Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming

Applied Microbiology and Biotechnology - Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are promising, eco-friendly substitutes for conventional chemical...     

Quartz crystal microbalance measurement of self-assembled micellar tubules of the amphiphilic decyl ester of D-tyrosine and their enzymatic polymerization.

Amphiphilic decyl derivatives of D-tyrosine self-assemble into long rodlike or tubular aggregate structures in aqueous buffered solution. In this report we demonstrate the novel use of the quartz crystal microbalance (QCM) to measure the presence in solution, and subequent enzymatic polymerization, of long rodlike monomer aggregates of the decyl ester of D-tyrosine (DEDT) as a function of their formation and increasing surface binding level as pH values increase from 3 to 7. From these data, using the Sauerbray equation to calculate the effective elastic mass surface binding of deprotonated DEDT aggregates, a pKapp of 8.3 is obtained for the DEDT alpha-NH2 group protonation-deprotonation and subsequent aggregation equilibrium. Furthermore, once aggregates are bound to the QCM surface, we initiate and subsequently monitor enzymatic polymerization of the DEDT monomers by horseradish peroxidase through the measurement of significant changes in the quartz crystal frequency and motional resistance. Following the onset of polymerization, the viscoelastic properties of the bound monomer aggregates change. A final polymerized state is achieved in which the altered physical properties of the polymerized rodlike aggregates make the solution immediately above the QCM surface-solution interface behave as a Newtonian fluid, producing a nearly pure viscosity-density energy dissipative effect on the measured crystal frequency and motional resistance values.     

Lipid A modifications characteristic of Salmonella typhimurium are induced by NH4VO3 in Escherichia coli K12. Detection of 4-amino-4-deoxy-L-arabinose, phosphoethanolamine and palmitate.

Two-thirds of the lipid A in wild-type Escherichia coli K12 is a hexa-acylated disaccharide of glucosamine in which monophosphate groups are attached at positions 1 and 4'. The remaining lipid A contains a monophosphate substituent at position 4' and a pyrophosphate moiety at position 1. The biosynthesis of the 1-pyrophosphate unit is unknown. Its presence is associated with lipid A translocation to the outer membrane (Zhou, Z., White, K. A., Polissi, A., Georgopoulos, C., and Raetz, C. R. H. (1998) J. Biol. Chem. 273, 12466-12475). To determine if a phosphatase regulates the amount of the lipid A 1-pyrophosphate, we grew cells in broth containing nonspecific phosphatase inhibitors. Na2WO4 and sodium fluoride increased the relative amount of the 1-pyrophosphate slightly. Remarkably, NH4VO3-treated cells generated almost no 1-pyrophosphate, but made six major new lipid A derivatives (EV1 to EV6). Matrix-assisted laser desorption ionization/time of flight mass spectrometry of purified EV1 to EV6 indicated that these compounds were lipid A species substituted singly or in combination with palmitoyl, phosphoethanolamine, and/or aminodeoxypentose residues. The aminodeoxypentose residue was released by incubation in chloroform/methanol (4:1, v/v) at 25 degrees C, and was characterized by 1H NMR spectroscopy. The chemical shifts and vicinal coupling constants of the two anomers of the aminodeoxypentose released from EV3 closely resembled those of synthetic 4-amino-4-deoxy-L-arabinose. NH4VO3-induced lipid A modification did not require the PhoP/PhoQ two-component regulatory system, and also occurred in E. coli msbB or htrB mutants. The lipid A variants that accumulate in NH4VO3-treated E. coli K12 are the same as many of those normally found in untreated Salmonella typhimurium and Salmonella minnesota, demonstrating that E. coli K12 has latent enzyme systems for synthesizing these important derivatives.     

