Immunohistochemical studies on S-100 cells in the anterior pituitary gland of Sprague Dawley rats and spontaneous dwarf rats

The S-100 cells in the pituitary glands of adult male Sprague Dawley rats (SDs) and spontaneous dwarf rats (SDRs) were immunohistochemically examined using anti-S-100 and anti-S-100 monoclonal antibodies. The immunoreactive cells against S-100 protein were divided into three subtypes on the basis of their immunore-activity against subunits of S-100 protein: S-100 dominant type (the -type cell), S-100 dominant type (the \-type cell) and immunoreactive against both S-100 and S-100 (the -type cell). In the SD, -type cells represented 26% of the total S-100 immunoreactive cells (S-100 cells) and were localized in the peripheral area of the ventral region of the pituitary gland. This type of cell was observed forming clusters, with more abundant cytoplasm than the -type cell. The proportion of -type cells was 53%. They were diffusely distributed throughout the gland, and their processes were thicker than those of the -type cell. In the SDR, the proportion of -type cells was 55%, and they were observed throughout the gland. In contrast, -type cells totalled 12% and were localized in small areas of the central and peripheral region of the gland. The proportion of -type cells was 21% in the SD and 33% in the SDR and they were observed forming small clusters in both animal groups. The proportion of -type cells compared with the total of S-100-immunoreactive cells was significantly higher (P < 0.05) in the SDR than in the SD, while the proportion of -type cells was markedly lower (P < 0.05).     

Erythromycin and cycloheximide sensitivities of protein and RNA Synthesis in sporulating cells of Saccharomyces cerevisiae: Environmentally induced modifications controlled by chromosomal and mitochondrial genes

Summary The purpose of the experiments reported below was to examine the response in sporulation medium of the three diploid cell types MAT MAT, MAT MAT (asporogenic diploids) and MAT MAT (sporogenic diploid) to erythromycin, a specific inhibitor of mitochondrial protein synthesis (MPS) in vegetative cultures, and cycloheximide, a specific inhibitor of cytosol protein synthesis (CPS) in vegetative cultures. When MAT MAT diploids are transferred to sporulation medium a significant fraction of total protein synthesis (CPS + MPS) becomes sensitive to erythromycin in contrast to the behavior of MATa MATa and MAT MAT diploids in which the resistance of CPS to erythromycin is maintained. The decompartmentalization of erythromycin sensitivity is thus cell type specific. Erythromycin stimulates total RNA synthesis of MAT MAT cells in sporulation medium but not of MAT MAT and MAT MAT cells. Cycloheximide inhibits protein synthesis and stimulates RNA synthesis in all three diploid cell types. An erythromycin resistant mutant, shown to be due to a mutation of the mitochondrial genome, exhibited only partial resistance of CPS to erythromycin in sporulation medium in the background of the MAT MAT mating type genotype. Total RNA synthesis in this mutant was not stimulated. The results reported indicate that mitochondrial functions during sporulation are not restricted to those involving respiratory metabolism.     

αv Integrins mediate adhesion and migration of breast carcinoma cell lines

Integrins with the _v subunit are involved in cell adhesion and cellular signaling. Some _v integrins have been associated with tumor progression and dissemination. The objective of this study was to assess the contribution of _v integrins to the adhesive and migratory behavior of cells derived from breast carcinoma (BCA). The expression and function of _v integrins was characterized in three BCA cell lines which exhibit different metastatic potentials. These include MCF-7 cells which metastasize inefficiently, MDA-MB-231 cells, which have a moderate metastatic potential, and MDA-MB-435 cells, which metastasize extensively. Each cell type displays a different repertoire of _v integrins on the cell surface. The complement of _v integrins on each cell type influences their ability to adhere and migrate. The most striking difference among these cell lines was the expression of the _{v3 3} integrin. The highly metastatic MDA-MB-435 cells express substantial levels of this receptor, whereas MDA-MB-231 and MCF-7 cells do not. The MDA-MB-435 cells showed a greater ability to adhere and to migrate and this functional difference can largely be attributed to the expression of _v3 integrin. This characterization is a first step toward determining the role of _v integrins in animal models of BCA metastasis, and lends support to the hypothesis that the _v3 integrin can be a contributing factor in metastatic disease. © Rapid Science 1998     

A comparative immunohistological study of cerebellar enolases

Summary A comparative immunohistological study of the neurone-specific enolase and enolase, demonstrates the exclusive neuronal localization of enolase and its absence from glial cells. In contrast, enolase is located in astroglial cells. The validity of enolase as a neuronal marker and enolase as an astrocytic marker, is confirmed both by a double labelling technique, using antibodies to and to revealed with fluorescence or peroxidase in the same tissue sections, and by immunoelectronmicroscopy.     

