Biomedical subjects

J Wu

Publications and source records attributed to J Wu.

At least 19 recordsLinked to original sources

PubMed2000

Crystal structure of recombinant trypsin-solubilized fragment of cytochrome b(5) and the structural comparison with Val61His mutant.

The crystal structure of the recombinant trypsin-solubilized fragment of the microsomal cytochrome b(5) from bovine liver has been determined at 1.9 A resolution and compared with the reported crystal structure of the lipase-solubilized fragment of the membrane protein cytochrome b(5). The two structures are similar to each other. However, some detailed structural differences are observed: the conformation of the segment Asn16-Ser20 is quite different, some helices around the heme and some segments between the helices are shifted slightly, the heme is rotated about the normal of the mean plane of heme, one of the propionates of the heme exhibits a different conformation. The average coordination distances between the iron and the two nitrogen atoms of the imidazole ligands are the same in the two structures. Most of the structural differences can be attributed to the different intermolecular interactions which result from the crystal packing. The wild-type protein structure is also compared with its Val61His mutant, showing that the heme binding and the main chain conformations are basically identical with each other except for the local area of the mutation site. However, when Val61 is mutated to histidine, the large side chain of His61 is forced to point away from the heme pocket toward the solvent region, disturbing the micro-environment of the heme pocket and influencing the stability and the redox potential of the protein.

Animals↗

PubMed2000

Roles of tissue transglutaminase in ethanol-induced inhibition of hepatocyte proliferation and alpha 1-adrenergic signal transduction.

The mechanisms by which ethanol inhibits hepatocyte proliferation have been a source of some considerable investigation. Our studies have suggested a possible role for tissue transglutaminase (tTG) in this process. Others have shown that tTG has two distinctly different functions: it catalyzes protein cross-linking, which can lead to apoptosis and enhancement of extracellular matrix stability, and it can function as a G protein (Galpha(h)). Under that circumstance, we speculated that the cross-linking activity would be decreased and that it would function to enhance hepatocyte proliferation in response to adrenergic stimulation. Ethanol treatment inhibited hepatocyte proliferation and led to enhanced tTG cross-linking activity, whereas treatment of hepatocytes with an alpha1 adrenergic agonist, phenylephrine, enhanced hepatocyte proliferation while decreasing tTG cross-linking. However, phenylephrine treatment of several hepatoma cell lines had no effect on cellular proliferation or tTG cross-linking activity, and of note, Northern blot analysis demonstrated that whereas primary hepatocytes had high levels of the alpha1beta adrenergic receptor (alpha1BAR) mRNA, the hepatoma cell lines did not have this mRNA. When the Hep G(2) cell line was stably transduced with an expression vector containing the alpha1BR cDNA, the cell line responded to phenylephrine treatment with enhanced proliferation and with decreased tTG cross-linking activity. Ethanol treatment of the alpha1BAR-transfected cells suppressed the phospholipase C-mediated signaling pathways, as detected in the phenylephrine-induced Ca(2+) response. These results suggest that phenylephrine stimulation of hepatocyte proliferation appears to be occurring through the alpha1BAR, which is known to be coupled with the tTG G protein moiety, Galpha(h), and that tTG appears to play a significant role in either enhancing or inhibiting hepatocyte proliferation, depending on its cellular location and on whether it functions as a cross-linking enzyme or a G protein.

Animals↗

PubMed2000

Focal adhesion kinase up-regulation and signaling in activated keratinocytes.

