Isolation and Molecular Characterization of Arsenite-Tolerant Alishewanella sp. GIDC-5 Originated from Industrial Effluents

An As-hypertolerant Alishewanella sp. GIDC-5 (Accession no. HQ659190) was isolated from an effluent treatment plant of the industrial area near Sachin, Gujarat (India). In vitro studies revealed that GIDC-5 can tolerate 18 mM of arsenite [As(III)] and 220 mM of arsenate [As(V)]. PCR analysis confirmed the presence of arsenite transporters [arsB and ACR3(1)] and arsenite oxidase gene [aioB]. Specific activities of arsenite oxidase and arsenate reductase, located on membrane and cytoplasmic fractions respectively, increased in dose dependent manner with arsenite concentration. Also, specific activities of antioxidant enzymes viz., catalase, ascorbate peroxidase, superoxide dismutase and glutathione S-transferase increased in presence of arsenite. Increased exposure to arsenite changes enzyme activities of the glycolysis, Krebs and glyoxylate branches dramatically. These results reveal that along with ars operon, metabolic adaptation and antioxidant activities participate in As(III) tolerance in Alishewanella sp. GIDC-5.     

Mechanism of zinc resistance in a plant growth promoting Pseudomonas fluorescens strain

Bacterial systems have evolved a number of mechanisms, both active and passive, to manage toxic concentrations of heavy metals in their environment. The present study is aimed at describing the zinc resistance mechanism in a rhizospheric isolate, Pseudomonas fluorescens strain Psd. The strain was able to sustain an external Zn²⁺ concentration of up to 5 mM in the medium. The strategy for metal management by the strain was found to be extracellular biosorption with a possible role of exopolysaccharides in metal accumulation. The attainment of equilibrium in biosorption reaction was found to be dependent on initial Zn²⁺ concentration, with the reaction reaching equilibrium faster (50 min) at high initial Zn²⁺ concentration. Biosorption kinetics of the process was adjusted to pseudo-first order rate equation. With the help of Langmuir and Freundlich adsorption isotherms, it was established that Zn²⁺ biosorption by the bacterium is a thermodynamically favourable process.     

Molecular Basis for Impaired DNA Damage Response Function Associated with the RAP80 ΔE81 Defect

Signal transduction within the DNA damage response is driven by the flux of protein-protein interaction cascades that ultimately recruit repair complexes to sites of damage. The protein RAP80 plays a central role in the damage response by targeting BRCA1/BRCA2 tumor suppressors to DNA damage foci through multivalent binding of Lys-63-linked polyubiquitin chains. Mutations within the high penetrance BRCA1/BRCA2 genes account for ∼20% of familial breast cancers. The genetic basis for the remaining cancers remains unknown, but may involve defects in binding partners for BRCA1 and BRCA2 that lead to impaired targeting to foci and a concomitant role in the pathogenesis of cancer. Recently, an in-frame deletion mutation (ΔE81) in a conserved region from the first ubiquitin interaction motif of RAP80 has been linked to an increase in chromosomal abnormalities. Using NMR spectroscopy, we demonstrate that the N-cap motif within the α-helix of the first ubiquitin interaction motif from ΔE81 undergoes a structural frameshift that leads to abolishment of multivalent binding of polyubiquitin chains. Loss of this single glutamate residue disrupts favorable electrostatic interactions between RAP80 and ubiquitin, establishing a plausible molecular basis for a potential predisposition to cancer unrelated to mutations within BRCA1/BRCA2 genes.     

Human intestinal bacteria as reservoirs for antibiotic resistance genes

Human intestinal bacteria have many roles in human health, most of which are beneficial or neutral for the host. In this review, we explore a more sinister side of intestinal bacteria; their role as traffickers in antibiotic resistance genes. Evidence is accumulating to support the hypothesis that intestinal bacteria not only exchange resistance genes among themselves but might also interact with bacteria that are passing through the colon, causing these bacteria to acquire and transmit antibiotic resistance genes.     

