Isolation and characterization of polymorphic microsatellite loci in yellowtail catfish, Pangasius pangasius (Hamilton, 1822)

A total of nine polymorphic microsatellite loci were obtained from a genomic library of Pangasius pangasius (order Siluriformes, family Pangasiidae). Samples from rivers Bhagirathi (n = 22) and Mahanadi (n = 20) were genotyped for each of the nine microsatellite loci to determine genetic variation. The mean number of alleles per locus was 5.22 in Bhagirathi and 5.78 in Mahanadi; and expected heterozygosity ranged from 0.567 (Bhagirathi) to 0.578 (Bhagirathi). Significant deviation (P < 0.003) from Hardy–Weinberg expectations was evident at three loci, Ppa2 (Bhagirathi), Ppa14 (Mahanadi) and Ppa28 (Bhagirathi and Mahanadi). The identified microsatellite loci were found to be promising for population genetics studies of P. pangasius.     

Molecular Cloning of Growth Hormone Complementary DNA in Barfin Flounder (Verasper moseri)

The olive flounder (family Paralichthidae; Paralichthys olivaceus) growth hormone (ofGH) appears to be the most derived among known growth hormones, with the deletion of 14 consecutive amino acids in the carboxy-terminal region. To ascertain if this deletion is common to all flounders, growth hormone complementary DNA of the barfin flounder (bfGH) (family Pleuronectidae; Verasper moseri) has been cloned. It was amplified by polymerase chain reaction using single-strand cDNA from the pituitary gland. Excluding the poly(A) tail, the bfGH cDNA is 919 nucleotides long and contains a 609-bp open reading frame encoding a putative signal peptide of 17 amino acids and a mature protein of 186 amino acids. Northern blot analysis detected 1.0 kb of bfGH messenger RNA in the pituitary gland, which is a reasonable value considering the poly(A) tail. The deduced amino acid sequence of bfGH has 78% identity with the sequence of ofGH. A major difference is the presence of a 14 amino acid segment (140–153) in bfGH, as in other growth hormones, suggesting that this deletion in the olive flounder occurred after the divergence of the Pleuronectoidae. Received May 7, 1999; accepted July 13, 1999.     

Microsatellite DNA markers for population-genetic studies of Labeo dyocheilus (McClelland, 1839)

Fifty‐four primers published for six cyprinid fishes were tested to amplify homologous microsatellite loci in Labeo dyocheilus. Fifteen primers yielded successful amplification and seven were polymorphic with 3–9 alleles. To evaluate utility of the identified loci in population genetic study, 84 samples were analysed. The samples were collected from four rivers viz. Beas, Satluj, Yamuna and Jiabharali. The four sample sets displayed different diversity levels, with observed heterozygosity from 0.34 to 0.53. Significant genotype heterogeneity (P < 0.001) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine‐scale population structure analysis of L. dyocheilus.     

Characterization of polymorphic microsatellite markers and genetic diversity in wild bronze featherback, Notopterus notopterus (Pallas, 1769)

Six polymorphic microsatellite DNA loci were identified in the primitive fish, bronze featherback, Notopterus notopterus for the first time and demonstrated significant population genetic structure. Out of the six primers, one primer (NN90) was specific to N. notopterus (microsatellite sequence within the RAG1 gene) and five primers were product of successful cross-species amplification. Sixty-four primers available from 3 fish species of order Osteoglossiformes and families Notopteridae and Osteoglossidae were tested to amplify homologous microsatellite loci in N. notopterus. Fifteen primer pairs exhibited successful cross-priming PCR product. However, polymorphism was detected only at five loci. To assess the significance of these six loci (including NN90) in population genetic study, 215 samples of N. notopterus from five rivers, viz Satluj, Gomti, Yamuna, Brahmaputra and Mahanadi were analyzed. The five sample sets displayed different diversity levels and observed heterozygosity ranged from 0.6036 to 0.7373. Significant genotype heterogeneity (P < 0.0001) and high F_ST (0.2205) over all loci indicated that the samples are not drawn from the same genepool. The identified microsatellite loci are promising for use in fine-scale population structure analysis of N. notopterus.     

