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Biomedical subjects

X Lu

Publications and source records attributed to X Lu.

At least 73 records · Page 4Linked to original sources
PubMed2001

[An application value of detecting K-ras and p53 gene mutation in the stool and pure pancreatic juice for diagnosis of early pancreatic cancer].

OBJECTIVE: To explore new methods for the early diagnosis of pancreatic cancer through detecting of K-ras, p53 mutations in pancreatic juice and stool. METHODS: 201 patients in PUMC Hospital from 1994-2000. 5 and 60-control individuals were enrolled. K-ras point mutations were detected by PCR-RFLP, however p53 mutation was detected by PCR-SSCP. RESULTS: K-ras mutations in pancreatic juice were found in 87.8% (36/41) of pancreatic cancer, 23.5% (4/17) of benign pancreatic disease. Of 261 stools specimens, amplification was successful in 235 (90.0%). K-ras mutation in stool were found in 88.0% (66/75) of pancreatic cancer 51.1% (24/47) of benign pancreatic disease, 19.6% (9/46) of normal individuals. p53 mutation in pancreatic juice were found in 47.4% (18/38), 12.5% (2/16) of benign pancreatic disease, p53 mutation in stool were found in 37.1% (23/62), 19.1% (4/12) of chronic pancreatitis. CONCLUSION: K-ras mutation in pancreatic juice has high sensitivity and specificity, so it can be used as a adjunct in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can be helpful to screen for pancreatic carcinoma. Combined with serum CA19-9 detection, it might increase the early diagnostic rate of pancreatic carcinoma.

Adult↗
PubMed2001

A novel phosphotyrosine mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp): exploitation in the design of nonpeptide inhibitors of pp60(Src) SH2 domain.

The novel phosphotyrosine (pTyr) mimetic 4'-carboxymethyloxy-3'-phosphonophenylalanine (Cpp) has been designed and incorporated into a series of nonpeptide inhibitors of the SH2 domain of pp60(c-Src) (Src) tyrosine kinase. A 2.2 A X-ray crystal structure of 1a bound to a mutant form of Lck SH2 domain provides insight regarding the structure-activity relationships and supports the design concept of this new pTyr mimetic.

Animals↗
PubMed2001

Increased inducible nitric oxide synthase expression contributes to myocardial dysfunction and higher mortality after myocardial infarction in mice.

BACKGROUND: Inducible nitric oxide synthase (iNOS) is expressed in the myocardium after myocardial infarction (MI) and in heart failure. Its pathophysiological role in these conditions, however, is not clear. We hypothesized that increased NO production from iNOS expression causes myocardial dysfunction and results in higher mortality after MI. METHODS AND RESULTS: MI was induced by left coronary artery ligation in iNOS(-/-) mutant and wild-type mice. Mortality was followed up for 30 days. MI resulted in a significant increase in mortality in both iNOS(-/-) and wild-type mice compared with sham operation (P<0.01). Mortality was significantly decreased and LV myocardial contractility was increased, however, in iNOS(-/-) mice compared with the wild-type mice (P<0.05). Five days after MI, myocardial iNOS mRNA expression, plasma nitrate and nitrite concentrations, and myocardial and plasma nitrotyrosine levels were significantly increased in wild-type compared with iNOS(-/-) mutant mice (P<0.05). Both basal LV +dP/dt and its response to dobutamine were significantly increased in iNOS(-/-) compared with the wild-type mice (P<0.05). CONCLUSIONS: Increased NO production from iNOS expression contributes to myocardial dysfunction and mortality after MI in mice.

Animals↗
PubMed2001

Fatty acids inhibit growth-factor-induced diacylglycerol kinase alpha activation in vascular smooth-muscle cells.

