Bi-PSSM: Position specific scoring matrix based intelligent computational model for identification of mycobacterial membrane proteins.

Mycobacterium is a pathogenic bacterium, which is a causative agent of tuberculosis (TB) and leprosy. These diseases are very crucial and become the cause of death of millions of people every year in the world. So, the characterize structure of membrane proteins of the protozoan play a vital role in the field of drug discovery because, without any knowledge about this Mycobacterium's membrane protein and their types, the scientists are unable to treat this pathogenic protozoan. So, an accurate and competitive computational model is needed to characterize this uncharacterized structure of mycobacterium. Series of attempts were carried out in this connection. Split amino acid compositions, Unbiased-Dipeptide peptide compositions (Unb-DPC), Over-represented tri-peptide compositions, compositions & translation were the few recent encoding techniques followed by different researchers in their publications. Although considerable results have been achieved by these models, still there is a gap which is filled in this study. In this study, an evolutionary feature extraction technique position specific scoring matrix (PSSM) is applied in order to extract evolutionary information from protein sequences. Consequently, 99.6% accuracy was achieved by the learning algorithms. The experimental results demonstrated that the proposed computational model will lead to develop a powerful tool for anti-mycobacterium drugs as well as play a promising rule in proteomic and bioinformatics.

Co-existence of extramammary Paget's disease and Bowen's disease of vulva.

Extramammary Paget's disease and Bowen's disease are histologically similar and immunohistochemistry is often required to make the diagnosis. We present a case of vulval Paget's disease with Bowen's disease in an elderly female. Strong positivity for cytokeratin 7, anti CAM 5.2, carcinoembryonic antigen (CEA) and periodic acid-Schiff (PAS) stain in clitoral, left labial and interface regions of the vulvectomy specimen confirmed the diagnosis of Paget's disease (PD) while positive staining for p63 in the right labial and interface regions helped in establishing the diagnosis of concurrent Bowen's disease (BD).

Modulation of lipid droplets by Mycobacterium leprae in Schwann cells: a putative mechanism for host lipid acquisition and bacterial survival in phagosomes.

The predilection of Mycobacterium leprae (ML) for Schwann cells (SCs) leads to peripheral neuropathy, a major concern in leprosy. Highly infected SCs in lepromatous leprosy nerves show a foamy, lipid-laden appearance; but the origin and nature of these lipids, as well as their role in leprosy, have remained unclear. The data presented show that ML has a pronounced effect on host-cell lipid homeostasis through regulation of lipid droplet (lipid bodies, LD) biogenesis and intracellular distribution. Electron microscopy and immunohistochemical analysis of lepromatous leprosy nerves for adipose differentiation-related protein expression, a classical LD marker, revealed accumulating LDs in close association to ML in infected SCs. The capacity of ML to induce LD formation was confirmed in in vitro studies with human SCs. Moreover, via confocal and live-cell analysis, it was found that LDs are promptly recruited to bacterial phagosomes and that this process depends on cytoskeletal reorganization and PI3K signalling. ML-induced LD biogenesis and recruitment were found to be independent of TLR2 bacterial sensing. Notably, LD recruitment impairment by cytoskeleton drugs decreased intracellular bacterial survival. Altogether, our data revealed SC lipid accumulation in ML-containing phagosomes, which may represent a fundamental aspect of bacterial pathogenesis in the nerve.

Immunogenic membrane-associated proteins of Mycobacterium tuberculosis revealed by proteomics.

Membrane-associated proteins of Mycobacterium tuberculosis offer a challenge, as well as an opportunity, in the quest for better therapeutic and prophylactic interventions against tuberculosis. The authors have previously reported that extraction with the detergent Triton X-114 (TX-114) is a useful step in proteomic analysis of mycobacterial cell membranes, and detergent-soluble membrane proteins of mycobacteria are potent stimulators of human T cells. In this study 1-D and 2-D gel electrophoresis-based protocols were used for the analysis of proteins in the TX-114 extract of M. tuberculosis membranes. Peptide mass mapping (using MALDI-TOF-MS, matrix assisted laser desorption/ionization time of flight mass spectrometry) of 116 samples led to the identification of 105 proteins, 9 of which were new to the M. tuberculosis proteome. Functional orthologues of 73 of these proteins were also present in Mycobacterium leprae, suggesting their relative importance. Bioinformatics predicted that as many as 73% of the proteins had a hydrophobic disposition. 1-D gel electrophoresis revealed more hydrophobic/transmembrane and basic proteins than 2-D gel electrophoresis. Identified proteins fell into the following major categories: protein synthesis, cell wall biogenesis/architecture and conserved hypotheticals/unknowns. To identify immunodominant proteins of the detergent phase (DP), 14 low-molecular-mass fractions prepared by continuous-elution gel electrophoresis were subjected to T cell activation assays using blood samples from BCG-vaccinated healthy donors from a tuberculosis endemic area. Analysis of the responses (cell proliferation and IFN-gamma production) showed that the immunodominance of certain DP fractions was most probably due to ribosomal proteins, which is consistent with both their specificity for mycobacteria and their abundance. Other membrane-associated proteins, including transmembrane proteins/lipoproteins and ESAT-6, did not appear to contribute significantly to the observed T cell responses.

