Biomedical subjects

L Zhong

Publications and source records attributed to L Zhong.

At least 91 records · Page 5Linked to original sources

PubMed1998

Tumour amplified kinase STK15/BTAK induces centrosome amplification, aneuploidy and transformation.

The centrosomes are thought to maintain genomic stability through the establishment of bipolar spindles during cell division, ensuring equal segregation of replicated chromosomes to two daughter cells. Deregulated duplication and distribution of centrosomes have been implicated in chromosome segregation abnormalities, leading to aneuploidy seen in many cancer cell types. Here, we report that STK15 (also known as BTAK and aurora2), encoding a centrosome-associated kinase, is amplified and overexpressed in multiple human tumour cell types, and is involved in the induction of centrosome duplication-distribution abnormalities and aneuploidy in mammalian cells. STK15 amplification has been previously detected in breast tumour cell lines and in colon tumours; here, we report its amplification in approximately 12% of primary breast tumours, as well as in breast, ovarian, colon, prostate, neuroblastoma and cervical cancer cell lines. Additionally, high expression of STK15 mRNA was detected in tumour cell lines without evidence of gene amplification. Ectopic expression of STK15 in mouse NIH 3T3 cells led to the appearance of abnormal centrosome number (amplification) and transformation in vitro. Finally, overexpression of STK15 in near diploid human breast epithelial cells revealed similar centrosome abnormality, as well as induction of aneuploidy. These findings suggest that STK15 is a critical kinase-encoding gene, whose overexpression leads to centrosome amplification, chromosomal instability and transformation in mammalian cells.

Aneuploidy↗

PubMed1998

Hormonal regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase messenger ribonucleic acid in the rat corpus luteum: induction by prolactin and placental lactogens.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,Zn-SOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

Animals↗

PubMed1998

Mta has properties of an RNA export protein and increases cytoplasmic accumulation of Epstein-Barr virus replication gene mRNA.

The Epstein-Barr virus (EBV) Zta and Mta regulatory proteins were previously found to be required for efficient replication of oriLyt in cotransfection-replication assays, but the contribution of Mta to the replication process was unknown. We now demonstrate that Mta regulates replication gene expression. Using the polymerase processivity factor BMRF1 as an example, we found that in transfected cells, total BMRF1 mRNA levels were unaffected by Mta but that the amounts of cytoplasmic BMRF1 RNA and protein were greatly increased in the presence of Mta. Mta also increased cytoplasmic accumulation of the BALF2, BALF5, BSLF1, and BBLF4 replication gene mRNAs but did not affect cytoplasmic levels of BBLF2/3 mRNA. Thus, five of the six core replication genes require Mta for efficient accumulation of cytoplasmic RNA. The contribution of Mta to posttranscriptional RNA processing was examined. Examination of Mta localization in transfected cells by indirect immunofluorescence revealed that Mta colocalized with the splicing factor SC35. We also found that Mta has RNA binding activity. Glutathione S-transferase-Mta bound to BMRF1 and BMLF1 transcripts but not to a control cellular gene RNA. Mta contains a consensus leucine-rich nuclear export signal. Such signal sequences are characteristic of proteins that undergo nuclear export. Examination of Mta localization in a heterokaryon assay provided evidence that Mta shuttles between the nucleus and the cytoplasm. Our experiments indicate that Mta functions in RNA processing and transport and mediates cytoplasmic accumulation of a number of EBV early mRNAs.

Amino Acid Sequence↗

PubMed1998

Ca2+ content and expression of an acidocalcisomal calcium pump are elevated in intracellular forms of Trypanosoma cruzi.

