The role of the active site residues in human galactokinase: implications for the mechanisms of GHMP kinases.

Galactokinase catalyses the phosphorylation of galactose at the expense of ATP. Like other members of the GHMP family of kinases it is postulated to function through an active site base mechanism in which Asp-186 abstracts a proton from galactose. This asparate residue was altered to alanine and to asparagine by site-directed mutagenesis of the corresponding gene. This resulted in variant enzyme with no detectable galactokinase activity. Alteration of Arg-37, which lies adjacent to Asp-186 and is postulated to assist the catalytic base, to lysine resulted in an active enzyme. However, alteration of this residue to glutamate abolished activity. All the variant enzymes, except the arginine to lysine substitution, were structurally unstable (as judged by native gel electrophoresis in the presence of urea) compared to the wild type. This suggests that the lack of activity results from this structural instability, in addition to any direct effects on the catalytic mechanism. Computational estimations of the pK(a) values of the arginine and aspartate residues, suggest that Arg-37 remains protonated throughout the catalytic cycle whereas Asp-186 has an abnormally high pK(a) value (7.18). Quantum mechanics/molecular mechanics (QM/MM) calculations suggest that Asp-186 moves closer to the galactose molecule during catalysis. The experimental and theoretical studies presented here argue for a mechanism in which the C-1-OH bond in the sugar is weakened by the presence of Asp-186 thus facilitating nucleophilic attack by the oxygen atom on the gamma-phosphorus of ATP.

Protein kinases C: potential targets for intervention in diabetic nephropathy

Protein kinases C are a family of serine threonine protein kinases that play key roles in extracellular signal transduction. Inappropriate activation of protein kinase C has been implicated in the pathophysiology of many diseases, including diabetes mellitus. Indeed, protein kinase C activation may contribute not only to the pathogenesis of diabetic complications such as nephropathy and retinopathy, but also to insulin resistance. Growing awareness that protein kinase C isoforms subserve specific subcellular functions has led to the development of isoform-specific inhibitors, which may be useful investigational tools and therapeutic agents for attenuating the effects of inappropriate protein kinase C activity. Here we review the role played by protein kinases C in diabetic nephropathy and the recent progress that has been made to modulate its activity using specific inhibitors. Curr Opin Nephrol Hypertens 7:563-570. (C) 1998 Lippincott Wiiliams & Wilkins.

Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN), In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo, Two different proliferative forms of anti-glomerular basal membrane (GEM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis, Administration of rabbit anti-rat GEM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GEM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN, Immunization of Wistar-Kyoto rats with bovine GEM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk, ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK), We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GEM CN, providing a possible mechanism of long-term activation of ERK in this disease model, In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN, Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

An updated moraine map of Far NE Russia

Barr and Clark published a series of maps depicting the distribution of end moraines across Far NE Russia. These
moraines outlined the former distribution and dimensions of glaciers, and were identified through the analysis of
Landsat ETM+ satellite images (15- and 30-m resolution). Now, a number of freely available digital elevation
model (DEM) datasets are available, which cover the entire 4 million km2 of Far NE Russia. These include
the 30-m resolution ASTER GDEM and the 90-m resolution Viewfinder Panorama DEM. Here we use these
datasets, in conjunction with Landsat ETM+ images, to complete the process of systematically and
comprehensively mapping end moraines. With the aid of the DEMs described above, here we present a total
dataset of 8414 moraines, which almost quadruples the inventory of Barr and Clark. This increase in the
number of moraines is considered to reflect the utility of the DEMs for mapping glacial landforms. In terms of
moraine distribution, the Barr and Clark map and the one presented here are comparable, with moraines found
to cluster in highland regions and upon adjacent lowlands, attesting to the former occupation of the region by
mountain-centred ice masses. This record is considered to reflect palaeoclimatic and topographic controls upon
the extent and dynamics of palaeoglaciers, as well as spatial variability in moraine preservation.

