Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K-i value of 0.06 muM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K-i value of 1 muM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions. (C) 2002 Elsevier Science Inc. All rights reserved.

Identification of Prorocentrum minimum and Takayama pulchella by fluorescence in situ hybridization through epifluorescence microscopy and flow cytometry

Partial rDNA sequences of Prorocentrum minimum and Takayama pulchella were amplified, cloned and sequenced. and these sequence data were deposited in the GenBank. Eight oligonucleotide probes (DNA probes) were designed based on the sequence analysis. The probes were employed to detect and identify P. minimum and T. pulchella in unialgal and mixed algal samples with a fluorescence in situ hybridization method using flow cytometry. Epifluorescence micrographs showed that these specific probes labeled with fluorescein isothiocyanate entered the algal cells and bound to target sequences, and the fluorescence signal resulting from whole-cell hybridization varied from probe to probe. These DNA probes and the hybridization protocol we developed were specific and effective for P. minimum and T. pulchella, without any specific binding to other algal species. The hybridization efficiency of different probes specific to P. minimum was in the order: PM18S02 > PM28S02 > PM28S01 > PM18S01, and that of the probes specific to T. pulchella was TP18S02 > TP28S01 > TP28S02 > TP18S01. The different hybridization efficiency of the DNA probes could also be shown in the fluorescent signals between the labeled and unlabeled cells demonstrated using flow cytometry. The DNA probes PM18S02, PM28S02; TP18S02 and TP28S01, and the protocol, were also useful for the detection of algae in natural samples.

Molecular cloning of growth hormone receptor (GHR) from common carp (Cyprinus carpio L.) and identification of its two forms of mRNA transcripts

The cDNA of growth hormone receptor (GHR) was cloned from the liver of 2-year common carp (Cyprinus carpio L.) by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE). Its open reading frame (ORF) of 1806 nucleotides is translated into a putative peptide of 602 amino acids, including an extracellular ligand-binding domain of 244 amino acids (aa), a single transmembrane domain of 24 aa and an intracellular signal-transduction domain of 334 aa. Sequence analysis indicated that common carp GHR is highly homologous to goldfish (Carassius auratus) GHR at both gene and protein levels. Using a pair of gene-specific primers, a GHR fragment was amplified from the cDNA of 2-year common carp, a 224 bp product was identified in liver and a 321 bp product in other tissues. The sequencing of the products and the partial genomic DNA indicated that the difference in product size was the result of a 97 bp intron that alternatively spliced. In addition, the 321 bp fragment could be amplified from all the tissues of 4-month common carp including liver, demonstrating the occurrence of the alternative splicing of this intron during the development of common carp. Moreover, a semi-quantitative RT-PCR was performed to analyze the expression level of GHR in tissues of 2-year common carp and 4-month common carp. The result revealed that in the tissues of gill, thymus and brain, the expression level of GHR in 2-year common carp was significantly tower than that of 4-month common carp.

An improved PCR method for direct identification of Porphyra (Bangiales, Rhodophyta) using conchocelis based on a RUBISCO intergenic spacer

An improved method of PCR in which the small segment of conchocelis is amplified directly without DNA extraction was used to amplify a RUBISCO intergenic spacer DNA fragment from nine species of red algal genus Porphyra (Bangiales, Rhodophyta), including Porphyra yezoensis (Jiangsu, China), P. haitanensis (Fujian, China), P. oligospermatangia (Qingdao, China), P. katadai (Qingdao, China), P. tenera (Qingdao, China), P. suborboculata (Fujian, China), P. pseudolinearis (Kogendo, Korea), P. linearis (Devon, England), and P. fallax (Seattle, USA). Standard PCR and the method developed here were both conducted using primers specific for the RUBISCO spacer region, after which the two PCR products were sequenced. The sequencing data of the amplicons obtained using both methods were identical, suggesting that the improved PCR method was functional. These findings indicate that the method developed here may be useful for the rapid identification of species of Porphyra in a germplasm bank. In addition, a phylogenetic tree was constructed using the RUBISCO spacer and partial rbcS sequence, and the results were in concordant with possible alternative phylogenies based on traditional morphological taxonomic characteristics, indicating that the RUBISCO spacer is a useful region for phylogenetic studies.

