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E P Smith

Publications and source records attributed to E P Smith.

At least 19 recordsLinked to original sources
PubMed1992

Mechanisms of Sertoli cell insulin-like growth factor (IGF)-binding protein-3 regulation by IGF-I and adenosine 3',5'-monophosphate.

FSH, which stimulates cAMP in the Sertoli cell, markedly lowers the concentration of insulin-like growth factor-binding protein-3 (IGFBP-3) in Sertoli cell-conditioned medium; in contrast, insulin-like growth factor-I (IGF-I) increases BP-3 expression. In this study, the mechanisms controlling the contrasting effects of cAMP and IGF-I were investigated. The abundance of BP-3 mRNA was dramatically lowered by (Bu)2cAMP, but was unaffected by IGF-I. Analyzed by ligand blot of conditioned medium, coincubation of (Bu)2cAMP and IGF-I largely eliminated the increase observed with IGF-I alone. Based on the following evidence, the effect of IGF-I appeared to be solely related to the capacity of IGF-I to interact directly with BP-3. 1) Insulin at micromolar concentrations failed to increase BP-3 abundance despite documentation by affinity cross-linking that insulin displaced [125I]IGF-I from the IGF-I receptor. 2) A synthetic IGF-I analog, [Leu24,1-62]IGF-I, which has reduced binding affinity for rat IGF-I receptor but displays high affinity for rat Sertoli cell-conditioned medium BPs, increased BP-3 abundance. 3) A synthetic IGF-I analog, B-chain mutant, which has reduced affinity for rat Sertoli cell BPs but displays normal affinity for the rat IGF-I receptor, failed to increase BP-3 abundance. 4) Human recombinant glycosylated [125I]BP-3 when added to cultured Sertoli cells was preserved in the medium when IGF-I or analogs with BP-3 affinity were present. 5) IGF-I, in dose-responsive manner, both retarded the disappearance from the medium of exogenously added human recombinant nonglycosylated BP-3 and decreased the amount of membrane-associated BP-3. These results indicate that whereas cAMP lowers BP-3 abundance in medium, most likely by markedly decreasing synthesis, IGF-I increases BP-3 accumulation by retarding its clearance by the Sertoli cell.

Animals↗
PubMed1991

Suppression of insulin-like growth factor I during epidermal growth factor-induced growth retardation.

Epidermal growth factor (EGF) attenuates growth when administered to rats less than 2 wk of age but lacks growth-retarding properties when given to older animals. Because the insulin-like growth factors (IGF) are postulated to be important regulators of somatic growth during the perinatal period, we examined the effect of exogenous EGF on serum and tissue concentrations of IGF-I and hepatic expression of mRNA for IGF-I and IGF-II. A single injection of EGF (500 ng/g body wt) produced a significant (P less than 0.01) decline in serum IGF-I concentration within 4 h in newborn rat pups [controls, 46.2 +/- 9.1 (SD) ng/ml; EGF treated, 29.4 +/- 4.0 ng/ml] but was ineffective in 2-wk-old animals (control IGF-I, 72.8 +/- 15.1 vs. 64.0 +/- 13.7 ng/ml). When the EGF was given on days 0-3 of life, circulating IGF-I concentrations were suppressed further (control, 61.4 +/- 8.6; EGF treated, 32.5 +/- 8.6 ng/ml). Despite the change in circulating IGF-I levels in the newborn rats, the amount of IGF-I extractable from liver and kidney of growth-retarded animals was not significantly different from control. Likewise, IGF-I and IGF-II mRNA expression in liver, as assessed by blot hybridization, was unchanged by the EGF treatment. The rapid decline in IGF-I concentration after EGF administration, coupled with the restriction of this phenomenon the first 2 wk of extrauterine life, implies that changes in IGF-I are involved in the pathogenesis of EGF-induced growth retardation.

