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L Lim

Publications and source records attributed to L Lim.

At least 145 records · Page 8Linked to original sources
PubMed1993

The CDC42 homologue from Caenorhabditis elegans. Complementation of yeast mutation.

A Caenorhabditis elegans cDNA encoding a homologue of the p21 ras-related CDC42, designated as CDC42Ce, was isolated from a nematode mixed stage cDNA library. The encoded protein of 188 amino acid residues has 85% identity to both human G25K and CDC42Hs and 79 and 76% identity to the yeast CDC42Sp and CDC42Sc proteins, respectively. The CDC42Ce cDNA maps to a position on C. elegans chromosome II in close proximity to lin-26, a cell lineage gene. The CDC42Ce cDNA hybridizes to 2- and 1.5-kilobase mRNAs. Their expression is developmentally regulated with highest levels at the embryonic stage, decreasing progressively during development except for an increase of the more abundant 1.5-kilobase mRNA at the L3 stage. The glutathione S-transferase/CDC42Ce fusion protein expressed in Escherichia coli displays both GTP binding and intrinsic GTPase activities. The GTPase activity of CDC42Ce is moderately stimulated by human n-chimaerin, a GTPase-activating protein for the related p21 rac1. The CDC42Ce protein complements the temperature-sensitive lethal mutation cdc42-1 in yeast Saccharomyces cerevisiae. These data suggest that CDC42Ce is the C. elegans homologue of the yeast CDC42. The developmental expression pattern of mRNA and is biochemical properties of its encoded protein which are closely related to CErac1 suggest that the two p21s might be involved in related biological processes.

Amino Acid Sequence↗
PubMed1993

A non-receptor tyrosine kinase that inhibits the GTPase activity of p21cdc42.

The Ras-related Rho subfamily of GTP-binding proteins (p21s), which includes Rho, Rac and Cdc42Hs, is implicated in different aspects of cytoskeletal organization. These proteins behave like Ras (p21ras) in that their active GTP-bound form is inactivated by intrinsic hydrolysis of the nucleotide gamma-phosphate, which can be stimulated by GTPase-activating proteins (GAPs). We have previously shown that there is a diversity of GAPs that recognize this subfamily, including n-chimaerin, which is enriched in the hippocampus; we also detected proteins that bind these p21 proteins and seem to inhibit GTP hydrolysis. We now report the characterization of a hippocampal complementary DNA encoding a tyrosine kinase that specifically binds Cdc42Hs in its GTP-bound form. This binding is mediated by a unique sequence of 47 amino acids C-terminal to an SH3 domain and inhibits both the intrinsic and GAP-stimulated GTPase activity of Cdc42Hs. Our findings indicate that there may be a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs and which is directly linked to a tyrosine phosphorylation pathway.

Amino Acid Sequence↗
PubMed1993

A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. The p21rac-GTPase activating protein n-chimaerin.

Phorbol esters are potent tumor promoters widely used for investigating mechanisms of cell transformation with protein kinase C (PKC) generally considered as being their only protein target. Lysophosphatidic acid (LPA) can act as a mitogen, affecting cell shape and the actin cytoskeleton. There is no identified functional target for LPA. We have isolated a cDNA encoding a protein n-chimaerin that is a high affinity phorbol ester receptor and a p21rac-GTPase activating protein (rac-GAP). p21rac is a member of the ras superfamily of small molecular weight GTP-binding proteins, which stimulates actin microfilament formation in Swiss 3T3 cells and superoxide production by the neutrophil oxidase. We now show that the rac-GAP activity of n-chimaerin is stimulated by phosphatidylserine (PS) and phosphatidic acid (PA) and that phorbol esters can synergize with PS and PA. LPA, in contrast, was found to inhibit n-chimaerin. The phospholipid/phorbol ester modulation of the rac-GAP activity requires the PKC-like cysteine-rich domain of n-chimaerin. Thus, n-chimaerin is a novel functional target (distinct from PKC) for both phorbol esters and LPA. These data suggest that the physiological role of n-chimaerin is to link events initiating at the cell surface/membrane with p21rac effector pathways.

Animals↗
PubMed1993

Alternating and intermittent regimens of zidovudine and dideoxycytidine in patients with AIDS or AIDS-related complex.

