Identification of peptides that bind with thymidylate synthase RNA using mRNA display technique

Using in vitro selection method to isolate nucleic acids, peptides and proteins has been studied intensively in recent years. In vitro mRNA display is a new and effective technique for peptides selection, and the rationale of this technique is that a synthetic mRNA with puromycin could covalently link with the protein that it encodes, thus an mRNA-protein fusion is formed. This approach has been used in identification of many functional peptides. The peptides binding with thymidylate synthase RNA were isolated using mRNA display technique from a large peptide library (>10(13) different sequences). The selection scheme was constructed, and the experimental conditions, including library synthesis, formation of RNA-peptide fusion and RNA immobilization were optimized. Eight cycles have been processed and the results confirmed that the selected peptides could bind with thymidylate synthase mRNA specifically. Compared the amino acid sequences of the selected peptides with those from the initial random library, the basic and aromatic residues in selected peptides were enriched significantly, suggesting these peptide regions may be important in the peptide-TS mRNA interaction. As a novel in vitro selection approach, mRNA display technique would be developed as a powerful tool for isolation of functional peptides and proteins that could interact with immobilized targets with high affinity and specificity.

Molecular cloning and expression analysis of Ch-penaeidin, an antimicrobial peptide from Chinese shrimp, Fenneropenaeus chinensis

A new member of antimicrobial peptide genes of the penaeidin family, Ch-penaeidin, has been cloned from the haemocytes of Chinese shrimp, Fenneropenaeus chinensis, by reverse transcription PCR (RT-PCR), 3'-rapid amplification of cDNA end (3'-RACE) and smart cDNA methods. The Ch-penaeidin cDNA was 655 bp and the open reading frame of the cDNA encoded a 71 amino acid peptide. Ch-penaeidin contained a putative NH2-terminal signal Sequence (1-19) followed by a mature peptide (20-71). The sequence identify with other penaeidins from Litopenaeus vannamei and Litopenaeus setiferus is between 48% and 71%. The signal sequence of Ch-penaeidin is almost completely identical to that of other penaeidins, while differing relatively in the N-terminal domain of the mature peptide. Ch-penaeidin was designated as a novel member of class penaeidin 3 according to phylogenetic analysis. The Mature peptide. with a predicted molecular weight of 5589.32 Da, and a pI of 9.77, has eight positively charged amino acids and no negatively charged amino acids. The expression and distribution of Ch-penaeidin in Unchallenged shrimps were studied by RT-PCR, Northern blot and in situ hybridisation. The results showed that the Ch-penaeidin transcripts were detected in haemocytes (granular haemocytes), heart, gill, intestine, and subcuticular epithelia of the shrimp. and that Ch-penaeidin was constitutively expressed mainly in haemocytes. (C) 2003 Elsevier Ltd. All rights reserved.

Preliminary study on a potential antibacterial peptide derived from histone H2A in hemocytes of scallop Chlamys farreri

Histone H2A is reported to participate in host defense response through producing novel antimicrobial peptides (AMPs) from its N-terminus in vertebrates and invertebrates, while the AMPs derived from H2A have not to our knowledge been reported in mollusca. In the present study, gene cloning, mRNA expression of H2A from scallop Chlamys farreri, and the recombinant expression of its N-terminus were conducted to investigate whether a similar mechanism exists in mollusca. The full-length DNA of H2A was identified by the techniques of homology cloning and genomic DNA walking, The full-length DNA of the scallop H2A was 696 bp long, including a 5'-terminal untranslated region (UTR) of 90 bp, a 3'-terminal UTR of 228 bp with a stem-loop structure and a canonical polyadenylation signal sequence AATAAA, and an open reading frame of 375 bp encoding a polypeptide of 125 amino acids. The mRNA expression of H2A in the hemocytes of scallop challenged by microbe was measured by semi-quantitative RT-PCR. The expression of H2A was not upregulated after stimulation, suggesting that H2A did not participate in immunity response directly. The DNA fragment of 117 bp encoding 39 amino acids corresponding to the N-terminus of scallop H2A, which was homologous to buforin I in vertebrates, was cloned into Pichia pastoris GS115. The transformants (His(+) Mut(+)) containing multi-copy gene insertion were selected with increasing concentration of antibiotic G418. The peptide of 39 amino acids was expressed by induction of 0.5% methanol. The recombinant product exerted antibacterial activity against both Gram-positive (G(+)) and Gram-negative (G(-)) bacteria. The antibacterial activity toward G(+) bacteria was 2.5 times more than that against G(-) bacteria. The results elucidated that N-terminus of H2A was a potential AMP and provided a promising candidate for a new antibiotic screening. However, whether H2A is really involved in scallop immune response mechanisms needs to be further investigated. (C) 2006 Elsevier Ltd. All rights reserved.

