Molecular genetics of dopa-responsive dystonia

The causative genes of two types of hereditary dopa-responsive dystonia (DRD) due to dopamine (DA) deficiency in the nigrostriatum DA neurons have been elucidated. Autosomal dominant DRD (AD-DRD) was originally described by Segawa as hereditary progressive dystonia with marked diurnal fluctuation (HPD). We cloned the human GTP cyclohydrolase I (GCH1) gene, and mapped the gene to chromosome 14q22.1-q22.2 within the HPD/DRD locus, which had been identified by linkage analysis. GCH1 isthe rate-limiting enzyme for the biosynthesis of tetrahydrobiopterin (BH4), the cofactor for tyrosine hydroxylase (TH), which is the first and rate-limiting enzyme of DA synthesis. We proved that the GCH1 gene is the causative gene for HPD/DRD based on the identification of mutations of the gene in the patients and decreases in the enzyme activity expressed in mononuclear blood cells to 2-20% of the normal value. About 60 different mutations (missense, nonsense, and frameshift mutations) in the coding region or in the exon-intron junctions of the GCH1 gene have been reported in patients with AD-DRD all over the world. Recent findings indicate that the decreased GCH1 activity in AD-DRD may be caused by the negative interaction of the mutated subunit with the wild-type one, i.e., a dominant negative effect, and/or by decreases in the levels of GCH1 mRNA and protein caused by inactivation of one allele of the GCH1 gene. Autosomal recessive DRD (AR-DRD) with Segawa's syndrome was discovered in Germany. The AR-DRD locus was mapped to chromosome 11p15.5 in the chromosomal site of the TH gene. In the AR-DRD with Segawa's syndrome, a point mutation in TH (Gln381Lys) resulted in a pronounced decrease in TH activity to about 15% of that of the wild type. Several missense mutations in the TH gene have been found in AR-DRD in Europe. The phenotype of AR-DRD with the Leu205Pro mutation in the TH gene, which produces a severe decrease in TH activity to 1.5% of that of the wild type, was severe, not dystonia/Segawa's syndrome, but early-onset parkinsonism. However, a marked improvement of all clinical symptoms with a low dose of L-dopa was reported in AR-DRD/parkinsonism patients. These findings on DRD indicate that the nigrostriatal DA neurons may be most susceptible to the decreases in GCH1 activity, BH4 level, TH activity, and DA level, and that DRD is the DA deficiency without neuronal death in contrast to juvenile parkinsonism or Parkinson's disease with DA cell death.     

Purification and further characterization of enteropeptidase from porcine duodenum

Enteropeptidase [EC 3.4.21.9] is a membrane-bound serine endopeptidase present in the duodenum that converts trypsinogen to trypsin. We previously cloned the cDNA of the porcine enzyme and deduced its entire amino acid sequence [M. Matsushima et al. (1994) J. Biol. Chem. 269, 19976-19982]. In the present study, we purified the porcine enzyme approximately 2,200-fold in a 12% yield from a duodenal mucosal extract to apparent homogeneity by an improved procedure comprising four steps of chromatography including benzamidine-Sepharose affinity chromatography. Lectin blotting analysis suggested that the enzyme is glycosylated mainly with N-linked carbohydrate chains of the tri- and/or tetraantennary complex type. The H and L chains of the enzyme were separated into two major bands upon SDS-PAGE under reducing conditions, suggesting that the enzyme mainly comprises two isoforms, a higher molecular weight form and a lower molecular weight form. The enzyme was also separated by lectin affinity chromatography into two major fractions, named isoforms I and II, which corresponded to the higher and lower molecular weight forms, respectively. These two isoforms appeared to be different only in the carbohydrate moiety, having essentially the same enzymatic properties. The enzyme was optimally active at pH 8.0 toward Gly-Asp-Asp-Asp-Asp-Lys-beta-naphthylamide, and was inhibited strongly by various serine proteinase inhibitors. Furthermore, it was also strongly inhibited by E-64 [L-trans-epoxysuccinyl-leucylamide-(4-guanido)-butane], a cysteine proteinase inhibitor. Substrate specificity studies involving various synthetic peptides indicated that acidic residues at the P2, P3, and/or P4 positions are especially favorable for maximal activity, but are not absolutely necessary, at least in the cases of peptide substrates.     

Modulation of control of muscle sympathetic nerve activity during orthostatic stress in humans

We tested the hypothesis that orthostatic stress would modulate the arterial baroreflex (ABR)-mediated beat-by-beat control of muscle sympathetic nerve activity (MSNA) in humans. In 12 healthy subjects, ABR control of MSNA (burst incidence, burst strength, and total activity) was evaluated by analysis of the relation between beat-by-beat spontaneous variations in diastolic blood pressure (DAP) and MSNA during supine rest (CON) and at two levels of lower body negative pressure (LBNP: -15 and -35 mmHg). At -15 mmHg LBNP, the relation between burst incidence (bursts per 100 heartbeats) and DAP showed an upward shift from that observed during CON, but the further shift seen at -35 mmHg LBNP was only marginal. The relation between burst strength and DAP was shifted upward at -15 mmHg LBNP (vs. CON) and further shifted upward at -35 mmHg LBNP. At -15 mmHg LBNP, the relation between total activity and DAP was shifted upward from that obtained during CON and further shifted upward at -35 mmHg LBNP. These results suggest that ABR control of MSNA is modulated during orthostatic stress and that the modulation is different between a mild (nonhypotensive) and a moderate (hypotensive) level of orthostatic stress.     

