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Biomedical subjects

P Xu

Publications and source records attributed to P Xu.

At least 73 records · Page 4Linked to original sources
PubMed2000

[Identification of telomerase activity in the experimental carcinogenesis of oral cavity].

OBJECTIVE: Telomerase is a ribonucleoprotein enzyme which synthesizes telomere DNA repeat sequences and maintain stably telomere length. The activity of telomerase may be necessary for the growth of immortalized cells overcoming cellular senescence. The researches have shown that telomerase activities are associated with most cancers. The purpose of the study is to detect the development of telomerase activity during golden hamster cheek pouch carcinogenesis induced by DMBA. METHODS: First, 52 golden hamsters were divided into 2 groups. Four of them were not done any special treatment and were killed after 3 days. The others were covered with DMBA on the surface of cheek pouch on one side in order to induce carcinogenesis, the other side of cheek pouch was treated as the control. Then, the 48 golden hamsters were divided into 4 groups and were killed in 7, 10, 14 and 20 weeks. The telomeric repeat amplification protocol (TRAP) based on PCR and ELISA was used to analysis the activity of telomerase. RESULTS: 1. The expression of telomerase activity existed in normal cheek pouch mucosa of golden hamsters, which meant that telomerase played an important role on controlling cell proliferation. 2. The level of telomerase activity gradually increased while hyperplasia and dysplasia was observed in the cheek pouch mucosa covered with DMBA. It reached its top at a later premalignant period and gradually decreased after that. 3. There was negative correlation between the degree the telomere length was shortened and the activity of telomerase (r = -0.9654), and there was positive correlation between the reduced rate of telomere length and the activity of telomerase (r = 0.9471). CONCLUSION: The activity of telomerase is one of important factors that can effect the stability of telomere length and plays a crucial role in the progression of oral cancer. So the activity of telomerase is an early molecular marker of carcinogenesis.

Animals↗
PubMed1999

Genetic, physical, and transcript map of the fld region on mouse chromosome 12.

The fatty liver dystrophy (fld) mutation is manifested in abnormalities of lipid and glucose metabolism and peripheral neuropathy. To identify the gene affected by this mutation, we generated a genetic map of the fld region on chromosome 12 by the analysis of F2 offspring from an intersubspecific cross between strains BALB/cByJ-fld and CAST/EiJ. The results localize fld to the 0.42-cM interval between the microsatellite markers D12Mit170 and D12Mit184. A contig of YACs and BACs covering the nonrecombinant genomic region has been constructed and used for the identification of genes. Expressed sequence tag mapping and exon trapping identified three transcripts within the critical interval: Ctla2b, which encodes a cysteine protease inhibitor, and mouse homologs of KIAA0188 and KIAA0575, two long human transcripts of unknown function. Expression analysis revealed that Kiaa0188 is expressed in wildtype but not in fld liver, implicating this gene as a candidate for harboring the fld mutation.

Animals↗
PubMed1999

Dynamic visualization of nervous system in live Drosophila.

We have constructed transgenic Drosophila melanogaster lines that express green fluorescent protein (GFP) exclusively in the nervous system. Expression is controlled with transcriptional regulatory elements present in the 5' flanking DNA of the Drosophila Na(+), K(+)-ATPase beta-subunit gene Nervana2 (Nrv2). This regulatory DNA is fused to the yeast transcriptional activator GAL4, which binds specifically to a sequence motif termed the UAS (upstream activating sequence). Drosophila lines carrying Nrv2-GAL4 transgenes have been genetically recombined with UAS-GFP (S65T) transgenes (Nrv2-GAL4+UAS-GFP) inserted on the same chromosomes. We observe strong nervous system-specific fluorescence in embryos, larvae, pupae, and adults. The GFP fluorescence is sufficiently bright to allow dynamic imaging of the nervous system at all of these developmental stages directly through the cuticle of live Drosophila. These lines provide an unprecedented view of the nervous system in living animals and will be valuable tools for investigating a number of developmental, physiological, and genetic neurobiological problems.

