecific translocation across the plasma membrane. These data suggest that the epitopes on TNF-alpha that mediate IR reside in two regions of the molecule spanning amino acid residues 69-100 and 133-157.
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- 1997
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12. An interleukin-1 receptor fragment inhibits spontaneous sleep and muramyl dipeptide-induced sleep in rabbits.
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Takahashi S, Kapás L, Fang J, Seyer JM, Wang Y, and Krueger JM
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- Acetylmuramyl-Alanyl-Isoglutamine administration & dosage, Animals, Fever chemically induced, Fever physiopathology, Humans, Injections, Interleukin-1 pharmacology, Male, Rabbits, Sleep Stages drug effects, Acetylmuramyl-Alanyl-Isoglutamine pharmacology, Peptide Fragments pharmacology, Receptors, Interleukin-1 chemistry, Sleep drug effects
- Abstract
Interleukin-1 (IL-1) is hypothesized to be involved in physiological sleep regulation and in sleep responses occurring during infectious disease. If this hypothesis is correct, then inhibition of endogenous IL-1 should reduce both normal sleep and N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP)-induced sleep. MDP is a somnogenic substance derived from bacterial cell walls. We report here the effects of a synthetic IL-1 receptor fragment corresponding to amino acid residues 86-95 of the human type I IL-1 receptor (IL-1RF) on spontaneous sleep and IL-1 beta- and MDP-induced sleep and fever in rabbits. Two doses of the IL-1RF (25 and 50 micrograms) were injected into normal rabbits intracerebroventricularly (icv). Both doses significantly decreased spontaneous non-rapid eye movement sleep (NREMS) across a 22-h recording period. Pretreatment of rabbits with 25 micrograms of IL-1RF blocked the somnogenic actions of 10 ng icv IL-1. Similarly, central pretreatment of animals with 25 micrograms IL-1RF significantly attenuated the NREMS-promoting and REMS-suppressive actions of 150 pmol MDP injected centrally. The increase in NREMS and decrease in REMS induced by systemic injection of 12.5 micrograms/kg MDP were also significantly suppressed by central administration of 50 micrograms IL-1RF. In contrast, the febrile response induced by either intracerebroventricularly or intravenously injected MDP were not significantly affected by IL-1RF. These results support the hypothesis that endogenous, brain-derived IL-1 contributes to the maintenance of normal sleep and may mediate sleep responses to systemic as well as central bacterial infections.
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- 1996
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13. Interleukin-1 beta peptides induce cerebral pial arteriolar dilation in anesthetized newborn pigs.
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Shibata M, Parfenova H, Zuckerman SL, Seyer JM, Krueger JM, and Leffler CW
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- 6-Ketoprostaglandin F1 alpha antagonists & inhibitors, 6-Ketoprostaglandin F1 alpha cerebrospinal fluid, Animals, Animals, Newborn, Arterioles drug effects, Dinoprostone antagonists & inhibitors, Dinoprostone cerebrospinal fluid, Indomethacin pharmacology, Receptors, Interleukin-1 physiology, Solubility, Swine, Cerebrovascular Circulation drug effects, Interleukin-1 pharmacology, Peptide Fragments pharmacology, Pia Mater blood supply, Vasodilation
- Abstract
Inflammatory cytokines may affect cerebral circulation under pathological conditions. Responses of cerebral pial arterioles to one such cytokine, interleukin (IL)-1 beta and its inhibitor [soluble IL-1 receptor (sIL-1R)] were examined in anesthetized newborn pigs using closed cranial windows. Levels of prostanoids and cyclic nucleotides in periarachnoid cerebral spinal fluid (CSF) were measured. To examine the structure-activity relationship of the parent IL-1 beta molecule, two IL-1 beta fragments with amino acid sequences of 187-204 [IL-1 beta-(187-204)] and 208-240 [IL-1 beta-(208-240)] were tested for their effects on pial arterioles. Diameter changes of pial arterioles were sequentially recorded every 5 min for 30 min after topical application of IL-1 peptides. CSF was sampled at the end of the 30 min. IL-1 beta dose dependently induced arteriolar dilation and increased prostaglandin E2 (PGE2), 6-keto-PGF1 alpha adenosine 3',5'-cyclic monophosphate (cAMP), and guanosine 3',5'-cyclic monophosphate (cGMP). Intravenous indomethacin blocked the IL-1 beta-induced vasodilation, the increased prostanoids, and the increased cAMP, but not the increased cGMP. Neither heat-inactivated IL-1 beta nor IL-1 beta vehicle affected arteriolar diameter or CSF levels of prostanoids. The sIL-1R blocked the IL-1 beta-induced vasodilation and the increased CSF prostanoids. IL-1 beta-(208-240) also induced pial arteriolar dilation; however, its vasodilatory potency was 1,000 times less than that of the whole IL-1 beta molecule. IL-1 beta-(187-204) did not induce pial arteriolar dilation even when its dose was increased to the level of IL-1 beta-(208-240). These results suggest that IL-1 beta, through the activation of membrane-bound IL-1 beta receptors, induces pial arteriolar dilation via mechanisms that involve prostanoids and cyclic nucleotides. The results also indicate that the 208-240 amino acid sequence of IL-1 beta has a sequence-specific physiological function.
- Published
- 1996
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14. Inhibition of tumor necrosis factor attenuates physiological sleep in rabbits.
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Takahashi S, Kapás L, Seyer JM, Wang Y, and Krueger JM
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- Amino Acid Sequence, Animals, Male, Molecular Sequence Data, Peptide Fragments, Polysomnography, Rabbits, Receptors, Tumor Necrosis Factor antagonists & inhibitors, Sleep Deprivation physiology, Sleep drug effects, Tumor Necrosis Factor-alpha antagonists & inhibitors
- Abstract
The involvement of tumor necrosis factor (TNF) in sleep regulation was investigated by blocking TNF using a synthetic TNF receptor fragment (TNFRF) in rabbits. Intracerebroventricular injection of 50 micrograms TNFRF decreased spontaneous non-rapid-eye-movement (NREM) sleep across the 21 h recording period. After 6 h of sleep deprivation (SD), both duration of NREM sleep and electroencephalographic (EEG) slow wave activity during NREM sleep were enhanced. TNFRF suppressed SD-enhanced NREM sleep and EEG slow wave activity. SD per se did not affect rapid-eye-movement (REM) sleep, but TNFRF treatment increased REM sleep after SD. These results are consistent with the hypothesis that TNF is involved in the regulation of physiological NREM sleep.
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- 1996
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15. Inhibition of tumor necrosis factor in the brain suppresses rabbit sleep.
- Author
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Takahashi S, Tooley DD, Kapás L, Fang J, Seyer JM, and Krueger JM
- Subjects
- Animals, Brain Chemistry drug effects, Electroencephalography drug effects, Injections, Intraventricular, Male, Rabbits, Receptors, Tumor Necrosis Factor chemistry, Recombinant Proteins antagonists & inhibitors, Sleep drug effects, Sleep, REM drug effects, Sleep, REM physiology, Tumor Necrosis Factor-alpha biosynthesis, Brain Chemistry physiology, Sleep physiology, Tumor Necrosis Factor-alpha antagonists & inhibitors
- Abstract
Tumor necrosis factor (TNF) is a cytokine that possesses many biological activities, including enhancement of non-rapid-eye-movement sleep (NREMS). The role of endogenous TNF in the regulation of spontaneous sleep is unknown. If TNF is involved in sleep regulation, then reduction of endogenous TNF should suppress spontaneous sleep. A soluble TNF-binding protein I (TNF-BP I) and a synthetic fragment of TNF-BP I, TNF-R-(159-178), that contains the biologically active region of TNF-BP I, were used. These substances bind TNF and possess TNF-inhibitory activity; their effects on rabbit sleep after intracerebroventricular injection were determined across a 6-h recording period. Two doses of TNF-BP I (0.05 micrograms and 0.5 micrograms) were administered; the higher dose of TNF-BP I significantly decreased NREMS. Four doses of TNF-R-(159-178) (0.25 micrograms, 2.5 micrograms, 25 micrograms and 50 micrograms) were used. The 25 micrograms and 50 micrograms doses significantly suppressed NREMS. The highest dose (50 micrograms) also decreased REM sleep. These results are consistent with the hypothesis that endogenous brain TNF is involved in the regulation of normal sleep.
- Published
- 1995
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16. Establishment by the rat lymph node method of epitope-defined monoclonal antibodies recognizing the six different alpha chains of human type IV collagen.
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Sado Y, Kagawa M, Kishiro Y, Sugihara K, Naito I, Seyer JM, Sugimoto M, Oohashi T, and Ninomiya Y
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- Amino Acid Sequence, Animals, Antibody Specificity, Blotting, Western, Collagen chemistry, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Epitope Mapping methods, Female, Fluorescent Antibody Technique, Indirect, Humans, Mice, Molecular Sequence Data, Peptides chemical synthesis, Peptides immunology, Rats, Rats, Inbred WKY, Vaccines, Synthetic, Antibodies, Monoclonal immunology, Collagen immunology, Epitopes immunology, Lymph Nodes immunology
- Abstract
A group of rat monoclonal antibodies recognizing the six different alpha chains of human type IV collagen have been established by our novel method. The method is designated the rat lymph node method in which enlarged medial iliac lymph nodes of a rat injected with an antigen emulsion via hind footpads are used as a source of B cells for cell fusion to produce hybridomas. The immunogens used were synthetic peptides having non-consensus amino acid sequences near the carboxyl termini of type IV collagen alpha chains. Hybridomas were screened both by ELISA with synthetic peptides and by indirect immunofluorescence with cryostat sections of human kidneys. Because the epitopes of all antibodies were determined by multipin-peptide scanning, they were confirmed to be isoform-specific. They are useful for identification of alpha chains of type IV collagen at the protein level in normal and abnormal conditions. The combined use of synthetic peptides as immunogens, the rat lymph node method as making monoclonal antibodies, and the multipin-peptide scanning as epitope mapping is found to be a strong tool for identification of peptides and proteins whose amino acid sequences are known or have been deduced.
