[Hanfeng Pre-reservoir Commissioning Time Variation Feature of the Hydrology and Water Quality in Three Gorges Reservoir].

Hanfeng Pre-reservoir is very rare in the world which is specially designed to reduce the impact of Fluctuating Zone, and it is formed in Hanfeng Lake of Three Gorges reservoir. The Hanfeng Pre-reservoir has many special hydrological characteristics and ecological environment features based on its unique "pre-reservoir" control mode, the wide seasonal wetland of Fluctuating Zone, the huge life pollution and agricultural pollution, and the pressure of huge city and excessive population. HanFeng Lake has a variety of morphological features such as lakes, rivers, and other backwater bay, for the effect of water level regulation in Three Gorges, since the successful commissioning of the Hanfeng Lake pre-dam system in 2015. The change of Hanfeng Lake hydrology and water quality during the commissioning was divided into four periods by combining Hydrological and Morphological Variation characteristics with Water quality indicators time clustering analysis:May to August as T1 (river period); January, March and November to December as T2 (lake period); February, April and September as T3 (water level fluctuation period); October as T4 (algal blooms period) in 2015. Principal component analysis and stepwise regression analysis showed that Eutrophication of Hanfeng Lake was dominated by different dominant components at different times and the water quality index factor which has a significant effect on the Chl-a was also different. Cumulative contribution rates of principal components were 82.93%, 77.61%, 78.32%, 88.40% for each period, respectively. The main water quality indicators of T1 (river period) were DP, TP, SD, pH and the significant influencing index of Chl-a was PC2, so Chl-a was mainly affected by water nitrogen content. The main water quality indicators of T2 (lake period) were TN, DN, DP, TP, NO3--N and the significant influencing index of Chl-a was PC1, so Chl-a was mainly affected by water eutrophication including nitrogen and phosphorus nutrient status. The main water quality indicators of T3 (water level fluctuation period) were SD, NH4+-N, DN, T and the significant influencing index of Chl-a was PC3. so Chl-a was mainly affected by water level fluctuation. The main water quality indicators of T4 (algal blooms period) were TN, DN, DO, NH4+-N, pH, permanganate index, H, NO3--N and the significant influencing index of Chl-a was PC3. so Chl-a was mainly affected by flow rate and hydrodynamic conditions. As mentioned in the review, the frequent and significant water level changes during the commissioning of Hanfeng Lake were the important factors influencing the change of hydrological and water quality characteristics.

[Hanfeng Pre-dam Commissioning Eutrophication Status and Control Evaluation in Three Gorges Reservoir].

To reduce the impact of Fluctuating Zone, the Three Gorges Reservoir pre-dam is rare in the world which is specially designed and is the largest artificial lake body in China. The ecological benefits of landscape, farmland and lake and the social benefits of livable city have been significantly enhanced since the successful commissioning of the Hanfeng Lake pre-dam system. The paper proposed the application of layered hydrology and water quality monitoring for analysis of Tributary runoff and lake body section in the pre-dam commissioning in the whole year, and a total of 17 measured indicators inlucding hydrological parameters such as v, H, etc, physical parameters such as T, pH, SD, DO, TSS etc. and chemical parameters such as permanganate index, Chl-a, TN, DN, NO3--N, NH4+-N, NO2--N, TP, DP, SRP etc. We found that the water quality was poor during beginning drain and impoundment period and was the worst in tributary inflow section of South River, while the best water quality was located in water section of regulating dam in February and October. The TLI Water Quality Evaluation and factor analyses performed have shown that the water body of Hanfeng Lake was slightly eutrophicated, and the main pollution indicators included DN, TN, NO3--N, TP. By the control of pre-dam in three Gorges to eutrophication in commissioning, we found that the average Chl-a reduction effect reached up to 57.73%, the average reduction rate of permanganate index was 28.12%, SRP, TP, TN, TSS, NO2--N, DN, DP etc. were on average cut down by 20.15%-22.81%, the average reduction rates of NH4+-N and NO3--N were 16.92%-18.74%, and the average eutrophic index of water body was reduced by 15.74%. The highest reduction average rate in lake form period appeared from January to March and October to December, and the lowest in river form period was during May to August.The analysis results showed that the commissioning of pre-dam was good and remarkable for controlling eutrophication, and cutting the concentrations of pollutant water storage in the Three Gorges.

