Biomedical subjects
Publications and source records attributed to Ying Li.
The concentration of alpha-oxygen which can oxidize methane to methanol and benzene to phenol at RT, increases linearly with the amount of introduced extraframework Al on Fe/ZSM-5 catalysts prepared by solid-state exchange of FeCl3 and AlCl3 with H-ZSM-5.
Tn7 transposition requires the assembly of a nucleoprotein complex containing four self-encoded proteins, transposon ends, and target DNA. Within this complex, TnsC, the molecular switch that regulates transposition, and TnsA, one part of the transposase, interact directly. Here, we demonstrate that residues 504-555 of TnsC are responsible for TnsA/TnsC interaction. The crystal structure of the TnsA/TnsC(504-555) complex, resolved to 1.85 A, illustrates the burial of a large hydrophobic patch on the surface of TnsA. One consequence of sequestering this patch is a marked increase in the thermal stability of TnsA as shown by differential scanning calorimetry. A model based on the complex structure suggested that TnsA and a slightly longer version of the cocrystallized TnsC fragment (residues 495-555) might cooperate to bind DNA, a prediction confirmed using gel mobility shift assays. Donor DNA binding by the TnsA/TnsC(495-555) complex is correlated with the activation of the TnsAB transposase, as measured by double-stranded DNA cleavage assays, demonstrating the importance of the TnsA/TnsC interaction in affecting Tn7 transposition.
Ultraviolet-induced DNA damage poses a lethal block to replication. To understand the structural basis for this, we determined crystal structures of a replicative DNA polymerase from bacteriophage T7 in complex with nucleotide substrates and a DNA template containing a cis-syn cyclobutane pyrimidine dimer (CPD). When the 3' thymine is the templating base, the CPD is rotated out of the polymerase active site and the fingers subdomain adopts an open orientation. When the 5' thymine is the templating base, the CPD lies within the polymerase active site where it base-pairs with the incoming nucleotide and the 3' base of the primer, while the fingers are in a closed conformation. These structures reveal the basis for the strong block of DNA replication that is caused by this photolesion.
Here, we characterize the actions of MK-0767, a dual ligand of the nuclear receptors peroxisome proliferator-activated receptor (PPAR)alpha and PPARgamma. In cell-based assays, MK-0767 produced potent activation of human PPARgamma and PPARalpha with a gamma:alpha potency ratio of approximately 2. The dual agonist induced high affinity interactions of PPARalpha and PPARgamma with the transcriptional coactivator CBP in vitro. In ob/ob mice, MK-0767 normalized hyperglycemia and hyperinsulinemia with equal or greater potency and efficacy than pioglitazone. Treatment of hamsters with MK-0767 produced substantial reductions in blood cholesterol and triglycerides. In dogs, MK-0767 reduced serum cholesterol levels with a potency more than 10-fold greater than simvastatin. The efficacies of MK-0767 and simvastatin were additive when given together. We conclude that MK-0767 is a potent dual PPARalpha/gamma agonist with robust insulin sensitizing and hypolipidemic activities.
Daphnetin (7,8-dihydroxycoumarin), one of the major bioactive components isolated from Daphne koreane Nakai, has been used in traditional Chinese medicine for the treatment of coagulation disorders. It is also a chelator, an antioxidant and a protein kinase inhibitor. In this paper, a combination of intrinsic fluorescence, Fourier transform infrared (FT-IR) spectroscopy and circular dichroic (CD) spectroscopy has been used to characterize the binding between daphnetin and human serum albumin (HSA) under physiological conditions with drug concentrations of 6.7 x 10(-6) - 2.3 x 10(-5) mol x L(-1), and a HSA concentration of 1.5 x 10(-6) mol x L(-1). Changes in the CD spectra and FT-IR spectra were observed upon ligand binding, and the degree of tryptophan fluorescence quenching did change significantly in the complexes. These data have proved the change in protein secondary structure accompanying ligand binding. The change in tryptophan fluorescence intensity was used to determine the binding constants. The thermodynamic parameters, the enthalpy change (DeltaH) and the entropy change (DeltaS) were calculated to be -12.45 kJ x mol(-1)and 52.48 J x mol(-1) x K(-1) according to the van't Hoff equation, which indicated that hydrophobic and electrostatic interactions played the main role in the binding of daphnetin to HSA, in accordance with the results of calculations performed on a Silicon Graphics Ocatane2 workstation. In addition, the binding distance between daphnetin and HSA was obtained (4.02 nm) based on the Forster energy transfer theory.
