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Biomedical subjects

H Li

Publications and source records attributed to H Li.

At least 505 records · Page 28Linked to original sources
PubMed2000

Polynitroxyl-albumin (PNA) plus tempol attenuate lung capillary leak elicited by prolonged intestinal ischemia and reperfusion(1).

Stable nitroxyl radicals (nitroxides) are potential antioxidant drugs, and we have previously reported that linking nitroxide to biological macromolecules can improve therapeutic activity in at least two ways. First, polynitroxylated compounds such as polynitroxyl human serum albumin (PNA) are a novel class of high molecular weight, extracellular antioxidants. Second, compounds such as PNA can prolong the half-life of free (unbound, low molecular weight) nitroxides such as 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl (Tempol) in vivo. Unlike PNA, Tempol can readily access the intracellular compartment. Thus PNA can act alone in the extracellular compartment, or in concert with Tempol, to provide additional antioxidant protection within cells. In this study, we compared the abilities of PNA, Tempol, and the combination of PNA + Tempol to prevent lung microvascular injury secondary to prolonged gut ischemia (I, 120 min) and reperfusion (R, 20 min) in the rat. Pulmonary capillary filtration coefficient (K(f,c)) and lung neutrophil retention (tissue myeloperoxidase activity, MPO) were measured in normal, isolated rat lungs perfused with blood harvested from I/R rats. Blood donor rats were treated with drug during ischemia. Gut I/R resulted in a marked increase in pulmonary capillary coefficient and lung MPO. PNA + Tempol, but not PNA alone or Tempol alone, at the doses used, prevented the development of lung leak. None of the treatments had an effect on lung neutrophil retention. Anti-inflammatory therapeutic activity appeared to correlate with blood Tempol level: in the presence of PNA, blood Tempol levels were maintained in the 50-100 microM range vs. essentially undetectable levels shortly after Tempol was administered alone. In this model of lung injury secondary to prolonged gut I/R, lung capillary leak was prevented when the membrane-permeable compound Tempol was maintained in its active, free radical state by PNA.

Albumins↗
PubMed2000

High-throughput single-molecule DNA screening based on electrophoresis.

In electrophoresis, the migration velocity is used for sizing DNA and proteins or for distinguishing molecules based on charge and hydrodynamic radius. Many protein and DNA assays relevant to disease diagnosis are based on such separations. However, standard protocols are not only slow (minutes to hours) but also insensitive (many molecules in a detectable band). We successfully demonstrated a high-throughput imaging approach that allows determination of the individual electrophoretic mobilities of many molecules at a time. Each measurement only requires a few milliseconds to complete. This opens up the possibility of screening single copies of DNA or proteins within single biological cells for disease markers without performing polymerase chain reaction or other biological amplification. The purpose is not to separate the DNA molecules but to identify each one on the basis of the measured electrophoretic mobility. We developed three different procedures to measure the individual molecular mobilities. The results correlate well with capillary electrophoresis (CE) experiments for the same samples (2-49 kb dsDNA) under identical separation conditions. The implication is that any electrophoresis protocols from slab gels to CE should be adaptable to single-molecule screening for disease diagnosis.

Buffers↗
PubMed2000

Exisulind induction of apoptosis involves guanosine 3',5'-cyclic monophosphate phosphodiesterase inhibition, protein kinase G activation, and attenuated beta-catenin.

Sulindac sulfone (exisulind), although a nonsteroidal anti-inflammatory drug derivative, induces apoptosis in tumor cells by a mechanism that does not involve cyclooxygenase inhibition. SW480 colon tumor cells contain guanosine 3',5'-monophosphate (cGMP) phosphodiesterase (PDE) isoforms of the PDE5 and PDE2 gene families that are inhibited by exisulind and new synthetic analogues. The analogues maintain rank order of potency for PDE inhibition, apoptosis induction, and growth inhibition. A novel mechanism for exisulind to induce apoptosis is studied involving sustained increases in cGMP levels and cGMP-dependent protein kinase (PKG) induction not found with selective PDE5 or most other PDE inhibitors. Accumulated beta-catenin, shown to be a substrate for PKG, is decreased by exisulind, suggesting a mechanism to explain apoptosis induction in neoplastic cells harboring adenomatous polyposis coli gene mutations.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed2000

Effects of Cu2+ on root growth, cell division, and nucleolus of Helianthus annuus L.

