Surfactant protein B: disulfide bridges, structural properties, and kringle similarities.

The disulfide bridges in porcine hydrophobic surfactant protein B (SP-B) were determined. Results show that three intrachain bridges link half-cystine residues 8 and 77, 11 and 71, and 35 and 46, respectively. This gives SP-B an appearance of three loops, a central big loop surrounded by two smaller ones. In the major form of SP-B, the remaining half-cystine, Cys-48, is probably interchain-linked to its counterpart in another molecule, compatible with the existence of dimeric molecules. A minor fraction, with monomeric SP-B but also lacking free thiols, could be due to polypeptides having Cys-57 (instead of Leu in the major form) and hence an additional intrachain bond (Cys-48-Cys-57). Notably, one of the three intrachain bonds common to all SP-B molecules is analogous to one of the disulfide linkages in the kringle structure of complex serine proteases. SP-B and kringles are also similar in size and in positions of half-cystine residues. SP-B and the kringle of coagulation factor XII exhibit 26% residue identity. This structural similarity of SP-B to a binding domain could reflect functional homology, compatible with the notion that SP-B interacts with surfactant anionic phospholipids, which is also in agreement with an SP-B excess of basic residues. Finally, weak similarities between the perform of SP-B and complex serine proteases are also found. This has implications on further possible relationships between kringles, serine proteases, and antiproteases.     

Reconstruction of the Evolutionary Dynamics of A(H3N2) Influenza Viruses Circulating in Italy from 2004 to 2012

Background

Influenza A viruses are characterised by their rapid evolution, and the appearance of point mutations in the viral hemagglutinin (HA) domain causes seasonal epidemics. The A(H3N2) virus has higher mutation rate than the A(H1N1) virus. The aim of this study was to reconstruct the evolutionary dynamics of the A(H3N2) viruses circulating in Italy between 2004 and 2012 in the light of the forces driving viral evolution.

Methods

Phylodinamic analyses were made using a Bayesian method, and codon-specific positive selection acting on the HA coding sequence was evaluated.

Results

Global and local phylogenetic analyses showed that the Italian strains collected between 2004 and 2012 grouped into five significant Italian clades that included viral sequences circulating in different epidemic seasons. The time of the most recent common ancestor (tMRCA) of the tree root was between May and December 2003. The tMRCA estimates of the major clades suggest that the origin of a new viral strain precedes the effective circulation of the strain in the Italian population by 6–31 months, thus supporting a central role of global migration in seeding the epidemics in Italy. The study of selection pressure showed that four codons were under positive selection, three of which were located in antigenic sites. Analysis of population dynamics showed the alternation of periods of exponential growth followed by a decrease in the effective number of infections corresponding to epidemic and inter-epidemic seasons.

Conclusions

Our analyses suggest that a complex interaction between the immune status of the population, migrations, and a few selective sweeps drive the influenza A(H3N2) virus evolution. Our findings suggest the possibility of the year-round survival of local strains even in temperate zones, a hypothesis that warrants further investigation.     

Effects of progressive and maximal exercise on plasma levels of adhesion molecules in athletes with sickle cell trait with or without alpha-thalassemia.

The aim of the study was to examine the effects of exercise on soluble vascular cell adhesion molecule-1 (sVCAM-1) and intercellular adhesion molecule-1 (sICAM-1) in sickle cell trait (SCT) athletes with or without alpha-thalassemia. Six athletes with SCT, seven athletes with both SCT and alpha-thalassemia (SCTAT), and seven control athletes (Cont) performed an incremental and maximal test on cycloergometer. Levels of sICAM-1 and sVCAM-1 were assessed at rest, immediately after the end of exercise, and 1, 2, and 24 h after exercise. Although Cont and SCTAT groups exhibited similar basal plasma levels of inflammatory and adhesion molecules, the SCT group had higher sVCAM-1 basal concentrations. Incremental exercise resulted in a significant increase of sVCAM-1 in all subjects, which remained elevated only in the SCT group during the recovery period. In conclusion, as sVCAM-1 increased with exercise and during the recovery period, our findings support the concept that SCT athletes might be at risk for microcirculatory disturbances and adhesive phenomena developing at rest and several hours after exercise. alpha-Thalassemia might be considered protective among exercising SCT subjects.     

