Search

The plasminogen activator (PA) system is thought to play a major role in the proteolytic events associated with spermatogenesis. The mechanisms controlling the expression of PA and of its major physiological inhibitor, plasminogen activator inhibitor type-1 (PAI-1), in the seminiferous epithelium are still unknown. In the present study we analyzed the expression of PA and PAI-1 in a murine Sertoli cell line (42GPA9) in response to stimulation by lipopolysaccharides (LPS) used to activate the phagocytic activity of these cells. Immortalized Sertoli cells cultured under basal conditions secreted predominantly tissue-type PA (tPA) as demonstrated by zymographic analysis and the presence of tPA transcripts. In zymographic experiments a larger molecular weight proteolytic band corresponding to the formation of PA-PAI-1 complex was also observed. The stimulation of immortalized Sertoli cells by LPS resulted in both alteration of the apparent tPA molecular weight to a higher form and transient increase in PAI-1 biosynthesis. The phorbol ester TPA stimulates similarly PAI-1 synthesis in the Sertoli cell line, while 8-bromo-cAMP has no effect. These results suggest for the first time the existence of a direct linkage between molecular events triggered by phagocytosis and regulation of tPA and PAI-1 in Sertoli cells., (Copyright 1999 Academic Press.)

To clarify the exact role of Sertoli cells in testicular intercellular communications, a murine Sertoli cell line (42GPA9) has recently been established. Electron-microscopy studies indicate that the morphology of these immortalized cells strongly resembles that of mouse Sertoli cells in vivo with an indentend nucleus, elongated mitochondria and numerous lysosome-like structures. Ultrastructure analysis has also revealed that 42GPA9 cells form gap junctions as demonstrated by the presence of small electron-dense bridges that connect the plasma membranes of adjacent cells. The gap junction protein connexin 43 (Cx43) has been identified in cultured 42GPA9 cells by immunofluorescence and Western blot analysis. No immunostaining is detected in the absence of apparent intercellular contact. The anti-Cx43 antibody labels the contacts between 42GPA9 cells at confluency. This specific staining appears as small dots forming isolated rows of dots or surrounding the entire cell, suggesting that Cx43 is assembled into membrane plaques. The gap junctional communication capacity of the 42GPA9 cell line has been demonstrated by the dye-transfer technique. Exposure of 42GPA9 cells for 24 h to cAMP and 12-O-tetradecanoylphorbol-13-acetate greatly reduces the Cx43 staining at cell-cell contacts and concomitantly increases the cytoplasmic staining, suggesting that these agents alter the trafficking of Cx43 to the plasma membrane. Thus, the 42GPA9 line may provide a useful in vitro model for studying gap junction communication between Sertoli cells.

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.

In the present study we report the isolation and characterization of a clonal Sertoli cell line (42GPA9) from sexually mature polyoma virus large T (PyLT) transgenic mice. The cells multiplied indefinitely and expressed large T antigen. The 42GPA9 cell line expressed biochemical features associated with normal Sertoli cells. Transferrin, sulfated glycoprotein-2, and the ligand of c-kit were detected by reverse transcription-polymerase chain reactions and Western blot analyses. Zymographic analysis indicated that the 42GPA9 cell line secreted tissue-type plasminogen activator. These cells also retained FSH receptors as suggested by their specific responsiveness to the gonadotropin (morphological and phagocytic changes, stimulation of cAMP production) and the detection of FSH receptor mRNAs. Another original aspect of the 42GPA9 cell line is its ability to form tight junctions at confluency as demonstrated by electron microscopic study and immunolocalization of the tight junction-associated protein zonula occludens 1. The 42GPA9 cell line, which has retained several important hallmarks of normal Sertoli cells, may prove useful for further studies on Sertoli cell behavior and on Sertoli-germ cell interactions in the mature testis.