Distributional records of Tor mahseer Tor tor (Hamilton, 1822) from Southern India

During exploratory surveys in the tributaries (Penganga and Satnala) of Godavari and (Bheema) Krishna basins, specimens of mahseer were collected. The morpho‐meristic characteristics of these specimens conformed to the taxonomic keys for Tor tor. The mitochondrial COI sequences of these specimens clustered with the T. tor specimens from the River Narmada and were distinct from the other mahseer such as T. khudree and T. mussullah, which are known to exist in the rivers of the region. This confirmed the distribution of T. tor in the rivers of peninsular India and indicated an extended distribution of the known range. The major predominating habitat characteristics of collection areas were cobbles mixed with gravel, and a riparian cover of shrubs and trees. The occurrence of fingerling size specimens in the river suggests that the species has adapted and is likely to have established self‐recruiting populations in these rivers.     

Development and characterization of a continuous macrophage cell line,LRTM, derived from thymus of Labeo rohita (Hamilton 1822)

A long-term thymic macrophage cell line from the thymus explants of Labeo rohita designated as LRTM (L. rohita thymic macrophages) was established, which has been maintained in culture for more than 1 yr. This cell line designated LRTM cells have been subcultured for 70 passages. The cells shape was initially long and elongated; with subsequent passages, the cells became short and epithelial like. The cells exhibited optimum growth in L-15 containing 10% fetal bovine serum and also in Dulbecco’s modified Eagle’s medium at 37°C with 5% CO₂ and showed 85+?% viability after 12 mo storage in liquid nitrogen. In addition, cells showed nonspecific esterase and surface expression of Fc receptors for immunoglobulin G and classes I and II major histocompatibility complex antigens. These observations confirmed that this cell line had the morphologic and functional features as a macrophage. The cells exhibited phagocytic activity by engulfing yeast cells as well as fluorescent latex beads, which was demonstrated by scanning electron microscopy and Giemsa staining. The long-term cultured cells show rapid production of reactive oxygen and nitrogen intermediates following stimulation with lipopolysaccharides and phorbol miristate acetate (PMA). Mostly, all the cells were alpha napthyl esterase acetate positive. After stimulation with PMA and lipopolysaccharide, cultured fish macrophages produced reactive oxygen and nitrogen intermediates. The karyotype analysis showed that these cells have a tetraploid karyotype with 100 chromosomes in each cell, indicating that they are normal L. rohita cells. Amplification, sequencing, and alignment of fragments of two mitochondrial genes 12S rRNA from rohu confirmed that the cell line originated from L. rohita. This cell line should be useful for studying the role of thymic macrophages in differentiation and maturation of thymocytes and can be source of macrophage-specific enzymes and cytokines. The macrophage cell line will be invaluable in studies of pathogen/macrophage interactions, the mechanisms of macrophage antimicrobial effector functions and the contribution of macrophages to the specific immune responses of teleosts.     

Cyclo(l-Pro-d-Arg): a new antibacterial and antitumour diketopiperazine from Bacillus cereus associated with a rhabditid entomopathogenic

In continuation of our search for new antimicrobial secondary metabolites from Bacillus cereus associated with rhabditid entomopathogenic nematode, a new microbial diketopiperazine, cyclo(l-Pro-d-Arg), was isolated from the ethyl acetate extract of fermented modified nutrient broth. The chemical structures of the isolated compounds were identified based on their 1D, 2D NMR and high-resolution electrospray ionisation–mass spectroscopy data. Antibacterial activity of the compound was determined by minimum inhibitory concentration and disc diffusion method against medically important bacteria, and the compound was recorded to have significant antibacterial activity against test bacteria. The highest activity was recorded against Klebsiella pneumoniae (1 μg/mL). Cyclo(l-Pro-d-Arg) was recorded to have significant antitumor activity against HeLa cells (IC₅₀ value 50 μg/mL), and this compound was recorded to have no cytotoxicity against normal monkey kidney cells (VERO) up to 100 μg/mL). To the best of our knowledge, this is the first time that cyclo(l-Pro-d-Arg) has been isolated from a microbial natural source.     

