Biomedical subjects

J Liu

Publications and source records attributed to J Liu.

At least 883 records · Page 49Linked to original sources

PubMed1999

The novel tyrosine kinase ZAK1 activates GSK3 to direct cell fate specification.

Inhibition of GSK3 by 7-TM Wnt/wg receptor signaling is critical for specifying embryonic cell fate patterns. In Dictyostelium, the 7-TM cAMP receptors regulate GSK3 by parallel, antagonistic pathways to establish a developmental body plan. We describe here a novel tyrosine kinase, ZAK1, downstream of 7-TM cAMP receptor signaling that is required for GSK3 activation during development. zak1-nulls have reduced GSK3 activity and are defective in GSK3-regulated developmental pathways. Moreover, recombinant ZAK1 phosphorylates and activates GSK3 in vitro. We propose that ZAK1 is a positive regulator of GSK3 activity required for cell pattern formation in Dictyostelium and speculate that similar mechanisms exist to antagonize Wnt/wg signaling for metazoan cell fate specification.

Amino Acid Sequence↗

PubMed1999

A 13-amino acid amphipathic alpha-helix is required for the functional interaction between the transcriptional repressor Mad1 and mSin3A.

Members of the Mad family of bHLHZip proteins heterodimerize with Max and function to repress the transcriptional and transforming activities of the Myc proto-oncogene. Mad:Max heterodimers repress transcription by recruiting a large multi-protein complex containing the histone deacetylases, HDAC1 and HDAC2, to DNA. The interaction between Mad proteins and HDAC1/2 is mediated by the corepressor mSin3A and requires sequences at the amino terminus of the Mad proteins, termed the SID, for Sin3 interaction domain, and the second of four paired amphipathic alpha-helices (PAH2) in mSin3A. To better understand the requirements for the interaction between the SID and PAH2, we have performed mutagenesis and structural studies on the SID. These studies show that amino acids 8-20 of Mad1 are sufficient for SID:PAH2 interaction. Further, this minimal 13-residue SID peptide forms an amphipathic alpha-helix in solution, and residues on the hydrophobic face of the SID helix are required for interaction with PAH2. Finally, the minimal SID can function as an autonomous and portable repression domain, demonstrating that it is sufficient to target a functional mSin3A/HDAC corepressor complex.

Amino Acid Sequence↗

PubMed1999

Smad3 inhibits transforming growth factor-beta and activin signaling by competing with Smad4 for FAST-2 binding.

Transcriptional regulation by transforming growth factor-beta and activin is mediated by interaction of Smad2 and Smad3 with specific transcription factors and/or DNA elements. However, Smad3 behaves differently from Smad2 in regulating transcription by a winged-helix transcription factor, FAST-2, on an activin-responsive element (ARE) in the Xenopus Mix.2 promoter. Smad3 alone was able to stimulate the ARE through FAST-2, but inhibited the ARE transactivation mediated by Smad2/Smad4 following receptor activation. We characterized the functional domains that are involved in these two activities of Smad3. Deletion of the MH1 domain as well as mutations of four lysine residues in the MH1 domain abrogated the inhibitory activity of Smad3, but did not compromise the self-stimulatory function. In contrast, deletion of the MH2 domain or a point mutation of glycine 379 within this domain obliterated the self-stimulatory activity of Smad3, but not the inhibitory activity. In an electrophoretic mobility shift assay, we found that Smad3 was able to associate with the FAST-2.ARE complex and that this association was dependent on FAST-2. In addition, Smad3 was not able to directly bind the ARE in a DNase I protection assay, in which FAST-2 binds the ARE around a motif (TGTGTATT) previously characterized to associate with the human FAST-1 protein. Interestingly, Smad4 was also able to directly associate with the FAST-2.ARE complex through binding with FAST-2. In a gel shift assay, the association of FAST-2 with Smad4 was mutually exclusive from the association with Smad3. Taken together, these data indicate that Smad3 exerts the inhibitory activity by competitive association with FAST-2.

Activin Receptors↗

PubMed1999

Heterodimeric tacrine-based acetylcholinesterase inhibitors: investigating ligand-peripheral site interactions.

