ASK2 Bioactive Compound Inhibits MDR Klebsiella pneumoniae by Antibiofilm Activity, Modulating Macrophage Cytokines and Opsonophagocytosis.

The emergence and spread of pathogens harboring extended spectrum beta-lactamase (ESBL) like carbapenem resistant Gram negative bacteria are the major emerging threat to public health. Of particular concern Klebsiella pneumoniae carbapenamase- producing strains have been recorded worldwide. Catheter associated urinary tract infections (CAUTI) caused by K. pneumoniae are significantly associated with morbidity and mortality. Hence the present work was aimed to develop a strategy for addressing these issues through an innovative approach of antibiofilm and immunomodulation. These two independent activities were analyzed in a Streptomyces derived ASK2 bioactive compound. While analysing the effect of sub-minimum inhibitory concentrations (sub-MICs), 0.5x of Minimum Inhibitory Concentration (MIC) was found to be more effective in preventing biofilm formation on coverslip and silicone catheter. The minimum biofilm eradication concentration (MBEC) was found to be 15-fold higher MIC with eradication of 75% of 3 day old biofilm. Apart from its antibiofilm potential, ASK2 also acts as an opsonin and enhances phagocytic response of macrophages against multidrug resistant K. pneumoniae. In addition, ASK2 resulted in elevated levels of nitric oxide generation by the macrophages and has a stimulating effect on IL-12, IFN-γ, and TNF-α proinflammatory cytokines. The opsonic role of ASK2 and its potential in modulating proinflammatory cytokines secreted by macrophages implies the importance of ASK2 in modulating cellular immune response of macrophages against MDR K. pneumoniae. The present study proposes ASK2 as a promising candidate for treating MDR K. pneumoniae infections with its dual properties of antibiofilm and immunomodulatory activities.

Immune response of anti-lectin Pjlec antibody in freshwater crab Paratelphusa jacquemontii.

Sialic acid specific lectin Pjlec isolated from serum of the freshwater crab Paratelphusa jacquemontii served as an antigen for the production of immunoglobulin (Ig) in Balb/c mice sera. Enzyme-linked immunosorbent assay (ELISA) of mice anti-sera with Pjlec lectin affirmed the induction and production of antibody. Anti-Pjlec antibody was isolated from the antisera of mice by Protein A Sepharose affinity chromatography and checked for purity by immunoblot with lectin. Mass spectrometry (MS/MS) of papain digethe peptide sequence of antigen binding fragment (Fab) and fragment crystallizable (Fc). Coatingsted anti-Pjlec revealed of anti-Pjlec to the target cell, rabbit erythrocyte failed to enhance in vitro phagocytosis in the crab. However, inoculation of anti-Pjlec in the hemolymph of the crab elicited in vitro phagocytosis. Proteins in hemocyte lysate supernatant (HLS) were separated by electrophoresis failed to immunoblot with Pjlec or anti-Pjlec. Peptide sequences of trypsin digested lectin protein appeared homologous to deuterostome chordate. The protostome crab that lack the ability to synthesize sialic acid however bind to sialic acid a deuterostome sugar to suggest the complexity in innate immune system of invertebrates. The application of lectin and its antibody require further study on application of pathological conditions associated with alterations in sialylated cell surface.

Hemocytes and hemocytic responses in the mole crab Emerita emeritus (Linnaeus 1767).

The mole crab, Emerita emeritus, collected from the sandy shores of a Chennai beach, was investigated for cellular immune responses based on the morphology and defensive reactions of the circulating haemocytes. Three haemocyte morphotypes were identified using light and electron microscopy, and separated in a discontinuous percoll gradient. A phagocytosis study using human B erythrocyte as a target cell under phase-contrast optics showed that granular and semi-granular haemocytes were phagocytic, and this response was enhanced by using serum (opsonin)-coated human B erythrocyte in unfractionated and fractionated haemocytes. Observation of TEM image of phagocytosis revealed that the initial recognition and binding of the target cell was restricted to granular and semigranular haemocytes, which were lacking with hyaline cells. However, the encapsulation of DEAE Sepharose CL 6B beads, either untreated or coated with serum (opsonin), was restricted to hyaline cells. This suggests the occurrence of two cell lines in haemocytes, based on the differences observed in the response of haemocytes to bind target cells for phagocytosis or encapsulation. The present study also differentiated the activation of PO in the plasma, serum, and haemocyte lysate supernatant (HLS).

