Outbreak of hirudiniasis in aquarium-reared albino red-bellied pacu Piaractus brachypomus.

We report hirudiniasis caused by the leech Hemiclepsis marginata asiatica Moore, 1924 in albino red-bellied pacu (pirapitinga) Piaractus brachypomus (Cuvier, 1818), constituting the first documentation of a freshwater fish species being affected in India. The outbreak occurred in a tank of an aquarium-fish retailer; infested fish appeared asphyxiated, unable to swim or swimming upside down, with cloudy eyes and body with thick mucus secretion. The prevalence and mortality was 100%, with a mean intensity of 81 leeches per fish. The histopathology of the morbid fish revealed degenerative necrosis, eosinophilic infiltration in the muscle tissue and haemorrhages in the fin membrane. The leech mitochondrial 18S rDNA and 12S rDNA genes were characterised and submitted to GenBank under accession numbers MN380443 (18S) and MK733282 (12S). A maximum likelihood tree was constructed using 12S rDNA gene sequences to demonstrate the phylogenetic position of Hemiclepsis marginata asiatica among its congeners.

The Influence of the Cholesterol Level in Cells on Endovanilloid Cytotoxicity.

The influence of the cellular cholesterol content on the cytotoxicity of endovanilloids acyldopamines was studied in MDA-MB-231 and MCF 10A cells. The activity of acyldopamines depends on the cellular cholesterol content, and a decrease in cholesterol content increases the cytotoxicity of acyldopamines.

Transport of a Sessile Aqueous Droplet over Spikes of Oil Based Ferrofluid in the Presence of a Magnetic Field.

Droplets can be used as carrier vehicles for the transportation of biological and chemical reagents. Manipulation of water- and oil-based ferromagnetic droplets in the presence of a magnetic field has been well-studied. Here, we elucidate the transport of a sessile aqueous (diamagnetic) droplet placed over spikes of oil-based ferrofluid (FF) in the presence of a nonuniform magnetic field. An oil-based FF droplet, dispensed over a rigid oleophilic surface, interacts with a magnetic field to get transformed into an array of spikes which then act as a carrier for the transportation of the aqueous droplet. Our study reveals that transportation phenomena is governed by the interplay of three different forces: magnetic force Fm, frictional force Ff, and interfacial tension force Fi, which is expressed in terms of the magnetic Laplace number ( Lam) and magnetic Bond number ( Bom) as Lam?1 = ( Ff1/ Fm, x) and Bom Lam?1 = ( Ff2/ Fi). Based on the values of the dimensionless numbers, three different regimes, steady droplet transport, spike extraction, and magnet disengagement, are identified. It is found that steady droplet transport is observed for Lam?1 ? 1 and Bom Lam?1 ? 1, whereas extraction of spikes is observed for Lam?1 ? 1 and Bom Lam?1 > 1 and magnet disengagement is observed for Lam?1 > 1. In the steady droplet transport regime, velocity of the aqueous droplet Uds was found to be dependent on the volumes of the aqueous droplet Vw and FF droplet VFF following Uds ? Vw?0.19 VFF0.36. A simple model is presented that accurately predicts the aqueous droplet velocity Uds within 5% of the corresponding experimental data. In the spike extraction regime, the spike extraction distance Lse was found to vary with Vw, VFF, and the magnet velocity Ums following Lse ? Vw?1.75 VFF0.75 Ums?1.56.

PTEN negatively regulates mTORC2 formation and signaling in grade IV glioma via Rictor hyperphosphorylation at Thr1135 and direct the mode of action of an mTORC1/2 inhibitor.

