A general synthesis of 5'-azido-5'-deoxy-2',3'-O-isopropylidene nucleosides.
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Indicators and Reagents↗
Biomedical subjects
F Liu
Publications and source records attributed to F Liu.
Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.
Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.
Agrobacterium tumefaciens↗
RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences.
External guide sequences (EGSs) are oligonucleotides that consist of a sequence complementary to a target mRNA and recruit intracellular RNase P for specific degradation of the target RNA. In this study, DNA-based EGS molecules were chemically synthesized to target the mRNA coding for the protease of human cytomegalovirus (HCMV). The EGS molecules efficiently directed human RNase P to cleave the target mRNA sequence in vitro. When EGSs were exogenously administered into HCMV-infected human foreskin fibroblasts, a reduction of about 80-90% in the expression level of the protease and a reduction of about 300-fold in HCMV growth were observed in the cells that were treated with a functional EGS, but not in cells that were not treated with the EGS or with a "disabled" EGS carrying nucleotide mutations that precluded RNase P recognition. Moreover, packaging of the viral DNA genome into the capsid was blocked in the cells treated with the functional EGS. These results indicate that HCMV protease is essential for viral DNA encapsidation. Moreover, our study provides direct evidence that exogenous administration of a DNA-based EGS can be used as a therapeutic approach for inhibiting gene expression and replication of a human virus.
Base Sequence↗
Synergistic outside-in regulation of platelet activation by GPIIb/IIIa ligand-induced conformation and oligomerization.
Full platelet activation with serotonin secretion and thromboxane A(2) (TxA(2)) formation induced by a low dose of thrombin receptor agonist peptide (TRAP) or high dose ADP requires platelet aggregation. This requirement can be replaced by pretreatment of platelets with a combination of reagents including: GPIIb/IIIa inhibitors yielding ligand-induced binding sites (LIBS), either arginine-glycine-aspartate-serine (RGDS) peptide or Ro 43-5054, cytochalasin to disrupt actin filaments and crosslinking by a GPIIb/IIIa mAb (pl-62). Crosslinking is required since Fab fragments of pl-62 do not support activation. Engagement of the Fc receptor by the mAb Fc domain is not required for pl-62 augmentation, since it is not blocked by the anti-Fc receptor mAb, IV-3. Another GPIIb/IIIa inhibitor, Ro 44-9883, not yielding LIBS epitopes, serves as a negative control and shows a requirement for LIBS in addition to crosslinking. Focal adhesion kinase tyrosine phosphorylation induced by TRAP is blocked by these GPIIb/IIIa antagonists, but restored by pl-62 crosslinking independent of LIBS induction. Tyrosine phosphorylation of a peptide comigrating with p38 MAP kinase is also inhibited by these antagonists and restored by pl-62 crosslinking. However, p38 MAP kinase activation by low dose TRAP is not affected by these aggregation inhibitors. Tyrosine phosphorylation of a 34-kDa phosphoprotein in the absence of aggregation or TxA(2) formation was uniquely augmented by Ro 43-5054 but not Ro 44-9883 under the above activation conditions.
Acetates↗
Insulin stimulates increased catalytic activity of phosphoinositide-dependent kinase-1 by a phosphorylation-dependent mechanism.
Phosphoinositide-dependent kinase-1 (PDK-1) is a serine-threonine kinase downstream from PI 3-kinase that phosphorylates and activates other important kinases such as Akt that are essential for cell survival and metabolism. Previous reports have suggested that PDK-1 has constitutive catalytic activity that is not regulated by stimulation of cells with growth factors. We now show that insulin stimulation of NIH-3T3(IR) cells or rat adipose cells may significantly increase the intrinsic catalytic activity of PDK-1. Insulin treatment of NIH-3T3(IR) fibroblasts overexpressing PDK-1 increased both phosphorylation of recombinant PDK-1 in intact cells and PDK-1 kinase activity in an immune-complex kinase assay. Insulin stimulation of rat adipose cells also increased catalytic activity of endogenous PDK-1 immunoprecipitated from the cells. Both insulin-stimulated phosphorylation and activity of PDK-1 were inhibited by wortmannin and reversed by treatment with the phosphatase PP-2A. A mutant PDK-1 with a disrupted PH domain (W538L) did not undergo phosphorylation or demonstrate increased kinase activity in response to insulin stimulation. Similarly, a PDK-1 phosphorylation site point mutant (S244A) had no increase in kinase activity in response to insulin stimulation. Thus, the insulin-stimulated increase in PDK-1 catalytic activity may involve PI 3-kinase- and phosphorylation-dependent mechanisms. We conclude that the basal constitutive catalytic activity of PDK-1 in NIH-3T3(IR) cells and rat adipose cells can be significantly increased upon insulin stimulation.
