Search PubMed⌕ Search

Biomedical subjects

A Hizi

Publications and source records attributed to A Hizi.

At least 73 records · Page 4Linked to original sources
PubMed1990

Illimaquinone, a selective inhibitor of the RNase H activity of human immunodeficiency virus type 1 reverse transcriptase.

We studied the effect of the natural marine substance illimaquinone on the catalytic activities of reverse transcriptase from human immunodeficiency virus type 1. Illimaquinone inhibited the RNase H activity of the enzyme at concentrations of 5 to 10 microgram/ml, whereas RNA-dependent DNA polymerase and DNA-dependent DNA polymerase activities were considerably less susceptible to this inhibition. Two synthetic derivatives of illimaquinone, in which the 6'-hydroxyl group at the ortho position to one of carbonyl groups of the quinone ring was modified, proved ineffective in inhibiting the human immunodeficiency virus type 1 reverse transcriptase RNase H function, suggesting involvement of the 6'-hydroxyl group in blocking the enzymatic activity.

Endoribonucleases↗
PubMed1989

Effects of small insertions on the RNA-dependent DNA polymerase activity of HIV-1 reverse transcriptase.

We have described a strain of Escherichia coli that expresses high levels of enzymatically active, soluble, HIV-1 reverse transcriptase (A. Hizi, C. McGill, and S. H. Hughes, Proc. Natl. Acad. Sci. USA, 85, 1218-1222, 1988). The clone can be used as a source of the enzyme and to generate and characterize mutations in the reverse transcriptase. We have made a series of small in-frame insertions in the region that encodes the reverse transcriptase. When the mutant plasmids are reintroduced into E. coli, they induce the synthesis of mutant forms of the enzyme. With one interesting exception, the reduction in RNA-dependent DNA polymerizing activity seen in the mutants correlates well with the degree of sequence conservation among the various reverse transcriptases. Insertions into regions that are evolutionarily conserved have a more profound effect on RNA-dependent DNA polymerase activity than do insertions into regions that are less conserved. The exception to this simple correlation is that a small insertion into the region encoding RNase H gives rise to a protein with essentially no RNA-dependent DNA polymerase activity. We suggest that this mutation may affect the ability of the reverse transcriptase to fold properly, which might explain our previous observation that small carboxyl terminal deletions profoundly affect RNA-dependent NAD polymerase activity.

Binding Sites↗
PubMed1989

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.

Adenocarcinoma↗
PubMed1989

Analysis of gag proteins from mouse mammary tumor virus.

Structural proteins designated p10gag, p21gag, p8gag, p3gag, p27gag, and p14gag from the C3H strain of mouse mammary tumor virus (MMTV) were purified by reversed-phase high-pressure liquid chromatography. The N- and C-terminal amino acid sequences and amino acid composition of each protein were determined and compared with the amino acids encoded by the proviral DNA sequences for the MMTV gag gene. The results show that each of the purified proteins is a proteolytic cleavage product derived from the predicted primary translational product of the gag gene (Pr77gag) and that their order in Pr77gag is p10-pp21-p8-p3-n-p27-p14 (where n represents 17 predicted residues that were not identified among the purified proteins). Purified p10gag lacks the initiator methionine and has a myristoyl group attached in amide linkage to the N-terminal glycine residue predicted by the second codon of the gag gene. The cleavage products are contiguous in the sequence of Pr77gag, and the C-terminal residue of p14gag is encoded by the last codon of the gag gene. By analogy with other retrovirus, p14gag is the viral nucleocapsid protein, p10gag is the matrix protein, and p27gag is the capsid protein of mature MMTV. Proteolytic cleavage sites in MMTV Pr77gag bear a striking resemblance to cleavage sites in the gag precursors of D-type retroviruses, suggesting that these viral proteases have similar specificities.

Amino Acid Sequence↗
PubMed1988

Expression in Escherichia coli of a Moloney murine leukemia virus reverse transcriptase whose structure closely resembles the viral enzyme.

