Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1.
(65/279)
We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA. (+info)
Cutinase-like enzyme from the yeast Cryptococcus sp. strain S-2 hydrolyzes polylactic acid and other biodegradable plastics.
(66/279)
A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (epsilon-caprolactone), and poly(3-hydroxybutyrate). (+info)
Hexanoate and octanoate inhibit transcription of the malic enzyme and fatty acid synthase genes in chick embryo hepatocytes in culture.
(67/279)
Hexanoate and octanoate inhibit the triiodothyronine (T3)-induced increases in the activities of malic enzyme and fatty acid synthase in chick embryo hepatocytes in culture. Butanoate was less effective as an inhibitor, and palmitate, stearate, and oleate had no effect or small stimulatory effects. Hexanoate and octanoate inhibited the lipogenic enzyme activities at a transcriptional step, and did so within 30 min of addition. Incubation for 2 h in the absence of fatty acid reversed the inhibition of transcription caused by hexanoate. The inhibitory effect of hexanoate was selective because DNA content and transcription of the glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes were not inhibited. Hexanoate-mediated inhibition of transcription rates of the lipogenic genes was not correlated with an inhibition of binding of T3 to its nuclear receptor. 2-Bromooctanoate and carnitine stimulated the T3-induced accumulation of the mRNAs for malic enzyme and fatty acid synthase. The presence of hexanoate stimulated by 2- to 3-fold the increase caused by carnitine, suggesting that hexanoate and carnitine may regulate lipogenic gene expression by a common pathway. Hexanedioate, acetoacetate, beta-hydroxybutyrate, branched chain fatty acids, and branched chain keto acids had little or no effect on abundance of the lipogenic mRNAs. We suggest that the active inhibitor is a metabolite derived from hexanoate or octanoate, possibly an intermediate derived from an acyl-CoA derivative. (+info)
Model-based development of gemcabene, a new lipid-altering agent.
(68/279)
The purpose of this study was to evaluate the value of model-based, quantitative decision making during the development of gemcabene, a novel lipid-altering agent. The decisions were driven by a model of the likely clinical profile of gemcabene in comparison with its competitors, such as 3-hydroxymethylglutaryl coenzyme A reductase inhibitors (statins), the cholesterol absorption inhibitor ezetimibe, and their combination. Dose-response models were developed for the lipid effects (low-density lipoprotein cholesterol [LDL-C] and high-density lipoprotein cholesterol); adverse effects, such as persistent alanine aminotransferase elevation and myalgia; tolerability issues, such as headache; and risk reduction for coronary artery disease-related events for 5 statins, ezetimibe, gemcabene, and their combinations. The integrated model was based on the joint analysis of publicly available summary-level data and proprietary patient-level data and included information from almost 10,000 patients. The model was made available and accessible to the development team by using the Pharsight Drug Model Explorer model visualization technology. The modeling greatly enhanced the understanding of the clinical profile of gemcabene when given alone or in combination with a statin. The interaction between statins and gemcabene for the LDL-C lowering effect was found to be significantly different from the interaction between statins and ezetimibe. Ezetimibe was found to have a pharmacological-independent interaction resulting in additional LDL-C lowering over the entire statin dose range. The gemcabene interaction was found to be less than independent, resulting in almost no additional LDL-C lowering at high-statin doses, although the drug has a significant LDL-C effect when administered alone or in combination with a low dose of a statin. The quick availability of the model after completion of the first phase II trial in the target patient population and the ability of the team to explore the potential clinical efficacy and safety of gemcabene in comparison with alternative treatment options facilitated a quick decision to stop development. (+info)
Preparation and characterization of chitosan and trimethyl-chitosan-modified poly-(epsilon-caprolactone) nanoparticles as DNA carriers.
(69/279)
The purpose of this research was to prepare poly-(epsilon-caprolactone) (PCL) particles by an emulsion-diffusion-evaporation method using a blend of poly-(vinyl alcohol) and chitosan derivatives as stabilizers. The chitosan derivatives used were chitosan hydrochloride and trimethyl chitosans (TMC) with varying degrees of quaternization. Particle characteristics-size, zeta potential, surface morphology, cytotoxicity, and transfection efficiency-were investigated. The developed method yields PCL nanoparticles in the size range of 250 to 300 nm with a positive surface charge (2.5 to 6.8 mV). The cytotoxicity was found to be moderate and virtually independent of the stabilizers' concentration with the exception of the highly quaternized TMC (degree of substitution 66%) being significantly more toxic. In immobilization experiments with gel electrophoresis, it could be shown that these cationic nanoparticles (NP) form stable complexes with DNA at a NP:DNA ratio of 3:1. These nanoplexes showed a significantly higher transfection efficiency on COS-1 cells than naked DNA. (+info)
Olfactory shifts parallel superspecialism for toxic fruit in Drosophila melanogaster sibling, D. sechellia.
