Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

Molecular Characterization of Viral G Gene in Emerging and Re-emerging Areas of Rabies in China, 2007 to 2011

In recent years (2007 to 2011), although the overall number of rabies cases in China has decreased, there is evidence of emerging or re-emerging cases in regions without previous rabies cases or with low incidence of rabies. To investigate the origin and the factors affecting the spread of rabies in China, specimens were collected from 2007 to 2011 from provinces with emerging and re-emerging cases and tested for the presence of the rabies virus. Positive specimens were combined with sequences from GenBank to perform comparisons of homology and functional sites, and to carry out phylogenetic analyses. Out of these regions, five provinces had 9 positive specimens from canine and cattle, and 34 canine or human specimens were obtained from previously high-incidence provinces. Complete sequences of G gene were obtained for these samples. Homology of the sequences of these 43 specimens was 87%-100% at the nucleotide level and 93.7% -100% at the amino acid level. These G gene sequences were combined with reference sequence from GenBank and used to construct a phylogenetic tree. The results showed that 43 specimens were all assigned to China clade I and clade II, with all specimens from emerging and re-emerging areas placed within clade I. Specimens isolated from Shanxi and Inner Mongolia in 2011 were distinct from previously-isolated local strains and had closer homology to strains from Hebei, Beijing and Tianjin whereas new isolates from Shanghai were tightly clustered with strains isolated in the 1990s. Finally, Shaanxi isolates were clustered with strains from adjacent Sichuan. Our results suggest that the rabies cases in emerging and re-emerging areas in China in the last 5 years are a consequence of the epidemic spreading from of neighboring provinces and regions experiencing a serious epidemic of rabies.

Preparation of protein samples for gel electrophoresis by sequential extraction

Since preparation and solubilization of protein samples are crucial factors in proteome research,the authors established a sequential extraction technique to prepare protein samples from the body wall of the 5th instar larvae of silkworm.Bombyx mori.Two kinds of protein samples were obtained from the body wall using the method.Between the two types of samples only about 15% proteins were identical;the majority were different,indicating that more species of proteins could be obtained with the sequential extraction method;which will be useful for preparation of protein samples for proteome study.

Surface plasmon resonance analysis to evaluate the importance of heparin sulfate groups＇ binding with human aFGF and bFGF

Human acidic and basic fibroblast growth factors (aFGF and bFGF) are classic and well characterized members of the heparin-binding growth factor family. Heparin is generally thought to play an extremely important role in regulating aFGF and bFGF bioactivities through its strong binding with them. In order to unravel the mechanism of the interactions between heparin and FGFs, and evaluate the importance of heparin sulfate groups' binding with FGFs, surface plasmon resonance analyses were performed using IAsys Cuvettes System. Heparin and its regioselectively desulfated derivatives were immobilized on the cuvettes, aFGF and bFGF solutions with different concentrations were pipetted into the cuvettes and the progress of the interaction was monitored in real-time by Windows-based software, yielding kinetic and equilibrium constants for these interactions. In addition, in order to reduce the delicate difference among the cuvettes, inhibition analyses of mixture of FGFs and immobilized native heparin by modified heparins were also done. The data from these two methods were similar, indicating that all sulfate groups at 2-0, 6-0 and N- in heparin were required for the binding to aFGF; and that their contribution to the binding was in the order 2-O, N- and 6-O-sulfate group.In contrast, definite contribution of the 6-O-sulfate group to the binding with bFGF was most apparent, while the other two sulfate groups appeared to be necessary in the order 2-O and N-sulfate group. These methods established here can be used for analysing the effect of sulfate groups in heparin on the binding with other human FGF members or other heparin-binding proteins.

Effect of polychlorinated biphenyls on spermatogenesis and testosterone secretion in adult cocks

The effects ofpolychlorinated biphenyls (PCBs) on reproduction of adult cocks were studied by gavaging peanut oil or PCBs (Aroclor 1254, 50 mg/kg) once a week for six consecutive weeks. Physiological parameters were recorded and gonads were removed at the end of experiment for histological examination. The results showed that there was no significant difference between the control and treatment group in body weight, respiration rate, heart rate, body temperature, and the numbers of red and white blood cells. However, there was a marked decrease in the testicular weight and serum testosterone level after PCB treatment. Morphological studies manifested severe damage of the seminiferous tubules by PCB. The number of the germ cells at the different developmental stages was decreased and condensed nuclei were observed in most of these cells. This study revealed that the reproductive function of the adult cocks is sensitive to PCBs, which inhibited mainly spermatogenesis and testosterone secretion.

