Regulation of Myelin P0 Glycoprotein Synthesis in Cultured Rat Schwann Cells and Continuous Rat PNS Cell Lines

We studied the effects of agents that raise intracellular cyclic AMP on synthesis of myelin components by cultured neonatal rat sciatic nerve Schwann cells and by continuous PNS cell lines derived from the fusion of neonatal rat sciatic nerve Schwann cells with rat RN22 Schwannoma. Treatment with N6,2'-O-dibutyryl cyclic AMP (dibutyryl cyclic AMP) caused a fourfold increase in Schwann cell incorporation of 35SO4 into sulfogalactosylceramide (sulfatide), and elicited a 10- to 20-fold increase in such incorporation by the continuous PNS cell lines; a similar effect on PNS cell line sulfatide radiolabelling was obtained with forskolin. Cultured Schwann cells expressed barely detectable levels of myelin P0 glycoprotein (P0) mRNA and myelin basic protein (MBP) mRNA. Treatment of the Schwann cells with axolemmal fragments or with dibutyryl cyclic AMP did not elicit a detectable increase in the levels of these mRNAs. The PNS cell lines constitutively expressed much higher levels of P0 mRNA than did the Schwann cells, and synthesized immunochemically demonstrable P0 glycoprotein, but did not express MBP. Treatment of the PNS cell lines with dibutyryl cyclic AMP markedly reduced expression of P0 mRNA and also diminished immunoreactive P0 glycoprotein. These PNS cell lines should prove useful for further studies of the control of Schwann cell differentiation.     

Autologous B lymphoblastoid cell lines and long-term cultured T cells as stimulators in the mixed lymphocyte reaction: analysis of the role of HLA class II antigens as stimulatory molecules

Human activated T cells, long-term cultured in the presence of interleukin 2 (IL 2), were compared with autologous Epstein Barr virus-transformed B lymphoblastoid cell lines for expression of human leukocyte (HLA)-HLA-DR and -DQ antigens and for ability to induce proliferative responses in autologous and allogeneic lymphocytes. Immunofluorescence analysis performed with a panel of monoclonal antibodies (mAb) specific for HLA-DR or -DQ antigens did not reveal any significant difference in the expression of HLA-DR antigens but revealed reduced expression of HLA-DQ antigens on two out of four T cell lines tested. No obvious difference could be detected in the two-dimensional gel electrophoretic profile of HLA-DR and -DQ beta-chains synthesized by the autologous pairs of B and T cell lines. In contrast with previous reports, the IL 2-dependent cell lines consistently induced alloproliferative responses in standard 6-day mixed lymphocyte cultures; however, these responses were severalfold lower than those elicited by the autologous B lymphoid lines. Both anti-HLA-DR and anti-HLA-DQ mAb blocked the proliferative responses induced by the B cell lines but did not affect those generated by the T cell lines, suggesting that the latter cells induce T lymphocyte activation via a mechanism independent of HLA-DR or -DQ antigen expression on their surface. Addition of IL 2 to the mixed cultures with B cell lines as stimulators did not affect the outcome of the proliferative responses but partially or completely reversed the blocking activity of the mAb. In contrast, IL 2 significantly enhanced the alloproliferation induced by the T lymphoblastoid cell lines, and the anti-HLA class II mAb partially antagonized this effect. Taken together, these data suggest that unlike the HLA-DR and -DQ gene products on B cells, those on IL 2-dependent long-term cultured T cells do not play a direct or primary stimulatory role in the mixed lymphocyte reaction; the reduced levels of alloproliferation induced by the T cell lines are, at least in part, due to a defective production of endogenous IL 2 by the responder lymphocytes rather than to a defective expression of IL 2 receptors by the alloproliferative T cell subset; and the anti-HLA class II mAb in these cultures act only at the responder cell level, since they can efficiently block the enhancement of T cell proliferation triggered by exogenous IL 2, but not the proliferative responses induced by T cell lines in standard conditions.     

v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.     

