Localization of epidermal growth factor (EGF) and its receptor (EGFR) during postnatal testis development in the alpaca (Lama pacos)

The objective of the present studies was to determine the localization of epidermal growth factor (EGF) and the epidermal growth factor receptor (EGFR) in testicular tissue collected from male alpacas at 12 and 24 months of age. In the testes of 12-month-old alpacas, positive staining for EGF was not detected. EGFR was localized to Leydig cells within the 12-month-old alpaca testis, but staining was absent within seminiferous tubules. At 24 months of age, EGF was localized to Leydig cells, peritubular myoid cells, Sertoli cells and germ cells of the alpaca testis, with a preferential adluminal compartment staining within the seminiferous tubules. EGFR was also localized to the Leydig cells, peritubular myoid cells, Sertoli cells and germ cells within the 24-month-old alpaca testis, but staining within the tubules was primarily within the basal compartment. Results indicate distinct temporal and spatial regulation of EGF and EGFR in the alpaca testis and support a potential role for EGF and its related ligands in alpaca testis development and spermatogenesis.     

Human breast microvascular endothelial cells retain phenotypic traits in long-term finite life span culture

Summary Attempts to study endothelial-epithelial interactions in the human breast have been hampered by lack of protocols for long-term cultivation of breast endothelial cells (BRENCs). The aim of this study was to establish long-term cultures of BRENCs and to compare their phenotypic traits with the tissue of origin. Microvasculature was localized in situ by immunohistochemitry in breast samples. From this tissue, collagen-rich stroma and adipose tissue were dissected mechanically and further disaggregated to release microvessel organoids BRENCs were cultured from these organoids in endothelial specific medium and characterized by staining for endothelial markers. Microvessels were a prominent feature of intralobular tissue as evidenced by immunostaining against endothelial specific markers such as CD31, VE-cadherin, and von Willebrand factor (VWF). Double staining against VE-cadherin and lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) showed that blood and lymphatic vessels could be distinguished. An antibody against CD31 was used to refine protocols for isolation of microvasculature from reduction mammoplasties. BRENCs retained critical traits even at high passage, including uptake of low-density lipoprotein, and had E-selectin induced upon treatment with tumor necrosis factor-α. The first signs of senescence in passage 14 were accompained by gain of trisomy 11. At passage 18 cells showed chromosomal aberrations and growth arrest as revealed by β-galactosidase staining. We demonstrate here that breast microvasculature may serve as a large-scale source for expansion of BRENCs with molecular and functional traits preserved. These cells will form the basis for studies on the role of endothelial cells in breast morphogenesis.     

Butyrate enhances the synthesis of interphotoreceptor retinoid-binding protein (IRBP) by Y-79 human retinoblastoma cells

The synthesis and secretion of interphotoreceptor retinoid-binding protein (IRBP) from Y-79 human retinoblastoma cells was investigated using immunocytochemistry and SDS-polyacrylamide gel electrophoresis. Indirect immunofluorescence of cells growing in monolayer culture for 11 and 13 days showed no significant IRBP staining although by SDS-polyacrylamide gel electrophoresis, a small amount of IRBP was detected in the culture medium, suggesting synthesis and extracellular secretion. Butyrate (2mM) treatment of cells starting on the eighth day of culture resulted in a dramatic increase of IRBP fluorescence 3-5 days after treatment. Treatment of cells in all conditions with 1 microM monensin for 3 h showed concentration of IRBP in the Golgi apparatus of about 10-20% of cells as proved by a double immunofluorescent technique, employing anti-IRBP antibody and wheat-germ agglutinin. Incubation of cells with either radiolabeled amino acids or glucosamine followed by analysis of cell cytosol and culture medium by SDS-polyacrylamide gel electrophoresis also confirmed that 1) IRBP is synthesized by the Y-79 cells and secreted into the medium and 2) its production is markedly increased by butyrate treatment. The enhancement of IRBP synthesis by butyrate suggests biochemical differentiation of Y-79 cells possibly into photoreceptor-like cells and offers a new system for studying the properties of this unique retinoid-binding protein and of factors that control its synthesis and secretion.     

