Alterations of mitochondrial protein assembly and jasmonic acid biosynthesis pathway in Honglian (HL)-type cytoplasmic male sterility rice.

It has been suggested that the mitochondrial chimeric gene orfH79 is the cause for abortion of microspores in Honglian cytoplasmic male sterile rice, yet little is known regarding its mechanism of action. In this study, we used a mass spectrometry-based quantitative proteomics strategy to compare the mitochondrial proteome between the sterile line Yuetai A and its fertile near-isogenic line Yuetai B. We discovered a reduced quantity of specific proteins in mitochondrial complexes in Yuetai A compared with Yuetai B, indicating a defect in mitochondrial complex assembly in the sterile line. Western blotting showed that ORFH79 protein and ATP1 protein, an F(1) sector component of complex V, are both associated with large protein complexes of similar size. Respiratory complex activity assays and transmission electron microscopy revealed functional and morphological defects in the mitochondria of Yuetai A when compared with Yuetai B. In addition, we identified one sex determination TASSELSEED2-like protein increased in Yuetai A, leading to the discovery of an aberrant variation of the jasmonic acid pathway during the development of microspores.

WITHDRAWN: Study on tolerance to ischemia-reperfusion injury and protection of ischemic preconditioning of type 1 diabetes rat heart.

The Publisher regrets that this article is an accidental duplication of an article that has already been published, http://dx.doi.org/10.1016/j.biomag.2010.09.004. The duplicate article has therefore been withdrawn.

Protective effects of rhu TNFR: Fc against the lipopolysaccharide induced intestinal damage of rats and its underlying mechanism / 药学学报

To investigate the protective effects of recombinant human tumor necrosis factor receptor II: IgG Fc fusion protein (rhu TNFR: Fc) against the lipopolysaccharide (LPS) induced intestinal damage of rats and its underlying mechanism. SD rats were randomly divided into four groups: control group, rhuTNFR: Fc group, LPS group and rhu TNFR: Fc + LPS group. Mean arterial pressure (MAP) was continuously monitored and the mortality rates were assessed. The levels of TNF-alpha and its bioactivity in the serum were assessed by ELISA and flow cytometry respectively. Pathologic changes of intestinal tissue were observed by HE staining. The rats of control and rhu TNFR: Fc group all survived with stable MAP, and the low level and bioactivity of TNF-alpha in the serum were maintained. While 83% of the rats in LPS group died by 6 h with the levels and bioactivity of TNF-alpha increasing significantly. In rhu TNFR: Fc + LPS group, the mortality rate of rats dropped to 33%. The TNF-alpha level increased compared with control group but its bioactivity decreased significantly compared with LPS group. The MPO activity and content of MDA decreased significantly. The status of pathological manifestation in the intestine was also ameliorated. These data suggest that rhu TNFR: Fc could protect rats from the acute intestine injury induced by LPS through ablating the rise in serum TNF-alpha level and bioactivity as well as anti-oxidation.

[Construction of T7 phage display library from the anther of Honglian hybrid line of rice].

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.

Effect of ryanodine receptor 2 gene silencing on ischemia-reperfusion injury of rat myocardial cells / 中华病理学杂志

<p><b>OBJECTIVES</b>To block the synthesis of ryanodine receptor 2 (RyR2) in myocardial cells by RNA interference and to investigate its biological impact on ischemia-reperfusion (I/R) in rat myocardial cells.</p><p><b>METHODS</b>Rat myocardial cells were isolated and cultured for an I/R model in vitro. RNA interference technique was used to block the synthesis of RyR2 in myocardial cells. Changes of LDH level, apoptosis, RyR2 mRNA expression and cytosolic Ca(2+) concentration were analyzed accordingly.</p><p><b>RESULTS</b>Myocardial cells after I/R manipolation were severely injuried (LDH leakage, 125 IU/L vs 12 IU/L, P < 0.05), apoptosis (60.1% vs 5.5%, P < 0.05), significant cytosolic Ca(2+) overload (21.2 vs 7.6, P < 0.05) and remarkable mitochondrial membrane potential loss (37.2 vs 85.1, P < 0.05). However, no visible change of RyR2 was observed (20.1 vs 22.7, P > 0.05). Pre-treatment with RyR2 specified siRNA demonstrated suppressed expression of RyR2 (6.8 vs 20.1, P < 0.05), increased mitochondrial membrane potential (55.8 vs 37.2, P < 0.05), attenuated cytosolic Ca(2+) overload (8.6 vs 21.2) and cellular apoptosis (31.2% vs 60.1%, P < 0.05).</p><p><b>CONCLUSION</b>RyR2 gene silencing enables to protect myocardial cells from I/R injury in vitro.</p>

[The editing sites in the transcripts of mitochondrial atp6 gene of maize sterile lines and maintainer line].

