Effect of rat soleus muscle overload on neuromuscular transmission efficacy during continuous and intermittent activation

Increased neuromuscular activity is known to provoke morphological and functional adaptations at the neuromuscular synapse. Most of these changes have been documented following endurance exercise training programmes. In this study, the effect of rat soleus muscle overload produced by tenotomy plus voluntary wheel-cage activity on neuromuscular transmission efficacy was investigated. The overload protocol increased miniature endplate potential (MEPP) and endplate potential (EPP) amplitudes by 17 and 19%, respectively (both P < 0.01), and increased MEPP frequency by 86% (P < 0.01). EPP amplitude rundown during continuous trains of activation was attenuated by approximately 10% in the overloaded group (P < 0.01). Also, during intermittent activation, the overload protocol attenuated EPP amplitude rundown, mainly by enhancing EPP amplitude recovery by approximately 10% during the quiescent periods (P < 0.01). Although the present results show that both the degree and direction of adaptation are similar to what has been observed at rat soleus neuromuscular junctions following an endurance training protocol, there are important nuances between the results, suggesting different mechanisms through which these changes may occur.     

Direct action of genistein on CFTR

 Human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels were expressed in oocytes from Xenopus laevis after injection of CFTR cRNA and studied with the two-electrode voltage-clamp and the giant patch techniques. The tyrosine kinase inhibitor genistein alone activated a small chloride current in whole oocytes expressing CFTR and substantially increased the chloride current obtained upon stimulation with forskolin and isobutyl methylxanthine (IBMX). In giant excised patches, genistein was unable to open protein-kinase-A-phosphorylated CFTR channels in the absence of ATP, but increased the ATP-induced CFTR channel currents by a factor of 3.8 ± 1.7. This genistein-mediated potentiation in excised patches is independent of protein phosphatase activity, as it is readily reversible, even after complete inhibition of protein kinase A activity. Involvement of protein tyrosine kinases also seems unlikely, because this effect of genistein is not antagonized by high concentrations of the tyrosine phosphatase inhibitor ortho-vanadate. We, therefore, propose a direct interaction of genistein with CFTR, probably at a nucleotide binding site, which leads to a higher open probability. Received: 10 March 1997 / Received after revision and accepted: 22 April 1997     

Specificity of microRNA target selection in translational repression

MicroRNAs (miRNAs) are a class of noncoding RNAs found in organisms as evolutionarily distant as plants and mammals, yet most of the mRNAs they regulate are unknown. Here we show that the ability of an miRNA to translationally repress a target mRNA is largely dictated by the free energy of binding of the first eight nucleotides in the 5' region of the miRNA. However, G:U wobble base-pairing in this region interferes with activity beyond that predicted on the basis of thermodynamic stability. Furthermore, an mRNA can be simultaneously repressed by more than one miRNA species. The level of repression achieved is dependent on both the amount of mRNA and the amount of available miRNA complexes. Thus, predicted miRNA:mRNA interactions must be viewed in the context of other potential interactions and cellular conditions.     

Acute dopamine/noradrenaline reuptake inhibition enhances human exercise performance in warm, but not temperate conditions

Nine healthy endurance-trained males were recruited to examine the effect of a dual dopamine/noradrenaline reuptake inhibitor on performance, thermoregulation and the hormonal responses to exercise. Subjects performed four trials, ingesting either a placebo (pla) or 2 × 300 mg bupropion (bup), prior to exercise in temperate (18°C) or warm (30°C) conditions. Trials consisted of 60 min cycle exercise at 55% W _max immediately followed by a time trial (TT). TT performance in the heat was significantly improved by bupropion (pla: 39.8 ± 3.9 min, bup: 36.4 ± 5.7 min; P = 0.046), but no difference between treatments was apparent in temperate conditions (pla: 30.6 ± 2.2 min, bup: 30.6 ± 1.9 min; P = 0.954). While TT power output was consistently lower in the heat when compared to temperate conditions, this decrement was attenuated by bupropion. At the end of the TT in the heat, both core temperature (pla 39.7 ± 0.3°C, bup 40.0 ± 0.3°C; P = 0.017) and HR (pla 178 ± 7 beats min⁻¹, bup 183 ± 12 beats min⁻¹; P = 0.039), were higher in the bupropion trial than in the placebo. Circulating pituitary and adrenal hormone concentrations increased throughout exercise in all trials. Circulating serum prolactin was elevated above temperate levels during exercise in a warm environment ( P < 0.001). These data indicate that performance in warm conditions is enhanced by acute administration of a dual dopamine/noradrenaline reuptake inhibitor. No such effect was apparent under temperate conditions. It appears that bupropion enabled subjects to maintain a greater TT power output in the heat with the same perception of effort and thermal stress reported during the placebo trial, despite the attainment of a higher core temperature.     