BACKGROUND: During wound healing keratinocytes undergo a process called "activation" that enables the cells to spread and migrate on wound matrix molecules. Focal adhesion kinase (FAK) is a key component of integrin-mediated intracellular signaling. We investigated the induction of FAK and its signaling activity during keratinocyte activation. MATERIALS AND METHODS: Keratinocytes were harvested from normal human skin. Previous work has shown that culture of keratinocytes causes activation in a manner similar to reepithelialization. Freshly isolated, unactivated cells were compared with cultured, activated cells. Activated cells were further examined either as growing colonies or after replating on type I collagen. FAK content was assessed by Western blotting. FAK distribution was shown using indirect immunofluorescence. FAK signaling activity was assessed using an antiphosphotyrosine antibody. RESULTS: FAK was not detectable by Western blotting in freshly isolated cells. In contrast FAK was detected in activated cells. FAK was up-regulated between Days 2 and 4 after cell isolation from skin. Immunostaining of activated, growing keratinocyte colonies in vitro showed a diffuse, cytoplasmic pattern. When these cells were replated on collagen, FAK became concentrated in focal adhesions. Lysates from replated cells showed increased tyrosine phosphorylation of FAK. CONCLUSIONS: In summary FAK is induced in keratinocytes in a time course comparable to that of activation. FAK is phosphorylated and undergoes redistribution to focal adhesions when cells are plated on the beta(1) integrin ligand collagen. These data suggest that induction of FAK and subsequent FAK-induced signaling may be responsible for changes in integrin-mediated behavior of activated keratinocytes during reepithelialization.

Cells, Cultured↗

PubMed2000

Requirement of SHP2 binding to Grb2-associated binder-1 for mitogen-activated protein kinase activation in response to lysophosphatidic acid and epidermal growth factor.

Grb2-associated binder-1 (Gab1) is a multisite docking protein containing a pleckstrin homology (PH) domain, multiple potential tyrosine phosphorylation sites, and several proline-rich sequences. Gab1 becomes tyrosine-phosphorylated in cells stimulated with growth factors, cytokines, and ligands for G protein-coupled receptors. A major Gab1-binding protein detected in cells treated with extracellular stimuli is the tyrosine phosphatase, SHP2. Although the role of SHP2-Gab1 interaction in cell signaling has not yet been characterized, SHP2 is known to mediate mitogen-activated protein (MAP) kinase activation induced by the epidermal growth factor (EGF). However, the mechanism by which the SHP2 phosphatase exerts a positive signaling role remains obscure. In this study, we prepared Gab1 mutants lacking the SHP2 binding site (Gab1Y627F), the phosphatidylinositol 3-kinase (PI3K) binding sites (Gab1DeltaPI3K), and the PH domain (Gab1DeltaPH). Expression of Gab1Y627F blocked the extracellular signal-regulated kinase-2 (ERK2) activation by lysophosphatidic acid (LPA) and EGF. Conversely, expression of the wild-type Gab1 in HEK293 cells augmented the LPA receptor Edg2-mediated ERK2 activation. Whereas the PH domain was required for Gab1 mediation of ERK2 activation by LPA, it was not essential for EGF-induced ERK2 activation. Expression of Gab1DeltaPI3K had no apparent effect on ERK2 activation by LPA and EGF in the cells that we have examined. These results establish a role for Gab1 in the LPA-induced MAP kinase pathway and clearly demonstrate that Gab1-SHP2 interaction is essential for ERK2 activation by LPA and EGF. These findings also suggest that the positive role of SHP2 in the MAP kinase pathway depends on its interaction with Gab1.

Animals↗

PubMed2000

Reaction rate, activation energy, and detection limit for the reaction of 5-furoylquinoline-3-carboxaldehyde with neurotransmitters in artificial cerebrospinal fluid.

The fluorogenic reagent 5-furoylquinoline-3-carboxaldehyde (FQ) has proven valuable in the analysis of proteins and small neurotransmitters. We monitored the reaction rate between this reagent and five neurotransmitters at 40 degrees and 65 degrees C in artificial cerebrospinal fluid. The reactions followed pseudo-first order kinetics. The activation energy for the reaction of FQ was 10.6, 10.7, 22.0, 31.4, and 34.4 kJ mol(-1) for alanine, taurine, gamma-aminobutyric acid (GABA), glutamine, and glutamic acid, respectively. At 65 degrees C, the reaction rate was quite similar for alanine, taurine, glutamine, and glutamic acid (1.8 x 10(-3) s(-1)) but was twice as fast for GABA. A reaction time of nearly 1 h was required to quantitatively convert these neurotransmitters to their fluorescent products at 65 degrees C. Detection limits for the labeled neurotransmitters were 10(-9)-10(-8) M, which corresponded to 0.3-7 amol injected onto the capillary.