Proper Restoration of Excitation-Contraction Coupling in the Dihydropyridine Receptor ��1-null Zebrafish Relaxed Is an Exclusive Function of the ��1a Subunit

The paralyzed zebrafish strain relaxed carries a null mutation for the skeletal muscle dihydropyridine receptor (DHPR) β_1a subunit. Lack of β_1a results in (i) reduced membrane expression of the pore forming DHPR α_1S subunit, (ii) elimination of α_1S charge movement, and (iii) impediment of arrangement of the DHPRs in groups of four (tetrads) opposing the ryanodine receptor (RyR1), a structural prerequisite for skeletal muscle-type excitation-contraction (EC) coupling. In this study we used relaxed larvae and isolated myotubes as expression systems to discriminate specific functions of β_1a from rather general functions of β isoforms. Zebrafish and mammalian β_1a subunits quantitatively restored α_1S triad targeting and charge movement as well as intracellular Ca²⁺ release, allowed arrangement of DHPRs in tetrads, and most strikingly recovered a fully motile phenotype in relaxed larvae. Interestingly, the cardiac/neuronal β_2a as the phylogenetically closest, and the ancestral housefly β_M as the most distant isoform to β_1a also completely recovered α_1S triad expression and charge movement. However, both revealed drastically impaired intracellular Ca²⁺ transients and very limited tetrad formation compared with β_1a. Consequently, larval motility was either only partially restored (β_2a-injected larvae) or not restored at all (β_M). Thus, our results indicate that triad expression and facilitation of 1,4-dihydropyridine receptor (DHPR) charge movement are common features of all tested β subunits, whereas the efficient arrangement of DHPRs in tetrads and thus intact DHPR-RyR1 coupling is only promoted by the β_1a isoform. Consequently, we postulate a model that presents β_1a as an allosteric modifier of α_1S conformation enabling skeletal muscle-type EC coupling.Excitation-contraction (EC)³ coupling in skeletal muscle is critically dependent on the close interaction of two distinct Ca²⁺ channels. Membrane depolarizations of the myotube are sensed by the voltage-dependent 1,4-dihydropyridine receptor (DHPR) in the sarcolemma, leading to a rearrangement of charged amino acids (charge movement) in the transmembrane segments S4 of the pore-forming DHPR α_1S subunit (1, 2). This conformational change induces via protein-protein interaction (3, 4) the opening of the sarcoplasmic type-1 ryanodine receptor (RyR1) without need of Ca²⁺ influx through the DHPR (5). The release of Ca²⁺ from the sarcoplasmic reticulum via RyR1 consequently induces muscle contraction. The protein-protein interaction mechanism between DHPR and RyR1 requires correct ultrastructural targeting of both channels. In Ca²⁺ release units (triads and peripheral couplings) of the skeletal muscle, groups of four DHPRs (tetrads) are coupled to every other RyR1 and hence are geometrically arranged following the RyR-specific orthogonal arrays (6).The skeletal muscle DHPR is a heteromultimeric protein complex, composed of the voltage-sensing and pore-forming α_1S subunit and auxiliary subunits β_1a, α₂δ-1, and γ₁ (7). While gene knock-out of the DHPR γ₁ subunit (8, 9) and small interfering RNA knockdown of the DHPR α₂δ-1 subunit (10-12) have indicated that neither subunit is essential for coupling of the DHPR with RyR1, the lack of the α_1S or of the intracellular β_1a subunit is incompatible with EC coupling and accordingly null model mice die perinatally due to asphyxia (13, 14). β subunits of voltage-gated Ca²⁺ channels were repeatedly shown to be responsible for the facilitation of α₁ membrane insertion and to be potent modulators of α₁ current kinetics and voltage dependence (15, 16). Whether the loss of EC coupling in β₁-null mice was caused by decreased DHPR membrane expression or by the lack of a putative specific contribution of the β subunit to the skeletal muscle EC coupling apparatus (17, 18) was not clearly resolved. Recently, other β-functions were identified in skeletal muscle using the β₁-null mutant zebrafish relaxed (19, 20). Like the β₁-knock-out mouse (14) zebrafish relaxed is characterized by complete paralysis of skeletal muscle (21, 22). While β₁-knock-out mouse pups die immediately after birth due to respiratory paralysis (14), larvae of relaxed are able to survive for several days because of oxygen and metabolite diffusion via the skin (23). Using highly differentiated myotubes that are easy to isolate from these larvae, the lack of EC coupling could be described by quantitative immunocytochemistry as a moderate ∼50% reduction of α_1S membrane expression although α_1S charge movement was nearly absent, and, most strikingly, as the complete lack of the arrangement of DHPRs in tetrads (19). Thus, in skeletal muscle the β subunit enables EC coupling by (i) enhancing α_1S membrane targeting, (ii) facilitating α_1S charge movement, and (iii) enabling the ultrastructural arrangement of DHPRs in tetrads.The question arises, which of these functions are specific for the skeletal muscle β_1a and which ones are rather general properties of Ca²⁺ channel β subunits. Previous reconstitution studies made in the β₁-null mouse system (24, 25) using different β subunit constructs (26) did not allow differentiation between β-induced enhancement of non-functional α_1S membrane expression and the facilitation of α_1S charge movement, due to the lack of information on α_1S triad expression levels. Furthermore, the β-induced arrangement of DHPRs in tetrads was not detected as no ultrastructural information was obtained.In the present study, we established zebrafish mutant relaxed as an expression system to test different β subunits for their ability to restore skeletal muscle EC coupling. Using isolated myotubes for in vitro experiments (19, 27) and complete larvae for in vivo expression studies (28-31) and freeze-fracture electron microscopy, a clear differentiation between the major functional roles of β subunits was feasible in the zebrafish system. The cloned zebrafish β_1a and a mammalian (rabbit) β_1a were shown to completely restore all parameters of EC coupling when expressed in relaxed myotubes and larvae. However, the phylogenetically closest β subunit to β_1a, the cardiac/neuronal isoform β_2a from rat, as well as the ancestral β_M isoform from the housefly (Musca domestica), could recover functional α_1S membrane insertion, but led to very restricted tetrad formation when compared with β_1a, and thus to impaired DHPR-RyR1 coupling. This impairment caused drastic changes in skeletal muscle function.The present study shows that the enhancement of functional α_1S membrane expression is a common function of all the tested β subunits, from β_1a to even the most distant β_M, whereas the effective formation of tetrads and thus proper skeletal muscle EC coupling is an exclusive function of the skeletal muscle β_1a subunit. In context with previous studies, our results suggest a model according to which β_1a acts as an allosteric modifier of α_1S conformation. Only in the presence of β_1a, the α_1S subunit is properly folded to allow RyR1 anchoring and thus skeletal muscle-type EC coupling.     