We have constructed an expression plasmid containing the portion of the Moloney murine leukemia virus genome encoding the reverse transcriptase (RT). When introduced into Escherichia coli this plasmid induces the synthesis of a 70-kDa protein. The RT made in E. coli differs from the viral protein only in that there are two new amino acids, methionine and glycine, substituted for the threonine found at the N terminus of the viral enzyme. Approximately half of the E. coli synthesized RT enzyme is soluble in cell extracts. This protein is active in an RT assay, and like the enzyme purified from virions, is more active in the presence of Mn2+ than Mg2+. We have also constructed a plasmid that induces the synthesis of an RT-integration protein fusion.

Escherichia coli↗
PubMed1988

Expression of soluble, enzymatically active, human immunodeficiency virus reverse transcriptase in Escherichia coli and analysis of mutants.

We have constructed a plasmid that, when introduced into Escherichia coli, induces the synthesis of large quantities of a protein with an apparent molecular mass of 66 kDa that differs from human immunodeficiency virus (HIV) RNA-dependent DNA polymerase (deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase or reverse transcriptase, EC 2.7.7.49) only in that it has two additional amino-terminal amino acids. This protein is soluble in E. coli extracts, is active in reverse transcriptase assays, and shows inhibition profiles with dideoxy-TTP and dideoxy-GTP that are indistinguishable from the viral enzyme. The deletion of 23 amino-terminal or carboxyl-terminal amino acids or the insertion of 5 amino acids at position 143 substantially decreases the polymerizing activity of the HIV reverse transcriptase made in E. coli. The properties of a 51-kDa reverse transcriptase-related protein made in E. coli suggests that the p51 found in the virion probably does not have substantial polymerizing activity. The full-length HIV reverse transcriptase and the various mutant proteins produced in E. coli should be quite useful for structural and biochemical analyses as well as for the production of antibodies.

Amino Acid Sequence↗
PubMed1988

Expression of the Moloney murine leukemia virus and human immunodeficiency virus integration proteins in Escherichia coli.

We have constructed expression plasmids containing the genes for the MuLV and the HIV integration proteins. When introduced into Escherichia coli, these plasmids cause the production of proteins of the expected molecular weight (43K for MuLV, 31K for HIV). The wild-type MuLV coding region induces the synthesis of large amounts of integration protein; to obtain large amounts of the full-length HIV integration protein in E. coli, it was necessary to modify the coding region to disrupt a sequence that promotes efficient internal translational initiation in E. coli. It is possible to disrupt this sequence without altering the encoded amino acids; the modified plasmid makes large amounts of the full-length protein and small amounts of the internally initiated protein. Both the MuLV and HIV integration proteins require SDS to be solubilized in E. coli extracts; however, following solubilization with SDS and transfer to a nitrocellulose filter, both integration proteins bind DNA.

Cloning, Molecular↗
PubMed1987

Human monoclonal antibodies derived from lymph nodes of a patient with breast carcinoma react with MuMTV polypeptides.

Nine human hybridoma cell lines were established from a fusion of axillary lymph node lymphocytes of a patient with breast ductal carcinoma with a human lymphoblastoid cell line. The human hybridoma nature of the fusion products was confirmed by chromosomal analysis and HLA typing. The hybridomas are stable over a year of growth, and can be frozen, thawed and regrown. The carcinoma cells of the patient harbor mouse mammary tumor virus (MuMTV) cross-reacting antigens. The patient's serum and the purified monoclonal antibodies reacted with MuMTV polypeptides. Radioimmunoprecipitation studies using labeled MuMTV confirmed the binding of the patient's serum to the viral proteins. None of the control immunoglobulins reacted with the virus. No binding of the hybridoma immunoglobulins was observed with two other retroviruses (avian myeloblastosis virus and simian sarcoma virus). The ligand binding characteristics of the monoclonal antibodies suggest binding to epitopes on the various structural virus polypeptides. These monoclonal antibodies may serve as a probe to analyze the MuMTV-human breast carcinoma relationship.

