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Biomedical subjects

A Woods

Publications and source records attributed to A Woods.

At least 55 records · Page 3Linked to original sources
PubMed1999

Homogeneous proximity tyrosine kinase assays: scintillation proximity assay versus homogeneous time-resolved fluorescence.

Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described.

Amino Acid Sequence↗
PubMed1999

Special-interest subgroups at the ASCB: Proteoglycans and cell adhesion.

The annual meeting of the American Society for Cell Biology (ASCB) is a large and diverse gathering. At last year's meeting**The American Society for Cell Biology 38th Annual Meeting, San Francisco, USA; 12-16 December, 1998. Program chair: Jennifer Lippincott-Schwartz., there were over 8000 attendees, and the topics discussed covered many areas of cell biology. It would be impossible to cover the entire meeting within a trends in CELL BIOLOGY report, so instead we are focusing on an aspect of it that provided some of the most interesting and fruitful discussions. On Saturday afternoon, before the main symposia began, there were 11 special-interest subgroup meetings. The atmosphere at these meetings was informal, and they encouraged open and frank discussion of data and issues. This report provides a brief summary of the discussions at seven of the special-interest subgroup meetings.

Journal Article↗
PubMed1999

Duration of apnoea with two different intubating doses of remifentanil.

We have studied the apnoea time after induction with two successful drug intubating regimes, both containing remifentanil. Group 1 (n = 20); propofol 2 mg kg-1 and remifentanil 2 micrograms kg-1 and group 2 (n = 20); propofol 2 mg kg-1, remifentanil 1 microgram kg-1 and lignocaine 1 mg kg-1. Intubation was possible in all 40 patients, and regarded as acceptable in 90% and 85% of patients, respectively. The median time to the return of spontaneous respiration was 487 s in group 1 and 270 s in group 2 (P < 0.05). Median end-tidal CO2 concentration at the time of the first spontaneous respiration was 7.37 kPa in group 1 and 6.3 kPa in group 2 (P < 0.05). Both groups had a decrease in heart rate after induction, but this failed to reach either clinical or statistical significance, and no patient required atropine. Similarly, there was a decrease in arterial pressure after induction in both groups (P < 0.05), but this was not deemed to be clinically significant. Only two patients in each group required ephedrine 6 mg before arterial pressure was restored to within 25% of the base-line. Intubating conditions were similar in both groups, but group 2 provided a significantly shorter apnoea time.

Adolescent↗
PubMed1999

The AMP-activated protein kinase is involved in the regulation of ketone body production by astrocytes.

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.

Aminoimidazole Carboxamide↗
PubMed1999

Syndecan-4 and integrins: combinatorial signaling in cell adhesion.

It is now becoming clear that additional transmembrane components can modify integrin-mediated adhesion. Syndecan-4 is a transmembrane heparan sulfate proteoglycan whose external glycosaminoglycan chains can bind extracellular matrix ligands and whose core protein cytoplasmic domain can signal during adhesion. Two papers in this issue of JCS demonstrate, through transfection studies, that syndecan-4 plays roles in the formation of focal adhesions and stress fibers. Overexpression of syndecan-4 increases focal adhesion formation, whereas a partially truncated core protein that lacks the binding site for protein kinase C(&agr;) and phosphatidylinositol 4, 5-bisphosphate acts as a dominant negative inhibitor of focal adhesion formation. Focal adhesion induction does not require interaction between heparan sulfate glycosaminoglycan and ligand but can occur when non-glycanated core protein is overexpressed; this suggests that oligomerization of syndecan-4 plays a major role in signaling from the extracellular matrix in adhesion.

Amino Acid Sequence↗
PubMed1999

Control of morphology, cytoskeleton and migration by syndecan-4.

Syndecan-4 is a widely expressed transmembrane heparan sulfate proteoglycan which localizes to focal adhesions. Previous studies showed that the syndecan-4 cytoplasmic domain can associate with and potentiate the activity of protein kinase C, which is required for focal adhesion formation. To examine further the role of syndecan-4 in cell adhesion, we expressed syndecan-4 cDNA constructs in CHO-K1 cells. Syndecan-2 transfection was used to confirm effects seen were specific for syndecan-4. Cells overexpressing full length syndecan-4 core protein exhibited a more flattened, fibroblastic morphology, with increased focal adhesion formation and decreased cell motility. Expression of a syndecan-4 core protein with either a partial or complete deletion of the cytoplasmic domain or of an antisense construct led to markedly decreased spreading and focal adhesion formation, a more epithelioid morphology, and decreased motility. Overexpression of syndecan-2 changed the adhesive phenotype, but did not markedly alter focal adhesion and microfilament bundle formation. The data suggest that syndecan-4 is a regulator of focal adhesion and stress fiber formation, and influences both morphology and migration.

