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Adiponectin, a novel adipose-derived adipocytokine, has beneficial effects not only on improvement of insulin sensitivity but also on mitigation of vascular damage. To evaluate whether adiponectin is implicated in the pathogenesis of diabetic nephropathy characterized by microvascular damage, we examined urinary and serum adiponectin levels in type 2 diabetic patients with different stages of nephropathy. We first confirmed adiponectin is excreted into urine through Western blot analysis, followed by measurements of urinary and serum adiponectin levels by radioimmunoassay. Interestingly, urinary adiponectin excretion levels were markedly increased in patient group with overt nephropathy relative to the groups without nephropathy and with incipient nephropathy. Surprisingly, serum adiponectin levels were also elevated in patient group with overt nephropathy. Increased urinary adiponectin excretion may result from elevations in circulating adiponectin levels and enhanced filtration of circulating adiponectin through the damaged kidney. Furthermore, adiponectin synthesis in adipose tissue and its secretion into circulating blood may be enhanced to mitigate microvascular damage in the advanced stage of diabetic nephropathy. [Copyright &y& Elsevier]

Group II A phospholipase A2 (PLA2) produces many inflammatory lipid mediators, and the elevation in the level during sepsis has been correlated positively with the decrease in the arterial blood pressure. We studied the effect of large-pore continuous venovenous hemodiafiltration (LP-CVVHDF) on the plasma PLA2 concentration and the clearance mechanism during septic acute renal failure. The subjects were 10 consecutive patients with septic acute renal failure receiving CVVHDF. Simultaneous samples of arterial, and filter inlet and outlet blood, and ultradiafiltrate were collected before starting CVVHDF (0 hr), and 4 hr, 12 hr and 24 hr after starting CVVHDF. PLA2 activity was measured in plasma and ultradiafiltrate. We eluted PLA2 bound to hemofilter from patient and the classification of PLA2 type of eluting solution and ultradiafiltrate was done using Western blot analysis. Plasma clearance (mL/min) was 28.1 ± 7.6 at 4 hr, 23.2 ± 8.9 at 12hr and 17.5 ± 8.0 at 24 hr. Plasma clearance at 4 hr was higher than that at either 12 hr or 24 hr. Plasma clearance mainly consisted of adsorption by LP-CVVHDF. The changes in arterial plasma PLA2 activity were not statistically significant. One mg/mL of heparin eluted PLA2 bound to the large-pore hemofilter. The PLA2 in eluting solution and in ultradiafiltrate were identified as an approximately 70 kD band in Western blot analysis using anti-human secretory II A-PLA2 monoclonal antibody. The results show that circulating PLA2 can be removed by adsorption with LP-CVVHDF to some extent and that plasma PLA2 activity is not significantly decreased. Because PLA2 clearance with LP-CVVHDF is estimated as < 1% of total body PLA2 clearance, LP-CVVHDF could not be a clinically efficient therapy to remove the circulating PLA2. [ABSTRACT FROM AUTHOR]

Lumican is a member of the small-leucine-rich proteoglycan (SLRP) family and is overexpressed during wound healing of the cornea, in ischemic and reperfused heart, and in several cancer tissues. Lumican is considered to regulate the collagen fibril diameter and interfibrillar spacing. However, the effect of lumican on cell growth has not been adequately examined. In the present study, we attempted to clarify whether lumican contributes to human embryonic kidney (HEK) 293 cell growth, using the morpholino antisense oligonucleotide (m-anti oligo) against lumican mRNA. M-anti oligo is a novel oligonucleotide and exhibits a higher antisense activity, higher water solubility, and greater resistance to nucleases in target cells than phosphorothioate types of oligonucleotide. After delivery of m-anti oligo against lumican mRNA, the fluorescein 5-isothiocyanate (FITC) conjugated oligonucleotides were observed in the cytoplasm and nucleus of HEK 293 cells at 24 h by confocal laser microscopy. M-anti oligo for lumican mRNA strongly inhibited the synthesis of lumican protein in the HEK 293 cells, and the HEK cell growth rate was higher than those in the control groups. These findings may indicate that lumican protein has an inhibitory effect on HEK 293 cell growth in vitro. [ABSTRACT FROM AUTHOR]

This study investigated the expression and cellular localization of neuronal nitric oxide synthase in the rat retina following optic nerve transection (ONT). In the normal rat retina, nNOS immunoreactivity was localized to amacrine cells and displaced amacrine cells. A few bipolar cells were also labeled. In the axotomized retina, ganglion cells showed nNOS immunoreactivity from 3 days after ONT, and these cells increased in number, peaking 5 days after ONT. Quantitative evaluation using immunoblotting confirmed that nNOS expression showed a peak value (255% of control levels) 5 days after ONT and decreased to 137% of controls by 28 days. These findings suggest that axotomized ganglion cells degenerate via NO-mediated excitotoxicity. [Copyright &y& Elsevier]

