• Volume 28, Issue 2

      March 2003,   pages  135-247

    • Clipboard: Avian origins revisited

      Dominique G Homberger

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    • Clipboard: Midwifery and assisted reproduction in Dictyostelium and Entamoeba

      Anuradha Lohia

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    • Commentary: Mysteries of the smallpox vaccine

      Sanjoy Bhattacharya

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    • Commentary: India’s Biological Diversity Act 2002: An act for the new millenium

      Madhav Gadgil

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    • Integrative microbiology – the third Golden Age

      Moselio Schaechter

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    • Sample size for collecting germplasms – a polyploid model with mixed mating system

      R L Sapra Prem Narain S V S Chauhan S K Lal B B Singh

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      The present paper discusses a general expression for determining the minimum sample size (plants) for a given number of seeds or vice versa for capturing multiple allelic diversity. The model considers sampling from a large 2 k-ploid population under a broad range of mating systems. Numerous expressions/results developed for germplasm collection/regeneration for diploid populations by earlier workers can be directly deduced from our general expression by assigning appropriate values of the corresponding parameters. A seed factor which influences the plant sample size has also been isolated to aid the collectors in selecting the appropriate combination of number of plants and seeds per plant. When genotypic multiplicity of seeds is taken into consideration, a sample size of even less than 172 plants can conserve diversity of 20 alleles from 50,000 polymorphic loci with a very large probability of conservation (0.9999) in most of the cases.

    • Molecular screening for Yq microdeletion in men with idiopathic oligozoospermia and azoospermia

      Rima Dada N P Gupta K Kucheria

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      Infertility affects 15% couples attempting pregnancy and in 40–50% of these cases the male partner has qualitative or quantitative abnormalities of sperm production. Microdeletions in the azoospermia factor (AZF) region on the long arm of the Y chromosome are known to be associated with spermatogenic failure and have been used to define three regions on Yq (AZFa, AZFb and AZFc) which are critical for spermatogenesis and are recurrently deleted in infertile males. Semen analysis was carried out on one hundred and twenty five infertile males with oligozoospermia and azoospermia. Cytogenetic analysis was done for all the cases and in all cytogenetically normal cases (𝑛 = 83) microdeletion analysis was carried out on DNA extracted from peripheral blood using PCR. The sequence tagged sites (STS) primers sY84, sY86 (AZFa); sY127, sY134 (AZFb); sY254, sY255 (AZFc) were used for each case. Eight of the eighty three cases (9.63%) showed deletion of at least one of the STS markers. Correlation of phenotype with microdeletion was done in each case to determine any phenotype association with deletion of particular AZF locus. Based on the present study, the frequency of microdeletion in the Indian population is 9.63%. This study emphasizes the need for PCR analysis for determining genetic aetiology in cases with idiopathic severe testiculopathy.

    • Sequence analysis corresponding to the PPE and PE proteins in Mycobacterium tuberculosis and other genomes

      Swathi Adindla Lalitha Guruprasad

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      Amino acid sequence analysis corresponding to the PPE proteins in H37Rv and CDC1551 strains of the Mycobacterium tuberculosis genomes resulted in the identification of a previously uncharacterized 225 amino acidresidue common region in 22 proteins. The pairwise sequence identities were as low as 18%. Conservation of amino acid residues was observed at fifteen positions that were distributed over the whole length of the region. The secondary structure corresponding to this region is predicted to be a mixture of 𝛼-helices and 𝛽-strands. Although the function is not known, proteins with this region specific to mycobacterial species may be associated with a common function. We further observed another group of 20 PPE proteins corresponding to the conserved C-terminal region comprising 44 amino acid residues with GFxGT and PxxPxxW sequence motifs. This region is preceded by a hydrophobic region, comprising 40–100 amino acid residues, that is flanked by charged amino acid residues. Identification of conserved regions described above may be useful to detect related proteins from other genomes and assist the design of suitable experiments to test their corresponding functions. Amino acid sequence analysis corresponding to the PE proteins resulted in the identification of tandem repeats comprising 41–43 amino acid residues in the C-terminal variable regions in two PE proteins (Rv0978 and Rv0980). These correspond to the AB repeats that were first identified in some proteins of the Methanosarcina mazei genome, and were demonstrated as surface antigens. We observed the AB repeats also in several other proteins of hitherto uncharacterized function in Archaea and Bacteria genomes. Some of these proteins are also associated with another repeat called the C-repeat or the PKD-domain comprising 85 amino acid residues. The secondary structure corresponding to the AB repeat is predicted mainly as 4 𝛽-strands. We suggest that proteins with AB repeats in Mycobacterium tuberculosis and other genomes may be associated as surface antigens. The M. leprae genome, however, does not contain either the AB or C-repeats and different proteins may therefore be recruited as surface antigens in the M. leprae genome compared to the M. tuberculosis genome.