Mechanisms of the Anti-Ischemic Effect of Angiotensin II AT 1 Receptor Antagonists in the Brain

SUMMARY 1. Circulating and locally formed Angiotensin II regulates the cerebral circulation through stimulation of AT₁ receptors located in cerebrovascular endothelial cells and in brain centers controlling cerebrovascular flow.2. The cerebrovascular autoregulation is designed to maintain a constant blood flow to the brain, by vasodilatation when blood pressure decreases and vasoconstriction when blood pressure increases.3. During hypertension, there is a shift in the cerebrovascular autoregulation to the right, in the direction of higher blood pressures, as a consequence of decreased cerebrovascular compliance resulting from vasoconstriction and pathological growth. In hypertension, when perfusion pressure decreases as a consequence of blockade of a cerebral artery, reduced cerebrovascular compliance results in more frequent and more severe strokes with a larger area of injured tissue.4. There is a cerebrovascular angiotensinergic overdrive in genetically hypertensive rats, manifested as an increased expression of cerebrovascular AT₁ receptors and increased activity of the brain Angiotensin II system. Excess AT₁ receptor stimulation is a main factor in the cerebrovascular pathological growth and decreased compliance, the alteration of the cerebrovascular eNOS/iNOS ratio, and in the inflammatory reaction characteristic of cerebral blood vessels in genetic hypertension. All these factors increase vulnerability to brain ischemia and stroke.5. Sustained blockade of AT₁ receptors with peripheral and centrally active AT₁ receptor antagonists (ARBs) reverses the cerebrovascular pathological growth and inflammation, increases cerebrovascular compliance, restores the eNOS/iNOS ratio and decreases cerebrovascular inflammation. These effects result in a reduction of the vulnerability to brain ischemia, revealed, when an experimental stroke is produced, in protection of the blood flow in the zone of penumbra and substantial reduction in neuronal injury.6. The protection against ischemia resulting is related to inhibition of the Renin–Angiotensin System and not directly related to the decrease in blood pressure produced by these compounds. A similar decrease in blood pressure as a result of the administration of β-adrenergic receptor and calcium channel blockers does not protect from brain ischemia.7. In addition, sustained AT₁ receptor inhibition enhances AT₂ receptor expression, associated with increased eNOS activity and NO formation followed by enhanced vasodilatation. Direct AT₁ inhibition and indirect AT₂ receptor stimulation are associated factors normalizing cerebrovascular compliance, reducing cerebrovascular inflammation and decreasing the vulnerability to brain ischemia.8. These results strongly suggest that inhibition of AT₁ receptors should be considered as a preventive therapeutic measure to protect the brain from ischemia, and as a possible novel therapy of inflammatory conditions of the brain.     

The Response of Antioxidant Enzymes in Cellular Organelles in Cucumber (Cucumis sativus L.) Leaves to Methyl Viologen-induced Photo-oxidative Stress

In order to clarify the response of antioxidant systems in various cellular organelles to photo-oxidative stress, the activities of superoxide dismutase (SOD) and enzymes of the ascorbate–glutathione (AsA-GSH) cycle were investigated in chloroplasts, mitochondria and cytosol of cucumber leaves subjected to methyl viologen (MV) treatment. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate (Pn) and increases in the ratio of the quantum efficiency of photosystem II (PSII), Φ_PSII to that of the quantum efficiency of CO₂ fixation (ΦCO₂), followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) except that the content of ascorbate (AsA) in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase (AO) in these organelles was independent of the MV treatment while the activity of l-galactono-1,4- lactone dehydrogenase (GLDH) in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA.     

Crystal structure of the C2 domain of class II phosphatidylinositide 3-kinase C2alpha

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3'-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel beta-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P(2) and PtdIns(4,5)P(2) and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.     

