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161.
Here, we describe a new approach for mutational scanning of PCR products through hybridization analysis between complementary oligonucleotides. Sets of overlapping probe oligonucleotides complementary to wild-type (WT) sequence are hybridized to microbead-immobilized PCR products under solution-like conditions. Mismatch-hybridization situations between a mutant sample and probe oligonucleotides result in higher remaining concentrations in solution of involved probe oligonucleotides. Post-hybridization supernatants are subsequently analyzed for their probe oligonucleotide compositions using surface plasmon resonance-based biosensor technology. Relative remaining probe oligonucleotide concentrations are monitored in real-time through hybridization analysis between probe oligonucleotides and their corresponding sensor-chip immobilized complementary counterparts. This allows for the construction of composition diagrams revealing the existence and approximate location of a mutation within an investigated sample DNA sequence. Applied on PCR products derived from clinical samples of microdissected tumor biopsies, single mutations in exons 6 and 7 of the human p53 tumor-suppressor gene were successfully detected and approximately localized. 相似文献
162.
A precultivation technique for microorganisms exhibiting overflow metabolism is presented. It is based on a low initial medium volume in the fermenter. The medium feed contains all the nutrients, but is diluted with respect to sugar, the concentration of which determines the biomass concentration at the end of the preculture. By this method, effects of varying activity of the inocula from shake flask cultures are minimised and the metabolic state, i.e. oxido-reductive, oxidative growth on sugar plus overflow metabolite or oxidative growth on sugar alone, can be controlled at the start of the main fermentation. 相似文献
163.
Pavlenko M Roos AK Leder C Hansson LO Kiessling R Levitskaya E Pisa P 《Cancer immunology, immunotherapy : CII》2004,53(12):1085-1092
The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8+ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines. 相似文献
164.
We have previously shown that protein kinase C (PKC) epsilon induces neurite outgrowth via its regulatory domain. This is accompanied by PKC-induced stress fibre loss. Here, we show that the regulatory domain (RD) of PKCepsilon induces processes also in NIH-3T3 fibroblasts, similar to what has been observed with p190RhoGAP. This study also shows that p190RhoGAP induces neurite outgrowth in SK-N-BE(2) neuroblastoma cells. We therefore investigated whether p190RhoGAP may be downstream of PKCepsilon. We could detect a co-localization of p190RhoGAP and PKCepsilon at membrane ruffles and an increased association between the proteins in fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate (TPA). The association is also seen in neuroblastoma cells, and nerve growth factor (NGF) treatment of SH-SYSY/TrkA cells decreases the interaction. However, overexpressed PKCepsilon did not coprecipitate overexpressed p190RhoGAP in CHO cells, indicating that the proteins do not interact directly. This raises the possibility that p190RhoGAP is involved in mediating the morphological effects of PKCepsilon. 相似文献
165.
Studies on gangliosides with affinity for Helicobacter pylori: binding to natural and chemically modified structures 总被引:1,自引:0,他引:1
Miller-Podraza H Johansson P Angström J Larsson T Longard M Karlsson KA 《Glycobiology》2004,14(3):205-217
Helicobacter pylori, like many other microbes, has the ability to bind to carbohydrate epitopes. Several sugar sequences have been reported as active for the bacterium, including some neutral, sulfated, and sialylated structures. We investigated structural requirements for the sialic acid-dependent binding using a number of natural and chemically modified gangliosides. We have chosen for derivatization studies two kinds of binding-active glycolipids, the simple ganglioside S-3PG (Neu5Ac alpha 3Gal beta 4GlcNAc beta 3Gal beta 4Glc beta 1Cer, sialylparagloboside) and branched polyglycosylceramides (PGCs) of human origin. The modifications included oxidation of the sialic acid glycerol chain, reduction of the carboxyl group, amidation of the carboxyl group, and lactonization. Binding experiments confirmed a preference of H. pylori for 3-linked sialic acid and penultimate 4-linked galactose. As expected, neolacto gangliosides (with Gal beta 4GlcNAc in the core structure) were active in our assays, whereas gangliosides with lacto (Gal beta 3GlcNAc) and ganglio (Gal beta 3GalNAc) carbohydrate chains were not. Negative binding results were also obtained for disialylparagloboside (with terminal NeuAc alpha 8NeuAc) and NeuAc alpha 6-containing glycolipids. Chemical studies revealed dependence of the binding on Neu5Ac and its glycerol and carboxyl side chains. Most of the derivatizations performed on these groups abolished the binding; however, some of the amide forms turned out to be active, and one of them (octadecylamide) was found to be an excellent binder. The combined data from molecular dynamics simulations indicate that the binding-active configuration of the terminal disaccharide of S-3PG is with the sialic acid in the anticlinal conformation, whereas in branched PGCs the same structural element most likely assumes the synclinal presentation. 相似文献
166.
