Identification of an integral plasma membrane pool of protein kinase C in mammalian tissues and cells

Protein kinase C (PKC) is a family of serine/threonine protein kinases that are pivotal in cellular regulation. Since its discovery in 1977, PKCs have been known as cytosolic and peripheral membrane proteins. However, there are reports that PKC can insert into phospholipids vesicles in vitro. Given the intimate relationship between the plasma membrane and the activation of PKC, it is important to determine whether such "membrane-inserted" form of PKC exists in mammalian cells or tissues. Here, we report the identification of an integral plasma membrane pool for all the 10 PKC isozymes in vivo by their ability to partition into the detergent-rich phase in Triton X-114 phase partitioning, and by their resistance to extractions with 0.2 M sodium carbonate (pH 11.5), 2 M urea and 2 M sodium chloride. The endogenous integral membrane pool of PKC in mouse fibroblasts is found to be acutely regulated by phorbol ester or diacylglycerol, suggesting that this pool of PKC may participate in cellular processes known to be regulated by PKC. At least for PKC(alpha), the C2-V3 region at the regulatory domain of the kinase is responsible for membrane integration. Further exploration of the function of this novel integral plasma membrane pool of PKC will not only shed new light on molecular mechanisms underlying its cellular functions but also provide new strategies for pharmaceutical modulation of this important group of kinases.     

Zinc Casting and Recycling (8 pp)

Aim, Scope and Background Metal die casting is a highly energy-intensive industry. In addition to that, the production of primary zinc by smelting consumes huge amounts of energy as well as generates many types of pollution. This paper uses LCA to investigate the environmental performance of a zinc cast product. The areas of environmental concern are focused on the direct and indirect air emissions that arise from the Zinc Smelting, Casting and Recycling, as well as transportation. Main Features The LCA case study employs a cradle-to-gate approach, which starts with the purchasing of primary zinc from abroad, casting, inspection, and ends when the scrap metal is sent back for recycling by truck. Based on a “generic” zinc casting product, the objective of the LCA was to compare the air emissions from the material cycle due to: i) the increased content of recycled metal in the final cast product; and ii) the choice of selecting between two Remelters (A and B), the first located near the company and the other in a neighbouring country, to send zinc scrap for recycling. The LCA SimaPro software (version 5.0) Eco-indicator “99 method is used to perform an impact assessment for Climate Change, Acidification, Ecotoxicity, Respiratory Inorganics, and Respiratory Organics was performed. Results The results from direct (process) and indirect (power plants) air emissions confirmed that the major air pollution occurs during Zinc Smelting, that is up to approximately 65–70%. Although an increase in recycling rates resulted in higher levels of air pollution from transportation as well as heavy metals from dross, these two issues were insignificant compared to the huge amount of energy consumed for primary metal production. Based on air pollution from transportation alone, a significant reduction of greenhouse gases and VOCs of 90% each was appreciated when Remelter A was selected. Conclusion The results verified that efforts to recycle zinc and consume the material in a more sustainable manner have become highly important. Also, a second LCA investigation that was made to compare zinc cast products that consists of: 100% primary zinc, mixtures of 50–50% and 40–60% primary-to-recycled zinc, and finally 100% recycled zinc; further emphasized the need for using recycled metal, as opposed to using primary metal.     

Synthesis and evaluation of bifunctional nitrocatechol inhibitors of pig liver catechol-O-methyltransferase

Bifunctional compounds were tested in vitro as potential inhibitors of pig liver catechol-O-methyltransferase (COMT) with respect to the catechol substrate 4-[(3,4-dihydroxyphenyl)azo]benzenesulfonate. The bifunctional compounds were a composite of either two nitrocatechols or one nitrocatechol and one phenol, linked by amide bonds to a spacer unit comprising two to five methylene groups. The unsymmetrical compounds N-[2-(4-hydroxybenzoylamine)ethyl]-3,4-dihydroxy-5-nitrobenzamide], N-[3-(4-hydroxybenzoyl-amine)propyl]-3,4-dihydroxy-5-nitrobenzamide] and N-[5-(4-hydroxybenzoylamine)pentyl]-3,4-dihydroxy-5-nitrobenzamide] demonstrated strong inhibitory action against COMT with K(i) values in the 100 nM range. In comparison, the monofunctional nitrocatechol analogues of these compounds had K(i) values that were significantly higher.     