Effect of Zinc Toxicity on Lymphoid Organs in Chickens

The experiment was conducted with the objective of studies on effects of zinc toxicity on lymphoid organs by the methods of experimental pathology and flow cytometry (FCM). 200 one-day-old Avian broilers were divided into four groups randomly, and fed on diets as follows: controls (Zn 100 mg kg-1)and zinc toxic (Zn 1 500 mg kg-1, zinc toxic group Ⅰ; Zn 2 000 mg kg-1, zinc toxic groupⅡ; Zn 2 500 mg kg-1, zinc toxic group Ⅲ) for seven weeks. The weight and growth index of the thymus, spleen and bursa of Fabricius were reduced in both zinc toxic groupⅡand zinc toxic group Ⅲ when compared with those of control group. The G0/G1 phase of the cell cycles of the lymphoid organs was higher, and S, G2+M phases lower in zinc toxic groups Ⅱand Ⅲ than in control group. Lymphocytes were depleted and degenerate in the lymphoid organs. The reticular cells of the bursa of Fabricius proliferated and the reticular cells of the thymus were also degenerate and necrotic, particularly in zinc toxic groups Ⅱand Ⅲ. The results demonstrated that more than 1 500 mg kg-1 impaired the progression of lymphocytes from the G0/Gl phase to S phase obviously, inhibited the development of lymphoid organs and caused marked pathological changes in the lymphoid organs. Potential mechanisms underlying these observations are also discussed.

Study on the Measures of Connectedness Between Herds

Pig breeding is generally conducted among many herds, so EBV comparisons across populationsare necessary. Genetic connectedness is required for reliable between-farm animal EBV comparisons.Five quantitative overall connectedness measures among populations have been proposed so far,coefficient of connectedness(γ*), coefficient of determination (CD) and overall indices ofprecision, connectedness rating, number of direct genetic links between subpopulations due tocommon sires and dams (GLt), and average genetic covariance (AGC) are reviewed and theirproperties are discussed in this paper. It is recommended to use AGC at present for measuringgenetic connectedness between herds.

Molecular Cloning and Sequence Analysis of IGF-Ⅰfrom Triangular Bream (Megalobrama terminalis)

The insulin-like growth factor Ⅰ(IGF-Ⅰ) gene of triangular bream (Megalobrama terminalis)(GenBank No.AY247412)(Tb)was cloned for the first time from liver by RT-PCR. Thenucleotide sequence analysis showed the Tb IGF-ⅠcDNA consisted of 486 nucleotides andencoded 117 amino acids including B, C, A, D and E five domains. Analysis of E-domainindicated that cloned Tb IGF-Ⅰbelonged to IGF-ⅠEa-2 subtype. Identity analysis showedthe IGF-Ⅰnucleotide sequence shared 99.8% homology with bluntnose bream, 88.8% withgrass carp, 85.8% with common carp; the pre-IGF-Ⅰamine acid sequence shared 99.4% withbluntnose bream, 88.8% with grass carp, 85.4% homology with common carp. In the CyprinusCarpio, the higher homology of nucleotide sequence and amino acid sequence in IGF-Ⅰshowed that the closer relationship the fishes have. These results could provide basicdata for the research on Tb germplasm and the development and utilization of biologicalfeed additives.

Influence of form and quantity of chromium on the development and survival of two silkworm(Bombyx mori L.)races

Growth inhibitory activity has proven important in Qiufeng×Baiyu and Qingsong×Haoyue silkworm larvae. The consumption of mulberry leaves was reduced in both silkworm races while Qiufeng×Baiyu larvae showed the higher reduction in leaf consumption. From the results obtained, it was revealed that even at low concentrations of 400 mg/L of either Cr(Ⅲ) or Cr(Ⅵ) ions, growth of Qiufeng × Baiyu and Qingsong × Haoyue was significantly depressed. Depression in relative growth index(RGI) and high death rate in both silkworm races indicated that the different concentrations of the two ions used caused their growth inhibitions. Through linear regression analysis, the EC 50 (concentration of the compound that caused 50% reduction) was interpolated for both tested compounds. EC 50 (mg/L) of Cr (Ⅲ) ions in Qiufeng × Baiyu and Qingsong × Haoyue were 800 and 600 respectively. EC 50 (mg/L) of Cr(Ⅵ) ions in Qiufeng × Baiyu and Qingsong × Haoyue was 600 and 316 respectively. According to the analysis of relative growth index, and an analysis of linear regression technique for measuring the growth of the silkworm races, it was indicated that the form of Cr affected growth rates, growth inhibition responses of the larvae, and toxicological effects. Thus, form and quantity of Cr accumulating in silkworms reared with contaminated leaves are likely to influence their population dynamics.

