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981.
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
982.
Mapping of antibody responses to the protective antigen of Bacillus anthracis by flow cytometric analysis 总被引:5,自引:0,他引:5
BACKGROUND: Different plant species vary as to the ratio of nucleotide base pairs of genomic DNA. A correlation between genome size and base pair ratio has been claimed. Base composition can be analyzed by base-specific dyes. METHODS: Genome size is determined by flow cytometry of suspensions of nuclei stained by the base independent dye, PI. For estimation of the AT frequency, the AT-specific dyes 4,6-diamidino-2-phenylindole, dihydrochloride (DAPI) and Hoechst 33342 (HO) were used. We define a dye factor (DF) as the ratio of the two estimates (peak ratios) of nuclear fluorescence intensities of sample relative to reference plant nuclei using a given dye and an intercalating fluorochrome. RESULTS: No significant correlation between genome size and the DF for DAPI was found when 54 plant species were investigated. However, similarities within and differences among the plant families were shown. The comparison of DAPI and HO DFs gave no consistent differences as would be predicted from the model of different binding site length of dyes. This result may be explained by the nonrandom distribution of base pairs. CONCLUSIONS: There is no general correlation between genome size and AT/GC ratio in higher plants. Similar AT/GC ratios within a plant family result from the general similarity of the DNA sequences within a family. The fluorescence of base-specific dyes is influenced by the nonrandom distribution of bases in the DNA molecule. 相似文献
983.
Telomere length measurement by fluorescence in situ hybridization and flow cytometry: tips and pitfalls 总被引:13,自引:0,他引:13
BACKGROUND: Telomeres containing noncoding DNA repeats at the end of the chromosomes are essential for chromosomal stability and are implicated in regulating the replication and senescence of cells. The gradual loss of telomere repeats in cells has been linked to aging and tumor development and methods to measure telomere length are of increasing interest. At least three methods for measuring the length of telomere repeats have been described: Southern blot analysis and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (flow-FISH). Both Southern blot analysis and Q-FISH have specific limitations and are time-consuming, whereas the flow-FISH technique requires relatively few cells (10(5)) and can be completed in a single day. A further advantage of the flow-FISH method is that data on the telomere length from individual cells and subsets of cells (lymphocytes and granulocytes) can be acquired from the same sample. In order to obtain accurate and reproducible results using the flow-FISH technique, we systematically explored the influence of various steps in the protocol on telomere length values and established an acceptable range for the most critical parameters. METHODS: Isolated leukocytes from whole blood are denatured by heat and 70%/75% formamide, then hybridized with or without a telomere-specific fluorescein isothiocyante (FITC)-conjugated peptide nucleic acid probe (PNA). Unbound telomere PNA is washed away, the DNA is counterstained, and telomere fluorescence is measured on a flow cytometer using an argon ion laser (488 nm) to excite FITC. For each sample, duplicates of telomere PNA-stained and unstained tubes are analyzed. RESULTS: Cell counts and flow-FISH telomere length measurements were performed on leukocytes and thymocytes of humans and other species. Leukocyte suspensions were prepared by two red blood cell lysis steps with ammonium chloride. Optimal denaturation of DNA was achieved by heating at 85-87 degrees C for 15 min in a solution containing 70%/75% formamide. Hybridization was performed at room temperature with a 0.3 microg/ml telomere-PNA probe for at least 60-90 min. Unbound telomere-PNA probe was diluted at least 4,000-40,000 times with wash steps containing 70%/75% formamide at room temperature. LDS 751 and DAPI were suitable as DNA counterstains as they did not show significant interference with telomere length measurement. CONCLUSIONS: The use of flow-FISH for telomere length measurements in nucleated blood cells requires tight adherence to an optimized protocol. The method described here can be used to determine rapidly the telomere length in subsets of nucleated blood cells. 相似文献
984.
985.