Salt-inducible multidrug efflux pump protein in the moderately halophilic bacterium Chromohalobacter sp

It has been known that halophilic bacteria often show natural resistance to antibiotics, dyes, and toxic metal ions, but the mechanism and regulation of this resistance have remained unexplained. We have addressed this question by identifying the gene responsible for multidrug resistance. A spontaneous ofloxacin-resistant mutant derived from the moderately halophilic bacterium Chromohalobacter sp. strain 160 showed a two- to fourfold increased resistance to structurally diverse compounds, such as tetracycline, cefsulodin, chloramphenicol, and ethidium bromide (EtBr), and tolerance to organic solvents, e.g., hexane and heptane. The mutant produced an elevated level of the 58-kDa outer membrane protein. This mutant (160R) accumulated about one-third the level of EtBr that the parent cells did. An uncoupler, carbonyl cyanide m-chlorophenylhydrazone, caused a severalfold increase in the intracellular accumulation of EtBr, with the wild-type and mutant cells accumulating nearly equal amounts. The hrdC gene encoding the 58-kDa outer membrane protein has been cloned. Disruption of this gene rendered the cells hypersusceptible to antibiotics and EtBr and led to a high level of accumulation of intracellular EtBr. The primary structure of HrdC has a weak similarity to that of Escherichia coli TolC. Interestingly, both drug resistance and the expression of HrdC were markedly increased in the presence of a high salt concentration in the growth medium, but this was not observed in hrdC-disrupted cells. These results indicate that HrdC is the outer membrane component of the putative efflux pump assembly and that it plays a major role in the observed induction of drug resistance by salt in this bacterium.     

Induction of uPA release in human peripheral blood lymphocytes by [deamino-Cysl,D-Arg8]-vasopressin (dDAVP)

[deamino-Cys(l),d-Arg(8)]-vasopressin (dDAVP), known to be an arginine vasopressin (AVP) V(2) receptor agonist, is an agent that increases fibrinolytic activity levels in plasma after its infusion into the human body. However, mechanisms underlying an increase and exact localization of the extrarenal dDAVP-responsive V(2) receptor remain unclarified. Two AVP receptors, V(1a) and V(2), and a related oxytocin (OT) receptor were found to be expressed in human lymphocytes. Furthermore, we found an increase of fibrinolytic activity in the medium of peripheral lymphocytes obtained from human volunteers less than 20 min after dDAVP infusion. The increased activity was also detected in the medium after incubating the lymphocytes in the presence of dDAVP in vitro, being highest at 20 min after the incubation. In accord with the increased fibrinolytic activity, the levels of urokinase-type plasminogen activator (uPA) in the medium were also increased. However, there was no significant difference of plasminogen activator inhibitor-1 (PAI-1), pro-uPA, and tissue-type plasminogen activator (tPA) concentrations in the medium between dDAVP treatment and control. When lymphocytes were preincubated with a V(2) receptor antagonist [Adamantaneacetyl(1),O-Et-d-Tyr(2),Val(4),Aminobutyryl(6),Arg(8,9)]-vasopressin, the dDAVP-induced uPA increase was diminished. In contrast, preincubation with a V(1) receptor antagonist, [beta-Mercapto-beta,beta-cyclopentamethylenepropionyl(1),O-Me-Tyr(2),Arg(8)]-vasopressin, prior to dDAVP treatment resulted in a greater increase of the uPA concentration in the medium than with the dDAVP treatment alone. Thus it was suggested that dDAVP may induce uPA release from human lymphocytes via V(2) receptor-mediated reaction, and also via cross-talk between V(1) and V(2) receptors.     

Accelerated biodegradation of poly(vinyl alcohol) by glycosidations of the hydroxyl groups or addition of sugars

Biodegradabilities of N-acetyl-d-glucosamine (GlcNAc)- (1) and chitobiose-substituted (2) poly(vinyl alcohol)s (PVA)s in a soil suspension (pH 6.5) were investigated at 25 degrees C for 40 days. Biochemical oxygen demand of 1 with a degree of substitution of 0.2-0.3 (DP = 430-480) was higher than that of PVA under the degradation condition. Size exclusion chromatography, (1)H NMR, and Fourier-transform infrared measurements of the recovered sample indicated that biodegradation of the PVA main chain was accelerated by partial glycosidation of hydroxyl groups in PVA. Similar acceleration was observed in a PVA/GlcNAc (50:50, w/w) mixture. Microbes which relate with degradation of the glycosidated polymers were grown in a culture medium including the soil suspension and the polymer as the carbon source. Polyacrylamide gel electrophoresis (SDS-PAGE) and IR measurements indicated that a cell-free extract derived from GlcNAc-substituted PVA was different from that in the PVA/GlcNAc mixture. The results suggested that the PVA main chain in GlcNAc-substituted PVA was cleaved by a different microorganism or via a mechanism different from that in the mixture. Chitobiose-substituted PVA 2 showed more enhanced acceleration, indicating that the sugar length influenced the degradability.     

