Characterization of BflI – A Thermostable,Co++-Requiring Isoschizomer of BsiYI from Anoxybacillus Flavithermus

A thermophile, isolated from geothermal areas in the northern Himalayan region of India, was identified by partial 16S rDNA sequence (GenBank accession # AF482430) analysis as Anoxybacillus flavithermus. The isolate produced BflI (REBASE # 4910), a Type II restriction endonuclease, which recognized the sequence 5′-CCNNNNN/NNGG-3′ and was the isoschizomer of BsiYI. The enzyme was purified to homogeneity by passing through Cibacron Blue F3GA agarose, DEAE-cellulose, heparin-agarose and MonoQ FPLC. The purified enzyme (MW 36 kDa) worked best at 60 °C in Promega's buffer C and preferentially required Co⁺⁺(0.4 mM) as cofactor followed by Mg⁺⁺(10 mM) and Mn⁺⁺(1 mM). The enzyme showed high specific activity and worked in the presence of high concentrations of β-mercaptoethanol (200 mM), Triton-X-100 (25%), urea (30%), formamide (6%) and guanidine (40 mM) and showed no star activity in the presence of 40% glycerol. In the absence of any stabilizing agent, BflI retained t _1/2 for at least 96 h at 37 °C, 6 h at 60 °C and 6 months at 4 °C. N-terminal sequencing showed that its first 10 amino acid residues were DFHEDKTIAR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Microbial and enzymatic improvement of anaerobic digestion of waste biomass

Metabolic activities of different microorganisms (Bacillus subtilis, B. licheniformis and Aspergillus niger) and hydrolytic enzymes (concentrations: 1 to 200 mg enzyme solids g^–1 feed) were studied individually and in combinations with respect to H₂ and methane production from damaged wheat grains. Bacillus subtilis, B. licheniformis and pre-existing hydrogen producers (control) produced 45 to 64 l H₂ kg^–1 total solids and subsequently, with the help of added methanogens, 155 to 220 l methane kg^–1 total solids could be produced. H₂ production from damaged wheat grains could be decreased to 28% or enhanced up to 152% with respect to control, by employing various microbial and enzymatic treatments. Similarly, it has been made possible to vary methane production capacities from as low as 17% to as high as 110% with respect to control.     

A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: Purification, characterization and indigo carmine degradation

γ-Proteobacterium JB, an alkali-tolerant soil isolate, produced laccase constitutively in unbuffered medium. The enzyme was purified to homogeneity by ammonium sulphate precipitation, DEAE-sepharose anion exchange chromatography and preparatory polyacrylamide gel electrophoresis. The purified enzyme was a monomeric polypeptide (MW 120 kDa) and absorbed at 590 nm indicating the presence of Type I Cu²⁺-centre. It worked optimally at 55 °C and showed different pH optima for different substrates. The enzyme was highly stable in the pH range 4–10 even after 60 days at 4 °C. K_m and V_max values for syringaldazine, catechol, pyrogallol, p-phenylenediamine, l-methyl DOPA and guaiacol substrates were determined. Inhibitors, viz. azide, diethyldithiocarbamate, thioglycollate and cysteine-hydrochloride all inhibited laccase non-competitively using guaiacol as substrate at pH 6.5. The enzyme degraded indigo carmine (pH 9, 55 °C) to anthranilic acid via isatin as determined spectrophotometrically and by HPLC analysis. Degradation was enhanced in the presence of syringaldehyde (571%), vanillin (156%) and p-hydroxybenzoic acid (91.6%) but not HOBT.     

Bench scale production of butyramide using free and immobilized cells of Bacillus sp. APB-6

Bioprocess and Biosystems Engineering - Butyramide is a commodity chemical having wide range of applications from material science to biological sciences including synthesis of therapeutic drugs,...     