Phenoloxidase activity in humoral plasma, hemocyanin and hemocyanin separated proteins of the giant freshwater prawn Macrobrachium rosenbergii.

Hemocyanin is a copper containing protein and its role in the immune function of phenoloxidase (PO) activity was investigated in the giant freshwater prawn Macrobrachium rosenbergii. Hemocyanin, sedimented by ultracentrifugation from the plasma appeared on polyacrylamide gel electrophoresis (PAGE 7%) on Coomassie Brilliant Blue and bathocuproine sulfonic acid stain as four copper containing proteins of molecular masses 50, 60, 114 and 325kDa. Accordingly, on diethylaminoethyl-cellulose anion exchange column hemocyanin separated into four proteins designated as MrHc1, MrHc2, MrHc3 and MrHc4 with electrophoretically (PAGE) determined molecular masses of 60, 114, 50 and 325kDa respectively. The reduction of proteins in sodium dodecyl sulphate (SDS)-PAGE revealed that MrHc1 and 3 were monomeric for 60and 50kDa respectively, MrHc2 dimeric of 56 and 58kDa subunits and MrHc4 appeared with three subunits of 74, 76 and 78kDa. The PO activity was determined in plasma, hemocyanin and the four separated hemocyanin proteins in vitro using L-3,4-dihydroxyphenylalanine (L-DOPA) at pH7.5, 25°C and appeared elicited by exogenous activators such as trypsin, SDS, cell wall components of bacteria and polysaccharide laminarin. This study clearly demonstrated hemocyanin as the major copper containing protein in the plasma of M. rosenbergii with potent PO activity.

Purification and characterisation of a pronase-inducible lectin isolated from human serum.

A new lectin was purified to electrophoretic homogeneity from pronase treated human serum by a single-step of affinity chromatography on concanavalin A-Sepharose 4B. The isolated lectin agglutinated five types of vertebrate RBC, with highest titer against hen RBC. This activity was independent of divalent cations, insensitive to EDTA and specific to mannosamine, glucosamine as well as galactosamine. Purified lectin gave a single symmetrical peak in its native form with a molecular mass estimate of 6kDa in FPLC analysis and 6.5kDa by MALDI-TOF MS. SDS-PAGE analysis of the lectin revealed that it is a homo-oligomer of a 3kDa subunit protein. Isolated lectin did possess both, hemagglutinating and phenoloxidase activities, but did not exhibit any antibacterial or antifungal activities. In addition, this lectin could oxidize all nine different phenolic substrates tested, with hydroquinone proving to be the best among them. Phenoloxidase inhibitors namely, phenylthiourea and tropolone inhibited this oxidation activity.

Activation of phenoloxidase activity by humoral lectin in hemocytes of freshwater crab Paratelphusa jacquemontii.

The lectin, Pjlec isolated from the hemolymph of the freshwater crab Paratelphusa jacquemontii hemagglutinated (HA) with mice, rabbit and rat erythrocytes. However, the lectin failed to agglutinate neraminidase treated asialylated erythrocytes showing its sialic acid specificity. The poyacyrlamide gel electrophoresis of lectin yielded 310kDa proteins, on sodium sulphate dodecyl (SDS) gel appeared as a tetramer with subunits of 76kDa. The observation of in vitro phagocytosis in granular hemocytes of lectin opsonized rabbit erythrocyte by Transmission electron microscopy (TEM) showed the release of lytic vesicles by exocytosis prior to engulfment. The Pjlec lectin also showed an ability to oxidize L-3, 4 dihydroxyphenylalanine (L-DOPA) and in hemocyte lysate preparation (HLS) was enhanced on reduction with SDS and on proteolytic cleavage with trypsin. The lectin appeared to have a regulatory role in activation of enzyme activity associated with phagocytosis and melanin formation.

Isolation and analysis of mannose/trehalose/maltose specific lectin from jack bean with antibruchid activity.

