Biomedical subjects

J Lu

Publications and source records attributed to J Lu.

At least 253 records · Page 14Linked to original sources

PubMed2000

[Determination of astragaloside IV in radix Astragali by HPLC with evaporative light scattering detector].

OBJECTIVE: To develop a new method for the determination of astragaloside IV in Radix Astragali. METHOD: A HPLC-ELSD method was set up, using Hypersil C18 column (150 mm x 4.6 mm), acetonitrile-water(1:2) as mobile phase with a flow rate of 1.0 ml.min-1. The parameters of drift tube and gas flow rate of the detector were set at 105 degrees C and 2.96 L.min-1 respectively. RESULT: The calibration curve was linear in the range of 2.02-10.12 micrograms. The average recovery was 100.5%. CONCLUSION: The active constituent astragaloside IV in Radix Astragali can be separated effectively. ELSD detection is appropriate for the determination of constituents with end absorption at low ultra-violet wavelengths.

Astragalus propinquus↗

PubMed2000

[Comparison of mechanical properties of laser-welded wrought and cast titanium].

OBJECTIVE: This study was aimed to compare the mechanical properties of laser-welded wrought and cast titanium. METHODS: According to relevant reports and clinical demands, the specimens of TA2 were machine milled or cast into certain sizes using LZ centrifuge-vacuum-pressure titanium casting machine. This experiment involved four groups including the laser-welded wrought titanium group, the laser-welded cast titanium group, the wrought titanium group and the cast titanium group. There were 6 complete specimens in each group and the specimens were ultrasonically cleaned with ethyl and distilled water before they were welded with home-made Nd:YAG apparatus, and comparisons of mechanical properties were made among these groups. All tensile specimens were mounted on the Instron universal testing machine to test tensile loads, yield loads and absolute elongations were measured. The cross section of fractured surface was observed under S-2700 Hitachi SEM and Vickers' microhardness values in different areas were detected with AKASHI microhardness tester. RESULTS: It was showed that the tensile load of laser-welded wrought titanium made no difference from that of the original wrought titanium, but the yield load of laser-welded wrought titanium was bigger than that of the original wrought titanium. The tensile load and yield load of cast titanium were slightly bigger than those of wrought titanium. The tensile load and yield load of laser-welded wrought titanium made no statistic difference from those of laser-welded cast titanium. The absolute elongations of cast or welded titanium were lower than those of wrought or non-welded titanium. The fractured surface of cast titanium under SEM appeared lath-like structure and that of welded cast titanium honeycomb-like structure. The honeycomb-like structure of wrought titanium was more obvious than that of laser-welded wrought titanium. The microhardness value of laser-welded cast titanium was slightly bigger than that of laser-welded wrought titanium. CONCLUSION: The mechanical property of laser-welded wrought titanium might be not apparently different from that of laser-welded cast titanium.

Dental Soldering↗

PubMed2000

[Influences of counterfeits on RAPD results of Semen Cuscutae].

Compared the differences of the RAPD results between pure Semen Cuscutae and 12 commercial samples, it comes into the conclusion that the exiting other sample influences the RAPD results when its content is over 60% and the results is same as its content being under 60%.

Cuscuta↗

PubMed1999

Metabolism of N-nitrosobenzylmethylamine by human cytochrome P-450 enzymes.

