Enzyme Thermistor Analysis of Penicillin in Standard Solutions and in Fermentation Broth

Immobilized penicillinase was applied in an enzyme thermistor for calorimetric analysis of samples containing penicillin G. Standard solutions as well as extracts from fermentation broth were analyzed. The enzyme was applied bound either to porous glass or, when dealing with crude preparations, to the inner surface of nylon tubing. In the fermentation system studied, high concentrations of penicillin were present, thus allowing dilution to reduce the influence of the composition of the medium on the analysis. The useful linear concentration range was from 0.1 to 100 mM. The coefficient of correlation between analytical results obtained with the present method and those from conventional assays was 0.997.     

Site-specific labeling of cytoplasmic peptide:N-glycanase by N,N'-diacetylchitobiose-related compounds

Peptide:N-glycanase (PNGase) is the deglycosylating enzyme, which releases N-linked glycan chains from N-linked glycopeptides and glycoproteins. Recent studies have revealed that the cytoplasmic PNGase is involved in the degradation of misfolded/unassembled glycoproteins. This enzyme has a Cys, His, and Asp catalytic triad, which is required for its enzymatic activity and can be inhibited by "free" N-linked glycans. These observations prompted us to investigate the possible use of haloacetamidyl derivatives of N-glycans as potent inhibitors and labeling reagents of this enzyme. Using a cytoplasmic PNGase from budding yeast (Png1), Man9GlcNAc2-iodoacetoamide was shown to be a strong inhibitor of this enzyme. The inhibition was found to be through covalent binding of the carbohydrate to a single Cys residue on Png1, and the binding was highly selective. The mutant enzyme in which Cys191 of the catalytic triad was changed to Ala did not bind to the carbohydrate probe, suggesting that the catalytic Cys is the binding site for this compound. Precise determination of the carbohydrate attachment site by mass spectrometry clearly identified Cys191 as the site of covalent attachment. Molecular modeling of N,N'-diacetylchitobiose (chitobiose) binding to the protein suggests that the carbohydrate binding site is distinct from but adjacent to that of Z-VAD-fmk, a peptide-based inhibitor of this enzyme. These results suggest that cytoplasmic PNGase has a separate binding site for chitobiose and other carbohydrates, and haloacetamide derivatives can irreversibly inhibit that catalytic Cys in a highly specific manner.     

Intravital microscopy of the murine pulmonary microcirculation.

Intravital microscopy (IVM) is considered as the gold standard for in vivo investigations of dynamic microvascular regulation. The availability of transgenic and knockout animals has propelled the development of murine IVM models for various organs, but technical approaches to the pulmonary microcirculation are still scarce. In anesthetized and ventilated BALB/c mice, we established a microscopic access to the surface of the right upper lung lobe by surgical excision of a window of 7- to 10-mm diameter from the right thoracic wall. The window was covered by a transparent polyvinylidene membrane and sealed with alpha-cyanoacrylate. Removal of intrathoracic air via a trans-diaphragmal intrapleural catheter coupled the lung surface to the window membrane. IVM preparations were hemodynamically stable for at least 120 min, with mean arterial blood pressure above 70 mmHg, and mean arterial Po(2) and arterial Pco(2) in the range of 90-100 Torr and 30-40 Torr, respectively. Imaged lungs did not show any signs of acute lung injury or edema. Following infusion of FITC dextran, subpleural pulmonary arterioles and venules of up to 50-microm diameter and alveolar capillary networks could be visualized during successive expiratory plateau phases over a period of at least 2 h. Vasoconstrictive responses to hypoxia (11% O(2)) or infusion of the thromboxane analog U-46619 were prominent in medium-sized arterioles (30- to 50-microm diameter), minor in small arterioles <30 microm, and absent in venules. The presented IVM model may constitute a powerful new tool for investigations of pulmonary microvascular responses in mice.     

Dependence of the flash-induced oxygen evolution pattern on the chemically and far red light-modulated redox condition in cyanobacterial photosynthetic electron transport

Flash-induced photosynthetic oxygen evolution was measured in cells and thylakoid preparations from the coccoid cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 and from the filamentous cyanobacterium Oscillatoria chalybea. The resulting characteristic flash patterns from these cyanobacteria can be chemically altered by addition of exogenously added substances like CCCP, DCPiP and inorganic salts. Potassium chloride, manganese sulfate and calcium chloride affected the sequences by specific increases in the flash yield and/or effects on the transition parameters. Chloride appeared to exert the strongest stimulatory effect on the oxygen yield. In comparison to chloride, both manganese and calcium did not significantly stimulate the flash amplitudes as such, but improved the functioning of the oxygen evolving complex by decreasing the miss parameter alpha. Particular effects were observed with respect to the time constants of the relaxation kinetics of the first two flash signals Y1/Y2 of the cyanobacterial patterns. In the presence of the investigated chemicals the amplitudes of the first two flash signals (Y2 in particular) were increased and the relaxation kinetics were enhanced so that the time constant became about identical to the conditions of steady state oxygen flash amplitudes. The results provide further evidence against a possible participation of either PS I or respiratory processes to Y1/Y2 of cyanobacterial flash patterns. Dramatic effects were observed when protoplasts from Oscillatoria chalybea or cells from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7942 were exposed to weak far red background illumination. Under these conditions, Y2 (and to a smaller extent Y1) of otherwise unchanged flash sequences were specifically modified. Y2 was substantially increased and again the relaxation kinetics were accelerated making the signal indistinguishable from a Y(SS) signal. From the mathematical fit of the sequences we conclude that S2 contributes to 10-20% of the S-state distribution (in comparison to 0% in the control). Thus, far red background illumination might represent a valuable means for photosynthetic investigations where high amounts of S2 are required like e. g. EPR measurements. In such experiments the corresponding EPR signals appeared substantially enhanced following far red preillumination (Ahrling and Bader, unpublished observations). Our results clearly show that the 'controversial results' from parts of the literature suggesting the participation of different mechanisms (net oxygen evolution, inhibited uptake processes etc.) are not required to explain the flash-induced oxygen evolution in cyanobacteria: the seemingly 'incompatible' conditions and conformations can be perfectly interconverted by different modulation techniques (chemicals, far red) of the respective redox condition within the water oxidation complex of photosynthesis.     