To investigate the role of PTEN (phosphatase and tensin homolog) in mammalian target of rapamycin complex 2 (mTORC2) signaling in glioblastoma multiforme (GBM), we found higher activation of mTORC2 in PTEN(mu) cells, as evidenced by enhanced phosphorylation of mTOR (Ser2481), AKT (Ser473) and glycogen synthase kinase 3 beta (GSK3ß) (Ser9) as compared with PTEN(wt) cells. In addition, PTEN(wt) cells upon PTEN depletion showed mTORC2 activation. The reduced mTORC2 signaling in PTEN(wt) cells was related to higher Rictor phosphorylation at Thr1135 residue. Phosphorylation of Rictor at Thr1135 inhibited its association with mTORC and thus there was a reduction in mTORC2 complex formation. In addition, PTEN(wt) cells expressing mutated Rictor in which Thr1135 was substituted with alanine, showed enhanced mTORC2 formation and signaling. This enhanced mTORC2 signaling promoted inactivation of GSK3ß. Thus, we established the reciprocal activation of mTORC2 and GSK3ß in GBM. To the best of our knowledge, this is the first report describing role of PTEN in mTORC2 formation by promoting Rictor phosphorylation (Thr1135) in GBM. Furthermore, the drug sensitivity of mTORC2 was evaluated. A newly identified carbazole alkaloid, mahanine, showed cytotoxicity in both PTEN(mu) and PTEN(wt) cells. It inhibited both mTORC1/2 and AKT completely in PTEN(mu) cells, whereas it inhibited only mTORC1 in PTEN(wt) cells. Cytotoxity and AKT-inhibitory activity of the mTORC1/2 inhibitor was increased either by depleting PTEN or in combination with phosphatidylinositol 3 kinase inhibitors in PTEN(wt) cells. In contrast, depletion of Rictor decreased the cytotoxicity of the mTORC1/2 inhibitor in PTEN(mu) cells. Thus, PTEN has an important role in mTORC2 formation and also influences the effectiveness of an mTORC1/2 inhibitor in GBM.

PCDHB14- and GABRB1-like nervous system developmental genes are altered during early neuronal differentiation of NCCIT cells treated with ethanol.

Ethanol (EtOH) exposure during embryonic development causes dysfunction of the central nervous system (CNS). Here, we examined the effects of chronic EtOH on gene expression during early stages of neuronal differentiation. Human embryonic carcinoma (NCCIT) cells were differentiated into neuronal precursors/lineages in the presence or absence of EtOH and folic acid. Gene expression profiling and pathway analysis demonstrated that EtOH deregulates many genes and pathways that are involved in early brain development. EtOH exposure downregulated several important genes, such as PCDHB14, GABRB1, CTNND2, NAV3, RALDH1, and OPN5, which are involved in CNS development, synapse assembly, synaptic transmission, and neurotransmitter receptor activity. GeneGo pathway analysis revealed that the deregulated genes mapped to disease pathways that were relevant to fetal alcohol spectrum disorders (FASD, such as neurotic disorders, epilepsy, and alcohol-related disorders). In conclusion, these findings suggest that the impairment of the neurological system or suboptimal synapse formation resulting from EtOH exposure could underlie the neurodevelopmental disorders in individuals with FASD.

Efficacy of Withania somnifera chemotypes NMITLI - 101R, 118R and Withaferin A against experimental visceral leishmaniasis.

The immunoprophylactic and therapeutic potentials of root extracts of Withania somnifera chemotypes (NMITLI-118, NMITLI-101) and pure withanolide-withaferin A was investigated against Leishmania donovani infection in hamsters. The naive animals, fed orally with immunostimulatory doses of chemotypes 101R, 118R (10 and 3 mg/kg) and withaferin A (9 and 3 mg/kg) for five consecutive days and challenged with Leishmania parasites on day 6, were euthanized on days 30 and 45 p.c. for the assessment of parasite clearance, real-time analysis of mRNAs of Th1/Th2 cytokines (IFN-γ, IL-12, TNF-α, iNOS/IL-4, IL-10 and TGF-ß), NO production, reactive oxygen species (ROS) generation, lymphocyte transformation test and antibody responses. By day 45 p.c., there was a significant increase in the mRNA expression of iNOS, IFN-γ, IL-12 and TNF-α but decrease in IL-4, IL-10 and TGF-ß, an enhanced Leishmania-specific LTT response as well as ROS, NO and antileishmanial IgG2 levels in 101R-treated hamsters followed by 118R- and withaferin A-treated ones, respectively. When these chemotypes were given to L. donovani-infected hamsters at different doses, there was moderate therapeutic efficacy of chemotype 101R (~50%) at 30 mg/kg × 5 followed by the other two. The results established that the 101R is the most potential chemotype and can be evaluated for combination therapy along with available antileishmanials.

