Biomedical subjects

Z Wu

Publications and source records attributed to Z Wu.

At least 181 records · Page 10Linked to original sources

PubMed2000

The insulin-like growth factor-phosphatidylinositol 3-kinase-Akt signaling pathway regulates myogenin expression in normal myogenic cells but not in rhabdomyosarcoma-derived RD cells.

Insulin-like growth factors (IGFs) can stimulate skeletal muscle differentiation. One of the molecular mechanisms underlying IGF-stimulated myogenesis is transcriptional induction of myogenin. The current work is aimed to elucidate the signaling pathways mediating the IGF effect on myogenin promoter in mouse C2C12 myogenic cells. We show that phosphatidylinositol 3-kinase (PI3K)/Akt and p70(S6K) are crucial signaling molecules mediating the stimulatory effect of IGFs on myogenin expression. We have identified three cis-elements, namely the E box, MEF2, and MEF3 sites, within the 133-base pair mouse proximal myogenin promoter that are under the control of the IGF/PI3K/Akt pathway. Simultaneous mutation of all three elements completely abolishes activation of the myogenin promoter by PI3K/Akt. We demonstrate that PI3K/Akt can increase both the MyoD and the MEF2-dependent reporter activity by enhancing the transcriptional activity of MyoD and MEF2. Interestingly, IGF1 does not enhance myogenin expression in Rhabdomyosarcoma-derived RD cells. Consistently, the constitutively active PI3K/Akt fail to activate the myogenic reporters, suggesting the IGF/PI3K/Akt pathway is defective in RD cells and the defect(s) is downstream to PI3K/Akt. This is the first time that a defect in the IGF/PI3K/Akt pathway has been revealed in RD cells which provides another clue to future therapeutic treatment of Rhabdomyosarcoma.

Amino Acid Sequence↗

PubMed2000

A high-affinity fluorenone-based beta 2-adrenergic receptor antagonist with a photoactivatable pharmacophore.

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.

Adenylyl Cyclase Inhibitors↗

PubMed2000

Determination of gestrinone in human serum by liquid chromatography--electrospray tandem mass spectrometry.

A rapid, sensitive and specific high-performance liquid chromatography-electrospray tandem mass spectrometric method has been developed for the determination of gestrinone (R 2323) in human serum using mifepristone (RU 486) as an internal standard. R 2323 was extracted from human serum by an ether extraction procedure. Multiple reaction monitoring was used to detect R 2323 and RU 486. The calibration curve was linear over the range of 3.5-177 ng/ml (r2 > or =0.99) with the limitation of detection of 0.8 ng/ml. The intra-day precision and accuracy, expressed as C.V. and RE, ranged from 2.3-13.7 to -4.8-3.0%. The inter-day precision and accuracy ranged from 5.5-14.8 to -6.7-3.1%. The mean recovery was 91.0% for R 2323, and 90.6% for the internal standard. The method was successfully applied to the pharmacokinetic study of R 2323.

Chromatography, High Pressure Liquid↗

PubMed2000

Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P. aeruginosa strain PAO1. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P. aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P. aeruginosa genome reflect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.

Bacterial Proteins↗

PubMed2000

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity.

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.

Carcinoma↗

PubMed2000

Identification and functional expression of four isoforms of ATPase II, the putative aminophospholipid translocase. Effect of isoform variation on the ATPase activity and phospholipid specificity.

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg(2+)-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

Amino Acid Sequence↗

PubMed2000

Kinetics of two-liquid-phase Taxus cuspidata cell culture for production of Taxol.

The effects of different organic solvents (paraffin, organic acid, alcohol and ester) and their volumetric fractions on the cell growth and Taxol production were studied in two-liquid-phase and the two-stage culture. A kinetic model, incorporated the effects of the toxicity of organic solvents was developed for two-liquid-phase culture of Taxus cuspidata in the two-stage Taxol production. The results showed that the proposed kinetic model could fit the experimental data satisfactorily. The results also showed that Taxol production could reach the optimal value when 10-logP was in the range of 2 to 5 and the volumetric fraction of the organic solvents at the corresponding the highest Taxol production should be lower when 10-logP was high.

Journal Article↗

PubMed2000

Purification of the newly found selenium-containing proteins in the arterial wall and brain of the rat.

Previously several selenium-containing proteins with different subunit molecular masses (M(r)) were detected in the arterial wall and brain of rats. In continuation of this work, after labeling of rats in vivo with [(75)Se]selenite, the new selenium-containing proteins of interest were purified on a Sephadex G-200 column followed by preparative isoelectric focusing. Nuclear analytical methods (gamma-counter and gamma-detector) were applied in the detection and identification of the (75)Se-labeled proteins. The two (75)Se-containing proteins from the arterial wall migrated as 15.0- and 67.0-kDa species on SDS-PAGE gels with pI values of 4.5 and 5.1, respectively. The three (75)Se-containing proteins from brain purified to homogeneity had M(r) values of 18.0, 30.0, and 42.9 kDa and pI values of 6.3, 6.5, and 6.0, respectively. Of these proteins, the 67.0-, 42.9-, and 30.0-kDa species may be yet not characterized selenoproteins with important biological functions.

