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991.
Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes 总被引:22,自引:0,他引:22
Lyamichev V Mast AL Hall JG Prudent JR Kaiser MW Takova T Kwiatkowski RW Sander TJ de Arruda M Arco DA Neri BP Brow MA 《Nature biotechnology》1999,17(3):292-296
Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. We have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, we provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA. 相似文献
992.
993.
Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous two-liquid-phase applications 总被引:1,自引:0,他引:1
Panke S de Lorenzo V Kaiser A Witholt B Wubbolts MG 《Applied and environmental microbiology》1999,65(12):5619-5623
Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts. 相似文献
994.
Dendritic GABA release depresses excitatory transmission between layer 2/3 pyramidal and bitufted neurons in rat neocortex 总被引:16,自引:0,他引:16
GABAergic, somatostatin-containing bitufted interneurons in layer 2/3 of rat neocortex are excited via glutamatergic excitatory postsynaptic potentials (EPSPs) by pyramidal neurons located in the same cortical layer. Pair recordings showed that short bursts of backpropagating dendritic action potentials (APs) reduced the amplitude of unitary EPSPs. EPSP depression was dependent on a rise in dendritic [Ca2+]. The effect was blocked by the GABA(B) receptor (GABA(B)-R) antagonist CGP55845A and was mimicked by the GABA(B)-R agonist baclofen. As presynaptic GABA(B)-Rs were activated neither by somatostatin nor by GABA released from axon collaterals of the bitufted cell, we conclude that GABA(B)-Rs were activated by a retrograde messenger, most likely GABA, released from the dendrite. Because synaptic depression was prevented by loading bitufted neurons with GDP-beta-S, it is likely to be caused by exocytotic GABA release from dendrites. 相似文献
995.
Relative digit lengths and testosterone levels in Guinea baboons 总被引:8,自引:0,他引:8
Roney JR Whitham JC Leoni M Bellem A Wielebnowski N Maestripieri D 《Hormones and behavior》2004,45(4):285-290
A growing body of literature suggests that the ratio of the lengths of the second to fourth digits (2D:4D) on human hands is sexually dimorphic and associated with prenatal exposure to gonadal hormones, circulating serum testosterone, and a number of psychological and behavioral measures. Little research has investigated digit ratios in nonhuman species. In the present study, we investigated sex differences in digit ratios and their possible association with serum testosterone in a captive group of Guinea baboons (Papio papio). Contrary to the sex difference typically reported in humans, male baboons exhibited a substantially larger 2D:4D than did female baboons. Consistent with the human data, however, lower 2D:4D was associated with higher serum testosterone among the males. The present findings suggest that the relationship between digit ratios and male gonadal hormones may be phylogenetically well-conserved, although they also suggest possible species differences in the causal relationships between developmental mechanisms and sex-differentiated digit length patterns. 相似文献
996.
Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia-reperfusion in vivo 总被引:6,自引:0,他引:6
Kaiser RA Bueno OF Lips DJ Doevendans PA Jones F Kimball TF Molkentin JD 《The Journal of biological chemistry》2004,279(15):15524-15530
The p38 branch of the mitogen-activated protein kinase (MAPK) signaling cascade has been implicated as a regulator of cardiomyocyte apoptosis in culture as well as in the adult heart. However, considerable disagreement persists as to the functional effects attributed to p38 signaling, given that both pro- and anti-apoptotic regulatory roles have been reported. To address this area of uncertainty in the literature, we investigated the cell death effects associated with p38 inactivation in both cultured neonatal cardiomyocytes and the adult heart. In vitro, adenoviral-mediated gene transfer of two different dominant-negative-encoding p38 vectors reduced apoptosis induced by 2-deoxyglucose treatment, whereas overexpression of wild-type p38alpha or an activated mitogen-activated protein kinase kinase (MKK)6 mutant each enhanced cell death. In vivo, transgenic mice expressing a dominant-negative MKK6 mutant or a dominant-negative p38alpha mutant were each significantly protected from ischemia-reperfusion injury, as assessed by infarct area measurements, DNA laddering, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, and functional assessment of ventricular performance. Similarly, transgenic mice overexpressing the p38-inactivating dual specificity phosphatase MAPK phosphatase-1 (MKP-1) were also partially protected, whereas MKP-1 gene-targeted mice showed greater injury after ischemia-reperfusion injury. Mechanistically, inhibition of p38 signaling promoted a dramatic up-regulation of Bcl-2 in the hearts of transgenic mice. In primary neonatal cardiomyocyte cultures, adenoviral-mediated gene transfer of a p38 inhibitory mutant up-regulated Bcl-2, whereas expression of an activated p38 mutant down-regulated Bcl-2 protein levels. Collectively, these results indicate that p38 functions as a pro-death signaling effector in both cultured myocytes as well as in the intact heart. 相似文献
997.