Metastatic mammary tumors in rats were treated by laser immunotherapy. Tumors injected by a laser-absorbing dye and an immunoadjuvant were irradiated non-invasively by a near-infrared laser. The successfully cured rats developed a long-term resistance to repeated tumor challenges. Using the sera from cured rats as the source of primary antibodies, immune responses induced by laser immunotherapy were observed in both cellular and molecular levels using histochemical assays and Western blot analysis. [ABSTRACT FROM AUTHOR]

Pregnancy results in attenuated baroreflex mediated sympathoexcitatory responses which may be due to potentiation of γ-aminobutyric acid (GABA) inhibition in the rostral ventrolateral medulla (RVLM). The major metabolite of progesterone, 3α-hydroxy-dihydroprogesterone (3α-OH-DHP), which is elevated in pregnancy, is a potent neurosteroid positive modulator of GABAA receptors, and sensitivity of GABAA receptors to 3α-OH-DHP is dependent on the receptor subunit composition. The purpose of this study was to evaluate the GABAA α1 and α2 receptor subunit mRNA and protein expression in the RVLM of nonpregnant and late term pregnant rats. Micropunches of RVLM were collected from nonpregnant and late term pregnant rats and the expression levels of GABAA α1 and α2 receptor subunits were analyzed using quantitative competitive reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblot techniques. The competitive RT-PCR analysis allows comparison of expression levels between different mRNA, and the mRNA expression level of GABAA α1 was several hundred fold greater than GABAA α2 in both groups. However, this relative distribution of GABAA α1 and α2 receptor subunits protein or mRNA expression was not altered in late term pregnant compared to nonpregnant rats. These data demonstrate, that within the RVLM of both nonpregnant and late term pregnant rats, the relative expression levels of GABAA α1,2 receptor subunits favor GABAA receptors susceptible to positive modulation by progesterone metabolites. [Copyright &y& Elsevier]

In kidney, nitric oxide (NO) synthesized by nitric oxide synthase (NOS) regulates sodium and water excretion, and renal medullary blood flow. The expression of constitutive NOS, endothelial NOS (eNOS) and neuronal NOS (nNOS), were assessed in kidney of the spontaneously hypertensive rat (SHR) and the normotensive Wistar Kyoto (WKY) rat by Western blot analysis and immunocytochemistry. Neuronal NOS expression was observed in the cortex and eNOS was detected only in the inner medulla of both WKY and SHR. In SHR, expression of eNOS was attenuated to 35.1 ± 10.8, while expression of nNOS was only 57.5 ± 35.74570360f the levels seen in WKY rat. Immunocytochemical studies revealed decreased staining of nNOS in the macula densa, collecting ducts and in the glomerulus of SHR compared to WKY rat. Endothelial NOS immunoreactivity was restricted to intima cells and smooth muscle cells, and was markedly SHR. The decreased renal blood flow observed in SHR expression of eNOS and nNOS, underscoring the importance pathophysiology and maintenance of genetic hypertension. [ABSTRACT FROM AUTHOR]

Objective: Expression of vascular endothelial growth factor (VEGF)-D by tumors is associated with metastasis to lymph nodes in mice. However, there are few reports concerning the clinical significance of VEGF-D protein in human carcinoma. Methods: After confirming production of VEGF-D by eight colorectal carcinoma cell lines, we investigated relationships between the expression of VEGF-D protein, lymph node metastasis and postoperative survival in 83 colorectal carcinoma patients. mRNA levels in cell lines were evaluated using the real-time reverse transcriptase-polymerase chain reaction, and protein was detected by Western blotting in cell lines and by immunohistochemistry in resected tissues using an antibody recognizing the processed form of the molecule. Results: Immunohistochemistry showed VEGF-D-positive staining in 26 of the 83 carcinomas (31%). There was a significant relationship between the presence of VEGF-D protein and the incidence of lymph node metastasis (p < 0.01). Multivariate logistic regression analysis revealed that VEGF-D protein expression was an independent factor affecting lymph node metastasis (p < 0.01). Nonetheless, the presence or absence of VEGF-D protein had no significant impact on the survival of the patients (p = 0.15). Conclusion: These results suggest that the expression of VEGF-D protein could be useful in predicting the nodal status of colorectal carcinoma patients.Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

We investigated c-Met expression in cultured astrocytes and their regulation by cytokines. Immunocytochemistry revealed that c-Met was expressed in cultured astrocytes. Western blotting revealed that acidic and basic fibroblast growth factor (FGF) enhanced and hepatocyte growth factor (HGF) reduced c-Met expression. Reverse transcription-polymerase chain reaction revealed that FGFs and HGF enhanced c-met expression. These findings suggest that c-Met expressed in astrocytes may have important roles during the nervous system regeneration. [Copyright &y& Elsevier]

