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Biomedical subjects

W Ma

Publications and source records attributed to W Ma.

At least 55 records · Page 3Linked to original sources
PubMed2001

[Influence of cytokine gene polymorphism on renal transplantation].

OBJECTIVE: To investigate the influence of cytokine gene polymorphism on renal transplantation. METHODS: The cytokine genotypes of cytokines TNF-alpha, IL-10, TGF-beta(1), and L-6 among 91 consecutive kidney recipients were detected by PCR-SSP. The distribution frequencies of different cytokine genotypes and the influence of cytokine gene polymorphism on renal transplantation were analyzed. RESULTS: Among the 91 renal recipients, the dominant genotypes of IL-10 and TNF-alpha were low producer genotype with the frequencies of 90.1% and 79.1% respectively; and the dominant genotypes of TGF-beta(1) and IL-6 were high producer genotypes (79.1% and 100% respectively). The kidney recipients with TNF-alpha high producer genotype and IL-10 high or intermediate producer genotype were associated with more acute rejection episodes than those with low producer genotypes. The acute rejection episodes in the kidney recipients with IL-10 high or intermediate producer genotype were mainly vascular acute rejection. CONCLUSION: The cytokine gene polymorphism has significant influence on incidence and severity of acute rejection after renal transplantation. Detecting the cytokine genotype before renal transplantation is beneficial to predict the outcome of transplantation accurately.

Adolescent↗
PubMed2001

Survival and neurite outgrowth of rat cortical neurons in three-dimensional agarose and collagen gel matrices.

To better understand interactions between neurons and extracellular matrix equivalents, embryonic day-18 rat cortical neurons were immobilized and maintained in culture for up to 24 days in agarose and type I collagen gels. Using live/dead staining, neuronal cultures in low density collagen gel lasted at least 3 weeks. At 14 days, over 50% of immobilized cells in collagen gel were found viable while in low density agarose gel no cells survived. In situ cell death detection showed that most, if not all, dead cells in either of the gels underwent apoptosis. The collagen-trapped neurons exhibited normal neuronal polarity and developed long neurites, estimated at over 500 microm. The results suggest that collagen, because it is a major extracellular matrix constituent, suppresses apoptosis and provides a suitable substrate for neuronal survival and differentiation.

Animals↗
PubMed2001

Regulation of the p21-activated kinase (PAK) by a human Gbeta -like WD-repeat protein, hPIP1.

The family of p21-activated protein kinases (PAKs) is composed of serine-threonine kinases whose activity is regulated by the small guanosine triphosphatases (GTPases) Rac and Cdc42. In mammalian cells, PAKs have been implicated in the regulation of mitogen-activated protein cascades, cellular morphological and cytoskeletal changes, neurite outgrowth, and cell apoptosis. Although the ability of Cdc42 and Rac GTPases to activate PAK is well established, relatively little is known about the negative regulation of PAK or the identity of PAK cellular targets. Here, we describe the identification and characterization of a human PAK-interacting protein, hPIP1. hPIP1 contains G protein beta-like WD repeats and shares sequence homology with the essential fission yeast PAK regulator, Skb15, as well as the essential budding yeast protein, MAK11. Interaction of hPIP1 with PAK1 inhibits the Cdc42/Rac-stimulated kinase activity through the N-terminal regulatory domains of PAK1. Cotransfection of hPIP1 in mammalian cells inhibits PAK-mediated c-Jun N-terminal kinase and nuclear factor kappa B signaling pathways. Our results demonstrate that hPIP1 is a negative regulator of PAK and PAK signaling pathways.

Amino Acid Sequence↗
PubMed2001

Leukocyte-specific adaptor protein Grap2 interacts with hematopoietic progenitor kinase 1 (HPK1) to activate JNK signaling pathway in T lymphocytes.