- Published
- 1995
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17. Identification of a chemotactic epitope in human transforming growth factor-beta 1 spanning amino acid residues 368-374.
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Postlethwaite AE and Seyer JM
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- Amino Acid Sequence, Epitope Mapping, Fibroblasts physiology, Humans, In Vitro Techniques, Molecular Sequence Data, Peptides chemistry, Chemotactic Factors chemistry, Chemotaxis, Leukocyte, Monocytes physiology, Neutrophils physiology, Transforming Growth Factor beta chemistry
- Abstract
TGF-beta 1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-beta 1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-beta 1 that contained residues 368-374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-beta 1 monomer were able to compete with [125I]hrTGF-beta 1 for binding to TGF-beta cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed.
- Published
- 1995
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18. Differential expression of two basement membrane collagen genes, COL4A6 and COL4A5, demonstrated by immunofluorescence staining using peptide-specific monoclonal antibodies.
- Author
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Ninomiya Y, Kagawa M, Iyama K, Naito I, Kishiro Y, Seyer JM, Sugimoto M, Oohashi T, and Sado Y
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- Adipocytes chemistry, Amino Acid Sequence, Antibodies, Monoclonal, Antibody Specificity, Blotting, Western, Fluorescent Antibody Technique, Gene Expression physiology, Humans, Kidney cytology, Kidney physiology, Liver chemistry, Molecular Sequence Data, Muscles chemistry, Nephritis, Hereditary pathology, Peptides immunology, Skin chemistry, Basement Membrane physiology, Collagen genetics
- Abstract
Genes for the human alpha 5(IV) and alpha 6(IV) collagen chains have a unique arrangement in that they are colocalized on chromosome Xq22 in a head-to-head fashion and appear to share a common bidirectional promoter. In addition we reported a novel observation that the COL4A6 gene is transcribed from two alternative promoters in a tissue-specific manner (Sugimoto, M., T. Oohashi, and Y. Ninomiya. 1994. Proc. Natl. Acad. Sci. USA. 91:11679-11683). To know whether the translation products of both genes are colocalized in various tissues, we raised alpha 5(IV) and alpha 6(IV) chain-specific rat monoclonal antibodies against synthetic peptides reflecting sequences near the carboxy terminus of each noncollagenous (NC)1 domain. By Western blotting alpha 6(IV) chain-specific antibody recognized 27-kD monomers and associated dimers of the human type IV collagen NC1 domain, which is the first demonstration of the presence in tissues of the alpha 6(IV) polypeptide as predicted from its cDNA sequence. Immunofluorescence studies using anti-alpha 6(IV) antibody demonstrated that in human adult kidney the alpha 6(IV) chain was never detected in the glomerular basement membrane, whereas the basement membranes of the Bowman's capsules and distal tubules were positive. The staining pattern of the glomerular basement membrane was quite different from that obtained with the anti-alpha 5(IV) peptide antibody. The alpha 5(IV) and alpha 6(IV) chains were colocalized in the basement membrane in the skin, smooth muscle cells, and adipocytes; however, little if any reaction was seen in basement membranes of cardiac muscles and hepatic sinusoidal endothelial cells. Thus, both genes are expressed in a tissue-specific manner, perhaps due to the unique function of the bidirectional promoter for both genes, which is presumably different from that for COL4A1 and COL4A2.
- Published
- 1995
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19. Intracellular phosphorylation of the Sendai virus P protein.
- Author
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Byrappa S, Hendricks DD, Pan YB, Seyer JM, and Gupta KC
- Subjects
- Animals, COS Cells, Cell-Free System, Phosphorylation, Virus Replication, Phosphoproteins physiology, Sendai virus physiology, Viral Proteins physiology
- Abstract
Phosphorylation status of the Sendai virus P protein was examined during virus infection and compared with cell-free phosphorylation. P protein from Sendai virus-infected (VI) and P/C gene-transfected (PT) mammalian cells and from purified virions (PV) was phosphorylated at only serine residues. In contrast, cell-free phosphorylation of the P protein with virion-associated protein kinase (VAPK) occurred at both threonine and serine. Tryptic phosphopeptide maps of the P protein from VI, PT, and PV showed that the phosphorylation was primarily localized on one peptide (TP1), while VAPK phosphorylated the P protein on several peptides. There was no change in the steady-state phosphopeptide map of the P protein during virus replication, indicating that the TP1 is constitutively phosphorylated. Inhibition of cellular phosphatases (PP1 and PP2A) by okadaic acid (OA) in virus-infected cells caused a sixfold increase in the P protein phosphorylation, solely at serine residues. The phosphopeptide map of the OA-P protein revealed that phosphorylation occurred on several peptides, but the OA-P map was significantly different from the VAPK-P map. However, additional phosphorylation of the P protein did not block its association with nucleocapsids. These results suggest that the Sendai virus P protein is constitutively phosphorylated primarily at one locus but has the potential for phosphorylation at additional sites. Further, our results do not show any correlations between the intracellular and cell-free phosphorylation of the P protein and, therefore, question the validity of cell-free phosphorylations.
- Published
- 1995
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20. Collagen-induced arthritis in B10.RIII mice (H-2r): identification of an arthritogenic T-cell determinant.
- Author
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Myers LK, Miyahara H, Terato K, Seyer JM, Stuart JM, and Kang AH
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- Amino Acid Sequence, Animals, Cattle, Chickens, Collagen chemistry, Mice, Mice, Inbred Strains, Molecular Sequence Data, Peptide Fragments immunology, Arthritis immunology, Autoimmune Diseases immunology, Collagen immunology, Epitopes immunology, T-Lymphocytes immunology
- Abstract
Susceptibility to collagen-induced arthritis (CIA), a murine model of autoimmune arthritis, is strongly linked to only two major histocompatibility complex (MHC) haplotypes, H-2q and H-2r. In order to identify the determinants of type II collagen (CII) required to induce arthritis in H-2r-bearing mice, B10.RIII mice were immunized with bovine, chick or human CII. Only bovine CII induced significant arthritis and autoantibodies. When the major CNBr peptides of bovine collagen were isolated and used for immunization, only mice immunized with CB8, representing CII 403-551, developed arthritis. To identify immunogenic epitope(s) within CB8, a panel of synthetic peptides representing overlapping sequences of the bovine peptide was generated. When each peptide was cultured with T cells from B10.RIII mice immunized with CII, one peptide, representing CII 430-466, contained a major T-cell epitope. By using an in vitro lymphokine production assay, the T-cell epitope was further narrowed to CII 442-456. These findings suggest that a T-cell determinant important for the initiation of arthritis in B10.RIII (H-2r) mice is located within a 15 amino acid sequence, residues 442-456 of bovine CII.
- Published
- 1995
21. Identification and characterization of a tolerogenic T cell determinant within residues 181-209 of chick type II collagen.
- Author
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Myers LK, Cooper SW, Terato K, Seyer JM, Stuart JM, and Kang AH
- Subjects
- Amino Acid Sequence, Amino Acids analysis, Animals, Arthritis, Rheumatoid prevention & control, Chickens, Epitopes chemistry, Immune Tolerance, Mice, Mice, Inbred DBA, Molecular Sequence Data, Peptides analysis, Collagen immunology
- Abstract
The murine model of collagen-induced arthritis is characterized by the development of an immune response against joint cartilage. Arthritis can be significantly suppressed by the administration of type II collagen (CII) or one of the CNBr peptides, CB11 (CII 124-402) as a tolerogen prior to immunization. We have previously shown that two synthetic peptides, representing sequences CII 260-270 and CII 181-209, are effective tolerogens. In this paper, we now characterize the T cell determinant with CII 181-209. A series of synthetic peptides overlapping CII 181-209 and analogs of chick CII 181-209 containing site-directed amino acid substitutions was developed and cultured with T cells from DBA/1 mice immunized with CII. Supernatants were collected and analyzed for the presence of the T cell lymphokine IFN-gamma. These data indicate the critical T cell determinant to be located within CII 190-200. This conclusion is further supported by the observation that an unodecapeptide representing CII 190-200 was just as effective as CII 181-209 in suppressing arthritis and anti-CII antibody response when tested as a tolerogen. Analogs containing single amino acid substitutions at residues 191, 194, 197, 198, or 200 were significantly less effective in inducing T cell responses. Each of these peptide analogs was then given as neonatal tolerogens to DBA/1 mice. Mice were subsequently immunized and observed for the development of arthritis. These studies identified residues 194, 197, 198, and 200, and probably residue 191, as critical for tolerance and the suppression of arthritis. Elucidation of the fine structures of T cell determinants which are critical for suppression of arthritis should allow these techniques to be used for developing specific immunotherapeutic approaches to autoimmune arthritis.
- Published
- 1995
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22. A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase.
- Author
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Sun H, Seyer JM, and Patel TB
- Subjects
- Amino Acid Sequence, Animals, Cattle, Cloning, Molecular, Cytosol metabolism, ErbB Receptors chemistry, Escherichia coli, GTP Phosphohydrolases metabolism, Guanosine 5'-O-(3-Thiotriphosphate) metabolism, Guanosine Triphosphate metabolism, Kinetics, Molecular Sequence Data, Phosphorylation, Recombinant Proteins metabolism, Adenylyl Cyclases metabolism, Brain metabolism, ErbB Receptors metabolism, GTP-Binding Proteins metabolism, Peptide Fragments pharmacology
- Abstract
Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs. Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs. A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs. This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein. Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi. Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs. Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein. Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function.
- Published
- 1995
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23. Collagen-induced arthritis in mice: synergistic effect of E. coli lipopolysaccharide bypasses epitope specificity in the induction of arthritis with monoclonal antibodies to type II collagen.
- Author
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Terato K, Harper DS, Griffiths MM, Hasty DL, Ye XJ, Cremer MA, and Seyer JM
- Subjects
- Animals, Antibodies, Monoclonal immunology, Antibody Specificity immunology, Collagen immunology, Escherichia coli immunology, Male, Metalloendopeptidases physiology, Mice, Mice, Inbred BALB C, Mice, Inbred DBA, Antibodies, Monoclonal toxicity, Arthritis, Experimental chemically induced, Arthritis, Experimental etiology, Collagen toxicity, Epitopes immunology, Lipopolysaccharides toxicity
- Abstract
DBA/1 mice develop a chronic peripheral arthritis after immunization with type II collagen termed collagen-induced arthritis. We have localized the main arthritogenic determinants of CB11, a CNBr-generated arthritogenic fragment of chick type II collagen (CII), using 3 smaller peptide fragments of CB11 generated by endoproteinase LysC, LysC1 (CII 124-290), LysC2 (CII 291-374) and LysC3 (CII 375-402) and a panel of monoclonal antibodies (mAb) specific to CB11. MAb specific to the arthritogenic region of CB11 were also used to study the synergistic effect of E. coli lipopolysaccharide (LPS) on antibody-mediated arthritis in naive DBA/1 mice. LysC2 contained a minimum essential arthritogenic fragment of type II collagen: LysC2 induced arthritis by active immunization, also, a combination of four mAb specific to LysC2 passively transferred arthritis to naive mice. A single i.p. injection of LPS (50 micrograms/mouse) reduced the threshold values of the arthritogenic dose of mAb from 1 mg to 50 micrograms/clone per mouse, and decreased the number of mAb required for inducing arthritis from 4 to 2 clones. These observations suggest that LysC2, an 84 amino acid residue fragment, contains the main arthritogenic determinants within chick CB11. Importantly, LPS, a strong inducer of pro-inflammatory cytokines, negates the required multiple epitope specificity of autoantibodies in the passive transfer model and acts synergistically in the induction of arthritis by autoantibody.
- Published
- 1995
- Full Text
- View/download PDF
24. Activation of Ito cells involves regulation of AP-1 binding proteins and induction of type I collagen gene expression.
- Author
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Armendariz-Borunda J, Simkevich CP, Roy N, Raghow R, Kang AH, and Seyer JM
- Subjects
- Animals, Base Sequence, Cells, Cultured, DNA metabolism, Gene Deletion, Genes, jun, Liver cytology, Molecular Sequence Data, Phenotype, Promoter Regions, Genetic physiology, Protein Binding, Rats, Rats, Wistar, Transcription Factor AP-1 metabolism, Transcription, Genetic, Transforming Growth Factor beta pharmacology, Collagen genetics, Gene Expression Regulation drug effects, Liver physiology, Transcription Factor AP-1 physiology
- Abstract
Activation of liver Ito cells is characterized by increased proliferation, fibrogenesis, loss of cellular retinoid and change of cell-shape. Here, we have described fundamental differences between freshly isolated Ito cells (FIC) and long-term cultured Ito cells (LTIC). This process of activation correlates with the absence of expression of Pro alpha 1(I) gene in FIC. LTIC expressed abundant transcripts of Pro alpha 1(I) gene. Nuclear run-off experiments showed the inability of FIC to support Pro alpha 1(I) RNA transcription while LTIC transcribed it greater than 5-fold as compared with FIC. Transforming growth factor beta (TGF beta)-treated LTIC had a preferential increase in the rate of Pro alpha 1(I) gene transcription as compared with control LTIC. A human collagen type I promoter-enhancer construct (pCOL-KT) [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549-2556] was readily expressed in LTIC but failed to be expressed in FIC. Furthermore, TGF beta treatment of LTIC resulted in an increased expression of pCOL-KT. The deletion of an activator protein-1 (AP-1) binding site (+598 to +604) in the 360 bp enhancer region of pCOL-KT (S360) caused decreased expression of the CAT reporter gene, suggesting that this bonafide AP-1 site can, at least in part, mediate the transactivation effect of TGF beta. Using DNAase I protection, we demonstrate a single foot-print located at +590 to +625 in the S360 fragment; nuclear extracts prepared from TGF beta-treated LTIC exhibited greater activity of these AP-1 binding proteins. Gel mobility assays corroborated and extended the footprinting observation. No AP-1-binding activity was found in the nuclear extracts of FIC. Double-stranded oligonucleotides containing the consensus AP-1 motif were able to compete out the binding; consensus NF-1 motif oligonucleotides failed to do so. The preincubation of nuclear extracts from control and TGF beta-treated LTIC with antibodies against c-jun and c-fos rendered a reduced binding of AP-1 proteins to the target S360 fragment.
- Published
- 1994
- Full Text
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25. Type XI collagen-induced arthritis in the Lewis rat. Characterization of cellular and humoral immune responses to native types XI, V, and II collagen and constituent alpha-chains.
- Author
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Cremer MA, Ye XJ, Terato K, Owens SW, Seyer JM, and Kang AH
- Subjects
- Animals, Antibody Formation, Female, Hypersensitivity, Delayed, Immunization, Immunohistochemistry, Rats, Rats, Inbred Lew, Arthritis immunology, Collagen immunology
- Abstract
Autoimmune arthritis can be induced in rats by immunization with cartilage-specific types II and XI collagen. Type XI collagen is composed of alpha 1(XI), alpha 2(XI), and alpha 3(XI) chains, in which alpha 3(XI) is essentially identical to alpha 1(II) of type II, and alpha 1(XI) and alpha 2(XI) are similar to alpha 1(V) and alpha 2(V) of type V collagen. To characterize the immune response to type XI collagen, Lewis rats were injected with bovine type XI collagen, and the cellular and humoral responses were compared with those of rats injected with types V and II collagen. Arthritis, IgG deposits in cartilage, and joint destruction were seen in rats immunized with types XI and II collagen. Type XI elicited strong cellular responses to rat types XI, V, and II; conversely, types II and V collagen elicited strong responses to rat type XI. Antitype XI Abs reacted with rat type XI, moderately with rat type V, but poorly with rat type II. Direct and inhibition ELISA showed that cross-reactions between types XI and V collagen resulted from recognition of determinants shared by their respective alpha 2(XI) and alpha 1(V) chains. Abs eluted from joints of rats immunized with type XI collagen, however, reacted only with native rat type XI collagen. These data demonstrate that type XI collagen induces diverse populations of Abs differing in collagen-type specificity, and suggest that only those Abs to native rat type XI collagen are central to the pathogenesis of type XI collagen-induced arthritis.
- Published
- 1994
26. An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.
- Author
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Wilkinson DA, Marion TN, Tillman DM, Norcum MT, Hainfeld JF, Seyer JM, and Carlson GM
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Monoclonal immunology, Binding Sites, Antibody, Female, Microscopy, Electron, Microscopy, Electron, Scanning Transmission, Molecular Sequence Data, Phosphorylase Kinase chemistry, Rabbits, Epitopes, Muscles enzymology, Phosphorylase Kinase immunology
- Abstract
An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.
- Published
- 1994
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27. Phosphorylation of the Sendai virus C proteins.
- Author
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Hendricks DD, Ono E, Seyer JM, and Gupta KC
- Subjects
- Animals, Autoradiography, Cell Line, Chlorocebus aethiops, Electrophoresis, Polyacrylamide Gel, Genes, Viral, Kidney, Molecular Weight, Phosphates metabolism, Phosphorus Radioisotopes, Phosphorylation, RNA, Messenger metabolism, Sulfur Radioisotopes, Transfection, Viral Proteins biosynthesis, Viral Proteins isolation & purification, Parainfluenza Virus 1, Human metabolism, Viral Proteins metabolism
- Abstract
We show that the Sendai virus C' and C proteins exist as both phosphorylated and nonphosphorylated forms, while the Y1 and Y2 proteins are not phosphorylated in virus-infected cells. Phosphorylation occurs primarily on serine residues, most likely at the N-terminus of the C' and C proteins. Phosphatases PP1 and PP2A significantly modulate the phosphorylation status of the C proteins as evidenced by okadaic acid inhibition of these phosphatases. The other Sendai virus proteins including the cotranslationally expressed P protein are not necessary for the appropriate phosphorylation of the C' and C proteins. Differential phosphorylation and potential for the modulation of phosphorylation suggests regulatory functions for the C proteins.
