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P Mak

Publications and source records attributed to P Mak.

At least 37 records · Page 2Linked to original sources
PubMed1996

Differential transcriptional regulation of the apoAI gene by retinoic acid receptor homo- and heterodimers in yeast.

Several members of the nuclear receptor superfamily including RXR (retinoid X receptor) bind to a specific retinoic acid response element (site A) of the apoAI promoter. However, transcriptional activation of the apoAI gene by different homo- and heterodimeric forms of RXR or RAR (retinoic acid receptors) cannot be evaluated in mammalian cells, which contain endogenous RXR or RAR. In order to circumvent this limitation, we assessed the DNA-binding activities and transcriptional activation of different homo- and heterodimers of these receptors in yeast. Electrophoretic mobility shift assays (EMSA) demonstrated that yeast expressed RARalpha does not bind to site A of the apoAl promoter, whereas binding of RARbeta to site A is ligand-dependent. Both RARalpha and RARbeta form heterodimers with RXRalpha and bind to site A with high affinity. These DNA-binding studies correlate with the transcriptional data, which indicated that RARbeta but not RARalpha activates transcription from site A in response equally well to 9-cis and all-trans retinoic acids. 9-cis RA is a more potent ligand than all-trans RA to activate transcription via RXR/RAR heterodimers. We conclude that this yeast expression system is a useful tool to elucidate the 'transactivation code' for apoAl site A via specific combinations of different homo and heterodimeric versions of RXR and RAR.

Apolipoproteins A↗
PubMed1996

Hamster antithrombin III: purification, characterization and acute phase response.

Antithrombin III was purified to homogeneity from hamster plasma by affinity chromatography on heparin-agrose, ion-exchange chromatography on Mono Q and size-exclusion chromatography on TSK G3000SWG column with 50% yield. The molecular mass of hamster antithrombin III was estimated at 62.5 kDa and the absorption coefficient (A280 nm 1%, 1 cm) at 6.48 (in 0.1 M sodium phosphate pH 7.0). Several isoforms of the inhibitor were detected with the pI in range of 4.95-5.25. The protein contains all residues characteristic for complex-type carbohydrate chains. The N-terminal amino acid sequence shows 84% of identity to mouse and 76% to human analogue. The hamster antithrombin III gives low immunological cross-reactivity with antibodies to human antithrombin III. Initiation of the acute phase response only slightly affected the plasma concentration of inhibitor (+/- 10% within 72-h period). The kinetic data suggest high efficiency in bovine and human thrombin inhibition. In summary, the study shows only similarities between hamster and other mammal antithrombins.

Acute-Phase Reaction↗
PubMed1996

Isolation, antimicrobial activities, and primary structures of hamster neutrophil defensins.

Hamster (Mesocricetus auratus) neutrophil granules contain at least four microbicidal peptides belonging to the defensin family. These compounds were purified from granule acid extracts by reverse-phase chromatography and termed HaNP-1 to -4 (hamster neutrophil peptide). HaNP-1 and HaNP-3 revealed the most bactericidal activity, with a 50% inhibitory concentration of 0.3 to 0.8 microg/ml for Staphylococcus aureus and Streptococcus pyogenes strains. The HaNP-4 was always isolated in concentrations exceeding about 10 times the concentrations of other hamster peptides, but its antibacterial activity as well as that of HaNP-2 was relatively lower, probably as a result of conserved Arg residue substitutions. Other microorganisms were also tested, and generally, hamster defensins exhibited less potency against gram-negative bacteria. The amino acid sequence of hamster defensins showed a high percentage of identity to the sequence of mouse enteric defensins, reaching about 60% identical residues in the case of HaNP-3 and cryptdin 3.

Amino Acid Sequence↗
PubMed1996

Evidence for the presence of different alpha-1-proteinase inhibitor genes products in mouse plasma.

The plasma alpha-1-proteinase inhibitor (API) of three mouse species Mus domesticus, M. caroli and M. pahori was isolated. Each of the species isoforms were then separated by chromatofocusing; however, no significant differences in association rate constants toward human neutrophil elastase and bovine chymotrypsin were observed. The amino-acid sequence of the P'1-P'15 C-terminal fragments of the API variants indicate that mouse plasma contains at least two different active API isoforms in the case of M. domesticus (five API genes) but only one active API isoform in M. pahori and M. caroli (one API gene).

