Experimental and computational studies investigating trehalose protection of HepG2 cells from palmitate-induced toxicity

Understanding the mechanism of saturated fatty acid-induced hepatocyte toxicity may provide insight into cures for diseases such as obesity-associated cirrhosis. Trehalose, a nonreducing disaccharide shown to protect proteins and cellular membranes from inactivation or denaturation caused by different stress conditions, also protects hepatocytes from palmitate-induced toxicity. Our results suggest that trehalose serves as a free radical scavenger and alleviates damage from hydrogen peroxide secreted by the compromised cells. We also observe that trehalose protects HepG2 cells by interacting with the plasma membrane to counteract the changes in membrane fluidity induced by palmitate. The experimental results are supported by molecular dynamics simulations of model cell membranes that closely reflect the experimental conditions. Simulations were performed to understand the specific interactions between lipid bilayers, palmitate, and trehalose. The simulations results reveal the early stages of how palmitate induces biophysical changes to the cellular membrane and the role of trehalose in protecting the membrane structure.     

Synthesis and evaluation of ether and halogenated derivatives of mannopeptimycin glycopeptide antibiotics

A number of 6-O-ether and 4-O-ether derivatives of mannopeptimycin-alpha with different steric bulk and lipophilicity were synthesized for structure-activity relationship study. Novel iodo and bromo mannopeptimycin-alpha were also prepared. These compounds were synthesized via electrophilic aromatic substitution. Many of the new ether derivatives exhibited potent antibacterial activity against Gram-positive resistant strains including VRE, MRSA, and PRSP.     

Cooperativity and oligomeric status of cardiac muscarinic cholinergic receptors

Muscarinic cholinergic receptors can appear to be more numerous when labeled by [(3)H]quinuclidinylbenzilate (QNB) than by N-[(3)H]methylscopolamine (NMS). The nature of the implied heterogeneity has been studied with M(2) receptors in detergent-solubilized extracts of porcine atria. The relative capacity for [(3)H]NMS and [(3)H]QNB was about 1 in digitonin-cholate, 0.56 in cholate-NaCl, and 0.44 in Lubrol-PX. Adding digitonin to extracts in cholate-NaCl increased the absolute capacity for both radioligands, and the relative capacity increased to near 1. The latency cannot be attributed to a chemically impure radioligand, instability of the receptor, an irreversible effect of NMS, or a failure to reach equilibrium. Binding at near-saturating concentrations of [(3)H]QNB in cholate-NaCl or Lubrol-PX was blocked fully by unlabeled NMS, which therefore appeared to inhibit noncompetitively at sites inaccessible to radiolabeled NMS. Such an effect is inconsistent with the notion of functionally distinct, noninterconverting, and mutually independent sites. Both the noncompetitive effect of NMS on [(3)H]QNB and the shortfall in capacity for [(3)H]NMS can be described quantitatively in terms of cooperative interactions within a receptor that is at least tetravalent; no comparable agreement is possible with a receptor that is only di- or trivalent. The M(2) muscarinic receptor therefore appears to comprise at least four interacting sites, presumably within a tetramer or larger array, and ligands appear to bind in a cooperative manner under at least some conditions.     

Fate of the (2Fe-2S)(2+) cluster of Escherichia coli biotin synthase during reaction: a Mössbauer characterization

Biotin synthase, the enzyme which catalyzes the last step of the biosynthesis of biotin, contains only (2Fe-2S)(2+) clusters when isolated under aerobic conditions. Previous results showed that reduction by dithionite or photoreduced deazaflavin converts the (2Fe-2S)(2+) to (4Fe-4S)(2+,+). However, until now, no detailed investigation concerning the fate of the (2Fe-2S)(2+) during reduction under assay conditions (NADPH, flavodoxin, flavodoxin reductase) has been realized. Here, we show by M?ssbauer spectroscopy on a partially purified fraction overexpressing the enzyme that, in the presence of a S(2)(-) source and Fe(2+), there is conversion of the predominant (2Fe-2S)(2+) clusters into a 1:1 mixture of (2Fe-2S)(2+) and (4Fe-4S)(2+). No change in this cluster composition was observed in the presence of the physiological reducing system. When the reaction was allowed to proceed by addition of the substrate dethiobiotin, the (4Fe-4S)(2+) was untouched whereas the (2Fe-2S)(2+) was degraded into a new species. This is consistent with the hypothesis that the reduced (4Fe-4S) cluster is involved in mediating the cleavage of AdoMet and that the (2Fe-2S)(2+) is the sulfur source for biotin.     

A local training and pruning approach for neural networks

The training of neural networks using the extended Kalman filter (EKF) algorithm is plagued by the drawback of high computational complexity and storage requirement that may become prohibitive even for networks of moderate size. In this paper, we present a local EKF training and pruning approach that can solve this problem. In particular, the by-products obtained along with the local EKF training can be utilized to measure the importance of the network weights. Comparing with the original global approach, the proposed local EKF training and pruning approach results in a much lower computational complexity and storage requirement. Hence, it is more practical in solving real world problems. The performance of the proposed algorithm is demonstrated on one medium- and one large-scale problems, namely, sunspot data prediction and handwritten digit recognition.     

Relaxation Effect of Abacavir on Rat Basilar Arteries

Background

The use of abacavir has been linked with increased cardiovascular risk in patients with human immunodeficiency virus infection; however, the mechanism involved remains unclear. We hypothesize that abacavir may impair endothelial function. In addition, based on the structural similarity between abacavir and adenosine, we propose that abacavir may affect vascular contractility through endogenous adenosine release or adenosine receptors in blood vessels.

Methods

The relaxation effect of abacavir on rat basilar arteries was studied using the myograph technique. Cyclic GMP and AMP levels were measured by immunoassay. The effects of abacavir on nucleoside transporters were studied using radiolabeled nucleoside uptake experiments. Ecto-5′ nucleotidase activity was determined by measuring the generation of inorganic phosphate using adenosine monophosphate as the substrate.

Results

Abacavir induced the relaxation of rat basilar arteries in a concentration-dependent manner. This relaxation was abolished when endothelium was removed. In addition, the relaxation was diminished by the nitric oxide synthase inhibitor, L-NAME, the guanylyl cyclase inhibitor, ODQ, and the protein kinase G inhibitor, KT5820. Abacavir also increased the cGMP level in rat basilar arteries. Abacavir-induced relaxation was also abolished by adenosine A2 receptor blockers. However, abacavir had no effect on ecto-5’ nucleotidase and nucleoside transporters. Short-term and long-term treatment of abacavir did not affect acetylcholine-induced relaxation in rat basilar arteries.

Conclusion

Abacavir induces acute endothelium-dependent relaxation of rat basilar arteries, probably through the activation of adenosine A2 receptors in endothelial cells, which subsequently leads to the release of nitric oxide, resulting in activation of the cyclic guanosine monophosphate/protein kinase G-dependent pathway in vascular smooth muscle cells. It is speculated that abacavir-induced cardiovascular risk may not be related to endothelial dysfunction as abacavir does not impair relaxation of blood vessels. The most likely explanation of increased cardiovascular risk may be increased platelet aggregation as suggested by other studies.