Biomedical subjects
Publications and source records attributed to F Liu.
We show that the equilibrium shape anisotropy of two-dimensional islands in heteroepitaxial growth depends on island size, a consequence of the presence of strain. Even in homoepitaxy, in which the island shape has conventionally been equated with the ratio of step energies, a substrate surface stress anisotropy can influence island shape.
A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the mRNA encoding human herpes simplex virus 1 (HSV-1) major transcription activator, ICP4. A reduction of more than 80% in the expression level of ICP4 and a reduction of about 1000-fold in viral growth were observed in cells that stably expressed the ribozyme. In contrast, a reduction of less than 10 % in ICP4 expression and viral growth was observed in cells that either did not express the ribozyme or produced a catalytically inactive ribozyme mutant. Thus, M1GS ribozyme is highly effective in inhibiting HSV-1 growth and can be used as a general gene-targeting agent for anti-HSV applications.
RNase P from Escherichia coli is a tRNA-processing enzyme and consists of a catalytic RNA subunit (M1 RNA) and a protein component (C5 protein). M1GS, a gene-targeting ribozyme derived from M1, can cleave a herpes simplex virus 1 mRNA efficiently in vitro and inhibit its expression effectively in viral-infected cells. In this study, the effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate were investigated by site-specific UV crosslink mapping. In the presence of the protein cofactor, the ribozyme regions crosslinked to the substrate sequence 3' immediately to the cleavage site were similar to those found in the absence of C5. Meanwhile, some of the ribozyme regions (e.g. P12 and J11/12) that were crosslinked to the leader sequence 5' immediately to the cleavage site in the presence of C5 were different from those regions (e.g. P3 and P4) found in the absence of the protein cofactor and were not among those that are believed to interact with a tRNA. Understanding how C5 affects the specific interactions between the ribozyme and its target mRNA may facilitate the development of gene-targeting ribozymes that function effectively in vivo, in the presence of cellular proteins.
The ansamycin antibiotics, herbimycin A (HA) and geldanamycin (GM), bind to a conserved pocket in heat shock protein 90 (Hsp90) and alter the function of this chaperone protein. Occupancy of this pocket results in the degradation of a subset of signaling molecules. These include proteins known to associate with Hsp90, e.g., the steroid receptors and Raf, as well as certain transmembrane tyrosine kinases, such as the ErbB receptor family. In a variety of tumor cell lines, treatment with HA potently inhibited cellular proliferation by inducing G1 arrest. This arrest was accompanied by hypophosphorylation of the retinoblastoma gene product (RB) and rapid down-regulation of cyclin D- and E-associated kinase activities. Inhibition of kinase activity was found to result from loss in expression of cyclins D1, D3, and E, as well as the associated cyclin-dependent kinases, cyclin-dependent kinase 4 and cyclin-dependent kinase 6. In addition, HA treatment also caused a late induction of p27(Kip1) protein. The loss of cyclin D preceded the other effects of HA, suggesting that it might be the primary cause of G1 arrest. To determine whether the effects of HA are mediated by selective inhibition of the cyclin D-RB pathway, HA was added to tumor cell lines lacking functional RB. HA treatment of Rb-negative tumor cell lines failed to elicit a G1 arrest. In addition, after release from synchronization with nocodazole, Rb-negative but not Rb-positive cell lines were able to progress through G1 into S phase in the presence of HA. Together, these findings suggest that induction of G1 arrest by HA results from down-regulation of cyclin D expression and its associated kinase activity. Furthermore, these findings imply that Hsp90 selectively regulates signaling pathways upstream of RB.
