Biomedical subjects

F Sinowatz

Publications and source records attributed to F Sinowatz.

At least 73 records · Page 4Linked to original sources

PubMed1995

Immunocytochemical characterization of porcine zona pellucida during follicular development.

Sections of bovine ovaries fixed in Bouin's fluid or methanol-acetic acid and embedded in paraffin were incubated with chicken polyclonal antibodies to HPLC-purified zona glycoproteins ZP3 alpha and ZP3 beta. Oocytes of primordial follicles as well as of primary follicles showed weak labelling with anti-ZP3 alpha and anti-ZP3 beta. No immunostaining could be observed in the follicle cells. The ZP of primary follicles displayed distinct immunoreactivity for both ZP3 alpha and ZP3 beta. In secondary follicles, distinct labelling with anti-ZP3 beta and weak labelling with anti-ZP3 alpha could be seen in the oocyte. The ZP showed immunoreactivity with antibodies to ZP3 alpha and ZP3 beta. Both antibodies labelled single follicle cells. In tertiary follicles, the oocytes were weakly labelled with anti-ZP3 alpha and anti-ZP3 beta. Some granulosa cells showed staining for ZP3 alpha and ZP3 beta. The ZP displayed strong immunoreactivity for ZP3 beta and ZP3 alpha. Cells of the corona radiata were strongly immunopositive for ZP3 alpha and ZP3 beta. Similar histotopography of immunoreactive cells could be seen in preovulatory follicles. The characteristic pattern observed for the distribution of ZP3 alpha and ZP3 beta strongly suggests that in the porcine ovary both the oocyte and the follicle cells contribute to the synthesis of the ZP, perhaps in sequence.

Animals↗

PubMed1995

Immunohistochemical localization of spermadhesin AWN in the porcine male genital tract.

Boar spermadhesin (AWN) is a 14-kDa multifunctional protein, attached to the surface of the spermatozoa and involved in sperm capacitation and zona pellucida binding. The cellular origin of AWN was previously unknown. Moreover, the region of the male genital tract in which AWN becomes attached to the surface of spermatozoa was also uncertain. By using monospecific polyclonal antibodies against AWN, the immunohistochemical distribution pattern of AWN epitopes has been investigated in tissue sections of the porcine male genital tract. Our study has revealed that AWN is synthesized in the rete testis and in the epithelium of the seminal vesicles. The latter are also the major contributors of seminal plasma AWN. In addition, immunoblotting analysis has shown that AWN is present on epididymal spermatozoa. Our results indicate that the cellular origin of spermadhesins is species-specific. The attachment of AWN to epididymal spermatozoa is probably important in developing the capacity for fertilization.

Animals↗

PubMed1995

Evidence of a direct role for growth hormone (GH) in mammary gland proliferation and lactation.

A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in ductal and alveolar epithelial cells from rat and rabbit mammary glands by immunohistochemistry. Intense immunoreactivity was present in membrane, cytoplasm and some nuclei of epithelial cells during proliferation and lactation. Receptor expression decreased during weaning and was absent or weak in regressive mammary glands. Immunoreactivity was weak in ductal epithelial cells from virgin adult animals. Pronounced expression of GH receptor/binding protein was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the existence of a soluble form on the GH receptor, namely the growth hormone binding protein derived from the membrane receptor by cleavage. Primary localization of the receptor in proliferating and lactating epithelial cells suggests that the rat and rabbit mammary gland is a GH target tissue. This finding is in contradiction to both classical GH action and the somatomedin hypothesis and challenges the widely held view that GH has no direct influence on mammary growth and function.

Animals↗

PubMed1995

Cloning and characterization of a second AP-2 transcription factor: AP-2 beta.

AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.

Amino Acid Sequence↗

PubMed1995

Affinity-purified antibodies against alpha-galactosyl residues from human serum: comparison of their binding in bovine testicular tissue with that of the Griffonia simplicifolia lectin (GSI-B4) and impact of labeling on epitope localization.