Z66 antigen-positive strains of Salmonella enterica serovar Typhi change flagellin expression in only one direction from the z66 antigen to the d or j antigen, which is different from the phase variation of S. enterica serovar Typhimurium. In the present study, we identified a new flagellin gene in z66 antigen-positive strains of S. enterica serovar Typhi. The genomic structure of the region containing this new flagellin gene was similar to that of fljBA operon of biphasic S. enterica serovars. A fljA-like gene was present downstream of the new flagellin gene. A rho-independent terminator was located between the new flagellin gene and the fljA-like gene. Hin-like gene was not present upstream of the new flagellin gene. We generated a mutant strain of S. enterica serovar Typhi, which carries a deletion of the new flagellin gene. Western blotting revealed that the 51-kDa z66 antigen protein was absent from the population of proteins secreted by the mutant strain. Southern hybridization demonstrated that the z66 antigen-positive strains of S. enterica serovar Typhi carried the new flagellin gene and fliC on two different genomic EcoRI fragments. When z66 antigen-positive strains were incubated with anti-z66 antiserum, the flagellin expression by S. enterica serovar Typhi changed from z66 antigen to j antigen. The new flagellin gene and the fljA-like gene were absent in the strain with altered flagellin expression. These results suggested that the new flagellin gene is a fljB-like gene, which encodes the z66 antigen of S. enterica serovar Typhi, and that deletion of fljBA-like operon may explain why S. enterica serovar Typhi alters the flagellin expression in only one direction from the z66 antigen to the d or j antigen.
BACKGROUND: Analysis of fetal DNA in maternal plasma has recently been introduced as a new method for noninvasive prenatal diagnosis, particularly for the analysis of fetal genetic traits, which are absent from the maternal genome, e.g., RHD or Y-chromosome-specific sequences. To date, the analysis of other fetal genetic traits has been more problematic because of the overwhelming presence of maternal DNA sequences in the circulation. We examined whether different biochemical properties can be discerned between fetal and maternal circulatory DNA. METHODS: Plasma DNA was examined by agarose gel electrophoresis. The fractions of fetal and maternal DNA in size-fractionated fragments were assayed by real-time PCR. The determination of paternally and maternally inherited fetal genetic traits was examined by use of highly polymorphic chromosome-21-specific microsatellite markers. RESULTS: Size fractionation of circulatory DNA indicated that the major portion of cell-free fetal DNA had an approximate molecular size of <0.3 kb, whereas maternally derived sequences were, on average, considerably larger than 1 kb. Analysis of size-fractionated DNA (</=0.3 kb) from maternal plasma samples facilitated the ready detection of paternally and maternally inherited microsatellite markers. CONCLUSIONS: Circulatory fetal DNA can be enriched by size selection of fragment sizes less than approximately 0.3 kb. Such selection permits easier analysis of both paternally and maternally inherited DNA polymorphisms.
A complete sequence of a chromosome centromere is necessary for fully understanding centromere function. We reported the sequence structures of the first complete rice chromosome centromere through sequencing a large insert bacterial artificial chromosome clone-based contig, which covered the rice chromosome 4 centromere. Complete sequencing of the 124-kb rice chromosome 4 centromere revealed that it consisted of 18 tracts of 379 tandemly arrayed repeats known as CentO and a total of 19 centromeric retroelements (CRs) but no unique sequences were detected. Four tracts, composed of 65 CentO repeats, were located in the opposite orientation, and 18 CentO tracts were flanked by 19 retroelements. The CRs were classified into four types, and the type I retroelements appeared to be more specific to rice centromeres. The preferential insert of the CRs among CentO repeats indicated that the centromere-specific retroelements may contribute to centromere expansion during evolution. The presence of three intact retrotransposons in the centromere suggests that they may be responsible for functional centromere initiation through a transcription-mediated mechanism.
Mutants of the Saccharomyces cerevisiae SRS2 gene are hyperrecombinogenic and sensitive to genotoxic agents, and they exhibit a synthetic lethality with mutations that compromise DNA repair or other chromosomal processes. In addition, srs2 mutants fail to adapt or recover from DNA damage checkpoint-imposed G2/M arrest. These phenotypic consequences of ablating SRS2 function are effectively overcome by deleting genes of the RAD52 epistasis group that promote homologous recombination, implicating an untimely recombination as the underlying cause of the srs2 mutant phenotypes. TheSRS2-encodedproteinhasasingle-stranded (ss) DNA-dependent ATPase activity, a DNA helicase activity, and an ability to disassemble the Rad51-ssDNA nucleoprotein filament, which is the key catalytic intermediate in Rad51-mediated recombination reactions. To address the role of ATP hydrolysis in Srs2 protein function, we have constructed two mutant variants that are altered in the Walker type A sequence involved in the binding and hydrolysis of ATP. The srs2 K41A and srs2 K41R mutant proteins are both devoid of ATPase and helicase activities and the ability to displace Rad51 from ssDNA. Accordingly, yeast strains harboring these srs2 mutations are hyperrecombinogenic and sensitive to methylmethane sulfonate, and they become inviable upon introducing either the sgs1Delta or rad54Delta mutation. These results highlight the importance of the ATP hydrolysisfueled DNA motor activity in SRS2 functions.