The involvement of bradykinin, 5-hydroxytryptamine, substance P and prostanoids in the hyperalgesia elicited by collagenase in rat paw was investigated. Collagenase (100 micrograms) induced a slight hyperalgesia in kininogen deficient rats in comparison with the behavioural response obtained in normal rats. Lisinopril (10(-5) M), and angiotensin-converting enzyme inhibitor, increased the duration of the hyperalgesia elicited in normal rats. Ondansetron (0.5 to 5 mumol/kg), a 5-HT3 antagonist, suppressed the hyperalgesia as did methysergide (1.1 to 11 mumol/kg), a mixed 5-HT1 and 5-HT2 receptor antagonist. However, the hyperalgesia was not modified by RP 67580 (1.8 to 18 mumol/kg), a NK1 receptor antagonist, and was only slightly delayed by indomethacin (2 mg/kg), a cyclo-oxygenase inhibitor. The oedema-promoting effect of 5-HT (6 nmol) was inhibited by methysergide but not by ondansetron. The swelling induced by collagenase in rat paw was reduced by methysergide but not by ondansetron. We conclude that the behavioural response induced by collagenase depends on an interactions between bradykinin and 5-HT. Prostanoids play a minor role in the beginning of the reaction whereas substance P is not significantly involved in this hyperalgesia.

The mechanism of the myostimulating activity of rat tissue kallikrein on rat uterus was re-examined using uterus from kininogen-deficient rats and HOE 140 (D-Arg[Hyp3, Thi5, D-Tic7, Oic8]bradykinin), a specific bradykinin receptor-B2 antagonist. The uterus from kininogen-deficient rats was 50 times less sensitive to rat kallikrein than that from normal rats. HOE 140 (6 to 60 nM) inhibited the contracting effects of bradykinin and of rat kallikrein. Porcine kallikrein had no effect on rat uterus. Bradykinin and rat kallikrein induced a relaxation of rat duodenum. The duodenum from kininogen-deficient rats was 100 times less sensitive to rat kallikrein than the duodenum from normal rats. HOE 140 (0.6 to 3 nM) inhibited the relaxing effects of bradykinin and of kallikrein. Preincubation of rat kallikrein with aprotinin (Trasylol) abolished the effects of kallikrein on smooth muscles. HOE 140 inhibited the amidolytic activity of tissue kallikrein with a Ki value of 220 microM. HOE 140, at micromolar concentrations, suppressed the kininogenase activity of tissue kallikrein. Plasma of deficient rats contained 0.7% of the normal levels of kininogens. After washing the blood vessels with saline, kininogens were present in uterine homogenates but not in duodenal homogenates from both rat strains.(ABSTRACT TRUNCATED AT 250 WORDS)

The large T antigen of polyomavirus (PyLT) efficiently immortalizes rodent fibroblasts, but, unlike SV40 T antigen, it is not sufficient to achieve complete oncogenic transformation. We analysed a series of transgenic mouse families that express the PyLT protein under control of the viral enhancer-promoter region. In all of them, the transgene was expressed in the seminiferous epithelium of the testis (Sertoli and germ cells), with no pathological consequences during most of the animals' lives. However, every old male developed large bilateral tumours of the testes, generated by the proliferation of Sertoli cell derivatives. Cell lines could be readily established both from the tumours and from the still apparently normal testis before the onset of tumoral growth. They retained in vitro morphological and ultrastructural features characteristic of Sertoli cells. But, in addition to this major Sertoli component, the maintenance of a cellular contingent of germinal origin was suggested by the expression of genes that are normally transcribed during the premeiotic and early meiotic stages of spermatogenesis (LDH-X, Hox1.4 and c-kit). The two cell types remained tightly associated, even at late passages in culture, and could not be separated by conventional cloning procedures. This association in culture of the two cell types whose interaction is critical for spermatogenesis may provide a useful tool for its molecular analysis.

The distribution in the nervous system of T-kininogen, the third kallikrein-resistant kininogen of the rat, was determined using bioassays and a radioimmunoassay system. In rat brain homogenates, trypsin released large amounts of a kinin-like myostimulating activity while urinary kallikrein released small amounts. The kinins released by trypsin were identified by HPLC as mostly T-kinin. Radioimmunoassays showed that a T-kininogen-like immunoreactive factor was uniformly distributed throughout the central nervous system. Higher levels were found in female rats than in male rats. Maximum levels were observed in newborn animals. A slight increase of T-kininogen content of the brain was observed after turpentine injection while T-kininogen level in liver was dramatically increased. T-kininogen plasma contamination to the nervous tissues was estimated by injecting 125I-labelled T-kininogen. The T-kininogen content of rat cultured cells and neurons was also examined. Highest levels were found in dorsal root ganglia neurons, lower levels in Schwann cells, phaeochromocytoma cells, mixed cells from spinal ganglion and in astrocytes. Immunocytochemistry showed the presence of T-kininogen in the cytoplasm of cultured dorsal root ganglia neurons and embryonic hippocampal neurons. The distribution of T-kininogen throughout the central and peripheral nervous system of the rat, the variations of its level during the life span suggest that T-kininogen would play the role of a cysteine proteinase inhibitor and not that of a T-kinin-releasing substrate in nervous tissues.

