Biomedical subjects
Publications and source records attributed to Ping Shen.
A carbapenem-resistant isolate of Klebsiella pneumoniae producing class A carbapenemase KPC-2 was identified in Zhejiang, China. The KPC-2 gene was located on an approximately 60-kb plasmid in a genetic environment partially different from that of blaKPC-2 in the isolates from the United States and Colombia.
A novel transformation system, in which neither a nonphysiological concentration of Ca2+ and temperature shifts nor electronic shocks were required, was developed to determine whether Escherichia coli is naturally transformable. In the new protocol, E. coli was cultured normally to the stationary phase and then cultured statically at 37 degrees C in Luria-Bertani broth. After static culture, transformation occurred in bacteria spread on Luria-Bertani plates. The protein synthesis inhibitor chloramphenicol inhibited this transformation process. The need for protein synthesis in plated bacteria suggests that the transformation of E. coli in this new system is regulated physiologically.
Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.
Autofluorescence has an advantage over the extrinsic fluorescence of an unperturbed environment during investigation, especially in complex system such as biological cells and tissues. NADH is an important fluorescent substance in living cells. The time courses of intracellular NADH autofluorescence in the process of yeast cells exposed to H(2)O(2) and ONOO(-) have been recorded in detail in this work. In the presence of different amounts of H(2)O(2) and ONOO(-), necrosis, apoptosis and reversible injury are initiated in yeast cells, which are confirmed by acridine orange/ethidum bromide and Annexin V/propidium iodide staining. It is found that intracellular NADH content increases momently in the beginning of the apoptotic process and then decreases continually till the cell dies. The most remarkable difference between the apoptotic and the necrotic process is that the NADH content in the latter case changes much more sharply. Further in the case of reversible injury, the time course of intracellular NADH content is completely different from the above two pathways of cell death. It just decreases to some degree firstly and then resumes to the original level. Based on the role of NADH in mitochondrial respiratory chain, the time course of intracellular NADH content is believed to have reflected the response of mitochondrial redox state to oxidative stress. Thus, it is found that the mitochondrial redox state changes differently in different pathways of oxidative injury in yeast cells.
A gene encoding an extracellular protease, sptA, was cloned from the halophilic archaeon Natrinema sp. J7. It encoded a polypeptide of 565 amino acids containing a putative 49-amino acid signal peptide, a 103-amino acid propeptide, as well as a mature region and C-terminal extension, with a high proportion of acidic amino acid residues. The sptA gene was expressed in Haloferax volcanii WFD11, and the recombinant enzyme could be secreted into the medium as an active mature form. The N-terminal amino acid sequencing and MALDI-TOF mass spectrometry analysis of the purified SptA protease indicated that the 152-amino acid prepropeptide was cleaved and the C-terminal extension was not processed after secretion. The SptA protease was optimally active at 50 degrees C in 2.5 M NaCl at pH 8.0. The NaCl removed enzyme retained 20% of its activity, and 60% of the activity could be restored by reintroducing 2.5 M NaCl into the NaCl removed enzyme. When the twin-arginine motif in the signal peptide of SptA protease was replaced with a twin-lysine motif, the enzyme was not exported from Hfx. volcanii WFD11, suggesting that the SptA protease was a Tat-dependent substrate.
A mediatorless microbial fuel cell based on the direct biocatalysis of Escherichia coli shows significantly enhanced performance by using bacteria electrochemically-evolved in fuel cell environments through a natural selection process and a carbon/PTFE composite anode with an optimized PTFE content.
OBJECTIVE: To study the role of herpes simplex virus type 1 ( HSV-1 ) in facial paralysis by developing an experimental animal model of viral facial paralysis. METHODS: Both sides of posterior auricular branch of facial nerve were anatomies and incised in 66 mice. The HSV-1 was inoculated into right ear branch and fetal bovine serum was inoculated into left ear branch as control. The symmetry of mouse face was observed and scored. The temporal bones were serially sectioned and stained with hematoxylin and eosin. The extratemporal facial nerves were stained with osmium tetroxide. HSV-1 DNA in bilateral facial nerve, brain stem, trigeminal ganglion and spinal cord was detected by the polymerase chain reaction. RESULTS: Twenty-eight (42. 42%) mice developed right facial paralysis between 2 and 5 days after inoculation. Continuing 3-6 days, the facial paralysis recovered spontaneously. Thirty-eight mice had no signs of facial paralysis. Compared with the left, nerve swelling, inflammatory cell infiltration were manifested in right temporal facial nerve of paralyzed mice. The ratio of the cross-sectional area of the facial nerve to the facial canal ( FN/FC ) was significantly higher than that on the control side (P < 0.01). Demyelinated nerve fibers were seen in the right extratemporal facial nerve. Not only in paralyzed mice, but also in non-paralyzed mice, HSV DNA was detected in some nerve tissues. CONCLUSIONS: Inoculating HSV-1 into posterior auricular branch of facial nerve can produce an acute and transient facial paralysis in mice. The possible pathophysiologic mechanism of the facial paralysis is viral invasion and transportation from distal branch to main trunk. Then the viral facial neuritis causes facial paralysis.