Animals↗
PubMed1999

Organization and transcriptional regulation of Drosophila Na(+), K(+)-ATPase beta subunit genes: Nrv1 and Nrv2.

Drosophila melanogaster has two Na(+),K(+)-ATPase beta subunit genes (Nervana 1 and 2; Nrv), with tissue-specific expression patterns. Nrv1 produces a single beta subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two different isoforms (2.1 and 2.2) expressed in the nervous system. We have determined the complete molecular genomic organization for both Nrv genes. Only 3kb of DNA separate the 3' end of Nrv2 from Nrv1. The cDNAs from all three forms of Nrv have been mapped onto the genomic structure and all intron-exon junctions have been confirmed by direct sequencing. The genomic DNA positioned in the 5' flanking region of each Nrv gene has also been tested for tissue-specific transcriptional regulatory activity. P-element transformation vectors were constructed, which contained either 7.7kb of Nrv2 or 3.5kb Nrv1 5' flanking DNA driving expression of a lacZ reporter gene. Multiple transgenic Drosophila lines were established for each construct and analyzed for their beta-galactosidase expression pattern. The tissue-specific expression of each Nrv gene is independently regulated by the cis-element(s) present in the 5' flanking region. The Nrv2 5' flanking DNA directs expression exclusively to the nervous system, whereas Nrv1 5' flanking DNA directs expression primarily in muscle tissue.

Animals↗
PubMed1999

Altered gene expression pattern in the fatty liver dystrophy mouse reveals impaired insulin-mediated cytoskeleton dynamics.

The mouse fatty liver dystrophy (fld) mutation is characterized by transient hypertriglyceridemia and fatty liver during the neonatal period, followed by development of a peripheral neuropathy. To uncover the metabolic pathway that is disrupted by the fld mutation, we analyzed the altered pattern of gene expression in the fatty liver of fld neonates by representational difference analysis of cDNA. Differentially expressed genes detected include a novel member of the Ras superfamily of small GTP-binding proteins, a novel Ser/Thr kinase, and several actin cytoskeleton-associated proteins including actin, profilin, alpha-actinin, and myosin light chain. Because these proteins have a potential functional link in the propagation of hormone signals, we investigated cytoskeleton dynamics in fld cells in response to hormone treatment. These studies revealed that preadipocytes from fld mice exhibit impaired formation of actin membrane ruffles in response to insulin treatment. These findings suggest that the altered mRNA expression levels detected in fld tissue represent a compensatory response for the nonfunctional fld gene and that the fld gene product may be required for development of normal insulin response.

Amino Acid Sequence↗
PubMed1999

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.

Animals↗
PubMed1999

Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface.

Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be essential for the functional interactions between these effector proteins and Cdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2 [PAK2(71-92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide binds to GTP-gammaS-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 microM, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92) with GTP-gammaS-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42 that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstrate structuring of PAK2(71-92) in the presence of GTP-gammaS-loaded Cdc42, through the observation of many nonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs show that the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the side chains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. The CRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and its effector proteins.

Amino Acid Sequence↗
PubMed1999

[Study on mutation of exon 8 of Wilson's disease gene]

OBJECTIVE: To analyze the frequency of mutation in exon 8 of Wilson's disease (WD) gene in Chinese people. METHODS: Screening for ATP7B gene mutation was conducted in 45 WD patients£® Mobility shift of exon 8 was analyzed by SSCP. Nucleotide sequence of exon 8 was analyzed, and the PCR products were cut by enzyme Msp I. The authors found G2273T mutation at codon 778, and according to this mutation sequence, made an analysis of enzyme cut by Msp I in all patients. 2 WD families were analyzed. RESULTS: No abnormality was found in 20 controls. In 45 patients, 2 were homozygous (4.4%) and 11 heterozygous (12.2%). The positive rate of mutation was 16.67%. The Arg778Leu mutation was validated by this study. CONCLUSION: The mutation in exon 8 of WD gene may play an important role in pathogenesis of Wilson's disease in Chinese.