We have previously shown that unsaturated fatty acids amplify platelet-derived-growth-factor (PDGF)-induced protein kinase C (PKC) activation in vascular smooth-muscle cells (VSMCs). Diacylglycerol-induced PKC activation is normally terminated by diacylglycerol kinases (DGKs). We thus hypothesized that fatty acids act by inhibiting a DGK. Fractionation of VSMC extracts demonstrated that the DGK alpha isoform was the major DGK activity present. PDGF markedly increased the DGK activity of cultured cells. An inhibitor selective for the DGK alpha isoform, R59949 [3-[2-[4-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-thioxo-4(1H)-quinazolinone], abolished the growth-factor-induced increase in DGK activity, but had little effect on basal activity. PDGF thus selectively activates DGKalpha. Epidermal growth factor and alpha-thrombin stimulated total DGK activity similarly to PDGF. Activation by epidermal growth factor was sensitive to R59949, again suggesting involvement of DGKalpha. However, the alpha-thrombin-induced activity was unaffected by this agent. Unsaturated fatty acids inhibited growth-factor-induced DGKalpha activation, but had no effect on basal activity. Fatty acids also amplified the PDGF-induced increase in cell diacylglycerol content. These results indicate that inhibition of DGKalpha contributes to fatty-acid-induced amplification of PKC activation. Increased levels of fatty acids in diabetes may thus contribute to chronic PKC activation associated with this disorder.

Animals↗
PubMed2001

Mapping the Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required for recruitment and activation of signalling proteins in T cells.

T-cell-receptor (TCR)-mediated LAT (linker for activation of T cells) phosphorylation is critical for the membrane recruitment of signalling complexes required for T-cell activation. Although tyrosine phosphorylation of LAT is required for recruitment and activation of signalling proteins, the molecular mechanism associated with this event is unclear. In the present study we reconstituted the LAT signalling pathway by demonstrating that a direct tyrosine phosphorylation of LAT with activated protein-tyrosine kinase Zap70 is necessary and sufficient for the association and activation of signalling proteins. Zap-70 efficiently phosphorylates LAT on tyrosine residues at positions 226, 191, 171, 132 and 127. By substituting these tyrosine residues in LAT with phenylalanine and by utilizing phosphorylated peptides derived from these sites, we mapped the tyrosine residues in LAT required for the direct interaction and activation of Vav, p85/p110alpha and phospholipase Cgamma1 (PLCgamma1). Our results indicate that Tyr(226) and Tyr(191) are required for Vav binding, whereas Tyr(171) and Tyr(132) are necessary for association and activation of phosphoinositide 3-kinase activity and PLCgamma1 respectively. Furthermore, by expression of LAT mutants in LAT-deficient T cells, we demonstrate that Tyr(191) and Tyr(171) are required for T-cell activation and Tyr(132) is required for the activation of PLCgamma1 and Ras signalling pathways.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Arg-Tyr-Asp (RYD) and Arg-Cys-Asp (RCD) motifs in dendroaspin promote selective inhibition of beta1 and beta3 integrins.

Arg-Gly-Asp (RGD) is a unique minimal integrin-binding sequence that is found within several glycoprotein ligands. This sequence has also been found in snake-venom anti-platelet proteins, including the disintegrins and dendroaspin, a natural variant of short-chain neurotoxins isolated from the venom of Dendroaspis jamesonii. In the present study, the motifs RYD and RCD were introduced into the dendroaspin scaffold to replace RGD. Both motifs in dendroaspin caused inhibition of ADP-induced platelet aggregation with IC(50) values of 200 and 300 nM respectively, similar to that of the wild-type RGD motif (170 nM). In comparison with wild-type dendroaspin, both RYD- and RCD-containing dendroaspins were more selective in the inhibition of the adhesion of K562 cells to laminin rather than to fibrinogen and fibronectin, even though they were 10-30-fold less potent at inhibiting K562 cell (containing alpha(5)beta(1) integrin) adhesion to laminin compared with wild-type. Interestingly, the RYD motif produced a similar IC(50) value to the RGD motif at inhibiting A375-SM cell (beta(3) integrin) adhesion to collagen, whereas the RCD motif was approx. 2-6-fold less potent compared with either RGD or RYD. These findings show that the selectivity of dendroaspin binding to beta(1) and beta(3) integrins can be modulated by the introduction of alternative cell recognition sequences.

