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1.
Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation. 相似文献
2.
Andrew W. Saxe Ji-Won Yoon Phillip Gorden Murray F. Brennan 《In vitro cellular & developmental biology. Plant》1982,18(10):884-890
Summary Dispersed cells from both fresh and cryopreserved human insulinoma have been maintained in cell culture. Initial yield of
viable cells was 50% for fresh and 25% for cryopreserved tissue. Viability of cells in culture was documented by increasing
numbers of cells (doubling time approximately 5 d initially and 2 d at the sixth subculture for both fresh and cryopreserved
tissue) and continued release of insulin over time (approximately 100 ng/ml per 105 cells at 10 d and 175 ng/ml per 105 cells at 30 d of culture for both fresh and cryopreserved tissue). Evidence that cells growing in culture were beta cells
was provided by: (a) recovery of intracellular and extracellular immunoreactive insulin (IRI), (b) electron microscopic morphology,
and (c) immunohistochemical staining. Cells from fresh insulinoma incubated with increasing concentrations of extracellular
glucose released increasing amounts of IRI up to approximately 15 mM glucose, which paralleled changes in plasma insulin obtained during a preoperative glucose tolerance test.
Under an Intergovernmental Personnel Act Exchange from the Department of Surgery, University of California, Davis, Sacramento
Medical Center. 相似文献
3.
Previous studies by us and others established that cell-cell adhesion is mediated by specific carbohydrate-to-carbohydrate
interaction (CCI). Those previous studies were based on various biochemical and biophysical approaches, including the use
of labeled glycosyl epitopes with fluorescent tag. However, these methods ideally require that the glycosyl epitope must be
fixed to a solid phase molecule, preferably with multivalency. The purpose of the present study is to establish a CCI process
using specific glycosyl residues conjugated to biotinylated diaminopyridine (BAP), and to observe: (i) clear occurrence of
homotypic CCI between “Os Fr.B” having 5–6 GlcNAc termini, vs. absence of such homotypic CCI between “Os Fr.1” having 2 GlcNAc termini; (ii) occurrence of heterotypic CCI between GM3
ganglioside and Os Fr.B, vs. absence of such heterotypic CCI between GM3 and Os Fr.1. Interaction between Os Fr.B-BAP conjugate and Os Fr.B-ceramide
mimetic (Os Fr.B-mCer) was demonstrated based on two experiments: (i) dose-dependent binding of Os Fr.B-BAP conjugate to polystyrene
plates coated with Os Fr.B-mCer was observed in the presence of bivalent cation, a prerequisite for all CCI processes, and
such binding was abolished by EDTA; (ii) binding between equal nanomolar Os Fr.B-BAP and Os Fr.B-mCer was inhibited by mM
concentration Os Fr.B without conjugate, in dose-dependent manner. Thus, cell adhesion processes based on homotypic CCI between
N-linked glycans having multiple GlcNAc termini, and heterotypic CCI between GM3 and such glycans, were clearly observed using
BAP conjugates of glycosyl epitopes. 相似文献
4.
Growth hormone induces two mRNA species of the serine protease inhibitor gene family in rat liver 总被引:2,自引:0,他引:2
In order to study the molecular actions of growth hormone on gene expression, we have cloned and characterized two unique, but related, cDNA sequences from rat liver, lambda Spi-1 and lambda Spi-2. These two cDNA sequences are complementary to rat hepatic mRNA species previously designated as Spots 3 and 20 when assayed by in vitro translation and two-dimensional gel electrophoresis. By Northern blot, the two mRNAs are both 1900 bases in length and growth hormone administered to hypophysectomized rats increases the levels of both of these mRNAs. In contrast, the combined administration of thyroxine, corticosterone, and dihydrotestosterone to hypophysectomized rats did not augment these mRNAs. The simultaneous administration of all four hormones resulted in a level greater than that observed for animals treated with growth hormone alone. Analysis of genomic DNA suggests the presence of two similar, but not identical, genes. DNA sequencing of lambda Spi-1 and lambda Spi-2 revealed that they were 90% homologous at the nucleotide level and 87% homologous at the amino acid sequence level. lambda Spi-2 has 78% homology with mouse contrapsin, 60% with human alpha 1-antichymotrypsin, and 51-55% with alpha 1-antitrypsins, all members of the serine protease inhibitor gene family. The nucleotide and deduced amino acid sequences of lambda Spi-1 and lambda Spi-2 which align with the reactive centers of known members of this family differ substantially from each other and from other members of the family. The difference in the reactive center suggests that the specificity or function of these proteins may differ from other members of serine protease inhibitor gene family. 相似文献
5.
