Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary functional bovine adrenal cortical cell cultures have been developed to study the factors controlling adrenal cell growth. Cells were prepared by the collagenase technique and maintained in F-12 medium containing fetal calf serum and horse serum. Cells contained abundant lipid as demonstrated by staining with Oil Red O and showed strongly positive staining for delta5,3beta-hydroxysteroid dehydrogenase. ACTH inhibited DNA synthesis and stimulated steriodogenesis in these cells. Fibroblast growth factor (FGF) was shown to be a potent stimulator of the growth of normal bovine adrenal cortical cells maintained in tissue culture. The minimal effective dose of FGF was 1 ng/ml with maximal effects being observed at 100 ng/ml. The effect of FGF was dependent on the serum concentration. Inclusion of FGF in F-12 medium containing serum permitted cloning of functional bovine adrenal cortical cells from cultures seeded at low density (4 cells/cm2). ACTH inhibited the mitogenic effects of FGF. In addition to its mitogenic action, FGF is a migratory factor for bovine adrenal cortical cells. Though ACTH inhibited the mitogenic effects of FGF, it did not block the migratory activity. Epidermal growth factor did not affect the growth of either normal bovine adrenal or functional mouse adrenal tumor cells (Y-1) in tissue culture. FGF is the first direct mitogen identified for adrenal cortical cells; ACTH opposes this mitogenic action and functions directly as a differentiate function signal.
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PMID:Control of bovine adrenal cortical cell proliferation by fibroblast growth factor. Lack of effect of epidermal growth factor. 18 90

The distribution of androgen metabolism in human skin was studied using tissues isolated either by direct dissection of axillary skin or by dissection of collagenase-digested forehead and axillary skin. All tissues (epidermis, sweat glands, sebaceous glands, hair follicles and dermis) were found to contain 17beta-, 3beta- and 3alpha-hydroxysteroid dehydrogenase (HSD) activities, 3beta-hydroxysteroid dehydrogenase-delta4--5 isomerase (delta5-3beta-HSD) activity and 5alpha-reductase activity. All tissues converted testosterone into 5alpha-dihydrotestosterone. In confirmation of previous histochemical studies, over 90% of the delta5-3beta-HSD of forehead skin was found in the sebaceous glands. In forehead skin, 40--66% of the 5alpha-reductase activity was in the sebaceous glands, while in axillary skin 50--70% was in the sweat glands, especially the apocrine glands. There was a more even distribution of 17beta-HSD activity in skin tissues than histochemical studies have indicated previously. Knowledge of the distribution of these enzymes has helped in the understanding of the function of androgen metabolism in skin.
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PMID:Distribution of androgen metabolizing enzymes in isolated tissues of human forehead and axillary skin. 21 24

The function of ovarian interstitial cells has been largely addressed using rat theca-interstitial cell culture. However, this preparation is primarily enriched with theca and secondary interstitial cells, which make it difficult to address selectively the function of the primary interstitial cells. We have developed an in vitro culture of hamster ovarian primary interstitial cells. Cells were isolated from postnatal hamster ovaries by collagenase digestion and purified over a Percoll gradient. The preparation contained 90% viable, pure interstitial cells, which anchored to the plastic and glass culture surface in the presence of fetal bovine serum. Cell proliferation was noted in the presence of serum dosages higher than 0.2%; however, reduction of serum concentration to 0.1% or complete serum starvation did not affect cell viability but almost completely abolished cell proliferation as determined by [3H]thymidine incorporation, labeling index, and DNA content of the culture. All cells exhibited active 3beta-hydroxysteroid dehydrogenase and P450 side chain cleavage immunoreactivity, which corresponded to basal progesterone and androstenedione accumulation. Replacement of serum to starving cells resulted in the induction of the "S" phase and "M" phase specific cyclins, and resumption of cell proliferation. Our results indicate that hamster primary interstitial cells can be cultured in vitro as a monolayer, and the anchorage and proliferation of these cells depend on serum supplement; however, a viable monolayer can be maintained for several days without serum. This model will be useful for addressing the mechanisms of differentiation of ovarian interstitial cells.
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PMID:In vitro culture of hamster ovarian primary interstitial cells: effect of serum. 978 Mar 26

