Lipid Content and Composition of Oocytes from Five Coral Species: Potential Implications for Future Cryopreservation Efforts

Given the previously documented importance of lipid concentration and composition in the successful cryopreservation of gorgonian corals, these parameters were assessed in oocytes of five species of scleractinian coral; Platygyra daedalea, Echinopora gemmacea, Echinophyllia aspera, Oxypora lacera and Astreopora expansa. Wax esters, phosphatidylethanolamine, phosphatidylcholine, and fatty acids were all measured at detectable levels, and the latter were produced at significantly elevated quantities in E. gemmacea, E. aspera, and O. lacera. On the other hand, phosphatidylethanolamine, phosphatidylcholine, and wax ester were found at significantly higher concentrations in A. expansa oocytes. Triacylglycerol was not present in any species. Interestingly, the total lipid content of oocytes from all five scleractinians was significantly lower than that of oocytes of two gorgonian species, Junceella juncea and Junceella fragilis. As higher total lipid concentrations may be correlated with greater degrees of cellular membrane fluidity at lower temperatures, it stands to reason that gorgonian coral oocytes may be more likely to survive the cryopreservation process than oocytes of scleractinian corals.     

Cloning and characterization of lactate dehydrogenase C4 from Ochotona curzoniae

Lactate dehydrogenase C4 (LDH-C4) is considered to be a good target protein for the development of contraceptive drugs. To develop contraceptive rodenticide against pika (Ochotona curzoniae) LDH-C4, the pika LDH-C gene was cloned and expressed in Escherichia coli. The recombinant protein was purified and characterized. The cDNA of pika LDH-C gene was cloned by the RACE method. The cDNA was 1498 bp in length and contained an ORF of 996 bp, which encoded a polypeptide of 332 amino acids. The ORF of pika LDH-C was introduced in E. coli and expressed with no fusion tags added. The recombinant LDH-C4 protein was purified by heating, affinity chromatography and ion-exchange chromatography. The recombinant pika LDH-C4 was a tetramer with a molecular weight of approximately 140 kDa, and it had temperature-dependent catalytic activity, as it was thermally stable up to 60°C. The optimal pH values in the forward and backward reactions were around 7.48 and 10.28, respectively. The apparent Michaelis constants for pyruvate and lactate were 51.2 ± 3.8 and 8568.8 ± 409 μM respectively. The inhibition constant for oxalic acid was 11.8 ± 3.5 mM. This study laid a solid foundation for contraceptive rodenticide development against pika LDH-C4.     

The Potential Neuroprotection Mechanism of GDNF in the 6-OHDA-Induced Cellular Models of Parkinson’s Disease

The glial cell line-derived neurotrophic factor (GDNF) potential as a therapeutic agent for the treatment of Parkinson’s Disease (PD) has been extensively explored. However, the mechanism of the GDNF neuroprotective effects is still unclear. In this study, the neuroprotective mechanism of the GDNF in the PD cellular models, which was obtained by the 6-hydroxydopamine (6-OHDA)-induced dopaminergic (DA) cell line MN9D damage was investigated by microarray. Interestingly, 54 constitutively increased or decreased genes were detected, 17 of which have not been reported previously. The expression of 5 up-regulated and 5 down-regulated genes which displayed the most obvious changes compared to the no GDNF treatment cells and was previously proven to be related to cell survival was validated by real-time PCR and western blot. Moreover, the up-regulated gene Ager and down-regulated gene Ccnl2 which were related to the PI-3K/Akt signaling pathway, but not researched in the neuron-cells, were investigated by overexpression and RNA interference. Overexpression of Ager or knockdown the expression of Ccnl2 decreased the damage to MN9D cells caused by 6-OHDA and reduced their apoptosis. All these results suggested that the protective effects of the GDNF on the 6-OHDA damaged MN9D cells could be understood by enhancing the expression of the apoptosis inhibiting genes and decreasing the expression of the apoptosis promoting genes. Thus, this study might provide a number of specific candidates and potential targets to investigate the protective mechanism of GDNF in DA neurons.     

Study of cell killing and morphology on S180 by ultrasound activating hematoporphyrin derivatives

The inhibition of ascitic S180 and induced sarcoma 180 in vivo was studied with the combination of hematoporphyrin derivatives (HpD) and ultrasound (US) at the frequency of 1.1 MHz and different intensities by light microscopy observation, electronic microscopy observation, cytochemical analysis and fluorescence labeling. The present study indicated that the injury of ascitic S180 increased as time passed and the inhibitory effect was stronger in US plus HpD group than that in other groups. Our results also indicated that the changes in cell structure, cytochrome C oxidase activity, the degradation and missing of DNA were the important factors that inhibited the tumor cell growth and even induced celldeath. The phenomenon of apoptosis of tumor cells indicated that cell death andinduced apoptosis exist in the treatment of sonodynamic therapy (SDT). Our study investigated the mechanism underlying the killing effect of S180 induced by USactivating HpD by the observation of cell morphology and dynamic changes from seminal injury to succeeded injury even to death. It would provide rich referencefor the study of SDT.     