Immune cell-specific adaptor proteins create various combinations of multiprotein complexes and integrate signals from cell surface receptors to the nucleus, modulating the specificity and selectivity of intracellular signal transduction. Grap2 is a newly identified adaptor protein specifically expressed in lymphoid tissues. This protein shares 40--50% sequence homology in the SH3 and the SH2 domain with Grb2 and Grap. However, the Grap2 protein has a unique 120-amino acid glutamine- and proline-rich domain between the SH2 and C-terminal SH3 domains. The expression of Grap2 is highly restricted to lymphoid organs and T lymphocytes. In order to understand the role of this specific adaptor protein in immune cell signaling and activation, we searched for the Grap2 interacting protein in T lymphocytes. We found that Grap2 interacted with the hematopoietic progenitor kinase 1 (HPK1) in vitro and in Jurkat T cells. The interaction was mediated by the carboxyl-terminal SH3 domain of Grap2 with the second proline-rich motif of HPK1. Coexpression of Grap2 with HPK1 not only increased the kinase activity of HPK1 in the cell, but also had an additive effect on HPK1 mediated JNK activation. Furthermore, over expression of Grap2 and HPK1 induced significant transcriptional activation of c-Jun in the JNK signaling pathway and IL-2 gene reporter activity in stimulated Jurkat T cells. Therefore, our data suggest that the hematopoietic specific proteins Grap2 and HPK1 form a signaling complex to mediate the c-Jun NH(2)-terminal kinase (JNK) signaling pathway in T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Structure and functionality of a designed p53 dimer.

P53 is a homotetrameric tumor suppressor protein involved in transcriptional control of genes that regulate cell proliferation and death. In order to probe the role that oligomerization plays in this capacity, we have previously designed and characterized a series of p53 proteins with altered oligomeric states through hydrophilc substitution of residues Met340 or Leu344 in the normally tetrameric oligomerization domain. Although such mutations have little effect on the overall secondary structural content of the oligomerization domain, both solubility and the resistance to thermal denaturation are substantially reduced relative to that of the wild-type domain. Here, we report the design and characterization of a double-mutant p53 with alterations of residues at positions Met340 and Leu344. The double-mutations Met340Glu/Leu344Lys and Met340Gln/Leu344Arg resulted in distinct dimeric forms of the protein. Furthermore, we have verified by NMR structure determination that the double-mutant Met340Gln/Leu344Arg is essentially a "half-tetramer". Analysis of the in vivo activities of full-length p53 oligomeric mutants reveals that while cell-cycle arrest requires tetrameric p53, transcriptional transactivation activity of monomers and dimers retain roughly background and half of the wild-type activity, respectively.

Binding Sites↗
PubMed2001

Activation of phosphatidylinositol-3 kinase (PI-3K) and extracellular regulated kinases (Erk1/2) is involved in muscarinic receptor-mediated DNA synthesis in neural progenitor cells.

Muscarinic acetylcholine receptor (mAChR), a member of the G-protein-coupled receptors (GPCRs) gene superfamily, has been shown to mediate the effects of acetylcholine on differentiation and proliferation in the CNS. However, the mechanism or mechanisms whereby mAChRs regulate cell proliferation remain poorly understood. Here we show that in vitro bFGF-expanded neural progenitor cells dissociated from rat cortical neuroepithelium express muscarinic acetylcholine receptor subtype mRNAs. We demonstrate that stimulation of these mAChRs with carbachol, a muscarinic agonist, activated extracellular-regulated kinases (Erk1/2) and phosphatidylinositol-3 kinase (PI-3K). This, in turn, stimulated DNA synthesis in neural progenitor cells. MEK inhibitor PD98059 and PI-3K inhibitors wortmannin and LY294002 inhibited a carbachol-induced increase in DNA synthesis. These findings indicate that the activation of both PI-3 kinase and MEK signaling pathways via muscarinic receptors is involved in stimulating DNA synthesis in the neural progenitor cells during early neurogenesis.

Animals↗
PubMed2001

Zirconium(IV) complexes of oxydiacetic acid in aqueous solution and in the solid state as studied by multinuclear NMR and X-ray crystallography.