The survival of a eukaryotic protozoan as an obligate parasite in the interior of a eukaryotic host cell implies its adaptation to an environment with a very different ionic composition from that of its extracellular habitat. This is particularly important in the case of Ca2+, the intracellular concentration of which is 3 orders of magnitude lower than the extracellular value. Ca2+ entry across the plasma membrane is a widely recognized mechanism for Ca2+ signaling, needed for a number of intracellular processes, and obviously, it would be restricted in the case of intracellular parasites. Here we show that Trypanosoma cruzi amastigotes possess a higher Ca2+ content than the extracellular stages of the parasite. This correlates with the higher expression of a calcium pump, the gene for which was cloned and sequenced. The deduced protein product (Tca1) of this gene has a calculated molecular mass of 121,141 Da and exhibits 34 to 38% identity with vacuolar Ca2+-ATPases of Saccharomyces cerevisiae and Dictyostelium discoideum, respectively. The tca1 gene suppresses the Ca2+ hypersensitivity of a mutant of S. cerevisiae that has a defect in vacuolar Ca2+ accumulation. Indirect immunofluorescence and immunoelectron microscopy analysis indicate that Tca1 colocalizes with the vacuolar H+-ATPase to the plasma membrane and to intracellular vacuoles of T. cruzi. These vacuoles were shown to have the same size and distribution as the calcium-containing vacuoles identified by the potassium pyroantimoniate-osmium technique and as the electron-dense vacuoles observed in whole unfixed parasites by transmission electron microscopy and identified in a previous work (D. A. Scott, R. Docampo, J. A. Dvorak, S. Shi, and R. D. Leapman, J. Biol. Chem. 272:28020-28029, 1997) as being acidic and possessing a high calcium content (i.e., acidocalcisomes). Together, these results suggest that acidocalcisomes are distinct from other previously recognized organelles present in these parasites and underscore the ability of intracellular parasites to adapt to the hostile environment of their hosts.

Amino Acid Sequence↗

PubMed1998

Differential expression of the estrogen receptors alpha and beta in the rat corpus luteum of pregnancy: regulation by prolactin and placental lactogens.

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ER alpha and ER beta) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER alpha and ER beta messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER alpha and ER beta) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER alpha mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ER beta mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER alpha and ER beta mRNA expression at the cellular level, by in situ hybridization, showed ER alpha expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ER beta mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypophysectomy, performed early in pregnancy, induced a sharp decline in ER alpha mRNA expression but a less-marked reduction in ER beta mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER alpha and ER beta mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ER beta mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ER beta mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.

Animals↗

PubMed1998

Isolation and identification of xylitol dehydrogenase gene from Trichoderma reesei.

A cDNA sub-library from the fungus Trichoderma reesei grown on xylan was constructed in S.cerevisiae recombinant strain H475 harboring a xylose reductase (XR) gene from Pichia stipitis. The xylitol dehydrogenase (XDH) gene was screened from sub-library on a SC selective medium in which xylose was used as a sole carbon source. The length of XDH gene, xdh1, was about 1.3 kb. Southern, Northern and Western blots were carried out, indicating that xdh1 has high affinity with T.reesei and the molecular weight of the xylitol dehydrogenase from T.reesei was about 40 kDa. The strain HX1 harboring both the XR gene from P.stipitis and the XDH gene from T.reesei was able to grow on the xylose medium and converted more than 90% of the xylose into xylitol, ethanol, and other by-products.

Cloning, Molecular↗

PubMed1998

[Generation of anti-rhEPO ScFv by using phage display technology].

In this paper, the recombinant phage antibody techniques was used to construct, clone, screen, and express anti-rhEPO single chain antibody ScFv. The variable region genes of antibody were amplified by PCR from a hybridoma cell line D3 which secreted monoclonal antibody to rhEPO. The ScFv gene fragments were successfully cloned into phagemid vector pCANTAB5E. The recombinant phages were panned by rhEPO which was coated on a microtiter plate. After three rounds of panning, 8 clones were determined specifically binding to rhEPO antigen. The positive recombinant phagemids were extracted and transformed into non-suppressed E. coli HB2151. The soluble single chain antibody was expressed, and the specificity of the expressed ScFv was determined by ELISA. Western blot and Dot-blot. The result of SDS-PAGE indicated that the apparent molecular weight of the target peptide which is mainly in culture supernatant is 32 kD. The DNA sequence data showed that the ScFv gene included 783 bp, encoding 261 amino acids.

Amino Acid Sequence↗

PubMed1998

[Research on the regulation of glucoamylase gene(glaA) expression in A. niger. II. Analysis of the function of 5'-regulatory region of A. niger T21 and 3.795 glaA gene].

