Complotypes in individuals of African origin: frequencies and possible extended MHC haplotypes

We analyzed the frequency distribution of 106 complotypes [four allele sets of the major histocompatibility complex (MHC) genes for the complement proteins factor B, C2, C4A, and C4B] from 32 Black families residing in Boston and Washington, DC. Twenty-five different complotypes were identified, among which there were four complotypes that had not been previously observed in our large database of complotypes compiled from family studies of Boston Caucasians and that are, presumably, unique to individuals of African origin. These four African-derived complotypes areFC(1,90)0, FC63, S1C2,17, andSC(3,2,90)0. The frequencies of two of these four unique Black complotypes,FC(1,90)0 andFC63, were increased significantly when compared to Caucasians (p_corr <0.00042, p_corr=0.00294, respectively). The complotypeFC(1,90)0 was in positive linkage disequilibrium withHLA-DR3 haplotypes containing theB locus antigens Bw42, Bw52, Bw53, and Bw58, whileFC63 was associated withHLA-Bw70,-DR5. These findings demonstrate the extensive polymorphism of complotypes in Blacks, and also suggest that it may be possible to define unique extended haplotypes of African origin.     

Loss-of-function and gain-of-function phenotypes of stomatocytosis mutant RhAG F65S

Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.     

Protection of Normal, Lysogenic, and Pyocinogenic Strains Against Ultraviolet Radiation by Bound Acriflavine

The presence of bound acriflavine protects bacteria against the lethal effects of ultraviolet (UV) light, presumably because pyrimidine dimer formation is inhibited. Although acriflavine present in plating medium usually results in reduced viable counts from irradiated bacteria, no enhancement of lethal effects is observed when acriflavine is added to irradiated bacteria left in suspending buffer for 45 min before plating. Acriflavine remaining bound to the deoxyribonucleic acid of irradiated bacteria at the time they are plated likewise does not affect their survival. Protection is precisely dose-modifying unless some killing of bacteria by UV results from induction of prophage, against which bound acriflavine is less protective, or from induction of pyocin, against which there is no protection at all. It is inferred that prophage induction proceeds in part, and pyocin induction wholly, by virtue of effects of UV other than pyrimidine dimerization. The response of Escherichia coli strain B to radiation has been postulated to be attributable in part to induction of a prophage or a lethal protein; but exact dose modification was observed for this strain, to about the same extent, whether or not the irradiated organisms were grown in conditions thought to enhance the expected contribution to killing if such a mechanism were involved. Our results support the hypothesis that the inhibition by acriflavine of dimer formation is attributable to energy transfer mechanisms. They fail to support the hypothesis that shapes of survival curves (in particular the manifestation of "shoulders") can be attributed to inactivation by radiation of repair enzymes.     

Hemodynamic changes in the kidney in a pediatric rat model of sepsis-induced acute kidney injury

Sepsis is a leading cause of acute kidney injury (AKI) and mortality in children. Understanding the development of pediatric sepsis and its effects on the kidney are critical in uncovering new therapies. The goal of this study was to characterize the development of sepsis-induced AKI in the clinically relevant cecal ligation and puncture (CLP) model of peritonitis in rat pups 17-18 days old. CLP produced severe sepsis demonstrated by time-dependent increase in serum cytokines, NO, markers of multiorgan injury, and renal microcirculatory hypoperfusion. Although blood pressure and heart rate remained unchanged after CLP, renal blood flow (RBF) was decreased 61% by 6 h. Renal microcirculatory analysis showed the number of continuously flowing cortical capillaries decreased significantly from 69 to 48% by 6 h with a 66% decrease in red blood cell velocity and a 57% decline in volumetric flow. The progression of renal microcirculatory hypoperfusion was associated with peritubular capillary leakage and reactive nitrogen species generation. Sham adults had higher mean arterial pressure (118 vs. 69 mmHg), RBF (4.2 vs. 1.1 ml·min(-1)·g(-1)), and peritubular capillary velocity (78% continuous flowing capillaries vs. 69%) compared with pups. CLP produced a greater decrease in renal microcirculation in pups, supporting the notion that adult models may not be the most appropriate for studying pediatric sepsis-induced AKI. Lower RBF and reduced peritubular capillary perfusion in the pup suggest the pediatric kidney may be more susceptible to AKI than would be predicted using adults models.     

Ribosomal DNA polymorphisms in the yeast Geotrichum candidum

The dimorphic yeast Geotrichum candidum (teleomorph: Galactomyces candidus) is commonly used to inoculate washed-rind and bloomy-rind cheeses. However, little is known about the phylogenetic lineage of this microorganism. We have sequenced the complete 18S, 5.8S, 26S ribosomal RNA genes and their internal transcribed spacers (ITS1) and ITS2 regions (5126 nucleotides) from 18 G. candidum strains from various environmental niches, with a focus on dairy strains. Multiple sequence alignments revealed the presence of 60 polymorphic sites, which is generally unusual for ribosomal DNA (rDNA) within a given species because of the concerted evolution mechanism. This mechanism drives genetic homogenization to prevent the divergent evolution of rDNA copies within individuals. While the polymorphisms observed were mainly substitutions, one insertion/deletion (indel) polymorphism was detected in ITS1. No polymorphic sites were detected downstream from this indel site, that is, in 5.8S and ITS2. More surprisingly, many sequence electrophoregrams generated during the sequencing of the rDNA had dual peaks, suggesting that many individuals exhibited intragenomic rDNA variability. The ITS1-5.8S-ITS2 regions of four strains were cloned. The sequence analysis of 68 clones revealed 32 different ITS1-5.8S-ITS2 variants within these four strains. Depending on the strain, from four to twelve variants were detected, indicating that multiple rDNA copies were present in the genomes of these G. candidum strains. These results contribute to the debate concerning the use of the ITS region for barcoding fungi and suggest that community profiling techniques based on rDNA should be used with caution.     

