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Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19+ purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.

Mantle-cell lymphoma (MCL) is a unique subtype of B-cell non-Hodgkin lymphoma (NHL) that behaves aggressively and remains incurable. In order to understand the pathogenesis of MCL and design new therapies, it is important to accurately analyze molecular changes in pathways dysregulated in MCL. We used antibody microarrays to compare patterns of protein expression between CD19+purified B lymphocytes from normal tonsil and 7 cases of histologically confirmed MCL. Protein overexpression was defined as a higher than 1.3-fold or 2-fold increase in at least 67% of tumor samples compared with normal B-cell control. Of the polypeptides, 77 were overexpressed using the higher than 1.3-fold cutoff, and 13 were overexpressed using the 2-fold cutoff. These included cell cycle regulators (regulator of chromosome condensation 1 [RCC1], murine double minute 2 [MDM2]), a kinase (citron Rho-interacting kinase [CRIK]), chaperone proteins (heat shock 90-kDa protein [Hsp90], Hsp10), and phosphatase regulators (A-kinase anchor protein 1 [AKAP149], protein phosphatase 5 [PP5], and inhibitor 2). The elevated expression of some of these polypeptides was confirmed by immunoblotting and immunohistochemistry, whereas elevated expression of others could not be confirmed, illustrating the importance of confirmatory studies. This study describes a novel technique that identifies proteins dysregulated in MCL.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and G-globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89–0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85–0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

Synovial fluid (SF) is a dynamic reservoir for proteins originating from serum, synovial tissue, and cartilage. The composition of the SF proteome may reflect the pathophysiological conditions affecting the circulatory system and cartilage. Our long-term goal is to identify reliable protein markers for osteoarthritis (OA) in SF. We first evaluated the pattern of SF proteins on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as a function of protein loading, pH range for isoelectric focusing, and concentration of acrylamide in SDS-PAGE. Removal of albumin and γ- globulins from the samples did not improve the detection of protein spots on 2D-PAGE. The repeatability of protein spot intensity was tested by triplicate 2D-PAGE of a given sample; these experiments showed low intrasample variability (correlation coefficients 0.89–0.95). Differences between multiple samples were tested by comparing the 2D-PAGE of four samples. These experiments showed slightly greater variation between samples (correlation coefficients 0.85–0.93) and a number of differentially expressed proteins. The intensity of 18 protein spots differed more than fivefold, and the intensity of nine protein spots differed more than 100-fold. These results show that 2D-PAGE can be used under standard conditions to screen SF samples and identify a small subset of proteins in SF that are potential markers associated with OA.

The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P< 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P< 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P< 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter.

Recently we reported on a novel H2E transgenic, IA-negative model of experimental autoimmune thyroiditis (EAT) that excludes reactivity to self in its susceptibility pattern to heterologous thyroglobulin (Tg). In conventional, susceptible mouse strains, EAT is inducible with both homologous and heterologous Tg; e.g., human (h)Tg shares conserved thyroiditogenic epitopes with mouse (m)Tg. However, when an H2Eak transgene is introduced into class II-negative B10.Ab0 mice, which express neither surface IA (mutant Aβ-chain) nor surface IE (nonfunctional Ea gene), the resultant H2Eb molecules are permissive for EAT induction by hTg, but not self mTg. Also, the hTg-primed cells do not cross-react with mTg.To explore this unique capacity of E+B10.Ab0 mice to distinguish self from nonself Tg, we have developed T cell lines to examine the T cell receptor repertoire and observed a consistent Vβ8+ component after repeated hTg stimulation. Enrichment and activation of Vβ8+ T cells by either superantigen staphylococcal entertoxin B or anti-Vβ8 in vitro enabled thyroiditis transfer to untreated A−E+ recipients, similar to hTg activation. Vβ8+ T cells isolated by FACS from hTg-immunized mice also proliferated to hTg in vitro. These studies support the contribution of Vβ8 genes to the pathogenicity of hTg in this H2A−E+ transgenic model.