Amino Acid Motifs↗
PubMed2001

Evaluation of the role of proline residues flanking the RGD motif of dendroaspin, an inhibitior of platelet aggregation and cell adhesion.

The effect of a panel of proline mutants of dendroaspin, an inhibitor of platelet aggregation and cell adhesion, including A(42)-dendroaspin, A(47)-dendroaspin, A(49)-dendroaspin, A(42,47)-dendroaspin and A(47,49)-dendroaspin, was investigated using platelet-aggregation and cell-adhesion assays. Here we show that a single alanine-for-proline substitution did not affect potency when measured as the ability either to inhibit platelet aggregation induced by ADP (IC(50) approximately 170 nM) or to block transfected A375-SM cell adhesion to fibrinogen in the presence of Mn(2+) as compared with wild-type dendroaspin. By comparison, double proline substitution with alanines significantly reduced the potency in both assays by approx. 5-8-fold. These observations, therefore, suggest that proline residues flanking the RGD motif in dendroaspin and other RGD-containing venom proteins, e.g. disintegrins, may contribute to maintaining a favourable conformation for the solvent-exposed RGD site for its recognition by integrin receptors.

Amino Acid Motifs↗
PubMed2001

Defining brain wiring patterns and mechanisms through gene trapping in mice.

The search to understand the mechanisms regulating brain wiring has relied on biochemical purification approaches in vertebrates and genetic approaches in invertebrates to identify molecular cues and receptors for axon guidance. Here we describe a phenotype-based gene-trap screen in mice designed for the large-scale identification of genes controlling the formation of the trillions of connections in the mammalian brain. The method incorporates an axonal marker, which helps to identify cell-autonomous mechanisms in axon guidance, and has generated a resource of mouse lines with striking patterns of axonal labelling, which facilitates analysis of the normal wiring diagram of the brain. Studies of two of these mouse lines have identified an in vivo guidance function for a vertebrate transmembrane semaphorin, Sema6A, and have helped re-evaluate that of the Eph receptor EphA4.

Alkaline Phosphatase↗
PubMed2001

Heat shock protein-90 and the catalytic activities of the 20 S proteasome (multicatalytic proteinase complex).

The effect of heat shock protein 90 (Hsp-90) and several other proteins on the catalytic activities of the 20 S proteasome (MPC) was examined. The chymotrypsin-like (ChT-L) and peptidylglutamyl-peptide hydrolyzing (PGPH) activities of the pituitary MPC were inhibited by Hsp-90 with IC50 values of 8 and 28 nM, respectively. Bovine serum albumin and two other proteins tested inhibited the same activities with much higher IC50 values. The trypsin-like and branched-chain amino-acid-preferring activities were not affected by any of the proteins. None of the activities of the bovine spleen MPC, an enzyme form in which the X, Y, and Z subunits are virtually completely replaced by the LMP2, LMP7, and LMP10 subunits, was affected by either Hsp-90 or the other proteins tested. Hsp-90 inhibited the degradation of the oxidized B-chain of insulin by the pituitary MPC but not by its spleen counterpart. The PA28 activator (11 S regulator; REG) of the proteasome abolished the inhibitory effect of Hsp-90 and other proteins on the ChT-L and PGPH activities of the pituitary MPC. It is suggested that Hsp-90 induces conformational changes that affect the ChT-L and PGPH activities expressed by the X and Y subunits, respectively, but does not affect the activities expressed by LMP subunits.

Animals↗
PubMed2001

Elevated AKT activity protects the prostate cancer cell line LNCaP from TRAIL-induced apoptosis.

