Immunodiagnosis of groundnut and watermelon bud necrosis viruses using polyclonal antiserum to recombinant nucleocapsid protein of Groundnut bud necrosis virus

In vitro gene expression strategy was used for the production of polyclonal antiserum to the nucleocapsid protein (NP) of Groundnut bud necrosis virus (GBNV). The GBNV NP gene from cowpea isolate was cloned into 6x His-tagged UA cloning vector and expressed in Escherichia coli [M15] cells. The fusion protein was detected in insoluble fraction and was purified by using Ni-NTA agarose resin. The purified 6x His-fusion protein (32 kDa) was used for immunisation to produce a high titre polyclonal antiserum. The antiserum to the NP of GBNV at 1:4000 dilution detected successfully natural infection of GBNV and Watermelon bud necrosis virus in a wide range of cucurbitaceous, leguminous and solanaceous hosts from different locations.     

Validity of self-report of recent opiate use in treatment setting

Self-report validity of recent drug use among heroin abusers depends on many factors including the population being studied and the setting in which the study is carried out. This study was conducted by the treating physicians to assess the self-report validity of recent heroin use by heroin dependent patients in the outdoor setting using 'thin layer chromatography' (TLC) and two highly sensitive methods of urinalysis viz. 'gas liquid chromatography' (GLC) and 'high performance liquid chromatography' (HPLC). Out of seventy-six heroin dependent patients who entered the study, 64 provided urine sample on the same day. Patients' self-report about recent opiate use was found to have a moderate agreement with urinalysis report. However, it is important to validate it with urinalysis during the treatment process because a substantial proportion of patients fails to report recent opiate use. It is recommended that all drug dependence treatment centres should be equipped with a sensitive urinalysis facility. Otherwise, the outcome of the treatment process should be considered with caution.     

Dystrophin gene deletions in South Indian Duchenne muscular dystrophy patients

66 unrelated patients from Southern India with Duchenne Muscular Dystrophy (DMD) were studied for intragenic deletion in 18 exons and Pm region of the DMD gene using multiplex PCR. Of these 41 (62.1%) showed intragenic deletions. 78% of the deletions were located at the distal hotspot region (44-55 exons) and 22% of the deletions were located at the proximal region (exon 2-19). Exon 50 is most frequently deleted. Deletions in isolated cases were significantly more compared to familial cases. The lower incidence reported from South India compared to North India, is suggestive of variations in the Southern and Northern population.     

Mortality trends in falciparum malaria--effect of gender difference and pregnancy

Falciparum malaria in pregnancy is a significant health problem in India. Pregnant women constitute an important high risk group for malaria infection which may cause abortions, stillbirths, intra-uterine growth retardation (IUGR) and premature labour. In this hospital based study on 602 admitted patients of falciparum malaria which included 314 males, 243 non-pregnant females and 45 pregnant females, there was significantly increased mortality rate in females (18.4%) in comparison to males (7.64%, p < 0.001). The mortality rate was highly significant in pregnant females (37.77%) in comparison to non-pregnant females (14.81%) and males (7.64%; p < 0.001). Severe anaemia with Hb < 5 gm% was observed more commonly in pregnant patients (20.0%) in comparison to non-pregnant patients (4.11%). Incidence of malaria infection was more in primi gravida and second gravida. Pregnancy related complications in the form of preterm live births, intra-uterine death (IUD), still births and abortions were more in primi parous than multiparous patients. As the pregnancy is associated with increased incidence and adverse outcome of P.falciparum malaria infection, chemoprophylaxis should be made an integral part of antenatal care along with antianaemia therapy to reduce the risk of serious maternal and fetal complications.     

Role of nitric oxide in the biology, physiology and pathophysiology of reproduction

Following its benchmark discovery, nitric oxide (NO) is nowknown to play important functional roles in a variety of physiologicalsystems. Within the vasculature, NO induces vasodilation, inhibitsplatelet aggregation, prevents neutrophil/platelet adhesionto endothelial cells, inhibits smooth muscle cell proliferationand migration, regulates programmed cell death (apoptosis) andmaintains endothelial cell barrier function. NO generated byneurons acts as a neurotransmitter, whereas NO generated bymacrophages in response to invading microbes acts as an antimicrobialagent. Because neurons, blood vessels and cells of the immunesystem are integral parts of the reproductive organs, and inview of the important functional role that NO plays in thosesystems, it is likely that NO is an important regulator of thebiology and physiology of the reproductive system. Indeed, inthe past 10 years, NO has established itself as a polyvalentmolecule which plays a decisive role in regulating multiplefunctions within the female as well as the male reproductivesystem. This review provides an overview of the role of NO invarious reproductive organs under physiological and pathologicalconditions.     