Introduction:Current standard electrocardiogram analysis algorithms cannot predict the presence and extent of coronary artery disease (CAD), especially in stable patients.Hypothesis:A novel artificial intelligence algorithm (ECGio) is able to predict the presence, location, and severity of coronary artery lesions in a minimally selected stable patient population as verified by coronary angiography.Methods:A cohort of 1659 stable outpatients were randomly divided into training (86%) and validation (14%) subsets, maintaining population characteristics. ECGio was trained and then validated using electrocardiograms paired with angiograms collected from electronic medical records. Coronary artery lesions were evaluated by a continuous measure of stenosis ranging from 0% - 100%. The prediction was then compared to the angiogram result (worst diameter stenosis in each vessel) with the error calculated per patient and per vessel.Results:In the primary analysis of the validation cohort, 22 had no angiographic CAD and were grouped with 56 patients with mild CAD (DS ≤30%), 31 had moderate CAD (DS 30-70%), and 113 had severe CAD (DS ≥70%). On a vessel level ECGio was able to predict stenosis severity with an overall average error of 16% as well as vessel specific errors of 18% in the LAD, 19% in the LCX, 18% in the RCA, and 8% in the Left Main. Absolute Error and Left Main error can be seen in the Figure.Conclusion:This validation study strongly suggests that it is possible to utilize an artificial intelligence algorithm to determine the presence and severity of CAD in stable patients using data from a standard 12- lead electrocardiogram.

Abstract:  Susceptibility to experimental autoimmune thyroiditis (EAT) is linked to H2-A class II genes;k and s haplotypes are susceptible, while b and f are resistant. EAT is inducible with thyroglobulins (Tgs) from several mammalian species which share portions of identical sequences. But cross-activation and cross-tolerance studies with mouse (m), human (h), and porcine (p) Tg have indicated mTg-unique T-cell epitope(s), in addition to conserved, in EAT induction. The recent introduction of the HLA-DRB1*0301 (DR3) transgene rendered major histocompatibility complex (MHC) class II-negative (Ab0) mice susceptible to EAT induction by both hTg and mTg, suggesting usage of conserved epitopes. Here, we introduced the H2-Ea k transgene into resistant B10 (H2 b ) or Ab0 mice with a defective Ea gene to provide functional surface H2E (b haplotype) expression. Surprisingly, both transgenic strains showed severe inflammation only after hTg, but not mTg, immunization, although the moderating influence of the A b gene in B10 was evident. In proliferative assays, hTg-primed cells did not respond to mTg, nor to conserved 12mer peptides from three primary hormonogenic sites, two of which can activate T cells for thyroiditis transfer and cytotoxicity. The vigorous response to hTg stimulation was reduced only by Eβb-specific monoclonal antibody. EAT induction with bovine and pTg showed responses similar to hTg, suggesting thyroiditogenic epitopes shared with hTg, but not mTg. This is the first demonstration of: (1) nonpermissiveness for EAT induction with mTg, normally the most thyroiditogenic Tg and the one with unique epitopes for susceptible mice, and (2) the separation of hTg from mTg in EAT induction in H2-E-transgenic mice.

The diagnosis of acquired myasthenia gravis (MG) in apparently seronegative individuals is aided by finding immunoglobulin complexed to acetylcholine receptors (AChR) and a reduction in the number of binding sites for a-bungarotoxin (α-BTx) in nerve-muscle biopsies. In this study, we found that anti-AChR antibodies in extracellular fluids can complex with cytoplasmic epitopes of AChR in the process of muscle extraction. When normal muscle was briefly exposed to antibodies (≥0.3 nmol/1) in the initial step of tissue homogenization (before detergent extraction), membranous AChR became complexed with IgG. This was so even with a nonmyasthenogenic monoclonal antibody specific for the α-subunit's presumptive cytoplasmic segment 366–389. We also found that antibodies reactive with AChR's α-BTx binding region can significantly lower apparent yields of α-BTx binding sites extracted from muscle. Thus, the finding of IgG complexed to AChR extracted from biopsied muscle does not necessarily reflect in vivo binding but, nevertheless, is a sensitive indicator of AChR seropositivity in patients suspected to have MG.