    • Expression of the neurotrophin receptors Trk A and Trk B in adult human astrocytoma and glioblastoma

      Shashi Wadhwa Tapas C Nag Anupam Jindal Rahul Kushwaha Ashok K Mahapatra Chitra Sarkar

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      Neurotrophins and their receptors of the Trk family play a critical role in proliferation, differentiation and survival of the developing neurons. There are reports on their expression in neoplasms too, namely, the primitive neuroectodermal tumours of childhood, and in adult astrocytic gliomas. The involvement of Trk receptors in tumour pathogenesis, if any, is not known. With this end in view, the present study has examined 10 tumour biopsy samples (identified as astrocytoma, pilocytic astrocytoma and glioblastoma) and peritumoral brain tissue of adult patients, for the presence of Trk A and Trk B receptors, by immunohistochemistry. The nature of the tumour samples was also confirmed by their immunoreactivity (IR) to glial fibrillary acidic protein. In the peritumoral brain tissue, only neurons showed IR for Trk A and Trk B. On the contrary, in the tumour sections, the IR to both receptors was localized in the vast majority of glia and capillary endothelium. There was an obvious pattern of IR in these gliomas: high levels of IR were present in the low-grade (type I and II) astrocytoma; whereas in the advanced malignant forms (WHO grade IV giant cell glioblastoma and glioblastoma multiforme) the IR was very weak. These findings suggest that Trk A and Trk B are involved in tumour pathogenesis, especially in the early stage, and may respond to signals that elicit glial proliferation, and thus contribute to progression towards malignancy.

    • Reduced meiotic fitness in hybrids with heterozygosity for heterochromatin in the speciating Mus terricolor complex

      Tikaram Sharma Amit Bardhan Min Bahadur

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      Mus terricolor I, II and III are the three chromosomal species which differ in stable autosomal short-arm heterochromatin variations established in homozygous condition. Analysis of meiosis in the laboratorygenerated F1 male hybrids from crosses (both ways) between M. terricolor I and II and between M. terricolor I and III shows high frequencies of pairing abnormalities at pachytene. The backcross (N3 generation) male hybrids between M. terricolor I and II have meiotic abnormalities as in the F1 male hybrids, though to a lesser extent. They show difference in pairing abnormalities in the different karyotypic forms; the backcross hybrids heterozygous for the heterochromatic short arms have more anomalies compared to the homokaryotypic hybrids. This suggests a negative influence of the heterochromatin heterozygosity in meiotic pairing. The results indicate a role for heterochromatin variations in the development of a reproductive barrier in the speciating M. terricolor complex.

    • Genetic analysis of Karnal bunt (Neovossia indica) resistance in wheat

      M Kumar O P Luthra V Chawla N R Yadav R Kumar A Khar

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      Embryos excised from seeds of six generations (P1, P2, F1, BC1, BC2 and F2) of a cross WH 283 × WH 533 were cultured on modified MS medium already inoculated with secondary sporidia of Neovossia indica. Significant variations for callusing response (CR) (54.55–75.55%) were observed among generations but the presence or absence of N. indicia did not affect callusing response. A clear inhibition zone (IZ) was formed around each embryo showing callusing. The diameter of IZ varied significantly among generations and was maximum in the resistant genotype, WH 283 (3.60 cm). Fresh weight and dry weight of calli, initiated from embryo cultured and inoculated with N. indica, varied significantly among generations. Coefficient of infection as well as percentage of infection reflected the overdominance of susceptibility. Generation mean analysis showed that the three parameter model was adequate for diameter of IZ only. Six-parameter model showed that additive (in presence of N. indica), additive and additive × dominance (in absence of N. indica) effects were also significant. Complementary type of epistasis for fresh weight of calli and dominance, and dominance × dominance effects for dry weight of calli were observed in the presence of N. indica. Magnitude of additive effects was higher for diameter of IZ in three parameter model. Therefore, selection might assist in improving this trait and thus indirectly help in attaining the resistance towards N. indica.

