Purification and assay of kinases that interact with Rac/Cdc42.
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Animals↗
Biomedical subjects
L Lim
Publications and source records attributed to L Lim.
Rapid identification of ApoE alleles by multiple-single-strand conformation polymorphism (SSCP) analysis.
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Alleles↗
Immunohistochemical and ultrastructural evidence of a modified microvasculature in the giant cell granuloma of the jaws.
A panel of five immunohistochemical markers, MB1, leukocyte common antigen, S-100 protein, smooth muscle specific actin, and factor VIII related antigen, were used to study 20 giant cell lesions. These included eight central giant cell granulomas, nine peripheral giant cell granulomas, and three giant cell tumors of bone. The multinucleated giant cells stained positively with MB1, the mononuclear round cells were positive to leukocyte common antigen and the spindle cells were unreactive to all the markers chosen in all the lesions. The most interesting finding was the staining pattern of the blood vessels to factor VIII related antigen in the giant cell granuloma. The blood vessels on the periphery of the lesions were strongly positive for this antibody. However, reaction product was not evident deeper in the lesion within the aggregations of giant cells. Two other endothelial cell markers, Ulex europaeus 1 lectin and QBend 10 were used to study 10 giant cell lesions and a similar pattern of staining was observed. Transmission electron microscopy was subsequently used to study the ultrastructure of the microvasculature of three peripheral giant cell granulomas, and the findings indicated that the reasons for the differential staining may lie in the differences in the structure of the microcirculation.
Actins↗
Patient assessment skills: assessing early cues of necrotizing enterocolitis.
A study is presented utilizing the critical decision method to elicit knowledge from neonatal intensive care unit nurses regarding the assessment of infants at risk for necrotizing enterocolitis. Researchers were successful in eliciting knowledge not available in current nursing texts, including a framework for considering necrotizing enterocolitis and problem-solving strategies used by experienced nurses. Implications for nurse instruction and potential applications of this information are discussed.
Cues↗
A randomised controlled trial of community based counselling among those discharged from hospital with ischaemic heart disease.
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Aged↗
Pheromone signalling in Saccharomyces cerevisiae requires the small GTP-binding protein Cdc42p and its activator CDC24.
Pheromone signalling in Saccharomyces cerevisiae is mediated by the STE4-STE18 G-protein beta gamma subunits. A possible target for the subunits is Ste20p, whose structural homolog, the serine/threonine kinase PAK, is activated by GTP-binding p21s Cdc42 and Rac1. The putative Cdc42p-binding domain of Ste20p, expressed as a fusion protein, binds human and yeast GTP-binding Cdc42p. Cdc42p is required for alpha-factor-induced activation of FUS1.cdc24ts strains defective for Cdc42p GDP/GTP exchange show no pheromone induction at restrictive temperatures but are partially rescued by overexpression of Cdc42p, which is potentiated by Cdc42p12V mutants. Epistatic analysis indicates that CDC24 and CDC42 lie between STE4 and STE20 in the pathway. The two-hybrid system revealed that Ste4p interacts with Cdc24p. We propose that Cdc42p plays a pivotal role both in polarization of the cytoskeleton and in pheromone signalling.
Amino Acid Sequence↗
The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts.
The Ras-related protein Cdc42 plays a role in yeast cell budding and polarity. Two related proteins, Rac1 and RhoA, promote formation in mammalian cells of membrane ruffles and stress fibers, respectively, which contain actin microfilaments. We now show that microinjection of the related human Cdc42Hs into Swiss 3T3 fibroblasts induced the formation of peripheral actin microspikes, determined by staining with phalloidin. A proportion of these microspikes was found to be components of filopodia, as analyzed by time-lapse phase-contrast microscopy. The formation of filopodia was also found to be promoted by Cdc42Hs microinjection. This was followed by activation of Rac1-mediated membrane ruffling. Treatment with bradykinin also promoted formation of microspikes and filopodia as well as subsequent effects similar to that seen upon Cdc42Hs microinjection. These effects of bradykinin were specifically inhibited by prior microinjection of dominant negative Cdc42HsT17N, suggesting that bradykinin acts by activating cellular Cdc42Hs. Since filopodia have been ascribed an important sensory function in fibroblasts and are required for guidance of neuronal growth cones, these results indicate that Cdc42Hs plays an important role in determining mammalian cell morphology.
