Molecular cloning and expression pattern of oriental river prawn (Macrobrachium nipponense) nitric oxide synthase.

Nitric oxide synthase (NOS) produces nitric oxide (NO) by catalyzing the conversion of l-arginine to l-citrulline, with the concomitant oxidation of nicotinamide adenine dinucleotide phosphate. Recently, various studies have verified the importance of NOS invertebrates and invertebrates. However, the NOS gene family in the oriental river prawn Macrobrachium nipponense is poorly understood. In this study, we cloned the full-length NOS complementary DNA from M. nipponense (MnNOS) and characterized its expression pattern in different tissues and at different developmental stages. Real-time quantitative polymerase chain reaction (RT-qPCR) showed the MnNOS gene to be expressed in all investigated tissues, with the highest levels observed in the androgenic gland (P < 0.05). Our results revealed that the MnNOS gene may play a key role in M. nipponense male sexual differentiation. Moreover, RT-qPCR revealed that MnNOS mRNA expression was significantly increased in post-larvae 10 days after metamorphosis (P < 0.05). The expression of this gene in various tissues indicates that it may perform versatile biological functions in M. nipponense.

Molecular cloning, characterization, and expression analysis of a Broad-Complex homolog during development in the oriental river prawn Macrobrachium nipponense.

Broad-Complex (BR-C) is an early ecdysone-responsive gene encoding a family of zinc-finger transcription factors. In this study, we isolated the full-length cDNA of a BR-C homolog from the testes of the oriental river prawn (Macrobrachium nipponense), according to established expressed sequence tag information, using the rapid amplification of cDNA ends technique. The homolog was designated as MnBR-C. The full-length cDNA of MnBR-C contained a 1095-bp open reading frame encoding a precursor protein of 365 amino acid residues. Comparative and bioinformatic analyses revealed that MnBR-C exhibited a high degree of homology with BR-C proteins, and contained the BTB and Zf-H2C2-2 domains. Real-time quantitative polymerase chain reaction (qPCR) analysis revealed that the MnBR-C expression level varied significantly in the developing embryo, postembryonic larva, and adult tissue. Real-time qPCR showed that the MnBR-C gene was expressed in all of the tissues investigated, with the highest level of expression in the brain. In addition, MnBR-C was more abundantly expressed in the testes than in the ovaries.

Gene expression profile analysis of testis and ovary of oriental river prawn, Macrobrachium nipponense, reveals candidate reproduction-related genes.

This study utilized high-throughput RNA sequencing technology to identify reproduction- and development-related genes of Macrobrachium nipponense by analyzing gene expression profiles of testis and ovary. More than 20 million 1 x 51-bp reads were obtained by Illumina sequencing, generating more than 7.7 and 11.7 million clean reads in the testis and ovary library, respectively. As a result, 10,018 unitags were supposed to be differentially expressed genes (DEGs) between ovary and testis. Compared to the ovary library, 4563 (45.5%) of these DEGs exhibited at least 6-fold upregulated expression, while 5455 (54.5%) DEGs exhibited at least 2-fold downregulated expression in the testis. The Gene Ontology (GO) enrichment analysis showed that 113 GO terms had potential molecular functions in reproduction. The Kyoto Encyclopedia of Genes and Genomes results revealed that the most important pathways may be relevant to reproduction and included 7 pathways. Forty-two genes were identified as reproduction-, development-, and sex-related genes based on GO classification and sequence comparison with other publications, including male reproductive-related LIM protein, spermatogenesis-associated protein, gametocyte-specific factor 1, VASA-like protein, vitellogenin, sex-determining protein fem-1, and other potential candidates. These results will advance research in the field of molecular genetics in M. nipponense and offer a valuable resource for further research related to reproduction in crustaceans.

Molecular cloning and expression analysis of female sterile homeotic gene (fsh) in the oriental river prawn Macrobrachium nipponense.