Adult↗
PubMed1987

Characterization of mouse mammary tumor virus gag-pro gene products and the ribosomal frameshift site by protein sequencing.

The synthesis of retroviral polyproteins that are the translational products of the genome-size mRNA is initiated in the upstream gag gene. The synthesis of the products of the protease gene (pro) and polymerase gene (pol) is regulated by translational suppression (in-frame read-through or frameshift) of termination codons as a strategy developed for controlling the level of replicative enzymes required only in catalytic amounts. In mouse mammary tumor virus (MMTV), three overlapping reading frames are utilized for the synthesis of gag-encoded Pr77, gag-pro-encoded Pr110, and gag-pro-pol-encoded Pr160 polyproteins. To characterize pro gene products and to determine the site of frameshift required for the synthesis of Pr110, we purified and sequenced three MMTV proteins: p14, p30, and p13. Sequence analysis showed that p14 is the basic nucleic acid-binding protein derived entirely from gag, and p13 is a product of the pro gene and has characteristic sequences of protease. A comparison of the amino acid sequences of p30 with the corresponding nucleotide sequence of proviral DNA allowed the delineation of the frameshift site utilized in vivo for the synthesis of the gag-pro-encoded fusion polyprotein Pr110. The results showed that: (i) the N-terminal 94 residues of p30 are translated from the gag frame; (ii) residue leucine-95 is specified by either the last UUG codon of gag or the overlapping CUU codon in the pro frame; and (iii) the elongation of the peptide chain from residue 96 continued to be encoded in the pro frame to the pro terminator. The possible mechanisms of frameshift and of the tRNAs involved are discussed.

Amino Acid Sequence↗
PubMed1987

Prognostic factors in breast cancer--a pathological and immunological study of patients with stage 1 breast cancer.

In order to analyze which features determine a poorer prognosis we undertook a study of 91 consecutive patients with pathological Stage I breast cancer operated on at Beth Israel Medical Center (1968-71). Tumor tissue slides were reviewed and features such as: tumor size, histologic type, nuclear grade, lymphocytic and perivenular lymphocytic infiltration, as well as sinus histiocytosis in the lymph nodes removed. Records were reviewed and classified according to age and ethnic background. Survival and recurrence data were recorded up to 14 years post-mastectomy. Also determined was the presence of an antigen, previously detected in certain human breast cancer tumor tissues, which has been found to cross-react immunologically with the 52,000 dalton major envelope protein of the mouse mammary tumor virus (MMTV--gp52). Ten-year cumulative disease-free survival was 0.65. Univariate analysis of survival within various factors revealed that the only statistically significant influencing factor was the presence or absence of the antigen. Factors such as perivenous lymphocytic infiltration, diffuse lymphoid infiltration and sinus histiocytosis in regional lymph nodes also showed trends in favor of improved survival but the sample may be too small for statistical significance. The presence or absence of the antigen was independent of the host immunological reaction. There was no relationship between localization of tumor, whether medial or lateral and survival, nor with presence or absence of antigen.

Antigens, Neoplasm↗
PubMed1985

Characterization of a protein, released by the T47D cell line, immunologically related to the major envelope protein of mouse mammary tumor virus.

The T47D human mammary adenocarcinoma cell line in vitro releases viral particles as well as soluble proteins. Both fractions were shown to contain antigens that immunologically crossreact with the major glycoprotein (gp52) of mouse mammary tumor virus. The crossreacting antigens are located on polypeptides with apparent molecular weights of about 68,000 and 60,000. The larger one is present in viral particles whereas both are found in the soluble fraction. Both proteins are glycosylated. The human tissue culture proteins differ from gp52 not only in molecular weight but also in charge heterogenity and in polypeptide profiles obtained after partial proteolysis. The results suggest that there is a restricted similarity between MMTV gp52 and the immunologically related T47D proteins.