Animals↗

PubMed2000

Control of energy transfer in oriented conjugated polymer-mesoporous silica composites

Nanoscale architecture was used to control energy transfer in semiconducting polymers embedded in the channels of oriented, hexagonal nanoporous silica. Polarized femtosecond spectroscopies show that excitations migrate unidirectionally from aggregated, randomly oriented polymer segments outside the pores to isolated, aligned polymer chains within the pores. Energy migration along the conjugated polymer backbone occurred more slowly than Forster energy transfer between polymer chains. The different intrachain and interchain energy transfer time scales explain the behavior of conjugated polymers in a range of solution environments. The results provide insights for optimizing nanostructured materials for use in optoelectronic devices.

Journal Article↗

PubMed2000

Compromised kidney graft rejection response in Vervet monkeys after withdrawal of immunosuppressants tacrolimus and sirolimus.

BACKGROUND: In nonprimates, organ allografts are often not rejected after withdrawal of immunosuppression. In this study, we examined whether such a phenomenon also occurs in primates. METHODS: Vervet monkeys were transplanted with renal allografts and treated for 60 days with tacrolimus, or tacrolimus plus sirolimus. The drugs were totally withdrawn on day 61. The survival of the monkeys was monitored, and their response to donor- or third party-derived alloantigens was examined in vivo and in vitro. RESULTS: The majority (80-100%) of the grafts survived for at least additional 30 days with no signs of acute rejection. The compromised rejection is donor-specific, because recipient monkeys failed to reject a donor-derived skin graft, but a third-party skin graft was rejected. In vitro mixed lymphocyte reaction and interleukin-2 production in the mixed lymphocyte reaction between the recipients and their donors or between the recipients and a third party had no discernable patterns, and thus did not reflect the in vivo status of the immune system. Although the recipients could not reject the graft acutely after drug withdrawal, the kidney grafts and the donor-derived skin grafts had pathological findings of chronic rejection. CONCLUSIONS: The rejection response of the monkeys to an established graft after withdrawal of immunosuppression is compromised. The compromised rejection is specific and is not due to a permanent alteration of the immune system by the initial drug treatment. The allografts are not inert but have low levels of interaction with the recipient immune system.

Animals↗

PubMed2000

Effect of tacrolimus (FK506) and sirolimus (rapamycin) mono- and combination therapy in prolongation of renal allograft survival in the monkey.

BACKGROUND: Our previous studies confirmed that tacrolimus (FK506) and sirolimus [rapamycin (RAPA)], in combination, are not antagonistic but are synergistic in the prolongation of heart and small bowel grafts in the rodent. The aim of this study was to confirm further the synergistic effect of combined FK506 and RAPA in the more clinically relevant model, kidney transplantation in monkeys. METHODS: A total of 60 male Vervet monkeys were randomly assigned to 10 groups (n> or =5). Monkeys with renal allografts were treated with different doses of FK506 and/or RAPA orally for 60 days. Graft survival, body weight, clinical biochemistry determinations, oral glucose tolerance test, trough levels of the two drugs, and histopathology were investigated. RESULTS: Low doses of FK506 (1 or 4 mg/kg) combined with RAPA (0.5 mg/kg) produced synergistic effect in the prolongation of renal graft survival [combination index (CI) = 0.292, 0.565]. There were no additive or synergistic drug-associated toxicities such as hyperglycemia, nephrotoxicity, and hyperlipidemia. There also was no pharmacological antagonism. CONCLUSION: Concomitant therapy of low-dose (drug-optimal) FK506 and RAPA produced a synergistic effect in the prolongation of kidney allograft survival in Vervet monkeys without additive drug-associated toxicities.

