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L Wu

Publications and source records attributed to L Wu.

At least 361 records · Page 20Linked to original sources
PubMed1998

Altering the nucleophile specificity of a protein-tyrosine phosphatase-catalyzed reaction. Probing the function of the invariant glutamine residues.

Protein-tyrosine phosphatases (PTPases) catalysis involves a cysteinyl phosphate intermediate, in which the phosphoryl group cannot be transferred to nucleophiles other than water. The dual specificity phosphatases and the low molecular weight phosphatases utilize the same chemical mechanism for catalysis and contain the same (H/V)C(X)5R(S/T) signature motif present in PTPases. Interestingly, the latter two groups of phosphatases do catalyze phosphoryl transfers to alcohols in addition to water. Unique to the PTPase family are two invariant Gln residues which are located at the active site. Mutations at Gln-446 (and to a much smaller extent Gln-450) to Ala, Asn, or Met (but not Glu) residues disrupt a bifurcated hydrogen bond between the side chain of Gln-446 and the nucleophilic water and confer phosphotransferase activity to the Yersinia PTPase. Thus, the conserved Gln-446 residue is responsible for maintaining PTPases' strict hydrolytic activity and for preventing the PTPases from acting as kinases to phosphorylate undesirable substrates. This explains why phosphoryl transfer from the phosphoenzyme intermediate in PTPases can only occur to water and not to other nucleophilic acceptors. Detailed kinetic analyses also suggest roles for Gln-446 and Gln-450 in PTPase catalysis. Although Gln-446 is not essential for the phosphoenzyme formation step, it plays an important role during the hydrolysis of the intermediate by sequestering and positioning the nucleophilic water in the active site for an in-line attack on the phosphorus atom of the cysteinyl phosphate intermediate. Gln-450 interacts through a bound water molecule with the phosphoryl moiety and may play a role for the precise alignment of active site residues, which are important for substrate binding and transition state stabilization for both of the chemical steps.

Bacterial Proteins↗
PubMed1998

Beta-chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK.

Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1 beta (MIP 1 beta) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the down-stream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/ SAPK activity. Thus, RAFTK appears to provide a functional "bridge" for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

Animals↗
PubMed1998

Analysis of PTEN/MMAC1 alterations in aerodigestive tract tumors.

PTEN/MMAC1 is a candidate tumor suppressor gene recently identified at chromosomal band 10q23. It is mutated in sporadic brain, breast, and prostate cancer and in the germ line of patients with hereditary Cowden disease. We searched for genetic alterations of the PTEN/MMAC1 gene in 39 primary head and neck cancers (HNSCCs), 42 primary non-small cell lung cancers (NSCLCs), 80 pancreatic cancer xenografts, and 37 cell lines and xenografts from colon, lung, and gastric cancers. Microsatellite analysis revealed loss of heterozygosity at markers near the gene in 41% of primary HNSCCs, 50% of NSCLCs, and 39% of the pancreatic cancers. Three cases of HNSCCs displayed homozygous deletion involving the gene. We sequenced the entire coding region of the PTEN/MMAC1 gene in the remaining tumors displaying loss of heterozygosity and found one terminating mutation in a HNSCC sample. Thus, a second inactivation event was observed in 4 of 39 primary HNSCC cases. By use of a protein truncation assay, one terminating mutation was also identified in one of eight NSCLC cell lines. Our results suggest that PTEN/MMAC1 gene inactivation plays a role in the genesis of some tumor types.

Carcinoma, Non-Small-Cell Lung↗
PubMed1998

Regional gene therapy with a BMP-2-producing murine stromal cell line induces heterotopic and orthotopic bone formation in rodents.

The ability to continuously deliver osteoinductive proteins to a specific anatomic site would facilitate the treatment of fracture nonunions and other clinical problems associated with bone loss. We have developed a murine model of regional gene therapy. A bone-marrow stromal cell line infected with an adenovirus expressing recombinant bone morphogenetic protein-2 cDNA secreted biologically active bone morphogenetic protein-2. These bone morphogenetic protein-2-producing cells were able to induce abundant heterotopic bone formation when implanted into the quadriceps muscle of severe combined immune deficient mice and also successfully healed large segmental femoral defects in nude rats. These studies demonstrate that regional gene therapy with continuous delivery of osteoinductive factors to a specific anatomic site can enhance the formation and repair of bone.