    • Role of matrix metalloprotease-2 in oxidant activation of Ca2+ ATPase by hydrogen peroxide in pulmonary vascular smooth muscle plasma membrane

      Malay Mandal Sudip Das Tapati Chakraborti Amritlal Mandal Sajal Chakraborti

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      Exposure of bovine pulmonary artery smooth muscle plasma membrane suspension with the oxidant H2O2 (1 mM) stimulated Ca2+ATPase activity. We sought to determine the role of matrix metalloprotease-2 (MMP-2) in stimulating Ca2+ATPase activity by H2O2 in the smooth muscle plasma membrane. The smooth muscle membrane possesses a Ca2+-dependent protease activity in the gelatin containing zymogram having an apparent molecular mass of 72 kDa. The 72 kDa protease activity was found to be inhibited by EGTA, 1 : 10-phenanthroline, 𝛼2-macroglobulin and tissue inhibitor of metalloprotease-2 (TIMP-2) indicating that the Ca2+-dependent 72 kDa protease is the MMP-2. Western immunoblot studies of the membrane suspension with polyclonal antibodies of MMP-2 and TIMP-2 revealed that MMP-2 and TIMP-2, respectively, are the ambient matrix metalloprotease and the corresponding tissue inhibitor of metalloprotease in the membrane.

      In addition to increasing the Ca2+ATPase activity, H2O2 also enhanced the activity of the smooth muscle plasma membrane associated protease activity as evidenced by its ability to degrade 14C-gelatin. The protease activity and the Ca2+ATPase activity were prevented by the antioxidant, vitamin E, indicating that the effect produced by H2O2 was due to reactive oxidant species(es). Both basal and H2O2 stimulated MMP-2 activity and Ca2+ATPase activity were inhibited by the general inhibitors of matrix metalloproteases: EGTA, 1 : 10-phenanthroline, 𝛼2-macroglobulin and also by TIMP-2 (the specific inhibitor of MMP-2) indicating that H2O2 increased MMP-2 activity and that subsequently stimulated Ca2+ATPase activity in the plasma membrane. This was further confirmed by the following observations:

      1. adding low doses of MMP-2 or H2O2 to the smooth muscle membrane suspension caused submaximal increase in Ca2+ATPase activity, and pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity;
      2. combined treatment of the membrane with low doses of MMP-2 and H2O2 augments further the Ca2+ATPase activity caused by the respective low doses of either H2O2 or MMP-2; and
      3. pretreatment with TIMP-2 prevents the increase in Ca2+ATPase activity in the membrane caused by the combined treatment of MMP-2 and H2O2.
    • Regulation of glucose utilization and lipogenesis in adipose tissue of diabetic and fat fed animals: Effects of insulin and manganese

      Najma Z Baquer M Sinclair S Kunjara Umesh C S Yadav P McLean

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      In order to evaluate the modulatory effects of manganese, high fat diet fed and alloxan diabetic rats were taken and the changes in the glucose oxidation, glycerol release and effects of manganese on these parameters were measured from adipose tissue. An insulin-mimetic effect of manganese was observed in the adipose tissue in the controls and an additive effect of insulin and manganese on glucose oxidation was seen when Mn2+ was added in vitro. The flux of glucose through the pentose phosphate pathway and glycolysis was significantly decreased in high fat fed animals. Although the in vitro addition of Mn2+ was additive with insulin when 14CO2 was measured from control animals, it was found neither in young diabetic animals (6–8 weeks old) nor in the old (16 weeks old). Both insulin and manganese caused an increased oxidation of carbon-1 of glucose and an increase of its incorporation into 14C-lipids in the young control animals; the additive effect of insulin and manganese suggests separate site of action. This effect was decreased in fat fed animals, diabetic animals and old animals. Manganese alone was found to decrease glycerol in both the control and diabetic adipose tissue in in vitro incubations. The results of the effects of glucose oxidation, lipogenesis, and glycerol release in adipose tissue of control and diabetic animals of different ages are presented together with the effect of manganese on adipose tissue from high fat milk diet fed animals.