Recognition of protein/gene names from text using an ensemble of classifiers

This paper proposes an ensemble of classifiers for biomedical name recognition in which three classifiers, one Support Vector Machine and two discriminative Hidden Markov Models, are combined effectively using a simple majority voting strategy. In addition, we incorporate three post-processing modules, including an abbreviation resolution module, a protein/gene name refinement module and a simple dictionary matching module, into the system to further improve the performance. Evaluation shows that our system achieves the best performance from among 10 systems with a balanced F-measure of 82.58 on the closed evaluation of the BioCreative protein/gene name recognition task (Task 1A).     

Computational model of blood flow in the aorto-coronary bypass graft

Background

Coronary artery bypass grafting surgery is an effective treatment modality for patients with severe coronary artery disease. The conduits used during the surgery include both the arterial and venous conduits. Long- term graft patency rate for the internal mammary arterial graft is superior, but the same is not true for the saphenous vein grafts. At 10 years, more than 50% of the vein grafts would have occluded and many of them are diseased. Why do the saphenous vein grafts fail the test of time? Many causes have been proposed for saphenous graft failure. Some are non-modifiable and the rest are modifiable. Non-modifiable causes include different histological structure of the vein compared to artery, size disparity between coronary artery and saphenous vein. However, researches are more interested in the modifiable causes, such as graft flow dynamics and wall shear stress distribution at the anastomotic sites. Formation of intimal hyperplasia at the anastomotic junction has been implicated as the root cause of long- term graft failure.Many researchers have analyzed the complex flow patterns in the distal sapheno-coronary anastomotic region, using various simulated model in an attempt to explain the site of preferential intimal hyperplasia based on the flow disturbances and differential wall stress distribution. In this paper, the geometrical bypass models (aorto-left coronary bypass graft model and aorto-right coronary bypass graft model) are based on real-life situations. In our models, the dimensions of the aorta, saphenous vein and the coronary artery simulate the actual dimensions at surgery. Both the proximal and distal anastomoses are considered at the same time, and we also take into the consideration the cross-sectional shape change of the venous conduit from circular to elliptical. Contrary to previous works, we have carried out computational fluid dynamics (CFD) study in the entire aorta-graft-perfused artery domain. The results reported here focus on (i) the complex flow patterns both at the proximal and distal anastomotic sites, and (ii) the wall shear stress distribution, which is an important factor that contributes to graft patency.

Methods

The three-dimensional coronary bypass models of the aorto-right coronary bypass and the aorto-left coronary bypass systems are constructed using computational fluid-dynamics software (Fluent 6.0.1). To have a better understanding of the flow dynamics at specific time instants of the cardiac cycle, quasi-steady flow simulations are performed, using a finite-volume approach. The data input to the models are the physiological measurements of flow-rates at (i) the aortic entrance, (ii) the ascending aorta, (iii) the left coronary artery, and (iv) the right coronary artery.

Results

The flow field and the wall shear stress are calculated throughout the cycle, but reported in this paper at two different instants of the cardiac cycle, one at the onset of ejection and the other during mid-diastole for both the right and left aorto-coronary bypass graft models. Plots of velocity-vector and the wall shear stress distributions are displayed in the aorto-graft-coronary arterial flow-field domain. We have shown (i) how the blocked coronary artery is being perfused in systole and diastole, (ii) the flow patterns at the two anastomotic junctions, proximal and distal anastomotic sites, and (iii) the shear stress distributions and their associations with arterial disease.

