Functional expression and characterization of Aedes aegypti dopachrome conversion enzyme.

A full-length mosquito dopachrome conversion enzyme (DCE) and its truncated form lacking the last 54 carboxyl-terminal amino acid residues are expressed using a baculovirus/insect cell expression system. The full-length recombinant DCE displayed multiple bands during native PAGE with substrate staining, but only one active band was detected when the truncated recombinant DCE was analyzed under identical analysis conditions. Our data suggest that the last 50 some carboxyl-terminal residues are involved in the polymerization of the DCE molecules and that the proposed DCE isozymes likely reflect the presence of multimers of the same DCE molecules. The significance of the recombinant DCE in accelerating the melanization pathway is demonstrated by a rapid production of melanin in a dopa and tyrosinase reaction mixture in the presence of recombinant DCE. The DCE sequence data obtained in our previous study, together with results of functional expression and biochemical characterization achieved in this study, provide a necessary reference for the study of other insect DCEs.     

Mitochondrial nitric oxide localization in H9c2 cells revealed by confocal microscopy.

This study shows the presence of all three nitric oxide synthases (NOSs) and NOS activity in H9c2 cells cultured under non-stimulated conditions. By using the 4,5 diaminofluoresceindiacetate (DAF-2DA) fluorimetric nitric oxide (NO(*)) detection system we observed NO(*) production in H9c2 cells. As revealed by confocal microscopy, NO(*) fluorescence colocalizes in mitochondria labeled with Mito-Tracker Red CM-H(2)Xros. Upon stimulation with acetylcholine (Ach), which increased NOS activity by 75%, the colocalization coefficient C(green) value, calculated as Pearson's correlation, increased from 0.07 to 0.10, demonstrating an augmented presence of NO(*) in mitochondria. Conversely, the presence of NO(*) in mitochondria decreased following cells pretreatment with l-MonoMethylArginine (L-NMMA), a competitive inhibitor of NOS activity, as indicated by the reduction of the C(green) value to 0.02. This work confirms that the presence of NO(*) in mitochondria can be modulated in response to different fluxes of NO(*).     

Molecular cloning and functional expression of a sodium bicarbonate cotransporter from guinea-pig parotid glands

We recently found that the concentration of HCO3- in guinea-pig saliva is very similar to that of human saliva; however, the entity that regulates HCO3- transport has not yet been fully characterized. In order to investigate the mechanism of HCO3- transport, we identified, cloned, and characterized a sodium bicarbonate (Na(+)/HCO3- cotransporter found in guinea-pig parotid glands (gpNBC1). The gpNBC1 gene encodes a 1079-amino acid protein that has 95% and 96% homology with human and mouse parotid NBC1, respectively. Oocytes expressing gpNBC1 were exposed to HCO3- or Na(+)-free solutions, which resulted in a marked change in membrane potentials (V(m)), suggesting that gpNBC1 is electrogenic. Likewise, a gpNBC1-mediated pH recovery was observed in gpNBC1 transfected human hepatoma cells; however, in the presence of 4, 4-diisothiocyanostilbene-2,2-disulfonic acid, a specific NBC1 inhibitor, such changes in V(m) and pH(i) were not observed. Together, the data show that the cloned guinea-pig gene is a functional, as well as sequence homologue of human NBC1.     

Metabolism of [6,7-3H, 35S] estradiol 17-sulfate in rats

To confirm whether or not the sulfo group of estradiol 17-sulfate (ES) is removed during in vivo metabolism in rats, the doubly labeled conjugate [6,7-3H, 35S] ES was injected into rats, and its biliary and urinary metabolites were determined by reverse isotope dilution method (RIDM). In male rats, the major radioactivity was detected in biliary disulfate fraction, which was composed of mainly ES and its two minor metabolites, 2-hydroxyestradiol 17-sulfate (2-OH-ES) and 2-methoxyestradiol 17-sulfate (2-MeO-ES). In female rats, in contrast, the radioactivity was dispersed into three fractions:biliary monosulfate, biliary disulfate, and urinary monosulfate fractions (Frs.) In both monosulfate Frs., 7beta-hydroxyestradiol 17-sulfate was detected as the major metabolite followed by 6alpha-, 6beta-, and 15beta-hydroxyestradiol 17-sulfates. Like male rats, 2-OH-ES and 2-Meo-ES as the minor products were detected in biliary disulfate fraction. The isotope ratios of ES and its metabolites in both sexes were essentially the same as that of the dose except that of 6alpha-hydroxylated metabolite, which may be derived from the loss of the tritium labeled at C6. These results confirm the occurrence of the direct metabolism of ES in rats.     