Amino Acid Sequence↗
PubMed1998

Disruption of the talin gene compromises focal adhesion assembly in undifferentiated but not differentiated embryonic stem cells.

We have used gene disruption to isolate two talin (-/-) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of beta1 integrin, although levels of alpha5 and alphaV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (-/-) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (-/-) ES cells were able to assemble talin-containing focal adhesions. Both talin (-/-) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (-/-) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the beta1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for beta1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

Actins↗
PubMed1998

AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform.

Mammalian AMP-activated protein kinase (AMPK) is the downstream component of a cascade that is activated by cellular stresses associated with ATP depletion. AMPK exists as heterotrimeric alphabetagamma complexes, where the catalytic subunit has two isoforms (alpha1 and alpha2) with different tissue distributions. The budding yeast homologue is the SNF1 kinase complex, which is essential for derepression of glucose-repressed genes, and seems to act by the direct phosphorylation of transcription factors in the nucleus. AMPK complexes containing the alpha2 rather than the alpha1 isoform have a greater dependence on AMP (approx. 5-fold stimulation compared with approx. 2-fold) both in direct allosteric activation and in reactivation by the upstream kinase. We have also examined their subcellular localization by using Western blotting of nuclear preparations, and by using two detection methods in the confocal microscope, i.e. indirect immunofluorescence of endogenous proteins and transfection of DNA species encoding green fluorescent protein-alpha-subunit fusions. By all three methods a significant proportion of alpha2, but not alpha1, is localized in the nucleus. Like SNF1, AMPK-alpha2 complexes could therefore be involved in the direct regulation of gene expression. The observed differences in the regulation of alpha1 and alpha2 complexes by AMP might result in differential responses to ATP depletion in distinct cellular and subcellular locations.

AMP-Activated Protein Kinases↗
PubMed1998

Solution structure of a syndecan-4 cytoplasmic domain and its interaction with phosphatidylinositol 4,5-bisphosphate.

Syndecan-4, a transmembrane heparan sulfate proteoglycan, is a coreceptor with integrins in cell adhesion. It has been suggested to form a ternary signaling complex with protein kinase Calpha and phosphatidylinositol 4,5-bisphosphate (PIP2). Syndecans each have a unique, central, and variable (V) region in their cytoplasmic domains, and that of syndecan-4 is critical to its interaction with protein kinase C and PIP2. Two oligopeptides corresponding to the variable region (4V) and whole domain (4L) of syndecan-4 cytoplasmic domain were synthesized for nuclear magnetic resonance (NMR) studies. Data from NMR and circular dichroism indicate that the cytoplasmic domain undergoes a conformational transition and forms a symmetric dimer in the presence of phospholipid activator PIP2. The solution conformations of both free and PIP2-complexed 4V have been determined by two-dimensional NMR spectroscopy and dynamical simulated annealing calculations. The 4V peptide in the presence of PIP2 formed a compact dimer with two twisted strands packed parallel to each other and the exposed surface of the dimer consisted of highly charged and polar residues. The overall three-dimensional structure in solution exhibits a twisted clamp shape having a cavity in the center of dimeric interface. In addition, it has been observed that the syndecan-4V strongly interacts not only with fatty acyl groups but also the anionic head group of PIP2. These findings reveal that PIP2 promotes oligomerization of syndecan-4 cytoplasmic domain for transmembrane signaling and cell-matrix adhesion.

Amino Acid Sequence↗
PubMed1998

Syndecan-4 proteoglycan cytoplasmic domain and phosphatidylinositol 4,5-bisphosphate coordinately regulate protein kinase C activity.

Phosphatidylinositol 4,5-bisphosphate (PIP2) is involved in the organization of the actin cytoskeleton by regulating actin-associated proteins. The transmembrane heparan sulfate proteoglycan syndecan-4 also plays a critical role in protein kinase C (PKC) signaling in the formation of focal adhesions and actin stress fibers. The cytoplasmic domain of syndecan-4 core protein directly interacts with and potentiates PKCalpha activity, and it can directly interact with the phos- phoinositide PIP2. We, therefore, investigated whether the interaction of inositol phosphates and inositol phospholipids with syndecan-4 could regulate PKC activity. Data from in vitro kinase assays using purified PKCalpha beta gamma show that in the absence of phosphatidylserine and diolein, PIP2 increased the extent of autophosphorylation of PKCalpha beta gamma and partially activated it to phosphorylate both histone III-S and an epidermal growth factor receptor peptide. This activity was dose-dependent, and its calcium dependence varied with PKC isotype/source. Addition of the cytoplasmic syndecan-4 peptide, but not equivalent syndecan-1 or syndecan-2 peptides, potentiated the partial activation of PKCalpha beta gamma by PIP2, resulting in activity greater than that observed with phosphatidylserine, diolein, and calcium. This study indicates that syndecan-4 cytoplasmic domain may bind both PIP2 and PKCalpha, localize them to forming focal adhesions, and potentiate PKCalpha activity there.

