Fucosylated protein of retinal cone photoreceptor outer segments: morphological and biochemical analyses

Cone outer segments (OS) of the goldfish retina are diffusely labeled after intravitreal injection of [(3)H]fucose while rod OS remain unlabeled. By electron microscopic radioautography, the OS of red- and blue-sensitive cones are heavily labeled while green- sensitive cone OS are lightly labeled. The time-course and pattern of OS labeling in all cone types from 30 min to 24 h resemble that of incorporation of other sugars into rhodopsin in rod OS. The nature of the cone OS-specific fucosylated component(s) was examined using biochemical techniques. Cone OS were prelabeled by intravitreal injection of [(3)H]fucose 24 h before sacrifice. Photoreceptor OS were isolated using a discontinuous sucrose density gradient and it was verified by electron microscopic radioautography that the only source of radioactivity in the preparations was cone OS. The different cone types could be recognized by the heaviness of labeling, characteristic membrane spacing, and 'staining' of green cone OS in vitro with horseradish peroxidase. After acid hydrolysis of prelabeled photoreceptor membranes, 90 percent of the counts were in the neutral sugar fraction which was analyzed by thin-layer chromatography. Approximately 70 percent of the radioactivity co-chromatographed with authentic fucose. SDS-PAGE/fluorography of prelabeled photoreceptor membranes revealed a single radioactive component that was lightly stained with coomassie blue and showed an apparent molecular weight of 33,000. This cone-derived band was separated from unlabeled rod opsin which was well stained and showed an apparent mol wt of 38,000. Isoelectric focusing under denaturing conditions produced two major and one minor band of radioactivity with isoelectric points of 8.2, 8.6, and 8.8 respectively. No radioactivity was found in association with a stained band corresponding in isoelectric point to that of bovine opsin (pl, 6.2). The fucosylated component was readily digested by pronase, indicating its protein nature. Washing of the isolated OS with isotonic and hypotonic buffers failed to extract major amounts of the radioactivity, suggesting that the fucosylated component is an integral membrane protein. The presence of a fucosylated protein thus represents a major difference between cone and rod OS in the goldfish and has enabled us to identify cone OS in preparations of isolated photoreceptor membranes and to demonstrate the separation of a cone-derived glycoprotein from rod opsin.     

First record of a spiral form of Aulacoseira,A. ambigua f. japonica (F.Meister) Tuji & D.M.Williams,in South African fresh waters

Water quality and algal populations in the Vaal River Barrage Reservoir have been monitored extensively for many decades, because of its importance as a water source for the most densely populated area in South Africa. Although Aulacoseira granulata (Ehrenberg) Simonsen is frequently found and at it times dominates algal assemblages in the Barrage, Aulacoseira ambigua (F.Meister) Tuji & D.M.Williams has never before been recorded at this locality. During a countrywide proficiency-testing scheme for algae counting, coordinated by Rand Water, spiral-shaped diatom colonies with distinctly curved cells were detected during May 2015. Upon investigation, it was found that the spiral colonies were Aulacoseira ambigua f. japonica, its presence in the fresh waters of South Africa being recorded for the first time. Since their first appearance during May 2015, colonies of Aulacoseira ambigua f. japonica have been constantly present in the surface waters of the Barrage. Their presence at this site can be linked to increasing eutrophication in the Vaal River, because the spiral form is known to prefer eutrophic conditions. Centric diatoms rarely form spiral colonies and there is some confusion in the literature as to the identity of this particular form, therefore in this paper we discuss this taxon, its nomenclature and ecological significance.     

An optimized microarray platform for assaying genomic variation in <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> field populations

We present an optimized probe design for copy number variation (CNV) and SNP genotyping in the Plasmodium falciparum genome. We demonstrate that variable length and isothermal probes are superior to static length probes. We show that sample preparation and hybridization conditions mitigate the effects of host DNA contamination in field samples. The microarray and workflow presented can be used to identify CNVs and SNPs with 95% accuracy in a single hybridization, in field samples containing up to 92% human DNA contamination.     

