Phosphorylation of myosin light chain during capping of mouse T-lymphoma cells.

Colchicine induces the clustering of at least three different T-lymphoma surface antigens (T200, Thy-1, and gp 69/71) into a cap structure in the absence of any external ligand. In addition, colchicine induces the intracellular accumulation of actin and myosin directly beneath the surface cap structure. We have discovered that myosin molecules (both heavy and light chains) are closely associated with the plasma membrane of T-lymphoma cells. Most importantly, we have found that the 20,000-dalton light chain of lymphocyte myosin is both phosphorylated and preferentially accumulated in the plasma membrane of colchicine-induced capped cells. It is proposed that myosin light chain is directly involved in the activation of membrane-associated actomyosin required for the collection of surface proteins into a cap structure (analogous to muscle cell sliding filament contraction).

A T-lymphoma transmembrane glycoprotein (gp180) is linked to the cytoskeletal protein, fodrin.

A major mouse T-lymphoma surface glycoprotein (gp180) has been identified by labeling cells with 125I and [3H]glucosamine. After ligand-induced receptor patching and/or capping, the amount of gp 180 in the membrane-associated cytoskeleton fraction increases in direct proportion to the percentage of patched/capped cells. There is a parallel increase in the amount of fodrin in the membrane-associated cytoskeleton fraction. Evidence is presented that gp180 is the same as or very similar to the T-lymphocyte-specific glycoprotein T-200. An immunobinding assay of Nonidet P-40-solubilized plasma membrane selectively co-isolates gp180 and fodrin. After induction of receptor rearrangement, double-label immunofluorescence reveals that fodrin accumulated directly beneath gp180 patches and caps. Membrane extraction with Triton X-114 followed by sucrose gradient centrifugation permits isolation of a gp180-fodrin complex with a 1:1 molar ratio and sedimentation coefficient(s) of approximately 20. This complex remains stable during isoelectric focusing and exhibits a pl in the range of 5.2-5.7. On the basis of our results we conclude that gp180, an integral membrane glycoprotein, and fodrin, a component of the membrane-associated cytoskeleton, are closely associated into a complex. Furthermore, we contend that, through fodrin's association with actin, this complex is of functional significance in ligand-induced patching and capping of gp180. We also propose that, through lateral interactions in the plane of the membrane, the gp180-fodrin complex might be responsible for linking other surface receptors to the intracellular microfilament network during lymphocyte patching and capping.

Association of myosin light chain kinase with lymphocyte membrane-cytoskeleton complex.

A specific antibody against myosin light chain kinase (MLCK) was used to identify the presence of a Ca2+-calmodulin-activated MLCK in mouse 1-lymphoma cells. With a double immunofluorescence technique, MLCK was determined to be accumulated directly under Con A-capped structures in a manner similar to that of previously described accumulation of actomyosin. The lymphocyte MLCK was phosphorylated in the uncapped cell and, by immunoprecipitation with a specific MLCK antibody, was shown to possess a Mr of 130,000. The MLCK was also found to constitute a major fraction of the phosphoproteins present in the plasma membrane associated-cytoskeleton. Myosin light chain kinase catalyzed the phosphorylation of both endogenous lymphocyte myosin light chains and those from smooth and skeletal muscle. The enzyme activity was dependent on the presence of Ca2+-calmodulin and was inhibited by the calmodulin-binding drug, trifluoperazine. These data suggest that the membrane-cytoskeleton-associated MLCK activity may be important in regulation of the actinmyosin contraction which is believed to be required for the collection of surface receptors into capped structures.

Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effects in rat Leydig cells.

