Functional aspects of co-variant surface charges in an antibody fragment

A mutational analysis of three co-variant pairs of residues, located at the surface of a single-chain fragment, variable (scFv), remote from the antigen-binding site, was performed to investigate the tolerance of these positions to amino acid changes. The replacements consisted of the elimination or addition of charges, or in their replacement by a charge of opposite sign. As measured by Biacore, antigen-binding kinetics and specificity were essentially unaffected by the mutations. The purified scFvs remained mostly 100% active for 14 h, and their sensitivity to guanidinium-chloride denaturation was similar. These observations indicate that the mutations did not affect antigen-binding properties and that protein folding was conserved. However, the various scFvs differed greatly in half-life in periplasmic extracts (<4 h to >16 h at 25 degrees C). The deleterious effect on half-life produced by single mutations could be reversed by introducing a second mutation that restores the natural combination of amino acids in the co-variant pair, indicating that the consequence of charge modifications at these locations depends on the sequence context. We propose that the differences in half-life result from differences in aggregation propensities with other periplasmic proteins, related to the presence of charged patches at the surface of the scFvs. The practical implication is that changes in surface charge may drastically affect the level of active molecules in complex protein mixtures, a potentially important consideration in engineering scFvs for biotechnological or medical purposes.     

Reevaluation of brush border motility: calcium induces core filament solation and microvillar vesiculation

The report that microvillar cores of isolated, demembranated brush borders retract into the terminal web in the presence of Ca(++) and ATP has been widely cited as an example of Ca(++)-regulated nonmuscle cell motility. Because of recent findings that microvillar core actin filaments are cross-linked by villin which, in the presence of micromolar Ca(++), fragments actin filaments, we used the techniques of video enhanced differential interference contrast, immunofluorescence, and phase contrast microscopy and thin-section electron microscopy (EM) to reexamine the question of contraction of isolated intestinal cell brush borders. Analysis of video enhanced light microscopic images of Triton- demembranated brush borders treated with a buffered Ca(++) solution shows the cores disintegrating with the terminal web remaining intact; membranated brush borders show the microvilli to vesiculate with Ca(++). Using Ca(++)/EGTA buffers, it is found that micromolar free Ca(++) causes core filament dissolution in membranated or demembranated brush borders, Ca(++) causes microvillar core solation followed by complete vesiculation of the microvillar membrane. The lengths of microvilli cores and rootlets were measured in thin sections of membranated and demembranated controls, in Ca(++)-, Ca(++) + ATP-, and in ATP-treated brush borders. Results of these measurements show that Ca(++) alone causes the complete solation of the microvillar cores, yet the rootlets in the terminal web region remain of normal length. These results show that microvilli do not retract into the terminal web in response to Ca(++) and ATP but rather that the microvillar cores disintegrate. NBD-phallicidin localization of actin and fluorescent antibodies to myosin reveal a circumferential band of actin and myosin in mildly permeabilized cells in the region of the junctional complex. The presence of these contractile proteins in this region, where other studies have shown a circumferential band of thin filaments, is consistent with the hypothesis that brush borders may be motile through the circumferential constriction of this “contractile ring,” and is also consistent with the observations that ATP-treated brush borders become cup shaped as if there had been a circumferential constriction.     

South African Papilionoid Legumes Are Nodulated by Diverse Burkholderia with Unique Nodulation and Nitrogen-Fixation Loci

The root-nodule bacteria of legumes endemic to the Cape Floristic Region are largely understudied, even though recent reports suggest the occurrence of nodulating Burkholderia species unique to the region. In this study, we considered the diversity and evolution of nodulating Burkholderia associated with the endemic papilionoid tribes Hypocalypteae and Podalyrieae. We identified distinct groups from verified rhizobial isolates by phylogenetic analyses of the 16S rRNA and recA housekeeping gene regions. In order to gain insight into the evolution of the nodulation and diazotrophy of these rhizobia we analysed the genes encoding NifH and NodA. The majority of these 69 isolates appeared to be unique, potentially representing novel species. Evidence of horizontal gene transfer determining the symbiotic ability of these Cape Floristic Region isolates indicate evolutionary origins distinct from those of nodulating Burkholderia from elsewhere in the world. Overall, our findings suggest that Burkholderia species associated with fynbos legumes are highly diverse and their symbiotic abilities have unique ancestries. It is therefore possible that the evolution of these bacteria is closely linked to the diversification and establishment of legumes characteristic of the Cape Floristic Region.     

Isolation and characterization of a manganese‐oxidizing bacterium from a biofiltration system for the treatment of borehole water in KwaZulu‐Natal (South Africa)

Using minimal salts medium containing vitamins and pyruvate (MSVP) plus added manganese sulfate, a manganese (II) oxidizing bacterial strain was isolated from water samples of a biofiltration system treating borehole water in KwaZulu‐Natal, South Africa. The nonmotile Gram‐negative and oxidase‐negative isolate was subsequently characterized microbiologically. Based on its morphological and physiological characteristics and on the analysis of its 16S rRNA gene sequence, the isolate was assigned to the genus Acinetobacter. Growth of the isolated strain in MSVP with added manganese sulfate gave rise to a drop in pH and a concomitant increase in oxidation–reduction potential, which was absent in controls, thus indicating manganese oxidation. The ability of Acinetobacter sp. strain LB1 to oxidize Mn (II) was further verified using the leucoberbelin blue dye assay as well as by energy dispersive X‐ray analysis of crystals formed in the medium. In addition, a biofilm assay indicated that this isolate can attach to solid surfaces such as a biofilter matrix, thus confirming its potential for biotechnological applications in the area of water purification.     