Double stranded RNA expression and its topical application for non-transgenic resistance to plant viruses

The discovery of RNA silencing has heralded a new phase in the understanding of nucleic acid metabolism and has provided researchers with alternative methods to define the biochemical, developmental and self-protection pathways in plants. Manipulation of these pathways to ultimately modify, induce or enhance favorable traits and repress or completely eliminate unfavorable qualities including disease susceptibility can be achieved by insertion or infiltration of transgenes encoding appropriate effectors. Remarkably, in contrast to transgene derived viral resistance, the RNA silencing mechanism can be readily induced by the topical application of dsRNA effector molecules. The current review summarises the various dsRNA production systems available for producing the large amounts of dsRNA required for topical application onto field grown crops. Also discussed are several investigations utilising a topical application of exogenous, sequence-specific dsRNA to induce the RNA silencing mechanism for viral resistance in plants. Currently, there are limitations for the spray-on application of dsRNA for field use. Further research to define a new phase in the non-transgenic delivery of RNA silencing for complete management of plant pathogens is crucial for its commercial application.     

Purification and Characterization of an Extracellular,Thermo-Alkali-Stable,Metal Tolerant Laccase from Bacillus tequilensis SN4

A novel extracellular thermo-alkali-stable laccase from Bacillus tequilensis SN4 (SN4LAC) was purified to homogeneity. The laccase was a monomeric protein of molecular weight 32 KDa. UV-visible spectrum and peptide mass fingerprinting results showed that SN4LAC is a multicopper oxidase. Laccase was active in broad range of phenolic and non-phenolic substrates. Catalytic efficiency (k _cat/K _m) showed that 2, 6-dimethoxyphenol was most efficiently oxidized by the enzyme. The enzyme was inhibited by conventional inhibitors of laccase like sodium azide, cysteine, dithiothreitol and β-mercaptoethanol. SN4LAC was found to be highly thermostable, having temperature optimum at 85°C and could retain more than 80% activity at 70°C for 24 h. The optimum pH of activity for 2, 6-dimethoxyphenol, 2, 2′-azino bis[3-ethylbenzthiazoline-6-sulfonate], syringaldazine and guaiacol was 8.0, 5.5, 6.5 and 8.0 respectively. Enzyme was alkali-stable as it retained more than 75% activity at pH 9.0 for 24 h. Activity of the enzyme was significantly enhanced by Cu²⁺, Co²⁺, SDS and CTAB, while it was stable in the presence of halides, most of the other metal ions and surfactants. The extracellular nature and stability of SN4LAC in extreme conditions such as high temperature, pH, heavy metals, halides and detergents makes it a highly suitable candidate for biotechnological and industrial applications.     

Characterization of a Spontaneous Retinal Neovascular Mouse Model

Background

Vision loss due to vascular disease of the retina is a leading cause of blindness in the world. Retinal angiomatous proliferation (RAP) is a subgroup of neovascular age-related macular degeneration (AMD), whereby abnormal blood vessels develop in the retina leading to debilitating vision loss and eventual blindness. The novel mouse strain, neoretinal vascularization 2 (NRV2), shows spontaneous fundus changes associated with abnormal neovascularization. The purpose of this study is to characterize the induction of pathologic angiogenesis in this mouse model.

Methods

The NRV2 mice were examined from postnatal day 12 (p12) to 3 months. The phenotypic changes within the retina were evaluated by fundus photography, fluorescein angiography, optical coherence tomography, and immunohistochemical and electron microscopic analysis. The pathological neovascularization was imaged by confocal microscopy and reconstructed using three-dimensional image analysis software.

Results

We found that NRV2 mice develop multifocal retinal depigmentation in the posterior fundus. Depigmented lesions developed vascular leakage observed by fluorescein angiography. The spontaneous angiogenesis arose from the retinal vascular plexus at postnatal day (p)15 and extended toward retinal pigment epithelium (RPE). By three months of age, histological analysis revealed encapsulation of the neovascular lesion by the RPE in the photoreceptor cell layer and subretinal space.

Conclusions

The NRV2 mouse strain develops early neovascular lesions within the retina, which grow downward towards the RPE beginning at p15. This retinal neovascularization model mimics early stages of human retinal angiomatous proliferation (RAP) and will likely be a useful in elucidating targeted therapeutics for patients with ocular neovascular disease.     

Five-Year Field Results and Long-Term Effectiveness of 20 mg/kg Liposomal Amphotericin B (Ambisome) for Visceral Leishmaniasis in Bihar,India

Background

Visceral Leishmaniasis (VL; also known as Kala-azar) is an ultimately fatal disease endemic in Bihar. A 2007 observational cohort study in Bihar of 251 patients with VL treated with 20 mg/Kg intravenous liposomal amphotericin B (Ambisome) demonstrated a 98% cure rate at 6-months. Between July 2007 and August 2012, Médecins Sans Frontières (MSF) and the Rajendra Memorial Research Institute (RMRI) implemented a VL treatment project in Bihar, India—an area highly endemic for Leishmania donovani—using this regimen as first-line treatment.