Analysis of nucleolar organizer constitution by fluorescent in situ hybridization (FISH) in diploid and artificially produced haploid sporophytes of the fernOsmunda japonica (Osmundaceae)

HaploidOsmunda japonica (2n = × = 22), produced in tissue culture by induced apogamy and acclimatized in vivo, was cytologically compared with the diploid sister plant. The haploid had smaller guard cells than the diploid and a numerically exactly reduced karyotype. Fluorescent in situ hybridization (FISH) using an rDNA probe showed four hybridization signals (one large and three small ones) in the haploid and eight signals (two large and six small ones) in the diploid plant. These results represent a cytological proof for the origin of the haploid plant by apogamy without recognizable chromosome aberrations.     

Immunohistochemical localization of atrial natriuretic factor (ANF) in the excretory system of the rabbit parotid gland

The immunohistochemical localization of atrial natriuretic factor (ANF) in the rabbit parotid gland was performed using an antibody against rabbit ANF and avidin-biotin or streptoavidin as detector. Results showed positivity in cuboidal and columnar cells of intralobular ducts and in basal cells of extralobular and main excretory duct. These data support the hypothesis that ANF produced by intralobular ducts could act through a paracrine mechanism; ANF produced by extralobular and main ducts may play a role in the regulation of salivary composition.     

Immunohistochemical and radioautographic evidence of monoamine-containing cells in bioluminescent elytra of the scale-worm Harmothoe imbricata (Polychaeta)

Summary Elytra of the scale-worm Harmothoe imbricata were examined for the presence of monoamine-like immunoreactivities and radioautographic reactions. Serotonin (5-HT)-like immunoreactivity was widely distributed among the cellular constituents of the elytra, being present in epithelial cells including photocytes, in elytral nerves, clear cells and the loose neuronal plexus of the middle compartment. The distribution of [³H]5-HT labelling coincided with that of the immunoreactivity except for an additional reactive band extending through the upper cuticle layer. Tyrosine hydroxylase (TH)-like immunoreactivity was detected in epithelial cells, sensory papillae and elytral ganglion and nerves, with little or no staining in clear cells and plexus neurons of the middle compartment. Radioautographic labelling with [³H]noradrenaline and [³H]adrenaline overlaid many epithelial cells, elytral nerves and sensory papillae, but not the loose neuronal plexus or, apparently, clear cells. It is concluded that monoaminergic systems are widely distributed and that they must play important roles as neuroactive and/or paracrine substances in the elytral neuroectoderm. The distribution of [³H]5-HT label in photocytes also suggests the involvement of serotonergic mechanisms in luminescence control, luminescence being the only known effector activity of elytra.     

Cells with morphological and immunohistochemical features of hepatic stellate cells (Ito cells) form an extralittoral (extrasinusoidal) compartment in the cirrhotic rat liver.

Systematic studies on hepatic stellate cells and myofibroblasts have so far mainly focused on cells located in the perisinusoidal space of Disse, the so-called littoral compartment. Here, these cells play a key role for intralobular fibrogenesis and sinusoidal capillarization. However, advanced hepatic fibrosis and cirrhosis are characterized by portal tract fibrosis and septal fibrosis, thus involving cells outside the perisinusoidal space. To study the question as to whether hepatic stellate cells occur and are expanded in an extralittoral (extrasinusoidal) compartment in cirrhogenesis, we systematically analyzed the distribution and density of desminreactive stellate cells in a rat model of hepatic fibrosis. Fibrosis and remodeling of the liver were induced by bile duct ligation, and stellate cells were identified by single and double immunohistochemistry. We can show that desmin-reactive cells are reproducibly detectable in extralittoral compartments of the normal and fibrotic rat liver. Periductular extralittoral stellate cells are significantly more frequent in cirrhosis, indicating that extralittoral stellate cells expand in concert with proliferating ductules. The findings suggest that ductular proliferation thought to represent a pacemaker of hepatic remodeling is accompanied by a population of cells exhibiting the same phenotype as perisinusoidal stellate cells.     