3-Phosphoinositide-Dependent Protein Kinases↗
Regulation of cyclin-dependent kinase 5 and casein kinase 1 by metabotropic glutamate receptors.
Cyclin-dependent kinase 5 (Cdk5) is a multifunctional neuronal protein kinase that is required for neurite outgrowth and cortical lamination and that plays an important role in dopaminergic signaling in the neostriatum through phosphorylation of Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, molecular mass 32 kDa). Casein kinase 1 (CK1) has been implicated in a variety of cellular functions such as DNA repair, circadian rhythm, and intracellular trafficking. In the neostriatum, CK1 has been found to phosphorylate Ser-137 of DARPP-32. However, first messengers for the regulation of Cdk5 or CK1 have remained unknown. Here we report that both Cdk5 and CK1 are regulated by metabotropic glutamate receptors (mGluRs) in neostriatal neurons. (S)-3,5-dihydroxyphenylglycine (DHPG), an agonist for group I mGluRs, increased Cdk5 and CK1 activities in neostriatal slices, leading to the enhanced phosphorylation of Thr-75 and Ser-137 of DARPP-32, respectively. The effect of DHPG on Thr-75, but not on Ser-137, was blocked by a Cdk5-specific inhibitor, butyrolactone. In contrast, the effects of DHPG on both Thr-75 and Ser-137 were blocked by CK1-7 and IC261, specific inhibitors of CK1, suggesting that activation of Cdk5 by mGluRs requires CK1 activity. In support of this possibility, the DHPG-induced increase in Cdk5 activity, measured in extracts of neostriatal slices, was abolished by CK1-7 and IC261. Treatment of acutely dissociated neurons with DHPG enhanced voltage-dependent Ca(2+) currents. This enhancement was eliminated by either butyrolactone or CK1-7 and was absent in DARPP-32 knockout mice. Together these results indicate that a CK1-Cdk5-DARPP-32 cascade may be involved in the regulation by mGluR agonists of Ca(2+) channels.
Animals↗
Search for the decay upsilon(1S) --> gammaeta(').
We report on a search for the radiative decay Upsilon(1S)-->gammaeta(') in 61.3 pb(-1) of data taken with the CLEO II detector at the Cornell Electron Storage Ring. Three decay chains were investigated, all involving eta(')-->pi(+)pi(-)eta, followed by eta-->gammagamma, eta-->pi(0)pi(0)pi(0), or eta-->pi(+)pi(-)pi(0). We find no candidate events in any of the three cases and set a combined upper limit of 1.6x10(-5) at 90% C.L., significantly smaller than the previous limit. We compare our result to other radiative Upsilon decays, to radiative J/psi decays, and to theoretical predictions.
Journal Article↗
Effect of PSC 833 liposomes and Intralipid on the transport of epirubicin in Caco-2 cells and rat intestines.
