A novel efflux system in inducibly erythromycin-resistant strains of Streptococcus pyogenes

Streptococcus pyogenes strains inducibly resistant (iMLS phenotype) to macrolide, lincosamide, and streptogramin B (MLS) antibiotics can be subdivided into three phenotypes: iMLS-A, iMLS-B, and iMLS-C. This study focused on inducibly erythromycin-resistant S. pyogenes strains of the iMLS-B and iMLS-C types, which are very similar and virtually indistinguishable in a number of phenotypic and genotypic features but differ clearly in their degree of resistance to MLS antibiotics (high in the iMLS-B type and low in the iMLS-C type). As expected, the iMLS-B and iMLS-C test strains had the erm(A) methylase gene; the iMLS-A and the constitutively resistant (cMLS) isolates had the erm(B) methylase gene; and a control M isolate had the mef(A) efflux gene. mre(A) and msr(A), i.e., other macrolide efflux genes described in gram-positive cocci, were not detected in any test strain. With a radiolabeled erythromycin method for determination of the intracellular accumulation of the drug in the absence or presence of an efflux pump inhibitor, active efflux of erythromycin was observed in the iMLS-B isolates as well as in the M isolate, whereas no efflux was demonstrated in the iMLS-C isolates. By the triple-disk (erythromycin plus clindamycin and josamycin) test, performed both in normal test medium and in the same medium supplemented with the efflux pump inhibitor, under the latter conditions iMLS-B and iMLS-C strains were no longer distinguishable, all exhibiting an iMLS-C phenotype. In conjugation experiments with an iMLS-B isolate as the donor and a Rif(r) Fus(r) derivative of an iMLS-C isolate as the recipient, transconjugants which shared the iMLS-B type of the donor under all respects, including the presence of an efflux pump, were obtained. These results indicate the existence of a novel, transferable efflux system, not associated with mef(A) or with other known macrolide efflux genes, that is peculiar to iMLS-B strains. Whereas the low-level resistance of iMLS-C strains to MLS antibiotics is apparently due to erm(A)-encoded methylase activity, the high-level resistance of iMLS-B strains appears to depend on the same methylase activity plus the new efflux system.     

Proteasome inhibitor-induced apoptosis in human monocyte-derived dendritic cells

Proteasome inhibitors possess potent antitumor activity against a broad spectrum of human malignancies. However, the effects of these compounds on the immune system still have to be clearly determined. In the present study, we have investigated the effects of proteasome inhibitors on dendritic cells (DC), antigen-presenting cells playing a key role in the initiation of immune responses. Exposure to the proteasome inhibitors bortezomib, MG132 or epoxomicin was found to promote apoptosis of human monocyte-derived DC and to reduce the yield of viable DC when given to monocytes early during differentiation to DC. DC apoptosis via proteasome inhibition was accompanied by mitochondria disruption and subsequent activation of the caspase cascade. Up-regulation and intracellular redistribution of Bcl-2-associated X protein (Bax), a pro-apoptotic Bcl-2 family protein, were observed in DC treated with these compounds and represent a suitable mechanism leading to activation of the intrinsic apoptotic pathway. Finally, active protein synthesis was found to represent an upstream prerequisite for DC apoptosis induced by proteasome inhibitors, since the translation inhibitor cycloheximide blocked all of the steps of the observed apoptotic response. In conclusion, induction of apoptosis in DC may represent a novel mechanism by which proteasome inhibitors affect the immune response at the antigen-presenting cell level.     

Effects of pink grapefruit juice on QT variability in patients with dilated or hypertensive cardiomyopathy and in healthy subjects

