Association of IL4 gene polymorphisms with asthma in North Indians

BACKGROUND: Asthma is a complex airway disorder, and a number of genetic loci have been found to be associated with asthma. The 5q31-33 region is one of the most important loci linked to asthma and atopic disorders. However, association studies with candidate genes in this region, such as IL4, were inconclusive, as both positive and negative results were obtained in several populations studied. The aim of our case-control study was to determine the association between IL4 and asthma in North Indians. PATIENTS AND METHODS: Polymorphisms in the promoter and a dinucleotide repeat in the 2nd intron in IL4 were genotyped by sequencing and GeneScan analysis, respectively, in ethnically matched, unrelated patients (n = 171) and controls (n = 128), following the guidelines of the American Thoracic Society. RESULTS: The proximal promoter region of the IL4 gene was found to be invariant. Previously reported polymorphisms, -590 C/T and +33 C/T, were found to be absent in our population. The chi2 test using only large expected cell counts (more than 5% of the sample size) showed a significant association between allele size and disease status (chi2 = 38.08, d.f. = 6, p < 0.05). In addition, a significant difference was observed for the allele and genotype frequencies (p < 0.0005 and p = 0.0009, respectively) in the patient and the control groups using the Fisher-Freeman-Halton test. CONCLUSION: Our studies indicate that the promoter of the IL4 gene is invariant in our population. The case-control studies on the CA repeat polymorphism in the 2nd intron of the IL4 gene have shown interesting results and indicate the need for further family-based studies.     

An improved method for utilization of peptide substrates for antibody characterization and enzymatic assays

Synthetic peptides have become an important tool in antibody production and enzyme characterization. The small size of peptides, however, has hindered their use in assays systems, such as Western blots, and as immunogens. Here, we present a facile method to improve the properties of peptides for multiple applications by ligating the peptides to intein-generated carrier proteins. The stoichiometric ligation of peptide and carrier achieved by intein-mediated protein ligation (IPL) results in the ligation product migrating as a single band on a SDS-PAGE gel. The carrier proteins, HhaI methylase (M.HhaI) and maltose-binding protein (MBP), were ligated to various peptides; the ligated carrier-peptide products gave sharp, reproducible bands when used as positive controls for antibodies raised against the same peptides during Western blot analysis. We further show that ligation of the peptide antigens to a different thioester-tagged carrier protein, paramyosin, produced immunogens for the production of antisera in rabbits or mice. Furthermore, we demonstrate the generation of a substrate for enzymatic assays by ligating a peptide containing the phosphorylation site for Abl protein tyrosine kinase to a carrier protein. This carrier-peptide protein was used as a kinase substrate that could easily be tested for phosphorylation using a phosphotyrosine antibody in Western blot analysis. These techniques do not require sophisticated equipment, reagents, or skills thereby providing a simple method for research and development.     

Molecular therapies for viral hepatitis

Current treatment modalities available for hepatitis B virus (HBV) or hepatitis C virus (HCV) infections are not efficient. The enormous disease burden caused by these two infections makes the development of novel therapies critical. For HCV, the development of an effective vaccine is urgent in view of the escalating number of infected individuals. Molecular therapies for HBV and HCV infection can be directed at reducing viral load by interfering with the life cycle of the viruses or at generating immune response against viral epitopes. The antiviral approaches consist of the delivery or expression of antisense RNAs, ribozymes or dominant negative proteins. Viral biology can be interrupted by attacking various potential targets within the two viruses. DNA-based vaccination strategies are being explored for both prevention and treatment of these diseases. Both non-viral and recombinant viral vectors are being developed for safe, effective and long-term gene transfer to the liver. Although no "ideal" vector is available at this time, the ingenuity of numerous investigators is leading to the improvement of the vector systems, promising successful application of gene therapy to the prevention and treatment of viral hepatitis in the foreseeable future.     