OBJECTIVE: To determine whether alternating regimens consisting of zidovudine and 2',3'-dideoxycytidine (ddC) reduce the toxicity and maintain or increase the antiretroviral effect associated with each drug alone. DESIGN: An unblinded, randomized (phase II) clinical trial in which seven treatment regimens were compared. SETTING: Outpatient clinics of 12 AIDS Clinical Trials Units. PATIENTS: One hundred thirty-one patients with the acquired immunodeficiency syndrome (AIDS) or AIDS-related complex and serum p24 antigenemia (> or = 70 pg/mL). INTERVENTION: Treatments included weekly or monthly alternating zidovudine (200 mg every 4 hours) and ddC (0.01 or 0.03 mg/kg body weight every 4 hours); weekly intermittent zidovudine, 200 mg every 4 hours, or ddC, 0.03 mg/kg every 4 hours; and continuous zidovudine. MEASUREMENTS: Toxicity, CD4 cell counts, serum p24 antigen levels, and clinical end points. Data were analyzed for the first 48 weeks of therapy (median follow-up, 40 weeks). RESULTS: Hematologic toxicity was significantly less frequent in patients who received zidovudine therapy every other week (11% to 15%) or every other month (11% to 14%) than in those who received continuous zidovudine therapy (33%) (P < 0.02). Weekly alternating therapy with zidovudine and ddC, 0.03 mg/kg, or intermittent therapy with ddC, 0.03 mg/kg, produced high rates of peripheral neuropathy (41% and 50%, respectively). Neuropathy occurred in 10% to 21% of patients in the other three alternating-therapy limbs and in 17% of patients receiving zidovudine alone (intermittently or continuously). Initial increases in CD4 cell counts were sustained in three alternating-therapy limbs, but counts returned to baseline by week 28 in the remaining limbs. The median weight gain at week 48 was significantly greater in patients treated with alternating regimens (0.9 to 3.8 kg) compared with those treated with continuous zidovudine therapy (-0.7 kg) (P = 0.008). Patients treated with alternating regimens and those treated with continuous zidovudine had similarly sustained decreases in p24 antigen levels. CONCLUSIONS: These findings suggest that alternating therapy with zidovudine and ddC reduces the toxicity associated with each drug alone while maintaining strong antiretroviral activity.

AIDS-Related Complex↗
PubMed1993

Germ cell beta-chimaerin, a new GTPase-activating protein for p21rac, is specifically expressed during the acrosomal assembly stage in rat testis.

Diverse GTPase-activating proteins (GAPs) for the p21rho subfamily were detected by a novel overlay assay (Manser, E., Leung, T., Monfries, C., Teo, M., Hall, C., and Lim, L. (1992) J. Biol. Chem. 267, 16025-16028), with some GAPs being tissue-specific. Using a PCR strategy exploiting conserved regions common to rho/rac-GAPs, we have isolated a rat testis cDNA encoding a 34-kDa rac-GAP termed beta-chimaerin, as it was highly related to n-chimaerin, containing both a GAP domain (77% identity) and the phorbol ester-binding region (93% identity). beta-Chimaerin mRNA is expressed exclusively in the testis at the onset of sexual maturation. In situ hybridization and cell fractionation analyses show beta-chimaerin mRNA expression to be stage-specific, paralleling acrosomal assembly at the late stage of spermatogenesis. A corresponding testis-specific 30-kDa rac-GAP was detected. The testis-specific and stage-dependent expression of this new member of the chimaerin family offer an alternative model system for investigating the functional role of this class of p21 GAPs, particularly in relation to cytoskeletal reorganization.

Acrosome↗
PubMed1993

A Drosophila gene encoding a protein with similarity to diacylglycerol kinase is expressed in specific neurons.

A Drosophila gene encoding a protein with similarity to diacylglycerol kinase (DGK) was isolated by screening a genomic DNA library with a human DGK cDNA under low-stringency hybridization conditions. This putative Drosophila DGK gene (dDGK) maps to 43E on the right arm of chromosome 2. A dDGK cDNA with an open reading frame encoding a 517 amino acid protein was obtained in a screen of a 3-12-h embryonic cDNA library. In a 236-amino-acid overlap at their C-termini there is an identity of 52.5% between the dDGK protein and human DGK. The N-terminus of the Drosophila protein is not similar to human DGK, and contains clusters of polar amino acids. dDGK is transcribed in the embryonic, pupal and adult stages, with little expression during the larval stages. Transcripts of 1.7-2.2 kb, 3.5 kb, 3.7 kb and 6.6 kb are seen, although most of the smaller transcripts may be from genes with similarity to dDGK. In stage-16 and stage-17 embryos, dDGK transcripts are limited to the central nervous system and head. There is a particularly high level of expression in the cell bodies of the larval photoreceptor organ, and in the cell bodies of the ventral unpaired median neurons. The dDGK protein may be involved in regulating signal transduction in these specific neurons.

