Inhibition of C3 Convertase Activity by Hepatitis C Virus as an Additional Lesion in the Regulation of Complement Components

We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response.     

Butyrate-induced cell differentiation of cell-cycle mutants and ''wild-type'' mastocytoma cells: Histamine, 5-hydroxytryptamine and metachromatic granules as independently regulated differentiation markers

In cultures of heat-sensitive (hs; arrested at 39.5 degrees C, multiplying at 33 degrees C) and cold-sensitive (cs; arrested at 33 degrees C, multiplying at 39.5 degrees C) cell-cycle mutants that had been isolated from the same subclone (K21) of the murine P-815-X2 mastocytoma line, the degree of cell differentiation was assessed by determining the cellular histamine and 5-hydroxytryptamine (5-HT) content as well as the number of metachromatic granules per cell. The findings were compared with those obtained for 'wild-type' K21 and P-815-X2 cells. The addition of butyrate to 'wild-type' cells or to mutant cells maintained at the respective permissive temperature resulted in a relative increase in the level of all three differentiation markers. In cs mutant cells, essentially the same pronounced increase in granule numbers was observed during butyrate treatment at 39.5 degrees C and during incubation at 33 degrees C without butyrate, thereby suggesting that butyrate induces morphological cell differentiation in cs mutants via the same mechanisms as exposure to the nonpermissive temperature. In contrast, the histamine and 5-HT levels reached in hs and cs mutant cells in the presence of butyrate were higher than those observed during incubation at the nonpermissive temperature. Large quantitative differences were detected with respect to the potential of individual cell lines to express the three differentiation parameters. High levels of histamine were characteristic of 'wild-type' P-815-X2 cells treated at 33 degrees C with butyrate, while low amine levels and small numbers of granules were observed in K21 cells (i.e., the parent line of hs and cs mutants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasopressin and angiotensin induce inositol lipid breakdown in rat adenohypophysial cells in primary culture

Adenohypophysial cells from female Wistar rats were dispersed and maintained for 4 days in primary culture in the presence of [3H]myoinositol. The effects of several releasing hormones, corticotropin-releasing factor (CRF), arginine vasopressin (AVP), angiotensin II (A II), thyrotropin-releasing hormone (TRH), and luteinizing hormone-releasing hormone (LHRH) on the liberation of labelled inositol phosphate (InsP), inositol-bisphosphate (InsP2), and inositol-trisphosphate (InsP3) from prelabelled inositol lipids were tested alone and in combination. Of the corticotropin (ACTH) secretagogues tested, AVP and A II produced a dose-dependent increase in inositol phosphate accumulation. CRF was inactive. The ED50 values of about 1 nM for both AVP and A II were close to the corresponding dissociation constants for binding to pituitary membranes: and, in the case of A II, close to the ED50 for A II-induced inhibition of pituitary membrane adenylate cyclase. The responses to A II and AVP could be inhibited by [Sar1,Ile8]A II and the AVP antagonist d(Et2)-VAVP, respectively. The magnitude of the maximal effect of AVP on accumulation of inositol phosphates was small (25% increase over basal value) suggesting that this effect was restricted to a minor subpopulation of pituitary cells (probably corticotrophes). CRF did not potentiate AVP-induced inositol phosphates accumulation. Maximal A II-induced increase in inositol phosphates accumulation represented 150% of the basal value and was partially additive with that of TRH suggesting that lactotrophes represent the main A II-sensitive subpopulation.     

Flow cytometric analysis and sorting of plant chromosomes from Petunia hybrida protoplasts

A method to obtain a high metaphase index and thereafter a plant chromosome suspension is described for Petunia hybrida (2n = 14). Mesophyll protoplast cultures have been used, giving easily disrupted cell walls and a high percentage of dividing cells after 42 h. On 2.5 mM colchicine-treated cells, metaphase indexes reaching 10% were routinely obtained. The lysis medium in which the protoplast-derived cells were disrupted was a simplified culture medium. After chromosome release, samples were stained with Hoechst 33342 dye and analysed by flow cytofluorometry. The histogram of fluorescence intensities included three peaks of metaphase chromosomes and a duplication of this flow karyotype provoked by "monochromatid chromosome." This interpretation was established after flow sorting; micronuclei could also be observed and sorted. Of the 7 chromosomes, only the largest formed a distinct peak while the others were incompletely resolved, due to the similar DNA content of various chromosomes. Model distributions of Petunia hybrida chromosomes have been computed according to the relative chromosome length. The theoretical histograms indicated that low variability is indispensable for resolving distinctive chromosome peaks. The experimental flow karyotype was consistent with one of the models having CV of 2.5%.     