Previous studies have shown that K562 chronic myelogenous leukemia cells are resistant to induction of apoptosis by a variety of agents, including the topoisomerase II (topo II) poison etoposide, when examined 4 to 24 hours after treatment with an initiating stimulus. In the present study, the responses of K562 cells and apoptosis-proficient HL-60 acute myelomonocytic leukemia cells to etoposide were compared, with particular emphasis on determining the long-term fate of the cells. When cells were treated with varying concentrations of etoposide for 1 hour and subsequently plated in soft agar, the two cell lines displayed similar sensitivities, with a 90% reduction in colony formation at 5 to 10 µmol/L etoposide. After treatment with 17 µmol/L etoposide for 1 hour, cleavage of the caspase substrate poly(ADP-ribose) polymerase (PARP), DNA fragmentation, and apoptotic morphological changes were evident in HL-60 cells in less than 6 hours. After the same treatment, K562 cells arrested in G2 phase of the cell cycle but otherwise appeared normal for 3 to 4 days before developing similar apoptotic changes. When the etoposide dose was increased to 68 µmol/L, apoptotic changes were evident in HL-60 cells after 2 to 3 hours, whereas the same changes were observed in K562 cells after 24 to 48 hours. This delay in the development of apoptotic changes in K562 cells was accompanied by delayed release of cytochrome c to the cytosol and delayed appearance of peptidase activity that cleaved the fluorogenic substrates Asp-Glu-Val-Asp-aminotrifluoromethylcoumarin (DEVD-AFC) and Val-Glu-Ile-Asp-aminomethylcoumarin (VEID-AMC) as well as an altered spectrum of active caspases that were affinity labeled with N-(Na-benzyloxycarbonylglutamyl-Ne-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone [z-EK(bio)D-aomk]. On the other hand, the activation of caspase-3 under cell-free conditions occurred with indistinguishable kinetics in cytosol prepared from the two cell lines. Collectively, these results suggest that a delay in the signaling cascade upstream of cytochrome c release and caspase activation leads to a long latent period before the active phase of apoptosis is initiated in etoposide-treated K562 cells. Once the active phase of apoptosis is initiated, the spectrum and subcellular distribution of active caspase species differ between HL-60 and K562 cells, but a similar proportion of cells are ultimately killed in both cell lines.

We describe a patient with Type I diabetes mellitus whose hemoglobin Ale (HbAlc) level was markedly elevated (45.3) when initially measured by routine low pressure ion exchange chromatography. Possible interference of a variant hemoglobin (Hb) was suspected, and subsequently the patient and his father were found to have two very rare Hb variants (Hb Raleigh and Hb Russ). Because of charge characteristics, Hb Raleigh cochromatographs with HbAlc, resulting in spuriously elevated assay values. Affinity chromatography of total glycosylated hemoglobin (GHb), a method that circumvents this interference, showed only slightly elevated GHb in the patient and a normal GHb in his nondiabetic father. This is the second family reported with both Hb Raleigh and Hb Russ and the fourth family where interference of Hb Raleigh with measurement of HbAlc resulted in detection of this Hb variant. More than 20 other Hb variants have been shown to interfere with estimation of HbAlc by ion exchange chromatography, the method most commonly employed in clinical laboratories. Clinicians should be aware of such potential interactions and request identification of variant Hb. Alternative methods of GHb measurement will allow reliable assessment of glycemic control in such patients

Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR‐1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia) and morphine (antinociception) were lost in a specific manner. Binding studies confirmed a large reduction in receptor sites. The loss of behavioral responses was long lasting but did fully recover. The implications of specifically and readily turning off gene expression in vivo are profound.

The specific high affinity binding of the avian oviduct progesterone receptor (PR) to target cell nuclei and chromatin has been shown to involve DNA complexed with specific chromatin acceptor proteins. One of these chromatin acceptor proteins has been partially purified and found to be a small hydrophobic protein with a broad pI of 5.0–6.0 [Goldberger and Spelsberg (1988),Biochem.27, 2103–2109]. Using western immunoblots with anti-RBF-1 polyclonal antibodies to monitor the purification, a 10 kD candidate acceptor protein, termed the Receptor Binding Factor-1 (RBF-1), has been purified to apparent homogeneity. RBF-1 has an amino acid composition consistent with a hydrophobic protein having an acidic pI and a unique N-terminal sequence. Two-dimensional polyacrylamide gel electrophoresis and high-performance capillary electrophoresis support the purity of a protein ≊10 kD in size, having an acidic pI, but with evidence of several differently charged isoforms. Phosphatase treatment provides evidence that charge heterogeneity may result from variable phosphorylation states. A role of this factor as a candidate “acceptor protein” in the chromatin acceptor sites for the avian oviduct PR is proposed.

To determine the nature and characteristics of a unique hemoglobin variant that causes a spurious increase in glycated hemoglobin (Hb Alc).

