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81.
Peytavi R Liu-Ying T Raymond FR Boissinot K Bissonnette L Boissinot M Picard FJ Huletsky A Ouellette M Bergeron MG 《BioTechniques》2005,39(1):89-96
The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms. 相似文献
82.
For sperm preservation, semen is generally diluted with extender containing egg yolk (EY), but the mechanisms of sperm protection by EY are unclear. The major proteins of bull seminal plasma (BSP proteins: BSP-A1/A2, BSP-A3, and BSP-30-kDa) bind to sperm surface at ejaculation and stimulate cholesterol and phospholipid efflux from the sperm membrane. Since EY low-density lipoprotein fraction (LDF) interacts specifically with BSP proteins, it is proposed that the sequestration of BSP proteins in seminal plasma by EY-LDF represents the major mechanism of sperm protection by EY. In order to gain further insight into this mechanism, we investigated the effect of seminal plasma, EY, and EY-LDF on the binding of BSP proteins to sperm and the lipid efflux from the sperm membrane. As shown by immunodetection, radioimmunoassays, and lipid analysis, when semen was incubated undiluted or diluted with control extender (without EY or EY-LDF), BSP proteins bound to sperm in a time-dependent manner, and there is a continuous cholesterol and phospholipid efflux from the sperm membrane. In contrast, when semen was diluted with extender containing EY or EY-LDF, there was 50%-80% fewer BSP proteins associated with sperm and a significant amount of lipid added to sperm membrane during incubation. In addition, sperm function analysis showed that the presence of EY or EY-LDF in the extender preserved sperm motility. These results show that LDF is the constituent of EY that prevents binding of the BSP proteins to sperm and lipid efflux from the sperm membrane and is beneficial to sperm functions during sperm preservation. 相似文献
83.
Hypoxia-induced ischemic tolerance in neonatal rat brain involves enhanced ERK1/2 signaling 总被引:10,自引:0,他引:10
Hypoxic preconditioning (HP) 24 h before hypoxic-ischemic (HI) injury confers significant neuroprotection in neonatal rat brain. Recent studies have shown that the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K) intracellular signaling pathways play a role in the induction of tolerance to ischemic injury in heart and brain. To study the role of MAPK (ERK1/2, JNK, p38MAPK) and PI3K/Akt/GSK3beta signaling pathways in hypoxia-induced ischemic tolerance, we examined the brains of newborn rats at different time points after exposure to sublethal hypoxia (8% O(2) for 3 h). Immunoblot analysis showed that HP had no effect on the levels of phosphorylated Akt, GSK3beta, JNK and p38MAPK. In contrast, significantly increased levels of phosphorylated ERK1/2 were observed 0.5 h after HP. Double immunofluorescence staining showed that hypoxia-induced ERK1/2 phosphorylation was found mainly in microvessels throughout the brain and in astrocytes in white matter tracts. Inhibition of hypoxia-induced ERK1/2 pathway with intracerebral administration of U0126 significantly attenuated the neuroprotection afforded by HP against HI injury. These findings suggest that activation of ERK1/2 signaling may contribute to hypoxia-induced tolerance in neonatal rat brain in part by preserving vascular and white matter integrity after HI. 相似文献
84.
OBJECTIVE: To validate the utilization of cervical specimens collected in the fixative liquid used in the CYTO-screen System (SEROA, Monaco) for oncogenic human papillomavirus (HPV) DNA detection by the Hybrid Capture II technique (HCII) (Digene, Gaithersburg, Maryland, U.S.A) by reference to cytologic and/or histologic results. STUDY DESIGN: A technical feasibility study was conducted on 3 modalities of sample preparation before HCII technique, 1 with a proteinase digestion, I with PBS washing and I using the Digene sample conversion kit recommended for ThinPrep medium preparation (Cytyc Corp., Boxborough, Massachusetts, U.S.A.). The stability of cells after storage at days 28, 60 and 90 was tested on 26 positive samples (13 with high initial indices and 13 with low initial indices). Results of HPV testing were compared to cytologic and histologic results on a sample of 98 smears already identified as high grade squamous intraepithelial lesion (HSIL) (48) or low grade squamous intraepithelial lesion (LSIL) (50). A retrospective analysis was then performed on 995 HPV tests perfornmed routinely in 2003 in terms of comparison with the corresponding cytologic and/or histologic results. RESULTS: The HCII technique after direct treatment by proteinase K appeared to be as effective as the Digene sample conversion kit. By using the first technique, all 26 positive cases remained positive at 60 days, but 4 of 13 (30%) with low indices became negative at 90 days. The sensitivity of HPV testing for detecting biopsy- proven cervical intraepithelial neoplasia (CIN) 2 or worse was 100% in the 50 LSIL and 98% in the 48 HSIL samples. In the retrospective study (n = 995), the cytologic diagnoses of atypical squamous cells of undetermined significance (ASC-US) (n=278), LSIL (n = 137) and HSIL (n = 28) were associated with a positive HPVtest in 44%, 75% and 96% of cases, respectively. On a subsample of 156 patients among 278 with a diagnosis of ASC- US, the sensitivity of HPV testingfor detecting CIN 2 or worse was 88%, specificity 57%, positive predictive value 10% and negative predictive value 99%. Performing HPV testing by the HCII technique for cervical specimens collected in the fixative liquid used in the CYTO-screen System is feasible in the context of an ASC-US cytologic diagnosis. 相似文献
85.