Synergistic effects of tumour necrosis factor-α and interferon-γ on feline haemopoietic progenitors

Pathogenic mechanisms that underlie feline leukaemia virus subgroup-C (FeLV-C) induced erythroid aplasia are unknown. FeLV-C infection is associated with higher serum levels of interferon- (IFN-) and tumour necrosis factor- (TNF-), which may act synergistically to cause haemopoietic suppression. In the present studies, the synergistic effects of TNF- and IFN- on feline bone marrow progenitors in vitro were evaluated. Bone marrow mononuclear cells from specific-pathogen-free cats were exposed to TNF- (100 and 200 pg/ml) and IFN- (100 or 200 units/ml), alone or in combination, for 2 h before plating for clonal assays of colony forming units. Our results show that TNF- and IFN- in combination caused marked suppression of feline colony forming units-erythroid (CFU-E), burst forming units-erythroid (BFU-E), and colony forming units-fibroblasts (CFU-F), whereas colony forming units-granulocyte/macrophage (CFU-GM) were minimally affected. The same concentrations of TNF- and IFN- alone had minimal effects on CFU-E, BFU-E and CFU-F. These results suggest that TNF- and IFN- may play a significant role in regulating haemopoiesis in cats and may be involved in the pathogenesis of erythroid aplasia in cats infected with feline leukaemia virus.     

Immunoreactivity of the JK-132 monoclonal antibody directed against basement membrane collagen in normal and diabetic glomeruli

The possible involvement of basement membrane-associated collagen (recognized by the monoclonal antibody JK-132) in the evolution of diabetic nephropathy was studied in kidney specimens from seven patients with noninsulin-dependent diabetes mellitus, and its distribution was compared with those of antibodies against 1 to 4 chains of type IV collagen. JK-132, a monoclonal antibody against basement membrane-associated collagen, reacted immunohistochemically exclusively with the mesangial matrix of the glomerular capillary. In contrast, antibodies to the 1 and 2 chains (IV) reacted strongly with mesangial matrix, and less strongly with the glomerular basement membrane (GBM). Antibodies to the 3 and 4 chains (IV) reacted mainly with GBM. In diabetes, JK-132 reacted most extensively with the expanded mesangial matrix, its staining intensity increasing with progression of the diabetic glomerulosclerosis. Antibodies to the 1 and 2 chains (IV) reacted prominently with the expanded mesangial matrix but less strongly with the GBM. Antibodies to the 3 and 4 chains reacted intensely with the thickened GBM. These results suggest that basement membrane-associated collagen differs from 1 to 4 chains of type IV collagen and that basement membrane-associated collagen is a good marker of mesangial expansion in diabetic nephropathy.     

Immunohistochemical distribution of S-100 protein and glial fibrillary acidic protein in normal and neoplastic salivary glands

Summary Immunohistochemical localization of S-100 protein, its and subunits, and glial fibrillary acidic protein (GFAP) in normal and neoplastic salivary glands was studied by the peroxidase-antiperoxidase method and immunoblot analysis. Positive immunostaining for S-100 protein was observed in pleomorphic adenoma, adenolymphoma, tubular adenoma, adenoid cystic carcinoma, acinic cell tumour, adenocarcinoma and carcinoma in pleomorphic adenoma. S-100 protein was localized in myoepithelial cells, epithelial cells of intercalated ducts and serous acinar cells of normal salivary gland. Both and subunits of S-100 protein showed almost identical distribution in normal and neoplastic salivary glands, but skeletal muscle cells were -positive/-negative whereas Schwann cells and fat cells were -negative/-positive in the stroma and neighbouring tissue. GFAP was only found in pleomorphic adenoma and its malignant counterpart. Immunoblot analysis showed that the GFAP-related antigen consisted of several polypeptide bands with a molecular weight ranging between 35,000 to 50,000 daltons.     