A new MSPQC system for rapid detection of pathogens in clinical samples

A new multi-channel series piezoelectric quartz crystal (MSPQC) system for detection of pathogens in clinical sample was proposed. Some factors, which affect the detection of pathogens by using MSPQC, were all investigated. A total of 650 clinical samples were detected by MSPQC and compared with licensed BACTEC 9120 system (Becton Dickinson Diagnostic Instrument Systems, Sparks, MD, USA) simultaneously in the Third Xiangya Hospital of Central South University, China. When the incubation period was 5 days, two systems had similar detected results: the MSPQC system detected 123 growth of 650 (18.92%) bottles while the BACTEC 9120 detected 125 growth of 650 (19.23%) bottles. The MSPQC had 2 false-positive signals and 2 false-negative signals. However, BACTEC 9120 had 3 false-positive signals and 0 false-negative signals. Further identifications of bacteria were run by VITEK-2 (bioMérieux China Ltd.), 5% sheep blood trypticase soy agar (SBA) and chocolate agar (CA). Comparing with BACTEC 9120, MSPQC system possesses following advantages: shorter average detection time, less blood volume needed, less false-positive results and low cost. It can also provide information in real time. So MSPQC has a wonderful perspective in clinical application.     

DNA damage evaluated by gammaH2AX foci formation by a selective group of chemical/physical stressors

It has been reported that the phosphorylated form of histone variant H2AX (gammaH2AX) plays an important role in the recruitment of DNA repair and checkpoint proteins to sites of DNA damage, particularly at double strand breaks (DSBs). Using gammaH2AX foci formation as an indicator for DNA damage, several chemicals/stress factors were chosen to assess their ability to induce gammaH2AX foci in a 24h time frame in a human amnion FL cell line. Two direct-acting genotoxins, methyl methanesulfonate (MMS) and N-ethyl-N-nitrosourea (ENU), can induce gammaH2AX foci formation in a time- and dose-dependent manner. Similarly, an indirect-acting genotoxin, benzo[a]pyrene (BP), also induced the formation of gammaH2AX foci in a time- and dose-dependent manner. Another indirect genotoxin, 2-acetyl-aminofluorene (AAF), did not induce gammaH2AX foci formation in FL cells; however, AAF can induce gammaH2AX foci formation in Chinese hamster CHL cells. Neutral comet assays also revealed the induction of DNA strand breaks by these agents. In contrast, epigenetic carcinogens azathioprine and cyclosporine A, as well as non-carcinogen dimethyl sulfoxide, did not induce gammaH2AX foci formation in FL cells. In addition, heat shock and hypertonic saline did not induce gammaH2AX foci. Cell survival analyses indicated that the induction of gammaH2AX is not correlated with the cytotoxic effects of these agents/factors. Taken together, these results suggest that gammaH2AX foci formation could be used for evaluating DNA damage; however, the different cell types used may play an important role in determining gammaH2AX foci formation induced by a specific agent.     

Trade-off between Multiple Constraints Enables Simultaneous Formation of Modules and Hubs in Neural Systems

The formation of the complex network architecture of neural systems is subject to multiple structural and functional constraints. Two obvious but apparently contradictory constraints are low wiring cost and high processing efficiency, characterized by short overall wiring length and a small average number of processing steps, respectively. Growing evidence shows that neural networks are results from a trade-off between physical cost and functional value of the topology. However, the relationship between these competing constraints and complex topology is not well understood quantitatively. We explored this relationship systematically by reconstructing two known neural networks, Macaque cortical connectivity and C. elegans neuronal connections, from combinatory optimization of wiring cost and processing efficiency constraints, using a control parameter , and comparing the reconstructed networks to the real networks. We found that in both neural systems, the reconstructed networks derived from the two constraints can reveal some important relations between the spatial layout of nodes and the topological connectivity, and match several properties of the real networks. The reconstructed and real networks had a similar modular organization in a broad range of , resulting from spatial clustering of network nodes. Hubs emerged due to the competition of the two constraints, and their positions were close to, and partly coincided, with the real hubs in a range of values. The degree of nodes was correlated with the density of nodes in their spatial neighborhood in both reconstructed and real networks. Generally, the rebuilt network matched a significant portion of real links, especially short-distant ones. These findings provide clear evidence to support the hypothesis of trade-off between multiple constraints on brain networks. The two constraints of wiring cost and processing efficiency, however, cannot explain all salient features in the real networks. The discrepancy suggests that there are further relevant factors that are not yet captured here.