Clinical applications of first-generation multidrug resistance (MDR) modulators, such as cyclosporin A (CsA) have been hampered because of their severe side effects in vivo. Recent investigations have led to the development of a more potent and less toxic modulator, PSC 833, which is a nonimmunosuppressive analogue of CsA. However, adverse pharmacokinetic interactions between anticancer drugs and PSC 833 have resulted in increased toxicity as compared to the individual toxicity. Our study evaluated the MDR reversing effect of PSC 833 in free, liposomal or Intralipid formulations on the uptake and transport of epirubicin in Caco-2 cells and everted gut sacs of rats. The results showed that PSC 833 in free or liposomal formulations significantly enhanced the intracellular accumulation of epirubicin in a dose-related manner in Caco-2 cells. The optimum in enhancement was observed at the concentration of 2 microM PSC 833. These formulations markedly increased the apical to basolateral absorption of epirubicin in Caco-2 cells and substantially improved the mucosal to serosal absorption of epirubicin in rat jejunum and ileum. PSC 833 in free, liposomal or Intralipid formulations all significantly reduced basolateral to apical efflux of epirubicin across Caco-2 monolayers. However, PSC 833 in liposomes showed greater enhancement than other formulations. In conclusion, PSC 833 and PSC 833 liposomes have the function as MDR reversing agents for the inhibition of intestinal P-glycoprotein. Liposomal preparations of PSC833 may provide a useful alternative dosage form for intravenous administration of PSC 833 to be combined with anticancer drugs to circumvent drug resistance in cancer chemotherapy.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Immunogene therapy of tumors with vaccine based on Xenopus homologous vascular endothelial growth factor as a model antigen.
Overcoming immune tolerance of the growth factors associated with tumor growth should be a useful approach to cancer therapy by active immunity. We used vascular endothelial growth factor (VEGF) as a model antigen to explore the feasibility of the immunogene tumor therapy with a vaccine based on a single xenogeneic homologous gene, targeting the growth factors associated with angiogenesis. To test this concept, we constructed a plasmid DNA encoding Xenopus homologous VEGF (XVEGF-p) and control vectors. We found that immunogene tumor therapy with a vaccine based on XVEGF was effective at both protective and therapeutic antitumor immunity in several tumor models in mice. VEGF-specific autoantibodies in sera of mice immunized with XVEGF-p could be found in Western blotting analysis and ELISA assay. The purified immunoglobulins were effective at the inhibition of VEGF-mediated endothelial cell proliferation in vitro, and at antitumor activity and the inhibition of angiogenesis by adoptive transfer in vivo. The elevation of VEGF in the sera of the tumor-bearing mice could be abrogated with XVEGF-p immunization. The antitumor activity and production of VEGF-specific autoantibodies, significantly elevated IgG1 and IgG2b, could be abrogated by the depletion of CD4(+) T lymphocytes. The observations may provide a vaccine strategy for cancer therapy through the induction of autoimmunity against the growth factors associated with tumor growth in a cross reaction with single xenogeneic homologous gene and may be of importance in the further exploration of the applications of other xenogeneic homologous genes identified in human and other animal genome sequence projects in cancer therapy.
Animals↗
Insulin receptor-mediated p62dok tyrosine phosphorylation at residues 362 and 398 plays distinct roles for binding GTPase-activating protein and Nck and is essential for inhibiting insulin-stimulated activation of Ras and Akt.
A GTPase-activating protein (GAP)-associated 60-kDa protein has been found to undergo rapid tyrosine phosphorylation in response to insulin stimulation. However, whether this protein is a direct in vivo substrate for the insulin receptor (IR) tyrosine kinase and whether the tyrosine phosphorylation plays a role in insulin signaling remain to be established. Here we show that the insulin-stimulated tyrosine phosphorylation of the GAP-associated protein, now identified as p62(dok), is inhibited by Grb10, an adaptor protein that binds directly to the kinase domain of the IR, both in vitro and in cells. Replacing Tyr(362) and Tyr(398) with phenylalanine greatly decreased the IR-catalyzed p62(dok) tyrosine phosphorylation in vitro, suggesting that these two residues are the major IR-mediated phosphorylation sites. However, mutations at Tyr(362) and Tyr(398) only partially blocked insulin-stimulated p62(dok) tyrosine phosphorylation in cells, indicating that p62(dok) is also a target for other cellular tyrosine kinase(s) in addition to the IR. Replacing Tyr(362) with phenylalanine abolished the interaction between p62(dok) and Nck. Mutations at Tyr(362/398) of p62(dok) disrupted the interaction between p62(dok) and GAP and decreased the inhibitory effect of p62(dok) on the insulin-stimulated activation of Ras and Akt, but not mitogen-activated protein kinase. Furthermore, the inhibitory effect of p62(dok) on Akt phosphorylation could be blocked by coexpression of a constitutively active Ras. Taken together, our findings indicate that p62(dok) is a direct substrate for the IR tyrosine kinase and that phosphorylation at Tyr(362) and Tyr(398) plays an essential role for p62(dok) to interact with its effectors and negatively regulate the insulin signaling pathway.