Two plasmid vectors pXH2 and pGH1 were constructed through the fusion of E. coli hph gene, the report gene and the 5' upstream regions of A. niger T21 and 3.795 respectively, as well as the terminator of A. nidulans trpC gene. The plasmid vectors were than used to transform A. niger T21 to functionally identify those different basic groups between the two 5' upstream regions responsible for high-level expression of the glaA gene. Southern analysis of two transformants XH2C and GH1C revealed that pXH2 and pGH1 were integrated respectively into the chromosome at same site with two copies in tandem array. The level resistant to HmB(3000 micrograms/ml) of XH2C was twice as high as that (1500 micrograms/ml) of GH1C, indicating that the changes of basic groups through mutation result in twice increase of functional level of region responsible for transcription and regulation of A. niger T21 glaA gene compared with that of 3.795.

Aspergillus niger↗

PubMed1998

[Subcloning and expression of coding region for cellulase binding domain of CBH I from P. janthinellum in E. coli].

The in vitro DNA manipulations, included the nested deletions, of cbh1 from P. janthinellum inserted into pUC18-181 were carried out. The two ends of fragments were modified into blunt ends and the fragments were self-ligated. Then, the encircled plasmids were transformed to E. coli JM109. Utilizing the characterization of CBD binding to crystalline cellulose, one catalytic domain deletion transformant producing active LacZ-CBD fusion protein was isolated from 24 transformants randomly picked from 400 transformants. The molecular weight of the LacZ-CBD fusion protein is 21 kD. The plasmid was designated pUC 18C. The LacZ-CBD fusion protein produced by JM109(pUC18C) was able to be purified by procedure of adsorption-desorption to cellulose. The pNPC activity of crude enzyme solution of JM109(pUC18C) induced by IPTG were zero, identified the JM109(pUC18C) has no CBHI activity.

Adsorption↗

PubMed1998

[Human trabeculer cells in culture and cell identification].

PURPOSE: To establish the method of culturing human trabecular cells(HTCs) in vitro and to determine identifyng main points concerning cultured HTCs. METHODS: Trabecular meshwork, corneal and scleral tissue were collected from sixty-eight eyeballs of thirty-four human donors less than six years of age within 24 hours after death. The tissues were primarily cultured and the cells subcultured. Cultured cells were observed by light and electron microscopes. Fibronectin(FN), laminin(LN) and IV collagen (IV C) in extracellular matrix(ECM) of the cultured cells were immunohistocytochemically stained by S-P method. RESULTS: HTCs easily grew in the event of young donors and short-time drawing materials. Trabecular meshwork should be accurately separated. Techniques in primary culture, and time and proportion in subculture should be suitabled. Experimental studies should select the third to fifth passages. The growing characteristics, morphological features of cultured HTCs under light and electron microscopes differed from those of corneal and scleral cells adjoining them. Ultramicrostucture distinguished HTCs from corneal interstitial cells. ECM stained by S-P method distinguished HTCs from scleral fibroblast. CONCLUSIONS: These studies suggest that it is not difficult to culture HTCs in general culturing condition as long as the main points about culturing them are known-well. The identification of cultured HTCs must be combined with three aspects: the growing characteristics and morphological features of cultured cells under light and electron microscopes, and the immunohistochemical stained peculiarities of FN, LN and IV C in ECM of cultured cells. To establish the method of culturing HTCs in vitro and to determine identifyng main points concerning cultured HTCs become an essential condition and an important guarantee in order to investigate the pathogenesis and mechanism on primary open angle glaucoma(POAG) deeply.

Cells, Cultured↗

PubMed1997

Mice lacking factor VII develop normally but suffer fatal perinatal bleeding.

Blood coagulation in vivo is initiated by factor VII (FVII) binding to its cellular receptor tissue factor (TF). FVII is the only known ligand for TF, so it was expected that FVII-deficient embryos would have a similar phenotype to TF-deficient embryos, which have defective vitello-embryonic circulation and die around 9.5 days of gestation. Surprisingly, we find that FVII-deficient (FVII-/-) embryos developed normally. FVII-/- mice succumbed perinatally because of fatal haemorrhaging from normal blood vessels. At embryonic day 9.5, maternal-fetal transfer of FVII was undetectable and survival of embryos did not depend on TF-FVII-initiated fibrin formation. Thus, the TF-/- embryonic lethal and the FVII-/- survival-phenotypes suggest a role for TF during embryogenesis beyond fibrin formation.

Animals↗

PubMed1997

Design, synthesis, and biological evaluation of new growth inhibitors of Trypanosoma cruzi (epimastigotes).

