Hepatopancreas but not ovary is the site of vitellogenin synthesis in female fresh water crab, Oziothelphusa senex senex

The objective of the present study was to explore the site of synthesis of vitellogenin (Vtg) in fresh water edible crab, Oziothelphusa senex senex. Vtg cDNA fragments were isolated from the hepatopancreas of female crabs using RT-PCR method, and the deduced amino acid sequence of O. senex senex showed more than 60% identity with other brachyuran Vtg sequences. RT-PCR analysis showed that Vtg mRNA can be detected only in hepatopancreas of female Oziothelphusa but not in other tissues including eyestalks, Y-organs, mandibular organs, thoracic ganglion, hypodermis and ovary. Antibodies were raised against vitellin purified from the ovary of O. senex senex. Immunoprecipitation analysis revealed the presence of Vtg in the hepatopancreas of vitellogenic stage I females and in the hemolymph, hepatopancreas and ovary extracts from vitellogenic stage II females but absent in hemolymph and hepatopancreas extract of males. These results suggest that Vtg is synthesized only in hepatopancreas but not in the ovaries of O. senex senex. In addition, Vtg synthesized in hepatopancreas is transported to ovary through hemolymph.     

Regeneration of doubled haploid plants by androgenesis of cucumber (<Emphasis Type="Italic">Cucumis sativus </Emphasis>L.)

Six experiments (including pretreatment, embryonic callus induction media, preculture conditions, embryo induction media, embryo germination media, and genotypic effects) were conducted to develop an efficient cucumber (Cucumis sativus L., 2n = 2x = 14) anther culture protocol. Pretreatment and embryo induction were key factors for successful anther culture. Suitable temperature stress depended on the ecotype, i.e., cucumbers from cold areas responded well to cold shock whereas those from temperate areas responded well to heat treatment. The best medium for embryonic callus induction was MS medium supplemented with 4.44 μM BA, 2.26 μM 2, 4-D, 4.64 μM KIN, 3% sucrose and 0.8% agar. For embryo induction, MS medium supplemented with 0.54 μM NAA, 13.32 μM BA, 3% sucrose and 0.8% agar was optimal, and for embryo germination MS medium containing 2.22 μM BA, 6% sucrose and 1.2% agar was best. Using this protocol, we produced callus from 16 genotypes and regenerated plants from three of 20 evaluated. Three embryos per anther and 42 DH per 45 anthers (93% success) were obtained for cv. Ningjia No. 1, which was an improved result over a previous report. The origin of regenerants from microspores was determined by cytological, morphological and AFLP analyses.     

Both infiltrating regulatory T cells and insufficient antigen presentation are involved in long-term cardiac xenograft survival

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.     

Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.     

Mass Spectrometrical Characterization of NDRG2 Protein (N-myc-Downstream Regulated Gene 2) and Description of Two Novel Phosphorylation Sites

Antidepressant-related protein (NDRG2) is a member of the N-myc downstream-regulated gene family and a role for differentiation and signaling has been proposed. Performing protein profiling we observed NDRG2 and decided to characterize this important biomolecule. Estrous cycle phases were determined in Sprague-Dawley rats and the hippocampus was taken. Proteins were extracted, run on two-dimensional gel electrophoresis with subsequent multi-enzyme digestion followed by MALDI-TOF-TOF and nano-LC-ESI-MS/MS analysis of spots. Spots identified as NDRG2 were quantified by specific software. Five spots were identified as NDRG2 and two novel phosphorylation sites (T330 and T334) were detected. Gender and estrous cycle-dependent NDRG2 levels were observed. Results are of importance for further qualitative and quantitative studies at the protein level as well as for the design of antibodies for immunochemical applications and for the interpretation of previous studies on NDRG2 that did not take into account different expression forms and posttranslational modifications.     

Stereoselective degradation of fungicide benalaxyl in soils and cucumber plants

The enantioselective degradation of benalaxyl has been investigated to elucidate its behavior in several agricultural soils and plants (cucumber). Racemic benalaxyl was fortified into five types of agricultural soils and sprayed leaves of cucumber plants, respectively. The degradation kinetics and the enantiomer fraction (EF) were determined by normal-phase high-performance liquid chromatography (HPLC) with diode array detection (DAD) on the chiral column filled cellulose-tri-(3,5-dimethylphenylcarbamate)-based chiral stationary phase (CDMPC-CSP). The process of the degradation of benalaxyl enantiomers followed pseudo-first-order kinetics in cucumber plant. However, the dissipation phases of benalaxyl enantiomers in soils were biphasic ("slow-fast-slow" process). It has been shown that the degradation of benalaxyl was stereoselective. The results indicated that the (+)-S-benalaxyl showed a faster degradation in plants, while the (-)-R-benalaxyl showed a faster degradation in Soils 3, 4, and 5. No stereoselective degradation was observed in other soils.