Amino Acid Sequence↗
PubMed1994

Transcriptional regulation of the apoAI gene by hepatic nuclear factor 4 in yeast.

Hepatocyte Nuclear Factor 4 (HNF-4), a liver-enriched orphan receptor of the nuclear receptor superfamily, is required for the expression of a wide variety of liver-specific genes including apoAI. To explore the possibility that site A of the apoAI gene enhancer might also be the target for HNF-4 without the interference of endogenous mammalian cell proteins that also bind to site A, we tested the ability of HNF-4 to activate transcription from site A in yeast cells. Electrophoretic mobility shift assays (EMSA) and Scatchard plot analysis demonstrated that yeast produced HNF-4 binds to site A with an affinity two times higher than that of yeast produced RXR alpha. Mapping analysis indicated that the 5' portion of site A containing two imperfect direct repeats (TGAACCCTTGACC) and the sequence of the trinucleotide spacer (CCT) between these imperfect repeats are critical determinants for selective binding and transactivation by HNF-4. Similar observations were obtained when these mutated versions of site A were evaluated by transient cotransfection assays in CV1 cells. We conclude that the unique structural determinants of site A in conjunction with the differential binding affinity of HNF-4 for site A may play a fundamental role in apoAI gene regulation.

Animals↗
PubMed1994

Retinoid X receptor homodimers function as transcriptional activators in yeast.

The possibility that different retinoids activate transcription from a specific retinoic acid (RA)-responsive element known as site A via different homo and heterodimeric versions of RA receptors cannot be evaluated in mammalian cells because they contain endogenous RA receptors (RAR). However, this limitation can be overcome by using yeast cells, which do not contain endogenous RAR, to study retinoid signaling pathways. Here, we describe heterologous expression of the human retinoid X receptor (RXR alpha) in yeast and hormone-dependent activation of a reporter construct containing site A upstream from a yeast promoter fused to the lacZ gene of Escherichia coli. Western blot analysis of yeast extracts containing RXR alpha revealed a distinct immunoreactive polypeptide co-migrating with the mammalian-produced RXR alpha. Electrophoretic mobility shift assays demonstrated that RXR alpha produced in yeast binds efficiently to site A in the absence of 9-cis-RA. However, transcription activation experiments showed that RXR alpha transactivates a yeast basal promoter linked to site A only in the presence of 9-cis-RA. We conclude that RXR alpha homodimers bind to site A in the absence of 9-cis-RA, but function as ligand-dependent transactivators in yeast cells. This retinoid-responsive transcription unit created in yeast cells provides a powerful genetic tool for the systemic unraveling of the synergistic interactions between RXR alpha and its heterodimeric partners.

Base Sequence↗
PubMed1989

Expression of functional chicken oviduct progesterone receptors in yeast (Saccharomyces cerevisiae).

The cDNAs encoding full-length chicken oviduct progesterone receptor B (PRB) and a truncated receptor (C1C2) lacking the amino-terminal domain were expressed in yeast (Saccharomyces cerevisiae) using a ubiquitin fusion system. The expression of the fusion protein is under the control of a copper-responsive yeast metallothionein promoter, and the fusion protein is subsequently cleaved by the yeast host enzyme to produce receptor protein. Western immunoblot analyses of yeast extracts containing full-length PRB revealed a polypeptide co-migrating with authentic chicken oviduct PRB. Using a polyclonal antibody (907) directed against the "hinge" region of the authentic chicken progesterone receptor, a 42-kDa polypeptide was detected by Western analysis in yeast extracts containing C1C2 receptors. Standard hormone binding assays indicated that these receptors produced in yeast cells exhibited steroid binding affinity and specificity characteristic of the authentic chicken progesterone receptor. To test for progesterone receptor-mediated activation of transcription in yeast, reporter plasmids were constructed to transform yeast cells expressing PRB or C1C2 receptors. The reporter gene contained two copies of a progesterone response element upstream of the yeast proximal CYC1 promoter fused to the beta-galactosidase gene of Escherichia coli. The induction of beta-galactosidase activity by PRB and C1C2 was strictly dependent on specific ligand and the presence of a progesterone response element. However, overproduced C1C2 receptors had an adverse effect on the transcription of the lacZ gene. It was found that when overproduced C1C2 was activated by progesterone, an inhibitory effect on normal yeast cell growth was evident. These observations suggest that C1C2 is a potent trans-acting factor in yeast and that the amino-terminal domain of the chicken progesterone receptor may play a role in selective modulation of target gene activation.