The transcriptional product in Halobacterium halobium R1 similar to eukaryotic gene rad25 was analyzed by RT-PCR. Using bgaH as the reporter genes, the promoter function of eukaryotic rad25-like DNA fragment in halophilic archaea was investigated by promoter probe analysis. The important functional regions, which could influence the promoter activity of rad25-like gene, were identified by deletion analysis of the promoter sequence in Haloferax volcanii. It is found that the DNA fragment of promoter similar to eukaryotic gene rad25 contains the typical characteristic sequence of archaeal promoter. These results indicate that rad25-like gene in Halobacterium halobium R1 is active and may play a role on the NER pathway as the eukaryotic pattern.
RM07 and RM13 DNA fragments could function as promoter in Escherichia coli, which were isolated from an archaeon Halobacterium halobium R1. In the present study, promoter activities of these two fragments were confirmed by beta-galactosidase activity analysis and microcalorimetric studies. They were cloned into promoter-probe vector pYLZ-2 respectively. Four recombinant strains TE07, TE07-2, TE131 and TE132 were obtained, and all fragments were found to be active in E. coli DH5alpha. The beta-galactosidase activity of TE132 was higher than that of TE07-2. Both TE07 and TE131 had weak beta-galactosidase activity. Then the heat output of E. coli DH5alpha and its transformants had been detected by a microcalorimetric method at 37 degrees C. Compared with E. coli DH5alpha, the growth rate constant of E. coli T2 (pYLZ-2), TE07, TE07-2, TE131 and TE132 strain was reduced 6.5%, 11%, 41.1%, 47.5% and 42.7% respectively. When IPTG was added to LB medium, beta-galactosidase activity and heat output had been enhanced slightly in all strains. The results suggested that there was close correspondence between promoter activity and microcalorimetric results, and the heat output of growth was mainly affected by gene expression in E. coli. The higher beta-galactosidase activity of E. coli was, the lower its growth rate constant was. At the meantime, Microcalorimetric studies implied that 700bps of RM13 ( RM131) fragment would have stronger promoter activity than RM13. Microcalorimetry may be used as a new approach for analyzing the regulation of foreign gene expression.
Influence of temperature, pH and organics on ANAMMOX activity was investigated through measuring the anaerobic ammonia oxidation rate of sludge. Results show that temperature and pH could influence ANAMMOX activity dramatically. The optimal temperature was between 30-35 degrees C , and the relationship between the anaerobic ammonia oxidation rate and temperature can be described by revised Arrhenius formula during 20-30 degreee C. The optimal pH was between 7.5 - 8.3, and the relationship between the anaerobic ammonia oxidation rate and pH can be described by two substrates and two inhibitors model during pH 7.0 - 9.0. There was substrata competition between the heterotrophic denitrification bacteria and ANAMMOX bacteria when organics in the
Strain sp-f was isolated, a siderophores over producing bacterium, using an improved universal Chrome Azurol S(CAS)-agar plate method from Donghu Lake. The result of the CAS solution siderophores quantitative determination showed the lowest As/Ar (OD680) ratio could be as low as 0.09 with Su (Siderophore Unit) of 90%. Some more experiments were made to make out the pertinence between its growth and siderophores production, indicating that its siderophores quantity reached maximum amount during the prophase of logarithmic growth. After then, siderophores concentration stopped accumulating and turned to be stable at stationary phase. Based on the characteristics of morphology, cultivation, physiology, (G + C) mol % content, 16S rDNA sequence and BIOLOG Station system analysis, it was identified as Pseudomonas fluorescens sp-f strain. RP-HPLC analysis showed there exist at least 3 kinds of catecholate siderophores, including fluorescent and non-fluorescent pyoverdins. But only fluorescent pyoverdin's excretion was completely repressed by the 200 micromol/L Fe2+ in the medium. And the non-pyoverdin siderophores excretion was induced at the same time, contrarily.
To identify the functional contributions of cooperations among transcription factors on regulatory DNA is critical for understanding transcription activation. But so far there is a great lack of effective identifying methods. Here we describe a novel strategy, based on comprehensively perturbed experiments and a computational model, to identify the cooperations among NF-kappaB (p65), CREB, and AP-1 in transcription activation of human cytomegalovirus major IE1 promoter/enhancer (MIEP). In this strategy, functional profiles of protein-MIEP association and RNA synthesis are achieved through comprehensively perturbing the association of p65, CREB or AP-1 with MIEP and then subjected to the computational model. Consequently, the 'real' cooperations contributing to MIEP activation are found to comprise five but not seven types of potential cooperations. Thus, our research provides a facile systematic approach to identifying the DNA-organized cooperations among transcription factors and understanding transcription activation.