Using maize (Zea mays L.) cytoplasmic male-sterile lines T Huang Zao Si, C Huang Zao Si, S Huang Zao Si and maintainer line N Huang Zao Si as the plant materials, editing sites in the conservative area of mitochondrial atp6 gene transcripts of the 4 experimental materials' tassels, of which microspores had developed to uni-nucleate stage, were analyzed. The results showed that DNA sequences of the T, C and S male-sterile cytoplasms were completely unanimous, while being compared with the N-cytoplasm, all the sequences were similar except for the 27th and 28th nucleotides. However, the cDNA sequences of each cytoplasm were not always the same. By comparing DNA and cDNA sequences, we found that within the conservative area of atp6 gene transcripts there were 19, 22, 20 and 19 editing sites in the N, T, C and S cytoplasms, respectively. The 4 cytoplasms also shared 18 sites. The majority of the editings occurred at the 1st or the 2nd position of codons, which might alter the amino acid type. Most the shared editings were fully editing, and the 1st and the 19th sites were partially edited in nearly all cytoplasms, except for the 19th sites editing in the N-cytoplasm. The specific editings in each cytoplasm occurred in the form of partially editing. Thus the editing of atp6 gene in maize was not only sequence specific but also affected by cytoplasmic background. Furthermore, plant RNA editing was inclined to improve the predicted protein's hydrophobicity and enhance the conservation among species.

[Distribution and SNPs of the rice CMS-related gene in AA-genome of Oryza species].

The moiety of a chimeric gene in mitochondrial genome, orf79 and orfH79, probably related to BT-type and HL-type CMS of rice respectively, has 98% homology and only 4 nucleotide variation in DNA sequence. Of which, the former comes from Oryza sativa L., and the latter originates from Oryza rufipogon Griff. That means the orf79/ orfH79 may widely exist in Oryza species with AA genome. In order to investigate the distribution and difference of orf79/ orfH79 in the Oryza species, 190 cultivated rice accessions (including O. sativa and O. glaberrima) and 104 accessions of AA-genome Oryza wild species (including O. nivara, O. rufipogon, O. barthii, O. longistaminlata, O. glumaepatula, and O. meridion-alis) were detected with PCR amplification. Of which, 31 accessions mainly from AA-genome Oryza species were found to share the special amplified fragment with the control of Yuetai A and Shijin A. The special amplified fragments were all recovered and sequenced. Phylogenetic analysis based on DNA sequences showed that the 31 accessions were fallen into two groups, correspondingly representing HL-type and BT-type cytoplasm group. Further, the results revealed that the HL-type cytoplasm distributed mainly in annual O. nivara, and the BT-type cytoplasm centered in cultivated varieties or perennial O. rufipogon.

[Cytological observation on pollen abortion of genetic male sterile mutant induced by space flight in maize].

In order to understand the cytological mechanism of pollen abortion of genetic male sterile mutant induced by space flight in maize, the sister cross population were used for sterility analysis and cytological observation. Intact anther observation, isolated cells observation and paraffin section were adopted in this research. The results showed that pollen abortion occured mostly in dyad stage of meiosis in genetic male sterile mutant. The dyad were degenerated with abnormal shape. In late anther developing stage, the tapetal cells were giant vacuolated and delayed degeneration. The pollen mother cells (PMC) began to dissolve and degenerate in a few anther before meiosis.

Identification, genetic characterization, GA response and molecular mapping of Sdt97: a dominant mutant gene conferring semi-dwarfism in rice (Oryza sativa L.).

