Association of BRCA1 expression with gonadotropin-responsive cells.
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Adult↗
Biomedical subjects
M Wan
Publications and source records attributed to M Wan.
Autoprocessing: an essential step for the activation of HIV-1 protease.
Human immunodeficiency virus type 1 (HIV-1) expresses its structural and functional proteins within Gag-Pol precursor polyproteins. Specific proteolytic processing of the precursors by the viral protease is critical for the maturation and infectivity of viral particles. To observe the influence of autoprocessing on the activation of recombinant HIV-1 protease, we constructed different HIV-1 protease forms, with or without the Phe-Pro bond directly upstream of the protease domain, and expressed them in Escherichia coli systems. We found that the presence of a short upstream sequence of the protease domain, which could generate the original N-terminus of the protease by autoproteolysis of the Phe-Pro bond, resulted in processing of active protease, whereas for a wild-type protease extended only with the initiator methionine, the proteolytic activity was not recovered. Our results suggested that autoprocessing of the direct upstream sequence of the protease domain is an essential step for the activation of recombinant HIV-1 protease in the E. coli expression system. Expression of HIV-1 protease as fusion proteins revealed that the existence of a fusion portion increased the accumulation of expressed protease by affecting its homotypic dimer formation.
Base Sequence↗
Potential role of the inactivated X chromosome in ovarian epithelial tumor development.
BACKGROUND: Ovarian epithelial tumors can be divided into subcategories often regarded as different stages of neoplastic transformation. Cystadenomas belong to the least aggressive subgroup and are noninvasive and nonmetastatic. Ovarian tumors of low malignant potential (LMP) are intermediate between cystadenomas and carcinomas and show markedly reduced invasive and metastatic abilities. Invasion and metastasis are the hallmarks of carcinomas, which constitute the most aggressive subgroup and can be further subdivided into different grades. PURPOSE: We performed comparative allelotype analyses of ovarian cystadenomas, LMP tumors, and carcinomas, reasoning that such analyses could provide clues about the molecular determinants of their phenotypic differences. Because we realized that allelic losses involving the X chromosome might be associated with LMP tumor development, we determined whether such losses were interstitial and whether they involved the active or the inactive X chromosome. METHODS: Frequencies of loss of heterozygosity (LOH) at specific loci in every chromosomal arm were determined in 16 ovarian cystadenomas, 23 ovarian LMP tumors, 15 low-grade ovarian carcinomas, and 35 high-grade ovarian carcinomas by use of either the polymerase chain reaction (PCR) or Southern blot analyses. We took advantage of the fact that DNA methylation is an important mechanism of X-chromosome inactivation to determine whether losses involving the X chromosome were in the active or the inactive copy. We analyzed the methylation status of retained alleles on the X chromosome by determining whether they could be amplified by PCR after digestion with the methylation-sensitive restriction endonuclease Hpa II. RESULTS: High-grade carcinomas contained frequent(>50%) LOH in four autosomal chromosome arms, i.e., 6q, 13q, 17p, and 17q. Except for 13q, these same chromosomal arms showed frequent LOH in low-grade carcinomas. LOH in autosomal chromosomes was comparatively rare in LMP tumors and was absent in cystadenomas. In contrast, half (eight of 16) of LMP tumors informative for a locus in the proximal portion of chromosome Xq showed LOH at that locus. These losses were the result of interstitial deletions in six of the eight cases and involved the inactive copy of the X chromosome exclusively. Similar losses in the X chromosome were not seen in either cystadenomas or low-grade carcinomas. CONCLUSIONS AND IMPLICATIONS: LOH at multiple loci is associated with the development of ovarian carcinomas but not with the development of cystadenomas and LMP tumors. However, the integrity of a locus in chromosome Xq that possibly escapes X-chromosome inactivation is important for the control of LMP tumor development. The fact that this locus does not appear to be involved in the genesis of low-grade carcinomas suggests that LMP tumors are not precursors of such carcinomas.
Alleles↗
Anti-HIV triterpene acids from Geum japonicum.
The methanol extract from the whole plant of Geum japonicum was found to inhibit the human immunodeficiency virus (HIV-1) protease. Through bioassay-directed fractionation of the extract, a new triterpene acid along with five known triterpene acids, ursolic acid, epipomolic acid, maslinic acid, euscaphic acid, and tormentic acid, were isolated. The structure of the new compound was determined by spectral means including 1H-1H COSY, HMQC, HMBC, and NOE experiments to be 2 alpha, 19 alpha-dihydroxy-3-oxo-12-ursen-28-oic acid (1). Of these compounds, 1, ursolic acid, and maslinic acid showed potent inhibitory activity against HIV-1 protease.
