Effects of low CO2 on NAD(P)H dehydrogenase,a mediator of cyclic electron transport around photosystem I in the cyanobacterium synechocystis PCC6803

The expression and activity of type-1 NAD(P)H dehydrogenase (NDH-1) was compared between cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO(2) conditions. Western analysis indicated that L-cells contain higher amounts of the NDH-1 subunits, NdhH, NdhI and NdhK. An NADPH-specific subcomplex of NDH-1 showed higher NADPH-nitroblue tetrazolium oxidoreductase activity in L-cells. The activities of both NADPH-menadione oxidoreductase and light-dependent NADPH oxidation driven by photosystem I were much higher in L-cells than in H-cells. The initial rate of re-reduction of P700(+) following actinic light illumination in the presence of DCMU under background far-red light was enhanced in L-cells. In addition, rotenone, a specific inhibitor of NDH-1, suppressed the relative rate of post-illumination increase in Chl fluorescence of L-cells more than that of H-cells, suggesting that the involvement of NDH-1 in cyclic electron flow around photosystem I was enhanced by low CO(2). Taken together, these results suggest that NDH-1 complex and NDH-1-mediated cyclic electron transport are stimulated by low CO(2) and function in the acclimation of cyanobacteria to low CO(2).     

ATP-sensitive potassium channels mediate hyperosmotic stimulation of NKCC in slow-twitch muscle

In mildly hyperosmotic medium, activation of the Na⁺-K⁺-2Cl^- cotransporter (NKCC) counteracts skeletal muscle cell water loss, and compounds that stimulate protein kinase A (PKA) activity inhibit the activation of the NKCC. The aim of this study was to determine the mechanism for PKA inhibition of NKCC activity in resting skeletal muscle. Incubation of rat slow-twitch soleus and fast-twitch plantaris muscles in isosmotic medium with the PKA inhibitors H-89 and KT-5720 caused activation of the NKCC only in the soleus muscle. NKCC activation caused by PKA inhibition was insensitive to MEK MAPK inhibitors and to insulin but was abolished by the PKA stimulators isoproterenol and forskolin. Furthermore, pinacidil [an ATP-sensitive potassium (K_ATP) channel opener] or inhibition of glycolysis increased NKCC activity in the soleus muscle but not in the plantaris muscle. Preincubation of the soleus muscle with glibenclamide (a K_ATP channel inhibitor) prevented the NKCC activation by hyperosmolarity, PKA inhibition, pinacidil, and glycolysis inhibitors. In contrast, glibenclamide stimulated NKCC activity in the plantaris muscle. In cells stably transfected with the Kir6.2 subunit of the of K_ATP channel, inhibition of glycolysis activated potassium current and NKCC activity. We conclude that activation of K_ATP channels in slow-twitch muscle is necessary for activation of the NKCC and cell volume restoration in hyperosmotic conditions. protein kinase A; glibenclamide; glycolysis; Na⁺-K⁺-2Cl^- cotransporter; Kir6.2     

Real-time quantitative assay of telomerase activity using the duplex scorpion primer

A novel real-time quantitative method for measuring telomerase activity is described in which the duplex scorpion primer is used to provide an intramolecular probing mechanism for specific detection of telomerase activity. Using this method, linearity from 10 to 10(4) cells expressing telomerase activity could be obtained (R2 = 0.994). The requirement of post-PCR steps is thus obviated.     

Involvement of NAD(P)H oxidase in the enhanced expression of cell adhesion molecules in the aorta of diabetic mice

The increased levels of cell adhesion molecules (CAM) have been identified in diabetic vasculatures, but the underlying mechanisms remain unclear. To determine the relationship among vascular production of superoxide, expression of CAM and diabetes, superoxide generation and expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E- and P-selectin in the aorta from control (C57BL/6J) and diabetic mice (ob/ob) were measured. In situ staining for superoxide using dihydroethidium showed an increased superoxide production in diabetic aorta in association with an enhanced NAD(P)H oxidase activity. Immunohistochemical analysis revealed that the endothelial expression of ICAM-1 (3.5 +/- 0.4) and VCAM-1 (3.8 +/- 0.3) in diabetic aorta was significantly higher than that in control aorta (0.9 +/- 0.5 and 1.6 +/- 0.3, respectively). Furthermore, there was a strong positive correlation (r = 0.89, p < 0.01 in ICAM-1 and r = 0.88, p < 0.01 in VCAM-1) between ICAM-1/VCAM-1 expression and vascular production of superoxide. The present data indicate that the increased production of superoxide via NAD(P)H oxidase may explain the enhanced expression of CAM in diabetic vasculatures.     