大鳞大马哈鱼垂体促甲状腺素和促性腺激素分离纯化的研究

本文报道应用ConA-Sepharose 4B.亲和层析、凝胶过滤层析、离子交换层析及免疫亲和层析等技术,首次从大鳞大马哈鱼垂体中分离纯化了促甲状腺素(sTSH)和促性腺激素(sGTH)。在TSH生物测定中,纯化的。sTSH制品具有明显促进虹鳟幼鱼甲状腺体内分泌甲状腺素(T4)的生物活性,而sGTH无此活性。在GTH生物测定中,制备的sGTH具有显著诱导虹鳟卵母细胞体外培养成熟(GVBD)及分泌17α、20β-二氢孕酮的能力,而sTSH无此活性。HPLC表明sTSH和sGTH的分子量分别为27500和38000道尔顿。作者用SDS聚丙烯酰胺凝胶电泳(SDS-PAGE)和等电聚焦检测了激素的纯度及等电点,用RIA或ELISA方法测定了几种垂体激素在sTSH终产物中的污染程度。     

Phosphorylation determines two distinct species of Tau in the central nervous system

The monoclonal antibody, Tau-1, which had previously been used to localize tau to the axonal compartment in brain has been reutilized for light and electron microscopic immunohistochemistry following phosphatase treatment of tissue. We report here that a significant quantity of tau in the central nervous system is phosphorylated in situ at or near the Tau-1 epitope, preventing the binding of the Tau-1 antibody. Upon removal of this/these phosphate group(s), however, Tau-1 was observed in the somatodendritic compartment of neurons as well as in axons. Furthermore, intense staining was also observed in astrocytes and in perineuronal glial cells. This immunoreactivity was present along the lengths of microtubules and on ribosomes (polysomes). Treatment of immunoblots of extracts of whole cerebral cortex with phosphatase confirmed the immunohistochemical results in that a 50-65% increase in Tau-1 binding to the tau region of the blot was noted. Moreover, a novel monoclonal antibody, Tau-2, was also used in these experiments. This antibody binds only to tau and localizes along microtubules in axons, somata, dendrites, and astrocytes and on ribosomes (polysomes) without phosphatase pretreatment.     

Effects of anabolic steroid (19-nortestosterone) on the secretion of testicular hormones in the stallion.

The aim of this study was to clarify the effect of anabolic steroids on the testicular endocrine function of mature stallions. Mature thoroughbred stallions were treated with 800 mg nandrolone decanoate every 3 weeks for 3 months. After the first treatment, plasma concentrations of LH, immunoreactive inhibin and testosterone decreased rapidly to the nadir. These hormones were maintained at significantly lower concentrations compared with concentrations in intact stallions. Histology of the testicular tissue indicated the arrest of advanced spermatogenesis in the seminiferous tubules and a severe depletion of the number of Leydig cells in the interstitial compartment as a result of treatment. Most of the immunopositive cells for the inhibin alpha-subunit and steroidogenesis enzymes in the interstitial compartment decreased below detectable amounts, whereas immunopositive reactions of inhibin alpha-subunit in the seminiferous tubules were clearly observed. In conclusion, the treatment of mature stallions with nandrolone decanoate caused a decrease in the secretion of ir-inhibin and testosterone from the testis, the depletion of the number of Leydig cells and a decrease below detectable amounts of inhibin alpha-subunit and steroidogenesis enzymes. The concentration of ir-inhibin in the peripheral blood may be a useful marker for the examination of testicular activity in stallions being treated with anabolic steroids.     

Distribution of aquaporin 1 in the rat pancreatic duct system examined with light- and electron-microscopic immunohistochemistry

The pancreatic duct is the major site for the secretion of pancreatic fluid, but the pathway of water transport in this system is not known. Recently, intense signal for mRNA of aquaporin 1 (AQP1) water channels was detected in isolated rat interlobular ducts. Therefore, we performed light- and electron-microscopic (EM) immunohistochemistry for AQP1 in the rat pancreatic ducts. AQP1 immunoproducts were not observed in the acinar cells, centroacinar cells or intercalated ducts. In the smaller intralobular ducts less than 10 microm in diameter (the lumen plus duct cells), most cells were immunonegative. AQP1-positive cells appeared in intralobular ducts 10-15 microm in diameter. In small and medium-sized interlobular ducts 15-70 microm in diameter surrounded by periductal connective tissue 2-40 microm thick, most cells were AQP1 positive with various degrees of immunoreactivity. In the larger interlobular ducts, the expression of AQP1 was variable, ranging from immunopositive to negative. In the main pancreatic duct, most cells were negative for AQP1. EM immunohistochemistry of the intralobular and small interlobular ductal epithelial cells showed that the AQP1 immunoproducts were more abundant in the basolateral membrane than in the apical membrane, though they were present in both membranes. In the medium-sized interlobular ducts, AQP1 immunoproducts were distributed densely along the apical, lateral interdigitation and basal membrane of the epithelial cells. In the various sizes of interlobular ducts, immunoproducts were associated not only with the plasma membrane, but also with the caveolae and vesicle-like structures. Secretin did not induce any significant difference in AQP1 expression and cellular and subcellular localization. These results indicate that the expression and subcellular localization of AQP1 vary considerably depending on the duct size, which may reflect water transport characteristics in the different divisions of the pancreatic duct system.     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.     

