Fusion of sphingomyelin vesicles induced by proteins from Taiwan cobra (Naja naja atra) venom. Interactions of zwitterionic phospholipids with cardiotoxin analogues

Egg sphingomyelin vesicles were used to assay aggregation/fusion activities of proteins from Taiwan (Naja naja atra) venom to avoid the problem of phospholipase A2 contamination during protein purification. It led to the identification of a new cardiotoxin (CTX) analogue protein (CTX V) with major aggregation/fusion, but few hemolysis, activities. On the contrary, cardiotoxin (CTX III) induced significant hemolysis of human red blood cells but exhibited few aggregation/fusion activities. To study the structure/activity relationship of these CTX-induced processes, the amino acid sequence of CTX V was determined and its aggregation/fusion activity was compared with that of CTX III by transmission electron microscopy, quasielastic laser light scattering, differential scanning calorimetry, and fluorescence spectroscopy. The results show that the CTX-induced fusion process at temperatures slightly above that of the gel to liquid-crystalline phase transition of sphingomyelin vesicles can ultimately convert small sonicated vesicles into large fused vesicles with sizes of 1-2 microns. The abilities of CTX V to induce the leakage of sphingomyelin vesicles content and to cause the fusion of vesicles are approximately 10-fold higher than those of CTX III. Based on the CTX structures determined in the present and other studies, it is suggested that the amino acid residue X within the well conserved sequence of -Cys-Pro-X-Gly-Lys-Gln-Leu-Cys- plays a role in the interaction of CTX with lipid molecules. The lipid phase transition could further enhance the protein-lipid interaction in the process leading to the fusion of vesicles.     

Biophysical correlates of lysophosphatidylcholine- and ethanol-mediated shape transformation and hemolysis of human erythrocytes. Membrane viscoelasticity and NMR measurement

The effects of monopalmitoylphosphatidylcholine (MPPC or lysophosphatidylcholine) and a series of short-chain primary alcohols (ethanol, 1-butanol and 1-hexanol) on cell shape, hemolysis, viscoelastic properties and membrane lipid packing of human red blood cells (RBCs) were studied. For MPPC, the effective membrane concentration to induce the formation of stage 3 echinocytes (8 x 10(6) molecules per cell) was one order of magnitude lower than that needed to induce 50% hemolysis (7 x 10(7) molecules per cell). In contrast, short-chain alcohols induced both shape changes and hemolysis within close concentration range (2.5 x 10(8) to 3.5 x 10(8) molecules per cell). Viscoelastic properties of the RBCs were studied by micropipette aspiration and correlated with shape change. Ethanol-treated RBCs showed a decrease in membrane elastic modulus and an increase in membrane viscosity in the recovery phase at the early stage of shape change. MPPC-treated cells showed the same type of viscoelastic changes, but these were not observed until the formation of stage 2 echinocytes. High-resolution solid-state 13C nuclear magnetic resonance technique was applied to study membrane lipid packing in the ghost membrane by following the chemical shift of hydrocarbon chains. Both MPPC and ethanol caused the 13C-NMR chemical shift to move upfield, indicating that membrane lipids were expanded due to the intercalation of these exogenous molecules. Using data obtained from model compounds, we convert values of chemical shift into a lipid packing parameter, i.e., number of gauche bonds for fatty acyl hydrocarbon chains. Approximately 10(8) interacting molecules per cell are required to induce a detectable change of lipid packing by both MPPC and ethanol. The results indicate that homolysis occurs at a smaller surface area for MPPC- than ethanol-treated RBCs. Our findings suggest that progressive changes in the molecular packing in the membrane lead eventually to hemolysis, but the mode responsible for shape transformation varies with these amphipaths.     

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK‐dependent Cyclooxygenase‐2

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47^phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG₂ receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.     

Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.     

Accumulation of lipid production in Chlorella minutissima by triacylglycerol biosynthesis-related genes cloned from Saccharomyces cerevisiae and Yarrowia lipolytica

Discovery of an alternative fuel is now an urgent matter because of the impending issue of oil depletion. Lipids synthesized in algal cells called triacylglycerols (TAGs) are thought to be of the most value as a potential biofuel source because they can use transesterification to manufacture biodiesel. Biodiesel is deemed as a good solution to overcoming the problem of oil depletion since it is capable of providing good performance similar to that of petroleum. Expression of several genomic sequences, including glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, and phospholipid:diacylglycerol acyltransferase, can be useful for manipulating metabolic pathways for biofuel production. In this study, we found this approach indeed increased the storage lipid content of C. minutissima UTEX 2219 up to 2-fold over that of wild type. Thus, we conclude this approach can be used with the biodiesel production platform of C. minutissima UTEX 2219 for high lipid production that will, in turn, enhance productivity.     

Spontaneous activation of circulating granulocytes in patients with acute myocardial and cerebral diseases.