Amino Acid Sequence↗
WAF1/CIP1 structural abnormalities do not contribute to cell cycle deregulation in ovarian cancer.
Mutations in the WAF1/CIP1 gene were not found in 36 ovarian carcinomas, including tumours with loss of heterozygosity at the WAF1/CIP1 locus and/or lacking p53 mutations. In addition, no association was demonstrable between a polymorphism in a conserved region of the WAF1/CIP1 gene and ovarian carcinoma.
Base Sequence↗
Active constituents against HIV-1 protease from Garcinia mangostana.
The ethanol extract of Garcinia mangostana L. (Guttiferae) showed potent inhibitory activity against HIV-1 protease. The activity-guided purification of the extract resulted in the isolation of two active, known compounds. The chemical structures of the isolated compounds were established by spectroscopic analyses as mangostin (IC50 = 5.12 +/- 0.41 microM) and gamma-mangostin (IC50 = 4.81 +/- 0.32 microM). The type of inhibition by both compounds is noncompetitive.
Amino Acid Sequence↗
[Cholecystokinin-octapeptide (CCK-8) reverses experimental endotoxin shock].
The effects of CCK-8 on the mean arterial pressure (MAP), blood components and path-morphological changes during endotoxin shock (endotoxin, 8 mg/kg b.w.iv.) in rats, as well as on the 24 h mortality rate (MR) of the lead-sensitized rats in endotoxin shock (endotoxin 1 microgram/100 g b.w.iv.) were observed. The results showed that injection of CCK in ES rats led to an increase in MAP, and a reduction of hematocrit and platelet as well as white cell count; the mortality rate decreased and the lesion in main organs lessened. It is suggested that release of endogenous CCK has important protective action during endotoxin shock.
Animals↗
Molecular alterations of the AKT2 oncogene in ovarian and breast carcinomas.
The AKT2 gene is one of the human homologues of v-akt, the transduced oncogene of the AKT8 virus, which induces lymphomas in mice. In previous studies, AKT2, which codes for a serine-threonine protein kinase, was shown to be amplified and overexpressed in some human ovarian carcinoma cell lines and amplified in primary tumors of the ovary. To confirm and extend these findings, we conducted a large-scale, multicenter study of AKT2 alterations in ovarian and breast cancer. Southern-blot analysis demonstrated AKT2 amplification in 16 of 132 (12.1%) ovarian carcinomas and in 3 of 106 (2.8%) breast carcinomas. No AKT2 alteration was detected in 24 benign or borderline tumors. Northern-blot analysis revealed overexpression of AKT2 in 3 of 25 fresh ovarian carcinomas which were negative for AKT2 amplification. The difference in the incidence of AKT2 alterations in ovarian and breast cancer suggests a specific role for this gene in ovarian oncogenesis. No significant association was found between AKT2 amplification and amplification of the proto-oncogenes MYC and ERBB2, suggesting that amplification of AKT2 defines an independent subset of breast and ovarian cancers. Ovarian cancer patients with AKT2 alterations appear to have a poor prognosis. Amplification of AKT2 was especially frequent in undifferentiated tumors (4 of 8, p = 0.019), suggesting that AKT2 alterations may be associated with tumor aggressiveness.
Adenocarcinoma↗
Genetic disparity between morphologically benign cysts contiguous to ovarian carcinomas and solitary cystadenomas.
BACKGROUND: Ovarian carcinomas occasionally contain large, histologically benign cysts contiguous to the clearly malignant areas (cystadenocarcinomas). The question of whether such cysts are remnants of pre-existing benign tumors (cystadenomas) or constitute integral components of the carcinomas is important in clarifying the role of cystadenomas in ovarian carcinogenesis. It is also important for our general understanding of tumor heterogeneity, a phenomenon thought to result from the gradual accumulation of genetic abnormalities in initially homogeneous tumors. This question is also pertinent to the clinical management of ovarian cystadenomas, which are frequent in women of childbearing age and are usually treated surgically based on the possibility that they may give rise to carcinomas. PURPOSE: Reasoning that molecular markers of ovarian malignancy would be confined to the histologically malignant portions of cystadenocarcinomas if the morphologically benign portions are in fact pre-existing typical cystadenomas, we sought to verify that mutations in the p53 tumor suppressor gene are markers of malignancy in ovarian tumors and to determine the distribution of such mutations in cystadenocarcinomas. METHODS: We used immunohistochemical and DNA-sequencing techniques to analyze 46 ovarian carcinomas, 21 ovarian tumors of low malignant potential, and 16 solitary cystadenomas for the presence of p53 mutations. We then used similar techniques to examine the distribution of such mutations in different portions of cystadenocarcinomas. The observed differences in mutation frequencies were analyzed by the two-tailed Fisher's exact test. RESULTS: Mutations in the p53 gene were present in 24 (52%) of the 46 carcinomas, but they were absent in the 21 tumors of low malignant potential (P < .0001) and the 16 solitary cystadenomas (P = .0002). Six of six cystadenocarcinomas with p53 mutations showed the presence of the same mutations in the adjacent, histologically benign cysts. The mutations were seen not only in cells immediately adjacent to the carcinomas, but also throughout the morphologically benign cysts. Twenty (83%) of the 24 cases showing mutation of one p53 allele also showed loss of genetic heterozygosity, suggesting that the other p53 allele was deleted. Such allelic loss, if present in morphologically malignant portions of cystadenocarcinomas, was also observed in the contiguous cysts. CONCLUSIONS: Ovarian carcinomas can be distinguished from ovarian cystadenomas and tumors of low malignant potential by p53 mutations. The fact that the mutations were present in histologically benign cysts contiguous to ovarian carcinomas suggests that such cysts are not typical cystadenomas and may carry a genetic predisposition to carcinogenesis that is not present in ordinary cystadenomas.