Amino Acid Sequence↗
PubMed1998

Induction of autoimmune arthritis in HLA-DR4 (DRB1*0401) transgenic mice by immunization with human and bovine type II collagen.

Although associations between the expression of particular HLA genes and the susceptibility to specific autoimmune diseases has been known for some time, the role that these HLA molecules play in the autoimmune response is unclear. Through the establishment of a chimeric HLA-DR/I-E transgene, we have examined the function of the rheumatoid arthritis (RA) susceptibility allele HLA-DR4 (DRB1*0401) in presenting antigenic peptides derived from the model Ag, type II collagen (CII), and in mediating an autoimmune response. As a transgene, the chimeric DR4 molecule conferred susceptibility to an autoimmune arthritis induced by immunization with human CII or bovine CII. These mice developed an inflammatory, autoimmune arthritis that was similar both histologically and in severity to that previously described for the collagen-induced arthritis model. The DR4-mediated autoimmune arthritis was accompanied by T cell and B cell responses to both the immunogen and the autoantigen, murine CII. The DR4-restricted T cell response to human CII was focused on an immunodominant determinant within CII263-270 and a minor determinant within CII286-300, the same CII determinants recently identified for yet another RA susceptibility allele, HLA-DR1 (DRB1*0101). Thus these data demonstrate that, like HLA-DR1, HLA-DR4 is capable of binding peptides derived from human CII and therefore probably plays a role in the autoimmune response to human CII observed in RA patients.

Amino Acid Sequence↗
PubMed1998

Syndecan proteoglycans and cell adhesion.

It is now becoming clear that a family of transmembrane proteoglycans, the syndecans, have important roles in cell adhesion. They participate through binding of matrix ligand to their glycosaminoglycan chains, clustering, and the induction of signaling cascades to modify the internal microfilament organization. Syndecans can modulate the type of adhesive responses induced by other matrix ligand-receptor interactions, such as those involving the integrins, and so contribute to the control of cell morphology, adhesion and migration.

Amino Acid Sequence↗
PubMed1998

Syndecans: synergistic activators of cell adhesion.

Cell-surface proteoglycans participate in cell adhesion, growth-factor signalling, lipase activity and anticoagulation. Until recently, only the roles of the glycosaminoglycan chains were investigated. Now, with molecular characterization of several core proteins, the roles of each individual proteoglycan species in cellular signalling pathways are being determined. This review describes some of the recent advances in our understanding of the major transmembrane group of heparan sulfate proteoglycans, the syndecans, including evidence that they play an important role as accessory signalling molecules modulating integrin-based adhesion.

Amino Acid Sequence↗
PubMed1998

Insulin-like growth factor-I and bone mineral density.

To assess the relationship between insulin-like growth factor-I (IGF-I) and bone mineral density (BMD) 201 healthy postmenopausal women (age 41-68 years) within 10 years of menopause were studied. In all subjects, BMD at the lumbar spine and left hip were measured using dual-energy X-ray absorptiometry and blood samples were obtained. In all subjects, serum IGF-I and parathyroid hormone (PTH) were measured. In a subgroup of these subjects serum concentrations of IGF-binding protein-3 (IGFBP-3), osteocalcin (OC), bone-specific alkaline phosphatase (BALP), tartrate-resistant acid phosphatase (TRAP), and carboxyterminal propeptide of type I procollagen (PICP) were also measured. Serum IGF-I correlated significantly with age (r = -0.159, p = 0.0241), serum OC (r = 0.226, p = 0.0131), BALP (r = 0.259, p < 0.0001), and TRAP (r = 0.261, p < 0.0015), but not with PICP, PTH, or BMD at any site. Although there was a strong correlation between IGF-I and IGFBP-3 (r = 0.559, p < 0.0001), there was no correlation between IGFBP-3 and any of the markers of bone turnover (OC, BALP, TRAP, or PICP) nor with PTH or BMD at any site. We conclude that IGF-I and markers of bone turnover are related, but there is no relationship between IGF-I and BMD.

Absorptiometry, Photon↗