During follicular development the proliferative and differentiated state of the epithelioid granulosa cells changes, and the movement of fluid across the follicular basal lamina enables the formation of an antrum. Type IV collagen is an important component of many basal laminae. Each molecule is composed of three alpha chains; however, six different type IV collagen chains have been identified. It is not known which of these chains are present in the follicular basal lamina and whether the type IV collagen composition of the basal lamina changes during follicular development. Therefore, we immunolocalized each of the six chains in bovine ovaries using antibodies directed to the nonconserved non-collagenous (NC) domains. Additionally, dissected follicles were digested with collagenase to release the NC domains, and the NC1 domains were then detected by standard Western immunoblot methods. The follicular basal lamina of almost all primordial and preantral follicles was positive for all type IV collagen alpha chains. Colocalization of type IV collagen and factor VIII-related antigen allowed for discrimination between the follicular and endothelial basal laminae. Type IV collagen alpha1, alpha2, alpha3, alpha4, and alpha5 chains were present within the follicular basal lamina of only a proportion of antral follicles (17 of 22, 20 of 21, 15 of 18, 14 of 28, and 12 of 23, respectively), and staining was less intense than in the preantral follicles. Staining for the alpha1 and alpha2 chains was diffusely distributed throughout the theca in regions not associated with recognized basal laminae. The specificity of this immunostaining for alpha1 and alpha2 chains of type IV collagen was confirmed by Western immunoblots. As well as being detected in the basal lamina of approximately half of the antral follicles examined, type IV collagen alpha4 also colocalized with 3beta-hydroxysteroid dehydrogenase-immunopositive cells in the theca interna. Type IV collagen alpha6 was detected in the basal lamina of only one of the 16 antral follicles examined. Thus, the follicular basal lamina changes in composition during follicular development, with immunostaining levels being reduced for all type IV collagen chains and immunoreactivity for type IV collagen alpha6 being lost as follicle size increases. Additionally, immunoreactivity for alpha1 and alpha2 appears in the extracellular matrix of the theca as it develops.
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PMID:Distribution of the alpha1 to alpha6 chains of type IV collagen in bovine follicles. 982 76

Estrogen sulfotransferase (EST) catalyzes the specific sulfonation and inactivation of estrogens. A common site for EST expression in mammalian species is the testicular Leydig cells. In previous in vivo studies, we have shown that testicular expression of EST is under the regulation of LH. Thus, EST expression in mouse Leydig cells was abolished by hypophysectomy, but could be restored by hCG injection. In this study, we have evaluated the downstream mechanisms by which LH exerts its regulatory effect on EST. Primary mouse Leydig cells were isolated and purified by collagenase digestion and Percoll density gradient centrifugation. They were cultured in serum-free medium at 32 C and treated with various agents for 24 or 48 h, and levels of EST messenger RNA and enzyme activity were determined. Consistent with the in vivo data suggesting an essential role of LH in regulating EST expression, treatment of primary mouse Leydig cells in vitro with 100 microM 8-bromo-dibutyryl cAMP [(Bu)2cAMP] increased EST expression 3- to 5-fold. The effect of (Bu)2cAMP was attenuated by the steroidogenesis inhibitor aminoglutethimide and was mimicked by the potent androgen 5alpha-dihydrotestosterone (5-DHT). The activity of 5-DHT in stimulating EST expression was blocked by the androgen receptor antagonist, hydroxyflutamide. These data suggested the involvement of androgen in (Bu)2cAMP-induced EST expression. Further evidence came from the study with interleukin-1beta, another agent known to suppress Leydig cell steroidogenesis by down-regulating P450c17 gene expression. Treatment of Leydig cells with 0.2 ng/ml interleukin-1beta inhibited (Bu)2cAMP-induced EST expression, which was overcome by the addition of 5-DHT. Finally, in the testis-feminized mouse (Tfm) in which the androgen receptor is nonfunctional due to a frameshift mutation, testicular EST expression is completely absent, whereas messenger RNAs of steroidogenic enzymes such as P450c17 and 3beta-hydroxysteroid dehydrogenase are relatively abundant. We conclude that, by acting as an autocrine or paracrine factor, androgen plays an essential role in the regulation of estrogen sulfotransferase expression in Leydig cell by LH and cAMP.
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PMID:Regulation of estrogen sulfotransferase expression in Leydig cells by cyclic adenosine 3',5'-monophosphate and androgen. 1006 24