Adaptor Proteins, Signal Transducing↗
Heterogeneous expression of Gap junction channels in the heart leads to conduction defects and ventricular dysfunction.
BACKGROUND: - Heterogeneous remodeling of gap junctions is observed in many forms of heart disease. The consequent loss of synchronous ventricular activation has been hypothesized to result in diminished cardiac performance. To directly test this hypothesis, we designed a murine model of heterogeneous gap junction channel expression. Methods and Results-- We generated chimeric mice formed from connexin43 (Cx43)-deficient embryonic stem cells and wild-type or genetically marked ROSA26 recipient blastocysts. Chimeric mice developed normally, without histological evidence of myocardial fibrosis or hypertrophy. Heterogeneous Cx43 expression resulted in conduction defects, however, as well as markedly depressed contractile function. Optical mapping of chimeric hearts by use of voltage-sensitive dyes revealed highly irregular epicardial conduction patterns, quantified as significantly greater negative curvature of the activation wave front (-1.86+/-0.40 mm in chimeric mice versus -0.86+/-0.098 mm in controls; P<0.01; n=6 for each group). Echocardiographic studies demonstrated significantly reduced fractional shortening in chimeric mice (26.6+/-2.3% versus 36.5+/-1.6% in age-matched 129/SvxC57BL/6F1 wild-type controls; P<0.05). CONCLUSIONS: - These data suggest that heterogeneous Cx43 expression, by perturbing the normal pattern of coordinated myocardial excitation, may directly depress cardiac performance.
Animals↗
Self-assembly of two-dimensional islands via strain-mediated coarsening.
We demonstrate two distinctive effects of strain-induced island-island interaction on island size and spatial distribution during coarsening of 2D islands. When coarsening proceeds via only mass transport between islands, the interaction broadens the island size distribution, leading to a power-law dependence of island size uniformity on island number density. When coarsening proceeds via island migration in addition to mass transport between islands, the interaction can effectively direct island motion through island edge diffusion, leading to self-organized formation of a regular array of islands with both uniform size and spacing.
Journal Article↗
[Perioperative observation of plasma endothelin in patients with congenital heart disease with pulmonary hypertension underwent open heart surgery].
UNLABELLED: To elucidate the correlation between endothelin(ET) and pulmonary hypertension, plasma ET was measured perioperatively by radioimmunoassay in 22 patients with pulmonary hypertension who underwent open heart surgery(PH group) in comparison with 16 patients without pulmonary hypertension(non-PH group) also underwent cardiopulmonary bypass(CPB) surgery. RESULTS: Plasma ET was significantly increased during CPB in both group, Plasma ET decreased rapisly and returned to preoperative level within 6 hours after operation in the non-PH group. In the PH group, however, plasma ET decreased slowly and did not return to preoperative level within 48 hours postoperation.
Cardiopulmonary Bypass↗
Cyclic AMP inhibits extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt pathways by inhibiting Rap1.