Transport and utilization of α-ketoglutarate by the rat kidney in vivo

In order to establish the characteristics of net renal transport and utilization of -ketoglutarate (-KG) in the rat, we have precisely quantified the renal blood flow, the urinary flow and the rates of -KG delivery, filtration, reabsorption or secretion, excretion, uptake or production by an in vivo rat kidney preparation. In normal rats, -KG uptake was higher than -KG reabsorption at both endogenous and elevated plasma -KG concentrations; thus, a net peritubular transport, which was the main supplier of -KG to the renal cells, took place. Saturation of reabsorption and peritubular transport of -KG occurred at blood -KG concentrations about 30 and 150 times above normal, respectively. Acute metabolic acidosis was found to have no effect on renal handling of -KG. At endogenous plasma -KG concentrations, alkalosis converted net renal uptake into net renal production of -KG resulting in addition of -KG by the renal cells both to blood and to the luminal fluid. Elevation of blood -KG concentration restored the renal uptake of -KG. This uptake, which was entirely accounted for by the peritubular transport of -KG, reached a maximum which was lower than that observed in normal and acidotic rats.     

An α-mating-type-specific mutation causing specific defect in sexual agglutinability in the yeast Saccharomyces cerevisiae

We have identified a recessive -mating-type-specific gene agl causing agglutinability defect without significant effects on other sexual activities. a cells carrying agl showed sexual agglutination with cells but cells carrying agl showed sexual agglutination with neither cells nor a cells. cells carrying agl produced pheromone and responded to a pheromone just like wild cells. cells carrying agl showed a little decreased but significant mating ability when tested on solid media or membrane filter. The agl mutant is different from any -specific ste mutants found so far in many sexual activities. The agl gene is recessive, and unlinked to the mating type locus. Biological significance of the mating type agglutinability is discussed based on the results obtained with the mutant.     

α2-Macroglobulin production by cultured human fibroblasts

Alpha₂-macroglobulin (₂M), a high-molecular-weight plasma protease inhibitor has been shown, by both immunological and functional methods, to be produced by cultured adult lung fibroblasts. Cultured skin fibroblasts synthesized approximately one tenth as much ₂M as lung fibroblasts. This quantitative difference in ₂M production was also demonstrated in fibroblasts of isogenic origin. There was no difference in the amount of ₂M produced between adult and fetal fibroblasts of the same tissue type (i.e., of lung or of skin origin). ₂M was produced in culture during log-phase growth as well as at confluence. Two other plasma protease inhibitors, C1-esterase inhibitor and a substance immunologically cross-reacting with human inter--trypsin inhibitor, were also made by the cultured fibroblasts. Plasma protease inhibitors not detectable in culture supernatants were ₁-antitrypsin, ₁-antichymotrypsin, and antithrombin III.     

Epidermal growth factor,transforming growth factor-α, and epidermal growth factor receptor content in normal and carcinomatous gastric and colonic tissue

Summary Epidermal growth factor (EGF) and transforming growth factor- (TGF-) are polypeptides which bind to the EGF receptor (EGFr) and may play a role in cell growth and carcinogenesis. Our study investigated the content of EGF, TGF-, and EGFr in tumors of the stomach and the colon in comparison with the sourrounding mucosa. EGF was detected in half of the stomach specimens with concentrations between 1 and 9 ng/g weight irrespective of histology. In the colon no EGF was found in the tumor or normal mucosa. In the stomach normal mucosa contained higher TGF- concentrations (mean 22.4 ng/g) than the tumors (mean 11.8 ng/g), but the difference was not statistically significant because of a wide variation in mucosal values. By contrast, the colon mucosa displayed significantly higher TGF- concentrations than the tumor tissues (33 ng/g versus 12 ng/g; P < 0.01). EGFr content in the gastric mucosa was lower compared to gastric carcinoma (48 fmol/g versus 75 fmol/g) yet not significantly different. In contrast, colorectal tumor specimens disclosed significantly higher concentrations than the mucosal tissues (mean of 155 fmol/g versus 80 fmol/g; P < 0.01). In conclusion, TGF- should not be considered a tumorigenic but a physiological growth factor in the stomach and colon. An elevated EGFr content in colorectal tumors in comparison with the normal mucosa could lead to a growth advantage by an autostimulating mechanism.Abbreviations EGF epidermal growth factor - EGFr epidermal growth factor receptor - TGF- transforming growth factor - ROC receiver operating characteristic Dedicated to Prof. Dr. G. Paumgartner on the occasion of his 60th birthday     