Genome-wide comparative analyses of domain organisation of repertoires of protein kinases of Arabidopsis thaliana and Oryza sativa

A comparative analysis on protein kinases encoded in the completely sequenced genomes of two plant species, namely Arabidopsis thaliana and Oryza sativa spp japonica cv. Nipponbare is reported in the current study. We have analysed 836 and 1386 kinases identified from A. thaliana and the O. sativa genomes respectively. Their classification into known subfamilies reveals selective expansions of the plant receptor kinase subfamily comprising of Ser/Thr receptor kinases. The presence of calcium dependent kinases, and potential absence of cyclic nucleotide-dependent protein kinase of the type found in other (non-plant) eukaryotes, are other notable features of the two plant kinomes described here. An analysis on domain organisation of each of the protein kinases encoded in the plant genome has been carried out. Uncommon composition of functional domains like nuclear translocation factor domain, redox sensor domain (PAS), ACT and lectin domains are observed in few protein kinases shared between the two plant species. Biochemical functions characteristic of the domains recruited in these protein kinase gene products suggest their mode of regulation by alternate cellular localisation, oxidation potential, amino acid flux and binding of carbohydrates. Occurrence of multi-functional kinases with diverse enzymatic modules, such as Transposases and peptidases, tethered to the kinase catalytic domain is another interesting feature of the protein kinase complement of the O. sativa genome. Co-occurrence of diverse nucleotide and carbohydrate binding domains with catalytic kinase domain containing gene products has also been observed. Putative homologues of protein kinases of A. thaliana that regulate plant-specific physiological processes like ethylene hormone response, somatic embryogenesis and pathogen defence have been identified in O. sativa genome as well.     

