Shivayogeeswar Neelagund

Thermostable lipase from Geobacillus sp. Iso5: Bioseparation, characterization and native structural studies

The extracellular thermoalkaline lipase from Geobacillus sp. Iso5 was purified to homogeneity by ultrafiltration, 6% cross‐linked agarose and Phenyl spehrose HIC column chromatography. The final purified lipase resulted in 8.7‐fold with 6.2% yield. The relative molecular weight of the enzyme was determined to be a monomer of 47 kDa by SDS–PAGE and MALDI‐TOF MS/MS spectroscopy. The purified enzyme exhibit optimum activity at 70 °C and pH 8.0. The enzyme retained above 90% activity at temperatures of 70 °C and about 35% activity at 85 °C for 2 h. However, the stability of the enzyme decreased at the temperature over 90 °C. The enzyme activity was promoted in the presence of Ca2+ and Mg2+ and strongly inhibited by HgCl2, PMSF, DTT, K+, Co2+, and Zn 2+. EDTA did not affect the enzyme activity. The secondary structure of purified lipase contains 36% α‐helix and 64% β‐sheet which was determined by Circular dichromism, FTIR, and Raman Spectroscopy.

Staged buccal mucosa urethroplasty in reoperative hypospadias

Introduction: Repeated attempts at surgical repair of serious complications involving either the partial or complete breakdown of the hypospadias repair are less likely to succeed because the penis is densely scarred, or significantly shortened, and the skin over the penis is immobile and hypovascular. Buccal mucosa (BM) has become the preferred material for reconstruction, whenever a child with skin-deficient hypospadias needs reoperation. We report the results of our surgical experience with staged reoperation using BM, in the repair of hypospadias in children with complications after multiple failed repairs. Materials and Methods: Children needing reoperation for hypospadias underwent a staged repair using buccal mucosa. The complications were noted. Results: Twenty-one children aged 3 – 16 years underwent this staged repair during the period May 2000 – April 2010. Two of these 21 children had a failed first stage. One child developed a urethro-cutaneous fistula following the second stage, which was corrected in an additional stage. Conclusions: The use of the buccal mucosa graft for urethral reconstruction in a child with hypospadias, needing a reoperation, is a successful method, with a low incidence of complications.

In vitro antioxidant and anticancer activity of Leea indica leaf extracts on human prostate cancer cell lines

Background To determine the phytochemical constituents, antioxidant, and anticancer activities of Leea indica leaf extracts on DU-145 and PC-3 human prostate cancer cell lines. Methods Leaf sample was subjected to Soxhlet extraction method with increasing polarity of solvents, namely, chloroform, ethyl acetate, methanol, ethanol, and aqueous. Phytochemical screening was done using different biochemical tests. Quantitative analysis for phenol was determined by Folin–Ciocalteu reagent method. The antioxidant activity was tested using 2,2-diphenyl-1-picrylhydrazyl, ferric ion reducing power assay, and phosphomolybdenum assay. In vitro anticancer activity on DU-145 and PC-3 human prostate cancer cell lines was evaluated by (3-(4, 5-dimethyl thiazole-2yl)-2, 5-diphenyl tetrazolium bromide) MTT assay. Results Phytochemical screening confirmed the presence of phyto-constituents like alkaloids, flavonoids, glycosides, phenols, lignins, saponins, sterols, tannins, anthraquinone, and reducing sugar. Methanol and ethanol extracts exhibited higher phenolic content as compare to aqueous extract. Antioxidant capacities were shown highest in methanol and ethanol extracts based on the test performed. The methanol and ethanol leaf extracts were found to be selectively cytotoxic in vitro to (DU-145 and PC-3) prostate cancer cell lines with IC50 values 529.44 ± 42.07 μg/mL and 677.11 ± 37.01 μg/mL for DU-145 and 547.55 ± 33.52 μg/mL and 631.99 ± 50.24 μg/mL for PC-3 respectively, while it had no cytotoxic effect on normal mice embryo fibroblast cells. Conclusion The results indicate that Leea indica was a promising antioxidant and anticancer agent for DU-145 and PC-3 human prostate cancer cell lines. However, further studies are needed to conclude its therapeutic use.