An Impact Of Various Shroud Bleed Slot Configurations And Cavity Vanes On Compressor Map Width And The Inducer Flow Field

This paper describes an investigation of map width enhancement and a detailed analysis of the inducer flow field due to various bleed slot configurations and vanes in the annular cavity of a turbocharger centrifugal compressor. The compressor under investigation is used in a turbocharger application for a heavy duty diesel engine of approximately 400 hp. This investigation has been undertaken using a computational fluid dynamics (CFD) model of the full compressor stage, which includes a manual multiblock-structured grid generation method. The influence of the bleed slot flow on the inducer flow field at a range of operating conditions has been analyzed, highlighting the improvement in surge and choked flow capability. The impact of the bleed slot geometry variations and the inclusion of cavity vanes on the inlet incidence angle have been studied in detail by considering the swirl component introduced at the leading edge by the recirculating flow through the slot. Further, the overall stage efficiency and the nonuniform flow field at the inducer inlet have been also analyzed. The analysis revealed that increasing the slot width has increased the map width by about 17%. However, it has a small impact on the efficiency, due to the frictional and mixing losses. Moreover, adding vanes in the cavity improved the pressure ratio and compressor performance noticeably. A detail analysis of the compressor with cavity vanes has also been presented.

Map Width Enhancement Technique for a Turbocharger Compressor

This paper presents an investigation of map width enhancement and the performance improvement of a turbocharger compressor using a series of static vanes in the annular cavity of a classical bleed slot system. The investigation has been carried out using both experimental and numerical analysis. The compressor stage used for this study is from a turbocharger unit used in heavy duty diesel engines of approximately 300 kW. Two types of vanes were designed and added to the annular cavity of the baseline classical bleed slot system. The purpose of the annular cavity vane technique is to remove some of the swirl that can be carried through the bleed slot system, which would influence the pressure
ratio. In addition to this, the series of cavity vanes provides a better guidance to the slot recirculating flow before it mixes with the impeller main inlet flow. Better guidance of the flow improves the mixing at the inducer inlet in the circumferential direction. As a consequence, the stability of the compressor is improved at lower flow rates and a wider map can be achieved. The impact of two cavity vane designs on the map width and performance of the compressor was highlighted through a detailed analysis of the impeller flow field. The numerical and experimental study revealed that an effective vane design can improve the map width and pressure ratio characteristic without an efficiency penalty compared to the classical bleed slot system without vanes. The comparison study between the cavity vane and noncavity vane configurations presented in this paper showed that the map width was improved by 14.3% due to a significant reduction in surge flow and the peak pressure ratio was improved by 2.25% with the addition of a series of cavity vanes in the annular cavity of the bleed slot system.

MAP kinase pathway signalling is essential for extracellular matrix determined mammary epithelial cell survival

Mammary epithelial cells in primary cell culture require both growth factors and specific extracellular matrix (ECM)-attachment for survival. Here we demonstrate for the first time that inhibition of the ECM-induced Erk 1/Erk 2 (p42/44 MAPK) pathway, by PD 98059, leads to apoptosis in these cells. Associated with this cell death is a possible compensatory signalling through the p38 MAP kinase pathway the inhibition of which, by SB 203580, leads to a more rapid onset of apoptosis. This provides evidence for a hitherto undescribed Erk 1/Erk 2 to p38 MAP kinase pathway 'cross-talk' that is essential for the survival of these cells. The cell death associated with inhibition of these two MAP kinase pathways however, occurred in the presence of insulin that activates the classical PI-3 kinase-dependent Akt/PKB survival signals and Akt phosphorylation. Cell death induced by inhibition of the MAP kinase pathways did not affect Akt phosphorylation and may, thus, be independent of PI-3 kinase signalling.

Defending the Irish Border: The Map of Watchful Architecture

This chapter offers an analysis of a map of the Irish border. The Map of Watchful Architecture charts the history of defensive structures in the border region.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.