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Biomedical subjects

D Wu

Publications and source records attributed to D Wu.

At least 109 records · Page 6Linked to original sources
PubMed2000

Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana.

Arabidopsis thaliana is an important model system for plant biologists. In 1996 an international collaboration (the Arabidopsis Genome Initiative) was formed to sequence the whole genome of Arabidopsis and in 1999 the sequence of the first two chromosomes was reported. The sequence of the last three chromosomes and an analysis of the whole genome are reported in this issue. Here we present the sequence of chromosome 3, organized into four sequence segments (contigs). The two largest (13.5 and 9.2 Mb) correspond to the top (long) and the bottom (short) arms of chromosome 3, and the two small contigs are located in the genetically defined centromere. This chromosome encodes 5,220 of the roughly 25,500 predicted protein-coding genes in the genome. About 20% of the predicted proteins have significant homology to proteins in eukaryotic genomes for which the complete sequence is available, pointing to important conserved cellular functions among eukaryotes.

Arabidopsis↗
PubMed2000

Adsorption of Amphiphilic Dimers at Surfaces.

Adsorption of amphiphilic dimers is analyzed in the framework of density functional Ono-Kondo theory. There are three configurations for dimers absorbed at a surface: one parallel to the surface and two perpendicular to the surface (AB and BA, with A or B touching the surface, respectively). Densities of molecules in each configuration are calculated from density functional theory and compared to Monte Carlo simulation data. There is good agreement between theory and simulations. It is shown that the parallel configuration is preferred over the perpendicular configuration, except when there are very strong asymmetries in intermolecular forces. In most cases, the parallel configuration is even preferred over the combination of the two perpendicular configurations. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Regulation of a novel human phospholipase C, PLCepsilon, through membrane targeting by Ras.

Phosphoinositide-specific phospholipase C (PI-PLC) plays a pivotal role in regulation of intracellular signal transduction from various receptor molecules. More than 10 members of human PI-PLC isoforms have been identified and classified into three classes beta, gamma, and delta, which are regulated by distinct mechanisms. Here we report identification of a novel class of human PI-PLC, named PLCepsilon, which is characterized by the presence of a Ras-associating domain at its C terminus and a CDC25-like domain at its N terminus. The Ras-associating domain of PLCepsilon specifically binds to the GTP-bound forms of Ha-Ras and Rap1A. The dissociation constant for Ha-Ras is estimated to be approximately 40 nm, comparable with those of other Ras effectors. Co-expression of an activated Ha-Ras mutant with PLCepsilon induces its translocation from the cytosol to the plasma membrane. Upon stimulation with epidermal growth factor, similar translocation of ectopically expressed PLCepsilon is observed, which is inhibited by co-expression of dominant-negative Ha-Ras. Furthermore, using a liposome-based reconstitution assay, it is shown that the phosphatidylinositol 4,5-bisphosphate-hydrolyzing activity of PLCepsilon is stimulated in vitro by Ha-Ras in a GTP-dependent manner. These results indicate that Ras directly regulates phosphoinositide breakdown through membrane targeting of PLCepsilon.

Amino Acid Sequence↗
PubMed2000

High-performance liquid chromatography with coulometric electrode array detector for the determination of quercetin levels in cells of the immune system.

There is mounting evidence emphasizing the importance of intracellular antioxidant levels for maintenance of the immune function. The flavonoid quercetin, a natural antioxidant, has been shown to modulate enzymes involved in the regulation of the inflammatory response. However, up to now, there have been no studies describing quercetin levels in cells of the immune system. A gradient reversed-phase HPLC technique to identify and quantify intracellular levels of quercetin and its application in mice splenocytes are described. Mobile phases were a 0.01 M sodium phosphate monobasic solution adjusted to pH 2.8 with 85% orthophosphoric acid (buffer, Solvent A) and methanol (Solvent B) with a flow rate of 1 ml/min. An eight-channel coulometric electrode array detector was used. In vitro supplementation with increasing concentration of quercetin (25, 50, and 100 microM) raises intracellular quercetin levels in a dose-dependent manner. The method has the required features of specificity and sensitivity for monitoring quercetin uptake in cells of the immune system.

Animals↗
PubMed2000

Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo.

