Proteogenomic analysis of Mycobacterium tuberculosis by high resolution mass spectrometry

The genome sequencing of H37Rv strain of Mycobacterium tuberculosis was completed in 1998 followed by the whole genome sequencing of a clinical isolate, CDC1551 in 2002. Since then, the genomic sequences of a number of other strains have become available making it one of the better studied pathogenic bacterial species at the genomic level. However, annotation of its genome remains challenging because of high GC content and dissimilarity to other model prokaryotes. To this end, we carried out an in-depth proteogenomic analysis of the M. tuberculosis H37Rv strain using Fourier transform mass spectrometry with high resolution at both MS and tandem MS levels. In all, we identified 3176 proteins from Mycobacterium tuberculosis representing ~80% of its total predicted gene count. In addition to protein database search, we carried out a genome database search, which led to identification of ~250 novel peptides. Based on these novel genome search-specific peptides, we discovered 41 novel protein coding genes in the H37Rv genome. Using peptide evidence and alternative gene prediction tools, we also corrected 79 gene models. Finally, mass spectrometric data from N terminus-derived peptides confirmed 727 existing annotations for translational start sites while correcting those for 33 proteins. We report creation of a high confidence set of protein coding regions in Mycobacterium tuberculosis genome obtained by high resolution tandem mass-spectrometry at both precursor and fragment detection steps for the first time. This proteogenomic approach should be generally applicable to other organisms whose genomes have already been sequenced for obtaining a more accurate catalogue of protein-coding genes.     

Head and neck lymphatic tumors and bony abnormalities: a clinical and molecular review

Lymphatic malformations and lymphatic-derived tumors commonly involve the head and neck, where they may be associated with bony abnormalities and other systemic symptoms. The reasons for the association between these disorders and local skeletal changes are largely unknown, but such changes may cause significant disease-related morbidity. Ongoing work in molecular and developmental biology is beginning to uncover potential reasons for the bony abnormalities found in head and neck lymphatic disease; this article summarizes current knowledge on possible mechanisms underlying this association.     

Structural basis for dual-inhibition mechanism of a non-classical Kazal-type serine protease inhibitor from horseshoe crab in complex with subtilisin

Serine proteases play a crucial role in host-pathogen interactions. In the innate immune system of invertebrates, multi-domain protease inhibitors are important for the regulation of host-pathogen interactions and antimicrobial activities. Serine protease inhibitors, 9.3-kDa CrSPI isoforms 1 and 2, have been identified from the hepatopancreas of the horseshoe crab, Carcinoscorpius rotundicauda. The CrSPIs were biochemically active, especially CrSPI-1, which potently inhibited subtilisin (Ki = 1.43 nM). CrSPI has been grouped with the non-classical Kazal-type inhibitors due to its unusual cysteine distribution. Here we report the crystal structure of CrSPI-1 in complex with subtilisin at 2.6 Å resolution and the results of biophysical interaction studies. The CrSPI-1 molecule has two domains arranged in an extended conformation. These two domains act as heads that independently interact with two separate subtilisin molecules, resulting in the inhibition of subtilisin activity at a ratio of 1:2 (inhibitor to protease). Each subtilisin molecule interacts with the reactive site loop from each domain of CrSPI-1 through a standard canonical binding mode and forms a single ternary complex. In addition, we propose the substrate preferences of each domain of CrSPI-1. Domain 2 is specific towards the bacterial protease subtilisin, while domain 1 is likely to interact with the host protease, Furin. Elucidation of the structure of the CrSPI-1: subtilisin (1∶2) ternary complex increases our understanding of host-pathogen interactions in the innate immune system at the molecular level and provides new strategies for immunomodulation.     

Oxidized chondroitin sulfate-cross-linked gelatin matrixes: a new class of hydrogels

A naturally occurring glycosaminoglycan such as chondroitin-6-sulfate was first converted in to its aldehyde derivative by periodate oxidation and used as a cross-linking agent for gelatin giving rise to a new class of hydrogels. Cross-linking was predominantly due to Schiff's base formation between the epsilon-amino groups of lysine or hydroxylysine side groups of gelatin and the aldehyde groups in oxidized chondroitin sulfate. The hydrogels were prepared from chondroitin sulfate with different degrees of oxidation and gelatin. They were characterized for degree of cross-linking, cross-linking density, equilibrium swelling, water vapor transmission rate, internal structure, and blood-compatibility. Degree of cross-linking of the gels determined by trinitrobenzene sulfonic acid assay showed that, the higher the degree of oxidation of the polysaccharide, the higher the degree of cross-linking. Examination of the internal structure by scanning electron microscopy showed that the hydrogels were highly porous in nature with interconnecting pores ranging from 50 to 200 mum. Equilibrium swelling showed that the gels retained about 90% water and did not undergo dehydration rapidly. The hydrogels were nontoxic and blood-compatible. Since an important phase of early wound healing has been shown to involve secretion of glycosaminoglycans such as chondroitin sulfate by fibroblasts which form a hydrophilic matrix suitable for remodeling during healing, this new class of hydrogels prepared from chondroitin sulfate and gelatin without employing any extraneous cross-linking agents are expected to have potential as wound dressing materials.     

