A comprehensive analysis of Trypanosoma brucei mitochondrial proteome

The composition of the large, single, mitochondrion (mt) of Trypanosoma brucei was characterized by MS (2‐D LC‐MS/MS and gel‐LC‐MS/MS) analyses. A total of 2897 proteins representing a substantial proportion of procyclic form cellular proteome were identified, which confirmed the validity of the vast majority of gene predictions. The data also showed that the genes annotated as hypothetical (species specific) were overpredicted and that virtually all genes annotated as hypothetical, unlikely are not expressed. By comparing the MS data with genome sequence, 40 genes were identified that were not previously predicted. The data are placed in a publicly available web‐based database (www.TrypsProteome.org). The total mitochondrial proteome is estimated at 1008 proteins, with 401, 196, and 283 assigned to the mt with high, moderate, and lower confidence, respectively. The remaining mitochondrial proteins were estimated by statistical methods although individual assignments could not be made. The identified proteins have predicted roles in macromolecular, metabolic, energy generating, and transport processes providing a comprehensive profile of the protein content and function of the T. brucei mt.     

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.     

Increased β-Catenin/Tcf Signaling in Pilocytic Astrocytomas: A Comparative Study to Distinguish Pilocytic Astrocytomas from Low-Grade Diffuse Astrocytomas

Although pilocytic and diffuse grade II astrocytomas considered as low-grade tumors, the distinction between them is still a major clinical problem. Previously we reported the activation of Wnt/β-catenin/Tcf signaling pathway in diffuse astrocytomas, however its role in pilocytic astrocytomas is not well understood. In this study, we investigated the Wnt/β-catenin/Tcf pathway in pilocytic astrocytomas and compared with diffuse astrocytomas. We observed the differential expression of β-catenin, Tcf4, Lef1 and c-Myc in astrocytomas particularly higher levels were observed in pilocytic astrocytomas and GBM while very little expression was documented in grade II tumors. Further, immunohistochemical analysis revealed the strong positivity of β-catenin, Tcf4, Lef1 and c-Myc in pilocytic astrocytomas than that of grade II tumors and also exhibited the strong positivity in vascular endothelial cells of pilocytic astrocytomas and GBM. Hence, Wnt/β-catenin/Tcf signaling pathway is differentially expressed in astrocytomas, activation of this pathway might be helpful in separating pilocytic astrocytomas from low-grade diffuse astrocytomas.     

Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing

Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3" exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3" pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5" phosphate of the 3" fragment, which is not required for U removal. The ability of the 3" U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3" exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (R_p)α-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.     

Modeling of molecular interaction between apoptin, BCR-Abl and CrkL--an alternative approach to conventional rational drug design

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin's action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.     

High ethylene production slackens seed filling in compact panicled rice cultivar

Change of plant type in rice resulting in increased compactness of the panicle, allows space for accommodation of a larger number of spikelets, but grain yield does not increase proportionately because of limitations in grain filling. The objective of this study was to evaluate potential causes of poor filling of spikelets by comparing the physiological processes that influence source and sink activities between a compact- (OR-1920-7) and a loose-panicled (Lalat) rice cultivars growing in the open field conditions in the farm of Regional Research and Technology Transfer Station, Orissa University of Agriculture and Technology, Chiplima, India during dry season of 2007. Although grain number per unit length of the branches was higher in the compact-panicled cultivar than the loose-panicled cultivar, average grain weight was lower on the primary and secondary branches at top, middle and basal positions of the panicle in the former compared to the corresponding positions of the panicle in the latter. Compared to Lalat, ethylene production rate was considerably higher in the boot of the flag leaf sheath of OR-1920-7 during the pre-anthesis period. Ethylene evolution rate correlated negatively with growth and cell division rates and starch concentration of the juvenile endosperm. Because spikelet growth was slower in OR-1920-7 than in Lalat, unused carbohydrates accumulated in the endosperm. The stomatal conductance of the flag leaf during this period was also lower in the former than that of the latter and it correlated negatively with ethylene evolution rate of the boot. It is concluded that high ethylene production slackened grain filling of compact-panicled rice cultivar OR-1920-7 because of its adverse influence on both source and sink activities.     

Biofouling and microbial corrosion problem in the thermo-fluid heat exchanger and cooling water system of a nuclear test reactor

This article discusses aspects of biofouling and corrosion in the thermo-fluid heat exchanger (TFHX) and in the cooling water system of a nuclear test reactor. During inspection, it was observed that >90% of the TFHX tube bundle was clogged with thick fouling deposits. Both X-ray diffraction and Mössbauer analyses of the fouling deposit demonstrated iron corrosion products. The exterior of the tubercle showed the presence of a calcium and magnesium carbonate mixture along with iron oxides. Raman spectroscopy analysis confirmed the presence of calcium carbonate scale in the calcite phase. The interior of the tubercle contained significant iron sulphide, magnetite and iron-oxy-hydroxide. A microbiological assay showed a considerable population of iron oxidizing bacteria and sulphate reducing bacteria (10⁵ to 10⁶ cfu g^?1 of deposit). As the temperature of the TFHX is in the range of 45–50°C, the microbiota isolated/assayed from the fouling deposit are designated as thermo-tolerant bacteria. The mean corrosion rate of the CS coupons exposed online was ～2.0 mpy and the microbial counts of various corrosion causing bacteria were in the range 10³ to 10⁵ cfu ml^?1 in the cooling water and 10⁶ to 10⁸ cfu ml^?1 in the biofilm.     

Immunization with outer-membrane protein(s) of Bacteroides fragilis: an experimental study in mice

A crude outer-membrane protein (OMP) preparation from a strain of Bacteroides fragilis, grown in supplemented brain-heart infusion broth, was tested for its protective effect against subcutaneous infection in mice. Immunization with six doses, each of 100, 150 or 200 g OMP preparation, gave some protection: abscesses completely disappeared 15 to 22 days after immunization. In non-immunized animals and animals immunized with doses of 10, 20, 40 or 80 g each, well demarcated abscesses were seen beyond day 22 post-immunization. Although crude OMP elicited good antibody response, with maximum titres on day 4 post-immunization, high titres could not be associated with healing of the abscesses.