Antioxidative responses of Salvinia (Salvinia natans Linn.) to aluminium stress and it's modulation by polyamine.

Antioxidative stress response of free-floating aquatic fern (Salvinia natans Linn.) was studied under increasing toxic amount of aluminium (Al) and its modulation by exogenous application of polymaine. Increased levels of superoxide (O2 (-)) and hydrogen peroxide (H2O2) species from affected tissues suggested that plants were undergoing oxidative stress and it was concominant with increased accumulation of Al in a dose dependent manner. Application of polyamine like putrescine (Put) led to a decrease in oxidative stress as revealed by reduced level of O2 (-) and H2O2. Al toxicity resulted into decreased biomass that was ameliorated by the application of Put. The changes observed in lipid peroxidation (MDA) and protein oxidation also indicated that plats are undergoing Al induced oxidative stress. In order to circumvent the oxidative stress resulting from Al toxicity, plants enzymatic and nonenzymatic antioxidant pathways were active. The ratio of both oxidized and reduced cellular glutathione exhibited significant variation in response to Al stress and was improved upon Put treatment. Peroxidase and glutathione were upregulated whereas catalse was downregulated under varying doses of Al. Isozyme profile of above enzymes also showed a trend with increasing amount of Al. The nuclear disintegration study using comet assay was indicative of Al induced oxidative stress. In the present study, we have explored the antioxidative response of aquatic fern Salvinia natans Linn in response to Al toxicity. The application of polyamine Put improved the overall antioxidative response and thus would make it a better candidate to be used as hyper accumulator of Al and other toxic metals.

Variations of antioxidative responses in two rice cultivars with polyamine treatment under salinity stress.

The rice varieties viz. Nonabokra and Swarna were evaluated on the basis of their responses for oxidative stress induced by sodium chloride (NaCl) and the effects of exogenously applied polyamine thereon. Rice seedlings were treated with 200 mM of NaCl supplemented with two dosages: 1 mM and 2 mM putrescine. Following treatments, plants were evaluated for accumulation of reactive oxygen species (ROS) like O2 (-), H2O2 etc. in tissues, lipid peroxidation, protein carbonylation, accumulation of flavonoids and anthocyanin, activities of different oxidative enzymes like guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR). Preliminary, oxidative stress out of salinity was ensured by plants from significantly higher accumulation of O2 (-) and H2O2 in the tissues of the NaCl treated varieties. Irrespective of varieties, there recorded a significant variation of the endogenous polyamine profiles under NaCl stress. Interestingly, exogenous application of putrescine had a close relationship on O2 (-) and H2O2 content for both the varieties. However, Nonabokra was evident as more respondent than Swarna to applied putrescine. The other effects of oxidative stress was impacted on plants as higher values of MDA content, enhanced rate of protein oxidation and putrescine recorded as an alleviating agent regardless of varieties with dose dependant manner. The generation of ROS and cellular disintegration was accompanied by up regulation of non-enzymatic and enzymatic antioxidation pathways with exogenous application of putrescine. For non-enzymatic antioxidant, it revealed that putrescine was highly effective for sustaining the anthocyanin and flavonoid content in both the varieties under salinity. Whereas, antioxidative enzyme, CAT showed its diminished activity; but activity of GPX and GR were significantly induced under salinity and it was according to the concentration of applied putrescine.

Disease-associated glycosylated molecular variants of human C-reactive protein activate complement-mediated hemolysis of erythrocytes in tuberculosis and Indian visceral leishmaniasis.

