Biomedical subjects

X Yan

Publications and source records attributed to X Yan.

At least 55 records · Page 3Linked to original sources

PubMed2001

[The effect of heparin on the deep second-degree burn in scalded rats].

OBJECTIVE: To investigate the effects of heparin on deep second-degree burn in scalded rats. METHODS: Thirty Sprague-Dawley rats inflicted with 20% TBSA deep second-degree scalding on the back were employed as the model and were randomly divided into two groups. i,e heparin (H) and normal saline groups (N). Heparin (100 U . kg(-1) . day(-1)) was injected subcutaneously to the rats in H group while normal saline to those in N group once a day. Wound healing rate and immunological indices were determined simultaneously. RESULTS: (1) The wound healing time was much shorter in H group than that in N group (P < 0.005). The granulation tissue growth condition examined by LM and EM was much better in H group that than in N group. (2) The lymphocytic transforming ratio in H group was evidently increased (P < 0.005). And the phagocytic function of the macrophages increased significantly (P < 0.05). But there was no difference between the two groups in terms of hemoltytic plaque test (P > 0.05). CONCLUSION: Injection of heparin subcutaneously could accelerate burn wound healing and strengthen the systemic cellular immune potential and the macrophage phagocytic capacity of the host, but exerted no effect on the humoral immunity.

Animals↗

PubMed2001

[Inhibition effect of soybean isoflavones on the oxidative modification of low-density lipoprotein].

In order to investigate the effect of soybean isoflavones(SI) on the oxidative modification to low-density lipoprotein(LDL) and to differentiate the effect of SI and alpha-tocopherol, in vitro and in vivo test were conducted. An in vitro model of LDL oxidative modification induced by copper-ion was established by monitoring the production of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes after SI or alpha-tocopherol was added. The in vivo test was conducted by feeding rats with a high fat diet supplemented with SI and measured the sensitivity of LDL oxidative modification mediated by Cu2+ in vitro. The results revealed that when SI was added into the in vitro LDL oxidation system, the content of TBARS or conjugated dienes in the system was much reduced with a dose-effect relationship, whether lipid oxidation being initiated or not by copper-ion at 37 degrees C. In comparison with SI, only a significant inhibiting effect on lipid oxidation while alpha-tocopherol was added before the initiation of oxidation. High fat diet induced a rising of LDL sensitivity of oxidative stress, and adding SI to the high fat diet could counteract the sensitivity of LDL oxidative modification significantly. It is concluded that SI is a valuable natural antioxidant different from alpha-tocopherol in inhibiting LDL oxidative modification both in vitro and inv vivo.

Animals↗

PubMed2001

[Speciation of metals and metalloids in biomolecules by hyphenated techniques].

This paper introduces briefly the analytical methods recently published in the literature on the speciation of arsenic, selenium and cadmium in biological samples using hyphenated techniques, HPLC-ICP-MS, HPLC-ES-MS/MS, and CE-ES-MS/MS. The coupling SEC-ICP-MS is the most popular technique for the first screening of unknown sample in view of the presence of macro molecular species of element. But due to its insufficient resolution, complementary chromatographic separations have to be carried out in order to guarantee the purity of the signal. CZE-ICP-MS is a powerful tool in trace metal speciation. In case standards and reference materials are not available, ES-MS/MS is the best technique in the characterization of metal complexes. During analysis care should be taken to avoid contamination of the sample and break-up of the original metal-protein bond during the separation process. Due to lack of standards and reference materials, the application of hyphenated technique has been looking for new species rather than determining a known compound. The difficult problems are whether a signal produced by a detector belongs to a particular compound and the identification of this compound.

Arsenic↗

PubMed2001

[Analysis of the parkin gene deletion mutations in Chinese patients with Parkinson's disease].