Hypocretin/orexin neurons play an important role in hypothalamic arousal. Synaptic glutamate input to hypocretin neurons regulates cell firing. We studied the actions of group III metabotropic glutamate receptors (mGluRs) in modulating the activity of hypocretin neurons using whole-cell voltage- and current-clamp recording in mouse whole hypothalamic slices or minislices consisting only of the lateral hypothalamus. Selective green fluorescent protein expression was used to detect live hypocretin neurons. The mGluR agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP-4) inhibited synaptic input to hypocretin neurons in a dose-dependent manner; both spontaneous glutamate and GABA-mediated synaptic currents were reduced in frequency. l-AP-4 also reduced the amplitude of postsynaptic potentials evoked by a stimulating electrode placed medial or lateral to the recorded cell. No postsynaptic effect of l-AP-4 was found relative to membrane potential, input resistance, or AMPA-evoked currents. l-AP-4 appeared to act by a presynaptic mechanism and reduced the frequency of both glutamate- and GABA-mediated miniature events recorded in the presence of tetrodotoxin, with no change in amplitude. (RS)-phosphonopentanoic acid (CPPG), a group III mGluR antagonist, suppressed the actions of l-AP-4. Of substantial interest, CPPG by itself increased synaptic activity recorded in hypocretin neurons, suggesting an ongoing inhibitory tone attributable to activation of group III mGluRs. Glutamatergic interneurons have been suggested to play a role in a positive feedback recruitment of hypocretin on hypocretin neurons. l-AP-4 blocked hypocretin-mediated increases in EPSCs and attenuated the hypocretin-mediated increase in spike frequency. Together, these data suggest that tonically active inhibitory mGluRs are expressed on local hypocretin-sensitive glutamate neurons within the lateral hypothalamus that modulate the output of the hypocretin arousal system.
Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.
OBJECTIVE: To explore the deep-frozen allograft strut in the rabbits for the sake of finding some evidence to support the clinical practice of cortical strut allograft. METHODS: Fifty deep-frozen allograft struts were implanted on the lateral side of the femur in 50 male New Zealand white rabbits. These rabbits were randomly divided into 5 groups. The specimen were retrieved at 2, 4, 8, 16, 24 weeks after surgeries. In each group histomorphology and biomechanic properties were studied. RESULTS: From the X ray photography the edge of the strut would turn blunt at the 4th week, union could be found at the 8th week, the inner side of the strut could be absorbed at the 24th week. In the early stage when the strut was implanted in the rabbit, the capillary would be found to grow into the cortical strut from the interior side of it. Then the capillaries were found to converge at the 16th week. This phenomenon is correspondent with the resorption of the dead bone and formation of the new bone. There are few neoformated capillaries on the lateral side of the strut. From the decalcificied section, allograft struts were found to unit to the host at 4 to 8th week. Osteoclast and the new bone formation could be seen at the 8th week, and 3% of the total area of what had been the allograft was replaced by the new bone at that time, 12% was replaced at the 16th week, 21% at the 24th week. There are few resorption or neoformated bone at the lateral side of the strut. The biomechanical property indicated that the strength of the strut decreased at the early time, and only 38% of its original strength remained at 16 weeks. CONCLUSIONS: The cortical allograft strut can unit to the host by the callus. The capillaries invading into the allograft from the interior side, new bone is formed from the interior side. The biomechanical strength of the strut decreased at early stage.
The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCF(HOS)) E3 ubiquitin ligase, which mediates ubiquitination and degradation of beta-catenin and the inhibitor of NFkappaB, IkappaB. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCF(HOS) complex in vitro, the addition of phosphorylated IkappaBalpha inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated IkappaBalpha) by activating IkappaB kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate.