Activation of Akt/protein kinase B overcomes a G(2)/m cell cycle checkpoint induced by DNA damage 总被引:20,自引:0,他引:20 下载免费PDF全文
Kandel ES Skeen J Majewski N Di Cristofano A Pandolfi PP Feliciano CS Gartel A Hay N 《Molecular and cellular biology》2002,22(22):7831-7841
Activation of Akt, or protein kinase B, is frequently observed in human cancers. Here we report that Akt activation via overexpression of a constitutively active form or via the loss of PTEN can overcome a G(2)/M cell cycle checkpoint that is induced by DNA damage. Activated Akt also alleviates the reduction in CDC2 activity and mitotic index upon exposure to DNA damage. In addition, we found that PTEN null embryonic stem (ES) cells transit faster from the G(2)/M to the G(1) phase of the cell cycle when compared to wild-type ES cells and that inhibition of phosphoinositol-3-kinase (PI3K) in HEK293 cells elicits G(2) arrest that is alleviated by activated Akt. Furthermore, the transition from the G(2)/M to the G(1) phase of the cell cycle in Akt1 null mouse embryo fibroblasts (MEFs) is attenuated when compared to that of wild-type MEFs. These results indicate that the PI3K/PTEN/Akt pathway plays a role in the regulation of G(2)/M transition. Thus, cells expressing activated Akt continue to divide, without being eliminated by apoptosis, in the presence of continuous exposure to mutagen and accumulate mutations, as measured by inactivation of an exogenously expressed herpes simplex virus thymidine kinase (HSV-tk) gene. This phenotype is independent of p53 status and cannot be reproduced by overexpression of Bcl-2 or Myc and Bcl-2 but seems to counteract a cell cycle checkpoint mediated by DNA mismatch repair (MMR). Accordingly, restoration of the G(2)/M cell cycle checkpoint and apoptosis in MMR-deficient cells, through reintroduction of the missing component of MMR, is alleviated by activated Akt. We suggest that this new activity of Akt in conjunction with its antiapoptotic activity may contribute to genetic instability and could explain its frequent activation in human cancers. 相似文献
986.
Savage MO Scommegna S Carroll PV Ho JT Monson JP Besser GM Grossman AB 《Hormone research》2002,58(Z1):39-43
Growth is disturbed by adrenal hypersecretion of androgens or cortisol. Androgen excess in virilizing adrenal tumours causes advanced growth and bone age. In 9 girls with virilizing tumours, mean heights at diagnosis and final heights were 1.23 +/- 0.42 and 1.3 +/- 0.37 SDS respectively. In poorly controlled CAH, excess androgens cause early epiphyseal fusion and adult short stature. Increased growth occurs only after 18 months of age, even in untreated CAH, i.e. hydrocortisone >10 mg/m(2)/day is not generally required and may suppress infantile growth, affecting childhood and adult height. Growth was studied in 19 patients, aged 6.4-17.8 years, with Cushing's disease (CD). At diagnosis, mean height SDS was -1.81 (1.2 to -4.17), 53% < -1.8 SDS, height velocity in 6 was 0.9-3.8 cm/year and mean BMI SDS 2.29 (0.7-5.06). From 1983 to 2001, CD was cured in 18 patients (61%) by transsphenoidal surgery (TSS) alone and 39% by TSS plus pituitary irradiation (RT). In 13 patients, growth hormone (GH) was assessed by ITT/glucagons at 1-108 months after cure. Four had severe GH deficiency (<9 mU/l), 7 subnormal (10-29 mU/l) and 2 normal (>30 mU/l) GH status. Subnormal GH was present in 7 subjects >2 years after TSS or RT cure. In 10 subjects, aged 12.9 +/- 3.4 years, growth after cure was studied for 9.1 +/- 5.0 years. Nine had no catch-up growth in the interval of 0.3-1.1 years after cure (mean HV 5.3 +/- 2.4 cm/year). All these had GH deficiency peak GH 0.5-20.9 mU/l, and received hGH 2.7 mg/m(2)/week, 3 with GnRHa. All 10 showed long-term catch-up growth with mean delta SDS at diagnosis (Ht SDS-target Ht SDS) -1.72 +/- 1.26 improving to -0.83 +/- 1.08 (p = 0.0005) at latest of final Ht. At diagnosis, virilization was present in 82% of 17 patients with CD. Mean SDS values of serum androstenedione, DHEA-S and testosterone were normal, i.e. 0.72 (-2.9 to 3.0), -0.8 (6.0 to 2.2), 0.7 (-7.9 to 9.5) respectively, whereas SHBG was reduced at -2.1 (-5.3 to 1.2), increasing free androgen levels. Bone age (BA) was delayed (mean 1.46 years) in 14/16 patients, suggesting cortisol excess contributed more then androgen effect to skeletal maturation. In conclusion, most paediatric patients with CD had subnormal linear growth with delayed BA. After cure by TSS or pituitary irradiation, GH deficiency was frequent and persisted for many years. Treatment with hGH induced significant long-term catch-up growth leading to reasonable final height. 相似文献
987.