Amino Acid Sequence↗
PubMed1993

A new member of the ras superfamily, the rac1 homologue from Caenorhabditis elegans. Cloning and sequence analysis of cDNA, pattern of developmental expression, and biochemical characterization of the protein.

A new member of the ras superfamily, designated CErac1 has been identified. The CErac1 cDNA clone was isolated from a Caenorhabditis elegans mixed stage library and encodes a protein of 191 amino acids with 82 and 79% identity to human rac1 and rac2 proteins, respectively. The CErac1 cDNA maps to a position on C. elegans chromosome IV in close proximity to cha-1, a choline acetyltransferase gene. The CErac1 cDNA hybridizes to two mRNAs (1.7 and 0.9 kilobases). Their expression is developmentally regulated, that of the more abundant 1.7 kilobases being highest at the embryonic stage and decreasing dramatically during development with 10% of the embryonic level in adult nematodes. The glutathione-S-transferase/CErac1 fusion protein expressed in Escherichia coli binds GTP and exhibits intrinsic GTPase activity. The GTPase activity of the CErac1 protein is stimulated by human n-chimaerin, a GTPase-activating protein for p21 rac1. These data suggest a role of CErac1 in C. elegans early development. The conserved biochemical properties indicate that further characterization of CErac1 by genetic analysis will be helpful in elucidating not only its role in the signal transduction, but also the biological function of its mammalian homologues.

Amino Acid Sequence↗
PubMed1993

Brain regional distributions of monoamine oxidase activities in postnatal development in normal and chronically manganese-treated rats.

To investigate the hypothesis that manganese toxicity may affect the development of the monoamine oxidases (MAOs) in brain, the regional distributions of the A (serotonin-oxidizing) and B (benzylamine-oxidizing) forms of MAO were determined in manganese-treated (1 mg or 10 mg of MnCl2.4H2O per ml of drinking water from conception onwards until the rats were used for experiments) and untreated male rats during various stages of postnatal development. The age-related variations in regional MAO-A activities (especially in pons and medulla and in cerebellum) were more marked than those in regional MAO-B activities. The MAO-A:MAO-B activity ratios decreased in all regions during development. Chronic manganese treatment (at the specified doses) did not significantly alter the age-related changes in regional MAO-A and MAO-B activities; nor were the A:B activity ratios affected. The results suggest that there is differential expression of MAO isoforms in various brain regions during postnatal development but the expression is not affected by chronic manganese toxicity.

Aging↗
PubMed1992

The Caenorhabditis elegans unc-13 gene product is a phospholipid-dependent high-affinity phorbol ester receptor.

The Caenorhabditis elegans unc-13 mutant is a member of a class of mutants that have un-coordinated movement. Mutations of the unc-13 gene cause diverse defects in C. elegans, including abnormal neuronal connections and modified synaptic transmission in the nervous system. unc-13 cDNA encodes a protein (UNC-13) of 1734 amino acid residues with a predicted molecular mass of 198 kDa and sequence identity to the C1/C2 regions but not to the catalytic domain of the ubiquitously expressed protein kinase C family [Maruyama & Brenner (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5729-5733]. To characterize the phorbol ester binding site of the UNC-13 protein, cDNA encoding the C1/C2-like regions (amino acid residues 546-940) was expressed in Escherichia coli and the 43 kDa recombinant protein was purified. Phorbol ester binding to the 43 kDa protein was zinc- and phospholipid-dependent, stereospecific and of high affinity (Kd 67 nM). UNC-13 specific antisera detected a protein of approx. 190 kDa in wild-type (N2) but not in mutant (e1019) C. elegans cell extracts. We conclude that UNC-13 represents a novel class of phorbol ester receptor.

Amino Acid Sequence↗
PubMed1992

Developmental regulation and neuronal expression of the mRNA of rat n-chimaerin, a p21rac GAP:cDNA sequence.

Human n-chimaerin is a GTPase-activating protein (GAP) for p21rac and a phorbol ester receptor. We have isolated rat n-chimaerin cDNA and investigated the cellular and developmental pattern of mRNA expression in the brain. There is extensive sequence conservation with human n-chimaerin in the coding region and the first 400 nucleotides of the 3'-untranslated region (UTR) (90% and 83% identity respectively). The rat cDNA encodes an additional 35 N-terminal amino acids compared with the reported human cDNA, which has a 5'-UTR sequence inversion and a 41-nucleotide deletion including the putative initiator methionine. The rat cDNA encodes a 334-amino acid protein (38200 M(r), pI 8.04) with 97% amino acid sequence identity with the human protein, after correction of the human 5'-DNA sequence. n-Chimaerin mRNA was detectable in embryonic rat brain at day 15 and increased in amount postnatally from birth to 20 days, coincident with cellular differentiation and synaptogenesis. n-Chimaerin mRNA is restricted to neurons, with highest concentrations in hippocampal pyramidal cells, granule cells of the dentate gyrus and cortical neurons. In the cerebellum the mRNA was detected only in Purkinje neurons. The pattern and specificity of mRNA expression suggests an important role for n-chimaerin in neuronal signal-transduction mechanisms.