Human C-reactive protein (CRP), as a mediator of innate immunity, removed damaged cells by activating the classical complement pathway. Previous studies have successfully demonstrated that CRPs are differentially induced as glycosylated molecular variants in certain pathological conditions. Affinity-purified CRPs from two most prevalent diseases in India viz. tuberculosis (TB) and visceral leishmaniasis (VL) have differential glycosylation in their sugar composition and linkages. As anemia is a common manifestation in TB and VL, we assessed the contributory role of glycosylated CRPs to influence hemolysis via CRP-complement-pathway as compared to healthy control subjects. Accordingly, the specific binding of glycosylated CRPs with erythrocytes was established by flow-cytometry and ELISA. Significantly, deglycosylated CRPs showed a 7-8-fold reduced binding with erythrocytes confirming the role of glycosylated moieties. Scatchard analysis revealed striking differences in the apparent binding constants (10(4)-10(5) M(-1)) and number of binding sites (10(6)-10(7)sites/erythrocyte) for CRP on patients' erythrocytes as compared to normal. Western blotting along with immunoprecipitation analysis revealed the presence of distinct molecular determinants on TB and VL erythrocytes specific to disease-associated CRP. Increased fragility, hydrophobicity and decreased rigidity of diseased-erythrocytes upon binding with glycosylated CRP suggested membrane damage. Finally, the erythrocyte-CRP binding was shown to activate the CRP-complement-cascade causing hemolysis, even at physiological concentration of CRP (10 microg/ml). Thus, it may be postulated that CRP have a protective role towards the clearance of damaged-erythrocytes in these two diseases.

9- O-acetylated sialic acids enhance entry of virulent Leishmania donovani promastigotes into macrophages.

SUMMARY: Distribution of 9-O-acetylated sialic acids (9-O-AcSA) on Leishmania donovani has been previously reported. Considering their role in recognition, the differential distribution of sialic acids especially 9-O-acetylated sialic acids in avirulent (UR6) versus virulent (AG83 and GE1) promastigotes of Leishmania donovani and its role in entry into macrophages was explored. Fluorimetric-HPLC, fluorimetric determination and ELISA revealed 14-, 8- and 5-fold lower sialic acids in UR6 as compared to AG83. Interestingly, on UR6, flow cytometry indicated lower (alpha2-->6)-linked sialoglycoproteins along with minimal 9-O-acetylated sialoglycoproteins by Scatchard analysis. Further, UR6 demonstrated a 9- and 14.5-fold lower infectivity and phagocytic index than AG83. Additionally, de-O-acetylation and de-sialylation of AG83 demonstrated a 3- and 1.5-fold reduced phagocytic index. The role of 9-O-AcSA in entry was further confirmed by pre-blocking the macrophage surface with a cocktail of sugars followed by microscopic quantification. The phagocytic index of AG83 exclusively through 9-O-AcSA was significantly high. Interestingly, AG83 produced higher metacyclic promastigotes containing increased 9-O-AcSA as compared to avirulent UR6 supporting its virulent nature. Taken together; our results conclusively demonstrate the increased presence of 9-O-acetylated sialic acid on promastigotes of virulent Leishmania donovani as compared to avirulent UR6 and their subsequent role in entry within macrophages.

Structural analysis of factor IX protein variants to predict functional aberration causing haemophilia B.

Factor IX (FIX) is a component protein of blood coagulation pathway, which activates factor X through interaction with factor VIII and Ca(2+). Defective FIX protein resulting from mutation in the corresponding gene causes an X-linked bleeding disorder known as haemophilia B. The aim of the present study was to speculate the potential detrimental effects of the FIX mutations upon the functionality of the protein, which could contribute to the comprehension of the mechanism underlying haemophilia B. In this report, we examined the effect of point mutation on the crystal structure of the native factor IX by measuring the change in the hydrogen-bonding pattern and electrostatic potential and explored the possibility of any correlation of the clinical severity of haemophilia B with the structural perturbation, by plotting the mutations of varying phenotype (severe and mild) on the crystal structure of FIX. Out of a total of 16 severe mutations 14 (88%) showed changes of hydrogen-bonding pattern to variable extent. Among the nine mild haemophilia B mutations, six (i.e. 66.66%) showed no change in hydrogen-bonding pattern. Our data suggest that there is a statistically significant correlation between the two groups of mutations as measured by change in the hydrogen-bonding pattern. Our study truly represents an initiation of an effort that would provide a framework for first evaluation of suspected mutations by in silico approaches, which might be further validated by other experimental techniques.