Alternative nucleophilic residues in intein catalysis of protein splicing

Protein-splicing inteins are widespread in nature and have found many applications in protein research and engineering. The mechanism of protein splicing typically requires a nucleophilic amino acid residue at both position 1 (first residue of intein) and position +1 (first residue after intein), however it was not clear whether or how the three different nucleophilic residues (Cys, Ser, and Thr) would work differently at these two positions. To use intein in a target protein of interest, one needs to choose an intein insertion site to have a nucleophilic residue at position +1, therefore it is desirable to know what nucleophilic residue(s) are preferred by different inteins. In this study we began with a statistical analysis of known inteins, which showed an unequal distribution of the three nucleophilic residues at positions 1 and +1, and then subjected six different mini-inteins to site-directed mutagenesis to systematically test the functionality of the three nucleophilic residues at the two positions. At position 1, most natural inteins had Cys and none had Thr. When the Cys at position 1 of the six inteins was mutated to Ser and Thr, the splicing activity was abolished in all except one case. At position +1, Cys and Ser were nearly equally abundant in natural inteins, and they were found to be functionally interchangeable in the six inteins of this study. When the two positions were studied as 1/+1 combination, the Cys/Ser combination was abundant in natural inteins, whereas the Ser/Cys combination was conspicuously absent. Similarly, all of the six inteins of this study spliced with the Cys/Ser combination, whereas none spliced with the Ser/Cys combination. These findings have interesting implications on the mechanism of splicing and the selection of intein insertion sites, and they also produced two rare mini-inteins that could splice with Thr at position +1.     

DNA甲基化在动植物遗传育种中的研究进展

DNA甲基化是真核生物表观遗传学重要的机制之一,对基因转录水平的表达具有重要的调控作用。近年来,DNA甲基化在动植物遗传育种领域的研究引起了人们广泛的关注。我们从DNA甲基化与基因的表达调控、动植物基因组的甲基化状态、甲基敏感扩增片段多态性方法、DNA甲基化与杂种优势,以及DNA甲基化与分子标记等方面,简要综述了国内外有关DNA甲基化在动植物遗传育种研究中的进展,着重于全基因组DNA甲基化模式在动植物遗传育种中的相关研究和应用。     

Locus Mapping,Molecular Cloning,and Expression Analysis of <Emphasis Type="Italic">rps6kb2</Emphasis>, a Novel Metamorphosis-Related Gene in Chinese Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9 cM corresponding to a 1.8 M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.     

Cloning and characterization of an acyl-CoA-dependent diacylglycerol acyltransferase 1 (DGAT1) gene from Tropaeolum majus, and a study of the functional motifs of the DGAT protein using site-directed mutagenesis to modify enzyme activity and oil content

SUMMARY: A full-length cDNA encoding a putative diacylglycerol acyltransferase 1 (DGAT1, EC 2.3.1.20) was obtained from Tropaeolum majus (garden nasturtium). The 1557-bp open reading frame of this cDNA, designated TmDGAT1, encodes a protein of 518 amino acids showing high homology to other plant DGAT1s. The TmDGAT1 gene was expressed exclusively in developing seeds. Expression of recombinant TmDGAT1 in the yeast H1246MATalpha quadruple mutant (DGA1, LRO1, ARE1, ARE2) restored the capability of the mutant host to produce triacylglycerols (TAGs). The recombinant TmDGAT1 protein was capable of utilizing a range of (14)C-labelled fatty acyl-CoA donors and diacylglycerol acceptors, and could synthesize (14)C-trierucin. Collectively, these findings confirm that the TmDGAT1 gene encodes an acyl-CoA-dependent DGAT1. In plant transformation studies, seed-specific expression of TmDGAT1 was able to complement the low TAG/unusual fatty acid phenotype of the Arabidopsis AS11 (DGAT1) mutant. Over-expression of TmDGAT1 in wild-type Arabidopsis and high-erucic-acid rapeseed (HEAR) and canola Brassica napus resulted in an increase in oil content (3.5%-10% on a dry weight basis, or a net increase of 11%-30%). Site-directed mutagenesis was conducted on six putative functional regions/motifs of the TmDGAT1 enzyme. Mutagenesis of a serine residue in a putative SnRK1 target site resulted in a 38%-80% increase in DGAT1 activity, and over-expression of the mutated TmDGAT1 in Arabidopsis resulted in a 20%-50% increase in oil content on a per seed basis. Thus, alteration of this putative serine/threonine protein kinase site can be exploited to enhance DGAT1 activity, and expression of mutated DGAT1 can be used to enhance oil content.