A lectin with insecticidal property against the stored product pest, Callosobruchus maculatus was successfully isolated from the seeds of Canavalia virosa using standard affinity chromatography. The isolated molecule typically behaved like a lectin in its characteristics. It agglutinated indicator red blood cells (RBC) in its native as well as enzyme treated conditions. The enzyme treated RBC types exhibited a very high hemagglutination (HA) titre values and this property of isolated molecule behaved like arcelin, the lectin-like molecules reported from several species of Phaseolus. As a characteristic feature of a lectin, the isolated molecule effectively inhibited the agglutination of indicator RBC types with simple and complex carbohydrates including glycoproteins. This nature of the isolated molecule also relate with characteristic feature of arcelin isoforms in inhibiting HA activity with complex glycoproteins as reported in many studies. Most interestingly, the present study disclosed trehalose as a potent inhibitor of C. virosa lectin. Therefore, feeding insect pests on the lectin like arcelin could serve as antibiosis factor/anti-insect activity. The molecular characteristics of this isolated molecule and its mass studies too revealed its homology with arcelin, arcelin-1, 2 and 6 isoforms of P. vulgaris and lectin from Canavalia cathartica, C. lineata and C. brasiliensis.

Opsonic function of sialic acid specific lectin in freshwater crab Paratelphusa jacquemontii.

The sialic acid specific humoral lectin, Pjlec of the freshwater crab Paratelphusa jacquemontii was investigated for its opsonin function with rabbit erythrocyte as target cell for phagocytosis by the crab's hemocyte. The untreated or trypsin treated erythrocyte induced lectin response after challenge however failed when treated with neuraminidase evidently indicating glycan dependency for elicited immune response. Our observation of in vitro phagocytosis of the erythrocyte untreated or coated with serum, clarified serum appeared to be recognized and engulfed by hemocytes but when coated with isolated lectin Pjlec, the response was elicited. Moreover, with trypsin treated erythrocyte the response remained unchanged but neuraminidase or O-glycosidase treatment eliminated the response reaction. This suggested the sialic acid specific reaction of lectin with the erythrocyte and was essential for recognition to allow the lectin Pjlec to act as an opsonin. The flowcytometry observation affirmed the enhancement of phagocytosis by Pjlec coated hemocyte. The efficiency of in vitro hemolysis of Pjlec coated erythrocyte with hemocyte when compared to untreated erythrocyte with or without hemocyte also established the opsonic function of the lectin. The mechanism of phagocytosis and induction were dependent on specific recognition of the erythrocyte by the multivalent binding site of the lectin protein, and the elicitation of the immune response was a function of the sialoglycan surface. The pathway of the challenge suggested that after entry of nonself recognition by lectin was followed by induction and activation of phagocytosis by opsonic binding of the lectin.

Synthesis of silver nanoparticles using Solanum trilobatum fruits extract and its antibacterial, cytotoxic activity against human breast cancer cell line MCF 7.

In the present study, we have synthesized silver nanoparticles by a simple and eco-friendly method using unripe fruits of Solanum trilobatum. The aqueous silver ions when exposed to unripe fruits extract were reduced and stabilized over long time resulting in biosynthesis of surface functionalized silver nanoparticles. The bio-reduced silver nanoparticles were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction (XRD). These biologically synthesized silver nanoparticles were tested for its antibacterial activity against few human pathogenic bacteria including Gram-positive (Streptococcus mutans, Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria. In addition, we also demonstrated anticancer activity of these nanoparticles in vitro against human breast cancer cell line (MCF 7) using MTT, nuclear morphology assay, Western blot and RT-PCR expression. These results taken together show the potential applications of biosynthesized silver nanoparticles using S. trilobatum fruits.

Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities.

The present study was aimed at biosynthesis of silver nanoparticles (AgNPs) using ethanolic extract of rose (Rosa indica) petals and testing their potential antibacterial activity using selective human pathogenic microbes, anticancer activity using human colon adenocarcinoma cancer cell line HCT 15 as well as anti-inflammatory activity using rat peritoneal macrophages in vitro. The biologically synthesized AgNPs were also characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The characterized AgNPs showed an effective antibacterial activity against Gram negative (Escherichia coli, Klebsiella pneumoniae) than Gram positive (Streptococcus mutans, Enterococcus faecalis) bacteria. MTT assay, analysis of nuclear morphology, mRNA expression of Bcl-2, Bax and protein expression of caspase 3 as well as 9, indicated potential anticancer activity. In addition, green synthesized AgNPs also attenuated cytotoxicity, nuclear morphology and free radical generation (O2(-) and NO) by rat peritoneal macrophages in vitro. The results of our study show the potential green synthesis of silver nanoparticles in mitigating their toxicity while retaining their antibacterial activities.