Animals↗

PubMed2000

Papain digestion of different mouse IgG subclasses as studied by electrospray mass spectrometry.

On-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) has been utilized to monitor the papain digestion of mouse monoclonal antibodies (mAb) of different subclasses (IgG1, IgG2a, IgG2b, and IgG3). The method is simple, rapid, sensitive, and allows for simultaneous determination of digestion products and the identification of microheterogenous fragments. The results provide important insight into the mechanism of papain digestion of mouse IgGs of different subclasses.

Animals↗

PubMed2000

Regulation and functional significance of utrophin expression at the mammalian neuromuscular synapse.

Duchenne muscular dystrophy (DMD) is caused by the absence of full-length dystrophin molecules in skeletal muscle fibers. In normal muscle, dystrophin is found along the length of the sarcolemma where it links the intracellular actin cytoskeleton to the extracellular matrix, via the dystrophin-associated protein (DAP) complex. Several years ago, an autosomal homologue to dystrophin, termed utrophin, was identified and shown to be expressed in a variety of tissues, including skeletal muscle. However, in contrast to the localization of dystrophin in extrajunctional regions of muscle fibers, utrophin preferentially accumulates at the postsynaptic membrane of the neuromuscular junction in both normal and DMD adult muscle fibers. Since it has recently been suggested that the upregulation of utrophin might functionally compensate for the lack of dystrophin in DMD, considerable interest is now directed toward the elucidation of the various regulatory mechanisms presiding over expression of utrophin in normal and dystrophic skeletal muscle fibers. In this review, we discuss some of the most recent data relevant to our understanding of the impact of myogenic differentiation and innervation on the expression and localization of utrophin in skeletal muscle fibers.

Animals↗

PubMed2000

Involvement of lipoxygenase in lysophosphatidic acid-stimulated hydrogen peroxide release in human HaCaT keratinocytes.

Although it is now recognized that low levels of reactive oxygen species (ROS) are required for the mitogenic response, mitogen-induced signalling pathways that regulate ROS generation in non-phagocytic cells remain largely uncharacterized. Using a real-time assay for measuring hydrogen peroxide (H(2)O(2)) formation, we analysed H(2)O(2) release in human HaCaT keratinocytes in response to lysophosphatidic acid (LPA), a mitogen for keratinocytes. LPA rapidly increased H(2)O(2) release in HaCaT cells. Unlike LPA-induced mitogen-activated protein (MAP) kinase activation, LPA-stimulated H(2)O(2) release was independent of the tyrosine kinase activity of the epidermal growth factor (EGF) receptor. Calcium chelators, phospholipase A(2) inhibitors, and lipoxygenase inhibitors effectively blocked LPA-stimulated H(2)O(2) release, whereas cyclooxygenase inhibitors were without effect. Addition of 5-lipoxygenase products 5-hydroperoxyeicosatetraenoic acid (5-HPETE) and leukotriene B(4), but not 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene C(4), restored LPA-stimulated H(2)O(2) release in cells treated with the lipoxygenase inhibitors nordihydroguaiaretic acid and Zileuton. These results suggest that the lipoxygenase products 5-HPETE and leukotriene B(4) are required for LPA-stimulated H(2)O(2) release in HaCaT cells.

Calcium↗

PubMed2000

Effective ring-opening reaction of aziridines with trimethylsilyl compounds: A facile access to beta-amino acids and 1,2-diamine derivatives

Ring-opening reactions of aziridines with trimethylsilyl compounds triggered by tetrabutylammonium fluoride give the corresponding products regioselectively in excellent yield. It provides a facile and efficient procedure for the ring-opening reactions of aziridines and affords a practical access to the synthesis of cyano-, azido-, or chloroamines because of its efficiency and simplicity. The products are easily transformed to vicinal diamines or beta-amino acids.

Journal Article↗

PubMed2000

Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review.