Previously we demonstrated that thyroxine (T4)-containing, 12-mer peptides from positions 5 (1–12) and 2553 (2549–2560), as well as thyronine (T0)-substituted 2553 peptide, derived from human (H) thyroglobulin (Tg) are capable of activating T cells that infiltrate the thyroid (thyroiditogenic). In contrast, peptides T4(2567) and T0(2567) (2559–2570) are not. To determine if these thyroiditogenic peptides, T4(5), T4(2553), and T0(2553), activated cytotoxic T cells (Tc) and served as target autoantigens when loaded onto indicator cells (BW5147 lymphoma,H2k), lymph node cells from CBA mice immunized with mouse (M) Tg were culturedin vitrowith MTg, HTg, or Tg peptide. After MTg or HTg activation, Tc were detected for both MTg- and HTg-loaded target cells in an 18-h,51Cr-release assay at an effector:target cell ratio of 50:1. These Tc also killed target cells labeled with T4(5), T4(2553), or T0(2553), but not the control peptide T4(2567). When MTg-primed lymphocytes were cultured with T4(5), T4(2553), or T0(2553), specific Tc were also generated against target cells labeled with the respective peptide. The data suggest that one of the thyroiditogenic properties of these peptides previously shown by adoptive transfer of thyroiditis is related to the generation of Tc. In addition, these conserved autoepitopes of Tg also serve as target antigens for Tc.

Twenty-nine overlapping synthetic peptides, twenty residues long, representing the entire extracellular sequence of the human thyroid stimulating hormone receptor (hTSHr), were used to test the epitope repertoire of CD4+T lymphocytes from patients with Graves' disease and from healthy subjects. The peptides were used to propagate and test short term CD4+T cell lines specific for hTSHr epitopes, and to directly test CD8+depleted, CD4+enriched peripheral blood lymphocytes. Analysis of the response of short-term CD4+T cell lines and CD8+depleted peripheral blood lymphocytes to the individual peptides revealed that 14 of the 15 patients and nine of the ten controls responded to at least one hTSHr peptide. There was no common response pattern, nor any region of the hTSHr sequence that was predominantly recognized. Several peptides were recognized by both patients and controls. These results support the notion that immunological tolerance to hTSHr is due to peripheral tolerance of potentially autoreactive CD4+T cells, not their clonal deletion. The presence of self-reactive, hTSHr-specific CD4+T cells in healthy individuals implies that these cells are not permanently anergized, since they can be activatedin vitro.

A comprehensive synthetic approach consisting of a series of consecutive, uniform overlapping peptides encompassing the entire protein chain was recently used to determine the full antigenic profile of the α-chain of human hemoglobin (Hb). The peptides synthesized enabled the localization of five major “continuous” antigenic regions within the α chain. The present findings describe the delineation of an antigenic site (site 2) residing within the region 41–65. Ten peptides representing the α-chain regions 41–55, 51–65, 45–54, 45–56, 45–58, 45–60, 48–56, 49–56, 50–56, and 51–56 were synthesized and purified. Quantitative radioimmunoadsorbent titrations were used to determine binding to peptide adsorbents of radioiodinated anti-Hb antibodies that were raised in rabbit, goat, and outbred mouse. In one set of peptides, the N-terminal was fixed while the C-terminal end was increased by increments of two residues from Gln-54 to Lys-60 (i.e., peptides 45–54, 45–45, 45–58, and 45–60). Binding studies revealed that maximum antibody activity resided in peptide 45–45, indicating that Lys-56 marks the C-terminal boundary of the site. In the second set of peptides, the C-terminal was fixed at Lys-56 while the peptides were elongated at their N-terminal by one-residue increments from Gly-51 to Leu-48. Antibody-binding studies with these peptides indicated that Ser-49 defines the N-terminal boundary of the site. Therefore, the antigenic site within region 41–65 of the α chain comprises residues 49–56. The relevance of these findings to the immune recognition of Hb and other proteins is discussed.

