Purification of tryptic peptides for mass spectrometry using polyvinylidene fluoride membrane.

A simple procedure for the purification of tryptic peptides, prior to mass spectrometric analysis, using polyvinylidene fluoride membrane (PVDF) is described. The sensitivity of mass spectrometric analysis is such that minor impurities in tryptic peptide digests suppress the signal obtained. However, we obtained useful signal, from a sample that did not yield any spectra earlier, by purifying the sample using PVDF membrane. For this, the tryptic peptide digest was first spotted on the membrane which was then air-dried and washed. Further, the membrane was extracted with trifluoroacetic acid (TFA) and acetonitrile and subjected to mass spectrometric analysis. This procedure enabled us to identify a cross-reactive D1 antigen on the neutrophil surface that bound antibodies that targeted 60 kD Ro autoantigen in systemic lupus erythematosus, an autoimmune disorder.     

Religion,ethnicity and politics: Hindu and Muslim Indian immigrants in the United States

This article focuses on the political struggles between Hindu and Muslim Indian immigrant groups in the United States over the definition of "Indianness". Hindu Indian American organizations define India as a Hindu society and are strong supporters of the Hindu nationalist movement in India. Muslim Indian American organizations, on the other hand, view India as a multi-religious and multicultural society. They are striving to safeguard India's secularism and towards this end, have entered into coalitional relationships with lower caste groups. Both types of organizations are working to influence American and Indian politics in line with their respective interests, leading to an exacerbation of the conflict between the two immigrant groups. This article examines the reasons for this development and its implications, both for the development of an Indian American community in the United States and for religion and politics in India.     

Mouse urine collection using clear plastic wrap

Qualitative urinalysis using Multistix reagent strips for the detection of urinary pH, protein, glucose, bilirubin, blood, ketone, urobilinogen and creatinine can be carried out with a few drops of mouse urine. The use of metabolic cages is not practical for such qualitative studies particularly when several animals are involved. Here we describe two different methods for collecting pure mouse urine. The single animal method (SAM) involves allowing a single mouse to urinate on Glad cling wrap outside of the animal cage. The multiple animal method (MAM) involves partitioning seven mice into seven different make-shift compartments laid out on top of the cling wrap and allowing them to urinate. The voided urine, in each case, is then aspirated into micro-centrifuge tubes using a Pipetman. Without coercion pure urine was obtained as early as 12 s. Volumes in the range of 10-250 microl were obtained. Modifications of the SAM could prove useful for rat or mouse urine collection under conditions of microgravity.     

Location of immunization and interferon-γ are central to induction of salivary gland dysfunction in Ro60 peptide immunized model of Sjögren's syndrome

Introduction

Anti-Ro antibodies can be found in the serum of the majority of patients with Sjögren''s syndrome (SS). Immunization with a 60-kDa Ro peptide has been shown to induce SS-like symptoms in mice. The aim of this study was to investigate factors involved in salivary gland (SG) dysfunction after immunization and to test whether the induction of SS could be improved.

Methods

Ro60 peptide immunization was tested in Balb/c mice, multiple antigenic peptide (MAP)-Ro60 and Pertussis toxin (PTX) were tested in SJL/J mice. In addition, two injection sites were compared in these two strains: the abdominal area and the tailbase. Each group of mice was tested for a loss of SG function, SG lymphocytic infiltration, anti-Ro and anti-La antibody formation, and cytokine production in cultured cells or homogenized SG extracts.

Results

Ro60 peptide immunization in the abdominal area of female Balb/c mice led to impaired SG function, which corresponded with increased Th1 cytokines (IFN-γ and IL-12) systemically and locally in the SG. Moreover, changing the immunization conditions to MAP-Ro60 in the abdominal area, and to lesser extend in the tailbase, also led to impaired SG function in SJL/J mice. As was seen in the Balb/c mice, increased IFN-γ in the SG draining lymph nodes accompanied the SG dysfunction. However, no correlation was observed with anti-MAP-Ro60 antibody titers, and there was no additional effect on disease onset or severity.

Conclusions

Effective induction of salivary gland dysfunction after Ro60 peptide immunization depended on the site of injection. Disease induction was not affected by changing the immunization conditions. However, of interest is that the mechanism of action of Ro60 peptide immunization appears to involve an increase in Th1 cytokines, resulting in the induction of SG dysfunction.     