Adiponectin and leptin are secreted through distinct trafficking pathways in adipocytes

Adiponectin and leptin are two adipokines secreted by white adipose tissue that regulate insulin sensitivity. Previously we reported that adiponectin but not leptin release depends on GGA-coated vesicle formation, suggesting that leptin and adiponectin may follow different secretory routes. Here we have examined the intracellular trafficking pathways that lead to the secretion of these two hormones. While adiponectin and leptin displayed distinct localization in the steady-state, treatment of adipocytes with brefeldin A inhibited both adiponectin and leptin secretion to a similar level, indicating a common requirement for class III ADP-ribosylating factors and an intact Golgi apparatus. Adiponectin secretion was significantly reduced by endosomal inactivation in both 3T3L1 and rat isolated adipocytes, whereas this treatment had no effect on leptin secretion. Importantly, endosomal inactivation completely abolished the insulin stimulatory effect on adiponectin release in rat adipocytes. Confocal microscopy studies revealed colocalization of adiponectin with endogenous rab11 a marker for the recycling endosome, and with expressed rab5-GFP mutant (rab5Q75L) a marker for the early endosome compartment. Colocalization of adiponectin and rab5Q75L was increased in endosome inactivated cells. Consistent with these findings adiponectin secretion was reduced in cells expressing mutants of Rab11 and Rab5 proteins. In contrast, expression of an inactive (kinase dead) mutant of Protein Kinase D1 moderately but significantly inhibited leptin secretion without altering adiponectin secretion. Taken together, these results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and that endosomal compartments are required for adiponectin but not for leptin secretion.     

Expression of Toll-like receptors (TLR) and responsiveness to TLR agonists by polarized mouse uterine epithelial cells in culture

The objective of the present study was to examine the expression of Toll-like receptors (TLRs) by mouse uterine epithelial cells and to determine if stimulation of the expressed TLR induces changes in cytokine and/or chemokine secretion. Using RT-PCR, the expression of TLRs 1-6 by mouse uterine epithelial cells was demonstrated, with TLRs 7-9 expressed only periodically. In the absence of pathogen-associated molecular patterns, polarized uterine epithelial cells constitutively secrete interleukin (IL) 1A, cysteine-cysteine ligand (CCL) 2, IL6, granulocyte-macrophage colony-stimulating factor 2 (CSF2), tumor necrosis factor A (TNFA), CSF3, and IL8 in vitro, with levels of cytokines/chemokines secreted into the apical compartment being significantly greater than those released into the basolateral compartment. When added to the apical surface for 48 h before analysis, the TLR2-agonist Pam3Cys-Ser-(Lys)4 and TLR1/6-agonist peptidoglycan increased epithelial cell apical secretion of IL1A, CCL2, and IL6 and apical/basolateral bidirectional secretion of CSF2, TNFA, CSF3, and IL8 when compared to controls. The TLR3-agonist poly (I:C) significantly increased bidirectional secretion of CCL2, IL6, TNFA, and CSF2 and basolateral secretion of CSF3. Lastly, the TLR4-agonist lipopolysaccharide increased bidirectional secretion CCL2, CSF2, TNFA, CSF3, and IL8 and apical secretion of IL6. These results indicate that mRNAs for Tlr1 through Tlr6 are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and proinflammatory cytokines that contribute to the defense of the uterus against potential pathogens.     

Effect of monensin on secretion of t-PA from melanoma (Bowes)

The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of 3H-mannose- and 35S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of 3H-mannose-related glycoprotein to 35S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation.     

