Fertile transgenic barley by particle bombardment of immature embryos

Transgenic, fertile barley (Hordeum vulgare L.) from the Finnish elite cultivar Kymppi was obtained by particle bombardment of immature embryos. Immature embryos were bombarded to the embryonic axis side and grown to plants without selection. Neomycin phosphotransferase II (NPTII) activity was screened in small plantlets. One out of a total of 227 plants expressed the transferred nptII gene. This plant has until now produced 98 fertile spikes (T₀), and four of the 90 T₀ spikes analyzed to date contained the nptII gene. These shoots were further analyzed and they expressed the transferred gene. From green grains, embryos were isolated and grown to plantlets (T₁). The four transgenic shoots of Toivo (the T₀ plant) produced 25 plantlets as T₁ progeny. Altogether fifteen of these T₁ plants carried the transferred nptII gene as detected with the PCR technique, fourteen of which expressed the nptII gene. The integration and inheritance of the transferred nptII gene was confirmed by Southern blot hybridization. Although present as several copies, the transferred gene was inherited as a single Mendelian locus into the T₂ progeny.     

4-Hydroxy-β-damascone and 4-Hydroxy-dihydro-β-damascone from Cigar Tobacco

The cDNA encoding a putative xylose reductase (xyrA) from Aspergillus oryzae was cloned and coexpressed in the yeast Saccharomyces cerevisiae with A. oryzae xylitol dehydrogenase cDNA (xdhA). XyrA exhibited NADPH-dependent xylose reductase activity. The S. cerevisiae strain, overexpressing the xyrA, xdhA, endogenous XKS1, and TAL1 genes, grew on xylose as sole carbon source, and produced ethanol.     

Different Methods Yielded Two-Fold Difference in Compliance with Physical Activity Guidelines on School Days

Introduction

The aim was to compare the average and the days method in exploring the compliance of children with physical activity guidelines and describe their physical activity patterns in different school day segments.

Methods

Physical activity was objectively measured in 472 children aged 6–13 for one school week. Children were compliant when fulfilling PA recommendations 1) on average over all measured days (average method) or 2) on at least four measured days (days method). To explore the difference in moderate to vigorous physical activity (MVPA) minutes between compliant and non-complaint children (using both the average and days method) in various day segments, linear mixed models was used.

Results

Compliance with physical activity guidelines was significantly higher with the average compared to the days method (51.7% and 23.7%, respectively). In segmented-day analysis, compliant children accrued more MVPA minutes in all day segments, especially during after-school. Gender differences appeared only during the in-school segments, where girls spent less time in MVPA (average method: -4.39 min, 95% CI = -5.36,-3.42, days method: -4.45 min, 95%CI = -5.46,-3.44). Older children accrued more MVPA minutes during physical education classes, but less during breaks, compared to younger children.

Conclusions

The used methods yielded remarkably different prevalence estimates for compliance to physical activity recommendations. To ensure comparability between studies, interventions and reports, there is a need for internationally agreed operationalization and assessment methods of physical activity guidelines. As non-compliant children had lower MVPA during all day segments, greater efforts should be made to provide physical activity opportunities both during and after school.     

Translocation of organic compounds in sunflower III. Effect of oligomycin, ouabain and phlorizin on translocation of sugars

The apical portions of intact sunflower leaves were infiltratedwith ¹⁴C-glucose, ¹⁴C-fructose or 3-O-methyl-¹⁴C-glucose andthe basal portions were treated with inhibitors. The effects of oligomycin, ouabain and phlorizin on translocationwere studied. Inhibition of translocation from the basal portionof the leaf to the stem was determined by experiments usingoligomycin. In other experiments, each leaf was divided intothree parts. The apical portion was fed with ¹⁴C-glucose andthe basal part treated with oligomycin. The effects of oligomycinon the distribution of ¹⁴C-glucose, ¹⁴C-sucrose, ¹⁴C-fructoseand ¹⁴C-sugar phosphate along the three parts of the leaf wereinvestigated. Inhibition of sucrose synthesis in the leavestreated with oligomycin was observed. Oligomycin inhibited ¹⁴Ctranslocation from the leaf. ¹Present address: Department of Biology, Faculty of Science,Science University of Tokyo, Kagurazaka, Shinjuku-ku, Tokyo,Japan. (Received August 17, 1978; )     