Role of HLA DRB1*15 and HLA DRB1*16alleles in the genetic susceptibility to develop systemic lupus erythematosus (SLE) after Chikungunya and Zika viruses infection in México

Systemic lupus erythematosus (SLE) is a clinically and genetically heterogeneous disease particularly prevalent in Mexico. Althoughits etiology is unknown, genetic factors strongly influence its presenceas well as triggering factors, such as viral infections, including Cytomegalovirus and Epstein-Barr virus. Here,the study presents the appearance of de novoSLE (patients who did not present SLE before de virus infection, corroborated by serological analysis and negative for antinuclear antibodies) cases in Mexicans who live near the southern border of Mexico, who presented clinical symptoms of arthritic, hematological, mucocutaneous and renal SLE, after Zika and/ or Chikungunya virus infection. Low resolution class Ⅱ HLA typing was performed, which found a significantly increased frequency of HLA DRB1*02 (15 and 16)when compared to a group of 99 healthy individuals (P =0.001, OR=4.5, IC95% 1.8~11.0). All the patients were diagnosed with SLE 1 to 3 years after being confirmed with the Zika, and/or Chikungunya infection. At the point of acute viral infection, none of the patients presented clinical signs or symptoms of autoimmunity or were negative for antinuclear antibodies. In genetically susceptible individuals, Zika and Chikungunya viral infection can trigger SLE.     

Aerobic fitness and performance in elite female futsal players

Despite its growing popularity, few studies have investigated specific physiological demands for elite female futsal. The aim of this study was to determine aerobic fitness in elite female futsal players using laboratory and field testing. Fourteen female futsal players from the Venezuelan National team (age =21.2±4.0 years; body mass =58.6±5.6 kg; height =161±5.0 cm) performed a progressive maximal treadmill test under laboratory conditions. Players also performed a progressive intermittent futsal-specific field test for endurance, the Futsal Intermittent Endurance Test (FIET), until volitional fatigue. Outcome variables were exercise heart rate (HR), VO₂, post-exercise blood lactate concentrations ([La]b) and running speeds (km · h^-1). During the treadmill test, VO₂max, maximal aerobic speed (MAS), HR and peak [La]b were 45.3±5.6 ml · kg^-1 · min^-1, 12.5±1.77 km · h^-1, 197±8 beats · min^-1 and 11.3±1.4 mmol · l^-1, respectively. The FIET total distance, peak running velocity, peak HR and [La]b were 1125.0±121.0 m, 15.2±0.5 km · h^-1, 199±8 beats · min^-1 and 12.5±2.2 mmol · l^-1, respectively. The FIET distance and peak speed were strongly associated (r= 0.85-87, p < 0.0001) with VO₂max and MAS, respectively. Peak HR and [La]b were not significantly different between tests. Elite female futsal players possess moderate aerobic fitness. Furthermore, the FIET can be considered as a valid field test to determine aerobic fitness in elite level female futsal players.     

Loss of phylogenetic information in chorion gene families of Bombyx mori gene conversion

The silkmoth chorion has provided a stimulating model for the study of evolution and developmental regulation of gene families. Previous attempts at inferring relationships among chorion sequences have been based on pairwise comparisons of overall similarity, a potentially problematic approach. To remedy this, we identified the alignable regions of low sequence variability and then analyzed this restricted database by parsimony and neighbor-joining methods. At the deepest level, the chorion sequence tree is split into two branches, called "alpha" and "beta." Within each branch, early- and late-expressing genes each constitute monophyletic groups, while the situation with middle-expressing genes remains uncertain. The HcB gene family appears to be the most basal beta-branch group, but this conclusion is qualified because the effect of gene conversion on branching order is unknown. Previous studies by Eickbush and colleagues have strongly suggested that ErA, HcA, and HcB families undergo gene conversion within a gene family, whereas the ErB family does not. The occurrence of conversion correlates with a particular tree structure; namely, branch lengths are much greater at the base of the family than at higher internodes and terminal branches. These observations raise the possibility that chorion gene families are defined by gene conversion events (reticulate evolution) rather than by descent with modification (synapomorphy).      