The effects of different concentrations (10-5 - 10-2 M) of copper sulfate on root growth, cell division and nucleoli in root-tip cells of Helianthus annuus L. were investigated. Cu had an inhibitory effect on the root growth at 10-4 - 10-2 M during the entire treatment. Cu has toxic effects on chromosomal morphology including anaphase bridges and chromosome stickiness and on nucleoli, causing some similar silver-stained particulate material scattered in the nuclei. Once the nucleolus is poisoned, showing some silver-stained particles scattered in the nucleus, the root growth of H. annuus is obviously inhibited. And once more silver-stained particles appear in the nucleus, the root growth is almost or completely stopped. The possible mechanism of the Cu poisoning of root-tip cells of H. annuus is also briefly discussed.

Cell Division↗
PubMed2000

Coordinate regulation of cadherin and integrin function by the chondroitin sulfate proteoglycan neurocan.

N-cadherin and beta1-integrins play decisive roles in morphogenesis and neurite extension and are often present on the same cell. Therefore, the function of these two types of adhesion systems must be coordinated in time and space to achieve the appropriate cell and tissue organization. We now show that interaction of the chondroitin sulfate proteoglycan neurocan with its GalNAcPTase receptor coordinately inhibits both N-cadherin- and beta1-integrin-mediated adhesion and neurite outgrowth. Furthermore, the inhibitory activity is localized to an NH(2)-terminal fragment of neurocan containing an Ig loop and an HA-binding domain. The effect of neurocan on beta1-integrin function is dependent on a signal originating from the cadherin cytoplasmic domain, possibly mediated by the nonreceptor protein tyrosine kinase Fer, indicating that cadherin and integrin engage in direct cross-talk. In the developing chick, neural retina neurocan is present in the inner plexiform layer from day 7 on, and the GalNAcPTase receptor becomes restricted to the inner nuclear layer and the ganglion cell layer (as well as the fiber layer), the two forming a sandwich. These data suggest that the coordinate inhibition of cadherin and integrin function on interaction of neurocan with its receptor may prevent cell and neurite migration across boundaries.

Amino Acid Sequence↗
PubMed2000

Combined effects of radiotherapy and angiostatin gene therapy in glioma tumor model.

The objective of the present study was to evaluate the antitumor effect of a defective adenovirus expressing a secretable angiostatin-like molecule (AdK3) in combination with radiotherapy in rat C6 gliomas s.c. preestablished into athymic mice. In vitro, the combination regimen was significantly (P < 0.001) more cytotoxic for human microcapillary endothelial cells than either treatment alone, whereas survival of C6 glioma cells was not affected in the conditions used. Radiotherapy and AdK3 gene delivery was then studied on well established C6 xenografts (165 +/- 70 mm(3)). In these tumors, AdK3 intratumoral injections had only a marginal effect. Interestingly, when experimental radiotherapy was added, significantly higher (P < 0.005), and possibly synergistic, antitumoral effects were observed that tightly correlated a marked decrease of intratumoral vascularization. The combination of radiotherapy and AdK3 intratumoral injections also revealed a significant (P < 0.05) inhibition of tumor growth as compared with either treatment alone for larger tumors (467 +/- 120 mm(3)). Altogether, these data emphasize the potential of combining a destructive strategy directed against the tumor cells with an anti-angiogenic approach to fight cancer.

Adenoviridae↗
PubMed2000

Atomic force microscopy reveals the mechanical design of a modular protein.

Tandem modular proteins underlie the elasticity of natural adhesives, cell adhesion proteins, and muscle proteins. The fundamental unit of elastic proteins is their individually folded modules. Here, we use protein engineering to construct multimodular proteins composed of Ig modules of different mechanical strength. We examine the mechanical properties of the resulting tandem modular proteins by using single protein atomic force microscopy. We show that by combining modules of known mechanical strength, we can generate proteins with novel elastic properties. Our experiments reveal the simple mechanical design of modular proteins and open the way for the engineering of elastic proteins with defined mechanical properties, which can be used in tissue and fiber engineering.

Biomechanical Phenomena↗
PubMed2000

Atomic force microscopy captures quantized plastic deformation in gold nanowires.