Animals↗
PubMed1998

Distribution of free seleno-amino acids in plant tissue of Melilotus indica L. grown in selenium-laden soils.

Accumulation of specific groups of seleno-amino acids in plant tissue reflects not only the Se tolerance of a plant species, but also Se toxicity to animals. The distribution of seleno-amino acids in a Se-tolerant grassland legume species (Melilotus indica L.) grown in Se-laden soils was studied using high-resolution gas chromatography- and gas chromatography-mass spectrometry. Five seleno-amino acids including selenocystine, selenomethionine, selenocysteine, Se-methylselenocysteine, and gamma-glutamyl-Se-methylselenocysteine were identified and measured for their plant tissue concentrations. Se-methylselenocysteine, a nonprotein seleno-amino acid, was found in the plant tissue. Its concentration ranged from 15.3 mumol kg-1 for the plants growing in soil of low Se concentration to 109.8 mumol kg-1 for the plants grown in soil of high Se concentration. Accumulation of the nonprotein seleno-amino acid in this species resembles that in Se accumulator plants. gamma-Glutamyl-Se-methylselenocysteine was detected in the plant. However, its concentration was very low. It might not become a toxic element in the food chain. Results of plant tissue Se accumulation analysis indicated that there was a five-fold increase in tissue selenocysteine concentration when the total tissue Se increased from 5.07 to 22.02 mg kg-1, but there was no further increase in tissue selenocysteine concentration when the tissue total Se concentration increased from 22.0 to 117.4 mg kg-1. Selenomethinone constituted more than 50% of the total seleno-amino acid in the plant. More research is needed to reveal whether the mechanisms limiting the accumulation of selenocysteine and preferential accumulation of selenomethionine found in this study play any role in Se tolerance in this species.

Amino Acids↗
PubMed1998

Recombinant production of cyanovirin-N, a potent human immunodeficiency virus-inactivating protein derived from a cultured cyanobacterium.

Here we describe the recombinant production and purification of a novel anti-human immunodeficiency virus (HIV) protein, cyanovirin-N (CV-N), in Escherichia coli. Initial attempts to express CV-N using a vector containing an ompA signal peptide sequence resulted in production of an intractable mixture of the full-length (101 amino acid residue) protein and a truncated form lacking the first two N-terminal amino acids. The truncated protein was observed regardless of the host cell line, culture conditions, or induction time. These observations suggested that an as yet unidentified protease or peptidase was responsible for proteolytic cleavage between the second and third N-terminal amino acids of CV-N when presented as an ompA-CV-N fusion protein. When the ompA signal peptide sequence was replaced by a pelB signal peptide sequence, CV-N was produced in high yield as a single, homogeneous protein. This was confirmed by electrospray ionization mass spectrometry and N-terminal sequencing. This expression system provides a basis for large-scale production of clinical grade CV-N for further research and development as an anti-HIV microbicide.

Amino Acid Sequence↗
PubMed1998

KRC transcripts: identification of an unusual alternative splicing event.

Mouse KRC is a large zinc finger protein that binds to the kappaB motif of gene transcription and to the recognition signal sequences for the somatic recombination of the immunoglobulin and T-cell receptor gene segments. The mouse KRC gene is more than 70 kilobases (kb) in size, and contains at least seven exons, with the largest transcript being approximately 9.5 kb. Multiple differentially spliced transcripts of KRC were identified in thymus and brain, which would result in the production of multiple KRC protein isoforms with different N-termini and number of DNA binding domains. Alternative splicing events leading to the production of these multiple transcripts have been elucidated. Of particular interest are the exclusions in some transcripts of sequences from a gigantic exon of 5487 base pairs (bp), or from an exon of 176 bp. Both potentially deleted exons code for zinc finger motifs that are essential components of the N-terminal and C-terminal DNA binding domains, respectively. Another intriguing phenomenon found in some KRC transcripts is the skipping of a 459 bp fragment within the gigantic exon that would code for the N-terminal DNA binding domain. Bacterial fusion proteins derived from this fragment bind specifically to KRC target DNAs. Apparently, distinct alternative splicing events could eliminate the N-terminal DNA binding domain of KRC.

