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Biomedical subjects

Y Wu

Publications and source records attributed to Y Wu.

At least 343 records · Page 19Linked to original sources
PubMed2000

Divergent regulation of the murine CC chemokine C10 by Th(1) and Th(2) cytokines.

Chemokines are typically found as products of acute stimulation of host defence cells. In contrast, the mouse CC chemokine C10 was previously shown to be a delayed, stably induced product of macrophages treated with interleukin 3 (IL-3), IL-4 or GM-CSF. We investigated the possibility that C10 is differentially regulated by cytokines associated with Th(1)and Th(2)cells. Northern blot analysis of bone marrow-derived macrophages showed that, in addition to IL-4, the Th(2)-specific cytokines IL-10 and IL-13 upregulated C10 over a 48-h period in a dose-dependent manner. In contrast, MIP-1alpha and MCP-1/JE were induced by IL-3 or GM-CSF at 48 h and this induction was inhibited by IL-4. Interferon gamma, a Th(1)-specific product, abolished the induction of C10 mRNA and protein by either IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF) in either bone marrow-derived or peritoneal macrophages. The inhibition of C10 production by interferon gamma was not NO dependent. Finally the GM-CSF-mediated induction of C10 in peritoneal macrophages was eliminated when these cells presented antigen to established T cells of Th(1)phenotype. The findings are consistent with a potential role for C10 in the modulation of immune reactions of Th(2)type.

Animals↗
PubMed2000

Changes in titin and collagen underlie diastolic stiffness diversity of cardiac muscle.

Small (N2B) and large (N2BA) cardiac titin isoforms are differentially expressed in a species-specific and heart location-specific manner. To understand how differential expression of titin isoforms may influence passive stiffness of cardiac muscle we investigated the mechanical properties of mouse left ventricular (MLV) wall muscle (expressing predominantly the small titin isoform), bovine left atrial (BLA) wall muscle (predominantly the large isoform), and bovine left ventricular (BLV) wall muscle (expressing small and large isoforms at similar levels). Results indicate that the overall passive muscle stiffness of the muscle types varies nearly ten-fold, with stiffness increasing in the following order: BLA, BLV and MLV. To investigate the basis of the variation in the overall muscle stiffness, the contributions of titin and collagen to muscle stiffness were determined. Results showed that increased muscle stiffness results from increases in both titin- and collagen-based passive stiffness, indicating that titin and collagen change in a co-ordinated fashion. The expression level of the small titin isoform correlates with titin's contribution to overall muscle stiffness, suggesting that differential expression of titin isoforms is an effective means to modulate the filling behavior of the heart.

Animals↗
PubMed2000

Characterization of a DNA region containing 5'-(CAAT)(n)-3' DNA sequences involved in lipooligosaccharide biosynthesis in Haemophilus somnus.

Repetitive tetranucleotide sequences of 5'-(CAAT)(n)-3' have been identified at the 5' end of an open reading frame (ORF) named lob1 from Haemophilus somnus strain 738. Based on sequence analysis, lob1 has 59% DNA homology to lex2B, which is involved in lipooligosaccharide (LOS) biosynthesis in H. influenzae. We now report that the number of 5'-CAAT-3' repeats in lob1 varied from 31-35, but that 94% of colonies contained 33 repeats of 5'-CAAT-3' downstream of two potential start codons, as determined by DNA sequence analysis of the 5'-CAAT-3' region from individual colonies. If transcription began with the start codon closest to the 5'-CAAT-3' repeats, a protein of 34.5 kDa would be encoded when 33 repeats were present. However, we could not establish a correlation between the number of 5'-CAAT-3' repeats in lob1 with a specific LOS electrophoretic profile or reactivity with two LOS monoclonal antibodies, indicating multiple genes control LOS phase variation in H. somnus. Complementation of strain 129Pt with lob1 containing 33 5 '-CAAT-3' repeats in shuttle vector pLS88 resulted in transformants 129Pt(pLSlob1-33A) and 129Pt(pLSlob1-33B), both of which demonstrated the same altered LOS electrophoretic profile. Unlike strain 129Pt, both transformants underwent limited LOS phase variation, which correlated with variation in the number of 5'-CAAT-3' repeats in pLSlob1-33. Nanoelectrospray-mass spectrometry of O-deacylated LOS indicated that transformant 129Pt(pLSlob1-33A) LOS was composed of a different distribution of glycoforms than LOS of the parent strain. The ratio of glucose to galactose changed from 1:2 in strain 129Pt LOS to 2:1 in transformant 129Pt(pLSlob1-33A) LOS, as determined by gas chromatography-mass spectrometry. Nuclear magnetic resonance spectroscopy confirmed and extended these observations. Transformant 129Pt(pLSlob1-33A) was constitutively more reactive in colony immunoblotting to polyclonal antiserum made to purified strain 738 LOS, and was more susceptible to complement-mediated killing in the presence of anti-738 LOS serum than parent strain 129Pt. Based on these results, Lob1 appears to be a phase variable galactosyl transferase involved in LOS biosynthesis in H. somnus.