Photodynamic action of C-phycocyanins obtained from marine and fresh water cyanobacterial cultures: a comparative study using EPR spin trapping technique

C-phycocyanins, major biliproteins of blue green algae (cyanobacteria), widely used as colourants in food and cosmetics are known for their antioxidant as well as therapeutic potential. Recent claims indicating phycobiliproteins exert stronger photodynamic action on tumor cells than clinically approved hematoporphyrin derivatives motivate us to investigate the photodynamic action of two newly isolated C-phycocyanins from Phormidium [PHR] and Lyngbya [LY] spp, respectively in comparison with known C-phycocyanin from Spirulina sp. [SPI]. Photolysis of air saturated solutions of PHR, LY and SPI in the presence of 2,2,6,6-Tetramethyl piperidinol (TEMPL) generated three line EPR spectrum characteristic of 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxyl (TEMPOL). The increase in intensity of the EPR spectrum with time of irradiation and decrease in intensity, in the presence of 1O2 quencher DABCO confirm the formation of 1O2. Photoirradiation in the presence of spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) generated EPR signal characteristic of O2(-) adduct. Efficiency of 1O2 generation is of the order LY > PHR> SPI. The yield of reactive oxygen species (ROS) generation is found to be 1O2>O2(-) indicating type II mechanism to be the prominent pathway for photosensitation by phycocyanins.     

Analyses of co-operative transitions in Plasmodium falciparum beta-ketoacyl acyl carrier protein reductase upon co-factor and acyl carrier protein binding

The type II fatty acid synthase pathway of Plasmodium falciparum is a validated unique target for developing novel antimalarials because of its intrinsic differences from the type I pathway operating in humans. beta-Ketoacyl-acyl carrier protein reductase is the only enzyme of this pathway that has no isoforms and thus selective inhibitors can be developed for this player of the pathway. We report here intensive studies on the direct interactions of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase with its cofactor, NADPH, acyl carrier protein, acetoacetyl-coenzyme A and other ligands in solution, by monitoring the intrinsic fluorescence (lambdamax 334 nM) of the protein as a result of its lone tryptophan, as well as the fluorescence of NADPH (lambdamax 450 nM) upon binding to the enzyme. Binding of the reduced cofactor makes the enzyme catalytically efficient, as it increases the binding affinity of the substrate, acetoacetyl-coenzyme A, by 16-fold. The binding affinity of acyl carrier protein to the enzyme also increases by approximately threefold upon NADPH binding. Plasmodiumbeta-ketoacyl-acyl carrier protein reductase exhibits negative, homotropic co-operative binding for NADPH, which is enhanced in the presence of acyl carrier protein. Acyl carrier protein increases the accessibility of NADPH to beta-ketoacyl-acyl carrier protein reductase, as evident from the increase in the accessibility of the tryptophan of beta-ketoacyl-acyl carrier protein reductase to acrylamide, from 81 to 98%. In the presence of NADP+, the reaction proceeds in the reverse direction (Ka=23.17 microM-1). These findings provide impetus for exploring the influence of ligands on the structure-activity relationship of Plasmodiumbeta-ketoacyl-acyl carrier protein reductase.