The gene female sterile homeotic (fsh) plays crucial roles in molecular function, including protein kinase activity and DNA binding, which are involved in biological processes such as terminal region determination and negative regulation of DNA-dependent transcription. Although fsh has been found in Drosophila melanogaster, little is known regarding its expression in crustaceans. In this study, a fsh gene homologue, designated as Mnfsh, was cloned and characterized from the testis of the oriental river prawn, Macrobrachium nipponense, by using EST analysis and the RACE approach for the first time. The full-length cDNA of Mnfsh was 2029 bp, consisting of a 5' UTR of 361 bp, a 3' UTR of 216 bp, and an ORF of 1452 bp encoding 484 amino acids. qRT-PCR analysis showed that the Mnfsh gene was expressed in the testis, ovary, muscle, heart, eyestalk, and abdominal ganglion, with the highest level of expression in the ovary and the lowest in the heart. qRT-PCR analyses showed that the expression levels of Mnfsh mRNA both significantly increased in the zoea stage, the VII larvae, and 1st day post-larvae after metamorphosis. In conclusion, the results of the present study indicate that Mnfsh is an arthropod fsh homologue and probably also plays important roles in embryogenesis, organogenesis, and morphological differentiation of M. nipponense.

Identification of androgenic gland microRNA and their target genes to discover sex-related microRNA in the oriental river prawn, Macrobrachium nipponense.

The oriental river prawn, Macrobrachium nipponense, is an important aquaculture species in China. The androgenic gland produces hormones that play crucial roles in the differentiation of crustaceans to the male sex. MicroRNA (miRNA) post-transcriptionally regulates many protein-coding genes, influencing important biological and metabolic processes. However, currently, there is no published data identifying miRNA in M. nipponense. In this study, we identified novel miRNA in the androgenic gland of M. nipponense. Using the high-throughput Illumina Solexa system, 1077 miRNA were identified from small RNA libraries by aligning with the de novo androgenic gland transcriptome of M. nipponense (obtained from RNA-Seq) and the sequences in the miRBase21 database. A total of 8,248, 76,011, and 78,307 target genes were predicted in the EST and SRA sequences provided in the NCBI database, and the androgenic gland transcriptome of M. nipponense, respectively. Some potential sex-related miRNA were identified based on the function of the predicted target genes. The results of our study provide new information regarding the miRNA expression in M. nipponense, which could be the basis for further genetic studies on decapod crustaceans.

Characterization of the male-specific lethal 3 gene in the oriental river prawn, Macrobrachium nipponense.

In this study, male-specific lethal 3 homolog (Mnmsl3) was cloned and characterized from the freshwater prawn Macrobrachium nipponense (Crustacea: Decapoda: Palaemonidae) by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnmsl3 showed high-sequence homology to the insect Msl3 and contained a conserved chromatin organization modifier domain and an MORF4-related gene domain. Real-time quantitative reverse transcription-polymerase chain reaction showed that the Mnmsl3 gene was expressed in all the investigated tissues, with the highest level of expression in the testis. The expression level of Mnmsl3 between males and females was different in the gonad (testis or ovary), abdominal ganglion, and heart. The results revealed that the Mnmsl3 gene might play roles in regulating chromatin and in dosage compensation of M. nipponense. Real-time quantitative reverse transcription-polymerase chain reaction also revealed that Mnmsl3 mRNA expression was significantly increased in both 5 and 20 days post-larvae after metamorphosis, suggesting that Mnmsl3 plays complex and important roles in the early embryonic development and sex differentiation of M. nipponense.

Genetic diversity analysis of oriental river prawn, Macrobrachium nipponense, in Yellow River using microsatellite marker.