Human CG (hCG) and insulin-like growth factor-I (IGF-I) have synergistic effects on Leydig cell function. Leydig cells express high affinity IGF-I receptors. The number of IGF-I receptors and IGF-I receptor mRNA levels can be up-regulated by hCG. The most abundant mRNA species of the IGF binding proteins (IGFBPs) in rat Leydig cells is IGFBP-2. In the present study, we investigated the effect of hCG on IGFBP-2 transcription, mRNA accumulation, and protein production/secretion. Biological effects of IGFBP-2 on Leydig cells were also examined. Rat Leydig cells were purified from testes using centrifugal elutriation followed by Percoll gradient centrifugation. Cells were cultured for 24 h and then treated with or without hCG (10 ng/ml) for 6 h. The expression of IGFBP-2 mRNA was decreased by hCG in a dose-dependent manner, and at a concentration of 10 ng/ml the expression of IGFBP-2 mRNA was reduced by 50%. As early as 2 h after the addition of hCG, there was a significant decrease in IGFBP-2 mRNA accumulation. To evaluate the mechanism(s) responsible for decreased IGFBP-2 gene expression by hCG, the effect of hCG on the rate of transcription and stability of the mRNA was determined. Human CG (10 ng/ml) reduced the IGFBP-2 transcription rate by 32%/h in comparison with the control, while the half-life (t1/2) of mRNA remained unaltered (hCG-treated cells, 0.58 h; control cells, 0.51 h). IGFBP-2 with a molecular size of 33 kilodaltons was detected as a major band in the Western ligand blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Human chorionic gonadotropin decreases insulin-like growth factor-I gene transcription in rat Leydig cells.

Insulin-like growth factor-I (IGF-I) and hCG have synergistic effects on Leydig cell steroidogenesis in primary culture. In the present study, we investigated the effects of hCG on IGF-I gene transcription in Leydig cells. Purified Leydig cells (8-10 x 10(6) cells/100-mm dish) obtained from 50- to 65-day-old male Sprague-Dawley rats were cultured for 24 h. After medium change, hCG (0.1-10 ng/ml) or 8-bromo-cAMP (0.1 mM) was added, and cultures were continued for varying periods of time. In response to stimulation with hCG, there was a marked increase in the expression of cholesterol side-chain cleavage cytochrome P450 mRNA. In contrast, hCG caused time- and dose-dependent decrements in IGF-I mRNA levels. Both large [7.5-kilobase (kb)] and small (0.8- to 1.2-kb) species of IGF-I mRNAs were markedly decreased 6 h after treatment with hCG. hCG in a concentration of 0.1 ng/ml did not alter IGF-I mRNA levels. Higher concentrations of hCG (1 and 10 ng/ml) markedly decreased both 7.5- and 0.8- to 1.2-kb IGF-I mRNAs (80% and 56% reductions, respectively). 8-Bromo-cAMP (0.1 mM) also markedly reduced IGF-I mRNA levels. Finally, we evaluated the effects of hCG on the stability and transcription rates of IGF-I mRNA. We found that t1/2 of IGF-I mRNA for control Leydig cells was 3.86 h, which was not significantly different from that of hCG-treated cells (t1/2 = 3.41 h). This indicates that treatment with hCG did not change the stability of IGF-I mRNA. The average transcription rate per h for IGF-I mRNA decreased from 1 (for control cells) to 0.74 (for hCG-treated cells). The t1/2 values and rates of transcription for beta-actin were 7.39 and 7.16 h, and 1 and 0.94 for control and hCG-treated cells, respectively, showing that RNA stability and rates of transcription did not change significantly for the beta-actin transcript. In conclusion, we have unequivocally demonstrated that hCG decreases the expression and transcription of IGF-I mRNA in Leydig cells.

Interleukin-1 induces insulin-like growth factor binding protein-3 gene expression and protein production by Leydig cells.

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side-chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4-, and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis.

Expression and regulation of insulin-like growth factor-binding protein-1, -2, -3, and -4 messenger ribonucleic acids in purified rat Leydig cells and their biological effects.

