[Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort.]

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and ß-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Molecular characterization of a chalcone synthase gene RhCHS from Rhododendron × hybridum Hort.

Chalcone synthase (CHS) plays a vital role in anthocyanin biosynthesis pathway, which is associated with petal color of flower. To date, lots of CHS genes have been obtained from plants, while few were from Rhododendron genus. In this study we got a new CHS gene named RhCHS (MW358095) from Rhododendron × hybridum Hort. It had a 2040 bp coding region consisting of two exons and one intron. By using the deduced RhCHS protein as a query sequence, 15 CHS homologous family genes with sequence similarity from 60% to 98%, designated as RgCHS-D(x), were retrieved from the genome assembly of Rhododendron griersonianum (RGv1.1) by TBlastN. 12 CHS family genes were found locating in No.9 chromosome arranged in clusters, while only 3 of them exhibited in No.1, 2, and 8 chromosomes, respectively. The results revealed gene duplication of CHS in evolutionary process. Multiple alignment of the deduced amino acid sequence of RhCHS showed high similarity of the active site, the catalytic residue, and the signature motif, the conserved characteristics of which were also exhibited in the tertiary structure prediction of the RhCHS, as well as the phylogenetic tree, all these demonstrated the RhCHS belonging to the type III PKS superfamily. HPLC-MS/MS of flower petals detected the total concentration of CC, DC, and PelC. These anthocyanidins showed an overall increasing trend during the flowering period and reached the peak in the full-blooming stage, which was consistence with the changeable rule of RhCHS expression level. The promoter, which was 1507 bp exhibiting high ß-glucuronidase (GUS) staining activity, was predicted containing many cis-acting elements, especially light and transcription factor such as bHLH, MYB, WRKY, Dof, and ERF. In short, this study may provide the help to Rhododendron × hybridum Hort. not only in the mechanism research of petals color exhibition, but also in molecular breeding of CHS practice value.

Transcriptomic and metabolomic data reveal key genes that are involved in the phenylpropanoid pathway and regulate the floral fragrance of Rhododendron fortunei.

BACKGROUND: To reveal the key genes involved in the phenylpropanoid pathway, which ultimately governs the fragrance of Rhododendron fortunei, we performed a comprehensive transcriptome and metabolomic analysis of the petals of two different varieties of two alpine rhododendrons: the scented R. fortunei and the unscented Rhododendron 'Nova Zembla'. RESULTS: Our transcriptomic and qRT-PCR data showed that nine candidate genes were highly expressed in R. fortunei but were downregulated in Rhododendron 'Nova Zembla'. Among these genes, EGS expression was significantly positively correlated with various volatile benzene/phenylpropanoid compounds and significantly negatively correlated with the contents of various nonvolatile compounds, whereas CCoAOMT, PAL, C4H, and BALDH expression was significantly negatively correlated with the contents of various volatile benzene/phenylpropanoid compounds and significantly positively correlated with the contents of various nonvolatile compounds. CCR, CAD, 4CL, and SAMT expression was significantly negatively correlated with the contents of various benzene/phenylpropanoid compounds. The validation of RfSAMT showed that the RfSAMT gene regulates the synthesis of aromatic metabolites in R. fortunei. CONCLUSION: The findings of this study indicated that key candidate genes and metabolites involved in the phenylpropanoid biosynthesis pathway may govern the fragrance of R. fortunei. This lays a foundation for further research on the molecular mechanism underlying fragrance in the genus Rhododendron.

[Identification of terpene synthase gene family members in Rhododendron and its relationship with terpenoid metabolism].

Terpene synthase (TPS) plays important roles in the synthesis of terpenoids which are the main fragrances in Rhododendron flowers. To understand the function of TPS genes in terpenoid metabolism in relation to flower aroma formation, we identified all TPS gene family members in Rhododendron by analyzing its genome database. We then used a transcriptomic approach to analyze the differential gene expression patterns of TPS gene family members in the scented flower Rhododendron fortunei compared to the non-scented flower Rhododendron 'Nova Zembla'. The contents of terpenoid compounds in petals of the above two Rhododendron species at different developmental stages were also measured by using qRT-PCR and head space-solid phase micro-extraction combined with gas chromatography-mass spectrometry. Our results showed that a total of 47 RsTPS members, with individual lengths ranged from 591 to 2 634 bp, were identified in the Rhododendron genome. The number of exons in RsTPS gene ranged from 3 to 12, while the length of each protein encoded ranged from 196 to 877 amino acids. Members of the RsTPS family are mainly distributed in the chloroplast and cytoplasm. Phylogenetic analysis showed that RsTPS genes can be clustered into 5 subgroups. Seven gene family members can be functionally annotated as TPS gene family since they were temporally and spatially expressed as shown in the transcriptome data. Notably, TPS1, TPS10, TPS12 and TPS13 in Rhododendron fortunei were expressed highly in flower buds reached the peak in the full blossoming. Correlation analysis between gene expression levels and terpenoid content indicates that the expression levels of TPS1, TPS4, TPS9, TPS10, TPS12 and TPS13 were positively correlated with the content of terpenoids in the petals of R. fortunei at all flower developmental stages, suggesting that these six genes might be involved in the aroma formation in R. fortunei.