Interleukin 2 receptors are expressed by alveolar macrophages during pulmonary sarcoidosis and are inducible by lymphokine treatment of normal human lung macrophages, blood monocytes, and monocyte cell lines

Expression of receptors for IL 2 was believed initially to be restricted to T cells after their activation by IL 1 and antigen. However, recently IL 2 receptors (IL 2R) were demonstrated on activated B cells by using an anti-IL 2R monoclonal antibody (anti-Tac). In this study, we examined the capacity of cultured human alveolar macrophages, blood monocytes, and myelomonocytic (HL-60) or monoblast (U937) cell lines to bind three different anti-IL 2R monoclonal antibodies before or after stimulation with the monocyte-activating agents IFN-gamma, LPS, phorbol ester, or lymphokine-containing conditioned medium. For each of the four cell populations examined, resting unstimulated cells bound little or no anti-IL 2R antibody, as shown independently by quantitative cell binding assay and by immunoperoxidase labeling. By contrast, incubation with recombinant IFN-gamma, conditioned medium, or to a lesser extent, native or recombinant IL 2 itself, resulted in a significant enhancement of anti-IL 2 receptor monoclonal antibody binding by all four populations, whereas LPS, PMA, or IL 1 had no effect. In addition, membrane binding of anti-Tac antibody, similar to that seen after stimulation of normal lung macrophages with IFN-gamma, was detected by using macrophages obtained by bronchoalveolar lavage of five patients with active pulmonary sarcoidosis. These findings are consistent with the expression of a functional IL 2R on activated cells of the monocyte lineage, since anti-Tac binding to IFN-gamma-treated HL-60 cells was inhibited by addition of excess IL-2; specific binding of anti-IL 2 monoclonal antibodies was detected in the presence of exogenous IL 2; and a 50 to 55 kD molecule was immunoprecipitated from both activated lung macrophages and T lymphoblasts by using anti-Tac antibody. We conclude that human mononuclear phagocytes can be induced by lymphokines to express IL 2R, and that such IL 2R+ macrophages can be detected in vivo during inflammation.     

The effects of platelet-activating factor on flow rate and eicosanoid release in the isolated perfused rat gastric vascular bed

In the isolated rat stomach perfused via the vasculature in situ under constant pressure bolus injections of platelet-activating factor (PAF, 3, 16, or 50 ng) induced dose-dependent, long-lasting reductions of flow rates and simultaneously significant increases in the release of cysteinyl-leukotrienes (cys-LT), thromboxane (TX) B2 and 6-keto-prostaglandin (PG) F1 alpha. Reversed phase high pressure liquid chromatography demonstrated the release of a mixture of comparable amounts of LTC4, LTD4 and LTE4 by PAF. Inhibition of cys-LT synthesis by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and L-651,896 did not significantly affect PAF-induced flow reduction indicating that endogenous cys-LT are of minor importance for the PAF effect on gastric vascular flow. This conclusion is supported by the fact that the cys-LT receptor antagonist FPL 55712 in a concentration (1 x 10(-6) M) that completely antagonized gastric flow reduction by exogenous LTC4 (1 x 10(-7) M) had no effect on the PAF-induced reduction of flow. The cyclooxygenase inhibitor indomethacin aggravated the PAF-induced flow reduction suggesting that the endogenous vasodilator PGI2 might act as a functional PAF antagonist in the rat gastric vascular bed. In contrast to FPL 55712 the PAF antagonist BN 52021 significantly and concentration-dependently antagonized the PAF effect on gastric vascular flow. The results demonstrate that PAF and LTC4 induce flow reductions in the rat gastric vascular bed by activating different receptors and that endogenous eicosanoids released by PAF do not contribute significantly to the PAF effect on gastric vascular flow.     

The 5'' noncoding region of the human leukemia-associated oncogene BCR/ABL is a potent inhibitor of in vitro translation.

The mRNA encoding the chimeric BCR/ABL oncogene, which is transcribed from the Philadelphia chromosome in human chronic myelogenous leukemia, has a 5' noncoding sequence greater than 500 bases in length which is highly GC rich and contains a short open reading frame. This untranslated sequence has a dramatic inhibitory effect upon translational efficiency in vitro. However, when BCR/ABL message is expressed in certain cell types such as the NIH 3T3 cell line, the 5' noncoding region has little inhibitory effect on translational efficiency.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday