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41.
Eukaryotic cells coordinate chromosome duplication by the assembly of protein complexes at origins of DNA replication by sequential binding of member proteins of the origin recognition complex (ORC), CDC6, and minichromosome maintenance (MCM) proteins. These pre-replicative complexes (pre-RCs) are activated by cyclin-dependent kinases and DBF4/CDC7 kinase. Here, we carried out a comprehensive yeast two-hybrid screen to establish sequential interactions between two individual proteins of the mouse pre-RC that are probably required for the initiation of DNA replication. The studies revealed multiple interactions among ORC subunits and MCM proteins as well as interactions between individual ORC and MCM proteins. In particular CDC6 was found to bind strongly to ORC1 and ORC2, and to MCM7 proteins. DBF4 interacts with the subunits of ORC as well as with MCM proteins. It was also demonstrated that CDC7 binds to different ORC and MCM proteins. CDC45 interacts with ORC1 and ORC6, and weakly with MCM3, -6, and -7. The three subunits of the single-stranded DNA binding protein RPA show interactions with various ORC subunits as well as with several MCM proteins. The data obtained by yeast two-hybrid analysis were paradigmatically confirmed in synchronized murine FM3A cells by immunoprecipitation of the interacting partners. Some of the interactions were found to be cell-cycle-dependent; however, most of them were cell-cycle-independent. Altogether, 90 protein-protein interactions were detected in this study, 52 of them were found for the first time in any eukaryotic pre-RC. These data may help to understand the complex interplay of the components of the mouse pre-RC and should allow us to refine its structural architecture as well as its assembly in real time. 相似文献
42.
Tumor vaccination after allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned tumor-bearing murine host 总被引:4,自引:0,他引:4
Zöller M 《Journal of immunology (Baltimore, Md. : 1950)》2003,171(12):6941-6953
Allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned host is supposed to provide an optimized platform for tumor vaccination. We recently showed that an allogeneic T cell-depleted graft was well accepted if the tumor-bearing host was NK depleted. Based on this finding, a vaccination protocol in tumor-bearing, nonmyeloablatively conditioned, allogeneically reconstituted mice was elaborated. Allogeneically reconstituted mice, bearing a renal cell carcinoma, received tumor-primed donor lymph node cells (LNC), which had or had not matured in the allogeneic host. Primed LNC were supported by tumor lysate-pulsed dendritic cells, which were donor or host derived. Optimal responses against the tumor were observed with host-tolerant, tumor-primed LNC in combination with host-derived dendritic cells. High frequencies of tumor-specific proliferating and CTLs were recorded; the survival time of tumor-bearing mice was significantly prolonged, and in >50% of mice the tumor was completely rejected. Notably, severe graft-vs-host disease was observed in reconstituted mice that received tumor-primed LNC, which had not matured in the allogeneic host. However, graft-vs-host was not aggravated after vaccination with tumor-primed, host-tolerant LNC. Thus, the LNC were tolerant toward the host, but not toward the tumor. The finding convincingly demonstrates the feasibility and efficacy of tumor vaccination after allogeneic reconstitution of the nonmyeloablatively conditioned host. 相似文献
43.
Support for association of schizophrenia with genetic variation in the 6p22.3 gene,dysbindin, in sib-pair families with linkage and in an additional sample of triad families 下载免费PDF全文
Schwab SG Knapp M Mondabon S Hallmayer J Borrmann-Hassenbach M Albus M Lerer B Rietschel M Trixler M Maier W Wildenauer DB 《American journal of human genetics》2003,72(1):185-190
Genetic variants in a gene on 6p22.3, dysbindin, have been shown recently to be associated with schizophrenia (Straub et al. 2002a). There is no doubt that replication in other independent samples would enhance the significance of this finding considerably. Since the gene is located in the center of the linkage peak on chromosome 6p that we reported earlier, we decided to test six of the most positive DNA polymorphisms in a sib-pair sample and in an independently ascertained sample of triads comprising 203 families, including the families for which we detected linkage on chromosome 6p. Evidence for association was observed in the two samples separately as well as in the combined sample (P=.00068 for SNP rs760761). Multilocus haplotype analysis increased the significance further to .00002 for a two-locus haplotype and to .00001 for a three-locus haplotype. Estimation of frequencies for six-locus haplotypes revealed one common haplotype with a frequency of 73.4% in transmitted, and only 57.6% in nontransmitted, parental haplotypes. All other six-locus haplotypes occurring at a frequency of >1% were less often transmitted than nontransmitted. Our results represent a first successful replication of linkage disequilibrium in psychiatric genetics detected in a region with previous evidence of linkage and will encourage the search for causes of schizophrenia by the genetic approach. 相似文献
44.
Miller MD Margot NA Hertogs K Larder B Miller V 《Nucleosides, nucleotides & nucleic acids》2001,20(4-7):1025-1028
The presence of the lamivudine-associated M184V RT mutation increases tenofovir susceptibility in multiple HIV genotypes. Tenofovir is uniquely active against multinucleoside-resistant HIV expressing the Q151M mutation, but shows reduced susceptibility to the T69S insertion mutations. HIV with common forms of zidovudine and lamivudine resistance are susceptible to tenofovir, corroborating phase II clinical results demonstrating the activity of tenofovir DF in treatment-experienced patients. 相似文献
45.
Comprehensive detection of genomic duplications and deletions in the DMD gene,by use of multiplex amplifiable probe hybridization 总被引:20,自引:0,他引:20
White S Kalf M Liu Q Villerius M Engelsma D Kriek M Vollebregt E Bakker B van Ommen GJ Breuning MH den Dunnen JT 《American journal of human genetics》2002,71(2):365-374
Duplications and deletions are known to cause a number of genetic disorders, yet technical difficulties and financial considerations mean that screening for these mutations, especially duplications, is often not performed. We have adapted multiplex amplifiable probe hybridization (MAPH) for the screening of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy. MAPH involves the quantitative recovery of specifically designed probes following hybridization to immobilized genomic DNA. We have engineered probes for each of the 79 exons of the DMD gene, and we analyzed them by using a 96-capillary sequencer. We screened 24 control individuals, 102 patients, and 23 potential carriers and detected a large number of novel rearrangements, especially small, one- and two-exon duplications. A duplication of exon 2 alone was the most frequently occurring mutation identified. Our analysis indicates that duplications occur in 6% of patients with DMD. The MAPH technique as modified here is simple, quick, and accurate; furthermore, it is based on existing technology (i.e., hybridization, PCR, and electrophoresis) and should not require new equipment. Together, these features should allow easy implementation in routine diagnostic laboratories. Furthermore, the methodology should be applicable to any genetic disease, it should be easily expandable to cover >200 probes, and its characteristics should facilitate high-throughput screening. 相似文献
46.
The initial events in bacterial adhesion are often explained as resulting from electrostatic and van der Waals forces between the cell and the surface, as described by DLVO theory (developed by Derjaguin, Landau, Verwey, and Overbeek). Such a theory predicts that negatively charged bacteria will experience greater attraction toward a negatively charged surface as the ionic strength of the medium is increased. In the present study we observed both smooth-swimming and nonmotile Escherichia coli bacteria close to plain, positively, and hydrophobically coated quartz surfaces in high- and low-ionic-strength media by using total internal reflection aqueous fluorescence microscopy. We found that reversibly adhering cells (cells which continue to swim along the surface for extended periods) are too distant from the surface for this behavior to be explained by DLVO-type forces. However, cells which had become immobilized on the surface did seem to be affected by electrostatic interactions. We propose that the "force" holding swimming cells near the surface is actually the result of a hydrodynamic effect, causing the cells to swim at an angle along the glass, and that DLVO-type forces are responsible only for the observed immobilization of irreversibly adhering cells. We explain our observations within the context of a conceptual model in which bacteria that are interacting with the surface may be thought of as occupying one of three compartments: bulk fluid, near-surface bulk, and near-surface constrained. A cell in these compartments feels either no effect of the surface, only the hydrodynamic effect of the surface, or both the hydrodynamic and the physicochemical effects of the surface, respectively. 相似文献
47.
RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-κB activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-κB and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-κB induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-κB activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-κB and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-κB-dependent pro-survival or pro-inflammatory signals. 相似文献
48.
49.