We find that the prostate cancer cell lines ALVA-31, PC-3, and DU 145 are highly sensitive to apoptosis induced by TRAIL (tumor-necrosis factor-related apoptosis-inducing ligand), while the cell lines TSU-Pr1 and JCA-1 are moderately sensitive, and the LNCaP cell line is resistant. LNCaP cells lack active lipid phosphatase PTEN, a negative regulator of the phosphatidylinositol (PI) 3-kinase/Akt pathway, and demonstrate a high constitutive Akt activity. Inhibition of PI 3-kinase using wortmannin and LY-294002 suppressed constitutive Akt activity and sensitized LNCaP cells to TRAIL. Treatment of LNCaP cells with TRAIL alone induced cleavage of the caspase 8 and XIAP proteins. However, processing of BID, mitochondrial release of cytochrome c, activation of caspases 7 and 9, and apoptosis did not occur unless TRAIL was combined with either wortmannin, LY-294002, or cycloheximide. Blocking cytochrome c release by Bcl-2 overexpression rendered LNCaP cells resistant to TRAIL plus wortmannin treatment but did not affect caspase 8 or BID processing. This indicates that in these cells mitochondria are required for the propagation rather than the initiation of the apoptotic cascade. Infection of LNCaP cells with an adenovirus expressing a constitutively active Akt reversed the ability of wortmannin to potentiate TRAIL-induced BID cleavage. Thus, the PI 3-kinase-dependent blockage of TRAIL-induced apoptosis in LNCaP cells appears to be mediated by Akt through the inhibition of BID cleavage.

Apoptosis↗
PubMed2001

AsMamDB: an alternative splice database of mammals.

The objective of database AsMamDB is to facilitate the systematic study of alternatively spliced genes of mammals. Version 1.0 of AsMamDB contains 1563 alternatively spliced genes of human, mouse and rat, each associated with a cluster of nucleotide sequences. The main information provided by AsMamDB includes gene alternative splicing patterns, gene structures, locations in chromosomes, products of genes and tissues where they express. Alternative splicing patterns are represented by multiple alignments of various gene transcripts and by graphs of their topological structures. Gene structures are illustrated by exon, intron and various regulatory elements distributions. There are 4204 DNAs, 3977 mRNAs, 8989 CDSs and 126 931 ESTs in the current database. More than 130 000 GenBank entries are covered and 4443 MEDLINE records are linked. DNA, mRNA, exon, intron and relevant regulatory element sequences are provided in FASTA format. More information can be obtained by using the web-based multiple alignment tool Asalign and various category lists. AsMamDB can be accessed at http://166.111.30.65/ASMAMDB.html.

Alternative Splicing↗
PubMed2001

Retrograde tracing techniques influence reported death rates of adult rat nigrostriatal neurons.

Injury often causes loss of neuronal markers and prior retrograde labeling can circumvent this problem of identification. We have previously used a time-consuming protocol for labeling all dopaminergic substantia nigra pars compacta neurons in adult rats by injecting the fluorescent tracer DiI into six sites throughout each neostriatum. Here, 2 weeks after injection of DiI into two central locations, only half of these nigrostriatal neurons were labeled. With six sites, more medial and lateral neurons were labeled, and also more in the midportion along the medial-lateral extent of the pars compacta. Less than 0.5% of the contralateral neurons were labeled. Two injections of Fluorogold also labeled fewer neurons, but their morphology was clearer. Two to 4 weeks after injection of the neurotoxin 6-OHDA into the two neostriatal sites, the total number of surviving neurons appeared greater with six sites of DiI than with two. However, within the middle region of the nigra, survival was lower with the six sites. This suggests that neurons that project outside the two central striatal tracer and 6-OHDA injection regions may be spared initially, but that those in the midportion that project to the central region are more vulnerable with the six-site protocol. Some reports suggest that Fluorogold prelabeling increases neuronal death. Here, survival after 6-OHDA or axotomy was similar with DiI or Fluorogold. These results suggest that because of a complex projection pattern of the nigrostriatal neurons, detailed quantification of neuronal survival should rely on extensive labeling. However, for drug screening purposes, faster labeling with Fluorogold using two sites is more suitable and should provide reliable data.