Animals↗

PubMed2000

Molecular basis for the polyamine-ompF porin interactions: inhibitor and mutant studies.

By testing the sensitivity of Escherichia coli OmpF porin to various natural and synthetic polyamines of different lengths, charge and other molecular characteristics, we were able to identify the molecular properties required for compounds to act as inhibitors of OmpF in the nanomolar range. Inhibitors require at least two amine groups to be effective. For diamines, the optimum length of the hydrocarbon spacer was found to be of eight to ten methylene groups. Triamine molecules based on a 12-carbon motif were found to be more effective that spermidine, an eight-carbon trivalent derivative. But differences in inhibition efficiencies were also found for trivalent compounds depending on the relative position of the internal secondary amine group with respect to the terminal groups. Finally, quaternary ammonium derivatives had no effect, suggesting that the nature of the terminal amine is important for the interaction. From these observations, we deduce that inhibition efficiency in the nanomolar range requires a 12-carbon chain triamine with terminal primary amine groups and replacement of the eighth methylene by a secondary amine. The need for this type of molecular architecture suggests that inhibition is governed by interactions between specific amine groups and protein residues, and that this is not simply due to the accumulation of charges into the pore. Together with previous observations from site-directed mutagenesis studies and inspection of the crystal structure of OmpF, these results allowed us to propose three residues (D113, D121 and Y294) as putative sites of interaction between the channel and spermine. Alanine substitution at each of these three residues resulted in a loss of inhibition by spermine, while mutations of only D113 and D121 affected inhibition by spermidine. Based on these observations, we suggest a model for the molecular determinants involved in the porin-polyamine interaction.

Amino Acid Substitution↗

PubMed2000

Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells.

We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.

Breast Neoplasms↗

PubMed2000

Induction of terminal differentiation by constitutive activation of p38 MAP kinase in human rhabdomyosarcoma cells.

MyoD inhibits cell proliferation and promotes muscle differentiation. A paradoxical feature of rhabdomyosarcoma (RMS), a tumor arising from muscle precursors, is the block of the differentiation program and the deregulated proliferation despite MyoD expression. A deficiency in RMS of a factor required for MyoD activity has been implicated by previous studies. We report here that p38 MAP kinase (MAPK) activation, which is essential for muscle differentiation, is deficient in RMS cells. Enforced induction of p38 MAPK by an activated MAPK kinase 6 (MKK6EE) restored MyoD function and enhanced MEF2 activity in RMS deficient for p38 MAPK activation, leading to growth arrest and terminal differentiation. Stress and cytokines could activate the p38 MAPK in RMS cells, however, these stimuli did not promote differentiation, possibly because they activated p38 MAPK only transiently and they also activated JNK, which could antagonize differentiation. Thus, the selective and sustained p38 MAPK activation, which is distinct from the stress-activated response, is required for differentiation and can be disrupted in human tumors.

Animals↗

PubMed2000

Transgenic mice neuronally expressing baculoviral p35 are resistant to diverse types of induced apoptosis, including seizure-associated neurodegeneration.

Apoptosis is a cell-suicide process that appears to play a central role not only during normal neuronal development but also in several neuropathological disease states. An important component of this process is a proteolytic cascade involving a family of cysteine proteases called caspases. Caspase inhibitors have been demonstrated to be effective in inhibiting neuronal cell death in various apoptotic paradigms. We have created transgenic mice that neuronally express the baculoviral caspase inhibitor p35. Neuronal expression of the p35 protein was found to confer functional caspase inhibitory activity and prevent apoptosis in isolated cerebellar granular cultures induced to undergo apoptosis either via staurosporine treatment or through withdrawal of extracellular potassium. Neuronal expression of p35 was also found to attenuate neurodegeneration associated with the excitotoxic glutamate analogue kainic acid (KA) in vitro and in vivo. Organotypic hippocampal cultures isolated from p35 transgenics demonstrated lowered caspase activity and decreased apoptosis compared with wild type when exposed to KA. In vivo injection of KA also produced decreased caspase activity and cell death in p35 transgenics vs. wild type. These results suggest that the presence of p35 in neurons in vivo is protective against various types of apoptosis, including seizure-related neurodegeneration, and that caspases may be attractive potential targets for preventing neuronal injury associated with diseases such as epilepsy. These mice also provide a valuable tool for exploring the role of caspases in other neuropathological conditions in which apoptosis has been implicated.

Animals↗

PubMed2000

Cloning and characterization of the bovine MHC class I-like Fc receptor.