Scanning probe microscopy has become a powerful tool to detect structural changes in small clusters of atoms. Herein, we use an atomic force microscope to measure the length of gold nanowire structures during extension and compression cycles. We have found that nanowires elongate under force in quantized steps of up to three integer multiples of 1.76 A and that they shorten spontaneously in steps of 1.52 A. Our results can be explained by the sliding of crystal planes within the gold nanowires creating stacking faults that change the local structure from face-centered cubic to hexagonal close packed. Our data also show that there can be up to three simultaneous slip events, in good agreement with the tetrahedral arrangement of slip planes in a gold crystal. These experiments provide direct evidence for the mechanism underlying the plastic deformation of a nanowire. A similar approach can be used to examine the atomic events underlying the plastic failure of other metals and their alloys.

Journal Article↗
PubMed2000

Three-dimensional structures of the free and antigen-bound Fab from monoclonal antilysozyme antibody HyHEL-63(,).

Antigen-antibody complexes provide useful models for studying the structure and energetics of protein-protein interactions. We report the cloning, bacterial expression, and crystallization of the antigen-binding fragment (Fab) of the anti-hen egg white lysozyme (HEL) antibody HyHEL-63 in both free and antigen-bound forms. The three-dimensional structure of Fab HyHEL-63 complexed with HEL was determined to 2.0 A resolution, while the structure of the unbound antibody was determined in two crystal forms, to 1.8 and 2.1 A resolution. In the complex, 19 HyHEL-63 residues from all six complementarity-determining regions (CDRs) of the antibody contact 21 HEL residues from three discontinuous polypeptide segments of the antigen. The interface also includes 11 bound water molecules, 3 of which are completely buried in the complex. Comparison of the structures of free and bound Fab HyHEL-63 reveals that several of the ordered water molecules in the free antibody-combining site are retained and that additional waters are added upon complex formation. The interface waters serve to increase shape and chemical complementarity by filling cavities between the interacting surfaces and by contributing to the hydrogen bonding network linking the antigen and antibody. Complementarity is further enhanced by small (<3 A) movements in the polypeptide backbones of certain antibody CDR loops, by rearrangements of side chains in the interface, and by a slight shift in the relative orientation of the V(L) and V(H) domains. The combining site residues of complexed Fab HyHEL-63 exhibit reduced temperature factors compared with those of the free Fab, suggesting a loss in conformational entropy upon binding. To probe the relative contribution of individual antigen residues to complex stabilization, single alanine substitutions were introduced in the epitope of HEL recognized by HyHEL-63, and their effects on antibody affinity were measured using surface plasmon resonance. In agreement with the crystal structure, HEL residues at the center of the interface that are buried in the complex contribute most to the binding energetics (DeltaG(mutant) - DeltaG(wild type) > 3.0 kcal/mol), whereas the apparent contributions of solvent-accessible residues at the periphery are much less pronounced (<1.5 kcal/mol). In the latter case, the mutations may be partially compensated by local rearrangements in solvent structure that help preserve shape complementarity and the interface hydrogen bonding network.

Amino Acid Sequence↗
PubMed2000

[Toxic effect of organic extracts of drinking-water on the culture luteal cells of rats].

A separation method of rat luteal cells was established. The results show that the survival rate of the cells is more than 95% during the whole operation. 14-20 days of pregnancy, 10(6) cells/ml and 4 hours of culture are the suitable conditions. The organic extracts of drinking-water produced significant dose-related increases(maximally three-fold) in progesterone concentration in the culture medium. Thus, the effects produced by the organic extracts of drinking-water in vitro may reflect the damage to the luteal cells.

Animals↗
PubMed2000

DNA melting and promoter clearance by eukaryotic RNA polymerase I.

Ribosomal RNA transcription initiation requires the melting of DNA to form an open complex, formation of the first few phosphodiester bonds, commencement of RNA polymerase I movement along the DNA, clearance of the promoter, and the formation of a steady-state ternary elongation complex. We examined DNA melting and promoter clearance by using potassium permanganate, diethylpyrocarbonate and methidiumpropylEDTA.Fe(II) footprinting. In combination, these methods demonstrated: (1) TIF-IB and RNA polymerase I are the only proteins required for formation of an initial approximately 9 base-pair open promoter region. This finding contradicts earlier results using diethylpyrocarbonate alone, which suggested an RNA synthesis requirement for stable melting. (2) DNA melting is temperature-dependent, with a tm between 15 and 20 degrees C. (3) Temperature-dependency of melting, as well as stalling the polymerase at sites close to the transcription start site revealed that the melted DNA region initially opens upstream of the transcription initiation site, and enlarges in a downstream direction coordinate with initiation, eventually attaining a steady-state transcription bubble of approximately 19 base-pairs. (4) The RNA-DNA hybrid protects the template DNA from single-strand footprinting reagents. The hybrid is 9 bp in length, consistent with the longer hybrid estimated by some for the Escherichia coli polymerase and with the hybrids estimated for eukaryotic polymerases II and III.

