A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

Drosophila MUS312 interacts with the nucleotide excision repair endonuclease MEI-9 to generate meiotic crossovers

MEI-9 is the Drosophila homolog of the human structure-specific DNA endonuclease XPF. Like XPF, MEI-9 functions in nucleotide excision repair and interstrand crosslink repair. MEI-9 is also required to generate meiotic crossovers, in a function thought to be associated with resolution of Holliday junction intermediates. We report here the identification of MUS312, a protein that physically interacts with MEI-9. We show that mutations in mus312 elicit a meiotic phenotype identical to that of mei-9 mutants. A missense mutation in mei-9 that disrupts the MEI-9-MUS312 interaction abolishes the meiotic function of mei-9 but does not affect the DNA repair functions of mei-9. We propose that MUS312 facilitates resolution of meiotic Holliday junction intermediates by MEI-9.     

Cyclic AMP is sufficient for triggering the exocytic recruitment of aquaporin-2 in renal epithelial cells

The initial response of renal epithelial cells to the antidiuretic hormone arginine vasopressin (AVP) is an increase in cyclic AMP. By applying immunofluorescence, cell membrane capacitance and transepithelial water flux measurements we show that cAMP alone is sufficient to elicit the antidiuretic cellular response in primary cultured epithelial cells from renal inner medulla, namely the transport of aquaporin-2 (AQP2)-bearing vesicles to, and their subsequent fusion with, the plasma membrane (AQP2 shuttle). The AQP2 shuttle is evoked neither by AVP-independent Ca²⁺ increases nor by AVP-induced Ca²⁺ increases. However, clamping cytosolic Ca²⁺ concentrations below resting levels at 25 nM inhibited exocytosis. Exocytosis was confined to a slow monophasic response, and readily releasable vesicles were missing. Analysis of endocytic capacitance steps revealed that cAMP does not decelerate the retrieval of AQP2 from the plasma membrane. Our data suggest that cAMP initiates an early step, namely the transport of AQP2-bearing vesicles towards the plasma membrane, and do not support a regulatory function for Ca²⁺ in the AQP2 shuttle.     

Role of rpoS in Stress Survival and Virulence of Vibrio cholerae

Vibrio cholerae is known to persist in aquatic environments under nutrient-limiting conditions. To analyze the possible involvement of the alternative sigma factor encoded by rpoS, which is shown to be important for survival during nutrient deprivation in several other bacterial species, a V. cholerae rpoS homolog was cloned by functional complementation of an Escherichia coli mutant by using a wild-type genomic library. Sequence analysis of the complementing clone revealed an 1.008-bp open reading frame which is predicted to encode a 336-amino-acid protein with 71 to 63% overall identity to other reported rpoS gene products. To determine the functional role of rpoS in V. cholerae, we inactivated rpoS by homologous recombination. V. cholerae strains lacking rpoS are impaired in the ability to survive diverse environmental stresses, including exposure to hydrogen peroxide, hyperosmolarity, and carbon starvation. These results suggest that rpoS may be required for the persistence of V. cholerae in aquatic habitats. In addition, the rpoS mutation led to reduced production or secretion of hemagglutinin/protease. However, rpoS is not critical for in vivo survival, as determined by an infant mouse intestinal competition assay.     

Vibrio cholerae Response Regulator VxrB Controls Colonization and Regulates the Type VI Secretion System

Two-component signal transduction systems (TCS) are used by bacteria to sense and respond to their environment. TCS are typically composed of a sensor histidine kinase (HK) and a response regulator (RR). The Vibrio cholerae genome encodes 52 RR, but the role of these RRs in V. cholerae pathogenesis is largely unknown. To identify RRs that control V. cholerae colonization, in-frame deletions of each RR were generated and the resulting mutants analyzed using an infant mouse intestine colonization assay. We found that 12 of the 52 RR were involved in intestinal colonization. Mutants lacking one previously uncharacterized RR, VCA0566 (renamed VxrB), displayed a significant colonization defect. Further experiments showed that VxrB phosphorylation state on the predicted conserved aspartate contributes to intestine colonization. The VxrB regulon was determined using whole genome expression analysis. It consists of several genes, including those genes that create the type VI secretion system (T6SS). We determined that VxrB is required for T6SS expression using several in vitro assays and bacterial killing assays, and furthermore that the T6SS is required for intestinal colonization. vxrB is encoded in a four gene operon and the other vxr operon members also modulate intestinal colonization. Lastly, though ΔvxrB exhibited a defect in single-strain intestinal colonization, the ΔvxrB strain did not show any in vitro growth defect. Overall, our work revealed that a small set of RRs is required for intestinal colonization and one of these regulators, VxrB affects colonization at least in part through its regulation of T6SS genes.     

