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.     

A nonerythropoietic peptide that mimics the 3D structure of erythropoietin reduces organ injury/dysfunction and inflammation in experimental hemorrhagic shock

Recent studies have shown that erythropoietin, critical for the differentiation and survival of erythrocytes, has cytoprotective effects in a wide variety of tissues, including the kidney and lung. However, erythropoietin has been shown to have a serious side effect-an increase in thrombovascular effects. We investigated whether pyroglutamate helix B-surface peptide (pHBSP), a nonerythropoietic tissue-protective peptide mimicking the 3D structure of erythropoietin, protects against the organ injury/ dysfunction and inflammation in rats subjected to severe hemorrhagic shock (HS). Mean arterial blood pressure was reduced to 35 ± 5 mmHg for 90 min followed by resuscitation with 20 mL/kg Ringer Lactate for 10 min and 50% of the shed blood for 50 min. Rats were euthanized 4 h after the onset of resuscitation. pHBSP was administered 30 min or 60 min into resuscitation. HS resulted in significant organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung). In rats subjected to HS, pHBSP significantly attenuated (i) organ injury/dysfunction (renal, hepatic, pancreas, neuromuscular, lung) and inflammation (lung), (ii) increased the phosphorylation of Akt, glycogen synthase kinase-3β and endothelial nitric oxide synthase, (iii) attenuated the activation of nuclear factor (NF)-κB and (iv) attenuated the increase in p38 and extracellular signal-regulated kinase (ERK)1/2 phosphorylation. pHBSP protects against multiple organ injury/dysfunction and inflammation caused by severe hemorrhagic shock by a mechanism that may involve activation of Akt and endothelial nitric oxide synthase, and inhibition of glycogen synthase kinase-3β and NF-κB.     

Ontogenetic de novo copy number variations (CNVs) as a source of genetic individuality: studies on two families with MZD twins for schizophrenia

Genetic individuality is the foundation of personalized medicine, yet its determinants are currently poorly understood. One issue is the difference between monozygotic twins that are assumed identical and have been extensively used in genetic studies for decades. Here, we report genome-wide alterations in two nuclear families each with a pair of monozygotic twins discordant for schizophrenia evaluated by the Affymetrix 6.0 human SNP array. The data analysis includes characterization of copy number variations (CNVs) and single nucleotide polymorphism (SNPs). The results have identified genomic differences between twin pairs and a set of new provisional schizophrenia genes. Samples were found to have between 35 and 65 CNVs per individual. The majority of CNVs (~80%) represented gains. In addition, ~10% of the CNVs were de novo (not present in parents), of these, 30% arose during parental meiosis and 70% arose during developmental mitosis. We also observed SNPs in the twins that were absent from both parents. These constituted 0.12% of all SNPs seen in the twins. In 65% of cases these SNPs arose during meiosis compared to 35% during mitosis. The developmental mitotic origin of most CNVs that may lead to MZ twin discordance may also cause tissue differences within individuals during a single pregnancy and generate a high frequency of mosaics in the population. The results argue for enduring genome-wide changes during cellular transmission, often ignored in most genetic analyses.     

Paraoxonase 1 (PON1) polymorphisms, haplotypes and activity in predicting cad risk in North-West Indian Punjabis

Background

Human serum paraoxonase-1 (PON1) prevents oxidation of low density lipoprotein cholesterol (LDL-C) and hydrolyzes the oxidized form, therefore preventing the development of atherosclerosis. The polymorphisms of PON1 gene are known to affect the PON1 activity and thereby coronary artery disease (CAD) risk. As studies are lacking in North-West Indian Punjabi''s, a distinct ethnic group with high incidence of CAD, we determined PON1 activity, genotypes and haplotypes in this population and correlated them with the risk of CAD.

Methodology/Principal Findings

350 angiographically proven (≥70% stenosis) CAD patients and 300 healthy controls were investigated. PON1 activity was determined towards paraoxon (Paraoxonase; PONase) and phenylacetate (Arylesterase; AREase) substrates. In addition, genotyping was carried out by using multiplex PCR, allele specific oligonucleotide –PCR and PCR-RFLP methods and haplotyping was determined by PHASE software. The serum PONase and AREase activities were significantly lower in CAD patients as compared to the controls. All studied polymorphisms except L55M had significant effect on PONase activity. However AREase activity was not affected by them. In a logistic regression model, after adjustment for the conventional risk factors for CAD, QR (OR: 2.73 (1.57–4.72)) and RR (OR, 16.24 (6.41–41.14)) genotypes of Q192R polymorphism and GG (OR: 2.07 (1.02–4.21)) genotype of −162A/G polymorphism had significantly higher CAD risk. Haplotypes L-T-G-Q-C (OR: 3.25 (1.72–6.16)) and L-T-G-R-G (OR: 2.82 (1.01–7.80)) were also significantly associated with CAD.