As a continuation of our project aimed at the search for new chemotherapeutic agents against Chagas' disease, several drugs structurally related to the insect growth regulator Fenoxycarb and the naturally occurring juvenile hormone of insects were designed, synthesized, and evaluated as antiproliferative agents against the parasite responsible of this disease. Isoprenoid derivatives (compounds 33, 34, 36, and 37) were potent growth inhibitors of Trypanosoma cruzi epimastigotes. In addition, taking into account the high activity observed for compound 30 and the inhibitory action of related compounds, the allyl ether moiety bonded at the polar extreme of these inhibitors proved to be a promising group for the design of new drugs.

Animals↗

PubMed1997

Prolactin-mediated inhibition of 20alpha-hydroxysteroid dehydrogenase gene expression and the tyrosine kinase system.

The rat luteal 20alpha-hydroxysteroid dehydrogenase plays a key role at catabolizing progesterone and at decreasing the level of this steroid secreted by the ovaries. Throughout pregnancy and before parturition neither the mRNA nor the protein for this enzyme could be detected. In this investigation we set to examine whether PRL and PRL-like hormone from placental origin silence the expression of this gene and whether PRL action involves tyrosine kinase activity and/or de novo protein synthesis. The results revealed that PRL and PRL-like hormone from rat placental origin (rPL-1 and rPL-2), but not rat growth hormone, caused a rapid and profound inhibition of 20alpha-HSD mRNA expression in highly luteinized granulosa cells. Immunoprecipition and western blot analysis indicate that PRL-R associates with JAK2 and Stat5, and this association is increased within 30 seconds with PRL treatment. Although both JAK2 and Stat5 were phosphorylated on tyrosine upon PRL treatment, the PRL mediated inhibition of 20alpha-HSD was not reversed by either tyrosine kinase inhibitors, AG18 and genistein, but was largely reversed by the protein synthesis inhibitor cycloheximide. In summary, results of this investigation indicate that although PRL can activate the JAK2/Stat5 system in the corpus luteum, the down regulation of 20alpha-HSD mRNA by PRL does not appear to involve tyrosine kinase activity but depends on de novo synthesis of protein(s).

20-Hydroxysteroid Dehydrogenases↗

PubMed1997

Intracellular Ca2+ pool content and signaling and expression of a calcium pump are linked to virulence in Leishmania mexicana amazonesis amastigotes.

Virulent and avirulent clones of Leishmania mexicana amazonensis promastigotes or amastigotes were loaded with the fluorescent reagent fura 2/AM to measure intracellular free calcium ([Ca2+]i). When the cells were treated with the calcium ionophore ionomycin in the nominal absence of extracellular Ca2+, there was an increase of [Ca2+]i that was further elevated by addition of either NH4Cl, nigericin, or the vacuolar H+-ATPase inhibitor bafilomycin A1. Similar results were obtained when the order of additions was reversed. Taking into account the relative importance of the ionomycin-releasable and the ionomycin plus NH4Cl-releasable Ca2+ pools, it is apparent that a significant amount of the Ca2+ stored in L. mexicana amazonensis promastigotes and amastigotes is present in an acidic compartment rich in Ca2+ (acidocalcisome). Results indicated that more releasable Ca2+ is stored intracellularly in virulent amastigotes than in virulent promastigotes or avirulent cells of both stages. This higher amount of releasable Ca2+ was correlated with the presence of Ca2+ signals in the virulent amastigotes during invasion of macrophages. Ca2+ signals and invasion were reduced by preloading the parasites with intracellular Ca2+ chelators (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM) and quin 2/AM) but not by a non-Ca2+-chelating analog (N-(2-methoxyphenyl)imidoacetic acid/AM). The gene encoding an organelle-type Ca2+-ATPase was cloned and sequenced and found overexpressed in virulent amastigotes as compared with all other forms. Together, these results demonstrate a significant link between expression of a Ca2+-ATPase, intracellular Ca2+ pool content and signaling, and virulence.

Amino Acid Sequence↗

PubMed1997

Assembly of complete, functionally active herpes simplex virus DNA replication compartments and recruitment of associated viral and cellular proteins in transient cotransfection assays.

