Pulsed nuclear magnetic resonance study of39K within Halobacteria

Summary The³⁹K contents of isolated pellets and supernatant solutions from suspensions ofHalobacterium halobium were studied at 21–22°C by pulsed NMR spectroscopy. The rates of transverse relaxation were measured directly from the free induction decay (FID). The rate of longitudinal relaxation was measured by studying the FID after pairs of pulses of approximately 90°. Care was exercised to minimize the effect of magnetic field inhomogeneity; its contribution to the FID was approximately 25–30 sec^–1. The transverse relaxation process was found to consist of at least two components, whose rates were 321–449 sec^–1 and 1,122–2,067 sec^–1. In one preparation where the longitudinal relaxation process was studied, the data could be well fit to a single exponential relaxing at 253±33 (mean ±95% confidence limits) sec^–1. Comparison of the relative intensities of the NMR signals with the results of atomic absorption photometric analyses indicated that the great bulk of the intracellular³⁹K was detected by the NMR techniques used. The data obtained from the current NMR ofH. halobium are consistent with: (1) fractional binding of <3% of the total intracellular K⁺, (2) a small ordering factor characterizing all of the intracellular K⁺, or (3) some combination of the two.     

A cluster pattern algorithm for the analysis of multiparametric cell assays.

The issue of multiparametric analysis of complex single cell assays of both static and flow cytometry (SC and FC, respectively) has become common in recent years. In such assays, the analysis of changes, applying common statistical parameters and tests, often fails to detect significant differences between the investigated samples. The cluster pattern similarity (CPS) measure between two sets of gated clusters is based on computing the difference between their density distribution functions' set points. The CPS was applied for the discrimination between two observations in a four-dimensional parameter space. The similarity coefficient (r) ranges between 0 (perfect similarity) to 1 (dissimilar). Three CPS validation tests were carried out: on the same stock samples of fluorescent beads, yielding very low r's (0, 0.066); and on two cell models: mitogenic stimulation of peripheral blood mononuclear cells (PBMC), and apoptosis induction in Jurkat T cell line by H2O2. In both latter cases, r indicated similarity (r < 0.23) within the same group, and dissimilarity (r > 0.48) otherwise. This classification and algorithm approach offers a measure of similarity between samples. It relies on the multidimensional pattern of the sample parameters. The algorithm compensates for environmental drifts in this apparatus and assay; it also may be applied to more than four dimensions.     

Protective autoimmunity: interferon-gamma enables microglia to remove glutamate without evoking inflammatory mediators

Glutamate in excessive amounts is a major contributor to neuronal degeneration, and its removal is attributed mainly to astrocytes. Traumatic injury to the central nervous system (CNS) is often accompanied by disappearance of astrocytes from the lesion site and failure of the remaining cells to withstand the ensuing toxicity. Microglia that repopulate the lesion site are the usual suspects for causing redox imbalance and inflammation and thus further exacerbating the neurotoxicity. However, our group recently demonstrated that early post-injury activation of microglia as antigen-presenting cells correlates with an ability to withstand injurious conditions. Moreover, we found that T cells reactive to CNS-specific self-antigens protected neurons against glutamate toxicity. Here, we show that antigen-specific autoimmune T cells, by tailoring the microglial phenotype, can increase the ability of microglia-enriched cultures to remove glutamate. This T-cell-mediated effect could not be achieved by the potent microglia-activating agent lipopolysaccharide (LPS), but was dose-dependently reproduced by the Th1 cytokine interferon (IFN)-gamma and significantly reduced by neutralizing anti-IFN-gamma antibodies. Under the same conditions, IFN-gamma had no effect on cultured astrocytes. Up-regulation of glutamate uptake induced by IFN-gamma activation was not accompanied by the acute inflammatory response seen in LPS-activated cultures. These findings suggest that T cells or their cytokines can cause microglia to adopt a phenotype that facilitates rather than impairs glutamate clearance, possibly contributing to restoration of homeostasis.     

Cellular Changes during Renal Failure-Induced Inflammatory Aortic Valve Disease

Background

Aortic valve calcification (AVC) secondary to renal failure (RF) is an inflammation-regulated process, but its pathogenesis remains unknown. We sought to assess the cellular processes that are involved in the early phases of aortic valve disease using a unique animal model of RF-associated AVC.

Methods

Aortic valves were obtained from rats that were fed a uremia-inducing diet exclusively for 2, 3, 4, 5, and 6 weeks as well as from controls. Pathological examination of the valves included histological characterization, von Kossa staining, and antigen expression analyses.

Results

After 2 weeks, we noted a significant increase in urea and creatinine levels, reflecting RF. RF parameters exacerbated until the Week 5 and plateaued. Whereas no histological changes or calcification was observed in the valves of any study group, macrophage accumulation became apparent as early as 2 weeks after the diet was started and rose after 3 weeks. By western blot, osteoblast markers were expressed after 2 weeks on the diet and decreased after 6 weeks. Collagen 3 was up-regulated after 3 weeks, plateauing at 4 weeks, whereas collagen 1 levels peaked at 2 and 4 weeks. Fibronectin levels increased gradually until Week 5 and decreased at 6 weeks. We observed early activation of the ERK pathway, whereas other pathways remained unchanged.