A survey of the hydrolytic enzymes present in pigeon dropping extracts (PDE) has shown that this material contains a variety of proteolytic and nonproteolytic activities. These enzymes were separated into their basic and acidic components by chromatography on DEAE-cellulose. Staining of immunoprecipitates with specific chromogenic substrates demonstrated the presence of antibodies in symptomatic breeders to several of the basic enzymes in PDE. Five distinct hydrolytic activities were isolated from the basic group of enzymes. Trypsin, elastase, and two forms of collagenase were the specific proteolytic activities isolated. A phospholipase was also purified from these preparations. The purified elastase consisted of a single polypeptide chain (Mr=22,000). The purified trypsin had a molecular weight (Mr=25,000) and charge similar to those reported for elastase and, like elastase, the trypsin from PDE appeared to be composed of a single polypeptide chain. Two molecular weight forms of collagenase were found; both hydrolyzed bovine collagen. The high-molecular-weight collagenase (Mr=51,000) was shown to be a glycoprotein consisting of two polypeptides (Mr=24,000). It was readily separated from the low-molecular-weight collagenase (Mr=15,000) by gel filtration. The phospholipase (Mr=99,000) appeared to be a dimer. The relevance of these enzymes to the development of pigeon breeder's disease is discussed.

The hydrolytic enzymes in pigeon dropping extracts (PDE) were separated into basic and acidic components by DEAE-cellulose chromatography. Six distinct hydrolytic activities were isolated from the acidic group of enzymes. Elastase, trypsin, and two forms of collagenase were the proteases identified. An esterase and a phospholipase were also detected. The sera of symptomatic pigeon breeders, analyzed by crossed immunoelectrofocusing techniques, were shown to contain antibodies to the enzymes trypsin, collagenase, and esterase in the acidic fractions of PDE by staining the immune precipitates with specific chromogenic substrates. These enzymes were purified by preparative isoelectric focusing, affinity chromatography, and gel filtration. The purified elastase hydrolyzed elastin and consisted of a single polypeptide chain (Mr=23,100). Trypsin hydrolyzed a synthetic arginine substrate, but not elastin or collagen. Its size (Mr=27,500) and subunit structure were similar to those reported here for elastase. Both enzymes were inhibited by α-1-antitrypsin,Nα-p-tosyl-L-lysine chloromethylketone, and phenylmethylsulfonyl fluoride. Two distinct collagenases were found; both cleaved bovine collagen. The high-molecular-weight (HMW) collagenase was a glycoprotein (Mr=117,500) and consisted of three polypeptide chains (Mr=37,100); a low-molecular-weight (LMW) collagenase (Mr=12,500) was also isolated. The HMW collagenase was inhibited byEDTA,p-chloromercuribenzoic acid, and phenylmethylsulfonyl fluoride, but not by α-1-antitrypsin. The source of these enzymes as well as their relationship to the basic hydrolytic enzymes of PDE are discussed.

A few synthetic peptides corresponding to amino acid sequences on human thyroglobulin (Tg) have been reported to induce moderate thyroiditis or activate mouse Tg (MTg)-primed T cells to transfer thyroiditis in mice susceptible to experimental autoimmune thyroiditis. Using three pairs of 12-mer peptides (1–12, 2549–2560, 2559–2570), with thyroxine (T4) or noniodinated thyronine (T0) at the conserved, hormonogenic site 5, 2553, or 2567 respectively, we reported that iodination was not required for a Tg hormonogenic site to be a thyroiditogenic autoepitope. To determine the relative importance of MHC class II and T cell receptor (TCR) repertoire, we compared two EAT-susceptiblekands(CBA and A.SW) haplotypes and their respective MHC-identical strain (C57BR and SJL) with ∼50% genomic deletion of TCR Vβ genes. Whereaskandsstrains develop MTg-induced EAT, vigorous immunization with peptides containing T4 or T0 at either 5 or 2553, but not at 2567, led to mild (10–20%) thyroiditis only in some mice of eitherkstrain. TCR Vβ gene differences played a minor role. T cell responses to all peptide pairs were quite similar in CBA and C57BR mice, and both hT0(2553) and hT4(2553) reciprocally primed and stimulated their T cells. In adoptive transfer, SJL mice were somewhat more responsive to peptide activation than A.SW but much weaker thankstrains. By comparing T4- and T0-containing peptides in different haplotypes, we show further that antigenicity of conserved hormonogenic sites is intrinsic, dependent more on amino acid sequence and binding to appropriate class II molecules and less on TCR repertoire or iodination of T0.

Healthy humans have CD4+T cells specific for self-components. Since autoreactive T cells in autoimmune patients may use a limited number of TCR V-region genes, we investigated here whether this also occurs for the potentially autoreactive CD4+cells present in healthy persons. We studied CD4+cells specific for human TSH receptor (TSHr) sequences, that are present with high frequency in healthy subjects and, as expected, in Graves’ disease (GD) patients.

