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901.
Kazutoshi Nishijima Mai Tanaka Yusuke Sakai Chihiro Koshimoto Masatoshi Morimoto Teruo Watanabe Jianglin Fan Shuji Kitajima 《Cryobiology》2014
We investigated the effects of antifreeze protein (AFP) III supplementation on the cryopreservation of rabbit sperm cells and embryos. Ejaculated semen was collected from male Japanese white (JW) rabbits and divided into four AFP-supplemented groups (0.1 μg/ml, 1 μg/ml, 10 μg/ml, 100 μg/ml) and one control group with no AFP-supplementation. The semen samples were treated with egg-yolk HEPES extender containing 6% acetamide before the sperm was cooled from room temperature to 5 °C, then packed into sperm straws. The straws were frozen in steam of liquid nitrogen (LN2) and then preserved in the LN2. The motility of the sperm after thawing in 37 °C water was analyzed. The percentage of rapidly motile sperm in the 1 μg/ml AFP group was significantly higher than in the control group. Morulae were collected from female JW rabbits and divided into three AFP-supplemented groups (100 ng/ml, 500 ng/ml, 1000 ng/ml) and one control group. The morulae, immersed in an embryo-freezing solution (M199-HEPES containing 20% ethylene glycol, 20% dimethylsulfoxide, 10% fetal bovine serum and 0.25 M sucrose), were packed into open pulled embryo straws and vitrified in LN2. The frozen embryos were thawed in the embryo-freezing solution, and the rates of embryo survival and development to blastocyte stage were analyzed after incubation for 72 h. The development rate of the embryos in the 500 ng/ml AFP group was significantly higher than in the control group, but that in the 1000 ng/ml AFP group was significantly lower. In conclusion, the appropriate dose of AFP III increased the number of rapidly motile sperm and embryo survival following freezing and thawing. The results suggest that supplementation with AFP III can increase the efficiency of cryopreservation of rabbit sperm cells and embryos. 相似文献
902.
Sumei Liao Marlise I. Klein Kyle P. Heim Yuwei Fan Jacob P. Bitoun San-Joon Ahn Robert A. Burne Hyun Koo L. Jeannine Brady Zezhang T. Wen 《Journal of bacteriology》2014,196(13):2355-2366
Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA. 相似文献
903.
Xiaofeng Bao ?sa Gylfe Gail L. Sturdevant Zheng Gong Shuang Xu Harlan D. Caldwell Mikael Elofsson Huizhou Fan 《Journal of bacteriology》2014,196(16):2989-3001
Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value. 相似文献
904.
Rapid and reliable detection of 11 food-borne pathogens using thin-film biosensor chips 总被引:1,自引:0,他引:1
Sulan Bai Jinyi Zhao Yaochuan Zhang Wensheng Huang Shi Xu Haodong Chen Liu-Min Fan Ying Chen Xing Wang Deng 《Applied microbiology and biotechnology》2010,86(3):983-990
Traditional methods for identifying food-borne pathogens are time-consuming and laborious, so it is necessary to develop innovative
methods for the rapid identification of food-borne pathogens. Here, we report the development of silicon-based optical thin-film
biosensor chips for sensitive detection of 11 food-borne pathogens. Briefly, aldehyde-labeled probes were arrayed and covalently
attached to a hydrazine-derivatized chip surface, and then, biotinylated polymerase chain reaction (PCR) amplicons were hybridized
with the probes. After washing and brief incubation with an antibiotin immunoglobulin G–horseradish peroxidase conjugate and
a precipitable horseradish peroxidase substrate, biotinylated chains bound to the probes were visualized as a color change
on the chip surface (gold to blue/purple). Highly sensitive and accurate examination of PCR fragment targets can be completed
within 30 min. This assay is extremely robust, sensitive, specific, and economical and can be adapted to different throughputs.
Thus, a rapid, sensitive, and reliable technique for detecting 11 food-borne pathogens was successfully developed. 相似文献
905.
J.‐M. Fan J. Luo G.‐P. Zhang L. Chen M. Teng M.‐F. Yang L. Wang C.‐Q. Wang 《Letters in applied microbiology》2010,51(1):11-17
Aims: Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine. Methods and Results: Genomic RNA was separated from JEV isolated strain Henan‐09‐03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT‐PCR and cloned into the pMD19‐T‐Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET‐32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column‐based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 ( AF495589 ), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific. Conclusions: The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit. Significance and Impact of the Study: As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody. 相似文献
906.
IscA is a key member of the iron-sulfur cluster assembly machinery in prokaryotic and eukaryotic organisms; however, the physiological function of IscA still remains elusive. In the present paper we report the in vivo evidence demonstrating the iron-binding activity of IscA in Escherichia coli cells. Supplement of exogenous iron (1 μM) in M9 minimal medium is sufficient to maximize the iron binding in IscA expressed in E. coli cells under aerobic growth conditions. In contrast, IscU, an iron-sulfur cluster assembly scaffold protein, or CyaY, a bacterial frataxin homologue, fails to bind any iron in E. coli cells under the same experimental conditions. Interestingly, the strong iron-binding activity of IscA is greatly diminished in E. coli cells under anaerobic growth conditions. Additional studies reveal that oxygen in medium promotes the iron binding in IscA, and that the iron binding in IscA in turn prevents formation of biologically inaccessible ferric hydroxide under aerobic conditions. Consistent with the differential iron-binding activity of IscA under aerobic and anaerobic conditions, we find that IscA and its paralogue SufA are essential for the iron-sulfur cluster assembly in E. coli cells under aerobic growth conditions, but not under anaerobic growth conditions. The results provide in vivo evidence that IscA may act as an iron chaperone for the biogenesis of iron-sulfur clusters in E. coli cells under aerobic conditions. 相似文献
907.
