Cloning and expression analysis of myogenin from flounder (Paralichthys olivaceus) and promoter analysis of muscle-specific expression

Myogenin is a bHLH transcription factor of the MyoD family. It plays a crucial role in myoblast differentiation and maturation. We report here the isolation of flounder myogenin gene and the characterization of its expression patterns. Sequence analysis indicated that flounder myogenin shared a similar structure and the conserved bHLH domain with other vertebrate myogenin genes. Flounder myogenin gene contains 3 exons and 2 introns. Sequence alignment and phylogenetic showed that flounder myogenin was more homologous with halibut (Hippoglossus hippoglossus) myogenin and striped bass (Morone saxatilis) myogenin. Whole-mount embryo in situ hybridization revealed that flounder myogenin was first detected in the medial region of somites that give rise to slow muscles, and expanded later to the lateral region of the somite that become fast muscles. The levels of myogenin transcripts dropped significantly in matured somites at the trunk region. Its expression could only be detected in the caudal somites, which was consistent with the timing of somite maturation. Transient expression analysis showed that the 546 bp flounder myogenin promoter was sufficient to direct muscle-specific GFP expression in zebrafish embryos. (c) 2007 Elsevier Inc. All rights reserved.

Identification and analysis of a CpG motif that protects turbot (Scophthalmus maximus) against bacterial challenge and enhances vaccine-induced specific immunity

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class II alpha, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. (C) 2010 Elsevier Ltd. All rights reserved.

attB site disruption in marine Actinomyces sp M048 via DNA transformation of a site-specific integration vector

An efficient conjugation method has been developed for the marine Actinomyces sp. isolate M048 to facilitate the genetic manipulation of the chandrananimycin biosynthesis gene cluster. A phi C31-derived integration vector pIJ8600 containing oriT and attP fragments was introduced into strain M048 by bi-parental conjugation from Escherichia coli ET12567 to strain M048. Transformation efficiency was (6.38 +/- 0.41) x 10(-5) exconjugants per recipient spore. Analysis of eight exconjugants showed that the plasmid pIJ8600 was stably integrated at a single chromosomal site (attB) of the Actinomyces genome. The DNA sequence of the attB was cloned and shown to be conserved. The results of antimicrobial activity analysis indicated that the insertion of plasmid pIJ8600 seemed to affect the biosynthesis of antibiotics that could strongly inhibit the growth of E. coli and Mucor miehei (Tu284). HPLC-MS analysis of the extracts indicated that disruption of the attB site resulted in the complete abolition of chandrananimycin A-C production, proving the identity of the gene cluster. Instead of chandrananimycins, two bafilomycins were produced through disruption of the attB site from the chromosomal DNA of marine Actinomyces sp. M048.

Thermal conductivity of graded spherical composites with contact resistance

The effective thermal conductivity of graded composites with contact resistance on the inclusion surface is investigated. As an example, we have considered the graded composite media with a spherical particle embedded in a homogeneous matrix, where the thermal conductivity of spherical inclusion is an exponential function k(i) = c exp(betar) (where r is the inside distance of a point in particle from the center of the spherical particle in a spherical coordinate). For both heat contact resistance and perfect contact cases, we have given a reasonable effective medium approximation to calculate the effective conductivity. (C) 2003 Elsevier B.V. All rights reserved.

Effective thermal conductivity of graded nonlinear composites with heat contact resistance

The perturbation expansion method is used to find the effective thermal conductivity of graded nonlinear composites having thermal contact resistance on the inclusion surface. As an example, we have studied the graded composites with cylindrical inclusions immersed in a homogeneous matrix. The thermal conductivity of the cylindrical inclusion is assumed to have a power-law profile of the radial distance r measured from its origin. For weakly nonlinear constitutive relations between the heat flow density q and the temperature field T, namely, q = -mu del T - chi vertical bar del T vertical bar(2) del T, in both the inclusion and the matrix regions, we have derived the temperature distributions using the perturbation expansion method. A nonlinear effective medium approximation of graded composites is proposed to estimate the effective linear and nonlinear thermal conductivities. by considering the temperature singularity on the inclusion surface due to the heat contact resistance. (c) 2006 Elsevier B.V. All rights reserved.