Characterization of two types of osteoclasts from human peripheral blood monocytes

The two osteoclastogenesis pathways, receptor activator nuclear factor (NF)-kappaB ligand (RANKL)-mediated and fusion regulatory protein-1 (FRP-1)-mediated osteoclastogenesis, have recently been reported. There were significant differences in differentiation and activation mechanisms between the two pathways. When monocytes were cultured with FRP-1 without adding M-CSF, essential for the RANKL system, TRAP-positive polykaryocyte formation occurred. FRP-1-mediated osteoclasts formed larger pits on mineralized calcium phosphate plates than RANKL+M-CSF-mediated osteoclasts did. Lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were inclined to be single and isolated. However, osteoclasts induced by RANKL+M-CSF made many connected pits on dentin surfaces as if they crawled on there. Interestingly, FRP-1 osteoclastogenesis was enhanced by M-CSF/IL-1alpha, while chemotactic behavior to the dentin slices was not effected. There were differences in pH and concentration of HCO3- at culture endpoint and in adherent feature to dentin surfaces. Our findings indicate there are two types of osteoclasts with distinct properties.     

Patterning, prestress, and peeling dynamics of myocytes

As typical anchorage-dependent cells myocytes must balance contractility against adequate adhesion. Skeletal myotubes grown as isolated strips from myoblasts on micropatterned glass exhibited spontaneous peeling after one end of the myotube was mechanically detached. Such results indicate the development of a prestress in the cells. To assess this prestress and study the dynamic adhesion strength of single myocytes, the shear stress of fluid aspirated into a large-bore micropipette was then used to forcibly peel myotubes. The velocity at which cells peeled from the surface, V(peel), was measured as a continuously increasing function of the imposed tension, T(peel), which ranges from approximately 0 to 50 nN/ micro m. For each cell, peeling proved highly heterogeneous, with V(peel) fluctuating between 0 micro m/s ( approximately 80% of time) and approximately 10 micro m/s. Parallel studies of smooth muscle cells expressing GFP-paxillin also exhibited a discontinuous peeling in which focal adhesions fractured above sites of strong attachment (when pressure peeled using a small-bore pipette). The peeling approaches described here lend insight into the contractile-adhesion balance and can be used to study the real-time dynamics of stressed adhesions through both physical detection and the use of GFP markers; the methods should prove useful in comparing normal versus dystrophic muscle cells.     

Effect of storage xyloglucans on peritoneal macrophages

Xyloglucans from seeds of Copaifera langsdorffii (XGC), Hymenaea courbaril (XGJ) and Mucuna sloanei (XGM) were obtained from milled and defatted cotyledons by aqueous extraction at 25 degrees C. The resulting fractions contained Glc, Xyl and Gal in molar ratios of 2.5: 1.5: 1.0 (XGC), 3.8: 2.6: 1.0 (XGJ) and 2.5: 1.6: 1.0 (XGM). HPSEC-MALLS/RI analysis showed that each polysaccharide fraction was homogeneous; M(w) values were 1.6 x 10(5), 2.0 x 10(5) and 1.5 x 10(5)g/mol, respectively. The effect of the xyloglucans on the production of O(2)*(-) and NO* and on the recruitment of macrophages to the mouse peritoneum was evaluated. All polysaccharides promoted an increase in the number of peritoneal macrophages in a dose-dependent manner. The largest increase, of 576% in comparison to the control group, was elicited by XGJ at 200 mg/kg. The effect of XGC, XGJ and XGM on O(2)*(-) production, in the presence or absence of phorbol 12-myristate 13-acetate (PMA), was not statistically significant. For NO(.) production, the lowest concentration of XGC (10 microg/ml) gave rise to an increase of 262% when compared to the control group; the effect was dose-dependent, reaching 307% at 50 microg/ml. On the other hand, XGJ at a concentration of 50 microg/ml enhanced NO* production by 92%. XGM did not affect NO* production significantly. The results indicate that xyloglucans from C. langsdorffii, H. courbaril and M. sloanei have immunomodulatory activity.     