Studies on interaction between hTNF-alpha and its two receptors with expressed hsTR55-preS1/hsTR75-preS1 fusion soluble receptors.

The gene encoding the N-terminal 2-50 amino acids of HBsAg-preS1 was amplified by PCR and fused to the 3'-end of two human soluble TNF receptor genes to form the hsTR55-preS1/hsTR75-preS1 fusion genes. The recombinant bicistronic expression vectors were further constructed, which contained one of the human soluble TNF receptor fusion genes and the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES), followed by the neomycin phosphotransferases as the selectable marker. BHK-21 cells transfected with those vectors by electroporation were selected with G-418, and the positive colonies expressing the protein of interest were obtained. All of the culture media of those transfects could fairly neutralize hTNFalpha-induced cytotoxicity to L929 cells. The expression of hsTR55-preS1/hsTR75-preS1 in those cells has been further demonstrated by RT-PCR and indirect ELISA at RNA transcription and protein translation levels. Their K(d) (dissociation constant) value has also been assayed with BIOSENSOR method. The results showed that the fused HBsAg-preS1 peptide did not affect the dissociation constant of hsTR55 or hsTR75 with hTNFalpha and its muteins. Thus, a novel nonradioactive ELISA method was developed for studies on interaction between hTNFalpha and its two receptors using those expressed fusion receptors.

Mitochondrial tRNA(Trp)s Imply a Eubacterial Origin.

Three mitochondrial tRNA(Trp)s genes from Oryza sativa, Homo sapiens and Saccharomyces serevisea were constructed. In vitro transcripts of these mitochondrial tRNA(Trp)s genes were able to be tryptophanylated by Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS), but could not be catalyzed by TrpRS from rat liver. Kinetic assay showed that B. subtilis TrpRS had half the binding capacity for mitochondrial tRNA(Trp)s than for wild type tRNA(Trp). While in the catalytic efficiency, the k(cat)/K(m) value of mitochondrial tRNA(Trp)s from O.sativa and S.serevisea by B.subtilis enzyme was reduced by 400-fold and 1 200-fold respectively as that of B.subtilis tRNA(Trp). Experimental results suggested that mitochondrial tRNA(Trp)s implied a eubacterial origin.

Expression in BHK Cells of Two Human TNF Receptors and Their Interaction with hTNF-alpha.

Two human tumor necrosis factor receptor genes were cloned into eukaryotic expression vectors pcDNA3, pDR2 and pXJ-41, respectively. Those vectors were transfected into BHK-21 cells and the expression of hTNFRs was identified and demonstrated by binding of (125)I hTNF-alpha and Scatchard analysis. The expression of two hTNFRs in BHK cells at the RNA transcriptional and the protein translation levels were also demonstrated by RT-PCR and indirect ELISA. However, hTNF-alpha did not appear cytotoxic to these cells. The competitive binding activity of mutant hTNF-alpha-R2K and wild type hTNF-alpha with these two receptors, respectively, were assayed. These results laid basis for further studies on structure-function relationships of these receptors as well as the hTNF-alpha.

Selection with SELEX Method of Small RNA Molecules Specifically Binding to Starch.

A 73-base DNA library with T7 promoter was chemically synthesized according to the sequence specified by the 2.5S RNA component of glycogen branching holoenzyme (EC 2.4.1.18) from rabbit muscle, in which 20 consecutive bases corresponding to a stem-loop structure were completely randomized. The in vitro transcribed RNA library was subjected to 9 successive rounds of selection with SELEX method, resulting in a sharp increase of the percentage of the starch-binding RNA from less than 0.01% for the first round pool to 31% for the ninth-round pool. This demonstrates the existence of small RNA species that can specifically bind to starch. Structural and functional analysis of them is in progress.

A Discriminator Base in tDNA(Trp).

The discriminator base G73 was one of the major identity elements of tRNA(Trp). Aminoacylation assay and CD spectrum of tDNA(Trp)-NCCrA demonstrated that under the same pH, the change of discriminator base affected tRNA conformation at the 3' terminus while tDNA(Trp) with the same discriminator base exhibited different conformation and aminoacylation activity under various pH conditions. Our results showed that it was the conformation of tRNA, but not the base itself, that determined the accurate recognition between tRNA and its cognate synthetase.

[Detection of 2'-O-ribose Methylation Sites on Rice 25 S rRNA].