1. Kinins were measured by a radioimmunoassay in the inflammatory exudates induced by carrageenin or zymosan in the peritoneal cavity of normal Wistar rats and of kininogen-deficient Brown Norway rats. 2. After administration of carrageenin to normal rats, levels of immunoreactive kinins showed a single peak during the first two hours and then decreased. The presence of kinins preceded and accompanied the exudation of 125I-labelled albumin. Kinins were identified as bradykinin by chromatography. 3. Captopril, an inhibitor of kininase 2, increased the level of kinins and the volume of the exudates after carrageenin treatment. In Brown Norway rats, the volume of the exudates was small and contained little or undetectable amounts of immunoreactive kinins. 4. During zymosan-induced peritonitis, the exudates were devoid of immunoreactive kinins in both species. The volume of the exudates was larger in kininogen-deficient rats than in normal rats. 5. We conclude that in rats, the kinin system is a major factor responsible for the development of the inflammatory reactions induced by carrageenin, but is not involved in the reactions induced by zymosan.

We studied the influence of aprotinin and soya bean trypsin inhibitor (SBTI) on the inflammatory reaction induced by the implantation of dry sponges in normal Wistar rats and in kininogen-deficient Brown Norway rats, during the first day after the implantation. In normal rats, aprotinin reduced the volume and total protein content of the exudates at 3 h but not thereafter. Aprotinin also markedly reduced the immunoreactive kinins and kallikrein in the exudates. Aprotinin did not modify the volume of the exudates of the Brown Norway rats. SBTI reduced the inflammatory reaction in both rat strains but did not significantly modify the formation of immunoreactive kinins. The inflammatory reaction developed more slowly in Brown Norway rats. The kinin system is thus involved during the first hours of the development of this acute inflammatory reaction. The anti-inflammatory effect of SBTI does not depend on the inhibition of kinin formation.

The volume of the peritoneal exudate induced in the rat by iota carrageenan is reduced by subcutaneous administration of indomethacin while the concentrations of three lysosomial enzymes in the exudate are slightly increased or not modified. Thus, the total enzymatic activities of the exudate are reduced by indomethacin. The leucocyte accumulation remains unchanged in the indomethacin treated rats. During the development of the peritoneal exudate, the circulating plasma displays a high degree of lysosomial enzymes activity which is suppressed by indomethacin at the dose of 4 mg/kg.

1. Rat serum levels in beta-glucuronidase and beta-galactosidase are higher than plasma levels. Rat platelets release these lysosomial enzymes during blood coagulation in vitro. 2. After anaphylactic shock, in the sensitized rat, there is no increase in beta-galactosidase and beta-glucuronidase plasma levels. The tissues of the sensitized rat do not release these enzymes during the antigen-antibody reaction. The blood platelet level is diminished after anaphylactic shock and the serum levels of the lysosomial enzymes are decreased. 3. In thrombopenic rat, anaphylactic shock is identical as in control animals. Rat platelets do not play a significant role in the anaphylactic shock.

Catechin dimers induce a large long-lasting oedema when injected in the paw of the rat. This oedema is not inhibited by methysergide, promethazine, indomethacin, phenidone, bromophenacyl bromide and colchicine. It is not modified in rats made leukopenic by methotrexate. It is slightly delayed in Brown Norway rats which were kallikrein-kininogen deficient. Similarly catechin dimers induce the formation of a large peritoneal exudate in the rat. The exudate contains insignificant levels of leucocytes and 5-hydroxytryptamine. It contains kinins but its PG content is very low. The exudate does not activate (14C)-arachidonic acid into PG. Catechin dimers induce kinin formation in rat plasma "in vitro". They inhibit the formation of PG and HETE-like compounds from (14C)-arachidonic acid by rat peritoneal cells "in vitro". Catechin dimers administered at sub-irritant doses reduced carrageenan-induced oedema. Catechin dimers at low doses have an anti-inflammatory effect which may depend on PG synthesis inhibition. At larger doses, they induce inflammatory responses which occur with almost complete lack of participation of PG.