Microbial Transport, Survival, and Succession in a Sequence of Buried Sediments

Abstract Two chronosequences of unsaturated, buried loess sediments, ranging in age from <10,000 years to >1 million years, were investigated to reconstruct patterns of microbial ecological succession that have occurred since sediment burial. The relative importance of microbial transport and survival to succession was inferred from sediment ages, porewater ages, patterns of abundance (measured by direct counts, counts of culturable cells, and total phospholipid fatty acids), activities (measured by radiotracer and enzyme assays), and community composition (measured by phospholipid fatty acid patterns and Biolog substrate usage). Core samples were collected at two sites 40 km apart in the Palouse region of eastern Washington State, near the towns of Washtucna and Winona. The Washtucna site was flooded multiple times during the Pleistocene by glacial outburst floods; the Winona site elevation is above flood stage. Sediments at the Washtucna site were collected from near surface to 14.9 m depth, where the sediment age was approximately 250 ka and the porewater age was 3700 years; sample intervals at the Winona site ranged from near surface to 38 m (sediment age: approximately 1 Ma; porewater age: 1200 years). Microbial abundance and activities declined with depth at both sites; however, even the deepest, oldest sediments showed evidence of viable microorganisms. Same-age sediments had equal quantities of microorganisms, but different community types. Differences in community makeup between the two sites can be attributed to differences in groundwater recharge and paleoflooding. Estimates of the microbial community age can be constrained by porewater and sediment ages. In the shallower sediments (<9 m at Washtucna, <12 m at Winona), the microbial communities are likely similar in age to the groundwater; thus, microbial succession has been influenced by recent transport of microorganisms from the surface. In the deeper sediments, the populations may be considerably older than the porewater ages, since microbial transport is severely restricted in unsaturated sediments. This is particularly true at the Winona site, which was never flooded.     

Prevalence of risk factors for cardiovascular disease in Canadians 55 to 74 years of age: results from the Canadian Heart Health Surveys, 1986-1992

BACKGROUND: By 2016, the proportion of Canadians older than 65 years of age will increase to 16%, and there will be an increase in the absolute number of cases of cardiovascular disease in older Canadians. The Canadian Heart Health Surveys database provides information about this population upon which health policy related to cardiovascular disease can be based. This paper presents for the first time population-based data on the risk factors for cardiovascular disease in older Canadians. METHODS: Canadians from all 10 provinces participated in surveys of cardiovascular risk factors; health insurance registries were used as sampling frames. In each province, probability samples of 2200 adults 18 to 74 years old not living in institutions, on reserves or in military camps were asked to participate in interviews and to undergo testing at clinics for major risk factors for cardiovascular disease. RESULTS: A total of 2739 men (response rate 70%) and 2617 women (response rate 66%) aged 55 to 74 years participated in the survey and also provided follow-up clinical measurements at the clinic. Overall, 52% of participants were hypertensive, 26% had isolated systolic hypertension, and 30% had a total blood cholesterol level of 6.2 mmol/L or greater. Rates of current smoking were lower in women than men (17% v. 22%). Overall, 87% of men and 78% of women who were current smokers smoked at least 10 cigarettes per day. Only slightly more than half of participants exercised at least once a week for at least 15 minutes, and almost half had a body mass index of 27 or greater. In only 4% was no major risk factor for cardiovascular disease detected. INTERPRETATION: Significant numbers of older Canadians have one or more major risk factors for cardiovascular disease. Many of these risk factors are amenable to modification.     

Mycobacterium tuberculosis curli pili (MTP) is associated with significant host metabolic pathways in an A549 epithelial cell infection model and contributes to the pathogenicity of Mycobacterium tuberculosis

Metabolomics - Diabetes mellitus is a serious metabolic disorder causing multiple organ damage in human. However, the lipidomic profiles in different organs and their associations are rarely...     

Effect of alkaline pre-treatment on enzyme synergy for efficient hemicellulose hydrolysis in sugarcane bagasse

This aim of this study was to investigate the effect of ammonium hydroxide (NH₄OH) and sodium hydroxide (NaOH) pre-treatment on the digestibility of sugarcane bagasse (SCB) by hemicellulase action. It was found that pre-treatment of SCB with NH₄OH removed a larger percentage of the SCB lignin and effectively increased SCB digestibility 13.13 fold. The greatest amount of reducing sugar (1194.88 μmol/min) and largest degree of synergy (2.85) was obtained using a combination of two enzymes (25% ManA and 75% XynA) with NH₄OH pre-treated SCB. In this study, NH₄OH therefore appeared to be a more effective pre-treatment step for subsequent hydrolysis by hemicellulases.     

Antibiotic resistance profiles of coagulase-positive and coagulase-negative staphylococci from pit latrine fecal sludge in a peri-urban South African community

The aim of this study was to assess pit latrine samples from a peri-urban community in KwaZulu-Natal (South Africa) for the presence of multidrug-resistant (MDR) Staphylococcus spp. Standard procedures were used to isolate Staphylococcus spp. from pit latrine fecal sludge samples, with confirmation at genus level by polymerase chain reaction (PCR). Sixty-eight randomly selected pit latrine Staphylococcus spp. isolates were further characterized by using established disk diffusion procedures. An average Staphylococcus spp. count of 2.1?×?10⁵ CFU per g fecal material was established using two randomly selected pit latrine samples. Of the 68-selected Staphylococcus spp. pit latrine isolates, 49% were identified as coagulase positive, 51% as coagulase negative and 65% (12 coagulase positive, 32 coagulase negative isolates) were categorized as MDR. The majority (66/68) of Staphylococcus spp. isolates displayed resistance to fusidic acid while only 5/68 isolates displayed resistance to chloramphenicol. The pit latrine samples analyzed in this study are a source of MDR Staphylococcus spp., highlighting the need for proper hygiene and sanitation regimes in rural communities using these facilities.