A selective inducible NOS dimerization inhibitor prevents systemic,cardiac, and pulmonary hemodynamic dysfunction in endotoxemic mice

Increased nitric oxide (NO) production by inducible NO synthase (NOS2), an obligate homodimer, is implicated in the cardiovascular sequelae of sepsis. We tested the ability of a highly selective NOS2 dimerization inhibitor (BBS-2) to prevent endotoxin-induced systemic hypotension, myocardial dysfunction, and impaired hypoxic pulmonary vasoconstriction (HPV) in mice. Mice were challenged with Escherichia coli endotoxin before treatment with BBS-2 or vehicle. Systemic blood pressure was measured before and 4 and 7 h after endotoxin challenge, and echocardiographic parameters of myocardial function were measured before and 7 h after endotoxin challenge. The pulmonary vasoconstrictor response to left mainstem bronchus occlusion, which is a measure of HPV, was studied 22 h after endotoxin challenge. BBS-2 treatment alone did not alter baseline hemodynamics. BBS-2 treatment blocked NOS2 dimerization and completely inhibited the endotoxin-induced increase of plasma nitrate and nitrite levels. Treatment with BBS-2 after endotoxin administration prevented systemic hypotension and attenuated myocardial dysfunction. BBS-2 also prevented endotoxin-induced impairment of HPV. In contrast, treatment with NG-nitro-l-arginine methyl ester, which is an inhibitor of all three NOS isoforms, prevented the systemic hypotension but further aggravated the myocardial dysfunction associated with endotoxin challenge. Treatment with BBS-2 prevented endotoxin from causing key features of cardiovascular dysfunction in endotoxemic mice. Selective inhibition of NOS2 dimerization with BBS-2, while sparing the activities of other NOS isoforms, may prove to be a useful treatment strategy in sepsis.     

Diazoxide triggers cardioprotection against apoptosis induced by oxidative stress

Although mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels have been reported to reduce the extent of apoptosis, the critical timing of mitoK(ATP) channel opening required to protect myocytes against apoptosis remains unclear. In the present study, we examined whether the mitoK(ATP) channel serves as a trigger of cardioprotection against apoptosis induced by oxidative stress. Apoptosis of cultured neonatal rat cardiomyocytes was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33342. Mitochondrial membrane potential (DeltaPsi) was measured by flow cytometry of cells stained with rhodamine-123 (Rh-123). Exposure to H(2)O(2) (500 microM) induced apoptosis, and the percentage of apoptotic cells increased progressively and peaked at 2 h. This H(2)O(2)-induced apoptosis was associated with the loss of DeltaPsi, and the time course of decrease in Rh-123 fluorescence paralleled that of apoptosis. Pretreatment of cardiomyocytes with diazoxide (100 microM), a putative mitoK(ATP) channel opener, for 30 min before exposure to H(2)O(2) elicited transient and mild depolarization of DeltaPsi and consequently suppressed both apoptosis and DeltaPsi loss after 2-h exposure to H(2)O(2). These protective effects of diazoxide were abrogated by the mitoK(ATP) channel blocker 5-hydroxydecanoate (500 microM) but not by the sarcolemmal K(ATP) channel blocker HMR-1098 (30 microM). Our results suggest for the first time that diazoxide-induced opening of mitoK(ATP) channels triggers cardioprotection against apoptosis induced by oxidative stress in rat cardiomyocytes.     

Mitochondrial reactive oxygen species reduce insulin secretion by pancreatic beta-cells

Pancreatic beta-cells exposed to hyperglycemia produce reactive oxygen species (ROS). Because beta-cells are sensitive to oxidative stress, excessive ROS may cause dysfunction of beta-cells. Here we demonstrate that mitochondrial ROS suppress glucose-induced insulin secretion (GIIS) from beta-cells. Intracellular ROS increased 15min after exposure to high glucose and this effect was blunted by inhibitors of the mitochondrial function. GIIS was also suppressed by H(2)O(2), a chemical substitute for ROS. Interestingly, the first-phase of GIIS could be suppressed by 50 microM H(2)O(2). H(2)O(2) or high glucose suppressed the activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme, and inhibitors of the mitochondrial function abolished the latter effects. Our data suggested that high glucose induced mitochondrial ROS, which suppressed first-phase of GIIS, at least in part, through the suppression of GAPDH activity. We propose that mitochondrial overwork is a potential mechanism causing impaired first-phase of GIIS in the early stages of diabetes mellitus.