Biomedical subjects

R Li

Publications and source records attributed to R Li.

At least 217 records · Page 12Linked to original sources

PubMed1999

Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility.

To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.

Actin-Related Protein 2↗

PubMed1999

Differential expression of trkC mRNA in the chicken embryo from gastrulation to development of secondary brain vesicles.

In situ hybridization revealed that mRNA for the neurotrophin receptor trkC first appears in the chicken embryo at stage 4 anterior to Hensen's node. At stages 6 and 8, trkC mRNA was restricted to the neural plate. By stage 11, trkC mRNA was absent from much of the prosencephalon, the entire mesencephalon and rhombomeres 1 and 4. At stage 15, trkC mRNA expression was limited to rhombomeres 3 and 5, and, by stage 18, there was no apparent expression of trkC mRNA in the hindbrain.

Animals↗

PubMed1999

The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene.

Narcolepsy is a disabling sleep disorder affecting humans and animals. It is characterized by daytime sleepiness, cataplexy, and striking transitions from wakefulness into rapid eye movement (REM) sleep. In this study, we used positional cloning to identify an autosomal recessive mutation responsible for this sleep disorder in a well-established canine model. We have determined that canine narcolepsy is caused by disruption of the hypocretin (orexin) receptor 2 gene (Hcrtr2). This result identifies hypocretins as major sleep-modulating neurotransmitters and opens novel potential therapeutic approaches for narcoleptic patients.

Amino Acid Sequence↗

PubMed1999

The events relating to lanthanide ions enhanced permeability of human erythrocyte membrane: binding, conformational change, phase transition, perforation and ion transport.

The binding and uptake of Gd3+ ions by human erythrocytes in vitro were studied by determining the Gd contents in membrane and in cytosol by means of particle-induced X-ray emission (PIXE) spectrometry. Results obtained from varied incubation time revealed that the Gd3+ ions bind to the membrane proteins and lipids at first. Gd3+ binding to the membrane lipids and proteins lasts 0 approximately 20 and 20 approximately 100 ms respectively, as shown by the stopped-flow studies. Then a fraction of Gd3+ ions diffuses through the membrane. The kinetics of Gd3+ binding indicates that the binding to phospholipids is prior to that to the membrane proteins, but a portion of the lipid-bound Gd3+ redistributed later to the proteins. PIXE studies showed that the entry of Gd3+ increased the influx of Ca2+ and Cl-. By monitoring the changes in fluorescence of proteins and that of the Ln3+, the uptake of La3+, Eu3+, Gd3+ and Tb3+ was shown to be a process comprising a series of events. Binding to the membrane molecules induces the phase transition of lipid bilayer and conformational changes and aggregation of membrane proteins. Conformational changes of the proteins were characterized by Fourier transform IR spectroscopy (FT-IR) deconvolved spectra, i.e. alpha-helix content decreases while beta-sheet increases. ESR spectra of MSL-labeled proteins reflect the aggregation state related with the conformational change. [31P]NMR spectra of membrane lipid bilayer revealed the Ln3+ ions induced hexagonal (H(II)) phase formation. Phase transition and aggregation of membrane proteins cause the formation of domain structure and perforation in the membrane. These alterations in membrane structure are responsible for the Ln3+ enhanced membrane permeability. Thus the previous Ln3+ binding will facilitate the across-membrane transport of other Ln3+ ions through the membrane.

Blood Proteins↗

PubMed1999

Induction of endocytic vesicles by exogenous C(6)-ceramide.