[Transcriptional analysis of grape in response to weak light stress].

Grape (Vitis vinifera L.) in production is frequently exposed to inadequate light, which significantly affects its agronomic traits via inhibiting their physiological, metabolic and developmental processes. To explore the mechanism how the grape plants respond to the weak light stress, we used 'Yinhong' grape and examined their physiology-biochemistry characteristics and transcriptional profile under different levels of weak light stress. The results showed that grape seedlings upon low intensity shading treatments were not significantly affected. As the shading stress intensity was strengthened, the epidermis cells, palisade tissue, and spongy tissue in the leaves were thinner, the intercellular space between the palisade tissue and spongy tissue was larger compared with that of the control, and the activities of superoxide dismutase, catalase and peroxidase were decreased gradually. Additionally, the soluble protein content increased and the free proline content decreased gradually. Compared with the control, significant changes in plant photosynthetic characteristics and physiology-biochemistry characteristics were observed under high intensity of shading (80%). RNA-seq data showed that the differentially expressed genes between CK and T2, CK and T4, T2 and T4 were 13 913, 13 293 and 14 943, respectively. Most of the enrichment pathways were closely related with the plant's response to stress. Several signaling pathways in response to stress-resistance, e.g. JA/MYC2 pathway and MAPK signal pathway, were activated under weak light stress. The expression level of a variety of genes related to antioxidation (such as polyphenol oxidase and thioredoxin), photosynthesis (such as phytochrome) was altered under weak light stress, indicating that 'Yinhong' grape may activate the antioxidation related pathways to cope with reactive oxygen species (ROS). In addition, it may activate the expression of photosynthetic pigment and light reaction structural protein to maintain the photosynthesis activity. This research may help better understand the relevant physiological response mechanism and facilitate cultivation of grape seedlings under weak light.

Comparison of the Evaluation of Combination of Ultrasonography of the Reproductive Tract With Hormone Administration on Dairy Cow Fertility.

Further optimization of reproduction management programs in dairy cows is a contemporary research topic. In this context, our study aimed to compare a hormone program, named "uterus-ovary monitoring and classified use of hormone program" (M+C), with the Pre-OvSynch program. The M+C was based on regular application of B-mode ultrasonography during a voluntary waiting period to monitor the uterus and ovaries, while using various treatments under different conditions. Results of the 30-33-day and 60-day pregnancy/artificial insemination after the first AI of M+C were significantly better than the Pre-OvSynch (p < 0.05). The pregnancy rates within 180 days in milk after M+C was significantly higher than that after Pre-OvSynch (p < 0.05). The total number of inseminations used for M+C was significantly lower than that for Pre-OvSynch (p < 0.01). The number of open days was fewer after M+C than after the Pre-OvSynch throughout the experimental period with highly significant differences (p < 0.01). In summary, the use of M+C enhances reproductive benefits and reduces the need for hormone drugs among cows.

[Cloning and functional analysis of the phenylalaninammo-nialyase gene from Rhododendron fortunei].