- Published
- 1993
- Full Text
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28. Collagen-platelet interaction: separate receptor sites for types I and III collagen.
- Author
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Chiang TM, Seyer JM, and Kang AH
- Subjects
- Animals, Blood Platelets metabolism, Cell Adhesion drug effects, Humans, Integrins drug effects, Integrins immunology, Integrins metabolism, Isoantibodies immunology, Molecular Weight, Platelet Aggregation drug effects, Platelet Membrane Glycoproteins immunology, Platelet Membrane Glycoproteins metabolism, Protein Binding, Rabbits, Receptors, Collagen, Blood Platelets drug effects, Collagen pharmacology, Integrins classification, Platelet Membrane Glycoproteins classification
- Abstract
We have isolated a platelet membrane protein of M(r) 47 kDa which is responsible for the interaction of platelets with type III collagen. The 47 kDa protein was purified to apparent homogeneity by type III collagen-Sepharose 2B column chromatography and preparative slab gel electrophoreses. The 47 kDa protein blocked the adhesion of platelets to type III but not to type I collagen. Polyclonal antibodies were obtained from rabbits immunized with the purified 47 kDa protein emulsified in complete Freund's adjuvant. The polyclonal antibodies inhibited the type III collagen but not type I collagen-induced platelet aggregation. The inhibitory effect of the antibodies on type III collagen-induced platelet aggregation was dose-dependent. Cross-inhibition on platelet aggregation studies showed that type I collagen receptor antibodies (M(r) 65 kDa) did not inhibit type III collagen-induced platelet aggregation and type III collagen receptor antibodies did not inhibit type I collagen induced platelet aggregation. These results suggest that type I and type III collagens interact with platelets at separate sites.
- Published
- 1993
- Full Text
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29. Transforming growth factor beta gene expression is transiently enhanced at a critical stage during liver regeneration after CCl4 treatment.
- Author
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Armendariz-Borunda J, Katai H, Jones CM, Seyer JM, Kang AH, and Raghow R
- Subjects
- Animals, Antibodies pharmacology, Cell Division drug effects, Cells, Cultured, DNA Replication, Immunohistochemistry, In Situ Hybridization, Liver drug effects, Liver metabolism, Liver pathology, Male, RNA, Messenger analysis, RNA, Messenger metabolism, Rats, Rats, Wistar, Reference Values, Thymidine metabolism, Transcription, Genetic, Transforming Growth Factor beta immunology, Transforming Growth Factor beta physiology, Carbon Tetrachloride toxicity, Gene Expression, Liver Regeneration drug effects, Transforming Growth Factor beta biosynthesis
- Abstract
Background: Transforming growth factor beta 1 (TGF beta 1) gene expression is increased in CCl4-injured rat livers. The biologic link of this increase and liver regeneration has not been established., Experimental Design: To explore the identity of the TGF beta 1-producing cells in the CCl4 regenerating liver, we hybridized untreated and CCl4-treated liver sections of TGF beta 1-specific riboprobes and immunolocalized TGF beta 1 protein, simultaneously. To assess the dynamics of cellular proliferation during hepatic regeneration, chronologic changes in the cellular DNA synthesis were also monitored by [3H]thymidine incorporation and autoradiography. In situ hybridization analyses were further extended by subfractionation of nonparenchymal cells into Kupffer cells/macrophages, endothelial and Ito cells, and determining TGF beta 1 mRNA levels in different cell types. Finally, we experimentally tested if the temporal relationship between the transient elevation of expression of TGF beta 1 and hepatic cell proliferation were casually related., Results: We observed that the rate of DNA synthesis was the highest around 36 hours post-treatment and preceded the time of enhanced accumulation of TGF beta 1 transcripts and protein, both of which peaked at approximately 48 hours and declined thereafter. Transient upregulation of TGF beta 1 gene expression was seen in the inflammatory cell infiltrates around the central vein and at less extent, in portal tracts, and in perisinusoidal cells near the zone of necrosis. Like TGF beta 1 transcripts, TGF beta 1 protein was also predominantly co-localized in and around the pericentral and periportal cells. Kupffer cells, that accumulate abundantly in the liver 48 hours after CCl4 administration, were the primary producers of TGF beta 1. The injection of neutralizing anti-TGF beta 1 antibodies into animals prevented both the decline in [3H]thymidine incorporation and cell division in the waning phases of hepatic regeneration at 72 hours., Conclusions: Based on our observations that (i) TGF beta 1 gene expression is triggered transiently during a crucial phase of liver regeneration, (ii) the exogenously added TGF beta 1 inhibits hepatic DNA synthesis and that (iii) the administration of TGF beta 1 antibodies extends the proliferative response of the regenerating liver, we conclude that TGF beta 1 plays a pivotal role in down regulating liver regeneration.
- Published
- 1993
30. T cell epitopes of type II collagen that regulate murine collagen-induced arthritis.
- Author
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Myers LK, Seyer JM, Stuart JM, Terato K, David CS, and Kang AH
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn immunology, Autoantigens chemistry, Epitopes, Immune Tolerance, Lymphocyte Activation, Mice, Mice, Inbred Strains, Molecular Sequence Data, Arthritis immunology, Autoantigens immunology, Autoimmune Diseases immunology, Collagen immunology, T-Lymphocytes immunology
- Abstract
Chick type II collagen (CII), a protein commonly found in joint cartilage, induces an autoimmune arthritis when administered to susceptible strains of mice. A cyanogen bromide fragment of CII, CB11, contains the requisite epitopes critical for inducing collagen-induced arthritis. If administered as a tolerogen, however, before immunization, CB11 prevents the onset of disease. Therefore, delineation of structural elements of CB11 that can regulate autoreactive T cells became the goal of this study. To delineate the structural elements of CB11 antigenic to T cells, 14 peptides containing overlapping sequences of CB11 were generated. Mononuclear cells from CII-immunized DBA/1 mice were cultured with these peptides and the resulting supernatants examined for the production of IFN-gamma. Two peptides, CII 181-209 and CII 245-270, generated the greatest responses. The ability of these two peptides to regulate arthritis was tested by administering them to neonatal DBA/1 mice as tolerogens before immunization with CII. Both peptides suppressed the incidence of arthritis whereas no other peptide used as a tolerogen significantly altered the course of the disease. T cells from four arthritis-resistant murine strains did not recognize either peptide when immunized with CII, whereas cells from the disease-susceptible B10.Q mice responded well to both. Thus, the coincidence of T cell responses to CII 181-209 and CII 245-270 in CIA-susceptible mice and the lack of response in disease-resistant strains or CII-tolerized mice identify these two peptides as containing important T cell epitopes that regulate CIA.
- Published
- 1993
31. A synthetic peptide analogue of a determinant of type II collagen prevents the onset of collagen-induced arthritis.
- Author
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Myers LK, Rosloniec EF, Seyer JM, Stuart JM, and Kang AH
- Subjects
- Amino Acid Sequence, Animals, Antigen-Presenting Cells immunology, Collagen chemistry, Histocompatibility Antigens Class II immunology, Immune Tolerance, Lymphocyte Activation, Mice, Mice, Inbred DBA, Molecular Sequence Data, Peptides chemistry, Peptides immunology, Arthritis, Experimental prevention & control, Collagen immunology, T-Lymphocytes immunology
- Abstract
The immunization of genetically susceptible strains of mice with type II collagen (CII) elicits a collagen-induced arthritis that resembles rheumatoid arthritis. Our laboratory previously identified a region of CII, CII-245-270, that contains a T cell epitope that is prominent in the immune response to CII. Residues critical to the I-Aq-restricted presentation of this determinant have been characterized. To produce synthetic peptides with the potential of disrupting I-Aq-restricted Ag presentation, synthetic analogue peptides were developed that contain site-directed substitutions in critical positions. One analogue peptide was found to be an efficient competitor for binding to I-Aq and to be capable of inhibiting T cell responses in vitro. When DBA/1 mice were coimmunized with CII and the analogue peptide, the incidence and severity of arthritis were greatly reduced, concordant with the humoral immune responses to CII.
- Published
- 1993
32. A pentapeptide from type I procollagen promotes extracellular matrix production.
- Author
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Katayama K, Armendariz-Borunda J, Raghow R, Kang AH, and Seyer JM
- Subjects
- Amino Acid Sequence, Animals, Cell Line, Cells, Cultured, Chick Embryo, Extracellular Matrix drug effects, Humans, Kinetics, Molecular Sequence Data, Transforming Growth Factor beta pharmacology, Extracellular Matrix physiology, Fibronectins biosynthesis, Peptide Fragments pharmacology, Procollagen biosynthesis, Procollagen pharmacology
- Abstract
The NH2 and COOH propieces of fibril-forming collagens are cleaved off extracellularly and have been implicated in feedback regulation of their own synthesis. Recently, we showed that a subfragment of the carboxyl-terminal propeptide of type I collagen (residues 197-241) dramatically augments extracellular matrix production in subconfluent fibroblasts. This stimulation of type I collagen, type III collagen, and fibronectin production occurred in a dose- and time-dependent manner with no effect on total protein synthesis or on the ratio of secreted proteins to cell-associated proteins (Katayama, K., Seyer, J.M., Raghow, R., and Kang, A.H. (1991) Biochemistry 30, 7097-7104). In the present report, we have extensively dissected this subfragment of the propeptide and found that the pentapeptide Lys-Thr-Thr-Lys-Ser (residues 212-216) is the minimum sequence necessary for potent stimulation of collagen and fibronectin production in a variety of mesenchymal cells. We postulate that the extracellular matrix production in fibroblasts may be subject to either positive or negative feedback regulation depending on the repertoire of specific proteases during postinflammatory tissue regeneration and fibrosis.