Most solid tumors possess unique pathophysiological characteristics that are not observed in normal tissues or organs, such as extensive angiogenesis and hence hypervasculature, defective vascular architecture, impaired lymphatic drainage/recovery system, and greatly increased production of a number of permeability mediators. The phenomenon now known as the enhanced permeability and retention (EPR) effect for lipid and macromolecular agents has been observed to be universal in solid tumors. Primarily, enhanced vascular permeability will sustain an adequate supply of nutrients and oxygen for rapid tumor growth. The EPR effect also provides a great opportunity for more selective targeting of lipid- or polymer-conjugated anticancer drugs, such as SMANCS and PK-1, to the tumor. In the present review, the basic characteristics of the EPR effect, particularly the factors involved, are described, as well as its modulation for improving delivery of macromolecular drugs to the tumor. Tumor-specific vascular physiology is also described.

Animals↗

PubMed2000

Mutations of the serine phosphorylated in the protein phosphatase-1-binding motif in the skeletal muscle glycogen-targeting subunit.

Cellular functions of protein phosphatase-1 (PP1) are determined by regulatory subunits that contain the consensus PP1-binding motif, RVXF. This motif was first identified as the site of phosphorylation by cAMP-dependent protein kinase (PKA) in a skeletal muscle glycogen-targeting subunit (G(M)). We reported previously that a recombinant fusion protein of glutathione S-transferase (GST) and the N-terminal domain of G(M) [GST-G(M)-(1-240)] bound PP1 in a pull down assay, and phosphorylation by PKA prevented PP1 binding. Here we report that substitution of either Ala or Val for Ser-67 in the RVS(67)F motif in GST-G(M)-(1-240) essentially eliminated PP1 binding. This was unexpected because other glycogen-targeting subunits have a Val residue at the position corresponding to Ser-67. In contrast, a mutation of Ser-67 to Thr (S67T) in GST-G(M)(1-240) gave a protein that bound PP1 the same as wild type and was unaffected by PKA phosphorylation. Full length G(M) tagged with the epitope sequence DYKDDDDK (FLAG) expressed in COS7 cells bound PP1 that was recovered by co-immunoprecipitation, but this association was prevented by treatment of the cells with forskolin. By comparison, PP1 binding with FLAG-G(M)(S67T) was not disrupted by forskolin treatment. Neither FLAG-G(M)(S67A) nor FLAG-G(M)(S67V) formed stable complexes with PP1 in COS7 cells. These results emphasise the unique contribution of Ser-67 in PP1 binding to G(M). The constitutive PP1-binding activity shown by G(M)(S67T) opens the way for studying the role of G(M) multisite phosphorylation in hormonal control of glycogen metabolism.

Amino Acid Motifs↗

PubMed2000

Linkage study of two polymorphisms at the dopamine D3 receptor gene and attention-deficit hyperactivity disorder.

Data from animal studies suggest that the dopamine D3 receptor gene may have a role in locomotion and behavioral regulation. Therefore, this gene has been suggested as a candidate for attention-deficit hyperactivity disorder (ADHD). The dopamine D3 receptor gene (DRD3) has two common polymorphisms, one in exon I that changes a Serine to Glycine (Ser9Gly) and alters the recognition site for the restriction enzyme MscI [Lannfelt et al., 1992]. The other common polymorphism is located in intron 5 and results in the change of a restriction site for MspI [Griffon et al., 1996]. We investigated the possibility of linkage of the dopamine D3 receptor gene in 100 small, nuclear families consisting of a proband with ADHD, their parents, and affected siblings. We examined the transmission of the alleles of each of these polymorphisms and the haplotypes of both polymorphisms using the transmission disequilibrium test [Spielman et al., 1993]. We did not observe biased transmission of the alleles at either polymorphism or any haplotype. Our findings using this particular sample do not support the role of the dopamine D3 gene in ADHD. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:114-117, 2000.