alpha-Galactosyl residues in the carbohydrate part of cellular glycoconjugates can serve as cell type-associated markers and are implicated in intercellular adhesion and biosignaling. This biological significance explains the interest to characterize probes with respective specificity as the Griffonia simplicifolia I-isolectin B4. Due to the documented occurrence of an alpha-galactoside-binding immunoglobulin G fraction in human serum we compared the extent of binding and its pattern for the lectin and the antibody using surface-immobilized extract proteins and fixed sections of bovine testicular tissue with known lectin reactivity. The antibody fractions were obtained either solely from affinity chromatography isolation on immobilized melibiose or after an additional step to deplete this fraction of galactoside-binding activities without pronounced specificity to the alpha-anomeric linkage. They yielded a rather indistinguishable reactivity in comparison to that of the lectin, when an indirect approach was used. Labeling of the antibodies with a hydrazide derivative of biotin did not affect the pattern of binding. However, significant differences were noted, when conjugation of label was targeted to amino groups via N-hydroxy-succinimide esters of biotin and digoxigenin despite performance of the modification under activity-preserving conditions. Notably, the apparent strong staining of Leydig cells and nuclei of primary spermatocytes, respectively, was not inhibitable by sugar. These differences were corroborated by a nonidentical response of the various probes in solid-phase assays with extract proteins. Thus, care should be exercised in the interpretation of histochemical data, obtained with this type of modified antibody. When these precautions are fulfilled, this immunoglobulin fraction from human serum has the potential as an alpha-galactosyl-specific histochemical tool.

Animals↗

PubMed1994

Galactose- binding sites in the acinar cells of the human accessory lacrimal gland.

This investigation for the first time has collected evidence of a specific glycoconjugate contribution of the acinar cells from accessory lacrimal glands to human tears. Amongst group III lectin binding glycoconjugates, monosaccharides seem to be more prominent than disaccharides. alpha (and less obvious beta) Galactose sugar moieties appear to be specifically important. A need for further differentiating investigations is outlined.

Acetylgalactosamine↗

PubMed1994

Differential distribution of immunoreactive S-100 protein in mammalian testis.

The present study deals with the immunohistochemical localization of S-100 protein in the testes of seven mammalian species including rat, cat, dog, pig, sheep, cattle and horse. Significant differences are demonstrated in the cellular distribution and intensity of immunoreaction for the protein. In bull, ram, boar and cat testes S-100 protein was localized in the cytoplasm and nuclei of Sertoli cells. A particularly intense staining was seen in the modified Sertoli cells of the terminal tubular segment. With the exception of the cat and horse S-100 protein immunoreactivity was additionally found in epithelial cells of the straight testicular tubules and in the epithelial cells of the rete testis. Endothelial cells of capillaries, veins and lymphatic vessels are regularly S-100 immunoreactive in ruminants. Leydig cells were found to be strongly positive for S-100 protein in the cat and rat testes and to a lower degree in pig and horse testes. Finally a distinct immunostaining of peritubular cells was restricted to the testis of dogs and rats. The remarkable species-specific variations of immunoreactivity for S-100 protein in different cell types of the testis support the hypothesis that S-100 protein is multifunctional protein and may have a different function in testicular physiology.

Animals↗

PubMed1994

Morphological evidence of sperm maturation in the ampulla ductus deferentis of the bull.

The present lectin histochemical comparison of cauda epididymal and ampullary bovine sperm was conducted to investigate whether ampullary secretions are involved in altering the plasma-membrane glycoconjugates of sperm. A marked redistribution of glycoconjugates between sperm from these two regions was indeed revealed on the basis of changes in binding patterns of the following fluoroscein-isothiocyanate (FITC)-labelled lectins: wheat-germ agglutinin (WGA), Maclura pomifera agglutinin (MPA), Griffonia simplicifolia I agglutinin (GS I) and Bauhinea purpurea agglutinin (BPA). This was evidenced in the first three cases by a relative reversal of staining intensity between the acrosomal and post-acrosomal regions, and by a pronounced increase in the staining of the midpiece. Changes in the distribution of BPA binding sites were limited to the latter phenomenon. The results are compared with previous findings, discussed in the context of the hypermotility characteristic of ampullary sperm and related to previously reported differences in the lectin-binding patterns of the luminal and glandular epithelia.

Animals↗

PubMed1994

Immunohistochemical localization of growth hormone receptor binding protein in the mammalian cerebellum.