Conclusions

We concluded that RF induces dramatic changes at the cellular level, including macrophage accumulation, activation of cell signaling pathway and extracellular matrix modification. These changes precede valve calcification and may increase propensity for calcification, and have to be investigated further.     

Clinical Escherichia coli isolates utilize alpha-hemolysin to inhibit in vitro epithelial cytokine production

Uropathogenic Escherichia coli is the primary cause of urinary tract infections, which affects over 60% of women during their lifetime. UPEC exhibits a number of virulence traits that facilitate colonization of the bladder, including inhibition of cytokine production by bladder epithelial cells. The goal of this study was to identify the mechanism of this inhibition. We observed that cytokine suppression was associated with rapid cytotoxicity toward epithelial cells. We found that cytotoxicity, cytokine suppression and alpha-hemolysin production were all tightly linked in clinical isolates. We screened a UPEC fosmid library and identified clones that gained the cytotoxicity and cytokine-suppression phenotypes. Both clones contained fosmids encoding a PAI II(J96)-like domain and expressed the alpha-hemolysin (hlyA) encoded therein. Mutation of the fosmid-encoded hly operon abolished cytotoxicity and cytokine suppression. Similarly, mutation of the chromosomal hlyCABD operon of UPEC isolate F11 also abolished these phenotypes, and they could be restored by introducing the PAI II(J96)-like domain-encoding fosmid. We also examined the role of alpha-hemolysin in cytokine production both in the murine UTI model as well as patient specimens. We conclude that E. coli utilizes alpha-hemolysin to inhibit epithelial cytokine production in vitro. Its contribution to inflammation during infection requires further study.     

Infantile cerebellar-retinal degeneration associated with a mutation in mitochondrial aconitase, ACO2

Degeneration of the cerebrum, cerebellum, and retina in infancy is part of the clinical spectrum of lysosomal storage disorders, mitochondrial respiratory chain defects, carbohydrate glycosylation defects, and infantile neuroaxonal dystrophy. We studied eight individuals from two unrelated families who presented at 2-6 months of age with truncal hypotonia and athetosis, seizure disorder, and ophthalmologic abnormalities. Their course was characterized by failure to acquire developmental milestones and culminated in profound psychomotor retardation and progressive visual loss, including optic nerve and retinal atrophy. Despite their debilitating state, the disease was compatible with survival of up to 18 years. Laboratory investigations were normal, but the oxidation of glutamate by muscle mitochondria was slightly reduced. Serial brain MRI displayed progressive, prominent cerebellar atrophy accompanied by thinning of the corpus callosum, dysmyelination, and frontal and temporal cortical atrophy. Homozygosity mapping followed by whole-exome sequencing disclosed a Ser112Arg mutation in ACO2, encoding mitochondrial aconitase, a component of the Krebs cycle. Specific aconitase activity in the individuals' lymphoblasts was severely reduced. Under restrictive conditions, the mutant human ACO2 failed to complement a yeast ACO1 deletion strain, whereas the wild-type human ACO2 succeeded, indicating that this mutation is pathogenic. Thus, a defect in mitochondrial aconitase is associated with an infantile neurodegenerative disorder affecting mainly the cerebellum and retina. In the absence of noninvasive biomarkers, determination of the ACO2 sequence or of aconitase activity in lymphoblasts are warranted in similarly affected individuals, based on clinical and neuroradiologic grounds.     

Familial mediterranean fever (FMF) in Moroccan Jews: Demonstration of a founder effect by extended haplotype analysis

Familial Mediterranean fever (FMF) is an autosomal recessive disease causing attacks of fever and serositis. The FMF gene (designated “MEF”) is on 16p, with the gene order 16cen–D16S80–MEF–D16S94–D16S283–D16S291–16pter. Here we report the association of FMF susceptibility with alleles at D16S94, D16S283, and D16S291 among 31 non-Ashkenazi Jewish families (14 Moroccan, 17 non-Moroccan). We observed highly significant associations at D16S283 and D16S291 among the Moroccan families. For the non-Moroccans, only the allelic association at D16S94 approached statistical significance. Haplotype analysis showed that 18/25 Moroccan FMF chromosomes, versus 0/21 noncarrier chromosomes, bore a specific haplotype for D16S94–D16S283–D16S291. Among non-Moroccans this haplotype was present in 6/26 FMF chromosomes versus 1/28 controls. Both groups of families are largely descended from Jews who fled the Spanish Inquisition. The strong haplotype association seen among the Moroccans is most likely a founder effect, given the recent origin and genetic isolation of the Moroccan Jewish community. The lower haplotype frequency among non-Moroccan carriers may reflect differences both in history and in population genetics.