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Biomedical subjects

A Hall

Publications and source records attributed to A Hall.

At least 163 records · Page 9Linked to original sources
PubMed1997

Rho, Rac and Cdc42 GTPases regulate the organization of the actin cytoskeleton.

Rho, Rac and Cdc42 are three Ras-related GTP-binding proteins that control the assembly and disassembly of the actin cytoskeleton in response to extracellular signals. During the past year, numerous candidate downstream targets for these GTPases have been identified using affinity chromatography and yeast two-hybrid techniques. These techniques have revealed that Rho regulates the myosin light chain phosphatase and that Rho and Rac control the synthesis of phosphatidylinositol 4,5-bisphosphate, two activities that might help to explain the effects of these GTPases on the actin cytoskeleton.

Actins↗
PubMed1997

The relative potencies of dendrotoxins as blockers of the cloned voltage-gated K+ channel, mKv1.1 (MK-1), when stably expressed in Chinese hamster ovary cells.

1. The mKv1.1 voltage-gated K+ channel has been expressed stably in Chinese hamster ovary cells and whole-cell currents recorded by the patch-clamp method. 2. A range of structurally related peptide toxins (dendrotoxins) from the venom of green mamba (Dendroaspis angusticeps) and black mamba (Dendroaspis polylepis polylepis) snakes were tested for mKv1.1 channel blocking activity. Their potencies were compared based on EC50s derived from their respective concentration-inhibition relationships. 3. The rank order of potency, thus determined was: Toxin K > 7-dendrotoxin(7-Dtx) > delta-Dtx > Toxin I = alpha-Dtx > beta-Dtx. 4. Block was independent of voltage and no effects of the toxins on the kinetics of activation were observed. These results are consistent with a mechanism involving the block of closed channels. 5. A wide range of activity was observed even between toxins with an extremely high degree of sequence homology. Toxin K, in particular was an exquisitely potent blocker of the mKv1.1 channel, having an EC50 of 30 pM compared with 1.8 nM for delta-Dtx in spite of 95% sequence identity.

Animals↗
PubMed1997

The reliability of self-reported blood in urine and schistosomiasis as indicators of Schistosoma haematobium infection in school children: a study in Muheza District, Tanzania.

The use of self-reported blood in urine and schistosomiasis by school children was investigated as a tool to estimate the prevalence of infection with Schistosoma haematobium and to identify infected individuals. A general questionnaire about common health problems, including questions about blood in urine and schistosomiasis, was administered by teachers to 25443 children in 137 primary schools in Muheza District, Tanzania. The prevalence of reported schistosomiasis was calculated for each school and used to select 15 schools across a range in prevalence. All children in the 15 schools (n = 2370) were interviewed again by a nurse and gave a urine sample which was subjected to a quantitative microscopical examination for the eggs of S. haematobium by filtration. The prevalence of reported schistosomiasis by the interview in the 15 schools correlated strongly with the prevalence reported during the questionnaire survey. The prevalence of reported schistosomiasis in the interview was strongly correlated with the prevalence of infection determined by microscopy and consistently under-estimated the latter by around 20% across a range in prevalence from 22% to 93%. The sensitivity of diagnosis by an interview increased almost linearly with the prevalence of infection, so that when the prevalence was high, more infected children reported schistosomiasis. The percentage of children who were correct in their self-diagnosis was independent of the prevalence of infection and of the mean concentration of eggs in urine, and averaged 75%. These findings suggest that self-reported schistosomiasis is a useful method to estimate the prevalence of infection in schools and might be used to identify infected individuals.

Adolescent↗
PubMed1997

Tyrosine hydroxylase phosphorylation in digitonin-permeabilized bovine adrenal chromaffin cells: the effect of protein kinase and phosphatase inhibitors on Ser19 and Ser40 phosphorylation.

The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [gamma-32P]ATP, in the presence or absence of 10 microM Ca2+, 1 microM cyclic AMP, 1 microM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273-302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5-22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19-31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.

Adrenal Glands↗
PubMed1997

Modification of cystatin C activity by bacterial proteinases and neutrophil elastase in periodontitis.