The structures of complexes of Zr(IV) and oxydiacetate (ODA2-) in aqueous solutions of pH 0-7 were investigated with the use of 1H, 13C, and 17O NMR spectroscopy. Equilibria of mononuclear [Zr(oda)]2+, [Zr(oda)2], and [Zr(oda)3]2- complexes have been observed. In all complexes ODA2- is bound in a tridentate fashion through the two carboxylate groups and the ether oxygen. No di- or oligonuclear species containing ODA2- were observed. An excess of free Zr(IV) remains in solution, probably as a result of weak electrostatic interactions between negatively charged Zr-ODA complexes or free ODA2- and a positively charged cyclic tetranuclear hydroxy zirconium complex. CP-MAS 13C NMR spectra of solid compounds isolated from the samples indicated that the structures of the [Zr(oda)2] and [Zr(oda)3]2- complexes in solution are similar to those in the solid state. This is corroborated by the single-crystal X-ray structure of Na2[Zr(oda)3] x 5.5 H2O, which was obtained from a solution containing exclusively the [Zr(oda)3]2- complex. In this structure Zr(IV) is nine-coordinate with the three ODA2- ligands bound in a tricapped trigonal prismatic geometry. The negative charge of this [Zr(oda)3]2complex is balanced by two Na+ ions, one of which is on a center of symmetry between delta and lambda enantiomers of [Zr(oda)3]2-. This Na+ is octahedrally coordinated to six (non Zr(IV)-bound) carboxylate oxygen atoms of six different [Zr(oda)3]2- units.

Journal Article↗
PubMed2001

Adaptive antioxidant response protects dermal fibroblasts from UVA-induced phototoxicity.

In response to the attack of reactive oxygen species (ROS) produced upon UV irradiation, the skin has developed a complex antioxidant defense system. Here we report that, in addition to the previously published induction of manganese superoxide dismutase (MnSOD) activity, single and, to a higher extent, repetitive low-dose UVA irradiation also leads to a substantial upregulation of glutathione peroxidase (GPx) activity. This concomitant adaptive response of two antioxidant enzymes acting in the same detoxification pathway coincided with the protection from high-UVA-dose-induced cytotoxicity conferred by low-dose UVA preirradiation. Whereas an interval of 24 h did not, an interval of 12 h did lead to the induction of MnSOD activity and, under selenium-supplemented conditions, of GPx activity as well, conferring definite cellular protection from UVA-induced phototoxicity. Moreover, under selenium-deficient conditions, which abrogate the UVA-mediated induction of GPx activity, adaptive protection against the cytotoxic effects of high UVA doses was significantly lower compared with selenium supplementation. Isolated 4.6-fold overexpression of MnSOD activity in stably transfected fibroblasts led to specific resistance from UVA-mediated phototoxicity under selenium-deficient conditions. Collectively, these data indicate that the concomitant induction of MnSOD and GPx activity is related to the optimal adaptive protection from photooxidative damage. This adaptive antioxidant protection clearly depends on the irradiation interval and a sufficient selenium concentration, findings that may have important implications for the improvement of photoprotective and phototherapeutic strategies in medicine.

Antioxidants↗
PubMed2001

UVA induces Ser381 phosphorylation of p90RSK/MAPKAP-K1 via ERK and JNK pathways.

UVA exposure plays an important role in the etiology of skin cancer. The family of p90-kDa ribosomal S6 kinases (p90(RSK)/MAPKAP-K1) are activated via phosphorylation. In this study, results show that UVA-induced phosphorylation of p90(RSK) at Ser(381) through ERKs and JNKs, but not p38 kinase pathways. We provide evidence that UVA-induced p90(RSK) phosphorylation and kinase activity were time- and dose-dependent. Both PD98059 and a dominant negative mutant of ERK2 blocked ERKs and p90(RSK) Ser(381) phosphorylation, as well as p90(RSK) activity. A dominant negative mutant of p38 kinase blocked UVA-induced phosphorylation of p38 kinase, but had no effect on UVA-induced Ser(381) phosphorylation of p90(RSK) or kinase activity. UVA-induced p90(RSK) phosphorylation and kinase activity were markedly attenuated in JnK1(-/-) and JnK2(-/-) cells. A dominant negative mutant of JNK1 inhibited UVA-induced JNKs and p90(RSK) phosphorylation and kinase activity, but had no effect on ERKs phosphorylation. PD169316, a novel inhibitor of JNKs and p38 kinase, inhibited phosphorylation of p90(RSK), JNKs, and p38 kinase, but not ERKs. However, SB202190, a selective inhibitor of p38 kinase, had no effect on p90(RSK) or JNKs phosphorylation. Significantly, ERKs and JNKs, but not p38 kinase, immunoprecipitated with p90(RSK) when stimulated by UVA and p90(RSK) was a substrate for ERK2 and JNK2, but not p38 kinase. These data indicate clearly that p90(RSK) Ser(381) may be phosphorylated by activation of JNKs or ERKs, but not p38 kinase.

