Gene cloning of porcine adiponectin gene from adipose tissue and construction of its eukaryotic expression vector

To clone adiponectin (ADPN) gene from Shaziling porcine adipocyte and construct its eukaryotic expression vector, total RNA was extracted from subcutaneous fatty tissue. One pair of specific primers was designed by Primer 5.0 software according to the sequence of ADPN gene of porcine available in GenBank. The ADPN gene was amplified by PCR from cDNA and cloned into pMD18‐T vector to construct recombinant clonal vector pMD‐ADPN, sequenced and analysed. A recombinant expression plasmid pPICZaA‐ADPN was constructed by subcloning the cloned ADPN gene into the linearized pPICZaA vector. Then, the plasmid pPICZaA‐ADPN was expressed in Pichia pastoris (GS115) by electrotransformation. Western blot and Bradford analysis were used to determine the target protein induced by methanol. Results showed that the genome size of ADPN was 732 bp and encoded 244 amino acid, the nucleotide sequence of ADPN shared 100% identity with that of porcine available in GenBank. Western blot and Bradford analysis showed that the recombinant ADPN was expressed in GS115 correctly and has certain immune activity. The expression level of ADPN was 28.5 μg/ml. In conclusion, the recombinant ADPN could express in eukaryotic expression vector pPICZaA‐ADPN constructed in this study effectively.     

Maternal Liver Damage Delays Meiotic Resumption in Bovine Oocytes through Impairment of Signalling Cascades Originated from Low p38MAPK Activity in Cumulus Cells

The main objective of the present study is to investigate the molecular mechanism underlying the delay in progression of nuclear maturation in oocytes derived from cows with damaged livers (DL cows), which was previously reported. In present study, delayed progression of nuclear maturation of oocytes derived from DL cows relative to oocytes derived from cows with healthy livers (HL cows) was accompanied by low maturation promoting factor (MPF) activity (0.43 fold, p < 0.05). When cumulus cells were removed from cumulus‐oocyte complexes and the denuded oocytes were cultured, there was no difference in the progression of nuclear maturation between the two liver conditions. In addition, gap junctional communication (GJC) between the oocyte and cumulus cells was higher in DL cows than in HL cows at 3 and 7 h of in vitro maturation (IVM) (p < 0.05). Supplementation of IVM medium with epidermal growth factor (EGF) increased the ratio of germinal vesicle breakdown (GVBD) of oocytes derived from DL cows to the level seen in oocytes derived from HL cows. Additionally, the level of p38MAPK phosphorylation at 0 h of IVM was significantly lower in cumulus cells derived from DL cows than in cumulus cells derived from HL cows (HL cows, 53.5%; DL cows, 28.9%; p < 0.05). Thus, a low level of p38MAPK phosphorylation in cumulus cells induced slow GJC closure between oocyte and cumulus cells, which resulted in slow meiotic maturation of oocytes derived from DL cows.     

Hanging Drop Monoculture for Selection of Optimal Antioxidants During In Vitro Maturation of Porcine Oocytes

We analysed the effect of three antioxidants that have different functional mechanisms on the in vitro maturation (IVM) of porcine oocytes. Single oocyte monoculture using the hanging drop (HD) system has some advantages such as improving analysis efficiency brought by the smaller number of samples than the number of oocytes cultured in one drop. Direct effects of ligands on single oocytes could also be detected without considering the effects of paracrine factors from other oocytes. After 22 h of pre‐culture, denuded oocytes were cultured for 22 h with 0.01 and 0.1 μg/ml of L‐carnitine (LC), lactoferrin (LF) or sulforaphane (SF) in the presence/non‐presence of oxidant stress induced by H₂O₂ supplementation to evaluate the reducing effects against oxidative stress on nuclear maturation. As a result, compared with LC and SF, LF showed effective reduction in oxidative stress at a lower concentration (0.01 μg/ml), suggesting that LF is a more effective antioxidant in porcine oocyte IVM. Additionally, LF also increased maturation rate even in culture without H₂O₂. Our results clearly suggest that the HD monoculture system is useful for screening the substances that affect porcine oocyte culture.     

Apoptosis‐Like Events and In Vitro Fertilization Capacity of Sex‐Sorted Bovine Sperm

This study utilized three staining assays (Annexin V, mitochondrial membrane potential (JC‐1) and TUNEL) for flow cytometric analysis of apoptosis in sex‐sorted sperm from four different bulls (A, B, C and D). Correlations between sperm quality and IVF efficiency were then assessed to determine which assay provided the best prediction of IVF efficiency. The results of the Annexin V assays, as well as measures of viable sperm, early apoptosis, necrotic sperm and mitochondrial membrane potential (?ψm) showed that the sex‐sorted sperm collected from bull A significantly differed from those of the other three bulls (p < 0.05). In addition, the levels of DNA fragmentation in sex‐sorted sperm from bull A were significantly lower than those from bulls B and C (p < 0.05). The percentage of cells reaching the cleavage and blastocyst stages in sex‐sorted sperm from bull A were significantly greater than those from the other bulls (p < 0.05). A significant positive correlation was observed between viable sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In contrast, a negative correlation was found between early apoptotic sperm and the percentage of cells at the cleavage or blastocyst stages (p < 0.05). In conclusion, these results indicated that the Annexin V assay was the most reliable technique for the prediction of the IVF success of sex‐sorted bovine sperm.     