A panel of monoclonal antibodies to the growth hormone (GH) receptor/binding protein was used to demonstrate the existence and detail the expression of GH receptors in the cerebellum of 2, 10, 28 days old postnatal and adult rats and 10, 20 days old and adult rabbits by immunohistochemistry to define potential targets for endogenous GH action in the cerebellum. Receptors were localized in membrane and cytoplasmic components of neurons and glial cells and expression decreased with age. Intense immunoreactivity was observed in the cytoplasm and dendrites of Purkinje cells and in cells of the cerebellar nuclei. Glial cells also showed receptor expression. Strong immunoreactivity was observed with two monoclonal antibodies and lesser reactivity was seen with others, paralleling their affinities for the receptor. The cytoplasmic presence of this putatively plasma membrane located GH receptor is accounted for by the high receptor content of endoplasmic reticulum and the existence of a soluble form of the GH receptor, namely the GH binding protein (BP) derived from the membrane receptor by cleavage, and receptor localization reported here correlate well with the distribution of insulin-like growth factor 1 (IGF-1) mRNA and immunoreactivity in cerebellar Purkinje cells and glial cells. Primary localization of the receptor in the cerebellum is in direct contradiction to both classical GH action and the somatomedin hypothesis and supports and extends the theory of genetically regulated macroneuronal maturation.

Animals↗

PubMed1994

Expression of insulin-like growth factor 1 (IGF-1) in the bovine oviduct during the oestrous cycle.

Growth factors are known to take part in the regulation of reproduction. Here we present evidence for the expression of insulin-like growth factor 1 (IGF-1) in the bovine oviduct. Two IGF-1 specific mRNA-transcripts of 1.5 and 4.4 kb were detected during the whole oestrous cycle, and showed increased expression after ovulation. Complete homology to the known IGF-1 sequence was achieved by specific RT-PCR (reverse transcription-polymerase chain reaction) amplification followed by dideoxysequencing of this 210 bp fragment. IGF-1 protein was localized in the secretory cells of the oviduct epithelium using immunohistochemical techniques. We suggest possible effects of IGF-1 during ovulation either on the oviductal cells or on the early embryo.

Animals↗

PubMed1994

Role of Mac-1 and ICAM-1 in ischemia-reperfusion injury in a microcirculation model of BALB/C mice.

The leukocyte beta 2-integrin Mac-1 (CD11b/CD18) and its endothelial ligand intercellular adhesion molecule 1 (ICAM-1) are involved in leukocyte adhesion to and macromolecular leakage from postcapillary venules during inflammatory reactions. Both events are also encountered after ischemia-reperfusion of striated muscle, suggesting a central role of both adhesion proteins in reperfusion injury. Using intravital fluorescence microscopy and a microcirculation model in awake BALB/C mice, we investigated the effects of monoclonal antibodies (MAb) and Fab fragments to Mac-1 and MAb to ICAM-1 on leukocyte-endothelium interaction and macromolecular leakage of fluorescein isothiocyanate-dextran (1.5 x 10(5) mol wt) in striated skin muscle after 3 h of ischemia followed by reperfusion. We demonstrated that administration of MAb and Fab to Mac-1 before reperfusion was as effective as administration of MAb to ICAM-1, which was found to be significantly upregulated in the postischemic tissue by immunohistochemical analysis, in preventing postischemic leukocyte adhesion to and macromolecular leakage from postcapillary venules, whereas postischemic leukocyte rolling was not affected after MAb administration. Postischemic capillary perfusion was efficiently preserved in animals treated with anti-Mac-1 and anti-ICAM-1 MAb compared with animals receiving the isotype-matched control antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Endothelin expression in normal human anterior pituitaries and pituitary adenomas.

Endothelins (ETs) are important regulators of growth and function in many endocrine tissues. This study was designed to verify the expression of ETs in a series of normal human pituitaries and pituitary adenomas. We examined 13 normal pituitaries and 58 pituitary adenomas for the presence of immunoreactive (ir) ET-1 and ET-3. No ir-ET-1 was detected in any of the 13 normal pituitaries, whereas ir-ET-3 was observed in 4 of 13 (31%) cases. In contrast, 48% (28 of 58) of pituitary adenomas display immunoreactivity for ET-1, whereas 31% (18 of 58) show immunoreactivity for ET-3. With respect to the type of tumors, staining was as follows: nonfunctioning adenomas: ET-1, 14 of 33; ET-3, 9 of 33; somatotropinomas: ET-1, 8 of 16; ET-3, 6 of 16; corticotropinomas: ET-1, 5 of 5; ET-3, 2 of 5; and prolactinomas; ET-1, 1 of 4; ET-3, 1 of 4. Using double immunostaining, we found the colocalization of ET-3 in normal pituitaries and of ET-1 and ET-3 in pituitaries adenomas in each hormone-secreting cell. In Cushing adenomas, ET-1 was coexpressed in corticotropic cells in all 5 cases (100%). In the same tumors, by reverse transcriptase polymerase chain reaction, we investigated the presence of ET-1 and ET-3 messenger ribonucleic acids and found that they are expressed, respectively, in 18 of 21 and 7 of 11 tumors examined. Our findings demonstrate that pituitary adenomas frequently display ET-1 as well as ET-3 immunoreactivity, in contrast to normal pituitaries, in which only ET-3 was found.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoma↗

PubMed1994

Immunohistochemical aspects of insulin-like growth factors I and II in the bovine corpus luteum.