AIM: To study the interaction between the human cysteine proteinase inhibitor, cystatin C, and proteinases of periodontitis associated bacteria. METHODS: Gingival crevicular fluid samples were collected from discrete periodontitis sites and their cystatin C content was estimated by enzyme linked immunosorbent assay (ELISA). The interaction between cystatin C and proteolytic enzymes from cultured strains of the gingival bacteria Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans was studied by measuring inhibition of enzyme activity against peptidyl substrates, by detection of break down patterns of solid phase coupled and soluble cystatin C, and by N-terminal sequence analysis of cystatin C products resulting from the interactions. RESULTS: Gingival crevicular fluid contained cystatin C at a concentration of approximately 15 nM. Cystatin C did not inhibit the principal thiol stimulated proteinase activity of P gingivalis. Instead, strains of P gingivalis and P intermedia, but not A actinomycetemcomitans, released cystatin C modifying proteinases. Extracts of five P gingivalis and five P intermedia strains all hydrolysed bonds in the N-terminal region of cystatin C at physiological pH values. The modified cystatin C resulting from incubation with one P gingivalis strain was isolated and found to lack the eight most N-terminal residues. The affinity of the modified inhibitor for cathepsin B was 20-fold lower (Ki 5 nM) than that of full length cystatin C. A 50 kDa thiol stimulated proteinase, gingipain R, was isolated from P gingivalis and shown to be responsible for the Arg8-bond hydrolysis in cystatin C. The cathepsin B inhibitory activity of cystatin C incubated with gingival crevicular fluid was rapidly abolished after Val10-bond cleavage by elastase from exudate neutrophils, but cleavage at the gingipain specific Arg8-bond was also demonstrated. CONCLUSIONS: The physiological control of cathepsin B activity is impeded in periodontitis, owing to the release of proteinases from infecting P gingivalis and neutrophils, with a contribution to the tissue destruction seen in periodontitis as a probable consequence.

Bacteria↗
PubMed1996

Faciogenital dysplasia protein (FGD1) and Vav, two related proteins required for normal embryonic development, are upstream regulators of Rho GTPases.

BACKGROUND: Dbl, a guanine nucleotide exchange factor (GEF) for members of the Rho family of small GTPases, is the prototype of a family of 15 related proteins. The majority of proteins that contain a DH (Dbl homology) domain were isolated as oncogenes in transfection assays, but two members of the DH family, FGD1 (the product of the faciogenital dysplasia or Aarskog-Scott syndrome locus) and Vav, have been shown to be essential for normal embryonic development. Mutations to the FGD1 gene result in a human developmental disorder affecting specific skeletal structures, including elements of the face, cervical vertebrae and distal extremities. Homozygous Vav-/- knockout mice embryos are not viable past the blastocyst stage, indicating an essential role of Vav in embryonic implantation. RESULTS: Here, we show that the microinjection of FGD1 and Vav into Swiss 3T3 fibroblasts induces the polymerization of actin and the assembly of clustered integrin complexes. FGD1 activates Cdc42, whereas Vav activates Rho, Rac and Cdc42. In addition, FGD1 and Vav stimulate the mitogen activated protein kinase cascade that leads to activation of the c-Jun kinase SAPK/JNK1. CONCLUSIONS: We conclude that FGD1 and Vav are regulators of the Rho GTPase family. Along with their target proteins Cdc42, Rac and Rho, FGD1 and Vav control essential signals required during embryonic development.

3T3 Cells↗
PubMed1996

Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.

Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.

3T3 Cells↗
PubMed1996

Phosphatidylinositol 3-kinase signals activate a selective subset of Rac/Rho-dependent effector pathways.