- Published
- 1993
33. Intercellular adhesion molecule-1 expression induced by interleukin (IL)-1 beta or an IL-1 beta fragment is blocked by an IL-1 receptor antagonist and a soluble IL-1 receptor.
- Author
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Hong L, Imeri L, Opp MR, Postlethwaite AE, Seyer JM, and Krueger JM
- Subjects
- Amino Acid Sequence, HLA-DR Antigens analysis, Humans, Intercellular Adhesion Molecule-1, Interferon-gamma pharmacology, Interleukin 1 Receptor Antagonist Protein, Molecular Sequence Data, Neuroblastoma chemistry, Recombinant Proteins, Tumor Cells, Cultured, Tumor Necrosis Factor-alpha pharmacology, Cell Adhesion Molecules analysis, Interleukin-1 pharmacology, Peptide Fragments pharmacology, Receptors, Interleukin-1 physiology, Sialoglycoproteins pharmacology
- Abstract
The effects of a recombinant human interleukin-1 (IL-1) receptor antagonist (IL-1ra) and a recombinant human soluble IL-1 receptor (sIL-1R) on cytokine-induced intercellular adhesion molecule-1 (ICAM-1) expression in a human glioblastoma cell line and a neuroblastoma cell line were determined. Cells were incubated with IL-1 beta, tumor necrosis factor (TNF) alpha and interferon (IFN) gamma. Cells were also tested under identical conditions with an IL-1 beta synthetic peptide fragment (IL-1 beta 208-240) previously shown to possess biological activity. IL-1 beta, TNF alpha and IFN gamma potentiated ICAM-1 expression in both cell lines in a dose-related manner. The IL-1 beta 208-240 fragments, corresponding to the rabbit, rat and human sequences, enhanced ICAM-1 expression in glioblastoma cells at high doses. ICAM-1 expression induced by IL-1 beta, rabbit IL-1 beta 208-240 and human IL-1 beta 208-240 was blocked by the IL-1ra, while TNF alpha- and IFN gamma-induced ICAM-1 expression were not. ICAM-1 expression induced by IL-1 beta and human IL-1 beta 208-240 was also blocked by the sIL-1R. Our findings suggest that IL1 beta 208-240 acts as an IL-1 beta agonist in enhancing ICAM-1 expression in vitro and that this effect is receptor-mediated.
- Published
- 1993
- Full Text
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34. Susceptibility of type I collagen containing mutated alpha 1(1) chains to cleavage by human neutrophil collagenase.
- Author
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Hasty KA, Wu H, Byrne M, Goldring MB, Seyer JM, Jaenisch R, Krane SM, and Mainardi CL
- Subjects
- Amino Acid Sequence, Animals, Collagen metabolism, Collagenases chemistry, Humans, Matrix Metalloproteinase 8, Mice, Molecular Sequence Data, Mutagenesis, Site-Directed, Sequence Alignment, Sequence Homology, Amino Acid, Substrate Specificity, Collagen genetics, Collagenases metabolism, Neutrophils enzymology
- Abstract
Two members of the matrix metalloproteinase family which can cleave native types I, II and III triple helical collagens are collagenases from fibroblasts and neutrophils. These enzymes are the products of different genes which share structural motifs but are only 57% identical. In this study, we determined the site of cleavage in the alpha 1(I) chains and showed that the neutrophil collagenase acted at the same site as the fibroblast collagenase. We also used collagens as substrates which were generated by site-directed mutagenesis of the murine Col1a1 gene and found that the pattern of susceptibility to cleavage by purified neutrophil collagenase was indistinguishable from that previously described for the fibroblast collagenase. Collagens containing substitutions of Pro for Ile-776 (P1) were not cleaved; whereas those containing substitutions of Met for Ile-776 were cleaved. Type I collagen which contained alpha 1(I) chains in which there were double substitutions of Pro for Gln-774 (P2) and Ala-777 (P2') were also not cleaved. These type I collagens contained wild type alpha 2(I) chains as well as mutant alpha 1(I) chains in the mixed helical trimers; the alpha 2(I) chain in the trimers containing the resistant alpha 1(I) chains were also not cleaved by the neutrophil collagenase.
- Published
- 1993
- Full Text
- View/download PDF
35. Identification of vicinal thiols of phosphoenolpyruvate carboxykinase (GTP).
- Author
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Lewis CT, Seyer JM, Cassell RG, and Carlson GM
- Subjects
- Alkylation, Amino Acid Sequence, Animals, Azides pharmacology, Chromatography, High Pressure Liquid, Cysteine analysis, Cysteine chemistry, Cystine analysis, Cystine chemistry, Cytosol enzymology, Disulfides chemistry, Dithionitrobenzoic Acid pharmacology, Guanosine Triphosphate analogs & derivatives, Guanosine Triphosphate pharmacology, Iodoacetates, Iodoacetic Acid, Molecular Sequence Data, Peptide Fragments chemistry, Peptide Fragments isolation & purification, Phosphoenolpyruvate Carboxykinase (GTP) chemistry, Phosphoenolpyruvate Carboxykinase (GTP) radiation effects, Rats, Trypsin metabolism, Liver enzymology, Phosphoenolpyruvate Carboxykinase (GTP) analysis, Sulfhydryl Compounds analysis
- Abstract
Phosphoenolpyruvate carboxykinase (PEPCK) from the cytosol of rat liver has 13 cysteines, at least one of which (Cys288) is known to be very reactive and critical for catalytic activity (Lewis, C. T., Seyer, J. M., and Carlson, G. M. (1989) J. Biol. Chem. 264, 27-33). Previous results provided evidence for the existence of at least 1 pair of vicinal cysteines within or near the active site of PEPCK (Lewis, C. T., Haley, B. E., and Carlson, G. M. (1989) Biochemistry 28, 9248-9255). An intramolecular cystine disulfide is induced to form upon treatment of PEPCK with equimolar 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) or upon irradiation of the enzyme in the presence of the photoaffinity probe 8-azidoGTP. In each case, modification is accompanied by a substantial loss in catalytic activity, and substrates protect against inactivation and modification. We now report the identification of these modified thiols by differential alkylation of cysteines and half-cystines with radioactive iodoacetate, followed by isolation and sequencing of the modified tryptic peptides. The results indicate that the disulfide formed by equimolar Nbs2 lies within a 15-residue region of the PEPCK sequence that includes Cys399, Cys407, and Cys413. In addition, Cys407 and/or Cys413 also appear to participate in formation of the disulfide induced by 8-azidoGTP. These thiols lie very near a consensus sequence that has been suggested to represent the binding site for the guanine ring of GTP.
- Published
- 1993
36. Isolation, characterization and immunolocalization of a 53-kDal dentin sialoprotein (DSP).
- Author
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Butler WT, Bhown M, Brunn JC, D'Souza RN, Farach-Carson MC, Happonen RP, Schrohenloher RE, Seyer JM, Somerman MJ, and Foster RA
- Subjects
- Amino Acid Sequence, Animals, Blotting, Western, Chromatography, Gel, Electrophoresis, Polyacrylamide Gel, Extracellular Matrix Proteins, Immunohistochemistry, Molecular Sequence Data, Molecular Weight, Phosphoproteins, Protein Precursors, Rats, Sialoglycoproteins chemistry, Sialoglycoproteins metabolism, Tissue Distribution, Sialoglycoproteins isolation & purification
- Abstract
We isolated a sialic-rich protein from rat dentin extracts and have named it dentin sialoprotein, DSP (formerly called 95K glycoprotein). DSP is rich in aspartic acid, glutamic acid, glycine and serine, but contains no cysteine or phosphate. The 30% carbohydrate content includes about 9% sialic acid and indicates that several N-glycosides and O-glycosides are present. Sedimentation equilibrium analysis gave a M(r) of 52,570. Based on this molecular weight we calculated that DSP contains about 350-amino acids and 75 monosaccharides. With automated Edman degradation the sequence of the first 8-amino acids was shown to be: Ile-Pro-Val-Pro-Gln-Leu-Val-Pro. The initial 3 residues of this sequence are identical to the first 3 in human osteopontin (OPN) and are closely similar to the Leu-Pro-Val sequences of OPN from other species, as well as at the beginning of bone acidic glycoprotein-75 (BAG-75). On Western immunoblots, purified polyclonal antibodies reacted only with DSP in dentin extracts and with none of the proteins from bone. Similarly, immunolocalization experiments showed the presence of DSP in dentin but not in enamel or alveolar bone. Along with immunohistochemical localization data reported elsewhere, these observations suggest that DSP may be an important marker for cells in the odontoblast lineage.