167.
Wahlund PO Gustavsson PE Izumrudov VA Larsson PO Galaev IY 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,807(1):121-127
The demand for highly purified plasmids in gene therapy and plasmid-based vaccines requires large-scale production of pharmaceutical-grade plasmid. Plasmid DNA was selectively precipitated from a clarified alkaline lysate using the polycation poly(N,N'-dimethyldiallylammonium) chloride which formed insoluble polyelectrolyte complex (PEC) with the plasmid DNA. Soluble PECs of DNA with polycations have earlier been used for cell transformation, but now the focus has been on insoluble PECs. Both DNA and RNA form stable PECs with synthetic polycations. However, it was possible to find a range of salt concentration where plasmid DNA was quantitatively precipitated whereas RNA remained in solution. The precipitated plasmid DNA was resolubilised at high salt concentration and the polycation was removed by gel-filtration. 相似文献
168.
Otterstedt K Larsson C Bill RM Ståhlberg A Boles E Hohmann S Gustafsson L 《EMBO reports》2004,5(5):532-537
The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. 相似文献
169.
White matter injury is the most frequently observed brain lesion in preterm infants. The etiology remains unclear, however, both cerebral hypoperfusion and intrauterine infections have been suggested as risk factors. We compared the neuropathological outcome, including the effect on oligodendrocytes, astrocytes, and microglia, following either systemic asphyxia or endotoxemia in fetal sheep at midgestation. Fetal sheep were subjected to either 25 minutes of umbilical cord occlusion or systemic endotoxemia by administration of Escherichia coli lipopolysaccharide (LPS O111:B4, 100 ng/kg, IV). Periventricular white matter lesions were observed in 2 of 6 asphyxiated fetuses, whereas the remaining animals showed diffuse injury throughout the subcortical white matter and neuronal necrosis in subcortical regions, including the striatum and hippocampus. LPS-treatment resulted in focal inflammatory infiltrates and cystic lesions in periventricular white matter in 2 of 5 animals, but with no neuron specific injury. Both experimental paradigms resulted in microglia activation in the white matter, damaged astrocytes, and loss of oligodendrocytes. These results show that the white matter at midgestation is sensitive to injury following both systemic asphyxia and endotoxemia. Asphyxia induced lesions in both white and subcortical grey matter in association with microglia activation, and endotoxemia resulted in selective white matter damage and inflammation. 相似文献
170.
Marchesan D Rutberg M Andersson L Asp L Larsson T Borén J Johansson BR Olofsson SO 《The Journal of biological chemistry》2003,278(29):27293-27300
We developed a microsome-based, cell-free system that assembles newly formed triglyceride (TG) into spherical lipid droplets. These droplets were recovered in the d = 1.055 g/ml fraction by gradient ultracentrifugation and were similar in size and appearance to those isolated from rat adipocytes and 3T3-L1 cells. Caveolin 1 and 2, vimentin, adipocyte differentiation-related protein, and the 78-kDa glucose regulatory protein were identified on the droplets from the cell-free system. The caveolin was soluble in 1% Triton X-100, as was the caveolin on lipid droplets from 3T3-L1 cells. The lipid droplets from the cell-free system, like those from 3T3-L1 cells, contained TG, diacylglycerol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The assembly of these TG-containing structures was dependent on the rate of TG biosynthesis and required an activator present in the 160,000 x g supernatant from homogenized rat adipocytes. The activator induced phospholipase D (PLD) activity, and its effect on the release of the TG-containing structures from the microsomes was inhibited by 1-butanol (but not 2-butanol) or 2,3-diphosphoglycerate. The activator could be replaced by a constitutively active PLD or phosphatidic acid. These results indicate that PLD and the formation of phosphatidic acid are important in the assembly of the TG-containing structures. 相似文献