    • Purification and characterization of a Ca2+ -dependent/calmodulin-stimulated protein kinase from moss chloronema cells

      Jacinta S D’souza Man Mohan Johri

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      We have demonstrated the presence of a Ca2+-dependent/calmodulin-stimulated protein kinase (PK) in chloronema cells of the moss Funaria hygrometrica. The kinase, with a molecular mass of 70,000 daltons (PK70), was purified to homogeneity using ammonium sulphate fractionation, DEAE-cellulose chromatography, and calmodulin (CaM)-agarose affinity chromatography. The kinase activity was stimulated at a concentration of 50 𝜇M free Ca2+, and was further enhanced 3–5-fold with exogenously added 3–1000 nm moss calmodulin (CaM). Autophosphorylation was also stimulated with Ca2+ and CaM. Under in vitro conditions, PK70 phosphorylated preferentially lysine-rich substrates such as HIIIS and HVS. This PK shares epitopes with the maize Ca2+-dependent/calmodulin-stimulated PK (CCaMK) and also exhibits biochemical properties similar to the maize, lily, and tobacco CCaMK. We have characterized it as a moss CCaMK.

    • Metabolic characteristics of an aerobe isolated from a methylotrophic methanogenic enrichment culture

      Stephen V Rapheal K R Swaminathan K Lalitha

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      An anaerobic methylotrophic methanogenic enrichment culture, with sustained metabolic characteristics, including that of methanation for over a decade, was the choice of the present study on interspecies interactions. Growth and methanation by the enrichment were suppressed in the presence of antibiotics, and no methanogen grown on methanol could be isolated using stringent techniques. The present study confirmed syntrophic metabolic interactions in this enrichment with the isolation of a strain of Pseudomonas sp. The organism had characteristic metabolic versatility in metabolizing a variety of substrates including alcohols, aliphatic acids, amino acids, and sugars. Anaerobic growth was favoured with nitrate in the growth medium. Cells grown anaerobically with methanol, revealed maximal nitrate reductase activity. Constitutive oxidative activity of the membrane system emerged from the high-specific oxygen uptake and nitrate reductase activities of the aerobically and anerobically grown cells respectively. Cells grown anaerobically on various alcohols effectively oxidized methanol in the presence of flavins, cofactor FAD and the methanogenic cofactor F420, suggesting a constitutive alcohol oxidizing capacity. In cells grown anaerobically on methanol, the rate of methanol oxidation with F420 was three times that of FAD. Efficient utilization of alcohols in the presence of F420 is a novel feature of the present study. The results suggest that utilization of methanol by the mixed culture would involve metabolic interactions between the Pseudomonas sp. and the methanogen(s). Methylotrophic, methanogenic partnership involving an aerobe is a novel feature hitherto unreported among anaerobic syntrophic associations and is of ecological significance.

    • Mycelial colonization by bradyrhizobia and azorhizobia

      Gamini Seneviratne H S Jayasinghearachchi

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      This study examines mycelial colonization of common soil fungi by bradyrhizobia and an azorhizobial strain, resulting in the forming of biofilms. The effects of the fungal exudates on a bradyrhizobial strain have also been investigated. Bradyrhizobia gradually colonized the mycelia for about 18 days, after which the biofilm structures collapsed with the release of the rhizobial cell clusters to the culture medium. The azorhizobial strain showed differential colonization of the mycelia. In general, there were no considerable mycotoxin effects of the fungal exudates on the bradyrhizobial strain used, instead the rhizobial strain utilized the exudates as a source of nutrition. This study indicates that the present microbial association with biofilm formation has important implications in the survival of rhizobia under adverse soil conditions devoid of vegetation. Further, it could have developed an as yet unidentified nitrogen fixing system that could have contributed to the nitrogen economy of soils.

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