To assess the genetic status of this species, the genetic diversity of wild Macrobrachium nipponense from seven geographic locations in the Yellow River basin were investigated using 20 polymorphic microsatellite DNA loci. The genetic diversity between populations was indicated by the mean number of alleles per locus and mean observed heterozygosity (H) and the expected H, which was arranged from 2 to 10, from 0.4705 to 0.5731, and from 0.5174 to 0.6146, respectively. Hardy-Weinberg equilibrium analysis indicated that a deficiency of heterozygotes existed in all seven populations. Both the F(ST) and AMOVA analyses showed that there is significant difference on population differentiation among populations. The UPGMA clustering tree demonstrated that their close relationship is consistent with their geographic proximity. The data suggest that this Yellow River population has a wide genetic base that is suitable for breeding.

Molecular cloning and expression analysis of two sex-lethal homolog genes during development in the oriental river prawn, Macrobrachium nipponense.

In this study, two Sxl gene homologs, designated as Mnsxl1 and Mnsxl2, were cloned and characterized from the freshwater prawn Macrobrachium nipponense by rapid amplification of cDNA ends. The deduced amino acid sequences of Mnsxl1 and Mnsxl2 showed high sequence homology to the insect Sxl and contained conserved domains in two RNA-binding motifs. Real-time quantitative reverse transcription-polymerase chain reaction (RT-QPCR) showed that the Mnsxl1 and Mnsxl2 genes were expressed in all investigated tissues, with the highest level of expression in the intestine and liver. RT-QPCR also revealed that Mnsxl1 and Mnsxl2 mRNAs expressions were both significantly increased at 5 and 20 days post-larvae after metamorphosis. Thus, the results of the present study imply that Mnsxl1 and Mnsxl2 play complex and important roles in the sex differentiation of M. nipponense.

Regions Important for Hemagglutination Activity and Serotypes of Avibacterium paragallinarum HMTp210 Protein.

Avibacterium paragallinarum is an important respiratory pathogen of domestic chickens. Avibacterium paragallinarum has been subtyped into three serogroups and nine serovars according to the Page and revised Kume schemes. The major hemagglutinin antigen of A. paragallinarum is HMTp210, which is a large protein of about 2000 amino acids (aa), including a 70-aa signal peptide at its N-terminal end. However, the regions important for the hemagglutination (HA) activity and serotypes of HMTp210 remain unclear. In this study we constructed a series of A. paragallinarum strains expressing HMTp210 in-frame deletion mutants and determined their HA titers to identify the regions important for the HA activity and serotypes of HMTp210. Two distinct types of HA activities were found in HMTp210. The type 1 HA activity resided in the region spanning the full-length HA (aa 71-2084), whereas the type 2 resided in the region spanning aa 1003-2084. The putative ligand binding of the type 1 HA activity was located at aa 176-360, which had a structure similar to YadA of Yersinia enterocolitica. The putative ligand binding site of the type 2 HA activity was located at aa 1003-1125, which had a structure similar to UspA1 from Moraxella catarrhalis. The type 1 HA activity appeared to be Page serogroup specific, whereas type 2 appeared to be Kume serovar specific. Finally, sequence analyses of the regions spanning aa 1-400 and aa 1100-1600 of HMTp210 could be useful for the molecular serotyping (the Page and revised Kume schemes) of A. paragallinarum isolates.