In vitro selected peptides bind with thymidylate synthase mRNA and inhibit its translation

Thymidylate synthase (TS), an essential enzyme for catalyzing the biosynthesis of thymidylate, is a critical therapeutic target in cancer therapy. Recent studies have shown that TS functions as an RNA-binding protein by interacting with two different sequences on its own mRNA, thus, repressing translational efficiency. In this study, peptides binding TS RNA with high affinity were isolated using mRNA display from a large peptide library (>10(13) different sequences). The randomized library was subjected up to twelve rounds of in vitro selection and amplification. Comparing the amino acid composition of the selected peptides (12th round, R12) with those from the initial random library (round zero, R0), the basic and aromatic residues in the selected peptides were enriched significantly, suggesting that these peptide regions might be important in the peptide-TS mRNA interaction. Categorizing the amino acids at each random position based on their physicochemical properties and comparing the distributions with those of the initial random pool, an obvious basic charge characteristic was found at positions 1, 12, 17 and 18, suggesting that basic side chains participate in RNA binding. Secondary structure prediction showed that the selected peptides of R12 pool represented a helical propensity compared with R0 pool, and the regions were rich in basic residues. The electrophoretic gel mobility shift and in vitro translation assays showed that the peptides selected using mRNA display could bind TS RNA specifically and inhibit the translation of TS mRNA. Our results suggested that the identified peptides could be used as new TS inhibitors and developed to a novel class of anticancer agents.

Construction and characterization of two bacterial artificial chromosome libraries of Zhikong scallop, Chlamys farreri Jones et Preston, and identification of BAC clones containing the genes involved in its innate immune system

Large-insert bacterial artificial chromosome (BAC) libraries are necessary for advanced genetics and genomics research. To facilitate gene cloning and characterization, genome analysis, and physical mapping of scallop, two BAC libraries were constructed from nuclear DNA of Zhikong scallop, Chlamys farreri Jones et Preston. The libraries were constructed in the BamHI and MboI sites of the vector pECBAC1, respectively. The BamHI library consists of 73,728 clones, and approximately 99% of the clones contain scallop nuclear DNA inserts with an average size of 110 kb, covering 8.0x haploid genome equivalents. Similarly, the MboI library consists of 7680 clones, with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1x. The combined libraries collectively contain a total of 81,408 BAC clones arrayed in 212 384-well microtiter plates, representing 9.1x haploid genome equivalents and having a probability of greater than 99% of discovering at least one positive clone with a single-copy sequence. High-density clone filters prepared from a subset of the two libraries were screened with nine pairs of Overgos designed from the cDNA or DNA sequences of six genes involved in the innate immune system of mollusks. Positive clones were identified for every gene, with an average of 5.3 BAC clones per gene probe. These results suggest that the two scallop BAC libraries provide useful tools for gene cloning, genome physical mapping, and large-scale sequencing in the species.

Construction of bacterial artificial chromosome libraries for Zhikong Scallop Chlamys farreri

Two Large-insert genomic bacterial artificial chromosome (BAC) libraries of Zhikong scallop Chlamys farreri were constructed to promote our genetic and genomic research. High-quality megabase-sized DNA was isolated from the adductor muscle of the scallop and partially digested by BamH I and Mbo I, respectively. The BamH I library consisted of 53 760 clones while the Mbo I library consisted of 7 680clones. Approximately 96 % of the clones in BamH I library contained nuclear DNA inserts in average size of 100 kb, providing a coverage of 5.3 haploid genome equivalents. Similarly, the Mbo I library with an average insert of 145 kb and no insert-empty clones, thus providing a genome coverage of 1.1 haploid genome equivalents.

PenBase, the shrimp antimicrobial peptide penaeidin database: Sequence-based classification and recommended nomenclature

Antimicrobial peptides play a major role in innate immunity. The penaeidins, initially characterized from the shrimp Litopenaeus vannamei, are a family of antimicrobial peptides that appear to be expressed in all penaeid shrimps. As of recent, a large number of penaeid nucleotide sequences have been identified from a variety of penaeid shrimp species and these sequences currently reside in several databases under unique identifiers with no nomenclatural continuity. To facilitate research in this field and avoid potential confusion due to a diverse number of nomenclatural designations, we have made a systematic effort to collect, analyse, and classify all the penaeidin sequences available in every database. We have identified a common penaeidin signature and subsequently established a classification based on amino acid sequences. In order to clarify the naming process, we have introduced a 'penaeidin nomenclature' that can be applied to all extant and future penaeidins. A specialized database, PenBase, which is freely available at http://www.penbase.immunaqua.com, has been developed for the penaeidin family of antimicrobial peptides, to provide comprehensive information about their properties, diversity and nomenclature. (c) 2005 Elsevier Ltd. All rights reserved.