Ceramide is a newly discovered second messenger that has been shown to cause cell growth arrest and apoptosis. Here, we present evidence that exogenously added C(6)-ceramide induces enlargement of late endosomes and lysosomes. 10 microM C(6)-ceramide caused the formation of numerous vesicles of varying sizes (2-10 micrometers) in fibroblasts (3T3-L1 and 3T3-F442A), without toxic effects. Vesicle formation induced by C(6)-ceramide was time- and dose-dependent, rapid, and reversible. Numerous small vesicles appeared within 8 h of treatment with 10 microM C(6)-ceramide. They enlarged with time, with large vesicles found in the perinuclear region and small ones observed at the cell periphery. Within 24 h of treatment, approximately 30% of the cells exhibited these vesicles. Removal of ceramide from the culture medium caused disappearance of the vesicles, which reappeared upon readdition of ceramide. Confocal immunofluorescence microscopic analysis using an anti-lysosome-associated membrane protein antibody identified the enlarged vesicles as late endosomes/lysosomes. The fluorescent C(6)-NBD-ceramide, a vital stain for the Golgi apparatus, did not stain these vesicles. The effect on vesicle formation was influenced by ceramide structure; D-erythro-C(6)-ceramide was the most active ceramide analogue tested. Short chain ceramide metabolites, such as sphingosine, sphingosine 1-phosphate, N-hexanoyl-sphingosylphosphorylcholine, N-acetylpsychosine, and C(2)-ceramide G(M3), (G(M3), N-acetylneuraminosyl-alpha(2, 3)-galactosyl-beta(1,4)-glucosylceramide), were inactive in causing vesicle formation when added exogenously. Together, these studies demonstrate that exogenous C(6)-ceramide induces endocytic vesicle formation and causes enlarged late endosomes and lysosomes in mouse fibroblasts.

3T3 Cells↗

PubMed1999

Genetic dissection of the budding yeast Arp2/3 complex: a comparison of the in vivo and structural roles of individual subunits.

In previous work, we identified the yeast Arp2/3 complex, which localizes to cortical actin patches and is required for their motility and integrity in vivo. This complex contains proteins homologous to each subunit of the Acanthamoeba and human Arp2/3 complex except for a 40-kDa subunit (p40), which was missing from the purified yeast complex. Here, we demonstrate by using immunoprecipitation and gel-filtration analysis that Arc40p, the homolog of p40 identified from the yeast genome database, associates with the yeast Arp2/3 complex. We have carried out gene disruptions of each subunit of the yeast Arp2/3 complex to study each subunit's role in the function of the complex. Surprisingly, we find that only ARC40 is fully essential for cell viability. Strains lacking each of the other subunits exhibit varying degrees of defects in cell growth and viability and in assembly and polarization of cortical actin patches. We have also examined each subunit's role in maintaining the structural integrity of the Arp2/3 complex. Arp2p, Arp3p, and Arc40p fall into the monomer pool in Deltaarc19 and Deltaarc35 cells, suggesting that Arc19p and Arc35p are the central scaffolding components of the complex. Arp2p and Arp3p do not have major roles in maintaining complex integrity, and Arc15p is required for association of Arp2p and Arc40p, but not other subunits, with the complex. These results provide evidence that each subunit contributes differently to the assembly and function of the Arp2/3 complex.

Acanthamoeba↗

PubMed1999

Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library.

A large insert canine genomic bacterial artificial chromosome (BAC) library was built from a Doberman pinscher. Approximately 166,000 clones were gridded on nine high-density hybridization filters. Insert analysis of randomly selected clones indicated a mean insert size of 155 kb and predicted 8.1 coverage of the canine genome. Two percent of the clones were nonrecombinant. Chromosomal fluorescence in situ hybridization studies of 60 BAC clones indicated no chimerism. The library was hybridized with dog PCR products representing eight genes (ADA, TNFA, GCA, MYB, HOXA, GUSB, THY1, and TOP1). The resulting positive clones were characterized and shown to be compatible with an eightfold redundant library.

Animals↗

PubMed1999

Activation of the yeast Arp2/3 complex by Bee1p, a WASP-family protein.