The actions of insulin-like growth factor-I (IGF-I) are modified by binding proteins [IGF-binding proteins (IGFBPs)]. Previously, we reported that IGF-I enhances Leydig cell steroidogenesis and that IGF-I mRNA is expressed in Leydig cells. In the present study, we evaluated the expression and regulation of IGFBP-1, -2, -3, and -4 in purified rat Leydig cells and their biological effects. We found that none of the testicular crude interstitial cells, purified Leydig cells, or seminiferous tubules expressed IGFBP-1 mRNA. This indicated that IGFBP-1 mRNA is not expressed in the testis in detectable amounts. In contrast, large amounts of IGFBP-2 with a size of 1.8 kilobases (kb) were expressed in purified Leydig cells, and lesser amounts in crude interstitial cells. Small amounts of IGFBP-2 mRNA were expressed in seminiferous tubules, but none could be detected in liver. IGFBP-3 mRNA was predominantly expressed in purified Leydig cells, crude interstitial cells, and liver, while appreciable amounts were not found in seminiferous tubules. Liver had the highest amounts of IGFBP-4 mRNA, whereas purified Leydig cells and crude interstitial cells had lesser amounts. We next evaluated the pituitary dependency of IGFBP mRNAs in Leydig cells. Purified Leydig cells were isolated from 50-day-old rats 5 days after hypophysectomy. IGFBP-2, -3, and -4 mRNA levels in Leydig cells decreased 22%, 80%, and more than 90%, respectively, after hypophysectomy. In the liver, however, IGFBP-2 mRNA levels increased, and IGFBP-3 mRNA levels decreased, while IGFBP-4 remained unchanged. As expected, hypophysectomy caused decrements in large (7.0- to 7.5-kb; a 75% reduction) and small (0.8- to 1.2-kb; a 90% reduction) IGF-I mRNA levels in Leydig cells. Hypophysectomy also reduced IGF-I mRNA expression in liver. Finally, the effects of IGFBP-2, -3, and -4 on Leydig cell testosterone formation were investigated. hCG in a concentration of 10 ng/ml increased testosterone formation from 0.6 +/- 0.01 to 27.4 +/- 1.01 ng/10(6) cells.h. In the presence of IGF-I (10 ng/ml), testosterone formation was further increased to 58.6 +/- 1.6 ng/10(6) cells.h (P < 0.01). IGFBP-3 (0.1, 1, and 2.5 pmol/ml) caused a dose-dependent inhibition of IGF-I- plus hCG-induced testosterone formation. IGFBP-3 in a concentration of 2.5 pmol/ml completely neutralized the effects of IGF-I on Leydig cell steroidogenesis. IGFBP-4 had a lesser effect, while IGFBP-2 had no effect on IGF-I- plus hCG-induced testosterone formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Human chorionic gonadotropin up-regulates insulin-like growth factor-I receptor gene expression of Leydig cells.

The effects of hCG, 8-bromo-cAMP, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, and forskolin on insulin-like growth factor-I (IGF-I) receptor gene expression of Leydig cells were studied. The treatment of purified Leydig cells with hCG caused a dose-dependent increase in [125I]IGF-I binding to Leydig cells without changes in binding affinity, indicating that the increased binding was due to increased receptor numbers and not to increased affinity. The minimal time required for hCG to induce IGF-I binding was 6 h, and it had reached a plateau at 16 h. 8-Bromo-cAMP (1 mM) increased IGF-I binding about 2-fold, and forskolin (10 microM) increased binding about 51%. Using the ribonuclease protection assay, we found that hCG and 8-bromo-cAMP could increase IGF-I receptor mRNA expression as early as 2 h before the increase in IGF-I binding. The induction by hCG was over 3.5-fold at 4 h and decreased to about 2-fold at 6 h. 4 beta-Phorbol 12 beta-myristate 13 alpha-acetate had a very small effect on IGF-I receptor mRNA levels (1.5-fold increase at 2 h and no changes at 4 and 6 h). In conclusion, IGF-I receptors can be up-regulated by hCG, 8-bromo-cAMP, and forskolin. The up-regulation of IGF-I receptor number is associated with transient increases in IGF-I receptor mRNA levels. This could be a mechanism by which hCG and IGF-I interact to enhance Leydig cell steroidogenesis.

Interleukin-1 inhibits cholesterol side-chain cleavage cytochrome P450 expression in primary cultures of Leydig cells.