Regiones importantes para la actividad de hemaglutinación y serotipos de la proteína HMTp210 de Avibacterium paragallinarum. La bacteria Avibacterium paragallinarum es un patógeno respiratorio importante de los pollos domésticos. Avibacterium paragallinarum se subtipificó en tres serogrupos y nueve serovares de acuerdo con los esquemas revisados de Page y Kume. El principal antígeno de la hemaglutinina de A. paragallinarum es la proteína HMTp210, que es una proteína grande de unos 2000 aminoácidos (aa), que incluye un péptido señal de 70 aminoácidos en su extremo N-terminal. Sin embargo, las regiones importantes para la actividad de hemaglutinación (HA) y de los serotipos de la proteína HMTp210 siguen sin estar determinados. En este estudio, se construyó una serie de cepas de A. paragallinarum que expresaban mutantes de deleción en marco de lectura de HMTp210 y se determinaron sus títulos de hemaglutinación para identificar las regiones importantes para la actividad de hemaglutinación y de los serotipos de HMTp210. Se encontraron dos tipos distintos de actividades hemaglutinación en la proteína HMTp210. La actividad de hemaglutinación de tipo 1 residía en la región que abarcaba la longitud completa (aminoácidos 71­2084), mientras que la de tipo 2 residía en la región que abarcaba entre los aminoácidos 1003­2084. El sitio supuesto de unión al ligando de la actividad de hemaglutinación tipo 1 se ubicó entre los aminoácidos 176­360, que tenía una estructura similar a la proteína YadA de Yersinia enterocolitica. El supuesto sitio de unión del ligando de la actividad de hemaglutinación tipo 2 se ubicó entre los aminoácidos 1003­1125, que tenía una estructura similar a la proteína UspA1 de Moraxella catarrhalis. La actividad de hemaglutinación tipo 1 parecía ser específica del serogrupo Page, mientras que la hemaglutinación tipo 2 parecía ser específica del serovar Kume. Finalmente, los análisis de secuencias de las regiones que abarcan los aminácidos 1­400 y aminoácidos 1100­1600 de HMTp210 podrían ser útiles para la serotipificación molecular (por el esquema revisado de Page y Kume revisado) de aislamientos de A. paragallinarum.

Absence of the adenosine A2A receptor confers pulmonary arterial hypertension and increased pulmonary vascular remodeling in mice.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by sustained elevation of pulmonary vascular resistance resulting from endothelial and smooth muscle cell dysfunction and collagen deposition in pulmonary vascular walls. In this study, we investigated the role of the adenosine A(2A) receptor (A(2A)R) in the development of PAH by determining the effect of genetic inactivation of A(2A)Rs on pulmonary vascular remodeling in mice. METHODS AND RESULTS: We characterized hemodynamic, histological and ultrastructural changes in pulmonary vascular remodeling in A(2A)R knockout (KO) mice compared with their wild-type (WT) littermates after exposure to normoxia and hypoxic conditions. After exposure to normoxia, compared to WT mice, A(2A)R KO mice displayed: (1) increased right ventricular systolic pressures and an elevated ratio of the right ventricle over left ventricle plus septum (Fulton index), (2) increased wall area and thickness as well as enhanced smooth muscle actin immunoreactivity in pulmonary resistance vessels, (3) increased proliferating cell nuclear antigen-positive cells in pulmonary resistance vessels and (4) increased smooth muscle cells hypertrophy and collagen deposition in the adventitia of pulmonary arteriole walls as revealed by electron microscope. By contrast, histological analysis revealed no features of hypertensive nephropathy in A(2A)R KO mice and there was no significant difference in systemic blood pressure, and left ventricular masses among the 3 genotypes. Furthermore, following chronic exposure to hypoxia, A(2A)R KO mice exhibited exacerbated elevation in right ventricular systolic pressure, hypertrophy of pulmonary resistance vessels and increased cell proliferation in pulmonary resistance vessels, compared to WT littermates. Thus, genetic inactivation of A(2A)Rs selectively produced PAH and associated increased smooth muscle proliferation and collagen deposition. CONCLUSIONS: Extracellular adenosine acting at A(2A)Rs represents an important regulatory mechanism to control the development of PAH and pulmonary vascular remodeling.

Relationship Between the Serotypes and Hemagglutinin Gene Sequences of Avibacterium paragallinarum.