Animals↗
PubMed2000

Mechanical properties of titin isoforms.

Titin is a giant filamentous polypeptide of multi-domain construction spanning between the Z- and M-lines of the sarcomere. As a result of differential splicing, length variants of titin are expressed in different skeletal and cardiac muscles. Here we first briefly review some of our previous work that has revealed that titin develops force in sarcomeres either stretched beyond their slack length (passive force) or shortened to below the slack length (restoring force) and that titin's force underlies a large fraction of the diastolic force of cardiac muscle. Next we present our mechanical and immunoelectron microscopical (IEM) studies of skeletal and cardiac muscles that express titin isoforms. The previously deduced molecular properties of titin were used to model titin's extensible region in the sarcomere as serially linked WLCs: rigid segments (containing folded Ig/Fn domains) and more flexible segments (PEVK segment). The model was tested on skeletal muscle fibers that express titin isoforms with tandem Ig and PEVK length variants. The model adequately predicts titin's behavior along a wide sarcomere length range in skeletal muscle, but at long sarcome lengths (SLs), predicted forces are much higher than those determined experimentally. IEM reveals that this may result from Ig domain unfolding. Experiments were also performed on cardiac myocytes from mouse and cow that express predominantly a small cardiac titin isoform (N2B titin) or a large isoform (N2BA titin), respectively. The passive tension-SL relation of myocytes was found to increase more steeply with SL in mouse than in cow. IEM revealed an additional source of extensibility within both of these cardiac titins: the unique N2B sequence (absent in skeletal muscle). Furthermore, the PEVK segment of the N2BA isoform extended to a maximal length of approximately 200 nm, as opposed to approximately 60 nm for the N2B isoform. We propose that, along the physiological SL range, the long PEVK segment found in N2BA titins results in a low PEVK fractional extension and that this underlies the lower passive tensions of N2BA-expressing cow myocytes.

Animals↗
PubMed2000

The influence of curcumin on the cell cycle of HL-60 cells and contrast study.

The mechanisms of curcumin inhibiting the proliferation of HL-60 cells were investigated. Acute myeloid leukemic cell line HL-60 was studied by using cell culture, NBT reduction, SABC method measuring BrdU incorporation rate, FCM measuring DNA contents and TUNEL method determining apoptotic cell percentage. Curcumin inhibited proliferation of HL-60 cells in a dose- and time-dependent manner. When the HL-60 cells were treated with 25 mumol/L curcumin for 48 h, the inhibitory rate was 60.71% +/- 1.20%. The study on BrdU incorporation rate and the distribution of DNA content and NBT reduction indicated that curcumin arrested the cells in G2/M phase of cell cycle at first, then in G0/G1 phase, the whole cell cycle progression was slowed down and DNA synthesis activities was halted. The arrested cells went to apoptosis instead of differentiation. The comparative study indicated that the ability of curcumin regulating the cell cycle of HL-60 cells was stronger than As2O3 and the acting points of curcumin were not completely consistent with those of VP-16. It was suggested that curcumin was able to regulate, to some extent, the G1/S and G2/M transmit checkpoints and disturb the HL-60 cell cycle to induce apoptosis.

Antineoplastic Agents↗
PubMed2000

Tbt-1, a new T-box transcription factor induced in activated Th1 and CD8+ T cells.