N-Nitrosobenzylmethylamine (NBzMA) is a potent esophageal carcinogen in rodents, and has been found as a dietary contaminant in certain areas of China where esophageal cancer is endemic. To determine which cytochrome P-450 enzymes in humans are primarily responsible for NBzMA metabolism, microsomes from lymphoblastoid cell lines expressing a panel of human cytochrome P-450s (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP2C9, CYP2C19, and CYP3A4) and a panel of 10 different human liver microsomal preparations were examined for their abilities to metabolize [3H]NBzMA. In addition, the ability of human liver microsomes to form various NBzMA metabolites was correlated with the abilities of these preparations to metabolize coumarin, ethoxyresorufin, chlorzoxazone, 7-ethoxy-4-trifluoromethylcoumarin, S-mephenytoin, and nifedipine. NBzMA metabolites were quantitated by reversed-phase high-performance liquid chromatography (HPLC) coupled with flow-through radioactivity detection. Major metabolites included benzaldehyde, benzyl alcohol, benzoic acid, and several uncharacterized radioactive peaks. Of the representative P-450 activities, only CYP2E1 and CYP2A6 catalyzed substantial metabolism of NBzMA. Compared to CYP2E1, CYP2A6 metabolized NBzMA more readily. NBzMA acted as a potent inhibitor of coumarin 7-hydroxylation in CYP2A6 microsomes. Human liver microsomes metabolized NBzMA readily. NBzMA metabolite formation was most highly correlated with coumarin 7-hydroxylase activity, a marker of CYP2A6 activity. 8-Methoxypsoralen substantially inhibited NBzMA metabolism in human hepatic microsomes. When the effects of the potent isothiocyanates PEITC and PHITC were analyzed on microsomes from cell lines expressing CYP2E1 and CYP2A6, it was found that PEITC inhibited both enzymes, PHITC was the more effective inhibitor of CYP2E1, and PHITC was an ineffective inhibitor of CYP2A6. Collectively, these data indicate that CYP2A6 and, to a lesser degree, CYP2E1 are important P-450 enzymes in the activation of NBzMA in human systems.

Benzylamines↗

PubMed1999

The interferon-induced double-stranded RNA-activated protein kinase PKR will phosphorylate serine, threonine, or tyrosine at residue 51 in eukaryotic initiation factor 2alpha.

The family of eukaryotic initiation factor 2alpha (eIF2alpha) protein kinases plays an important role in regulating cellular protein synthesis under stress conditions. The mammalian kinases PKR and HRI and the yeast kinase GCN2 specifically phosphorylate Ser-51 on the alpha subunit of the translation initiation factor eIF2. By using an in vivo assay in yeast, the substrate specificity of these three eIF2alpha kinases was examined by substituting Ser-51 in eIF2alpha with Thr or Tyr. In yeast, phosphorylation of eIF2 inhibits general translation but derepresses translation of the GCN4 mRNA. All three kinases phosphorylated Thr in place of Ser-51 and were able to regulate general and GCN4-specific translation. In addition, both PKR and HRI were found to phosphorylate eIF2alpha-S51Y and stimulate GCN4 expression. Isoelectric focusing analysis of eIF2alpha followed by detection using anti-eIF2alpha and anti-phosphotyrosine-specific antibodies demonstrated that PKR and HRI phosphorylated eIF2alpha-S51Y on Tyr in vivo. These results provide new insights into the substrate recognition properties of the eIF2alpha kinases, and they are intriguing considering the potential for alternate substrates for PKR in cellular signaling and growth control pathways.

Alleles↗

PubMed1999

Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding.

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.

Alanine↗

PubMed1999

Nitric oxide participates in the recovery of normal jejunal epithelial ion transport following exposure to the superantigen, Staphylococcus aureus enterotoxin B.

Bacterial superantigens (SAgs) are potent T cell activators. Mice treated 4 h previously with the SAg, Staphylococcus aureus enterotoxin B (SEB), display reduced ion transport (assessed by short circuit current) responses to prosecretory stimuli, which normalize 24 h posttreatment. Here, mice were treated with SEB alone or in combination with an inhibitor of the inducible form of NO synthase (iNOS), l -NIL. Subsequently, jejunal iNOS expression was detected by immunohistochemistry, ion transport was evaluated in Ussing chambers, and serum levels of TNF-alpha and IFN-gamma were measured by ELISA. SEB-treated mice had increased epithelial iNOS immunoreactivity, and numerous iNOS-positive CD3+ T cells occurred in their mucosa and submucosa. Concomitant treatment with l -NIL did not affect the reduced short circuit current responsiveness to electrical nerve stimulation or the prosecretory agents, carbachol and forskolin, that occurred 4 h post-SEB (5 microgram) treatment. However, Isc responses in l -NIL- plus SEB-treated mice were still significantly reduced 24 h posttreatment, indicating a role for NO in the restoration of normal ion transport following exposure to SAgs. The prolongation of epithelial ion transport abnormalities correlated with elevated serum levels of TNF-alpha and IFN-gamma in mice treated 24 h previously with l -NIL plus SEB compared with those in controls and SEB-only-treated mice. Additionally, mice treated with l -NIL plus SEB and TNF-alpha- or IFN-gamma-neutralizing Abs displayed normal jejunal ion transport characteristics 24 h posttreatment. We conclude that NO mobilization is important in the homeostatic recovery response following immune stimulation by SAgs and that the beneficial effect of NO in this model system is probably via regulation of TNF-alpha and IFN-gamma production.

