Reduction of Hexavalent Chromium by Cell-Free Extract of Bacillus sphaericus AND 303 Isolated from Serpentine Soil

Cell-free extracts (CFEs) of chromium-resistant bacterium Bacillus sphaericus AND 303 isolated from serpentine soil of Andaman, India reduced Cr(VI) in in vitro condition, and the reductase activity was solely localized in the soluble cell-fractions (S₁₂, S₃₂, and S₁₅₀). The enzyme was constitutive as the CFEs from cells grown in Cr(VI)-free and Cr(VI)-containing media reduced a more or less equal amount of Cr(VI). Optimum Cr(VI) reductase activity was obtained at an enzyme (S₁₅₀) concentration equivalent to 4.56 mg protein/mL, 300 μM Cr(VI) and pH 6.0 after 30 min incubation at 30°C. The enzyme was heat labile; 80% of its activity was lost when exposed at 70°C for 15 min. Kinetics of Cr(VI) reductase activity fit well with the linearized Lineweaver-Burk plot and showed a V_max of 1.432 μmol Cr(VI)/mg protein/min and K_m of 158.12 μM Cr(VI). The presence of additional electron donors accelerated Cr(VI) reductase activity of CFE, and an increase of 28% activity over control was recorded with 1.0 μM NADH. Heavy metal ions such as Ni(II), Cu(II), and Cd(II) were strong inhibitors of Cr(VI) reductase unlike that of 100 μM Co(II), which retained 93% activity over control.     

Secretion of cellobiase is mediated via vacuoles in Termitomyces clypeatus

The majority of cellobiase activity in Termitomyces clypeatus was localized in vacuolar fractions of the fungus under secretory and nonsecretory conditions of growth. Activities of marker proteins for subcellular organelles, e.g., vacuoles, cytosol, ER, and mitochondria, in mycelial extracts from the secreting conditions increased by approximately 20, 12, 5, and 2.5 times, respectively, as compared to those obtained from mycelium grown in nonsecreting conditions. The average size and concentration of vacuoles visualized by electron microscopy were also increased in secreting conditions in the fungus. The specific activity of cellobiase in vacuoles isolated in Ficoll-sucrose gradient, as obtained from mycelial growth in secretory medium, was more than 40 times higher in comparison to that found from nonsecretory medium. The results indicated that subcellular localization of cellobiase in vacuoles is regulated by the cellular signaling prevailing in the fungus. Mycelial extraction of intracellular proteins by hand grinding and by bead-beater from cells frozen in the presence or absence of liquid nitrogen was also compared. Maximum recovery of intracellular protein was obtained with the bead-beater under aerobic conditions in the absence of nitrogen. Highest recovery of vacuoles up to 85% was obtained by single-step ultracentrifugation of the mycelial extract of the fungus in Ficoll-sucrose gradient. The method appeared to be useful for separation of other subcellular organelles in filamentous fungi.     

Lipid-regulated sterol transfer between closely apposed membranes by oxysterol-binding protein homologues

Sterols are transferred between cellular membranes by vesicular and poorly understood nonvesicular pathways. Oxysterol-binding protein–related proteins (ORPs) have been implicated in sterol sensing and nonvesicular transport. In this study, we show that yeast ORPs use a novel mechanism that allows regulated sterol transfer between closely apposed membranes, such as organelle contact sites. We find that the core lipid-binding domain found in all ORPs can simultaneously bind two membranes. Using Osh4p/Kes1p as a representative ORP, we show that ORPs have at least two membrane-binding surfaces; one near the mouth of the sterol-binding pocket and a distal site that can bind a second membrane. The distal site is required for the protein to function in cells and, remarkably, regulates the rate at which Osh4p extracts and delivers sterols in a phosphoinositide-dependent manner. Together, these findings suggest a new model of how ORPs could sense and regulate the lipid composition of adjacent membranes.     

A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

Background

A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.

Methodology/Principal Findings

We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins are natively folded and functional. This screen also identified two novel protein-protein interactions, between P12 and PVX_110945, and between MSP3.10 and MSP7.1, the latter of which was confirmed by surface plasmon resonance.

Conclusions/Significance

We produced a new library of recombinant full-length P. vivax ectodomains, established that the majority of them contain tertiary structure, and used them to identify predicted and novel protein-protein interactions. As well as identifying new interactions for further biological studies, this library will be useful in identifying P. vivax proteins with vaccine potential, and studying P. vivax malaria pathogenesis and immunity.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00663546","term_id":"NCT00663546"}}NCT00663546     

Layered electrophoretic transfer - A method for pre-analytic processing of histological sections

Current technologies for measuring protein expression across a tissue section are based on MS or in situ detection such as immunohistochemistry. However, due to the inherent molecular complexity of tissue samples and the large dynamic range of protein expression in cells, current approaches are often unable to measure moderate- and low-abundant proteins. In addition, they do not provide information on the physico-chemical properties of the proteins studied. To address these problems, we are developing a new pre-analytic methodology termed layered electrophoretic transfer (LET) that selectively separates and processes proteins from an intact tissue section without compromising important two-dimensional histological information. LET offers two potential advantages over standard techniques: (i) A reduced complexity of the tissue proteome for subsequent analysis; (ii) An opportunity to assess the biochemical status of proteins as they exist in situ. As an initial proof-of-concept, we demonstrate here that the protein content from a mixture of molecular weight standards, human tissue lysates, and tissue sections can be successfully transferred and separated using LET, and further demonstrate that the method can be coupled with immunoblotting or MS for downstream measurements. LET technology represents a new pre-analytic tool for interrogating the proteome in tissue sections while preserving valuable spatial information.     