Animals↗
PubMed1991

Characterization of an insulin-like growth factor binding protein (IGFBP-4) produced by the B104 rat neuronal cell line: chemical and biological properties and differential synthesis by sublines.

A previous report from our laboratory described an approximately 30 kilodalton (kDa) insulin-like growth factor binding protein (IGFBP) that inhibited the binding of insulin-like growth factor I (IGF-I) by its receptor and was secreted by a subline of the B104 rat neuronal cell line. To better understand the biology of this IGFBP, it was purified from media conditioned by these B104 cells, and the chemical and biological properties of the protein were examined. The IGFBP existed as a 24 kDa form and a 28 kDa form when the conditioned media were analyzed by ligand blot. Deglycosylation studies indicated the 28 kDa species was the N-linked glycosylated form of the 24 kDa IGFBP. Multiple forms at both mol wts were found using two-dimensional electrophoresis, suggesting that there were posttranslational modifications in addition to glycosylation. The amino acid sequence of the 12 amino-terminal residues was identical to that of rat IGFBP-4. Increased synthesis of IGFBP-4 by the subline contrasted with neglible production by other B104 cells. Blot hybridization with rat IGFBP-4 complementary DNA showed differential expression of a 2.6 kilobase transcript among B104 cell lines that correlated with quantities of IGFBP-4 secreted in media. The difference persisted even when the cells were xenografted into athymic nude mice. Purified IGFBP-4 inhibited the binding of [125I]IGF-I by its receptor and blunted stimulation of [3H]thymidine incorporation by IGF-I. These findings suggest a role for IGFBP-4 in neural cell function and indicate the B104 cell lines may be a useful model for further examination of IGFBP-4 biology.

Amino Acid Sequence↗
PubMed1990

Gene expression in cells infected with gammaherpesvirus saimiri: properties of transcripts from two immediate-early genes.

During productive infections of cells with the gammaherpesvirus, herpesvirus saimiri (HVS), a polyadenylated RNA of 2.2-2.4 kb accumulates to form a large fraction of virus-specified RNA. This transcript is from the virus thymidylate synthase (TS) gene and its synthesis, like that of late mRNAs encoding the virus structural proteins, is sensitive to an inhibitor of virus DNA synthesis (phosphonoacetic acid, PAA). Transcription which is insensitive to PAA occurs from many parts of the HVS genome, including the EcoRI-D, EcoRI-E, EcoRI-I, and HindIII-G fragments. A 1.6-kb RNA from EcoRI-I/E and a 1.3-kb RNA from HindIII-G accumulate in HVS-infected cells incubated in the continuous presence of cycloheximide, and thus represent immediate-early (IE) class transcripts. The 1.3-kb message from HindIII-G is the predominant stable RNA under these conditions; accumulation of the 1.6-kb transcript from EcoRI-I/E (which encodes the previously characterized 52-kDa IE phosphoprotein) is markedly more dependent on the multiplicity of infection. The sequence of a 2.5-kbp region of the HindIII-G fragment has been determined and a single major open reading frame is present within the boundaries of the 1.3-kb IE RNA. Comparison of the amino acid sequence of the encoded protein (IE-G) with current databases of protein sequences failed to demonstrate significant similarities with herpesvirus proteins, but did detect a significant similarity with a region of the protein specified by an open reading frame in the LTR of mouse mammary tumor virus. The function of the IE gene in HindIII-G and the basis for the distinctive multiplicity dependence of IE transcription from the 52-kDa gene remain to be established.

Amino Acid Sequence↗
PubMed1990

Insulin-like growth factor binding protein-3 secretion from cultured rat sertoli cells: dual regulation by follicle stimulating hormone and insulin-like growth factor-I.