Crystal structure of oxidized cytochrome c(6A) from Arabidopsis thaliana

Compared with algal and cyanobacterial cytochrome c(6), cytochrome c(6A) from higher plants contains an additional loop of 12 amino acid residues. We have determined the first crystal structure of cytochrome c(6A) from Arabidopsis thaliana at 1.5 Angstrom resolution in order to help elucidate its function. The overall structure of cytochrome c(6A) follows the topology of class I c-type cytochromes in which the heme prosthetic group covalently binds to Cys16 and Cys19, and the iron has octahedral coordination with His20 and Met60 as the axial ligands. Two cysteine residues (Cys67 and Cys73) within the characteristic 12 amino acids loop form a disulfide bond, contributing to the structural stability of cytochrome c(6A). Our model provides a chemical basis for the known low redox potential of cytochrome c(6A) which makes it an unsuitable electron carrier between cytochrome b(6)f and PSI.     

Binding of arachidonic acid to myeloid-related proteins (S100A8/A9) enhances phagocytic NADPH oxidase activation

Activation of the O(2)(-) generating NADPH oxidase of phagocytes results from the assembly of the membrane-bound flavocytochrome b(558) with cytosolic proteins, p67(phox), p47(phox), and Rac. However, it has been recently reported that the arachidonic acid- and calcium-binding heterodimer S100A8/A9, abundant in neutrophil cytosol, influences the activation process. In a semi-recombinant system comprising neutrophil membranes, recombinant proteins, p67(phox), p47(phox), GTPgamma S-loaded Rac2, and arachidonic acid (AA), both the rate and the extent of the oxidase activation were increased by S100A8/A9, provided it was preloaded with AA. Binding of [(14)C]AA to S100A8/A9 was potentiated by recombinant cytosolic phox proteins and GTPgammaS, suggesting the formation of a complex, comprising oxidase activating proteins and S100A8/A9, with a greater affinity for AA. The rate constant of oxidase activation was not increased by AA-loaded S100A8/A9, whereas the maximal oxidase activity elicited was twice as high. AA-loaded S100A8/A9 increases oxidase activation probably by decreasing the deactivation rate.     

Oxidative stress reduces transintestinal transports and (Na+, K+) -ATPase activity in rat jejunum

Because oxidative stress is a component of gastrointestinal injury, we investigated the effect of H(2)O(2) on transintestinal transport using isolated rat jejunum incubated in vitro. Millimolar concentrations of H(2)O(2) inhibited all the tested parameters without inducing any cytotoxic effect. Electrophysiological experiments indicated that H(2)O(2) decreases significantly both short circuit current and transepithelial electrical potential difference without affecting transepithelial resistance. The possibility that H(2)O(2) could influence (Na+, K+) -ATPase activity was explored using isolated basolateral membranes. Besides H(2)O(2), free radicals (O(2)(*-), HO*) were generated using different iron-dependent and independent systems; (Na+, K+) -ATPase activity was inhibited after membrane exposure to all ROS tested. The inhibition was prevented by allopurinol, superoxide dismutase or desferrioxamine. Western blot analysis showed a decreased expression of the alpha(1)-subunit of (Na+, K+) -ATPase. We conclude that H(2)O(2) may be a modulator of jejunal ion and water transport by multiple mechanisms, among which a significant inhibition of the basolateral (Na+, K+) -ATPase.     

Effects of cellulase on the modification of cellulose

Multicomponent cellulases, purified endoglucanases and cellobiohydrolases were assayed and shown to modify pure natural cellulose (softwood pulp). Changes in structure and properties of the cellulose caused by enzymatic treatment depend on the composition, the type of enzyme, and the treatment conditions. The reactivity of cellulose for some dissolving and derivatization processes may be improved by enzymatic hydrolysis. Endoglucanases decreased the average degrees of polymerization (DP) and improved the alkaline solubility of cellulose most efficiently. The variation in the supramolecular structure estimated from the infrared spectra of the cellulose samples was found to be correlated with the reactivity and might represent wide variations in conformation caused by the breakdown of the hydrogen bonds.     

A divergent synthesis of [1-14C]-mono-E isomers of fatty acids

A convenient synthesis of [1-14C]-mono-trans fatty acid using olefin inversion as a key-step is described. This methodology allows for a facile synthesis of [1-14C]-labelled mono-trans analogues of oleic, linoleic and linolenic acids. As an example, only eleven steps were necessary to obtain the [1-14C]-mono-E isomers of linolenic acid from its commercial all-Z form. In the first step, Barton's decarboxylation procedure yielded a bromo intermediate. Epoxidation of this compound resulted in the formation of three monoepoxides, which could be separated by HPLC. After identification by 1H NMR and MS, the pure monoepoxides were then subjected to inversion consisting of a stereospecific deoxygenation followed by a beta-elimination step. Finally, the labelling was introduced by substitution of the bromine by a [14C]-cyano group followed by hydrolysis.     