- Published
- 1992
- Full Text
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37. Somnogenic, pyrogenic, and anorectic activities of tumor necrosis factor-alpha and TNF-alpha fragments.
- Author
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Kapás L, Hong L, Cady AB, Opp MR, Postlethwaite AE, Seyer JM, and Krueger JM
- Subjects
- Animals, Body Temperature drug effects, Brain drug effects, Brain physiology, Cell Adhesion Molecules analysis, Cell Adhesion Molecules chemistry, Cell Adhesion Molecules immunology, Eating drug effects, HLA-DR Antigens analysis, Intercellular Adhesion Molecule-1, Male, Rabbits, Rats, Rats, Sprague-Dawley, Tumor Cells, Cultured, Anorexia chemically induced, Fever chemically induced, Peptide Fragments pharmacology, Sleep drug effects, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Exogenously administered tumor necrosis factor-alpha (TNF-alpha) elicits several symptoms of generalized infections such as fever, increased sleep, and anorexia. The aim of the present work was to localize these effects of TNF-alpha to specific amino acid sequences of the parent molecule by characterizing the in vivo and in vitro activities of several synthetic TNF-alpha fragments. Intracerebroventricular injection of TNF-alpha elicited dose-dependent fevers and increases in non-rapid-eye-movement sleep (NREMS) in rabbits. Four fragments also promoted NREMS and five elicited monophasic fevers. All of the somnogenic fragments share the amino acid sequence 31-36. In rats, TNF-alpha and one of the fragments [TNF-alpha-(69-100)] suppressed 12-h food intake. Furthermore, TNF-alpha increased the expression of the intercellular adhesion molecule-1 and enhanced interferon-gamma-induced HLA-DR expression in human glioblastoma cell line. In contrast, none of the fragments possessed these in vitro activities. Our in vivo results support the concept that there are biologically active regions in the TNF-alpha molecule.
- Published
- 1992
- Full Text
- View/download PDF
38. Characterization of a tolerogenic T cell epitope of type II collagen and its relevance to collagen-induced arthritis.
- Author
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Myers LK, Terato K, Seyer JM, Stuart JM, and Kang AH
- Subjects
- Amino Acid Sequence, Animals, Animals, Newborn, Collagen chemistry, Epitopes, Mice, Molecular Sequence Data, Peptides chemistry, Peptides immunology, Arthritis immunology, Collagen immunology, Immune Tolerance, T-Lymphocytes immunology
- Abstract
A synthetic peptide representing sequences of type II collagen, (CII 245-270), has previously been used to induce tolerance and suppress arthritis in DBA/1 mice. To determine important residues, a series of peptides, each containing one or two site-directed substitutions, was generated. Mononuclear cells from DBA/1 mice immunized with CII were cultured in the presence of each peptide and the T cell response determined by measuring IFN-gamma in culture supernatant fluids. Substitutions within the region CII 260-270 led to significant decreases in IFN-gamma responses, identifying this sequence as a T cell epitope. To determine the effects of substitutions within this epitope on arthritis, substituted peptides were administered to neonatal mice as tolerogens. Five site-directed substitutions, four of which included the insertion of a residue found in type I collagen to replace its type II counterpart, abrogated the ability of the peptides to induce tolerance and suppress arthritis. These substitutions were located at residues 260, 261, 263, 264, and 266. Two patterns of T cell reactivity were observed. Peptides containing individual substitutions at positions 261, 264, or 266 were capable of generating a significant T lymphokine response, although those containing substitutions at residues 260 or 263 were ineffective Ag. Systematic analysis of the fine structures of T cell determinants important for autoimmune arthritis can lead to strategies for therapeutic intervention.
- Published
- 1992
39. Transcriptional mechanisms of type I collagen gene expression are differentially regulated by interleukin-1 beta, tumor necrosis factor alpha, and transforming growth factor beta in Ito cells.
- Author
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Armendariz-Borunda J, Katayama K, and Seyer JM
- Subjects
- Animals, Blotting, Northern, Cell Nucleus drug effects, Cell Nucleus physiology, Cells, Cultured, Collagen biosynthesis, Extracellular Matrix drug effects, Extracellular Matrix physiology, Liver cytology, Liver drug effects, Poly A genetics, Poly A isolation & purification, Proline metabolism, RNA, Messenger genetics, RNA, Messenger isolation & purification, RNA, Messenger metabolism, Rats, Rats, Inbred Strains, Collagen genetics, Gene Expression Regulation drug effects, Interleukin-1 pharmacology, Liver physiology, Transcription, Genetic drug effects, Transforming Growth Factor beta pharmacology, Tumor Necrosis Factor-alpha pharmacology
- Abstract
Regulation of the procollagen type I (Pro alpha 1) gene in cultured Ito cells by diverse cytokines was studied. Specifically, we have examined the effect of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), and transforming growth factor beta (TGF beta) on collagen biosynthesis, levels of Pro alpha 1 (I) mRNA, and rate of transcription of Pro alpha 1 (I) gene. TGF beta stimulated procollagen synthesis at least 2-fold at every concentration tested (5-20 ng/ml), whereas TNF alpha inhibited it at the same concentrations. In contrast to what occurs in dermal fibroblasts, IL-1 beta (5-20 units/ml) preferentially inhibited procollagen production as measured by [3H]proline incorporation. A similar pattern was obtained when total protein synthesis was analyzed by [25S]methionine radiolabeling. Interestingly, while TGF beta-treated cells exhibited greater than 3-fold increase in steady-state levels of Pro alpha 1 (I) mRNA, the treatment with IL-1 had no effect on procollagen mRNA levels. TNF alpha treatment resulted in a 2-fold decrease in the amount of collagen mRNA. The treatment with combinations of cytokines indicated that collagen gene expression in Ito cells is differentially regulated by these cytokines. Furthermore, nuclear run-off transcription experiments were performed. The results obtained suggest that TGF beta regulates increasing collagen type I gene expression at transcriptional levels, and TNF alpha inhibits the transcriptional rate of Pro alpha 1 (I) gene. It is noteworthy that IL-1 beta acts on collagen type I gene regulation by a separate mechanism at a posttranscriptional level.
- Published
- 1992
40. Photochemical cross-linking of guanosine 5'-triphosphate to phosphoenolpyruvate carboxykinase (GTP).
- Author
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Lewis CT, Seyer JM, and Carlson GM
- Subjects
- Adenosine Monophosphate chemistry, Amino Acid Sequence, Chromatography, High Pressure Liquid, Cross-Linking Reagents, Guanosine Diphosphate chemistry, Inosine Triphosphate chemistry, Molecular Sequence Data, Serine Endopeptidases metabolism, Guanosine Triphosphate chemistry, Phosphoenolpyruvate Carboxykinase (GTP) chemistry
- Abstract
Mammalian phosphoenolpyruvate carboxykinase (PEPCK) specifically requires a guanosine or inosine nucleotide as a substrate; however, the structural basis for this nucleotide specificity is not yet known. Because affinity labels derived from guanosine have not yielded a stable, modified peptide in quantities sufficient for sequence analysis, we have investigated the utility of direct photochemical cross-linking of GTP to PEPCK in order to identify the nucleotide binding site. UV irradiation at a distance of 2 cm by a Mineralight lamp (330 microW/cm2) results in the attachment of [alpha-32P]GTP to PEPCK via a stable, covalent linkage in a reaction that is dependent upon GTP concentration and duration of irradiation. After 10 min of irradiation, more than 0.2 mol of [alpha-32P] GTP is incorporated per mole of PEPCK; under these conditions the GTP concentration required for half-maximal labeling is 69 microM. The substrates phosphoenolpyruvate, ITP, and GDP provide protection against photolabeling, as do Mn2+ and Mg2+. One major and one minor radioactive peptide derived from proteolytic digests of photolabeled PEPCK have been isolated and identified. The major modified peptide has been provisionally assigned to an acidic region near the C-terminus, and the minor peptide has been identified as Ser462-Lys471.
- Published
- 1992
- Full Text
- View/download PDF
41. Further characterization of the loop structure of platelet glycoprotein IIIa: partial mapping of functionally significant glycoprotein IIIa epitopes.
- Author
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Kouns WC, Newman PJ, Puckett KJ, Miller AA, Wall CD, Fox CF, Seyer JM, and Jennings LK
- Subjects
- Amino Acid Sequence, Antibodies, Monoclonal, Blood Platelets chemistry, Blotting, Western, Chymotrypsin metabolism, Epitopes chemistry, Flow Cytometry, Humans, Immunoblotting, Kinetics, Molecular Sequence Data, Molecular Weight, Peptide Fragments chemistry, Peptide Fragments metabolism, Peptide Mapping, Platelet Membrane Glycoproteins immunology, Platelet Membrane Glycoproteins metabolism, Protein Conformation, Platelet Membrane Glycoproteins chemistry
- Abstract
Glycoprotein (GP) IIb-IIIa serves as the platelet fibrinogen receptor. Studies of the tertiary structure of GPIIIa have shown that the protein has a large loop structure of at least 325 amino acids in length. To further characterize this loop structure, intact platelets were digested with alpha-chymotrypsin. Digestion products were examined using the anti-GPIIIa monoclonal antibodies (MoAbs) AP3, D3GP3, and C5GP3, as well as the human alloantibody, anti-PLA1. AP3 recognized GPIIIa digestion products of 109, 95, and 68 Kd. D3GP3 and C5GP3 recognized an additional band of 51 Kd. Time course digestions demonstrated that the 51-Kd fragment was generated by proteolysis of the 68-Kd peptide. Sequence analysis of the reduced 51-Kd peptide showed that this fragment began at amino acid 422. The nonreduced 51-Kd peptide was reactive with antibodies directed against the first 13 amino acids of GPIIIa, demonstrating the presence of a covalently attached N-terminal peptide. These data suggest that: (1) the minimum length of the loop structure is at least 384 amino acids; (2) the AP3 epitope is formed at least in part by a determinant contained within residues 348 to 421; and (3) the D3GP3 and C5GP3 epitopes are contained within amino acids 422 to 692 of GPIIIa, a region that may be flexible and involved in conformational changes that occur after ligand binding.
