Fluorescence resonance energy transfer analysis of protein translocase. SecYE from Thermus thermophilus HB8 forms a constitutive oligomer in membranes

SecY and SecE are the two principal translocase subunits that create a channel-like pathway for the transit of preprotein across the bacterial cytoplasmic membrane. Here we report the cloning, expression, and purification of the SecYE complex (TSecYE) from a thermophilic bacterium, Thermus thermophilus HB8. Purified TSecYE can be reconstituted into proteoliposomes that function in T. thermophilus SecA (TSecA) dependent preprotein translocation. After the mixing of TSecYE derivatives labeled with either a donor or an acceptor fluorophore during reconstitution, fluorescence resonance energy transfer experiments demonstrated that 2 or more units of TSecYE in the lipid bilayer associate to form a largely non-exchangeable oligomeric structure.     

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.     

RICS,a novel GTPase-activating protein for Cdc42 and Rac1, is involved in the beta-catenin-N-cadherin and N-methyl-D-aspartate receptor signaling

Cadherin adhesion molecules are believed to be important for synaptic plasticity. beta-Catenin, which links cadherins and the actin cytoskeleton, is a modulator of cadherin adhesion and regulates synaptic structure and function. Here we show that beta-catenin interacts with a novel GTPase-activating protein, named RICS, that acts on Cdc42 and Rac1. The RICS-beta-catenin complex was found to be associated with N-cadherin, N-methyl-d-aspartate receptors, and postsynaptic density-95, and localized to the postsynaptic density. Furthermore, the GTPase-activating protein activity of RICS was inhibited by phosphorylation by Ca(2+)/calmodulin-dependent protein kinase II. These results suggest that RICS is involved in the synaptic adhesion- and N-methyl-d-aspartate-mediated organization of cytoskeletal networks and signal transduction. Thus, RICS may regulate dendritic spine morphology and strength by modulating Rho GTPases.     

In vivo monitoring of norepinephrine and its metabolites in skeletal muscle

Although skeletal muscle sympathetic nerve activity plays an important role in the regulation of vascular tone and glucose metabolism, relatively little is known about regional norepinephrine (NE) kinetics in the skeletal muscle. With use of the dialysis technique, we implanted dialysis probes in the adductor muscle of anesthetized rabbits and examined whether dialysate NE and its metabolites were influenced by local administration of pharmacological agents through the dialysis probes. Dialysate dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were measured as two major metabolites of NE. The skeletal muscle dialysate NE, DHPG and MHPG were 11.7+/-1.2, 38.1+/-3.2, and 266.1+/-28.7 pg/ml, respectively. Basal dialysate NE levels were suppressed by tetrodotoxin (Na(+) channel blocker, 10 microM) (5.1+/-0.6 pg/ml), and augmented by desipramine (NE uptake blocker, 100 microM) (25.8+/-3.2 pg/ml). Basal dialysate DHPG levels were suppressed by pargyline (monoamine oxidase blocker, 1mM) (24.3+/-4.6 pg/ml) and augmented by reserpine (vesicle NE transport blocker, 10 microM) (75.8+/-2.7 pg/ml). Basal dialysate MHPG levels were not affected by pargyline, reserpine, or desipramine. Addition of tyramine (sympathomimetic amine, 600 microM), KCl (100 mM), and ouabain (Na(+)-K(+) ATPase blocker, 100 microM) caused brisk increases in dialysate NE levels (200.9+/-14.2, 90.6+/-25.7, 285.3+/-46.8 pg/ml, respectively). Furthermore, increases in basal dialysate NE levels were correlated with locally administered desipramine (10, 100 microM). Thus, dialysate NE and its metabolite were affected by local administration of pharmacological agents that modified sympathetic nerve endings function in the skeletal muscle. Skeletal muscle microdialysis with local administration of a pharmacological agent provides information about NE release, uptake, vesicle uptake and degradation at skeletal muscle sympathetic nerve endings.     

Mechanisms of enhanced apoptosis in HL-60 cells by UV-irradiated n-3 and n-6 polyunsaturated fatty acids

We examined the effects of arachidonic acid (AA), eicosapentaenoic acid (EPA), and their ultraviolet (UV)-irradiated products on HL-60 cells and isolated mitochondria to explore the following four obscure points in the mechanism of polyunsaturated fatty acids (PUFAs)-induced apoptosis: (i). the role of reactive oxygen species, (ii). the interaction of PUFAs and their metabolites with mitochondria in situ, (iii). the cyclosporine A (CsA)-sensitivity in PUFA-induced membrane permeability transition, (iv). the specificity of oxidized n-3 PUFAs in the induction of apoptosis in cancer cells. UV-oxidized PUFAs contained conjugated dienes and thiobarbituric acid reactive substances (TBARS). The apoptotic effects of PUFAs on HL-60 cells were increased by UV-irradiation whereas the swelling effect of PUFAs on isolated mitochondria was decreased. Both oxidized n-3 and n-6 PUFAs induced increased depolarization, ferricytochrome c release, the activation of various caspases, and DNA-fragmentation in a CsA-insensitive mechanism concomitant with a slight increase in the value of TBARS in cells. Furthermore, there were no significant differences in the mechanism of apoptosis induced by either oxidized AA or oxidized EPA. On the basis of these results, it was concluded that both oxidized n-3 or n-6 PUFAs induced apoptosis in HL-60 cells by a similar mechanism in a CsA-insensitive manner and also that oxidized products of PUFAs, but not the cellular oxidation process itself, play an important role in the mechanism of apoptosis in HL-60 cells.     

