Alternative Crassulacean Acid Metabolism Modes Provide Environment-Specific Water-Saving Benefits in a Leaf Metabolic Model

Crassulacean acid metabolism (CAM) evolved in arid environments as a water-saving alternative to C₃ photosynthesis. There is great interest in engineering more drought-resistant crops by introducing CAM into C₃ plants. However, it is unknown whether full CAM or alternative water-saving modes would be more productive in the environments typically experienced by C₃ crops. To study the effect of temperature and relative humidity on plant metabolism in the context of water saving, we coupled a time-resolved diel (based on a 24-h day-night cycle) model of leaf metabolism to an environment-dependent gas-exchange model. This combined model allowed us to study the emergence of CAM as a trade-off between leaf productivity and water saving. We show that vacuolar storage capacity in the leaf is a major determinant of the extent of CAM. Moreover, our model identified an alternative CAM cycle involving mitochondrial isocitrate dehydrogenase as a potential contributor to initial carbon fixation at night. Simulations across a range of environmental conditions show that the water-saving potential of CAM strongly depends on the daytime weather conditions and that the additional water-saving effect of carbon fixation by isocitrate dehydrogenase can reach 11% total water saving for the conditions tested.     

Archaea in the Gulf of Aqaba

Using a polyphasic approach, we examined the presence of Archaea in the Gulf of Aqaba, a warm marine ecosystem, isolated from major ocean currents and subject to pronounced seasonal changes in hydrography. Catalyzed reported deposition FISH analyses showed that Archaea make up to >20% of the prokaryotic community in the Gulf. A spatial separation between the two major phyla of Archaea was observed during summer stratification. Euryarchaeota were found exclusively in the upper 200 m, whereas Crenarchaeota were present in greater numbers in layers below the summer thermocline. 16S rRNA gene-based denaturing gradient gel electrophoresis confirmed this depth partitioning and revealed further diversity of Crenarchaeota and Euryarchaeota populations along depth profiles. Phylogenetic analysis showed pelagic Crenarchaeota and Euryarchaeota to differ from coral-associated Archaea from the Gulf, forming distinct clusters within the Marine Archaea Groups I and II. Endsequencing of fosmid libraries of environmental DNA provided a tentative identification of some members of the archaeal community and their role in the microbial community of the Gulf. Incorporation studies of radiolabeled leucine and bicarbonate in the presence of different inhibitors suggest that the archaeal community participates in autotrophic CO₂ uptake and contributes little to the heterotrophic activity.     

Adherence ability of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis to two different epithelial cell lines

The ability of 59 wild-type strains of Pseudomonas aeruginosa to adhere to the HeLa and Buffalo Green Monkey Kidney (BGMK) cells was investigated. Twenty strains were isolated from sputa of cystic fibrosis patients, while 19 strains were isolated from tracheal aspirates and 20 from bronchial secretions of patients without cystic fibrosis, and they were used as a control group of strains. The statistically significant difference between adherence ability of strains was observed (p < 0.01). While most of the tracheal and bronchial isolates were hyperadhesive (51-110 bacteria per cell) most of the cystic fibrosis isolates adhered poorly to the HeLa and BGMK cells (1-10 bacteria per cell). The bacterial binding to the cells was blocked when bacteria were incubated at 80 degrees C for 20 min before the adherence assay. These results indicate that alginate is not involved in the adherence of P. aeruginosa to the used epithelial cell lines, and, because of that, mucoid strains isolated from persistently colonized cystic fibrosis patients showed poor adherence ability.     

Mathematical modeling of vascular endothelial layer maintenance: the role of endothelial cell division, progenitor cell homing, and telomere shortening

Maintenance of the endothelial cell (EC) layer of the vessel wall is essential for proper functioning of the vessel and prevention of vascular disorders. Replacement of damaged ECs could occur through division of surrounding ECs. Furthermore, EC progenitor cells (EPCs), derived from the bone marrow and circulating in the bloodstream, can differentiate into ECs. Therefore, these cells might also play a role in maintenance of the endothelial layer in the vascular system. The proliferative potential of both cell types is limited by shortening of telomeric DNA. Accelerated telomere shortening might lead to senescent vascular wall cells and eventually to the inability of the endothelium to maintain a continuous monolayer. The aim of this study was to describe the dynamics of EC damage and repair and telomere shortening by a mathematical model. In the model, ECs were integrated in a two-dimensional structure resembling the endothelium in a large artery. Telomere shortening was described as a stochastic process with oxidative damage as the main cause of attrition. Simulating the model illustrated that increased cellular turnover or elevated levels of oxidative stress could lead to critical telomere shortening and senescence at an age of 65 yr. The model predicted that under those conditions the EC layer could display defects, which could initiate severe vascular wall damage in reality. Furthermore, simulations showed that 5% progenitor cell homing/yr can significantly delay the EC layer defects. This stresses the potential importance of EPC number and function to the maintenance of vascular wall integrity during the human life span.     

Anticancer activity for 4,4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) analogues and their abilities to interact with lymphoendothelial cell surface markers

The structure of the anticancer agent 4,4'-dihydroxybenzophenone-2,4-dinitrophenylhydrazone (A-007) has been modified through SAR and by incorporating barbituric acid, pyridine, quinoline, and alkylcarboxylic acids into A-007's moieties. Analogue anticancer activity and interacting with CD surface markers on a T-cell leukemia cell line were evaluated and the correlation between SAR and biological properties are discussed.     

Co- and/or post-translational modifications are critical for TCH4 XET activity

TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana . XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo ; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus β-glucanases, this region may be the active site of XET enzymes.     

N-acetylation of amino sugar methyl glycosides for gas-liquid chromatographic analysis.

A procedure is described for rapid and quantitative N-acetylation of amino sugars, particularly suitable for gas-liquid chromatographic analysis of constituent carbohydrates in glycoproteins.     

In vitro activities of four xyloglucan endotransglycosylases from Arabidopsis

Xyloglucan endotransglycosylases (XETs) are encoded by a gene family in Arabidopsis thaliana. These enzymes modify a major structural component of the plant cell wall, xyloglucan, and therefore may influence plant growth and development. We have produced four Arabidopsis XETs (TCH4, Meri-5, EXGT and XTR9) using the baculovirus/insect cell system and compared their biochemical activities. TCH4, as previously demonstrated, and the other three proteins are capable of carrying out transglycosylation of xyloglucans. The K(m) for XLLGol acceptor oligosaccharide is in the range of 20-40 microM for all the XETs except XTR9, which has a Km of 5 microM and is significantly inhibited by high levels of XLLGol. All four enzymes are most active between pH 6.0 and 6.5. TCH4 and XTR9 have temperature optima of 18 degrees C, whereas Meri-5 and EXGT are most active at 28 and 37 degrees C, respectively. Although the activity levels of three of the XETs are not influenced by the presence of fucose on the xyloglucan polymer, XTR9 has a clear preference for non-fucosylated xyloglucan polymer. The four XETs show a marked preference for XLLGol over either XXFGol or XXXGol as acceptor oligosaccharide. All four XETs are glycosylated; however, only the activities of TCH4 and Meri-5 are affected by the removal of the N-glycan with PNGase F. These four enzymes most likely function solely as transglycosylases because xyloglucan endoglucanase activity was not apparent. Subtle differences in biochemical activities may influence the physiological functions of the distinct XETs in vivo.