Induction of parturition in the mare with prostaglandin F2α

Thirty-one mares of Quarter Horse and Thoroughbred breeding were utilized in two experiments to evaluate the efficacy of prostaglandin F₂α (PGF₂α)_for induction of equine parturition and to monitor the effects of this treatment on viability of the resulting foals.Three of five mares given 5 mg PGF₂α (im) on day 338 of gestation foaled 19.6 ± 8.2 hr postinjection. In the second experiment immediately following 3 daily injections of 10 mg estradiol cypionate (ECP) given on days 326, 327 and 328 of gestation, seven mares were infused (iv) with PGF₂α at the rate of 1.3 mg/hr for 24 hr or until parturition occurred. Four of the seven mares foaled in 8.8 ± 1.8 hr after the start of infusion. Side effects including sweating, hypothermia, increased respiration rate and diarrhea were evident in both injected and infused mares, but effects were transient. Neither the injection, nor infusion route of administration of PGF₁α adversely affected the viability of foals. However, some mares induced to foal 12 days prior to expected parturition had foals with slightly weaker pasterns than those of control mares.     

A Naturally Occurring rev1-vpu Fusion Gene Does Not Confer a Fitness Advantage to HIV-1

Background

Pandemic strains of HIV-1 (group M) encode a total of nine structural (gag, pol, env), regulatory (rev, tat) and accessory (vif, vpr, vpu, nef) genes. However, some subtype A and C viruses exhibit an unusual gene arrangement in which the first exon of rev (rev1) and the vpu gene are placed in the same open reading frame. Although this rev1-vpu gene fusion is present in a considerable fraction of HIV-1 strains, its functional significance is unknown.

Results

Examining infectious molecular clones (IMCs) of HIV-1 that encode the rev1-vpu polymorphism, we show that a fusion protein is expressed in infected cells. Due to the splicing pattern of viral mRNA, however, these same IMCs also express a regular Vpu protein, which is produced at much higher levels. To investigate the function of the fusion gene, we characterized isogenic IMC pairs differing only in their ability to express a Rev1-Vpu protein. Analysis in transfected HEK293T and infected CD4+ T cells showed that all of these viruses were equally active in known Vpu functions, such as down-modulation of CD4 or counteraction of tetherin. Furthermore, the polymorphism did not affect Vpu-mediated inhibition of NF-кB activation or Rev-dependent nuclear export of incompletely spliced viral mRNAs. There was also no evidence for enhanced replication of Rev1-Vpu expressing viruses in primary PBMCs or ex vivo infected human lymphoid tissues. Finally, the frequency of HIV-1 quasispecies members that encoded a rev1-vpu fusion gene did not change in HIV-1 infected individuals over time.

Conclusions

Expression of a rev1-vpu fusion gene does not affect regular Rev and Vpu functions or alter HIV-1 replication in primary target cells. Since there is no evidence for increased replication fitness of rev1-vpu encoding viruses, this polymorphism likely emerged in the context of other mutations within and/or outside the rev1-vpu intergenic region, and may have a neutral phenotype.     

A Multistep High-Content Screening Approach to Identify Novel Functionally Relevant Target Genes in Pancreatic Cancer

In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of in vivo and in vitro models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of ‘reverse transfection array’ technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from “druggable” gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented.     