Distribution of the nonlinear random ocean wave period

Because of the intrinsic difficulty in determining distributions for wave periods, previous studies on wave period distribution models have not taken nonlinearity into account and have not performed well in terms of describing and statistically analyzing the probability density distribution of ocean waves. In this study, a statistical model of random waves is developed using Stokes wave theory of water wave dynamics. In addition, a new nonlinear probability distribution function for the wave period is presented with the parameters of spectral density width and nonlinear wave steepness, which is more reasonable as a physical mechanism. The magnitude of wave steepness determines the intensity of the nonlinear effect, while the spectral width only changes the energy distribution. The wave steepness is found to be an important parameter in terms of not only dynamics but also statistics. The value of wave steepness reflects the degree that the wave period distribution skews from the Cauchy distribution, and it also describes the variation in the distribution function, which resembles that of the wave surface elevation distribution and wave height distribution. We found that the distribution curves skew leftward and upward as the wave steepness increases. The wave period observations for the SZFII-1 buoy, made off the coast of Weihai (37A degrees 27.6' N, 122A degrees 15.1' E), China, are used to verify the new distribution. The coefficient of the correlation between the new distribution and the buoy data at different spectral widths (nu=0.3-0.5) is within the range of 0.968 6 to 0.991 7. In addition, the Longuet-Higgins (1975) and Sun (1988) distributions and the new distribution presented in this work are compared. The validations and comparisons indicate that the new nonlinear probability density distribution fits the buoy measurements better than the Longuet-Higgins and Sun distributions do. We believe that adoption of the new wave period distribution would improve traditional statistical wave theory.

The effects of random surface waves on the steady Ekman current solutions

The response of near-surface current profiles to wind and random surface waves are studied based on the approach of Jenkins [1989. The use of a wave prediction model for driving a near surface current model. Dtsch. Hydrogr. Z. 42,134-149] and Tang et al. [2007. Observation and modeling of surface currents on the Grand Banks: a study of the wave effects on surface currents. J. Geophys. Res. 112, C10025, doi:10.1029/2006JC004028]. Analytic steady solutions are presented for wave-modified Ekman equations resulting from Stokes drift, wind input and wave dissipation for a depth-independent constant eddy viscosity coefficient and one that varies linearly with depth. The parameters involved in the solutions can be determined by the two-dimensional wavenumber spectrum of ocean waves, wind speed, the Coriolis parameter and the densities of air and water, and the solutions reduce to those of Lewis and Belcher [2004. Time-dependent, coupled, Ekman boundary layer solutions incorporating Stokes drift. Dyn. Atmos. Oceans. 37, 313-351] when only the effects of Stokes drift are included. As illustrative examples, for a fully developed wind-generated sea with different wind speeds, wave-modified current profiles are calculated and compared with the classical Ekman theory and Lewis and Belcher's [2004. Time-dependent, coupled, Ekman boundary layer solutions incorporating Stokes drift. Dyn. Atmos. Oceans 37, 313-351] modification by using the Donelan and Pierson [1987. Radar scattering and equilibrium ranges in wind-generated waves with application to scatterometry. J. Geophys. Res. 92, 4971-5029] wavenumber spectrum, the WAM wave model formulation for wind input energy to waves, and wave energy dissipation converted to currents. Illustrative examples for a fully developed sea and the comparisons between observations and the theoretical predictions demonstrate that the effects of the random surface waves on the classical Ekman current are important, as they change qualitatively the nature of the Ekman layer. But the effects of the wind input and wave dissipation on surface current are small, relative to the impact of the Stokes drift. (C) 2008 Elsevier Ltd. All rights reserved.

Second-order solutions for random interfacial waves under steady uniform currents

In the present research, the study of Song (2004) for random interfacial waves in two-layer fluid is extended to the case of fluids moving at different steady uniform speeds. The equations describing the random displacements of the density interface and the associated velocity potentials in two-layer fluid are solved to the second order, and the wave-wave interactions of the wave components and the interactions between the waves and currents are described. As expected, the extended solutions include those obtained by Song (2004) as one special case where the steady uniform currents of the two fluids are taken as zero, and the solutions reduce to those derived by Sharma and Dean (1979) for random surface waves if the density of the upper fluid and the current of the lower fluid are both taken as zero.

