Laboratory analogue of a supersonic accretion column in a binary star system.

Astrophysical flows exhibit rich behaviour resulting from the interplay of different forms of energy-gravitational, thermal, magnetic and radiative. For magnetic cataclysmic variable stars, material from a late, main sequence star is pulled onto a highly magnetized (B>10 MG) white dwarf. The magnetic field is sufficiently large to direct the flow as an accretion column onto the poles of the white dwarf, a star subclass known as AM Herculis. A stationary radiative shock is expected to form 100-1,000 km above the surface of the white dwarf, far too small to be resolved with current telescopes. Here we report the results of a laboratory experiment showing the evolution of a reverse shock when both ionization and radiative losses are important. We find that the stand-off position of the shock agrees with radiation hydrodynamic simulations and is consistent, when scaled to AM Herculis star systems, with theoretical predictions.

Genetic risk markers related to diabetes-associated autoantibodies in young patients with type 1 diabetes in berlin, Germany.

AIMS: To determine the prevalence of genetic risk markers of type 1 diabetes (T1D) in children diagnosed at a single centre in Germany and to assess their relation to diabetes-associated autoantibodies. METHODS: Blood samples from 243 paediatric patients were genotyped for the high-risk HLA haplotypes DR3-DQ2 (DQA1*05-DQB1*02) and DR4-DQ8 (DRB1*0401/2/4/5-DQB1*0302) and PTPN22 C1858 T polymorphism. The patients (51.4% male) were diagnosed with T1D at a median age of 8.6 y. The T1D-related autoantibodies GADA, IAA and IA-2A were analysed at diagnosis. RESULTS: 166 patients (68.6%) carried the DR3-DQ2, 114 (47.1%) the DR4-DQ8 haplotype, while 41 (16.9%) patients were negative for both. The PTPN22 CC genotype was detected in 177 (72.8%), CT in 58 (23.9%) and TT in eight (3.3%) patients, respectively. The prevalence of T1D-related autoimmunity was 77.0% for IA-2A, 71.6% for GADA and 43.6% for IAA. There were no differences between patients with and without the 1858 T allele in terms of the frequency, levels or number of autoantibodies, but the former were younger at diagnosis than the latter (p=0.002), IA-2A were positively related to HLA DR4-DQ8 (p=0.004) and inversely associated with HLA DR3-DQ2 (p=0.002). GADA-positive patients were older than those without GADA (p=0.004). In multivariate logistic regression analysis including gender and age as confounding variables, DR4-DQ8 (OR 2.56, 95%CI 1.35-4.86) and DR3-DQ2 (OR 0.36, 95%CI 0.19-0.68) were the only independent predictors of IA-2A positivity. CONCLUSION: The prevalence of genetic risk markers in Berlin children with T1D is found to be comparable to other Caucasian T1D populations. The presence of IA-2A at diagnosis is strongly associated with the HLA risk haplotypes, but not with PTPN22 polymorphism.

Secretion of protease nexin-1 by C6 glioma cells is under the control of a heterotrimeric G protein, Go1.

Heterotrimeric Go proteins have recently been described as regulators of vesicular traffic. The Goalpha gene encodes, by alternative splicing, two Goalpha polypeptides, Go1alpha and Go2alpha. By immunofluorescence and electron microscopy, we detected Go1alpha on the membrane of small intracellular vesicles in C6 glioma cells. After stable transfection of these cells, overexpression of Go1alpha but not Go2alpha was followed by a rise in the secretion of a serine protease inhibitor, protease nexin-1 (PN-1). This secretion was enhanced as a function of the amount of expressed Go1alpha. Metabolic cell labeling indicated that this increase in PN-1 secretion was not the result of an enhancement in PN-1 biosynthesis or a decrease in its uptake, but revealed a potential role of Go1alpha in the regulation of vesicular PN-1 trafficking. Furthermore, activators of Go proteins, mastoparan and a peptide derived from the amino terminus of the growth cone-associated protein GAP43, increased PN-1 secretion in parental and Go1alpha-overexpressing cells. Brefeldin A, an inhibitor of vesicular traffic, inhibited both basal and mastoparan-stimulated PN-1 secretions. These results indicate, that in C6 glioma cells, PN-1 secretion could be regulated by both Go1alpha expression and activation.

