Erratum

We studied the structural characteristics of the littoral epilithon in nine lentic water bodies at Hope Bay, Antarctic Peninsula, during summer 2002. At each site we measured the main physical and chemical variables and took epilithic samples for the analysis of dry weigh, ash, ash-free dry weight and chlorophyll a concentration. Distance from the sea of each water body was also considered. One site was selected for sampling lakes and ponds, except for Boeckella lake, where two sampling sites (A and B) were selected due to the heterogeneity of its littoral zone. Three stones for chlorophyll a analysis and another three to estimate dry weight, ash and ash-free dry weight, were taken randomly about 1 m away from the shore-line of each sampling site. Measurements of physical and chemical characteristics were obtained similarly. Water samples for chemical analysis were taken sub-superficially. Lakatoss system of classification and the Autotrophic Index were used to make functional inferences about the epilithic communities. Two PCA analyses were made to classify the water bodies according to environmental features and epilithic variables. In the latter, major patterns in data of epilithon were subsequently interpreted based on environmental data using external validation. Pingüi pond, located in the middle of the penguin rookery, was considered as a passive sample in both PCA analyses due to its extreme characteristics. Limnological features of the studied water bodies were similar to those of other Maritime Antarctic lakes. According to the Lakatoss index, 60% of the sampled lakes had high epilithic mass and a same proportion showed an inorganic type of epilithon The fact that 40% of the water bodies were autotrophic confirmed the importance of benthos as primary producer. According to environmental features, the well-defined groups of lakes emerged from the PCA were mainly determined by distance from the sea, pH, conductivity and salinity, and corresponded to the principal hydrological basins found in the region. Based on the results of the second PCA, littoral epilithon was affected by nitrate concentration and conductivity. In this ordination, water bodies from the same hydrological basin were separated probably as a result of a very complex inter-play of factors with a site-specific response to particular microhabitat characteristics.     

Anaerobiospirillum succiniciproducens phosphoenolpyruvate carboxykinase: mutagenesis at metal site 2

Phosphoenolpyruvate (PEP) carboxykinases harbor two divalent metal-binding sites. One cation interacts with the enzyme (metal binding site 1) to elicit activation, while a second cation (metal binding site 2) interacts with the nucleotide to serve as the metal nucleotide substrate. Mutants of Anaerobiospirillum succiniciproducens PEP carboxykinase have been constructed where Thr249 and Asp262, two residues of metal binding site 2 of the enzyme, were altered. Binding of the 3'(2')-O-(N-methylantraniloyl) derivative of ADP provides a test of the structural integrity of these mutants. The conservative mutation (Asp262Glu) retains a significant proportion of the wild type enzymatic activity. Meanwhile, removal of the OH group of Thr249 in the Thr249Ala mutant causes a decrease in V(max) by a factor of 1.1 x 10(4). Molecular modeling of wild type and mutant enzymes suggests that the lower catalytic efficiency of the Thr249Ala enzyme could be explained by a movement of the lateral chain of Lys248, a critical catalytic residue, away from the reaction center.     

Homofermentative lactate production cannot sustain anaerobic growth of engineered Saccharomyces cerevisiae: possible consequence of energy-dependent lactate export

Due to a growing market for the biodegradable and renewable polymer polylactic acid, the world demand for lactic acid is rapidly increasing. The tolerance of yeasts to low pH can benefit the process economy of lactic acid production by minimizing the need for neutralizing agents. Saccharomyces cerevisiae (CEN.PK background) was engineered to a homofermentative lactate-producing yeast via deletion of the three genes encoding pyruvate decarboxylase and the introduction of a heterologous lactate dehydrogenase (EC 1.1.1.27). Like all pyruvate decarboxylase-negative S. cerevisiae strains, the engineered strain required small amounts of acetate for the synthesis of cytosolic acetyl-coenzyme A. Exposure of aerobic glucose-limited chemostat cultures to excess glucose resulted in the immediate appearance of lactate as the major fermentation product. Ethanol formation was absent. However, the engineered strain could not grow anaerobically, and lactate production was strongly stimulated by oxygen. In addition, under all conditions examined, lactate production by the engineered strain was slower than alcoholic fermentation by the wild type. Despite the equivalence of alcoholic fermentation and lactate fermentation with respect to redox balance and ATP generation, studies on oxygen-limited chemostat cultures showed that lactate production does not contribute to the ATP economy of the engineered yeast. This absence of net ATP production is probably due to a metabolic energy requirement (directly or indirectly in the form of ATP) for lactate export.     

Crataegus special extract WS 1442 improves cardiac function and reduces infarct size in a rat model of prolonged coronary ischemia and reperfusion

In Germany, hydroalcoholic extracts from hawthorn (Crataegus spp.) leaves with flowers are approved drugs for the treatment of mild forms of heart insufficiency. Besides cardiotonic effects these herbal remedies have been shown to possess cardioprotective properties. We now evaluated if treatment of rats with the Crataegus special extract WS 1442 also improves cardiac function and prevents myocardial infarction during prolonged ischemia and reperfusion lasting for 240 and 15 min, respectively. Oral administration of WS 1442 (10 or 100 mg x kg(-1) x day(-1)) for 7 days before ligation of the left coronary artery dose-dependently suppressed the decrease of the pressure rate product. WS 1442 treatment also attenuated the elevation of the ST-segment in the ECG, diminished the incidence of ventricular fibrillations (control: 67%; 10 mg x kg(-1): 64%; 100 mg x kg(-1): 27%) and reduced the mortality rate (control: 47%; 10 mg.kg(-1): 27%; 100 mg x kg(-1): 9%). Furthermore, the area of myocardial infarction within the ischemic zone was significantly smaller in treated rats (10 mg x kg(-1): 64.3 +/- 5.1%; 100 mg x kg(-1): 42.8 +/- 4.1%) when compared with controls (78.4 +/- 2.6%). It is suggested that these pharmacological effects are accounted for by the combined antioxidative, leukocyte elastase inhibiting and endothelial nitric oxide (NO) synthesis enhancing properties of WS 1442.     