Statistical distribution of depth-integrated local horizontal momentum for second-order random ocean waves in finite water depth

Based on the second-order random wave solutions of water wave equations in finite water depth, statistical distributions of the depth- integrated local horizontal momentum components are derived by use of the characteristic function expansion method. The parameters involved in the distributions can be all determined by the water depth and the wave-number spectrum of ocean waves. As an illustrative example, a fully developed wind-generated sea is considered and the parameters are calculated for typical wind speeds and water depths by means of the Donelan and Pierson spectrum. The effects of nonlinearity and water depth on the distributions are also investigated.

Statistical distribution of wave-surface elevation for second-order random directional ocean waves in finite water depth

Based on the second-order random wave solutions of water wave equations in finite water depth, a statistical distribution of the wave-surface elevation is derived by using the characteristic function expansion method. It is found that the distribution, after normalization of the wave-surface elevation, depends only on two parameters. One parameter describes the small mean bias of the surface produced by the second-order wave-wave interactions. Another one is approximately proportional to the skewness of the distribution. Both of these two parameters can be determined by the water depth and the wave-number spectrum of ocean waves. As an illustrative example, we consider a fully developed wind-generated sea and the parameters are calculated for various wind speeds and water depths by using Donelan and Pierson spectrum. It is also found that, for deep water, the dimensionless distribution reduces to the third-order Gram-Charlier series obtained by Longuet-Higgins [J. Fluid Mech. 17 (1963) 459]. The newly proposed distribution is compared with the data of Bitner [Appl. Ocean Res. 2 (1980) 63], Gaussian distribution and the fourth-order Gram-Charlier series, and found our distribution gives a more reasonable fit to the data. (C) 2002 Elsevier Science B.V. All rights reserved.

Second-order solutions for random interfacial waves in N-layer density-stratified fluid with steady uniform currents

In the present paper, the random inter facial waves in N-layer density-stratified fluids moving at different steady uniform speeds are researched by using an expansion technique, and the second-order a symptotic solutions of the random displacements of the density interfaces and the associated velocity potentials in N-layer fluid are presented based on the small amplitude wave theory. The obtained results indicate that the wave-wave second-order nonlinear interactions of the wave components and the second-order nonlinear interactions between the waves and currents are described. As expected, the solutions include those derived by Chen (2006) as a special case where the steady uniform currents of the N-layer fluids are taken as zero, and the solutions also reduce to those obtained by Song (2005) for second-order solutions for random interfacial waves with steady uniform currents if N=2.

Statistical distribution of nonlinear random wave height

A statistical model of random wave is developed using Stokes wave theory of water wave dynamics. A new nonlinear probability distribution function of wave height is presented. The results indicate that wave steepness not only could be a parameter of the distribution function of wave height but also could reflect the degree of wave height distribution deviation from the Rayleigh distribution. The new wave height distribution overcomes the problem of Rayleigh distribution that the prediction of big wave is overestimated and the general wave is underestimated. The prediction of small probability wave height value of new distribution is also smaller than that of Rayleigh distribution. Wave height data taken from East China Normal University are used to verify the new distribution. The results indicate that the new distribution fits the measurements much better than the Rayleigh distribution.

Probability distribution of random wave-current forces

Based on the second-order solutions obtained for the three-dimensional weakly nonlinear random waves propagating over a steady uniform current in finite water depth, the joint statistical distribution of the velocity and acceleration of the fluid particle in the current direction is derived using the characteristic function expansion method. From the joint distribution and the Morison equation, the theoretical distributions of drag forces, inertia forces and total random forces caused by waves propagating over a steady uniform current are determined. The distribution of inertia forces is Gaussian as that derived using the linear wave model, whereas the distributions of drag forces and total random forces deviate slightly from those derived utilizing the linear wave model. The distributions presented can be determined by the wave number spectrum of ocean waves, current speed and the second order wave-wave and wave-current interactions. As an illustrative example, for fully developed deep ocean waves, the parameters appeared in the distributions near still water level are calculated for various wind speeds and current speeds by using Donelan-Pierson-Banner spectrum and the effects of the current and the nonlinearity of ocean waves on the distribution are studied. (c) 2006 Elsevier Ltd. All rights reserved.