Lumican is a member of a small leucine-rich proteoglycan family. We previously found that lumican mRNA and its protein were ectopically and highly expressed in acinar cells in chronic pancreatitis (CP)-like lesions close to pancreatic cancer cells. CP-like lesions are characterized by acinar and ductal-ductular cell proliferation with expanding fibrosis. This finding suggests that lumican is ectopically synthesized by acinar cells under chronic inflammatory conditions and plays a role in fibrosis of the pancreas. However, the expression and role of lumican in acute inflammatory changes of the pancreas are not completely elucidated. In the present study, we aim to clarify whether lumican mRNA and its protein are expressed in exocrine or endocrine components in acute pancreatitis (AP). For experimental AP, Wistar rats received an intraperitoneal injection of l-arginine. Western blot analysis showed an intense 50-kDa band corresponding to the lumican protein ín normal and l-arginine-treated rat pancreas. After l-arginine injection, three intense bands at 42, 57, and 92 kDa were detected on day 1. Immunohistochemically, the lumican protein was localized in ductal and a few centroacinar cells in the normal pancreas. After l-arginine injection, an immature fibrosis with fragmented and loose collagen fibers was observed in AP on day 4 and lumican immunoreactivity was detected in the collagen fibers. Lumican mRNA was faintly detected in islet cells in the normal pancreas, but it was strongly expressed in acinar and islet cells on day 1. Furthermore, lumican mRNA was expressed in many proliferating fibroblasts on day 4 by in situ hybridization. These findings indicate that lumican is transiently synthesized by acinar cells and fibroblasts in AP. Lumican proteins synthesized by acinar cells, islet cells, and fibroblasts may contribute to immature and transient fibrosis of AP. [Copyright &y& Elsevier]

The coding region of guinea pig peroxisome proliferator activated receptor gamma1 (gpPPARγ1) cDNA was successfully cloned from adipose tissue by reverse transcription polymerase chain reaction (RT-PCR) using the designated primers based on the conserved regions of the other mammalian PPARγ1 sequence. From RT-PCR, a combination of three cDNA fragments that comprised of the full length coding region PPARγ1 cDNA gene were amplified, with the size of 498, 550 and 557 bp, respectively. All three fragments were then successfully assembled by utilising the internal restriction sites present at the overlapping regions to give rise to the full-length coding region of gpPPARγ1 with the size of 1428 bp and consisting of 475 amino acids. Guinea pig PPARγ1 is highly conserved with those of other species at protein and nucleotide levels. Gene expression studies showed that gpPPARγ mRNA was predominantly expressed in adipose tissue followed by lung and spleen. However, at the protein level, PPARγ was also found to be expressed in skeletal muscle. [Copyright &y& Elsevier]

ABSTRACT: We previously cloned cDNA of type V/XI collagen α1 chain (ColVa1) gene from cultured cells derived from red sea bream embryo. We raised an antibody against the deduced C-telopeptide of ColVa1 in order to detect the translation products of this cDNA and their degradation products in red sea bream muscle. To improve its specificity, the antibody was purified from rabbit antiserum by use of an affinity column cross-linked with recombinant C-terminal peptide of ColVa1 produced by Epicurian coli. The purified antibody recognized a band corresponding to the α chain of type V/XI collagen in western blot analysis of the extract of cultured cells. The antibody also recognized two bands in acid-soluble and pepsin-solubilized collagens, indicating that the translation products of the ColVa1 gene are present in muscle and that bands correspond to α and β chains of type V/XI collagen. A band corresponding to a molecular weight of approximately 65 k was detected in the NaOH extracts of muscle, suggesting that type V/XI collagen α1 chain is restrictedly digested in red sea bream muscle. [ABSTRACT FROM AUTHOR]

Several neuronal cell lines were screened for endogenous expression of neuronal nitric oxide synthase (nNOS) protein using Western blot analysis. Detectable levels of the nNOS protein were evident in the SK-N-SH, SH-SY5Y, and N1E-115 neuroblastoma cell lines, as well as the NG108-15 neuroblastoma × glioma hybrid. Only trace amounts were visible in Neuro2A human neuroblastoma cells. The presence of endogenously expressed nNOS in these cells may allow for the study of the interaction between nNOS and the endogenous receptor systems expressed in the same cells. [Copyright &y& Elsevier]

The active form of human blood coagulation factor X (FXa, EC 3.4.21.6) showing N-α-Benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine- p-nitroanilide (S-2222) hydrolyzing activity was first detected in human semen (seminal plasma) by affinity chromatography using anti-human coagulation factor X, and this enzyme activity was inhibited by anti-human FX. This enzyme has been associated with the human coagulation factor X (FX) in human semen (seminal plasma) by Western blot analysis, and the molecular mass of mature FX was also estimated to be 59 KDa by SDS-PAGE and Western blot analysis. [ABSTRACT FROM AUTHOR]

We investigated the effects of NG-nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor, on retinal development in the postnatal rat by immunocytochemistry and immunoblotting using antisera against neuronal nitric oxide synthase (nNOS) or growth-associated protein 43 (GAP-43). An nNOS-immunoreactive band of 155 kDa and a GAP-43-immunoreactive band of 48 kDa were present in the extracts of both control and l-NAME-treated rat retinas. The intensity of the nNOS-immunoreactive band was much weaker in the treated rats, whereas the intensity of the GAP-43-immunoreactive band of 48 kDa was much stronger in the treated rats. Much stronger GAP-43 immunoreactivity was visible in the inner plexiform layer (IPL) of the treated retinas at P10, P14 and P21. Our findings suggest that NO may play an important role in the maturation of the IPL in the developing rat retina. [Copyright &y& Elsevier]