Attention Deficit Disorder with Hyperactivity↗

PubMed2000

Influence of alcohol concentration on lipase-catalyzed enantioselective esterification of racemic naproxen in isooctane: under controlled water activity*

According to a previous study, alcohol concentration influences the enantiomeric ratio and initial rate of Candida cylindracea lipase-catalyzed enantioselective esterification of racemic 2-(6-methoxy-2-naphthyl) propionic acid in microaqueous isooctane in a similar manner. Such an influence might be attributed to the different water partitioning coefficients. In this work, we performed enzymatic resolutions in controlled water activity atmospheres, thereby separating the influence of alcohol concentrations from the effect of water partitioning. Despite this constant water activity condition, a similar dependence of enantiospecificity and initial rate on the concentration of acyl acceptor (n-butanol) was also found. This finding suggests that the alcohol concentration influences enzyme performance, but not because it strips water from the enzyme. The unexpected observations imply that an undetermined factor must be considered when the enzymatic resolution is performed in nonconventional media.

Journal Article↗

PubMed2000

Tumor-targeting chemotherapy by a xanthine oxidase-polymer conjugate that generates oxygen-free radicals in tumor tissue.

Xanthine oxidase (XO) mediates anticancer activity because of its ability to generate cytotoxic reactive oxygen species (ROS), including superoxide anion radical and hydrogen peroxide. However, the high binding affinity of XO to blood vessels would cause systemic vascular damage and hence limits the use of native XO in clinical settings. We demonstrate here that chemical conjugation of XO with poly(ethylene glycol) (PEG; the conjugates hereafter referred to as PEG-XO) significantly enhanced the tumor-targeting efficacy and the antitumor activity of XO. By using a succinimide-activated PEG derivative, PEG was conjugated to epsilon-amino groups of lysine residues of XO, which play a crucial role in binding of XO to blood vessels. PEG-XO administered i.v. showed a 2.8-fold higher accumulation in solid tumor compared with that of native XO 24 h after injection, whereas a slight or negligible increase in accumulation of PEG-XO was observed in normal organs. The highest PEG-XO enzyme activity was detected in tumor compared with normal organs or tissues except blood; enzyme activity in tumor was 5.0, 3.9, and 9.4 times higher than that in liver, kidney, and spleen, respectively. Intratumor activity remained high for >48 h. Administration of hypoxanthine, a substrate of XO, at 33 mg/kg body weight i.p. 12 h after the administration of PEG-XO (0.6 unit/mouse, i.v.) resulted in significant suppression of tumor growth (P < 0.001), with no tumor growth even after 52 days. However, either PEG-XO or hypoxanthine alone, or native XO with hypoxanthine, showed no effect on the inhibition of tumor growth under present experimental conditions. These findings suggest that PEG-XO, which accumulates preferentially in tumor tissue, warrants further investigation as a novel anticancer agent.

Animals↗

PubMed2000

Functional analysis of six novel ORFs on the left arm of chromosome XII in Saccharomyces cerevisiae reveals two essential genes, one of which is under cell-cycle control.

Six novel Open Reading Frames (ORFs) located on the left arm of chromosome XII (YLL044w, YLL042c, YLL040c, YLL038c, YLL035w and YLL034c) have been analysed using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YLL035w and YLL034c are essential for cell growth; yll035w spores arrested after two or three cell divisions, while the majority of yll034c spores stopped growth within two cell cycles after germination. Complementation of the yll035w deletion with its cognate clone, and a promoter-substitution experiment, indicated that the promoter of YLL035w may lie within the adjacent ORF, YLL036c. Transcriptional analysis demonstrated that YLL035w is under cell-cycle regulation. Bioinformatic analyses produced significant matches between YLL034c and mammalian valosin and many other ATPases. The standard EUROFAN growth tests failed to reveal obvious phenotypes resulting from deletion of any of the four non-essential ORFs. Replacement cassettes, comprising the kanMX marker flanked by each ORF's promoter and terminator regions, were cloned into pUG7. All the cognate clones, except for YLL040c, were generated using direct PCR products amplified from genomic DNA or using gap-repair. All clones and strains produced have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt).

Cell Cycle↗