On the regulation of the expressed L-type calcium channel by cAMP-dependent phosphorylation

The Ca²⁺ channel subunits _1C-a and _1C-b were stably expressed in Chinese hamster ovary (CHO) and human embryonic kidney (HEK) 293 cells. The peak Ba²⁺ current (I _Ba) of these cells was not affected significantly by internal dialysis with 0.1 mM cAMP-dependent protein kinase inhibitor peptide (mPKI), 25 M cAMP-dependent protein kinase catalytic subunit (PKA), or a combination of 25 M PKA and 1 M okadaic acid. The activity of the _1C-b channel subunit expressed stably in HEK 293 cells was depressed by 1 M H 89 and was not increased by superfusion with 5 M forskolin plus 20 M isobutylmethylxanthine (IBMX). The _1C-a·₂·₂/ complex was transiently expressed in HEK 293 cells; it was inhibited by internal dialysis of the cells with 1 M H 89, but was not affected by internal dialysis with mPKI, PKA or microcystin. Internal dialysis of cells expressing the _1C-a·₂·₂/ channel with 10 M PKA did not induce facilitation after a 150-ms prepulse to +50 mV. The Ca²⁺ current (I _Ca) of cardiac myocytes increased threefold during internal dialysis with 5 M PKA or 25 M microcystin and during external superfusion with 0.1 M isoproterenol or 5 M forskolin plus 50 M IBMX. These results indicate that the L-type Ca²⁺ channel expressed is not modulated by cAMP-dependent phosphorylation to the same extent as in native cardiac myocytes.     

Defective NF-κB Signaling in Dedifferentiated Hepatoma Cells

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1 pathway. We examined induction of NF-B, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-B induction. IB- levels, but not NF-B subunit p65 or IB- levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-B was found to be independent of degradation of IB- or IB-. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.     

Structure of the liver as a possible factor in the regulation of α-fetoprotein synthesis

During regeneration of the mouse liver after poisoning with CCl₄ and paracetamol, hepatocytes containing -fetoprotein (FP) lose their membrane antigen that served as marker for biliary capilaries. Both during regeneration and in early postnatal development, cessation of FP synthesis by the cells coincides with the appearance of an antigen marking the biliary capillaries on their surface. It is suggested that cessation of FP synthesis is the result of establishment of a system of contacts characteristic of the definitive hepatic column.Laboratory of Immunochemistry and Diagnosis of Tumors, Oncologic Scientific Center, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR L. M. Shabad.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 87, No. 6, pp. 576–579, June, 1979.     

Influence of catecholamines on thein vivo release of histamine in the hypothalamus

The hypothalamus of conscious, freely moving rats was superfused with artificial CSF through push-pull cannulae and the release of endogenous histamine was determined radioenzymatically in the superfusate. Superfusion with potassium chloride enhanced the release rate of histamine. The effect of potassium chloride was abolished by -fluoromethylhistidine. Noradrenaline (₁- and ₁-agonist) and clonidine (₂-agonist) decreased the release rate of histamine and inhibited the potassium-induced histamine release. In preliminary experiments, yohimbine (₂-agonist) seemed to increase the release rate of histamine in the superfusate. -Agonists and antagonists (isoprenaline, salbutamol, propranolol) did not influence the release of histamine.This work was supported by the Fonds zur Förderung der wissenschaftlichen Forschung.     

Poly ADP Ribose-Polymerase Inhibitors Prevent the Upregulation of ICAM-1 and E-selectin in Response to Th1 Cytokine Stimulation

We studied the role of poly-ADP-ribose polymerase (PARP) in the mobilization of ICAM-1, VCAM-1, and E-selectin by TNF- and IL-1 in cultured human endothelial cells. Enzyme linked immunosorbent analysis (ELISA) was used to assess if ICAM-1, VCAM-1, and E-selectin were expressed at the cell surface, and if PARP inhibition (using the selective PARP inhibitor GPI 6150) blocked the induced expression. Endothelial cell adhesion molecule expression was evaluated at 4 and at 24 h after cytokine stimulation. At 4 h ICAM-1 and E-selectin, but not VACM-1, were stimulated by both IL-1 and TNF-. Blocking PARP via GPI 6150 only affected TNF- induced E-selectin expression at 4 hours. ICAM-1, VCAM-1, and E-selectin expression were all stimulated by both IL-1 and TNF- in the 24 h assays. PARP inhibition with GPI 6150 blocked the IL-1 mediated stimulation of both ICAM-1 and E-selectin expression, and blocked TNF- stimulation of ICAM-1 expression at 24 h. These experiments suggest that specific PARP inhibition may provide a novel method of controlling leukocyte dependent inflammation through the reduction of ICAM-1 and E-selectin expression in endothelial cells in response to cytokines.     

