Characterization and expression of a cDNA, AmphiSDHD,encoding the amphioxus cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase

In this study, an amphioxus cDNA, AmphiSDHD, encoding the cytochrome b small subunit in mitochondrial succinate-ubiquinone oxidoreductase, was isolated from the gut cDNA library of amphioxus Branchiostoma belcheri tsingtauense. It is 1429 bp in length, with an open reading frame of 465 bp coding for a protein of 154 amino acids. The deduced protein contains a mitochondrial targeting presequence of 65 amino acids rich in basic residues like arginine and hydroxy residues such as serine and threonine. Alignment of the amino acid sequences of AmphiSDHD and other eukaryotic SDHD proteins showed that AmphiSDHD has three transmembrane segments, and includes two histidine residues in the second transmembrane segment that are the putative binding sites for the heme b molecule. The phylogenetic tree constructed suggests that AmphiSDHD appears more closely related to vertebrate SDHD proteins than invertebrate ones. Northern blotting demonstrated that AmphiSDHD is ubiquitously expressed in amphioxus, being in line with the fact that SDHD is a house-keeping protein.     

Cloning, Characterization, and Expression Analysis of Calreticulin from Pearl Oyster Pinctada fucata

Calreticulin is a unique calcium-binding protein with multiple functions mostly located in the sarcoplasmic/endoplasmic reticulum. A large amount of calcium is absorbed from the medium and transported to mineralization sites during biomineralization in pearl oyster. This paper describes the cloning of the full-length cDNA of calreticulin from Pinctada fucata, namely PCRT. PCRT encodes a deduced 414-amino acid protein, which includes a predicted 17- amino acid signal peptide and an endoplasmic reticulum retrieval sequence HDEL. The protein shows 63%-76% sequence identity and shares some common characteristics with calreticulins from other species. Semi-quantitative RT-PCR indicates that PCRT is ubiquitously expressed in all tissues tested with the highest expression in the hemolymph and the mantle. In situ hybridization analysis of PCRT in the mantle showed strong signals in the inner fold, the inner side of middle fold, and the inner side of outer fold of the mantle epithelium, All these results suggest PCRT might be involved in Ca^2＋ transport and storage during oyster biomineralization.     

Insights into the subunit interactions of the chloroplast ATP synthase

Subunit interactions of the chloroplast F0F1- ATP synthase were studied using the yeast two-hybrid system. The coding sequences of all the nine subunits of spinach chloroplast ATP synthase were cloned in two-hybrid vectors. The vectors were transformed into the yeast strains HF7c and SFY526 by various pairwise combinations, and the protein interactions were analyzed by measuring the yeast growth on minimal SD medium without serine, lucine and histidine. Interactions of γ Subunit with wild type or two truncated mutants of γ sununit, △εN21 and △εC45, which lose their abilities to inhibit the ATP hydrolysis, were also detected by in vitro and in vivo binding assay. The present results are largely accordant to the common structure model of F0F1-ATP synthase. Different from that in the E. Coli F0F1-ATP synthase, the δ subunit of chloroplast ATP syn- thase could interact with β,γ,ε and all the CF0 subunits in the two-hybrid system. These results suggested that though the chloroplast ATP synthase shares the similar structure and composition of subunits with the enzyme from E. Coli, it may be different in the subunit interactions and con- formational change during catalysis between these two sources of ATP synthase. Based on the present results and our knowledge of structure model of E. Coli ATP synthase, a deduced structure model of chloroplast ATP synthase was proposed.     

Effects of truncated mutants of the ε subunit of chloroplast ATP synthase on the fast phase of millisecond delayed light emission of chloroplast and its ATP synthesis ability

The ε subunit of the chloroplast ATP synthase and the truncated ε mutants which lack some amino acid residues from the N-terminus or C-terminus were overexpressed in E. coil When the ε subunit or the truncated ε proteins was added to the spinach chloroplast suspension, both the intensity of the fast phase of millisecond delayed light emission (ms-DLE) and the cyclic and noncyclic photophosphorylation activity of chloroplast were enhanced. With an increase in the number of residues deleted from the N-terminus, the enhancement effect of the N-terminal truncated proteins decreased gradually. For the C-terminal truncated proteins, the enhancement effect increased gradually with an increase in the number of residues deleted from the C-terminus. Besides, the ATP synthesis activity of ε-deficient membrane reconstituted with the ε subunit or the truncated ε proteins was compared. The ATP synthesis activity of reconstituted membrane with the N-terminal truncated proteins decreased gradually as the number of residues deleted from the N-terminus increased. For the C-terminal truncated proteins, the ATP synthesis activity of reconstituted membrane increased gradually with an increase in the number of residues deleted from the C-terminus, but was still lower than that of the wild type ε protein. These results suggested that: (a) the N-terminal domain of the ε subunit of the chloroplast ATP synthase could affect the ATP synthesis activity of ATP synthase by regulating the efficiency of blocking proton leakage of ε subunit; and (b) the C-terminal domain of the ε subunit of the chloroplast ATP synthase had a subtle function in modulating the ATP synthesis ability of ATP synthase.     