Structure and Evolution of the Archaeal Lipid Synthesis Enzyme sn-Glycerol-1-phosphate Dehydrogenase

One of the most critical events in the origins of cellular life was the development of lipid membranes. Archaea use isoprenoid chains linked via ether bonds to sn-glycerol 1-phosphate (G1P), whereas bacteria and eukaryotes use fatty acids attached via ester bonds to enantiomeric sn-glycerol 3-phosphate. NAD(P)H-dependent G1P dehydrogenase (G1PDH) forms G1P and has been proposed to have played a crucial role in the speciation of the Archaea. We present here, to our knowledge, the first structures of archaeal G1PDH from the hyperthermophilic methanogen Methanocaldococcus jannaschii with bound substrate dihydroxyacetone phosphate, product G1P, NADPH, and Zn²⁺ cofactor. We also biochemically characterized the enzyme with respect to pH optimum, cation specificity, and kinetic parameters for dihydroxyacetone phosphate and NAD(P)H. The structures provide key evidence for the reaction mechanism in the stereospecific addition for the NAD(P)H-based pro-R hydrogen transfer and the coordination of the Zn²⁺ cofactor during catalysis. Structure-based phylogenetic analyses also provide insight into the origins of G1PDH.     

Comparison of Small- and Large-scale Expression of Selected Pyrococcus furiosus Genes as an Aid to High-throughput Protein Production

As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.     

Phosphorylation and stabilization of ATP binding cassette transporter A1 by synthetic amphiphilic helical peptides

To investigate structural requirement of helical apolipoprotein to phosphorylate and stabilize ATP-binding cassette transporter A1 (ABCA1), synthetic peptides (Remaley, A. T., Thomas, F., Stonik, J. A., Demosky, S. J., Bark, S. E., Neufeld, E. B., Bocharov, A. V., Vishnyakova, T. G., Patterson, A. P., Eggerman, T. L., Santamarina-Fojo, S., and Brewer, H. B. (2003) J. Lipid Res. 44, 828-836) were examined for these activities. L37pA, an L amino acid peptide that contains two class-A amphiphilic helices, and D37pA, the same peptide with all D amino acids, both removed cholesterol and phospholipid from differentiated THP-1 cells more than apolipoproteins (apos) A-I, A-II, and E. Both peptides also mediated lipid release from human fibroblasts WI-38 similar to apoA-I. L2D37pA, an L-peptide whose valine and tyrosine were replaced with D amino acids also promoted lipid release from WI-38 but less so with THP-1, whereas L3D37pA, in which alanine, lysine, and asparatic acid were replaced with D amino acids was ineffective in lipid release for both cell lines. ABCA1 protein in THP-1 and WT-38 was stabilized against proteolytic degradation by apoA-I, apoA-II, and apoE and by all the peptides tested except for L3D37pA, and ABCA1 phosphorylation closely correlated with its stabilization. The analysis of the relationship among these parameters indicated that removal of phospholipid triggers signals for phosphorylation and stabilization of ABCA1. We thus concluded that an amphiphilic helical motif is the minimum structural requirement for a protein to stabilize ABCA1 against proteolytic degradation.     

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4⁺CD45RO⁺ T cells from peripheral blood, the percentage of ICOS⁺ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [³H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.     

Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.     

Ablation of estrogen receptor alpha (ERalpha) prevents upregulation of POMC by leptin and insulin

Diabetic Akita male mice are more hyperphagic because of downregulation of proopiomelanocortin (POMC) caused by hypoleptinemia. We investigated the role of estrogen receptor α (ERα) in the regulation of the hypothalamic POMC in females. ERaKOAkt mice consumed 30% greater food (g/3 weeks) than the Akita diabetic controls. Ovariectomized diabetic (AFO) and nondiabetic (B6FO) mice had significantly lower food intake and elevated serum leptin levels. ERaKOAkt and ERaKO mice also increased serum leptin concentrations, while hypoinsulinemia was observed in ERaKOAkt and hyperinsulinemia in ERaKO mice. RT-PCR showed a significant attenuation of POMC expression in both ERaKOAkt and ERaKO mice, irrespective of the elevated leptin serum levels or hyperinsulinemia, while elevated serum leptin levels in AFO and B6FO mice upregulated POMC gene expression. These results indicate that ERα plays an essential role in leptin- and insulin-stimulated upregulation of the POMC gene. This action of ERα is likely mediated in a ligand-independent manner.