Differentiation of CD4+ T cells into T helper (Th) 1 or Th2 cells requires the cytokines interleukin (IL)-12 and IL-4, respectively. However, transcription factors that regulate expression of Th1 or Th2 cell-specific genes remain largely unclear. In the present study, a new Th1-specific transcription factor, named Tbt-1 (T-box protein expressed in T lymphocytes), was identified. Tbt-1 is a novel member of the T-box family, which is characterized by a conserved T-box DNA-binding domain. Unlike other known T-box proteins that regulate embryo development and organogenesis, Tbt-1 expression is restricted to adult lymphoid organs. Tbt-1 mRNA is only detected in peripheral lymphoid tissues such as spleen, lymph nodes, and blood leukocytes, but not in thymus or bone marrow. Tbt-1 mRNA is not detected in resting T cells but is strongly induced in differentiating Thl cells and CD8+ cytotoxic effector cells. In contrast, Tbt-1 expression was not observed in the entire process of Th2 cell differentiation. In addition, phylogenetic analyses indicate that Tbt-1 co-evolved with adaptive immune responses. Thus, Tbt-1 is the first T-box transcription factor shown to be specific for Th1 cell differentiation.

Amino Acid Sequence↗
PubMed2000

Functional loss of ABCA1 in mice causes severe placental malformation, aberrant lipid distribution, and kidney glomerulonephritis as well as high-density lipoprotein cholesterol deficiency.

Tangier disease (TD) and familial HDL deficiency (FHA) have recently been linked to mutations in the human ATP-binding cassette transporter 1 (hABCA1), a member of the ABC superfamily. Both diseases are characterized by the lowering or lack of high-density lipoprotein cholesterol (HDL-C) and low serum cholesterol. The murine ABCA1-/- phenotype corroborates the human TD linkage to ABCA1. Similar to TD in humans, HDL-C is virtually absent in ABCA1-/- mice accompanied by a reduction in serum cholesterol and lipid deposition in various tissues. In addition, the placenta of ABCA1-/- mice is malformed, resulting in severe embryo growth retardation, fetal loss, and neonatal death. The basis for these defects appears to be altered steroidogenesis, a direct result of the lack of HDL-C. By 6 months of age, ABCA1-/- animals develop membranoproliferative glomerulonephritis due to deposition of immunocomplexes followed by cardiomegaly with ventricular dilation and hypertrophy, ultimately succumbing to congestive heart failure. This murine model of TD will be very useful in the study of lipid metabolism, renal inflammation, and cardiovascular disease and may reveal previously unsuspected relationships between them.

ATP Binding Cassette Transporter 1↗
PubMed2000

Extensibility of isoforms of cardiac titin: variation in contour length of molecular subsegments provides a basis for cellular passive stiffness diversity.

Titin is a giant polypeptide that spans between the Z- and M-lines of the cardiac muscle sarcomere and that develops force when extended. This force arises from titin's extensible I-band region, which consists mainly of three segment types: serially linked immunoglobulin-like domains (Ig segments), interrupted by the PEVK segment, and the N2B unique sequence. Recently it was reported that the myocardium of large mammals co-expresses small (N2B) and large (N2BA) cardiac isoforms and that the passive stiffness of cardiac myocytes varies with the isoform expression ratio. To understand the molecular basis of the differences in passive stiffness we investigated titin's extensibility in bovine atrium, which expresses predominantly N2BA titin, and compared it to that of rat, which expresses predominantly N2B titin. Immunoelectron microscopy was used with antibodies that flank the Ig segments, the PEVK segment, and the unique sequence of the N2B element. The extension of the various segments was then determined as a function of sarcomere length (SL). When slack sarcomeres of bovine atrium were stretched, the PEVK segment extended much more steeply and the unique N2B sequence less steeply than in rat, while the Ig segments behaved similarly in both species. However, the extensions normalized with the segment's contour length (i.e., the fractional extensions) of Ig, PEVK, and unique sequence segments all increase less steeply with SL in cow than in rat. Considering that fractional extension determines the level of entropic force, these differences in fractional extension are expected to result in shallow and steep passive force-SL curves in myocytes that express high levels of N2BA and N2B titin, respectively. Thus, the findings provide a molecular basis for passive stiffness diversity.