Combination of two mutant alpha spectrin alleles underlies a severe spherocytic hemolytic anemia.

We studied a patient with a severe spherocytic hemolytic anemia without family history of spherocytosis. Analysis of patient's erythrocyte membrane proteins revealed spectrin deficiency and a truncated alpha spectrin protein. We determined that the patient is a compound heterozygote with two mutations in alpha spectrin gene. Mutation in the paternal allele, designated alpha spectrin(PRAGUE), is a transition A to G in the penultimate position of intron 36 that leads to skipping of exon 37, frameshift, and production of the truncated alpha spectrin protein. The maternal allele, designated alpha spectrin(LEPRA), contains transition C-->T in position -99 of intron 30. This mutation enhances an alternative acceptor splice site 70 nucleotides upstream from the regular site. The alternative splicing causes a frameshift and premature termination of translation leading to a significant decrease in alpha spectrin production. The alpha(LEPRA) mutation is linked to a spectrin alphaIIa marker that was found to be associated with recessive or nondominant spectrin-deficient hereditary spherocytosis in approximately 50% of studied families. We conclude that the alpha(LEPRA) mutation combined in trans with the alpha(PRAGUE) mutation underlie the severe hemolytic anemia in the proband. We suggest that allele alpha spectrin(LEPRA) may be frequently involved in pathogenesis of recessive or nondominant spectrin-deficient hereditary spherocytosis.

Relationship between host histones and armadillo-derived Mycobacterium leprae.

A major protein previously recognized as being primarily associated with the cell walls of Mycobacterium leprae, major wall protein (MWP), is now identified as histoprotein H2b based on N-terminal amino-acid sequencing, electrophoretic comparisons, and several other properties. An avid association between several host/armadillo-derived histones and M. leprae was demonstrated. Since such armadillo-derived M. leprae are the basis of several ongoing vaccine trials, a simple procedure that permits the prompt solubilization and quantification of histones in M. leprae preparations is described. The quantity of histones associated with M. leprae is significant, ranging from 0.6 to 4.8 micrograms of histoprotein H2b per mg of bacteria.

Immunochemical characterization of a protein associated with Mycobacterium leprae cell wall.

A panel of nine monoclonal antibodies to Mycobacterium leprae were used to characterize a protein antigen of the bacillus. Two monoclonal antibodies (IVD8 and IIIE9) were specific for M. leprae and reacted with an epitope (CWPa) present on a protein molecule associated with the cell wall fraction of M. leprae. This protein, designated cell wall-associated protein (CWP), lost its immunoreactivity upon treatment with trypsin and had an apparent molecular weight of 65,000, though additional lower-molecular-weight forms of the protein were observed by immunoblotting. Four other cross-reactive epitopes (CWPb, CWPc, CWPd, and CWPe) were defined on the same molecule using seven independent monoclonal antibodies. Therefore, M. leprae possesses a trypsin-sensitive, heat-stable protein associated with the cell wall which contains at least one species-specific and four cross-reactive antigenic determinants.

Immunochemical and structural integrity of surface protein antigens of mycobacteria during separation from armadillo liver tissue.

Surface proteins of Mycobacterium smegmatis were iodinated using the lactoperoxidase method. Sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated two major surface proteins in the radiolabelled M. smegmatis. Both surface proteins were released from M. smegmatis using the nonionic detergent Triton X-100. The major surface component was sensitive to pronase digestion and contained no detectable carbohydrate. The second radiolabelled component was found to be of low molecular weight, resistant to pronase digestion and stained positive for carbohydrate by the periodic acid/Schiff method. Triton X-100 solubilized radiolabelled surface proteins were antigenic as assessed by a radioimmune precipitation test. When surface labelled M. smegmatis was mixed with armadillo liver tissue and separated from tissue using a method formerly employed by the World Health Organization Immunology of Leprosy Program for the purification of M. leprae, as much as 50% of the surface proteins of M. smegmatis was either released or destroyed. In addition, another twenty distinct proteins were released from M. smegmatis after treatment with Triton X-100. Similar losses of proteins from M. leprae may also occur using this procedure for M. leprae purification. Separation techniques employing surfactants and enzymatic treatment should be carefully evaluated since proteins lost during these procedures may prove relevant to human immune responses to M. leprae.