Journal Article↗
PubMed1999

Embryonic silk gland development in Bombyx: molecular cloning and expression of the Bombyx trachealess gene.

We describe embryonic development of the Bombyx silk gland. To extend the analysis further we isolated a Bombyx counterpart gene of the Drosophila trachealess (trh) gene. Bombyx trh encodes a protein of 849 amino acids. When compared with the amino acid sequence of Drosophila trh, the identity of Bombyx bHLH, PAS-A and PAS-B domains is 100%, 97%, and 80%, respectively. Northern blot analysis revealed the presence of a single Bombyx trh transcript of 5.4 kb. We analyzed the expression pattern of the Bombyx trh transcript during embryogenesis by in situ hybridization. Bombyx trh mRNA was first detected in the tracheal primordial cells at around embryonic stage 18. Thereafter levels of Bombyx trh mRNA increased, and the high expression level was maintained until hatching. At embryonic stage 19 the transcript was also detected in the posterior basal region of the labial segment from where the silk gland invaginates. By the blastokinesis stage (around stage 23), the silk gland was lengthened, and, interestingly, the Bombyx trh transcript was restricted to the anterior silk gland. These results suggest that Bombyx trh plays a role in the formation of the trachea and the anterior silk glands.

Animals↗
PubMed1999

Preparation and characterization of new electrocardiogram electrodes.

A new generation of electrocardiogram (ECG) electrodes have been fabricated and superficially modified by a special controlled precorrosion process. The electrodes have been characterized by scanning electron microscopy (SEM), resistance-capacitance measurements (RCM) and simulated defibrillation recovery (SDR). The resulting surface of the Sn alloys was rough, and, consequently, a large active surface area was available. As a result, a higher electrocardiogram electrode performance, including low alternating current (a.c.) impedance, excellent time stability and improved SDR, was achieved. Such new design can surely find important future applications in clinical diagnosis.

Journal Article↗
PubMed1999

A 30-residue fragment of the carp granulin-1 protein folds into a stack of two beta-hairpins similar to that found in the native protein.

Upon air oxidation, a peptide corresponding to the 30-residue N-terminal subdomain of carp granulin-1 spontaneously formed the disulfide pairing observed in the native protein. Structural characterization using NMR showed the presence of a defined secondary structure within this peptide. The chemical shifts for most of the alphaCH protons of the peptide and the protein are very similar, and the observed NOE contacts of the peptide strongly resemble those in the protein. A structure calculation of the peptide using NOE distance constraints indicates that the peptide fragment adopts the same conformation as formed within the native protein. The 30-residue N-terminal peptide of carp granulin-1 is the first example of an independently folded stack of two beta-hairpins reinforced by two interhairpin disulfide bonds. Two key areas of the structure show a clustering of hydrophobic residues that may account for its exceptional conformational stability.

Amino Acid Sequence↗
PubMed1999

The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond.

A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.

Amino Acid Sequence↗
PubMed1999

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells.

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.

Adaptor Proteins, Signal Transducing↗
PubMed1999

[Study on mutation of exon 8 of Wilson's disease gene].

OBJECTIVE: To analyze the frequency of mutation in exon 8 of Wilson's disease (WD) gene in Chinese people. METHODS: Screening for ATP7B gene mutation was conducted in 45 WD patients. Mobility shift of exon 8 was analyzed by SSCP. Nucleotide sequence of exon 8 was analyzed, and the PCR products were cut by enzyme Msp I. The authors found G2273T mutation at codon 778, and according to this mutation sequence, made an analysis of enzyme cut by Msp I in all patients. 2 WD families were analyzed. RESULTS: No abnormality was found in 20 controls. In 45 patients, 2 were homozygous (4.4%) and 11 heterozygous (12.2 ). The positive rate of mutation was 16.67%. The Arg778Leu mutation was validated by this study. CONCLUSION: The mutation in exon 8 of WD gene may play an important role in pathogenesis of Wilson's disease in Chinese.