Animals↗
PubMed2000

Single strand conformation polymorphism analysis of K-ras gene mutations by capillary electrophoresis with laser-induced fluorescence (LIF) detector.

Mutations of K-ras gene play an important role in neoplastic progression. The capillary electrophoresis-single strand conformation polymorphism (CE-SSCP) technique is available for the detection of gene mutations. Using an automated capillary electrophoresis with short-chain linear polyacrylamide, after denaturation of PCR products, injections were performed at reverse polarity of 5 kV for 15 s and the separations were carried out under a constant voltage of 8 kV. Of 16 specimens of lung cancer tissue, two specimens were found to have abnormal peaks in the electrophoretogram. CE-SSCP is rapid, automated, and has high performance.

Electrophoresis, Capillary↗
PubMed2000

Incidence of RET mutations in patients with Hirschsprung's disease.

BACKGROUND: RET mutations have been reported variously to affect 7% to 41% of Hirschsprung's disease (HSCR) patients depending on familial or sporadic occurrence of the disease, length of aganglionosis and possible association with other disease phenotypes. The authors report a study of the incidence of RET mutations in unselected HSCR patients from two regional centers and correlate their genotypes and phenotypes. METHODS: The records of HSCR patients treated in 2 regional centers with a combined population of 5 million were reviewed, and blood samples were obtained from 57 patients. During the same period, 39 patients with similar demographic data refused or provided insufficient blood for study. DNA was extracted, and the 21 exons of the RET protooncogene were screened for mutations using denaturing gradient gel electrophoresis (DGGE). RESULTS: Of 57 patients, 48 were sporadic, and 9 were familial. Lengths of aganglionosis were total colonic, 4; long, 11; short, 39; ultrashort, 1; unclassified, 2. Associated anomalies were present in 20. Causative mutations were identified in 4 (7%): missense or "silent" in 3 (exons 5, 11, 13) and deletion in 1. The silent mutation of exon 11 recently has been shown to have effects on correct RET mRNA splicing. One mutation occurred in total colonic aganglionosis (25%), 1 in long segment dysganglionosis (9%), and 2 in short segment aganglionosis (5%). Surprisingly, all these mutations occurred in sporadic cases (10%). Five patients (9%) had rare polymorphic alleles at exon 14 (n = 1) and exon 18 (n = 4). Fifty patients (88%) showed common polymorphic alleles (sequence variants) in 1 or more exons (> 4, n = 5). CONCLUSIONS: RET mutation as a primary cause for Hirschsprung's disease in the general surgical population is less frequent than previously thought. This observation is compatible with the hypothesis that HSCR could be a polygenic disease caused by additive subclinical effects of more than one gene, including RET.

Drosophila Proteins↗
PubMed2000

Beta-agkistrodotoxin inhibits fast and Ca2+-activated K+ currents recorded from mouse motor nerve terminals.

Beta-agkistrodotoxin (beta-AgTx), a polypeptide purified from the venom of Agkistrodon blomhoffii brevicaudus, is a presynaptic blocker acting on neurotransmitter release. In this work, perineural recording technique was employed to study the effects of beta-AgTx on sodium, potassium and calcium currents of mouse motor nerve terminals. The results showed that beta-AgTx selectively inhibited Ca2+-dependent (I(K,Ca)) and fast (I(K,f) K+ currents, but did not affect slow K+ current (I(K,s)), sodium and calcium currents. However there are other components in A. blomhoffii brevicaudus venom which inhibit perineural sodium current. The present data have provided additional evidence that the site of action of beta-AgTx is different from that of beta-bungarotoxin.

Animals↗
PubMed2000

Chemical anatomy of striatal interneurons in normal individuals and in patients with Huntington's disease.