PURPOSE: To examine the relationships of total and cause-specific mortality to serum cholesterol in four diverse populations. METHODS: Chinese, Polish, Russian, and US population-based samples were studied. The relationship between cholesterol levels and mortality was assessed by Cox proportional hazard regression with restricted piecewise cubic splines. RESULTS: The cholesterol and total mortality relationship was statistically significantly J-shaped for all men combined. In country-specific relationships, cholesterol was significantly, linearly, and positively related to total mortality in Russian and US men. For women, the relationship was non-linear, but not statistically significant, and became statistically significant upon adjustment for other risk factors. For Polish women, a statistically significant inverse relationship existed. CHD mortality and cardiovascular disease (CVD) mortality increased linearly with cholesterol in Polish, Russian, and US men and the aggregate of men, but there was no relationship for women. Cancer mortality was not related to cholesterol except for the Polish cohort and Russian women, where there was an inverse relationship. CONCLUSIONS: Serum cholesterol was a strong, consistent predictor of CHD and CVD mortality in Polish, Russian, and US men despite their social diversity. In contrast to CHD mortality, the relation of cholesterol to total mortality and non-CVD mortality varied by country and gender.
A single fourth lumbar dorsal rootlet was transected at the entry point into the spinal cord. The nerve fibres were labelled with biotin dextran injected into the rootlet. An endogenous matrix containing olfactory-ensheathing cells (OECs) labelled with green fluorescent protein was applied to the opposing cut surfaces of the rootlet and the spinal cord, which were then brought into apposition and held in place by fibrin glue. Two weeks later, a ladderlike bridging structure has been formed by astrocytic processes growing out for about 200-300 microm from the spinal cord. The transplanted cells remained largely confined to this area. They were elongated along the nerve axis but did not enter the spinal cord itself. Labelled dorsal root axons crossed the repaired dorsal root entry zone in alignment with the bridging astrocytic processes and the transplanted cells and then proceeded beyond the transplant to enter the grey matter of the dorsal horn and send axons both rostrally and caudally for at least 10 mm in the white matter of the ascending dorsal columns.
Targeted gene delivery to neurons is crucial to effective gene therapy of neurodegenerative diseases. Several types of viral gene vectors may target neurons through retrograde axonal transport to somas of projection neurons after viral internalization at axon terminal fields. In this report we demonstrate for the first time that recombinant baculovirus vectors could migrate by axonal transport to cell bodies, resulting in transgene expression in projection neurons. After stereotaxic injection of Cy3-labeled baculovirus vectors into the rat striatum, retrograde axonal transport of the baculovirus vectors was observed along the corticostriatal pathway and nigrostriatal pathway. Furthermore, after intra-vitreous body injection, anterograde axonal transport and transsynaptic transport of the virus particles were observed in defined connections of the visual system, from the retina to the optic nerve, the lateral geniculate body, the superior colliculus, and the primary visual cortex. PCR analysis confirmed the existence of transported viral DNA in the tissue samples collected from projection fields. Driven by a neuron-specific promoter, transgene expression from the recombinant baculovirus vectors was detectable in target regions remote from injection sites. The attributes of baculovirus vectors in the bidirectional axonal transport and transneuronal transport in neural circuits of the central nervous system could be utilized for targeted gene delivery.
It is very important to determine if recombinant adenoviral vector (Ad) can damage the auditory hair cells in guinea pig cochlea after transgene expression. In this study, the scanning electron microscope was used to determine if there was loss of the auditory hair cells after Ad.LacZ (Ad5 containing Escherichia coli galactosidase) was inoculated into the cochlea through the round window membrane. Seven days later all inner and outer hair cells were found to express the LacZ gene. Except for the sparse loss of outer hair cells in the basal turn and the second turn, there was insignificant loss in the other turns. All inner hair cells were present. The damage to auditory hair cells resulting from intracochlear inoculation of Ad is limited, and this vector can be used as one of the ideal delivery tools in gene therapy of the cochlea.
Studies on bladder cancer cell lines have shown that low adenoviral (Ad) infectivity is associated with low-level coxsackie adenovirus receptor (CAR) expression. Recently, we and others demonstrated a tumor stage- and grade-dependent downregulation of CAR expression in a large series of clinical bladder cancer specimens. Here, we demonstrate adenoviral gene transfer can be markedly enhanced in bladder cancer cells by upregulation of CAR through the use of certain differentiating agents, including the histone deacetylase inhibitors (HDACI) trichostatin A and sodium phenylbutyrate. CAR upregulation to supraphysiologic levels was demonstrated by quantitative rt-PCR, Western blotting, flow cytometry and adenoviral gene transfer. Normal urothelial cells and CAR-positive papilloma cells (RT4) failed to demonstrate upregulation under the same conditions. Upregulation was cell cycle dependent, associated with increased adenoviral gene transfer and persisted for at least 7 days after a single treatment. Such upregulation, however, appears to be tumor cell specific, as other CAR-negative cell lines failed to demonstrate enhanced adenoviral gene transfer with the same treatments. These results provide a rational basis for combining HDACI therapy with gene therapy as a method of augmenting activity in bladder cancer, but this strategy may not be universally applicable to other cell types.