In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine-DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine epsilon-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (+/-) ratios. Use of excess cationic charge in the polyplexes (+/- 2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.

Animals↗
PubMed2000

Identification of novel circulating human embryonic blood stem cells.

Using murine models, primitive hematopoietic cells capable of repopulation have been shown to reside in various anatomic locations, including the aortic gonad mesonephros, fetal liver, and bone marrow. These sites are thought to be seeded by stem cells migrating through fetal circulation and would serve as ideal targets for in utero cellular therapy. In humans, however, it is unknown whether similar stem cells exist. Here, we identify circulating hematopoeitic cells present during human in utero development that are capable of multilineage repopulation in immunodeficient NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Using limiting dilution analysis, the frequency of these fetal stem cells was found to be 1 in 3.2 x 10(5), illustrating a 3- and 22-fold enrichment compared with full-term human cord blood and circulating adult mobilized-peripheral blood, respectively. Comparison of in vivo differentiation and proliferative capacity demonstrated that circulating fetal stem cells are intrinsically distinct from hematopoietic stem cells found later in human development and those derived from the fetal liver or fetal bone marrow compartment at equivalent gestation. Taken together, these studies demonstrate the existence of unique circulating stem cells in early human embryonic development that provide a novel and previously unexplored source of pluripotent stem cell targets for cellular and gene-based fetal therapies. (Blood. 2000;96:1740-1747)

ADP-ribosyl Cyclase↗
PubMed2000

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Alleles↗
PubMed2000

Characterisation of calcitonin gene-related peptide receptors in rat atrium and vas deferens: evidence for a [Cys(Et)(2, 7)]hCGRP-preferring receptor.

The present study was performed in order to characterise calcitonin gene-related peptide (CGRP) receptor subtypes in rat left atrium and vas deferens by using [R-(R*, S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl]pentyl ]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide (BIBN4096BS), a novel CGRP receptor antagonist. When CGRP was used as an agonist, BIBN4096BS exhibited an almost 10-fold higher affinity for CGRP receptors in rat left atrium compared to those in the vas deferens, indicating that CGRP acts through different CGRP receptor subtypes in these two tissues. In addition, BIBN4096BS was almost 10-fold more potent in antagonizing [Cys(Et)(2,7)]hCGRPalpha and human adrenomedullin-induced responses than CGRP-induced responses in rat vas deferens. This might indicate receptor heterogeneity in rat vas deferens. Accordingly, the present work provides first experimental evidence that the rat vas deferens contains two CGRP-like receptor subtypes. Namely, the CGRP(2) receptor and a "novel" receptor that possesses low efficacy for CGRP and that is selectively stimulated by [Cys(Et)(2,7)]hCGRP or adrenomedullin and which can be blocked with high affinity by BIBN4096BS.

Animals↗
PubMed2000

Protection against ischemic injury in primary cultured mouse astrocytes by bis(7)-tacrine, a novel acetylcholinesterase inhibitor [corrected].

The effects of bis(7)-tacrine, a novel acetylcholinesterase inhibitor, on ischemia-induced cell death and apoptosis were investigated in primary cerebral cortical astrocytes of mice. Following a 6 h in vitro ischemic incubation of the cultures, a marked decrease in the percentage of viable cells was observed by lactate dehydrogenase (LDH) release assay. Furthermore, using bisbenzimide staining, we determined that approximately 65% of the cells underwent apoptosis. Treatment with bis(7)-tacrine (1-10 nM) during ischemic incubation effectively inhibited the ischemia-induced apoptosis, as demonstrated by morphological and biochemical tests. Our results demonstrated that bis(7)-tacrine could protect astrocytes against ischemia-induced cell injury, indicating that the drug might be beneficial for the treatment of vascular dementia, in addition to Alzheimer's disease.

Alzheimer Disease↗
PubMed2000

Primary structure and functional characterization of a soluble, alternatively spliced form of B7-1.

