The CD81 tetraspanin facilitates instantaneous leukocyte VLA-4 adhesion strengthening to vascular cell adhesion molecule 1 (VCAM-1) under shear flow

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.     

Deletion of the copper transporter CaCCC2 reveals two distinct pathways for iron acquisition in Candida albicans

Efficient iron acquisition is an essential requirement for growth of pathogenic organisms in the iron-poor host environment. In Saccharomyces cerevisiae, high-affinity iron import depends on the multicopper ferroxidase ScFet3. ScFet3 biogenesis in the trans-Golgi compartment requires a copper-transporting P-type ATPase, ScCcc2. Here, we describe the isolation by functional complementation of a Ccc2 homologue from the pathogenic yeast Candida albicans. CaCcc2 is functionally distinct from a previously described C. albicans copper-transporting P-type ATPase, CaCrp1, which appears to be specifically involved in copper detoxification. Regulation of CaCCC2 and the phenotype of the homozygous CaCCC2 deletion indicate that it is required for high-affinity iron import, making it the bona fide CCC2 homologue of C. albicans. Remarkably, in a mouse model of systemic infection, the Caccc2Delta strain displayed robust proliferation and no significant reduction in pathogenicity, suggesting the existence of alternative mechanisms of iron uptake from host tissues. We identify haemin and haemoglobin as potential iron sources that can be used by C. albicans in a CaCcc2-independent manner.     

Membrane binding and permeation by indolicidin analogs studied by a biomimetic lipid/polydiacetylene vesicle assay

Membrane binding and relative penetration of indolicidin analogs were studied using lipid/polydiacetylene (PDA) chromatic biomimetic membranes. Colorimetric and fluorescence analyses determined that an indolicidin analog with a proline and tryptophan residue substituted with lysines showed more pronounced bilayer surface interactions, while indolicidin and particularly an indolicidin analog in which all prolines were replaced with alanine residues exhibited deeper insertion into the lipid bilayer. The colorimetric data demonstrated that more pronounced blue-red transitions were observed when the chromatic vesicles incorporated lipopolysaccharide (LPS) within the lipid bilayer, indicating that LPS promoted preferred binding and incorporation of the peptides at the lipid/water interface. The fluorescence quenching experiments further confirmed this outcome. The results indicate that the antibacterial activity of indolicidin most likely requires initial binding to the LPS moieties within bacterial membranes, as well as disruption of the bilayer interface. The degree of hemolysis induced by the analogs, on the other hand, correlated to the extent of penetration into the hydrophobic core of the lipid assembly.     

Effect of hypothermia on brain multi-parametric activities in normoxic and partially ischemic rats

Hypothermia, as well as anesthesia, are known to protect the brain against ischemia, hypoxia and other pathological damages. One of the mechanisms of this improvement could be by lowering brain function, and thereby lowering oxygen demand. We examined the effect of hypothermia on brain function and blood supply in awake and anesthetized rats and studied the interaction between partial ischemia and the responses to hypothermia. The brain function multiprobe (BFM) used enabled simultaneous measurements of cerebral blood flow (CBF), mitochondrial NADH redox state, extracellular K(+) concentration, DC potential and ECoG from the cerebral cortex in rats whose brain temperature was lowered by 5 degrees C. Hypothermia was induced in awake, anesthetized and brain ischemic-anesthetized rats. In anesthetized and ischemic-anesthetized rats, the time required for lowering the brain temperature by 5 degrees C was five times less than in the normal awake animals. No significant changes in CBF and NADH levels were found in response to hypothermia in the awake animals. In contrast, a significant decrease in extracellular K(+) concentration was recorded under hypothermia, probably due to the lower rate of depolarization. Hypothermia in anesthetized and in ischemic-anesthetized rats did not significantly affect the levels of mitochondrial NADH, CBF and extracellular K(+). Hypothermia under ischemia was expected to be more effective.     

Cardiomyocytes generated from CPVTD307H patients are arrhythmogenic in response to β-adrenergic stimulation