The Arp2/3 complex is a highly conserved cytoskeletal component that has been implicated in the nucleation of actin filament assembly. Purified Arp2/3 complex has a low intrinsic actin nucleation activity, leading to the hypothesis that an unidentified cellular activator is required for the function of this complex. We showed previously that mutations in the Arp2/3 complex and in Bee1p/Las17p, a member of the Wiskott-Aldrich syndrome protein(WASP) family, lead to a loss of cortical actin structures (patches) in yeast. Bee1p has also been identified as an essential nucleation factor in the reconstitution of actin patches in vitro. Recently, it was reported that WASP-like proteins might interact directly with the Arp2/3 complex through a conserved carboxy-terminal domain. Here, we have shown that Bee1p and the Arp2/3 complex co-immunoprecipitate when expressed at endogenous levels, and that this interaction requires both the Arc15p and Arc19p subunits of the Arp2/3 complex. Furthermore, the carboxy-terminal domain of Bee1p greatly stimulated the nucleation activity of purified Arp2/3 complex in vitro, suggesting a direct role for WASP-family proteins in the activation of the Arp2/3 complex. Interestingly, deletion of the carboxy-terminal domain of Bee1p neither abolished the localization of the Arp2/3 complex, as had been suggested, nor resulted in a severe defect in cortical actin assembly. These results indicate that the function of Bee1p is not mediated entirely through its interaction with the Arp2/3 complex, and that factors redundant with Bee1p might exist to activate the nucleation activity of the Arp2/3 complex.

Actin-Related Protein 2↗

PubMed1999

The growth-dependent expression of the adenine nucleotide translocase-2 (ANT2) gene is regulated at the level of transcription and is a marker of cell proliferation.

The adenine nucleotide translocator-2 (ANT2) gene is expressed in growth-activated cells together with the early-immediate genes. We have studied the mechanism of ANT2 expression during the serum-induced transition from G0 to G1 and during reentry into G0 as cells approach confluence. Actinomycin D completely blocked ANT2 expression of serum-induced quiescent NIH3T3. In addition, no serum-dependent changes were observed in the stability of ANT2 transcripts in cells activated by serum or during the breakdown of transcripts caused by serum removal and reentry into G0. Thus, all changes in ANT2 transcript levels appear to be regulated predominantly at the level of transcription. Using cells permanently transfected with deletion constructs of the ANT2 promoter, we identified a suppressor region that is responsible for decreased expression of ANT2 in cells leaving the growth cycle at confluence. Thus, ANT2 is expressed during the proliferation state via a mechanism that most probably includes transcription repression/derepression.

3T3 Cells↗

PubMed1999

Bifurcation of the mitotic checkpoint pathway in budding yeast.

The coordination of mitotic events is ensured through the spindle assembly checkpoint. BFA1 is required for this checkpoint in budding yeast because its disruption abolishes the mitotic arrest when spindle assembly is inhibited. Analysis of the genetic interaction of BFA1 with known mitotic checkpoint genes suggest that Bfa1 functions in the same pathway with Bub2 but not with Mad1 or Mad2. Both Bfa1 and Bub2 localize to spindle poles, and overexpression of Bfa1 arrests the cell cycle in anaphase. These findings suggest a bifurcation of the spindle assembly checkpoint: whereas one branch of the pathway, consisting of Mad1-3, Bub1 and 3, and Mps1, may prevent premature disjunction of sister chromosomes, the other, consisting of Bfa1 and Bub2, may function at spindle poles to prevent cytokinesis before the completion of chromosome segregation.

Cell Cycle Proteins↗

PubMed1999

The p42 variant of ETS1 protein rescues defective Fas-induced apoptosis in colon carcinoma cells.