Application of electrophoretic methods for detection of protein-porphyrin complexes

Simple methods for detection and isolation of protein-porphyrin complexes were elaborated in our laboratory. They are based on the separation of protein-porphyrin complexes in native polyacrylamide gel and measurement of their fluorescence, with the use of two detection systems: the commercially available Gel Doc(TM) 2000 system, and a system specially designed for the purpose of these investigations, concerning protein-porphyrin interactions. The fluorescent complexes can be electro-transferred from the gel onto PVDF membrane, eluted and analyzed in order to identify the protein interacting with porphyrins.     

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.     

Development of an immunomagnetic separation-polymerase chain reaction (IMS-PCR) assay specific for Enterocytozoon bieneusi in water samples

AIMS: Microsporidia have become widely recognized as important human pathogens. Among Microsporidia, Enterocytozoon bieneusi is responsible for severe gastrointestinal disease. To date, no current therapy has been proven effective. Their mode of transmission and environmental occurrence are poorly documented because of the lack of detection methods that are both species-specific and sensitive. In this study, we developed a sensitive and specific molecular method to detect E. bieneusi spores in water samples. METHODS AND RESULTS: The molecular assay combined immunomagnetic separation (IMS) and polymerase chain reaction (PCR) amplification to detect E. bieneusi spores. A comparison was made of IMS magnetic beads coated with two different monoclonal antibodies, one specific for the Encephalitozoon genus that cross-reacts with E. bieneusi and the other specific only for the E. bieneusi species itself. CONCLUSIONS: Immunotech beads coated with the antibody specific for E. bieneusi were found to be the most effective combination. SIGNIFICANCE AND IMPACT OF THE STUDY: The highly specific IMS-PCR assay developed in this study provides a rapid and sensitive means of screening water samples for the presence of E. bieneusi spores.     

Human liver catalase: cloning,expression and characterization of monoclonal antibodies

We isolated a cDNA encoding liver catalase from a human liver cDNA library. The cDNA had a high degree of sequence similarity to the corresponding enzyme from other sources. It was expressed in E. coli using the pET15b vector. The protein produced was enzymatically active after purification, and its kinetic parameters closely resembled those of other mammalian catalases. Monoclonal antibodies were generated against the purified catalase; six antibodies recognizing different epitopes were obtained, one of which inhibited the enzyme. The cross reactions of the antibodies with brain catalases from human and other mammalian tissues were investigated, and all the immunoreactive bands obtained on Western blots had molecular masses of about 58 kDa. Similarly fractionated extracts of several mammalian cell lines all gave a single band of molecular mass 58 kDa. These results indicate that mammalian livers and human cell lines contain only one major type of immunologically reactive catalase, even though some of catalases have been previously reported to differ in certain properties.     

Refolding from denatured inclusion bodies,purification to homogeneity and simplified assay of MGDG synthases from land plants

In plant cells, the synthesis of monogalactosyldiacylglycerol (MGDG) is catalyzed within plastid envelope membranes by MGD proteins. MGDG synthesis was also reported in apicomplexan parasites, a phylum of protists harbouring a plastid that proved essential for the parasite survival. MGD activity is therefore a potent target for herbicidal and anti-parasitic molecules. In this study, we describe a detailed in vitro refolding protocol for denatured recombinant MGD accumulated in inclusion bodies from transformed Escherichia coli. The refolding process was dependent on CHAPS detergent and lipids, such as diacylglycerol and phosphatidylglycerol, as well as bivalent metals. Owing to this refolding procedure, the recombinant MGD protein from spinach was purified to homogeneity, allowing a definite characterization of its non-processivity and an investigation of its dimerization using cross-linking reagents. Additionally, using the portion of recombinant enzyme that accumulates in an active form in bacterial membranes, we developed a miniature assay for high-throughput screening for inhibitors.