Cellular Destruction Following Transmyocardial Laser Revascularization (TMR)

Summary During transmyocardial revascularization, cellular destruction of cardiomyocytes occurs as a result of the high-energy laser. However, the features of myocardial cellular destruction are unclear. The present study was undertaken to examine the structural characteristics of cell death in the myocardium following transmyocardial revascularization. Myocardial specimens from 3 male patients who had died within 11 days following laser revascularization were collected within 1 h of death and were analyzed by immunohistochemistry and electron microscopy. For immunohistochemistry, antibodies to pro-apoptotic proteins CPP32 and BAX were used. Immunohistochemical examination demonstrated the presence of cells expressing both CPP32 and BAX along the laser channel. Electron microscopic analysis revealed that the lining surface of laser channels consisted of condensed acellular debris and dead cells. No endothelialization of channels was noted. The lumen of laser channels were surrounded by a rim of acellular debris with several outer concentric rims of cardiomyocytes showing features of cellular destruction. The present study identified features of both necrotic and apoptotic cellular death following laser revascularization.     

Finger joint impedance during tapping on a computer keyswitch

We studied the dynamic behavior of finger joints during the contact period of tapping on a computer keyswitch, to characterize and parameterize joint function with a lumped-parameter impedance model. We tested the hypothesis that the metacarpophalangeal (MCP) and interphalangeal (IP) joints act similarly in terms of kinematics, torque, and energy production when tapping. Fifteen human subjects tapped with the index finger of the right hand on a computer keyswitch mounted on a two-axis force sensor, which measured forces in the vertical and sagittal planes. Miniature fiber-optic goniometers mounted across the dorsal side of each joint measured joint kinematics. Joint torques were calculated from endpoint forces and joint kinematics using an inverse dynamic algorithm. For each joint, a linear spring and damper model was fitted to joint torque, position, and velocity during the contact period of each tap (22 per subject on average). The spring-damper model could account for over 90% of the variance in torque when loading and unloading portions of the contact were separated, with model parameters comparable to those previously measured during isometric loading of the finger. The finger joints functioned differently, as illustrated by energy production during the contact period. During the loading phase of contact the MCP joint flexed and produced energy, whereas the proximal and distal IP joints extended and absorbed energy. These results suggest that the MCP joint does work on the interphalangeal joints as well as on the keyswitch.     

Hemoglobin site-mutants reveal dynamical role of interhelical H-bonds in the allosteric pathway: time-resolved UV resonance Raman evidence for intra-dimer coupling

The dynamical effect of eliminating specific tertiary H-bonds in the hemoglobin (Hb) tetramer has been investigated by site-directed mutagenesis and time-resolved absorption and ultraviolet resonance Raman (UVRR) spectroscopy. The Trp alpha 14...Thr alpha 67 and Trp beta 15...Ser beta 72 H-bonds connect the A and E helices in the alpha and beta chains, and are proposed to break in the earliest protein intermediate (Rdeoxy) following photo-deligation of HbCO, along with a second pair of H-bonds involving tyrosine residues. Mutation of the acceptor residues Thr alpha 67 and Ser beta 72 to Val and Ala eliminates the A-E H-bonds, but has been shown to have no significant effect on ligand-binding affinity or cooperativity, or on spectroscopic markers of the T-state quaternary interactions. However, the mutations have profound and unexpected effects on the character of the Rdeoxy intermediate, and on the dynamics of the subsequent steps leading to the T state. Formation of the initial quaternary contact (RT intermediate) is accelerated, by an order of magnitude, but the locking-in of the T state is delayed by a factor of 2. These rate effects are essentially the same for either mutation, or for the double mutation, suggesting that the alpha beta dimer behaves as a mechanically coupled dynamical unit. Further evidence for intra-dimer coupling is provided by the Rdeoxy UVRR spectrum, in which either or both mutations eliminate the tyrosine difference intensity, although only tryptophan H-bonds are directly affected. A possible mechanism for mechanical coupling is outlined, involving transmission of forces through the alpha(1)beta(1) (and alpha(2)beta(2)) interface. The present observations establish that quaternary motions can occur on the approximately 100 ns time-scale. They show also that a full complement of interhelical H-bonds actually slows the initial quaternary motion in Hb, but accelerates the locking in of the T-contacts.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

The narrow substrate specificity of human tyrosine aminotransferase--the enzyme deficient in tyrosinemia type II

Human tyrosine aminotransferase (hTATase) is the pyridoxal phosphate-dependent enzyme that catalyzes the reversible transamination of tyrosine to p-hydrophenylpyruvate, an important step in tyrosine metabolism. hTATase deficiency is implicated in the rare metabolic disorder, tyrosinemia type II. This enzyme is a member of the poorly characterized Igamma subfamily of the family I aminotransferases. The full length and truncated forms of recombinant hTATase were expressed in Escherichia coli, and purified to homogeneity. The pH-dependent titration of wild-type reveals a spectrum characteristic of family I aminotransferases with an aldimine pK(a) of 7.22. I249A mutant hTATase exhibits an unusual spectrum with a similar aldimine pK(a) (6.85). hTATase has very narrow substrate specificity with the highest enzymatic activity for the Tyr/alpha-ketoglutarate substrate pair, which gives a steady state k(cat) value of 83 s(-1). In contrast there is no detectable transamination of aspartate or other cosubstrates. The present findings show that hTATase is the only known aminotransferase that discriminates significantly between Tyr and Phe: the k(cat)/K(m) value for Tyr is about four orders of magnitude greater than that for Phe. A comparison of substrate specificities of representative Ialpha and Igamma aminotransferases is described along with the physiological significance of the discrimination between Tyr and Phe by hTATase as applied to the understanding of the molecular basis of phenylketonuria.