Acanthamoeba↗
PubMed2000

Targeting of lung cancer mutational hotspots by polycyclic aromatic hydrocarbons.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in combustion products of organic matter, including cigarette smoke. Metabolically activated diol epoxides of these compounds, including benzo[a]pyrene diol epoxide (B[a]PDE), have been suggested as causative agents in the development of lung cancer. We previously mapped the distribution of B[a]PDE adducts within the p53 tumor suppressor gene (also known as TP53), which is mutated in 60% of human lung cancers, and found that B[a]PDE adducts preferentially form at lung cancer mutational hotspots (codons 154, 157, 158, 245, 248, and 273). Other PAHs may be important in lung cancer as well. METHODS: Here we have mapped the distribution of adducts induced by diol epoxides of additional PAHs: chrysene (CDE), 5-methylchrysene (5-MCDE), 6-methylchrysene (6-MCDE), benzo[c]phenanthrene (B[c]PDE), and benzo[g]chrysene (B[g]CDE) within exons 5, 7, and 8 of the p53 gene in human bronchial epithelial cells. RESULTS: CDE exposure produced only low levels of adducts. Exposure of cells to the other activated PAHs resulted in DNA damage patterns similar to those previously observed with B[a]PDE but with some distinct differences. 5-MCDE, 6-MCDE, B[g]CDE, and B[c]PDE efficiently induced adducts at guanines within codons 154, 156, 157, 158, and 159 of exon 5, codons 237, 245 and 248 of exon 7, and codon 273 of exon 8, but the relative levels of adducts at each site varied for each compound. B[g]CDE, B[c]PDE, and 5-MCDE induced damage at codon 158 more selectively than 6-MCDE or B[a]PDE. The sites most strongly involved in PAH adduct formation were also the sites of highest mutation frequency (codons 157, 158, 245, 248, and 273). CONCLUSION: The data suggest that PAHs contribute to the mutational spectrum in human lung cancer.

Base Sequence↗
PubMed2000

Pinacidil pretreatment extends ischemia tolerance of neonatal rabbit hearts.

OBJECTIVES: Activating ATP-sensitive potassium (K(ATP)) channels improves ischemia tolerance of adult rabbit hearts. We hypothesize that (a) endogenous activation of the K(ATP) channel accounts for better ischemia tolerance of neonatal hearts and (b) exogenous K(ATP) channel activation with pinacidil further improves the neonatal heart's tolerance to cardioplegic ischemia. METHODS: Study 1: Seven (control) neonatal rabbits received intraperitoneal saline, whereas five others (Glib) received 0.3 mg/kg glibenclamide 10 min before sacrifice. They were perfused on Langendorff with Krebs-Henseleit buffer (KHB). Baseline left ventricle (LV) performance and coronary flow (CF) were measured. After 20 min of 37 degrees C ischemia and 10 min of reperfusion, recovery was measured. Study 2: Ten (control) neonatal hearts underwent 90 min of normothermic ischemia with St. Thomas' cardioplegia (STCP) solution administered every 30 min. Ten others were pretreated with a 10-min infusion of 1 microM pinacidil in KHB and received 1 microM pinacidil-enriched STCP. Recovery of LV performance and CF were measured after 60 min of reperfusion. RESULTS: Study 1: Glib significantly reduced preischemia LV performance by 28%* compared to control hearts. Recovery of Glib-treated hearts was significantly less (67%*) than controls (81%*). Study 2: Pinacidil-treated hearts had significantly better recovery of LV performance (39%*) and CF (78%*) compared to 23 and 52%, respectively, in untreated controls (*P < 0.05 vs control hearts). CONCLUSIONS: Endogenous K(ATP) channel activation in neonatal hearts contributes to their better tolerance to ischemia. Exogenous K(ATP) channel activation by pinacidil pretreatment and cardioplegic enrichment significantly improved the neonatal rabbit heart's tolerance to cardioplegic ischemia. This may be an important addition to myocardial protection during pediatric cardiac surgery.

Animals↗
PubMed2000

Microglia-like cells in rat organ of Corti following aminoglycoside ototoxicity.