Early during the herpes simplex virus (HSV) lytic cycle or in the presence of DNA synthesis inhibitors, core viral replication machinery proteins accumulate in intranuclear speckled punctate prereplicative foci, some of which colocalize with numerous sites of host cellular DNA synthesis initiation known as replisomes. At later times, in the absence of inhibitors, several globular or large irregularly shaped replication compartments are formed; these compartments also contain progeny viral DNA and incorporate the IE175(ICP4) transcription factor together with several cellular proteins involved in DNA replication and repair. In this study, we demonstrate that several forms of both prereplication foci and active viral replication compartments that display an appearance similar to that of the compartments in HSV-infected cells can be successfully assembled in transient assays in DNA-transfected cells receiving genes encoding all seven essential HSV replication fork proteins together with oriS target plasmid DNA. Furthermore, bromodeoxyuridine (BrdU)-pulse-labeled DNA synthesis initiation sites colocalized with the HSV single-stranded DNA-binding protein (SSB) in these replication compartments, implying that active viral DNA replication may be occurring. The assembly of complete HSV replication compartments and incorporation of BrdU were both abolished by treatment with phosphonoacetic acid (PAA) and by omission of any one of the seven viral replication proteins, UL5, UL8, UL9, UL42, UL52, SSB, and Pol, that are essential for viral DNA replication. Consistent with the fact that both HSV IE175 and IE63(ICP27) localize within replication compartments in HSV-infected cells, the assembled HSV replication compartments were also able to recruit both of these essential regulatory proteins. Blocking viral DNA synthesis with PAA, but not omission of oriS, prevented the association of IE175 with prereplication structures. The assembled HSV replication compartments also redistributed cotransfected cellular p53 into the viral replication compartments. However, the other two HSV immediate-early nuclear proteins IE110(ICP0) and IE68(ICP22) did not enter the replication compartments in either infected or transfected cells.

Animals↗

PubMed1997

Expression, purification and characterization of the rat luteal 20 alpha-hydroxysteroid dehydrogenase.

The enzyme, rat ovarian 20 alpha-hydroxysteroid dehydrogenase (20 alpha HSD), plays a central role in luteolysis and parturition. It catalyzes the reduction of progesterone, leading to the formation of progestationally inactive steroid, 20 alpha-hydroxypregn-4-ene-3-one (20 alpha-hydroxyprogesterone). Recently, we reported the cloning, sequencing, and deduced amino acid sequence of the rat luteal 20 alpha HSD. To further investigate whether phosphorylation and/or glycosylation affect the activity of 20 alpha HSD and to study its kinetic and biochemical properties, we established both bacterial and insect expression systems for obtaining large quantities of enzyme. The recombinant (rec) 20 alpha HSD expressed as glutathione-S-transferase-20 alpha HSD fusion protein was purified from bacterial lysates by affinity binding to glutathione-Sepharose beads followed by thrombin digestion, whereas the rec enzyme expressed in baculovirus-insect cell system was purified to apparent homogeneity by ion exchange chromatography, followed by dye affinity chromatographies. Both rec preparations of 20 alpha HSD demonstrated a single polypeptide chain of 37 kDa with similar K(m) values for 20 alpha-hydroxyprogesterone and NADP, although the corresponding maximum velocity values were slightly lower for the rec 20 alpha HSD expressed in the insect cells. The rec 20 alpha-HSD showed preference for progesterone/20 alpha-hydroxyprogesterone. 17 alpha-Hydroxyprogesterone was only 30% as effective. The enzyme also used various substrates specific for aldo-keto reductases, although with much less efficiency. The rec enzyme preparations showed an absolute requirement for NADP(H). In vitro phosphorylation of rec bacterial enzyme with either protein kinase A or protein kinase C had no demonstrable effect on its activity. Finally, no differences in enzyme activity were noted between glycosylated (expressed in insect cells) and nonglycosylated (expressed in bacteria) forms of the enzyme. In conclusion, these studies demonstrate that rat luteal 20 alpha HSD can be prepared in large amounts from either bacterial or insect expression systems in a catalytically active form. Indirect evidence also suggests that the catalytic activity of 20 alpha HSD may be independent of phosphorylation and glycosylation states of the enzyme protein, i.e. posttranslational modification of 20 alpha HSD may not be required for the maximal expression of enzyme activity.