Phenylalaninammo-nialyase (PAL) is a key enzyme in the synthesis of methyl benzoate - a plant aroma compound. In order to understand the function of this enzyme in the formation of fragrance in the scented Rhododendron species-Rhododendron fortunei, we cloned a gene encoding this enzyme and subsequently examined the gene expression patterns and the profile of enzyme activity during development in various tissues. The full length of RhPAL gene was cloned by reverse transcription-PCR (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques. The expression levels of RhPAL gene were measured by real-time quantitative reverse transcription PCR (qRT-PCR) and the amount of phenylalanine and cinnamic acid were assayed with LC-MS. The results showed that the ORF sequence of RhPAL gene amplified from the cDNA templates of flower buds had 2 145 bp, encoding 715 amino acids, and shared 90% homology to the PAL amino acid sequences from other species. qRT-PCR analysis showed that the expression of RhPAL in petals during flowering kept in rising even until the flowers wilted. The expression of RhPAL in pistil was much higher than that in stamen, while the expression in the younger leaves was higher than in old leaves. However, the expression level was relatively lower in petal and stamen compared to that in leaves. We also measured the PAL activity by Enzyme-linked immuno sorbent assay in the petals of flowers at different flowering stages. The results showed that PAL activity reached the highest at the bud stage and then decreased gradually to the lowest when the flowers wilted, which followed a similar trend in the emission of the flower fragrance. The phenylalanine and cinnamic acid contents measured by LC-MS were highly correlated to the expression level of RhPAL in various tissues and at different flowering stages, implying that RhPAL plays an important role in the formation of the flower fragrance. This work may facilitate the breeding and improvement of new fragrant Rhododendron cultivars.

Analysis on the risk of respiratory virus transmission by air conditioning system operation based on experimental evidence.

It is important to know whether SARS-CoV-2 is spread through the air conditioning systems. Taking the central air conditioning system as an example, we analyze the mechanism and potential health risk of respiratory virus transmission in air-conditioned rooms and propose a method to study the risk of virus transmission in central air conditioning systems by investigating the data from medical experiments. The virus carrying capacity and the decay characteristics of indoor pathogen droplets are studied in this research. Additionally, the effects of air temperature and relative humidity on the virus survival in the air or on surfaces are investigated. The removal efficiency of infectious droplet nuclei by using an air conditioning filter was then determined. Thus, the transmission risk during the operation of the centralized air conditioning system is evaluated. The results show that the indoor temperature and humidity are controlled in the range of 20-25 °C and 40-70% by central air conditioning during the epidemic period, which not only benefits the health and comfort of residents, but also weakens the vitality of the virus. The larger the droplet size, the longer the viruses survive. Since the filter efficiency of the air conditioning filter increases with the increase in particle size, increasing the number of air changes of the circulating air volume can accelerate the removal of potential pathogen particles. Therefore, scientific operation of centralized air conditioning systems during the epidemic period has more advantages than disadvantages.

COVID-19 epidemic prediction and the impact of public health interventions: A review of COVID-19 epidemic models.

The coronavirus disease outbreak of 2019 (COVID-19) has been spreading rapidly to all corners of the word, in a very complex manner. A key research focus is in predicting the development trend of COVID-19 scientifically through mathematical modelling. We conducted a systematic review of epidemic prediction models of COVID-19 and the public health intervention strategies by searching the Web of Science database. 55 studies of the COVID-19 epidemic model were reviewed systematically. It was found that the COVID-19 epidemic models were different in the model type, acquisition method, hypothesis and distribution of key input parameters. Most studies used the gamma distribution to describe the key time period of COVID-19 infection, and some studies used the lognormal distribution, the Erlang distribution, and the Weibull distribution. The setting ranges of the incubation period, serial interval, infectious period and generation time were 4.9-7 days, 4.41-8.4 days, 2.3-10 days and 4.4-7.5 days, respectively, and more than half of the incubation periods were set to 5.1 or 5.2 days. Most models assumed that the latent period was consistent with the incubation period. Some models assumed that asymptomatic infections were infectious or pre-symptomatic transmission was possible, which overestimated the value of R0. For the prediction differences under different public health strategies, the most significant effect was in travel restrictions. There were different studies on the impact of contact tracking and social isolation, but it was considered that improving the quarantine rate and reporting rate, and the use of protective face mask were essential for epidemic prevention and control. The input epidemiological parameters of the prediction models had significant differences in the prediction of the severity of the epidemic spread. Therefore, prevention and control institutions should be cautious when formulating public health strategies by based on the prediction results of mathematical models.

[Effects of seeding pattern and phosphorus application on population structure, photosynthe-tic characteristics and yield of winter wheat]. / æ’­ç§æ–¹å¼å’Œæ–½ç£·å¯¹å†¬å°éº¦ç¾¤ä½“ç»“æž„ã€å ‰åˆç‰¹æ€§å’Œäº§é‡çš„å½±å“.

