Biomedical subjects

W Lu

Publications and source records attributed to W Lu.

At least 73 records · Page 4Linked to original sources

PubMed2001

Stabilization of the MDM2 oncoprotein by mutant p53.

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.

Humans↗

PubMed2001

Monitoring the response to antiretroviral therapy in HIV-1 group O infected patients using two new RT-PCR assays.

Failure to recognise infection caused by human immunodeficiency virus type 1 (HIV-1) group O variants has been described using both serological and genetic procedures. Moreover, monitoring the response to antiretroviral therapy is a difficult task in patients infected with HIV-1 group O since commercial tests are not available so far for the quantitation of this virus. In this study, the virological response to antiretroviral therapy were assessed in five HIV-1 group O-infected patients living in Spain by using two new and different RT-PCR methods (MUPROVAMA and LCx). Twenty-four plasma samples belonging to these five patients were selected. As reference, p24 antigenaemia levels and CD4+ cell counts were used. All samples yielded positive viral load values using MUPROVAMA (range: 138 to 595,500 HIV-RNA copies/ml) and 23 of 24 using LCx (range: < 178 to 98,356 HIV-RNA copies/ml). Overall, the results obtained using both assays showed a good correlation among themselves, and in respect to p24 antigenaemia and CD4+ cell counts. However, the values provided by LCx were significantly lower (0.33 logs on average) than those provided by MUPROVAMA. In conclusion, both the highly sensitive MUPROVAMA and LCx Quantitative assays might represent an useful tool for guiding the decision on when start treatment and for monitoring the response to antiretroviral therapy in HIV-1 group O-infected patients.

Adolescent↗

PubMed2001

The study on intramyocardial calcium overload and apoptosis induced by coxsackievirus B3.

The isolated cardiac myocytes of rats were immediately infected by cosackievirus B3 (CVB3) to investigate the effects of such procedure on the cell cycle, apoptosis and intracellular ionized calcium (Ca2+ i) of cardiac myocytes. Newborn Balb/c murine cardiac myocytes were cultivated, then infected by CVB3. Intracellular Ca2+ i was measured by flow cytometer. The calcium in the medium for culturing cardiac myocytes was detected by using atom absorb spectrum test. It was found that CVB3 could markedly inhibit the differentiation and proliferation of the infected cardiac myocytes and induce the apoptosis. The intracellular Ca2+ i level in the infected group was significantly higher than in the control group (P < 0.01). The calcium concentration in the medium for culturing cardiac myocytes in the infected group was significantly lower than in the control group (P < 0.05). It was suggested that the apoptosis and intracellular calcium overload of the CVB3-affected cardiac myocytes are likely to play an important role in the pathogenesis of viral myocarditis.

Animals↗

PubMed2001

Effect of ligustrazine on hematopoiesis in bone marrow transplantation mice.

The effect of Ligustrazine on the hematopoiesis after bone marrow transplantation (BMT) in allogenic BMT mice was investigated. After the typical mice model of allogenic BMT had been established, the mice were randomly divided into three groups: BMT group, Ligustrazine group and normal group. The BMT group was given normal saline (0.2 ml, twice a day) through gastric tube, while the Ligustrazine group was given Ligustrazine through gastric tube (0.2 ml, twice a day). At the 1st, 7th and 14th day after BMT, we observed the peripheral blood cells and bone marrow nuclear cells (BMNC), as well as the expression level of Heparan Sulfate (HS) and stromal cell derived factor-1 (SDF-1) on bone marrow sections by using immunohistochemistry (SABC-AP), the expression of CXCR4 on the BMNC. The results showed that on the 7th and 14th day, the peripheral blood white cells, platelets, BMNC and the expression levels of CXCR4, HS and SDF-1 were significantly higher in Ligustrazine group than in the BMT group (P < 0.05). It was concluded that Ligustrazine could promote hematopoiesis at the early stage of hematopoietic reconstitution after BMT.

Animals↗

PubMed2001

Synthesis of polyhydroxysterols (I): synthesis of 24-methylenecholest-4-en-3beta,6beta-diol, a cytotoxic natural hydroxylated sterol.

Starting from stigmasterol (2), 24-methylenecholest-4-en-3beta, 6beta-diol (1), a cytotoxic natural dihydroxylated sterol, was synthesized via 10 steps in 20% overall yield. The introduction of a side-chain of sterol was achieved by solid-liquid phase-transfer Wittig reaction using (3-methyl-2-oxo)butyltriphenylarsonium bromide (12) and K(2)CO(3). Construction of the steroidal nucleus was finished by the addition of 3beta-acetoxycholest-5,6-en-24-one (7) with NBA in dioxane under ambient temperature and by the elimination of 3beta, 6beta-diacetoxy-5a-bromocholestane-24-one (9). The spectral data of the synthetic product (1) are completely consistent with those of the natural compound (1).

