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71.
Having an effective means to cryopreserve human oocytes would offer more flexibility in healthcare services for infertility patients, and obviate cryopreservation of preimplantation embryos. It is essential to establish good animal models for human oocyte cryopreservation and the rabbit is a good candidate. Attempts to improve oocyte cryopreservation are often empirical, with results often being irreproducible. Cryopreservation protocols may be optimized by modeling the changes in oocyte volume and the associated damages incurred during the addition and dilution of cryoprotective agents (CPA). The objectives of the current study were to determine cryobiological properties of rabbit oocytes, including the isotonic volume, osmotically inactive cell fraction (Vb), hydraulic conductivity (Lp), permeability (Ps) to dimethylsulfoxide (Me2SO), ethylene glycol (EG), and glycerol (GLY) and to examine the correlation between cell volume excursions and viability. This has led to the development of the accumulative osmotic damage (AOD) model associated with the processes of CPA addition/dilution. Mature rabbit oocytes were perfused with 15% (V/V) CPA medium (dissolved in 1× PBS). The osmotic responses of the oocytes were videotaped. A two-parameter model was fit to the experimental data to determine the values of Lp and Ps. Oocyte volumes reached upon equilibration with 285, 600, 900, and 1200 mOsm (milliosmolal) solutions of non-permeating compounds were plotted in a Boyle van’t Hoff plot. The average radius of rabbit oocytes in an isotonic solution was determined to be 55.7 ± 1.2 μm (n = 16). The rabbit oocyte exhibited an “ideal” osmotic response in the range from iso-osmolity to 1200 mOsm. The Vb was determined to be 20% of the isotonic value with r2 = 0.97. The values of Lp were determined to be 0.79 ± 0.26, 0.82 ± 0.22, and 0.64 ± 0.16 μm min−1 atm−1 and the Ps values were determined to be 2.9 ± 1.3, 2.7 ± 1.3, and 0.27 ± 0.18 × 10−3 cm min−1 for Me2SO, EG and GLY, respectively. There were no significant differences (p > 0.05) between values for Lp and PS in the presence of the Me2SO and EG. However, these values were significantly different from the values in presence of GLY. We calculated the AOD values of those oocytes that experienced the process of CPA additions/dilutions and found that these values were highly correlated to the development rates of these oocytes after parthenogenetic activation (r = −0.98). 相似文献
72.
Agnes?Csanadi Ildiko?Faludi Andras?MiczakEmail author 《Molecular biology reports》2009,36(8):2341-2344
The MSMEG_4626 gene was cloned from Mycobacterium smegmatis MC2 155. It codes for a protein of 1,037 amino acids, identified as ribonuclease E by matching to the protein family HMM
TIGR00757. The protein was expressed and purified. Although its calculated molecular weight is 112.7 kDa, it has an aberrant
mobility in SDS-polyacrylamide gels, like other ribonuclease E enzymes (it migrates as a 180 kDa protein). The central part
of the protein displays high similarity to the catalytic domains of other RNase E enzymes. Mass spectrometric analysis revealed
the presence of the chaperonin GroEL, ribosomal proteins, a negative regulator of genetic competence and GTP pyrophosphokinase
in the affinity-purified preparation. It is a very unstable protein; despite the use of protease inhibitors in addition to
the full-length RNase E its proteolytic fragments were detected. 相似文献
73.
In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed. 相似文献
74.
Rajesh Nair Jinfeng Liu Ta-Tsen Soong Thomas B. Acton John K. Everett Andrei Kouranov Andras Fiser Adam Godzik Lukasz Jaroszewski Christine Orengo Gaetano T. Montelione Burkhard Rost 《Journal of structural and functional genomics》2009,10(2):181-191
The Protein Structural Initiative (PSI) at the US National Institutes of Health (NIH) is funding four large-scale centers
for structural genomics (SG). These centers systematically target many large families without structural coverage, as well
as very large families with inadequate structural coverage. Here, we report a few simple metrics that demonstrate how successfully
these efforts optimize structural coverage: while the PSI-2 (2005-now) contributed more than 8% of all structures deposited
into the PDB, it contributed over 20% of all novel structures (i.e. structures for protein sequences with no structural representative
in the PDB on the date of deposition). The structural coverage of the protein universe represented by today’s UniProt (v12.8)
has increased linearly from 1992 to 2008; structural genomics has contributed significantly to the maintenance of this growth
rate. Success in increasing novel leverage (defined in Liu et al. in Nat Biotechnol 25:849–851, 2007) has resulted from systematic
targeting of large families. PSI’s per structure contribution to novel leverage was over 4-fold higher than that for non-PSI
structural biology efforts during the past 8 years. If the success of the PSI continues, it may just take another ~15 years
to cover most sequences in the current UniProt database. 相似文献
75.
