Preferential induction of a 9-lipoxygenase by salt in salt-tolerant cells of Citrus sinensis L. Osbeck

Recent findings in our laboratory suggested that in citrus cells the salt induction of phospholipid hydroperoxide glutathione peroxidase, an enzyme active in cellular antioxidant defense, is mediated by the accumulation of hydroperoxides. Production of hydroperoxides occurs as a result of non-enzymatic auto-oxidation or via the action of lipoxygenases (LOXs). In an attempt to resolve the role of LOX activity in the accumulation of peroxides we analyzed the expression of this protein under stress conditions and in cells of Citrus sinensis L. differing in sensitivity to salt. Lipoxygenase expression was induced very rapidly only in the salt-tolerant cells and in a transient manner. The induction was specific to salt stress and did not occur with other osmotic-stress-inducing agents, such as polyethylene glycol or mannitol, or under hot or cold conditions, or in the presence of abscisic acid. The induction was eliminated by the antioxidants dithiothreitol and kaempferol, thus once more establishing a correlation between salt and oxidative stresses. Analyses of both in vitro and in vivo products of LOX revealed a specific 9-LOX activity, and a very fast reduction of the hydroperoxides to the corresponding hydroxy derivatives. This suggests that one of the metabolites further downstream in the reductase pathway may play a key role in triggering defense responses against salt stress. Received: 3 February 2000 / Accepted: 13 June 2000     

Conformational changes and loose packing promote E. coli Tryptophanase cold lability

Background  

Oligomeric enzymes can undergo a reversible loss of activity at low temperatures. One such enzyme is tryptophanase (Trpase) from Escherichia coli. Trpase is a pyridoxal phosphate (PLP)-dependent tetrameric enzyme with a Mw of 210 kD. PLP is covalently bound through an enamine bond to Lys270 at the active site. The incubation of holo E. coli Trpases at 2°C for 20 h results in breaking this enamine bond and PLP release, as well as a reversible loss of activity and dissociation into dimers. This sequence of events is termed cold lability and its understanding bears relevance to protein stability and shelf life.     

Biological Control of Fusarium-wilt in Carnation With Serratia liquefaciens and Hafnia alvei Isolated from Rhizosphere of Carnation

Serratia liquefaciens provided better protection of carnations from infection by Fusarium oxysporum f. sp. dianthi, than did Hafnia alvei. The protection occurred when the bacterial isolates were applied to the cuttings before rooting, but not when applied to the root system of rooted cuttings. S. liquefaciens was recovered from carnation stem segments along the stem up to the top after 60 days but after 120 days they were recovered only up to 2.5cm. Zones of inhibition of Fusarium-conidia sprayed on agar plates previously incubated with stem segments appeared around the bacterial colonies of S. liquifaciens after additional incubation for 24—48h.     

Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell''s pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways.     

Bone Marrow and Nonbone Marrow Toll Like Receptor 4 Regulate Acute Hepatic Injury Induced by Endotoxemia

Background

Toll-like receptors (TLRs) are expressed in immune cells and hepatocytes. We examined whether hepatic Toll-like receptor 4 (TLR4) is involved in the acute hepatic injury caused by the administration of lipopolysaccharide (LPS) (septic shock model).

Methods

Wild type (WT), TLR4-deficient and chimera mice underwent myeloablative bone marrow transplantation to dissociate between TLR4 expression in the liver or in the immune-hematopoietic system. Mice were injected with LPS and sacrificed 4 hours later.

Results

Compared to TLR4 deficient mice, WT mice challenged with LPS displayed increased serum liver enzymes and hepatic cellular inflammatory infiltrate together with increased serum and hepatic levels of interleukin 1β (IL-1β), tumor necrosis factor α (TNFα) ,Up-regulation of hepatic mRNA encoding TLR4, IκB and c-jun expressions. TLR4 mutant mice transplanted with WT bone marrow were more protected than WT chimeric mice bearing TLR4 mutant hemopoietic cells from LPS, as seen by IL-1β and TNFα levels. We then used hepatocytes (Huh7) and macrophages from monocytic cell lines to detect TLR mRNA expression. Macrophages expressed a significantly higher level of TLR4 mRNA and TLR2 (more than 3000- and 8000-fold respectively) compared with the hepatocyte cell line. LPS administration induced TLR4 activation in a hepatocyte cell line in a dose dependent manner while TLR2 mRNA hardly changed.

Conclusions

These results suggest that TLR4 activation of hepatocytes participate in the immediate response to LPS induced hepatic injury. However, in this response, the contribution of TLR4 on bone marrow derived cells is more significant than those of the hepatocytes. The absence of the TLR4 gene plays a pivotal role in reducing hepatic LPS induced injury.     

Proteomics‐level analysis of myelin formation and regeneration in a mouse model for Vanishing White Matter disease

Vanishing white matter (VWM) is a recessive neurodegenerative disease caused by mutations in translation initiation factor eIF2B and leading to progressive brain myelin deterioration, secondary axonal damage, and death in early adolescence. Eif2b5^R132H/R132H mice exhibit delayed developmental myelination, mild early neurodegeneration and a robust remyelination defect in response to cuprizone‐induced demyelination. In the current study we used Eif2b5^R132H/R132H mice for mass‐spectrometry analyses, to follow the changes in brain protein abundance in normal‐ versus cuprizone‐diet fed mice during the remyelination recovery phase. Analysis of proteome profiles suggested that dysregulation of mitochondrial functions, altered proteasomal activity and impaired balance between protein synthesis and degradation play a role in VWM pathology. Consistent with these findings, we detected elevated levels of reactive oxygen species in mutant‐derived primary fibroblasts and reduced 20S proteasome activity in mutant brain homogenates. These observations highlight the importance of tight translational control to precise coordination of processes involved in myelin formation and regeneration and point at cellular functions that may contribute to VWM pathology.

     

Chromosome cohesion: ring around the sisters?

Sister chromatid cohesion is a key aspect of accurate chromosome transmission during mitosis, yet little is known about the structure of cohesin, the protein complex that links the two sister chromatids. Recent studies shed light on the structure of the cohesin complex, leading to intriguing models that could explain how sister chromatids are held together.