Production and purification of monoclonal antibodies to Pisum and Avena phytochrome

At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Regulation of the activity and synthesis of malic enzyme in 3T3-L1 cells

Malic enzyme activity in differentiated 3T3-L1 cells was about 20-fold greater than activity in undifferentiated cells. A new steady-state level was achieved about 8 days after initiating differentiation of confluent cultures with a 2-day exposure to dexamethasone, isobutylmethylxanthine, and insulin. This increase in enzyme activity resulted from an increase in the mass of malic enzyme as detected by immunotitration of enzyme activity with goat antiserum directed against purified rat liver malic enzyme. Malic enzyme synthesis was undetectable in undifferentiated cells and increased to about 0.2% of soluble protein in differentiated cells, suggesting that the increase in enzyme mass was due primarily to an increase in enzyme synthesis. Thyroid hormone, a potent stimulator of malic enzyme activity in hepatocytes in culture and in liver and adipose tissue in intact animals, decreased or increased malic enzyme activity in differentiating 3T3-L1 cells by about 40% when it was removed or added to the medium, respectively. Insulin, another physiologically important regulator of malic enzyme activity in vivo, had no effect on the initial rate of accumulation of malic enzyme activity in the differentiating cells and caused a 30 to 40% decrease in the final level of enzyme activity in the fully differentiated cells. Cyclic AMP, a potent inhibitor of malic enzyme synthesis in hepatocytes in culture, inhibited this process in 3T3-L1 cells by 30%. Malic enzyme is like several other enzymes in that the large increase in its concentration which accompanies differentiation of 3T3-L1 cells is due to increased synthesis of enzyme protein. However, the hormonal modulation of malic enzyme characteristic of liver and adipose tissue in intact animals does not appear to occur in differentiated 3T3-L1 cells, suggesting that differentiated 3T3-L1 cells may not be an appropriate model system in which to study the hormonal modulation of malic enzyme that occurs in liver and adipose tissue of intact animals.     

酸性矿山废水污染的水稻田土壤中重金属的微生物学效应

采样调查了广东大宝山地区受酸性采矿废水长期污染的亚热带水稻田的土壤理化性质 ,重金属 Cu、Pb、Zn、Cd的全量及其 DTPA浸提量 ,以及微生物生物量及其呼吸活性等指标。利用主成分和逐步回归分析了影响土壤重金属的有效性及其微生物学效应的因素。结果表明 :土壤高含硫 ,强酸性 ,有机碳、全氮较低 ,4种金属的全量普遍超标。DTPA可提取态金属含量较高 ,不仅与其全量呈显著正相关 ,而且与土壤酸度和粘粒含量正相关 ,和 Mn含量负相关。过量的金属显著降低了土壤微生物生物量 C、N、微生物商、生物量 N/全 N比 ,并抑制了微生物呼吸强度和对有机碳的矿化率 ,导致了土壤 C/N比的升高。同时 ,金属对微生物群落及生理代谢指标 ,如微生物生物量 C/N比和代谢商的影响不显著。 DTPA可提取态金属 ,特别是 DTPA- Cu是导致微生物生物量和活性指标变化的主要因素。以有机碳 (或全氮 )为基数的复合微生物指标降低了土壤性质差异造成的干扰 ,较单一指标更能准确指示微生物对金属胁迫的反应。土壤硫没有对金属有效性和微生物指标产生明显影响 ,但其氧化过程可能引起酸化和金属离子的释放     

Partial modelling of the perennial crop cycle misleads LCA results in two contrasted case studies

Purpose

As highlighted in recent reviews, there is a need to harmonise the way life cycle assessment (LCA) of perennial crops is conducted. In most published LCA on perennial crops, the modelling of the agricultural production is based on data sets for just one productive year. This may be misleading since performance and impacts of the system may greatly vary year by year. The purposes of this study are to analyse how partial modelling of the perennial cycle through non-holistic data collection may affect LCA results and to make recommendations.

Methods

Three modelling choices for the perennial crop cycle were tested in parallel in two contrasted LCA case studies: oil palm fruits from Indonesia, and small citrus from Morocco. Modelling choices tested were as follows: (i) a chronological modelling over the complete crop cycle of orchards, (ii) a 3-year average from the productive phase, and (iii) various single years from the productive phase. In both case studies, the system boundary was a cradle-to-farm gate with a functional unit of 1 kg fresh fruits. LCA midpoint impacts were calculated with ReCiPe 2008 in Simapro©V.7. We first analysed how inputs, yields and potential impacts varied over time. We then analysed process contributions in the baseline model, i.e. the chronological modelling, and finally compared LCA results for the various perennial modelling choices.

Results and discussion

Agricultural practices, yields and impacts varied over the years especially during the first 3–9 years depending on the case study. In both case studies, the modelling choices to account or not for the whole perennial cycle drastically influenced LCA results. The differences could be explained by the inclusion or not of the yearly variability and the accounting or not of the immature phase, which contributed to 7–40 or 6.5–29 % of all impact categories for oil palm fruit and citrus, respectively.

Conclusions

The chosen approach to model the perennial cycle influenced the final LCA results for two contrasted case studies and deserved specific attention. Although data availability may remain the limiting factor in most cases, assumptions can be made to interpolate or extrapolate some data sets or to consolidate data sets from chronosequences (i.e. modular modelling). In all cases, we suggest that the approach chosen to model the perennial cycle and the representativeness of associated collected data should be made transparent and discussed. Further research work is needed to improve the understanding and modelling of perennial crop functioning and LCA assessment.

     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

Phosphocaveolin-1 is a mechanotransducer that induces caveola biogenesis via Egr1 transcriptional regulation

Maintenance of epithelial cell adhesion is crucial for epidermal morphogenesis and homeostasis and relies predominantly on the interaction of keratins with desmosomes. Although the importance of desmosomes to epidermal coherence and keratin organization is well established, the significance of keratins in desmosome organization has not been fully resolved. Here, we report that keratinocytes lacking all keratins show elevated, PKC-α–mediated desmoplakin phosphorylation and subsequent destabilization of desmosomes. We find that PKC-α activity is regulated by Rack1–keratin interaction. Without keratins, desmosomes assemble but are endocytosed at accelerated rates, rendering epithelial sheets highly susceptible to mechanical stress. Re-expression of the keratin pair K5/14, inhibition of PKC-α activity, or blocking of endocytosis reconstituted both desmosome localization at the plasma membrane and epithelial adhesion. Our findings identify a hitherto unknown mechanism by which keratins control intercellular adhesion, with potential implications for tumor invasion and keratinopathies, settings in which diminished cell adhesion facilitates tissue fragility and neoplastic growth.