Detection of cyclin D1 in B cell lymphoproliferative disorders by flow cytometry

AIMS: To describe and revise a flow cytometric assay for evaluating cyclin D1 overexpression in B cell lymphoproliferative disorders (B-LPDs). METHODS: Cyclin D1 expression was evaluated in 11 healthy controls and 51 patients with B-LPD by flow cytometry using the 5D4 monoclonal antibody. In 25 cases, experiments were repeated up to four times with mononuclear cells (MNC) fixed in ethanol for 1-120 days to evaluate the consistency of cyclin D1 expression. Flow cytometry results were compared with fluorescence in situ hybridisation (FISH) for the t(11;14) translocation in 19 patients and with immunohistochemistry (IHC) using the DCS-6 monoclonal antibody in nine patients. RESULTS: A mean fluorescence intensity ratio (MFIR) of 4.8 was defined as the cut off point for positivity based on cyclin D1 expression in healthy controls (mean + 3 SD). Ten patients overexpressed cyclin D1 by flow cytometry. These included five of eight patients with mantle cell lymphoma, four of 19 with chronic lymphocytic leukaemia, and one with follicular lymphoma. MFIR in the repeat experiments differed less than 25% in 20 of 25 patients and in no cases did it cross the cut off point. There was a good correlation between cyclin D1 expression by flow cytometry and FISH for t(11;14) in 15 of 19 patients and six of nine had concordant results with flow cytometry, FISH, and IHC. CONCLUSION: Cyclin D1 expression remains fairly stable once MNC are fixed in ethanol and the flow cytometric assay can be used for the routine screening of B-LPD. Further comparisons between flow cytometry, IHC, and FISH may be needed to ascertain the diagnostic value of the flow cytometric assay.     

Differential host range of viruses of feline leukemia-sarcoma complex.

Selected strains of feline leukemia and sarcoma viruses of subgroups A, B, and C show a differential pattern in their ability to cross species barrier and productively infect cells of heterologous host species. A virus of subgroup B showed the widest host range; it caused productive infection of cells of diverse host species including cells from cat, human, monkey, dog, bovine, pig, and hamster species. Two virus strains of subgroup A showed the narrowest host range; of the cells of several mammalian species examined, they only caused productive infection of cat and dog cells. Preliminary studies indicated that certain other strains of subgroup A viruses cause productive infection of whole human embryo cells. One strain of subgroup C virus examined showed a host range that was intermediate between that of A and B subgroup viruses. This strain caused productive infection of cat, dog, and certain human cells. In addition, the subgroup C virus caused productive infection of guinea pig cells found to be resistant to subgroup A as well as subgroup B virus strains examined.Preliminary studies suggested that certain, but not all, virus mixtures of A and B viruses can be purified into B type by passage of virus in heterologous human cells.The factor(s) that may govern the differential susceptibilities of heterologous host cells to the described strains of feline viruses are discussed.     

Comparative analysis of the polycystic kidney disease 1 (PKD1) gene reveals an integral membrane glycoprotein with multiple evolutionary conserved domains

PKD1 is the major locus of the common genetic disorder autosomal dominant polycystic kidney disease (ADPKD). Analysis of the predicted protein sequence of the human PKD1 gene, polycystin, shows a large molecule with a unique arrangement of extracellular domains and multiple putative transmembrane regions. The precise function of polycystin remains unclear with a paucity of mutations to define key structural and functional domains. To refine the structure of this protein we have cloned the genomic region encoding the Fugu PKD1 gene. Fugu PKD1 spans 36 kb of genomic DNA and has greater complexity with 54 exons compared with 46 in man. Comparative analysis of the predicted protein sequences shows a lower level of homology than in similar studies with identity of 40 and 59% similarity. However key structural motifs including leucine rich repeats (LRR), a C-type lectin and LDL-A like domains and 16 PKD repeats are maintained. A region of homology with the sea urchin REJ protein was also confirmed in Fugu but found to extend over 1000 amino acids. Several highly conserved intra- and extra- cellular regions, with no known sequence homologies, that are likely to be of functional importance were detected. The likely structure of the membrane associated region has been refined with similarity to the PKD2 protein and voltage gated Ca2+ and Na+ channels highlighted over part of this area. The overall protein structure has therefore been clarified and this comparative analysis derived structure will form the basis for the functional study of polycystin and its individual domains.      

Fully automatic quantification of microarray image data

DNA microarrays are now widely used to measure expression levels and DNA copy number in biological samples. Ratios of relative abundance of nucleic acids are derived from images of regular arrays of spots containing target genetic material to which fluorescently labeled samples are hybridized. Whereas there are a number of methods in use for the quantification of images, many of the software systems in wide use either encourage or require extensive human interaction at the level of individual spots on arrays. We present a fully automatic system for microarray image quantification. The system automatically locates both subarray grids and individual spots, requiring no user identification of any image coordinates. Ratios are computed based on explicit segmentation of each spot. On a typical image of 6000 spots, the entire process takes less than 20 sec. We present a quantitative assessment of performance on multiple replicates of genome-wide array-based comparative genomic hybridization experiments. By explicitly identifying the pixels in each spot, the system yields more accurate estimates of ratios than systems assuming spot circularity. The software, called, runs on Windows platforms and is available free of charge for academic use.