As a first step in understanding the changes in protein synthesis that occur in renal cell carcinoma, we have prepared poly(A)+ RNA from surgically removed tumors and from their normal tissue ...counterpart. These RNAs were then translated in vitro in the rabbit reticulocyte lysate system and the synthesized labeled polypeptides were separated by one- and two-dimensional gel electrophoresis. A major 25-kDa primary translation product was observed with all renal cell carcinomas. The synthesis of this protein was barely detectable with the RNA from normal tissue adjacent to the tumor. To determine if this protein could be further processed (removal of signal peptide and (or) core glycosylation), canine pancreatic microsomal membranes were added to the system. This addition resulted in the formation of a vertical row of three additional spots, with the same isoelectric point as the primary translation product and with molecular masses ranging from 27 to 31 kDa. The 31-kDa protein was retained on Concanavalin A. After digestion with endoglycosidase H, it was no longer visible on sodium dodecyl sulfate gels and a new 27-kDa band was generated suggesting that the mature protein was indeed a glycoprotein. Future experiments will be aimed at identifying this protein and examining its potential value as a marker of renal cell carcinoma.
In addition to other known markers of the human prostate, it was shown that the prostatic fraction of the split ejaculate was rich in a 16‐kDa protein with properties not described previously. This ...protein was purified from human seminal plasma using ammonium sulfate precipitation, DEAE‐Sepharose CL‐6B ion exchange chromatography, and gel filtration on Sephadex G‐100. The purified protein showed a single prominent spot on two‐dimensional gel electrophoresis. The sequence of the first 40 amino acids that could be positively identified was identical to that of a prostatic secretory protein of 94 amino acids (PSP94) previously designated as β‐inhibin. Antibodies produced in rabbits against the purified protein were used to develop a radioimmunoassay. These antibodies appeared to recognize only the NH2‐terminal portion of the native molecule since they did not react with a synthetic peptide composed of the 28 C‐terminal residues. The radioimmunoassay showed that the concentration of the protein was 1320 ± 183 μg/ml in the seminal plasma of adult fertile men and 1134 ± 136 μg/ml in vasectomized patients. In hypertrophic and adenocarcinomatous prostates, the concentrations were 326 ± 156 and 104 ± 23 μg/ml, respectively, while values were lower than 0.060 μg/ml in the testis, epididymis, vas deferens and liver. The blood plasma concentration was 0.019 ± 0.002 μg/ml in 23 asymptomatic men 45 to 65 years old and 0.115 ± 0.036 μg/ml in eight patients with prostate cancer. These results show that PSP94 is a major protein in prostatic secretions that warrants further study in the monitoring of prostate cancer patients.
The dramatic increase in metabolic disorders such as obesity and Type 2 diabetes emphasizes the urgent need to understand better the mechanisms that coordinate body composition and energy metabolism. ...The insulin receptor PTP: PTP1B plays a critical role in these disorders. In this regard, this chapter focuses on the biological function of PTP1B, and clinical applications that use PTP1B as a drug target in human diseases. Landmark studies have demonstrated a major role for PTP1B as a regulator of insulin sensitivity and body weight. Furthermore, findings on the role of PTP1B in muscle and liver have contributed to validating PTP1B as an outstanding target for the prevention and treatment of Type 2 diabetes. Preclinical studies utilizing antisense oligonucleotides (ASO) that are complementary to the PTP1B messenger RNA and that subsequently reduce translation of PTP1B proteins have confirmed PTP1B as a new class of therapeutic target. Also, the expanding clinical problems of obesity and diabetes and the relative absence of other more promising avenues have thus far kept PTP1B as one of the most promising targets of the pharmaceutical industry. Data from research and studies have defined PTP1B as a novel target for therapeutic interventions in the modulation of the insulin receptor and Type 2 diabetes. It is anticipated that the coming time will provide further advances in defining the regulation of PTP1B, in identifying genetic markers associated with ptpn1 in the population at risk, and in improved therapeutics.
We have investigated whether PBMC of HIV-1-seropositive subjects are as susceptible to in vitro infection by HIV-1 as are PBMC from seronegative controls. Accordingly, stimulated PBMC from 19 ...HIV-1-infected subjects were inoculated with four different variants of HIV-1. None of these cultures produced either detectable quantities of viral reverse transcriptase activity or p24 Ag following inoculation with HIV-1. In contrast, in five of six cases in which these PBMC were depleted of B cells by antibody plus complement prior to viral inoculation, the presence of viral reverse transcriptase and p24 Ag was detected. The presence of normal levels of CD4-Ag at the surface of the CD4+ cells in these populations was established by flow cytometry. Analysis by an immunoblot assay revealed that anti-HIV antibodies were present in the sera obtained from these infected donors; in addition, 7 of 10 culture fluids derived from the nondepleted PBMC were shown to contain virus-neutralizing antibodies. Cultures which were depleted of B cells did not contain detectable levels of antiviral antibodies. Confirmation that the virus produced by the PBMC which had been depleted of B cells was of the strain used to infect the cultures, rather than that which initially caused patient infection, was provided on the basis of differential susceptibility to antibody neutralization. These results suggest that antibodies produced by B cells in cultures of PBMC from seropositive donors may restrict infection by HIV-1 of such cultures under laboratory conditions.
Infection of a newly described human T lymphoid cell line, CEM-CL10, with three different variants of HIV-1 resulted in cytopathic effects followed by cell lysis. Following primary lytic infection, ...proviral DNA could not be detected by Southern blot analysis in the outgrowth of the surviving CEM-CL 10 cells 60 days after initial exposure to HIV-1. These surviving cells could be further grown as a separate line, derived from CEM-CL10, and were found to be resistant to subsequent infection by HIV-1. A marked decrease in CD4 antigen expression was observed in these latter cells but not of the CD3 and transferrin receptor antigens. This decline in cell surface CD4 expression was correlated with both an absence of specific CD4 mRNA and with changes in structure of the CD4 gene. Both the HIV-1-sensitive CEM-CL10 cell line and its CD4(-), HIV-1-resistant derivative line, will be made available to interested investigators.
The precursor to a seminal plasma protein reported to have inhibin-like activity was characterized through cDNA cloning and sequencing. It is a 114-amino-acid polypeptide which differs from its ...seminal plasma derivative mainly by the presence of a 20-residue amino-terminal extension, a putative signal sequence, carrying a possible N-glycosylation site. The protein is specified by a single gene per haploid genome. Its mRNA is detectable in the prostate but not in the testis, which suggests that it is primarily a prostatic secretory protein.
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