Classification and selection of biomarkers in genomic data using LASSO

High-throughput gene expression technologies such as microarrays have been utilized in a variety of scientific applications. Most of the work has been done on assessing univariate associations between gene expression profiles with clinical outcome (variable selection) or on developing classification procedures with gene expression data (supervised learning). We consider a hybrid variable selection/classification approach that is based on linear combinations of the gene expression profiles that maximize an accuracy measure summarized using the receiver operating characteristic curve. Under a specific probability model, this leads to the consideration of linear discriminant functions. We incorporate an automated variable selection approach using LASSO. An equivalence between LASSO estimation with support vector machines allows for model fitting using standard software. We apply the proposed method to simulated data as well as data from a recently published prostate cancer study.     

Congenital methemoglobinemia due to NADH-methemoglobin reductase deficiency in three Indian families

Congenital methemoglobinemia is a relatively rare clinical disorder characterized by life-long cyanosis, caused by either an inherited mutant hemoglobin (Hb-M) or deficiency of physiologically active NADH-dependent methemoglobin reductase (NADH-MR). NADH-MR deficiency leads to two different types of recessive congenital methemoglobinemia. In type I, cyanosis is the only major symptom and NADH-MR deficiency is restricted only to the red blood cells. In type II, cyanosis is associated with severe mental retardation and neurological impairment. The objective of this study is to establish the cause of cyanosis in our cases of congenital methaemoglobinemia. Erythrocyte NADH-MR activity was assayed spectrophotometrically. Spectral analysis of the hemolysate treated with potassium ferricyanide was recorded between 400-700 nm and Hb electrophoresis on starch gel at pH 7.0 was done to rule out the presence of Hb-M. NADH-MR deficiency was detected in 3 families. There was a history of consanguinity in one of these cases. The three propositi presented with breathlessness, fever and peripheral cyanosis. There was no history of cardiac illness or exposure to drugs and chemicals. There were no signs and symptoms of mental retardation. The presence of Hb-M was ruled out. Hb-A2, Hb-F, G6PD activity and reduced glutathione levels were normal. NADH-MR activity in all the cases ranged from 4.1 to 9.2 IU/g Hb (normal range 7.0-24.0 IU/g Hb). We describe NADH-MR deficiency in three unrelated cases (age 4 months to 6 years) where the activity of the enzyme was 30-40% of normal. These three cases of congenital methemoglobinemia are due to type-I NADH-MR deficiency without mental retardation.     

Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey

The ultrastructural characteristics of peri-implantation stage embryos recovered on day 6 after ovulation from rhesus monkeys with or without mifepristone (RU486) treatment during the early luteal phase were examined in the present study. Monkeys were randomly allocated to two groups; group 1 animals were injected s.c. with 2 ml vehicle (1:4, benzyl benzoate: olive oil, v/v, n = 21) and group 2 animals received a single dose of mifepristone (2 mg/kg body weight, w/v, n = 30) in the same volume of vehicle on day 2 after ovulation in mated cycles. On day 6 after ovulation, female monkeys of both groups were laparotomized and their reproductive tracts were flushed to retrieve preimplantation stage embryos. Embryos that showed frank degeneration or desynchrony on gross microscopical examination were not included in the present study. Preimplantation embryo growth on day 6 after ovulation was significantly (P < 0.05) affected in the morula-blastocyst transition stage in mifepristone-treated monkeys compared with that in the control group of monkeys. Ultrastructurally, administration of mifepristone on day 2 after ovulation depressed preimplantation stage embryo development, characterized by loss of cell polarity, lack of mitochondrial maturity, and lack of differentiation in trophoblast cells. Furthermore, preimplantation embryos from mifepristone-treated animals displayed a higher occurrence of inter-blastomere space, intra-cytoplasmic vacuoles, myelinoid bodies, accumulation of lipid droplets, lysosomes, lipofuscins, autophagosomes and multivesicular bodies. Collectively, it appears that the developmental potential of preimplantation embryos was significantly compromised in mifepristone-treated cycles.