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. We have reported that IL-1 inhibited hCG-induced cAMP and testosterone formation. In the present study we evaluated the effect of IL-1 on Leydig cell cholesterol side-chain cleavage cytochrome P450 (P450scc) mRNA levels. P450scc is the rate-limiting enzyme for Leydig cell steroidogenesis. Highly purified Leydig cells were prepared from adult Sprague-Dawley male rats (55-65 day-old) using the combination of elutriation and Percoll gradient. Purified Leydig cells were then cultured with or without IL-1 beta (1-100 ng/ml) and recombinant human monocyte-derived IL-1 receptor antagonist (250 ng/ml) for 24 h. hCG (10 ng/ml), 8-bromo-cAMP (0.1 mM), or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate was then added, and cultures were continued for an additional 6 h. P450scc mRNA levels of Leydig cells were very low to undetectable after 24 h in culture and could be stimulated by the addition of either hCG (10 ng/ml) or 8-bromo-cAMP (0.1 mM), but the addition of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate had no effect. P450scc mRNA levels increased as early as 2 h after the addition of hCG. Furthermore, cycloheximide (1 microgram/ml) markedly blocked hCG-induced P450scc mRNA expression. This indicates that synthesis of a labile new protein(s) is required for the induction of P450scc mRNA by hCG. IL-1 beta inhibited hCG-stimulated testosterone formation and P450scc mRNA expression in a dose-dependent manner. The inhibitory effects of IL-1 beta could be reversed by the concomitant addition of IL-1 receptor antagonist. Our results suggest that P450scc mRNA levels of Leydig cells are modulated by IL-1. This may be one mechanism that could explain the inhibitory effects of IL-1 on Leydig cell steroidogenesis.

Interleukin-6 secretion from rat Leydig cells in culture.

Recent evidence indicates that interleukin-6 (IL-6) acts on Sertoli cells to modulate secretory function. IL-6 is also detected in medium bathing tissue or cells from the seminiferous tubule, suggesting a testicular regulatory role. Because other cytokines found to be active in testicular function have more than one site of production, we examined whether Leydig cells may serve as an alternate source of IL-6. Purified Leydig cells were cultured with or without modulatory substances, and the medium was subjected to the 7TD1 bioassay for IL-6. Northern analysis using an IL-6 cDNA probe was performed on companion cell preparations. Incubation with either hCG or IL-1 beta increased the levels of bioactive IL-6 released into the medium and IL-6 mRNA detected in the cells in a dose-related manner. When used together, these agents had an additive stimulatory influence on both the release of IL-6 bioactivity and the amount of IL-6 mRNA. Our results demonstrate that IL-6 is secreted from enriched preparations of Leydig cells and that its release is under the control of at least two modulators of testicular function. Identification of interstitial cells as a site of IL-6 production coupled with reports of IL-6 release and action in seminiferous tubular cell preparations suggest that IL-6 may serve a role in signal integration or communication from one testicular location to another.

Down-regulation of Leydig cell insulin-like growth factor-I gene expression by interleukin-1.

We have reported previously that insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) is expressed in Leydig cells and that IGF-I can enhance androgen production, whereas interleukin-1 (IL-1) is a potent inhibitor of Leydig cell steroidogenesis. Molecular cloning studies have confirmed the existence of at least two species of IL-1: IL-1 alpha and IL-1 beta. Both IL-1 alpha and beta bind to the same receptors and have the same spectrum of biological activities. The purpose of the present study was to elucidate the molecular mechanisms of the interaction between IGF-I and IL-1 both in vivo and in vitro. Adult Sprague-Dawley rats (55-65 days old) were treated with three injections of human recombinant IL-1 beta (1 microgram/rat ip) at 12-h intervals. Rats were killed 2 h after the last injection of IL-1 beta. Purified Leydig cells were isolated and RNA extracted for Northern blot analyses. For in vitro studies, highly purified Leydig cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 0.1% fetal calf serum with or without IL-1 beta (1-100 ng/ml) for 24 h. RNA was then extracted from these cells. For time course studies, purified Leydig cells were initially cultured for 24 h. Fresh medium was then added with or without IL-1 beta (10 ng/ml), and the cultures were continued for an additional 2, 4, or 6 h. In vivo administration of IL-1 beta inhibited IGF-I mRNA expression in Leydig cells (a 40% reduction, P less than 0.05). IL-1 beta also suppressed IGF-I mRNA expression in Leydig cells in vitro in a time- and dose-dependent fashion. Inhibitory effects of IL-1 beta (10 ng/ml) could be demonstrated as early as 2 h and reached a nadir at 6 h (a 60% reduction, P less than 0.05). IL-1 beta (100 ng/ml) inhibited IGF-I mRNA expression to about 10% of the controls (P less than 0.01). Moreover, the inhibitory effect of IL-1 beta could be reversed by the addition of IL-1 receptor antagonist. In conclusion, IL-1 beta could directly inhibit the mRNA expression in Leydig cells for IGF-I, an important autocrine modulator of Leydig cell function. This suggests that the effect of IL-1 beta on Leydig cell function is, at least in part, achieved by down-regulation of IGF-I mRNA levels.