Treatment of cells with insulin and protein tyrosine phosphatase inhibitors such as vanadate and pervanadate resulted in the tyrosine phosphorylation of Grb10, a Src homology 2 (SH2) and pleckstrin homology domain-containing adaptor protein which binds to a number of receptor tyrosine kinases including the insulin receptor (IR). Although Grb10 binds directly to the kinase domain of the IR, our data show that Grb10 is not a direct substrate for the IR tyrosine kinase. Consistent with this finding, Grb10 tyrosine phosphorylation in cells was inhibited by herbimycin A, a relatively specific inhibitor for members of the Src tyrosine kinase family, and by the expression of dominant negative Src or Fyn. In addition, Grb10 tyrosine phosphorylation was stimulated by expression of constitutively active Src or Fyn in cells and by incubation with purified Src or Fyn in vitro. The insulin stimulated or Src/Fyn-mediated tyrosine phosphorylation in vivo was significantly reduced when Grb10 tyrosine 67 was changed to glycine. This mutant form of Grb10 bound with higher affinity to the IR in cells than that of the wild-type protein, suggesting that tyrosine phosphorylation of Grb10 may normally negatively regulate its binding to the IR. Our data show that Grb10 is a new substrate for members of the Src tyrosine kinase family and that the tyrosine phosphorylation of the protein may play a potential role in cell signaling processes mediated by these kinases. Oncogene (2000).
SMAD proteins can mediate transforming growth factor beta (TGF-beta)-inducible transcriptional responses. Whereas SMAD can recognize specific DNA sequences, it is usually recruited to a promoter through interaction with a DNA-binding partner. In an effort to search for TGF-beta-inducible genes, we used a subtractive screening method and identified human Smad7, which can antagonize TGF-beta signaling and is rapidly up-regulated by TGF-beta. In this report, we show that TGF-beta can stabilize Smad7 mRNA and activate Smad7 transcription. The Smad7 promoter is the first TGF-beta responsive promoter identified in vertebrates that contains the 8-bp palindromic SMAD-binding element (SBE), an optimal binding site previously identified by a PCR-based selection from random oligonucleotides by using recombinant Smad3 and Smad4. We demonstrate that on TGF-beta treatment, endogenous SMAD complex can bind to a Smad7 promoter DNA as short as 14 or 16 bp that contains the 8-bp SBE in gel mobility shift and supershift assays. Our studies provide strong evidence that SMAD proteins can bind to a natural TGF-beta responsive promoter independent of other sequencespecific transcription factors. We further show that, whereas recombinant Smad3 binds to the SBE, endogenous or even transfected Smad3 cannot bind to the SBE in the absence of Smad4. These findings have important implications in the identification of target genes of the TGF-beta/SMAD signaling pathways.
9-Benzylpurines with a variety of substituents in the 2-, 6- and/or 8-position have been prepared and screened for antimycobacterial effects. High inhibitory activity against Mycobacterium tuberculosis was found for 9-benzylpurines carrying a phenylethynyl-, trans-styryl or aryl substituents in the 6-position and generally chlorine in the 2-position tends to increase activity.
A sequence-specific ribozyme (M1GS RNA) derived from the catalytic RNA subunit of RNase P from Escherichia coli was used to target the overlapping exon 3 region of the mRNAs encoding the major transcription regulatory proteins IE1 and IE2 of human cytomegalovirus. A reduction of more than 80% in the expression levels of IE1 and IE2 and a reduction of about 150-fold in viral growth were observed in human cells that stably expressed the ribozyme. In contrast, a reduction of less than 10% in the IE1/IE2 expression and viral growth was observed in cells that either did not express the ribozyme or produced a "disabled" ribozyme that carried mutations that abolished its catalytic activity. Examination of the expression of several other viral early and late genes in the cells that expressed the M1GS ribozyme further revealed an overall reduction of at least 80% in their expression. These results are consistent with the notion that the antiviral effects in these cells are due to the fact that the ribozyme specifically inhibits the expression of IE1 and IE2 and, consequently, abolishes the expression of viral early and late genes as well as viral growth. Our study is the first, to our knowledge, to use M1GS ribozyme for inhibiting human cytomegalovirus replication and demonstrates the utility of this ribozyme for antiviral applications.