A gradient of development consisting of successive zones of cell division, cell elongation and cell maturation occurs along the longitudinal axis of elongating leaf blades of tall fescue (Festuca arundinacea Schreb.), a C3 grass. An increase in specific leaf weight (SLW; dry weight per unit leaf area) in the maturation region has been hypothesized to result from deposition of secondary cell walls in structural tissues. Our objective was to measure the transverse cell wall area (CWA) associated with the increase in SLW, which occurs following the cessation of leaf blade elongation at about 25 mm distal to the ligule. Digital image analysis of transverse sections at 5, 15, 45, 75 and 105 mm distal to the ligule was used to determine cell number, cell area and protoplast area of structural tissues, namely fibre bundles, mestome sheaths and xylem vessel elements, along the developmental gradient. Cell diameter, protoplast diameter and area, and cell wall thickness and area of fibre bundle cells were calculated from these data. CWA of structural tissues increased in sections up to 75 mm distal to the ligule, confirming the role of cell wall deposition in the increase in SLW (r2 = 0.924; P < or = 0.01). However, protoplast diameter of fibre cells did not decrease significantly as CWA increased, although mean thickness of fibre cell walls increased by 95 % between 15 and 105 mm distal to the ligule. Therefore, secondary cell wall deposition in fibre bundles of tall fescue leaf blades resulted in continued radial expansion of fibre cells rather than in a decrease in protoplast diameter. 相似文献
988.
Christian SL McDonough J Liu Cy CY Shaikh S Vlamakis V Badner JA Chakravarti A Gershon ES 《Genomics》2002,79(5):635-656
The human 13q32-q33 region has been linked to both bipolar disorder and schizophrenia. Before completion of the draft sequences, we developed an approximately 15-Mb comprehensive map for the region extending from D13S1300 to ATA35H12. This map was assembled using publicly available mapping data and sequence-tagged site (STS)-based PCR confirmation. We then compared this map with the NCBI, Celera Genomics, and UCSC Golden Path data in February, June, and September 2001. All data sets showed gaps, misassignment of STSs, and errors in orientation and marker order. Surprisingly, the completed sequences of many bacterial artificial chromosomes (BACs) had been truncated. Of 21 gaps that were detected, 4 were present in both the NCBI and Celera databases. All gaps could be filled using 1-2 BAC clones. A total of 39 loci mapped to additional sites within the human genome, providing evidence of segmental duplications. Additionally, 61 unique cDNA clones were sequenced to increase available transcribed sequence, and 11,353 reference single-nucleotide polymorphisms (SNPs) with an average density of 1 SNP/3720 bases were identified. Overall, integration of the data from multiple sources is still needed for complete assembly of the 13q32-q33 region. (c) 相似文献
989.
PRL activates the cyclin D1 promoter via the Jak2/Stat pathway 总被引:12,自引:0,他引:12
990.
Stage-specific expression of Smad2 and Smad3 during folliculogenesis 总被引:10,自引:0,他引:10
Paracrine and autocrine growth factors can affect many different aspects of ovarian follicle development. Many members of the transforming growth factor beta (TGFbeta) family of growth factors and their receptors are expressed in developing follicles. However, the presence and function of the family of the TGFbeta signaling molecules known as Smads have not been evaluated during follicle development. We have demonstrated that two Smad family members that function as mediators for both activin and TGFbeta are expressed in granulosa cells of preantral follicles but not in large antral follicles. Smad2 expression, but not Smad3 expression, returns in luteal cells. Both Smad2 and Smad3 are translocated to the nucleus of granulosa cells in response to treatment with either TGFbeta or activin. However, Smad2 is more responsive to activin stimulation, and Smad3 is more responsive to TGFbeta stimulation. Stage-specific expression and differing ligand sensitivity of signaling molecules may work together to allow different effects of TGFbeta family ligands using the same signaling pathways over the course of follicular development. 相似文献