The repair process of neomycin induced cochlear damage in the postnatal developing rat was investigated in the present study. The results showed that electron dense atypical cells with a cluster of microvilli on their apical surface, resembling early stage of embryonic hair cell, were observed in the former hair cell region. The striking finding was that microglia-like cells appeared and replaced OHCs in the injured auditory sensory epithelium. Using Brdu immunohistochemistry, cell proliferation was found in the area of inner and outer spiral sulcus but not in the hair cells and supporting cells. It is proposed that microglia-like cells play a role in eliminating waste products from the organ of Corti and may participate in direct structural repair.

Animals↗
PubMed2000

Analysis of tetramethylpyrazine in Ephedrae herba by gas chromatography-mass spectrometry and high-performance liquid chromatography.

A simple and reliable HPLC method was developed for the determination of 2,3,5,6-tetramethylpyrazine (TMP) in Ephedrae herba. Further identification of TMP was achieved using GC-MS. The mobile phase used was methanol-water-35% acetic acid (35:65:0.5, v/v/v) at a flow-rate of 0.8 ml/min. The detection wavelength was set at 290 nm. The linear range of the peak area calibration curve of TMP was 2.64-264 mg/l (r=0.9987) and the recovery for TMP in Ephedrae herba extracts was 101.1-106.9%. The relative standard deviations of retention time and peak area were 0.18 and 1.5% (n=6), respectively. The detection limit of TMP was 0.03 mg/l. The contents of TMP in Ephedrae herba could easily be determined within 10 min.

Calibration↗
PubMed2000

Identification of an enhancer sequence within the first intron required for cartilage-specific transcription of the alpha2(XI) collagen gene.

Type XI collagen, a heterotrimer composed of alpha1(XI), alpha2(XI) and alpha3(XI), is primarily synthesized by chondrocytes in cartilage and is also present in some other tissues. Type XI collagen plays a critical role in collagen fibril formation and skeletal morphogenesis. We investigated a tissue-specific transcriptional enhancer in the first intron of the alpha2(XI) collagen gene (Col11a2). Transient transfection assays using reporter gene constructs revealed that a 60-base pair (bp) segment within intron 1 increased promoter activity of Col11a2 in rat chondrosarcoma cells but not in either BalB/3T3 cells or undifferentiated ATDC5 cells, suggesting that it contained cell type-specific enhancer activity. In transgenic mice, this 60-bp fragment was also able to target beta-galactosidase expression to cartilage including the limbs and axial skeleton, with similar localization specificity as the full-length intron 1 fragment. Competition experiments in gel shift assays using mutated oligonucleotides showed that recombinant Sox9 bound to a 7-bp sequence, CTCAAAG, within the 60-bp segment. Anti-Sox9 antibodies supershifted the complex of the 60-bp segment with recombinant Sox9 or with rat chondrosarcoma cell extracts, confirming the binding of Sox9 to the enhancer. Moreover, a site-specific mutation within the 7-bp segment resulted in essentially complete loss of the enhancer activity in chondrosarcoma cells and transgenic mice. These results suggest that the 7-bp sequence within intron 1 plays a critical role in the cartilage-specific enhancer activity of Col11a2 through Sox9-mediated transcriptional activation.

3T3 Cells↗
PubMed2000

Genomic, transcriptional and phenotypic analysis of ftsE and ftsX of Neisseria gonorrhoeae.

Although ftsE and ftsX are not universally present in bacteria, they are present in various Neisseria species as determined by Southern hybridization. The ftsE and ftsX genes of Neisseria gonorrhoeae (Ng) CH811 were cloned, sequenced and were shown to be co-transcribed from two promoters (P(E)1 and P(E)2) which were identified upstream of ftsE(Ng) by primer extension. Sequence analysis of FtsE(Ng) and alignment with other FtsE indicated that it contained the conserved motifs of ABC domains while sequence alignment of FtsX(Ng) with other published FtsX sequences predicted that they all contain four transmembrane segments and a conserved motif (Leu-hydrophobic aa-Gly-Ala/Gly) which may prove to be important for FtsX function. The viability of ftsE(Ng) and ftsX(Ng) mutants that were constructed by insertional inactivation indicated that these genes are not essential. The role of FtsE and FtsX is controversial. Analysis of ftsE(Ng) and ftsX(Ng) mutants by transmission electron microscopy showed that both exhibited morphological abnormalities indicative of defective division sites and in some cases aberrant condensation of DNA.

ATP-Binding Cassette Transporters↗