Interleukin inhibition by a parasite proteinase inhibitor, taeniaestatin

A proteinase inhibitor, taeniaestatin, isolated from the larval stage of the cestode Taenia taeniaeformis inhibits endogenous IL 2 generation in murine lymphocytes and IL 1 induced proliferation of murine thymocytes in a dose-dependent manner. However, taeniaestatin does not inhibit exogenous IL 2-induced proliferation of an IL 2-dependent cell line at any dose tested. These data indicate that the lack of IL 2 generation may be due in part to inhibition of a crucial cell-associated proteinase subsequent to cellular activation, or the lack of an effective IL 2 signal for differentiation. Our results are novel findings concerning molecular pathways for parasite inhibition of host immune responses, and suggest that selected proteinase inhibitors may be useful in clinical situations in which IL 1 or IL 2 are elevated.     

Attachment of Entamoeba histolytica to glass in a defined maintenance medium: specific requirement for cysteine and ascorbic acid

Cysteine and ascorbic acid were previously shown to be required by Entamoeba histolytica trophozoites for attachment to glass, elongation, and ameboid movement as well as for short-term (12-24 h) survival in a balanced salt solution containing bovine serum albumin and a vitamin solution (Maintenance Medium 1). If the only function of cysteine and ascorbate was to decrease the redox potential, other reducing agents should be effective. However, the requirement for cysteine in the presence of ascorbic acid was highly specific. Equally effective were D- and L-cysteine; however, of many other compounds tested, only thioglycolic acid, ascorbic acid, or L-cystine (in decreasing order) were somewhat active. Under N2 atmosphere, cysteine and ascorbic acid were still required, although their concentrations could be halved. The ability to attach in the maintenance medium was irreversibly lost after only 5 min of cysteine-ascorbic acid deprivation; however, there was no decrease in viability when the amebae were transferred to growth medium within 30 min. Cysteine thiol groups in the medium were oxidized rapidly regardless of the concentration of ascorbic acid or the presence of amebae; however, ascorbic acid prolonged attachment of amebae.     

Properties of aminoglycoside-3'-phosphotransferases determined by kanamycin transposones Tn 601 and Tn 5

Aminoglycoside-3'-phosphotransferase I and II (APT-3'-I and APT-3'-II) has been purified to homogenity from the cells of E. coli containing the plasmids R6 and JR67, respectively. The purification procedure involved competitive affinity chromatography on neomycin-sepharose and gel-filtration on Sephadex G-100. The specific activity of APT-3'-I with the substrates--lividomycin A, neomycin B, paromycin, ribostamycin, kanamycins A and B--are 4.3, 2.8, 2.1, 1.6, 0.9 and 0.8 mol/min. mg protein, respectively. The specific activity of APT-3'-II with the substrates--ribostamycin, paromycin, kanamycins A and B, neomycin B--are 8.0, 7.2, 4.0, 4.5 and 3.6, respectively. Mg2+ is required for the activity of both enzymes. Co2+, Zn2+ and Mn2+ are active in case of APT-3'-I; however, these cations are less active than Mg2+. The pH-optimum of APT-3'-I and APT-3'-II is 7.0--7.5. High ionic strength is required for the activity of both enzymes. The molecular weights of APT-3'-I and APT-3'-II are about 36 000 and 26 000, respectively. The amino acid composition of APT-3'-I and APT-3'-II was determined. Both enzymes contain tryptophane residues whose fluorescence intensity decreased when ATP, but not amino-glycoside antibiotics, is added. The interrelationship between the molecular weights of these enzymes and the sizes of the loops of transposones Tn 601 and Tn 5, encoding APT-3'-I and APT-3'-II, is discussed.     

Multicenter evaluation of the performance and clinical utility in longitudinal monitoring of the Bayer Immuno 1 complexed PSA assay.