Synthetic peptides are valuable tools for determining sites of interaction between hormones and their receptors. We have learned much about linear receptor binding regions of the glycoprotein hormone human choriogonadotropin (hCG) using synthetic peptides corresponding to its primary sequence. Of paramount importance in any study using synthetic peptides as a tool to investigate protein structure are an efficient means of peptide purification and a definitive measure of peptide purity and composition. Purification is easily achieved for all but the most hydrophobic peptides using preparative reverse-phase HPLC. Of the methods available for analysis of peptide purity, mass spectrometry is perhaps the most useful and often most rapid approach. Other essential components of studies involving synthetic peptides and hormone binding are reproducible hormone labeling, receptor preparations, and bioassays. The ability of peptides to compete with hCG for binding to specific receptors is tested in radioreceptor binding assays and bioassays with transformed Leydig cell lines. These cells express the lutropin receptor that is coupled to a measurable endpoint such as cAMP or steroid production. The conditions for these assays are optimized for rapid and accurate measurement of peptide activity. Since the three-dimensional structure of hCG is not known, a systematic approach to the identification of potential receptor binding sites is advocated. First, a comprehensive analysis using synthetic overlapping peptides of uniform length that span the entire sequence of the α-subunit is employed. This approach is an effective means for surveying the entire subunit for receptor binding sites. Next, the boundaries of the active regions are delimited by a series of nested peptides sequentially shortened in length. The importance of each residue within the delimited binding regions is then studied using a series of peptides containing single alanine substitutions. Finally, modifications to enhance activity of the synthetic peptides are further made on the basis of data from alanine substitution studies, circular dichroic analysis, and molecular modeling. These studies provide valuable information to aid in the design of synthetic hormone analogs and for further analysis of the structure–function of hCG via site-directed mutagenesis.Copyright 1993, 1999 Academic Press

Recently, the rat neurotensin receptor and the two human neurotensin receptor clones (differing by one amino acid residue) have been isolated. We present results with 33 newly synthesized neurotensin analogs. We have evaluated their binding potency at the three neurotensin receptor clones by determining equilibrium dissociation constants and coupling to phosphatidylinositol turnover. Our work focused on position 8 and 9 substitutions as well as position 11 of the neurotensin hexamer NT8-13. The results presented include: 1) the development of a compound that is species selective, with a binding potency at the rat receptor that is 20-fold more potent than at the human receptor; 2) the development of a pair of stereoselective compounds with the L-isomer exhibiting 190-700-fold more potency than the D-isomer; and 3) the development of an agonist that has a Kdof 0.3 and 0.2 nM at the human and rat neurotensin receptor, respectively, ranking it as among the most potent tested. Also, we present the first evidence that 1) the effect of pi electrons at position 11 (L-Tyr) are important for binding to the neurotensin receptor, and 2) the length of the side chain on position 9 (L-Arg) changes binding potency.

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The ∗∗∗glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common α subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are α26 to 46 and α75‐92. Two synthetic disulfide loop peptides from the hCGβ subunit β38‐57 and β93‐100 also block binding of hCG to its receptor. In addition, the β38‐57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the α and βsubunits, and that the α subunit sites are similar for several hormones.—Ryan, R. J.; Charlesworth, M. C.; McCormick, D. J.; Milius, R. P.; Keutmann, H. T. The glycoprotein hormones: recent studies of structure‐function relationships. FASEB J.2: 2661‐2669; 1988.

Percutaneous balloon valvuloplasty was performed on a patient with pulmonary stenosis. Right to left shunting through a patent foramen ovale during balloon inflation was documented by contrast two-dimensional echocardiography. Right and left ventricular pressures recorded during balloon inflation showed a decrease in left ventricular end-diastolic pressure and equilibration with right ventricular end-diastolic pressure. Systemic hypotension was minimal during balloon inflation, possibly due to persistent filling of the left ventricle via the patent foramen ovale. Persistent right ventricular systolic hypertension immediately after valvuloplasty may have been due to infundibular narrowing and resolved on restudy 2 weeks later.