Conclusion

The computed results have revealed that (i) maximum perfusion of the occluded artery occurs during mid-diastole, and (ii) the maximum wall shear-stress variation is observed around the distal anastomotic region. These results can enable the clinicians to have a better understanding of vein graft disease, and hopefully we can offer a solution to alleviate or delay the occurrence of vein graft disease.

     

How the turtle forms its shell: a paracrine hypothesis of carapace formation

We propose a two-step model for the evolutionary origin of the turtle shell. We show here that the carapacial ridge (CR) is critical for the entry of the ribs into the dorsal dermis. Moreover, we demonstrate that the maintenance of the CR and its ability to attract the migrating rib precursor cells depend upon fibroblast growth factor (FGF) signaling. Inhibitors of FGF allow the CR to degenerate, with the consequent migration of ribs along the ventral body wall. Beads containing FGF10 can rearrange rib migration in the chick, suggesting that the CR FGF10 plays an important role in attracting the rib rudiments. The co-ordinated growth of the carapacial plate and the ribs may be a positive feedback loop (similar to that of the limbs) caused by the induction of Fgf8 in the distal tips of the ribs by the FGF10-secreting mesenchyme of the CR. Once in the dermis, the ribs undergo endochrondral ossification. We provide evidence that the ribs act as signaling centers for the dermal ossification and that this ossification is due to bone morphogenetic proteins secreted by the rib. Thus, once the ribs are within the dermis, the ossification of the dermis is not difficult to achieve. This relatively rapid means of carapace formation would allow for the appearance of turtles in the fossil record without obvious intermediates.     

Proteomic analysis of hepatitis B virus-associated hepatocellular carcinoma: Identification of potential tumor markers

Hepatocellular carcinoma (HCC) is a malignancy of both underdeveloped and developing countries. Proteomes of ten pairs of clinical hepatitis B virus associated HCC tissue samples were obtained by high resolution two-dimensional gel electrophoresis. Comprehensive analyses of proteins associated with B-type HCC were focused on total differentially expressed proteins (> or = two-fold increase or decrease, Student's t-test, p < 0.05) from one pair of samples. Protein identification was done by peptide mass fingerprinting with matrix assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography-tandem mass spectrometry. Comparative analyses of proteins associated with B-type HCC included repeat statistics in ten cases. A total of 100 protein spots, corresponding to 80 different gene products, were identified. Proteins whose expression levels were different by more than 2-fold in at least 50% of the cases (five of ten cases) were further analyzed and 45 proteins were selected out as candidates for HCC-associated proteins. Western blotting further validated up-regulated expressions of two candidate proteins in tumor tissues: proliferating cell antigen and stathmin 1. This comprehensive and comparative analyses of proteins associated with B-type HCC could provide useful molecular markers for diagnostics and prognostics and for therapeutic targets. The physiological significance of the differential expressions for several candidate proteins are discussed.     

Sequence analysis and structural prediction of the severe acute respiratory syndrome coronavirus nsp5

The non-structural proteins (nsp or replicase proteins) of coronaviruses are relatively conserved and can be effective targets for drugs. Few studies have been conducted into the function of the severe acute respiratory syndrome coronavirus (SARS-CoV) nsp5. In this study, bioinformatics methods were employed to predict the secondary structure and construct 3-D models of the SARS-CoV GD strain nsp5. Sequencing and sequential comparison was performed to analyze the mutation trend of the polymerase nsp5 gene during the epidemic process using a nucleotide-nucleotide basic local alignment search tool (BLASTN) and a protein-protein basic local alignment search tool (BLASTP). The results indicated that the nsp5 gene was steady during the epidemic process and the protein was homologous with other coronavirus nsp5 proteins. The protein encoded by the nsp5 gene was expressed in COS-7 cells and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This study provided the foundation for further exploration of the protein‘s biological function, and contributed to the search for anti-SARS-CoV drugs.