Antineoplastic Agents↗

PubMed2001

Expression of AMPA receptor flip and flop mRNAs in the nucleus accumbens and prefrontal cortex after neonatal ventral hippocampal lesions.

Many lines of evidence implicate dysfunctional excitatory amino acid (EAA) transmission in schizophrenia. The present study examined alpha-amino-3-hydroxy-5-methylisoxazole- 4-priopionate (AMPA) receptor expression in the prefrontal cortex (PFC) and nucleus accumbens (NAc) using a rat model of schizophrenia in which excitotoxic lesions of ventral hippocampus (VH) on postnatal day (PD) 7 lead to the postpubertal emergence of behavioral abnormalities that resemble schizophrenic symptoms. In situ hybridization histochemistry with 35S-labeled oligonucleotide probes was used to quantify mRNA levels for GluR1-3 flip and flop variants at prepubertal and postpubertal time-points (PD 35 and 60, respectively). Comparisons of PD 35 and PD 60 groups suggested that there may be changes in the relative expression of flip and flop isoforms during normal development. The most pronounced change was an apparent increase in the flip/flop ratio for GluR1 from PD 35 to PD 60 in both the PFC and the NAc. Neonatal VH lesions did not significantly alter GluR1-3 flip and flop mRNA levels in the NAc at either time-point. However, in the PFC, GluR3 flop mRNA levels were significantly decreased in lesioned rats at PD 60. AMPA receptors incorporating flop variants exhibit faster desensitization, so a shift towards flip incorporation might lead to increased neuronal excitability in the PFC, thereby influencing subcortical DA systems regulated by PFC efferents. In summary, an early developmental injury can predispose rats to abnormal development of EAA transmission in the PFC. This may contribute to the postpubertal onset of symptoms after neonatal VH lesions.

Animals↗

PubMed2001

Activation of synaptic NMDA receptors induces membrane insertion of new AMPA receptors and LTP in cultured hippocampal neurons.

Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.

Animals↗

PubMed2001

CAKbeta/Pyk2 kinase is a signaling link for induction of long-term potentiation in CA1 hippocampus.

Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.

Animals↗

PubMed2001

Site-specific incorporation of a phosphotyrosine mimetic reveals a role for tyrosine phosphorylation of SHP-2 in cell signaling.

The regulation of protein tyrosine phosphatase (PTPase) SHP-2 is proposed to involve tyrosine phosphorylation on two tail tyrosine residues. Using "expressed protein ligation", nonhydrolyzable phosphotyrosine analogs were introduced at known phosphorylation sites in SHP-2. Biochemical analysis suggests that a phosphonate at Tyr542 interacts intramolecularly with the N-terminal SH2 domain to relieve basal inhibition of the PTPase, whereas a phosphonate at Tyr-580 stimulates the PTPase activity by interaction with the C-terminal SH2 domain. Microinjection experiments indicate that a single phosphorylation of Tyr-542 of SHP-2 is sufficient to activate the MAP kinase pathway in living cells. These studies support a novel mechanism explaining how tyrosine phosphorylation of a PTPase is important in signal transduction.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Adenovirus-mediated interferon-beta gene therapy suppresses growth and metastasis of human prostate cancer in nude mice.

The purpose of this study was to determine the effects of interferon-beta (IFN-beta) gene transfer on the growth of PC3MM2 human prostate cancer cells in nude mice. Intralesional delivery of an adenoviral vector encoding murine IFN-beta (AdIFN-beta), but not a vector encoding bacterial beta-galactosidase (AdLacZ), suppressed PC3MM2 tumors in a dose-dependent manner. At the highest dose (2x10(9) plaque-forming units, PFU), a single injection of AdIFN-beta (but not AdLacZ) suppressed orthotopic PC3MM2 tumors and development of metastasis by 80%, and eradicated the tumors in 20% of mice. Immunohistochemical staining showed that AdIFN-beta-treated tumors contained fewer microvessels, fewer proliferating cells, and more apoptotic cells than did the control tumors. Compared with controls, tumors injected with AdIFN-beta expressed higher levels of IFN-beta and inducible nitric oxide synthase (iNOS) and lower levels of basic fibroblast growth factor (bFGF) and transforming growth factor beta1 (TGF-beta1). In vitro analysis indicated that expression of bFGF and TGF-beta1 in PC3MM2 cells could be suppressed by the nitric oxide donor sodium nitroprusside. These data suggest that intratumoral delivery of the IFN-beta gene with adenoviral vectors could be an effective therapy for prostate cancer and that tumor suppression by AdIFN-beta correlated with up-regulation of iNOS and down-regulation of angiogenesis.