Seven cross-bred dogs were inoculated with Angiostrongylus vasorum and serum samples were analyzed using the enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). ELISA detected specific antibodies anti-A. vasorum, from 14 to 28 days after inoculation (DAI) and persisted throughout the experiment. Using WB, the main antigens detected had molecular weight of approximately 115, 102, 86, 76, 69, 56, 41, 32, 28, 20–22 and 10 kDa. [Copyright &y& Elsevier]

Abstract Expression patterns of phospholipase D1 (PLD1) in the developing rat retina were investigated using immunocytochemistry and Western blot analysis and compared with the expression patterns of glutamine synthetase. PLD1 immunoreactivity appeared first in a few neuroblasts in the middle of the mantle zone of the primitive retina by embryonic (E) day 13. PLD1-immunoreactive primitive ganglion cells were characterized in the ganglion cell layer by E17. Faint immunoreactivity at E17 profiled radially orientated cells and this pattern appeared up to postnatal (P) day 7. In the ganglion cell layer at P3, displaced amacrine cells and ganglion cells were classified. At P5, presumptive horizontal cells and amacrine cells were identified. By P7, a thin outermost layer of newly formed segments of the photoreceptor cells was also PLD1 immunoreactive. PLD1 immunoreactivity at P8 was limited to radial Müller cells and the outer segment layer of the photoreceptor cells, and the expression pattern was conserved to adulthood. Western blot analysis showed relatively high amounts of PLD1 protein at E17 and P3, a decrease at P7, and moderate amounts from P8 onward. Co-expression of PLD1 with glutamine synthetase in the retina appeared first after birth in differentiating neurons and in Müller cells by P8; thereafter the pattern was maintained. The expression pattern of the PLD1 during development of the retina suggests that PLD1 plays important roles in glutamate-associated differentiation of both specific neurons and radial glial cells, and in glutamate-mediated cellular signalling in Müller cells. [ABSTRACT FROM AUTHOR]

Antigens recognized by host species in response to ectoparasite infestation have been widely reported. Although differences in the immune responses of different host species have been described, only a very few of these studies compare the range of antigens recognized by different host species in response to infestation. We used Western blot analysis to investigate antigenic responses of different host species that were repeatedly infested with Ixodes ricinus ticks. Antigenic profiles of larval and nymphal whole tick homogenates were compared with the respective salivary gland extract (SGE) samples using sera from rabbits repeatedly infested with either adults, nymphs or larvae. SGE samples were also analysed using sera from hamsters infested with adults, nymphs or larvae. Sera from BALB/C mice, Apodemus flavicollis (yellow-necked mouse) or Clethrionomys glareolus (bank vole) repeatedly infested with larvae were used to compare the antigenic profiles of SGE and larval homogenate samples. We also investigated different sources of tick antigens, using rabbit sera, by comparing midgut extracts from female adult ticks and SGE from unfed ticks and from ticks throughout the 6-day feeding period with whole tick homogenates of female and male adults, nymphs and larvae. The pattern of antigenic tick-molecules recognized by infested host species varies with the period of feeding, developmental stage and the particular host species parasitized. [ABSTRACT FROM AUTHOR]

Several lines of evidence including reverse transcription polymerase chain reaction, immunoreactivity and their ability to efflux rhodamine 123 have implied the existence of P-glycoprotein in natural killer (NK) cells. It has been a natural tendency to assume that NK-cell P-glycoprotein is identical to the P-glycoprotein of multidrug resistant (MDR) cell lines, however, the present study uncovered major differences. Functionally, NK cells demonstrated a restricted substrate profile, being unable to transport daunorubicin and calcein acetoxymethylester while efficiently transporting other P-glycoprotein substrates. Furthermore, physical differences in NK-cell P-glycoprotein were established by differential reactivity with P-glycoprotein antibodies. NK cells demonstrated strong reactivity with C494 and JSB-1, but did not react appreciably with C219. In addition, NK cells were unable to bind to the antibody MM4·17 unless they had been fixed and permeabilized, yet this antibody normally recognizes an extracellular epitope of P-glycoprotein. These differences culminated in the demonstration using Western analysis that NK cells did not express detectable levels of 170 kDa P-glycoprotein. Instead, NK cells expressed small-molecular-weight ‘mini P-glycoprotein’ products, of approximately 70 and 80 kDa. Collectively, these data indicate that the predominant P-glycoprotein species of NK cells are novel mini P-glycoproteins and not the classic P-glycoprotein of MDR models. [ABSTRACT FROM AUTHOR]