Semi-dwarfism is an important agronomic trait in rice breeding programmes. sd-1, termed the 'Green Revolution gene', confers semi-dwarf stature, increases harvest index, improves lodging resistance, and is associated with increased responsiveness to nitrogen fertilizer. It has contributed substantially to the significant increase in rice production. In this paper, a novel semi-dwarf mutant in rice is reported. Genetic analysis revealed that only a single dominant gene locus non-allelic to sd-1, temporarily designated Sdt97, is involved in the control of semi-dwarfism of the mutant. The semi-dwarfism of the mutant could be partly restored to the tall wild-type by application of exogenous GA3, suggesting that the mutant gene Sdt97 may be involved in the gibberellin (GA) synthesis pathway and not the GA response pathway in rice. A residual heterozygous line (RHL) population derived from a recombinant inbred line (RIL) was developed. Simple sequence repeat (SSR) and bulked segregation analysis (BSA) combined with recessive class analysis (RCA) techniques were used to map Sdt97 to the long arm of chromosome 6 at the interval between two STS markers, N6 and TX5, with a genetic distance of 0.2 cM and 0.8 cM, respectively. A contig map was constructed based on the reference sequence aligned by the Sdt97 linked markers. The physical map of the Sdt97 locus was defined to a 118 kb interval, and 19 candidate genes were detected in the target region. This is the first time that a dominant semi-dwarf gene has been reported in rice. Cloning and functional analysis of gene Sdt97 will help us to learn more about molecular mechanism of rice semi-dwarfism.

[Improvement of two-dimensional gel electrophoresis of total proteins from rice anthers].

This paper reported an improvement in 2-D gel electrophoresis of the proteome in Honglian cytoplasmic male sterile rice. An IPGphor unit with immobile pH gradient strips was used as the first dimension and SDS-PAGE as the second. The total anther proteins were extracted using TCA/acetone and then were washed 5-6 times with acetone till the proteins were white and clean, and then tributylphosphine and DTT were added into the rehydration buffer to improve the solubility of the proteins. The 2-D gel was stained by both methods of coomassie blue G-250 and silver. Extraction of proteins, pH of the strips and rehydration of the strips were optimized and compared. Higher repeatability and better separating protein pattern could be gained by this technique.

[A novel method of the genome-wide prediction for the target genes and its application].

Based on the protein databases of several model species, this study developed a new method of the Genome-wide prediction for the target genes, using Hidden Markov model by Perl programming. The advantages of this method are high throughput, high quality and easy prediction, especially in the case of multi-domains proteins families. By this method, we predicted the PPR and TPR proteins families in whole genome of several model species. There were 536 PPR proteins and 199 TPR proteins in Oryza sativa ssp. japonica, 519 PPR proteins and 177 TPR proteins in Oryza sativa L. ssp. indica, 735 PPR proteins and 292 TPR proteins in Arabidopsis thaliana, 6 PPR proteins and 32 TPR proteins in Cyanidioschyzon merolae. Synechococcus and Thermophilic archaebacterium did not have PPR proteins. By contrast, 10 TPR proteins were found in Synechococcus and 4 TPR proteins were found in Thermophilic archaebacterium. Moreover, of these results, some further bioinformatics analyses were conducted.

RFLP analysis for mitochondrial genome of CMS-rice.

Restriction fragment length polymorphism (RFLP) was used to analyze mitochondrial (mt) genome of cytoplasmic male sterility (CMS) rice. Differences were observed among mitochondrial genomes of the sterile line (A) and maintain line (B) of nine types of CMS rice; Mitochondrial genomic differences were also detected between A and B in many functional gene regions. Even the materials with the same nucleic background have differences in their mtDNA. This provides molecular evidence for the cytoplasmic heterogeneity and the CMS mechanism research.

[Preliminary proteomics analysis of the total proteins of HL Type cytoplasmic male sterility rice anther].