Previous studies from this laboratory employing a comprehensive synthetic overlapping peptide strategy showed that the α-chain of human hemoglobin (Hb) contains a single haptoglobin (HP) binding region residing within residues α121–135. The present study describes a precise delineation of this Hp-binding site on the α-chain. Two overlapping peptides (α111–125 and α121–135) spanning this region and a panel of five peptides decreasing at the C-terminal from residue 135 by decrements of two residues (α119–135, α119–133, α119–131, α119–129, and α119–127) were synthesized, purified, and characterized. Quantitative radiometric titration of125I-labeled human HP (type 2-1) with adsorbents of each of these synthetic peptides showed that the peptide α119–127 retained a Hp-binding activity equivalent to that of peptide α121–135. This finding indicated that Lys-127 marked the C-terminal boundary of the binding site. Another panel of eight peptides was then synthesized, which had their C-terminus fixed at Lys-127 and increased at the N-terminus by one-residue increments from residue 122 up to residue 115 (α122–127, α121–127, α120–127, α119–127, α118–127, α117–127, α116–127, and α115–127). The binding of125I-Hp to adsorbents of these peptides demonstrated that the N-terminal boundary of the site did not extend beyond Valine 121. It is, therefore, concluded that the Hp-binding site on the α-chain of human Hb comprises residues α121–127.

Peptide nucleic acids (PNAs) are DNA analogs that can hybridize to complementary sequences with high affinity and stability. Here, we report the first evidence of intracellular delivery of PNAs in vivo. Two CNS receptors, an opioid (mu) and a neurotensin (NTR-1), were targeted independently by repeated microinjection of PNAs into the periaqueductal gray. Behavioral responses to neurotensin (antinociception and hypothermia) and morphine (antinociception) were lost in a specific manner. Binding studies confirmed a large reduction in receptor sites. The loss of behavioral responses was long lasting but did fully recover. The implications of specifically and readily turning off gene expression in vivo are profound.

Recent genetic and biochemical studies have implicated cysteine-dependent aspartate-directed proteases (caspases) in the active phase of apoptosis. In the present study, three complementary techniques were utilized to follow caspase activation during the course of etoposide-induced apoptosis in HL-60 human leukemia cells. Immunoblotting revealed that levels of procaspase-2 did not change during etoposide-induced apoptosis, whereas levels of procaspase-3 diminished markedly 2-3 h after etoposide addition. At the same time, cytosolic peptidase activities that cleaved DEVD-aminotrifluoromethylcoumarin and VEID-aminomethylcoumarin increased 100- and 20-fold, respectively; but there was only a 1.5-fold increase in YVAD-aminotrifluoromethylcoumarin cleavage activity. Affinity labeling with N-(Nα-benzyloxycarbonylglutamyl-Nε-biotinyllysyl)aspartic acid [(2,6-dimethylbenzoyl)oxy]methyl ketone indicated that multiple active caspase species sequentially appeared in the cytosol during the first 6 h after the addition of etoposide. Analysis on one- and two-dimensional gels revealed that two species comigrated with caspase-6 and three comigrated with active caspase-3 species, suggesting that several splice or modification variants of these enzymes are active during apoptosis. Polypeptides that comigrate with the cytosolic caspases were also labeled in nuclei of apoptotic HL-60 cells. These results not only indicate that etoposide-induced apoptosis in HL-60 cells is accompanied by the selective activation of multiple caspases in cytosol and nuclei, but also suggest that other caspase precursors such as procaspase-2 are present but not activated during apoptosis.

A patient with cardiac sarcoidosis proved by biopsy specimen and no history of sudden death or clinical sustained ventricular tachycardia prophylactically received an implantable cardioverter defibrillator (ICD) that later reversed an episode of near syncope. The patient was supported with the ICD until heart transplantation. The physiology and treatment of arrhythmias associated with cardiac sarcoidosis is described. Consideration for use of the ICD in asymptomatic patients and as bridge therapy until heart transplantation is discussed.