Phospholipase C (PLC)-γ1 is known to play a central role in ligand-mediated signal transduction for cell proliferation. The purpose of this study was to elucidate the distribution of PLC-γ1 in deep meatal skin, retroauricular skin and cholesteatoma matrix of experimental animals. We induced canal ligation cholesteatoma using Mongolian gerbils and investigated the expression of PLC-γ1 in this model by using Western blot analysis and immunohistochemistry. By Western blot analysis, considerably higher levels of PLC-γ1 protein were detectable in experimental cholesteatoma. On immunohistochemistry, experimental cholesteatoma showed more intense immunolabeling of PLC-γ1 protein than deep meatal skin and retroauricular skin. In conclusion, overexpression of PLC-γ1 may in part contribute to abnormal proliferation and differentiation of experimental cholesteatoma in Mongolian gerbils. [ABSTRACT FROM AUTHOR]

Several annual and perennial species in the genus Glycine Willd., including G. soja, long-pod G. tomentella, short-pod G. tomentella and G. tabacina, collected in Taiwan and nearby islands were studied for variations of their seed proteins. SDS-PAGE and Western blotting were used to analyze the total proteins, the heat soluble proteins, six seed maturation proteins (GmPMs) and one seed storage protein. The various species had different patterns of seed heat soluble proteins. In addition,each species of Glycine collected in Taiwan exhibited unique seed maturation protein patterns. They had several cross-reactive polypeptides recognized by specific antibodies against GmPM1, GmPM2 and GmPM8, but only one polypeptide recognized by antibodies against GmPM4, GmPM5 and MP130. The long pod G. tomentella, which has been suggested as a new species and renamed as G. dolichocarpa, could be distinct from the short pod G. tomentella on the basis of the analysis of these biochemical markers. It is also indicated that these GmPM antibodies may be used to distinguish between and within other Glycine species. [ABSTRACT FROM AUTHOR]

The relationship between the density of ionic currents through major two channels, voltage-dependent L-type Ca2+ channels (L-type VDCC) and large-conductance, Ca2+-activated K+ channels (BKC), and the mRNA expression levels of α1C subunit of L-type VDCC (α1C) and α/β subunits of BKC (αBK/βBK) were compared in smooth muscle cells (SMC) of rabbit aorta and vas deferens using whole cell-voltage clamp and reverse transcription-polymerase chain reaction (RT-PCR) analyses. The density of the currents through VDCC (ICa) and BKC (IK,Ca) at +10 mV in aortic SMC was approximately one-seventh and one-sixth respectively of that in vas deferens. Whilst application of the Ca2+ channel agonist Bay K 8644 increased ICa by 75–90% in these SMC, the increase in IK,Ca was far greater in aorta than in vas deferens. The expression of the α1C transcript in vas deferens was approximately 3.5 times higher than that in aorta. In contrast, expression of αBK/βBK was almost identical in both tissues, indicating the dissociation of IK,Ca density from the expression levels of BKC transcripts in aorta. The results were supported by Western blot and immunocytochemical analyses using subunit-specific antibodies. The lower Ca2+ influx through VDCC in aorta activates only a very limited fraction of BKC compared with that in vas deferens. The greater expression of BKC than of VDCC in aortic SMC contributes to a strong negative feed-back mechanism that minimizes membrane depolarization and acts as a safety margin to maintain low membrane excitability. [ABSTRACT FROM AUTHOR]

The expression of phosphatidylinositol-3 kinase in tumors and homologous tissues from 29 patients with lung cancer, 5 patients with lung metastases of various tumors, and some non-tumorous pulmonary diseases was studied by Western blot analysis. The expression of phosphatidylinositol-3 kinase was increased in these tumors in comparison with histologically intact lung tissue in 5 patients with non-small-cell cancer. In 20 patients expression of phosphatidylinositol-3 kinase was the same as in homologous tissue and in 4 patients it was decreased. No relationship between phosphatidylinositol-3 kinase expression and clinical and morphological characteristics of lung cancer was revealed. [ABSTRACT FROM AUTHOR]

The localization of nitric oxide synthase (NOS) was investigated in the developing rat retina by immunocytochemistry and western blot analysis, using an antiserum directed against neuronal NOS. NOS-labeled cells were first detected at postnatal day 5 (P5) in the inner row of the neuroblastic layer. These cells were considered to correspond to the type 1 cell of the adult rat retina. Type 2 cells, characterized by a small soma and weak immunoreactivity, and a class of displaced amacrine cells were detected at P9 and P7, respectively. By P14 or P15, the time of eye opening, NOS immunoreactivity appeared in some bipolar cells. NOS was first expressed at the protein level at P9. Thereafter, quantitative evaluation by immunoblotting confirmed that the intensity of the immunoreactive bands increased abruptly, reaching the same value as is found in the adult retina at P21. Our results demonstrate that differentiation of NOS-labeled cells follows a discrete developmental pattern and is most active during the 2nd postnatal period in the rat retina. [ABSTRACT FROM AUTHOR]

