PubMed Health⌕ Search

Biomedical subjects

K A West

Publications and source records attributed to K A West.

At least 19 recordsLinked to original sources
PubMed2003

Importance of peak PRA in predicting the kidney transplant survival in highly sensitized patients.

Do patients with high historic peak panel-reactive antibodies (PRA) remain high risk if their PRA levels fall before transplantation? We examined retrospectively 406 first and repeat kidney recipients with a peak PRA of >50%, who were transplanted from our center between January 1990 and December 2001. Univariate analysis by log-rank test was performed for variables that affect graft survival. The factors tested included current PRA, peak PRA, difference between peak and current PRA (DeltaPRA), HLA mismatch, gender, age, transplant number, and donor source. Receiver operator characteristic curves (ROC) were generated to obtain the best cutpoints for current PRA and DeltaPRA. Current PRA (P < .0001), peak PRA (P = .0004), and DeltaPRA (P = .0015) were significant predictors by univariate analysis. However, in a multivariate model, peak PRA was not significant. Current PRA (P < .0001) was significantly associated with graft survival, while DeltaPRA showed a strong trend to significance (P = .05). Current PRA of <26% and DeltaPRA of >37% were the best cutpoints for separating good and poor outcomes. This study shows that current PRA and DeltaPRA impact on graft survival in highly sensitized (>50%) patients. Sensitized patients with peak PRA >50% who subsequently have a drop in PRA to <26% are at lower risk of graft loss than those with a persistently high PRA. A fall in peak PRA of >37% at the time of transplant appears to be of benefit only in those patients who achieve a current PRA of <26%.

Analysis of Variance↗
PubMed2002

Adequate early cyclosporin exposure is critical to prevent renal allograft rejection: patients monitored by absorption profiling.

This study used receiver operating characteristic analysis to investigate the properties of area under the concentration-time curve during the first 4h after cyclosporin-microemulsion dosing (AUC0-4) and cyclosporin (CyA) levels immediately before and at 2 and 3h after dosing (C0, C2 and C3) to predict the risk of biopsy-proven acute rejection (AR) at 6 months. Ninety-eight kidney transplant recipients treated with CyA-microemulsion-based triple therapy immunosuppression were studied on post-transplant days 3, 5, and 7, and at increasing intervals thereafter. The most sensitive and specific predictor of AR was AUC0-4. Of the single time-point measurements, the measurement properties of C2 were closest to those of AUC0-4, and superior to those of C3. The relationship between C0 and subsequent AR was weak and did not reach statistical significance. On day 3, CyA AUC0-4 > or = 4,400 ng.h/mL and C2 > or = 1,700 ng/mL were each associated with a 92% negative predictive value for rejection in the first 6months. Pharmacokinetic measurements on or after day 5, and measurements on day 3 in patients with delayed graft function, were not predictive of AR. Adequate exposure within the first 3days post transplantation may be critically important in preventing subsequent rejection.

Adult↗
PubMed2001

Proteomic method identifies proteins nitrated in vivo during inflammatory challenge.

Inflammation in asthma, sepsis, transplant rejection, and many neurodegenerative diseases associates an up-regulation of NO synthesis with increased protein nitration at tyrosine. Nitration can cause protein dysfunction and is implicated in pathogenesis, but few proteins that appear nitrated in vivo have been identified. To understand how this modification impacts physiology and disease, we used a proteomic approach toward targets of protein nitration in both in vivo and cell culture inflammatory disease models. This approach identified more than 40 nitrotyrosine-immunopositive proteins, including 30 not previously identified, that became modified as a consequence of the inflammatory response. These targets include proteins involved in oxidative stress, apoptosis, ATP production, and other metabolic functions. Our approach provides a means toward obtaining a comprehensive view of the nitroproteome and promises to broaden understanding of how NO regulates cellular processes.

Animals↗
PubMed2001

The LD4 motif of paxillin regulates cell spreading and motility through an interaction with paxillin kinase linker (PKL).