The proteins of HL type cytoplasmic male sterility rice anther of YTA (CMS) and YTB (maintenance line) were separated by two-dimensional electrophoresis with immobilized ph (3-10 non-linear) gradients as the first dimension and SDS-PAGE as the second. The silver-stained proteins spots were analyzed using Image Master 2D software, there were about 1800 detectable spots on each 2D-gel, and about 85 spots were differential expressed. With direct MALDI-TOF mass spectrometry analysis and protein database searching, 9 protein spots out of 16 were identified. Among those proteins, there were Putative nucleic acid binding protein, glucose-1-phosphate adenylyltransferase (ADP-glucose pyrophosphorylase, AGPase) (EC: 2.7.7.27) large chain, UDP-glucuronic acid decarboxylase, putative calcium-binding protein annexin, putative acetyl-CoA synthetase and putative lipoamide dehydrogenase etc. They were closely associated with metabolism, protein biosynthesis, transcription, signal transduction and so on, all of which are cell activities that are essential to pollen development. Some of the identified proteins, i.e. AGPase, putative lipoamide dehydrogenase and putative acetyl-CoA synthetase were deeply discussed on the relationship to CMS. AGPase catalyzes a very important step in the biosynthesis of alpha 1,4-glucans (glycogen or starch) in bacteria and plants: synthesis of the activated glucosyl donor, ADP-glucose, from glucose-1-phosphate and ATP. The lack of the AGPase in male sterile line might directly result in the reduction of starch, and the synthesis of starch was the most important processes during the development of pollen. In present research, the descent or reduction of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase seemed involved in pollen sterility in rice. The degeneration and formation of various tissues during pollen development may impose high demands for energy and key biosynthetic intermediates. Under such conditions, the TCA cycle needs to operate fully, because the TCA cycle is an important source for many intermediates required for biosynthetic pathways, in addition to performing an oxidative, energy-producing role. Thus, it seemed reasonable to infer that the decrease of putative lipoamide dehydrogenase and putative acetyl-CoA synthetase in anther might prevent the conversion of pyruvate into acetyl-CoA, and as a result, the TCA cycle could no longer operate at a sufficient rate to meet all requirements in anther cells, leading to pollen sterility. This study gave new insights into the mechanism of CMS in rice and demonstrated the power of the proteomic approach in plant biology studies.

Effect of high glucose on expression of MIP-2 and MCP-1 in macrophages cultured in vitro / 上海第二医科大学学报

0.05).The protein and mRNA expression of MIP-2 in high glucose group significantly increased after culture for 4 h,and guadually decreased then.The protein and mRNA expression of MCP-1 began to increase significantly after culture for 8 h,reached peak at 12 h,and slightly decreased after culture for 24 and 48 h. Conclusion High glucose promotes the protein and mRNA expression of MIP-2 and MCP-1 from mouse peritoneal macrophages cultured in vitro,which indicates that high glucose may delay the wound healing by increasing the expression of chemokines in diabetic mice.

Effect of folic acid on plasma homocysteine in patients with sudden sensorineural hearing loss / 上海第二医科大学学报

Objective To study the effect of folic acid on decreasing level of plasma total homocysteine(tHcy)in patients with sudden sensorineural hearing loss(SSHL) and the optimal dosage of folic acid. Methods Ten randomized controlled trials involving treatment data on 210 patients with SSHL were retrospectively studied.They were divided into seven groups according to the daily dosage of folic acid: group A to group G,0.2 mg,0.4 mg,0.8 mg,2.0 mg,5.0 mg,10.0 mg and 15.0 mg,respectively.Besides oral administration of folic acid,Vitamine B6 and B12were supplemented,and other routine treatment were performed.Fluorescence polarization immunoassay was employed to detect the plasma tHcy before and 3 months after the treatment.And the data of plasma tHcy of 210 patients without SSHL were collected and served as controls.The levels of plasma tHcy were statistically analysed between the SSHL group and control group and among group A to group G. Results The level of plasma tHcy in the SSHL group was significantly higher than that in the control group,(18.07?1.58)?mol/L vs(13.63?1.33) ?mol/L(P0.05). Conclusion The levels of plasma tHcy are significantly increased in SSHL.Folic acid may play an important role in decreasing the levels of tHcy in patients with SSHL,and a dosage of 10 mg/d for oral adminstration is well suggested.

Effect of Myocardial Ischemic Preconditioning on Ischemic/Reperfusion Injury in High Blood Fat Rat / 上海第二医科大学学报

Objective To study the effect of myocardial ischemic preconditioning on activity of ATPase and creatine kinase(CK) in high blood fat rat. Methods High blood fat rat mode was established from SD rats.The rats were randomly divided into three groups: ischemic preconditioning(IPC), ischemic/reperfusion(I/R) and control group.The activity of CK in coronary outflow,the activity of malonyldialdehyde(MDA),superoxide dismutase(SOD),glutathione perodxidase(GSH-Px) and ATPase in myocardium were dectected. Results CK and MDA were significantly less in IPC group than those in I/R group.In IPC group,the activity of SOD,GSH-Px,Na~(+)-K~(+)-ATPase,Ca~(2+)-ATPase and Ca~(2+)-Mg~(2+)-ATPase were much higher than those in I/R group. Conclusion Myocardial ischemic preconditioning can protect high blood fat rat from ischemic/reperfusion injury.