Genetic lesions associated with Muller's ratchet in an RNA virus.

The molecular basis of Muller's ratchet has been investigated using the important animal pathogen foot-and-mouth disease virus (FMDV). Clones from two FMDV populations were subjected to serial plaque transfers (repeated bottleneck events) on host BHK-21 cells. Relative fitness losses were documented in 11 out of 19 clones tested. Small fitness gains were observed in three clones. One viral clone attained an extremely low plating efficiency, suggesting that accumulation of deleterious mutations had driven the virus near extinction. Nucleotide sequence analysis revealed unique genetic lesions in multiply transferred clones that had never been seen in FMDVs isolated in nature or subjected to massive infections in cell culture. In particular, a frequent internal polyadenylate extension has identified a mutational hot spot on the FMDV genome. Furthermore, amino acid residue substitutions in internal capsid sites which are severely restricted during FMDV evolution, amounted to half of capsid replacements in the transferred clones. In addition, a striking dominance of non-synonymous replacements fixed upon large population infections of FMDV was not observed upon serial plaque transfers. The nucleotide sequence of the entire genome of a severely debilitated clone suggests that very few mutations may be sufficient to drive FMDV near extinction. The results provide an account of the molecular basis of Muller's ratchet for an RNA virus, and insight into the types of genetic variants which populate the mutant spectra of FMDV quasispecies.

Long-term, large-population passage of aphthovirus can generate and amplify defective noninterfering particles deleted in the leader protease gene.

During serial undiluted passage of a clonal population of foot-and-mouth disease virus (FMDV C-S8c1) in BHK-21 cells, two species of defective RNA were generated and selected. Sequence analysis revealed that they included deletions within the L-coding region, and retained the correct reading frame for viral protein synthesis. These deleted RNAs directed the synthesis of capsid protein VP1, were packaged in particles sedimenting with standard virus, required homologous infectious helper virus in order to produce viral particles, but did not interfere with the replication of helper virus. While detection of defective particles in FMDV required more than 100 serial passages, once produced, these defective RNAs could be stably maintained upon further passages in the FMDV C-S8c1 quasispecies. Furthermore, a high fitness, monoclonal-antibody-resistant virus was able to replace the standard virus and support the amplification of the deleted particles. This is the first description of naturally occurring, defective particles of FMDV.

Transfected Go1 alpha inhibits the calcium dependence of beta-adrenergic stimulated cAMP accumulation in C6 glioma cells.

Increasing evidence indicates that heterotrimeric G proteins, and in particular Go, regulate ionic channel activities. In order to investigate the role of Go proteins in the modulation of the Ca2+ influx, C6 glioma cells were stably transfected with alpha o1 cDNA. Expression of the Go1 alpha protein was checked by Bordetella pertussis toxin-catalyzed ADP-ribosylation and Western blots using one- and two-dimensional gel analyses. Three clones were selected based on their degree of Go1 alpha expression. In alpha o1-transfected cells, cAMP accumulations, in response to isoproterenol or forskolin, were lower than in control cells. This inhibitory effect was a function of the amount of expressed Go1 alpha. In contrast, Go1 alpha expression was not followed by a significant inhibition of isoproterenol- or forskolin-stimulated adenylyl cyclase activities in particulate fractions. In C6 parental cells, 50-60% of the isoproterenol-induced cAMP accumulation was dependent on external Ca2+ concentration. This Ca(2+)-dependent cAMP accumulation was related to an induced transient Ca2+ influx. In transfected cells, expression of Go1 alpha inhibited the Ca2+ influx and the Ca(2+)-dependent component of isoproterenol-induced cAMP accumulation. In conclusion, beta-adrenergic agonists stimulate an entry of Ca2+ which exerts a positive feedback on cAMP production, and Go1 alpha blocks this positive feedback by inhibiting the Ca2+ influx.