β-Catenin acts as a key mediator of the Wnt/Wingless signaling pathway involved in cell proliferation, differentiation and survival. Recent studies have shown that an unstable interaction between β-catenin and the mutant presenilin-1 induces neuronal apoptosis, and that β-catenin levels are decreased in the brains of patients with Alzheimer’s disease (AD). Since activated microglia and astrocytes play a role in the process of neuronal degeneration in AD, the cytokine/growth factor-regulated expression of β-catenin in human neural cell lines, including NTera2 teratocarcinoma-derived differentiated neurons (NTera2-N), IMR-32 neuroblastoma, SKN-SH neuroblastoma and U-373MG astrocytoma, was studied quantitatively following exposure to epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, interferon (IFN)-γ, transforming growth factor (TGF)-β1, dibutyryl cyclic adenosine 3′,5′-cyclic monophosphate (cAMP) (dbcAMP) or phorbol 12-myristate 13-acetate (PMA). β-Catenin mRNA expressed constitutively in all of these cell lines was unaffected by treatment with any factors examined. In contrast, β-catenin protein levels were reduced markedly in NTera2-N cells by exposure to dbcAMP, EGF or bFGF, and in U-373MG cells by treatment with dbcAMP or PMA, but were unaffected in any cell lines by BDNF, TNF-α, IL-1β, IL-6, IFN-γ or TGF-β1. These results indicate that β-catenin is expressed constitutively in human neural cells and downregulated at a protein level by a set of growth factors in a cell type-specific manner. [ABSTRACT FROM AUTHOR]

We isolated a rat glia maturation factor-λ (rGMFG) cDNA and examined the tissue distribution of GMFG in rat by Northern and Western blot analyses. Sequence analysis of the entire cDNA revealed an open reading frame of 426 nucleotides with a deduced protein of 142 amino acid residues. The deduced amino acid sequence of the putative product is highly homologous (78.9%) to rat glia maturation factor-β (rGMFB). Northern blot analysis indicated that a 0.9-kb mRNA is predominantly expressed in rat thymus, testis, and spleen. GMFG showed a different tissue distribution from GMFB, being present predominantly in proliferative and differentiative organs. [ABSTRACT FROM AUTHOR]

This chapter presents a protocol for studying the effects of curcumin in a colorectal cell line and a mouse model of colitis-associated colon carcinogenesis. The protocol using the CT26 cell line incorporates cell proliferation, migration, invasion, spheroid formation, cell cycle, polymerase chain reaction (PCR), and western blot analyses. For the mouse model, this involved a macroscopic and histological examination of the colon and assays for oxidative damage markers., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This study aimed to evaluate the interspecies difference in metabolism of mulberrin and examine the interaction between mulberrin and CYP enzymes or recombinant human uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) enzymes. Liver microsomes from human (HLMs), Beagle dog (DLMs), minipig (PLMs), monkey (MLMs), rabbit (RLMs), rat (RAMs), and mouse (MIMs) were used to investigate metabolic diversity among different species. Additionally, recombinant human supersomes were used to confirm that metabolic enzymes are involved in the biotransformation of mulberrin. We also evaluated the influence of mulberrin on protein expression by Western blot analysis. Mulberrin metabolism showed significant interspecies differences. We found four and two metabolites in phase I and II reaction systems, respectively. In phase I metabolism profiles of mulberrin for HLMs, PLMs and MLMs conformed to the classic Michaelis-Menten kinetics, RAMs and MIMs followed biphasic kinetics; phase II reaction of mulberrin in HLMs, DLMs, PLMs, MLMs, RLMs, RAMs and MIMs followed biphasic kinetics. UGT1A1 were the major CYP isoforms responsible for the metabolism of mulberrin. Mulberrin showed potent inhibitory effects against CYP3A4, CYP2C9, CYP2E1, UGT1A1, UGT1A3 and UGT2B7 with IC 50 values of 54.21, 9.93, 39.12, 3.84, 2.01, 16.36 µM, respectively. According to Western blot analysis, mulberrin can upregulate the protein expression of CYP2C19, and downregulate the expression levels of CYP3A5 and CYP2C9 in HepG2 cells as concentration increased. The interspecies comparisons can help find other species with metabolic pathways similar to those in humans for future in vivo studies.

We performed Western blot analysis to detect anticerebellar antibodies in the serum of patients with Miller Fisher syndrome (MFS). We studied 7 MFS patients, 6 Guillain-Barré syndrome (GBS) patients and 10 normal healthy persons as controls. Six MFS patients (86%) had IgG antibodies against mouse cerebellar protein, whereas 3 GBS patients (50%) and 4 healthy controls (40%) had antibodies. The mean number of antibodies in the serum of patient with MFS was 2.43, which was significantly more than that of GBS patients (mean 0.67) and healthy control (mean 0.70). This finding suggests variability and complexity of target in nervous systems that suffer aberrant immunity in MFS, and may also reflect the variability and heterogeneity of the pathogenesis of this syndrome.Copyright©1999S.KargerAG,Basel [ABSTRACT FROM AUTHOR]