Molecular phylogeny and species identification of pufferfish of the genus Takifugu (Tetraodontiformes, Tetraodontidae)

The phylogenetic relationships and species identification of pufferfishes of the genus Takifugu were examined by use of randomly amplified polymorphic DNA (RAPD) and sequencing of the amplified partial mitochondrial 16S ribosomal RNA genes. Amplifications with 200 ten-base primers under predetermined optimal reaction conditions yielded 1962 reproducible amplified fragments ranging from 200 to 3000 bp. Genetic distances between 5 species of Takifugu and Lagocephalus spadiceus as the outgroup were calculated from the presence or absence of the amplified fragments. Approximately 572 bp of the 16S ribosonial RNA gene was amplified, using universal primers, and used to determine the genetic distance values. Topological phylogenic trees for the 5 species of Takifugu and outgroup were generated from neighbor-joining analysis based on the data set of RAPD analysis and sequences of mitochondrial 16S rDNA. The genetic distance between Takifugu rubripes and Takifugu pseudommus was almost the same as that between individuals within cacti species, but much smaller than that between T. rubripes, T. pseudommus, and the other species. The molecular data gathered from both analysis of mitochondria and nuclear DNA strongly indicated that T. rubripes and T. pseudommus should be regarded as the same species. A fragment of approximately 900 bp was amplified from the genome of all 26 T. pseudommus individuals examined and 4 individuals of intermediate varieties between T. rubripes and T. pseudommus. Of the 32 T. rubripes individuals, only 3 had the amplified fragment. These results suggest that this fragment may be useful in distinguishing between T. rubripes and T. pseudommus.

Toxin-screening and identification of bacteria isolated from highly toxic marine gastropod Nassarius semiplicatus

Bacteria isolated from a highly toxic sample of gastropod Nassarius semiplicatus in Lianyungang, Jiangsu Province in July 2007, were studied to probe into the relationship between bacteria and toxicity of nassariid gastropod. The toxicity of the gastropod sample was 2 x 10(2) mouse unit (MU) Per gram Of tissue (wet weight). High concentration of tetrodotoxin (TTX) and its analogues (TTXs) were found in the digestive gland and muscle of the gastropod, using high performance liquid chromatography coupled with mass chromatography (LC-MS). Bacterial strains isolated from the digestive gland were cultured and screened for TTX with a competitive ELISA method. Tetrodotoxin was detected in a proportion of bacterial strains, but the toxin content was low. Partial 16S ribosomal DNA (rDNA) of the TTX-producing strains was then sequenced and compared with those published in the GenBank to tentatively identify the toxic strains. It was found that most of the toxic strains were closely affiliated with genus Vibrio, and the others were related to genus Shewanella, Marinomonas, Tenacibaculum and Aeromonas. These findings suggest that tetrodotoxin-producing bacteria might play an important role in tetrodotoxin accumulation/production in N. semiplicatus. (C) 2008 Elsevier Ltd. All rights reserved.

Identification of nonlinear dynamic systems: Time-frequency filtering and skeleton curves

The nonlinear behavior varying with the instantaneous response was analyzed through the joint time-frequency analysis method for a class of S. D. O. F nonlinear system. A masking operator an definite regions is defined and two theorems are presented. Based on these, the nonlinear system is modeled with a special time-varying linear one, called the generalized skeleton linear system (GSLS). The frequency skeleton curve and the damping skeleton curve are defined to describe the main feature of the non-linearity as well. Moreover, an identification method is proposed through the skeleton curves and the time-frequency filtering technique.

An Amino Acid Substitution Matrix for Protein Conformation Identification

Amino acid substitution matrices play an essential role in protein sequence alignment, a fundamental task in bioinformatics. Most widely used matrices, such as PAM matrices derived from homologous sequences and BLOSUM matrices derived from aligned segments of PROSITE, did not integrate conformation information in their construction. There are a few structure-based matrices, which are derived from limited data of structure alignment. Using databases PDB_SELECT and DSSP, we create a database of sequence-conformation blocks which explicitly represent sequence-structure relationship. Members in a block are identical in conformation and are highly similar in sequence. From this block database, we derive a conformation-specific amino acid substitution matrix CBSM60. The matrix shows an improved performance in conformational segment search and homolog detection.

Identification of Nonlinear Systems Through Time-Frequency Filtering Technique

In the previous paper, a class of nonlinear system is mapped to a so-called skeleton linear model (SLM) based on the joint time-frequency analysis method. Behavior of the nonlinear system may be indicated quantitatively by the variance of the coefficients of SLM versus its response. Using this model we propose an identification method for nonlinear systems based on nonstationary vibration data in this paper. The key technique in the identification procedure is a time-frequency filtering method by which solution of the SLM is extracted from the response data of the corresponding nonlinear system. Two time-frequency filtering methods are discussed here. One is based on the quadratic time-frequency distribution and its inverse transform, the other is based on the quadratic time-frequency distribution and the wavelet transform. Both numerical examples and an experimental application are given to illustrate the validity of the technique.