Exons↗
PubMed1999

Dosimetry of room-air germicidal (254 nm) radiation using spherical actinometry.

A method is demonstrated for measuring germicidal (254 nm) ultraviolet radiation (GUVR) in an omnidirectional manner. This method has application for those situations in which GUVR from more than one direction or source impinges on a three-dimensional object, and when it is of interest to determine the radiant fluence experienced by the object. For this purpose spherical quartz vessels were constructed varying from 0.8 to 1.25 cm in outside diameter. The vessels were filled with an actinometric solution consisting of KI and KIO3. This solution is optically opaque at 254 nm and is insensitive to room light. Upon exposure to GUVR this solution, which counts all of the 254 nm photons, forms triiodide. Following radiation, the contents of the vessel are removed, the absorbance of triiodide measured and the fluence rate determined based on a relationship that takes the volume to cross-sectional area of the actinometer into account. It is demonstrated that, in accordance with theory, the area of the sphere that intercepts the radiant energy is equal to the cross-sectional area of the sphere. A demonstration of the utility of this method was carried out in a test facility 90 m3 equipped with five GUVR lamps, one in each corner and one in the center of the room, arranged so as to irradiate the air in the upper portion of the room. Twenty spherical actinometers were evenly distributed around the room and exposed for either 30 or 90 min. The fluence rate varied from one part of the room to another, peaking near the GUVR lamps at 65-85 microW per cm2. By averaging over all 20 points, a pseudoaverage fluence rate for the room was found to be 42 and 44 microW per cm2 for the 30 and 90 min exposures, respectively. The similarities in the values for this metric demonstrates that the actinometric response is linear over this exposure range.

Disinfection↗
PubMed1999

[Genetic complementation group analysis of xeroderma pigmentosum patients in China].

OBJECTIVE: To establish skin fibroblast cell strains derived from Chinese xeroderma pigmentosum(XP) patients and to analyze the genetic complementation group and distribution. METHODS: From four XP patients, skin fibroblast cell strains were derived and used for the complementation group analysis by autoradiography and cell-fusion technique. RESULTS: Of the four XP patients from apparently independent families, three were assigned to group C, and one belonged to group E. This is the first report on XP-E patient in China. CONCLUSION: Based on the data from the previous 7 cases and the present 4 cases, the distribution of complementation group in Chinese XP patients known at present is: nine XP-C, one XP-F and one XP-E. Although the number of cases is still limited, XP-C appears to be more frequent in China. This forms a marked contrast to Japanese XP patients, who are dominated by XP-A with infrequent presence of XP-C.

Adolescent↗
PubMed1999

Effects of the configuration of a multi-epitope chimeric malaria DNA vaccine on its antigenicity to mice.

OBJECTIVE: Four B and Th cell epitopes were selected from conservative domain of Plasmodium falciparum antigens to construct two groups of chimeric malaria DNA vaccines with different configurations and their antigenicities were studied. METHODS: The partially synthesized oligonucleotide was annealed, PCR amplified and cloned into a mammalian cell expression vector. By using a pair of isocaudamers on the vector, different single copies of B epitopes were multiplied and were tenderly stringed into two groups of chimeric DNA vaccine with different configurations. BALB/c mice were immunized with these DNA plasmids by either intramuscular or intradermal injections. RESULTS: The antisera from the immunized mice tested by ELISA showed that only the configuration which had a single copy of universal T helper cell epitope, CS. T3, located at the C terminal of the multi-copy B cell epitopes induced a high antibody response. The T helper cell epitope at any other position of the peptide, or the double T helper cell epitopes configured with the B cell epitopes did not enhance antibody response, and some configurations even decreased the humoral response to a B cell epitope. CONCLUSION: This study demonstrated that both combination and configuration of the epitope may affect the antigenicity of a chimeric multiple antigen.

Animals↗