Animals↗

PubMed1999

The endoplasmic reticulum chaperone glycoprotein GRP94 with Ca(2+)-binding and antiapoptotic properties is a novel proteolytic target of calpain during etoposide-induced apoptosis.

GRP94 is a 94-kDa chaperone glycoprotein with Ca(2+)-binding properties. We report here that during apoptosis induced by the topoisomerase II inhibitor etoposide, a fraction of GRP94 associated with the endoplasmic reticulum membrane undergoes specific proteolytic cleavage, coinciding with the activation of the caspase CPP32 and initiation of DNA fragmentation. In vivo, inhibitors of caspases able to block etoposide-induced apoptosis can only partially protect GRP94 from proteolytic cleavage, whereas complete inhibition is observed with calpain inhibitor I but not with the proteasome inhibitor. In vitro, GRP94 is not a substrate for CPP32; rather, it can be completely cleaved by calpain, a Ca(2+)-regulated protease. The cleavage of GRP94 by calpain is Ca(2+)-dependent and generates a discrete polypeptide of 80 kDa. In contrast, calpain has no effect on other stress proteins such as GRP78 or HSP70. Further, immunohistochemical staining reveals specific co-localization of GRP94 with calpain in the perinuclear region following etoposide treatment. We further showed that reduction of GRP94 by antisense decreased cell viability in etoposide-treated Jurkat cells. Our studies provide new evidence that the cytoprotective GRP94, as in the case of the antiapoptotic protein Bcl-2, can be targets of proteolytic cleavage themselves during the apoptotic process.

Apoptosis↗

PubMed1999

An alternating selection strategy for cloning phage display antibodies.

Phage display is a powerful technique that can be used to develop antibodies to target molecules. One approach for antibody phage display is to select phage from a large naive library of antibody immunoglobulin variable region fragments (Fv) expressed on the surface of phage. Phage that display antibody fragments of interest are selected by their ability to bind the target antigen immobilized on a solid support surface. A major difficulty often encountered with this approach is that phage that bind to additional antigens that are present during the phage selection steps are also selected. We have developed an alternating selection approach to minimize selection of unwanted phage. In the alternating selection approach, two selection methods are used. Each selection method contains different contaminating antigens. This approach was used to select phage that bind a phosphoryated form of the E47 transcription factor. Phage were selected based on their ability to bind a phospho-peptide in solution and alternatively a phospho-protein coated on a polyvinyl micortiter plate. This approach proved significantly better than selection with only one method. With one selection technique, 2 of 48 (4%) selected clones bound to the target antigen. With another selection technique, 15 of 48 (31%) selected clones bound to the target antigen. With alternating selection, 71 of 93 (76%) of the clones bound to the target antigen.

Antibodies↗

PubMed1999

Partial molar volumes of some alpha-amino acids in aqueous sodium acetate solutions at 308.15 K.