Identification of serum component involved in generation of neo-lectin with agglutinating and phenoloxidase activities in human serum.

Human serum albumin (HSA) was identified as the component involved in generation of neo-lectin molecules with both lectin and phenoloxidase activities. Pronase treated HSA was able to agglutinate hen RBC and oxidize hydroquinone. Sodium dodecyl sulphate (SDS) treated HSA agglutinated both hen and sheep RBC as well as oxidized dopamine. The hemagglutinating activities of pronase/SDS treated HSA observed against hen RBC were dosimetric. The oxidation of pronase/SDS treated HSA with hydroquinone/dopamine, respectively, was inhibitable by inhibitors of phenoloxidase, namely, phenylthiourea and tropolone. Very low concentrations of HSA could generate these humoral neo-lectin molecules.

Luteolin induces growth arrest in colon cancer cells through involvement of Wnt/ß-catenin/GSK-3ß signaling.

Cancer is a multistep process that typically occurrs over an extended period of time, beginning with initiation followed by promotion and progression. Colon cancer is the leading cause of morbidity and mortality worldwide. For a variety of reasons, patients prefer naturally occurring dietary substances over synthetic agents to prevent cancer. Luteolin, a bioflavonoid, possesses antioxidant, anti-inflammatory, and antiproliferative effects. We analyzed the in vitro anticancer and apoptosis-inducing property of luteolin using HCT-15 colon adenocarcinoma cells. Cell viability was assessed using trypan blue assay at different concentrations. Luteolin at a concentration of 100 µM (IC50) decreased the expressions of non-P-ß-catenin, phosphorylated (inactive) glycogen synthase kinase-3ß, and cyclin D1 expressions in HCT-15 cells, which were confirmed by Western blot analysis. Luteolin also promoted substantial cell cycle arrest at the G2/M phase in HCT-15 cells, and it induces apoptosis in HCT-15 cells, as revealed by flow cytometric analysis. Furthermore, Western blot analysis showed that luteolin treatment enhanced the expression of Bax and caspase-3, whereas the expression of Bcl-2 was suppressed. Together, the results of this study revealed that luteolin can act as a potent inhibitor of HCT-15 proliferation and can be used as an agent against colon cancer.

Protective effect of ferulic acid and resveratrol against alloxan-induced diabetes in mice.

The present study was to investigate the effect of ferulic acid and resveratrol on alloxan-induced diabetic mice, through analysis of basic biochemical parameters, enzymic as well as non-enzymic activities, lipid peroxidation and immunohistochemical studies. Alloxan was administered as a single dose (75 mg/kg body weight) to induce diabetes in mice. A dose of ferulic acid (10 mg/kg body weight) and resveratrol (20 mg/kg body weight) were administrated orally, to the alloxan-induced diabetic mice. The levels of basic biochemical markers and lipid peroxidation were significantly (P<0.05) increased in alloxan-induced diabetic mice. The levels of antioxidants were significantly (P<0.05) decreased in liver, kidney and serum. Immunohistochemical studies in alloxan induced mice demonstrated a marked increase in the immunoreactivity of nuclear transcription factor (NF-κB). Treating the diabetic mice with doses of ferulic acid and resveratrol restored the changes in the above parameters analyzed. The present study, showed that ferulic acid and resveratrol exerted antioxidant as well as anti-diabetic effects, consequently alleviate liver, kidney and pancreas damage caused by alloxan-induced diabetes, probably through inhibition of the proinflammatory factor, NF-κB.

Detection of genetic variability in various isolates of cattle tick, Boophilus microplus from Tamil Nadu, India using PCR-RAPD analysis.

The genomic DNA from ten isolates of the cattle tick, Boophilus microplus collected in and around Chennai, India, was analyzed by random amplified polymorphic DNA (RAPD) using PCR. Selected five random primers were used for the study of genetic variability among different isolates of B. microplus. A high degree of genetic polymorphism with a different pattern of RAPD profiles for each tick isolate was detected with all these random primers. This variability was also confirmed by similarity coefficient values and dendrogram which were performed using mean RAPD profiles for all the primers between various isolates of ticks. The findings suggest the existence of a complex genotypic diversity of the tick B. microplus in an endemic region such as Chennai.