Trends and changing roles in emergency medicine.
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Economic Competition↗
Biomedical subjects
B Shen
Publications and source records attributed to B Shen.
[The application of microwave irradiation to derivatization].
The methods using microwave irradiation for rapid preparation of acetyl, trifluoroacetyl, pentafluoropyl, heptafluorobutyl and MSTFA derivatives of Morphine, AMP and MAMP were developed. Conventional techniques for the reaction mixture to be heated need 30 min at 60-70 degrees C to form derivatizations, but by microwave irradiation, only 2-3 min is needed. The mass spectral fragmentation patterns and gas chromatographic retention times of the derivatives obtained by both microwave irradiation and conventional heating are similar.
Acetylation↗
[Transfer and expression of rh-SCF gene in human hematopoietic stem/progenitor cells].
OBJECTIVE: To explore the transfer and expression of recombinant human stem cell factor (rh-SCF) gene in human hematopoietic stem/progenitor cell. METHODS: pLXSN-SCF, a recombinant retrovirus vector of soluble human SCF gene, was constructed by recombinant gene technique. The recombinant plasmid was introduced into virus packaging cells psi2 and PA317 by lipofectin-mediated DNA transfection, and PA317/SCF, the recombinant virus producing cell, was obtained by G418 screening [the virus potency was (2.4-8.5) x 10(5) CFU/ml]. Then, human hematopoietic stem/progenitor cells were transfected with PA317/SCF. The transfer and expression of human SCF gene were assayed by PCR, APAAP immunohistochemical staining and chemoluminance-direct ELISA. RESULTS AND CONCLUSION: retrovirus-mediated rh-SCF gene was successfully transferred and expressed in human hematopoietic stem/progenitor cells.
Animals↗
[The role of MPO mRNA, IgH mRNA and CD3epsilon mRNA expressions in classification of acute leukemias].
OBJECTIVE: To compare the role of genotypic and immunophenotypic markers in classification of acute leukemia. METHODS: Expression of myeloperoxidase (MPO), CD3epsilon chain and immunoglobulin heavy chain (IgH) gene in 69 cases of acute leukemia were studied by in situ hybridization with streptavidin-gold (ISH-SAG). RESULTS AND CONCLUSION: MPO mRNA and CD3epsilon mRNA expressions were specific for early diagnosis of AML and T-ALL, respectively; and IgH mRNA expression did for early B cell leukemia. Combination of genotypic and immunophenotypic markers assured accurate lineage diagnosis of leukemias.
CD3 Complex↗
[Studies of bone marrow T lymphocytes in patients with aplastic anemia].
OBJECTIVE: To study the role of immunological factors in the pathogenesis of aplastic anemia(AA). METHODS: Phenotype of T lymphocytes in bone marrow and CFU-GM yields of bone marrow mononuclear cells with or without T cells were assayed with APAAP and semi-solid culture method, respectively. RESULTS: The percentage of lymphocytes in bone marrow of AA patients was higher than that of normal controls, and the lymphocytes were mainly CD8 and CD25 positive cells. Changes of T lymphocyte subsets in peripheral blood of AA patients did not completely reflect the changes in the bone marrow. In some AA patients, increased percentage of CD8+ cells was found in bone marrow, while it was normal in peripheral blood. The CFU-GM yields of bone marrow cells of AA patients were much lower than that of normal controls, and were markedly improved by removing T cells before culture. CONCLUSION: The abnormalities of cell-mediated immune response played important roles in the pathogenesis of AA. The changes of T lymphocytes in bone marrow were more meaningful than that in peripheral blood. It is suggested that bone marrow T lymphocyte and its subsets should be routinely examined in all of the AA patients diagnosed so as to assess the indication and predict the effectiveness of immunosuppresive therapy.
Adolescent↗
[Analysis and assessment of meperidine and normeperidine concentrations in urine].
In this study, urine concentrations of meperidine and normeperidine after a single therapeutic dose of meperidine in 5 healthy volunteers have been measured and compared the results to those in meperidine addicts. The results showed that there was a significant difference between two groups in the ratio of metabolite to parent drug. If can be concluded that the ratio should be aid in making a cause of meperidine injection.
Adult↗
Mechanism of tracking and cleavage of adduct-damaged DNA substrates by the mammalian 5'- to 3'-exonuclease/endonuclease RAD2 homologue 1 or flap endonuclease 1.