Cyclic AMP inhibited both ERK and Akt activities in rat C6 glioma cells. A constitutively active form of phosphatidylinositol 3-kinase (PI3K) prevented cAMP from inhibiting Akt, suggesting that the inactivation of Akt by cAMP is a consequence of PI3K inhibition. Neither protein kinase A nor Epac (Exchange protein directly activated by cAMP), two known direct effectors of cAMP, mediated the cAMP-induced inhibition of ERK and Akt phosphorylation. Cyclic AMP inhibited Rap1 activation in C6 cells. Moreover, inhibition of Rap1 by a Rap1 GTPase-activating protein-1 also resulted in a decrease in ERK and Akt phosphorylation, which was not further decreased by cAMP, suggesting that cAMP inhibits ERK and Akt by inhibiting Rap1. The role of Rap1 in ERK and Akt activity was further demonstrated by our observation that an active form of Epac, which activated Rap1 in the absence of cAMP, increased ERK and Akt phosphorylation. Inhibition of ERK and/or PI3K pathways mediated the inhibitory effects of cAMP on insulin-like growth factor-I (IGF-I) and IGF-binding protein-3 gene expression. Moreover, cAMP, as well as ERK and PI3K inhibitors produced equivalent stimulation and inhibition, respectively, of p27(Kip1) and cyclin D2 protein levels, potentially explaining the observation that cAMP prevented C6 cells from entering S phase.
Animals↗
Synthesis of a new class of 5'-functionalized adenosines using a rh(ii)-catalyzed 1,3-dipolar cycloaddition.
[reaction: see text] Chemically protected adenosine was functionalized at the 5' position to generate novel dipolarophiles and mesoionic dipoles. These species were found to undergo facile 1,3-dipolar cycloaddition to afford a new series of adenosine derivatives that contain a point of diversification at the 5' position of adenosine.
Adenosine↗
Regulation of differentiation- and proliferation-inducers on Lewis antigens, alpha-fucosyltransferase and metastatic potential in hepatocarcinoma cells.
The expressions of Lewis (Le) antigens, alpha-1,3/1,4 fucosyltransferases (alpha-1,3/1,4 FuTs), and metastatic potential after the treatment of 2 differentiation inducers, all- trans retinoic acid (ATRA), 8-bromo-cyclic 3',5'adenosine monophosphate (8-Br-cAMP); and 2 proliferation inducers, epidermal growth factor (EGF) and phobol-12-myristate-13-acetate (PMA), on 7721 human hepatocarcinoma cell line were studied. Cell adhesion to human umbilical vein endothelial cells (HUVEC), cell migration through transwell and invasion through matrigel were selected as the indexes of metastatic potential-related phenotypes. Using fluorescence-labelled antibodies and flow-cytometric analysis, it was found that 7721 cells mainly expressed sialyl Lewis X (SLe(x)) and a less amount of sialyl dimeric Lewis X (SDLe(x)) antigens on the cell surface. Their expressions were down-regulated by ATRA, and up-regulated by EGF. SLe(x)antigen was also decreased and increased by the treatment of 8-Br-cAMP and PMA respectively. With Northern blot to detect the mRNAs of alpha-1,3/1,4 FuTs, the main enzymatic basis for the change in SLe(x)expression was found to be the alteration of the expression of alpha-1,3 FuT-VII. It was evidenced by the observations that alpha-1,3 FuT-VII was the main alpha-1,3/1,4 FuT in 7721 cells, while alpha-1,3/1,4 FuT-III and alpha-1,3 FuT-VI were expressed rather low. The changes in the expressions of SLe(x)antigen and alpha-1,3 FuT-VII resulted in the altered cell adhesion to tumour necrosis factor-alpha stimulated HUVEC, since only the monoclonal antibody of the SLe(x), but not other monoclonal antibodies blocked the adhesion of 7721 cells to HUVEC. The migration and invasion of 7721 cells were also reduced by the treatment of ATRA or 8-Br-cAMP, and elevated by EGF or PMA. The above findings indicate that the metastatic potential of 7721 cells is suppressed by differentiation-inducers and promoted by proliferation-inducers.
8-Bromo Cyclic Adenosine Monophosphate↗
Diminished cardioprotective response to inhibition of angiotensin-converting enzyme and angiotensin II type 1 receptor in B(2) kinin receptor gene knockout mice.
