Germicide capacity of macrophages (MØ) in the Antarctic fish Notothenia coriiceps (Richardson, 1844) at 0°C

The germicide capability of the macrophage (MØ) of the Antarctic fish Notothenia coriiceps is demonstrated using fluorescence microscopy for the first time. The MØs were able to kill microorganisms by intracellular mechanism and this killing can be stimulated by oyster-derived glycogen. Although the phagocytosis index is lower than in temperate water fish species, this work demonstrates that non-specific defence mechanism plays an important role in the polar environment. There are some studies on inflammation in N. coriiceps [Silva et al. (1998) Polar Biol 20:206–212], parasite–host relation [Silva et al. (1999) Polar Biol 22:417–424] and phagocytosis [Silva et al. (2002) J Fish Biol 60:466–478]. These previous studies have shown that the MØ were able to identify biotic and abiotic factors. However, it can be of interest to study the activity of MØ in microorganism killing, and this work adds new insights of this fundamental process under Antarctic temperatures.     

Adeno-associated virus: from defective virus to effective vector

Infection by human hepatitis C virus (HCV) is the principal cause of post-transfusion hepatitis and chronic liver diseases worldwide. A reliable in vitro culture system for the isolation and analysis of this virus is not currently available, and, as a consequence, HCV pathogenesis is poorly understood. We report here the first robust in vitro system for the isolation and propagation of HCV from infected donor blood. This system involves infecting freshly prepared macrophages with HCV and then transmission of macrophage-adapted virus into freshly immortalized B-cells from human fetal cord blood. Using this system, newly isolated HCV have been replicated in vitro in continuous cultures for over 130 weeks. These isolates were also transmitted by cell-free methods into different cell types, including B-cells, T-cells and neuronal precursor cells. These secondarily infected cells also produced in vitro transmissible infectious virus. Replication of HCV-RNA was validated by RT-PCR analysis and by in situ hybridization. Although nucleic acid sequencing of the HCV isolate reported here indicates that the isolate is probably of type 1a, other HCV types have also been isolated using this system. Western blot analysis shows the synthesis of major HCV structural proteins. We present here, for the first time, a method for productively growing HCV in vitro for prolonged periods of time. This method allows studies related to understanding the replication process, viral pathogenesis, and the development of anti-HCV drugs and vaccines.     

Photosynthesis and carbon metabolism in plantain (Musa AAB) plantlets growing in temporary immersion bioreactors and during ex vitro acclimatization

Summary The photosynthetic capacity changes and the main enzymatic systems related to carbon metabolism were investigated during the in vitro culture of plantain shoots (Musa AAB cv. CEMSA 3/4) in temporary immersion bioreactors (TIB) and their subsequent acclimatization. The maximal rate of photosynthesis (Pn), transpiration, and the activity of the carbon metabolism enzymes phosphoenolpyruvate carboxylase (PEPC), acid invertase (AI), pyruvate kinase (PK) and sucrose phosphate synthase (SPS) were measured every 7 d during the 21 d of elongation in TIB, and the following 42 d of acclimatization. Sucrose content in the liquid medium and in the leaves was also determined. The most significant changes in plant growth were observed during acclimatization. During the in vitro stage photosynthesis was limited (4–6 μmol CO₂m⁻²s⁻¹); the photosynthetic rate however increases rapidly and significantly as soon as in vitro culture is over during acclimatization. PEPC activity increased during the whole evaluation period. The highest levels were achieved around days 42 and 56. PK and SPS activities were optimal during the first weeks in acclimatization (28–35 d), while AI increased at the beginning of the elongation phase (7 d), and later at the end of the acclimatization (49–63 d). The relationships between morphological parameters, photosynthetic capacity of the plantlets and the carbon metabolism enzymes during both phases of the culture are discussed.     

Comparing and evaluating process-based ecosystem model predictions of carbon and water fluxes in major European forest biomes

Process‐based models can be classified into: (a) terrestrial biogeochemical models (TBMs), which simulate fluxes of carbon, water and nitrogen coupled within terrestrial ecosystems, and (b) dynamic global vegetation models (DGVMs), which further couple these processes interactively with changes in slow ecosystem processes depending on resource competition, establishment, growth and mortality of different vegetation types. In this study, four models – RHESSys, GOTILWA+, LPJ‐GUESS and ORCHIDEE – representing both modelling approaches were compared and evaluated against benchmarks provided by eddy‐covariance measurements of carbon and water fluxes at 15 forest sites within the EUROFLUX project. Overall, model‐measurement agreement varied greatly among sites. Both modelling approaches have somewhat different strengths, but there was no model among those tested that universally performed well on the two variables evaluated. Small biases and errors suggest that ORCHIDEE and GOTILWA+ performed better in simulating carbon fluxes while LPJ‐GUESS and RHESSys did a better job in simulating water fluxes. In general, the models can be considered as useful tools for studies of climate change impacts on carbon and water cycling in forests. However, the various sources of variation among models simulations and between models simulations and observed data described in this study place some constraints on the results and to some extent reduce their reliability. For example, at most sites in the Mediterranean region all models generally performed poorly most likely because of problems in the representation of water stress effects on both carbon uptake by photosynthesis and carbon release by heterotrophic respiration (R_h). The use of flux data as a means of assessing key processes in models of this type is an important approach to improving model performance. Our results show that the models have value but that further model development is necessary with regard to the representation of the some of the key ecosystem processes.     

Submerged versus air-exposed intertidal macrophyte productivity: from physiological to community-level assessments

The photosynthetic productivity of the intertidal communities dominated by the seagrass Zostera noltii and the cordgrass Spartina maritima was assessed in two contrasting situations during a tidal cycle, i.e., air exposure and water immersion. Two complementary methods were used: infra red gas analysis of CO₂ flux measurements in whole communities and chlorophyll a fluorescence measurements of individual plants photosynthetic activity. Higher photosynthetic rates of Z. noltii in air were observed both at the individual plants response level determined by chlorophyll fluorescence and at the community level measured as gas exchange (CO₂ uptake). S. maritima plants consistently showed low photosynthetic response when immersed. Gross community production (GCP) measured as carbon dioxide uptake was always higher in air than in water for both communities. When immersed, the GCP of both communities was similar. However, when exposed to the air, the GCP of the S. maritima community was higher than the one of Z. noltii's. The key factor in CO₂ assimilation by air-exposed Z. noltii was the retention of water in sediment microdepressions. During low tide, depressions in the sediment retain a considerable amount of water, enough to maintain leaf hydration. In these conditions, rapid air-water CO₂ diffusion occurs, making it readily available to plants. The community gas exchange measurements compared well with the fluorescence indications. Both Z. noltii and S. maritima were shown to be responsible for the overall pattern of photosynthetic carbon fixation within their respective communities, both during submersion and emersion periods. The short-term incubations method described in this report proved to be a valuable tool for field measurements of intertidal lagoon productivity. It provides fast and precise values of carbon dioxide fixation, both in submerged and air-exposed communities.     

Purification and characterization of a methanol dehydrogenase derived fromMethylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) fromMethylomicrobium sp. HG-1, which belongs to the type 1 group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in anα ₂ β ₂ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and 60°C, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.     

The first crystal structure of class III superoxide reductase from Treponema pallidum

Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl₂ and diffracted beyond 1.55 Å resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the β-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)₄(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.This work is dedicated to the memory of Prof. Frank Rusnak.Coordinates and observed structure factor amplitudes have been deposited in the Protein Data Bank under the accession code 1Y07.