The apparent molar volumes V(2,phi) have been determined for glycine, DL-alpha-alanine, DL-alpha-amino-n-butyric acid, DL-valine and DL-leucine in aqueous solutions of 0.5, 1.0, 1.5 and 2.0 mol kg(-1) sodium acetate by density measurements at 308.15 K. These data have been used to derive the infinite dilution apparent molar volumes V(0)(2,phi) for the amino acids in aqueous sodium acetate solutions and the standard volumes of transfer, Delta(t)V(0), of the amino acids from water to aqueous sodium acetate solutions. It has been observed that both V(0)(2,phi) and Delta(t)V(0) vary linearly with increasing number of carbon atoms in the alkyl chain of the amino acids. These linear correlations have been utilized to estimate the contributions of the charged end groups (NH(3)(+), COO(-)), CH(2) group and other alkyl chains of the amino acids to V(0)(2,phi) and Delta(t)V(0). The results show that V(0)(2,phi) values for (NH(3)(+), COO(-)) groups increase with sodium acetate concentration, and those for CH(2) are almost constant over the studied sodium acetate concentration range. The transfer volume increases and the hydration number of the amino acids decreases with increasing electrolyte concentrations. These facts indicate that strong interactions occur between the ions of sodium acetate and the charged centers of the amino acids. The volumetric interaction parameters of the amino acids with sodium acetate were calculated in water. The pair interaction parameters are found to be positive and decreased with increasing alkyl chain length of the amino acids, suggesting that sodium acetate has a stronger dehydration effect on amino acids which have longer hydrophobic alkyl chains. These phenomena are discussed by means of the co-sphere overlap model.

Journal Article↗

PubMed1999

Somatic mutations in the BRCA1 gene in Chinese sporadic breast and ovarian cancer.

Inherited mutations in the BRCA1 gene confer increased susceptibility to breast and ovarian cancer. Its role in sporadic carcinogenesis is not well defined. Somatic mutations in breast cancers have not been reported and to date there are only three reports of somatic mutations in sporadic ovarian cancers. To investigate the contribution of BRCA1 mutations to sporadic breast and ovarian cancer in the Chinese population, we analysed 62 samples from Chinese women using the protein truncation test. There were 40 cases of breast cancer under age 50 and 22 cases of ovarian cancer, all unselected for family history. There was no age selection for the ovarian cancers. We found two somatic BRCA1 mutations in exon 11, one in a breast cancer and the other in an ovarian cancer, both of which result in truncated proteins. Our results indicate that somatic BRCA1 mutations, like somatic mutations in the BRCA2 gene, though very rare, can be found in both breast and ovarian cancers and support a tumor suppressor function for BRCA1 in sporadic tumors.

Adult↗

PubMed1999

Comparison of chromatin remodeling and transcriptional activation of the mouse mammary tumor virus promoter by the androgen and glucocorticoid receptor.

We examined the interaction between the androgen (AR) and glucocorticoid receptor (GR) at the transcriptional level using mouse fibroblast cell lines harboring an integrated mouse mammary tumor virus (MMTV) promoter. We found that the AR, after induction with dihydrotestosterone (DHT), caused a progressive increase in MMTV-CAT reporter activity over 72 h which was correlated to an increase in chromatin remodeling of the MMTV promoter in the vicinity of the hormone response element (HRE). In contrast, stimulation of the GR by the synthetic glucocorticoid dexamethasone (Dex) caused a transient increase in MMTV transcriptional activity which returned to basal levels after 72 h. These changes were correlated to a transient increase in chromatin remodeling in the region of the HRE. Neither cotreatment nor pretreatment with Dex affected the DHT response. In fact, there was a more than additive effect of the two hormones on transcription at early time points. This suggests that the inability of GR to remodel chromatin, after 24 h of hormone treatment, is most likely related to changes in the GR itself and not the chromatin remodeling process. Consistent with this, nuclear GR levels dropped by greater than 50% after Dex treatment whereas the AR was induced fourfold after 24 h of DHT treatment. We conclude that a promoter with an ordered chromatin structure can still respond to androgens even after its glucocorticoid responsiveness is lost. This may be one mechanism cells utilize to establish target gene specificity for nuclear receptors that recognize identical DNA sequences.

Animals↗

PubMed1999

The events relating to lanthanide ions enhanced permeability of human erythrocyte membrane: binding, conformational change, phase transition, perforation and ion transport.