Specific antibodies against Go isoforms reveal the early expression of the Go2 alpha subunit and appearance of Go1 alpha during neuronal differentiation.

We have previously identified two isoforms of Go alpha in membranes of N1E-115 neuroblastoma cells, using an antibody raised against the purified Go alpha subunit; one isoform of the Go alpha subunit (pI 5.80) is present in undifferentiated cells, whereas a more acidic isoform (pI 5.55) appears during differentiation [J. Neurochem. 54:1310-1320 (1990)]. Recently, the Go alpha gene has been shown to encode, by alternative splicing, two polypeptides, Go1 alpha and Go2 alpha, which differ only in their carboxyl-terminal part. To determine unambiguously whether the two Go alpha subunits detected in neuroblastoma cells were actually the products of different mRNAs, rabbit polyclonal antibodies were generated against synthetic peptides (amino acids 291-302) of both sequences. Specificity of the two affinity-purified antipeptide antibodies was assessed on Western blots by comparing their immunoreactivities with those of other G alpha antibodies. On a blotted mixture of purified brain guanine nucleotide-binding proteins, the anti-alpha o1 and anti-alpha o2 peptide antibodies only recognized the 39-kDa Go alpha subunit. Furthermore, the immunological recognition of brain membranes from 15-day-old mouse fetuses by antipeptide antibodies could be specifically blocked by addition of the corresponding antigen. When membrane proteins from differentiated neuroblastoma cells and mouse fetus brain were blotted after two-dimensional gel electrophoresis, the anti-alpha o1 and anti-alpha o2 peptide antibodies labeled a 39-kDa subunit focused at a pI value of 5.55 or 5.80, respectively. Study of the ontogenesis of both Go alpha subunits revealed the predominance of Go2 alpha in the frontal cortex at day 15 of gestation. Thereafter, there was a progressive decline of the Go2 alpha polypeptide to a very low level, concomitant with an increase in the Go1 alpha protein, which plateaued about 15 days after birth to a level 8 times higher than at gestational day 15. Similarly, on neuroblastoma cells, the Go2 alpha subunit was almost exclusively present in undifferentiated cells, and differentiation induced the appearance of the Go1 alpha subunit, with a reduction in the amount of Go2 alpha polypeptide. Thus, the evolution of the two Go alpha subunits during cell differentiation, unambiguously identified with specific antibodies, suggests that neuronal differentiation is responsible for the on/off switch of the expression of the Go alpha isoforms and indicates that Go1 alpha, rather than Go2 alpha, is involved in neurotransmission.

On concanavalin A-treated striatal neurons quisqualate clearly behaves as a partial agonist of a receptor fully activated by kainate.

In cultured striatal neurons, maximal [3H]GABA release stimulated by quisqualate (QA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) was 10-20 times smaller than that stimulated by kainate (KA), and we have previously reported that QA or AMPA competitively inhibited KA-evoked GABA release. Since the lectin concanavalin A (Con A) has been shown to inhibit QA receptor desensitization, the interaction between QA and KA was further studied in Con A-treated neurons. Con A dose-dependently and specifically potentiated QA- or AMPA-evoked [3H]GABA release, so that maximal responses of QA or AMPA were half of that of KA. The responses of these agonists were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with similar apparent Ki values, indicating that they resulted from non-NMDA receptor activation. In Con A-treated neurons, QA and AMPA competitively inhibited the KA-induced GABA release. The apparent affinities of QA and AMPA in inhibiting the KA response were identical to their affinities in stimulating GABA release. Moreover, the maximal KA response measured in the presence of QA or AMPA was identical to that measured with KA alone. These results clearly indicate that to stimulate GABA release from Con A-treated striatal neurons, QA and AMPA behave as partial agonists of a receptor fully activated by KA. These results further support the hypothesis that QA, AMPA and KA act on a common receptor type in striatal neurons.