Avibacterium paragallinarum has been subtyped into three serogroups (A, B, and C) and nine serovars (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C-3, and C-4) according to the Page and Kume schemes. Both schemes use the hemagglutination inhibition test for serotyping. However, the relationship between the hemagglutinin gene (HMTp210) sequences and serotypes of A. paragallinarum is still unclear. This problem is partly due to the lack of information on the complete HMTp210 sequence from the formal reference strain of Page serogroup B (strain 0222 or Spross). In this study, we determined the complete HMTp210 sequence of strain Spross. The sequence of Spross and those of other HMTp210 sequences retrieved from GenBank were used to conduct phylogenetic analyses to investigate the relationship between the serotypes and HMTp210 sequences of A. paragallinarum. Four phylogenetic clusters, designated clusters A-1, A-2, B, and C, were identified. Clustering based on complete HMTp210 sequences correlates with serotyping based on hemagglutination inhibition tests. Serovar A-2 was found to contain a chimeric HMTp210 gene that might have resulted from recombination between serovar A-1 and serovar C-1. In addition, phylogenetic analysis based on partial sequences (approximately nucleotides 1-1200) of HMTp210 was sufficient to discriminate between serogroups A, B, and C. These findings could be valuable for developing a molecular method for serotyping of A. paragallinarum.

Relación entre los serotipos y las secuencias génicas de hemaglutinina de Avibacterium paragallinarum. Avibacterium paragallinarum se ha subtipificado en tres serogrupos (A, B y C) y nueve serovares (A-1, A-2, A-3, A-4, B-1, C-1, C-2, C- 3 y C-4) de acuerdo con los esquemas Page y Kume. Ambos esquemas utilizan la prueba de inhibición de la hemaglutinación para la serotipificación. Sin embargo, la relación entre las secuencias del gene de la hemaglutinina (HMTp210) y los serotipos de A. paragallinarum aún no está clara. Este problema se debe en parte a la falta de información sobre la secuencia completa del gene HMTp210 de la cepa de referencia formal del serogrupo B de Page (cepa 0222 o Spross). En este estudio, se determinó la secuencia completa de HMTp210 de la cepa Spross. La secuencia de Spross y las de otras secuencias del gene HMTp210 obtenidas de GenBank se utilizaron para realizar análisis filogenéticos para investigar la relación entre los serotipos y las secuencias de HMTp210 de A. paragallinarum. Se identificaron cuatro agrupaciones filogenéticas, denominadas grupos A-1, A-2, B y C. La agrupación basada en las secuencias completas del gene HMTp210 se correlaciona con la serotipificación basada en pruebas de inhibición de la hemaglutinación. Se encontró que el serovar A-2 contenía un gene HMTp210 quimérico que podría haber resultado de la recombinación entre el serovar A-1 y el serovar C-1. Además, el análisis filogenético basado en secuencias parciales (aproximadamente nucleótidos 1-1200) del gene HMTp210 fue suficiente para discriminar entre los serogrupos A, B y C. Estos hallazgos podrían ser valiosos para desarrollar un método molecular para la serotipificación de A. paragallinarum.

Transport stress in broilers. II. Superoxide production, adenosine phosphate concentrations, and mRNA levels of avian uncoupling protein, avian adenine nucleotide translocator, and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha in skeletal muscles.

The effect of transport stress on superoxide production and adenosine phosphate concentration in addition to avian uncoupling protein (avUCP), avian adenine nucleotide translocator, and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA levels of skeletal muscles in broilers was investigated. Arbor Acres chicks (n = 360, 46 d old, males) were randomly allotted to 1 of 5 treatments: unstressed control, 45-min (short-term) transport with 45-min (short-term) recovery, 45-min transport with 3-h (long-term) recovery, 3-h (long-term) transport with 45-min recovery, and 3-h transport with 3-h recovery. Each treatment consisted of 6 replicates with 12 birds each. All birds (except control group) were transported according to a designed protocol. Transport time affected reactive oxygen species production in the thigh muscle (P < 0.05), adenosine triphosphate (ATP) content and energy charge (EC) in both breast and thigh muscles (P < 0.05 for all 4 comparisons), ATP:adenosine diphosphate (ADP) ratio in the breast muscle (P < 0.05), and avUCP mRNA levels in the thigh muscle (P < 0.05). Long-term transport increased (P < 0.05) reactive oxygen species production, ATP content, ATP:ADP ratio, and EC in the thigh muscle, but it decreased ATP content, ATP:ADP ratio, and EC in the breast muscle. Long-term transport increased avUCP mRNA in the thigh muscle (P < 0.05). Long-term recovery increased the ATP (P < 0.05) and ADP (P < 0.05) concentrations, avian adenine nucleotide translocator mRNA (P < 0.05), and avian peroxisome proliferator-activated receptor-gamma coactivator-1alpha mRNA (P < 0.05) in the thigh muscle, whereas EC decreased (P < 0.05) in the breast muscle. There were interactions between transport and recovery time on ATP (P < 0.05), EC (P < 0.05), and avUCP mRNA level (P < 0.05) in the thigh muscle. This study suggests that long-term transport accelerates muscle energy metabolism and lipid peroxidation. A long-term recovery may help alleviate cellular damage and maintain meat quality by reducing the rate of energy metabolism and scavenging of free radicals formed.