Study on Bombyx mori silk treated by oxygen plasma

Study of the morphology, aggregation structure and properties ofBombyx mori silk treated by low temperature oxygen plasma showed that slight flutes appeared on the surface of Bombyx mori silk fiber and that its surface structure changed after plasma treatment. The conformation also changed and crystalline degree decreased. The stannic filling rate of treated fiber was improved. Because of etching, the weight of the fiber decreased but the breaking strength changed little after short-time treatment.

The Study on Correlation Analysis of Single Nucleotide Polymorphism of IGF2 Gene and Body Fatness Traits in Chicken

Insulin-like growth factor Ⅱhas profound effects on the growth and differentiation of animal embryo. Some researches indicated that it affects the fat metabolism of poultry. This study was designed to investigate the effect of IGF2 on chicken fatness traits. Broiler, Hyline Brown layer and three native breeds (Shiqiza, Beijing You, Baier) were used in this research. Body weight and body composition traits were measured in broiler line at the age of 7 weeks. Primers for exon2 in IGF2 were designed from database of chicken genomic sequence. Polymorphisms were detected by PCR-SSCP and DNA sequencing. The total χ2 test results showed that there was a significant difference (P < 0.01) in the frequency of genotype among breeds. A C/G mutation at base position 139 was found among individuals in broiler line and the least square analysis showed that BB genotype birds had significant lower (P <0.05) abdominal fat weight and percentage of abdominal fat than AA or AB genotype birds. From the results we can conclude putatively that IGF2 gene is the major gene affecting the fatness traits of chicken or it links with the major gene, and the mutation could be used as the molecular genetic marker to select the chicken for low abdominal fat.

Effect of vertebrate hormones and prostaglandins on growth, silk quality and metabolic activities of Bombyx mori L

The economic importance of silkworm has moved biologists to explore various intricate mechanisms of the action of vertebrate hormones. The dietary administration of several vertebrate hormones and prostaglandins enhanced both developmental and metabolic processes of silkworm, Bombyx mori L. The main objective of sericulture research is to apply the results to achieve superior quality silk and greater output, to apply lab findings to achieve desirable ecenomic results.

QTL Detection on Chromosome 6 in Landrace×Lantang Pig Resource Population

A resource population constructed by F2 design with Landrace and Chinese indigenous Lantangpigs was used in this study. Seven microsatellite DNA markers on chromosome 6 and USDA2.6 piggenetic linkage map were used for interval QTL mapping. The results revealed that at theposition of 38-41 cM there was a chromosome-wide highly significant QTL affecting carcassbackfat A thickness (P < 0.01), which was closely linked with MN007 and the ratio of QTL addi-tive variance to F2 phenotypic variance was 5.90%. At the position of 60-70 cM there were twochromosome-wide significant QTLs affecting carcass lean percentage (P < 0.01) and skin and fatpercentage (P < 0.05), which were closely linked with MN003 and the ratio of QTL additivevariance to F2 phenotypic variance were 18.44 and 3.75%, respectively. At the same position,there was a single-point QTL also closely linked with MN003 and highly significantly (P < 0.01)affecting carcass lean. In addition, there were two chromosome-wide highly significant (P < 0.01)QTLs affecting meat color and marbling, which were closely linked with MN13 at the positionof 70-75 cM and the ratio of QTL additive variance to F2 phenotypic variance were 14.05and 1.77%, respectively.

A Study on the Inhibitory Potency of Protease Inhibitorsin Porcine Colostrum

Porcine colostrum and milk were separated into the acid-soluble fraction(SF)and casein fraction(CF)by centrifuge. Trypsin and chymotrypsin inhibitory capacity in porcine colostrum, milk and their components were determined by incubating bovine trypsin or chymotrypsin in a medium. The inhibition of insulin-like growth factor I(IGF-I)and epidermal growth factor(EGF)degradation in pig small intestinal contents by porcine colostrum was measured by incubating iodinated IGF-I or EGF. Degradation of labeled IGF-I or EGF was determined by monitoring the generation of radioactivity soluble in 30% trichloroacetic acid(TCA). The results showed that porcine colostrum had high levels of trypsin and chymotrypsin inhibitory activity and increased the stability of IGF-I and EGF in pig intestinal contents. The SF was higher in inhibitory potency than CF. The present study revealed that the protease inhibitors in porcine colostrum, milk-derived and colostrum-specific, existed mainly in SF.