The aim of this study was to answer the question whether gonadotropins are able to stimulate the synthesis of delta4 gestagens and androgens in adrenals by the same way as in gonads. Adrenal cells of male guinea pigs (n=12) and adrenocortical cells of sows (n=2) were isolated with collagenase 1A and DNA-se and used in two separate experiments. Cell suspensions divided in quadruplicate number of aliquots for each test were preincubated (1 h) and then incubated (h) with high purity pLH-USDA, pLH-GPZ (this was used only in one experiment), pFSH-NIH (the residual contamination of this preparation with ACTH was not excluded) and ACTH1-24. The concentrations of progesterone (P), 17alpha-hydroxyprogesterone (OH-P), androstenedione (A), testosterone (T) and cortisol (F) in the incubated cells were estimated by RIA. The stimulatory effect of two high purity pLH preparations on P, A and T synthesis in guinea pig adrenal cells and pig adrenocortical cells was demonstrated. Moreover, the synthesis of OH-P in pig adrenocortical cells was also stimulated. It may be concluded that these results are specific for LH, since the used pLH-USDA was deprived of any residual ACTH contamination and pLH-GPZ was chromatographically homogenous. These preparations also showed indirect evidences of the activation of steroid 3beta-hydroxysteroid dehydrogenase/isomerase (3beta HSD) which catalyses the synthesis of these four delta4 steroids from their delta5 path precursors. The LH dependent activation of this enzyme in adrenals, which was demonstrated in this work, supported the well known observations of its independence of ACTH. As high as 6-times increase of P synthesis and 2-5 times increase of OH-P synthesis under the influence of pLH in pig adrenocortical cells (consistent with the species of LH) needs the induction of the labile protein i synthesis, since the cholesterol transport into mitochondria and the extent of pregnenolone and its derivatives synthesis depends on that protein. The influence of LH on adrenal steroidogenesis indicates that adrenal cells are the target not only for ACTH but also for LH. The influence of the used pFSH specimen on adrenal steroidogenesis resembles that of ACTH, including the increase of cortisol synthesis. Due to this similarity and lack of evidences of excluding residual ACTH contamination of such pFSH specimen, these results are considered nonspecific. Thus, the problem of FSH influence on adrenal steroidogenesis is still open. Regardless of that, the presented demonstration of specific LH effect appears to be an original contribution to the basic knowledge on adrenal steroidogenesis.
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PMID:Specific Stimulatory Effect of LH on the Synthesis of delta4 Gestagens and Androgens in Adrenocortical Cells in vitro. 1040 64

Gap junctions are intercellular protein channels which provide a pathway for the exchange of ions and small molecules. This exchange of materials allows metabolic coupling of cells. Gap junction channels are made up of connexins, integral membrane proteins encoded by a multigene family. Rat testes contain mRNAs for at least five different connexins: Cx26, Cx32, Cx33, Cx37 and Cx43. Immunocytochemical studies have shown that Cx43 assembles gap junctions between Leydig cells. The present study investigated the expression and regulation of the Cx43 gene in rat Leydig cells. Purified Leydig cells were obtained from 40- to 80-day-old Sprague-Dawley rats using a combination of arterial perfusion, collagenase digestion, centrifugal elutriation and Percoll gradient centrifugation. Leydig cells from 20- and 30-day-old rats were isolated without arterial perfusion or centrifugal elutriation. Cx43 mRNA was present in 20-day-old rat Leydig cells, reached a plateau at day 40, and remained at high levels in 65- and 80-day-old rat Leydig cells. To evaluate the regulation of Cx43 gene expression, Leydig cells were cultured overnight and then treated with human chorionic gonadotropin (hCG) for variable periods of time. Addition of hCG (10 ng/ml) increased cytochrome P450 side-chain cleavage and steroidogenic acute regulatory protein mRNA levels and testosterone formation. However, Cx43 mRNA levels were inhibited by hCG in a time- and dose-dependent manner. Cx43 mRNA levels decreased 27% as early as 2 h after the addition of hCG and decreased 60% by 24 h. Treatment of Leydig cells with 8-bromo-cAMP (0.1 mM) for 6 and 24 h also reduced Cx43 mRNA levels by 36 and 56% respectively. Primary cultured Leydig cells stained strongly positive with anti-Cx43 monoclonal antibody. Treatment with hCG for 24 h reduced Cx43 signals and caused Cx43 to redistribute to the periphery of the cells. To evaluate the regulation of Cx43 in vivo, rats were treated with hCG (300 ng i.p.) and testes were removed 24 h later. Frozen section of testes revealed that these interstitial cells stained positive for 3beta-hydroxysteroid dehydrogenase (3beta-HSD) by histochemical staining and were positive for Cx43 by immunofluorescence staining. The adjacent seminiferous tubules stained only weakly positive for Cx43. Twenty-four hours after hCG treatment, 3beta-HSD activity increased while Cx43 immunostaining of Leydig cells was reduced. In conclusion, gap junction channels of Leydig cells are regulated by hCG both in vivo and in vitro. hCG increased Leydig cell steroidogenesis and steroidogenic enzyme mRNA levels but caused a redistribution of Cx43.
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PMID:Expression and regulation of connexin43 in rat Leydig cells. 1092 34