998.
IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi 总被引:2,自引:0,他引:2
Ferreira IA Eybrechts KL Mocking AI Kroner C Akkerman JW 《The Journal of biological chemistry》2004,279(5):3254-3264
Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i). 相似文献
999.
Amyloid beta-induced changes in nitric oxide production and mitochondrial activity lead to apoptosis 总被引:9,自引:0,他引:9
Keil U Bonert A Marques CA Scherping I Weyermann J Strosznajder JB Müller-Spahn F Haass C Czech C Pradier L Müller WE Eckert A 《The Journal of biological chemistry》2004,279(48):50310-50320
Increasing evidence suggests an important role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease. Thus, we investigated the effects of acute and chronic exposure to increasing concentrations of amyloid beta (Abeta) on mitochondrial function and nitric oxide (NO) production in vitro and in vivo. Our data demonstrate that PC12 cells and human embryonic kidney cells bearing the Swedish double mutation in the amyloid precursor protein gene (APPsw), exhibiting substantial Abeta levels, have increased NO levels and reduced ATP levels. The inhibition of intracellular Abeta production by a functional gamma-secretase inhibitor normalizes NO and ATP levels, indicating a direct involvement of Abeta in these processes. Extracellular treatment of PC12 cells with comparable Abeta concentrations only leads to weak changes, demonstrating the important role of intracellular Abeta. In 3-month-old APP transgenic (tg) mice, which exhibit no plaques but already detectable Abeta levels in the brain, reduced ATP levels can also be observed showing the in vivo relevance of our findings. Moreover, we could demonstrate that APP is present in the mitochondria of APPsw PC12 cells. This presence might be directly involved in the impairment of cytochrome c oxidase activity and depletion of ATP levels in APPsw PC12 cells. In addition, APPsw human embryonic kidney cells, which produce 20-fold increased Abeta levels compared with APPsw PC12 cells, and APP tg mice already show a significantly decreased mitochondrial membrane potential under basal conditions. We suggest a hypothetical sequence of pathogenic steps linking mutant APP expression and amyloid production with enhanced NO production and mitochondrial dysfunction finally leading to cell death. 相似文献
1000.
This study compared domestic guinea pigs (Cavia aperea f. porcellus; DGP) and two different populations of the wild cavy (Cavia aperea), its ancestor, to examine whether rearing of wild mammals in captivity affects their behavior and physiological stress responses. One population of wild cavies consisted of wild-trapped animals and their first laboratory-reared offspring (WGP-1). The animals of the other population were reared in captivity for about 30 generations (WGP-30). The spontaneous behavior of each of six groups of WGP-1 and WGP-30 and nine groups of DGP, each consisting of one adult male and two adult females, was analyzed quantitatively. Blood samples of the males were taken to determine cortisol, epinephrine, and norepinephrine concentrations. In addition, the exploratory behavior of 60-day-old male WGP-1, WGP-30, and DGP was investigated in an exploration apparatus. The domesticated animals displayed significantly less aggression, but significantly more sociopositive and male courtship behavior than their wild ancestors. In addition, DGP were much less attentive to their physical environment. Surprisingly, no behavioral difference was found between WGP-1 and WGP-30. Basal cortisol concentrations did not differ between wild and domestic guinea pigs. Catecholamine concentrations, however, as well as the challenge values of cortisol, were distinctly reduced in the DGP. WGP-1 and WGP-30 did not differ with respect to their endocrine stress responses. In the exploration apparatus both forms of wild cavies were much more explorative than the domestic animals. These data suggest that the long-term breeding and rearing of wild guinea pigs in captivity do not result in significant changes in behavior and hormonal stress responses. It appears to take much longer periods of time and artificial selection by humans to bring about characters of domestication in wild animals. 相似文献