ATPG is required for the accumulation and function of chloroplast ATP synthase in Arabidopsis

The subunit Ⅱ of chloroplast ATP synthase is one of the two peripheral stalks, which associates the catalytic CF1 with mem-brane-spanning CFo . Although the structural and functional roles of chloroplast ATP synthase have been extensively examined, the physiological significance of subunit Ⅱ in vivo is still unclear. In this work, we identified one Arabidopsis T-DNA insertion mutant of atpG gene encoding the subunit Ⅱ of chloroplast ATP synthase. The atpg null mutant displayed an albino lethal pheno-type, as it could not grow photoautotrophically. Transmission electron microscopy analysis showed that chloroplasts of atpg lacked the organized thylakoid membranes. Loss of subunit Ⅱ affected the accumulation of CF1-CFo complex, however, it did not seem to have an effect on the CF1 assembly. The light induced ATP formation of atpg was significantly reduced compared with the wild type. Based on these results, we suggested that ATPG was essential for the accumulation and function of chloroplast ATP synthase.     

Partial Purification and Properties of an Acid Phosphatase from Pearl Oyster Pinctada Fucata

IntroductionAcid phosphatase ( ACP,EC 3.1 .3.2 ) andalkaline phosphatase ( ALP,EC 3.1 .3.1 ) havebeen found to exist widely in the biosphere frombacteria to plants,animals and humans[18] .Thetwo enzymes catalyze the hydrolysis of variousphosphate containing compounds and act astransphosphorylases at acid- and alkaline- p Hvalue.In our lab,an alkaline phosphatase fromthe pearl oyster Pinctada fucata has been purifiedand some of its kinetics has been studied[9] .　Acid phosphatases actas ma…     

Cloning and characterization of 37 kD laminin receptor precursor in pearl oyster, Pinctada fucata

A 1063 bp cDNA clone encoding a putative 37 kDa laminin receptor precursor （37 kDa LRP） is isolated from the mantle tissue of pearl oyster, Pinctadafucata. The amino acid sequence predicted from the cDNA sequence is 301 residues long, with a calculated molecular mass of 33.5 kDa. RT-PCR analysis shows that 37 kDa LRP mRNA is especially highly expressed in the mantle while widely expressed in several tissues. In situ hybridization analysis reveals that 37 kDa LRP is expressed in the outer epithelial ceils of the mantle edge, suggesting its involvement in cell proliferation and secretion in P. fucata. The identification and characterization of 37 kDa LRP in the pearl oyster will help us to further understand the signal transduction in the processes of mantle epithelial cell proliferation and tissue formation.     

Extraction and Purification of Matrix Protein from the Nacre of Pearl Oyster Pinctada fucata

A soluble matrix protein P14 with an apparent molecular mass of 14.5 kDa was isolated from fragmented nacre of pearl oysters （Pinctada fucata） treated with 10% NaOH solution to investigate the nacre matrix proteins and their effect on the CaCO3 crystal. The protein was characterized by gel exclusion chromatography and reversed-phase high performance liquid chromatography after demineralization by 10% acetic acid. The X-ray diffraction pattern of P14 crystals indicates that P14 plays an important role in nacre biomineralization. P14 can induce aragonite formation, stimulate CaCO3 crystal formation, and accelerate aragonite precipitation. Heating of the acid insoluble nacre residue, which was named conchiolin, in 10% sodium dodecyl sulfate solution supplemented with 10% β-mercaptoethanol solution for 10-20 min at about 100℃ gave two other soluble proteins having molecular masses of 19.4 kDa and 25.0 kDa. The present study suggests that these two proteins are linked to the insoluble organic matrix by disulfide bridges because the extraction yield increases when β-mercaptoethanol is added to the medium.     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).     