Partial-Dislocation-Mediated Processes in Nanocrystalline Ni with Nonequilibrium Grain Boundaries

The partial-dislocation-mediated processes have so far eluded high-resolution transmission electron microscopy studies in nanocrystalline nc Ni with nonequilibrium grain boundaries. It is revealed that the nc Ni deformed largely by twinning instead of extended partials. The underlying mechanisms including dissociated dislocations, high residual stresses, and stress concentrations near stacking faults are demonstrated and discussed.

Surface Tension and Its Temperature Coefficient of Molten Tin Determined with the Sessile Drop Method at Different Oxygen Partial Pressures

The surface tension of molten tin has been determined by the sessile drop method at The surface tension of molten tin has been determined by the sessile drop method at temperatures ranging from 523 to 1033 K and in the oxygen partial pressure (P-O2) range from 2.85 x 10(-19) to 8.56 x 10(-6) MPa, and its dependence on temperature and oxygen partial pressure has been analyzed. At P-O2 = 2.85 x 10(-19) and 1.06 x 10(-15) MPa, the surface tension decreases linearly with the increase of temperature and its temperature coefficients are -0.151 and -0.094 mNm(-1) K-1, respectively. However, at high P-O2 (3.17 x 10(-10), 8.56 x 10(-6) MPa), the surface tension increases with the temperature near the melting point (505 K) and decreases above 723 K. The surface tension decrease with increasing P-O2 is much larger near the melting point than at temperatures above 823 K. The contact angle between the molten tin and the alumina substrate is 158-173degrees, and the wettability is poor.

Predictions for Partial-Dislocation-Mediated Processes in Nanocrystalline Ni by Generalized Planar Fault Energy Curves: An Experimental Evaluation

Generalized planar fault energy (GPFE) curves have been used to predict partial-dislocation-mediated processes in nanocrystalline materials, but their validity has not been evaluated experimentally. We report experimental observations of a large quantity of both stacking faults and twins in nc Ni deformed at relatively low stresses in a tensile test. The experimental findings indicate that the GPFE curves can reasonably explain the formation of stacking faults, but they alone were not able to adequately predict the propensity of deformation twinning.

Some advances in study of crack identification problems

In the present paper, the crack identification problems are investigated. This kind of problems belong to the scope of inverse problems and are usually ill-posed on their solutions. The paper includes two parts: (1) Based on the dynamic BIEM and the optimization method and using the measured dynamic information on outer boundary, the identification of crack in a finite domain is investigated and a method for choosing the high sensitive frequency region is proposed successfully to improve the precision. (2) Based on 3-D static BIEM and hypersingular integral equation theory, the penny crack identification in a finite body is reduced to an optimization problem. The investigation gives us some initial understanding on the 3-D inverse problems.

Designing Of Partial Similarity Models And Evaluation Method In Polymer Flooding Experiment

Based on the scaling criteria of polymer flooding reservoir obtained in our previous work in which the gravity and capillary forces, compressibility, non-Newtonian behavior, absorption, dispersion, and diffusion are considered, eight partial similarity models are designed. A new numerical approach of sensitivity analysis is suggested to quantify the dominance degree of relaxed dimensionless parameters for partial similarity model. The sensitivity factor quantifying the dominance degree of relaxed dimensionless parameter is defined. By solving the dimensionless governing equations including all dimensionless parameters, the sensitivity factor of each relaxed dimensionless parameter is calculated for each partial similarity model; thus, the dominance degree of the relaxed one is quantitatively determined. Based on the sensitivity analysis, the effect coefficient of partial similarity model is defined as the summation of product of sensitivity factor of relaxed dimensionless parameter and its relative relaxation quantity. The effect coefficient is used as a criterion to evaluate each partial similarity model. Then the partial similarity model with the smallest effect coefficient can be singled out to approximate to the prototype. Results show that the precision of partial similarity model is not only determined by the number of satisfied dimensionless parameters but also the relative relaxation quantity of the relaxed ones.

Partial-mediated slips in nanocrystalline Ni at high strain rate

Previous experiments on nanocrystalline Ni were conducted under quasistatic strain rates (similar to 3x10(-3)/s), which are much lower than that used in typical molecular dynamics simulations (>3x10(7)/s), thus making direct comparison of modeling and experiments very difficult. In this study, the split Hopkinson bar tests revealed that nanocrystalline Ni prefers twinning to extended partials, especially under higher strain rates (10(3)/s). These observations contradict some reported molecular dynamics simulation results, where only extended partials, but no twins, were observed. The accuracy of the generalized planar fault energies is only partially responsible, but cannot fully account for such a difference. (C) 2007 American Institute of Physics.