OBJECTIVE: Clarify parkin gene deletion mutations at exons 3 to 7 in Chinese patients with Parkinson's disease (PD) and analyze them together with the clinical features of PD. METHODS: DNA was extracted from peripheral blood of 33 sporadic PD patients and 6 familial PD patients. Deletion mutations of parkin gene were identified by PCR amplification and agarose gel electrophoresis. The clinical data were analyzed together with the above information. RESULTS: Of the 33 sporadic PD patients, 1 had exons 5 and 7 deletion, 2 had exon 7 deletion. The age of onset was 46, 48, 50 respectively. In the 6 familial PD patients, 1 had exon 5 deletion. The inheritance pattern in this patient was autosomal recessive and onset of the patient was 60. All of the patients who had parkin gene deletion mutations had tremor, rigidity and bradykinesia, but athetosis was not found. In addition, deletion mutations of parkin gene at exons 3, 4 and 6, were not found. CONCLUSION: There are deletion mutations of parkin gene at exons 5 and 7 in Chinese PD patients.

Adolescent↗

PubMed2000

Carrier-mediated uptake of rhodamine 123: implications on its use for MDR research.

We have examined the effects of verapamil and PSC 833 on cellular uptake and release of rhodamine 123 (R123) in two human cancer cell lines. Both verapamil and PSC 833 were able to increase R123 accumulation in the multidrug resistant (MDR) MV522/Q6 and KB-8-5 lines in the release study. However, the effects of these drugs on R123 accumulation during accumulation study were quite different. Incubation with PSC 833 increased R123 accumulation in both MDR lines. By contrast, incubation with verapamil only increased R123 accumulation in the KB-8-5 line. The failure of verapamil to increase R123 accumulation in the MV522/Q6 cells can be attributed to the presence of a carrier system in the parent MV522 cells that recognizes both R123 and verapamil, but not PSC 833, as substrates. These results imply that performing R123 accumulation study without first ascertaining possible role of a carrier system for cellular uptake of R123 and putative P-gp modulators might inadvertently lead one to draw improper conclusions on P-gp activity.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Construction and structural modeling of a single-chain Fv-asparaginase fusion protein resistant to proteolysis.

In this study, we construct a fusion protein composed of L-asparaginase (ASNase; from Escherichia coli AS 1.357) and a protective single-chain Fv (scFv), which was selected from a phage-display scFv library from our previous studies. The antibody moiety of the fusion protein was fused to the N-terminus of the enzyme moiety via a linker peptide, (Gly(4)Ser)(6). Recombinant plasmid pET-SLA was constructed to express scFv-ASNase fusion to high levels in E. coli and the expressed product was found to form inclusion bodies. We obtained a soluble fusion protein by refolding and purification. The soluble fusion protein exhibited about 82% of the enzymatic activity of the native ASNase at the same molar concentration, and had a K(m) value similar to that of the native enzyme for the substrate L-asparagine. Importantly, the fusion protein was more stable than native ASNase. In addition: (1) following treatment with trypsin, alpha-chymotrypsin, and rennet, at 37 degrees C for 30 min, scFv-ASNase fusion retained 94.0%, 88.8%, and 84.5% of its original activity, respectively, whereas native ASNase became inactive; and (2) ScFv-ASNase fusion had a much longer in vitro half-life (9 h) in serum than the native enzyme (2 h). The three-dimensional structure of the fusion protein was obtained by modeling with the Homology and Discover modules of the INSIGHT II software package. On the basis of the structural evidence and biochemical properties, we propose that the scFv moiety of the fusion protein may confer ASNase moiety resistance to proteolysis as a result of both steric hindrance and a change in the electrostatic surface of the enzyme.

Asparaginase↗

PubMed2000

Development of a mechanism-based, DNA staining protocol using SYTOX orange nucleic acid stain and DNA fragment sizing flow cytometry.