Adenoviridae↗

PubMed2001

The pharmacokinetics of remifentanil in patients undergoing coronary artery bypass grafting with cardiopulmonary bypass.

UNLABELLED: Remifentanil is a potent opioid with a short duration of action. It has the potential for large-dose opioid anesthesia without an obligatory prolonged period of mechanical ventilation. However, because of high clearance and rapid tissue distribution, cardiopulmonary bypass (CPB) may influence its pharmacokinetics and alter drug requirements. We administered remifentanil by continuous infusion to 68 patients having coronary artery bypass graft surgery during CPB with hypothermia to describe the effects of these interventions on its pharmacokinetics. Remifentanil concentrations were measured before, during, and after CPB. Disposition was best described by a two-compartment model. The volume of distribution increased by 86% with institution of CPB and remained increased after CPB. Elimination clearance decreased by 6.37% for each degree Celsius decrease from 37 degrees C. IMPLICATIONS: Remifentanil concentrations decrease with the institution of cardiopulmonary bypass because of an increase in the volume of distribution. The decrease in elimination clearance with hypothermia results in increased total remifentanil concentrations during cardiopulmonary bypass if the infusion rate is not altered. More constant blood remifentanil levels may be obtained by reducing remifentanil infusion rate by 30% for each 5 degrees C decrease in temperature.

Analgesics, Opioid↗

PubMed2001

Performance of the applied biosystems ViroSeq human immunodeficiency virus type 1 (HIV-1) genotyping system for sequence-based analysis of HIV-1 in pediatric plasma samples.

The ViroSeq HIV-1 Genotyping System is a commercially available, integrated sequence-based system for analysis of human immunodeficiency virus type 1 (HIV-1) drug resistance. We evaluated the performance of this system by analyzing HIV-1 in pediatric plasma samples. Plasma samples from children 4 months to 17 years of age were obtained from a clinical trial protocol (PACTG 377). Children in PACTG 377 were randomized to four treatment arms, including different combinations of antiretroviral drugs. HIV-1 genotyping was performed using samples collected prior to antiretroviral therapy (baseline) and at the time of virologic failure. Performance of the genotyping system was compared in three university laboratories. A total of 196 samples were analyzed, including 135 baseline and 61 failure samples. Plasma volumes ranged from 0.05 to 0.5 ml, and viral loads ranged from 1,084 to 3,484,991 copies/ml. PCR products suitable for sequencing were obtained for 192 of the 196 samples. Complete sequences for protease and reverse transcriptase were obtained for all of these 192 samples. For 180 samples, data were obtained from both DNA strands for the entire region analyzed. There was no evidence of sample cross-contamination based on phylogenetic analysis of HIV-1 sequences. Performance of the genotyping system was similar in three laboratories. This genotyping system performs well for analysis of HIV-1 in pediatric plasma samples, including those with low volume and low viral load. The availability of this system should facilitate studies of HIV-1 drug resistance.

Adolescent↗

PubMed2001

In vitro human immunodeficiency virus eradication by autologous CD8(+) T cells expanded with inactivated-virus-pulsed dendritic cells.

Despite significant immune recovery with potent highly active antiretroviral therapy (HAART), eradication of human immunodeficiency virus (HIV) from the bodies of infected individuals represents a challenge. We hypothesized that an inadequate or inappropriate signal in virus-specific antigen presentation might contribute to the persistent failure to mount efficient anti-HIV immunity in most HIV-infected individuals. Here, we conducted an in vitro study with untreated (n = 10) and HAART-treated (n = 20) HIV type 1 (HIV-1) patients which showed that pulsing of monocyte-derived dendritic cells (DC) with aldrithiol-2-inactivated autologous virus resulted in the expansion of virus-specific CD8(+) T cells which were capable of killing HIV-1-infected cells and eradicating the virus from cultured patient peripheral blood mononuclear cells independently of the disease stages and HAART response statuses of the patients. This in vitro anti-HIV effect was further enhanced by the HIV protease inhibitor indinavir (at a nonantiviral concentration), which has been shown previously to be able to up-regulate directly patient T-cell proliferation following immune stimulation. However, following a 2-day treatment with culture supernatant derived from immune-activated T cells (which mimics an in vivo environment of HIV-disseminated and immune-activated lymphoid tissues), DC lost their capacity to present de novo inactivated-virus-derived antigens. These findings provide important information for understanding the establishment of chronic HIV infection and indicate a perspective for clinical use of DC-based therapeutic vaccines against HIV.