Phenotypic characteristics of lewis lung carcinoma cells

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLe^x [Neu5Ac2-3Gal1-4(Fuc1-3) GlcNAc] and SiaLe^a [Neu5Ac2-3Gal1-3(Fuc1-4)GlcNAc] and trisaccharide HSO₃Le^x [HSO₃2-3Gal1-4(Fuc1-3)GlcNAc] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Ac2-6Gal1-4Glc], 6-HSO₃LacNAc, and A-di [GalNAc 1-3Gal] and trisaccharides H-type 1 [Fuc1-2Gal1-3GlcNAc and Le^x [Fuc1-3(Gal 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLe^x, SiaLe^a, and HSO₃Le^x play a role in the progress of tumor process and metastasizing.     

Monocyte chemoattractant protein (MCP)-1 production via functionally reconstituted Fcα receptor (CD89) on glomerular mesangial cells

Background: Fc alpha receptor (FcR; CD89) is the receptor for Fc portion of IgA in various cells, and displays various immunological responses on binding. It is important to analyze the mesangial functions via FcR in the pathogenesis of IgA nephropathy. However, it is still controversial whether FcR is expressed on mesangial cells. To assess biological functions of FcR on the mesangial cells, we established mesangial transfectants that expressed FcR with or without FcR chain that is a common signaling molecule of FcRs. The production of monocyte chemoattractant protein-1 (MCP-1) by mesangial cells is known to contribute to cellular infiltration into glomeruli and subsequent glomerular injuries. Methods: Murine mesangial cell lines (SV40 MES 13) were transfected with cDNA of the human FcR. Furthermore, we co-transfected some of the FcR transfectants with cDNA of human FcR chain. The tyrosine phosphorylation of the intra-mesangial proteins after FcR cross-linking was examined by immunoprecipitation. MCP-1 production from each transfectant stimulated with heat aggregated IgA was determined by sandwich ELISA. Results: Two kinds of mesangial transfectants stably expressed human FcR with or without FcR chain (FcR⁺, FcR⁺/⁺). Phosphorylation of FcR chain and syk kinase was detected in FcR⁺ and FcR⁺/⁺ cells, but not in untransfected cells. Aggregated IgA induced significantly higher MCP-1 production in FcR⁺/⁺ than those in FcR⁺ or untransfected control. Conclusions: Present study demonstrated that FcR and FcR chain could be reconstituted in mesangial cells and mediated MCP-1 production by aggregated IgA in a dose-dependent manner. Current data would argue that FcR can be activated in mesangial cells through their own machinery, although underlying mechanisms for FcR induction in mesangial cells remain unclear.Received 10 February 2003; returned for revision 14 May 2003; accepted by M. J. Parnham 16 June 2003     

Regional location of α1-antichymotrypsin and α1-antitrypsin genes on human chromosome 14

The human protease inhibitor genes ₁ antitrypsin (₁-PI) and ₁-antichymotrypsin (₁-ACT) are acute-phase proteins which are induced in response to inflammation. These inhibitors function to limit the activity of serine proteases in vivo. ₁-PI acts as an inhibitor of neutrophil elastase to protect the elastin fibers of the lung. Genetic deficiencies of ₁-PI result in development of chronic pulmonary emphysema. The physiologic role of ₁-ACT has not been clearly defined, but it also appears to function in the maintenance of protease-protease inhibitor equilibrium in the lung. Nucleic acid and protein sequence homologies detected between ₁-PI and ₁ t-ACT suggested an evolutionary relationship. Gene mapping experiments were performed to determine if these protease inhibitor genes reside at the same chromosomal locus in man. In situ hybridization data demonstrate that both ₁-PI and ₁-ACT map to the same region, q31–q32.3, on chromosome 14.