Amino Acid Sequence↗
PubMed1992

Diversity and versatility of GTPase activating proteins for the p21rho subfamily of ras G proteins detected by a novel overlay assay.

The p21ras superfamily, involved in diverse processes including cell growth and intracellular trafficking, possesses intrinsic GTPase activity and cycles between GTP-bound active and GDP-bound quiescent states. This intrinsic activity, which results in down-regulation, is accelerated by GTPase activating proteins (GAPs). Other proteins regulating the GDP/GTP cycle include exchange proteins and dissociation inhibitors. The p21s rho, rac, and cdc42Hs constitute a subfamily implicated in cytoskeletal organization. BCR and n-chimaerin are prototypes of a new GAP family for these p21s. To investigate proteins modulating GTP hydrolysis of the three p21s, we developed a novel overlay assay applicable to tissue extracts. Diverse GAPs with different specificities were identified in all rat tissues. Brain contained rac1 GAPs of 45, 50, 85, 100, and 150 kDa. The p50 and p150 GAPs also act on rhoA and cdc42Hs and are ubiquitous, while the p45-GAP, n-chimaerin, is brain- and testis-specific and acts preferentially on rac1; the p100 GAP acts on both rac1 and cdc42Hs and is brain-specific. A new class of p21-interacting proteins was also identified. This diversity, versatility, and tissue specificity of GAPs may be required for fine control of the down-regulation of GTP-bound p21s and the suggested specific downstream effects of individual GAPs, which could involve "cross-talk" between GAPs and p21s.

Blotting, Western↗
PubMed1992

Neurochemical changes in rats chronically treated with a high concentration of manganese chloride.

Several neurochemical parameters were studied in brain regions of rats chronically treated with a high concentration of manganese chloride (20 mg MnCl2.4H2O per ml. of drinking water) throughout development until adulthood. Large increases in Mn accumulation were found in all brain regions (hypothalamus, +530%; striatum, +479%; other regions, +152 to +250%) of Mn-treated adult rats. In these animals, Ca levels were decreased (-20 to -46%) in cerebellum, hypothalamus, and cerebral cortex but were increased (+186%) in midbrain. Mg levels were decreased (-12 to -32%) in pons and medulla, midbrain, and cerebellum. Fe levels were increased (+95%) in striatum but were decreased (-28%) in cerebral cortex. Cu levels were increased (+43 to +100%) in pons and medulla and striatum but Zn levels were decreased (-30%) in pons and medulla. Na levels were increased (+22%) in striatum but those of K and Cl remained unchanged. Type A monoamine oxidase activities were decreased (-13 to -16%) in midbrain, striatum, and cerebral cortex, but type B monoamine oxidase activities decreased (-13%) only in hypothalamus. Acetylcholinesterase activities were increased (+20 to +22%) in striatum and cerebellum. The results are consistent with our hypothesis that chronic manganese encephalopathy not only affects brain metabolism of Mn but also that of other metals.

Acetylcholinesterase↗
PubMed1992

Novel brain-specific bovine cDNA for a developmentally regulated mRNA encoding a putative new member of the leucine-rich glycoprotein (LRG) family.

We have isolated a bovine cDNA which hybridized to a 2 kb mRNA specifically expressed in the rat and human brain. The mRNA was abundantly expressed in adult but not 21-week old human brain. In the rat brain, there was very little of the transcript in 15-day old fetus but it increased in abundance with development, being most abundant in the adult and expressed in all brain regions. Genomic analysis showed that the sequence is single copy and conserved in all vertebrates examined, including chicken. The 702 bp partial cDNA encoded an amino acid sequence for a putative member of the leucine-rich glycoprotein (LRG) family known to be involved in cell adhesion/recognition. The predicted polypeptide displayed sequence identity with that recently reported for the human oligodendrocyte-myelin glycoprotein. This cDNA should prove useful in further investigations on brain-specific cell-cell interactions.

Amino Acid Sequence↗
PubMed1992

Differential effects of metal ions on type A and type B monoamine oxidase activities in rat brain and liver mitochondria.