BACKGROUND: Phosphatidylinositol 3'-hydroxyl kinase (PI 3-kinase) is activated by many growth factor receptors and is thought to exert its cellular functions through the elevation of phosphatidylinositol (3,4,5)-triphosphate levels in the cell. PI 3-kinase is required for growth-factor induced changes of the actin cytoskeleton which are mediated by the GTPases Rac and Rho. Recently, a role for Rac and Rho in regulating gene transcription has become evident. RESULTS: Here, we show that membrane targeting of the p110 catalytic subunit, but not the p85 regulatory subunit, of PI 3-kinase generates a constitutively active enzyme that allows us to assess the relative contribution of PI 3-kinase activation to a particular cellular response. Expression of this active PI 3-kinase induced actin reorganization in the form of Rac-mediated lamellipodia and focal complexes, and Rho-mediated stress fibres and focal adhesions. However, expression of active PI 3-kinase did not induce the Ras/Rac/Rho signalling pathways that regulate gene transcription controlled by the c-fos promoter, the c-fos serum response element or the transcription factors Elk-1 and AP-1. CONCLUSIONS: Our results demonstrate that PI 3-kinase induces a selective subset of cellular responses, but is not sufficient to stimulate the full repertoire of Rac- or Rho-mediated responses.

3T3 Cells↗
PubMed1996

Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease.

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.

Biological Transport↗
PubMed1996

Sam68 from an immortalised B-cell line associates with a subset of SH3 domains.

The binding of proteins from an immortalised B-cell line to a panel of SH3 domains was investigated in vitro. One of the most prominent SH3 domain binding proteins was a 68 kD polypeptide which strongly associated with the SH3 domains of c-src, p85a and p47phox and weakly with the SH3 domain of PLCgamma and n-src with undetectable binding to the other SH3 domains tested. Immunoblotting identified this protein as human Sam68. The ability of proline-rich peptides homologous to the Sam68 primary sequence to inhibit the binding of Sam68 to SH3 domains was investigated. Only one peptide inhibited binding of Sam68 to the p85alpha SH3 domain, whereas several peptides inhibited binding of Sam68 to c-src SH3 domain, suggesting that Sam68 uses different proline-rich motifs to bind to different SH3 domains. A peptide derived from residues 32-44 of Sam68 which fits the class II SH3 domain binding consensus sequence inhibited binding of Sam68 to both p85alpha SH3 domain and c-src SH3 domain, but with differential potency, suggesting a differential affinity of these SH3 domains for this proline-rich motif.

Amino Acid Sequence↗
PubMed1996

Tyrosine hydroxylase phosphorylation in bovine adrenal chromaffin cells. Clonidine stimulates basal but inhibits nicotinic receptor evoked phosphorylation.

Clonidine inhibited the uptake of calcium and the overall phosphorylation of tyrosine hydroxylase induced by nicotinic receptor activation in bovine adrenal medullary chromaffin cells in culture. However, clonidine did not inhibit the increase in these parameters that accompanied K+ depolarisation of the cells. There was also no effect of clonidine on the overall phosphorylation of tyrosine hydroxylase when cells were stimulated by muscarine. Nicotinic receptor activation increased the phosphorylation of Ser-19, Ser-31, and Ser-40 on tyrosine hydroxylase, and this was inhibited by clonidine in a concentration-dependent manner. On the other hand, clonidine had no effect on calcium uptake, yet increased the phosphorylation of Ser-19 under basal conditions. Using calcium and calmodulin-stimulated protein kinase II obtained from rat brain clonidine increased the autophosphorylation of the alpha-subunit of the kinase by 37%, and also its activity against an exogenous peptide substrate by 29%. These data are consistent with the hypothesis that clonidine inhibits nicotinic receptor-induced tyrosine hydroxylase phosphorylation by decreasing calcium influx into chromaffin cells, perhaps by an action at the nicotinic receptor. Clonidine also increases the basal phosphorylation of tyrosine hydroxylase at Ser-19, perhaps by directly activating calcium and calmodulin-stimulated protein kinase II.

Adrenal Medulla↗
PubMed1996

Role of guanine nucleotide-binding proteins--ras-family or trimeric proteins or both--in Ca2+ sensitization of smooth muscle.