Omnivores, including humans, have an inborn tendency to avoid noxious or unfamiliar foods. Such defensive foraging behaviors are modifiable, however, in response to physiological needs. Here we describe a method for assessing risk-sensitive food acquisition in Drosophila melanogaster. Food-deprived fly larvae become more likely to feed on noxious foods (adulterated with quinine) as the duration of deprivation increases. The neuropeptide F receptor NPFR1, a mammalian neuropeptide Y (NPY) receptor homolog, centrally regulates the response to noxious food in D. melanogaster. Overexpression of NPFR1 was sufficient to cause nondeprived larvae to more readily take in noxious food, whereas loss of NPFR1 signaling led to the opposite phenotype. Moreover, NPFR1 neuronal activity may be directly regulated by the insulin-like signaling pathway. Upregulation of insulin-like receptor signaling in NPFR1 cells suppressed the feeding response to noxious food. Our results suggest that the coordinated activities of the conserved NPY- and insulin-like receptor signaling systems are essential for the dynamic regulation of noxious food intake according to the animal's energy state.
Hunger elicits diverse, yet coordinated, adaptive responses across species, but the underlying signaling mechanism remains poorly understood. Here, we report on the function and mechanism of the Drosophila insulin-like system in the central regulation of different hunger-driven behaviors. We found that overexpression of Drosophila insulin-like peptides (DILPs) in the nervous system of fasted larvae suppressed the hunger-driven increase of ingestion rate and intake of nonpreferred foods (e.g., a less accessible solid food). Moreover, up-regulation of Drosophila p70/S6 kinase activity in DILP neurons led to attenuated hunger response by fasted larvae, whereas its down-regulation triggered fed larvae to display motivated foraging and feeding. Finally, we provide evidence that neural regulation of food preference but not ingestion rate may involve direct signaling by DILPs to neurons expressing neuropeptide F receptor 1, a receptor for neuropeptide Y-like neuropeptide F. Our study reveals a prominent role of neural Drosophila p70/S6 kinase in the modulation of hunger response by insulin-like and neuropeptide Y-like signaling pathways.
Using water/AOT/n-octane reversed micelle as the medium, the optical signal of the reactive intermediate of laccase-catalyzed oxidation of o-phenylenediamine, which was indetectable in aqueous solutions, was successfully captured. Thus online kinetic studies of the intermediate were accomplished. Two-way kinetic spectral data were acquired with stopped-flow technique. By resolving the data with global analysis software, both the kinetic curves and the absorption spectra of the components involved in the reaction process were simultaneously obtained. The whole reaction in the reversed micelle was proved to be composed of two successive steps, an enzymatic generation of the intermediate and a following nonenzymatic decay of the intermediate. A consecutive first-order kinetic model of the whole reaction was confirmed. The influences of microenvironmental factors of the medium (such as the pH value of the water pool and the water/AOT ratio) on the detection of the intermediate were also investigated.
Alcohol is likely to affect neurons nonselectively, and the understanding of its action in the CNS requires elucidation of underlying neuronal circuits and associated cellular processes. We have identified a Drosophila signaling system, comprising neurons expressing neuropeptide F (NPF, a homolog of mammalian neuropeptide Y) and its receptor, NPFR1, that acutely mediates sensitivity to ethanol sedation. Flies deficient in NPF/NPFR1 signaling showed decreased alcohol sensitivity, whereas those overexpressing NPF exhibited the opposite phenotype. Furthermore, controlled functional disruption of NPF or NPFR1 neurons in adults rapidly confers resistance to ethanol sedation. Finally, the NPF/NPFR1 system selectively mediates sedation by ethanol vapor but not diethyl ether, indicating that the observed NPF/NPFR1 activity reflects a specialized response to alcohol sedation rather than a general response to intoxication by sedative agents. Together, our results provide the molecular and neural basis for the strikingly similar alcohol-responsive behaviors between flies and mammals.
Microcalorimetric method was successfully used to study the effect of nucleotide mutations on promoter activity and identify the important nucleotide necessary for the promoter function in Escherichia coli. The thermokinetic parameters, such as k, I and IC(50), were calculated from the metabolic power-time curves obtained by microcalorimetric measurement using the TAM air Isothermal Microcalorimeter (manufactured by Thermometric AB company of Sweden). Analysis of these data revealed that different nucleotide mutations in -10 box sequence of RM07 fragment had different effect on the promoter activity. Our research also suggest that the microcalorimetric method is a very sensitive and easily performed method for investigation of promoter mutation.
Estrogens maintain female sexual health. The hormone also drives the growth of estrogen receptor (ER) positive breast tumors, and ER modulators, like tamoxifen, are used to reduce tumor recurrence. To identify phytoestrogens with possible health benefits, we screened several Traditional Chinese Medicines and encountered an extract from the leaves of Epimedium brevicornum (EB), with strong (EC50: 1.3 microg/mL) and specific ER-stimulatory activity. It increased estrogen-responsive human breast cancer cell proliferation at low doses, but paradoxically caused profound inhibition of growth at higher doses. Using bioassay-guided fractionation, we isolated and characterized a new prenylflavone, breviflavone B, which exerted biphasic stimulatory and inhibitory effects on breast cancer cell proliferation, mimicking the effects of EB. In contrast to estradiol and genistein, high doses (> 2 microM) of breviflavone B almost eliminated ERalpha protein; a process that may be mediated through increased proteasome degradation. Pre-clinical studies are needed to explore whether these prenylflavones are of value in estrogen-deficiency states and for prophylaxis of breast cancer.