Procedures for purification of Leydig cells have facilitated studies of their regulatory biology. A multistep procedure that includes a filtration with nylon mesh (100-micron pore size) to separate interstitial cells from the seminiferous tubules, combining centrifugal elutriation and Percoll density gradient sedimentation, has been used to obtain a 95% enrichment of rat Leydig cells. However, the number of recovered Leydig cells by this procedure represents only a small fraction of the 25 million, on average, that exist in the adult rat testis. The objective of this study was to test whether the yield of purified Leydig cells might be enhanced by substitution of unit-gravity sedimentation (S method) for the filter step (F method). We also asked whether a greater number of Leydig cell clusters, macrophages, or both would be recovered by this new method, and if the presence of Leydig cell clusters is associated with increased capacity for testosterone production in vitro. The number of purified Leydig cells was 1.9-fold higher for the S method than for the F method, with no differences in purity assessed by 3beta-hydroxysteroid dehydrogenase histochemical staining. Leydig cell clusters were also found in greater numbers with the S method both after collagenase dispersion and at the end of the purification. No differences were seen in testosterone production or in the number of macrophages present in the Leydig cells that were prepared by the 2 methods. These results indicate that the new method recovers greater numbers of Leydig cells by collecting clustered Leydig cells that are systematically eliminated when a filtration step is used.
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PMID:Purification of rat leydig cells: increased yields after unit-gravity sedimentation of collagenase-dispersed interstitial cells. 1145 64

The factors regulating the dynamic expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the primate corpus luteum (CL) during the menstrual cycle are unknown. We hypothesized that LH or progesterone (P) regulate interstitial-collagenase (MMP-1), the gelatinases (MMP-2 and -9), TIMP-1, and TIMP-2 in the CL. Hormone ablation/replacement was performed in rhesus monkeys on Days 9-11 of the luteal phase in five treatment groups (n = 4/group): control (no treatment), antide (GnRH antagonist), antide + LH; antide + LH + trilostane (TRL; 3beta-hydroxysteroid dehydrogenase inhibitor), and antide + LH + TRL + R5020 (nonmetabolizable progestin). On Day 12, the CL was removed and the RNA and protein isolated for real-time polymerase chain reaction and immunoassays, respectively. The MMP-1 mRNA increased 20-fold with antide, whereas LH replacement maintained MMP-1 mRNA at control levels. Likewise, TRL increased MMP-1 mRNA 54-fold, and R5020 prevented this effect. Immunodetectable MMP-1 protein also increased with antide or TRL; these increases were abated with LH or R5020. Gelatinase mRNA and/or protein levels increased with antide (e.g., 3-fold, MMP-2 mRNA), and LH replacement reduced protein levels (e.g., 11-fold, MMP-2). The TRL increased MMP-9, but not MMP-2, expression; however, R5020 replacement had no effect on mRNA or protein levels. The LH treatment increased TIMP-1 and -2 mRNA and TIMP-1 protein expression compared to controls and antide groups, whereas R5020 enhanced only immunodetectable TIMP-1. These data strongly suggest that LH suppresses MMP-1 in the primate CL via P and that it also suppresses gelatinases, either at the mRNA (MMP-2) or protein (MMP-2 and -9) levels, perhaps in part via steroids, including P. In contrast, LH promotes TIMP expression, perhaps via steroids, including P.
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PMID:Gonadotropin and steroid regulation of matrix metalloproteinases and their endogenous tissue inhibitors in the developed corpus luteum of the rhesus monkey during the menstrual cycle. 1367 8

Placental progesterone synthesis in humans prevents abortion of the fetus by maintaining uterine quiescence and low myometrial excitability. In rodents, a transient steroidogenic output is observed in the trophoblast giant cells during mid-pregnancy. Although the exact role of this locally produced progesterone is not clear, rodent trophoblast giant cells are an important cell model for studying the regulation of placental steroidogenesis. This chapter describes the methods we developed to analyze the regulation of genes involved in progesterone biosynthesis in miniature cultures of primary trophoblast cells from rodents. These genes include cholesterol side chain cleavage cytochrome P450 (P450scc) and its accessory proteins, steroidogenic acute regulatory protein (StAR) and 3beta-hydroxysteroid dehydrogenase/isomerase (3betaHSD). To obtain giant cells, uterine implantation sites are sliced in half, and the trophoblast giant cell layers are separated from the surrounding decidua by scraping. Cells can subsequently be separated by gentle enzymatic digestion with trypsin, or collagenase, and plated for further study in vitro. This chapter provides instructions, insights, and comments instrumental for performing in situ visualization of giant cell mRNA and proteins, analyzing enzyme activities, and conducting promoter analyses with a limited number of cells.
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PMID:Analysis of trophoblast giant cell steroidogenesis in primary cultures. 1651 89


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