Methods and Principal Findings

Intravenous Ambisome 20 mg/kg was administered in four doses of 5 mg/kg over 4–10 days, depending on the severity of disease. Initial clinical cure at discharge was defined as improved symptoms, cessation of fever, and recession of spleen enlargement. This observational retrospective cohort study describes 8749 patients with laboratory-confirmed primary VL treated over a 5-year period: 1396 at primary healthcare centers, 7189 at hospital, and 164 at treatment camps. Initial clinical cure was achieved in 99.3% of patients (8692/8749); 0.3% of patients (26/8749) defaulted from treatment and 0.4% (31/8749) died. Overall, 1.8% of patients (161/8749) were co-infected with HIV and 0.6% (51/8749) with tuberculosis. Treatment was discontinued because of severe allergic reactions in 0.1% of patients (7/8749). Overall, 27 patients (0.3%) were readmitted with post Kala-azar dermal leishmaniasis (PKDL). Risk factors for late presentation included female sex, age >15 years and being from a scheduled caste.In 2012, a long-term efficacy survey in the same area of Bihar determined relapse rates of VL after 5 years'' intervention with Ambisome. Of 984 immunocompetent patients discharged between September 2010 and December 2011, 827 (84.0%) were traced in order to determine their long-term outcomes. Of these, 20 patients (2.4%) had relapsed or received further treatment for VL. Of those completing 6, 12, and 15 month follow-up, 0.3% (2/767), 3.7% (14/383), and 2.4% (4/164), respectively, had relapsed. The mean ±SD time-to-relapse was 9.6±3.0 months.

Significance

This is the largest cohort of VL patients treated with 20 mg/kg Ambisome worldwide. The drug has high initial and long-term efficacy, and a low rate of adverse reactions when administered under field conditions in Bihar, India. Although challenging, its use as first line treatment in rural settings in Bihar is safe and feasible.     

Immunogenicity of Outer Membrane Proteins VirB9-1 and VirB9-2, a Novel Nanovaccine against Anaplasma marginale

Anaplasma marginale is the most prevalent tick-borne livestock pathogen and poses a significant threat to cattle industry. In contrast to currently available live blood-derived vaccines against A. marginale, alternative safer and better-defined subunit vaccines will be of great significance. Two proteins (VirB9-1 and VirB9-2) from the Type IV secretion system of A. marginale have been shown to induce humoral and cellular immunity. In this study, Escherichia coli were used to express VirB9-1 and VirB9-2 proteins. Silica vesicles having a thin wall of 6 nm and pore size of 5.8 nm were used as the carrier and adjuvant to deliver these two antigens both as individual or mixed nano-formulations. High loading capacity was achieved for both proteins, and the mouse immunisation trial with individual as well as mixed nano-formulations showed high levels of antibody titres over 10⁷ and strong T-cell responses. The mixed nano-formulation also stimulated high-level recall responses in bovine T-cell proliferation assays. These results open a promising path towards the development of efficient A. marginale vaccines and provide better understanding on the role of silica vesicles to deliver multivalent vaccines as mixed nano-formulations able to activate both B-cell and T-cell immunity, for improved animal health.     

Expression of the Cellulomonas fimi cellulase genes cex and cenA from the divergent tet promoters of transposon Tn10

A cartridge was constructed which contained the divergent tet promoters of transposon Tn10 between an exoglucanase gene (cex) and an endoglucanase gene (cenA) of Cellulomonas fimi. When carried in a broad-host-range vector, the cartridge gave expression of cex and cenA in Escherichia coli, Rhodobacter capsulatus and Klebsiella pneumoniae.Abbreviations CM-cellulose carboxymethyl cellulose - MUC methylumbelliferyl--d-cellobioside - pNPC p-nitrophenyl--d-cellobioside - TBE 89 mM Tris-borate-89 mM boric acid-8 mM EDTA, pH 8.0 CFB and RAJW dedicate this paper to John L. Ingraham, stimulating teacher, wise counsel, and good friend, on the occasion of his retirement