Actin Cytoskeleton↗
A dominant inhibitory version of the small GTP-binding protein Rac disrupts cytoskeletal structures and inhibits developmental cell shape changes in Drosophila.
The Rho subfamily of Ras-related small GTP-binding proteins is involved in regulation of the cytoskeleton. The cytoskeletal changes induced by two members of this subfamily, Rho and Rac, in response to growth factor stimulation, have dramatic effects on cell morphology. We are interested in using Drosophila as a system for studying how such effects participate in development. We have identified two Drosophila genes, DRacA and DRacB, encoding proteins with homology to mammalian Rac1 and Rac2. We have made transgenic flies bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure which are due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin thought to be driving epidermal cell shape change. Thus the Rho subfamily may be generating localized changes in the cytoskeleton during Drosophila development in a similar fashion to that seen in mammalian and yeast cells. The Rho subfamily is likely to be participating in a wide range of developmental processes in Drosophila through its regulation of the cytoskeleton.
Actins↗
The Caenorhabditis elegans small GTP-binding protein RhoA is enriched in the nerve ring and sensory neurons during larval development.
p21 Ras has been implicated in vulval differentiation in Caenorhabditis elegans. We now describe the characteristics during nematode development of the related p21 RhoA which has been ascribed a morphological role in mammals. The CeRhoA cDNA isolated in this study encodes a sequence of 192 amino acids residues with 87.6% identity to human RhoA. Genomic Southern analysis indicates the presence of a single Rho gene in C. elegans. Its 2-kilobase mRNA is expressed at the highest levels during embryogenesis and decreases gradually thereafter. However, the level of the 24-kDa protein detected by the anti-CeRhoA antibody is high at the larval stages but low in embryos. The glutathione S-transferase/CeRhoA fusion protein expressed in Escherichia coli displays conserved biochemical activities. Unlike its counterpart in mammalian cells which is predominantly cytosolic, most of CeRhoA is associated with the membrane and the cytoskeleton throughout development. Indirect immunofluorescence analysis indicates an ubiquitous expression of CeRhoA throughout development with a particular enrichment at larval stages in the pharyngeal nerve ring and at the tip of the head containing chemosensory and mechanosensory neurons. This suggests a stage-specific role for p21 RhoA in mediating the signaling pathway underlying the sensory circuitry in C. elegans post-embryonic development.
Amino Acid Sequence↗
Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis.
The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin. At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr. The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships. Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain. Here, we present a mutational analysis of this domain using n-chimaerin sequences. Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified. Seven of the mutants, including deletions, still possessed GAP activity for Rac1. Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1. IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants. Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues.
Amino Acid Sequence↗
Cerebellar beta 2-chimaerin, a GTPase-activating protein for p21 ras-related rac is specifically expressed in granule cells and has a unique N-terminal SH2 domain.
beta-Chimaerin, a 30-kDa GTPase-activating protein (GAP) for the p21 Ras-related Rac, is expressed specifically in late stage spermatocytes (Leung, T., How, B.-E., Manser, E., and Lim, L. (1993) J. Biol. Chem. 268, 3813-3816). Using antibodies raised against beta-chimaerin, we detected a major 46-kDa RacGAP in the rat cerebellum. With beta-chimaerin cDNA as a probe and using polymerase chain reaction, cDNAs from both human and rat cerebellum were isolated. The human and rat cDNAs encoded sequences containing cysteine-rich and GAP domains identical to those of testis beta-chimaerin. The cDNAs also encoded an additional N-terminal SH2 (Src homology 2) domain, probably derived from the beta-chimaerin gene by alternate splicing. This SH2 domain of the predicted 54-kDa protein was strikingly similar to that of alpha 2-chimaerin, including replacement by glutamic acid of the invariant tryptophan present at the start of other SH2 domains. The SH2 domains of alpha- and beta-chimaerin thus represent a subset of SH2 domains. The cerebellar beta-chimaerin (beta 2-) is expressed mainly in granule cells and exhibits postnatal developmental increases. beta 2-Chimaerin was enriched in particulate/synaptosomal fractions. In the mouse weaver mutant lacking mature granule cells, there is a corresponding decrease in beta 2-chimaerin, which could well serve as a marker of granule cell differentiation.