Novel Rumen Bacterial Diversity in Two Geographically Separated Sub-Species of Reindeer

Svalbard reindeer (Rangifer tarandus platyrhynchus) live under austere nutritional conditions on the high-arctic archipelago of Svalbard, while semi-domesticated Norwegian reindeer (R. tarandus tarandus) migrate between lush coastal summer pastures and inland winter pastures with lichens on mainland Norway. Svalbard reindeer are known to have high rumen concentrations of cellulolytic bacteria, ranging from 15% of the viable population in summer to 35% in winter, compared to only 2.5% in Norwegian reindeer. Their rumen bacterial diversity was investigated through comparative analyses of 16S rRNA gene sequences (∼1.5 kb in length) generated from clone libraries (n = 121) and bacterial isolates (n = 51). LIBSHUFF comparisons of the composition of the two 16S rRNA libraries from Norwegian reindeer showed a significant effect of artificial feeding compared to natural pasture, but failed to yield significant differences between libraries from Norwegian reindeer and Svalbard reindeer. The combined sequences from reindeer were not significantly different from those reported in wild Thompson’s gazelle in Kenya but did differ from those reported in domestic cattle in Japan. A total of 90 distinct operational taxonomic units (OTUs) were identified by employing a criterion of 97% similarity, while the Chao1 index estimated the reindeer bacterial rumen population richness at 698 OTUs. The majority of the clone library sequences (92.5%) represented novel strains with <97% identity to any known sequence in the public database, most of them affiliated with the bacterial phylum Firmicutes (low G+C Gram-positives) related to the order Clostridiales (76.7%), while Gram-negative bacteria in the Bacteriodales (Prevotella–Bacteroides group) contributed to 22.5%. Also, six of the isolates were putatively novel strains, possibly representing new species in the Clostridium subphylum (cluster XIVa), Actinomyces and Butyrivibrio.     

In Central Obesity,Weight Loss Restores Platelet Sensitivity to Nitric Oxide and Prostacyclin

Central obesity shows impaired platelet responses to the antiaggregating effects of nitric oxide (NO), prostacyclin, and their effectors—guanosine 3′,5′‐cyclic monophosphate (cGMP) and adenosine 3′,5′‐cyclic monophosphate (cAMP). The influence of weight loss on these alterations is not known. To evaluate whether a diet‐induced body‐weight reduction restores platelet sensitivity to the physiological antiaggregating agents and reduces platelet activation in subjects affected by central obesity, we studied 20 centrally obese subjects before and after a 6‐month diet intervention aiming at reducing body weight by 10%, by measuring (i) insulin sensitivity (homeostasis model assessment of insulin resistance (HOMA_IR)); (ii) plasma lipids; (iii) circulating markers of inflammation of adipose tissue and endothelial dysfunction, and of platelet activation (i.e., soluble CD‐40 ligand (sCD‐40L) and soluble P‐selectin (sP‐selectin)); (iv) ability of the NO donor sodium nitroprusside (SNP), the prostacyclin analog Iloprost and the cyclic nucleotide analogs 8‐bromoguanosine 3′,5′‐cyclic monophosphate (8‐Br‐cGMP) and 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP) to reduce platelet aggregation in response to adenosine‐5‐diphosphate (ADP); and (v) ability of SNP and Iloprost to increase cGMP and cAMP. The 10 subjects who reached the body‐weight target showed significant reductions of insulin resistance, adipose tissue, endothelial dysfunction, and platelet activation, and a significant increase of the ability of SNP, Iloprost, 8‐Br‐cGMP, and 8‐Br‐cAMP to reduce ADP‐induced platelet aggregation and of the ability of SNP and Iloprost to increase cyclic nucleotide concentrations. No change was observed in the 10 subjects who did not reach the body‐weight target. Changes of platelet function correlated with changes of HOMA_IR. Thus, in central obesity, diet‐induced weight loss reduces platelet activation and restores the sensitivity to the physiological antiaggregating agents, with a correlation with improvements in insulin sensitivity.     