The insulin-like growth factors (IGFs) are found in extracellular fluids bound to carrier proteins which influence the biological activity of the IGFs. Three structurally different binding proteins (BPs) have been isolated and cloned; each has distinct tissue specific expression and unique properties. We report here that testicular cells synthesize a specific subset of these binding proteins. Ligand blot analysis and RNA blot hybridization indicates that cultured peritubular cells synthesize primarily IGFBP-2. In contrast, as determined by ligand blot, RNA blot hybridization and N-linked deglycosylated studies, IGFBP-3 is predominantly synthesized by the Sertoli cell. In a dose dependent fashion, FSH markedly reduces the levels of IGFBP-3 in Sertoli cell conditioned medium. Similarly, isoproterenol, (Bu)2cAMP and cholera toxin also markedly reduce the abundance of IGFBP-3 in conditioned media. In contrast, IGF-I increases the concentrations of IGFBP-3 with the concentration required for half-maximal stimulation, approximately 20 ng/ml. Consistent with a peritubular cell origin, IGFBP-2 may be the predominant species found in interstitial fluid. In summary, our data reveal that the IGFBPs are expressed in a cell type specific manner in the testis. The opposing effects of FSH and IGF-I on Sertoli cell IGFBP-3 expression suggests a mechanism by which the IGF-I biological activity on Sertoli cell might be influenced.

Animals↗
PubMed1990

Alternative splicing produces messenger RNAs encoding insulin-like growth factor-I prohormones that are differentially glycosylated in vitro.

Rat insulin-like growth factor-I (IGF-I) cDNA sequences predict two prohormones that differ in the carboxy-terminal extension peptide (E-peptide) as a result of the inclusion or exclusion of the 52-basepair exon 4 sequence. In the absence of exon 4, the sequence codes for the IGF-Ia prohormone, whose E region contains two potential N-glycosylation sites. With differential splicing and the inclusion of exon 4, the resultant mRNA codes for IGF-Ib, with a longer E-region sequence. In addition, as a consequence of a frame shift, both potential glycosylation sites are lost in the IGF-Ib peptide. We used an in vitro translation system supplemented with canine pancreatic microsomal membranes to analyze cotranslational processing of the IGF-I propeptides. We have demonstrated that IGF-Ia prohormone, which contains two potential N-glycosylation sites in the E region, can be N-glycosylated in vitro, and that both glycosylation sites are probably used. As expected, the IGF-Ib preprohormone is processed by microsomes, but is not glycosylated.

Animals↗
PubMed1989

A form of basic fibroblast growth factor is secreted into the adluminal fluid of the rat coagulating gland.

Rat coagulating gland and its secreted fluid contain a mitogenic substance that, by numerous criteria, appears to be similar to basic fibroblast growth factor (basic FGF). It is a potent competence factor in the BALB/c3T3 mouse embryo fibroblast assay, elutes from heparin-agarose at concentrations of NaCl greater than 1.0 M, and has an isoelectric point (pI) between 8 and 9. In coagulating gland fluid, its molecular weight was 20,000 +/- 5,000, as indicated by gel filtration on Sephadex G-200. Western blot analysis of purified factor from coagulating gland cytosol and fluid, respectively, revealed immunoreactive bands of 16,000 and 17,000 relative molecular weight similar to that of purified bovine basic FGF.

Animals↗
PubMed1989

Multivariate measures of similarity and niche overlap.

Niche overlap measures are used to assess the similarity in resource use by two species. Recently researchers have used niche overlap measures as summary measures and for making inferences, typically about competition for resources. The problem of estimating niche overlap when the niches are multivariate normal distributions with equal covariance matrices has previously been studied. In this work, the assumption of equal covariance matrices is relaxed. Two general measures of similarity are evaluated assuming general multivariate normal distributions. Commonly used measures of overlap are given as special cases of these two general measures. The question of bias in estimating these measures is discussed and shown to be a potential problem, especially when there are many redundant variables or if sample sizes are small.

Animals↗
PubMed1989

Regulation of c-fos messenger ribonucleic acid by fibroblast growth factor in cultured Sertoli cells.