Recent evidence shows that pink grapefruit juice, which is a recommended dietary addition that contains high amounts of the antioxidant flavonoid naringenin, prolongs the corrected QT (QT(c)), a noninvasive electrophysiological marker of spatial myocardial repolarization, and does so by inhibiting the rapid component of the delayed rectifier K+ current (I(Kr)). Prompted by the observation that all class III antiarrhythmic drugs inhibit this current, thereby sometimes provoking torsades de pointes, we compared the effects of a liter of freshly squeezed pink grapefruit juice with those of 2 commonly used class III antiarrhythmics amiodarone and sotalol on the major noninvasive markers of temporal variability in myocardial repolarization used to stratify the risk of sudden death from malignant ventricular arrhythmias. In 32 subjects, 10 with postischemic dilated cardiomyopathy, 12 with hypertensive cardiomyopathy, and 10 healthy, we assessed QT(c) and QT variability index (QTVI) after administration of fresh pink grapefruit juice, placebo, amiodarone, or sotalol. After pink grapefruit juice and sotalol, all these indexes increased significantly from values observed after placebo (P<0.05) and from values after amiodarone (P<0.05). Conversely, after amiodarone, QT(c), but not QTVI, increased significantly from values after placebo (P<0.05). Presumably because of its high naringenin glycoside content, pink grapefruit juice prolongs cardiac repolarization and concurrently increases temporal cardiac repolarization dispersion. The potential proarrhythmic actions of pink grapefruit juice might be of concern in patients with major myocardial structural disorders.     

Neonatal Williams syndrome presenting as an isolated supravalvular pulmonary stenosis

An infant with normal facies and none of the extracardiac anomalies usually associated with Williams syndrome presented at birth with an echocardiographic pattern of supravalvular pulmonary stenosis and displastic pulmonary valve. A clinical reappraisal was planned at 3 months of age, but the girl died suddenly at home at 2 months of age. At autopsy, both ventricles were hypertrophic, and the valves showed mild dysplasia. The walls of the great arteries were thick, with a "washed leather" consistency, but there was no gross evidence of discrete stenosis. The histologic mosaic appearance of the media of the great arteries, due to elastosis and extreme disarray of the elastic lamellae, prompted a postmortem diagnosis of supravalvar aortic stenosis and suggested a diagnosis of Williams syndrome, which was subsequently confirmed by fluorescence in situ hybridization. Pediatricians and pathologists should be alerted that Williams syndrome in the newborn may present as an isolated supravalvular pulmonary stenosis, whereas supravalvular aortic stenosis becomes clinically significant only a few months later.     

Genetic variance contributes to ingestive processes: a survey of 2-deoxy-D-glucose-induced feeding in eleven inbred mouse strains

The feeding response following administration of the anti-metabolic glucose analogue, 2-deoxy-d-glucose (2DG), is conceptualized as an experimental model of glucoprivation, which may contribute to the understanding of inter-individual differences in glucose and carbohydrate intake and, ultimately, obesity. Although variation in the intake of several nutrients as well as food and water are known to be associated with genetic variation, it is not known whether 2DG-induced feeding is similarly genotype dependent. The present study therefore examined 2DG-induced feeding in mice of 11 inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL6/J, C57BL10/J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) strains across a wide range of previously determined effective 2-DG doses (200, 400, 600, 800 mg/kg) and test times (1-4 h). Orderly dose-dependent increases in 2DG-induced feeding occurred after all four doses in outbred CD-1 and inbred DBA/2J mice, across the three highest doses for BALB/cJ, SJL/J and 129P3/J mice, and across the two highest doses for CBA/J and AKR/J mice. Limited instances of 2DG-induced feeding were noted only at the highest dose in A/J and C3H/HeJ mice, or at a moderate dose in C57BL/6J mice. Further, the full 2DG dose range failed to alter food intake in C57BL/10J mice, and produced significant reductions in food intake in SWR/J mice. Food intake after 2DG doses of 200-600 mg/kg, but not 800 mg/kg, displayed significant cross-correlation, suggesting that large 2DG doses may recruit non-specific effects upon food intake. There was no correlation between food intake in the absence (vehicle baseline) of and presence of 2DG, suggesting that the regulation of glucose intake in non-challenged mice does not predict subsequent responses to glucoprivic challenge. The data demonstrate genotype-dependent variability in this glucoprivic response, and may provide the basis for the subsequent identification of trait-relevant genes.     