Anti-HIV Agents↗

PubMed2001

Spectral karyotyping (SKY) of mouse meiotic chromosomes.

The spectral karyotyping procedure of in situ hybridization with chromosome-specific probes assigns a unique colour code to each of the 21 mouse mitotic chromosomes. We have adapted this procedure to meiotic prophase chromosomes, and the results show that each of the pachytene or metaphase I bivalents can be identified. This technique has the potential to recognize synaptic anomalies and chromosome-specific structural and behavioural characteristics. We confirm these potentials by the recognition of the heterologous synapsis of the X and Y chromosomes and by the variances of synaptonemal complex lengths for each of the colour-coded bivalents in eight prophase nuclei.

Animals↗

PubMed2001

An integrated genetic and physical map of the 650-kb region containing the congenital polycystic kidney (cpk) locus on mouse chromosome 12.

Mice homozygous for the congenital polycystic kidney (cpk) mutation develop a rapidly progressive form of polycystic kidney disease. We report an integrated genetic and physical map of the 650-kb region containing the cpk locus and the exclusion of Rrm2 and Idb2 as candidate cpk genes. Our study establishes the requisite foundation for positional cloning of the cpk gene.

Animals↗

PubMed2001

Re-defining the chromatin loop domain.

It is commonly accepted that the loop domain represents the basic structural unit of eukaryotic chromatin associated with DNA replication, gene expression and higher order packaging. However, molecular-cytological information defining the loop domain is lacking. There are gaps in our knowledge of the loop structure and how it regulates gene expression. The combination of new data/reagents from the Human Genome Project plus the use of novel molecular cytological technology will provide answers. Here we briefly review the status of chromatin loop research and pose questions that need to be addressed. New experimental systems are also presented to target some long-standing issues regarding the structure and function of the chromatin loop domain and its relationship with the nuclear matrix. This new knowledge will have a profound impact for modern genetics and molecular medicine.

Animals↗

PubMed2001

The combination of SKY and specific loci detection with FISH or immunostaining.

Spectral karyotyping (SKY) represents an effective tool to detect individual chromosomes and analyze major karyotype abnormalities within an entire genome. We have tested the feasibility of combining SKY and FISH/protein detection in order to combine SKY's unique abilities with specific loci detection. Our experimental results demonstrate that various combined protocols involving SKY, FISH and immunostaining work well when proper procedures are used. This combined approach allows the tracking of key genes or targeted chromosome regions while monitoring changes throughout the whole genome. It is particularly useful when simultaneously monitoring the behavior of both protein complexes and DNA loci within the genome. The details of this methodology are described and systematically tested in this communication.

Animals↗

PubMed2001

Indirect arterial blood pressure measurement at the wrist using a pad-type square cuff and volume-oscillometric method.

Using theoretical and experimental approaches, we examined whether blood pressure at the wrist can be accurately measured by a volume-oscillometric method using a small pad-type square cuff placed above the radial artery (RA). Nuclear magnetic resonance imaging of the wrist allowed us to determine the geometry of two-dimensional (2-D) finite-element models. Finite-element method (FEM) analysis predicted that the pressure transmission ratio (calculated tissue pressure over externally applied pressure; square cuff to RA) was 98.8% for a cuff with a bladder sidelength 0.25 times the wrist diameter placed on the skin surface between the tendons of the brachioradialis muscle (Ta) and the flexor capri radialis muscle (Tb) and over the site (L) at which RA crosses the most protuberant spot on the volar aspect of the distal end of the radius. In addition, FEM analysis using a 3-D finite-element model (constructed by extending the 2-D finite-element model at site L in the longitudinal direction) showed that for all square cuffs with bladder sidelengths greater than or equal to 0.25 times the wrist diameter, the external pressure was transmitted almost completely to RA beneath the cuff center. Moreover, when the bladder sidelength was 0.44 times the wrist diameter, but not 0.29 times the wrist diameter, the mean blood pressure measured at site L in human subjects was similar to that measured at the upper arm. Taken together, the theoretical and the experimental results suggest that (i) blood pressure at the wrist can be measured accurately using a pad-type square cuff placed on the skin surface between Ta and Tb at site L, and (ii) the minimum bladder sidelength for accurate readings is somewhere between one-third and one-half of the wrist diameter.

Adult↗