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.     

Tracing paternal ancestry in mice, using the Y-linked, sex-determining locus, Sry

The molecular evolution of mammalian Y-linked DNA sequences is of special interest because of their unique mode of inheritance: most Y- linked sequences are clonally inherited from father to son. Here we investigate the use of Y-linked sequences for phylogenetic inference. We describe a comparative analysis of a 515-bp region from the male sex- determining locus, Sry, in 22 murine rodents (subfamily Murinae, family Muridae), including representatives from nine species of Mus, and from two additional murine genera--Mastomys and Hylomyscus. Percent sequence divergence was < 0.01% for comparisons between populations within a species and was 0.19%-8.16% for comparisons between species. Our phylogenetic analysis of 12 murine taxa resulted in a single most- parsimonius tree that is highly concordant with phylogenies based on mitochondrial DNA and allozymes. A total evidence tree based on the combined data from Sry, mitochondrial DNA, and allozymes supports (1) the monophyly of the subgenus Mus, (2) its division into a Palearctic group (M. musculus, M. domesticus, M. spicilegus, M. Macedonicus, and M. spretus) and an Oriental group (M. cookii++, M. cervicolor, and M. caroli), and (3) sister-group relationships between M. spicilegus and M. macedonicus and between M. cookii and M. cervicolor. We argue that Y- chromosome DNA sequences represent a valuable new source of characters for phylogenetic inference.      

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.     

Le ionophore A23187

The acrosomic status of spermatozoa prepared for IVF has been evaluated by means of immunofluorescence test from Fenichel and Hsi using calcium A 23187 ionophore as inductor of acrosome reaction (AR). The spontaneous AR remains slight, even after 6 hour-incubation in Menezo B2 (6,8+2,7%). The response to ionophore, moderate before (11,2+9%), frankly increases after a 6h-capacitation (28,9+8,3%) in a group of 25 IVF couples (tubal indication, normal semen, positive fertilization). Nevertheless, it remains slight or null in 4 cases of unexplained repeated failure of fertilization. The response to ionophore A 23187 allows to explore the kinetics of capacitation of spermatozoa and their ability to perform AR. Its significance in terms of fecondance remains to be precised.     

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.     

HIV-1 Neutralizing Antibody Signatures and Application to Epitope-Targeted Vaccine Design

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Long-term creatine supplementation does not significantly affect clinical markers of health in athletes

Creatine has been reported to be an effective ergogenic aid for athletes. However, concerns have been raised regarding the long-term safety of creatine supplementation. This study examined the effects of long-term creatine supplementation on a 69-item panel of serum, whole blood, and urinary markers of clinical health status in athletes. Over a 21-month period, 98 Division IA college football players were administered in an open label manner creatine or non-creatine containing supplements following training sessions. Subjects who ingested creatine were administered 15.75 g/day of creatine monohydrate for 5 days and an average of 5 g/day thereafter in 5–10 g/day doses. Fasting blood and 24-h urine samples were collected at 0, 1, 1.5, 4, 6, 10, 12, 17, and 21 months of training. A comprehensive quantitative clinical chemistry panel was determined on serum and whole blood samples (metabolic markers, muscle and liver enzymes, electrolytes, lipid profiles, hematological markers, and lymphocytes). In addition, urine samples were quantitatively and qualitative analyzed to assess clinical status and renal function. At the end of the study, subjects were categorized into groups that did not take creatine (n = 44) and subjects who took creatine for 0–6 months (mean 4.4 ± 1.8 months, n = 12), 7–12 months (mean 9.3 ± 2.0 months, n = 25), and 12–21 months (mean 19.3 ± 2.4 months, n = 17). Baseline and the subjects' final blood and urine samples were analyzed by MANOVA and 2 × 2 repeated measures ANOVA univariate tests. MANOVA revealed no significant differences (p = 0.51) among groups in the 54-item panel of quantitative blood and urine markers assessed. Univariate analysis revealed no clinically significant interactions among groups in markers of clinical status. In addition, no apparent differences were observed among groups in the 15-item panel of qualitative urine markers. Results indicate that long-term creatine supplementation (up to 21-months) does not appear to adversely effect markers of health status in athletes undergoing intense training in comparison to athletes who do not take creatine.     

Behavioural and chemical mechanisms behind a Mediterranean ant–ant association

1. Interspecific competition among ants is common, and so is competitive exclusion among dominant ant species. In contrast, specific associations between non‐parasitic ant species are rare, especially in the temperate zones. As an exception, the subordinate ant Camponotus lateralis frequently co‐occurs with the dominant Crematogaster scutellaris but rarely with other dominant ants. 2. This association is one of various associations between Camponotus and Crematogaster species across the world. However, the mechanisms behind these co‐occurences are largely unknown. 3. In the present study, we therefore investigated the association of Ca. lateralis and Cr. scutellaris. We studied the spatial association of the nests, interspecific aggression, both species' cuticular hydrocarbon profiles, and their propensity to follow the other species' pheromone trails. 4. Crematogaster scutellaris usually attacked and displaced the generally submissive Ca. lateralis, but was significantly less aggressive at jointly used trails. Camponotus nests were always in close proximity to Crematogaster nests. 5. The cuticular hydrocarbons of both species consisted of alkanes with chain lengths between C21 and C35. The two species had 25 hydrocarbons in common, including mono‐, di‐, and tetramethyl alkanes. Despite this qualitative similarity, however, the quantitative hydrocarbon composition differed between the two species. 6. Camponotus lateralis followed artificial trails containing trail pheromones of Cr. scutellaris, but the latter did not follow Ca. lateralis trail pheromones. Interspecific trail‐following by Camponotus, but not vice versa, has been observed in another Camponotus–Crematogaster association and may be a more general mechanism that facilitates associations between the two ant genera.     

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.