The small GTPases of the Rho family are intimately involved in integrin-mediated changes in the actin cytoskeleton that accompany cell spreading and motility. The exact means by which the Rho family members elicit these changes is unclear. Here, we demonstrate that the interaction of paxillin via its LD4 motif with the putative ARF-GAP paxillin kinase linker (PKL) (Turner et al., 1999), is critically involved in the regulation of Rac-dependent changes in the actin cytoskeleton that accompany cell spreading and motility. Overexpression of a paxillin LD4 deletion mutant (paxillinDeltaLD4) in CHO.K1 fibroblasts caused the generation of multiple broad lamellipodia. These morphological changes were accompanied by an increase in cell protrusiveness and random motility, which correlated with prolonged activation of Rac. In contrast, directional motility was inhibited. These alterations in morphology and motility were dependent on a paxillin-PKL interaction. In cells overexpressing paxillinDeltaLD4 mutants, PKL localization to focal contacts was disrupted, whereas that of focal adhesion kinase (FAK) and vinculin was not. In addition, FAK activity during spreading was not compromised by deletion of the paxillin LD4 motif. Furthermore, overexpression of PKL mutants lacking the paxillin-binding site (PKLDeltaPBS2) induced phenotypic changes reminiscent of paxillinDeltaLD4 mutant cells. These data suggest that the paxillin association with PKL is essential for normal integrin-mediated cell spreading, and locomotion and that this interaction is necessary for the regulation of Rac activity during these events.

ADP-Ribosylation Factors↗
PubMed2001

The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts.

SU5416 and SU6668 are potent antiangiogenic small-molecule inhibitors of receptor tyrosine kinases, including those of the vascular endothelial growth factor and platelet-derived growth factor receptor families. The stem cell factor (SCF) receptor, c-kit, is structurally related to these receptors and, although not expressed on mature peripheral blood cells, is expressed in leukemic blasts derived from 60% to 80% of acute myeloid leukemia (AML) patients. The c-kit kinase inhibitory activity of SU5416 and SU6668 was evaluated in MO7E cells, a human myeloid leukemia cell line. Tyrosine autophosphorylation of the receptor, induced by SCF, was inhibited in these cells by SU5416 and SU6668 in a dose-dependent manner (inhibitory concentration of 50% [IC(50)] 0.1-1 microM). Inhibition of extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, a signaling event downstream of c-kit activation, was also inhibited in a dose-dependent manner. Both compounds also inhibited SCF-induced proliferation of MO7E cells (IC(50) 0.1 microM for SU5416; 0.29 microM for SU6668). Furthermore, both SU5416 and SU6668 induced apoptosis in a dose- and time-dependent manner as measured by the increase in activated caspase-3 and the enhanced cleavage of its substrate poly(ADP-ribose) polymerase. These findings with MO7E cells were extended to leukemic blasts from c-kit(+) patients. In patient blasts, both SU5416 and SU6668 inhibited SCF-induced phosphorylation of c-kit and ERK1/2 and induced apoptosis. These studies indicate that SU5416 and SU6668 inhibit biologic functions of c-kit in addition to exhibiting antiangiogenic properties and suggest that the combination of these activities may provide a novel therapeutic approach for the treatment of AML.

Angiogenesis Inhibitors↗
PubMed2001

The dendritic cell cytoskeleton is critical for the formation of the immunological synapse.

The binding of a T cell to an APC results in T cell actin cytoskeletal rearrangement leading to the formation of an immunological synapse. The APC cytoskeleton has been thought to play a passive role in this process. In this study, we demonstrate that dendritic cells (DC), unlike other APC, actively polarize their actin cytoskeleton during interaction with T cells. DC cytoskeletal rearrangement was critical for both the clustering and the activation of resting T cells. This study provides compelling evidence that the APC cytoskeleton plays an active role in the immunological synapse and may explain the unique ability of DC to activate resting T cells.

Actins↗
PubMed2001

Fascin is involved in the antigen presentation activity of mature dendritic cells.

Maturation of dendritic cells (DC) is critical to their development into potent APCs. Upon maturation, DC up-regulate the expression of MHC class II as well as costimulatory and adhesion molecules, all of which are important in Ag presentation. In addition, they undergo structural changes characterized by the expression of numerous long dendrites. Fascin is an actin-bundling protein that has been reported to be important for the development of dendrites. In this study, we evaluated fascin expression and function during DC maturation into potent APC. In vitro, treatment of bone marrow-derived DC (BM-DC) with GM-CSF resulted in increased levels of fascin expression. This increase correlated directly with an increase in MHC class II and B7-2 expression. Fascin expression was decreased by the addition of TGF-ss and increased by the addition TNF-alpha to the culture. These cytokines suppress or enhance DC maturation, respectively. Increased levels of fascin expression were found to correlate with increased APC activity in a one-way MLR. Specific inhibition of fascin expression, using antisense oligonucleotides, markedly reduced this APC allostimulatory activity. These data demonstrate that fascin expression correlates with DC maturation into APC, and it plays a significant role in the ability of DC to function as APC. This observation is the first evidence linking fascin-mediated dendrite formation with the APC activity of DC.

Actins↗
PubMed2001

Proteome survey of proliferating and differentiating rat RPE-J cells.