Alternative Splicing↗
PubMed1998

Segmentation and classification of EEG during epileptic seizures.

We present a method for the automatic comparison of epileptic seizures in EEG, allowing the grouping of seizures having similar overall patterns. Each channel of the EEG is first broken down into segments having relatively stationary characteristics. Features are then calculated for each segment and all segments of all channels of the seizures of one patient are grouped into clusters of similar morphology. This clustering allows labeling of every EEG segment. Methods derived from string matching procedures are then used to obtain an overall edit distance between two seizures, a distance that represents how the two seizures, taken in their entirety and including the channels not actually involved in the discharge, resemble each other. Examples from 5 patients, 3 with intracerebral electrodes and two with scalp electrodes, illustrate the ability of the method to group seizures of similar morphology.

Algorithms↗
PubMed1998

Increased fiber capillarization in flight muscle of finch at altitude.

We examined fiber capillarization and ultrastructure in the highly aerobic flight muscle of six gray crowned rosy finches (Leucosticte arctoa; mass 22.9 +/- 0.5 (SE) g) living at altitude (A; White Mountains of Eastern California; 4000 m) compared to eight sea-level (SL) house finches (Carpodacus mexicanus, mass, 19.8 +/- 0.6 g) of the same subfamily, Carduelinae. Capillary length per fiber volume (A, 10,400 +/- 409 mm(-2); SL, 7513 +/- 423; P < 0.001) and capillary-to-fiber ratio (A, 2.32 +/- 0.07; SL, 1.85 +/- 0.06; P < 0.001) were significantly greater in A, with no difference in fiber cross-sectional area compared to SL. Capillary geometry was significantly different in A, yielding a greater contribution of tortuosity and branching to capillary length than in SL. Capillary-to-fiber surface ratio and fiber mitochondrial volume were both greater in A, but their ratio was similar to SL, indicating a proportional increase in the size of the capillary to fiber interface and fiber mitochondrial volume in A to sustain high levels of aerobic capacity while living at altitude.

Adaptation, Physiological↗
PubMed1998

Impaired basal sympathetic tone and alpha1-adrenergic responsiveness in association with the hypotensive effect of melatonin in spontaneously hypertensive rats.

Early investigations have suggested a relationship between hypertension and melatonin, a pineal hormone. The aims of this study were to evaluate the implication of the sympathetic nervous system in the acute effect of melatonin on blood pressure in conscious 12-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY), and to determine whether the hypotensive effect of melatonin is associated with alterations in pre- or postsynaptic mechanisms. Melatonin, 10 mg/kg, produced a sustained time-dependent decrease of mean arterial pressure only in SHR without changes in heart rate in both groups. Until 20 min after melatonin administration, plasma epinephrine (EPI) levels were reduced by about 60% in both groups, but norepinephrine (NE) levels were decreased only in SHR by about 30%. The nitroprusside-induced hypotension responses and the associated increases in heart rate were similar in both groups before or after administration of melatonin. Unexpectedly, the sympathetic reactivity to nitroprusside, evaluated by the increases in NE and EPI, was markedly enhanced after melatonin treatment in both WKY and SHR. The stimulation induced [3H]-norepinephrine release from isolated atria was not altered by melatonin in SHR. In cultured aortic vascular smooth muscle cells, the basal and phenylephrine induced inositol phosphate formations were greater in SHR, and the melatonin pretreatment dose dependently attenuated the phenylephrine responses in cells from both WKY and SHR. Therefore the hypotensive action of melatonin appears to be associated with an inhibition of basal sympathoadrenal tone and could also be mediated partly by the blockade of postsynaptic alpha1-adrenergic receptor-induced inositol phosphate formation.