Sex Identification of Red-crowned Crane by the Polymerase Chain Reaction

Sex determining gene primers of Oriental White Stork were used to amplify sex-linked gene of the Red-crowned Crane′s W chromosome-specific by PCR for sex identification. The sexes of 7 couples of grown Red-crowned Cranes and 15 youngs were identified. Through DNA sequence analysis, the identity is 94.77% between Red-crowned Crane and Oriental White Stork. The results of this study suggest that the application of the polymerase chain reaction technique is practicable for determining sex in the Red-crowned Crane.

A Study on Heat Resistance and Initial Characterization of Protease Inhibitors in Porcine Colostrum

Porcine colostrum was separated into the acid soluble fraction (SF) and casein fraction (CF) by acidifying followed by centrifuge. SF was further separated by liquid chromatography and anisotropic membrane filtration. Capacities of the SF or CF of porcine colostrum, to inhibit trypsin and chymotrypsin activity and to inhibit the epidermal growth factor (EGF) degradation in pig small intestinal contents, were determined under different heat treatments. The study showed that trypsin inhibitors in porcine colostrum survived heat treatments of 100℃ water bath for up to 10 min, but exposure to boiling water bath for 30 min significantly decreased the inhibitory activity. Compared with the trypsin inhibitors, the chymotrypsin inhibitors were more heat sensitive. SF was more heat sensitive than CF. Separation of the SF of porcine colostrum by liquid chromatography and anisotropic membrane filtration revealed that the porcine colostrum protease inhibitors, those had the capacity to inhibit the trypsin chymotrypsin activity and enhanced the stability of EGF in the gastrointestinal(GI) lumen of weaned pigs, existed mainly in SF, milk derived, were a group of heat labile small proteins with molecular weight of 10 00050 000.

Effects of different nuclear recipients on developmental potential of mouse somatic nuclear transfer embryos

In order to investigate the effects of different kinds of nuclear recipients from Kunming (KM) mouse on developmental potential of somatic nuclear transfer em- bryos, the enucleated MⅡ oocytes, enucleated zygotes and 2-cell blastomere were used to produce cloned mouse em-bryos. Using fibroblast deriving from C57/BL6 ear tissue as nuclear donor, we produced cloned embryos by transferring the fibroblast nuclei into enucleated KM mouse oocytes (sin-gle nuclear transfer, SNT), transferring pronuclei from the SNT embryos into enucleated KM zygotes (nuclear into zy-gote, NZ), and 2-cell blastomere nuclei from SNT embryos into enucleated KM mouse oocytes (nuclear into oocytes, NO); tetraploid embryos (tetraploid embryos, TE) were ob-tained by fusing two blastomeres, one is from the SNT cloned embryos, and the other from normal 2-cell KM mouse em-bryos. In group SNT, the cloned embryos could not develop beyond 8-cell stage and the rate of 8-cell stage is only 0.3%; in group NO, the reconstructed embryos could develop to morula stage, the rate of 8-cell stage was significantly greater than that of SNT group (P < 0.05); in group NZ, the devel-opment rate was further improved, and the reconstructed embryos could develop into blastocyst stage, the rate of blastocyst was 1.9%; in group TE, as high as 62.3% of the reconstructed embryos could develop into blastocyst. Results suggested that different nuclear recipients could significantly affect the developmental potential of cloned mouse embryos; KM MⅡ oocyte cytoplasm was not so effective as zygotes to reprogram the mouse somatic cell nuclei; serial nuclear transfer could improve the developmental potential of cloned mouse embryos.

Immunomodulating Steroidal Glycosides from the Roots of Stephanotis Mucronata

1 Introduction The plants beonging to the Asclepiadaceae family are reported to be rich in pregnane and cardiac glycosides.In recent years, the pregnanes and their glycosides hale been shown to possess antitumor, antiepilepsy, and antifertility activities. The dried roots of Stephanotis mncronata(BLANCO) Meer.