Intramuscular adaptations to eccentric exercise and antioxidant supplementation

Prophylactic supplementation of N-acetyl-cysteine (NAC) and epigallocatechin gallate (EGCG) was studied for physiological and cellular changes in skeletal muscle after eccentric muscle contractions. Thirty healthy, active males (20.0 ± 1.8 years, 160 ± 7.1 cm, 76.1 ± 17.0 kg) ingested for 14 days either 1,800 mg of NAC, 1,800 mg of EGCG, or 1,000 mg of fiber (glucomannan) placebo (PLC) in a double blind, prophylactic fashion. Subjects completed one eccentric exercise bout (100 repetitions at 30°/s) using the dominant knee extensors. Strength and soreness were assessed, and blood and muscle samples obtained before and 6, 24, 48, and 72 h with no muscle sample being collected at 72 h. Separate mixed factorial repeated measures ANOVA (P < 0.05) were used for all statistical analysis. All groups experienced significantly reduced peak torque production after 6 and 24 h, increased soreness at all time points from baseline [with even greater soreness levels 24 h after exercise in PLC when compared to EGCG and NAC (P < 0.05)], increased lactate dehydrogenase at 6 h, and increased creatine kinase 6, 24 and 48 h after exercise. No significant group × time interaction effects were found for serum cortisol, neutrophil counts, and the neutrophil:lymphocyte ratio; although, all values experienced significant changes 6 h after exercise (P < 0.05), but at no other time points. At 48 h after the exercise bout the Neu:Lym ratio in EGCG was significantly less than NAC (P < 0.05), whereas there was a trend (P = 0.08) for the EGCG values to be less when compared to PLC at this time point. Markers of intramuscular mitochondrial and cytosolic apoptosis were assessed (e.g., bax, bcl-2, cytochrome C, caspase-3 content/enzyme activity, and total DNA content). Significant increases (P < 0.05) in muscle levels of bax and bcl-2 were observed in all groups with no significant differences between groups, whereas no changes (P > 0.05) were reported for cytochrome C, caspase-3 content, caspase-3 enzyme activity, and total DNA. Caspase-3 enzyme activity was significantly greater in all groups 48 h after exercise when compared to baseline (P < 0.05) and 6 h (P < 0.05) after exercise. An eccentric bout of muscle contractions appears to significantly increase muscle damage, markers of mitochondrial apoptosis, apoptotic enzyme activity, and whole-blood cell markers of inflammation with no changes in oxidative stress. While soreness ratings were blunted in the two supplementation groups 24 h after exercise when compared to PLC values, more research is needed to determine the potential impact of EGCG and NAC supplementation on changes related to oxidative stress, apoptosis, and eccentric exercise.     

Pleiotropic effects resulting from mutations in genes for ribosomal proteins: analysis of revertants from streptomycin dependence

In order to study the functions of the individual ribosomal proteins and their interaction, a group of revertants from streptomycin dependence to independence was analyzed. Reversion from dependence resulted from a number of different mutational events, all resulting in altered ribosome function. The mutants selected for study exhibited extensive pleiotropy—in addition to the elimination of the requirement for streptomycin for growth, the strains differed from the dependent parent and each other in growth rate, level of streptomycin resistance, effect of antibiotics on viability, rate of subunit assembly in vivo, affinity of isolated ribosomes for streptomycin and functionality of ribosomes in various cell-free assays.There appear to be strong correlations between the level of resistance to streptomycin in growing cells and the ability of the isolated ribosomes to bind streptomycin, the effect of antibiotic on cell-free protein synthesis programmed with natural message (but not poly(U)) and the degree of translational fidelity. There seems to be no relation between level of antibiotic resistance and the overall growth rate, the presence of a defect in ribosome assembly or the ribosomal protein altered by the mutation. Mutations in genes for 30 S proteins S4 and S5 can result in the same phenotype, while different changes in S4 in otherwise isogenic strains result in widely varying phenotypes.The wide variety of effects resulting from single mutational events suggests that each of these changes in a ribosomal protein changes the conformation of the ribosome or its ability to undergo configurational changes.     

Ribosomal Proteins Involved in the Suppression of Streptomycin Dependence in Escherichia coli

Suppression of streptomycin dependence in Escherichia coli strain K-114, a spectinomycin-sensitive strain, is correlated with modification of 30S ribosomal protein P4, the component modified in spectinomycin-resistant mutants. The mutant is unusual in that reversion from dependence has previously been correlated only with modification in 30S protein P4a. Introduction into K-114 of another mutation conferring spectinomycin resistance results in a further alteration in protein P4.