Stress relaxation behaviour of trabecular bone specimens

The present study defines several conditions under which stress relaxation tests can be performed and investigates the viscoelastic behaviour of trabecular bone in compression through a series of stress relaxation tests at three strain levels and in three loading directions of each cubic specimen. A visoelastic model is proposed to characterize the behaviour of trabecular bone and a spectrum of relaxation times is determined. Trabecular bone from the femoral head is non-linearly viscoelastic and displays anisotropic behaviour, which cannot be more symmetric elastically than orthotropic.     

An evaluation of different enzymatic cleavage methods for recombinant fusion proteins, applied on des(1–3)insulin-like growth factor I

Different enzymatic methods for cleavage of recombinant fusion proteins were compared. To find an efficient cleavage method, five different fusion proteins were produced. The fusion proteins differed only in the linker region between the fusion partner and the desired product, human des(1–3)insulin-like growth factor I. A cleavage study was performed with enterokinase, plasmin, thrombin, urokinase, and recombinant H64A subtilisin. Significant cleavage was obtained using thrombin, H64A subtilisin, and enterokinase. Thrombin cleavage was studied on a larger scale and des(1–3)IGF-I was recovered at a final yield of 3 mg/L growth medium. Thrombin and enterokinase were also studied as immobilized proteases and they cleaved the fusion proteins with retained activity. To further improve thrombin cleavage, a continuous reactor was constructed, consisting of a closed system with a thrombin column and an ion exchange column in series. Here, the fusion protein circulated while free des(1–3)IGF-I was bound to the ion exchange column after release from the fusion protein. In the reactor, thrombin was as efficient as the free enzyme but gave a diminished rate of product degradation.     

Human immunodeficiency viral protease is catalytically active as a fusion protein: characterization of the fusion and native enzymes produced in Escherichia coli

Processing of the gag and pol gene precursor proteins of retroviruses is essential for the production of mature infectious virions. The processing is directed by a viral protease that itself is part of these precursors and is presumed to cleave itself autocatalytically. To facilitate study of this process, the protease was produced as a fusion protein in Escherichia coli. In this construct, the 10,793-Da protease was preceeded by two copies of a modified IgG binding domain derived from protein A. The IgG binding domain was linked to the protease by an Asp-Pro peptide bond which could not be cleaved by the viral protease. A dimer of the 25,400-Da fusion protein was catalytically active, specifically cleaving a substrate peptide at the correct Tyr-Pro bond. Thus, the fusion protein could serve as a model of the viral gag-pol polyprotein. The finding that the fusion protein was catalytically active supports the suggestion that a gag-pol dimer can initiate a proteolytic cascade after budding of the immature virus. The fusion protein also provided a source of authentic protease. The protease was released from the fusion construct by incubation with formic acid, cleaving the Asp-Pro linkage which had been inserted between the IgG binding domain and the protease.     

The rat brain hippocampus proteome

The hippocampus is crucial in memory storage and retrieval and plays an important role in stress response. In humans, the CA1 area of hippocampus is one of the first brain areas to display pathology in Alzheimer's disease. A comprehensive analysis of the hippocampus proteome has not been accomplished yet. We applied proteomics technologies to construct a two-dimensional database for rat brain hippocampus proteins. Hippocampus samples from eight months old animals were analyzed by two-dimensional electrophoresis and the proteins were identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The database comprises 148 different gene products, which are in the majority enzymes, structural proteins and heat shock proteins. It also includes 39 neuron specific gene products. The database may be useful in animal model studies of neurological disorders.     

Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase

1,4-beta-D-Xylan is the major component of plant cell-wall hemicelluloses. beta-D-Xylosidases are involved in the breakdown of xylans into xylose and belong to families 3, 39, 43, 52, and 54 of glycoside hydrolases. Here, we report the first crystal structure of a member of family 39 glycoside hydrolase, i.e. beta-D-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. This study also represents the first structure of any beta-xylosidase of the above five glycoside hydrolase families. Each monomer of T. saccharolyticum beta-xylosidase comprises three distinct domains; a catalytic domain of the canonical (beta/alpha)(8)-barrel fold, a beta-sandwich domain, and a small alpha-helical domain. We have determined the structure in two forms: D-xylose-bound enzyme and a covalent 2-deoxy-2-fluoro-alpha-D-xylosyl-enzyme intermediate complex, thus providing two snapshots in the reaction pathway. This study provides structural evidence for the proposed double displacement mechanism that involves a covalent intermediate. Furthermore, it reveals possible functional roles for His228 as the auxiliary acid/base and Glu323 as a key residue in substrate recognition.