Dimeric acetylcholinesterase (AChE) inhibitors containing a single 9-amino-1,2,3,4-tetrahydroacridine (tacrine) unit were constructed in an effort to further delineate structural requirements for optimal binding to the AChE peripheral site. Basic amines of differing hydrophobicity were selected as peripheral site ligands, and in each case, improvements in inhibitory potency and selectivity were seen relative to tacrine itself. AChE IC(50) values of the optimum dimers decrease significantly as the peripheral site ligand was permuted in the series ammonia > dimethylamine > 4-aminopyridine > 4-aminoquinoline > tacrine. Calculated desolvation free energies of the optimum dimers match the trend in IC(50) values, suggesting the importance of ligand hydrophobicity for effective cation-pi interaction with the peripheral site.

Acetylcholinesterase↗

PubMed1999

A novel role for 3-O-sulfated heparan sulfate in herpes simplex virus 1 entry.

Herpes simplex virus type 1 (HSV-1) binds to cells through interactions of viral glycoproteins gB and gC with heparan sulfate chains on cell surface proteoglycans. This binding is not sufficient for viral entry, which requires fusion between the viral envelope and cell membrane. Here, we show that heparan sulfate modified by a subset of the multiple D-glucosaminyl 3-O-sulfotransferase isoforms provides sites for the binding of a third viral glycoprotein, gD, and for initiation of HSV-1 entry. We conclude that susceptibility of cells to HSV-1 entry depends on (1) presence of heparan sulfate chains to which virus can bind and (2) 3-O-sulfation of specific glucosamine residues in heparan sulfate to generate gD-binding sites or the expression of other previously identified gD-binding receptors.

Amino Acid Substitution↗

PubMed1999

Differential roles of Ca(2+)/calmodulin-dependent protein kinase II and mitogen-activated protein kinase activation in hippocampal long-term potentiation.

The roles of Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) and mitogen-activated protein kinase (MAPK) in long-term potentiation (LTP) were investigated in the CA1 area of hippocampal slices, using electrophysiological and biochemical approaches. A brief high-frequency stimulation, but not low-frequency stimulation, delivered to Schaffer collateral/commissural afferents produced a stable LTP and activated both CaM kinase II and 42 kDa MAPK. Different from the activity of CaM kinase II, the increase in MAPK activity was transient. At a concentration of 50 microM, but not of 30 microM, PD098059, a potent inhibitor of MAPK kinase, markedly inhibited the induction of LTP. Although the two concentrations had similar inhibitory effects on MAPK activity, only 50 microM PD098059 suppressed the activation of CaM kinase II. Application of calmidazolium, an antagonist of calmodulin, blocked both CaM kinase II activation and the LTP induction without affecting the increase in 42 kDa MAPK activity. Application of neurotrophin brain-derived neurotrophic factor (BDNF) promoted the induction of LTP, with concomitant activation of CaM kinase II. Under the same conditions, BDNF failed to activate MAPK in hippocampal slices. These results indicate that, although the LTP induction is accompanied by increases in two kinase activities, only CaM kinase II activation is required for this event.

Animals↗

PubMed1999

[Preparation and identification of anti-fumonisin B1 monoclonal antibody].

Spleen cells from Balb/c mice immunized with an artificially KLH-glutaraldehyde fumonisin B1(FB1) conjugate were fused with murine Sp2/0 myeloma cells. One hybridoma cell line secreting monoclonal antibody against FB1 was established after the fusion cells subcloned for 4 cycles. This antibody was designated as FB(1)4G4. The titer of the antibody from ascites was 1:1 x 10(7).

Animals↗

PubMed1999

[Progress in detecting methods on paralytic shellfish poisons].

Paralytic shellfish poisons have led to uncountable economic losses, and done potential harm to human health now. In order to overcome the limits of the traditional bioassay with mouse, many novel detecting methods have been developed, which made it possible to detect the paralytic shellfish poisons with sensitive, high effective and rapid methods. This paper systematically introduces the progress in research on paralytic shellfish poisons detection.

Biological Assay↗

PubMed1999

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.

Animals↗

PubMed1999

Binding of correolide to K(v)1 family potassium channels. Mapping the domains of high affinity interaction.