Airborne Fungi Survey of Some Residences in Durham,North Carolina,USA

Gravity settling culture (GSC) plate collections of endogenous fungi were made at four homes in Durham, North Carolina. During frost-free periods (May-August), the most frequently isolated genera included Mucor, Cladosporium (Hormodendrum), Aspergillus, Penicillium, Rhizopus, Alternaria, Cunninghamella, Aureobasidium, Fusarium, Heterosporium, Amblyosporium, and other (unidentified) fungi. Higher numbers of mold isolates were associated with high shade and high levels of organic debris near the home and poor landscaping and landscape maintenance. Lower concentrations of mold isolates were associated with the presence of a central electrostatic filtration system and good compliance with dust controls. The viable mold spore levels were lower in homes where the electrostatic filtration unit was operated continuously rather than intermittently. These findings reflect the ease with which outdoor spore clouds may penetrate structures and obscure evidence of internal fungus source. The data also imply that because of size-related undersampling, open plates can exclude abundant types of recovery. No statistically significant correlations could be made between indoor mold isolates and any of the following: number and age of the occupants, age and size of home, month of survey or the presence of plants. Attempts have been made to facilitate the diagnosis of fungal allergy by gathering together examples of places and materials which are most likely to be a risk for mold sensitive patients.     

Sequence recombination improves target specificity in a redesigned collagen peptide abc‐type heterotrimer

Stability of the collagen triple helix is largely governed by its imino acid content, namely the occurrence of proline and 4R‐hydroxyproline at the X and Y positions, respectively, of the periodic (Gly‐X‐Y)_n sequence. Although other amino acids at these positions reduce stability of the triple helix, this can be partially compensated by introducing intermolecular side‐chain salt bridges. This approach was previously used to design an abc‐type heterotrimer composed of one basic, one acidic, and one neutral imino acid rich chain (Gauba and Hartgerink, J Am Chem Soc 2007;129:15034–15041). In this study, an abc‐type heterotrimer was designed to be the most stable species using a sequence recombination strategy that preserved both the amino acid composition and the network of interchain salt bridges of the original design. The target heterotrimer had the highest T_m of 50°C, 7°C greater than the next most stable species. Stability of the heterotrimer decreased with increasing ionic strength, consistent with the role of intermolecular salt bridges in promoting stability. Quantitative meta‐analysis of these results and published stability measurements on closely related peptides was used to discriminate the contributions of backbone propensity and side‐chain electrostatics to collagen stability. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Effects of cell entrapment on nucleic acid content and microbial diversity of mixed cultures in biological wastewater treatment

The effects of entrapment on nucleic acid content and microbial diversity of mixed cultures in biological municipal wastewater treatment were investigated. Deoxyribonucleic acid content increased 1.6-5.5 times more in alginate entrapped cells than in free and polyvinyl alcohol (PVA) entrapped cells. PVA entrapment resulted in 1.1- to 5.9-fold more increases in ribonucleic acid content compared to that experienced by free and alginate entrapped cells. Entrapment in carrageenan changed the bacterial community structure more than the alginate and PVA entrapments (35-80% versus 0-35%) as determined by single-strand conformation polymorphism analyses. The change in the bacterial community structure of alginate entrapped cells was less time dependent than that of PVA entrapped cells. This study enhances understandings on the physiology of entrapped cells and their community evolution in wastewater treatment environments.     

Aggregation of poly(γ-benzyl-α,L-glutamate)

The aggregation of poly(γ-benzyl-α,L -glutamate) and its enantiomer in toluene has been investigated by following the viscosity as a function of temperature, concentration, molecular weight, molecular-weight distribution, helix chirality, and shear rate. The temperature and concentration data for a 138,000-molecular-weight sample was fitted to an open, reversible end-to-end aggregation model. The aggregation numbers resulting from this fit were consistent with the sudden onset in non-Newtonian flow resulting from only a 0.2-wt% increase in concentration. The association equilibrium constant was then used to predict viscosity for comparison with other data, in particular, the effect of molecular weight and molecular-weight distribution. A mixture of right-and left-handed helices showed the aggregation was not chiral selective. The stiffness of end-to-end aggregated (hydrogen-bonded) molecules differed little from their covalent counterparts, at least below a molecular weight of ～10⁶. We conclude that polybenzylglutamate aggregation in toluene can be described by an open end-to-end aggregation model.