The data reported here demonstrate that basic FGF and Sertoli-cell-derived FGF have rapid stimulatory effects on c-fos mRNA levels. Basic FGF appears to utilize a signal transduction pathway that is distinct from that used by FSH and serum but similar in its potency and transiency. This is consistent with other reports in the literature on FGFs mechanism of action. For example, a monoclonal antibody to phosphatidyl 4,5-biphosphate will block the effects of PDGF on thymidine incorporation into NIH 3T3 cells but has no effect on basic FGF. Our data support the emerging possibility of a novel pathway mediating FGF actions. A similar precedent has been established for serotonin-induced DNA synthesis in smooth muscle cells. The rat Sertoli cell, with both FSH and FGF rapidly inducing c-fos, is an excellent model system for addressing the mechanism of action of basic FGF, the specificity of the c-fos response and the role of FGF in the reproductive system. Stimulation of c-fos mRNA by basic FGF in the cultured Sertoli cell presents questions regarding the role of FGF and c-fos in the male reproductive system. Basic FGF has been shown to stimulate cell division and plasminogen activator activity in cultured immature porcine Sertoli cells. Plasminogen activator may play a critical role in the tissue remodeling required for spermiogenesis. Interestingly, fos has been shown to be expressed preferentially in pachytene spermatocytes in the mouse. These observations taken together with the finding that basic FGF mRNA levels in Xenopus oocytes are markedly elevated, suggests that basic FGF may be important for Sertoli cell function, spermatogonial cell proliferation and subsequent meiotic and sperm maturational events.

Animals↗
PubMed1989

A rat Sertoli cell factor similar to basic fibroblast growth factor increases c-fos messenger ribonucleic acid in cultured Sertoli cells.

We have shown that the cultured Sertoli cell from the immature rat contains a fibroblast growth factor (FGF)-like factor. It behaves as a cationic peptide, is a potent competence factor for BALB/c3T3 mouse embryo fibroblasts, and displays a high affinity for heparin. Both bovine basic FGF and Sertoli cell FGF-like factor rapidly increase c-fos mRNA in cultured Sertoli cells. FSH, serum, and phorbol esters individually stimulate c-fos in cultured Sertoli cells whereas platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor-I have little affect. However, unlike FSH, basic FGF does not stimulate an increase in cAMP and unlike either serum or phorbol esters, basic FGF does not stimulate phosphoinositol turnover or intracellular calcium changes. When Sertoli cell protein kinase C activity is suppressed by preexposure to phorbol ester, basic FGF continues to be a potent stimulator of c-fos, indicating that the calcium/phospholipid pathway is not involved in FGF induction. Basic FGF and FSH also increase jun-B mRNA levels in cultured Sertoli cells. In response to FGF, jun-B is more transiently increased than c-fos. In contrast, in response to FSH, jun-B persists longer than c-fos. These results indicate that cultured Sertoli cells contain a FGF-like factor that increases c-fos mRNA via a mechanism not involving cAMP and the calcium/phospholipid pathways. The different responsiveness of c-fos and jun-B to FSH and basic FGF may explain differences in the ultimate actions of these two ligands.

Animals↗
PubMed1988

An improved method for the purification of Bacillus subtilis glucose dehydrogenase cloned in Escherichia coli.

An improved and simplified purification procedure has been developed for the isolation of the Bacillus subtilis glucose dehydrogenase which has resulted in a 10 fold higher yield of pure enzyme. The purification procedure utilizes gene cloning and an additional ammonium sulfate step to facilitate the removal of contaminating proteins. The procedure requires fewer chromatographic steps than previously reported, thus simplifying the procedure. This improved and simplified purification of B. subtilis glucose dehydrogenase will facilitate further structure-function studies of this sporulation specific enzyme.

Bacillus subtilis↗
PubMed1987

Isolation of rat testis cDNAs encoding an insulin-like growth factor I precursor.