Genetic elements associated with antimicrobial resistance in enteropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (EPEC) from Brazil

Background  

We recently observed an association of resistance with a certain enteropathogenic Escherichia coli (EPEC) serotypes and identified a conjugative plasmid, similar to plasmid pED208, that was conserved among archival O111:H2/NM and O119:H2 strains of diverse geographical origin. In this study, we sought to determine the prevalence and distribution of this plasmid among a collection of EPEC isolates from Brazil, as well as to study the susceptibilities of these isolates to antimicrobial agents.     

Immunohistochemical localization of TLR2 and CD14 in gingival tissue of healthy individuals and patients with chronic periodontitis

We used immunohistochemistry to quantify and compare the expression of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14) in gingival tissues of both healthy individuals and patients with chronic periodontitis. We also correlated the expression of TLR2 and CD14 with the histological grades of chronic periodontitis. We examined 30 gingival specimens from chronic periodontitis patients and 10 from healthy individuals. Tissues from both groups were immunostained with antibodies against TLR2 and CD14. TLR2 and CD14 were expressed by endothelial cells, fibroblasts, lymphocytes and plasma cells. The immunohistochemical expression of TLR2 and CD14 was significantly greater in inflammatory cells of the chronic periodontitis group than in healthy individuals. Expression of these molecules was greater in the inflammatory cells of connective tissue adjacent to pocket epithelium in both groups. The expression of TLR2 and CD14 was greatest in the periodontitis group that was classified as severe grade, followed by moderate and mild grades, which suggests a role of TLR2 and CD14 in the pathogenesis of chronic periodontitis. The positive correlation of TLR2 and CD14 expression levels with the severity grades of chronic periodontitis suggests that they are correlated also with disease severity; therefore, they may be useful for predicting disease progression. Our findings are consistent with the possibility that CD14 acts as a co-receptor for TLR2.     

Anticonvulsant mechanisms of piperine,a piperidine alkaloid

Piperine, a natural compound isolated from the fruits of Piper, is known to modulate several neurotransmitter systems such as serotonin, norepinephrine, and GABA, all of which have been linked to the development of convulsions. Fruits of Piper species have been suggested as means for managing seizure disorders. The present study was designed to elucidate the anticonvulsant effect of piperine and its mechanisms of action using in-silico, in-vivo and in-vitro techniques.PASS software was used to determine its possible activity and mechanisms. Furthermore the latency for development of convulsions and mortality rate was recorded in different experimental mouse models of epilepsy (pentylenetetrazole, maximal electroshock, NMDA, picrotoxin, bicuculline, BAYK-8644, strychnine-induced convulsions) after administration of various doses of piperine (5, 10 and 20 mg/kg, i.p.). Finally, the effect of piperine on Na⁺ and Ca²⁺ channels were evaluated using the whole cell patch clamp techniqueOur results revealed that piperine decreased mortality in the MES-induced seizure model. Moreover, piperine (10 mg/kg) delayed the onset of tonic clonic convulsions in the pentylenetetrazole test and reduced associated mortality. Furthermore, an anticonvulsant dose of piperine also delayed the onset of tonic clonic seizures in strychnine, picrotoxin and BAY K-8644. Complete protection against mortality was observed in BAYK-8644 induced convulsions. Finally, whole cell patch clamp analysis suggested an inhibitory effect of piperine on Na⁺ channels. Together, our data suggest Na⁺ channel antagonist activity as a contributor to the complex anticonvulsant mechanisms of piperine.