Amino Acid Sequence↗
Characterization of a promiscuous GTPase-activating protein that has a Bcr-related domain from Caenorhabditis elegans.
Human breakpoint cluster region (bcr) gene product is a member of a group of GTPase-activating proteins that act exclusively on members of the Ras-related Rho subfamily. A complementary DNA was isolated from Caenorhabditis elegans that encoded a polypeptide of 1438 amino acid residues, CeGAP, which contains a domain with sequence similarity to the COOH-terminal segment (GTPase-activating protein region) of Bcr and other known GTPase-activating proteins of the Rho subfamily. It also contains a "pleckstrin homology" motif, present in many signaling proteins including GTPase-activating proteins and nucleotide exchange factors. The Bcr-like domain of CeGAP exhibited activity not only on members of the C. elegans and human Rho subfamily but surprisingly also on C. elegans Ras protein (let-60), human Ras, and Rab3A. CeGAP is therefore the first GTPase-activating protein acting on Ras-related proteins across different subfamilies. Together with the presence of the pleckstrin homology motif, our finding suggests a central and integrative role for CeGAP in a signaling pathway common to Ras and related proteins.
Amino Acid Sequence↗
A brain serine/threonine protein kinase activated by Cdc42 and Rac1.
A new brain serine/threonine protein kinase may be a target for the p21ras-related proteins Cdc42 and Rac1. The kinase sequence is related to that of the yeast protein STE20, implicated in pheromone-response pathways. The kinase complexes specifically with activated (GTP-bound) p21, inhibiting p21 GTPase activity and leading to kinase autophosphorylation and activation. Autophosphorylated kinase has a decreased affinity for Cdc42/Rac, freeing the p21 for further stimulatory activities or downregulation by GTPase-activating proteins. This bimolecular interaction provides a model for studying p21 regulation of mammalian phosphorylation signalling pathways.
Amino Acid Sequence↗
Linear plasmids, pLm9 and pLm10, can be isolated from the phytopathogenic ascomycete Leptosphaeria maculans by pulsed-field gel electrophoresis.
Two linear DNA plasmids (pLm9 and pLm10, sized 9 and 10 kb respectively) were isolated from the phytopathogenic ascomycete Leptosphaeria maculans, using pulsed-field gel electrophoresis. pLm9 and pLm10 are found only in aggressive isolates of L. maculans but, because aggressive and non-aggressive strains appear to be different species, these plasmids are probably not involved in pathogenicity. pLm9 and pLm10 copurify with a mitochondrially-enriched cell fraction, and do not hybridise to chromosomal or mitochondrial DNA, or to each other. Exonuclease digestions suggest that both these molecules contain covalently-bound proteins at their 5' termini. pLm9 hybridises to the RNA polymerase of a linear plasmid from the ascomycete Podospora anserina, and pLm10 hybridises to the DNA polymerase from the same P. anserina plasmid, suggesting that pLm9 and pLm10 encode their own replication and transcription enzymes.
Ascomycota↗
Heterogeneity of monoamine oxidase activities in synaptic and non-synaptic mitochondria derived from three brain regions: some functional implications.
The heterogeneity of monoamine oxidase (MAO; EC 1.4.3.4) activities was studied in two fractions of synaptic mitochondria (SM & SM2) and one fraction of non-synaptic ("free") mitochondria (M) isolated from three rat brain regions (cerebral cortex, striatum, and pons & medulla) by the Lai and Clark (1979, 1989) method in order to elucidate the heterogeneity of MAO at the subcellular and brain regional levels. The activities toward serotonin (MAO-A), benzylamine (MAO-B), and dopamine (MAO-DA) in SM2 from all three regions were different from the corresponding values in SM. In addition, the various MAO activities in SM and SM2 showed heterogeneous distribution with respect to the three brain regions investigated. Both the distribution of MAO-A and MAO-B in non-synaptic mitochondria (M) did not show marked regional differences although MAO-DA in M varied depending on the region. These results clearly demonstrate that the distribution of MAO activities toward different substrates is heterogeneous both at the subcellular and the brain regional levels. The MAO-A:MAO-B ratios in the various mitochondrial fractions also showed trends that are consistent with this hypothesis. Furthermore, in fraction SM of synaptic mitochondria, this ratio was consistently higher than values in the other two mitochondrial fractions (SM2 & M) irrespective of the region from which they were isolated. In view of the functional importance of MAO in the regulation and compartmentation of amine metabolism, the heterogeneity of MAO at subcellular and regional levels may assume pathophysiological importance in neurological diseases (e.g., Parkinsonism) with which altered amine metabolism is associated.