Human choriogonadotropin (hCG) consists of an α subunit and a β subunit. The existing evidence from various studies using truncation, substitution, synthetic hormone peptides, and hCG crystals suggests that the C-terminal region of the α subunit contacts the luteinizing hormone/choriogonoadotropin (LH/CG) receptor and is involved in receptor activation. Despite a deluge of the speculation and the important role of the αC-terminal region, direct evidence for its interaction with the receptor has been elusive. Because of the significant biological activity, it is imperative to prove the interaction of the αC-terminal region. For this purpose, decamer peptides corresponding to the α subunit sequence from His83to Ser92(α83-92) were derivatized with the N-hydroxysuccinimide ester of 4-azidobenzoylglycine (ABG) and radioiodinated. The resulting ABG-125I-α83-92was capable of binding and activating the LH/CG receptor. Furthermore, UV-sensitive ABG-125I-α83-92exclusively photoaffinity-labeled an ~86-kDa molecule. This labeled molecule was shown to be the LH/CG receptor by various methods including immunoprecipitation by anti-LH/CG receptor antiserum. In addition, evidence is presented that the amino group of αLys91of α83-92is in such close proximity to a carboxyl group of the receptor that this pair is cross-linked to form an amide, a zero length cross-link. This low affinity contact of α83-92and the receptor is sufficient for receptor activation and is crucial for the full understanding of the mechanistics of the receptor activation steps.

Phosphorylation of G protein-coupled receptors is an established mechanism for desensitization in response to agonist stimulation. We previously reported phosphorylation of the pancreatic acinar cell cholecystokinin (CCK) receptor and the establishment of two-dimensional phosphopeptide mapping of its sites of phosphorylation (Ozcelebi, F., and Miller, L. J.(1995) J. Biol. Chem. 270, 3435-3441). Here, we use similar techniques to map sites of phosphorylation of the same receptor expressed on a stable receptor-bearing Chinese hamster ovary (CHO)-CCKR cell line. Like the native cell, the CHO-CCKR cell receptor was phosphorylated in response to agonist stimulation in a concentration-dependent manner; however, the time course was quite different. CHO-CCKR cell receptor phosphorylation increased progressively to a plateau after 15 min, while in the acinar cell it peaks within 2 min and returns to baseline over this interval. There were distinct qualitative and quantitative differences in the sites of phosphorylation of the two receptor systems. One site previously attributed to action of a staurosporine-insensitive kinase in the acinar cell was absent in the CHO-CCKR cell. Site-directed mutagenesis was utilized to eliminate predicted sites of protein kinase C action, but only two of four such sites affected the phosphopeptide map of this receptor. Chemical and radiochemical sequencing were performed on these and other phosphopeptides which were present in both the CHO-CCKR cells and agonist-stimulated pancreatic acinar cells to provide direct evidence for the phosphorylation sites actually utilized. Thus, these data support the usefulness and limitations of a model cell system in studying receptor phosphorylation and desensitization.

Aims Cenderitide is a novel dual natriuretic peptide (NP) receptor chimeric peptide activator, which targets the particulate guanylyl cyclase B (pGC-B) receptor and pGC-A unlike native NPs. Cenderitide was engineered to retain the anti-fibrotic properties of C-type natriuretic peptide (CNP)/pGC-B with renal-enhancing actions facilitated by fusion to the carboxyl terminus of Dendroaspis NP (DNP), a pGC-A agonist, to CNP. Here, we address significance of the DNP carboxyl terminus in dual pGC receptor activation and actions of cenderitide compared with CNP on renal function and cyclic guanosine monophosphate (cGMP) in vivo and ex vivo in normal canines. Methods and results In vitro, only cenderitide and not CNP or three CNP-based variants was a potent dual pGC-A/pGC-B activator of cGMP production (from 5 to 237 pmol/mL) in human embryonic kidney (HEK) 293 cells overexpressing human pGC-A while in pGC-B overexpressing cells cenderitide increased cGMP production (from 4 to 321 pmol/mL) while the three CNP-based variants were weak agonists. Based upon our finding that the DNP carboxyl terminus is a key structural requirement for dual pGC-A/pGC-B activation, we defined in vivo the renal-enhancing actions of cenderitide compared with CNP. Cenderitide increased urinary cGMP excretion (from 989 to 5977 pmol/mL), net generation of renal cGMP (821–4124 pmol/min), natriuresis (12–242 μEq/min), and glomerular filtration rate (GFR) (37–51 mL/min) while CNP did not. We then demonstrated the transformation of CNP ex vivo into a renal cGMP-activating peptide which increased cGMP in freshly isolated glomeruli eight-fold greater than CNP. Conclusion The current study establishes that dual pGC-A and pGC-B activation with CNP requires the specific carboxyl terminus of DNP. In normal canines in vivo and in glomeruli ex vivo, the carboxyl terminus of DNP transforms CNP into a natriuretic and GFR-enhancing peptide. Future studies of cenderitide are warranted in cardiorenal disease states to explore its efficacy in overall cardiorenal homeostasis.