- Published
- 1991
42. Kupffer cells from carbon tetrachloride-injured rat livers produce chemotactic factors for fibroblasts and monocytes: the role of tumor necrosis factor-alpha.
- Author
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Armendariz-Borunda J, Seyer JM, Postlethwaite AE, and Kang AH
- Subjects
- Animals, Antibodies physiology, Carbon Tetrachloride, Cells, Cultured, Chemical and Drug Induced Liver Injury, Chemotactic Factors antagonists & inhibitors, Chemotactic Factors isolation & purification, Culture Media, Liver Diseases pathology, Lymphotoxin-alpha immunology, Rats, Tumor Necrosis Factor-alpha immunology, Tumor Necrosis Factor-alpha physiology, Chemotactic Factors biosynthesis, Fibroblasts physiology, Kupffer Cells metabolism, Liver metabolism, Liver Diseases metabolism, Monocytes physiology
- Abstract
Conditioned media from cultured Kupffer and mononuclear macrophagic cells obtained 48 hr after CCl4 administration to rats contains chemotactic factors for human skin fibroblasts and human monocytes. The chemotactic mediator for fibroblasts was approximately 17 kD and was more prominent at early stages of culture. It induced a dose-dependent chemotactic response in fibroblasts. Although the conditioned medium from cultured Kupffer cells of normal rats also contained detectable biological activity, it was significantly less than that in conditioned medium from cultured Kupffer cells from CCl4-treated rats. The activity obtained after purification by high-performance liquid chromatography was completely ablated by incubation with tumor necrosis factor-alpha antibody. Transforming growth factor-beta antibody diminished biological activity by 20%. Human recombinant tumor necrosis factor-alpha and transforming growth factor-beta used in the assay as control showed significant chemotactic activity. The chemotactic activity present in whole normal conditioned medium was only present after 24 and 48 hr of culture. Furthermore, this activity was not neutralized by human recombinant tumor necrosis factor-alpha or transforming growth factor-beta antibodies. Incubation of whole 6-hr conditioned medium with human recombinant tumor necrosis factor-alpha and transforming growth factor-beta antibodies demonstrated and confirmed that tumor necrosis factor-alpha plays a major role in inducing the chemotactic response. On acidification of this supernatant, we found a notable increase in the biological response that could be neutralized by transforming growth factor-beta antibody. Thus tumor necrosis factor-alpha and transforming growth factor-beta may sequentially provide important signals for fibroblast and monocyte recruitment in vivo at initial stages of liver injury.
- Published
- 1991
- Full Text
- View/download PDF
43. Processing of TCP pilin by TcpJ typifies a common step intrinsic to a newly recognized pathway of extracellular protein secretion by gram-negative bacteria.
- Author
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Kaufman MR, Seyer JM, and Taylor RK
- Subjects
- Amino Acid Sequence, Base Sequence, Blotting, Southern, Cloning, Molecular, DNA genetics, DNA isolation & purification, Endopeptidases metabolism, Escherichia coli genetics, Genetic Complementation Test, Models, Structural, Molecular Sequence Data, Mutagenesis, Insertional, Protein Conformation, Protein Sorting Signals metabolism, Restriction Mapping, Sequence Homology, Nucleic Acid, Vibrio cholerae ultrastructure, Fimbriae, Bacterial metabolism, Genes, Bacterial, Membrane Proteins, Serine Endopeptidases, Vibrio cholerae genetics
- Abstract
Biogenesis of the Vibrio cholerae toxin-coregulated pilus (TCP) requires the activities of at least seven accessory proteins. We demonstrate that a portion of this pathway involves a novel processing step in which a hydrophilic leader peptide is proteolytically removed from TcpA by the gene product characterized in this report, TcpJ, to yield the mature, export-competent form of the pilin. Cleavage of the pilin leader peptide is independent of known signal peptidases as demonstrated by pilin-processing profiles in Escherichia coli strains conditionally defective for production of leader peptidase or grown in the presence of the antibiotic globomycin. Additionally, pilin cleavage did not rely on the SecA protein, as evidenced by TcpA processing in azide-treated cells. These results suggest that TcpJ is representative of a new class of proteins involved in SecA-independent proteolytic cleavage of a set of atypical leader peptides during extracellular export.
- Published
- 1991
- Full Text
- View/download PDF
44. Regulation of extracellular matrix production by chemically synthesized subfragments of type I collagen carboxy propeptide.
- Author
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Katayama K, Seyer JM, Raghow R, and Kang AH
- Subjects
- Amino Acid Sequence, Cells, Cultured, Collagen chemical synthesis, DNA biosynthesis, Electrophoresis, Polyacrylamide Gel, Extracellular Matrix Proteins biosynthesis, Fibronectins analysis, Humans, Lung metabolism, Molecular Sequence Data, Peptide Fragments chemical synthesis, Peptide Fragments physiology, Procollagen chemical synthesis, RNA Processing, Post-Transcriptional, RNA, Messenger analysis, Sequence Homology, Nucleic Acid, Collagen physiology, Extracellular Matrix metabolism, Procollagen physiology
- Abstract
The complete COOH-propeptide of human alpha 1(I) procollagen was chemically synthesized as a series of overlapping subfragments which were then tested for their effect on extracellular matrix protein production by subconfluent human lung fibroblasts (HFL-1). One peptide (R11; residues 197-241) stimulated production of both collagen and fibronectin by 6-8-fold while a second peptide with a partial overlap with R11 (R9; residues 182-216) enhanced collagen accumulation. The peptide R12 (residues 197-216), which has a sequence common to both R9 and R11, also stimulated collagen production, suggesting that this 20-residues peptide alone contains the required structure for activity. The other synthetic peptides, R1-R13, were inactive in their ability to alter collagen or fibronectin production. Consistent with previously published data, the COOH-terminal peptide, R14, inhibited extracellular matrix production [Aycock, R.A., Raghow, R., Stricklin, G.P., Seyer, J.M., & Kang, A.H. (1986) J. Biol. Chem. 261, 14355-14360]. Both R9 and R11 preferentially stimulated production of collagen types I and III and fibronectin in dose-dependent manner. Elevated collagen and fibronectin production was evident at 4-h posttreatment, and maximal enhancement was seen at 8 h after exposure to peptides. Interestingly, subconfluent cultures of HFL-1 fibroblasts responded vigorously to the stimulatory action of R9 and R11 while confluent cells failed to show any response. Steady-state levels of messenger RNAs encoding type I procollagen and fibronectin were not measurably altered by treatment with R9 or R11, suggesting that the regulation of procollagens and fibronectin by these peptides involves posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)
- Published
- 1991
- Full Text
- View/download PDF
45. Immunity to type XI collagen in mice. Evidence that the alpha 3(XI) chain of type XI collagen and the alpha 1(II) chain of type II collagen share arthritogenic determinants and induce arthritis in DBA/1 mice.
- Author
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Cremer MA, Terato K, Seyer JM, Watson WC, O'Hagan GO, Townes AS, and Kang AH
- Subjects
- Animals, Antibodies analysis, Female, Hypersensitivity, Delayed, Immunization, Immunoglobulin G analysis, Male, Mice, Mice, Inbred DBA, Arthritis etiology, Collagen immunology, Epitopes analysis
- Abstract
To determine whether native bovine type XI collagen (BXI) is arthritogenic, five strains of inbred mice were immunized with BXI/CFA. Arthritis was not observed in any of these strains, though it was prevalent in DBA/1 and B10.RIII controls immunized with bovine type II collagen (BII). Antisera from BXI-immunized mice reacted with mouse type XI collagen (MsXI), weakly with the alpha-chains of BXI, and minimally with mouse type II collagen (MsII). However, antisera to BII reacted with MsII and MsXI, indicating antibodies to conformation-independent epitopes shared by alpha 1(II) and alpha 3(XI). Mice immunized with BXI containing a small amount of BII developed arthritis much like those immunized with BII; sera from these mice reacted with MsXI and MsII. Delayed-type hypersensitivity responses differed from IgG responses, i.e., BXI elicited responses to alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II); BII, to alpha 3(XI) and alpha 1(II) exclusively. To determine whether alpha 1(XI), alpha 2(XI), alpha 3(XI), and alpha 1(II) are arthritogenic, DBA/1J mice were immunized with each alpha-chain. Arthritis was seen in mice injected with alpha 3(XI) or alpha 1(II). Sera to both alpha-chains reacted similarly with MsII and peptide fragment alpha 1(II)-CB11. Epitope mapping using polyclonal and mAb to type II collagen revealed that all polyclonal and 11 of 14 mAb reacted with alpha 3(XI) and alpha 1(II), whereas three mAb reacted only with alpha 1(II). In conclusion, BXI is immunogenic but not arthritogenic in five strains of mice, whereas alpha 3(XI) and alpha 1(II) are arthritogenic and immunogenic in DBA/1 mice and share greater than or equal to 11 epitopes recognized by autoantibody.