Protective effect of partially purified 35 kDa protein from silk worm (Bombyx mori) fecal matter against carbon tetrachloride induced hepatotoxicity and in vitro anti-viral properties

Context: It has been found that many proteins from silkworm (Bombyx mori L.) fecal matter have been active against human immunodeficiency virus, Sendai virus, herpes simplex virus type-1, and nuclear polyhedrosis virus. Objective: A partially purified 35 kDa protein from silkworm was screened for its hepatoprotective activity, and in vitro antioxidant, and antiviral properties against camelpox and goatpox viruses. Materials and methods: The study investigated the efficiency of the partially purified 35kDa protein from silk worm fecal matter against CCl4-induced liver damage measured in terms of enzyme levels such as aspartate aminotransferase (AST), alanine amino transferase(ALT), alkaline phosphatase (ALP) and total bilirubin, which maintain liver integrity. In vitro antioxidant potential of this protein was determined based on its ability to scavenge 2, 2-diphenylpicrylhydrazyl (DPPH) and superoxide anions scavenging activity. Further, in vitro cytotoxic effect on Vero cells and antiviral activity against goatpox and camelpox viruses were also studied. Results: The protein had significant hepatoprotection against CCl4-induced liver damage and scavenging of DPPH radical and superoxide anion activity. However, the protein did not inhibit the multiplication of either virus tested at its maximum non-toxic concentration (MNTC) in vitro. Discussion and conclusion: The partially purified 35 kDa protein from silk worm Bombyx mori L fecal matter possessed protective effect against CCl4-induced oxidative stress in rat model. The protein was found to be ineffective against camelpox and goatpox viruses at its MNTC in vitro.

A novel bioassay based gold nanoribbon biosensor to aid the preclinical evaluation of anticancer properties

In this work, we report a microbial biosensor fabricated for the preclinical assay of anticancer compounds. Gold nanoribbons were used as a transducer for mounting the microbe. For the synthesis of these unique Au nanostructures, quercetin stabilized gold nanoparticles (Q-AuNPs) were synthesized as a first step using onion peel. Later, dityrosine peptide was used as a sacrificial template for the synthesis of the gold nanoribbons (AuNRs). The structural morphology of the as-synthesized Au nanomaterial was examined using UV spectroscopy, XRD, SEM and TEM. The AuNRs were found to be <10 nm in diameter, which provided a good biocompatible environment and effective protection for the immobilization of Agrobacterium tumefaciens (At), a causative agent of crown gall disease. At is reported to cause tumors in plants through a tumorigenic mechanism similar to that of humans. Inhibition of At indicates that the inhibitory compound being screened exhibits anticancer activity. Clitoria ternatea (Ct) is traditionally used to cure many diseases and is known to possess anticancer activity. Therefore, we have used a Ct flower extract in the preclinical study of its anticancer activity against At by fabricating a simple electrochemical sensor. We have employed electrochemical techniques such as CV and EIS for the characterization of the developed microbial biosensor. Moreover, the as-synthesized AuNRs behave as an ideal transducer and platform, thus improving the electrode surface area and providing good biocompatibility for the immobilization of At. In contrast to other immobilization techniques and biosensors that often require elaborate procedures, cross-linking agents and rigorous chemical reactions, At was directly adsorbed onto the electrode under optimum conditions without any mediators. The results show that the developed biosensor is useful in the pre-clinical analysis of anticancer properties. Indeed the study examines the use of electrochemistry, demonstrating the rapid response and high sensitivity of the proposed sensor in contrast to bioassay procedures. In conclusion, the experimental results indicate that the developed biosensor accentuates the excellent properties of the synthesized AuNRs, which promises to be a novel avenue in designing biosensors.

Ganoderma lucidum : A Source for Novel Bioactive Lectin

Ganoderma lucidum is known for its high medicinal value, clinically used in treatment for various diseases. We have selected this mushroom for isolation of novel bioactive lectin. The isolation procedure comprised of ion-exchange chromatography on DEAE- cellulose and affinity chromatography on Affi-gel blue gel. Purified lectin was monomer with a molecular mass of 15 kDa, determined by SDS-PAGE, Gel filtration, MALDI-ToF. It showed hemagglutinating activity against both human and animal erythrocytes. The hemagglutination activity was not inhibited by simple sugars but inhibited by glycoproteins. The activity was maximal at pH range 4.0-9.0 and at temperature up to 60° C. The hemagglutination activity was stable even in the presence of 10mM EDTA and other divalent metal cations such as CaCl2, MgCl2, ZnCl2, and MnCl2. Lectin was shown antifungal activity against following pathogens Fusarium oxysporium, Penicillium chrysogenum, Aspergillus Niger, Colletotrichum musae, Botrytis cinerea, Trichophyton rubrum, Trichophyton tonsurans, Trichophyton interdigitale, Epidermophyton floccosum and Microsporum canis.