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121.
Powierska-Czarny J Miścicka-Sliwka D Czarny J Grzybowski T Wozniak M Drewa G Czechowicz W Sir J 《Acta biochimica Polonica》2003,50(4):1195-1203
Analysis of microsatellite instability (MI) and loss of heterozygosity (LOH) is recommended for screening patients with sporadic and hereditary malignancies. This study shows an application of a fluorescent hexaplex PCR system for microsatellite typing on A.L.F. DNA Sequencer (Pharmacia Biotech). This technique detects changes in microsatellites providing a time-efficient, reliable and accurate method for MI and LOH analyses. The Fragment Manager software was used for automated size calculation and quantitation of DNA fragments, enabling rapid and precise measurement of allelic ratios. We examined 70 breast cancer and 70 control DNA specimens, classified all the patterns of microsatellite alterations, and set up MI and LOH assessment criteria for the automated multiplex fluorescent method. 相似文献
122.
Genetic rescue of an endangered mammal by cross-species nuclear transfer using post-mortem somatic cells 总被引:28,自引:0,他引:28
Since the advent of procedures for cloning animals, conservation biologists have proposed using this technology to preserve endangered mammals. Here we report the successful cloning of a wild endangered animal, Ovis orientalis musimon, using oocytes collected from a closely related, domesticated species, Ovis aries. We injected enucleated sheep oocytes with granulosa cells collected from two female mouflons found dead in the pasture. Blastocyst-stage cloned embryos transferred into sheep foster mothers established two pregnancies, one of which produced an apparently normal mouflon. Our findings support the use of cloning for the expansion of critically endangered populations. 相似文献
123.
Correlations between amide proton temperature coefficients (HN/T) and hydrogen bonds were investigated for a data set of 793 amides derived from 14 proteins. For amide protons showing temperature gradients more positive than –4.6 ppb/K there is a hydrogen bond predictivity value exceeding 85%. It increases to over 93% for amides within the range between –4 and –1 ppb/K. Detailed analysis shows an inverse proportionality between amide proton temperature coefficients and hydrogen bond lengths. Furthermore, for hydrogen bonds of similar bond lengths, values of temperature gradients in -helices are on average 1 ppb/K more negative than in -sheets. In consequence, a number of amide protons in -helices involved in hydrogen bonds shorter than 2 Å show HN/T < –4.6 ppb/K. Due to longer hydrogen bonds, 90% of amides in 310 helices and 98% in -turns have temperature coefficients more positive than –4.6ppb/K. Ring current effect also significantly influences temperature coefficients of amide protons. In seven out of eight cases non-hydrogen bonded amides strongly deshielded by neighboring aromatic rings show temperature coefficients more positive than –2 ppb/K. In general, amide proton temperature gradients do not change with pH unless they correspond to conformational changes. Three examples of pH dependent equilibrium showing hydrogen bond formation at higher pH were found. In conclusion, amide proton temperature coefficients offer an attractive and simple way to confirm existence of hydrogen bonds in NMR determined structures. 相似文献
124.
2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose 1-sulfate have been synthesized by treatment of 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose with pyridine-sulfur trioxide complex. Direct hydrolysis of the isopropylidene group at C-4, C-5 gave 2,3-O-isopropylidene-beta-D-fructopyranose 1-sulfate. The crystal and molecular structures of ammonium (1a) and potassium (1b) salts of diisopropylidene derivative and ammonium (2) salt of monoisopropylidene derivative were determined by X-ray crystallography. Data for 1a and 1b were collected in 120 K and in 150 K for 2. All salts crystallized in P2(1)2(1)2(1) space group. There are three independent anions in asymmetric unit in 1b. Pyranose rings in the diisopropylidene derivative salts studied adopt 2S(0) twist boat conformation, whereas in the monoisopropylidene exists in a slightly distorted chair conformation (4C(1)). A staggered conformation is preferred by the sulfate group as indicated by values of C-(ester)-S-O(terminal) torsion angles: -173.2(4) degrees in 1a, 175.1(6) degrees in anion A of 1b, 170.8(6) degrees in anion C of 1b and 177.9(2) degrees in 2. However, strong interactions such as potassium-oxygen and H-bonds may affect the geometry: in anion B of 1b the value of the torsion angle is 139.4(6) degrees. 相似文献
125.