Recent animal studies have suggested that there exists an activated subpopulation of circulating granulocytes which plays an important part in microvascular sequestration and tissue injury during shock and ischemia. In this respect, spontaneous granulocyte activation in form of pseudopod formation, a manifestation of actin polymerization, is a high risk for microvascular entrapment. The present investigation was carried out to determine if there is a significant difference in pseudopod formation in vitro between granulocytes obtained from healthy volunteers without symptoms and patients with acute cardiovascular illnesses. Blood samples from 25 healthy volunteers, 12 patients with acute myocardial infarction (AMI) and 12 patients with acute cerebral infarction (ACI) to determine spontaneous pseudopod formation in granulocytes with a high resolution light microscope over a period of several hours. The results revealed that the mean percentage of cells with pseudopod formation in the control group was below 10% in the first 3 hours, and increased to about 50% at 12 hours. In AMI patients, the level of activation within the first hour was not significantly different from the controls, but it rose rapidly to 90% in 4 to 5 hours. Patients with cerebral infarction, however, showed no significant difference from the control group. When the granulocytes of healthy subjects were incubated in plasma of AMI, the cells were activated similar to AMI granulocytes in their own plasma. When AMI plasma was serially diluted with Ringer's solution, the activation curve fell successively. These results indicate that AMI patients' blood contains plasma factor(s) which can activate granulocytes at a more rapid rate than controls.     

Rapid enrichment of phosphopeptides and phosphoproteins from complex samples using magnetic particles coated with alumina as the concentrating probes for MALDI MS analysis

In this study, we used nanocomposite magnetic particles coated with alumina as the affinity probes to selectively concentrate phosphorylated peptides and proteins from a low volume of sample solution. Tryptic digest products of phosphoproteins including alpha and beta-caseins, human protein phosphatase inhibitor 1, nonfat milk, egg white, and a cell lysate were used as the samples to demonstrate the feasibility of this approach. In only 30 and 90 s, phosphopeptides and phosphoproteins sufficient for characterization by MALDI-MS were enriched by the particles, respectively. Proteins trapped on the particles could be directly digested on the particles. The same particles in the digest solution were employed for enrichment of phosphopeptides. We estimated the required time for performing the enrichment of phosphopeptides from complex samples and characterization by MALDI MS was within 5 min. A small volume (50 microL) and a low concentration (5 x 10(-10) M) of tryptic digest product of a phosphoprotein sample could be dramatically enriched and characterized using this approach.     

Effectiveness of a Government-Organized and Hospital-Initiated Treatment for Multidrug-Resistant Tuberculosis Patients-A Retrospective Cohort Study

Background

In contrast to the conventional model of hospital-treated and government directly observed treatment (DOT) for multidrug-resistant tuberculosis (MDR-TB) patient care, the Taiwan MDR-TB Consortium (TMTC) was launched in May 2007 with the collaboration of five medical care groups that have provided both care and DOT. This study aimed to determine whether the TMTC provided a better care model for MDR-TB patients than the conventional model.

Methods and Findings

A total of 651 pulmonary MDR-TB patients that were diagnosed nation-wide from January 2000-August 2008 were enrolled. Of those, 290 (45%) MDR-TB patients whose initial sputum sample was taken in January 2007 or later were classified as patients in the TMTC era. All others were classified as patients in the pre-TMTC era. The treatment success rate at 36 months was better in the TMTC era group (82%) than in the pre-TMTC era group (61%) (p<0.001). With multiple logistic regressions, diagnosis in the TMTC era (adjusted odds ratio (aOR) 2.8, 95% confidence interval (CI) 1.9–4.2) was an independent predictor of a higher treatment success rate at 36 months. With the time-dependent proportional hazards method, a higher treatment success rate was still observed in the TMTC era group compared to the pre-TMTC era group (adjusted hazard ratio 6.3, 95% CI 4.2–9.5).

Conclusion

The improved treatment success observed in the TMTC era compared to the pre-TMTC era is encouraging. The detailed TMTC components that contribute the most to the improved outcome will need confirmation in follow-up studies with large numbers of MDR-TB patients.     

Impacts of ISCO Persulfate,Peroxide and Permanganate Oxidants on Soils: Soil Oxidant Demand and Soil Properties

The consumption of in-situ chemical oxidation (ISCO) oxidant by soil oxidizable matter (OM), termed the soil oxidant demand (SOD), is an essential factor when designing treatments for successful remediation at an ISCO site. This study aims to assess the impact of different oxidants on SOD and the soil itself, using the Taguchi experimental design. Five oxidation systems, including persulfate (PS), hydrogen peroxide (HP), permanganate (PM), Fe²⁺ activated PS and Fe²⁺ activated HP, and four factors including oxidant concentration, activator concentration, reaction time, and pH were investigated. The results of the Taguchi analysis in this study show that oxidant concentration had the greatest effect on the SOD. Other factors also affected the SOD and the optimum conditions for achieving a lower SOD were determined using the Taguchi design method. Additionally, original and oxidized soils were analyzed using a scanning electron microscope equipped with an X-ray energy dispersive spectrometer to determine the surface morphology and chemical composition of the samples. Variations in soil organic carbon levels and total soil bacterial counts were recorded and the speciation of soil minerals (Fe, Mn, Cu, and Zn) was analyzed.