Animals↗
PubMed1989

Mutational analysis of the chicken progesterone receptor.

Oligonucleotide-directed site mutagenesis was used to prepare a series of chicken progesterone receptor deletion mutants in an attempt to elucidate structure-function relationships of the receptor. These mutants spanned the entire 659-amino acid coding region of the A form of the receptor. The ability of these mutants to bind progesterone was analyzed following in vitro transcription and translation. Results obtained indicate that a large portion of the protein ranging from amino acid 420 to the extreme carboxyl terminus is necessary to maintain the protein in a conformation which is capable of binding hormone. Following transient cotransfection of mutant receptor proteins into CV-1 cells along with a reporter gene containing an authentic GRE/PRE (PRE-TK-CAT), our results indicated that any deletion throughout the entire molecule results in a decrease in transcriptional activation. Most of these decreases result from an inability of the mutant receptor proteins to bind DNA or hormone. However, two areas of the receptor have been identified which are unrelated to either DNA or hormone binding but markedly affect the ability of the receptor to transactivate target genes.

Animals↗
PubMed1989

Regulation of spermatogenesis: the shark testis model.

Spermatogenesis is a unique developmental sequence dependent on FSH and androgen. Due to the complex organization of the mammalian testis, however, mechanistic details of regulation are largely unknown. Using the dogfish shark (Squalus acanthias) in which there is a cystic mode of spermatogenesis and a topographic separation of different germ cell stages within the testis, we have obtained new information of general relevance on stage-related biochemical and morphological changes and have proposed a model in which steroids serve as parahormonal regulators of the spermatogenic progression. In addition, techniques developed for culturing staged spermatocysts (intact Sertoli/germ cell units) and isolated, staged Sertoli cells demonstrate the usefulness of this model for studying spermatogenic regulation under defined conditions in vitro.

Animals↗
PubMed1987

A novel steroid-binding protein in the testis of the dogfish Squalus acanthias.

In an earlier study, we identified and characterized a testicular estrogen receptor in the spiny dogfish (Squalus acanthias); however, in this species estrogen receptors were located exclusively in nuclear subfractions. We describe here a nonreceptor, sex hormone-binding protein (SBP) present in Squalus testicular cytosol and distinguishable in its physicochemical characteristics from both the estrogen receptor and serum SBP of the same species. Cytosol (100,000g supernatant) was prepared by differential centrifugation of testicular homogenates and incubated with [3H]estradiol (E2) or [3H]testosterone (T) (10-15 nM +/- 100-fold excess radioinert competitor) to determine total and nonspecific bound radioactivity. The testicular SBP had a broad specificity: E2 = T greater than progesterone greater than 5 alpha-dihydrotestosterone greater than estrone, but diethylstilbestrol was not an effective competitor. It displayed a high affinity for both E2 and T (Kd = 2.2-2.5 X 10(-9) M), sedimented at 8-10 S in both low- and high-salt sucrose gradients, and migrated more slowly than BSA during polyacrylamide disc gel electrophoresis. The testicular SBP-E2 complex was relatively stable (t1/2 = 160 min) compared to the serum SBP-E2 complex (t1/2 = less than 30 min). The testicular SBP was not found in nuclear subfractions nor did it bind to DNA-cellulose affinity columns. Its intratesticular distribution was stage-dependent: Zone III (mature spermatids) greater than Zone II (spermatocytes) greater than Zone I (stem cells and spermatogonia). Moreover, increased binding activity corresponded exactly to the hypertrophy and differentiation of Sertoli cells in the same zones, pointing to this cell as the possible site of SBP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Effects of short days on aromatization and accumulation of nuclear estrogen receptors in the hamster brain.