Iduronate-2-sulfatase gene mutations in 16 patients with mucopolysaccharidosis type II (Hunter syndrome)

Mutations of the iduronate-2-sulfatase gene were identifiedin 16 patients with mucopolysaccharidosis type II (Hunter syndrome).Together with another 10 cases reported by us earlier it emergesthat about 20% of the patients have deletions of the whole geneor other major structural alterations. One, two or three basepair deletions are found in about 23% of the cases while theremaining about 57% carry point mutations predicting amlno acidreplacement, premature termination of translation, or aberrantsplicing. Molecular analysis of mRNA in splice site mutantsshowed that these latter defects frequently resulted in useof cryptic splice sites in exons or introns. 62% of the smalldeletions and point mutations have occurred in 3 of the 9 iduronate-2-sulfatasegene exons. Knowledge of the primary genetic defect allows fastand reliable carrier detection and prenatal diagnosis as wellas insight into the relationship between genotype and phenotype.     

In vivo depletion of CD11c+ dendritic cells abrogates priming of CD8+ T cells by exogenous cell-associated antigens

Cytotoxic T lymphocytes (CTL) respond to antigenic peptides presented on MHC class I molecules. On most cells, these peptides are exclusively of endogenous, cytosolic origin. Bone marrow-derived antigen-presenting cells, however, harbor a unique pathway for MHC I presentation of exogenous antigens. This mechanism permits cross-presentation of pathogen-infected cells and the priming of CTL responses against intracellular microbial infections. Here, we report a novel diphtheria toxin-based system that allows the inducible, short-term ablation of dendritic cells (DC) in vivo. We show that in vivo DC are required to cross-prime CTL precursors. Our results thus define a unique in vivo role of DC, i.e., the sensitization of the immune system for cell-associated antigens. DC-depleted mice fail to mount CTL responses to infection with the intracellular bacterium Listeria monocytogenes and the rodent malaria parasite Plasmodium yoelii.     

Molecular characterization of a serotype O121:H19 clone,a distinct Shiga toxin-producing clone of pathogenic Escherichia coli

Most illnesses caused by Shiga toxin-producing Escherichia coli (STEC) have been attributed to E. coli serotype O157:H7, but non-O157 STEC infections are now increasingly recognized as public health problems worldwide. The O121:H19 serotype is being isolated more frequently from clinical specimens and has been implicated in one waterborne outbreak. We used multilocus virulence gene profiling, a PCR-based assay, to characterize the virulence gene content of 24 isolates of serotype O121:H19 and nonmotile variants. We also performed multilocus enzyme electrophoresis and multilocus sequencing to establish the clonal relatedness of O121 isolates and to elucidate the relationship of O121 to common STEC clones. The 24 isolates were found to represent a single bacterial clone, as there was no allelic variation across 18 enzyme loci among the isolates. The complete nucleotide sequence of the intimin gene differed by four substitutions from that of the epsilon (Int- epsilon ) allele of O103:H2 strain PMK5. The typical O121 virulence gene profile was similar to the profiles of enterohemorrhagic E. coli (EHEC) clones of E. coli: it included a Shiga toxin 2 gene (stx(2)), two genes on the EHEC plasmid (toxB and ehxA), and the gene encoding intimin (eae). Despite the similarities, putative virulence genes distributed on O islands-large chromosomal DNA segments present in the O157:H7 genome-were useful for discriminating among STEC serotypes and the O121:H19 clone had a composite profile that was distinct from the profiles of the other major EHEC clones of pathogenic E. coli. On the basis of sequencing analysis with 13 housekeeping genes, the O121:H19 clone did not fall into any of the four classical EHEC and enteropathogenic E. coli groups but instead was closely related to two eae-negative STEC strains.     

Interstitial deletion of 20q in a patient with Waldenström macroglobulinemia following chemotherapy

A 76-year-old male with a history of renal insufficiency was found to have anemia, an IgM kappa paraprotein on serum immunofixation studies, absence of lytic bone lesions, and findings in the bone marrow consistent with Waldenstr?m macroglobulinemia (WM). Cytogenetic studies including fluorescence in situ hybridization (FISH) on the post-treatment bone marrow revealed the karyotype 46,XY,del(20)(q13.1q13.3). Less than 70 cases of karyotypic abnormalities in patients with WM have been reported, which have shown no abnormalities specific to WM. Monosomy or trisomy of chromosome 20 has been reported in approximately eight cases, but to our knowledge this is the first case report of an interstitial deletion of 20q, confirmed by FISH using chromosome 20 subtelomeric specific probes. Interstitial deletions of 20q are known to occur in polycythemia vera and other hematological malignancies, especially those of myeloid origin.