Kinetic resolution of 1-chloro-3-(1-naphthyloxy)-2-propanol,an intermediate in the synthesis of beta-adrenergic receptor blockers

(R)- and (S)-1-chloro-3-(1-naphthyloxy)-2-propanol are intermediates in the synthesis of β-adrenergic blocking agents and antihypertensive drugs such as propranolol and nadoxolol. Herein, improvement in the preparation of racemic 1-chloro-3-(1-naphthyloxy)-2-propanol generated from 1-naphthol and epichlorohydrin are reported. In addition, kinetic resolution studies have been conducted to obtain both (R) and (S)-1-chloro-3-(1-naphthyloxy)-2-propanol. These compounds were obtained in highly optically pure form by the stereoselective hydrolysis of its acyl derivatives using whole cell preparations containing enzymes from native sources. The results were compared with those obtained using commercial lipases.     

Halorespiring bacteria-molecular characterization and detection

Recently, a rapidly increasing number of bacteria has been isolated that is able to couple the reductive dehalogenation of various halogenated aromatic and aliphatic compounds like chlorophenols and tetrachloroethene to energy conservation by electron-transport-coupled phosphorylation. The potential of these halorespiring bacteria for innovative clean-up strategies of polluted anoxic environments has greatly stimulated efforts to unravel the molecular basis of the novel respiratory chains they possess. The thorough characterization of halorespiratory key components at the physiological, biochemical and molecular genetic level has revealed both structural and functional similarity of chloroaryl- and chloroalkyl-respiratory chains from different phylogenetically distinct microorganisms. The reductive dehalogenases from halorespiring bacteria were found to comprise a novel class of corrinoid-containing Fe/S-proteins. Sensitive molecular methods for monitoring both presence and fate of halorespiring bacteria have been developed, which will be instrumental for the design and maintenance of optimised in situ bioremediation processes.     

Plant endemism in two forests in southern Bahia, Brazil

An important factor in determining species rarity is the geographic distribution of species. Estimates were made of the level of endemism of the flora of two sites in the southern Bahian wet forest zone. Estimates were made for endemism in the Atlantic forest biome and for the much more restricted area of southern Bahia and northern Espi´rito Santo and are derived from analyses of the distributions of the species known from each area. The species checklist for each area is based on identified specimens resulting from intensive collecting in a forest near Serra Grande (40km north of Ilhe´us) and the Una Biological Reserve (40km south of Ilhe´us). Slightly less than half of the species (45.2% at Una and 47.7% at Serra Grande) have widespread distributions and 7.4% at each site are disjunct between the coastal forests and Amazonia. In the Una Reserve, 44.1% of the species are endemic to the coastal forest and 28.1% endemic to southern Bahia and northern Espi´rito Santo. At Serra Grande, 41.6% of the species are endemic to the coastal forest and 26.5% endemic to southern Bahia and northern Espi´rito Santo.     

In vitro antifungal activity of hydroxychavicol isolated from Piper betle L

Background

Hydroxychavicol, isolated from the chloroform extraction of the aqueous leaf extract of Piper betle L., (Piperaceae) was investigated for its antifungal activity against 124 strains of selected fungi. The leaves of this plant have been long in use tropical countries for the preparation of traditional herbal remedies.

Methods

The minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of hydroxychavicol were determined by using broth microdilution method following CLSI guidelines. Time kill curve studies, post-antifungal effects and mutation prevention concentrations were determined against Candida species and Aspergillus species "respectively". Hydroxychavicol was also tested for its potential to inhibit and reduce the formation of Candida albicans biofilms. The membrane permeability was measured by the uptake of propidium iodide.

Results

Hydroxychavicol exhibited inhibitory effect on fungal species of clinical significance, with the MICs ranging from 15.62 to 500 μg/ml for yeasts, 125 to 500 μg/ml for Aspergillus species, and 7.81 to 62.5 μg/ml for dermatophytes where as the MFCs were found to be similar or two fold greater than the MICs. There was concentration-dependent killing of Candida albicans and Candida glabrata up to 8 × MIC. Hydroxychavicol also exhibited an extended post antifungal effect of 6.25 to 8.70 h at 4 × MIC for Candida species and suppressed the emergence of mutants of the fungal species tested at 2 × to 8 × MIC concentration. Furthermore, it also inhibited the growth of biofilm generated by C. albicans and reduced the preformed biofilms. There was increased uptake of propidium iodide by C. albicans cells when exposed to hydroxychavicol thus indicating that the membrane disruption could be the probable mode of action of hydroxychavicol.