Oxidatively modified autoantigens in autoimmune diseases

Free radical-mediated oxidative damage and consequent protein modification by the end products of oxidative damage are important mediators of cell toxicity and disease pathogenesis. Aldehydic products, mainly the 4-hydroxy-2-alkenals, form adducts with proteins and make them highly immunogenic. Oxidative modification of proteins has been shown to elicit antibodies in a variety of diseases including systemic lupus erythematosus (SLE), alcoholic liver disease, diabetes mellitus (DM), and rheumatoid arthritis (RA). Oxidatively modified DNA (8-oxodeoxyguanine) and low-density lipoproteins (LDL) occur in SLE, a disease in which premature atherosclerosis is a serious problem. In addition, immunization with 4-hydroxy-2-nonenal (HNE)-modified 60-kDa Ro autoantigen elicits an accelerated epitope spreading in an animal model of SLE. Advanced glycation end product (AGE) pentosidine and AGE-modified IgG have been shown to correlate with RA disease activity. Oxidatively modified glutamic acid decarboxylase is important in type 1 DM, while autoantibodies against oxidized LDL are prevalent in Behcet's disease. The fragmentation of scleroderma-specific autoantigens occurs as a result of oxidative modification and is thought to be responsible for the production of autoantibodies through the release of cryptic epitopes. In the face of overwhelming evidence for the involvement of oxidative damage in autoimmunity the administration of antioxidants is a viable untried alternative for preventing or ameliorating autoimmune disease, although results in cardiovascular disease are disappointing.     

Principal component analysis and artificial neural network analysis of oral tissue fluorescence spectra: classification of normal premalignant and malignant pathological conditions

Pulsed laser-induced autofluorescence spectroscopic studies of pathologically certified normal, premalignant, and malignant oral tissues were carried out at 325 nm excitation. The spectral analysis and classification for discrimination among normal, premalignant, and malignant conditions were performed using principal component analysis (PCA) and artificial neural network (ANN) separately on the same set of spectral data. In case of PCA, spectral residuals, Mahalanobis distance, and scores of factors were used for discrimination among normal, premalignant, and malignant cases. In ANN, parameters like mean, spectral residual, standard deviation, and total energy were used to train the network. The ANN used in this study is a classical multiplayer feed-forward type with a back-propagation algorithm for the training of the network. The specificity and sensitivity were determined in both classification schemes. In the case of PCA, they are 100 and 92.9%, respectively, whereas for ANN they are 100 and 96.5% for the data set considered.     

Identification of lanthionine synthase C-like protein-1 as a prominent glutathione binding protein expressed in the mammalian central nervous system

Proteomic experiments were performed to identify novel glutathione (GSH) binding proteins expressed in the mammalian central nervous system. Bovine brain lysate was affinity purified using an immobilized glutathione-Sepharose column. Proteins that bound the immobilized glutathione were eluted with free glutathione and identified by one- and two-dimensional electrophoresis coupled with mass spectrometric analysis of tryptic fragments. Major proteins purified by this technique were glutathione S-transferase-mu (GST-mu) and GST-pi and lanthionine synthase C-like protein-1 (LanCL1). LanCL1 is a mammalian homologue of a prokaryotic enzyme responsible for the synthesis of thioether (lanthionine) cross-links within nascent polypeptide chains, yielding macrocyclic proteins with potent microbicidal activity. An antibody against LanCL1 was generated and applied to immunochemical studies of spinal cord tissue from SOD1G93A transgenic mice, a model for amyotrophic lateral sclerosis (ALS), wherein LanCL1 expression was found to be increased at presymptomatic stages of the disease. These results indicate LanCL1 is a glutathione binding protein possibly significant to neurodegenerative disease.     

Risk Factors for MDR and XDR-TB in a Tertiary Referral Hospital in India

Background

India has a high burden of drug resistant TB, although there are few data on XDR-TB. Although XDR-TB has existed previously in India, the definition has not been widely applied, and surveillance using second line drug susceptibility testing has not been performed. Our objective was to analyze clinical and demographic risk factors associated with isolation of MDR and XDR TB as compared to susceptible controls, at a tertiary center.

Methodology/Findings

Retrospective chart review based on positive cultures isolated in a high volume mycobacteriology laboratory between 2002 and 2007. 47 XDR, 30 MDR and 117 susceptible controls were examined. Drug resistant cases were less likely to be extrapulmonary, and had received more previous treatment regimens. Significant risk factors for XDR-TB included residence outside the local state (OR 7.43, 3.07-18.0) and care costs subsidized (OR 0.23, 0.097-0.54) in bivariate analysis and previous use of a fluoroquinolone and injectable agent (other than streptomycin) (OR 7.00, 95% C.I. 1.14-43.03) and an initial treatment regimen which did not follow national guidelines (OR 5.68, 1.24-25.96) in multivariate analysis. Cavitation and HIV did not influence drug resistance.

Conclusions/Significance

There is significant selection bias in the sample available. Selection pressure from previous treatment and an inadequate initial regimen increases risk of drug resistance. Local patients and those requiring financial subsidies may be at lower risk of XDR-TB.