Vitreous TIMP-1 levels associate with neovascularization and TGF-β2 levels but not with fibrosis in the clinical course of proliferative diabetic retinopathy

In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and CCN2 (connective tissue growth factor; CTGF) cause blindness by neovascularization and subsequent fibrosis. This angio-fibrotic switch is associated with a shift in the balance between vitreous levels of CCN2 and VEGF in the eye. Here, we investigated the possible involvement of other important mediators of fibrosis, tissue inhibitor of metalloproteinases (TIMP)-1 and transforming growth factor (TGF)-β2, and of the matrix metalloproteinases (MMP)-2 and MMP-9, in the natural course of PDR. TIMP-1, activated TGF-β2, CCN2 and VEGF levels were measured by ELISA in 78 vitreous samples of patients with PDR (n = 28), diabetic patients without PDR (n = 24), and patients with the diabetes-unrelated retinal conditions macular hole (n = 10) or macular pucker (n = 16), and were related to MMP-2 and MMP-9 activity on zymograms and to clinical data, including degree of intra-ocular neovascularization and fibrosis. TIMP-1, CCN2 and VEGF levels, but not activated TGF-β2 levels, were significantly increased in the vitreous of diabetic patients, with the highest levels in PDR patients. CCN2 and the CCN2/VEGF ratio were the strongest predictors of degree of fibrosis. In diabetic patients with or without PDR, activated TGF-β2 levels correlated with TIMP-1 levels, whereas in PDR patients, TIMP-1 levels, MMP-2 and proMMP-9 were associated with degree of neovascularization, like VEGF levels, but not with fibrosis. We confirm here our previous findings that retinal fibrosis in PDR patients is significantly correlated with vitreous CCN2 levels and the CCN2/VEGF ratio. In contrast, TIMP-1, MMP-2 and MMP-9 appear to have a role in the angiogenic phase rather than in the fibrotic phase of PDR.     

Ethidium monoazide and propidium monoazide for elimination of unspecific DNA background in quantitative universal real-time PCR

Unspecific background DNA in quantitative universal real-time PCR utilizing a hydrolysis probe was completely suppressed by the addition of EMA or PMA to the PCR mix via cross-linking of the dyes to DNA during 650 W visible light exposure. The proposed procedure had no effect on the sensitivity of the real-time PCR reaction.     

Overwintering and prevalence of Neozygites floridana (Zygomycetes: Neozygitaceae) in hibernating females of Tetranychus urticae (Acari: Tetranychidae) under cold climatic conditions in strawberries

To evaluate overwintering strategies of the fungus Neozygites floridana, an important natural enemy of Tetranychus urticae, hibernating T. urticae females were investigated for the presence of fungal structures throughout one winter (October 12, 2006 to February 19, 2007) in field-grown strawberries in a cold climate in Norway (min. ambient temp -15.3 degrees C). Neozygites floridana was present as hyphal bodies inside live, hibernating females in T. urticae populations throughout the sampling period. The lowest percentages of hibernating females with hyphal bodies were found at the two first dates of sampling at 5.5 and 0% on October 12 and 19, respectively. The prevalence then increased and peaked at 54.4% on January 14. Resting spores (immature) were also found in live hibernating females at some dates, but at lower prevalence than for hyphal bodies and predominantly only until November 8. Prevalence of resting spores in live hibernating females ranged from 2.5 to 13.8%. Total number of T. urticae was also recorded, and most mites of all four categories (nymphs, males, non-hibernating and hibernating females) were found at the first sampling date. At this date non-hibernating females were the most abundant. A sharp decrease in non-hibernating females, nymphs and males was, however, seen from mid-October to mid-November; also numbers of hibernating females decreased, but not as fast. The relative abundance of hibernating females compared to non-hibernating females increased from 32.2% at the first collection (October 12) to 97.7% at the last collection (February 2). This study confirms that N. floridana survives the winter as a semi-latent hyphal body infection, protected inside live hibernating females. It is therefore ready to develop and sporulate as soon as climatic conditions permit, resulting in early season infection of T. urticae.     

Bioactive lipopeptides of ice-nucleating snow bacterium Pseudomonas syringae strain 31R1

The production of secondary metabolite lipopeptides by ice-nucleating Pseudomonas syringae strain 31R1 was investigated. Pseudomonas syringae strain 31R1 is a rifampicin-resistant derivative of P. syringae no. 31 used for the commercial production of snow. It is shown that P. syringae strain 31R1 produces antifungal lipodepsipeptides, syringomycins E and G, and, in addition, a novel and unique lipopeptide, peptin31. Spectroscopic and spectrometric analyses revealed that peptin31 is a linear undecalipopeptide with sequence identities to N- and C-terminal portions but lacking 11 amino acids of known lipodepsipeptide syringopeptin SPPhv. Peptin31 displayed antifungal activities against Rhodotorula pilimanae, Rhizoctonia solani, and Trichoderma harzianum and also hemolytic and antibacterial activities. Extracts of P. syringae strain 31R1 grown in medium with chloride were fungicidal, but not when grown without chloride. The latter extracts lacked peptin 31 and contained des-chloro forms of syringomycins E and G with low antifungal activities. Thus, the three lipopeptides account for the fungicidal properties of P. syringae 31R1 extracts. The occurrence of these bioactive metabolites should be considered when P. syringae no. 31 and its derivatives are used in products for making artificial snow.     