Enzyme Activation↗
PubMed2001

Cellular and molecular responses of the uterus to embryo implantation can be elicited by locally applied growth factors.

The implantation of a blastocyst into a receptive uterus is associated with a series of events, namely the attachment reaction followed by decidualization of the stroma. Previous studies established that the gene encoding heparin-binding EGF-like growth factor (HB-EGF) is expressed in the luminal epithelium solely at the site of blastocyst apposition preceding the attachment reaction. We report here the expression during implantation of 21 genes encoding other signaling proteins, including those belonging to the Bone morphogenetic protein (BMP), fibroblast growth factor (FGF), WNT, and Hedgehog (HH) pathways. We find that the attachment reaction is associated with a localized stromal induction of genes encoding BMP-2, FGF-2, and WNT-4. Despite efforts by many investigators, a simple in vitro model of implantation is not yet available to study either the hierarchy of the events triggered in the uterus by the embryo or the function of individual signaling proteins. We have therefore approached these questions by introducing beads loaded with purified factors into the receptive uterus. We show that beads soaked in HB-EGF or insulin-like growth factor-1 (IGF-1), but not other proteins, induce many of the same discrete local responses elicited by the blastocyst, including increased localized vascular permeability, decidualization, and expression of Bmp2 at the sites of the beads. By contrast, the expression domains of Indian hedgehog (Ihh), patched, and noggin become restricted as decidualization proceeds. Significantly, beads containing BMP-2 do not themselves elicit an implantation response but affect the spacing of implantation sites induced by blastocysts cotransferred with the beads.

Animals↗
PubMed2001

The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages.

Interleukin-10 (IL-10), a pleiotropic cytokine that inhibits inflammatory and cell-mediated immune responses, is produced by a wide variety of cell types including T and B cells and monocytes/macrophages. Regulation of pro- and anti-inflammatory cytokines has been suggested to involve distinct signaling pathways. In this study, we investigated the regulation of the human IL-10 (hIL-10) promoter in the human monocytic cell line THP-1 following activation with lipopolysaccharide (LPS). Analysis of hIL-10 promoter sequences revealed that DNA sequences located between base pairs -652 and -571 are necessary for IL-10 transcription. A computer analysis of the promoter sequence between base pairs -652 and -571 revealed the existence of consensus sequences for Sp1, PEA1, YY1, and Epstein-Barr virus-specific nuclear antigen-2 (EBNA-2)-like transcription factors. THP-1 cells transfected with a plasmid containing mutant Sp1 abrogated the promoter activity, whereas plasmids containing the sequences for PEA1, YY1, and EBNA-2-like transcription factors did not influence hIL-10 promoter activity. To understand the events upstream of Sp1 activation, we investigated the role of p38 and extracellular signal-regulated kinase mitogen-activated protein kinases by using their specific inhibitors. SB202190 and SB203580, the p38-specific inhibitors, inhibited LPS-induced IL-10 production. In contrast, PD98059, a specific inhibitor of extracellular signal-regulated kinase kinases, failed to modulate IL-10 production. Furthermore, SB203580 inhibited LPS-induced activation of Sp1, as well as the promoter activity in cells transfected with a plasmid containing the Sp1 consensus sequence. These results suggest that p38 mitogen-activated protein kinase regulates LPS-induced activation of Sp1, which in turn regulates transcription of the hIL-10 gene.