Using B(2) kinin receptor gene knockout mice (B(2)(-/-)), we tested the hypothesis that (l) lack of B(2) receptors may affect blood pressure and cardiac function and aggravate cardiac remodeling after myocardial infarction (MI), and (2) kinins partially mediate the cardiac beneficial effect of angiotensin-converting enzyme inhibitors (ACEi) or angiotensin II type 1 receptor antagonists (AT(1)-ant), whereas lack of B(2) receptors may diminish this cardioprotective effect. Chronic heart failure (HF) was induced by MI, which was caused by coronary artery ligation in both B(2)(-/-) and 129/SvEvTac mice (wild-type control, B(2)(+/+)). An ACEi (ramipril, 2.5 mg/kg/d) or AT(1)-ant (L-158809, 3 mg/kg/d) was given 1 week after MI and was continued for 12 weeks. Left ventricular (LV) ejection fraction, cardiac output (CO), diastolic LV dimension (LVDd), and LV mass were evaluated by echocardiography. Myocyte cross-sectional area and interstitial collagen fraction were studied histopathologically. We found that basal blood pressure and cardiac function were similar in B(2)(+/+) and B(2)(-/-) mice. After MI, development of HF and remodeling were also similar between the 2 strains. The ACEi improved cardiac function and remodeling in both strains; however, its effects were attenuated in B(2)(-/-) mice (respective values for B(2)(+/+) versus B(2)(-/-) mice: overall increase in ejection fraction, 64+/-10% versus 21+/-5% [P<0.01]; increase in CO, 69+/-17% versus 23+/-9% [P<0.01]; overall decrease in LVDd, -24+/-3% versus -7+/-4% [P<0.01]; and decrease in LV mass, -38+/-3% versus -6+/-6% [P<0.01]). AT(1)-ant had a beneficial cardiac effect similar to that produced by ACEi, and this effect was also diminished in B(2)(-/-) mice (respective values for B(2)(+/+) versus B(2)(-/-) mice: overall increase in ejection fraction, 46+/-10% versus 25+/-9% [P<0.01]; increase in CO, 44+/-14% versus 15+/-5% [P<0.01]; overall decrease in LVDd, -14+/-4% versus -6+/-3% [P<0.01]; and decrease in LV mass, -33+/-4 versus -16+/-7% [P<0.01]). The effect of ACEi or AT(1)-ant on myocyte cross-sectional area was similar between strains; however, their effect on the interstitial collagen fraction was diminished in B(2)(-/-) mice. We concluded that (1) lack of B(2) kinin receptors does not affect cardiac phenotype or function, either under normal physiological conditions or during the development of HF; and (2) kinins acting via the B(2) receptor play an important role in the cardioprotective effect of ACEi and AT(1)-ant.
Angiotensin Receptor Antagonists↗
[Keratin 17 gene mutation in patients with steatocystoma multiplex].
OBJECTIVE: To study the relationship between steatocystoma multiplex (SCM) and keratin 17 gene mutation. METHODS: The keratin 17 gene mutation in the cDNA of cystic tissue of 5 patients of SCM and in the DNA in peripheral blood of 25 patients with SCM from a SCM family was studied by direct sequencing of the RT-PCR products, nested PCR, and restricted fraction length polymorphism (RFLP) analysis. Thirty-nine blood specimens from the unaffected members of that family were collected and tested too. Ten DNA pool specimens and other 2000 DNA pool specimens of normal individuals outside that SCM family were used as controls. RESULTS: In the base 428, 94(th) codon in keratin 17 gene in the cDNA of patients' cystic tissue, R94C mutation, a G-->A mutation, was detected Nested PCR, and restricted enzyme Acil polymorphism analysis showed that in the DNA specimens of peripheral blood of patients a mutated allele lacking enzyme cutting locus was detected, thus causing an uncut band with 200 bp while the corresponding allele was cut and caused two bands with 108 bp and 92 bp. In the DNA pool specimens of normal controls only these two bands with 108 bp and 92 bp were observed. CONCLUSION: The R94C mutation in keratin 17 gene is one of the genetic bases of SCM in Chinese. The results of this study provide scientific data for genetic diagnosis and counseling of SCM.
Adolescent↗