Réale D Garant D Humphries MM Bergeron P Careau V Montiglio PO 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》2010,365(1560):4051-4063
The pace-of-life syndrome (POLS) hypothesis specifies that closely related species or populations experiencing different ecological conditions should differ in a suite of metabolic, hormonal and immunity traits that have coevolved with the life-history particularities related to these conditions. Surprisingly, two important dimensions of the POLS concept have been neglected: (i) despite increasing evidence for numerous connections between behavioural, physiological and life-history traits, behaviours have rarely been considered in the POLS yet; (ii) the POLS could easily be applied to the study of covariation among traits between individuals within a population. In this paper, we propose that consistent behavioural differences among individuals, or personality, covary with life history and physiological differences at the within-population, interpopulation and interspecific levels. We discuss how the POLS provides a heuristic framework in which personality studies can be integrated to address how variation in personality traits is maintained within populations. 相似文献
86.
Queiroz-Claret C Jolivet P Chardot T Bergeron E Meunier JC 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》2000,323(3):257-266
In the growth course of the lipolytic yeast Yarrowia lipolytica, the activities of protein phosphatase 2A (PP2A) and glycogen synthase (GS) rise during the exponential phase and concomitantly glycogen storage occurs in the cells. There is also an increase in the independence ratio (RI) indicating a shift from an inactive phosphorylated GS form to an active dephosphorylated GS form. During the early stationary phase, an increase in protein kinase CK2 (CK2) activity, a reversion of RI variation and a glycogen content decrease are observed. GS activity proved to be a good indicator of early culture growth phase. Experiments carried out with enzymes purified from Y. lipolytica show strong RI variations upon the action of CK2 and PP2Ac, and 32P incorporation into GS protein through phosphorylation by CK2. GS activity would be controlled by the sequential action of PP2A and CK2. 相似文献
87.
Lu Z Bohn J Bergeron R Deng Q Ellsworth KP Geissler WM Harris G McCann PE McKeever B Myers RW Saperstein R Willoughby CA Yao J Chapman K 《Bioorganic & medicinal chemistry letters》2003,13(22):4125-4128
A new class of diacid analogues that binds at the AMP site not only are very potent but have approximately 10-fold selectivity in liver versus muscle glycogen phosphorylase (GP) in the in vitro assay. The synthesis, structure, and in vitro and in vivo biological evaluation of these liver selective glycogen phosphorylase inhibitors are discussed. 相似文献
88.
Leisch H Shi R Grosse S Morley K Bergeron H Cygler M Iwaki H Hasegawa Y Lau PC 《Applied and environmental microbiology》2012,78(7):2200-2212
A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members. 相似文献
89.
ABSTRACT: BACKGROUND: There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA) in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. RESULTS: In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30degreesC to 42degreesC. However, using Escherichia coli DH5alpha as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30degreesC, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42degreesC. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5alpha[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42degreesC. CONCLUSIONS: Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production. 相似文献
90.
G De Serres MC Gariépy B Coleman I Rouleau S McNeil M Benoît A McGeer A Ambrose J Needham C Bergeron C Grenier K Sleigh A Kallos M Ouakki N Ouhoummane G Stiver L Valiquette A McCarthy J Bettinger;PHAC-CIHR influenza Research Network 《PloS one》2012,7(7):e38563