Exposure of hamsters to short days increases sensitivity to the negative feedback effects of testosterone (T) but decreases responsiveness to the behavioral effects of the hormone. Since T is metabolized in the brain to 5 alpha-dihydrotestosterone (DHT) and estradiol, which differentially affect gonadotropin secretion and sex behavior, it is reasonable to postulate that daylength can modulate neural responses by quantitative or qualitative alterations in T metabolism and subsequent receptor binding of active hormone. Experiments reported here focused on aromatization and the nuclear accumulation of estrogen receptors. Adult male hamsters were maintained for 6-12 wk in long (14:10 LD) or short (8:16 LD) daily photoperiods. Both intact and castrated animals were used to assess direct effects of short days versus changes due to short-day-induced testicular regression. Discretely dissected regions of the brain (preoptic area, POA; hypothalamus, HTH; and corticomedial amygdala, CMA) or limbic blocks (LIM) comprised of all three regions were assayed for estrogen-synthesizing activity (aromatase) and estrogen-binding activity (receptors). Aromatase was estimated in vitro by conversion of [7-(3)H] androstenedione to [3H] estrogen and in vivo by measuring increases in nuclear estrogen receptor levels after injection of aromatizable androgen. Receptor-binding activity was assayed in crude cytosolic and nuclear extracts by incubating samples with [3H] estradiol +/- 100-fold excess inert estradiol, and separating free and bound steroids by Sephadex LH-20 gel filtration. When aromatase was assayed in homogenates prepared from discrete brain regions of individual hamsters, significantly lower activity was found in the HTH of short-day animals than in long-day controls. This effect was seen in both intact and castrated animals, which indicates that it was not mediated by the testis. Decreased enzyme activity in the POA and CMA of short-day hamsters was not significant, nor was there an effect of castration independent of short days. Low levels of nuclear estrogen receptors were present in LIM of intact males, but these were reduced after castration or concomitant with testicular regression after short-day exposure. This suggests that the hamster testis normally secretes estrogen or aromatizable androgen. A single injection of estradiol or aromatizable androgen (T or androstenedione) increased nuclear receptors in LIM of castrated animals. Cytosolic receptors were not different in short-day vs. long-day hamsters, nor were there differences in nuclear receptor levels after a single estradiol injection.(ABSTRACT TRUNCATED AT 400 WORDS)

Androgens↗
PubMed1986

Tissue and species specificity of unmasked nuclear acceptor sites for the estrogen receptor of Squalus testes.

The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.

Animals↗
PubMed1985

Characterization of estrogen receptors in the hamster brain.

Although the hamster is frequently used as an experimental animal for studying reproductive neuroendocrinology and sex behavior, estrogen receptors (ER) in the central nervous system have not been fully characterized. Using Sephadex LH-20 gel filtration and DNA-cellulose affinity chromatography, estrogen binding macromolecules having the physicochemical properties of classical ER were identified in cytosolic and nuclear extracts of brain tissues. These receptors exhibited high affinity for estradiol (Kd = 10(-9) M), limited capacity (30-50 fmol/g tissue), and estrogen specificity; however, competition studies indicate that brain and uterine ER have different binding kinetics. The neuroanatomic distribution of ER was similar in males and females with highest levels in the limbic brain and consistently low levels in remaining forebrain and mid/hindbrain. No sex differences in receptor number or other binding parameters were evident. Sucrose gradient centrifugation showed that cytosolic ER sedimented in the 7-8S region of a 5-20% linear gradient (no salt), whereas nuclear ER had a sedimentation coefficient of 5S under high ionic strength. On DNA-cellulose affinity columns, these receptors had an elution maximum of 0.18 M NaCl. After a single injection of estradiol, nuclear ER increased and cytosolic ER were depleted. The lower estradiol binding affinity and receptor levels in hamster brain as compared to the rat are consistent with observed species differences in neural sensitivity to estrogen. We expect these data in hamsters, a markedly photosensitive species, to provide a basis for future studies examining the role of receptors in mediating the effects of day-length on steroid dependent feedback and behavioral responses.

Animals↗
PubMed1985

Exclusive nuclear location of estrogen receptors in Squalus testis.