Carcinoma↗
Regulation of metallothionein gene expression in Cd- or Zn-adapted RK-13 cells.
We explored the molecular genetics underlying the massive induction of isoMTs by Zn2+ or Cd2+ in metal tolerant rabbit kidney (RK-13) sub-line cells, using band shift assays and Southern blotting analysis. In sub-line cells accommodated to intermediate metal concentrations (100 microM Zn2+; 1-20 microM Cd2+) evidence suggested that the increase in the capacity for isoMT synthesis is brought about by an increased binding activity of the nuclear transcription factors MTF-1 and Sp1. Using quantitative band shift analysis with a mouse MRE-d oligonucleotide probe, the binding of both transcription factors was found to be enhanced two to three times over the binding activity measured in the unexposed parental RK-13 cells. Their increase in binding activity is probably the cause of the overexpression of MT genes and the development of metal tolerance in these cells. In cells tolerant to the highest concentrations of metal the analysis of Southern blot signals revealed MT gene amplification to be the most probable cause of the increased MT production. Thus, in cells of sub-lines growing in the presence of 350 microM Zn2+, two of the isoMT genes were coordinately triplicated and in cells tolerant to 150 microM Cd2+ one isoMT gene was amplified two-fold.
Animals↗
Primary structures of seven metallothioneins from rabbit tissue.
Metallothionein from tissues of rabbits exposed to cadmium chloride was separated into seven distinct isoforms by reverse-phase liquid chromatography and their complete amino acid sequences were determined. Five of the seven isometallothioneins showed structural features so far not identified in other mammalian metallothioneins. Thus, two isoproteins contain a polypeptide with a chain length of 62 rather than 61 amino acid residues. Two isoforms are characterized by an additional positive charge and one by the presence of an isopeptide bond between aspartic acid and serine in the N-terminal half of the protein. The isoproteins characterized were identified from different sources: rabbit liver and kidney and a rabbit kidney cell-line (RK-13). In all three, the structural characteristics of the individual isoforms are retained, indicating that in the different tissues the same mechanisms control the synthesis and the stability of the different cadmium-induced isoMTs.
Amino Acid Sequence↗
[Protective effect of endothelium-derived relaxing factor on ischemic (hypoxic) and reperfused (reoxygenated) myocardium].
The present study is undertaken to investigate the effects of NO, its inhibitor L-NNA and its procursor L-Arg on the status of myocardial tissue during ischemia (hypoxia) and reperfusion (reoxygenation) in two different models, i.e. Langendorff heart and cultured heart cells of rat. The results were as follows: (1) When heart perfusion was stopped for 30 min and reinstitued for 20 min with K-H buffer containing NO, the coronary flow rate (CFR), left ventricular pressure (LVP) and +/- dp/dtmax increased significantly. When NO was replaced by L-NNA opposite effects were observed. L-Arg alone was without effect on CFR, LVP and +/- dp/dtmax, but attenuated the decreasing effect of L-NNA on CFR. NO decreased MDA and NAGase content of myocardium while L-NNA increased them. (2) When cultured ventricular myocytes were subjected to hypoxia for 30 min and reoxygenated for 20 min, none of the substances under investigation showed any effects on Ca2+ content of heart cells, but all of them decreased MDA, NAGase content of the culture tissue after reoxygenation. The above findings show that NO plays an important role in protecting myocardium from ischemic and reperfused injury by improving blood supply of reperfused myocardium and attenuation of oxygen free radical injury.