Arabidopsis Proteins↗
PubMed2001

Suppression of cell transformation and induction of apoptosis by caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE), which is derived from the propolis of honeybee hives, has been shown to block tumor promotion and to have toxic effects on several cancer cells. The mechanism of the anti-tumor promotion activity of CAPE is unclear, however. In this study, we found that CAPE suppressed 12-O-tetradecanoylphorbol-13-acetate-induced cell transformation and induced apoptosis in mouse epidermal JB6 Cl 41 cells. No difference in induction of apoptosis was observed between normal lymphoblasts and sphingomyelinase-deficient cell lines. Although CAPE treatment of two p53 mutant tumor cell lines, NCI-H358 and SK-OV-3, and p53-deficient (p53(-/-)) cells caused the cleavage of caspase-3 as well as DNA fragmentation, caspase-3 cleavage was seen early (at 6 h) only in cells expressing wild-type p53 (p53(+/+)) and Cl 41 cells. These results suggested that p53 may be involved in the early stage of CAPE-induced apoptosis. The p53-dependent transcription activation occurred 2 h after treatment with CAPE and reached a maximum at 6 h in Cl 41 p53 DNA-binding sequence stable transfectant cells. In addition, phosphorylation of p53 at serine 15 and serine 392 was induced in Cl 41 cells within 6 h after treatment with CAPE. Therefore, CAPE may induce apoptosis through p53-dependent and p53-independent pathways and its anti-tumor promotion activity may have occurred through the induction of apoptosis.

Animals↗
PubMed2001

The effect of aging on glutathione peroxidase-i knockout mice-resistance of the lens to oxidative stress.

Populations of control, C, and glutathione peroxidase-1 (GPx-1) knockout mice, K, were studied over a period of 2 years. No significant difference was observed between the C and K populations with respect to longevity, vitality, weight, lens biochemistry or morphology based on light and electron microscopy. It was concluded that under normal animal room barrier facilities, GPx-1 is not required. Furthermore, C and K lenses placed in organ culture and observed over a 24 hr period were indistinguishable. Organ cultured C lenses degraded medium H(2)O(2)levels at only a slightly greater rate than K lenses and this did not appear to change with age. However, tertiary butyl hydroperoxide (TBHP) was degraded less effectively by K lenses and this deficiency increased with age. No indication of change in redox non-protein SH (equivalent to GSH) status was observed between C and K whole lenses or epithelial cell fractions. With H(2)O(2)stress, the drop in C and K non-protein SH was comparable and there was little change with age. Examination of the impact of photochemical stress with 1.5 microM riboflavin and 4% O(2)upon choline transport indicated considerable damage with both C and K lenses, but little difference between the two populations until 1 or 2 years of age when the K lenses appear more vulnerable. With TBHP, the detrimental effect on the K lenses is greater and is observed earlier than with photochemical stress suggesting that the K lens membrane function is more susceptible to phospholipid hydroperoxide stress than are C lenses. Light and electron microscopy of the oxidative stressed lenses indicates significant damage which was generally somewhat greater in the K lenses. TBHP was a more potent oxidant than photochemically generated oxidants particularly at the anterior pole. The overall results suggest that under normal conditions, at any age, the lens does not require the presence of GPx-1 but depending on the type of oxidative stress, the enzyme may significantly contribute to its defense and this dependency may increase with age.

Aging↗
PubMed2001

Improved immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) by flow cytometry.

BACKGROUND: The immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) is of particular importance in the differential diagnosis of interstitial lung disorders. The standard method of lymphocyte phenotyping is peroxidase-anti-peroxidase technique (PAP). However, it was time-consuming and experience-dependent. Flow cytometric (FCM) analysis of BALF lymphocytes was introduced to overcome these disadvantages. Unfortunately, when the number of cells counted was small, FCM could not distinguish lymphocytes from other cells and particles in BALF by light scattering. METHODS: We established a tri-color flow cytometric approach to phenotyping of lymphocytes in BALF. FITC-CD45/PE-CD14 antibodies were used to gate lymphocytes and exclude other contamination. Propidium iodide (PI) was introduced to distinguish lymphocytes from debris. Forty-three BALF species were tested by flow cytometer as well as peripheral blood as a control group by conventional PAP method. RESULTS: The variation of FCM (CV<1.0%) was much lower that that of PAP method (CV>9.8%). Meanwhile, we found that BALF had more clinical significance than peripheral blood in T subset analysis (p<0.01). There were characteristic changes in some lung diseases. Both CD3 and CD4 were significantly increased with decreasing CD8 in sarcoidosis (n=14). Idiopathic pulmonary fibrosis (n=16) demonstrated the reverse tendency: CD8 rose but both CD3 and CD4 dropped. As for lung cancer (n=7), CD3 was normal but the CD4/CD8 ratio declined. Tuberculosis of the lungs (n=6) showed a normal CD3, CD4 and CD8. CONCLUSIONS: The high precision and reliability of tri-color flow cytometric approach to phenotyping of lymphocytes in BALF suggested that it should be used as a routine test, especially of BALF, which was often contaminated by inorganic particles.