Christ ER Carroll PV Albany E Umpleby AM Lumb PJ Wierzbicki AS Sönksen PH Russell-Jones DL 《American journal of physiology. Endocrinology and metabolism》2002,282(5):E1154-E1162
Abnormal lipid metabolism may be related to the increased cardiovascular risk in type 1 diabetes. Secretion and clearance rates of very low density lipoprotein (VLDL) apolipoprotein B100 (apoB) determine plasma lipid concentrations. Type 1 diabetes is characterized by increased growth hormone (GH) secretion and decreased insulin-like growth factor (IGF) I concentrations. High-dose IGF-I therapy improves the lipid profile in type 1 diabetes. This study examined the effect of low-dose (40 microg.kg(-1).day(-1)) IGF-I therapy on VLDL apoB metabolism, VLDL composition, and the GH-IGF-I axis during euglycemia in type 1 diabetes. Using a stable isotope technique, VLDL apoB kinetics were estimated before and after 1 wk of IGF-I therapy in 12 patients with type 1 diabetes in a double-blind, placebo-controlled trial. Fasting plasma triglyceride (P < 0.03), VLDL-triglyceride concentrations (P < 0.05), and the VLDL-triglyceride-to-VLDL apoB ratio (P < 0.002) significantly decreased after IGF-I therapy, whereas VLDL apoB kinetics were not significantly affected by IGF-I therapy. IGF-I therapy resulted in a significant increase in IGF-I and a significant reduction in GH concentrations. The mean overnight insulin concentrations during euglycemia decreased by 25% after IGF-I therapy. These results indicate that low-dose IGF-I therapy restores the GH-IGF-I axis in type 1 diabetes. IGF-I therapy changes fasting triglyceride concentrations and VLDL composition probably because of an increase in insulin sensitivity. 相似文献
50.
Hovorka R Shojaee-Moradie F Carroll PV Chassin LJ Gowrie IJ Jackson NC Tudor RS Umpleby AM Jones RH 《American journal of physiology. Endocrinology and metabolism》2002,282(5):E992-1007
We have separated the effect of insulin on glucose distribution/transport, glucose disposal, and endogenous production (EGP) during an intravenous glucose tolerance test (IVGTT) by use of a dual-tracer dilution methodology. Six healthy lean male subjects (age 33 +/- 3 yr, body mass index 22.7 +/- 0.6 kg/m(2)) underwent a 4-h IVGTT (0.3 g/kg glucose enriched with 3-6% D-[U-(13)C]glucose and 5-10% 3-O-methyl-D-glucose) preceded by a 2-h investigation under basal conditions (5 mg/kg of D-[U-(13)C]glucose and 8 mg/kg of 3-O-methyl-D-glucose). A new model described the kinetics of the two glucose tracers and native glucose with the use of a two-compartment structure for glucose and a one-compartment structure for insulin effects. Insulin sensitivities of distribution/transport, disposal, and EGP were similar (11.5 +/- 3.8 vs. 10.4 +/- 3.9 vs. 11.1 +/- 2.7 x 10(-2) ml small middle dot kg(-1) small middle dot min(-1) per mU/l; P = nonsignificant, ANOVA). When expressed in terms of ability to lower glucose concentration, stimulation of disposal and stimulation of distribution/transport accounted each independently for 25 and 30%, respectively, of the overall effect. Suppression of EGP was more effective (P < 0.01, ANOVA) and accounted for 50% of the overall effect. EGP was suppressed by 70% (52-82%) (95% confidence interval relative to basal) within 60 min of the IVGTT; glucose distribution/transport was least responsive to insulin and was maximally activated by 62% (34-96%) above basal at 80 min compared with maximum 279% (116-565%) activation of glucose disposal at 20 min. The deactivation of glucose distribution/transport was slower than that of glucose disposal and EGP (P < 0.02) with half-times of 207 (84-510), 12 (7-22), and 29 (16-54) min, respectively. The minimal-model insulin sensitivity was tightly correlated with and linearly related to sensitivity of EGP (r = 0.96, P < 0.005) and correlated positively but nonsignificantly with distribution/transport sensitivity (r = 0.73, P = 0.10) and disposal sensitivity (r = 0.55, P = 0.26). We conclude that, in healthy subjects during an IVGTT, the two peripheral insulin effects account jointly for approximately one-half of the overall insulin-stimulated glucose lowering, each effect contributing equally. Suppression of EGP matches the effect in the periphery. 相似文献