This paper reviews the major anatomical and chemical features of the various types of interneurons in the human striatum, as detected by immunostaining procedures applied to postmortem tissue from normal individuals and patients with Huntington's disease (HD). The human striatum harbors a highly pleomorphic population of aspiny interneurons that stain for either a calcium-binding protein (calretinin, parvalbumin or calbindin D-28k), choline acetyltransferase (ChAT) or NADPH-diaphorase, or various combinations thereof. Neurons that express calretinin (CR), including multitudinous medium and a smaller number of large neurons, are by far the most abundant interneurons in the human striatum. The medium CR+ neurons do not colocalize with any of the known chemical markers of striatal neurons, except perhaps GABA, and are selectively spared in HD. Most large CR+ interneurons display ChAT immunoreactivity and also express substance P receptors. The medium and large CR+ neurons are enriched with glutamate receptor subunit GluR2 and GluR4, respectively. This difference in AMPA GluR subunit expression may account for the relative resistance of medium CR+ neurons to glutamate-mediated excitotoxicity that may be involved in HD. The various striatal chemical markers display a highly heterogeneous distribution pattern in human. In addition to the classic striosomes/matrix compartmentalization, the striosomal compartment itself is composed of a core and a peripheral region, each subdivided by distinct subsets of striatal interneurons. A proper knowledge of all these features that appear unique to humans should greatly help our understanding of the organization of the human striatum in both health and disease states.

Corpus Striatum↗
PubMed2000

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.

Animals↗
PubMed2000

A new developmentally regulated gene family in Leishmania amastigotes encoding a homolog of amastin surface proteins.

The ability of Leishmania to survive within the phagolysosomes of mammalian macrophages is heavily dependent on the developmental regulation of a number of genes. Characterization of genes preferentially expressed during the parasite's intracellular growth would help to elucidate the mechanisms controlling stage-specific gene regulation and the intracellular life of the parasite in general. Using a genomic approach based on the differential hybridization screening of high-density filters, we have identified a new developmentally regulated gene in Leishmania, which is part of a multigene family and encodes a highly hydrophobic protein that shares homology with the Trypanosoma cruzi amastin proteins. The fusion of the Leishmania amastin gene homolog with the green fluorescent protein and analysis by confocal microscopy suggested a surface localization for this protein. The amastin gene homolog is expressed predominantly in the amastigote form of several Leishmania species and is strictly regulated by acidic pH at the post-transcriptional level. Its developmental expression involves sequences within the 3'-untranslated region.

Amino Acid Sequence↗
PubMed2000

The organization of the striatal output system: a single-cell juxtacellular labeling study in the rat.

The aim of the present study was to provide a detailed account of the axonal branching pattern of striatal projection neurons in the rat. Seventy-seven striatofugal neurons were singly labeled following juxtacellular injection of biotin dextran amine. Their axons were entirely reconstructed along the sagittal plane with the help of a light microscope equipped with a camera lucida. The major findings of this study can be summarized as follows, (1) the striatofugal system originates from medium-sized spiny neurons that project only to globus pallidus (GP, type I, 36.4%), to both GP and substantia nigra pars reticulata (SNr, type II, 26%), or to globus pallidus, entopeduncular nucleus (EP) and SNr (type III, 37.6%); (2) the striatofugal system displays a high degree of axonal collateralization; about two-thirds of its axons arborize into two or three striatal target structures; (3) virtually all striatofugal axons send collaterals to the GP and none project exclusively to the EP and or SNr; (4) the three types of striatal projection neurons share similar somatodendritic morphology and have no preferential distribution in the dorsal striatum. These data, together with those of previous investigations, indicate that the striatofugal system can no longer be considered to be a simple dual (direct indirect) projection system. Instead, it stands out as a complex and widely distributed neuronal network whose elements are endowed with a highly patterned set of axon collaterals, which allows them to control in an exquisitely precise manner the flow of information along the main axis of the basal ganglia.

Animals↗
PubMed2000

A DPDPE-induced enhancement of inward rectifier potassium current via opioid receptor in neuroblastomaxglioma NG108-15 cells.

The effect of a delta-selective opioid agonist, DPDPE([D-Pen(2, 5)]-enkephalin), on the inward rectifier potassium current (I(KIR)) of NG108-15 cell was studied by whole cell voltage-clamp technique. It was found that microM DPDPE increased the amplitude and delayed the activation and inactivation of I(KIR) rapidly and reversibly. These effects could be reversed by naloxone, but were still obtained in pertussis toxin (PTX) preincubated cells or when using GDP-betas (guanosine 5'-o-[2-thio] diphoaphate) containing electrodes to block the G-protein coupled events. The above results suggest that DPDPE-induced change of I(KIR) is mediated by delta-opioid receptor but does not involve G-proteins.

Animals↗