An Investigation of the Ability of the Glutaraldehyde Test to Distinguish between Acute and Chronic Inflammatory Disease in Horses

The Glutaraldehyde test (GT), a rapid and inexpensive test, has been utilized empirically for many years in bovine practice for diagnosing inflammatory diseases. GT is used primarily to demonstrate increased serum concentrations of fibrinogen and globulin. Glutaraldehyde binds with free amino groups in fibrinogen and immunoglobulin to create a clot in a first degree chemical reaction. The clotting time of the GT estimates the content of proteins produced in response to inflammation. The applicability of GT for diagnosing inflammation in the horse has never been investigated. The objective of this study was to determine the ability of GT to distinguish between acute and chronic inflammatory disease in horses. Thirty-seven horses with suspected inflammatory diseases were evaluated using the GT, history, complete clinical examination and routine blood analysis. GT-times, laboratory results and clinical outcome were compared statistically. Horses that were determined to be acutely affected (based on history, clinical examination and routine blood analysis) tended to have a negative GT (75%). Results of the GT did not correlate with blood fibrinogen concentration. Positive GT also predicted a fatal outcome in 69% of the clinical cases. The results of this trial indicate that GT can be a useful screening test to distinguish between acute and chronic inflammatory disease in horses.     

Exosites 1 and 2 are essential for protection of fibrin-bound thrombin from heparin-catalyzed inhibition by antithrombin and heparin cofactor II

Assembly of ternary thrombin-heparin-fibrin complexes, formed when fibrin binds to exosite 1 on thrombin and fibrin-bound heparin binds to exosite 2, produces a 58- and 247-fold reduction in the heparin-catalyzed rate of thrombin inhibition by antithrombin and heparin cofactor II, respectively. The greater reduction for heparin cofactor II reflects its requirement for access to exosite 1 during the inhibitory process. Protection from inhibition by antithrombin and heparin cofactor II requires ligation of both exosites 1 and 2 because minimal protection is seen when exosite 1 variants (gamma-thrombin and thrombin Quick 1) or an exosite 2 variant (Arg93 --> Ala, Arg97 --> Ala, and Arg101 --> Ala thrombin) is substituted for thrombin. Likewise, the rate of thrombin inhibition by the heparin-independent inhibitor, alpha1-antitrypsin Met358 --> Arg, is decreased less than 2-fold in the presence of soluble fibrin and heparin. In contrast, thrombin is protected from inhibition by a covalent antithrombin-heparin complex, suggesting that access of heparin to exosite 2 of thrombin is hampered when ternary complex formation occurs. These results reveal the importance of exosites 1 and 2 of thrombin in assembly of the ternary complex and the subsequent protection of thrombin from inhibition by heparin-catalyzed inhibitors.     

Mechanism of catalysis of inhibition of factor IXa by antithrombin in the presence of heparin or pentasaccharide

Because of the homology between factor IXa and factor Xa (f.IXa and f.Xa, respectively), and the critical upstream position of f.IXa in the coagulation cascade, the contribution of the heparin-derived pentasaccharide to antithrombin-mediated inhibition of f.IXa was investigated. Pentasaccharide promotes inhibition of both f.IXa and f.Xa generated in recalcified plasma. This result demonstrates that antithrombin is the predominant inhibitor of f.IXa in plasma, and that the activity of antithrombin is promoted by pentasaccharide. Kinetic experiments reveal that pentasaccharide increases the rates of antithrombin-mediated inhibition of both f.IXa and f.Xa by 2 orders of magnitude. These findings indicate that pentasaccharide-induced conformational changes in antithrombin enhance its capacity to inhibit both f.IXa and f.Xa. In the presence of Ca2+, full-length heparin produces an additional approximately 10-fold increase in the rates of inhibition of both enzymes, consistent with a template role of heparin. Heparin binding to f.Xa was previously shown to be promoted in the presence of Ca2+. Binding studies with f.IXa reveal a 10-fold higher affinity for heparin in the presence of Ca2+ compared with its absence. Thus, Ca2+ promotes heparin-catalyzed inhibition of f.IXa and f.Xa by antithrombin by augmenting the template mechanism. These results indicate that heparin-mediated catalysis of f.IXa inhibition by antithrombin reflects both pentasaccharide-induced conformational changes and heparin-mediated bridging of antithrombin to f.IXa. Furthermore, our data suggest that the efficacy of pentasaccharide for prevention and treatment of thrombotic disorders may reflect its action at two sites in the coagulation system.