Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.     

TAK-778 enhances osteoblast differentiation of human bone marrow cells

TAK-778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK-778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK-778 on human bone marrow cells. Cells were cultured in 24-well culture plates at a cell density of 2 x 10(4) cells/well in culture medium containing TAK-778 (10(-7), 10(-6), and 10(-5) M, each) or vehicle. During the culture period, cells were incubated at 37 degrees C in a humidified atmosphere of 5% CO(2) and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone-like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK-778 did not affect cell attachment and viability. Cell number was reduced by TAK-778 in all time period evaluated in a dose-dependent way. The effect of TAK-778 on total protein content, ALP activity and bone-like formation was a dose-dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK-778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone-like formation are enhanced by TAK-778 in a time and dose-dependent way. It means that TAK-778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery.     

TROLL--tandem repeat occurrence locator

SUMMARY: Tandem Repeat Occurrence Locator (TROLL), is a light-weight Simple Sequence Repeat (SSR) finder based on a slight modification of the Aho-Corasick algorithm. It is fast and only requires a standard Personal Computer (PC) to operate. We report running times of 127 s to find all SSRs of length 20 bp or more on the complete Arabdopsis genome--approx. 130 Mbases divided in five chromosomes--using a PC Athlon 650 MHz with 256 MB of RAM. AVAILABILITY: TROLL is an open source project and is available at http://finder.sourceforge.net.     

Ion regulatory patterns of mosquito larvae collected from breeding sites in the Amazon rain forest

We examined the ion composition of mosquito breeding sites located in the Amazon rain forest and the ion regulatory patterns of larvae from these habitats. We found larvae of Toxorhynchites haemorroidalis, Limatus durhamii, Culex (Carrollia) bonnei, and Culex (Culex) sp. residing in fallen palm bracts, leaves, and tree holes that were filled with water. These breeding sites had micromolar levels of Na(+) (1.6-99 micromol L(-1)), but K(+) and Cl(-) concentrations were higher and varied over a large range (231-17,615 micromol L(-1) K(+); 355-2,700 micromol L(-1) Cl(-)). Despite the variability in environmental ion levels and ratios, all four species maintain high hemolymph NaCl levels (80-120 mmol L(-1) Na(+); 60-80 mmol L(-1) Cl(-)). However, the species differed in the means by which they maintain hemolymph ion balance, as indicated by the range of unidirectional Na(+) and Cl(-) uptake rates. Toxorhynchites haemorroidalis had extremely low rates of Na(+) uptake and undetectable Cl(-) uptake, whereas L. durhamii had high rates of uptake for both ions. This variability in rates of uptake may reflect species differences in rates of diffusive ion loss (i.e., permeability). We observed the same curious pattern of Na(+) inhibition and Cl(-) stimulation by low-pH exposure in all four species of mosquitoes, as has been documented in other mosquitoes and aquatic insects. Kinetic analyses of Na(+) and Cl(-) uptake in C. bonnei larvae revealed an unusual pattern of Na(+) uptake that increases linearly (nonsaturable) to extremely high rates, while Cl(-) uptake is a low-affinity, low-capacity system. This pattern contrasts with L. durhamii and Culex (Culex) sp. larvae, which had large increases in both Na(+) and Cl(-) uptake when external NaCl levels were increased. Our results suggest that although these rain forest mosquito larvae are residing in habitats with similar low Na(+), high Cl(-) composition and maintain similar hemolymph NaCl levels, the underlying mechanisms of ion regulation differ among the species.     

Liquid-liquid extraction of xylitol dehydrogenase from Candida guilliermondii homogenate by reversed micelles

The intracellular enzyme xylitol dehydrogenase (XD, EC 1.1.1.9) from Candida guilliermondii, grown in sugarcane bagasse hydrolysate, was separated by reversed micelles of BDBAC [N-benzyl-N-dodecyl-N-bis (2-hydroxyethyl) ammonium chloride] cationic surfactant. An experimental design was employed to evaluate the influence of the following factors on the enzyme separation: temperature, co-solvent concentration and surfactant concentration. The results showed that just the temperature did not show significant effect on XD recovery. A model was used to represent the activity recovery and fit the experimental data. Under optimized conditions, the recovery of total activity was about 121%, and the purity increased 2.3-fold.     

Protection by natural blackwater against disturbances in ion fluxes caused by low pH exposure in freshwater stingrays endemic to the Rio Negro

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.     

Fine‐scale Beta‐diversity Patterns Across Multiple Arthropod Taxa Over a Neotropical Latitudinal Gradient

Documenting how diversity patterns vary at fine‐ and broad scales may help answer many questions in theoretical and applied ecology. However, studies tend to compare diversity patterns at the same scale and within the same taxonomic group, which limits the applicability and generality of the results. Here, we have investigated whether vegetation‐dwelling arthropods from different trophic ranks and with distinct life histories (i.e., ants, caterpillars, cockroaches, and spiders) have different beta‐diversity patterns at multiple scales. Specifically, we compared their beta diversity across architecturally distinct plant species (fine‐scale process) and a latitudinal gradient of sites (broad‐scale process) along 2040 km of coastal restinga vegetation in the Neotropics. Over 50 percent of the compositional changes (β‐diversity) in ants, caterpillars, and spiders and 41 percent of those in cockroaches were explained by plant identity within each site. Even groups that do not feed on plant tissues, such as omnivores and predators, were strongly affected by plant identity. Fine‐scale variation was more important than large‐scale processes for all studied groups. Performing a cross‐scale comparison of diversity patterns of groups with distinct life histories helps elucidate how processes that act at regional scales, such as dispersal, interact with local processes to assemble arthropod communities.     

Culture of osteogenic cells from human alveolar bone: a useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of approximately 120 kDa that could be solubilized by phospholipase C or Polidocanol.     

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MW(r) of about 120 kDa and specific PNPP activity of 1200 U/mg. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 U/mg), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and beta-glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function.