Histochemical localization of oxidative enzymes in the hepatopancreas of scylla serrata (forskål) (brachyura: decapoda)

A histochemical study on the distribution of mitochondrial and oxidative enzymes viz., succinic, NAD and NADP-linked isocitrate and malic dehydrogenases and cytochrome oxidase in the hepatopancreas of Scylla serrata (Forskål) shows that the R cells possess the highest concentration of them at the apical parts; F and B cells showed poor staining reactions whereas E cells and connective tissue exhibited trace staining reactions. A moderate staining for these was obtained in the cells lining the main ducts.

The bilateral removal of eyestalks resulted in the stimulation of succinic and NAD-linked malic dehydrogenases and cytochrome oxidase, observed 4 h after the operation; after 24 h, however, these enzymes showed a slight reduction in the activity. A significant increase in the activity of NADP-linked isocitrate and malic dehydrogenases was noted 24 h after eyestalk ablation. The major alterations were noticed in the R cells.

After injecting eyestalk extract into destalked or intact crabs, a fall in the staining intensity of succinic, NAD-linked malic and isocitrate dehydrogenases in the R cells was apparent 2–4 h after the treatment but was subsequently re-established after 24 h.

It seems from the present results that there may be a factor(s) in the eyestalk of S. serrata which regulates the oxidative metabolism in the hepatopancreas. The physiological significance of oxidative enzymes in various cytologic elements is discussed.     

Zinc-Specific N-(6-Methoxy-8-Quinolyl)-Para-Toluenesulfonamide as a Selective Nontoxic Fluorescence Stain for Pancreatic Islets

Separation of the endocrine from the exocrine pancreatic tissue by fluorescence activated sorting has been limited by the lack of an ideal fluorescent label for islet tissue. Our studies indicates the zinc-specific stain N-(6-methoxy-8-quinolyl)-para-toluenesulfonamide (TSQ), has characteristics ideal for use as a fluorescent label for islet tissue. Dispersed rat pancreas cells stained with TSQ produced bright blue fluorescence when excited by UV light [peak emission wavelength at 480 nm. maximal excitation at 365 nm). The fluorescence was specific for islet tissue as confirmed by counterstaining with the islet-specific stain dithizone and there was minimal background staining of exocrine tissue. Stained tissue remained brightly fluorescent for 2 hr. with some fading by 4 hr. Injection of TSQ into rats at a concentration sufficient to produce staining of islets produced no toxicity discernible at 4 months. The viability of isolated rat islets stained with TSQ was maintained as shown by supravital staining, in vitro secretion of insulin, and reversal of diabetes after transplantation of stained islets into diabetic syngeneic recipients.     

Oxidative stress caused by pyocyanin impairs CFTR Cl(-) transport in human bronchial epithelial cells

Pyocyanin (N-methyl-1-hydroxyphenazine), a redox-active virulence factor produced by the human pathogen Pseudomonas aeruginosa, is known to compromise mucociliary clearance. Exposure of human bronchial epithelial cells to pyocyanin increased the rate of cellular release of H(2)O(2) threefold above the endogenous H(2)O(2) production. Real-time measurements of the redox potential of the cytosolic compartment using the redox sensor roGFP1 showed that pyocyanin (100 microM) oxidized the cytosol from a resting value of -318+/-5 mV by 48.0+/-4.6 mV within 2 h; a comparable oxidation was induced by 100 microM H(2)O(2). Whereas resting Cl(-) secretion was slightly activated by pyocyanin (to 10% of maximal currents), forskolin-stimulated Cl(-) secretion was inhibited by 86%. The decline was linearly related to the cytosolic redox potential (1.8% inhibition/mV oxidation). Cystic fibrosis bronchial epithelial cells homozygous for DeltaF508 CFTR failed to secrete Cl(-) in response to pyocyanin or H(2)O(2), indicating that these oxidants specifically target the CFTR and not other Cl(-) conductances. Treatment with pyocyanin also decreased total cellular glutathione levels to 62% and cellular ATP levels to 46% after 24 h. We conclude that pyocyanin is a key factor that redox cycles in the cytosol, generates H(2)O(2), depletes glutathione and ATP, and impairs CFTR function in Pseudomonas-infected lungs.