Accurate measurement of single DNA fragments by DNA fragment sizing flow cytometry (FSFC) depends upon precise, stoichiometric DNA staining by the intercalating dye molecules. In this study, we determined the binding characteristics of a commercially available 532 nm wavelength-excitable dye and used this information to develop a universal DNA staining protocol for DNA FSFC using a compact frequency-doubled Nd:YAG laser excitation source. Among twelve 532 nm wavelength-excitable nucleic acid staining dyes tested, SYTOX Orange stain showed the highest fluorescence intensity along with a large fluorescence enhancement upon binding to double-stranded DNA ( approximately 450-fold). Furthermore, using SYTOX Orange stain, accurate fragment-size-distribution histograms were consistently obtained without regard to the staining dye to base pair (dye/bp) ratio. A model describing two binding modes, intercalation (primary, yielding fluorescence) and external binding (secondary, involving fluorescence quenching), was proposed to interpret the performance of the dyes under different dye/bp ratios. The secondary equilibrium dissociation constant was found to be the most critical parameter in determining the sensitivity of each fluorophore to the staining dye/bp ratio. The measurements of both equilibrium dissociation constants provided us with a theoretical framework for developing a universal protocol which was successfully demonstrated over a wide range of DNA concentrations on a compact flow cytometer equipped with a frequency-doubled, diode-pumped, solid-state Nd:YAG laser for rapid and sensitive DNA fragment sizing.

Bacteriophage lambda↗

PubMed2000

Subpicosecond electro-optic measurement of relativistic electron pulses

Time-resolved measurements of the transverse electric field associated with relativistic electron bunches are presented. Using an ultrafast electro-optic sensor close to the electron beam, the longitudinal profile of the electric field was measured with subpicosecond time resolution and without time-reversal ambiguity. Results are shown for two cases: inside the vacuum beam line in the presence of wake fields, and in air behind a beryllium window, effectively probing the near-field transition radiation. Especially in the latter case, reconstruction of the longitudinal electron bunch shape is straightforward.

Journal Article↗

PubMed2000

Contractile reserve and intracellular calcium regulation in mouse myocytes from normal and hypertrophied failing hearts.

Mouse myocyte contractility and the changes induced by pressure overload are not fully understood. We studied contractile reserve in isolated left ventricular myocytes from mice with ascending aortic stenosis (AS) during compensatory hypertrophy (4-week AS) and the later stage of early failure (7-week AS) and from control mice. Myocyte contraction and [Ca(2+)](i) transients with fluo-3 were measured simultaneously. At baseline (0.5 Hz, 1.5 mmol/L [Ca(2+)](o), 25 degrees C), the amplitude of myocyte shortening and peak-systolic [Ca(2+)](i) in 7-week AS were not different from those of controls, whereas contraction, relaxation, and the decline of [Ca(2+)](i) transients were slower. In response to the challenge of high [Ca(2+)](o), fractional cell shortening was severely depressed with reduced peak-systolic [Ca(2+)](i) in 7-week AS compared with controls. In response to rapid pacing stimulation, cell shortening and peak-systolic [Ca(2+)](i) increased in controls, but this response was depressed in 7-week AS. In contrast, the responses to both challenge with high [Ca(2+)](o) and rapid pacing in 4-week AS were similar to those of controls. Although protein levels of Na(+)-Ca(2+) exchanger were increased in both 4-week and 7-week AS, the ratio of SR Ca(2+)-ATPase to phospholamban protein levels was depressed in 7-week AS compared with controls but not in 4-week AS. This was associated with an impaired capacity to increase sarcoplasmic reticulum Ca(2+) load during high work states in 7-week AS myocytes. In hypertrophied failing mouse myocytes, depressed contractile reserve is related to an impaired augmentation of systolic [Ca(2+)](i) and SR Ca(2+) load and simulates findings in human failing myocytes.

Animals↗

PubMed2000

Characterization of l-asparaginase fused with a protective ScFv and the protection mechanism.

A fusion protein of the protective scFv linked to the C-terminus of ASNase via (Gly(4)Ser)(6) peptide was constructed. The ASNase-scFv fusion protein expressed in Escherichia coli exists mainly in the form of inclusion bodies, and a small amount of it was soluble. The soluble form was purified by four-step purification and it has been demonstrated that ASNase-scFv fusion exists as a dimer. By assay of the stability against proteolysis, the ASNase-scFv fusion was found to be more stable than native ASNase but less stable than scFv-ASNase fusion. The results of immunological assay indicated that the immunogenicity of the fusion proteins increased while their binding capacity with the anti-ASNase serum decreased by comparison to the native ASNase. Moreover, here the comparison of the basic physical and chemical properties of the ASNase-scFv fusion, scFv-ASNase fusion, and native ASNase is presented. Based on the structural evidence and the biochemical analysis described in this paper, the protection mechanism proposed in our previous study was further supported. The scFv moiety of the fusion protein may confer the ASNase moiety resistance to proteolysis as a result of both steric hindrance such as blocking the cleavage sites of trypsin and a change in the electrostatic potential surface of the enzyme.