Animals↗
PubMed1998

CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of alpha-CaMKII mRNA at synapses.

Long-term changes in synaptic efficacy may require the regulated translation of dendritic mRNAs. While the basis of such regulation is unknown, it seemed possible that some features of translational control in development could be recapitulated in neurons. Polyadenylation-induced translation of oocyte mRNAs requires the cis-acting CPE sequence and the CPE-binding protein CPEB. CPEB is also present in the dendritic layers of the hippocampus, at synapses in cultured neurons, and in postsynaptic densities of adult brain. alpha-CaMKII mRNA, which is localized in dendrites and is necessary for synaptic plasticity and LTP, contains two CPEs. These CPEs are bound by CPEB and mediate polyadenylation-induced translation in injected Xenopus oocytes. In the intact brain, visual experience induces alpha-CaMKII mRNA polyadenylation and translation, suggesting that this process likely occurs at synapses.

Animals↗
PubMed1998

Comparative evaluation of three recombinant antigen-based enzyme immunoassays for detection of IgM and IgG antibodies to human parvovirus B19.

BACKGROUND: Diagnosis of acute and past infection with parvovirus B19 is based on detection of IgM and IgG antibodies. OBJECTIVES: To evaluate two commercial recombinant antigen-based enzyme immunoassay (EIA) test kits for detection of IgM and IgG antibodies to parvovirus B19 and to compare the commercial EIAs to in-house EIA test procedures. STUDY DESIGN: A panel of 121 sera was used to compare the three IgM EIAs. The panel included 84 sera submitted for parvovirus B19 testing and 37 sera that were IgM positive for other viral pathogens. The same serum panel plus an additional 14 sera submitted for B19 testing was used to compare the three IgG EIAs. The commercial EIAs were performed according to manufacturers' instructions. Using the in-house EIA test procedures as the reference, sensitivity and specificity for each of the commercial EIAs was determined. RESULTS: The commercial B19 IgM EIAs showed agreements of 95.0 and 93.4% to the in-house IgM EIA. Compared to the in-house B19 IgM EIA, the commercial B19 IgM EIAs were 97.4 and 97.5% sensitive, respectively. Specificities were 93.5 and 91.4%, respectively. Sensitivities for the commercial IgG EIAs, compared to in-house IgG EIA, were 88.0 and 85.2%, respectively, and specificities were 94.1 and 98.0%. CONCLUSION: We found that the commercial parvovirus B19 IgM and IgG EIAs are comparable to standard in-house EIAs and are suitable for testing for B19 antibodies in human sera.

Antibodies, Viral↗
PubMed1998

Structure-based design and synthesis of small molecule protein-tyrosine phosphatase 1B inhibitors.

Protein-tyrosine phosphatase (PTP) inhibitors are attractive as potential signal transduction-directed therapeutics which may be useful in the treatment of a variety of diseases. We have previously reported the X-ray structure of 1,1-difluoro-1-(2-naphthalenyl)methyl] phosphonic acid (4) complexed with the human the protein-tyrosine phosphatase 1B (PTP1B) and its use in the design of an analogue which binds with higher affinity within the catalytic site (Burke, T. R., Jr. et al. Biochemistry 1996, 35, 15989). In the current study, new naphthyldifluoromethyl phosphonic acids were designed bearing acidic functionality intended to interact with the PTP1B Arg47, which is situated just outside the catalytic pocket. This residue has been shown previously to provide key interactions with acidic residues of phosphotyrosyl-containing peptide substrates. Consistent with trends predicted by molecular dynamics calculations, the new analogues bound with 7- to 14-fold higher affinity than the parent 4, in principal validating the design rationale.

Drug Design↗
PubMed1998

RelB is essential for the development of myeloid-related CD8alpha- dendritic cells but not of lymphoid-related CD8alpha+ dendritic cells.