Relationships among porcine and human P[6] rotaviruses: evidence that the different human P[6] lineages have originated from multiple interspecies transmission events

Porcine rotavirus strains (PoRVs) bearing human-like VP4 P[6] gene alleles were identified. Genetic characterization with either PCR genotyping or sequence analysis allowed to determine the VP7 specificity of the PoRVs as G3, G4, G5 and G9, and the VP6 as genogroup I, that is predictive of a subgroup I specificity. Sequence analysis of the VP8* trypsin-cleavage product of VP4 allowed PoRVs to be characterized further into genetic lineages within the P[6] genotype. Unexpectedly, the strains displayed significantly higher similarity (up to 94.6% and 92.5% at aa and nt level, respectively) to human M37-like P[6] strains (lineage I), serologically classifiable as P2A, or to the atypical Hungarian P[6] human strains (HRVs), designated as lineage V (up to 97.0% aa and 96.1% nt), than to the porcine P[6] strain Gottfried, lineage II (<85.1% aa and 82.2 nt), which is serologically classified as P2B. Interestingly, no P[6] PoRV resembling the original prototype porcine strain, Gottfried, was detected, while Japanase P[6] PoRV clustered with the atypical Japanase G1 human strain AU19. By analysis of the 10th and 11th genome segments, all the strains revealed a NSP4B genogroup (Wa-like) and a NSP5/6 gene of porcine origin. These findings strongly suggest interspecies transmission of rotavirus strains and/or genes, and may indicate the occurrence of at least 3 separate rotavirus transmission events between pigs and humans, providing convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses.     

Frataxin, iron-sulfur clusters, heme, ROS, and aging

A deficiency in mitochondrial frataxin causes an increased generation of mitochondrial reactive oxygen species (ROS), which may contribute to the cell degenerative features of Friedreich's ataxia. In this work the authors demonstrate mitochondrial iron-sulfur cluster (ISC) defects and mitochondrial heme defects, and suggest how both may contribute to increased mitochondrial ROS in lymphoblasts from human patients. Mutant cells are deficient in the ISC-requiring mitochondrial enzymes aconitase and succinate dehydrogenase, but not in the non-ISC mitochondrial enzyme citrate synthase; also, the mitochondrial iron-sulfur scaffold protein IscU2 co-immunoprecipitates with frataxin in vivo. Presumably as a consequence of the iron-sulfur cluster defect, cytochrome c heme is deficient in mutants, as well as heme-dependent Complex IV. Mitochondrial superoxide is elevated in mutants, which may be a consequence of cytochrome c deficiency. Hydrogen peroxide, glutathione peroxidase activity, and oxidized glutathione (GSSG) are each elevated in mutants, consistent with activation of the glutathione peroxidase pathway. Mutant status blunted the effects of Complex III and IV inhibitors, but not a Complex I inhibitor, on superoxide production. This suggests that heme defects late in the electron transport chain of mutants are responsible for increased mutant superoxide. The impact of ISC and heme defects on ROS production with age are discussed.     

Outbreak of Saccharomyces cerevisiae subtype boulardii fungemia in patients neighboring those treated with a probiotic preparation of the organism

We report an outbreak of Saccharomyces cerevisiae subtype boulardii fungemia among three intensive care unit roommates of patients receiving lyophilized preparations of this fungus. The fungemia was probably due to central venous catheter contamination and resolved after fluconazole treatment. The need for stringent application of proper hygiene when using a probiotic preparation of this organism is emphasized.     

Intercellular adhesion molecule-1 K469E gene polymorphism and Alzheimer's disease

Inflammatory processes are considered important in the pathogenesis of Alzheimer's disease (AD). Intercellular adhesion molecule-1 (ICAM-1) is an important mediator of inflammatory response and immune cell activation, is expressed on cerebrovascular endothelium and neuritic plaques in brain of AD patients, and seems to be implicated in the process of neuro-degeneration. A common polymorphism of the ICAM-1 gene (K469E) has been recently reported. In this case-control study, we evaluated the distribution of E/K alleles and genotypes of the ICAM-1 gene in 98 patients affected by sporadic AD and 115 age- and sex-matched controls. The frequency of the EE genotype was significantly higher in AD patients (P<0.01). Logistic regression analysis indicated that the presence of EE genotype significantly increased the risk of AD (odds ratio 3.01 [1.1-8.0], P<0.05). This study shows for the first time an association between ICAM-1 E/K gene polymorphism and AD, suggesting that polymorphisms of the ICAM-1 gene may be clinically important and confirming that inflammatory mechanisms may be crucial in the pathophysiology of neuro-degenerative diseases.