The mechanisms that regulate hematopoietic progenitor cell (HPC) mobilization from the bone marrow to blood have not yet been defined. HPC mobilization by granulocyte colony-stimulating factor (G-CSF), cyclophosphamide (CY), or interleukin-8 but not flt-3 ligand is markedly impaired in G-CSF receptor-deficient (G-CSFR-deficient) mice. G-CSFR is expressed on mature hematopoietic cells, HPCs, and stromal cells, which suggests that G-CSFR signals in one or more of these cell types was required for mobilization by these agents. To define the cell type(s) responsible for G-CSF-dependent mobilization, a series of chimeric mice were generated using bone marrow transplantation. Mobilization studies in these chimeras demonstrated that expression of the G-CSFR on transplantable hematopoietic cells but not stromal cells is required for CY- or G-CSF-induced mobilization. Moreover, in irradiated mice reconstituted with both wild type and G-CSFR-deficient bone marrow cells, treatment with CY or G-CSF resulted in the equal mobilization of both types of HPCs. This result held true for a broad spectrum of HPCs including colony-forming cells, CD34(+) lineage(-) and Sca(+) lineage(-) cells, and long-term culture initiating cells. Collectively, these data provide the first definitive evidence that expression of the G-CSFR on HPCs is not required for their mobilization by G-CSF and suggest a model in which G-CSFR-dependent signals act in trans to mobilize HPCs from the bone marrow.
Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr(774) in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Czeta, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.
A new panel of steroidal cationic lipids has been synthesized for gene delivery. Using commercially available vitamin D2 (calciferol) or vitamin D3 (cholecalciferol) as hydrophobic motifs and a variety of cationic head groups as binding sites for negatively charged phosphate groups in DNA, we demonstrated that the transfection activity of the synthetic vitamin D-based cationic lipids 1d, 2d formulated with dioleoylphosphatidylethanolamine (DOPE) as a co-lipid is comparable to that of 3-(-[N-N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol). These synthetic lipids are effective in transfecting a variety of cell lines. These results suggest that vitamin D-based cationic lipids are useful transfection reagents for in vitro gene transfer studies.
An in vitro selection procedure was used to select RNase P ribozyme variants that efficiently cleaved the sequence of the mRNA encoding thymidine kinase of herpes simplex virus 1. Of the 45 selected variants sequenced, 25 ribozymes carried a common mutation at nucleotides 224 and 225 of RNase P catalytic RNA from Escherichia coli (G(224)G(225) --> AA). These selected ribozymes exhibited at least 10 times higher cleavage efficiency (k(cat)/K(m)) than that derived from the wild type ribozyme. Our results suggest that the mutated A(224)A(225) are in close proximity to the substrate and enhance substrate binding of the ribozyme. When these ribozyme variants were expressed in herpes simplex virus 1-infected cells, the levels of thymidine kinase mRNA and protein were reduced by 95-99%. Our study provides the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results demonstrate the potential for using RNase P ribozymes as gene-targeting agents against any mRNA sequences, and using the selection procedure as a general approach for the engineering of RNase P ribozymes.
The C-terminal domain of G protein-coupled receptor kinases (GRKs) consists of a conserved region and a variable region, and the variable region has been shown to direct the membrane translocation of cytosolic enzymes. The present work has revealed that the C-terminal domain may also be involved in kinase-receptor interaction that is primarily mediated by the conserved region. Truncation of the C-terminal domain or deletion of the conserved region in this domain of GRK2 resulted in a complete loss of its ability to phosphorylate rhodopsin and in an obvious decrease in its sensitivity to receptor-mediated phosphorylation of a peptide substrate. On the contrary, deletion of the betagamma subunit binding region in the C-terminal domain of GRK2 did not significantly alter the ability of the enzyme to phosphorylate rhodopsin. In addition, the recombinant proteins that represent the C-terminal domain and the conserved region of GRK2 could inhibit GRK2-mediated phosphorylation of rhodopsin and receptor-mediated activation of GRK2 but not GRK2-mediated phosphorylation of the peptide substrate. Furthermore, the conserved region as well as the C-terminal domain could directly bind rhodopsin in vitro. These results indicate that the C-terminal domain, or more precisely, the conserved region of this domain, is important for enzyme-receptor interaction and that this interaction is required for GRK2 to catalyze receptor phosphorylation.