(70/279)
Olfaction in the fruit fly Drosophila melanogaster is increasingly understood, from ligand-receptor-neuron combinations to their axonal projection patterns into the antennal lobe . Drosophila thus offers an excellent opportunity to study the evolutionary and ecological dynamics of olfactory systems. We compared the structure and function of the generalist D. melanogaster with that of specialist D. sechellia, which oviposits exclusively on morinda fruit . Our analyses show that whereas the fruit's headspace was dominated by acids, antennae responded most strongly to hexanoates. D. sechellia exhibited an extraordinarily strong response to methyl hexanoate (MeHex). Behaviorally, D. sechellia was much more attracted to these morinda fruit volatiles than was D. melanogaster. The high sensitivity to MeHex was paralleled by a 2.5x-3 x overrepresentation of MeHex neurons on the antenna and a concordant 2.9 x increase in volume of the corresponding glomerulus as compared to D. melanogaster. In addition, the MeHex neuron exhibited an extreme sensitivity down to femtograms of its ligand. In contrast, no peripherally mediated shift was found paralleling D. sechellia's increased attraction to acids. These findings are a demonstration of evolution acting at several levels in the olfactory circuitry in mediating a fruit fly's unique preference for fruit toxic to its sibling species . (+info)
Glial cells dilate and constrict blood vessels: a mechanism of neurovascular coupling.
(71/279)
Neuronal activity evokes localized changes in blood flow. Although this response, termed neurovascular coupling, is widely used to monitor human brain function and diagnose pathology, the cellular mechanisms that mediate the response remain unclear. We investigated the contribution of glial cells to neurovascular coupling in the acutely isolated mammalian retina. We found that light stimulation and glial cell stimulation can both evoke dilation or constriction of arterioles. Light-evoked and glial-evoked vasodilations were blocked by inhibitors of cytochrome P450 epoxygenase, the synthetic enzyme for epoxyeicosatrienoic acids. Vasoconstrictions, in contrast, were blocked by an inhibitor of omega-hydroxylase, which synthesizes 20-hydroxyeicosatetraenoic acid. Nitric oxide influenced whether vasodilations or vasoconstrictions were produced in response to light and glial stimulation. Light-evoked vasoactivity was blocked when neuron-to-glia signaling was interrupted by a purinergic antagonist. These results indicate that glial cells contribute to neurovascular coupling and suggest that regulation of blood flow may involve both vasodilating and vasoconstricting components. (+info)
Effectively assessing subtle hepatic metabolic functions by novel non-invasive tests might be of clinical utility in scoring NAFLD (non-alcoholic fatty liver disease) and in identifying altered metabolic pathways. The present study was conducted on 39 (20 lean and 19 obese) hypertransaminasemic patients with histologically proven NAFLD {ranging from simple steatosis to severe steatohepatitis [NASH (non-alcoholic steatohepatitis)] and fibrosis} and 28 (20 lean and eight overweight) healthy controls, who underwent stable isotope breath testing ([(13)C]methacetin and [(13)C]ketoisocaproate) for microsomal and mitochondrial liver function in relation to histology, serum hyaluronate, as a marker of liver fibrosis, and body size. Compared with healthy subjects and patients with simple steatosis, NASH patients had enhanced methacetin demethylation (P=0.001), but decreased (P=0.001) and delayed (P=0.006) ketoisocaproate decarboxylation, which was inversely related (P=0.001) to the degree of histological fibrosis (r=-0.701), serum hyaluronate (r=-0.644) and body size (r=-0.485). Ketoisocaproate decarboxylation was impaired further in obese patients with NASH, but not in patients with simple steatosis and in overweight controls. NASH and insulin resistance were independently associated with an abnormal ketoisocaproate breath test (P=0.001). The cut-off value of 9.6% cumulative expired (13)CO(2) for ketoisocaproate at 60 min was associated with the highest prediction (positive predictive value, 0.90; negative predictive value, 0.73) for NASH, yielding an overall sensitivity of 68% and specificity of 94%. In conclusion, both microsomal and mitochondrial functions are disturbed in NASH. Therefore stable isotope breath tests may usefully contribute to a better and non-invasive characterization of patients with NAFLD. (+info)