The mammalian 5'- to 3'-exonuclease/endonuclease, called RAD2 homologue 1 or flap endonuclease 1, has a unique cleavage activity, dependent on specific substrate structure. On a primer-template, in which the primer has an unannealed 5'-tail, endonucleolytic cleavage near the annealing point releases the tail intact. Entering at the 5'-end, the nuclease tracks along the entire tail to the point of cleavage. Genetic analyses suggest that this nuclease removes DNA adducts in vivo (Sommers, C. H., Miller, E. J., Dujon, B., Prakash, S., and Prakash, L. (1995) J. Biol. Chem. 270, 4193-4196). Micrococcal nuclease footprinting shows that after tracking the nuclease protects a region of the tail 25 nucleotides long, adjacent to the cleavage site. Substrates with adducts at specific locations were used to assess the mechanism of RAD2 homologue 1 nuclease tracking and its ability to cleave modified DNA. Either a conventional cis-diamminedichloroplatinum (II) (CDDP) or a bulky CDDP derivative was placed within or beyond the region protected by the nuclease. The nuclease cleaved the tail of both substrates. In contrast, a CDDP adduct just adjacent to the expected cleavage point was inhibitory. A CDDP adduct at the very 5'-end of the tail was also cleaved. The nuclease could remove tails containing adducts on the sugar-phosphate backbone. Apparently, the nuclease is designed to slide over various types of damage on single stranded DNA and then cut past the damaged site.
Animals↗
Identification and characterization of an eight-cysteine repeat of the latent transforming growth factor-beta binding protein-1 that mediates bonding to the latent transforming growth factor-beta1.
Most cultured cell types secrete small latent transforming growth factor-beta (TGF-beta) as a disulfide-bonded complex with a member of the latent TGF-beta binding protein (LTBP) family. Using the baculovirus expression system, we have mapped the domain of LTBP-1 mediating covalent association with small latent TGF-beta1. Coexpression in Sf9 cells of small latent TGF-beta1 with deletion mutants of LTBP-1 showed that the third eight-cysteine repeat of LTBP-1 is necessary and sufficient for covalent interaction with small latent TGF-beta1. Analysis by mass spectrometry of this eight-cysteine repeat, produced as a recombinant peptide in Sf9 cells, confirmed that it was N-glycosylated, as expected from the primary sequence. No other post-translational modifications of this domain were detected. Alkylation of the recombinant peptide with vinyl pyridine failed to reveal any free cysteines, indicating that, in the absence of small latent TGF-beta, the eight cysteines of this domain are engaged in intramolecular bonds. These data demonstrate that the third LTBP-1 eight-cysteine repeat recognizes and associates covalently with small latent TGF-beta1 through a mechanism that does not require any specific post-translational modification of this domain. They also suggest that this domain adopts different conformations depending on whether it is free or bound to small latent TGF-beta.
Animals↗
Human RAD2 homolog 1 5'- to 3'-exo/endonuclease can efficiently excise a displaced DNA fragment containing a 5'-terminal abasic lesion by endonuclease activity.
Repair of abasic lesions, one of the most common types of damage found in DNA, is crucial to an organism's well-being. Studies in vitro indicate that after apurinic-apyrimidinic endonuclease cleaves immediately upstream of a baseless site, removal of the 5'-terminal sugar-phosphate residue is achieved by deoxyribophosphodiesterase activity, an enzyme-mediated beta-elimination reaction, or by endonucleolytic cleavage downstream of the baseless sugar. Synthesis and ligation complete repair. Eukaryotic RAD2 homolog 1 (RTH1) nuclease, by genetic and biochemical evidence, is involved in repair of modified DNA. Efficient endonucleolytic cleavage by RTH1 nuclease has been demonstrated for annealed primers that have unannealed 5'-tails. In vivo, such substrate structures could result from repair-related strand displacement synthesis. Using 5'-tailed substrates, we examined the ability of human RTH1 nuclease to efficiently remove 5'-terminal abasic residues. A series of upstream primers were used to increasingly displace an otherwise annealed downstream primer containing a 5'-terminal deoxyribose-5-phosphate. Until displacement of the first annealed nucleotide, substrates resisted cleavage. With further displacement, efficient cleavage occurred at the 3'-end of the tail. Therefore, in combination with strand displacement activity, RTH1 nucleases may serve as an important alternative to other pathways in repair of abasic sites in DNA.
DNA↗
Kinetic analysis of human flap endonuclease-1 by flow cytometry.
Human flap endonuclease-1 (FEN-1) is a structure-specific endonuclease and exonuclease which is essential for DNA replication and repair. We have cloned a human FEN-1 gene, overexpressed it in Escherichia coli, purified the recombinant protein to near homogeneity, and characterized its cleavage of a flap DNA structure using a novel analytical approach based on flow cytometry. With this approach, we were able to measure continuously the kinetics of DNA cleavage by FEN-1 and to separate experimentally the binding and catalysis functions of the enzyme. When the reaction was initiated by the addition of FEN-1, the cleavage kinetics were dependent on enzyme concentration and appeared to saturate at high concentrations. When enzyme and substrate were preincubated in the presence of EDTA and the reaction initiated by the addition of Mg2+, rapid kinetic flow cytometry measurements showed that cleavage is fast (t1/2 approximately 6 s, k = 0.10 s-1). Using the single-turnover kinetics as a measure of the amount of enzyme-substrate complex present, we estimated the Kd for the FEN-1-flap DNA substrate to be 7.5 nM in the absence of Mg2+ and the rate constant for dissociation of the enzyme-substrate complex to be 0.07 s-1. Computer fitting of the experimental data to a kinetic model confirms these estimates for the individual steps and suggests some interesting features of enzymology using a surface-bound substrate.