The binding and uptake of Gd3+ ions by human erythrocytes in vitro were studied by determining the Gd contents in membrane and in cytosol by means of particle-induced X-ray emission (PIXE) spectrometry. Results obtained from varied incubation time revealed that the Gd3+ ions bind to the membrane proteins and lipids at first. Gd3+ binding to the membrane lipids and proteins lasts 0 approximately 20 and 20 approximately 100 ms respectively, as shown by the stopped-flow studies. Then a fraction of Gd3+ ions diffuses through the membrane. The kinetics of Gd3+ binding indicates that the binding to phospholipids is prior to that to the membrane proteins, but a portion of the lipid-bound Gd3+ redistributed later to the proteins. PIXE studies showed that the entry of Gd3+ increased the influx of Ca2+ and Cl-. By monitoring the changes in fluorescence of proteins and that of the Ln3+, the uptake of La3+, Eu3+, Gd3+ and Tb3+ was shown to be a process comprising a series of events. Binding to the membrane molecules induces the phase transition of lipid bilayer and conformational changes and aggregation of membrane proteins. Conformational changes of the proteins were characterized by Fourier transform IR spectroscopy (FT-IR) deconvolved spectra, i.e. alpha-helix content decreases while beta-sheet increases. ESR spectra of MSL-labeled proteins reflect the aggregation state related with the conformational change. [31P]NMR spectra of membrane lipid bilayer revealed the Ln3+ ions induced hexagonal (H(II)) phase formation. Phase transition and aggregation of membrane proteins cause the formation of domain structure and perforation in the membrane. These alterations in membrane structure are responsible for the Ln3+ enhanced membrane permeability. Thus the previous Ln3+ binding will facilitate the across-membrane transport of other Ln3+ ions through the membrane.

Blood Proteins↗

PubMed1999

Determination of residues involved in ligand binding and signal transmission in the human IFN-alpha receptor 2.

The human IFN-alpha receptor (hIFNAR) is a complex composed of at least two chains, hIFNAR1 and hIFNAR2. We have performed a structure-function analysis of hIFNAR2 extracellular domain regions using anti-hIFNAR2 mAbs (1D3, 1F3, and 3B7) and several type I human IFNs. These mAbs block receptor activation, as determined by IFN-stimulated gene factor 3 formation, and block the antiviral cytopathic effects induced by type I IFNs. We generated alanine substitution mutants of hIFNAR2-IgG and determined that regions of hIFNAR2 are important for the binding of these blocking mAbs and hIFN-alpha2/alpha1. We further demonstrated that residues E78, W101, I104, and D105 are crucial for the binding of hIFN-alpha2/alpha1 and form a defined protrusion when these residues are mapped upon a structural model of hIFNAR2. To confirm that residues important for ligand binding are indeed important for IFN signal transduction, we determined the ability of mouse L929 cells expressing hIFNAR2 extracellular domain mutants to mediate hIFN signal. hIFN-alpha8, previously shown to signal a response in L929 cells expressing hIFNAR1, was unable to signal in L929 cells expressing hIFNAR2. Transfected cells expressing hIFNAR2 containing mutations at residues E78, W101, I104, or D105 were unresponsive to hIFN-alpha2, but remained responsive to hIFN-beta. In summary, we have identified specific residues of hIFNAR2 important for the binding to hIFN-alpha2/1 and demonstrate that specific regions of the IFNAR interact with the subspecies of type I IFN in different manners.

Amino Acid Substitution↗

PubMed1999

Inhibition of angiogenesis and breast cancer xenograft tumor growth by VEGI, a novel cytokine of the TNF superfamily.

Recently, we reported a novel protein of the tumor necrosis factor (TNF) superfamily, named vascular endothelial cell growth inhibitor (VEGI), which is expressed predominantly in endothelial cells. When a secreted form of this new protein was overexpressed in mouse colon cancer cells, the growth of tumors formed by these cells in black mice was inhibited. We now report that recombinant VEGI inhibits the proliferation of endothelial cells but not that of other types of cells examined. The protein also inhibits formation of capillary-like structures by endothelial cells in collagen gels, and the growth of capillaries into collagen gels placed on the chick chorioallantoic membrane. The anticancer potential of VEGI was examined in a breast cancer xenograft tumor model in which the cancer cells were co-injected with Chinese hamster ovary cells overexpressing a secreted form of the protein. The co-injection resulted in potent inhibition of xenograft tumor growth. Our findings are consistent with the view that VEGI is an endothelial cell-specific negative regulator of angiogenesis.