Summary The effects of phosphate on -amylase fermentation byBacillus amyloliquefaciens were investigated. It was observed through batch culture that optimal phosphate level which maximizes -amylase biosynthesis exists. High concentration of phosphate level promotes maltose uptake and growth of the microorganism, while high maltose uptake rate in the microorganism at the same time represses the enzyme biosynthesis presumably due to catabolite repression inside the microorganism. In continuous cultivation, a steady state of -amylase biosynthesis was obtained by maintaining phosphate level at a certain level. In fed-batch culture, by intermittant feeding of phosphate as well as maltose, higher activity of -amylase in the broth was obtained compared to the result from single nutrient feeding. 相似文献
6.
Heat generation in laser irradiated tissue 总被引:1,自引:0,他引:1
A J Welch J A Pearce K R Diller G Yoon W F Cheong 《Journal of biomechanical engineering》1989,111(1):62-68
Many medical applications involving lasers rely upon the generation of heat within the tissue for the desired therapeutic effect. Determination of the absorbed light energy in tissue is difficult in many cases. Although UV wavelengths of the excimer laser and 10.6 microns wavelength of the CO2 laser are absorbed within the first 20 microns of soft tissue, visible and near infrared wavelengths are scattered as well as absorbed. Typically, multiple scattering is a significant factor in the distribution of light in tissue and the resulting heat source term. An improved model is presented for estimating heat generation due to the absorption of a collimated (axisymmetric) laser beam and scattered light at each point r and z in tissue. Heat generated within tissue is a function of the laser power, the shape and size of the incident beam and the optical properties of the tissue at the irradiation wavelength. Key to the calculation of heat source strength is accurate estimation of the light distribution. Methods for experimentally determining the optical parameters of tissue are discussed in the context of the improved model. 相似文献
7.
Identification of an unusual deletion within homologous repeats of human reticulocyte beta-spectrin and probable peptide polymorphism 总被引:1,自引:0,他引:1
We screened two different human reticulocyte cDNA libraries with beta-spectrin(beta Sp)-specific polyclonal antibodies and with our original radiolabeled human BSP cDNA probe (encoding beta Sp). Of the 20 independent clones, the largest had about a 2.5-kb insert corresponding to the deduced amino acid (aa) sequence of the beta-7 to beta-14 repetitive segments. Among these segments, segment 12 was 7 aa shorter than the other repetitive segments. We showed that this truncation was not a result of (i) cloning artifact, (ii) alternate splicing, or (iii) common genomic polymorphism by additional examination of 14 individual human chromosomes. Recently, another laboratory described the BSP nucleotide (nt) sequence overlapping partially with our sequence. These overlapping sequences were homologous with the exception of two nt differences at the positions 1342 and 1514. The discrepancy at nt 1342 changes the His to Arg. This newly derived probe has been used to find an additional example of BSP restriction fragment length polymorphism. 相似文献
8.
9.
R Dahiya B Boyle B C Goldberg W H Yoon B Konety K Chen T S Yen W Blumenfeld P Narayan 《Biochimie et biologie cellulaire》1992,70(7):548-554
Metastatic variants of human prostatic adenocarcinoma cell lines (DU-145, LNCaP, and ND-1) were studied by using soft agar colony forming efficiency, nude mice tumorigenicity, in vitro invasion assay, and type IV collagenase assay. The DU-145 and ND-1 cell line showed higher metastatic potential than LNCaP. Lipids from DU-145, ND-1, and LNCaP cells were extracted and analyzed by thin-layer chromatography and gas-liquid chromatography. The major lipids were phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, fatty acids, and cholesterol. The sphingomyelin level was significantly higher in highly metastatic cells (DU-145 and ND-1) compared with the lower metastatic variant (LNCaP). The increase in the synthetic pathway and decrease in degradation pathway of sphingomyelin in microsomal fractions was sufficient to account for the measured increase in sphingomyelin in DU-145 cells compared with LNCaP cells. The major fatty acids of these lipids were palmitic (16:0), stearic (18:0), oelic (18:1), and arachidonic acid (20:4). The arachidonic acid level was significantly decreased in DU-145 and ND-1 compared with LNCaP cells. Electron microscopic studies showed no significant changes in the morphology of DU-145, ND-1, and LNCaP cells. The results of these investigations demonstrate for the first time that sphingomyelin and arachidonic acid contents are different in high and low metastatic variants of human prostatic adenocarcinoma cell lines. 相似文献
10.
We describe dideoxy fingerprinting (ddF), a hybrid between dideoxy sequencing and SSCP that can detect the presence of single base and other sequence changes in PCR-amplified segments. As implemented herein, ddF involves a Sanger sequencing reaction with one dideoxynucleotide followed by nondenaturing gel electrophoresis. When ddF was used to examine segments of the human factor IX gene, 84 of 84 different mutations were detected with a very low rate of false positive signals. The approximate locations of the sequence changes could be determined from the ddF pattern and samples with different sequence changes had different fingerprints. In addition, large segments could be amplified and rapidly screened by ddF in multiple smaller subsegments. The patterns observed with ddF are instructive in that they suggest an inherent limitation in the detection of certain mutations by SSCP. 相似文献