Base Sequence↗
Deciphering the mechanism for the assembly of aromatic polyketides by a bacterial polyketide synthase.
Aromatic polyketides are assembled by a type 11 (iterative) polyketide synthase (PKS) in bacteria. Understanding the enzymology of such enzymes should provide the information needed for the synthesis of novel polyketides through the genetic engineering of PKSs. Using a previously described cell-free system [B.S. & C.R.H. (1993) Science 262, 1535-1540], we studied a PKS enzyme whose substrate is not directly available and purified the TcmN polyketide cyclase from Streptomyces glaucescens. TcmN is a bifunctional protein that catalyzes the regiospecific cyclization of the Tcm PKS-bound linear decaketide to Tcm F2 and the 0-methylation of Tcm D3 to Tcm B3. In the absence of TcmN, the decaketide formed by the minimal PKS consisting of the TcmJKLM proteins undergoes spontaneous cyclization to form some Tcm F2 as well as SEK15 and many other aberrant shunt products. Addition of purified TcmN to a mixture of the other Tcm PKS components both restores and enhances Tcm F2 production. Interestingly, Tcm F2 but none of the aberrant products was bound tightly to the PKS. The results described support the notion that the polyketide cyclase, not the minimal PKS, dictates the regiospecificity for the cyclization of the linear polyketide intermediate. Furthermore, because the addition of TcmN to the TcmJKLM proteins results in a significant increase of the total yield of decaketide, interactions among the individual components of the Tcm PKS complex must give rise to the optimal PKS activity.
Bacterial Proteins↗
Essential amino acids for substrate binding and catalysis of human flap endonuclease 1.
Human flap endonuclease 1 (FEN-1) is a member of the structure-specific endonuclease family and is involved in DNA repair. Eight restrictively conserved amino acids in FEN-1 have been converted individually to an alanine to elucidate their roles in specific DNA substrate binding and catalysis. Flap endonuclease activity of the wild type and mutant enzymes was measured by kinetic flow cytometry. Mutants D34A, D86A, and D181A lost their cleavage activity completely but retained substrate binding ability, as measured by their ability to inhibit the wild type enzyme in a competition assay. This indicates that these amino acids contribute to integrity of the enzyme active site. Loss of both binding and cleavage competency for the flap substrate by mutants E156A, G231A, and D233A suggests that these amino acids are involved in substrate binding. Mutants R103A and D179A retained wild type-like enzyme activity.
Amino Acid Sequence↗
Radioprotective effects of antioxidative plant flavonoids in mice.
Radioprotective effects of tea infusions and plant flavonoids were investigated by using the micronucleus test for anticlastogenic activity and the thiobarbituric acid assay for antioxidative activity. A single gastric intubation of rooibos tea (Aspalathus linearis) infusion at 1 ml per mouse 2 h prior to gama-ray irradiation (1.5 Gy) reduced the frequency of micronucleated reticulocytes (MNRETs). After the fractionation of rooibos tea infusion, the flavonoid fraction was found to be most anticlastogenic and antioxidative. From this fraction, luteolin was isolated as an effective component. Then, anticlastogenic effects of 12 flavonoids containing luteolin and their antioxidative activities against lipid peroxidation by Fenton's reagent were examined. A good correlation (r=0.717) was observed between both activities. Luteolin showed the most effective potency. A gastric intubation of luteolin (10 micromoles/kg) 2 h prior to gamma-ray irradiation (6 Gy) suppressed lipid peroxidation in mouse bone marrow and spleen and a trend of protective effect of luteolin against the decrease of endogenous ascorbic acid in mouse bone marrow after gamma-ray irradiation (3 Gy) was observed. These results suggest that plant flavonoids, which show antioxidative potency in vitro, work as antioxidants in vivo and their radioprotective effects may be attributed to their scavenging potency towards free radicals such as hydroxyl radicals. Therefore, the flavonoids contained in tea, vegetables and fruits seem to be important as antioxidants in the human diet.
Animals↗
Modeling the spontaneous reactivation of experience-specific hippocampal cell assembles during sleep.