We have characterized rat testis cDNAs encoding insulin-like growth factor I (IGF-I) precursor to facilitate studies of IGF-I expression in the male reproductive system. Two clones, P2 and P3, with inserts of 786 and 1200 bp, respectively, were isolated from a lambda gt11 library of rat testis cDNAs. The longest open reading frame of cDNA P2 predicts a 153-amino-acid residue IGF-I precursor that has only 11 amino acid substitutions compared with a human IGF-IA precursor encoded by a human liver mRNA. Three substitutions are within the predicted rat IGF-I sequence: a Pro for Asp in the B domain, an Ile for Ser in the C domain, and Thr for Ala in the D domain. Only two substitutions distinguish the predicted rat sequence from a mouse liver IGF-IA precursor: Thr for Ala in the signal peptide and Ala for Ser in the D domain. P2 hybridizes with poly(A)+ mRNAs of 7.5, 4.7, 1.7, and 1.2-0.9 kb in rat liver and testis. The other testis cDNA, P3, appears to represent a partially processed rat IGF-I mRNA precursor. By comparing the sequence of cDNA P2 with that of cDNA P3 and a 2.3-kb rat IGF-I genomic fragment, we predict exon splice sites within the codon for residue 26 and between residues 86-87 of the rat IGF-I precursor. Both of the predicted splice sites align with exon-intron junctions in the human IGF-I gene. We conclude, therefore, that IGF-I is synthesized as a precursor in the rat testis and that the structure of IGF-I genes, mRNAs, and precursors are highly conserved across species.

Animals↗
PubMed1987

Partial characterization of a somatomedin-like peptide from the medium of cultured rat Sertoli cells.

A peptide that is recognized by antibodies to human somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) has been partially purified from cultured Sertoli cells prepared from sexually immature rats. The mol wt of this peptide is about 25,000, as determined by gel filtration chromatography and immunoblot analysis of samples resolved by polyacrylamide gel electrophoresis. Isoelectric focusing indicated that the isoelectric point of this peptide was near neutrality. However, a smaller peptide of mol wt 8,000 that cross-reacted with antibodies to Sm-C/IGF-I, was released after gel filtration in acetic acid. Similarly, reverse phase HPLC on a C18 column under acidic conditions released a Sm-C/IGF-I immunoreactive peptide of 8,000 mol wt. This smaller species apparently resulted from the dissociation of this peptide from a binding protein. Unlike the larger neutral form, the isoelectric point of the smaller peptide was 9.8. This pI is similar to the GH-dependent Sm-C/IGF-I peptide isolated from rat serum. The small peptide, unlike the larger form, reacted in a parallel manner to human Sm-C/IGF-I in the Sm-C/IGF-I RIA and radioreceptor assays. In addition, the 8,000 mol wt peptide behaved as a progression factor in the BALB/c-3T3 assay and competed with [125I]Sm-C/IGF-I for binding to the type I Sm-C/IGF-I receptor from cultured rat Sertoli cells. In summary, results of this study demonstrate that rat Sertoli cells in culture secrete a peptide that is the rat equivalent of human Sm-C/IGF-I.

Age Factors↗
PubMed1987

Somatomedins/insulin-like growth factors, their receptors and binding proteins are present during mouse embryogenesis.