Ribose methylation is a widespread type of nucleotide modification in rRNA. In order to map the methylation sites of rice 25 S rRNA, a series of primers complementary to both yeast 28 S and rice 25 S rRNA simultaneously were synthesized. Primer extensions at different dNTP concentrations were carried out to detect the methylation sites of both yeast and rice rRNAs. The data showed that over 80% of the methylation sites in yeast 28 S rRNA was also detected in rice. In addition, compared with the known methylation sites of Arabidopsis 25 S rRNA, other 54 sites probably methylated in rice were found in Arabidopsis. Thus, there are 85 methylation sites detected altogether; the distribution of the methyl sites in rice 25 S rRNA was determined. The results show that most of the sites are conserved among different species, especially between closely related species. And remarkably, there are much more ribose methylation sites in plant rRNA, and the propinquous methylation sites in plants are more frequent than those in other eukaryotes. Moreover, the data provide the most important clue for searching new box C/D snoRNAs.

Properties of Recombinant Aeromonas punctata Prolyl Endopeptidase.

The properties of recombinant Aeromonas punctata prolyl endopeptidase(apPEP) were studied using specific substrate and peptides. Results show that the optimum catalytic temperature and pH was 34 degrees and 8.4, the stability of the apPEP was in the range of 4-32 degrees and pH 6.0-10.0, and its K(m) was 0.03 mmol/L based on the Z-Gly-Pro-betaNA. The apPEP was not sensitive to PMSF, TLCK, TPCK, Trypsion inhibitor, EDTA, tetrathionate and some metal ions, but was sensitive to SDS and Zn(2 ), and was completely inhibited by DFP. Oxytocin and calcitonin could be specifically hydrolyzed by apPEP at the carboxyl site of proline residue, but the hydrolysis efficiency of calcitonin by the enzyme was less than for oxytocin and for Z-Gly-Pro-betaNA.

SELEX Screening and Characterization of Small RNA Molecules That Specifically Bind the Reactive Blue Dye.

A 73-base single-stranded DNA library with a T7 promoter was chemically synthesized, in which 20 consecutive bases were completely randomized. After PCR amplification and T7 RNA polymerase transcription of the DNA random library, the in vitro transcribed RNA random library was subjected to 8 successive rounds of reactive blue dye column selection by SELEX method, resulting in a sharp increase of the percentage of the transcribed RNA pool that could bind reactive blue dye, from less than 0.03% in the first round pool to 22.4% in the eighth-round pool. Cloning and sequencing of the enriched RNAs pool led to three groups of RNA molecules in terms of molecular lengths. The correlation between the binding capacities and their corresponding secondary structures of the screened RNAs suggests that the RNA helix is the main motif in the interaction between RNAs and reactive blue dye. These results demonstrate that it is possible to find RNA candidates with a given function in a shorter RNA random library so that it is easier to follow the structure and function relationship. In addition, longer RNA sequences could theoretically be derived from short RNA sequences through a process of motif combination in the course of molecular evolution.

[Identification of a snoRNA47 gene cluster in Oryza sativa].

Small nucleolar RNAs (snoRNAs) are required for ribose 2'-O-methylation of eukaryotic ribosomal RNA. By researching in international rice genome databases, a snoRNA gene cluster, consisting of three box C/D snoRNA gene candidates, was found on chromosome 6. All of snoRNA coding sequences in this cluster exhibited the characteristic structure of box C/D antisense snoRNA. They shared conserved box C and box D motifs, a stable terminal stem formed by a 4-6 nt-long inverted repetition sequence located upstream of box C and downstream of box D. All candidates had a 12 nt-long sequence complementary to the region from 621 nt to 6 32 nt in rice 18 S rRNA (nt numbering according to GenBank accession No. X00755), and might mediate the ribose 2'-O-methylation of A(623) in rice 18 S rRNA. Comparison of these sequences showed that these three rice snoRNAs were homologue of yeast snR47. These three rice snoRNAs were named as OSsnR47.1, OSsnR47.2 and OSsnR47.3, respectively. Primer extension assay showed that these three snoRNAs were transcribed in vivo and determined the 5' ends of each snoRNA. RT-PCR detected transcripts containing linked snoRNA47s and this suggested that the snoRNAs encoded in the cluster might be transcribed as a polycistronic transcript under the control of a single upstream promoter. The gene sequences encoding these three snoRNAs had been deposited in GenBank under accession number of AF453504 AF453503 an d AF453502.