We conducted a multicenter evaluation of the analytical and clinical performance of the automated Bayer Immuno 1 complexed PSA (cPSA) assay, and compared assay performance to the Bayer Immuno 1 PSA assay. We sought to determine whether measurements of cPSA could be of clinical utility in the management of patients with prostate cancer. Results of the 10-day imprecision across three evaluation sites produced total CV < 2.50% and an analytical sensitivity of 0.02 microgram/L. There was an increased trend in clinical sensitivity for prostate cancer with increasing stage of disease (71-86%). Clinical specificity for patients with benign urogenital disease was 74.8%, and for other nonprostate diseases ranged from 91.1-100%. Retrospective serial monitoring of 155 patients with prostate cancer demonstrated concordance of cPSA measurements to clinical status for 97% of the patients analyzed. Results from the clinical studies using the Bayer Immuno 1 cPSA assay were comparable to results obtained with the Bayer Immuno 1 PSA assay. The Bayer Immuno 1 cPSA assay demonstrates analytical performance and clinical effectiveness in the management of prostate cancer patients during the course of disease and therapy.     

Delayed nucleoid segregation in Escherichia coli

To study the role of cell division in the process of nucleoid segregation, we measured the DNA content of individual nucleoids in isogenic Escherichia coli cell division mutants by image cytometry. In pbpB(Ts) and ftsZ strains growing as filaments at 42 degrees C, nucleoids contained, on average, more than two chromosome equivalents compared with 1.6 in wild-type cells. Because similar results were obtained with a pbpB recA strain, the increased DNA content cannot be ascribed to the occurrence of chromosome dimers. From the determination of the amount of DNA per cell and per individual nucleoid after rifampicin inhibition, we estimated the C and D periods (duration of a round of replication and time between termination and cell division respectively), as well as the D' period (time between termination and nucleoid separation). Compared with the parent strain and in contrast to ftsQ, ftsA and ftsZ mutants, pbpB(Ts) cells growing at the permissive temperature (28 degrees C) showed a long D' period (42 min versus 18 min in the parent) indicative of an extended segregation time. The results indicate that a defective cell division protein such as PbpB not only affects the division process but also plays a role in the last stage of DNA segregation. We propose that PbpB is involved in the assembly of the divisome and that this structure enhances nucleoid segregation.     

Paclitaxel enhances macrophage IL-12 production in tumor-bearing hosts through nitric oxide.

Tumor-induced macrophages (Mphis) mediate immunosuppression, in part, through increased production of factors that suppress T cell responsiveness and underproduction of positive regulatory cytokines. Pretreatment of tumor-bearing host (TBH) Mphis with the anticancer agent paclitaxel (Taxol) partially reverses tumor-induced Mphi suppressor activity, suggesting that paclitaxel may restore TBH Mphi production of proimmune factors. Because paclitaxel demonstrates LPS-mimetic capabilities and increased production of the LPS-induced immunostimulatory cytokine IL-12 could account for enhanced T cell responsiveness, we investigated whether paclitaxel induces Mphi IL-12 production. Tumor growth significantly down-regulated Mphi IL-12 p70 production through selective dysregulation of IL-12 p40 expression. LPS stimulation failed to overcome tumor-induced dysregulation of p40 expression. In contrast, paclitaxel significantly enhanced both normal host and TBH Mphi IL-12 p70 production in vitro, although TBH Mphi IL-12 production was lower than that of similarly treated normal host Mphis. Paclitaxel enhanced p40 expression in a dose-dependent manner. Through reconstituted Mphi IL-12 expression, paclitaxel pretreatment relieved tumor-induced Mphi suppression of T cell alloreactivity. Blocking Mphi NO suppressed paclitaxel's ability to induce IL-12 production. This suggests that paclitaxel-induced activities may involve a NO-mediated autocrine induction pathway. Collectively, these data demonstrate that paclitaxel restores IL-12 production in the TBH and ascribe a novel immunotherapeutic component to the pleiotropic activities of NO. Through its capacity to induce IL-12 production, paclitaxel may contribute to the correction of tumor-induced immune dysfunction.