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Understanding cell proliferation regulation in hormone refractory prostate cancer may provide answers for novel solutions. Protein tyrosine phosphatases have been thought to have key roles in regulating cell proliferation and be involved in oncogenesis, although to our knowledge their functional roles in human prostate cancer remain unknown. Human prostatic acid phosphatase (PAcP), a major phosphatase in prostate epithelium, has been shown to function as a neutral protein tyrosine phosphatase in these cells. We evaluated the biological significance of cellular prostatic acid phosphatase expression in human prostate cancer cells.Immunohistochemical testing of human prostate cancer archival specimens was done to evaluate the expression of cellular PAcP. Immunoprecipitation and immunoblotting were performed to determine cellular PAcP and SH2 domain-bearing tyrosine phosphatase-1 levels as well as tyrosine phosphorylation of c-ErbB-2/neu in different human prostate cancer cells. The biological behavior of LNCaP derivative sublines was characterized in vitro and in vivo by soft agar analysis and xenograft animal inoculation.Immunohistochemical staining of human prostate clearly showed that cellular levels of PAcP significantly decreases in prostate cancer cells (p0.001). The results of biochemical characterization revealed that the cellular level of PAcP but not SHP-1, another differentiation associated protein tyrosine phosphatase, consistently correlated negatively with the growth of several human prostate cancer cell lines. Reintroducing cellular PAcP activity in prostate cancer cells by PAcP complementary DNA transfection resulted in decreased tyrosine phosphorylation of c-ErbB-2/neu, decreased proliferation rates in culture as well as decreased anchorage independent growth in soft agar. The xenograft animal model demonstrated that a higher tumor growth rate as well as larger size is associated with a lower level of cellular PAcP.Cellular PAcP can down-regulate prostate cancer cell growth, at least partially by dephosphorylating c-ErbB-2/neu. Therefore, decreased cellular PAcP expression in cancer cells may be involved in prostate cancer progression.

Recent biochemical and genetic approaches have clearly defined the functional role of critical components in tyrosine phosphorylation-dependent signal transduction. These signaling modulators often exhibit evolutionarily conserved functions across various species. It has been proposed that if protein tyrosine kinases (PTKs), protein tyrosine phosphatases (PTPs) and thousands of their substrates could be identified and characterized, it would significantly advance our understanding of the underlying mechanisms that control animal development and physiological homeostasis. The fruit fly Drosophila melanogester has been used extensively as a model organism for investigating the developmental processes but the state of its tyrosine phosphorylation is poorly characterized. In the current study, we used advanced mass spectrometry (MS)-based shotgun analyses to profile the tyrosine phosphoproteome of Drosophila S2 cells. Using immunoaffinity isolation of the phosphotyrosine (pTyr) ubproteome from cells treated with pervanadate followed by enrichment of phosphopeptides, we identified 562 non-redundant pTyr sites in 245 proteins. Both this pre-defined pTyr proteome subset and the total cell lysates were then used as sample sources to identify potential substrates of dPTP61F, the smallest member in terms of amino acid number and molecular weight in the Drosophila PTP family and the ortholog of human PTP1B and T Cell-PTP, by substrate trapping. In total, 20 unique proteins were found to be specifically associated with the trapping mutant form of dPTP61F, eluted by vanadate (VO4 3), and identified by MS analyses. Interestingly, several potential substrates were previously doi:10.4172/jpb.s1000024

To determine the effect of zidovudine (ZDV) on the pharmacokinetic disposition of recombinant soluble CD4 immunoglobulin G (rCD4-IgG) and to evaluate the safety and preliminary activity of concurrent administration of ZDV with rCD4-IgG, we undertook an open-label, dose-escalating, 12-week study. The regimens of intravenous rCD4-IgG and oral ZDV we used were (a) 300 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day, (b) 300 micrograms/kg rCD4-IgG twice per week and 600 mg ZDV per day, (c) 1,000 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day, (d) 1,000 micrograms/kg rCD4-IgG twice per week and 600 mg ZDV per day, and (e) 3,000 micrograms/kg rCD4-IgG twice per week and 300 mg ZDV per day. Subjects were recruited from three AIDS clinical trials units. Forty-one patients with HIV infection who had CD4 cell countsor = 500 cells/mm3 and120 days of previous ZDV therapy participated. Pharmacokinetic interactions were assessed with the second regimen. Mean calculated peak serum rCD4-IgG concentrations were 5.47 micrograms/ml with ZDV and 8.28 micrograms/ml without ZDV, with serum half-lives of 34.2 and 32.0 h, respectively. Antibodies to rCD4-IgG were not detected. Seven episodes of severe adverse events occurred in five patients: one episode each of severe nausea, fever, or abnormal liver function tests and four episodes of severe neutropenia. Mean hemoglobin and neutrophil counts decreased, and mean platelet counts increased in all regimens, but there were no significant differences among regimens, rCD4-IgG dose, or ZDV dose.(ABSTRACT TRUNCATED AT 250 WORDS)