Animals↗

PubMed1999

Mpl ligand enhances the transcription of the cyclin D3 gene: a potential role for Sp1 transcription factor.

Cyclin D3 plays a major role in the development of polyploidy in megakaryocytes. The expression of cyclin D3 gene and the level of cyclin D3 protein are increased by the Mpl ligand in the Y10/L8057 megakaryocytic cell line, as indicated by Northern and Western blot analyses, and by nuclear run-on assays and transfection experiments with cyclin D3 promoter constructs. DNase I footprinting of the promoter region showed protected segments, at -75 to -60 bp and at -134 to -92 bp, which display binding sites for the Sp family of transcription factors. Gel mobility shift assay and supershifts with specific antibodies indicate that Sp1 binds to these regions in the cyclin D3 promoter and that Sp1 binding activity is significantly increased by Mpl ligand. Mutation of either Sp1 site both decreases the basal promoter activity and eliminates the induction by Mpl ligand. We find that the nonphosphorylated form of SP1 has greater affinity for the cyclin D3 promoter and that the majority of Sp1 in the cells is nonphosphorylated. Mpl ligand treatment results in increased levels of Sp1 protein, which also appears as nonphosphorylated. Okadaic acid, which inhibits protein phosphatase 1 (PP1) and shifts Sp1 to a phosphorylated form, decreases cyclin D3 gene expression and suppresses Mpl ligand induction. Our data point to the potential of Mpl ligand to activate at once several Sp1-dependent genes during megakaryopoiesis.

Animals↗

PubMed1999

Developmental regulation of the neuronal-specific isoform of K-Cl cotransporter KCC2 in postnatal rat brains.

We examined the expression of the KCC2 isoform of the K-Cl cotransporter in the developing and adult brain, using an affinity-purified antibody directed against a unique region of the KCC2 protein. Expression was shown to be limited to neurons at the cell bodies and cell processes in the hippocampus and cerebellum. Expression seemed to be the highest at the end of processes that originated from the CA1 pyramidal cells. Developmental up-regulation of KCC2 expression was demonstrated in the entire rat brain by Northern and Western blot analyses, and in the hippocampus by immunofluorescence. Level of KCC2 expression was minimal at birth and increased significantly during postnatal development. This pattern of expression was opposite to the one of the Na-K-2Cl cotransporter that is highly expressed in immature brain and decreases during development. The up-regulation of the K-Cl cotransporter expression is consistent with the developmental down-regulation of the intracellular Cl- concentration in neurons. The level of intracellular Cl-, in turn, determines the excitatory versus inhibitory response of the neurotransmitter gamma-aminobutyric acid in the immature versus mature brain. Finally, KCC2 expression was shown in dorsal root ganglion neurons, demonstrating that expression of the cotransporter is not strictly confined to central nervous system neurons.

Animals↗

PubMed1999

The structural characteristics and mechanical behaviors of nonstoichiometric apatite coatings sintered in air atmosphere.

Two types of nonstoichiometric apatite coatings on Ti6Al4V substrates were prepared in our laboratory by electrodeposition and hydrothermal synthesis. One was composed of highly pure needle-like calcium-deficient hydroxyapatite, and the other consisted of needle-like calcium-deficient hydroxyapatite and plate-like brushite. In this paper, the morphology, phase transition, and bonding strength of these coatings sintered at 450( degrees ) approximately 980 degrees C in an air atmosphere were studied. In addition, the residual stresses in pure calcium-deficient hydroxyapatite coatings and coatings sintered at 800 degrees C were measured by a hole-drilling method. The results show that after being sintered at a higher temperature, the coating/Ti6Al4V samples exhibited structural characteristics such as biphasic [hydroxyapatite + beta-Ca3(PO4)2] coatings and dense TiO2 film/Ti6Al4V in which the residual stresses in the coatings slightly increased. In addition, the results indicate that bonding strength was improved.

Air↗