Conclusions

In conclusion this study shows that CAD patients had lower PONase and AREase activities as compared to the controls. The coding Q192R polymorphism, promoter −162A/G polymorphism and L-T-G-Q-C and L-T-G-R-G haplotypes are all independently associated with CAD.     

Antibody-targeted chemotherapy of B-cell lymphoma using calicheamicin conjugated to murine or humanized antibody against CD22

Antibody-targeted chemotherapy with immunoconjugates of calicheamicin is a clinically validated strategy in cancer therapy. This study describes the selection of a murine anti-CD22 mAb, m5/44, as a targeting agent, its conjugation to a derivative of calicheamicin (CalichDM) via either acid-labile or acid-stable linkers, the antitumor activity of CalichDM conjugated to m5/44, and its subsequent humanization by CDR grafting. Murine IgG1 mAb m5/44 was selected based on its subnanomolar affinity for CD22 and ability to be internalized into B cells. CalichDM conjugated to m5/44 caused potent growth inhibition of CD22⁺ human B-cell lymphomas (BCLs) in vitro. The conjugate of m5/44 with an acid-labile linker was more potent than an acid-stable conjugate, a nonbinding conjugate with a similar acid-labile linker, or unconjugated CalichDMH in inhibiting BCL growth. CalichDM conjugated to m5/44 caused regression of established BCL xenografts in nude mice. In contrast, both unconjugated m5/44 and a nonbinding conjugate were ineffective against these xenografts. Based on the potent antitumor activity of m5/44-CalichDM conjugates, m5/44 was humanized by CDR grafting to create g5/44, an IgG4 anti-CD22 antibody. Both m5/44 and g5/44 bound CD22 with subnanomolar affinity. Competitive blocking with previously characterized murine anti-CD22 mAbs suggested that g5/44 recognizes epitope A located within the first N-terminal Ig-like domain of human CD22. Antitumor efficacy of CalichDM conjugated to g5/44 against BCL xenografts was more potent than its murine counterpart. Based on these results, a calicheamicin conjugate of g5/44, CMC-544, was selected for further development as a targeted chemotherapeutic agent for the treatment of B-cell malignancies.Abbreviations AcBut 4-(4-Acetylphenoxy) butanoic acid - AcPAc (3-Acetylphenyl) acetic acid - ATC Antibody-targeted chemotherapy - BCL B-cell lymphoma - CalichDM N-Acetyl--calicheamicin dimethyl disulfide derivative(s) - CalichDMA CalichDM acid - CalichDMH CalichDM hydrazide - CDR Complementarity determining region - NHL Non-Hodgkins lymphoma - PBMC Peripheral blood mononuclear cell - TAA Tumor-associated antigen     

Immunological studies on peroxynitrite modified human DNA

Peroxynitrite (ONOO(-)) is a strong and potent oxidizing and nitrating agent, formed by rapid reaction of two highly reactive, nitric oxide and superoxide anion. The action of peroxynitrite generated by synergistic action of diethylamine NONOate (a nitric oxide donor) and 1,4-hydroquinone (a superoxide donor), on human placental DNA was monitored by ultraviolet and fluorescence spectroscopy, melting temperature studies, S1 nuclease digestibility and alkaline agarose electrophoresis. The peroxynitrite modified human DNA (ONOO(-)-DNA) was found to be highly immunogenic in rabbits inducing high titre immunogen specific antibodies. However, the induced antibodies exhibited appreciable cross-reactivity with various polynucleotides and nucleic acids. The data demonstrate that the antibodies, though cross-reactive, preferentially bind ONOO(-)-modified epitopes on DNA. Visual detection of immune complex formation with native and ONOO(-)-DNA reiterated preferential binding with modified human DNA. DNA modified by ONOO(-) presents unique epitopes which may be one of the factors for the induction of autoantibodies in cancer patients.     

Genetic transformation technology: Status and problems

Summary Transfer of genes from heterologous species provides the means of selectively introducing new traits into crop plants and expanding the gene pool beyond what has been available to traditional breeding systems. With the recent advances in genetic engineering of plants, it is now feasible to introduce into crop plants, genes that have previously been inaccessible to the conventional plant breeder, or which did not exist in the crop of interest. This holds a tremendous potential for the genetic enhancement of important food crops. However, the availability of efficient transformation methods to introduce foreign DNA can be a substantial barrier to the application of recombinant DNA methods in some crop plants. Despite significant advances over the past decades, development of efficient transformation methods can take many years of painstaking research. The major components for the development of transgenic plants include the development of reliable tissue culture regeneration systems, preparation of gene constructs and efficient transformation techniques for the introduction of genes into the crop plants, recovery and multiplication of transgenic plants, molecular and genetic characterization of transgenic plants for stable and efficient gene expression, transfer of genes to elite cultivars by conventional breeding methods if required, and the evaluation of transgenic plants for their effectiveness in alleviating the biotic and abiotic stresses without being an environmental biohazard. Amongst these, protocols for the introduction of genes, including the efficient regeneration of shoots in tissue cultures, and transformation methods can be major bottlenecks to the application of genetic transformation technology. Some of the key constraints in transformation procedures and possible solutions for safe development and deployment of transgenic plants for crop improvement are discussed.     