Characterization of threerice CCoAOMT genes

Caffeoyl-Coenzyme A 3-O-Methyltransferase (CCoAOMT) is a key enzyme in lignin biosynthesis pathway. Three rice CCoAOMT genes were identified and designated as OsCOAI, OsCOA20 and OsCOA26. OsCOAI contains four exons and three introns, while the other two have three exons and two introns. The deduced amino acid sequences of these rice genes share a high identity (75.43%) with other plant CCoAOMT proteins and contain the CCoAOMT specific motifs. Phylogenetic analysis indicates that OsCOAI has the closest evolutionary relationship to maize CCoAOMT. In contrast, OsCOA20 and OsCOA26 belong to another clade.Northern blot analyses and in situ hybridization studies indicate that the three rice CCoAOMT genes are highly expressed in developing sclerenchyma cells and vessel bundles of young leaves, suggesting that they are probably involved in constitutive lignification.     

Localization of calmodulin and calmodulin-like protein and their functions in biomineralization in P. fucata

Calmodulin （CAM） and calmodulin-like protein （CaLP） are two proteins involved in biomineralization. Their localizations in Pinctada fucata mantle epithelia were studied by Western blot （WB） analysis of the nuclear/cytosol fraction of primary cultured P. fucata mantle cells and immunogold electron microscopy. The results showed a completely different distribution of these two proteins at the subcellular level. CaM was distributed throughout both the nucleus and cytoplasm of the mantle epithelium but CaLP was distributed only in the cytoplasm. The functions of these two proteins in biomineralization were investigated by shell regeneration. During this pro- cess, the expressions of CaM and CaLP were greatly enhanced in different organelles of the mantle epithelium. Overexpression of these two proteins and a mutant of calmodulin-like protein （M-CaLP） that lacks an extra C-terminal tail in MC3T3-E1 promoted the mRNA expression of osteopontin, a biomineralization marker for osteoblasts. All of the results indicated that CaM and CaLP have completely different distributions in the mantle epithelium and affect the biomineralization process at different levels. The extra C-terminal tail of CaLP is important for its functions in biomineralization in P. fucata.     

Remote sensing of spatial patterns of urban renewal using linear spectral mixture analysis: A case of central urban area of Shanghai (1997―2000)

It is very important to integrate remote sensing with urban geography that the spectral mixture analysis technique is applied to urban land cover evolvement and its eco-environmental effect. Urban land cover is mainly composed of complicated artificial materials, which is the key factor to limit the development of the spectral mixture analysis technique. There are two main aspects in which the technique of spectral mixture analysis is applied to urban geography： one is to calculate vegetation fraction; the other is to build quantitative model of the urban impervious surface obtained from the combination between high albedo fraction and low albedo fraction. The technique of spectral mixture analysis is firstly applied to study urban renewal pattern, scale and mode which happened in Shanghai City from 1997 to 2000,     

Remarks on Psychological Analyses of Sons and Lovers

Sons and Lovers is considered to be D.H.Lawrene's first masterpiece.Social critique and psychological exploration are two props of creative works of D.H.Lawrence.There are two points of view in literature world about the novel Sons and Lovers:One is its critical function which criticizes the destroy of capitalistic industrial civilization to human body and mind.The other is psychological analyses.The tragedy of the characters attributes to the Oedipus complex.     

Mutagenesis of Ser24 of Cytochrome b559 α Subunit Affects PSII Acitivies in Chlamydomonas reinhardtii

In order to study the functions of cytochrome b559 (Cyt b559) in photosystem two (PSII) activity, mutant S24F of Chlamydomonas reinhardtii was constructed using site directed mutagenesis, in which Serine24 (Ser24) locating downstream of Histidine23 (His23) in α subunit of Cyt b559 was replaced by Phenylalanine (Phe). Physiological and biochemical analysis showed that mutant S24F could be grown photoautotrophically or photoheterotrophically. However, their growth rate was slower either on HSM or TAP medium than that of the control; Analysis of PSII activity revealed that its oxygen evolution was about 71% of wild type (WT); The Photochemical efficiency of PSII (Fv/Fm) of S24F was reduced 0.23 compared with WT; S24F was more sensitive to strong light irradiance than the wild type; Furthermore, SDS-PAGE and Western-blotting analysis indicated that the expression levels of α subunit of Cyt b559, LHCII and PsbO of S24F were a little less than those of the wild type. Overall, these data suggests that Ser24 plays a significant role in making Cyt b559 structure maintain PSII complex activity of oxygen evolution although it is not directly bound to heme group.