Whole-body amino acid pattern of F-4 human growth hormone gene-transgenic red common carp (Cyprinus carpio) fed diets with different protein levels

F-4 generation of human growth hormone (hGH) gene-transgenic red common carp, and the non-transgenic controls were fed for 8 weeks on purified diets with 20%, 30% or 40% protein. Analysis of whole-body amino acids showed that the proportions of lysine, leucine, phenylalanine, valine and alanine, as percentages of body protein, increased significantly, while those of arginine, glutamic acid and tyrosine decreased, with increases in dietary protein level in at least one strain of fish. Proportions of the other amino acids were unaffected by the diets. The proportions of lysine and arginine were significantly higher, while those of leucine and alanine were lower in the transgenics than in the controls in at least one diet group. Proportions of the other amino acids were unaffected by strain. The results suggest that the whole-body amino acid profile of transgenic carp, when expressed as proportions of body protein, was in general, similar to that of the non-transgenic controls. (C) 2000 Elsevier Science B.V. All rights reserved.

Differential proteomic analysis of neonatal cardiomyocytes in response to beta-adrenergic receptor stimulation

beta-Adrenoceptors(beta-ARs) play a critical role in regulating cardiac functions under both physiological and pathological conditions. To further explore the mechanisms through which beta-ARs perform its actions, proteomic approaches were adopted to study the global protein patterns in cultured neonatal rat cardiomyocytes exposed to isoproterenol (ISO). A modified method, "Mirror Images in One Gel", was used to improve the reproducibility and resolution power of two-dimensional electrophoresis. A 2-DE map with a good reproducibility was obtained in which 1281 70 spots were detected and about 1191 +/- 54 spots were matched, with an average matching rate of 92.9%. Nine proteins with significant changes were identified by using peptide mass fingerprinting(PMF) data obtained via MALDI-MS.

First-passage time distribution and non-Markovian diffusion dynamics of protein folding

We study the kinetics of protein folding via statistical energy landscape theory. We concentrate on the local-connectivity case, where the configurational changes can only occur among neighboring states, with the folding progress described in terms of an order parameter given by the fraction of native conformations. The non-Markovian diffusion dynamics is analyzed in detail and an expression for the mean first-passage time (MFPT) from non-native unfolded states to native folded state is obtained. It was found that the MFPT has a V-shaped dependence on the temperature. We also find that the MFPT is shortened as one increases the gap between the energy of the native and average non-native folded states relative to the fluctuations of the energy landscape. The second- and higher-order moments are studied to infer the first-passage time distribution. At high temperature, the distribution becomes close to a Poisson distribution, while at low temperatures the distribution becomes a Levy-type distribution with power-law tails, indicating a nonself-averaging intermittent behavior of folding dynamics. We note the likely relevance of this result to single-molecule dynamics experiments, where a power law (Levy) distribution of the relaxation time of the underlined protein energy landscape is observed.

Temperature dependence of the distribution of the first passage time: Results from discontinuous molecular dynamics simulations of an all-atom model of the second beta-hairpin fragment of protein G

More than 22 000 folding kinetic simulations were performed to study the temperature dependence of the distribution of first passage time (FPT) for the folding of an all-atom Go-like model of the second beta-hairpin fragment of protein G. We find that the mean FPT (MFPT) for folding has a U (or V)-shaped dependence on the temperature with a minimum at a characteristic optimal folding temperature T-opt*. The optimal folding temperature T-opt* is located between the thermodynamic folding transition temperature and the solidification temperature based on the Lindemann criterion for the solid. Both the T-opt* and the MFPT decrease when the energy bias gap against nonnative contacts increases. The high-order moments are nearly constant when the temperature is higher than T-opt* and start to diverge when the temperature is lower than T-opt*. The distribution of FPT is close to a log-normal-like distribution at T* greater than or equal to T-opt*. At even lower temperatures, the distribution starts to develop long power-law-like tails, indicating the non-self-averaging intermittent behavior of the folding dynamics. It is demonstrated that the distribution of FPT can also be calculated reliably from the derivative of the fraction not folded (or fraction folded), a measurable quantity by routine ensemble-averaged experimental techniques at dilute protein concentrations.

A fibrinogen-related protein from bay scallop Argopecten irradians involved in innate immunity as pattern recognition receptor

The family of fibrinogen-related proteins (FREPs) is a group of proteins with fibrinogen-like domains. Many members of this family play important roles as pattern recognition receptors in innate immune responses. The cDNA of bay scallop Argopecten irradians FREP (designated as AiFREP) was cloned by rapid amplification of cDNA ends (RACE) method based on the expressed sequence tag (EST). The full-length cDNA of AiFREP was of 990 bp. The open reading frame encoded a polypeptide of 251 amino acids, including a signal sequence and a 213 amino acids fibrinogen-like domain. The fibrinogen-like domain of AiFREP was highly similar to those of mammalian ficolins and other FREPs. The temporal expression of AiFREP mRNA in hemolymph was examined by fluorescent quantitative real-time PCR. The mRNA level of scallops challenged by Listonella anguillarum was significantly up-regulated, peaked to 9.39-fold at 9 h after stimulation, then dropped back to 4.37-fold at 12 h, while there was no significant change in the Micrococcus luteus challenged group in all periods of treatment. The function of AiFREP was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli Rosetta gami (DE3). The recombinant AiFREP (rAiFREP) agglutinated chicken erythrocytes and human A, B, O-type erythrocytes. The agglutinating activities were calcium-dependent and could be inhibited by acetyl group-containing carbohydrates. rAiFREP also agglutinated Gram-negative bacteria E. coli JM109, L anguillarum and Gram-positive bacteria M. luteus in the presence of calcium ions. These results collectively suggested that AiFREP functions as a pattern recognition receptor in the immune response of bay scallop and contributed to nonself recognition in invertebrates, which would also provide clues for elucidating the evolution of the lectin pathway of the complement system. (C) 2008 Elsevier Ltd. All rights reserved.

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Protein phosphatase inhibition assay for detection of diarrhetic shellfish poison in oyster

A method based on protein phosphatase enzyme activity inhibition for the detection of diarrhetic shellfish poison (DSP) was used to analyze the DSP toxicity in three oyster samples. Based on the standard dose-effect curve developed with a series of okadaic acid (OA) standard solutions, the DSP toxicity of the three oyster samples collected were screened, and the results showed that there were no OA and dinophysis toxins ( DTXs) in the samples without hydrolization. However, the OA toxicity could be detected in two of the hydrolyzed samples, and the OA toxicity of the two samples were 1.81 and 1.21 mu g OA eq./kg oyster, respectively.