The transcription factor RelB had been shown to be important for dendritic cell (DC) development, but the type of DC involved was not clear. Here, we report that RelB mRNA is expressed strongly in CD8alpha- DEC-205- DC but only weakly in CD8alpha+ DEC-205+ DC. In addition, CD8alpha+ DEC-205+ DC are present and functional in RelB null mice, the DC deficiency being mainly in the CD8alpha- DEC-205- population. By constructing bone-marrow chimeric mice, we demonstrate that the partial deficiency in RelB null thymic DC is a secondary effect of disrupted thymic architecture. However, the deficiency in splenic CD8alpha- DEC-205- DC is a direct, stem cell intrinsic effect of the RelB mutation. Thus, RelB selectively regulates a myeloid-related DC lineage.

Animals↗
PubMed1998

Role of the Ped gene and apoptosis genes in control of preimplantation development.

PURPOSE: The properties of the mouse Ped gene and the genes that mediate apoptosis in mediating preimplantation embryonic survival were reviewed. METHODS: Preimplantation mouse oocytes and embryos were evaluated microscopically and biochemically for rate of development, degree of fragmentation, and gene expression to correlate these characteristics with embryo mortality, Biochemical assays included PCR for DNA analysis, RT-PCR for mRNA analysis, immuno-PCR for protein analysis, and TUNEL assay for assessment of apoptosis. RESULTS: Using the mouse as a model system we have identified a gene that controls the rate of development, the Ped gene. The Ped gene product is a class Ib major histocompatibility complex protein called the Qa-2 antigen. Research to understand the molecular mechanisms of Ped gene action and to identify the human homologue of the Ped gene is under way. We have also shown using the mouse model, that fragmented embryos show the morphological and biochemical characteristics of apoptosis. Genes in the two major gene families that regulate apoptosis, the caspase and Bcl-2 families, are expressed in mouse oocytes and preimplantation embryos. CONCLUSIONS: Preimplantation embryonic survival depends on two major morphological parameters: rate of development and degree of fragmentation. A fast rate of development and a low degree of fragmentation lead to a better chance of producing live offspring. Both rate of development and degree of fragmentation are genetically controlled, the former by the Ped gene and the latter most likely by genes that mediate apoptosis. It seems probable that regulation of apoptosis will prove to be a major mechanism that mediates oocyte and preimplantation embryonic survival.

Animals↗
PubMed1998

Preparation and hybridization analysis of DNA/RNA from E. coli on microfabricated bioelectronic chips.

Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.

Biosensing Techniques↗
PubMed1998

Kinin B2 receptor-mediated contraction of tail arteries from normal or streptozotocin-induced diabetic rats.

1. The vasoactive effects of bradykinin (BK) are mediated by different subtypes of kinin receptors, of which the expression varies among different tissues. In rat tail artery tissues, BK elicited a concentration-dependent vasoconstriction (EC50, 25.9+/-2.4nM; Emax, 0.39+/-0.01 g; n=16). This effect of BK was endothelium independent and indomethacin insensitive. The BK-induced contraction of tail artery tissues, however, depended on both membrane potential-sensitive extracellular Ca2+ entry and thapsigargin-sensitive intracellular Ca2+ release. 2. Kinin B1 receptor antagonist or agonist did not affect the basal tension or the BK-induced contraction of tail artery tissues in the absence or presence of endothelium (P>0.05). In contrast, the BK-induced vasoconstriction was inhibited by kinin B2 receptor antagonists. Pretreatment of vascular tissues with Hoe 140 (1 nM) significantly changed EC50 of the BK-induced vasoconstriction from 25.5+/-7.4 nM to 82.6+/-16.8nM (n=8, P<0.01) and Emax from 0.43+/-0.03g to 0.16+/-0.01 g (n=8, P<0.01). 3. In the tail artery tissues from streptozotocin-induced diabetic rats, the BK-elicited vasoconstriction was significantly reduced (EC50, 67.8+/-11 nM: Emax, 0.19+/-0.01 g) compared to their counterparts from normal rats. The decreased vasoconstrictive effects of BK on diabetic arteries were endothelium independent and indomethacin insensitive. 4. Our study demonstrated that the contraction of rat tail arteries induced by BK was mediated by B2 receptors located on vascular smooth muscles. The altered B2 receptor-mediated vascular activity may play an important role in the vascular complications of diabetes.

Animals↗