The purpose of this study was to identify guanine nucleotide-binding proteins (G proteins) involved in the agonist- and guanosine 5'-[gamma-thio]triphosphate (GTP[gamma-S])-induced increase in the Ca2+ sensitivity of 20-kDa myosin light chain (MLC20) phosphorylation and contraction in smooth muscle. A constitutively active, recombinant val14p21rhoA.GTP expressed in the baculovirus/Sf9 system, but not the protein expressed without posttranslational modification in Escherichia coli, induced at constant Ca2+ (pCa 6.4) a slow contraction associated with increased MLC20 phosphorylation from 19.8% to 29.5% (P < 0.05) in smooth muscle permeabilized with beta-esein. The effect of val14p21rhoA.GTP was inhibited by ADP-ribosylation of the protein and was absent in smooth muscle extensively permeabilized with Triton X-100. ADP-ribosylation of endogenous p21rho with epidermal cell differentiation inhibitor (EDIN) inhibited Ca2+ sensitization induced by GTP [in rabbit mesenteric artery (RMA) and rabbit ileum smooth muscles], by carbachol (in rabbit ileum), and by endothelin (in RMA), but not by phenylephrine (in RMA), and only slowed the rate without reducing the amplitude of contractions induced in RMA by 1 microM GTP[gamma-S] at constant Ca2+ concentrations. AlF(4-)-induced Ca2+ sensitization was inhibited by both guanosine 5'-[beta-thio]diphosphate (GDP[beta-S]) and by EDIN. EDIN also inhibited, to a lesser extent, contractions induced by Ca2+ alone (pCa 6.4) in both RMA and rabbit ileum. ADP-ribosylation of trimeric G proteins with pertussis toxin did not inhibit Ca2+ sensitization. We conclude that p21rho may play a role in physiological Ca2+ sensitization as a cofactor with other messengers, rather than as a sole direct inhibitor of smooth muscle MLC20 phosphatase.

Animals↗
PubMed1996

Two GTPases, Cdc42 and Rac, bind directly to a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome.

BACKGROUND: Members of the Rho family of small GTPases play an essential role in controlling the motile behaviour of animal cells. Specifically, Cdc42 and Rac have been shown to induce the formation of filopodia and lamellipodia, respectively, at the cell periphery of Swiss 3T3 fibroblasts. In addition, both GTPases are required for progression through G1 phase of the cell cycle, possibly by regulating the activity of the Jun N-terminal kinase (JNK) signalling pathway. In order to examine more closely the mechanisms underlying the diverse functions of Rho GTPases in mammalian cells, we searched for downstream targets of these proteins. RESULTS: A yeast two-hybrid screen for proteins interacting with the human Cdc42 GTPase identified WASP, a protein implicated in the immunodeficiency disorder Wiskott-Aldrich syndrome (WAS). Recombinant WASP, expressed in Escherichia coli, also bound to Cdc42 and weakly to Rac, but not at all to Rho. The Cdc42/Rac-binding domain was identified in a region between amino acids 201-321 of WASP, and binding was dependent on Cdc42 and Rac being in the GTP-bound conformation. Furthermore, WASP did not catalyze GTPase activation or nucleotide exchange activity on Cdc42. CONCLUSIONS: Positional cloning has implicated WASP in causing WAS, and the protein is defective in patients suffering from the disease. WASP is expressed exclusively in cells of hematopoietic lineage, and lymphocytes from WAS patients have a distorted cell-surface and exhibit reduced proliferative capacity. WASP has recently been found to bind to the Src-homology 3 (SH3) domain of the adapter protein Nck. This observation, and the results presented here, suggest that WAS is the result of defects in signal transduction pathways regulated by Cdc42/Rac and Nck.

Amino Acid Sequence↗
PubMed1996

Rho: a connection between membrane receptor signalling and the cytoskeleton.

The Rho family of GTP-binding proteins has yielded fresh insights into cell signalling in relation to motility, shape and the control of the actin cytoskeleton. Rho itself is probably near the top of several diverse signalling cascades and has been implicated in cell adhesion, actin filament organization, control of mitogen-activated protein kinase pathways and phospholipid synthesis and turnover. As a member of the Ras superfamily, Rho is regulated by GDP-GTP exchange factors (GEFs) that have homology to the dbl oncogene, and by GTPase-activating proteins (GAPs). These proteins regulate the nucleotide (GDP or GTP) bound to Rho, thus determining the activity of Rho and the interactions of Rho with many of its downstream targets. In the past year, many new targets of Rho have been identified, which hopefully will uncover molecular connections among the diverse downstream effects of Rho activation.

Journal Article↗