Bio-amelioration of alkali soils through agroforestry systems in central Indo-Gangetic plains of India

A long-term field study was initiated during 1995 at Central Soil Salinity Research Institute, Regional Research Station, Lucknow(26°47′58′′ N and 80°46′24′′ E) to analyze the effect of agroforestry systems on amelioration of alkali soils. Three agroforestry systems(pastoral, silvipastoral and silvicultural) were compared with the control where no agroforestry system was introduced. Tree-based silvicultural and silvipastoral systems were characterized by tree species Prosopis juliflora and Acacia nilotica along with grass species Leptochloa fusca, Panicum maximum, Trifolium alexandrium and Chloris gayana. Growth of ten-year-old Prosopis juliflora and Acacia nilotica planted in combination with grasses was significantly higher over the silviculture system with the same species. Tree biomass yields of P. juliflora(77.20 t?ha-1) and A. nilotica(63.20 t?ha-1) planted under silvipastoral system were significantly higher than the sole plantation of(64.50 t?ha-1 and 52.75 t?ha-1). Fodder yield under the pastoral system was significantly higher than the silvipastoral system during initial years but it was at par with that of silvipastoral systems after eight years of plantation. The microbial biomass carbon in the soils of silvipastoral systems was significantly higher than in soils under sole plantation of trees and control systems. The Prosopis-based silvipastoral system proved more effective in reducing soil pH, displacing Na+ from the exchange complex, increasing organic carbon and available N, P and K. Improvement in soil physical properties such as bulk density, porosity, soil moisture and infiltration rate was higher in the Prosopis-based silvipastoral system than in the silviculture system or control. On the basis of biomass production and improvement in soil health due to tree + grass systems, silvipastoral agroforestry system could be adopted for sustainable reclamation of highly alkali soils.     

Transgenic woody plants for biofuel

Transgenic trees as a new source for biofuel have brought a great interest in tree biotechnology. Genetically modifying forest trees for ethanol production have advantages in technical challenges, costs, environmental concerns, and financial problems over some of crops. Genetic engineering of forest trees can be used to reduce the level of lignin, to produce the fast-growing trees, to develop trees with higher cellulose, and to allow the trees to be grown more widely. Trees can establish themselves in the field with less care of farmers, compared to most of crops. Transgenic crops as a new source for biofuel have been recently reviewed in several reviews. Here, we overview transgenic woody plants as a new source for biofuel including genetically modified woody plants and environment; main focus of woody plants genetic modifications; solar to chemical energy transfer; cellulose biosynthesis; lignin biosynthesis; and cellulosic ethanol as biofuel.     

Salix tetradenia Hand.‐Mazz: a new natural plant host of ‘Candidatus phytoplasma’

This article reports Salix tetradenia Hand.‐Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer‐simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI‐B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.     

Morphological changes in carp epithelial cells infected with Aeromonas hydrophila

The in vitro interaction of Aeromonas hydrophila with epithehoma papillosum cells of carp (EPC) was studied. All the virulent and type strains invaded and multiplied inside EPC cells. Morphological changes were induced by the virulent and type strains during the processes of invasion and intracellular replication. Among the virulent group, strain PPD134/91 required the shortest time (30 min post-infection) to induce cytopathic effects in the EPC monolayers. The EPC cells infected with PPD 134/91 became retracted and condensed, lost their attachment abilities, and eventually disintegrated. Confocal microscopy revealed that tubular structures measuring 0.77 ± 0.19 μm appeared to connect the retracted EPC cells. The cytopathic effect was attributed to the growing and metabolically active bacteria. Avirulent strains such as L37 did not multiply nor induce cytopathic effects in the EPC monolayers.     

Salmon gonadotropes secrete free form alpha (α)-subunit: quantitative analysis by the sandwich cell immunoblot assay

The sandwich cell immunoblot assay confirmed that salmon LH-cells secreted the free form α-subunit along with the dimeric LH, but lower in amounts than the LH.     

Cloning and seasonal changes in ovarian expression of a TSH receptor in the channel catfish, Ictalurus punctatus

The cDNA encoding the thyroid stimulating hormone (TSH) receptor was isolated from the ventral aorta (thyroid follicles) and in both gonads of the channel catfish. The seasonal changes in ovarian expression of this receptor seem to be correlated to reproduction. TSH, but not LH or FSH, activated the recombinant TSH receptor expressed in COS cells. These results indicate that the isolated cDNA encodes a functional and highly specific TSH receptor. The seasonally regulated expression in the catfish ovary suggests a role for TSH in gametegenesis.