Bacterial roles in the formation of high-molecular-weight dissolved organic matter in estuarine and coastal waters: Evidence from lipids and the compound-specific isotopic ratios

High-molecular-weight dissolved organic matter (HMW-DOM, > 1,000 Daltons) is actively involved in the global biogeochemical cycling of many elements, but its carbon sources and detailed formation pathways are still not well understood. In this study, we measured bulk stable carbon and nitrogen isotopic ratios, lipid composition, and compound-specific carbon isotopic ratios of HMW-DOM samples collected from four U.S. estuaries (Boston Harbor/Massachusetts Bay, Delaware/Chesapeake Bay, San Diego Bay, and San Francisco Bay). Analytical results show (1) a fraction of HMW-DOM (lipid associated) in estuarine and coastal waters is derived from bacteria and phytoplankton; (2) this fraction of HMW-DOM is formed by various release processes of bacterial membrane components and bacterial reworking of phytoplankton-derived material; (3) this fraction of HMW-DOM is generally present in all samples from different coastal systems despite variable organic matter inputs and environmental conditions, suggesting an important bacterial role in HMW-DOM formation.

Identification of Crassostrea ariakensis and related oysters by multiplex species-specific PCR

Genetic markers are needed for rapid and reliable identification of oysters. In this study, we developed multiplex genus- and species-specific PCR markers for the identification of oysters from China. We used the mitochondrial cytochrome oxidase I (COI) and nuclear 28S ribosomal RNA genes for marker development. DNA sequences from different species were obtained from GenBank or by direct sequencing. Sequences were aligned, and genus- and species-specific nucleotides were identified. Primers were designed for genus/species-specific amplification to generate fragments of different sizes. A multiplex set of genus- and species-specific primers from the 28S gene was able to separate C. ariakensis and C. hongkongensis from other species and assign oysters to four genera. A set of species-specific COI primers provided positive identification of all five Crassostrea species from China, C. ariakensis, C. hongkongensis, C. angulata, C. gigas, and C. sikamea in a single PCR. The multiplex PCR assays do not require fluorescence-labeling or post-PCR enzyme digestion, providing a simple, fast and reliable method for the identification of oysters from China.

Selective and specific detection of sulfate-reducing bacteria using potentiometric stripping analysis

A fast, sensitive and reliable potentiometric stripping analysis (PSA) is described for the selective detection of the marine pathogenic sulfate-reducing bacterium (SRB). Desulforibrio caledoiensis. The chemical and electrochemical parameters that exert influence on the deposition and stripping of lead ion, such as deposition potential, deposition time and pH value were carefully studied. The concentration of SRB was determined in acetate buffer solution (pH 5.2) under the optimized condition (deposition potential of -1.3 V. deposition time of 250 s, ionic strength of 0.2 mol L-1 and oxidant mercury (II) concentration of 40 mg L-1). A linear relationship between the stripping response and the logarithm of the bacterial concentration was observed in the range of 2.3 x 10 to 2.3 x 10(7) cfu mL(-1). In addition, the potentiometric stripping technique gave a distinct response to the SRB, but had no obvious response to Escherichia coli. The measurement system has a potential for further applications and provides a facile and sample method for detection of pathogenic bacteria. (C) 2010 Elsevier B.V. All rights reserved.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.