Rainfall manipulation experiments as simulated by terrestrial biosphere models: Where do we stand?

Changes in rainfall amounts and patterns have been observed and are expected to continue in the near future with potentially significant ecological and societal consequences. Modelling vegetation responses to changes in rainfall is thus crucial to project water and carbon cycles in the future. In this study, we present the results of a new model‐data intercomparison project, where we tested the ability of 10 terrestrial biosphere models to reproduce the observed sensitivity of ecosystem productivity to rainfall changes at 10 sites across the globe, in nine of which, rainfall exclusion and/or irrigation experiments had been performed. The key results are as follows: (a) Inter‐model variation is generally large and model agreement varies with timescales. In severely water‐limited sites, models only agree on the interannual variability of evapotranspiration and to a smaller extent on gross primary productivity. In more mesic sites, model agreement for both water and carbon fluxes is typically higher on fine (daily–monthly) timescales and reduces on longer (seasonal–annual) scales. (b) Models on average overestimate the relationship between ecosystem productivity and mean rainfall amounts across sites (in space) and have a low capacity in reproducing the temporal (interannual) sensitivity of vegetation productivity to annual rainfall at a given site, even though observation uncertainty is comparable to inter‐model variability. (c) Most models reproduced the sign of the observed patterns in productivity changes in rainfall manipulation experiments but had a low capacity in reproducing the observed magnitude of productivity changes. Models better reproduced the observed productivity responses due to rainfall exclusion than addition. (d) All models attribute ecosystem productivity changes to the intensity of vegetation stress and peak leaf area, whereas the impact of the change in growing season length is negligible. The relative contribution of the peak leaf area and vegetation stress intensity was highly variable among models.     

Conversion of AFLP markers to high-throughput markers in a complex polyploid,sugarcane

The application of DNA markers linked to traits of commercial value in sugarcane may increase the efficiency of sugarcane breeding. The majority of markers generated for quantitative trait locus mapping in sugarcane have been single sequence repeats or AFLPs (amplified fragment length polymorphisms). Since AFLP markers are not adapted for large-scale implementation in plant breeding, our objective was to assess the feasibility of converting AFLP markers to fast, cheap and reliable PCR-based assays in a complex polyploid, sugarcane. Three AFLP markers were selected on the basis of an association to resistance to the fungal pathogen Ustilago scitaminea, the causal agent of smut in sugarcane. We developed an approach which enabled the identification of polymorphisms in these AFLP markers. Towards this goal, we employed GenomeWalking and 454 sequencing to isolate sequences adjacent to the linked AFLP markers and identify SNP (single nucleotide polymorphisms) haplotypes present in the homo(eo)logous chromosomes of sugarcane. One AFLP marker was converted to a cleavage amplified polymorphic sequence marker, another to a SCAR (sequence characteristered amplified region) marker and the final AFLP marker to a SNP PCR-based assay. However, validation of each of the markers in 240 genotypes resulted in 99, 90 and 60% correspondence with the original AFLP marker. These experiments indicate that even in a complex polyploid such as sugarcane, polymorphisms identified by AFLP can be converted to high-throughput marker systems, but due to the complexity this would only be carried out for high-value markers. In some cases, the polymorphisms identified are not transferable to more sequence-specific PCR applications.     

Ring-based versus disc-based separation of spatial scales: a case study on the impact of arable land proportions on invertebrates in freshwater streams

The impact of different land-use types on species is traditionally estimated by correlating landscape proportions recorded in buffer areas around focal points with species data observed at these sites. If a high proportion of a specific land-use type exists within a small radius, it will be accumulated in larger buffers and may confound the interpretation at larger scales. We sampled freshwater invertebrates in ten streams using cages with artificial substrate and compared the effects of arable land proportions calculated in disc-shaped buffers of increasing radius versus areas calculated from non-overlapping rings of increasing radius. We hypothesize that (1) the accumulative disc-based approach leads to confounding effects across increasing buffer size and that (2) the use of ring-based methods facilitates the identification of relevant scales for conservation measures. The abundance of crustaceans showed a positive relationship with arable land proportions, but Plecoptera abundance and the taxonomic richness of Ephemeroptera and Plecoptera decreased with increasing arable land proportions in the surrounding landscape. Our results further support the presence of confounding effects in disc-based analyses, as correlations between arable land proportions and Crustacea, or Plecoptera, respectively, were affected by the accumulation of small-scale area proportions. The distance at which arable land proportions significantly affected benthic fauna in freshwater streams was consistently shorter if calculated from rings rather than from discs. Although an a priori definition of ring width introduces new challenges, a combined use of disc- and ring-based techniques for the estimation of land-use effects may substantially improve the realization of conservation and protection measures in terrestrial and aquatic systems.