Immobilized Tryptophanyl-tRNA Synthetase from Bacillus subtilis.

In order to investigate the recognition mechanism and the relationship between structure and function of tRNA(Trp) with tryptophanyl-tRNA synthetase (TrpRS), TrpRS from Bacillus subtilis was purified and immobilized on CNBr-activated Sepharose 4B. Protein recovery and activity recovery of the immobilization were 95.5% and 31.3%, respectively. Properties of immobilized TrpRS were studied in detail. The thermal stability and the shelf stability of immobilized TrpRS were much higher than those of the native TrpRS. Besides these, the immobilized TrpRS, with good operation stability, had increased optimum temperature and optimum pH. A 56-base single-stranded RNA library containing 20 consecutive completely randomized bases was subjected to 3 successive rounds of immobilized TrpRS column selection with SELEX method, resulting in a sharp increase of the percentage of the RNA pool that could bind immobilized TrpRS from 4.3% for the first round pool to 14.7% for the third-round pool. After sequencing the third-round RNA pool, a RNA secondary structure resembling the structure of the acceptor stem in tRNA(Trp) was obtained though the selection. All the results indicated that immobilized TrpRS could be used as an affinity chromatography matrix and was qualified for the SELEX of a RNA pool simulating tRNA(Trp) molecule.

Efficient Secretion of Proteins Expressed from Insect Cells Directed by PAM Signal Peptide.

Signal and leading peptide sequences of rat PAM was inserted into the baculovirus transfer vector, and secretion expression plasmids pBACPAG2 and pBacPAI for the fusion gene PABC-hGRF and PABC-IGF-I were constructed, respectively. By cotransfection with linear genomic DNA of modified Autographa californica nuclear polyhedrosis virus (BacPAK6) and homologous recombination, the recombinant AcNPV, BacPAG and BacPAI, were obtained and identified. Fusion proteins PABC-hGRF and PABC-IGF-I were secreted efficiently from Sf21 cells infected with BacPAG and BacPAI, respectively, and those fusion protein could be purified efficiently by IgG affinity column.

Overexpressed Human TNF Receptor-75 Can Independently Mediate hTNFalpha-induced Cytotoxicity in BHK-21 Cells.

A recombinant bicistronic expression vector was constructed containing human TNF receptor-75 gene and encephalomyocarditis virus(EMCV)internal ribosome entry site(IRES), followed by a neomycin phosphotransferase gene as selectable marker. BHK-21 cells were transfected with this vector using electroporation method and positive clones overexpressing the genes of interest were obtained after selection with G-418. The expression of two hTNFRs in those cells at RNA transcriptional and the protein translation levels had been proved by RT-PCR and indirect ELISA. It was found that the hTR75 could mediate cytotoxicity independently in BHK-21 cells after those cells were treated by hTNFalpha. Furthermore, hTR55 did not display the synergistic activity on this function of hTR75. These results put forward a new way for further studies on structure and function relationships of hTR75 and the interactions between two hTNFRs.

Construction and Expression of hTNFalpha-hTGF3 Fused Gene.

Human tumor necrosis factor was fused with hTGF3, the third loop region of hTGFalpha specifically binding to EGF receptor, through a flexible linker by recombinant DNA techniques. The constructed plasmid containing hTNFalpha-hTGF3 fused gene, pSB92-TLT, was expressed under the control of P(L) promoter and after the induction at 42 degrees it gave a high level of expression of the fused gene, ranging in 50%-60% of total bacterial protein. 95% of the expressed product was inclusion body, and only 5% was soluble. When the fused gene was constructed into the constitutive plasmid pLC, a new plasmid controlled by P(L) promoter but without cIts857, and expressed at 30 degrees, the expression yield reached 50% and a high proportion of soluble protein, 35% of total bacterial protein, was obtained. Western blot analysis indicated that the fusion protein could specifically bind to anti-hTNFalpha antibody. Comparing to original hTNFalpha, the fused hTNFalpha derivative showed about equal cytotoxicity to L929 cell line and higher cytotoxicity to some human tumor cell lines. This shows that the fusion protein TLT may be promising in application.

Isolation and Identification of Membrane Proteins Binding to Transfer RNA.