To ascertain whether combination therapy with zidovudine (AZT) and zalcitabine (ddC) delayed the emergence of AZT resistance, isolates of human immunodeficiency virus (HIV) were evaluated from 15 previously untreated patients with advanced HIV disease who received combination therapy. Eighteen sequential viral isolates were available from 15 patients who receivedor = 6 months of combination therapy. Isolates from eight (73%) of 11 patients obtained between 24 and 48 weeks of therapy were AZT resistant [50% inhibitory concentration (IC50)or = 0.45 microM; range, 0.45-2.0 microM]. Four of these eight patients yielded virus isolates that were highly AZT resistant (IC501.0 microM). No changes in ddC susceptibility were discerned. The median IC50 for ddC was 0.2 microM and ranged from 0.07 to 0.5 microM. The CD4 cell counts of patients with AZT-sensitive virus tended to have higher median areas under the curve (AUC) and greater increases compared with patients who had AZT-resistant virus. They also had higher means and ranges of the average CD4 cell counts at week 36 (p = 0.01) and week 48 (p = 0.04). These data would indicate that the previously described more sustained CD4 cell responses conferred by the addition of ddC to AZT therapy cannot be ascribed to delayed emergence of AZT resistance with combination therapy.

When polymorphonucleocytes are incubated in proteinaceous fluid, they cause extensive protein degradation, which leads to accumulation of free amino acids. The authors tested whether these free amino acids, particularly valine and leucine, also accumulate in human abscess fluids, but not in other body fluids, and thus could be a specific and distinguishing marker for the presence of an abscess.Thirty fluids, obtained by percutaneous drainage from 28 patients, were lyophilized and reconstituted in 2H2O before in vitro 1H magnetic resonance (MR) spectroscopy. Concentrations of valine and leucine were determined by comparison of spectra before and after addition of known amounts of valine and leucine. Two chart reviewers, blinded to the spectroscopic results, categorized cases as abscess (n = 14), non-abscess (n = 15), or infection but not abscess (n = 1).The concentration of valine and leucine was significantly higher in the abscess fluids, 2.57 +/- 1.90 mM than in the non-abscess fluids, 0.25 +/- 0.33 mM (P.001). The one infected fluid which was not an abscess had no amino acids. Using 0.8 mM as the threshold concentration of valine and leucine necessary for the diagnosis of abscess resulted in a sensitivity rate of 86% and a specificity rate of 94%.The authors conclude that identification of high concentrations of valine and leucine by 1H MR spectroscopy may be a specific test for the diagnosis of abscess. This technique merits further investigation in vivo.

To determine the quantitative microbiologic response and the clinical response of patients with Mycobacterium avium complex bacteremia and AIDS to an oral antimycobacterial regimen.A phase II, multicenter clinical trial.Four university-affiliated medical centers.Forty-one patients with HIV infection who had at least two consecutive blood cultures positive for M. avium complex and who had not received previous antimycobacterial therapy were enrolled in the study. Thirty-one patients were evaluable with regard to the efficacy of the oral regimen.Patients received a combination of orally administered rifampin (600 mg), ethambutol (15 mg/kg body weight), clofazimine (100 mg once daily), and ciprofloxacin (750 mg twice daily) for 12 weeks. Parenterally administered amikacin, 7.5 mg/kg daily for 4 weeks after the first 4 weeks of oral therapy, was used at the discretion of the individual investigator.Clinical symptoms, Karnofsky scores, and adverse events were monitored. Colony counts for M. avium complex were determined.The mean logarithmic (log) baseline colony count decreased from 2.1 to 0.7 after 4 weeks of oral therapy (P less than 0.001). Suppression of bacteremia was sustained throughout therapy. Thirteen patients (42%) became culture negative during therapy. The mean duration of treatment was 9.7 weeks. Nineteen evaluable patients (61%) completed 12 weeks of therapy. Adverse reactions to one or more agents were common.A rapid reduction in symptoms and bacteremia can be achieved as early as week 2 of therapy using an oral regimen of rifampin, ethambutol, clofazimine, and ciprofloxacin. Colony counts rose dramatically after therapy was discontinued, suggesting that more prolonged periods of therapy are necessary to eradicate systemic infection.

