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101.
102.
A simple protocol for transfecting human mesenchymal stem cells 总被引:1,自引:0,他引:1
Talita Giacomet de Carvalho Felipe Matheus Pellenz Alvaro Laureano Lucia Mariano da Rocha Silla Roberto Giugliani Guilherme Baldo Ursula Matte 《Biotechnology letters》2018,40(3):617-622
Objectives and results
Mesenchymal stromal cells (MSCs) are potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and have significant expansion capability. These characteristics make them strong candidates for delivering genes and restoring organ systems function. However, as other primary cells, MSCs are difficult to transfect. In order to standardize a simple protocol for transfection of MSCs, we conducted a series of experiments and achieved a protocol that does not require the use of viral particles or specific expensive equipment.Conclusion
MSCs transfection at early passages using a ratio lipid/DNA of 3.0 µL/µg with Lipofectamine 3000® yields good transfection efficiencies for human MSCs (up to 26%) and is rapid, simple, and safe.103.
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Marie-Paule Otte Josée Gagnon Yves Comeau Nathalie Matte Charles W. Greer Réjean Smason 《Applied microbiology and biotechnology》1994,40(6):926-932
Soil activation, a concept based on the cultivation of biomass from a fraction of a comtaminated soil for subsequent use as an inoculum for bioaugmentation of the same soil, was studied as a method for the aerobic biodegradation of pentachlorophenol (PCP) and polycyclic hydrocarbons (PAH) in contaminated soils. A microbial consortium able to degrade PCP and PAH in contaminated soil from wood-preserving facilities was isolated and characterized for PCP degradation and resistance. To obtain an active consortium from the contaminated soil in a fed-batch bioreactor, the presence of soil as a support or source of nutrients was found to be essential. During the 35 days of bioreactor operation, residual PCP in solution remained near zero up to a loading rate of 700mg/l per day. The PCP meneralization rate increased from 70 mg/l per day when no PCP was added to the bioreactor to 700 mg/l per day at the maximum loading rate. The consortium tolerated a PCP concentration of 400 mg/l in batch experiments. Production of a PCP-degrading consortium in a fed-batch slurry bioreactor enhanced the activity of PCP biodegradation by a factor of ten. PAH biodegradation increased, during the same time period, by a factor of 30 and 81 for phenanthrene and pyrene, respectively. Preliminary laboratory-scale results indicated that a significant reduction in the time required for degradation of PCP and PAH in contaminated soil could be achieved using activated soil as an inoculum.Issued as NRC 33861
correspondence to: R. Samson 相似文献
106.
Molecular Analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 Strains: Clonal Relationships between Clinical and Environmental Isolates 总被引:6,自引:0,他引:6
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D. V. Singh Maria H. Matte G. R. Matte Sunny Jiang F. Sabeena B. N. Shukla S. C. Sanyal A. Huq R. R. Colwell 《Applied microbiology》2001,67(2):910-921
A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains. 相似文献
107.
Schoenhofen IC Lunin VV Julien JP Li Y Ajamian E Matte A Cygler M Brisson JR Aubry A Logan SM Bhatia S Wakarchuk WW Young NM 《The Journal of biological chemistry》2006,281(13):8907-8916
Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-alpha-D-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-beta-L-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the beta-L-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway. 相似文献
108.
Structural snapshots of Escherichia coli histidinol phosphate phosphatase along the reaction pathway
Rangarajan ES Proteau A Wagner J Hung MN Matte A Cygler M 《The Journal of biological chemistry》2006,281(49):37930-37941
HisB from Escherichia coli is a bifunctional enzyme catalyzing the sixth and eighth steps of l-histidine biosynthesis. The N-terminal domain (HisB-N) possesses histidinol phosphate phosphatase activity, and its crystal structure shows a single domain with fold similarity to the haloacid dehalogenase (HAD) enzyme family. HisB-N forms dimers in the crystal and in solution. The structure shows the presence of a structural Zn(2+) ion stabilizing the conformation of an extended loop. Two metal binding sites were also identified in the active site. Their presence was further confirmed by isothermal titration calorimetry. HisB-N is active in the presence of Mg(2+), Mn(2+), Co(2+), or Zn(2+), but Ca(2+) has an inhibitory effect. We have determined structures of several intermediate states corresponding to snapshots along the reaction pathway, including that of the phosphoaspartate intermediate. A catalytic mechanism, different from that described for other HAD enzymes, is proposed requiring the presence of the second metal ion not found in the active sites of previously characterized HAD enzymes, to complete the second half-reaction. The proposed mechanism is reminiscent of two-Mg(2+) ion catalysis utilized by DNA and RNA polymerases and many nucleases. The structure also provides an explanation for the inhibitory effect of Ca(2+). 相似文献
109.
Ten sheep were used to define the anatomical basis for the feeding systems sensitive to gamma-aminobutyric acid, by using intrahypothalamic microinjections of the gamma-aminobutyric acid agonist, muscimol. In satiated sheep, 1 microL of muscimol (0.5 nmol/microL) elicited feeding when injected into paraventricular, ventromedial, and anterior hypothalamic areas. Similar injections into 39 sites tested in 6-h fasted sheep failed to decrease feed intake. The data suggest that neurons sensitive to gamma-aminobutyric acid in medial hypothalamus may be involved in the initiation of feeding. 相似文献
110.