The cellular distribution of insulin-like growth factors I and II (IGF-I and -II) was examined in bovine corpora lutea at different stages of the luteal phase using specific antibodies and the avidin-biotin immunoperoxidase technique. At the cellular level, intense immunostaining for IGF-I was exclusively observed in large and small luteal cells and in a limited number of endothelial cells. Positive IGF-I immunoreactivity in luteal cells was thereby distributed in a distinct topographical, lobule-specific manner. Immunoreactivity in central areas of luteal lobules was most abundant in large luteal cells, whereas in peripheral zones significantly (P < 0.05) more small luteal cells exhibited IGF-I immunoreactivity. This distribution pattern was evident from day 4 of the cycle onwards and occurred at all the stages investigated. The percentage of positive small (SLC) and large (LLC) luteal cells revealed by semiquantitative analysis depended on the stage of the cycle as follows: days 4-7: 34% LLC versus 21.3% SLC in central areas and 25.1% LLC versus 32.7% SLC in peripheral zones; days 8-12: 42.9% LLC versus 19.9% SLC in central areas and 23.5% LLC versus 35.2% SLC in peripheral zones; days 13-16: 47.7% LLC versus 19.4% SLC in central areas and 19.2% LLC versus 41.4% SLC in peripheral zones. In contrast to IGF-I, no expression of IGF-II immunoreactivity was seen in large or small luteal cells. Positive immunoreactivity was restricted to the perivascular fibroblasts of large blood vessels and to the pericytes of capillaries.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Potent stimulation of monocytic endothelin-1 production by HIV-1 glycoprotein 120.

Monocytes/macrophages play a critical role in the pathogenesis of HIV infection, both as targets for virus replication and as sources of production of multifunctional cytokines. Endothelins, peptides with potent vasoconstricting activities originally isolated from endothelial cells, are also produced and secreted by macrophages in a manner similar to that of other cytokines. In an attempt to explore the potential role of endothelins in HIV-infection, we investigated the effect of the HIV-1 envelope glycoprotein, glycoprotein 120, on monocytic endothelin-1 production. This glycoprotein has been identified as a potent stimulator of monokines such as TNF-alpha and IL-6, which have been implicated as potential mediators of HIV-encephalopathy. We found that glycoprotein 120, similar to LPS, stimulates the secretion of endothelin-1, as well as TNF-alpha, from macrophages in a concentration-dependent manner. Using reverse transcriptase polymerase chain reaction, we found that circulating monocytes in HIV-infected individuals show a distinct expression of the endothelin-1 gene that is not detectable in healthy controls, indicating chronic activation of this gene in HIV-infection. In addition, cerebral macrophages in patients with HIV-encephalopathy were strongly positive for endothelin. Thus, monocytic endothelins appear to be stimulated during HIV infection. Their potent vasoactive properties render them potential candidates for mediating alterations in the cerebral perfusion pattern associated with the AIDS dementia complex.

AIDS Dementia Complex↗

PubMed1993

Tissue-specific gene expression as an indicator of epididymis-specific functional status in the boar, bull and stallion.

cDNA probes derived from genes expressed specifically in the human epididymis were used to examine gene expression in the epididymides of boar, bull and stallion by Northern hybridization. Two probes for the HE1 and HE4 gene products were found to recognize tissue-specific transcripts in all three species, with a regionally differential distribution within the epididymis. Additionally, antibodies recognizing the HE4 protein were shown to react specifically in the epididymis of the boar and bull. An extensive study of the boar showed that, whereas mRNA for the HE1-homologue was up-regulated markedly only at puberty, the HE4-homologue was already present at moderate levels prepubertally. The distribution of the HE1-homologue changed at sexual maturity from a maximum in the cauda epididymis in the 3-4-week-old pig, to a maximum in the corpus/caput region in the adult, while the shift was in the opposite direction for the HE4-homologue. Evidently, gene expression is not fixed regionally through epididymal development in this species. The abdominal epididymis of a hemicryptorchid pig also showed a differential change in expression for the two gene products by comparison with the scrotal testis from the same animal. The results suggest that the HE1 and HE4 gene homologues may be sensitive markers for physiological changes within the mammalian epididymis, and that the boar could prove a useful model to examine the regulation of these human epididymal transcripts.