Photoproduction of Hydrogen by the Marine Heterocystous Cyanobacterium Anabaena Species TU37-1 Under a Nitrogen Atmosphere

Hydrogen production rates by Anabaena sp. strain TU37-1 obtained after an initial 1-day incubation period were approximately 70 to 80 and 3 to 9 µmol (mg chl)^–1 h^–1 under argon and nitrogen atmospheres, respectively. Hydrogen production under argon was not enhanced by addition of carbon dioxide, but was enhanced to some extent under nitrogen by increasing the initial carbon dioxide concentration. Rates of hydrogen and oxygen production during the initial 7-hour period were 15 and 220 µmol (mg chl)^–1 h^–1, respectively, in vessels with 18.5% initial carbon dioxide. Hydrogen production under nitrogen was enhanced by addition of carbon monoxide (1%). The rate obtained from the initial 1-day incubation period was about 40 µmol (mg chl)^–1 h^–1, which corresponded to about 60% of that under argon. On the basis of these observations, a possible strategy for hydrogen production by nitrogen-fixing cyanobacteria under nitrogen in the presence of carbon monoxide is indicated.     

DNA polymerases eta and kappa are responsible for error-free translesion DNA synthesis activity over a cis-syn thymine dimer in Xenopus laevis oocyte extracts

In translesion synthesis (TLS), specialized DNA polymerases (pols) facilitate progression of replication forks stalled by DNA damage. Although multiple TLS pols have been identified in eukaryotes, little is known about endogenous TLS pols and their relative contributions to TLS in vivo because of their low cellular abundance. Taking advantage of Xenopus laevis oocyte cells, with their extraordinary size and abundant enzymes involved in DNA metabolism, we have identified and characterized endogenous TLS pols for DNA damage induced by ultraviolet (UV) irradiation. We designed a TLS assay which monitors primer elongation on a synthetic oligomer template over a single UV-induced lesion, either a cys-syn cyclobutane pyrimidine dimer (CPD) or a pyrimidine (6-4) pyrimidone photoproduct. Four distinct TLS activities (TLS1-TLS4) were identified in X. laevis oocyte extracts, using three template/primer (T/P) DNA substrates having various sites at which primer extension is initiated relative to the lesion. TLS1 and TLS2 activities appear to be sequence-dependent. TLS3 and TLS4 extended the primers over the CPD in an error-free manner irrespective of sequence context. Base insertion opposite the CPD of the T/P substrate in which the 3'-end of the primer is placed one base upstream of the lesion was observed only with TLS3. TLS3 and TLS4 showed primer extension with similar efficiencies on the T/P substrate whose 3'-primer terminal dinucleotide (AA) was complementary to the CPD lesion. Investigations with antibodies and recombinant pols revealed that TLS3 and TLS4 were most likely attributable to pol eta and pol kappa, respectively. These results indicate that error-free insertion in CPD bypass is due mainly to pol eta (TLS3) in the extracts, and suggest that pol kappa (TLS4) may assist pol eta (TLS3) in error-free extension during CPD bypass.     

Synthesis and biological activities of novel aryl indole-2-carboxylic acid analogs as PPARgamma partial agonists

A series of novel aryl indole-2-carboxylic acids has been identified as potent selective PPARgamma modulators. Their chemical synthesis and in vitro activities are discussed. Compound 5 was selected for in vivo testing in the db/db mouse model of type 2 diabetes and resulted in reduction of hyperglycemia at comparable plasma exposure when compared to rosiglitazone.     

Synthesis and biological activity of sulphostin analogues, novel dipeptidyl peptidase IV inhibitors

The structure of sulphostin (1), a novel dipeptidyl peptidase IV (DPP-IV) inhibitor, is consisted of three key functional groups, including a characteristic amino(sulfoamino)phosphinyl group, on a piperidine ring. To examine the relationship between its structure and the inhibitory activity against DPP-IV, various analogues were synthesized and their activities were investigated. These results indicated that all of the functional groups on the piperidine ring were crucial to the DPP-IV inhibitory activity of sulphostin, and that the sulfonic acid group, which constructed the amino(sulfoamino)phosphinyl group, contributed to the stability of the compound. Moreover, those functional groups should be adjoined on the piperidine ring for the inhibitory activity. The size of the nitrogen-containing heterocyclic ring including piperidine appeared to scarcely affect the activity. In the present study, the sulfonic acid-deficient five-membered ring analogue 27a showed the strongest inhibitory activity (IC50=11 nM).