Transport stress in broilers: I. Blood metabolism, glycolytic potential, and meat quality.

The effect of transport stress on blood metabolism, glycolytic potential, and meat quality in broilers was investigated. Arbor Acres chicks (n = 360, 1 d old, males) were randomly allotted to 1 of 5 treatments: unstressed control, 45-min (short-term) transport with 45-min (short-term) recovery, 45-min transport with 3-h (long-term) recovery; 3 h (long-term) transport with 45-min recovery, and 3-h transport with 3-h recovery. Each treatment consisted of 6 replicates with 12 birds each. On d 46, all birds (except the control group) were transported according to a designed protocol. Transport time affected plasma glucose level (P<0.05) and glycogen level (P=0.06) in breast muscle as well as the area (P<0.01) and density (P<0.01) of IIa fibers. Glucose concentration increased slightly during the first 45 min of transport and then decreased dramatically in the long-term transported broilers (P<0.05). Long-term transport decreased the concentration of breast glycogen (P=0.06) and affected the size of IIa fibers in tibialis anterior by decreasing the area (P<0.01) with an increase in density (P<0.01). However, a long-term recovery after transport contributed to the homeostasis of blood corticosterone (CORT, P=0.05) and low levels of glycogen (P<0.05), lactate (P<0.01), and glycolytic potential (P<0.01) in thigh muscles. Interactions existed between transport and recovery time on area (P<0.05) and density (P<0.01) of IIa fibers. Furthermore, plasma nonesterified fatty acids increased significantly in the 3-h transport with 3-h recovery group (P<0.05) in comparison with the control. These results suggested that transport induced the release of plasma CORT and glycopenia, which affected the contractive status of muscle fibers by changing their area and density, and enhanced glycolysis and even lipolysis. A long-term recovery after transport was beneficial in lowering plasma CORT levels and reducing muscle glycolysis, which might improve broiler meat quality.

Effects of human umbilical cord mesenchymal stem cells-derived exosomes on fracture healing in rats through the Wnt signaling pathway.

OBJECTIVE: To investigate the role of human umbilical cord mesenchymal stem cell (hucMSC)-derived exosomes in the Wnt signaling pathway and their effects on fracture healing in rats. MATERIALS AND METHODS: A total of 24 healthy male Sprague-Dawley (SD) rats were randomly divided into 3 groups, of which the experimental groups were injected with Phosphate-Buffered Saline (PBS) and hUCMSC-derived exosomes, respectively, at the fracture site, and a blank control group was set. At 2 and 3 w after treatment, respectively, the healing condition at the fracture site in the rats was detected by micro-computed tomography (CT). The protein expressions of ß-catenin and Wnt3a of the Wnt signaling pathway in the bone tissue were measured via Western blotting (WB) assay. Quantitative Real Time-fluorescence Polymerase Chain Reaction (qRT-PCR) was performed to determine the expressions of osteogenic marker genes [collagen type I (COL-1), osteopontin (OPN) and runt-related transcription factor 2 (RUNX2)]. RESULTS: The results of the micro-CT scan showed that the rats treated with exosomes had better apposition of the fracture site, and the appearance of cortical bone was continuous. The fracture sites in the blank control group and PBS injection group were not healed, and the appearance of cortical bone was discontinuous, with significant fracture lines. According to the WB results, the protein expression levels of ß-catenin and Wnt3a in exosome treatment group were significantly higher than those in the blank control group and PBS injection group (p<0.01). The qRT-PCR results indicated that the expression levels of COL-1, OPN and RUNX2 in exosome treatment group were increased evidently compared with those in the other two groups (p<0.01). CONCLUSIONS: HucMSC-derived exosomes are probably involved in the repair of fracture in rats through the Wnt signaling pathway.