Animals↗

PubMed1993

Localization and concentration of a new bioactive acetic seminal fluid protein (aSFP) in bulls (Bos taurus).

The concentration of the new bioactive acetic seminal fluid protein (aSFP) was determined in body fluids of bulls. An aSFP-specific radioimmunoassay was elaborated and established for routine measurement. No crossreaction of the aSFP antibody was found with other seminal plasma proteins and known growth factors. The 14 kDa aSFP was detected in secretions of ampulla and seminal vesicles by immunoblot analysis, but not in testis, epididymis or blood. Large quantities of aSFP were measured by radioimmunoassay in the fluid from the ampulla (2.6 +/- 0.3 mg ml-1) and seminal vesicles (3.0 +/- 0.4 mg ml-1). Immunohistochemistry techniques demonstrated that aSFP was localized mainly in the secretory epithelium of ampulla and seminal vesicles. Large amounts of aSFP (4.0 +/- 0.4 mg ml-1) were present in seminal plasma of bulls within a range of 1-7 mg ml-1, but aSFP could not be found on membranes of spermatozoa. Concentrations vary considerably among individuals, suggesting that aSFP may have a role in bovine reproduction.

Animals↗

PubMed1993

Reverse lectin histochemistry: design and application of glycoligands for detection of cell and tissue lectins.

Plant and invertebrate lectins are valuable cyto- and histological tools for the localization of defined carbohydrate determinants. The well-documented ubiquitous occurrence of sugar receptors encourages functional considerations. Undoubtedly, analysis of the presence of vertebrate lectins in tissues and cells is required to answer the pertinent and tempting question on the physiological relevance of protein (lectin)-carbohydrate recognition in situ. Carrier-immobilized glycoligands, derived from custom-made chemical synthesis, enable the visualization of respective binding sites. Histochemically inert proteins or synthetic polymers with appropriate functional groups are suitable carrier molecules for essential incorporation of ligand and label. The resulting neoglycoconjugates can track down tissue receptors that are neither impaired by fixation procedures nor blocked by endogenous high-affinity ligands. Lectins, especially the receptors of the tissue under investigation (endogenous lectins), and appropriately tailored immobilized glycoligands or lectin-specific antibodies (when available) are complementary tools to test the attractive hypothesis that diverse, functionally relevant glycobiological processes within or between cells are operative. Concomitant evaluation of both sides of lectin histochemistry, namely lectins as tools and lectins as functionally important molecules in situ, will indubitably render desired progress amenable in our often still fragmentary understanding of the importance of tissue lectin and glycoconjugate expression and its regulation.

Animals↗

PubMed1992

Opioid-controlled adenylate cyclase in the guinea-pig myenteric plexus is confined to nerve somata.

Previous studies with the electrically stimulated longitudinal muscle-myenteric plexus preparation of the guinea-pig ileum suggested that opioid control of adenylate cyclase is confined to nerve somata. No indication was found for an opioid effect on the enzyme at nerve terminals of the neuro-muscular junction. The aim of the present investigation was to directly study the effect of opioids on cAMP generation in nerve fragments associated with somata or terminals of the myenteric plexus. Employing the ultracentrifugation technique an enrichment of cell organelles in fractions relating to either somata or terminals was achieved. Opioid binding studies revealed specific mu-receptors which in both fractions were regulated by GTP. Challenge of these fractions with forskolin and prostaglandin E1, respectively, resulted in an increased production of cAMP regardless of their neuroanatomical assignment. Examining the response of neuronal material to the selective mu-opioid DAMGO ([D-Ala2, MePhe4, Gly-ol5]enkephalin) revealed an inhibitory action on cAMP synthesis in somata-enriched fractions. No effect of DAMGO was observed in material linked to nerve terminals, although the presence of mu-opioid receptors and adenylate cyclase has been demonstrated. We conclude that opioid control of adenylate cyclase in the myenteric plexus of the guinea pig is confined to nerve somata.

Adenylyl Cyclases↗