Here we determined NMR solution structures of two mutants of bovine pancreatic trypsin inhibitor (BPTI) to reveal structural reasons of their decreased thermodynamic stability. A point mutation, A16V, in the solvent-exposed loop destabilizes the protein by 20 degrees C, in contrast to marginal destabilization observed for G, S, R, L or W mutants. In the second mutant introduction of eight alanine residues at proteinase-contacting sites (residues 11, 13, 17, 18, 19, 34, 37 and 39) provides a protein that denatures at a temperature about 30 degrees C higher than expected from additive behavior of individual mutations. In order to efficiently determine structures of these variants, we applied a procedure that allows us to share data between regions unaffected by mutation(s). NOAH/DYANA and CNS programs were used for a rapid assignment of NOESY cross-peaks, structure calculations and refinement. The solution structure of the A16V mutant reveals no conformational change within the molecule, but shows close contacts between V16, I18 and G36/G37. Thus, the observed 4.3kcal/mol decrease of stability results from a strained local conformation of these residues caused by introduction of a beta-branched Val side-chain. Contrary to the A16V mutation, introduction of eight alanine residues produces significant conformational changes, manifested in over a 9A shift of the Y35 side-chain. This structural rearrangement provides about 6kcal/mol non-additive stabilization energy, compared to the mutant in which G37 and R39 are not mutated to alanine residues. 相似文献
126.
Bacich DJ Ramadan E O'Keefe DS Bukhari N Wegorzewska I Ojeifo O Olszewski R Wrenn CC Bzdega T Wroblewska B Heston WD Neale JH 《Journal of neurochemistry》2002,83(1):20-29
Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N-acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/Folh1 were generated by inserting into the genome a targeting cassette in which the intron-exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT-PCR analysis of RNA from the brains of -/- mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane-bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were Vmax = 45 and 3 pmol/mg/min and Km = 2650 nm and 2494 nm, respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4'-phosphinicobis-(butane-1,3 dicarboxilic acid) (FN6) and 2-(phosphonomethyl)pentanedioic acid (2-PMPA) inhibited the residual activity. The IC50 value for 2-PMPA was about 1 nm for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 nm for the activity in brain membranes of the null mutants. 相似文献
127.
128.
Heyduk T 《Current opinion in biotechnology》2002,13(4):292-296
Fluorescence resonance energy transfer (FRET) provides a unique means of measuring interatomic distances in biological molecules in real time. Recent advances have been made in the application of this technique to studies of conformational changes in proteins. New ways of introducing fluorescence probes into proteins, newly developed fluorescence probes, and progress in the technologies for fluorescence signal detection have greatly expanded the range of applications of FRET. In particular, studies of conformational changes in proteins at a single molecule level and in the native in vivo context of a living cell are now possible. 相似文献
129.
Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress. 相似文献
130.
Folliculogenesis was studied daily in the 18 oestrous cycles in six prolific Olkuska ewes from October to December using transrectal ultrasonography to record the number and size of all ovarian follicles > or =2 mm in diameter. Blood samples were taken once a day and were analyzed for concentrations of FSH, LH, estradiol and progesterone. Follicular and hormonal data were analyzed for associations between different stages of development of the follicular waves and concentrations of FSH and estradiol. The first wave during which at least one follicle reached maximum diameter of > or =4 mm after ovulation, was defined as a wave 1, and the following waves were numbered sequentially. Waves 1, 2, 3, 4 and the ovulatory one emerged on days: -2 to 4, 4 to 8, 6 to 11, 10 to 12 and 11 to 15, respectively. The mean number of follicles per wave that reached diameter of > or =4 mm was 4.15 +/- 1.1 and 16.62 +/- 8.6 follicles per estrous cycle of a total 299 follicles were observed. Significantly more follicles (p> or =0.05) emerged on days 2, 8 and 13 than in other days. Serum FSH concentrations fluctuated from 0.11 ngml(-1) on day 2 to preovulatory maximum 1.81 ngml(-1) on day 17 of the estrous cycle. The emergence of follicular waves was associated with elevations of FSH concentrations in blood serum. The mean increase in FSH concentration was followed by the recruitment of follicles of the next wave. The mean daily FSH concentration and the mean number of follicles emerging each day were negatively correlated. The length of the interwave interval (4.4 +/- 1.6 days) did not differ significantly from the interval between pulses of FSH (4.8 +/- 0.3 days). The mean serum estradiol concentrations showed fluctuations until day 14 and then gradually increased from 5.47 +/- 0.3 pgml(-1) to reach a peak 13.14 +/- 0.2 pgml(-1) on the day before ovulation. To summarize, the growth of ovarian follicles during the estrous cycle in high fecundity Olkuska sheep exhibited a distinct wave-like pattern. Ovarian follicles emerged from the pool of 2 mm follicles. The preovulatory follicles originated from the large follicle population were present in the ovary at the time of luteal regression. The initial stages of the growth of the largest follicles appears to be controlled primarily by increases in FSH secretion. 相似文献