A new cell secreting insulin.

The pancreatic beta-cell is the only cell in animals that expresses the insulin gene and secretes insulin protein. We have found copious release of immunoreactive and bioactive insulin into the medium from the primary culture of carp adipocytes. Glucose augmented this release to more than 2-fold, and glucose transporter, Glut2, was detected in these cells. These all reflect characteristics of a pancreatic beta-cell. The expression of the adipocyte-specific flotillin gene, the presence of peroxisomal proliferator-activated receptor gamma and Glut4, and the colocalization of insulin and leptin confirmed the identity of these cells as adipocytes. Purified carp adipocyte insulin (AdpInsl) comigrated with porcine and bovine insulin in SDS-PAGE, indicating the similarity of their molecular sizes (5.5 kDa). AdpInsl strongly reduced hyperglycemia in streptozotocin-induced diabetic rats. It also stimulated significantly higher glucose uptake in carp and hamster adipocytes than porcine insulin. Adipocyte RNA hybridized with rat and zebrafish insulin cDNA showing the expression of the insulin gene in this cell. Using oligonucleotide primers designed on the basis of conserved insulin domain, AdpInsl cDNA was reverse transcribed, cloned, and sequenced. The deduced amino acid sequence of AdpInsl A and B chain exhibited 98% homology with zebrafish and more than 70% homology with human, porcine, and murine insulin. To understand the structure-function relationship between AdpInsl and mammalian beta-cell insulin, we have analyzed the amino acid sequences and three-dimensional structure of AdpInsl. In the critical determinant segment for receptor binding, AdpInsl has His at the A8 position instead of Thr in human and porcine insulin, and this attributed greater biological activity to AdpInsl. Our results show that carp adipocyte is a unique cell. As an insulin target cell it can express the insulin gene and secrete highly active insulin protein; thus, it may serve as a natural alternative to pancreatic beta-cell insulin.

Wastewater quality status of the Tally nulla in Calcutta.

The study of the Tally nulla wastewater system showed that the entire canal system is completely anoxic and unsuitable for sustaining aquatic life. Color and odor have exceeded the threshold limit. Tidal exchange is seemed to take place only up to six km. from the confluence zone of the river hooghly. Beyond this, comparatively higher ionic load in the water mass is encountered in both seasons. The fresh water during rainy season decreases the chemical load by dilution along with an increase in bacterial populations in the system indicating significant contamination with disease causing bacteria and pollution with excreta. This may become environmental hazards of the public health in course of any type of interaction with the waste water.

Studies of pharmacognostical profiles of cleome viscosa L (family:capparidaceae).

The macroscopic characters of the whole plant, physical constant values, extractive values, behavior on treatment with different chemical reagents, fluorescence characters under ultra violet light after treatment with different reagents of the powered entire plant of Cleome viscose Linn.(Capparidaceae) were studied to fix some pharmacognostical parameters. Preliminary phytochemical screening on the methanol extract of the plant also performed. These studies will help in identification of this plant for further research.

Acute phase response of C-reactive protein of Labeo rohita to aquatic pollutants is accompanied by the appearance of distinct molecular forms.

Different forms of C-reactive proteins have been purified to electrophoretic homogeneity by calcium dependent affinity chromatography on a phosphorylcholine (PC)-Sepharose column from the sera of Labeo rohita confined in fresh water (CRP(N)) and water polluted with sublethal doses of cadmium (CRP(Cd)), mercury (CRP(Hg)), phenol (CRP(Ph)), and hexachlorocyclohexane (CRP(Hx)), which elevate serum CRP levels by three- to fivefold. On native PAGE, induced forms of CRP show remarkable differences in their electrophoteric mobility indicating differences in molecular mass, charge, and/or shape. Kinetic studies reveal the appearance of a pollutant specific molecular variant, which replaces the normal form at the peak of induction. Studies on amino acid and carbohydrate compositions, isoelectric focusing, binding to PC, C-polysaccharide (CPS) & lectins, and secondary structures of the purified CRPs, indicate, that, they differ significantly from each other, but grossly share the common properties of a CRP, including pentraxin, structure revealed by electron microscopy.