Synthesis and evaluation of arylaminoethyl amides as noncovalent inhibitors of cathepsin S. Part 3: heterocyclic P3

A series of N(alpha)-2-benzoxazolyl-alpha-amino acid-(arylaminoethyl)amides were identified as potent, selective, and noncovalent inhibitors of cathepsin S. Structure-activity relationships including strategies for modulating the selectivities among cathepsins S, K, and L, and in vivo pharmacokinetics are discussed. A X-ray structure of compound 3 bound to the active site of cathepsin S is also reported.     

Genetic polymorphism of the sixth component (C6) of rat complement

The complement protein C6 has been shown to be genetically polymorphic in the rat. Isoelectric focusing of plasma samples from 19 inbred strains demonstrated two electrophoretically distinguishable migration patterns, each consisting of three bands. Breeding studies with the use of the BN and DA strains showed that the C6 patterns were inherited in a manner consistent with the co-dominant autosomal expression of two alleles (C6 A and C6 B). The distribution of the C6 alleles in a backcross mating was compared with eight independently segregating marker genes: RT1.A, RT2, Gdc -1, Igk-1, Hbb, Svp-1, Fh-1, and Es-6. There was no detectable linkage between C6 and any of these eight loci.     

Alkylation damage causes MMR-dependent chromosomal instability in vertebrate embryos

S(N)1-type alkylating agents, like N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU), are potent mutagens. Exposure to alkylating agents gives rise to O(6)-alkylguanine, a modified base that is recognized by DNA mismatch repair (MMR) proteins but is not repairable, resulting in replication fork stalling and cell death. We used a somatic mutation detection assay to study the in vivo effects of alkylation damage on lethality and mutation frequency in developing zebrafish embryos. Consistent with the damage-sensing role of the MMR system, mutant embryos lacking the MMR enzyme MSH6 displayed lower lethality than wild-type embryos after exposure to ENU and MNU. In line with this, alkylation-induced somatic mutation frequencies were found to be higher in wild-type embryos than in the msh6 loss-of-function mutants. These mutations were found to be chromosomal aberrations that may be caused by chromosomal breaks that arise from stalled replication forks. As these chromosomal breaks arise at replication, they are not expected to be repaired by non-homologous end joining. Indeed, Ku70 loss-of-function mutants were found to be equally sensitive to ENU as wild-type embryos. Taken together, our results suggest that in vivo alkylation damage results in chromosomal instability and cell death due to aberrantly processed MMR-induced stalled replication forks.     

Immunolocalization of AE2 anion exchanger in rat and mouse epididymis.

A low-bicarbonate concentration and an acidic pH in the luminal fluid of the epididymis and vas deferens are important for sperm maturation. These factors help maintain mature sperm in an immotile but viable state during storage in the cauda epididymidis and vas deferens. Two proton extrusion mechanisms, an Na(+)/H(+) exchanger and an H(+)ATPase, have been proposed to be involved in this luminal acidification process. The Na(+)/H(+) exchanger has not yet been localized in situ, but we have reported that H(+)ATPase is expressed on the apical membrane of apical (or narrow) and clear cells of the epididymis. These cells are enriched in carbonic anhydrase II, indicating the involvement of bicarbonate in the acidification process and suggesting that the epididymis is a site of bicarbonate reabsorption. Previous unsuccessful attempts to localize the Cl/HCO(3) anion exchanger AE1 in rat epididymis did not investigate other anion exchanger (AE) isoforms. In this report, we used a recently described SDS antigen unmasking treatment to localize the Cl/HCO(3) exchanger AE2 in rat and mouse epididymis. AE2 is highly expressed in the initial segment, intermediate zone, and caput epididymidis, where it is located on the basolateral membrane of epithelial cells. The cauda epididymidis and vas deferens also contain basolateral AE2, but in lower amounts. The identity of the AE2 protein was further confirmed by the observation that basolateral AE2 expression was unaltered in the epididymis of AE1-knockout mice. Basolateral AE2 may participate in bicarbonate reabsorption and luminal acidification, and/or may be involved in intracellular pH homeostasis of epithelial cells of the male reproductive tract.     

Prazosin unmasks a renin response to intravenous para-chloroamphetamine

When injected intraperitoneally, p-chloroamphetamine (PCA) causes the acute release of catecholamines and serotonin, increases mean arterial pressure (MAP) and increases plasma renin activity (PRA) in rats. Experiments were designed to determine the dose-response and time-course for the effect of PCA administered intravenously on PRA in conscious, unrestrained rats. It was found initially that intravenous doses of PCA ranging from 0.3 - 6.0 mg/kg caused rapid and marked hypertension, but produced variable effects on PRA for up to 30 minutes after injection. In a second study PCA (0.3 - 6.0 mg/kg) did not alter PRA at 30 or 60 minutes after intravenous injection, but did increase PRA 60 minutes after 10 mg/kg, intraperitoneally. When the hypertension elicited by intravenous PCA was abolished by pretreatment with the alpha 1-adrenoceptor antagonist prazosin (100 micrograms/kg, iv), PCA produced marked elevations in PRA from 15 - 60 minutes. Thus it appeared that the renin response to intravenous PCA was masked by an elevation in MAP; when the vascular response to PCA was blocked, a large increase in PRA was observed.