[The discovery of a specific DNA fragment associated with maize cytoplasmic male sterility and its differential display].

Three pairs of PCR primers were designed according to the mitochondrial DNA sequence. PCR amplification was applied to 3 sets of isonuclear alloplasm materials and 3 sets of isoplasm allonuclear materials. Multiplex PCR and general PCR protocol were adopted with total genomic DNA. As for the primers having detected polymorphsim between male sterility and its maintainers, differential display was conducted with mRNA from different development stage of microspore. The results showed as follows: with total genomic DNA template, primer P1-P2 has amplified a specific fragment only in all the male sterile materials, primer P5-P6 has amplified a specific fragment only in maintainer Huangzaosi, primer P3-P4 has no amplification in all the experiment materials. So primer P1-P2 can be used to distinguish male sterile cytoplasm and normal cytoplasm. RT-PCR was conducted with primer P1-P2 in inbred line huangzaosi and 48-2 with male sterile cytoplasm and normal cytoplasm, mRNA was separately isolated from tetrad stage, uninucleate stage and binucleate stage of microspore development, cDNA was obtained with random hexanucleotide primers. With the cDNA template, specific amplified fragments were also detected by primer P1-P2 in the male sterile materials at different development stage of microspore, but there was no amplification by primer P1-P2 in the 2 maintainer lines. This result indicated that primer P1-P2 can be transcripted at 3 development stages of microspore in all male sterile materials, and same transcript was produced by primer P1-P2 among all male sterile materials include 3 sets of isonuclear alloplasm and 3 sets of isoplasm allonuclear. It was suggested from this experiment that the specific DNA sequence detected by primer P1-P2 in all male sterile material total genomic DNA might be related to the cytoplasmic male sterile character.

[Isolation and analysis of isolation of differentially-methylated fragment CIDM7 in rice induced by cold stress].

By using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites, AFLP analysis is carried out to analyse differences of the methylation state of anonymous CCGG sequences between cold-treated(5 degrees for 2 d) and control 9311(Oryza sativa L.) leaves DNA. Demethylation or De novo methylation induced by cold stress are found in some CCGG sites. Some differentially-methylated encoding sequences are isolated, of which a fragment named CIDM7 (cold-inducing differential methylation) is sequenced and executed BLAST against Nipponbare cDNA data, thus we obtain its full length cDNA of 1422 bp encoding a hypothetical F-box protein(425 aa).CIDM7 is demethylated induced by cold stress. Northern blot analysis confirms that CIDM7 gene expression is up-regulated by cold stress. CIDM7 is single copy and lacated on the Nipponbare chromosome 10 (12.56 Mb-12.57 Mb).

[Genetic polymorphism of bluegrass cultivars detected by RAPDs].

Kentucky bluegrass (Poa pratensis L.) is a hardy, persistent forage and turf grass adapted to a wide range of soils and climates. Its ever-increasing adoption in highly cared-for sports fields has attracted the attention of many seed companies. However in the past, the breeding of elite varieties was often hampered by the extreme complexity of the genome. The polymorphism is important for broading the genetic basis and may be exploited for application of heterosis. The genetic relationship of 16 bluegrass cultivars, including 15 accessions Kentucky bluegrass cultivars and 1 entries Canada bluegrass (Poa compressa L.) cultivar from different breeding company were analyzed using 25 RAPD markers. 25 RAPD primers generated 218 bands, of which 196 bands (89.91%) were polymorphism. It showed that the Canada Bluegrass was separated from other Kentucky Bluegrass and genetic polymorphism in the Kentucky Bluegrass cultivars was low, the genetic similarity among the cultivars fell between 66%-98%. Dendrogram obtained using these molecular markers were partly in agreement with their separated morphologic character. Cultivars from the same company were not clustered in one group.

[RAPD Identification of Three Types of Three-lines Hybrid Rice with Defferent Male Sterile Cytoplasms and Their Parent Lines.].

The RAPD analysis was conducted on genome DNA from 21 sets of rice, including 6 three-line hybrid rice combinations, separately derived from three different kinds of cytoplasmic male sterile (CMS) lines and their related parents. Out of 264 random primers screened first, 25 primers displayed well in polymorphisms. It was shown that only 7 bands amplified respectively from 7 primers were enough to discriminate the different types of CMS, the hybrid combinations and their parents.