Under Xinjiang winter wheat seeding pattern, in order to sort out proper phosphorus application (PA) and find out the effects and mechanism of PA on population structure, photosynthesis characteristics and yield and provide reliable evidence for PA management of winter wheat, we arranged a two-factor complete split-plot design of wheat variety "Xindong 22". The main area consisted of two seeding ways: drill seeding pattern (D) and uniform seeding pattern (U), while in the sub-area there were four levels of PA(P2O5): 0, 60, 120, and 180 kg·hm-2(represented by P0, P60, P120 and P180 for those treatments, respectively). The results showed that the earbearing percentage in U was 15.9% higher than that in D, and the other features (PAR interception rate, extinction coefficient, leaf area index, SPAD and photosynthetic parameters) were more optimal in 120 kg·hm-2 treatment. Our results showed that the 120 kg·hm-2 treatment in U would be the optimal option with respect to population structure, photosynthetic characteristics, and yield.

Registration of Panoramic/Fish-Eye Image Sequence and LiDAR Points Using Skyline Features.

We propose utilizing a rigorous registration model and a skyline-based method for automatic registration of LiDAR points and a sequence of panoramic/fish-eye images in a mobile mapping system (MMS). This method can automatically optimize original registration parameters and avoid the use of manual interventions in control point-based registration methods. First, the rigorous registration model between the LiDAR points and the panoramic/fish-eye image was built. Second, skyline pixels from panoramic/fish-eye images and skyline points from the MMS’s LiDAR points were extracted, relying on the difference in the pixel values and the registration model, respectively. Third, a brute force optimization method was used to search for optimal matching parameters between skyline pixels and skyline points. In the experiments, the original registration method and the control point registration method were used to compare the accuracy of our method with a sequence of panoramic/fish-eye images. The result showed: (1) the panoramic/fish-eye image registration model is effective and can achieve high-precision registration of the image and the MMS’s LiDAR points; (2) the skyline-based registration method can automatically optimize the initial attitude parameters, realizing a high-precision registration of a panoramic/fish-eye image and the MMS’s LiDAR points; and (3) the attitude correction values of the sequences of panoramic/fish-eye images are different, and the values must be solved one by one.

Nonlinear spectral mixture effects for photosynthetic/non-photosynthetic vegetation cover estimates of typical desert vegetation in western China.

Desert vegetation plays significant roles in securing the ecological integrity of oasis ecosystems in western China. Timely monitoring of photosynthetic/non-photosynthetic desert vegetation cover is necessary to guide management practices on land desertification and research into the mechanisms driving vegetation recession. In this study, nonlinear spectral mixture effects for photosynthetic/non-photosynthetic vegetation cover estimates are investigated through comparing the performance of linear and nonlinear spectral mixture models with different endmembers applied to field spectral measurements of two types of typical desert vegetation, namely, Nitraria shrubs and Haloxylon. The main results were as follows. (1) The correct selection of endmembers is important for improving the accuracy of vegetation cover estimates, and in particular, shadow endmembers cannot be neglected. (2) For both the Nitraria shrubs and Haloxylon, the Kernel-based Nonlinear Spectral Mixture Model (KNSMM) with nonlinear parameters was the best unmixing model. In consideration of the computational complexity and accuracy requirements, the Linear Spectral Mixture Model (LSMM) could be adopted for Nitraria shrubs plots, but this will result in significant errors for the Haloxylon plots since the nonlinear spectral mixture effects were more obvious for this vegetation type. (3) The vegetation canopy structure (planophile or erectophile) determines the strength of the nonlinear spectral mixture effects. Therefore, no matter for Nitraria shrubs or Haloxylon, the non-linear spectral mixing effects between the photosynthetic / non-photosynthetic vegetation and the bare soil do exist, and its strength is dependent on the three-dimensional structure of the vegetation canopy. The choice of linear or nonlinear spectral mixture models is up to the consideration of computational complexity and the accuracy requirement.

HSP90 expression correlation with the freezing resistance of bull sperm.

To date, there has been little improvement in cryopreservation of bull sperm due to lack of understanding of the freezing mechanisms. Therefore, this study set out to investigate expression levels of fertility-associated proteins in bull sperm, and in particular the relationship between the 90 kDa heat-shock protein (HSP90) and the sperm characteristics after freezing-thawing. Semen was collected from eight Holstein bulls by artificial vagina. Characteristics of these fresh semen, including sperm motility, morphology, viability and concentration, were evaluated. Sperm quality was also assessed after freezing-thawing. Eight ejaculates were divided into two groups based on freezing resistance and sperm motility. Sperm proteins were extracted and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and western blotting were performed. SDS-PAGE results showed that there was substantial diversity in 90 kDa proteins in the frozen-thawed sperm and HSP90 was confirmed as one of the 90 kDa proteins by western blot. This study indicated that HSP90 expression correlated positively with sperm quality. The amount of expressed 90 kDa proteins in the high freezing resistance (HFR) group was significantly higher than that in the low freezing resistance (LFR) group (P < 0.05). Thus, higher expression of HSP90 could probably lead to the higher motility and freezing resistance of sperm found after freezing-thawing. Therefore, we concluded that level of HSP90 expression could be used to predict reliably and simply the freezing resistance of bull sperm.