Effects of vitamin A supplementation on growth performance, carcass characteristics and meat quality in Limosin×Luxi crossbreed steers fed a wheat straw-based diet.

Three experiments were conducted to examine the effects of dietary vitamin A supplementation on performance and carcass parameters in Limosin×Luxi crossbreed finishing steers fed a wheat straw-based diet. Sixteen 12-month old (301±22kg) steers, 16 12-month old (309±15kg) steers and 16 24-month old (411±20kg) steers were used in experiment 1 for 6 months feeding period, in experiment 2 for three months feeding period and in experiment 3 for three months feeding period, respectively. Sixteen steers of each experiment were randomly divided into the four groups of four animals. Treatments consisted of four vitamin A supplementation levels (0, 1100, 2200 and 4400IU/kg DM). The growth rate was affected by dietary vitamin A level in experiment 1 and 2, revealing that the suitable amount of vitamin A supplementation increased the growth rate; excessive vitamin A in the ration decreased the growth rate of 12-month-old finishing steers. The marbling deposition decreased with the increment of vitamin A supplementation level, but possibly associated with vitamin A supplementing duration. Furthermore, the suitable dietary vitamin A level probably decreased lipid and pigment oxidation, and increased the tenderness of beef meat. Vitamin A supplementation had no significant effect on chemical composition of gluteus medius muscle and longissimus dorsi muscle.

Relaxin inhibits cardiac fibrosis and endothelial-mesenchymal transition via the Notch pathway.

BACKGROUND: Relaxin (RLX) can prevent cardiac fibrosis. We aimed to investigate the possible mechanism and signal transduction pathway of RLX inhibiting cardiac fibrosis. METHODS: Isoproterenol (5 mg·kg(-1)·d(-1)) was used to establish the cardiac fibrosis model in rats, which were administered RLX. The cardiac function, related targets of cardiac fibrosis, and endothelial-mesenchymal transition (EndMT) were measured. Transforming growth factor ß (TGF-ß) was used to induce EndMT in human umbilical vein endothelial cells, which were pretreated with RLX, 200 ng·mL(-1), then with the inhibitor of Notch. Transwell cell migration was used to evaluate cell migration. CD31 and vimentin content was determined by immunofluorescence staining and Western blot analysis. Notch protein level was examined by Western blot analysis. RESULTS: RLX improved cardiac function in rats with cardiac fibrosis; it reduced the content of collagen I and III, increased the microvascular density of the myocardium, and suppressed the EndMT in heart tissue. In vitro, RLX decreased the mobility of human umbilical vein endothelial cells induced by TGF-ß, increased the expression of endothelial CD31, and decreased vimentin content. Compared to TGF-ß and RLX co-culture alone, TGF-ß + RLX + Notch inhibitor increased cell mobility and the EndMT, but decreased the levels of Notch-1, HES-1, and Jagged-1 proteins. CONCLUSION: RLX may inhibit the cardiac fibrosis via EndMT by Notch-mediated signaling.

Interaction of berbamine compound E6 and calmodulin-dependent myosin light chain kinase.