Adenocarcinoma↗
PubMed1983

Hamster endogenous retrovirus (HaER)--distinct properties of structural proteins and DNA polymerase.

The structural proteins as well as some features of the RNA-dependent DNA polymerase of the hamster endogenous retrovirus (HaER) were examined. The polypeptide pattern of this virus is substantially different from that of other known retroviruses in containing major polypeptides with molecular weights of 68,000, 59,000, 27,000 and 24,000 daltons. Double antibody competitive radioimmunoassays showed that the HaER particles do not share any detectable antigenic relatedness with the murine viruses' p30, but manifest a considerable relatedness with the feline leukemia virus p27 and a slight cross-reactivity with the rat virus major protein. The RNA-dependent DNA polymerase of HaER virus has a molecular size of approximately 73,000 daltons and in contrast to other mammalian retroviruses shows no significant preference for Mn2+ over Mg2+. Apart from the lack of antigenic relatedness between the HaER virus proteins and the p30 protein of murine viruses, there is also no antigenic relatedness between HaER and murine viruses insofar as their DNA polymerase is concerned.

Animals↗
PubMed1982

A viral antigen as a marker for the prognosis of human breast cancer.

An antigen present in human breast tumor cells, and which is immunologically related to the envelope protein (gp52) of murine mammary tumor virus, was used as a marker for the detection of breast cancer in an Israeli population. The results show that the antigen was detectable in 128 of 204 breast carcinomas tested (62.7%). The immunological reaction was not detected in normal breast tissue, benign breast tumors, ductal hyperplasia or in primary malignancies in other organs. A significantly higher percentage of cases with demonstrable antigen was found in Israeli women born in North Africa (78%) as compared to women of European origin (60.6%). The frequency of detection of the antigen was higher in stage IV (80%) as compared to stage I (15%), suggesting that the gp52 cross-reacting antigen is a marker for the severity of the disease. Moreover, a retrospective study of 97 cases of stage II breast cancer shows that if the antigen is detected at the time of mastectomy, one can usually predict an unfavorable prognosis. Survival data analysis indicates that patients without detectable antigen survived significantly longer than those with a detectable antigen.

Adult↗
PubMed1982

DNA-processing activities associated with the purified alpha, beta 2, and alpha beta molecular forms of avian sarcoma virus RNA-dependent DNA polymerase.

The RNA-dependent DNA polymerase purified from B77 avian sarcoma virus exhibited two distinct DNA-processing activities. The alpha and beta 2 isoenzymes possessed an endodeoxyribonuclease activity capable of nicking simian virus 40 superhelical DNA, whereas the alpha beta isoenzyme performed as an untwisting topoisomerase. Both activities associated with the three molecular forms of the retroviral DNA polymerase were dependent on the presence of either Mn2+ or Mg2+ ions. From analysis of the denaturated DNA products, it is apparent that the alpha and beta 2 isoenzymes introduced two nicks, one per each strand in the superhelical simian virus 40 DNA molecules, whereas the alpha beta polymerase converted these supercoiled molecules to the relaxed covalently closed circular form. The notion that the DNA-processing activities are located on the DNA polymerase molecules was supported by the following: (i) the three isoenzymes were of a high purity; (ii) the activities cosedimented in glycerol gradients with the DNA polymerase activities of the alpha, beta 2, and alpha beta molecular forms; and (iii) immunoglobulin directed against the purified polymerase immunoprecipitated the DNA-processing activities. Chemical treatments of the DNA polymerase molecules (with pyridoxalphosphate, iodoacetamide, and sulfhydryl reagents), which inhibited the polymerase activity, also suppressed the endonucleolytic and topoisomerase activities, suggesting that cystein and amino groups play an important role in the active sites of the DNA-processing activities as well.

Alpharetrovirus↗