<Emphasis Type="Italic">Saturnispora serradocipensis</Emphasis> sp. nov. and <Emphasis Type="Italic">Saturnispora gosingensis</Emphasis> sp. nov., two ascomycetous yeasts from ephemeral habitats

Two novel ascomycetous yeast species, Saturnispora serradocipensis and Saturnispora gosingensis, were isolated from leaf detritus in a tropical stream of Southeastern Brazil and a mushroom collected in Taiwan, respectively. Analysis of the D1/D2 domains of the large-subunit of the rRNA gene of these strains showed that these species are related to Saturnispora hagleri, their closest relative. Saturnispora serradocipensis and S. gosingensis differed from S. hagleri, respectively, by seven nucleotide substitutions and two indels and three nucleotide substitutions and three indels in D1/D2 rRNA sequences. The two new species differ from each another by four nucleotide substitutions and one indel in D1/D2 rRNA sequences. However, the ITS sequences of S. serradocipensis, S. gosingensis and S. hagleri were quite divergent, showing that they are genetically separate species. The type strain of S. serradocipensis is UFMG-DC-198^T (=CBS 11756^T = NRRL Y-48717^T), and of S. gosingensis GA4M05^T is (CBS 11755^T = NRRL Y-48718^T).     

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimer's diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.     

Perturbation of cytochrome P450, generation of oxidative stress and induction of DNA damage in Cyprinus carpio exposed in situ to potable surface water

Epidemiological evidence suggests a link between consumption of chlorinated drinking water and various cancers. Chlorination of water rich in organic chemicals produces carcinogenic organochlorine by-products (OBPs) such as trihalomethanes and haloacetic acids. Since the discovery of the first OBP in the 1970s, there have been several investigations designed to determine the biological effects of single chemicals or small artificial OBP combinations. However, there is still insufficient information regarding the general biological response to these compounds, and further studies are still needed to evaluate their potential genotoxic effects. In the current study, we evaluated the effect of three drinking water disinfectants on the activity of cytochrome P450 (CYP)-linked metabolizing enzymes and on the generation of oxidative stress in the livers of male and female Cyprinus carpio fish (carp). The fish were exposed in situ for up 20 days to surface water obtained from the Trasmene lake in Italy. The water was treated with 1-2 mg/L of either sodium hypochlorite (NaClO) or chlorine dioxide (ClO2) as traditional disinfectants or with a relatively new disinfectant product, peracetic acid (PAA). Micronucleus (MN) frequencies in circulating erythrocytes from the fish were also analysed as a biomarker of genotoxic effect. In the CYP-linked enzyme assays, a significant induction (up to a 57-fold increase in the deethylation of ethoxyresorufin with PAA treatment) and a notable inactivation (up to almost a 90% loss in hydroxylation of p-nitrophenol with all disinfectants, and of testosterone 2beta-hydroxylation with NaClO) was observed in subcellular liver preparations from exposed fish. Using the electron paramagnetic resonance (EPR) spectroscopy radical-probe technique, we also observed that CYP-modulation was associated with the production of reactive oxygen species (ROS). In addition, we found a significant increase in MN frequency in circulating erythrocytes after 10 days of exposure of fish to water treated with ClO2, while a non-significant six-fold increase in MN frequency was observed with NaClO, but not with PAA. Our data suggest that the use of ClO2 and NaClO to disinfect drinking water could generate harmful OBP mixtures that are able to perturb CYP-mediated reactions, generate oxidative stress and induce genetic damage. These data may provide a mechanistic explanation for epidemiological studies linking consumption of chlorinated drinking water to increased risk of urinary, gastrointestinal and bladder cancers.     