20-Hydroxysteroid Dehydrogenases↗

PubMed1997

The different forms of the prolactin receptor in the rat corpus luteum: developmental expression and hormonal regulation in pregnancy.

The corpora lutea of pregnancy in the rat are highly dependent on the action of PRL and PRL-like hormones to hypertrophy and to produce progesterone needed for the maintenance of gestation. Two forms of the PRL receptor (PRL-R), designated as long (PRL-RL) and short (PRL-RS), have been described in rat tissues. To determine whether both forms are present in the corpus luteum during pregnancy and to examine the developmental and hormonal regulation of their expression, total RNA isolated from corpora lutea at different stages of pregnancy and from highly luteinized granulosa cells subjected to different hormonal treatments were analyzed by semiquantitative RT-PCR. Immunoblotting of luteal proteins from early and late pregnancy was also performed to determine if the pattern of PRL-R proteins follows that of PRL-R messenger RNA (mRNA) expression. In addition, the correlation between the well characterized PRL-regulated gene, 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), and PRL-R gene expression was investigated during the time of luteolysis. Both PRL-RL and PRL-RS mRNA and protein were expressed in corpora lutea of pregnancy, with the long form being the most dominant at all stages. Whereas no changes in mRNA level of either PRL-RL or PRL-RS were found until day 20 of gestation, a profound decline in PRL-R mRNA and protein for both receptor types occurred at the end of pregnancy. This drop in PRL-R expression was accompanied by a sharp and abrupt expression of 20alpha-HSD mRNA. Studies performed in vivo and in luteinized cells in culture indicate that PRL can up-regulate the expression of the PRL-RL mRNA, an effect prevented by the tyrosine kinase inhibitor, genistein. PRL-RL mRNA was also selectively increased by cAMP. In summary, the results of this investigation have established that: 1) the corpus luteum of pregnancy expresses both the short and long forms of the PRL-R with the long form being more abundant; 2) the mRNA for both forms of the PRL-R remains at constant levels throughout pregnancy but drops before parturition; 3) the decline in PRL-R mRNA at the end of pregnancy is accompanied by a dramatic rise in 20alpha-HSD; 4) PRL is able to increase the expression of PRL-R mRNA; and that 5) both A kinase and tyrosine kinase mediated pathways appear to participate in the up-regulatory mechanism involved in PRL-R mRNA expression.

20-Hydroxysteroid Dehydrogenases↗

PubMed1997

PRAP, a prolactin receptor associated protein: its gene expression and regulation in the corpus luteum.

We have recently identified, characterized, and cloned a luteal microsomal 32-kDa phosphoprotein that we named PRAP (for PRL-receptor associated protein), and we have demonstrated that PRAP binds to the intracellular domain of the short but not the long form of the PRL receptor. In this study, we used PRAP cDNA to examine the tissue specificity, the developmental expression, and the hormonal regulation of PRAP gene expression. Northern blot analysis revealed that in the corpus luteum, PRAP cDNA hybridized to multiple transcripts (5.5 kb, 4.3 kb, and 1.8 kb), with the smallest transcript (1.8 kb) corresponding to the size of the cDNA clone. However, none of these transcripts were detected in any other tissues examined. PRAP appears to be tightly regulated by steroids and PRL. When pregnant rats were treated with aminoglutethimide, a steroid synthesis inhibitor, all three PRAP transcripts became barely detectable. Similar results were obtained when all luteotropic support was removed by hypophysectomy and hysterectomy. Estradiol up-regulated PRAP expression and, more specifically, the two lower transcripts. PRL had no stimulatory effect on PRAP messenger RNA (mRNA) expression but caused a substantial increase in the level of PRAP protein when administered to hypophysectomized pregnant rat, suggesting that PRL may stabilize this protein. Similar dissociation between levels of mRNA and protein were observed during luteal development. Although both PRAP mRNA and protein were barely detectable in early pregnancy, their expression increased abruptly from midpregnancy; however, whereas levels of PRAP mRNA declined from day 18, those of the protein remained elevated until parturition. In summary, results of this study have defined the tissue specificity and developmental expression of PRAP mRNA during pregnancy. The data have also revealed that the gene expression of this protein is up-regulated by estradiol, suggesting a pivotal role for PRAP in the synergistic action of estradiol and PRL on the function of the rat corpus luteum.

Aminoglutethimide↗