Animals↗
PubMed2001

Imprinting and evolution of two Kruppel-type zinc-finger genes, ZIM3 and ZNF264, located in the PEG3/USP29 imprinted domain.

We have isolated Kruppel-type (C2H2) zinc-finger genes, ZIM3 (zinc-finger gene 3 from imprinted domain) and ZNF264, located downstream of human and mouse USP29 genes (encoding ubiquitin-specific processing protease 29). In human, both ZIM3 and ZNF264 encode zinc-finger proteins with Kruppel-associated box (KRAB) A and B domains at the amino-terminal regions of the predicted proteins. In contrast, mouse Zim3 and Zfp264 seem to have lost protein-coding capability based on the lack of open reading frames (ORFs) in their cDNA sequences. In particular, the 3' end of the Zim3 transcript overlaps with the coding region of the adjacent gene Usp29 in an antisense orientation, indicating the conversion of mouse Zim3 into an antisense transcript gene for Usp29. The expression patterns of ZIM3 and ZNF264 have been largely conserved between human and mouse, with testis-specific expression of ZIM3 and ubiquitous expression of ZNF264, but high expression levels in adult testes in both species. Our studies also demonstrate that both mouse genes are imprinted with maternal expression of Zim3 in adult testes and paternal expression of Zfp264 in neonatal and adult brain. The reciprocal imprinting of two neighboring mouse genes, Zim3 and Zfp264, is consistent with a pattern observed frequently in other imprinted domains, and suggests that the imprinting of these two genes might be coregulated.

Amino Acid Sequence↗
PubMed2001

The influence of phorbol ester on the effect of tamoxifen in breast cancer cells.

To investigate the correlation between the activity of kinases in the growth factor signal transduction pathway and the development of resistance of breast cancer to tamoxifen, reporter gene regulated by the regulating fragment of CCD1 was transfected into the MCF-7 cells, and the influence of tamoxifen on the reporter gene expression was examined under different conditions of TPA treatment. Our results showed that the reporter gene expression was inhibited by tamoxifen and promoted by TPA. Furthermore, tamoxifen exerts an agonist effect on the reporter gene expression when the cells was treated by TPA previously for 12 h. It is concluded that TPA could induce estrogen-like effect of tamoxifen on estrogen receptor positive breast cancer cells and it may be one of the mechanisms responsible for the development of tamoxifen resistance.

Antineoplastic Agents, Hormonal↗
PubMed2001

The murine perilipin gene: the lipid droplet-associated perilipins derive from tissue-specific, mRNA splice variants and define a gene family of ancient origin.

The Perilipins are a family of intracellular neutral lipid droplet storage proteins that are responsive to acute protein kinase A-mediated, hormonal stimulation. Perilipin (Peri) expression appears to be limited to adipocytes and steroidogenic cells, in which intracellular neutral lipid hydrolysis is regulated by protein kinase A. We have isolated cDNA sets and overlapping genomic fragments of the murine Peri locus and mapped chromosomal location, transcription start sites, polyadenylylation sites, and intron/exon junctions. Data confirm that the Perilipins are encoded by a single-copy gene, with alternative and tissue-specific, mRNA splicing and polyadenylylation yielding four different protein species. The Perilipin proteins have identical approximately 22-kDa amino termini with distinct carboxyl terminal sequences of varying lengths. These genomic and transcriptional maps of murine Perilipin are also essential for evaluating presumptive endogenous and targeted mutations within the locus. The N-terminal identity region of the Perilipins defines a sequence motif, which we term PAT, that is shared with the ADRP and TIP47 proteins; additionally, the PAT domain may represent a novel, conserved pattern for lipid storage droplet (LSD) proteins of vertebrates and invertebrates alike. Comparative genomics suggest the presence of related LSD genes in species as diverse as Drosophila and Dictyostelium.