ETS1 is a cellular homologue of the product of the viral ets oncogene of the E26 virus, and it functions as a tissue-specific transcription factor. It plays an important role in cell proliferation, differentiation, lymphoid cell development, transformation, angiogenesis, and apoptosis. ETS1 controls the expression of critical genes involved in these processes by binding to ets binding sites present in the transcriptional regulatory regions. The ETS1 gene generates two proteins, p51 and a spliced variant, p42, lacking exon VII. In this paper we show that p42-ETS1 expression bypasses the damaged Fas-induced apoptotic pathway in DLD1 colon carcinoma cells by up-regulating interleukin 1beta-converting enzyme (ICE)/caspase-1 and causes these cancer cells to become susceptible to the effects of the normal apoptosis activation system. ICE/caspase-1 is a redundant system in many cells and tissues, and here we demonstrate that it is important in activating apoptosis in cells where the normal apoptosis pathway is blocked. Blocking ICE/caspase-1 activity by using specific inhibitors of this protease prevents the p42-ETS1-induced apoptosis from occurring, indicating that the induced ICE/caspase-1 enzyme is responsible for killing the cancer cells. p42-ETS1 activates a critical alternative apoptosis pathway in cancer cells that are resistant to normal immune attack, and thus it may be useful as an anticancer therapeutic.

Apoptosis↗

PubMed1999

[Detection of apoptotic cells by comet assay].

Comet assay is a method measuring DNA strand beaks on single cell level, which has been developed for about 10 years. Theoretically, apoptotic cells with extensive DNA internucleosomal fragments can be detected by this method. On the basis that apoptosis in immature mice thymocytes could be induced by dexamethasone(DEX), the detection of apoptotic cells by comet assay was studied and compared with agarose gel electrophoresis, morphological observations with Giemsa staining and TUNEL assay. Apoptotic cells showed a comet feature with a very small head, and a large and wide tail. The optimal conditions for electrophoresis in comet assay was 20 volt in 15 minutes. Comet assay is sensitive, valid, rapid and simple and is highly correlated with Giemsa staining and TUNEL assay in detecting apoptotic cells.

Animals↗

PubMed1999

Chromatin remodeling and activation of chromosomal DNA replication by an acidic transcriptional activation domain from BRCA1.

An increasing number of transcription factors have been shown to activate DNA replication. However, the underlying mechanism remains to be elucidated. Here it is shown that when tethered to a cellular replication origin, the acidic transcriptional activation domain of the breast cancer protein BRCA1 alters the local chromatin structure and stimulates chromosomal DNA replication. Cancer-predisposing mutations in BRCA1 that abolish transcriptional activation also prevent chromatin remodeling and activation of replication. Chromatin remodeling occurs even in the absence of a functional replication origin. Thus, increasing chromatin accessibility may be an important mechanism used by transcription factors to facilitate multiple nuclear processes.

BRCA1 Protein↗

PubMed1999

Identification of TAXREB107 as an erythropoietin early response gene.

The protooncogenes c-myc and c-myb are erythropoietin (Epo)-regulated early response genes. They appear to play distinct roles in the growth- and differentiation-inducing signals of the hormone. Using a subtractive hybridization strategy, we have identified the murine homolog of the TAX response element-binding protein TAXREB107 as an Epo early response gene in Rauscher murine erythroleukemia cells. This was confirmed by Northern blot analyses, which showed a fourfold increase in TAXREB107 mRNA after 1 hr of erythropoietin treatment. After 3 hr the transcript had decreased to approximately twofold above control levels. Inhibition of this induction with antisense oligodeoxynucleotides increased Epo-induced hemoglobinization 2.5-fold. The results implicate TAXREB107 in erythropoiesis and support the hypothesis that the TAXREB proteins have functions outside the context of HTLV-I infection.

Animals↗

PubMed1999

Phylogenetic relationships between toxic and non-toxic strains of the genus Microcystis based on 16S to 23S internal transcribed spacer sequence.

16S to 23S ribosomal DNA internal transcribed spacer sequences of 47 strains of the genus Microcystis were determined. Derived maximum likelihood and DNA distance trees indicated that Microcystis can be divided into three clusters. The first cluster included toxic and non-toxic strains, the second only toxic ones, and the third only non-toxic ones. The tree topologies were not necessarily correlated with morphospecies distinction or phycobilin pigment composition, and one genotype may have more than one morphotype. Phylogenetic analysis based on intergenic spacer sequences was thought to be effective for understanding relationships among closely related species and strains.