Sudden cardiac death caused by ventricular arrhythmias is a disastrous event, especially when it occurs in young individuals. Among the five major arrhythmogenic disorders occurring in the absence of a structural heart disease is catecholaminergic polymorphic ventricular tachycardia (CPVT), which is a highly lethal form of inherited arrhythmias. Our study focuses on the autosomal recessive form of the disease caused by the missense mutation D307H in the cardiac calsequestrin gene, CASQ2. Because CASQ2 is a key player in excitation contraction coupling, the derangements in intracellular Ca(2+) handling may cause delayed afterdepolarizations (DADs), which constitute the mechanism underlying CPVT. To investigate catecholamine-induced arrhythmias in the CASQ2 mutated cells, we generated for the first time CPVT-derived induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts from skin biopsies of two patients, and demonstrated that the iPSCs carry the CASQ2 mutation. Next, iPSCs were differentiated to cardiomyocytes (iPSCs-CMs), which expressed the mutant CASQ2 protein. The major findings were that the β-adrenergic agonist isoproterenol caused in CPVT iPSCs-CMs (but not in the control cardiomyocytes) DADs, oscillatory arrhythmic prepotentials, after-contractions and diastolic [Ca(2+) ](i) rise. Electron microscopy analysis revealed that compared with control iPSCs-CMs, CPVT iPSCs-CMs displayed a more immature phenotype with less organized myofibrils, enlarged sarcoplasmic reticulum cisternae and reduced number of caveolae. In summary, our results demonstrate that the patient-specific mutated cardiomyocytes can be used to study the electrophysiological mechanisms underlying CPVT.     

Missense mutations in the APOL1 gene are highly associated with end stage kidney disease risk previously attributed to the MYH9 gene

MYH9 has been proposed as a major genetic risk locus for a spectrum of nondiabetic end stage kidney disease (ESKD). We use recently released sequences from the 1000 Genomes Project to identify two western African-specific missense mutations (S342G and I384M) in the neighboring APOL1 gene, and demonstrate that these are more strongly associated with ESKD than previously reported MYH9 variants. The APOL1 gene product, apolipoprotein L-1, has been studied for its roles in trypanosomal lysis, autophagic cell death, lipid metabolism, as well as vascular and other biological activities. We also show that the distribution of these newly identified APOL1 risk variants in African populations is consistent with the pattern of African ancestry ESKD risk previously attributed to MYH9. Mapping by admixture linkage disequilibrium (MALD) localized an interval on chromosome 22, in a region that includes the MYH9 gene, which was shown to contain African ancestry risk variants associated with certain forms of ESKD (Kao et al. 2008; Kopp et al. 2008). MYH9 encodes nonmuscle myosin heavy chain IIa, a major cytoskeletal nanomotor protein expressed in many cell types, including podocyte cells of the renal glomerulus. Moreover, 39 different coding region mutations in MYH9 have been identified in patients with a group of rare syndromes, collectively termed the Giant Platelet Syndromes, with clear autosomal dominant inheritance, and various clinical manifestations, sometimes also including glomerular pathology and chronic kidney disease (Kopp 2010; Sekine et al. 2010). Accordingly, MYH9 was further explored in these studies as the leading candidate gene responsible for the MALD signal. Dense mapping of MYH9 identified individual single nucleotide polymorphisms (SNPs) and sets of such SNPs grouped as haplotypes that were found to be highly associated with a large and important group of ESKD risk phenotypes, which as a consequence were designated as MYH9-associated nephropathies (Bostrom and Freedman 2010). These included HIV-associated nephropathy (HIVAN), primary nonmonogenic forms of focal segmental glomerulosclerosis, and hypertension affiliated chronic kidney disease not attributed to other etiologies (Bostrom and Freedman 2010). The MYH9 SNP and haplotype associations observed with these forms of ESKD yielded the largest odds ratios (OR) reported to date for the association of common variants with common disease risk (Winkler et al. 2010). Two specific MYH9 variants (rs5750250 of S-haplotype and rs11912763 of F-haplotype) were designated as most strongly predictive on the basis of Receiver Operating Characteristic analysis (Nelson et al. 2010). These MYH9 association studies were then also extended to earlier stage and related kidney disease phenotypes and to population groups with varying degrees of recent African ancestry admixture (Behar et al. 2010; Freedman et al. 2009a, b; Nelson et al. 2010), and led to the expectation of finding a functional African ancestry causative variant within MYH9. However, despite intensive efforts including re-sequencing of the MYH9 gene no suggested functional mutation has been identified (Nelson et al. 2010; Winkler et al. 2010). This led us to re-examine the interval surrounding MYH9 and to the detection of novel missense mutations with predicted functional effects in the neighboring APOL1 gene, which are significantly more associated with ESKD than all previously reported SNPs in MYH9.     

Unusual pathological condition in the lower extremities of a skeleton from ancient Israel.

We report here an unusual pathology in a 1,500-year-old skeleton recovered from Bet Guvrin, Israel. The pathological changes in the lower extremities and the diagnostic difficulties are presented. We attribute this condition to Madura foot, found primarily in tropical and subtropical regions, although other diagnoses are possible and are noted. We discuss its appearance in the region in light of the historical context.