The 14-3-3 protein belongs to a family of 30-kD proteins originally identified by two-dimensional analysis of brain protein extracts. Recently, the detection of the 14-3-3 protein in the cerebrospinal fluid (CSF) is utilized as a highly reliable test for the premortem diagnosis of prion diseases such as Creutzfeldt-Jakob disease. For the initial step, to clarify the biological implication of the CSF 14-3-3 protein in these diseases, its expression was investigated in neural tissues and cultures and CSF samples from patients with a variety of neurological diseases by Western blot analysis and immunocytochemistry. The constitutive expression of the 14-3-3 protein was identified in all neural and nonneural tissues examined. It was expressed in all neurons, astrocytes, oligodendrocytes, and microglia in culture with its location in both cytoplasmic and nuclear regions. The 14-3-3 protein was detected in the CSF of 8 out of 71 patients, including 1 Gerstmann-Sträussler-Scheinker disease patient and 7 patients with prion-unrelated neurological diseases, such as meningoencephalitis of viral, bacterial, or tuberculous origin, multiple sclerosis, and mitochondrial myopathy, encephalopathy, lactic acidosis, and strokelike episodes. These results suggest that the 14-3-3 protein expressed constitutively at substantial levels in both neurons and glial cells might be released into the CSF as a disease-nonspecific consequence of the extensive brain damage and indicate that the analysis of the 14-3-3 protein in the CSF is not useful as a screening test for prion diseases. [ABSTRACT FROM AUTHOR]

The flavonoid 3′,5′-hydroxylase (F3′,5′H) gene is in the NADPH-Cytochrome P450 reductase family. A partial F3′,5′H cDNA was cloned by RT-PCR from corollas of Gentiana scabra. Activity of F3′,5′H was observed during flower development, and activity was highest at the stage prior to flower opening. The enzyme activity was increased with the addition of SO μM gibberellic acid (GA3) immediately prior to flower opening. Western blot analysis showed that purified F3′,5′H protein from G. scabra was cross-reacted with rabbit anti-Gentiana triflora NADPH-Cyt P450 reductase antibodies. Only one 80 to 82 kDa protein was detected by western blot analysis of fresh microsomal extracts. The enzyme activity was not distinctly changed by adding 50 μM GA3 to corollas. Therefore, we conclude that GA3 controls the expression of the F3′,5′H gene at the transcriptional level, not at the translational level. [ABSTRACT FROM AUTHOR]

An H+-K+-adenosinetriphosphatase (H+-K+-ATPase) contributes to potassium reabsorption by the collecting ducts of the rat kidney. mRNAs for two isoforms of the H+-K+- ATPase, HKa1 and HKa2, have been found in the rat kidney. To evaluate whether the HKa1 and HKa2 proteins are present in the rat kidney, microsomes enriched in HKa1 or HKa2 were isolated using the MiniMac magnetic separation system with antibodies directed against either HKa1 (HK 12.18) or HKa2 (AS 31.7). Immunoblots of rat kidney microsomal protein isolated with HK 12.18 revealed a band ~94 kDa in size that comigrated with the Gl fraction of the stomach. Immunoblots of rat kidney microsomal protein isolated with AS 31.7 revealed a band slightly greater than 94 kDa that comigrated with a band obtained from rat colonic microsomal protein. To examine the effect of perturbations in potassium metabolism, the abundance of the HKa1 and HKa2 isoforms was compared in rats fed a normal or potassium-deficient diet. A lowpotassium diet increased the abundance of HKa2, whereas that of HKa1 was not altered. These data suggest that HKa2 might be the isoform responsible for potassium conservation by the kidney. [ABSTRACT FROM AUTHOR]

We investigated the presence of retroviral protein in the pancreatic islets of non-obese diabetic mice to prove that the virus-like particle observed specifically in the pancreatic Beta cell of these mice was retrovirus. Western blot analysis probed with anti-retrovirus antibody demonstrated the existence of retroviral gag (group specific antigen) protein p30 in the islets of female non-obese diabetic mice. Islets of non-obese diabetic mice which were treated with cyclophosphamide, known to accelerate the development of insulitis and diabetes mellitus, have shown both a significantly increased number of retrovirus-like particles (type C) and enhanced expression of gag protein p30, compared to those of mice not treated with cyclophosphamide. These results confirmed the presence of type C retrovirus in non-obese diabetic mouse Beta cells and suggest a role for retrovirus in the development of insulitis and diabetes in these mice. [ABSTRACT FROM AUTHOR]

Both in histologically proven (N = 3) and clinically diagnosed (N = 3) definite (peri)myocarditis in children, as well as in probable (N = 6) and possible inflammatory perimyocardial disease (N = 31), antibodies directed against the sarcolemma and myolemma (AMLAs) were demonstrated. In the acute phase AMLAs of the IgM class and complement fixation prevailed in the serum and the biopsy specimens. Complement dependent cytolysis by the patients' sera in the acute phase may simulate in vitro what could also be operative in vivo: a secondary immunopathogenesis due to cytolytic antimembrane antibodies. [ABSTRACT FROM PUBLISHER]

