Genomic structure,cloning and expression of two phospholipase D isoenzymes from white cabbage

Phospholipase D (PLD) from cabbage is interesting as biocatalyst in phospholipid transformation. To provide the basis for genetic engineering of the enzyme, gene cloning and sequencing were carried out. We have recently identified two isoenzymes, PLD1 and PLD2, on the basis of their cDNAs, here we describe their genomic structure consisting of 3404 and 3614 bp, respectively. Based on their sequence, PLD1 and PLD2 can be assigned to the α‐type of plant PLDs, they contain two HKD motifs and the C2 domain with a phosphatidylinositol 4,5‐bisphosphate (PIP₂) binding motif. Starting from pld1 cDNA, expression studies were carried out. Whereas expression using constructs with StrepTactin or Glutathion S‐transferase tags was not successful, soluble active enzyme was produced from constructs without tag. pld2 was expressed accordingly. Both enzymes were purified by Ca²⁺‐mediated hydrophobic interaction chromatography to high purity. N‐terminal sequencing of PLD1 and PLD2 revealed that the Met‐free N‐termini of both enzymes correspond to sequences derived from the coding region of the pld1 and pld2 genes, respectively. Both recombinant enzymes showed highest hydrolytic activities at pH 5.5 to 5.6, independent of Ca²⁺ concentration (10—100 mM). The optimum Ca²⁺ concentration was 45 mM for PLD1 and PLD2. Both enzymes showed comparable activities in hydrolysis and transphosphatidylation of phospholipids.     

Effect of purified Trichoderma reesei cellulases on formation of cotton powder from cotton fabric

The mode of action of monocomponent purified Trichoderma reesei cellobiohydrolases (CBHI and CBHII) and endoglucanases (EGI and EGII) on cotton fabrics was studied by analyzing the weight loss of the fabric, the reducing sugars, the soluble oligosaccharides and the molecular weight of the cotton powder formed. The impact of mechanical action on these factors was also evaluated. EGI and EGII released the highest amounts of reducing sugars and soluble oligosaccharides in both treatments with or without additional mechanical action. After cellulase treatment without additional mechanical action, all of the cellulases were found to have reduced the molecular weight of cotton poplin powder. When mechanical action was combined with enzyme treatments, only EGII reduced the molecular weight. The weight loss of EG‐treated fabrics was clearly higher than the weight loss of CBH‐treated fabrics with both low and high mechanical action levels. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 1917–1922, 2003     

Effect of ground rubber powder and devulcanizates on the properties of natural rubber compounds

Ground rubber powder (GRP) and devulcanized rubber (Dev.R) waste through a continuous shear‐flow reaction were used as fillers for virgin natural rubber. A Horikx plot and the gel fraction proved that both main‐chain scission and crosslink scission occurred during the devulcanization. The cure characteristics, swelling behavior, crosslink density, and mechanical properties were studied. GRP and Dev.R, generated from passenger car and light truck tires, showed applicable mechanical properties in the new rubber compounds, and the compounds containing Dev.R demonstrated better properties than those containing GRP. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 97: 208–217, 2005     

Improvement of mechanical properties of rubber compounds using waste rubber/virgin rubber

Ground rubber powder (GRP) and devulcanized rubber waste obtained using the process of continuous shear flow reaction were used as both fillers and a part of rubber in virgin natural rubber (NR). The cure characteristics, swelling behavior, cross‐link density, mechanical properties, and morphology were studied in our laboratory. The GRP and devulcanized rubber, which were generated from passenger car and light truck tires, showed applicable mechanical properties in new rubber compounds, and the compounds that contained devulcanized rubber showed better properties than those with GRP. In particular, devulcanized rubber as a part of rubber obtained through a cross‐link reaction exhibited excellent mechanical properties, with a strong potential for future applications. POLYM. ENG. SCI., 45:1239–1246, 2005. © 2005 Society of Plastics Engineers     

4‐Biphenylalanine‐ and 3‐Phenyltyrosine‐Derived Hydroxamic Acids as Inhibitors of the JumonjiC‐Domain‐Containing Histone Demethylase KDM4A

Overexpression of the histone lysine demethylase KDM4A, which regulates H3K9 and H3K36 methylation states, has been related to the pathology of several human cancers. We found that a previously reported hydroxamate‐based histone deacetylase (HDAC) inhibitor (SW55) was also able to weakly inhibit this demethylase with an IC₅₀ value of 25.4 μm . Herein we report the synthesis and biochemical evaluations, with two orthogonal in vitro assays, of a series of derivatives of this lead structure. With extensive chemical modifications on the lead structure, also by exploiting the versatility of the radical arylation with aryldiazonium salts, we were able to increase the potency of the derivatives against KDM4A to the low‐micromolar range and, more importantly, to obtain demethylase selectivity with respect to HDACs. Cell‐permeable derivatives clearly showed a demethylase‐inhibition‐dependent antiproliferative effect against HL‐60 human promyelocytic leukemia cells.     