Reducing GBA2 Activity Ameliorates Neuropathology in Niemann-Pick Type C Mice

The enzyme glucocerebrosidase (GBA) hydrolyses glucosylceramide (GlcCer) in lysosomes. Markedly reduced GBA activity is associated with severe manifestations of Gaucher disease including neurological involvement. Mutations in the GBA gene have recently also been identified as major genetic risk factor for Parkinsonism. Disturbed metabolism of GlcCer may therefore play a role in neuropathology. Besides lysosomal GBA, cells also contain a non-lysosomal glucosylceramidase (GBA2). Given that the two β-glucosidases share substrates, we speculated that over-activity of GBA2 during severe GBA impairment might influence neuropathology. This hypothesis was studied in Niemann-Pick type C (Npc1 ^-/-) mice showing secondary deficiency in GBA in various tissues. Here we report that GBA2 activity is indeed increased in the brain of Npc1 ^-/- mice. We found that GBA2 is particularly abundant in Purkinje cells (PCs), one of the most affected neuronal populations in NPC disease. Inhibiting GBA2 in Npc1 ^-/- mice with a brain-permeable low nanomolar inhibitor significantly improved motor coordination and extended lifespan in the absence of correction in cholesterol and ganglioside abnormalities. This trend was recapitulated, although not to full extent, by introducing a genetic loss of GBA2 in Npc1 ^-/- mice. Our findings point to GBA2 activity as therapeutic target in NPC.     

Processive cytoskeletal motors studied with single-molecule fluorescence techniques

Processive cytoskeletal motors from the myosin, kinesin, and dynein families walk on actin filaments and microtubules to drive cellular transport and organization in eukaryotic cells. These remarkable molecular machines are able to take hundreds of successive steps at speeds of up to several microns per second, allowing them to effectively move vesicles and organelles throughout the cytoplasm. Here, we focus on single-molecule fluorescence techniques and discuss their wide-ranging applications to the field of cytoskeletal motor research. We cover both traditional fluorescence and sub-diffraction imaging of motors, providing examples of how fluorescence data can be used to measure biophysical parameters of motors such as coordination, stepping mechanism, gating, and processivity. We also outline some remaining challenges in the field and suggest future directions.     

Structural, technological and productivity analyses of rainbow trout (Oncorhynchus mykiss) farms in the Marmara region, Turkey

This study analyses the structural, technological performance components and the overall productivity of inland rainbow trout ( Oncorhynchus mykiss ) farms in the Marmara region, Turkey. Of 81 active farms, 36 were small scale (1–10 t year⁻¹), 32 medium-sized (11–30 t year⁻¹) and 13 large production units (over 30 t year⁻¹). Sufficient data for this study were collected from 70 farms for inclusion in the analyses; 59.3% were so-called combined farms (hatchery and grow-out), 37.9% on-growing farms (for market production) and 2.9% producing juvenile fish only for stocking or for supply to grow-out farms. Juvenile fish production was largely conducted on large farms. Capacity utilization (102.8%) of the large farms was most similar to their projected capacity. Many large farms (43.9%) use high-tech utilities and tools. Small farms mostly use concrete ponds whereas most medium and large-scale farms use fiberglass tanks. Medium and large-scale farms were found to be more successful in broodstock management, fertilization, hatching success and survival rate of juvenile fish. Fish stocking density in medium-sized farms (21.8 kg m⁻³) was higher than in small-scale (14.5 kg m⁻³) and large-scale farms (15.5 kg m⁻³). Overall feed conversion ratio for all farms was estimated as 1.2. From the survey results it appears that production capacity planning was calculated more accurately in the projection phase for small and large-scale farms rather than for medium-sized farms. Medium and large-scale farms were more successful in terms of performance (good experience, good structural and technological capabilities).