Conclusions

The antifungal activity exhibited by this compound warrants its use as an antifungal agent particularly for treating topical infections, as well as gargle mouthwash against oral Candida infections.     

Characterization of an anion-exchange porous polypropylene hollow fiber membrane for immobilization of ABL lipase

Hollow fiber membrane offers the advantage to integrate catalytic conversion, product separation and catalyst recovery into a single separation process compared to conventional systems. Polypropylene (PP) hollow fiber membrane is a chemically inert and stable membrane with high potential for enzyme immobilization. The surface properties of polypropylene have been modified by radiation induced graft polymerization. Samples were prepared by grafting of glycidylmethacrylate (GMA) using gamma radiation, at different monomer concentrations and irradiation dose. The resulting epoxy was converted into a diethylamino group as an anion-exchange medium to bind the lipase molecules. Surface properties of the grafted and amine treated samples were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM) and contact angle measurements. AFM revealed higher surface roughness for grafted samples than that of virgin polymer. SEM micrographs illustrated that the porous network was retained at high degree of grafting. Contact angle measurements showed excellent wetting properties with water for the grafted and amine treated membranes. Thermal properties were studied using differential scanning calorimeter (DSC) and thermogravimetic analysis (TGA). It was observed that grafting occurred mainly in the amorphous region of the membranes. Activity and operational stability of ABL lipase, isolated from Arthobacter sp. were assayed after immobilizing it to the modified PP hollow fiber. Immobilized lipase retained 20U/g activity after ten hydrolysis cycles and 68% residual activity after 12 weeks of storage.     

Hydrated conidia of Metarhizium anisopliae release a family of metalloproteases

Metarhizium anisopliae spores release isoforms of metalloprotease during hydration over a 4-day incubation period. The isoforms were identified and characterized by using one-dimensional native PAGE (1-DE nPAGE) and one-dimensional SDS non-dissociating (1-DE nSDS-PAGE) zymography. The ability of these isozymes to degrade gelatin varied as revealed by 2-D spot densitometry. 1-DE nPAGE zymography revealed five isoforms of gelatinase from Tween wash of conidia. Where as, one to three activities with different intensities appeared on gel from washing of conidia to incubation in water till day 4. The relative migrations of these activities on 1-DE nPAGE zymograms appeared as fast, medium and slow on gel. The 2-D spot densitometry of zymograms indicated isoforms have different proteolytic activity as quantified by pixel intensities. SDS-PAGE zymography indicated the release of two isozymes of Mr 103 and 12 kDa during Tween treatment of conidia. However, during the first washing step with water and incubation of spores at day 2 and 3, respectively, only 12 kDa protein was evident. Majority of these proteases were inhibited by EDTA, but stimulated by CaCl(2), and MgCl(2). The presence of isozymes in conidia and their release during hydration must have functional significance for fungi and in this case it should provide advantages to M. anisopliae in its saprobic or pathogenic modalities. To our knowledge this is the first report describing release of metalloprotease isozymes from conidia.     

The HC fragment of tetanus toxin forms stable, concentration-dependent dimers via an intermolecular disulphide bond

Protein oligomerisation is a prerequisite for the toxicity of a number of bacterial toxins. Examples include the pore-forming cytotoxin streptolysin O, which oligomerises to form large pores in the membrane and the protective antigen of anthrax toxin, where a heptameric complex is essential for the delivery of lethal factor and edema factor to the cell cytosol. Binding of the clostridial neurotoxins to receptors on neuronal cells is well characterised, but little is known regarding the quaternary structure of these toxins and the role of oligomerisation in the intoxication process. We have investigated the oligomerisation of the receptor binding domain (H(C)) of tetanus toxin, which retains the binding and trafficking properties of the full-length toxin. Electrophoresis, size exclusion chromatography and mass spectrometry were used to demonstrate that H(C) undergoes concentration-dependent oligomerisation in solution. Reducing agents were found to affect H(C) oligomerisation and, using mutagenesis, Cys869 was shown to be essential for this process. Furthermore, the oligomeric state and quaternary structure of H(C) in solution was assessed using synchrotron small-angle X-ray scattering. Ab initio shape analysis and rigid body modelling coupled with mutagenesis data allowed the construction of an unequivocal model of dimeric H(C) in solution. We propose a possible mechanism for H(C) oligomerisation and discuss how this may relate to toxicity.     