Animals↗
A postmarketing surveillance study of zopiclone in insomnia.
Zopiclone, a novel cyclopyrrolone compound has recently become available for the treatment of insomnia in Singapore. Pharmacodynamic and pharmacokinetic studies have shown it to be an effective hypnotic with a good safety profile and minimal side-effects. The efficacy of Zopiclone was studied in a group of patients with insomnia (n = 40). The study group comprised patients with and without a psychiatric diagnosis. About two-thirds (65%) of the patients had been on hypnotics previously without relief of the insomnia. There was improvement in the various sleep parameters studied: sleep latency, sleep duration, night awakening, sleep quality, dreams, "day-after" effects. The drug was well-tolerated and only four patients experienced minor side-effects.
Adult↗
Prevalence and patterns of use of concomitant medications among participants in three multicenter human immunodeficiency virus type I clinical trials. AIDS Clinical Trials Group (ACTG).
Data on the prevalence and patterns of use of concomitant medications among participants in three large phase III clinical trials of zidovudine (ZDV) in human immunodeficiency virus type 1 (HIV-1) infection were analyzed. Overall, 2,801 patients reported 43,331 uses of concomitant medications. Over 85% of clinical trial participants used one or more concomitant medications at some point during the study. Patients with acquired immune deficiency syndrome (AIDS) used an average of 7.1 drugs per month. Patients with AIDS-related complex (ARC) or who were asymptomatic used relatively fewer drugs: 3.1 and 2.7 per month, respectively. Fourteen percent of patients with AIDS used more than 10 concomitant medications per month. The three most commonly utilized classes of drugs were antiinfectives (57%), analgesics or antipyretics (55%), and vitamins (47%). A total of 17% of patients overall and 30% of AIDS patients used acyclovir while on trial. Consumption of prescription drugs was greater, and "over-the-counter" drugs less, among AIDS patients. Reported use of agents not approved by the Food and Drug Administration or approved drugs used for off-label indications was infrequent. Overall use of concomitant medications did not differ across demographic subgroups when corrected for disease stage at the time of enrollment. White, non-Hispanic, homosexual and bisexual men consumed significantly more antivirals and vitamins than other trial participants. Women in all three protocols took more analgesics or antipyretics than did men.(ABSTRACT TRUNCATED AT 250 WORDS)
AIDS-Related Complex↗
The human active breakpoint cluster region-related gene encodes a brain protein with homology to guanine nucleotide exchange proteins and GTPase-activating proteins.
GTPase-activating proteins (GAPs) modulate the activity of the ras superfamily of proteins by converting active GTP-bound to inactive GDP-bound p21s. Employing a novel GAP overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), we demonstrated a diversity of proteins with GAP activities in different tissues. Using a polymerase chain reaction strategy exploiting conserved residues in the GAP domains of n-chimaerin and the product of the breakpoint cluster region gene (BCR), we isolated a human brain 5.3-kilobase cDNA containing a 486-base pair region with complete identity to a previously reported active BCR-related (ABR) gene sequence on human chromosome 17. The brain cDNA encoded a 98-kDa protein (ABR) resembling BCR (68% identity), containing both the oncogene dbl-related domain at the N terminus and the GAP domain at the C terminus; however, it lacks the N-terminal BCR protein kinase domain. The ABR GAP domain expressed as an Escherichia coli fusion protein was active against Rac1 and Cdc42 of the rho subfamily. The ABR mRNA is highly enriched in the brain. ABR probably corresponds to the brain-enriched 100-kDa GAP for Rac and Cdc42Hs previously detected. The relationship of ABR to Miller-Dieker syndrome, a neurological disorder co-mapping to 17p13.3, is discussed.
Amino Acid Sequence↗