Interleukin-1 beta induces interleukin-1 alpha messenger ribonucleic acid expression in primary cultures of Leydig cells.

Previously, we have reported that interleukin-1 (IL-1) can modulate Leydig cell steroidogenesis. Recently, IL-1-like material has been shown to be present in the testis; however, the cellular source of this material remains unclear. In the present study we found that human recombinant IL-1 beta (1-100 ng/ml) caused dose-dependent increases in IL-1 alpha mRNA expression in Leydig cells. Similar to that reported in other tissues, IL-1 alpha mRNA from Leydig cells is mainly 2.2 kilobases. IL-1 alpha mRNA expression in Leydig cells was detectable as early as 2 h after the addition of IL-1 beta (10 ng/ml) and persisted for up to 24 h. Lipopolysaccharide also stimulated IL-1 alpha mRNA expression in these cells, but phorbol ester had no effect. Our results indicate that Leydig cells are a potential source of IL-1, which has both autocrine and paracrine effects.

Expression and regulation of interferon-gamma-inducible protein 10 gene in rat Leydig cells.

In the present study, we report the cloning of a gene that is differentially expressed in normal adult rat Leydig cells and whose expression is inhibited by hCG but is induced by interferon-gamma (IFNgamma). DNA sequence analysis identified this gene as rat IFNgamma-inducible protein 10 (IP-10), a member of the -C-X-C- chemokine superfamily of proinflammatory cytokines. High levels of IP-10 messenger RNA (mRNA) were constitutively expressed in freshly isolated and primary cultured Leydig cells. hCG inhibited this expression in a dose-dependent manner. The addition of 1 ng/ml hCG inhibited IP-10 mRNA levels more than 80%. Conversely, IP-10 mRNA levels were markedly increased in response to murine interleukin-1alpha, murine tumor necrosis factor-alpha, and murine IFNgamma by 3.3-, 10-, and 26-fold, respectively. Concomitant addition of murine interleukin-1alpha, murine tumor necrosis factor-alpha, and murine IFNgamma synergistically increased IP-10 mRNA levels by 58-fold. Furthermore, in addition to one previously described rat IP-10 mRNA transcript (1.5 kb), another larger transcript (2.7 kb) was identified by Northern blot in rat Leydig cells. After screening a rat testis complementary DNA library, we obtained a partial structural gene and an intron sequence, which possibly originated from the larger transcript of rat IP-10 mRNA. Histochemical and immunocytochemical staining revealed that purified cells were positive for 3beta-hydroxysteroid dehydrogenase and IP-10, confirming that IP-10 is indeed present in Leydig cells. IP-10 antisense oligonucleotides enhanced basal and hCG-induced testosterone formation. This suggests that endogenous IP-10 has an inhibitory effect on Leydig cell steroidogenesis. In conclusion, IP-10 is expressed in rat Leydig cells and may have paracrine and autocrine effects on testicular function.

Phagocytic properties of human keratocyte cultures.

The uptake of polystyrene latex beads (approximately 0.75 micrometers) and glutaraldehyde-treated erythrocytes by human corneal stromal keratocytes maintained in culture has been studied. Combined phase-contrast and scanning electron microscopic observations on individual cells after exposure to either beads or erythrocytes demonstrated that the majority of these particles were present intracellularly. Transmission electron microscopy revealed that the beads were membrane-bound within the cytoplasm of these cells. Thus the human keratocytes in culture are phagocytic and able to internalize particles ranging in size from approximately 0.75 to 6 micrometer. Kinetic studies showed continuous uptake of the polystyrene latex beads for at least 72 hr, with an approximate linear uptake rate between 4 and 48 hr. The extent of bead uptake was dependent on the initial bead concentration. The human keratocyte cultures were markedly more phagocytic than human skin fibroblasts or rabbit chondrocytes. It was also found that after extensive bead uptake the normal growth pattern of the keratocytes was affected. It is suggested that the phagocytic ability of the human keratocytes is involved in the turnover of the corneal stromal matrix as well as in the initial response of this avascular tissue to injury or bacterial infection.