O-acetyl sialic acid specific IgM in childhood acute lymphoblastic leukaemia.

Initial studies have revealed an enhanced surface expression of O-acetylated sialoglycoconjugates (O-AcSGs) on lymphoblasts concomitant with high titres of IgG in childhood Acute Lymphoblastic Leukaemia (ALL) (Mandal C, Chatterjee M, Sinha D, Br J Haematol 110, 801-12, 2000). In our efforts to identify disease specific markers for ALL, we have affinity-purified IgM directed against O-AcSGs that reacts with three disease specific O-AcSGs present on membrane proteins derived from peripheral blood mononuclear cells (PBMC) of ALL patients. Antibody specificity towards O-AcSGs was confirmed by selective binding to erythrocytes bearing surface O-AcSGs, decreased binding with de-O-acetylated BSM and following pretreatment with O-acetyl esterase. Competitive inhibition ELISA demonstrated a higher avidity of IgM for O-AcSG than IgG. Flow cytometry demonstrated the diagnostic potential of purified O-AcSA IgM as binding was specific with ALL patients and minimal with other haematological disorders and normal individuals. It therefore may be adopted as a non-invasive approach for detection of childhood ALL. Taken together, the data indicates that carbohydrate epitopes having terminal O-AcSA alpha2 --> 6 GalNAc determinants induce disease specific IgG and IgM, potentially useful molecular markers for childhood ALL.

Glycosylated molecular variants of C-reactive proteins from the major carp Catla catla in fresh and polluted aquatic environments.

Elevated level of pollutant specific glycosylated molecular variants of C-reactive protein have been purified to electrophoretic homogeneity from the sera of major carp, Catla catla confined in freshwater (CRP(N)) and water polluted with nonlethal doses of cadmium (CRP(Cd)), mercury (CRP(Hg)), phenol (CRP(Ph)) and hexachlorocyclohexane (CRP(Hex)). These CRPs differ amongst themselves in electrophoretic mobility, and in their carbohydrate content ranging from 20-50%. CRPs interact with pneumococcal C-polysaccharide (CPS) showing different binding constants. Both phosphorylcholine (PC) and calcium are indispensable for binding. Studies on amino acid compositions, electrophoretic analysis, isoelectric focusing, binding to PC & CPS and secondary structures of the purified CRPs indicate, that, they differ from each other. However, they share the common properties of a CRP, including pentraxin structure revealed by electron microscopy. Taken together, our results provide a new structural insight regarding the connection between the presence of unique molecular variants and probably the toxicity therein combated.

Region-specific attenuation of a trypsin-like protease in substantia nigra following dopaminergic neurotoxicity by 1-methyl-4-phenyl-1,2, 3,6-tetrahydropyridine.

We analysed apoptosis, caspase-1 and -3, and trypsin-like protease activity in the nigrostriatal pathway during 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP injected (30 mg/kg, i.p., twice, 16 h apart) mice were sacrificed on 1, 2 and 7 days. DNA extracted from nucleus caudatus putamen (NCP) and substantia nigra (SN) was subjected to agarose gel electrophoresis. Typical apoptotic-like DNA cleavage was absent in SN or NCP after this dose of MPTP. A trypsin-like protease activity was significantly decreased in SN and not in NCP. While caspase-3 activity in the whole brain was increased significantly, caspase-1 activity was unaffected. Striatal dopamine content was decreased to 75% by 7 days. The absence of typical DNA 'ladder' when there was severe striatal dopamine depletion suggests that in vivo MPTP-mediated dopaminergic neurotoxicity may not involve apoptotic cell death, and explains why in mice MPTP-induced dopamine depletion is transient. The region-specific decrease in trypsin-like protease activity and absence of caspase-3 activation in SN signify the importance of trypsin-like protease in the regulation of apoptosis in MPTP-neurotoxicity in mice.