Recent studies have suggested that soluble forms of B7-1 and B7-2 may exist, but transcripts that code for these molecules have not been previously described. In this study, we report the cloning and characterization of an alternatively spliced soluble form of porcine B7-1 (sB7-1) that lacks exons coding for both the transmembrane and cytoplasmic domains. Northern blot analysis of RNA from alveolar macrophages revealed an approximate 3:1 ratio of the transmembrane form of B7-1 mRNA relative to sB7-1 mRNA. Porcine B7-1 was present on the surface of both B and T cells following stimulation with PMA/ionomycin. A histidine-tagged form of porcine sB7-1 (sB7-1-His) interacted with both CD28 and CTLA-4, and effectively blocked IL-2 production from human responder cells stimulated with PHA and either porcine or human stimulator cells. In addition, sB7-1-His inhibited human T cell proliferation in response to porcine or human peripheral blood leukocytes. This study is the first report of an alternatively spliced form of B7 that codes for a soluble protein. Furthermore, these data demonstrate that porcine B7-1 interacts with the human receptors CD28 and CTLA-4, suggesting a potential role for this molecule in pig to human xenotransplantation. Possible physiological functions for the soluble form of B7-1 are discussed.

Abatacept↗
PubMed2000

[Toxic effect of organic extracts of drinking-water on the culture luteal cells of rats].

A separation method of rat luteal cells was established. The results show that the survival rate of the cells is more than 95% during the whole operation. 14-20 days of pregnancy, 10(6) cells/ml and 4 hours of culture are the suitable conditions. The organic extracts of drinking-water produced significant dose-related increases(maximally three-fold) in progesterone concentration in the culture medium. Thus, the effects produced by the organic extracts of drinking-water in vitro may reflect the damage to the luteal cells.

Animals↗
PubMed2000

Role of Raf-1 conserved region 2 in regulation of Ras-dependent Raf-1 activation.

Full activation of Raf-1 requires the interaction of its CRD with Ras. The serine/threonine-rich region, CR2, of Raf-1 was implicated in Raf-1 regulation, but the underlying mechanism was unclear. Here we show that CRD loses its Ras-binding activity when expressed in connection with CR2, suggesting that CR2 masks CRD. This masking effect is abolished by substitution of Asp or Ala for Ser-259, a growth factor- and TPA-induced phosphorylation site in CR2. Treatment of COS-7 cells expressing Ha-Ras(Val-12) and Raf-1 with TPA enhances the Ha-Ras(Val-12)-dependent Raf-1 kinase activity. In contrast, the Ha-Ras(Val-12)-dependent activities of the Raf-1(S259D) and Raf-1(S259A) mutants are comparable to that of wild-type Raf-1 stimulated by both Ha-Ras(Val-12) and TPA and cannot be further stimulated by TPA treatment. These results suggest that the in vivo phosphorylation of Ser-259 may comprise a crucial step for Ras-dependent Raf-1 activation by unmasking CRD and promoting its association with Ras.

Animals↗
PubMed2000

Bis(1-acetonylpyridinium) pyridinium hexaiodobismuth(III).

The crystal structure of the title complex, (C(8)H(10)N)(2)(C(5)H(6)N)[BiI(6)], contains discrete [BiI(6)](3-) anions, and (HNC(5)H(5))(+) and (CH(3)COCH(2)NC(5)H(5))(+) cations separated by normal van der Waals contacts. The [BiI(6)](3-) anion has the Bi atom on an inversion centre. The (HNC(5)H(5))(+) cation also lies about an inversion centre and is disordered. The (CH(3)COCH(2)NC(5)H(5))(+) cation lies in a general position.

Journal Article↗
PubMed2000

In vitro supplementation with different tocopherol homologues can affect the function of immune cells in old mice.