Expression and regulation of glucose transporter 8 in rat Leydig cells.

Basal and LH/human chorionic gonadotropin (hCG)-stimulated testosterone formation by Leydig cells is dependent on ambient glucose levels. Inhibition of glucose uptake is associated with decreased testosterone formation. Recently, glucose transporter 8 (GLUT8) has been shown to be highly expressed in the testis. In the present study, we have investigated the expression and regulation of the GLUT8 gene in rat Leydig cells. Primers were designed by using sequences that are not conserved in GLUT1 to GLUT5 and that contain the glycosylation region of GLUT8. This yielded an amplicon of 186 bp. The tIssue-specific expression experiments in adult rat (55- to 65-day-old) tIssues revealed that GLUT8 is expressed predominantly in the testis, in smaller amounts in heart and kidney, and in negligible amounts in liver and spleen. Furthermore, GLUT8 mRNA was found to be highly expressed in crude interstitial cells, Leydig cells and testicular and epididymal germ cells. In prepubertal rat (20-day-old) tIssues, GLUT8 expression was comparatively much lower than in the adult rat tIssues. By comparative RT-PCR, hCG caused dose- and time-dependent increases of GLUT8 mRNA levels. hCG and IGF-I had synergistic effects on GLUT8 mRNA and protein expression. GLUT1 and GLUT3 were also found to be expressed in Leydig cells. However, neither GLUT1 nor GLUT3 were affected by treatments with hCG, IGF-I or hCG and IGF-I combined. The addition of murine interleukin-1alpha (mIL-1alpha; 10 ng/ml), murine tumor necrosis factor-alpha (mTNF-alpha; 10 ng/ml), murine interferon-gamma (mIFN-gamma; 500 U/ml) separately or in combination decreased hCG-induced GLUT8 mRNA levels significantly. In conclusion, GLUT8 mRNA in Leydig cells was positively regulated by hCG and IGF-I and down-regulated by cytokines, mIL-1alpha, mTNF-alpha and mIFN-gamma. These results indicate that hCG, growth factors and cytokines affect Leydig cell steroidogenesis by modulating GLUT8 expression.

Human chorionic gonadotropin induces interleukin-1 gene expression in rat Leydig cells in vivo.

Luteinizing hormone (LH)/human chorionic gonadotropin (hCG) causes inflammatory-type responses in the testis. In the present study, we evaluated the effects of hCG on Leydig cell interleukin-1 (IL-1) gene expression. Using monoclonal antibody (ED2) staining for macrophages, our Leydig cell preparations had no significant contamination with macrophages. When purified Leydig cells from normal rats were cultured for 24 h and then treated with IL-1 beta (1-100 ng/ml) for 6 h, IL-1 beta induced dose-dependent increases in IL-1 beta mRNA levels. IL-1 beta also induced IL-1 alpha mRNA accumulation; however, the level of IL-1 alpha mRNA was much lower than that of IL-1 beta mRNA. When rats were treated with either saline or hCG (5 units i.p.), hCG markedly induced IL-1 beta mRNA accumulation in purified Leydig cells at 6 h which persisted for over 24 h. However, hCG had no direct effect on purified Leydig cell or crude interstitial cell IL-1 mRNA levels. Our results suggest that inflammatory effects of hCG in vivo may be mediated by increased IL-1 gene expression in Leydig cells.

Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells.

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-alpha (TNF-alpha) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 +/- 3.1 ng/10(6) cells/h (mean + SE). TNF-alpha in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-alpha almost completely inhibited testosterone formation (p < 0.001). TNF-alpha (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-alpha (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 +/- 9 ng/10(6) cells/h to 4.9 ng/10(6) cells/h. This indicates that the major effect of TNF-alpha is at steps beyond LH receptor site. To further evaluate the site(s) of action of TNF-alpha, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-alpha on hCG-induced testosterone formation. TNF-alpha had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-alpha is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-alpha had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1beta. In the present study, we demonstrated that both murine TNF-alpha and human IL-1beta were potent inhibitors of hCG-induced testosterone formation. IL-1beta alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-alpha (10 ng/ml), IL-1beta in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-alpha on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-alpha and IL-1beta inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-alpha on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-alpha inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-alpha is a potent inhibitor of Leydig cell function. TNF-alpha inhibited both P450scc and IGF-I mRNA gene expression.