云南岩石圈流变学及多震层孕震环境

The Study on rheology of the lithosphere and the environments of the seismogenic layer is currently the basic project of the international earthquake research. Yunnan is the ideal place for studying this project. Through the multi-disciplinary comprehensive study of petrology, geophysics, seismo-geology, rock mechanics, etc., the depth-strength profiles of the lithosphere have been firstly constructed, and the seismogenic layer and its geophysical and tectonic environments in Yunnan have been systematically expounded in this paper. The related results achieved are of the important significances for further understanding the mechanism of strong earthquake generation, dividing the potential foci and exposing recent geodynamical processes in Yunnan. Through the comprehensive contrast of the metamorphic rocks in early and middle Proterozoic outcropping on the surface, DSS data and experimental data of rock seismic velocity under high temperature and high pressure, the petrological structure of the crust and upper mantle has been studied on Yunnan: the upper, middle and lower crust is composed of the metamorphic rocks of greenschist, amphibolite and granulite facies, respectively or granitoids, diorites and gabbros, respectively, and the upper mantle composed of the peridotites. Through the contrast studies of the heat flow and epicenters of the strong earthquakes, the distribution of the geotemperature and the data of focal depth, the relationship of between seismicity and geothermal structure of the lithosphere in Yunnan has been studied: the strong earthquakes with magnitude M ≥ 6.0 mainly take place at the geothermal gradient zone, and the seismic foci densely distribute between 200～500 ℃ isogeotherms. On the basis of studies of the rock properties and constituents of the crust and upper mantle and geothermal structure of the lithosphere, the structure of the rheological stratification of the lithosphere has been studied, and the corresponding depth-strength profiles have been constructed in Yunnan. The lithosphere in majority region of Yunnan has the structure of the rheological stratification, i.e. the brittle regime in the upper crust or upper part of the upper crust, ductile regime in the middle crust or lower part of the upper crust to middle crust, ductile regime in the lower crust and ductile regime in the subcrustal lithosphere. The rheological stratification has the quite marked lateral variations in the various tectonic units. The distributions of the seismogenic layer have been determined by using the high accurate data of focal depth. Through the contrast of the petrological structure, the structure of seismic velocity, electric structure, geotemperature structure, and rheological structure and the study of the focal mechanism in the seismogenic layer, the geophysical environments of the seismogenic layer in Yunnan have been studied. The seismogenic layer in Yunnan is located at the depths of 3 ～ 20 km; the rocks in the seismogenic layer are composed of the metamorphic rocks of greenschist to amphibolite facies (or granites to diorites); the seismogenic layer and its internal focal regions of strong earthquakes have the structure of medium properties with the relatively high seismic velocity, high density and high resistivity; there exists the intracrustal low seismic velocity and high conductivity layer bellow the seismogenic layer, the geotemperature is generally 100～500 ℃ in the depth range in which the seismogenic layer is located. The horizontal stress field predominates in the seismogenic layer, the seismogenic layer corresponds to the brittle regime of the upper crust or brittle regime of the upper crust to semibrittle regime of the middle crust. The formation of the seismogenic layer, preparedness and occurrence of the strong earthquakes is the result of the comprehensive actions of the source fault, rock constituent, structure of the medium properties, distribution of the geotemperature, rheological structure of the seismogenic layer and its external environments. Through the study of the structure, active nature, slip rate, segmentation of the active faults, and seismogenic faults, the tectonic environments of the seismogenic layer in Yunnan have been studied. The source faults of the seismogenic layer in Yunnan are mainly A-type ones and embody mainly the strike slip faults with high dip angle. the source faults are the right-lateral strike slip ones with NW-NNW trend and left-lateral strike slip ones with NE-NEE trend in Southwestern Yunnan, the right-lateral strike slip ones with NNW trend and left-lateral strike slip ones with NNE trend (partially normal ones) in Northwestern Yunnan, the right-lateral strike slip ones with NWW trend in Central Yunnan and left-lateral strike slip ones with NW-NNW trend in Eastern Yunnan. Taking Lijiang earthquake with Ms = 7.0 for example. The generating environments of the strong earthquake and seismogenic mechanical mechanism have been studied: the source region of the strong earthquake has the media structure with the relatively high seismic velocity and high resistivity, there exists the intracrustal low velocity and high conductivity layer bellow it and the strong earthquakes occur near the transitional zone of the crustal brittle to ductile deformation. These characteristics are the generality of the generating environments of strong earthquakes. However, the specific seismogenic tectonic environments and action of the stress field of the seismic source in the various regions, correspondingly constrains the dislocation and rupture mechanical mechanism of source fault of strong earthquake.

Low temperature specific heat and thermal stability of Fe-B ultrafine amorphous alloy particles

Fe-B ultrafine amorphous alloy particles (UFAAP) were prepared by chemical reduction of Fe3+ with NaBHO4 and confirmed to be ultrafine amorphous particles by transmission electron microscopy and X-ray diffraction. The specific heat of the sample was measured by a high precision adiabatic calorimeter, and a differential scanning calorimeter was used for thermal stability analysis. A topological structure of Fe-B atoms is proposed to explain two crystallization peaks and a melting peak observed at T=600, 868 and 1645 K, respectively.