Alpha-tocophorel (T) is the most common form of vitamin E inplasma and tissues. Alpha-T is also believed to be superior to its homologues beta-T, gamma-T, and delta-T in antioxidant activity. Biological activity of alpha-T has been intensively studied in a number of bodily systems. In contrast, the other homologues have received little attention beyond the evaluation of their relative antioxidant activity. We as well as others have previously shown that alpha-T can enhance cell- mediated immune function of aged animals and humans. Gamma-T is a principal form of vitamin E in the American diet and some cooking oils contain substantial amount of beta-T and delta-T. Thus it is of public health interest to compare their biological effects with than of alpha-t in various systems. In this study, we used an in vitro supplementation protocol to determine immunologic effects of these T homologues on murine splenocytes. The results showed that all four T homologues enhance both spontaneous and mitogen-stimulated lymphocyte proliferation (LP) and the maximal enhancement produced by them was of the same magnitude. The dose range to produce maximal enhancement varied with different homologues. The efficiency was in the order of beta-T approximately delta-T > alpha-T. Interestingly, at 50 (optimal for alpha-T) and 150 micromol/L, while alpha-T enhanced LP, all the other homologues inhibited LP. This inhibition was found to be due to their cytotoxicity at these levels. T homologues had a differential effect on interleukin (IL)-2 and prostaglandin (PG)E(2) production. IL-2 production by mouse splenocytes was not affected by alpha-T or beta-T, but was increased by gamma-T and delta-T. All T homologues, except for beta-T, inhibited PGE(2) alpha-T. Thus, all the T homologues enhance LP. However, the dose required to reach maximal enhancement varies among the homologues. On the other hand, they have a differential effect on IL-2 and PGE(2) production. The difference in nature and magnitude of the effect on immune function does not correlate with their reported relative antioxidant activity and might be due to minor differences in their structure important to their other biological activities.

Animals↗
PubMed2000

Roles of PLC-beta2 and -beta3 and PI3Kgamma in chemoattractant-mediated signal transduction.

The roles of phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC) in chemoattractant-elicited responses were studied in mice lacking these key enzymes. PI3Kgamma was required for chemoattractant-induced production of phosphatidylinositol 3,4,5-trisphosphate [PtdIns (3,4,5)P3] and has an important role in chemoattractant-induced superoxide production and chemotaxis in mouse neutrophils and in production of T cell-independent antigen-specific antibodies composed of the immunoglobulin lambda light chain (TI-IglambdaL). The study of the mice lacking PLC-beta2 and -beta3 revealed that the PLC pathways have an important role in chemoattractant-mediated production of superoxide and regulation of protein kinases, but not chemotaxis. The PLC pathways also appear to inhibit the chemotactic activity induced by certain chemoattractants and to suppress TI-IglambdaL production.

Animals↗
PubMed2000

Drug delivery systems based on trimethyl lock lactonization: poly(ethylene glycol) prodrugs of amino-containing compounds.

A novel methodology for the synthesis of poly(ethylene glycol) (PEG) prodrugs of amino-containing compounds has been developed which is based on the trimethyl lock lactonization reaction. These PEG-modified double prodrugs are water soluble, and by selective modification of the specifier or trigger, plasma half-lives can be adjusted at will to result in a wide range of values. Facile syntheses of ester, carbonate, and carbamate functionalities were accomplished and combined with greater or lesser degrees of steric hindrance in the spacer group, or on the aromatic framework, to achieve predictable ranges of drug concentration in plasma. In vivo screening of PEG prodrugs was done using a M109 syngeneic solid mouse tumor model. One of the PEG-daunorubicin prodrugs, with a half-life of 2 h, was evaluated in an in vivo solid tumor panel and found to be more efficacious against ovarian tumors (SKOV3) than equivalent amounts of daunorubicin.

Animals↗
PubMed2000

Normal isotype switching in B cells lacking the I mu exon splice donor site: evidence for multiple I mu-like germline transcripts.

Ig class switch recombination (CSR) in activated B cells is preceded by the generation of "switch" transcripts from the heavy chain constant region (CH) genes targeted for rearrangement. Switch transcripts include a sterile "I" exon spliced onto the first CH exon. Targeted mutations disrupting the expression or splicing of I exons severely hamper CSR to all tested CH loci, except mu. However, all mu switch transcript mutations tested so far have left the I mu exon splice donor site intact. To test the possibility that the residual CSR activity in I mu mutants could be due to splicing of a truncated I mu exon, we generated new mutants specifically lacking the I mu splice donor site. Surprisingly, normal CSR was observed in the I mu splice donor mutants even in the absence of detectable spliced I mu transcripts. In a search for potential alternative sources of switch-like transcripts in the mu locus, we identified two novel exons which map just upstream of the Smu region and splice onto the C mu 1 exon. Their expression is detectable from early B cell developmental stages, and, at least in hybridomas, it does not require the Emu enhancer. These studies highlight a unique structure for the mu locus I exon region, with multiple nested switch transcript-like exons mapping upstream of Smu. We propose that all of these transcripts directly contribute to mu class switching activity.

Animals↗