Antigens, CD↗
PubMed2001

The aggregation behavior of collagen in aqueous solution and its property of stabilizing liposomes in vitro.

The aim of this study is to acquire the relationship between the aggregation behavior of collagen in aqueous solution and its property of stabilizing liposomes in vitro. Firstly, we used an intramolecular charge transfer compound 3-methoxy-4'-N,N-dimethylamino flavone derivative (DMMF) as a fluorescence probe to detect collagen association in aqueous solution. Results showed that, when collagen concentration was increased to 0.5 mg/ml or above, the fluorescence intensity of DMMF at 465 nm increased dramatically. This indicated that collagen could associate in aqueous solution. The subsequent fluorescence polarization study on the same collagen solutions also proved that the aggregation behavior existed in the aqueous solution of collagen. Secondly. to evaluate the in vitro stability of small unilamellar vesicles (SUV), made from soybean phosphatidylcholine (PC) and coated with collagen, 5(6)-carboxyfluorescein was used as a fluorescence probe to determine the change of their permeability. We found that the coating with a collagen/PC weight ratio of 2:1, significantly decreased the permeability of liposomal membranes. The likely weight ratio of collagen and PC was mainly dependent on the critical aggregation concentration of collagen in aqueous solution. In addition, the fluorescence polarization method was also used to study the influence of a coating with collagen at the above ratio on the fluidity of liposomal membranes, employing DMMF as a fluorescence probe. The fluidity of liposomal membranes coated with collagen showed no obvious change, indicating that SUV coated with collagen at an appropriate ratio, could significantly improve the in vitro stability of liposomal membranes without disturbing their fluidity. It is thus clear that collagen self-association behavior is closely correlated to its coating liposomes to improve their stability.

Animals↗
PubMed2001

Neurophysiologic effects of chemical agent hydrolysis products on cortical neurons in vitro.

The neurophysiologic effects of chemical agent hydrolysis products were examined on cultured cortical neurons using multielectrode array (MEA) recording and the whole-cell patch clamp technique. Measurement of neuronal network extracellular potentials showed that the primary hydrolysis product of soman, pinacolyl methylphosphonic acid (PMPA), inhibited network mean burst and spike rates with an EC50 of approximately 2 mM. In contrast, the degradation product of sarin, isopropyl methylphosphonic acid (IMPA), and the final common hydrolysis product of both soman and sarin, methylphosphonic acid (MPA), failed to affect neuronal network behavior at concentrations reaching 5 mM. Closer examination of the effects of PMPA (2 mM) on discriminated extracellular units revealed that mean spike amplitude was slightly diminished to 95 +/- 1% (mean +/- S.E.M., n = 6, P < 0.01) of control. Whole-cell patch clamp records under current clamp mode also showed a PMPA-induced depression of the firing rate of spontaneous action potentials (APs) to 36 +/- 6% (n = 5, P < 0.001) of control. In addition, a minor depression with exposure to PMPA was observed in spontaneous and evoked AP amplitude to 93 +/- 3% (n = 5, P < 0.05) of control with no change in either the baseline membrane potential or input resistance. Preliminary voltage clamp recordings indicated a reduction in the occurrence of spontaneous inward currents with application of PMPA. These findings suggest that PMPA, unlike MPA or IMPA, may more readily interfere with one or more aspects of excitatory synaptic transmission. Furthermore, the data demonstrate that the combination of extracellular microelectrode array and patch clamp recording techniques facilitates analysis of compounds with neuropharmacologic effects.

Action Potentials↗