Base Sequence↗

PubMed1999

Auditory-vocal cholinergic pathway in zebra finch brain.

The two main song control nuclei in the zebra finch forebrain, the higher vocal center (HVC) and the robust nucleus of the archistriatum (RA), receive cholinergic innervation from the ventral paleostriatum (VP) of the basal forebrain which may play a key role in song learning. By injecting two kinds of neuroanatomical tracers, we found that a topographically segregated pathway from nucleus ovoidalis (Ov) and nucleus dorsomedialis posterior thalami (DMP) to VP and further to RA and HVC. Ov is known as a major relay in the main ascending auditory pathway. The results suggest that cholinergic neurons in the VP which are responsible for song learning are regulated by auditory information from the Ov.

Acetylcholine↗

PubMed1999

Family history of coronary heart disease and pre-clinical carotid artery atherosclerosis in African-Americans and whites: the ARIC study: Atherosclerosis Risk in Communities.

The association between family history of coronary heart disease (CHD) and morbidity and mortality due to atherosclerotic sequelae, although well documented in population-based samples of whites, has been little studied in African Americans. Less is known about the relationship between a family history of CHD and pre-clinical atherosclerosis. We report the relation between family history of CHD, summarized in a family risk score (FRS), and asymptomatic atherosclerosis at the extracranial carotid arteries, measured by B-mode ultrasound. The FRS was assessed in relatives of 3,034 African Americans and 9,048 white probands aged 45 to 64 years, in the four community-based cohorts of the ARIC Study. The analyses were restricted to individuals free of clinically manifest CHD. The distribution of CHD FRS by ethnic-gender groups was right skewed, with slightly higher mean values for white than African-American males, and for African-American than white females. In a series of multivariate linear regression models with mean carotid artery intima-media wall thickness (IMT) as the dependent variable, FRS was associated positively with IMT in white and African-American women and white men. In a multiple regression model, approximately one-half of the quantitative statistical relationship of the CHD FRS with IMT in whites was statistically explained by the major risk factors considered as intervening, explanatory variables in this analysis. This association in African-American women was fully explained by the major risk factors. The FRS was not, however, associated with atherosclerosis or major risk factors in African-American males, in the ARIC Study.

Arteriosclerosis↗

PubMed1999

The transport kinetics of lanthanide species in a single erythrocyte probed by confocal laser scanning microscopy.

A novel method has been developed to visualize and follow the temporal course of lanthanide transport across the membrane into a single living erythrocyte. By means of confocal scanning microscopy and the optical section technique, the entry of lanthanide ions was followed by the fluorescence quenching of fluorescein isothiocyanate (FITC)-labeled membrane and cytosol. From the difference of the quenching kinetics of the whole section and the central area, the time for diffusion through the membrane and the diffusion in the extracellular and intracellular media can be deduced. To clarify the mechanism of lanthanide-induced fluorescence quenching of FITC-labeled erythrocytes and to ensure that this reaction can be used in this method, the reaction was investigated by steady-state fluorescence techniques. The results showed that the lanthanides strongly quenched the florescence emitted by FITC covalently bound to membrane proteins and cytosolic proteins. The static quenching mechanism is responsible for the fluorescence quenching of FITC-labeled proteins by Ln species. The quenching mechanism is discussed on the basis of complex formation. The dependence of fluorescence quenching on both ion size and the total orbital angular momentum L supports the complexation mechanism. The transport time across the membrane is strikingly correlated with Ln species and extracellular concentration. For a given concentration, the transport time of [Ln(cit)2]3- is much shorter than that of Ln3+, since they enter the cells via the anion channel. This is supported by the inhibition effect of 4,4'-diisothiocyanato-2,2'-stilbenendisulfonate on the transport of [Ln(cit)2]3-. On the other hand, the transport of free Ln3+ might be attributed to the enhanced permeability of erythrocytes owing to Ln3+ binding. These findings strongly demonstrate the existence of the non-internalization mechanism of Ln species uptake by erythrocytes.

Animals↗