Animals↗

PubMed2000

Direct absorption of gas-phase atomic hydrogen by si(100): A narrow temperature window

Absorption of thermal-energy gaseous hydrogen atoms by Si(100), exceeding by far the dopant and other impurity concentrations, occurs within a narrow substrate temperature (T(s)) window centered at approximately 460 K. The absorbed hydrogen persists in the crystalline bulk as highly mobile species before migrating out and desorbing as molecular hydrogen at T(s) as high as 900 K, well above the recombinative desorption temperatures of surface-adsorbed H. Developing and sustaining atomic-scale surface roughness, by H-induced silicon etching, is a prerequisite for H absorption and determines the T(s) window.

Journal Article↗

PubMed2000

The specificity of the protein kinase C alpha, betaII and gamma isoforms as assessed by an unnatural alcohol-appended peptide library.

Previous studies using conventional peptide-based libraries have demonstrated that homologous protein-processing enzymes [e.g. the alpha, betaII and gamma isoforms of protein kinase (PKC)] typically display identical amino acid consensus sequences. These observations have hampered the acquisition of selective synthetic substrates for the individual members of these enzyme families. We describe here a parallel synthesis strategy, readily adaptable to the preparation of large libraries, that has led to the emergence of the first examples of selective substrates for the conventional PKC isoforms. In addition, we have found that a wide variety of structurally diverse N-appended alcohol-containing residues, including tyrosine, serve as substrates for the PKC alpha, betaII and gamma isoforms. This broad active-site substrate specificity with respect to both natural and unnatural residues may prove to be especially applicable to the construction of transition-state analogues and suicide substrates, species that often require the presence of structurally elaborate functionality.

Amino Acid Sequence↗

PubMed2000

Left ventricular hypertrophy in ascending aortic stenosis mice: anoikis and the progression to early failure.

BACKGROUND: To determine potential mechanisms of the transition from hypertrophy to very early failure, we examined apoptosis in a model of ascending aortic stenosis (AS) in male FVB/n mice. METHODS AND RESULTS: Compared with age-matched controls, 4-week and 7-week AS animals (n=12 to 16 per group) had increased ratios of left ventricular weight to body weight (4.7+/-0.7 versus 3.1+/-0.2 and 5. 7+/-0.4 versus 2.7+/-0.1 mg/g, respectively, P<0.05) with similar body weights. Myocyte width was also increased in 4-week and 7-week AS mice compared with controls (19.0+/-0.8 and 25.2+/-1.8 versus 14. 1+/-0.5 microm, respectively, P<0.01). By 7 weeks, AS myocytes displayed branching with distinct differences in intercalated disk size and staining for beta(1)-integrin on both cell surface and adjacent extracellular matrix. In vivo left ventricular systolic developed pressure per gram as well as endocardial fractional shortening were similar in 4-week AS and controls but depressed in 7-week AS mice. Myocyte apoptosis estimated by in situ nick end-labeling (TUNEL) was extremely rare in 4-week AS and control mice; however, a low prevalence of TUNEL-positive myocytes and DNA laddering were detected in 7-week AS mice. The specificity of TUNEL labeling was confirmed by in situ ligation of hairpin oligonucleotides. CONCLUSIONS: Our findings indicate that myocyte apoptosis develops during the transition from hypertrophy to early failure in mice with chronic biomechanical stress and support the hypothesis that the disruption of normal myocyte anchorage to adjacent extracellular matrix and cells, a process called anoikis, may signal apoptosis.

Animals↗

PubMed2000

Marked differences between two isoforms of human pyruvate dehydrogenase kinase.

Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors.

Acetyl Coenzyme A↗

PubMed2000

NMR studies of Bacillus subtilis tRNA(Trp) hyperexpressed in Escherichia coli. Assignment of imino proton signals and determination of thermal stability.