Jing-Jing Li Ai-Qing Wang Jan-Christer Janson Andras Ballagi Jing Chen Yong-Dong Liu Guang-Hui Ma Zhi-Guo Su 《Process Biochemistry》2009,44(3):277-282
A new protein refolding technique based on the use of the non-charged detergent Triton X-100 immobilized to the cross-linked agarose gel Sepharose High Performance has been developed. The new solid phase was used in combination with soluble β-cyclodextrin (β-CD) to refold recombinant Green Fluorescent Protein fused to Tobacco Etch Virus protease (GFPTEVP) expressed as inclusion bodies in E. coli. Previous attempts to refold recombinant GFPTEVP by dilution had failed. In the new procedure a column packed with Triton X-100-coupled Sepharose High Performance was used to capture unfolded GFPTEVP followed by elution using an increasing β-CD concentration gradient. The yield of properly refolded GFPTEVP was 46% at a protein concentration of 380 μg/ml. In contrast, dilution refolding of GFPTEVP at 200 μg/ml refolding buffer resulted in only 4.7% of native protein. 相似文献
76.
Bobkov AA Muhlrad A Pavlov DA Kokabi K Yilmaz A Reisler E 《Journal of molecular biology》2006,356(2):325-334
Using site-specific fluorescence probes and cross-linking we demonstrated that cofilin (ADF), a key regulator of actin cellular dynamics, weakens longitudinal contacts in F-actin in a cooperative manner. Differential scanning calorimetry detected a dual nature of cofilin effects on F-actin conformation. At sub-stoichiometric cofilin to actin ratios, cofilin stabilized sterically and non-cooperatively protomers at the points of attachment, and destabilized allosterically and cooperatively protomers in the cofilin-free parts of F-actin. This destabilizing effect had a long range, with one cofilin molecule affecting more than 100 protomers, and concentration-dependent amplitude that reached maximum at about 1:2 molar ratio of cofilin to actin. In contrast to existing models, our results suggest an allosteric mechanism of actin depolymerization by cofilin. We propose that cofilin is less likely to sever actin filaments at the points of attachment as thought previously. Instead, due to its dual structural effect, spontaneous fragmentation occurs most likely in cofilin-free segments of filaments weakened allosterically by nearby cofilin molecules. 相似文献
77.
Lim J Menon V Bitzer M Miller LM Madrid-Aliste C Weiss LM Fiser A Angeletti RH 《Analytical biochemistry》2011,(1):78-84
Differential detergent fractionation (DDF) is frequently used to partition fresh cells and tissues into distinct compartments. We have tested whether DDF can reproducibly extract and fractionate cellular protein components from frozen tissues. Frozen kidneys were sequentially extracted with three different buffer systems. Analysis of the three fractions with liquid chromatography–tandem mass spectrometry (LC–MS/MS) identified 1693 proteins, some of which were common to all fractions and others of which were unique to specific fractions. Normalized spectral index (SIN) values obtained from these data were compared to evaluate both the reproducibility of the method and the efficiency of enrichment. SIN values between replicate fractions demonstrated a high correlation, confirming the reproducibility of the method. Correlation coefficients across the three fractions were significantly lower than those for the replicates, supporting the capability of DDF to differentially fractionate proteins into separate compartments. Subcellular annotation of the proteins identified in each fraction demonstrated a significant enrichment of cytoplasmic, cell membrane, and nuclear proteins in the three respective buffer system fractions. We conclude that DDF can be applied to frozen tissue to generate reproducible proteome coverage discriminating subcellular compartments. This demonstrates the feasibility of analyzing cellular compartment-specific proteins in archived tissue samples with the simple DDF method. 相似文献
78.
79.
Bartoszewski R Brewer JW Rab A Crossman DK Bartoszewska S Kapoor N Fuller C Collawn JF Bebok Z 《The Journal of biological chemistry》2011,286(48):41862-41870
80.