To investigate the hypothesis that neurotoxic metals can exert their toxicity through the direct inhibition of monoamine oxidases (MAOs), the effects of several neurotoxic metal ions on type A (MAO-A) and type B (MAO-B) monoamine oxidase activities in rat forebrain nonsynaptic mitochondria and rat liver mitochondria were studied. At pathophysiological levels (10-100 microM), Cu2+ and Cd2+ are good inhibitors of brain mitochondrial MAO-A and, to a lesser extent, liver mitochondrial MAO-A. The inhibition of MAO-B activities in brain and liver mitochondria by Cu2+ and Cd2+ is only detected at the higher end of the concentration range (i.e., 50-100 microM). At the pathophysiological level of 0.5 mM, Al3+ only inhibits brain mitochondrial MAO-A but at the higher level of 2.5 mM, it inhibits both forms of MAO in brain as well as liver mitochondria. Even at toxic levels (e.g., 5 mM), neither Mn2+ nor Li+ inhibits the activities of MAO-A and MAO-B in brain and liver mitochondria. Our results are consistent with the hypothesis that some neurotoxic metals can exert their toxicity through the direct inhibition of the isoforms of MAO. Our data also suggest that the selective inhibition of brain MAO-A by Cu2+ and Cd2+ may assume pathophysiological importance in the neurotoxicity of copper and cadmium.

Animals↗
PubMed1992

Changes in brain regional manganese and magnesium levels during postnatal development: modulations by chronic manganese administration.

Manganese (Mn) and magnesium (Mg) levels in hypothalamus, cerebellum, pons and medulla, striatum, midbrain, and cerebral cortex of control and Mn-treated (10 mg MnCl2.4H2O per ml of drinking water) rats during postnatal development were studied using instrumental neutron activation analysis. The age-dependent Mn accumulation showed regional variation: at day 5, this accumulation was most marked in striatum (12.05 micrograms/g wet weight) but least marked in cerebral cortex (0.85 micrograms/g wet weight). By day 10, pons and medulla, and hypothalamus were regions with, respectively, the highest (4.73 micrograms/g wet weight) and the lowest (0.52 micrograms/g wet weight) Mn levels. By contrast, brain regional Mn variations were less pronounced in weanling and adult rats. The age-dependent Mg accumulation showed regional variation at day 5, being most marked in pons and medulla (720 micrograms/g wet weight) and least marked in cerebral cortex (295 micrograms/g wet weight). Mg levels in all regions decreased after day 5; by day 120, only Mg level in cerebral cortex was lower than levels in other regions (the latter being very similar). In general, the age-related decreases in Mn and Mg levels paralleled the decreases in water content and increases in tissue weight, suggesting that the maturation of the blood-brain barrier may play important role(s) in brain Mn and Mg homeostasis. Chronic Mn-treatment from conception onwards altered the regional Mn and Mg distribution patterns during development. Our results are consistent with the hypothesis that chronic Mn toxicity exerts modulatory effects on brain regional metabolism and homeostasis of Mn and other metals during development.

Aging↗
PubMed1992

The human heat-shock genes HSPA6 and HSPA7 are both expressed and localize to chromosome 1.

HSPA6 is a member of the human heat-shock protein gene family, encoding a basic 70-kDa protein, with unique induction characteristics (Leung et al., 1990, Biochem. J. 267: 125-132). Hybridization analyses with a somatic cell hybrid DNA panel localized the gene to chromosome 1q. The highly related HSPA7 DNA sequence (Voellmy et al., 1985, Proc. Natl. Acad. Sci. USA 82: 4949-4953) colocalized. Both HSPA6 and HSPA7 represent functional genes, as determined by analyses of mRNA from heat-shocked human cells using specific oligonucleotides, although their pattern of expression differed. Neither mRNA was detected in the absence of heat stress. A BamHI polymorphism in the HSPA7 gene was present in a predominantly Asian population.

Base Sequence↗
PubMed1992

Discrepancy between self- and observer ratings of performance in social phobics.

Twenty-eight subjects meeting Diagnostic and Statistical Manual of Mental Disorders (rev. 3rd ed.; American Psychiatric Association, 1987) criteria for social phobia and without a comorbid affective disorder and 33 nonclinical controls were asked to present a brief, impromptu speech to a small audience. Speakers themselves, as well as members of the audience, rated each speaker on a public speaking questionnaire that included both specific items (e.g., voice shook) and global items (e.g., appeared confident). For global items, no significant difference was indicated between the two groups on observers' ratings of public speaking performance. However, social phobics rated their own performance worse than did nonclinical controls, and there was a significantly greater discrepancy between self and other ratings for social phobics than controls. Fear of negative evaluation was the only significant predictor of the self-other discrepancy on global items.

Adult↗