The suitability of the rat derived SV-40T immortalized RPE-J cell line for identifying proteome changes associated with RPE differentiation was evaluated by surveying changes in protein expression levels. Rat RPE-J cells were induced to undergo differentiation in culture by growth at the nonpermissive temperature of 40 degrees C in the presence of retinoic acid. Total proteins were extracted from cells grown under proliferating or differentiating conditions and separated by 1D and 2D gel electrophoresis. Gel spots were excised, digested in situ with trypsin, and analysed by mass spectrometry to identify proteins. Computer assisted image analysis was used to align gel patterns and quantify spot intensities. Neither proliferating nor differentiating RPE-J cell cultures exhibited detectable levels of cellular retinaldehyde-binding protein, RPE65, 11- cis -retinol dehydrogenase or lecithin retinol acyl transferase, suggesting that RPE-J cells are not appropriate for visual cycle studies. About 18% of the 61 identified proteins appear to change expression levels with the cell growth conditions. Seven proteins appeared to be up-regulated and four proteins down-regulated when the cells were changed from proliferating to differentiating culture conditions. The majority of the apparent changes in protein expression levels were associated with stress response genes. Significant changes in the apparent mass and charge properties of proteins were also observed and for select proteins, the modifications appeared to be correlated with cell growth conditions. The results demonstrate that proteome differences in RPE-J cells associated with growth conditions can be identified and support the suitability of RPE-J cells for more targeted and/or more global proteome analysis of RPE differentiation.

Animals↗
PubMed2001

Paxillin-ARF GAP signaling and the cytoskeleton.

Several new families of ARF GTPase activating proteins (ARF GAPs) have been described recently that associate with paxillin and other cytoskeletal and signaling proteins. Important insights have been gained regarding their subcellular distribution, enzymatic specificity and protein scaffold function. Evidence suggests an important role for ARF GAPs in mediating changes in the cell's actin cytoskeleton in response to adhesion and growth factor stimulation.

ADP-Ribosylation Factors↗
PubMed2000

Identification of the vitamin K-dependent carboxylase active site: Cys-99 and Cys-450 are required for both epoxidation and carboxylation.

The vitamin K-dependent carboxylase modifies and renders active vitamin K-dependent proteins involved in hemostasis, cell growth control, and calcium homeostasis. Using a novel mechanism, the carboxylase transduces the free energy of vitamin K hydroquinone (KH(2)) oxygenation to convert glutamate into a carbanion intermediate, which subsequently attacks CO(2), generating the gamma-carboxylated glutamate product. How the carboxylase effects this conversion is poorly understood because the active site has not been identified. Dowd and colleagues [Dowd, P., Hershline, R., Ham, S. W. & Naganathan, S. (1995) Science 269, 1684-1691] have proposed that a weak base (cysteine) produces a strong base (oxygenated KH(2)) capable of generating the carbanion. To define the active site and test this model, we identified the amino acids that participate in these reactions. N-ethyl maleimide inhibited epoxidation and carboxylation, and both activities were equally protected by KH(2) preincubation. Amino acid analysis of (14)C- N-ethyl maleimide-modified human carboxylase revealed 1.8-2.3 reactive residues and a specific activity of 7 x 10(8) cpm/hr per mg. Tryptic digestion and liquid chromatography electrospray mass spectrometry identified Cys-99 and Cys-450 as active site residues. Mutation to serine reduced both epoxidation and carboxylation, to 0. 2% (Cys-99) or 1% (Cys-450), and increased the K(m)s for a glutamyl substrate 6- to 8-fold. Retention of some activity indicates a mechanism for enhancing cysteine/serine nucleophilicity, a property shared by many active site thiol enzymes. These studies, which represent a breakthrough in defining the carboxylase active site, suggest a revised model in which the glutamyl substrate indirectly coordinates at least one thiol, forming a catalytic complex that ionizes a thiol to initiate KH(2) oxygenation.

Amino Acid Sequence↗
PubMed1999

Pseudechetoxin: a peptide blocker of cyclic nucleotide-gated ion channels.