Application of Pneumatic Fracturing to Enhance In Situ Bioremediation

A field pilot demonstration integrating pneumatic fracturing and in situ bioremediation was carried out in a gasoline-contaminated, low permeability soil formation. A pneumatic fracturing system was used to enhance subsurface air flow and transport rates, as well as to deliver soil amendments directly to the indigenous microbial populations. An in situ bioremediation zone was established and operated for a period of 50 weeks, which included periodic subsurface injections of phosphate, nitrate, and ammonium salts. Off-gas data indicated the formation of a series of aerobic, denitrifying, and methanogenic microbial degradation zones. Based on soil samples recovered from the site, 79% of soil-phase benzene, toluene, and xylenes (BTX) was removed by the integrated technology. From mass balance calculations, accounting for all physical losses, it was estimated that 85% of the total mass of BTX removed (based on mean concentration levels) was attributable to biodegradation.     

Über Funktionen des Mangans im Stoffwechsel der höheren Pflanze

Ohne ZusammenfassungD 11.     

Mutational analysis and homology modelling of SyrC, the aminoacyltransferase in the biosynthesis of syringomycin

SyrC, a component of the multienzyme system of syringomycin biosynthesis, has been shown to shuttle Thr/4-Cl-Thr between the thiolation domains SyrB1-T1 and SyrE-T8,9 by transiently linking it to Cys224 in the enzyme active site. We present data on the structure-function relationship in vivo of this protein and an in silico model of its three-dimensional structure. The biosynthetic activity of SyrC was not influenced when either Asp348 or His376 that together with Cys224 form a putative catalytic triad, were replaced with Ala, but it was abolished by the exchange Cys224 with Ser. The presence of the FLAG peptide on either the N- or C-terminus of the protein did not affect activity, whereas the deletion of the first 16 amino acids at the N-terminus or the insertion of Maltose Binding Protein abolished the production of syringomycin. We present the model of the three-dimensional structure of SyrC suggesting a homodimeric structure for the protein and biochemical data that are supportive of this model.     

Importance of the gradient in photosynthetically active radiation in a vegetation stand for leaf nitrogen allocation in two monocotyledons

Carex acutiformis and Brachypodium pinnatum were grown with a uniform distribution of photosynthetic photon flux density (PFD) with height, and in a vertical PFD gradient similar to the PFD gradient in a leaf canopy. Distribution of organic leaf N and light-saturated rates of photosynthesis were determined. These parameters were also determined on plants growing in a natural vegetation stand. The effect of a PFD gradient was compared with the effect of a leaf canopy. In Brachypodium, plants growing in a vegetation stand had increasing leaf N with plant height. However, distribution of leaf N was not influenced by the PFD gradient treatment. The gradient of leaf N in plants growing in a leaf canopy was not due to differences within the long, mostly erect, leaves but to differences between leaves. In Carex, however, the PFD gradient caused a clear increase of leaf N with height in individual leaves and thus also in plants. The leaf N gradient was similar to that of plants growing in a leaf canopy. Leaf N distribution was not affected by nutrient availability in Carex. In most cases, photosynthesis was positively related to leaf N. Hence, lightsaturated rates of photosynthesis increased towards the top of the plants growing in leaf canopies in both species and, in Carex, also in the PFD gradient, thus contributing to increased N use efficiency for photosynthesis of the whole plant. It is concluded that in Carex the PFD gradient is the main environmental signal for leaf N allocation in response to shading in a leaf canopy, but one or more other signals must be involved in Brachypodium.     

Antimycin A and lipopolysaccharide cause the leakage of superoxide radicals from rat liver mitochondria

Here we show that both Antimycin A, a respiratory chain inhibitor inducing apoptosis, and endotoxic shock, a syndrome accompanied by both necrosis and apoptosis, cause not only an increase but also the leakage of superoxide radicals (O(2)(*-)) from rat heart mitochondria (RHM), while O(2)(*-) generated in intact RHM do not escape from mitochondria. This was shown by a set of O(2)(*-)-sensitive spin probes with varying hydrophobicity. The levels of O(2)(*-) detected in intact RHM gradually increase as the hydrophobicity of spin probes increases and were not sensitive to superoxide dismutase (SOD) added to the incubation medium. Both Antimycin A and endotoxic shock elevated O(2)(*-) levels. Elevated O(2)(*-) levels became sensitive to SOD but in a different manner. The determination of O(2)(*-) with water-soluble PPH was fully sensitive to SOD, while the determination of O(2)(*-) with the more hydrophobic CMH and CPH was only partially sensitive to SOD, suggesting the release of a portion of O(2)(*-) into the surrounding medium.