Immunomodulatory activity of an extract of the novel fungal endophyte Entrophospora infrequens isolated from Nothapodytes foetida (Wight) Sleumer

A novel camptothecin-producing endophytic fungus viz., Entrophospora infrequens was isolated from an important Indian medicinal plant Nothapodytes foetida. The present study reports evaluation ofbioactivities of two novel extracts viz., chloroform (CEEI) and methanolic (MEEI) extracts of Entrophospora infrequens with respect to their immunomodulatory potential in vitro and in vivo (in Balb/c mice). The endophyte E. infrequens was found to synthesize camptothecin, which tested positive in CEEI. The immunomodulatory potential of CEEI and MEEI was compared with standard camptothecin (CPT). Doses of the chloroform extract (CEEI) ranging from 12.5-100 mg/kg body weight, significantly (p < 0.05) stimulated the humoral and cell-mediated immune responses in a dose-dependent manner. MEEI on the other hand significantly (p < 0.05) stimulated the delayed type hypersensitivity (DTH) reaction (by nearly 80%), plaque forming cell (PFC) assay (33%), phagocytic response (38%) and haemagglutination antibody (HA) titre [IgM by 79.07% and IgG by 62.05%] at a dose of 12.5 mg/kg body weight. The present study is the first report of the immunomodulatory potential of this neoteric camptothecin-producing endophyte from Nothapodytes foetida.     

A cold-active esterase of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> A3(2): from genome sequence to enzyme activity

The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C₂–C₁₀) with maximum activity towards the acetate ester (C₂). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of 1.1–1.9).     

Differential Expression of Apoptotic Proteins Following Hypoxia-induced CREB Phosphorylation in the Cerebral Cortex of Newborn Piglets

The present study investigates the correlation between the hypoxia-induced phosphorylation of cyclic AMP response element binding protein and the expression of apoptotic proteins (proapoptotic proteins Bax and Bad and antiapoptotic proteins Bcl-2 and Bcl-xl) during hypoxia in the cerebral cortex of newborn piglets. Piglets were divided into normoxic (Nx) and hypoxic (Hx, FiO₂ = 0.06 for 1 h) groups. Cerebral tissue hypoxia was documented by ATP and phosphocreatine (PCr) levels. Ser¹³³ phosphorylation of cyclic AMP response element binding (CREB) protein was determined by Western blot analysis using a specific anti-phosphorylated Ser¹³³-CREB protein antibody. The expression of apoptotic proteins was determined by using specific anti-Bax, anti-Bad, anti-Bcl-2 and anti-Bcl-xl antibodies. ATP and PCr values (μmoles/g brain) in Hx were significantly different from Nx (ATP: 4.40 ± 0.39 in Nx vs. 1.19 ± 0.44 in Hx, P < 0.05 vs. Nx; PCr: 3.60 ± 0.40 in Nx vs. 0.70 ± 0.31 in Hx, P < 0.05 vs. Nx). Ser¹³³ phosphorylated CREB protein (OD × mm²) was 74.55 ± 4.75 in Nx and 127.13 ± 19.36 in Hx (P < 0.05 vs. Nx). The expression of proapoptotic proteins Bax and Bad increased and strongly correlated with the increase in CREB protein phosphorylation (correlation coefficient r = 0.82 and r = 0.85, respectively). The expression of antiapoptotic proteins Bcl-2 and Bcl-xl did not show correlation with CREB protein phosphorylation. We conclude that cerebral hypoxia results in differential regulation of CREB protein-mediated expression of proapoptotic and antiapoptotic proteins in the cerebral cortex of newborn piglets. We propose that the increased expression of proapoptotic vs antiapoptotic genes will lead to an increased potential for apoptotic programmed cell death in the Hx newborn brain.