In anesthetized dog, intravenous infusion of NA does not modify the relationship between [3H]-(+/-)-NA urinary clearance/GFR. Urinary excretion of NA is directly related to glomerular ultrafiltration.

Urinary elimination of conjugated catecholamines (adrenaline and noradrenaline) is increased during normal pregnancy (60.5 % in the normal ; 70 % in the pregnants).

Conjugated noradrenaline (NA) has been identified as a constituant of the walls of a Vespid wasp: Vespula germanica Linne. Concentrations range between 1,8 mug/g (external wall) and 18 mug/g (internal structure). Probably NA originates from the saliva of the Hymenoptera.

The influence of serum from normal and turpentine-treated rats on prostaglandin synthesis by peritoneal cells has been investigated. Both types of serum had the same inhibitory effect on the formation of myostimulating prostaglandin-like substances and of 14C-PGE2 by rat peritoneal cells in the presence of 82 or 100 mumol of arachidonic acid. On guinea-pig peritoneal cells, the serum from turpentine-treated rats had a smaller inhibitory effect than normal serum on 14C-PGE2 and 14C-HETE formation from 2 mumol of arachidonic acid. Thus the acute inflammatory reaction produced by turpentine did not increase the inhibitory effect of rat serum on prostaglandin synthesis. It is suggested that the anti-inflammatory effect of counter irritation by turpentine does not depend on prostaglandin synthesis inhibition but has to be attributed to some other mechanisms.

A rapid and convenient three-step purification scheme has been developed for the purification of T-kininogen (alpha 1-cysteine proteinase inhibitor) from rat plasma. The purification process includes chromatography on hydroxyapatite, immunoaffinity chromatography and gel filtration. This procedure is applied to plasma from the brown Norway rat which is known to be deficient in high and low molecular weight kininogens. The method furnished large amounts of T-kininogen from turpentine-treated Wistar rats as well as from untreated and turpentine-treated deficient brown Norway rats. The amino acid and hexose content of the three T-kininogens has been determined. While the composition of the molecules isolated from both injured rats was similar, the neutral sugar content of T-kininogen purified from untreated brown Norway rats was lower and its amino acid composition showed slight differences. The three molecules have identical behaviour and similar physicochemical and immunological properties when analysed by SDS electrophoresis, isoelectrofocusing and two-dimensional immunoelectrophoresis.

The urinary clearance for DL-[3H] noradrenaline has been measured in four human subjects. The mean ratio of noradrenaline clearance over glomerular filtration rate was 0.93, the confidence limit for the mean at the 5% level being 0.81-1.06. These results are in perfect agreement with those obtained in dogs with the same experimental procedure. It is concluded that the urinary excretion of noradrenaline is strictly proportional to the glomerular filtration rate.

Antibodies raised in rabbits against rat T-kininogen (alpha 1-cysteine proteinase inhibitor) were used to develop a radioimmunoassay and a nephelometric quantification for T-kininogen. These assays were specific and analytically reliable. We also described a radioimmunoassay for kinin measurement. These immunological methods have been used to study the behaviour of T-kininogen during inflammatory processes and specify the two properties of this kind of kininogen: its inhibitory capacity towards cysteine proteinases and its activity as precursor of T-kinin. Control plasma level of T-kininogen in male rats was lower than that of female rats. The maximum level was observed in plasma, liver, kidney and uterus of female rats during metestrus. After turpentine injection, T-kininogen level increased not only in plasma but also in liver and kidney. In carrageenan-induced peritoneal exudates, we found a large accumulation of T-kininogen and of immunoreactive kinins, these latter being identified by HPLC as bradykinin.

In dogs, the urinary clearance of IPNA-3H is very near GFR (ratio CIPNA-3H/GFR = 0,92). This ratio is not modified by cocaine. In our experimental conditions, we observe no tubulary secretion of IPNA-3H.

Conjugated urinary catecholamines are increased during normal human pregnancy. The modification in the proportions of conjugated fractions are particularly significant for norepinephrine. They depend on the foetoplacental unit since delivery is followed by a reduction of the urinary concentrations of these conjugated fractions. Such a modification of catecholamine metabolism must be kept in mind when the level of sympathetic nervous system activity is studied during pregnancy.