Induction of transplantation tolerance in non-human primate preclinical models

Short-term outcomes following organ transplantation have improved considerably since the availability of cyclosporine ushered in the modern era of immunosuppression. In spite of this, many of the current limitations to progress in the field are directly related to the existing practice of relatively non-specific immunosuppression. These include increased risks of opportunistic infection and cancer, and toxicity associated with long-term immunosuppressive drug exposure. In addition, long-term graft loss continues to result in part from a failure to adequately control the anti-donor immune response. The development of a safe and reliable means of inducing tolerance would ameliorate these issues and improve the lives of transplant recipients, yet given the improving clinical standard of care, the translation of new therapies has become appropriately more cautious and dependent on increasingly predictive preclinical models. While convenient and easy to use, rodent tolerance models have not to date been reliably capable of predicting a therapy's potential efficacy in humans. Non-human primates possess an immune system that more closely approximates that found in humans, and have served as a more rigorous preclinical testing ground for novel therapies. Prior to clinical adaptation therefore, tolerance regimens should be vetted in non-human primates to ensure that there is sufficient potential for efficacy to justify the risk of its application.     

Alpha-synuclein and parkin contribute to the assembly of ubiquitin lysine 63-linked multiubiquitin chains

Mutations in alpha-synuclein, Parkin, and UCH-L1 cause heritable forms of Parkinson disease. Unlike alpha-synuclein, for which no precise biochemical function has been elucidated, Parkin functions as a ubiquitin E3 ligase, and UCH-L1 is a deubiquitinating enzyme. The E3 ligase activity of Parkin in Parkinson disease is poorly understood and is further obscured by the fact that multiubiquitin chains can be formed through distinct types of linkages that regulate diverse cellular processes. For instance, ubiquitin lysine 48-linked multiubiquitin chains target substrates to the proteasome, whereas ubiquitin lysine 63-linked chains control ribosome function, protein sorting and trafficking, and endocytosis of membrane proteins. It is notable in this regard that ubiquitin lysine 63-linked chains promote the degradation of membrane proteins by the lysosome. Because both Parkin and alpha-synuclein can regulate the activity of the dopamine transporter, we investigated whether they influenced ubiquitin lysine 63-linked chain assembly. These studies revealed novel biochemical activities for both Parkin and alpha-synuclein. We determined that Parkin functions with UbcH13/Uev1a, a dimeric ubiquitin-conjugating enzyme, to assemble ubiquitin lysine 63-linked chains. Our results and the results of others indicate that Parkin can promote both lysine 48- and lysine 63-linked ubiquitin chains. alpha-Synuclein also stimulated the assembly of lysine 63-linked ubiquitin chains. Because UCH-L1, a ubiquitin hydrolase, was recently reported to form lysine 63-linked conjugates, it is evident that three proteins that are genetically linked to Parkinson disease can contribute to lysine 63 multiubiquitin chain formation.     

Disease-related modifications in tau affect the interaction between Fyn and Tau

Microtubule-associated protein tau is the major component of the neurofibrillary tangles of Alzheimer disease (AD) and is genetically linked to frontotemporal dementias (FTDP-17). We have recently shown that tau interacts with the SH3 domain of Fyn, an Src family non-receptor tyrosine kinase, and is tyrosine-phosphorylated by Fyn on Tyr-18. Also, tyrosine-phosphorylated tau is present in the neuropathology of AD. To determine whether alterations in the tau-Fyn interaction might correlate with disease-related factors in AD and FTDP-17, we have performed real-time surface plasmon resonance studies on a panel of 21 tau constructs with Fyn SH3. We report that the interaction between Fyn SH3 and 3R-tau was 20-fold higher than that with 4R-tau. In addition, the affinity between 4R-tau and Fyn SH3 was increased 25-45-fold by phosphorylation-mimicking mutations or by FTDP-17 mutations. In vitro kinase reactions show that tau, with lower affinity SH3 interactions, exhibited a lower level of Tyr-18 phosphorylation under our reaction conditions. Lastly, we have demonstrated that tau is phosphorylated on Tyr-18 in the tau P301L mouse model for tauopathy (JNPL3). In summary, our results suggest that disease-related phosphorylation and missense mutations of tau increase association of tau with Fyn. Because these effects are mediated through the 4R component of the tau population, these results also have implications for the FTDP-17 diseases caused by increased expression of 4R-tau. Our data support a role for the Fyn-tau interaction in neurodegeneration.