Thioredoxin h2 contributes to the redox regulation of mitochondrial photorespiratory metabolism

Thioredoxins (TRXs) are important proteins involved in redox regulation of metabolism. In plants, it has been shown that the mitochondrial metabolism is regulated by the mitochondrial TRX system. However, the functional significance of TRX h2, which is found at both cytosol and mitochondria, remains unclear. Arabidopsis plants lacking TRX h2 showed delayed seed germination and reduced respiration alongside impaired stomatal and mesophyll conductance, without impacting photosynthesis under ambient O₂ conditions. However, an increase in the stoichiometry of photorespiratory CO₂ release was found during O₂-dependent gas exchange measurements in trxh2 mutants. Metabolite profiling of trxh2 leaves revealed alterations in key metabolites of photorespiration and in several metabolites involved in respiration and amino acid metabolism. Decreased abundance of serine hydroxymethyltransferase and glycine decarboxylase (GDC) H and L subunits as well as reduced NADH/NAD⁺ ratios were also observed in trxh2 mutants. We further demonstrated that the redox status of GDC-L is altered in trxh2 mutants in vivo and that recombinant TRX h2 can deactivate GDC-L in vitro, indicating that this protein is redox regulated by the TRX system. Collectively, our results demonstrate that TRX h2 plays an important role in the redox regulation of mitochondrial photorespiratory metabolism.     

Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method

Shoot tips obtained from in vitro Rosa plants (three cultivars) were successfully cryopreserved by a combined droplet vitrification method and subsequently shoots regenerated. The excised shoot tips (1–4 mm long) were incubated in a liquid MS medium supplemented with 2.5 mg l⁻¹ thiamine, 0.2 mg l⁻¹ biotin, 0.2 mg l⁻¹ pyridoxine, 0.25 mg l⁻¹ 6-benzylaminopurine (BAP), 0.5 mg l⁻¹ gibberellic acid (GA₃) and 0.08 M sucrose, for 24 h. Following that incubation shoot tips were pre-cultured in this MS medium containing 0.1 till 1.0 M sucrose for 24 and 48 h, respectively. Pre-cultured shoot tips were dehydrated with concentrated PVS2 cryoprotective solution for 10–30 min at room temperature, prior to a direct plunge in liquid nitrogen. After rapid rewarming in the above mentioned liquid medium shoot tips were plated on a modified MS medium (5 g l⁻¹ agar) supplemented with vitamins and plant growth regulators as mentioned above for regrowth. Cryopreserved shoot tips resumed growth within 10 days and regenerated shoots within 3 weeks. The highest numbers of regrowing shoot tips were 64.44% for cv. Kardinal, 67.73% for cv. Fairy and 57.57% for cv. Maidy.     

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.     

Isoprene emission protects photosynthesis in sunfleck exposed Grey poplar

In the present study, we combined transient temperature and light stress (sunfleck) and comparably analyzed photosynthetic gas exchange in Grey poplar which has been genetically modified in isoprene emission capacity. Overall, we demonstrate that for poplar leaves the ability to emit isoprene is crucial to maintain photosynthesis when exposed to sunflecks. Net CO₂ assimilation and electron transport rates were strongly impaired in sunfleck-treated non-isoprene emitting poplars. Similar impairment was not detected when the leaves were exposed to high light (lightflecks) only. Within 10 h non-isoprene emitting poplars recovered from sunfleck-related impairment as indicated by chlorophyll fluorescence and microarray analysis. Unstressed leaves of non-isoprene emitting poplars had higher ascorbate contents, but also higher contents of malondialdehyde than wild-type. Microarray analyses revealed lipid and chlorophyll degradation processes in the non-isoprene emitting poplars. Thus, there is evidence for an adjustment of the antioxidative system in the non-isoprene emitting poplars even under normal growth conditions.     

Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma

3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.     

Pseudomonas aeruginosa virulence genes identified in a Dictyostelium host model

The human pathogen Pseudomonas aeruginosa has been shown previously to use similar virulence factors when infecting mammalian hosts or Dictyostelium amoebae. Here we randomly mutagenized a clinical isolate of P. aeruginosa , and identified mutants with attenuated virulence towards Dictyostelium . These mutant strains also exhibited a strong decrease in virulence when infecting Drosophila and mice, confirming that P. aeruginosa makes use of similar virulence traits to confront these very different hosts. Further characterization of these bacterial mutants showed that TrpD is important for the induction of the quorum-sensing circuit, while PchH and PchI are involved in the induction of the type III secretion system. These results demonstrate the usefulness and the relevance of the Dictyostelium host model to identify and analyse new virulence genes in P. aeruginosa .     

A novel type of detergent-resistant membranes may contribute to an early protein sorting event in epithelial cells

One sorting mechanism of apical and basolateral proteins in epithelial cells is based on their solubility profiles with Triton X-100. Nevertheless, apical proteins themselves are also segregated beyond the trans-Golgi network by virtue of their association or nonassociation with cholesterol/sphingolipid-rich microdomains (Jacob, R., and Naim, H. Y. (2001) Curr. Biol. 11, 1444-1450). Therefore, extractability with Triton X-100 does not constitute an absolute criterion of protein sorting. Here, we investigate the solubility patterns of apical and basolateral proteins with other detergents and demonstrate that the mild detergent Tween 20 is adequate to discriminate between apical and basolateral proteins during early stages in their biosynthesis. Although the mannose-rich forms of the apical proteins sucrase-isomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV reveal similar solubility profiles comprising soluble and nonsoluble fractions, the basolateral proteins, vesicular stomatitis virus G protein, major histocompatibility complex class I, and CD46 are entirely soluble with this detergent. The insoluble Tween 20 membranes are enriched in phosphatidylinositol and phosphatidylglycerol compatible with their synthesis in the endoplasmic reticulum and the existence of a novel class of detergent-resistant membranes. The association of the mannose-rich biosynthetic forms of the apical proteins, sucraseisomaltase, lactase-phlorizin hydrolase, aminopeptidase N, and dipeptidylpeptidase IV with the Tween 20-resistant membranes suggests an early polarized sorting mechanism prior to maturation in the Golgi apparatus.     

Anaerobic bacteria in bronchoalveolar lavage fluid (BAL) after thoracic surgery

Purpose of this study was to find out what kind of anaerobic bacteria were in lower respiratory tract and how often they were present there considering patients after thoracic surgery. Also, what is susceptibility of bacteria to antibiotics. Research covered 30 patients after operation. Material for research was bronchoalveolar lavage (BAL) taken during bronchoscopy. Collected sample was cultivated in anaerobic and aerobic conditions. Anaerobic bacteria were found in 28 samples (93%). Totally there were 100 anaerobic bacteria strains. The most common Gram-negative rods were from genus Prevotella (24 strains, 24%) and Bacteroides (15 strains, 15%). Gram-negative bacteria except Bacteroides characterised biggest susceptibility to imipenem, piperacillin/tazobactam, amoxicillin/clavulanate, ampicillin/sulbactam, piperacillin, clindamycin and metronidazol. Bacteroides were susceptible to imipenem, piperacillin/tazobactam and metronidazol. Among Gram-positive anaerobic bacteria mostly were isolated from cocci Peptostreptococcus (18 strains, 18%) and were susceptible to all antibiotics. Gram-positive rods were in most cases represented by Actinomyces (12 strains 12%) and were highly susceptible to all antibacterial means except metronidazol (100% is resistant).