A transposon derived from Escherichia coli Tn3 was introduced into the genome of murine cytomegalovirus (MCMV) to generate a pool of viral mutants. We analyzed three of the constructed recombinant viruses that contained the transposon within the M25, M27, and m155 open reading frames. Our studies provide the first direct evidence to suggest that M25 and M27 are not essential for viral replication in mouse NIH 3T3 cells. Studies in cultured cells and Balb/c mice indicated that the transposon insertion is stable during viral propagation both in vitro and in vivo. Moreover the virus that contained the insertion mutation in M25 exhibited a titer similar to that of the wild-type virus in the salivary glands, lungs, livers, spleens, and kidneys of the Balb/c mice that were intraperitoneally infected with these viruses. These results suggest that M25 is dispensable for viral growth in these organs and the presence of the transposon sequence in the viral genome does not significantly affect viral replication in vivo. The Tn3-based system can be used as a mutagenesis approach for studying the function of MCMV genes in both tissue culture and in animals.
GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.
A variety of flavonoids, lignans, an alkaloid, a bisbenzyl, coumarins and terpenes isolated from Chinese herbs was tested for antioxidant activity as reflected in the ability to inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis. The pro-oxidant activities of the aforementioned compounds were assessed by their effects on bleomycin-induced DNA damage. The flavonoids baicalin and luteolin-7-glucuronide-6'-methyl ester, the lignan 4'-demethyldeoxypodophyllotoxin, the alkaloid tetrahydropalmatine, the bisbenzyl erianin and the coumarin xanthotoxol exhibited potent antioxidative activity in both lipid peroxidation and hemolysis assays. The flavonoid rutin and the terpene tanshinone I manifested potent antioxidative activity in the lipid peroxidation assay but no inhibitory activity in the hemolysis assay. The lignan deoxypodophyllotoxin, the flavonoid naringin and the coumarins columbianetin, bergapten and angelicin slightly inhibited lipid peroxidation in brain and kidney homogenates. It is worth stressing that the compounds with antioxidant effects in this assay, with the exception of tetrahydropalmatin and tanshinone I, have at least one free aromatic hydroxyl group in structure. Obviously, the aromatic hydroxyl group is very important for antioxidative effects of the compounds. None of the compounds tested exerted an obvious pro-oxidant effect.
The antioxidative and superoxide- and hydroxyl radical-scavenging activities and pro-oxidant effect of twelve selected medicinal herbs were studied. The aqueous extracts of Coptis chinensis, Paeonia suffruticosa, Prunella vulgaris and Senecio scandens exhibited the highest potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates. The aforementioned four herbs also demonstrated strong superoxide- and hydroxyl radical-scavenging activity, but exerted only a slight pro-oxidant effect.
Reactive oxygen species have been shown to be involved in the mutagenicity, clastogenicity, and apoptosis of mammalian cells treated with arsenic or cadmium. As these endpoints require several hours of cellular processing, it is not clear that reactive oxygen species damage DNA directly or interfere with DNA replication and repair. Using single-cell alkaline electrophoresis, we have detected DNA strand breaks (DSBs) in bovine aortic endothelial cells by a 4-h treatment with sodium arsenite (As) and cadmium chloride (Cd) in sublethal concentrations. As-induced DSBs could be decreased by nitric oxide (NO) synthase inhibitors, superoxide scavengers, and peroxynitrite scavengers and could be increased by superoxide generators and NO generators. Treatment with As also increased nitrite production. These results suggest that As-increased NO may react with O2*- to produce peroxynitrite and cause DNA damage. The results showing that Cd increased cellular H2O2 levels and that Cd-induced DSBs could be modulated by various oxidant modulators suggest that Cd may induce DSBs via O2*-, H2O2, and *OH. Nevertheless, the DSBs in both As- and Cd-treated cells seem to come from the excision of oxidized bases such as formamidopyrimidine and 8-oxoguanine, as the Escherichia coli enzyme formamidopyrimidine-DNA glycosylase (Fpg) increased DSBs in cells treated with As, 3-morpholinosydnonimine (a peroxynitrite-generating agent), Cd, or H2O2.