Ion channels activated by the binding of cyclic nucleotides first were discovered in retinal rods where they generate the cell's response to light. In other systems, however, it has been difficult to unambiguously determine whether cyclic nucleotide-dependent processes are mediated by protein kinases, their classical effector enzymes, or cyclic nucleotide-gated (CNG) ion channels. Part of this difficulty has been caused by the lack of specific pharmacological tools. Here we report the purification from the venom of the Australian King Brown snake of a peptide toxin that inhibits current through CNG channels. This toxin, which we have named Pseudechetoxin (PsTx), was purified by cation exchange and RP-HPLC and has a molecular mass of about 24 kDa. When applied to the extracellular face of membrane patches containing the alpha-subunit of the rat olfactory CNG channel, PsTx blocked the cGMP-dependent current with a Ki of 5 nM. Block was independent of voltage and required only a single molecule of toxin. PsTx also blocked CNG channels containing the bovine rod alpha-subunit with high affinity (100 nM), but it was less effective on the heteromeric version of the rod channel (Ki approximately 3 microM). We have obtained N-terminal and partial internal sequence data and the amino acid composition of PsTx. These data indicate that PsTx is a basic protein that exhibits some homology with helothermine, a toxin isolated from the venom of the Mexican beaded lizard. PsTx promises to be a valuable pharmacological tool for studies on the structure and physiology of CNG channels.

Amino Acids↗
PubMed1998

Cellular retinaldehyde-binding protein ligand interactions. Gln-210 and Lys-221 are in the retinoid binding pocket.

Cellular retinaldehyde-binding protein (CRALBP) carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the retinal pigment epithelium (RPE) and Müller cells of the retina and has been linked with autosomal recessive retinitis pigmentosa. Ligand interactions determine the physiological role of CRALBP in the RPE where the protein is thought to function as a substrate carrier for 11-cis-retinol dehydrogenase in the synthesis of 11-cis-retinal for visual pigment regeneration. However, CRALBP is also present in optic nerve and brain where its natural ligand and function are not yet known. We have characterized the interactions of retinoids with native bovine CRALBP, human recombinant CRALBP (rCRALBP) and five mutant rCRALBPs. Efforts to trap and/or identify a Schiff base in the dark, under a variety of reducing, denaturing, and pH conditions were unsuccessful, suggesting the lack of covalent interactions between CRALBP and retinoid. Buried and solvent-exposed lysine residues were identified in bovine CRALBP by reductive methylation of the holoprotein followed by denaturation and reaction with [3H]acetic anhydride. Radioactive lysine residues were identified by Edman degradation and electrospray mass spectrometry following proteolysis and purification of modified peptides. Human rCRALBP mutants K152A, K221A, and K294A were prepared to investigate possible retinoid interactions with buried or partially buried lysines. Two other rCRALBP mutants, I162V and Q210R, were also prepared to identify substitutions altering the retinoid binding properties of a random mutant. The structures of all the mutants were verified by amino acid and mass spectral analyses and retinoid binding properties evaluated by UV-visible and fluorescence spectroscopy. All of the mutants bound 11-cis-retinal essentially like the wild type protein, indicating that the proteins were not grossly misfolded. Three of the mutants bound 9-cis-retinal like the wild type protein; however, Q210R and K221A bound less than stoichiometric amounts of the 9-cis-isomer and exhibited lower affinity for this retinoid relative to wild type rCRALBP. Residues Gln-210 and Lys-221 are located within a region of CRALBP exhibiting sequence homology with the ligand binding cavity of yeast phosphatidylinositol-transfer protein. The data implicate Gln-210 and Lys-221 as components of the CRALBP retinoid binding cavity and are discussed in the context of ligand interactions in structurally or functionally related proteins with known crystallographic structures.

Amino Acid Sequence↗
PubMed1998

Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein.

Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.

Amino Acid Sequence↗
PubMed1998

Ratings of perceived exertion to determine intensity during outdoor running.

A paucity of data exists related to the usefulness of Ratings of Perceived Exertion (RPE) to set exercise intensity in non-laboratory settings. The purpose of this study was to determine if RPE could be used on an outdoor track to generate blood lactate and heart rate (HR) responses similar to those obtained on a treadmill (tm) run. Nine experienced runners (6 males, 3 females; VO2peak = 56.2 +/- 6.7 ml.kg-1.min-1) completed a horizontal, incremental tm test. HR, RPE, and lactate were measured for each stage. Subsequently, subjects ran for 30 min on an outdoor track at the RPE corresponding with 2.5 mM lactate during the tm run. Repeated measures ANOVA compared lactate and HR values at 2.5 mM lactate on the tm run and values obtained during the track run. Lactate during the track run was significantly higher (p < .05) than 2.5 mM throughout the 30 min (6.9 +/- 2.9, 6.3 +/- 2.9, and 5.8 +/- 3.0 mM at 10, 20, and 30 min, respectively). HR at 2.5 mM lactate during the tm run (173 +/- 6.1 bpm) was significantly lower (p < .05) than at min 10 and 20 of the track run (182.6 +/- 9.3 and 182.9 +/- 8.0 bpm, respectively) but not different from min 30 (181.3 +/- 10.6 bpm). In summary, it is difficult to generate specific physiological responses using RPE.