Since Giardia lamblia trophozoites are exposed to high concentrations of fatty acids in their human small intestinal milieu, we determined the pattern of incorporation of [3H]palmitic acid and myristic acid into G. lamblia proteins. The pattern of fatty acylation was unusually simple since greater than 90% of the Giardia protein biosynthetically labeled with either [3H]palmitate or myristate migrated at approximately 49 kDa (GP49) in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis during both growth and differentiation. GP49, which partitions into the Triton X-114 detergent phase, is localized on the cell surface since it is 125I-surface-labeled. GP49 was also biosynthetically labeled with [14C]ethanolamine and [3H]myoinositol, suggesting that it has a glycosylphosphatidylinositol (GPI) anchor. Moreover, phospholipase A2 (PLA2) or mild alkaline treatment released free fatty acids, indicating a diacylglycerol moiety with ester linkages. Finally, a 3H- and 14C-labeled species was released by nitrous acid deamination from [14C]palmitate- and [3H]myoinositol-labeled GP49. The GPI anchor of GP49 is unusual, however, because purified GP49 was cleaved by Bacillus cereus phosphatidylinositol (PI)-specific PLC, but not by Staphylococcus aureus PI-PLC, or plasma PLD, and did not react with antibody against the variant surface glycoprotein cross-reactive determinant. Moreover, the double-labeled deaminated GP49 anchor migrated faster than authentic PI in TLC and produced [3H]glycerophosphoinositol after deacylation. In contrast to the variable cysteine-rich G. lamblia surface antigens described previously, GP49 was identified in Western blots of every isolate tested, as well as in subclones of a single isolate which differ in expression of a major cysteine-rich 85/66-kDa surface antigen, which does not appear to be GPI-anchored. These observations suggest that GP49, the first common surface antigen to be described in G. lamblia, may play an important role in the interaction of this parasite with its environment.

Experiments were designed to examine the effects of pregnancy-associated growth factor (PAGF), a substance found in commercial preparations of crude human chorionic gonadotropin (hCG), on unfractionated human cord blood cells (CBC) and adult peripheral blood lymphocytes (PBL) cultured in 5% fetal calf serum (FCS). Comparisons of PAGF-induced [3H]TdR incorporation in nine pairs of simultaneously cultured CBC and PBL with phytohemagglutinin (PHA) and tetanus toxoid (TT) showed that all CBC and PBL responded to PAGF and PHA whereas all PBL and one CBC responded to TT. The rank order of potency for CBC and PBL was PHA greater than PAGF greater than TT. To examine phenotypic changes induced by PAGF, flow cytometry was performed on precultured cells, control cultures, and PAGF-stimulated cultures at 2, 5, 7, and 9 days. The monoclonal antibodies (mAbs) included T3, T4, and T8 (T cells), T9 (transferrin receptor), Tac (IL-2 receptor), 12 (Ia or DR-framework antigen), and T10 (putative activation and/or maturation antigen). PAGF-stimulated cultures had statistically significant increased percentages of T3, T4, T9, T10, and Tac but not T8 when compared to precultured cells and control cultures. PAGF also increased PBL but not CBC Ia. In PAGF-stimulated cultures, CBC had more T3 and T4 cells with increased fluorescence intensity than PBL. Maximal expression of phenotypes usually occurred at Days 7 and 9, 2 days after maximal [3H]TdR incorporation. In comparison to PAGF, PHA-stimulated PBL had earlier expression of these phenotypes but included T8. These data indicate PAGF induces proliferation, activation antigens, and T3 expansion predominantly confined to the T4 subset in both CBC and PBL.