The interaction of the berbamine compound E6 and calmodulin (CaM)-dependent myosin light chain kinase (MLCK) has been studied. The experimental results showed that the inhibition of MLCK activity was increased with increasing amounts of E6 and was overcome completely by the addition of excessive CaM. The stimulatory activity of MLCK induced by CaM was gradually inhibited by the increasing concentrations of compound E6, showing that the inhibition of MLCK activity by compound E6 was concentration dependent; and the Ki was 0.95 microM. Compound E6 diminished the fluorescence intensity of dansyl-labeled CaM and the intensity was increased gradually by the addition of different amounts of CaM. Compound E6 had no effect on the activity of MLCK fragments produced by limited trypsinization, and it is a novel and considerably potent calmodulin antagonist.

Chromatographic isolation and characterization of a novel peroxidase from large lima legumes.

A novel peroxidase with antifungal activity was isolated from the large lima bean (Phaseolus limensis) seeds. The procedure entailed extraction, ammonium sulfate precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and gel filtration on Sephadex G-75. The enzyme was adsorbed on Affi-gel blue gel and SP-Toyopearl, and possessed a molecular weight of 34 kDa in SDS-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. There was an almost 110-fold increase in specific activity of the purified peroxidase compared with that of the crude extract. The enzyme exhibited a pI of 8.6 by isoelectric focusing electrophoresis, indicating that it is a basic protein. The optimum pH and the optimum temperature were 5.5 and 30 degreeC, respectively. The enzyme was stable up to 55 degreeC. It potently suppressed mycelial growth in Fusarium solani, Mycosphaerella arachidicola, and Pythium aphanidermatum with an IC50 of 76, 103, and 119 microM, respectively.

Retroviral gene transfer of tissue-type plasminogen activator targets thrombolysis in vitro and in vivo.

Patients usually have serious complications of thrombosis and bleeding by eating anticoagulation medicine for their residual lives after mechanical valve replacement operation. Tissue-type plasminogen activator (tPA) could target thrombolysis by activating plasminogen to fibrinolysin. In this study, we recombined a retroviral vector pLEGFP-N1-tPA and cultured purified packaging cells PT67/pLEGFP-N1-tPA to produce high-titer retrovirus. In vitro, two target cells, endothelial cell of umbilical vein (ECUV) 304 and heart muscle cell (HMC) that consist of endocardium and heart muscle, were infected by pLEGFP-N1-tPA. The results demonstrated that exogenous tPA was successfully transferred into ECUV304 and HMC. tPA in the two cells shows significant thrombolysis in plasma plate and the activity and content of tPA were high. Furthermore, in vivo, no thrombus was seen on the surface of Dacron patches (the same material making up a ring of mechanical valve) by tPA locally transferring around Dacron patches that were transplanted in the inferior caval veins of rabbits. tPA was successfully transferred into the local inferior caval vein. Activity and content of tPA were high in local tissue and blood and thrombolysis was effectively demonstrated by tPA rapidly, efficiently and long expressing. This laid the foundation for study and appliance of the tPA gene valve.

Effect of 17beta-oestradiol and ginsenoside on osteoporosis in ovariectomised rats.

To study the anti-osteoporosis effects and mechanism of action of oestradiol (E2) and ginsenoside (tR), we measured the bone mineral densities (BMD) of lumbar vertebra and tibia and analysed the tibia histological morphological data, as well observed the activity and the number of osteoblasts and the activity of alkaline phosphatase (ALP) and the concentration of cAMP. Results showed that E2 (400 microg kg- 1 week- 1) and tR (10, 20, 30 mg kg- 1 day- 1) were able to countervail the decreasing in BMDs of lumbar vertebra and tibia induced by OVX in rats (P<0.05); E2 (0.1 micromol l- 1) and ginsenoside Rg1 (1 micromol l- 1 and 10 micromol l- 1) were able to increase the number of osteoblasts, the activity of ALP and the concentration of intercellular cAMP in cultured osteoblast cells. The present findings suggest that E2 and tR have an anti-osteoporosis effect in ovariectomised rats.