Correolide, a novel nortriterpene natural product, potently inhibits the voltage-gated potassium channel, K(v)1.3, and [(3)H]dihydrocorreolide (diTC) binds with high affinity (K(d) approximately 10 nM) to membranes from Chinese hamster ovary cells that express K(v)1.3 (Felix, J. P., Bugianesi, R. M., Schmalhofer, W. A., Borris, R., Goetz, M. A., Hensens, O. D., Bao, J.-M., Kayser, F. , Parsons, W. H., Rupprecht, K., Garcia, M. L., Kaczorowski, G. J., and Slaughter, R. S. (1999) Biochemistry 38, 4922-4930). Mutagenesis studies were used to localize the diTC binding site and to design a high affinity receptor in the diTC-insensitive channel, K(v)3.2. Transferring the pore from K(v)1.3 to K(v)3.2 produces a chimera that binds peptidyl inhibitors of K(v)1.3 with high affinity, but not diTC. Transfer of the S(5) region of K(v)1.3 to K(v)3.2 reconstitutes diTC binding at 4-fold lower affinity as compared with K(v)1.3, whereas transfer of the entire S(5)-S(6) domain results in a normal K(v)1.3 phenotype. Substitutions in S(5)-S(6) of K(v)1.3 with nonconserved residues from K(v)3.2 has identified two positions in S(5) and one in S(6) that cause significant alterations in diTC binding. High affinity diTC binding can be conferred to K(v)3.2 after substitution of these three residues with the corresponding amino acids found in K(v)1.3. These results suggest that lack of sensitivity of K(v)3.2 to diTC is a consequence of the presence of Phe(382) and Ile(387) in S(5), and Met(458) in S(6). Inspection of K(v)1.1-1.6 channels indicates that they all possess identical S(5) and S(6) domains. As expected, diTC binds with high affinity (K(d) values 7-21 nM) to each of these homotetrameric channels. However, the kinetics of binding are fastest with K(v)1.3 and K(v)1.4, suggesting that conformations associated with C-type inactivation will facilitate entry and exit of diTC at its binding site. Taken together, these findings identify K(v)1 channel regions necessary for high affinity diTC binding, as well as, reveal a channel conformation that markedly influences the rate of binding of this ligand.

Amino Acid Sequence↗

PubMed1999

TTID: A novel gene at 5q31 encoding a protein with titin-like features.

A deletion of the long arm of chromosome 5 is a recurring abnormality in malignant myeloid disorders. In previous studies, we identified an approximately 1-Mb segment in 5q31 that was deleted in all patients examined. As part of a positional cloning project to identify transcribed sequences in this region, we identified and characterized the TTID gene. This gene contains 10 exons that extend over 19 kb. The composite cDNA is approximately 2.3 kb and encodes a protein of 498 amino acids, with a predicted molecular mass of 55 kDa. The C-terminal half of this putative protein contains an internally repeated domain of 43 amino acids, which resembles the N-terminal half of an immunoglobulin domain from the immense skeletal muscle protein titin. The TTID gene is expressed in multiple muscle tissue types as well as in thyroid gland and bone marrow. We evaluated the gene as a candidate tumor suppressor gene by searching for mutations in malignant myeloid disorders with abnormalities of chromosome 5. However, we detected no inactivating mutations. A single nucleotide change (G to A) was identified at nucleotide position 1889 in the untranslated region of the mRNA, which may represent a polymorphism. Therefore, TTID is unlikely to be the candidate tumor suppressor gene involved in malignant myeloid disorders.

Amino Acid Sequence↗

PubMed1999

Metallothionein-null mice are highly susceptible to the hematotoxic and immunotoxic effects of chronic CdCl2 exposure.

Cadmium (Cd) is an environmental pollutant. Chronic exposure of humans to Cd results in various maladies, including anemia and altered immune function. Metallothionein (MT) has been proposed to play an important role in Cd detoxication. Thus, we hypothesized that intracellular MT protects against Cd-induced hematotoxicity and immunotoxicity. Control and MT-I/II knock-out (MT-null) mice were given s.c. injections of CdCl2 over a wide range of doses, 6 times/week for up to 10 weeks. Cd-induced anemia was evident after 5 weeks of exposure and progressed with time. MT-null mice were about 10 times more susceptible to Cd-induced anemia, as evidenced by decreased erythrocytes (25%), hemoglobin concentration (30%), and hematocrit (35%) after 10 weeks of Cd injections. Cd produced dose- and time-dependent increases in neutrophils (7x), along with a marked elevation of serum IL-1 beta (6x) and TNF-alpha (20x) levels. MT-null mice were more susceptible than controls to Cd-induced alterations in peripheral leukocytes and cytokine levels. Chronic exposure to Cd also produced dose- and time-dependent splenomegaly (5x), with loss of lymphoid structure, inflammation, hyperplasia, appearance of giant cells, and fibrosis. Thymus weights were decreased by Cd in a dose-dependent manner (60%). MT-null mice were also approximately 10 times more susceptible than controls to these lesions. In conclusion, the present study demonstrates that repeated injections of Cd produces hematotoxic and immunotoxic effects, and intracellular MT protects against these chronic Cd-induced effects.