Liyue Wang Fan Gong Xiaoyan Dong Wei Zhou Qiutang Zeng 《Molecular and cellular biochemistry》2010,337(1-2):159-166
Endothelium-derived nitric oxide (NO) is a cytoprotective molecule to prevent endothelial cells (ECs) from apoptosis. CREB-binding protein (CBP) is involved in the apoptotic pathway in several tumor cells, however, little is known whether CBP is associated with apoptosis in ECs and the apoptotic effect of CBP on ECs is regulated by NO. Therefore, the purpose of the present study was to investigate whether silencing CBP expression could affect the sensitivity of ECs toward apoptotic stimuli and determined the role of NO. In this study, we found that when CBP expression was silenced by RNA interference, ECs were more prone to apoptosis under serum deprivation, whereas the apoptosis was not significantly induced in the serum-containing condition. The increased apoptosis is paralleled by a reduction of NO, and the apoptosis was reversed by NO donors, suggesting an important role of NO. Furthermore, CBP silencing decreased NO production by downregulating the endothelial NO synthase (eNOS) expression in a dose-dependent manner. These results indicated that CBP silencing is associated with decreased eNOS expression and NO production, and therefore concomitantly increased the sensitivity of ECs toward apoptosis. 相似文献
908.
Z. W. Wang X. Y. Li Z. L. Tang S. L. Yang Z. Z. Ying T. Fu B. Fan Y. L. Mu H. Ao K. Li 《Molecular biology reports》2010,37(7):3393-3400
F-box proteins are quite significant ubiquitin-proteasome pathway regulators in eukaryotic cells. FBXO40, a member of this large family, alters its expression pattern in muscle atrophy. Here we isolated most of the verified porcine
FBXO40 coding sequence (CDS) (2258 bp) and assigned it to the porcine chromosome 13q4.1-4.6 by using the INRA-Minnesota porcine
radiation hybrid panel, and we also explored the tissue expression distributions, which is relatively high in longissimus
dorsi muscle, heart, low in kidney, small intestine, brain, hypophysis, lymphonode, thymus, spleen, large intestine, ovary,
stomach, and undetectable in testis, liver, uterus and thyroid gland. Inferring phylogenetic tree was constructed to study
the evolutionary implications. Moreover, a HindII (HincII)-RFLP (A/C) polymorphism in 3′-untranslated region (3′-UTR) of porcine FBXO40 gene was demonstrated by sequencing and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis. Statistical analysis
result of this polymorphism showed that the allele A was predominant in all detected indigenous breeds, but C in western introduced
commercial breeds. The SNP was further analyzed in our experimental pig population including Tongcheng, Landrace, Large White,
and crossbreds of Large White × (Landrace × Tongcheng) and Landrace × (Large White × Tongcheng). The association analysis
results indicated that the A/C base substitution was associate with some hematological indexes, the hemoglobin concentration
(P < 0.0001), mean corpuscular volume hemoglobin concentration (P = 0.0002) and mean corpuscular volume (P = 0.0138). 相似文献
909.
Yaping Xin Linsen Zan Yongfeng Liu Hongyu Liu Wanqiang Tian Yueyuan Fan Lei Huang 《Molecular biology reports》2010,37(6):3043-3049
Six Y-STR loci (UMN0929, UMN0108, UMN0920, INRA124, UMN2404 and UMN0103) were analyzed using 576 healthy and unrelated males
and 10 females of the Qinchuan cattle population in Chinese Shaanxi Province. Allele frequency, gene diversity, the polymorphic
information content, and the number of effective gene were calculated. All loci were in accordance with the Hardy–Weinberg
equilibrium (P > 0.05). The population data were compared with published data of other cattle breeds, suggesting that Qinchuan cattle were
originated primarily from Bos Taurus. Results are valuable for individual identification, paternity testing, and origin analysis of Qinchuan cattle breed. 相似文献
910.
Vallisneria natans and Vallisneria spinulosa are two morphologically very similar and sympatrically dominant submerged macrophytes in lakes of the middle-lower reaches of the Yangtze River. Genetic variation was compared based on a total of 196 individuals from six V. natans populations and 201 individuals from seven V. spinulosa populations. Using eight ISSR primers, a total of 139 and 129 DNA fragments were generated with 121 being polymorphic in V. natans and 99 in V. spinulosa. The two species maintained higher genetic variation both at the species and population levels in comparison with other aquatic macrophytes. A higher level of genetic diversity among populations was found in V. natans than in V. spinulosa: the percentage of polymorphic loci (PPL) in V. natans was 52-62% vs. 38-47% in V. spinulosa; gene diversity (H) was 0.21 in V. natans vs. 0.17 in V. spinulosa.Both an analysis of molecular variance (AMOVA) and F-estimation (FST) showed that most of the total genetic variation resided within populations of both species (AMOVA: 85% and 80%; FST: 0.132 and 0.202), indicating low genetic differentiation between populations. Principal coordinates analysis (PCA) indicated evident gene flow between populations of both species. The outcrossing reproductive mode and pervasive gene flow might have played important roles in maintaining high genetic diversity and in shaping low population differentiation of the two Vallisneria species, while the extent of clonal growth might account for the different levels of population divergence between them. 相似文献