Inhibition of HIV-1 Infection by Human α-Defensin-5, a Natural Antimicrobial Peptide Expressed in the Genital and Intestinal Mucosae

Background

α-defensin-5 (HD5) is a key effector of the innate immune system with broad anti-bacterial and anti-viral activities. Specialized epithelial cells secrete HD5 in the genital and gastrointestinal mucosae, two anatomical sites that are critically involved in HIV-1 transmission and pathogenesis. We previously found that human neutrophil defensins (HNP)-1 and -2 inhibit HIV-1 entry by specific bilateral interaction both with the viral envelope and with its primary cellular receptor, CD4. Despite low amino acid identity, human defensin-5 (HD5) shares with HNPs a high degree of structural homology.

Methodology/Principal Findings

Here, we demonstrate that HD5 inhibits HIV-1 infection of primary CD4⁺ T lymphocytes at low micromolar concentration under serum-free and low-ionic-strength conditions similar to those occurring in mucosal fluids. Blockade of HIV-1 infection was observed with both primary and laboratory-adapted strains and was independent of the viral coreceptor-usage phenotype. Similar to HNPs, HD5 inhibits HIV-1 entry into the target cell by interfering with the reciprocal interaction between the external envelope glycoprotein, gp120, and CD4. At high concentrations, HD5 was also found to downmodulate expression of the CXCR4 coreceptor, but not of CCR5. Consistent with its broad spectrum of activity, antibody competition studies showed that HD5 binds to a region overlapping with the CD4- and coreceptor-binding sites of gp120, but not to the V3 loop region, which contains the major determinants of coreceptor-usage specificity.

Conclusion/Significance

These findings provide new insights into the first line of immune defense against HIV-1 at the mucosal level and open new perspectives for the development of preventive and therapeutic strategies.     

Effects of Clostridium perfringens phospholipase C in mammalian cells

Clostridium perfringens phospholipase C (Cp-PLC), the major virulence factor in the pathogenesis of gas gangrene, is a Zn(2+) metalloenzyme with lecithinase and sphingomyelinase activities. Its structure shows an N-terminal domain containing the active site, and a C-terminal Ca(2+) binding domain required for membrane interaction. Although the knowledge of the structure of Cp-PLC and its interaction with aggregated phospholipids has advanced significantly, an understanding of the effects of Cp-PLC in mammalian cells is still incomplete. Cp-PLC binds to artificial bilayers containing cholesterol and sphingomyelin or phosphatidylcholine (PC) and degrades them, but glycoconjugates present in biological membranes influence its binding or positioning toward its substrates. Studies with Cp-PLC variants harboring single amino-acid substitutions have revealed that the active site, the Ca(2+) binding region, and the membrane interacting surface are required for cytotoxic and haemolytic activity. Cp-PLC causes plasma membrane disruption at high concentrations, whereas at low concentrations it perturbs phospholipid metabolism, induces DAG generation, PKC activation, Ca(2+) mobilization, and activates arachidonic acid metabolism. The cellular susceptibility to Cp-PLC depends on the composition of the plasma membrane and the capacity to up-regulate PC synthesis. The composition of the plasma membrane determines whether Cp-PLC can bind and acquire its active conformation, and thus the extent of phospholipid degradation. The capacity of PC synthesis and the availability of precursors determine whether the cell can replace the degraded phospholipids. Whether the perturbations of signal transduction processes caused by Cp-PLC play a role in cytotoxicity is not clear. However, these perturbations in endothelial cells, platelets and neutrophils lead to the uncontrolled production of intercellular mediators and adhesion molecules, which inhibits bacterial clearance and induces thrombotic events, thus favouring bacterial growth and spread in the host tissues.