Anemia↗

PubMed1999

Structure of d(CGCGAATTCGCG) in the presence of Ca(2+) ions.

The dodecamer d(CGCGAATTCGCG) was the first oligonucleotide to be crystallized as a B-DNA duplex. Its structure was analyzed in detail in the early 1980s. Here we show that, in the presence of Ca(2+), it crystallizes in a different way (R3 space group). The dodecamers form parallel columns of straight duplexes with ten base pairs in the B form. The terminal cytosines in each molecule are disordered, whereas the terminal guanines are placed in the minor groove of neighbor duplexes. The central GAATTC region is practically identical to that found in the classic structure of the same dodecamer crystallized in the P2(1)2(1)2(1) space group in the presence of Mg(2+) and spermine. Its structure is thus independent of the crystallization conditions which have been used.

Calcium↗

PubMed1999

Two modes of PriA binding to DNA.

The role of PriA, required for the assembly of the phiX174-type primosome on DNA, in cellular DNA replication has been unclear since its discovery. Recent evidence, based on the phenotypes of strains carrying priA null mutations, has led to proposals that the primosome assembly activity of PriA was required to load replication forks at intermediates such as D loops during homologous recombination. McGlynn et al. (McGlynn, P., Al-Deib, A. A., Liu, J., Marians, K. J., and Lloyd, R. G. (1997) J. Mol. Biol. 270, 212-221) demonstrated that PriA could, in fact, bind D loops. We show here that there are two modes of stable binding of PriA to DNA. One mode, in which the enzyme binds 3'-single-stranded extensions from duplex DNAs, presumably reflects the 3' --> 5' DNA helicase activity of PriA. The D loop DNA binding activity of PriA can be accounted for by the second mode, where the enzyme binds bent DNA at three strand junctions.

Adenosine Triphosphatases↗

PubMed1999

PriA-directed assembly of a primosome on D loop DNA.

Escherichia coli strains carrying null mutations in priA are chronically induced for the SOS response and are defective in homologous recombination, repair of UV damaged DNA, double-strand break repair, and both induced and constitutive stable DNA replication. This led to the proposal that PriA directed replication fork assembly at D loops formed by the homologous recombination machinery. The demonstration that PriA specifically recognized and bound D loop DNA supported this hypothesis. Using DNA footprinting as an assay, we show here that PriA also directs the assembly of a varphiX174-type primosome on D loop DNA. The ability to load a complete primosome on D loop DNA is a step necessary for replication fork assembly.

Adenosine Triphosphatases↗

PubMed1999

Gas chromatographic-mass spectrometric analysis of dichlorobenzene isomers in human blood with headspace solid-phase microextraction.

Headspace solid-phase microextraction (HS-SPME) was utilized for the determination of three dichlorobenzene isomers (DCBs) in human blood. In the headspace at 30 degrees C, DCBs were absorbed for 15 min by a 100-micron polydimethylsiloxane (PDMS) fiber. They were then analyzed by capillary column gas chromatography-mass spectrometry (GC-MS). By setting the initial column oven temperature at 20 degrees C, the three isomers were resolved at the baseline level. p-Xylene-d10 was used as the internal standard (I.S.). For quantitation, the molecular ion at m/z 146 for each isomer and the molecular ion at m/z 116 for I.S. were selected. For day-to-day precision, relative standard deviations in the range 3.2-10.7% were found at blood concentrations of 1.0 and 10 micrograms/ml. Each compound was detectable at a level of at least 0.02 microgram per 1 g of whole blood (by full mass scanning). HS-SPME-GC-MS, when performed at relatively low temperatures, was found to be feasible in toxicological laboratories. Using this method, the plasma levels of one patient who had drunk a pesticide-like material were measured.

Adult↗

PubMed1999

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.

Crystallization↗

PubMed1999

Potent, easily synthesized huperzine A-tacrine hybrid acetylcholinesterase inhibitors.

Hybrid acetylcholinesterase inhibitors composed of a key fragment of huperzine A and an intact tacrine unit were prepared. The syntheses are quite direct, proceeding in a maximum of 4 linear steps from commercially available starting materials. The optimum hybrid inhibitor (+/-)-9g is 13-fold more potent than (-)-huperzine A, and 25-fold more potent than tacrine.

Acetylcholinesterase↗