An estrogen (E)-binding molecule having both occupied and unoccupied sites is restricted to nuclear subfractions in the testis of the spiny dogfish (Squalus acanthias). We investigated the hypothesis that a species characterized by high body-fluid osmolarity (1010 mosM) has an estrogen receptor (ER) that binds to chromatin with high affinity and consequently resists redistribution during tissue processing. Although the steroid binding and sedimentation properties of the Squalus nuclear ER conformed to those of classical ER, its elution maximum from DNA-cellulose was unusually high (0.55 M NaCl). A tendency to adhere tightly to cell nuclei was reflected in the high salt concentration (0.43 M KCl) required to extract 50% of the receptors from the nuclear compartment during homogenization and in the stability of the nuclear ER population in the presence of high concentrations of a nonionic solute (urea) or increased buffer volume. Mixing and redistribution experiments showed that nuclear ER could be quantitatively and qualitatively measured in cytosolic extracts, ruling out the possibility that soluble receptors were being masked. Although Squalus oviduct ER was similar to that of testis, ER in the testis and liver of a related elasmobranch (Potamotrygon) that maintains osmotic equilibrium at 300 mosM more closely resembled mammalian ER in its elution maximum from DNA-cellulose (0.22 M NaCl) and cytosolic/nuclear ratios in low-salt buffers. We conclude that Squalus testis has a single ER pool located exclusively in the nuclear compartment. These observations support a revised concept of steroid action and further indicate that the chromatin affinity of the hormone-ER complex is an important factor in determining subfractional distribution during tissue processing.

Animals↗
PubMed1985

Stage-dependent changes in steroidogenic enzymes and estrogen receptors during spermatogenesis in the testis of the dogfish, Squalus acanthias.

In the spiny dogfish (Squalus acanthias), germ cells and adjacent steroidogenic elements are topographically segregated within the testis according to stage of development. In the experiments reported here, we have taken advantage of this favorable anatomical arrangement to demonstrate stage-specific variations in steroidogenic enzymes and estrogen receptor number. The testes of Squalus collected in July-November were sectioned transversely and further subdivided into three zones as follows: I) germinal bed plus immature lobules with spermatogonia; II) lobules with primary or secondary spermatocytes; III) lobules with spermatids or mature spermatozoa. The morphology of these zones was verified by light microscopy and, in a separate study, by electron microscopy. Through the course of spermatogenesis, Sertoli cells increased dramatically in size and in the abundance of steroidogenic organelles. By contrast, interstitial tissue was sparse in all stages of development, and only relatively undifferentiated Leydig-like cells were present. Microsomes prepared from each zone were incubated with [3H] progesterone, [3H]17 alpha-hydroxyprogesterone, or [3H]androstenedione to evaluate androgen and estrogen biosynthetic potentials. Based on product formation, 17 alpha-hydroxylase and C-17,20-lyase activities increased progressively from less mature to more mature regions (zone III greater than II greater than I), whereas aromatase was greatest in regions undergoing meiosis or early spermiogenesis (zone II). These enzymes were not detected in semen, although C21 substrates were converted to unidentified polar metabolites in high yield. Estrogen receptors were concentrated in immature zones (zone I greater than II greater than III), and the percentage of occupied receptors revealed the same distribution. Semen and epigonal tissues were receptor negative. We conclude that Sertoli cells are responsible for steroidogenesis in Squalus testis and that hormone production is keyed to the spermatogenic cycle. The data are consistent with an important role for androgens during spermatid maturation and/or after sperm release, but reveal that estrogen actions are expressed primarily during early spermatogenic stages. This report demonstrates the usefulness of unconventional animal models for obtaining new information of general relevance.

17-alpha-Hydroxyprogesterone↗
PubMed1983

Estradiol binding activity in epididymal cytosol of the turtle, Chrysemys picta.

Estrogen binding activity was investigated in the epididymis of the turtle, Chrysemys picta using DNA-cellulose affinity chromatography. A component binding estradiol-17 beta specifically with high affinity (Kd:8.0 X 10(-10) M) and limited capacity (20 fmol/mg protein) was demonstrated in the epididymal cytosol. In addition, binding of estradiol-17 beta was sensitive to excess (100-fold) diethylstilbestrol or natural estrogens (estradiol-17 beta, estrone, and estriol) but not to progesterone or androgens (testosterone and 5 alpha-dihydrotestosterone). The specific estrogen binding macromolecules eluted from DNA-cellulose columns sedimented at 4-5 S in linear 5-20% sucrose gradients. These characteristics suggest the presence of an estrogen receptor in this androgen target organ.

Animals↗