15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.

Anticodon↗

PubMed2000

Characterization and gene structure of a novel retinoblastoma-protein-associated protein similar to the transcription regulator TFII-I.

Retinoblastoma protein (Rb) is an important regulator of vertebrate cell cycle and development. It functions through a direct interaction with protein factors involved in cell cycle progression and differentiation. In the present study we characterized a novel Rb-associated protein, Cream1, which bound to Rb specifically through a C-terminal region. Cream1 contained 959 amino acid residues and migrated as a protein of approx. 120 kDa on SDS/PAGE. It was a widely expressed nuclear protein with a nuclear localization signal resembling that of the large T antigen of simian virus 40. Its primary sequence was characteristic of five direct repeats that were similar to, but distinct from, those of TFII-I, a multifunctional transcription regulator. Three additional regions were also highly conserved in both proteins. Cream1 exhibited an activation activity that was attributed to its N-terminal portion when assayed in yeast. Its relationship with the muscle-enhancer-binding protein MusTRD1 further suggests a role in regulating gene expression. The structural gene, CREAM1, contained 27 exons and spanned more than 150 kb. It was located at human chromosome 7q11.23 in a region deleted for Williams' syndrome, a neurodevelopmental disease with multisystem abnormalities, implying its involvement in certain disorders. Taken together, our results suggest that Cream1 might serve as a positive transcription regulator under the control of Rb.

Amino Acid Sequence↗

PubMed2000

Matrix metalloproteinases and tumor necrosis factor alpha in glaucomatous optic nerve head.

OBJECTIVE: To study expression and location of matrix metalloproteinases (MMPs) and tumor necrosis factor alpha (TNF-alpha) in glaucomatous optic nerve heads, which are known to be secreted in response to a variety of neuronal injury. METHODS: Four postmortem eyes from patients with primary open-angle glaucoma, 7 eyes from patients with normal-pressure glaucoma, and 4 eyes from age-matched normal donors were studied by immunohistochemistry. The sections of the optic nerve heads were examined after immunostaining with antibodies to MMPs (MMP-1, MMP-2, and MMP-3), TNF-alpha, or TNF-alpha receptor 1. RESULTS: The intensity of the immunostaining and the number of stained cells for MMPs, TNF-alpha, or TNF-alpha receptor 1 were greater in the glaucomatous optic nerve heads, particularly in eyes with normal-pressure glaucoma compared with age-matched controls. Positive immunostaining was observed in all regions of the glaucomatous optic nerve heads, but most prominently in the postlaminar region. Immunostaining was observed mainly in glial cells and their processes around the axons and blood vessels and in pial septae. CONCLUSION: There is increased immunostaining for MMPs, TNF-alpha and TNF-alpha receptor 1 in the glaucomatous optic nerve head, which suggests increased expression of these proteins in glaucoma and thereby implies a role in the tissue remodeling and degenerative changes seen in glaucomatous optic nerve heads. CLINICAL RELEVANCE: The MMPs and TNF-alpha may be components of astroglial activation that occurs in glaucomatous optic nerve heads. The biological alterations in the expression of these proteins may play a role in the progression of glaucomatous optic neuropathy.

Aged↗

PubMed2000

Determination of internucleotide (h)J(HN) couplings by the modified 2D J(NN)-correlated [(15)N, (1)H] TROSY.

Recent developments in the direct observation of J couplings across hydrogen bonds in proteins and nucleic acids provide additional information for structure and function studies of these molecules by NMR spectroscopy. A J(NN)-correlated [(15)N, (1)H] TROSY experiment proposed by Pervushin et al. (Proc. Natl. Acad. Sci. USA 95, 14147-14151, 1998) can be applied to measure (h)J(HN) in smaller nucleic acids in an E.COSY manner. However, it cannot be effectively applied to large nucleic acids, such as tRNA(Trp), since one of the peaks corresponding to a fast relaxing component will be too weak to be observed in the spectra of large molecules. In this Communication, we proposed a modified J(NN)-correlated [(15)N, (1)H] TROSY experiment which enables direct measurement of (h)J(HN) in large nucleic acids.

Bacillus subtilis↗