The rolC gene of Agrobacterium rhizogenes codes for a peptide with an apparent molecular weight of approximately 20 kDa. Immunolocalization of the rolC peptide, in leaves of transgenic plants which are genetic mosaics for the expression of the rolC gene, is restricted to the phenotypically altered sectors. Subcellular fractionation of homogenates from 35S- rolC transgenic leaves shows the cytosolic localization of the rolC peptide. [ABSTRACT FROM AUTHOR]

Accumulated evidence implicates immunological alterations in endometriosis. The purpose of this study was to look for variations in antibodies to distinct antigens in peritoneal fluid of women with and without endometriosis. Peritoneal fluid was aspirated from 17 women undergoing laparoscopy for tubal ligation and 37 patients complaining of symptoms of pain and/or infertility. Peritoneal fluid antibodies to a standard preparation of peritoneal fluid antigens were detected by Western blot analysis using peroxidase-labelled anti-human immunoglobulin G antibodies specific to the Fc region. Antibodies to distinct antigens were quantified by estimating the ratio of the relative optical density between samples and a standard amount of antibodies. Marked changes were found in the antibody detection to two antigens having apparent molecular weights of 22 and 18 kDa. The intensity of the antibody signal was significantly weaker in the peritoneal fluid from endometriosis patients (0.36 ± 0.06 and 0.46 ± 0.06) compared with that in women without endometriosis (0.62 ± 0.08 and 0.75 ± 0.06). It was also weaker in patients without endometriosis presenting with infertility (036 ± 0.07 and 0.47 ± 0.08), but only the 18 kDa antigen result was significant After adjusting for infertility, the P values for the 18 and 22 kDa bands were 0.03 and 0.28 (not significant) respectively in the group of endometriosis patients. These changes were not related to the phase of the menstrual cycle. These data suggest an alteration in the immune response to two distinct antigens in the peritoneal fluid from women with endometriosis and infertility. Further evaluation of these two antigens and their antibodies would be of interest to help understand endometriosis and its associated infertility. [ABSTRACT FROM PUBLISHER]

Immunological cross-reactivity between Mycobacterium bovis BCG stress protein (heat shock protein: HSP64) and Meth A tumour cells was analysed by using anti-BCG HSP64 mAb whose recognition epitopes were characterized against BCG HSP64 peptides. By indirect immunofluorescence analysis (IIFA), it was found that one of seven anti-BCG HSP64 mAb, XVIIIG1, bound to the cell surface of Meth A in BALB/c mice. This result was further confirmed by western blot analysis, demonstrating the presence of a 64 kDa protein which reacted with mAb XVIIIG1 that recognizes the 110-123 amino acid peptide of the BCG HSP64. Comparison of the amino acid sequence between the mouse HSP65 and BCG HSP64 recognized by mAb XVIIIG1 revealed 50% amino acid sequence homology. It was concluded from these results that Meth A tumour cells continuously express the stress protein HSP64 as a kind of tumour-associated antigen on the surface of tumour cells. [ABSTRACT FROM AUTHOR]

Allergic bronchopulmonary aspergillosis patients (ten in the age group 5-13) were studied with respect to clinical and immunodiagnostic tests. All the patients showed wheal and flare reaction with A. fumigatus antigens. Clinical features included bilateral infiltration and hilar lymphadenopathy. Eosinophilic count in these patients was observed to be in the range of 300-2500/mm3. Sera of six patients indicated precipitin reaction with A. fumigatus antigens. An elevated total serum IgE was noticed in all the patients. A. fumigatus specific IgG and IgE antibodies were increased in these patients. Optical density of the sera of all the patients against controls were observed to be in the range of (0.426 to 1.8 for IgG and 0.147 to 0.562 for IgE) by ELISA. Western blot analysis indicated that there may be correlation between the clinical stages of the disease and immunological reactivity of the sera with various antigenic components. [ABSTRACT FROM AUTHOR]

Four isoforms of the extracellular lignin peroxidase of the ligninolytic actinomycete Streptomyces viridosporus T7A (ALip-P1, P2, P3, and P4) were individually purified by ultrafiltration and ammonium sulfate precipitation, followed by electro-elution using polyacrylamide gel electrophoresis. Three of the purified peroxidases were compared for their immunologic relatedness by Western blot analysis using a polyclonal antibody preparation produced in rabbits against pure isoform P3. The anti-P3 antibody was also tested for its reactivity towards a lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium and another ligninolytic actinomycete Streptomyces badius 252. Results showed that peroxidases ALip-P1 through ALip-P3 are immunologically related to one another. The peroxidases of S. badius, but not the peroxidase of P. chrysosporium, also reacted with the antibody, thus indicating that the lignin peroxidases of S. viridosporus and S. badius are immunologically related. Based upon its specific affinity, lignin peroxidase isoform ALip-P3 of S. viridosporus was readily purified using an anti-P3 antibody affinity column. [ABSTRACT FROM AUTHOR]