Alternative NMR method for quantitative determination of acyl positional distribution in triacylglycerols and related compounds

High-resolution 13C NMR spectroscopy has been used to analyze the positional distribution of fatty acids in model triacylglycerols. A novel method for quantitative determination of the positional distribution of unsaturated chains in triacylglycerols simultaneously with the ratio of saturated/unsaturated acyl chains has been proposed, utilizing the chemical shift differences of the aliphatic atoms C4, C5, and C6. The use of HSQC-TOCSY spectra allows unequivocal proof of the position of the unsaturated chain as well as complete assignment of the 13C NMR signals in tripalmitin.     

Synthesis of (20S)-[7,7,21,21-2H4]-3beta-(tert-butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol,a useful intermediate for the preparation of deuterated isotopomers of sterols

(20S)-[7,7,21,21-2H(4)]-3beta-(tert-Butyldimethylsilanyloxy)-20-methyl-pregn-5-en-21-ol, an intermediate for the preparation of deuterated isotopomers of sterols to be used as standards for biomedical studies, was prepared by reduction with dichloroaluminum deuteride of ethyl (20S)-3beta-(tert-butyldimethylsilanyloxy)-7-oxo-pregn-5-en-20-carboxylate. Using controlled experimental conditions, it has also been shown that the dichloroaluminum hydride reduction of a 7-keto steroid affords the corresponding 7beta-hydroxy derivative in a highly stereoselective manner.     

Diuretic factors and second messengers stimulate secretion of the organic cation TEA by the Malpighian tubules of Drosophila melanogaster

This study showed that four factors which stimulate transepithelial fluid secretion and inorganic ion transport across the main segment of the Malpighian tubules of Drosophila melanogaster also stimulate transepithelial secretion of the prototypical organic cation tetraethylammonium (TEA). TEA fluxes across the Malpighian tubules and gut were measured using a TEA-selective self-referencing (TEA-SeR) microelectrode. TEA flux across isolated Malpighian tubules was also measured using a TEA-selective microelectrode positioned in droplets of fluid secreted by tubules set up in a modified Ramsay assay. TEA flux was stimulated by the intracellular second messengers cAMP and cGMP, which increase the lumen-positive transepithelial potential (TEP), and also by tyramine and leucokinin-I (LK-I), which decrease TEP. The largest increase was measured in response to 1 micromol l-1 LK-I which increased transepithelial TEA flux by 72%. TEA flux in the lower tubule was stimulated slightly (13%) by 1 micromol l-1 tyramine but not by any of the other factors. TEA flux across the midgut was unaffected by cAMP, cGMP or tyramine. This is the first study to demonstrate the effects of insect diuretic factors and second messengers on excretion of organic cations.     

Application of HN(C)N to rapid estimation of 1J(N-C(alpha)) coupling constants correlated to psi torsion angles in proteins: implication to structural genomics

We recently described a triple resonance experiment, HN(C)N, for sequential correlation of H(N) and 15N atoms in (15N, 13C) labeled proteins [J. Biomol. NMR. 20 (2001) 135]. Here, we describe an approach based on this experiment for estimation of one bond N-C(alpha) J-couplings in medium size labeled proteins, which seem to show good correlations with psi torsion angles along the protein backbone. The approach uses the ratio of the intensities of the sequential and diagonal peaks in the F(2)-F(3) planes of the HN(C)N spectrum. The reliability of the approach has been demonstrated using a short peptide wherein the coupling constants have been measured by the present method and also independently from peak splittings in HSQC spectra. The two results agree within 10%. The applicability of the procedure to proteins has been demonstrated using doubly labeled FK506 binding protein (FKBP, molecular mass approximately 12 kDa). Coupling constant estimates have been obtained for 62 out of 100 non-proline residues and they show a correlation with psi torsion angles, as has been reported before. This semi-quantitative application of HN(C)N extends the significance of the experiment especially, in the context of structural genomics, since the single experiment, not only provides a great enhancement in the speed of resonance assignment, but also provides quantitative structural information.     

Pressure equilibrium and jump study on unfolding of 23-kDa protein from spinach photosystem II

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20 degrees C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (DeltaG(u)) and volume change (DeltaV(u)) for the unfolding are 5.9 kcal/mol and -160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in DeltaG(u) but also with a decrease in DeltaV(u). The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (-66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.