Proteins Marking the Sequence of Genotoxic Signaling from Irradiated Mesenchymal Stromal Cells to CD34+ Cells

Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.     

Effects of the Anti-Tumorigenic Agent AT101 on Human Glioblastoma Cells in the Microenvironmental Glioma Stem Cell Niche

Glioblastoma (GBM) is a barely treatable disease due to its profound chemoresistance. A distinct inter- and intratumoral heterogeneity reflected by specialized microenvironmental niches and different tumor cell subpopulations allows GBMs to evade therapy regimens. Thus, there is an urgent need to develop alternative treatment strategies. A promising candidate for the treatment of GBMs is AT101, the R(-) enantiomer of gossypol. The present study evaluates the effects of AT101, alone or in combination with temozolomide (TMZ), in a microenvironmental glioma stem cell niche model of two GBM cell lines (U251MG and U87MG). AT101 was found to induce strong cytotoxic effects on U251MG and U87MG stem-like cells in comparison to the respective native cells. Moreover, a higher sensitivity against treatment with AT101 was observed upon incubation of native cells with a stem-like cell-conditioned medium. This higher sensitivity was reflected by a specific inhibitory influence on the p-p42/44 signaling pathway. Further, the expression of CXCR7 and the interleukin-6 receptor was significantly regulated upon these stimulatory conditions. Since tumor stem-like cells are known to mediate the development of tumor recurrences and were observed to strongly respond to the AT101 treatment, this might represent a promising approach to prevent the development of GBM recurrences.     

An analysis of hydrogen production from renewable electricity sources

Three aspects of producing hydrogen via renewable electricity sources are analyzed to determine the potential for solar and wind hydrogen production pathways: a renewable hydrogen resource assessment, a cost analysis of hydrogen production via electrolysis, and the annual energy requirements of producing hydrogen for refueling. The results indicate that ample resources exist to produce transportation fuel from wind and solar power. However, hydrogen prices are highly dependent on electricity prices. For renewables to produce hydrogen at $2 kg⁻¹, using electrolyzers available in 2004, electricity prices would have to be less than $0.01 kWh⁻¹. Additionally, energy requirements for hydrogen refueling stations are in excess of 20 GWh/year. It may be challenging for dedicated renewable systems at the filling station to meet such requirements. Therefore, while plentiful resources exist to provide clean electricity for the production of hydrogen for transportation fuel, challenges remain to identify optimum economic and technical configurations to provide renewable energy to distributed hydrogen refueling stations.     

Investments into forest biorefineries under different price and policy structures

Increasing scarcity of oil reserves and the high CO₂ emissions from using oil have contributed to the development of renewable biofuels. Pulp and paper mill integrated forest biorefineries offer one important means to increase biofuel production. This study analyzes the effects of policies to support biofuel production in the pulp and paper sector. We study the relative effectiveness of three biofuel supporting policy instruments, namely production subsidy, input subsidy and investment subsidy. We present a partial equilibrium pulp and paper market model with a biorefinery investment option. A numerical model is used to evaluate the impacts of policy instruments on wood prices, as well as input choices and investment strategies of pulp and paper industries. The data represent the Finnish pulp and paper sector. We evaluate the values and direct costs of the policy instruments in a situation of exogenous biofuel production targets. The direct costs of input and investment subsidies are higher than those of a production subsidy. With all the policy instruments, Finnish pulp and paper mills would invest in wood-gasifying technology, instead of black liquor based one. The number of biorefinery units is dependent on the subsidy type — investment and input subsidies are likely to result in more numerous but smaller biofuel production units than a production subsidy. With all the policy instruments the demand for wood increases in Finland leading to higher wood prices. This, in turn, could reflect negatively on the profitability of the pulp and paper industries. To a significant degree, the model and the results can be generalized to other countries and markets where integrated pulp and paper mills are operating.