- Published
- 1991
46. cDNA cloning and expression of platelet p24/CD9. Evidence for a new family of multiple membrane-spanning proteins.
- Author
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Lanza F, Wolf D, Fox CF, Kieffer N, Seyer JM, Fried VA, Coughlin SR, Phillips DR, and Jennings LK
- Subjects
- Amino Acid Sequence, Animals, Base Sequence, Cricetinae, Cricetulus, Electrophoresis, Polyacrylamide Gel, Female, Gene Expression Regulation, Humans, Molecular Sequence Data, Open Reading Frames, Polymerase Chain Reaction, Precipitin Tests, Restriction Mapping, Sequence Alignment, Tetraspanin 29, Transcription, Genetic, Xenopus, Antigens, CD genetics, Antigens, Differentiation genetics, Blood Platelets metabolism, DNA genetics, Membrane Glycoproteins
- Abstract
This study was designed to clone, sequence, and express the full-length cDNA for the human platelet p24/CD9 antigen. A 1.3-kilobase cDNA clone was identified that has an open reading frame encoding a mature protein of 228 amino acids (approximately 25,400 Da) containing 10 cysteine residues and four putative transmembrane domains. The identity of the clone was confirmed by: (i) its predicted size, (ii) identity to four peptide sequences from the isolated protein including the NH2 terminus, and (iii) expression of the isolated clone in Xenopus oocytes and Chinese hamster ovary cells. p24/CD9 has sequence identity (24-34%) to four other cell-surface proteins: ME491, a melanoma antigen; CO-029, a carcinoma antigen; CD37, a leukocyte antigen; and SM23, an antigen of the parasitic helminth Schistosoma mansoni. The five proteins have a similar number of amino acids and are characterized by the presence of four putative transmembrane domains. These data indicate the presence of a new family of surface antigens that may function in cellular activation and differentiation.
- Published
- 1991
47. Fibroblast chemotaxis induction by human recombinant interleukin-4. Identification by synthetic peptide analysis of two chemotactic domains residing in amino acid sequences 70-88 and 89-122.
- Author
-
Postlethwaite AE and Seyer JM
- Subjects
- Amino Acid Sequence, Antibodies, Humans, In Vitro Techniques, Molecular Sequence Data, Peptides chemistry, Peptides pharmacology, Receptors, Interleukin-4, Receptors, Mitogen physiology, Recombinant Proteins, Structure-Activity Relationship, Chemotaxis, Fibroblasts physiology, Interleukin-4 physiology
- Abstract
Interleukin-4 is a T lymphocyte- and mast cell-derived cytokine with pleiotropic properties with biological effects on a variety of target cells including B and T lymphocytes, macrophages, hematopoietic cells, mast cells, and fibroblasts. In addition to the proliferation effect of IL-4 on fibroblasts, which has been previously described, in this report the chemotactic properties of IL-4 for fibroblasts is described. Human recombinant IL-4 induced the chemotactic migration of dermal fibroblasts in vitro in modified Boyden-type chambers at concentrations between 10(-12) and 10(-11) M. The chemotactic activity of IL-4 was neutralized by anti-human recombinant IL-4 IgG antibodies. Oligopeptides representing the complete deduced amino acid sequence of human IL-4 were synthesized by the Merrifield technique and tested for their ability to induce fibroblast chemotaxis. Two peptides representing residues 70-88 and 89-122 induced fibroblast migration. Peptide 70-88 was the more potent of the two causing chemotaxis of fibroblasts at 10(-8)-10(-6) M while peptide 89-129 induced migration at 10(-7)-10(-5) M. Although the mechanism by which IL-4 and these two peptides induce fibroblast chemotaxis is unknown, each of these three compounds were able to chemotactically desensitize fibroblasts to the chemotactic effects of the other two but not to a structurally unrelated chemotactic cytokine, transforming growth factor beta-1. These studies suggest that IL-4 might function in vivo to induce the accumulation of fibroblasts at sites of tissue injury, inflammatory and immune reactions in which T lymphocytes and mast cells participate.
- Published
- 1991
- Full Text
- View/download PDF
48. Identification of the rat bone 60K acidic glycoprotein as alpha 2HS-glycoprotein.
- Author
-
Mizuno M, Farach-Carson MC, Pinero GJ, Fujisawa R, Brunn JC, Seyer JM, Bousfield GR, Mark MP, and Butler WT
- Subjects
- Amino Acid Sequence, Animals, Blood Proteins chemistry, Blood Proteins isolation & purification, Blotting, Western, Electrophoresis, Polyacrylamide Gel, Femur chemistry, Fluorescent Antibody Technique, Isoelectric Point, Kidney chemistry, Liver chemistry, Molecular Sequence Data, Molecular Weight, Proteins analysis, Rats, Tibia chemistry, alpha-2-HS-Glycoprotein, Blood Proteins analysis, Bone and Bones chemistry
- Abstract
Previous reports have described an Mr 60,000-64,000 glycoprotein present in guanidium chloride (GdmCl)/EDTA extracts of bovine and rat bone. We have purified this protein from the long bones of rats and have raised polyclonal antibodies to the purified protein. The 60K glycoprotein has amino acid and carbohydrate compositions that are similar to those reported for the 60-64K protein(s). Several lines of evidence indicate that the 60K bone glycoprotein is the rat homologue of human alpha 2HS-glycoprotein. First, immunochemical data demonstrated that the 60K bone glycoprotein was present in serum as well as in EDTA/GdmCl extracts of bone. Second, immunolocalization and metabolic labelling experiments showed that the 60K protein is synthesized in liver and not in bone cells, although it is sequestered in vascularized regions of bone matrix. Finally, the NH2-terminal sequence for the rat 60K bone glycoprotein was highly similar to that of the human alpha 2HS-glycoprotein A chain. A surprising finding was that small amounts of contaminating 60K/alpha 2HS-glycoprotein were found in several protein fractions purified by ion-exchange chromatography of bone EDTA/GdmCl extracts. Because this protein was found to be highly immunogenic, the presence of anti-60K antibodies in anti-sera prepared against purified bone proteins should be considered as a potential problem.
- Published
- 1991
- Full Text
- View/download PDF
49. Genomic organization of the human procollagen alpha 1(II) collagen gene.
- Author
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Huang MC, Seyer JM, Thompson JP, Spinella DG, Cheah KS, and Kang AH
- Subjects
- Base Sequence, Cloning, Molecular, Collagen genetics, Exons, Genetic Vectors, Humans, Introns, Molecular Sequence Data, Plasmids, Restriction Mapping, Genes, Procollagen genetics
- Abstract
The nucleotide sequence of the human procollagen alpha 1(II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N-propeptide and part of the triple-helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N-propeptide domain. Although the genomic structure of the human procollagen alpha 1(II) gene is quite different from the rat procollagen alpha 1(II) gene, the nucleotide coding sequences are 89% identical.
- Published
- 1991
- Full Text
- View/download PDF
50. Increase in the number of atrial natriuretic hormone receptors in regenerating rat liver.
- Author
-
Nair BG, Steinke L, Yu YM, Rashed HM, Seyer JM, and Patel TB
- Subjects
- Animals, Atrial Natriuretic Factor metabolism, Atrial Natriuretic Factor pharmacology, Cell Membrane metabolism, Guanylate Cyclase metabolism, Hepatectomy, Kinetics, Male, Rats, Rats, Inbred Strains, Receptors, Atrial Natriuretic Factor, Reference Values, Liver metabolism, Liver Regeneration, Receptors, Cell Surface metabolism
- Abstract
Forty-eight hours after partial (approximately 67%) hepatectomy the activity of the particulate guanylate cyclase was increased by 2-fold in the regenerating rat liver. This increase was not an artifact of membrane isolation procedures, and as determined by 125I-labeled Tyr-28 atrial natriuretic hormone-(1-28) ANF binding, was accompanied by a 2-fold increase in the number of ANF receptors. The Kd of the receptors in membranes of regenerating livers was not significantly different from the Kd of the receptors in livers of sham-operated rats. The linear synthetic descysteine analog of ANF, analog I, which binds only to the 66-kDa receptors, displaced approximately 40% of the specifically bound 125I-ANF in liver membranes from both hepatectomized and sham-operated (control) animals. Affinity cross-linking studies with 125I-ANF confirmed the increase in the 116-kDa ANF receptor in membranes of regenerating livers. In perfused livers derived from control and hepatectomized animals, the basal rates of cGMP production were not significantly different. However, atriopeptin II-stimulated cGMP production was twice as great in regenerating livers as compared with controls. These data demonstrate that the increase in particulate guanylate cyclase activity observed during liver regeneration is due to an increase in the 116-kDa ANF receptor-associated activity. Additionally, our data demonstrate that the regenerating rat liver may be a valuable model with which to study the role of the hepatic ANF receptor/particulate guanylate cyclase.
- Published
- 1991
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