5' Untranslated Regions↗
PubMed2001

The combined effect of inhibiting both ACAT and HMG-CoA reductase may directly induce atherosclerotic lesion regression.

We hypothesized that coadministration of avasimibe and simvastatin would limit size, composition and extent of atherosclerotic lesions and potentially promote lesion regression, since bioavailable ACAT inhibitors decrease monocyte-macrophage enrichment and HMG-CoA reductase inhibitors limit smooth muscle cell migration and proliferation. Male New Zealand white rabbits were sequentially fed a 0.5% cholesterol, 3% peanut oil, 3% coconut oil diet for 9 weeks and a chow-fat diet for 6 weeks prior to drug administration. A time zero control group was necropsied prior to drug administration and the progression control was fed various diets but untreated. Avasimibe (10 mg/kg), simvastatin (2.5 mg/kg) or combination of avasimibe (10 mg/kg) with simvastatin (2.5 mg/kg) were administered in the chow-fat diet for 8 weeks. Plasma total cholesterol exposure was unchanged by avasimibe but was reduced 21% by both simvastatin alone and in combination with avasimibe. Combination of avasimibe and simvastatin decreased VLDL-cholesterol exposure by 56%. VLDL+IDL lipid composition was similar in the progression control and simvastatin-treated animals. Administration of avasimibe alone or in combination with simvastatin reduced the cholesteryl ester fraction and increased the triglyceride fraction to comparable extents. Relative to the progression control, avasimibe plus simvastatin markedly decreased thoracic aortic cholesteryl ester content and lesion coverage by 50% and aortic arch lesion size and macrophage area by 75 and 73%, respectively. With respect to lesion regression, avasimibe+simvastatin decreased aortic arch lesion size by 64% and monocyte-macrophage area by 73% when compared to time zero. Based on these data, we conclude that despite changes in plasma total and lipoprotein cholesterol exposure and lipoprotein composition comparable to monotherapy, inhibition of both ACAT and HMG-CoA reductase may not only directly blunt lesion progression but also promote regression of pre-established atherosclerotic lesions.

Acetamides↗
PubMed2001

Regional distribution of axial strain and circumferential residual strain in the layered rabbit oesophagus.

The oesophagus is subjected to large axial strains in vivo and the zero-stress state is not a closed cylinder but an open circular cylindrical sector. The closed cylinder with no external loads applied is called the no-load state and residual strain is the difference in strain between the no-load state and zero-stress state. To understand oesophageal physiology and pathophysiology, it is necessary to know the distribution of axial strain, the zero-stress state, the stress-strain relations of oesophageal tissue, and the changes of these states and relationships due to biological remodeling of the tissue under stress. This study is addressed to such biomechanical properties in normal rabbits. The oesophagi were marked on the surface in vivo, photographed, excised (in vitro state), photographed again, and sectioned into rings (no-load state) in an organ bath containing calcium-free Kreb's solution with dextran and EGTA added. The rings were cut radially to obtain the zero-stress state for the non-separated wall and further dissected to separate the muscle and submucosa layers. Equilibrium was awaited for 30min in each state and the specimens were photographed in no-load and the zero-stress states. The oesophageal length, circumferences, layer thicknesses and areas, and openings angle were measured from the digitised images. The oesophagus shortened axially by 35% after excision. The in vivo axial strain showed a significant variation with the highest values in the mid-oesophagus (p<0.001). Luminal area, circumferences, and wall and layer thicknesses and areas varied in axial direction (in all tests p<0.05). The residual strain was compressive at the mucosal surface and tensile at the serosal surface. The dissection studies demonstrated shear forces between the two layers in the non-separated wall in the no-load and zero-stress states. In conclusion, our data show significant axial variation in passive morphometric and biomechanical properties of the oesophagus. The oesophagus is a layered composite structure with nonlinear and anisotropic mechanical behaviour.

Animals↗