Animals↗
Expression and purification of active form of HIV-1 protease from E.coli.
We have subcloned an N-terminal extended protease gene of human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame into expression vector pGEX-KG. A relatively high level of expression of recombinant HIV-1 protease (PR) was achieved with isopropyl beta-D-thiogalactoside (IPTG) induction and glucose supplement. An isolation method consisting of denaturation of protein and followed by refolding was developed for releasing this recombinant HIV-1 PR into the soluble phase since most of the expressed protease was initially present in insoluble inclusion bodies. High purity of this recombinant HIV-1 PR was obtained by sequential purification using Sephadex G-50 gel filtration and CM-23 cellulose cation exchange chromatography, yielding the protease more than 1 mg per liter culture. N-terminal amino acid sequence analysis showed that the recombinant HIV-1 PR underwent autocleavage from the fusion protein during expression. SDS-PAGE indicated that the molecular weight of this recombinant HIV-1 PR is 11 kDa. This recombinant HIV-1 PR showed proteolytic activity for the synthetic peptide substrates corresponding to the sequence at the Gag MA/CA and Pol p6*/PR junctions. The purified enzyme whose specific activity for the heptapeptide SQNYPIV was 848.7 nmol*min-1*mg protease-1 also processed recombinant polyprotein Gag41 as its substrate.
Amino Acid Sequence↗
Comparison of HIV-1 protease expression in different fusion forms.
Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)
ATP-Binding Cassette Transporters↗
[Tissue culture and rapid propagation of Pinellia ternata (Thunb) Breit].
In this papar, selection of the explaints and morphogenesis of the plantlet involved in the tissue culture of Pinellia ternata have been investigated, the effect of different combinations of growth-regulating substances on the morphogenesis has been studied, and the result of the establishment of clonal rapid propagation system and the transplanting of test-tube Pinellia seedlings has also been reported.
Culture Techniques↗
Histologically benign or low-grade malignant tumors adjacent to high-grade ovarian carcinomas contain molecular characteristics of high-grade carcinomas.
It is presently not clear if ovarian carcinomas arise de novo or from benign precursors (cystadenomas) and if high-grade malignant tumors (carcinomas) develop from preexisting low-grade carcinomas. The presence of allelic losses on chromosome 11p15.5 distinguishes high-grade ovarian carcinomas from either low-grade carcinomas or cystadenomas. We therefore examined the distribution of such losses in different parts of heterogeneous tumors showing mixed histological grades or showing adjacent large histologically benign neoplasms. The results showed that all neoplastic areas, including those that were histologically benign or compatible with low-grade carcinomas, contained allelic losses at the above locus. This suggests that the morphologically less aggressive portions of these heterogeneous tumors were not typical cystadenomas or low-grade carcinomas and contained molecular abnormalities indicative of at least a predisposition to the high-grade carcinoma phenotype.
Alleles↗
Induction of metallothionein synthesis by cadmium and zinc in cultured rabbit kidney cells (RK-13).
The effects of increasing concentrations of Zn(II) and Cd(II) on the expression of the four isometallothioneins (isoMTs), namely MT-1a, MT-2a, MT-2d and MT-2e, in rabbit kidney cells (RK-13) and the development of cellular tolerance to these metal ions were studied. The results showed that, whereas in parental cells MT concentration was low and composed nearly exclusively of MT-2a and MT-1a, all four isoMTs increased massively in abundance when the cells were exposed to toxic concentrations of Zn(II) or Cd(II), the relative increase being largest in the two minor isoforms MT-2d and MT-2e. While the response of the four isoMTs to the challenge by Zn(II) or Cd(II) was qualitatively comparable, there were differences in sensitivity and delay time, Cd(II) being the more efficient inducer and much faster in eliciting the onset of isoMT synthesis. An even larger production of isoMTs resulted when RK-13 cells were cultured in the presence of a series of metal concentrations yielding sub-lines of increased metal tolerance. In this instance too, there were marked differences in the response to Cd(II) and Zn(II). Thus, in cells of sub-lines selected for tolerance to moderate concentrations of Cd(II) the kinetic analysis of isoMT accretion gave indications of a saturable induction process while no such evidence was forthcoming for Zn(II). In sub-line cells selected for tolerance to the highest concentrations of Cd(II) or Zn(II) isoMT formation was increased by another order of magnitude, reaching for some isoforms a 100- to 1000-fold augmentation over the amounts measured in cells of the unexposed parental cells. A potentiation of this magnitude goes beyond the range of ordinary regulation of gene expression. It is to be viewed instead as an enlargement of the capacity of isoMT synthesis acquired by a variety of mechanisms in the surviving cells.
Animals↗