Quantitative analysis of neutral and acidic sugars in whole bacterial cell hydrolysates using high-performance anion-exchange liquid chromatography-electrospray ionization tandem mass spectrometry.

A procedure for analysis of a mixture of neutral and acidic sugars in bacterial whole cell hydrolysates using high-performance anion-exchange liquid chromatography-electrospray ionization tandem mass spectrometry (HPAEC-ESI-MS-MS) is described. Certain bacteria (including bacilli), grown under phosphate-limited conditions, switch from producing a teichoic acid (containing ribitol) to a teichuronic acid (characterized by glucuronic acid content). Bacterial cells were hydrolyzed with sulfuric acid to release sugar monomers. The solution was neutralized by extraction with an organic base. Hydrophobic and cationic contaminants (including amino acids) were removed using C18 and SCX columns, respectively. HPAEC is well established as a high-resolution chromatographic technique, in conjunction with a pulsed amperometric detector. Alternatively, for more selective detection, sugars (as M-H- ions) were monitored using ESI-MS. In HPAEC, the mobile phase contains sodium hydroxide and sodium acetate, which are necessary for chromatographic separation of mixtures of neutral and acidic sugars. Elimination of this high ionic content prior to entry into the ESI ion source is vital to avoid compromising sensitivity. This was accomplished using an on-line suppressor and decreasing post-column flow-rates from 1 ml to 50 microliters/min. In the selected ion monitoring mode, background (from the complex sample matrix as well as the mobile phase) was eliminated, simplifying chromatograms. Sugar identification was achieved by MS-MS using collision-induced dissociation.

Protein and glycoprotein components of phagosome membranes derived from mouse L cells.

1. Sodium dodecylsulfate--polyacrylamide gel electrophoresis has been employed to analyze the protein and glycorprotein components of phagosome membranes prepared from mouse L cells by the polystyrene latex bead method. 2. These experiments showed that phagosome membranes contain at least 20 major membrane protein species having apparent molecular weights between 27,000 and 250,000; the most abundant proteins have molecular weights of 112,000, 103,000, 76,000, 68,000, 62,000, 42,000 and 36,000. 3. Phagosome membrane glycoproteins, which were detected on the gels by staining with periodic acid-Schiff's reagent, were found to migrate in two broad zones centered at positions on the gel corresponding to proteins of mol. wt 140,000 and 85,000. 4. Comparison of the phagosome membrane results with the results of similar experiments carried out with cell surface membranes revealed a high degree of similarity between the two. All major protein and glycoprotein components present in phagosome membranes could be identified in both of two types of cell surface membrane preparations analyzed. 5. These results strongly support the view that phagosome membranes contain a representative, not a highly selected, sample of surface membrane proteins and glycoproteins.

Expression of the RAD1 and RAD3 genes of Saccharomyces cerevisiae is not affected by DNA damage or during the cell division cycle.

The RAD1 and RAD3 genes of Saccharomyces cerevisiae are required for excision repair of UV damaged DNA. In addition, the RAD3 gene is essential since rad3 deletions are recessive lethals. We have examined the induction of the RAD1 and RAD3 genes by DNA damage and during the cell division cycle. We have made fusions of the RAD1 and RAD3 genes with the Escherichia coli lacZ gene encoding beta-galactosidase. Beta-galactosidase activity was measured in a Rad+ yeast strain containing the RAD1-lacZ or the RAD3-lacZ fusion, either in a multicopy replicating plasmid or as a single copy integrant resulting from transformation with an integrating plasmid which transforms yeast by homologous recombination in the yeast genome. No induction of beta-galactosidase activity occurred after ultraviolet light (UV) or 4-nitroquinoline-1-oxide (NQO) treatment. Haploid cells of mating type a were synchronized by treatment with alpha factor and beta-galactosidase activity was determined during different cell cycle stages. No change in beta-galactosidase activity was observed in the strain containing the RAD1-lacZ or the RAD3-lacZ fusion integrated in the yeast genome.