In the cow, maternal immunity is exclusively mediated by colostral Igs, but the receptor responsible for the IgG transport has not yet been identified. The role of an IgG-Fc receptor (FcRn) that resembles a class I MHC Ag in transporting IgGs through epithelial cells was recently shown in selected species. We now report the cloning and characterization of the bovine FcRn (bFcRn). The cDNA and deduced amino acid sequences show high similarity to the FcRn in other species, and it consists of three extracellular domains, a hydrophobic transmembrane region, and a cytoplasmic tail. Despite the high similarity of the extracellular domains with other species, the bovine cytoplasmic tail is the shortest thus far analyzed. Aligning the known FcRn sequences, we noted that the bovine protein shows a 3-aa deletion compared to the rat and mouse sequences in the alpha1 loop. Furthermore, we found a shorter transcript of the bFcRn reflecting an exon 6-deleted mRNA, which results from an inadequate splice acceptor site in intron 5 and produces a transmembrane-deficient molecule, as was previously demonstrated in the related MHC class I gene family in mouse and humans. The presence of bFcRn transcripts in multiple tissues, including the mammary gland, suggests their involvement both in IgG catabolism and transcytosis.

Amino Acid Sequence↗

PubMed2000

Alkylation of aromatic aldehydes with boron halide derivatives

[reaction: see text] Aryl aldehydes react with alkylboron chloride derivatives in the presence of base to generate the corresponding arylalkylmethanols in good yields. A variety of alkylboron chlorides can be utilized in the new alkylation reaction.

Journal Article↗

PubMed2000

Grafted supports in solid-phase synthesis.

Solid-phase synthesis is greatly dependent on the solid phase. We are interested in the development of a "pellicular" type of solid support where a more mobile polymer is grafted to rigid plastics. Compared to low cross-linked microporous beads that dominate the field, this approach allows great flexibility of design, as plastics are available as sheets, films, or threads, or can be molded into any shape, as required. Many different polymers or copolymers can be grafted onto any particular shape to give a wide choice of options in the physicochemical characteristics of the actual solid support. As an example of such a solid support, we report on polystyrene-grafted polypropylene in a particular shape that we have called "Lanterns." Its synthesis characteristics are compared to the commonly available low cross-linked polystyrene resins. As well, the handling advantages of these types of supports in multiple synthesis are highlighted.

Chemical Engineering↗

PubMed2000

Specific PCR primers for Cryptosporidium parvum with extra high sensitivity.

One pair of high-sensitive polymerase chain reaction (PCR) primers for Cryptosporidium parvum was constructed based on the sequence of random amplified polymorphic DNA. PCR with this primer pair amplified only the DNA of C. parvum, not the control DNA including Cryptosporidium muris. This primer pair had most advantageous in its sensitivity over the six pairs of primers reported elsewhere. The minimum amount of template DNA required to produce visible bands after gel electrophoresis and ethidium bromide staining was 0.156 pg of C. parvum or just a single oocyst in the PCR tube.

Adult↗

PubMed2000

DNA fingerprints of Trichinella as revealed by restriction fragment length polymorphism and single-strand conformational polymorphism (RFLP-SSCP).

A method was developed for gene fingerprinting, combining the principles of restriction fragment length polymorphism and single-strand conformational polymorphism (PCR-RFLP-SSCP). Taking advantage of this method, we analysed the genotypes of 20 isolates from five species of Trichinella (Trichinella spiralis, Trichinella britovi, Trichinella nativa, Trichinella murrelli and Trichinella pseudospiralis) and two uncertain genotypes (Trichinella T6 and Trichinella T8). Target genes for the analysis included three kinds of random amplified polymorphic DNA (RAPD), the gene encoding 43 kDa excretory-secretory protein, the gene encoding mitochondrial cytochrome c -oxidase subunit I and the gene of 18 S rRNA. The genotype revealed by this method was in good concordance with the taxonomy of Trichinella (six species and two uncertain genotypes which is currently accepted based on morphology, isozyme pattern and reproductive isolation). More important, this method revealed intraspecies polymorphism among geographical isolates of T. spiralis and T. britovi.

Animals↗

PubMed2000

Immunohistochemical study of NGF and its receptors in the synovial membrane of the ankle joint of adjuvant-induced arthritic rats.

To study the role of nerve growth factor (NGF) in local inflammation, we investigated the expression of NGF and its receptors, trkA and p75, in the ankle joints of adjuvant-induced arthritic rats. Infiltrated mononuclear cells revealed a positive immunoreactivity for NGF and trkA; they were also positive for immunostaining for W3/25 and ED1, which mainly stain T cells and macrophages, respectively. Changes in the ratios of NGF-positive cells to mononuclear cells showed a relatively similar pattern for trkA-positive cells, which peaked at weeks 2 to 3 after the adjuvant injection. In double-immunofluorescence staining, 80% and 65% of NGF-positive cells stained for W3/25 and ED1, respectively. Similarly, 67% and 80% of trkA-positive cells also corresponded to W3/25- and ED1-positive cells, respectively. However, p75 immunoreactivity localized on the nerve fibers but not on the cells of the ankle joints. Dense meshworks of p75-positive nerve fibers with numerous terminal varicosities were observed at weeks 2 to 4. The present findings suggest that infiltrated mononuclear cells may secrete NGF in an autocrine or paracrine manner in the inflamed synovium. An upregulation of NGF in these mononuclear cells and an increase in density of the synovial nerve fibers may be involved in the development of adjuvant arthritis in rats.

Animals↗