Somatomedins/insulin-like growth factors (Sm/IGFs) are considered to have important roles in regulating fetal growth; however, because of limited quantities of tissue, few studies have been performed on their effects on embryonic growth. To assess a potential role for these factors, we evaluated mouse embryonic tissues for the presence of Sm/IGF and insulin receptors and Sm/IGF-binding proteins by chemical affinity labelling. In addition, we measured extractable Sm-C/IGF-I radioimmunoactivity in mouse embryonic tissues. Finally, we compared these data with those from the embryonal carcinoma cell line, PC13. All embryos from day 9 (3-4 somites) to day 12 (45 somites) possessed both Sm-C/IGF-I and IGF-II receptors in apparent greater abundance than insulin receptors. The visceral yolk sac appeared to have proportionally more insulin receptors than the corresponding embryonic tissue. Extracts from the embryos contained immunoreactive Sm-C/IGF-I and binding proteins of 30-45 X 10(3) Mr. PC13 cells possessed all three receptors and the apparent abundance of the insulin and IGF-II receptors was reduced after differentiation was induced with retinoic acid. PC13 cells released both immunoreactive Sm-C/IGF-I- and Sm-C/IGF-I-binding proteins into their medium. When differentiated, the binding proteins resembled the native ones extracted from the intact embryos. The presence of Sm/IGF activity, receptors and binding proteins in early embryogenesis suggests a role for these factors in embryonic growth. The PC13 cell line appears to only partially reflect normal development.

Affinity Labels↗
PubMed1986

Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures.

Sertoli-spermatogenic cell co-cultures prepared from sexually immature rats (20-22 days old) and maintained in serum-free, hormone/growth factor-supplemented medium were used to determine the cell-specific localization of the growth factor somatomedin-C (SM-C). SM-C localization studies were carried out by indirect immunofluorescence using a monoclonal antibody (sm-1.2) to SM-C. In cultured rat hepatocytes, Sertoli and testicular peritubular cells, SM-C immunoreactivity was observed as a diffuse distribution of discrete immunofluorescent granules. Radio-immunoassay experiments using a rabbit antibody against human SM-C showed that testicular peritubular cells and Sertoli cells in primary culture accumulated SM-C in the medium. In spermatogenic cells co-cultured with subjacent Sertoli cells, immunoreactive SM-C was associated with pachytene spermatocytes but not with spermatogonia or early meiotic prophase spermatocytes (leptotene or zygotene). Both Sertoli cells and pachytene spermatocytes displayed binding sites for exogenously added SM-C. SM-C6 binding to spermatocytes reaching an advanced stage of meiotic prophase suggests a possible role of this growth factor in the meiotic process.

Animals↗
PubMed1986

A monoclonal antibody recognizes a form of intermediate filament protein in rat Sertoli cells that is not present in seminiferous peritubular cells.

We extracted intermediate filament proteins (IFP) that were insoluble in in detergent from cultured rat Sertoli and peritubular cells after labeling with [35S] methionine and resolved them by two-dimensional polyacrylamide gel electrophoresis, autoradiography and Western blotting. We found that vimentin was the predominant IFP in both Sertoli and peritubular cells. However, while two isoelectric variants of vimentin were observed in Sertoli cells, only one was detected in peritubular cells. In addition, vimentin in Sertoli cells generated a large number of breakdown products, many differing in molecular weight and isoelectric point when compared to peritubular cells. These findings suggested that a Ca2+-activated protease, usually found in cells containing vimentin, was capable of generating proteolytic fragment patterns that were specific to cell type. Monoclonal antibodies were generated against IFP extracted from cultured Sertoli cells and their reactivity monitored by indirect immunofluorescence using Sertoli cells, peritubular cells and intact testis. One monoclonal antibody, designated IFP-SC, was an immunoglobulin (IgM) that produced a vimentin-like immunofluorescent pattern in cultured Sertoli cells. This pattern consisted of an extensive filamentous network that extended throughout the cell and surrounded the nucleus. In cultured peritubular cells, IFP-SC generated a diffuse, nonfilamentous immunoreactivity. IFP-SC reacted with components of seminiferous tubules and displayed immunoreactivity associated with Sertoli cells but not with elements of the seminiferous peritubular cell wall. Because immunofluorescent patterns were distinctly specific to cell type in cultured cells and in the intact tissue, monoclonal antibody IFP-SC is useful for the quantitative evaluation of cell cross-contamination of Sertoli and peritubular cells and, therefore, allows a precise characterization of functional events specific to cell type and monitoring of the differentiation state of cells with time in culture.

Animals↗