During slow-wave sleep (SWS) following periods of spatial activity, hippocampal place cells that were temporally correlated, by virtue of the overlap of their place fields, exhibit enhanced temporal correlations, even though the animal sleeps in a different location (Wilson and McNaughton [1994] Science 267:676-679). The discharge of cells with overlapped place fields is more correlated in subsequent sleep, particularly during sharp waves, than in sleep episodes prior to the behavior, or than cell pairs with non-overlapped place fields. The reactivated correlated states appear during hippocampal sharp waves (SPWs), and are weak or absent in the inter-SPW interval. A simple conceptual hypothesis for this phenomenon is developed, based on the idea that hippocampal place fields reflect a two-dimensional distribution of continuously overlapping dynamic attractors in which each location is represented by the self-sustaining activity of a small subset of neurons with overlapping place fields. A numerical simulation of this hypothesis, based on a simplified representation of the CA8 recurrent network, accounts qualitatively for the main observations, including SPW-like dynamics. It is shown that, under conditions in which the connection patterns have been previously established, either associative or nonassociative mechanisms might underlie the reactivation of recently experienced states. These two alternatives appear, under at least some conditions (e.g., sparse coding), to be indistinguishable.
Animals↗
Partial hippocampal kindling affects retention but not acquisition and place but not cue tasks on the radial arm maze.
The performance of rats that were partially kindled in the hippocampus was assessed on an 8-arm radial arm maze with 4 baited arms. In rats first trained and then kindled, deficits were found on a place task in which rats reached the goal arms of the maze using salient extramaze spatial cues, but not on an intramaze cue task in which rats reached the goal arms using salient intramaze cues. Acquisition of a new place task on the maze was not different between kindled and control rats. In conclusion, partial hippocampal kindling disrupted the retention but not the acquisition of a spatial or place task; retention of a nonspatial cue task was not disrupted.
Animals↗
The DNA-binding and enhancer-blocking domains of the Drosophila suppressor of Hairy-wing protein.
Mutations in the suppressor of Hairy-wing [su(Hw)] gene of Drosophila melanogaster can cause female sterility and suppress mutations that are insertions of the gypsy retrotransposon. Gypsy binds the protein (SUHW) encoded by su(Hw), and SUHW prevents enhancers promoter-distal to gypsy from activating gene transcription. SUHW contains 12 zinc fingers flanked by acidic N- and C-terminal domains. We examined the roles of each of the 12 zinc fingers in binding gypsy DNA and classified them into four groups: essential (fingers 6 through 10); beneficial but nonessential (fingers 1, 2, 3, and 11); unimportant (fingers 5 and 12); and inhibitory (finger 4). Because finger 10 is not required for female fertility but is essential for binding gypsy, these results imply that the SUHW-binding sites required for oogenesis differ in sequence from the gypsy-binding sites. We also examined the functions of the N- and C-terminal domains of SUHW by determining the ability of various deletion mutants to support female fertility and to alter expression of gypsy insertion alleles of the yellow, cut, forked, and Ultrabithorax genes. No individual segment of the N- and C-terminal domains of SUHW is absolutely required to alter expression of gypsy insertion alleles. However, the most important domain lies between residues 737 and 880 in the C-terminal domain. This region also contains the residues required for female fertility, and the fertility domain may be congruent with the enhancer-blocking domain. These results imply that SUHW blocks different enhancers and supports oogenesis by the same or closely related molecular mechanisms.
Amino Acid Sequence↗
[Granulocyte-macrophage colony-stimulating factor (GM-CSF), preclinical and phase I clinical investigations].
OBJECTIVE: To conduct preclinical studies and phase I trial of the recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). METHODS: Pharmacodynamics, pharmacokinetic and toxicology of the rhGM-CSF were studied in animal models, and the safety was also evaluated in humans. RESULTS: The human bone marrow cells could be stimulated by purified rhGM-CSF to form multilineage colonies (CFU-GM and BFU-E). The rhGM-CSF administered for 7 days to Beagle dogs and monkeys subjected to 60Co r-ray irradiation was shown to induce both rapid and sustained increase in circulating leukocyte counts. Toxicology testing showed that the LD50 (i.v) was over 5000 micrograms/kg, and LD50 (i.p) over 10000 micrograms/kg in mice. Administration of the rhGM-CSF in excess of four times as much as clinical dosages was not associated with severe chronic toxicities. Most injected rhGM-CSF was excreted from urine, and did not accumulate in the body. In the phase I clinical trial, injecting 2.5-7.5 micrograms/day of rhGM-CSF was safe. CONCLUSION: It is effective and safe to use rhGM-CSF in the treatment of leukocytopenia.
Adolescent↗
[Pathogenesis of Guillain-Barre syndrome and its relation with Campylobacter jejuni].
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Antibodies↗