Adult↗
PubMed1997

Identification and characterization of glycosylation sites in human serum clusterin.

Clusterin is a ubiquitous, heterodimeric glycoprotein with multiple possible functions that are likely influenced by glycosylation. Identification of oligosaccharide attachment sites and structural characterization of oligosaccharides in human serum clusterin has been performed by mass spectrometry and Edman degradation. Matrix-assisted laser desorption ionization mass spectrometry revealed two molecular weight species of holoclusterin (58,505 +/- 250 and 63,507 +/- 200). Mass spectrometry also revealed molecular heterogeneity associated with both the alpha and beta subunits of clusterin, consistent with the presence of multiple glycoforms. The data indicate that clusterin contains 17-27% carbohydrate by weight, the alpha subunit contains 0-30% carbohydrate and the beta subunit contains 27-30% carbohydrate. Liquid chromatography electrospray mass spectrometry with stepped collision energy scanning was used to selectively identify and preparatively fractionate tryptic glycopeptides. Edman sequence analysis was then used to confirm the identities of the glycopeptides and to define the attachment sites within each peptide. A total of six N-linked glycosylation sites were identified, three in the alpha subunit (alpha 64N, alpha 81N, alpha 123N) and three in the beta subunit (beta 64N, beta 127N, and beta 147N). Seven different possible types of oligosaccharide structures were identified by mass including: a monosialobiantennary structure, bisialobiantennary structures without or with one fucose, trisialotriantennary structures without or with one fucose, and possibly a trisialotriantennary structure with two fucose and/or a tetrasialotriantennary structure. Site beta 64N exhibited the least glycosylation diversity, with two detected types of oligosaccharides, and site beta 147N exhibited the greatest diversity, with five or six detected types of oligosaccharides. Overall, the most abundant glycoforms detected were bisialobiantennary without fucose and the least abundant were monosialobiantennary, trisialotriantennary with two fucose and/or tetrasialotriantennary. Clusterin peptides accounting for 99% of the primary structure were identified from analysis of the isolated alpha and beta subunits, including all Ser- and Thr-containing peptides. No evidence was found for the presence of O-linked or sulfated oligosaccharides. The results provide a molecular basis for developing a better understanding of clusterin structure-function relationships and the role clusterin glycosylation plays in physiological function.

Amino Acid Sequence↗
PubMed1997

Changes in plasma tryptophan/branched chain amino acid ratio in responses to training volume variation.

The major symptoms of overtraining including decreased exercise performance, altered mood states, and depleted muscle glycogen stores closely resemble the effects of brain serotonin, the level of which is dependent on the plasma ratio of tryptophan to branched-chain amino acids (BCAA). To examine the relation between plasma amino acids and overtraining, ten highly-trained endurance runners underwent two weeks of base training (normal training) before increasing their training volume by 40% for two weeks to achieve a state of short-term overtraining (or overreaching). The overtraining period was followed by two weeks of recovery in which training volume was reduced by 41% of the base training. For the whole group, no significant changes were observed in running economy and maximum oxygen uptake. There were no changes in resting heart rate, blood pressure, resting metabolic rate, and serum cortisol level in response to the changes in training volume. The runners experienced a significant increase (p < 0.05) in fatigue score for the profile of mood states when the training volume was increased. The elevated fatigue score returned to baseline when the training volume was reduced. Plasma free or total tryptophan, BCAA, and the tryptophan/BCAA ratio were not significantly altered throughout the course of this study. We concluded that proposed physiological markers of overtraining, including plasma tryptophan and BCAA levels, were unchanged despite a 40% increase in training volume.

Adult↗
PubMed1995

Differential expression of HLA-DQB1 alleles in a heterozygous cell line.

Regulation of HLA class II transcription is complex, with both locus-specific and allele-specific polymorphisms associated with consensus regulatory region promoter elements. In order to evaluate the potential function of allele-specific elements, HLA-DQB1 transcripts and in vivo footprinting of the DQB1 promoter were studied in human cell lines inducible for HLA class II expression. In the heterozygous melanoma cell line Me9229/18, differential expression was observed for two DQB1 alleles. This variation was reflected in both steady-state and inducible transcripts, although methylation patterns of the promoter elements were similar for both alleles. This in vivo allelic difference expression, which correlates with previous studies of in vitro reporter gene transcription, indicates the potential for functionally important differences in tissue-specific and inducible expression attributable to promoter region polymorphism in HLA genes.

Alleles↗