Glycocerebroside bearing a novel long-chain base from Sagina japonica (Caryophyllaceae).

A new glycocerebroside (1), along with one reported one (2), was isolated from the ethanol extract of Sagina japonica (Caryophyllaceae) and was fully characterized. The structures of two compounds were identified as (2S, 3S, 4R, 8E)-1-(beta-D-glucopyranosyl-3, 4-dihydroxy-2-[(R)-2'- hydroxypalmitoyl]amino-8-heptadecaene (1) and (2S, 3R, 8E)-1-(beta-D-glucopyranosyl-3-hydroxy-2-[(R)-2'-hydroxypalmitoyl]amino-8-octadecaene (2) by using spectroscopic methods ((1)H, (13)C, and 2D NMR, MS) and chemical degradation.

The cryoprotective effect of low-density lipoproteins in extenders on bull spermatozoa following freezing-thawing.

Egg low-density lipoprotein (LDL) was added at concentrations of 7-10% to the extenders used to freeze bull semen and its effects on the motility, mitochondria activity, acrosome integrity, membrane integrity and DNA integrity of frozen-thawed sperm were assessed. Analysis of data showed that the motility and characteristics of spermatozoa movement were higher with LDL in the extender, as compared to the extender containing 20% egg yolk. The results indicated that 8% LDL supplementation provided the highest sperm motility (55.8%) and movement characteristics (VSL, straight linear velocity: 33.8 microm/s; VCL, curvilinear velocity: 50.2 microm/s; LIN, linearity index: 56.5%; STR, mean coefficient: 76.7%; VAP, average path velocity: 35.9 microm/s; WOB, wobble coefficient: 63.9%). A concentration of 10% LDL resulted in a significant decline in the VSL, LIN, VAP and WOB values (P<0.05). Supplementation of LDL at 8% LDL resulted in significantly higher spermatozoa mitochondrial activity, acrosome integrity, membrane integrity and DNA integrity (P<0.05). According to all measured parameters, the extender containing 8% LDL showed beneficial cryoprotective effects on frozen-thawed bull spermatozoa. In conclusion, our results indicated that the extender containing 8% LDL extracted from egg yolk could be used successfully in the cryopreservation of bull semen with an efficacy that would be greater than present extenders containing 20% egg yolk.

Purification and characterization of a new L-amino acid oxidase from Daboia russellii siamensis venom.

A new L-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom L-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic L-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates--L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 microg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 microM) and TMVA (55.0 nM) with an IC(50) value of 32.8 microg/ml and 32.3 microg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 microg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 microg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 microg/ml.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

Jerdonase, a novel serine protease with kinin-releasing and fibrinogenolytic activity from Trimeresurus jerdonii venom.

A novel kinin-releasing and fibrin(ogen)olytic enzyme termed jerdonase was purified to homogeneity from the venom of Trimeresurus jerdonii by DEAE Sephadex A-50 anion exchange, Sephadex G-100 (superfine) gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). Jerdonase migrated as a single band with an approximate molecular weight of 55 kD under the reduced conditions and 53 kD under the non-reduced conditions. The enzyme was a glycoprotein containing 35.8% neutral carbohydrate. The N-terminal amino acid sequence of jerdonase was determined to be IIGGDECNINEHPFLVALYDA, which showed high sequence identity to other snake venom serine proteases. Jerdonase catalyzed the hydrolysis of BAEE, S-2238 and S-2302, which was inhibited by phenylmethylsulfonyl fluoride (PMSF), but not affected by ethylenediaminetetraacetic acid (EDTA). Jerdonase preferentially cleaved the A alpha-chain of human fibrinogen with lower activity towards B beta-chain. Moreover, the enzyme hydrolyzed bovine low-molecular-mass kininogen and releasing bradykinin. In conclusion, all results indicated that jerdonase was a multifunctional venom serine protease.

Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus.

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating unit of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/-0.3) unit (mg/L), and TmF (5.0 x10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x10(-5 )mg/kg) with approximate descent value of (14 +/-2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin II, 2 x10(-3) mg of TmF caused 50% inhibition (IC(50)) of angiotensin- converting enzyme (ACE) hydrolyzing activity to bradykinin.