Crude extract of total membrane proteins of yeast was obtained by a method of phase separation in Triton X-114. The membrane extract was applied to the affinity column on which yeast total tRNAs were covalently coupled to hydrazinyl Sepharose 4B. By eluting with increasing concentration gradient of (NH(4))(2)SO(4), eluted proteins were found between concentrations of (NH(4))(2)SO(4) 0.1 mol/L to 0.3 mol/L. By gel mobility-shift assays, it was also observed that there were more than two mobility-shift bands on the gel electropherogram after incubation of sample of (32)P-labeled tRNA with the eluent proteins and the reaction mixtures were analyzed by non-denaturing polyacrylamide gel electrophoresis. These results confirm that these eluted proteins contain two major tRNA specific binding proteins.

Cloning and Expression of the cDNA Coding for Rat Peptidylglycine alpha-Amidating Monooxygenase.

The dDNA of genes encoding rat peptidylglycine alpha-aminating monooxygenase(rPAM) were cloned and their expression in E. coli studied. Three DNA fragments were isolated from the rat brain cDNA library using the methods of plaque hybridization and PCR. DNA sequencing showed that they contained the total coding sequence for rPAM-2. By using the sited-directed mutation and PCR recombination methods, we obtained intact genes coding for the rPHM domain, the rPAL domain and rPAM, respectively. Different plasmids of these genes controlled under T(7) or P(L) promoter were constructed and transformed into E. coli. The high-level expression of rPAM-N260 in E. coli was first observed and its antiserum was prepared for the immunoassay of natural or recombinant PAMs. By the analysis with SDS-PAGE and Western blot, the products of rPHM and rPAM in E. coli were detected, and the amount of rPHM reached over 10% of the total bacterial proteins. It was found that low temperature and copper ion obviously increased the stability and solubility of the rPHM expressed in E. coli.

Analysis of C-terminal Amidating Structure and Determination of Amidating Enzyme Activity by Using Capillary Electrophoresis.

Based on the difference in charge and hydrophobicity between amidating moiety and the carboxyl group in a given buffer solution, a new method to analyze the C-terminal amidating structure and the amidating enzyme activity by using capillary electrophoresis was established.

[Comparison of the electrophysiological features between the rhythmic cells of the aortic vestibule and the sinoatrial node in the rabbit].

The purpose of this study was to clarify the characteristics of the pacemaker cells in the left ventricular outflow tract (aortic vestibule) and compare them with those of the cells in the sinoatrial node (SAN). By using conventional intracellular microelectrode technique to record their action potentials, some ionic channel blockers were used to observe their electrophysiological effects on the two types of pacemaker cells in the rabbit, especially on the ionic movement during phase 0 and phase 4. The results obtained are as follows. (1) Perfusion with 1 micromol/L verapamil (VER) resulted in a significant reduction in the amplitude of action potential (APA), maximal rate of depolarization (V(max)), absolute value of the maximal diastolic potential (MDP), velocity of diastolic depolarization (VDD) and rate of pacemaker firing (RPF), and also a prolongation of the 90% of the duration of action potential (APD(90)) in the pacemaker cells of the SAN and aortic vestibule (P<0.05). (2) Perfusion with 180 micromol/L nickel chloride (NiCl2) resulted in a decrease in VDD in the two types of the pacemaker cells (P<0.01). APA, V(max) and RPF fell notably, and the APD(90) prolonged in the sinoatrial node cells (P<0.05). (3) 2 mmol/L 4-aminopyridine (4-AP) led to a increase in VDD in both types of pacemaker cells (P<0.01). At the same time the absolute values of MDP, APA and V(max) decreased significantly, and APD(90) prolonged notably (P<0.05). During the perfusion, RPF in SAN increased markedly, while RPF in aortic vestibule exhibited no significant change. (4) 2 mmol/L cesium chloride (CsCl) led to a decrease in VDD and RPF in the two types of the pacemaker cells (P<0.05).These results suggested: (1) the ion currents in phase 0 and phase 4 of depolarization and repolarization of slow-response activity in aortic vestibule are similar to those in dominant pacemaker cells of sinoatrial node; (2) for the pacemaker cells in the left ventricular outflow tract, Ca(2+) current is the main depolarizing ion current of the phase 0, K(+) current is the main factor responsible for the repolarization. Attenuation of K(+) current is responsible for the phase 4 spontaneous depolarization. In addition, it seems that I(Ca-T), I(Ca-L) and I(f ) play some role in the pacemaker currents.