In situ proteolysis for protein crystallization and structure determination

We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work in progress; so far we determined structures of 9 bacterial proteins and the human aminoimidazole ribonucleotide synthetase (AIRS) domain.     

Demography and modelling of clonal fragments in the pseudoannual plant shape Trientalis europaea L.

The clonal growth pattern and demography of clonal fragments (aggregation of ramets derived from a common parent ramet) in the pseudoannual plant Trientalis europaea were studied in field conditions from 1991 to 1993. During this period the population of clonal fragments declined, with a half-life of 7.4 years. Number and size of the clonal progeny and stolon length were positively related to the size of the mother ramet. Survival rates of ramets and tubers increased with size. The rate of clonal growth was low: after three years, about 70% of the clonal fragments had only one ramet. This suggests that the pseudoannual growth habit in T. europaea is more important as mechanism of perennation than of ramet multiplication.Field data were used in a simulation model of architecture and population dynamics of clonal fragments. About 10% of the clonal fragments survived to the end of the simulation (15 years) and the mean survival was 4.7 years. The model predicted a positive correlation between persistence of the clonal fragment and number of ramets produced. Sensitivity analysis showed that the production of a daughter ramet of at least the same size as the parent ramet was the most important pathway for the survival and the number of ramets of the clonal fragment, whereas the production of secondary ramets had a very small effect. This confirms the interpretation of the pseudoannual life-cycle as a mechanism of ramet replacement in this species. Sensitivity analysis also revealed that changes in survival probabilities of the smallest ramets had the largest impact on clonal fragment dynamics. This reflects the important role of the smallest size class of ramets as a source of new vegetative propagules, maintaining a hierarchy in the size structure of the population.     

Decreased PLTP mass but elevated PLTP activity linked to insulin resistance in HTG: effects of bezafibrate therapy

Hypertriglyceridemia (HTG) is associated with insulin resistance, increased cholesteryl ester transfer (CET), and low HDL cholesterol. Phospholipid transfer protein (PLTP) may be involved in these relationships. Associations between CET, lipids, insulin resistance, CETP and PLTP activities, and PLTP mass were investigated in 18 HTG patients and 20 controls. Effects of 6 weeks of bezafibrate treatment were studied in HTG patients. HTG patients had higher serum triglycerides, insulin resistance, free fatty acid (FFA), and CET, lower levels of HDL cholesterol (-44%) and PLTP mass (-54%), and higher CETP (+20%) and PLTP activity (+48%) than controls. Bezafibrate reduced triglycerides, CET (-37%), insulin resistance (-53%), FFA (-48%), CETP activity (-12%), PLTP activity (-8%), and increased HDL cholesterol (+27%), whereas PLTP mass remained unchanged. Regression analysis showed a positive contribution of PLTP mass (P = 0.001) but not of PLTP activity to HDL cholesterol, whereas insulin resistance positively contributed to PLTP activity (P < 0.01). Bezafibrate-induced change in CET and HDL cholesterol correlated with changes in CETP activity and FFAs, but not with change in PLTP activity. Bezafibrate-induced change in PLTP activity correlated with change in FFAs (r = 0.455, P = 0.058). We propose that elevated PLTP activity in HTG is related to insulin resistance and not to increased PLTP mass. Bezafibrate-induced diminished insulin resistance is associated with a reduction of CET and PLTP activity.     

Carbon emission and sequestration by agricultural land use: a model study for Europe

A model was developed to calculate carbon fluxes from agricultural soils. The model includes the effects of crop (species, yield and rotation), climate (temperature, rainfall and evapotranspiration) and soil (carbon content and water retention capacity) on the carbon budget of agricultural land. The changes in quality of crop residues and organic material as a result of changes in CO₂ concentration and changed management were not considered in this model. The model was parameterized for several arable crops and grassland. Data from agricultural, meteorological, soil, and land use databases were input to the model, and the model was used to evaluate the effects of different carbon dioxide mitigation measures on soil organic carbon in agricultural areas in Europe. Average carbon fluxes under the business as usual scenario in the 2008–2012 commitment period were estimated at 0.52 tC ha^?1 y^?1 in grassland and ?0.84 tC ha^?1 y^?1 in arable land. Conversion of arable land to grassland yielded a flux of 1.44 tC ha^?1 y^?1. Farm management related activities aiming at carbon sequestration ranged from 0.15 tC ha^?1 y^?1 for the incorporating of straw to 1.50 tC ha^?1 y^?1 for the application of farmyard manure. Reduced tillage yields a positive flux of 0.25 tC ha^?1 y^?1. The indirect effect associated with climate was an order of magnitude lower. A temperature rise of 1 °C resulted in a ?0.05 tC ha^?1 y^?1 change whereas the rising CO₂ concentrations gave a 0.01 tC ha^?1 y^?1 change. Estimates are rendered on a 0.5 × 0.5° grid for the commitment period 2008–2012. The study reveals considerable regional differences in the effectiveness of carbon dioxide abatement measures, resulting from the interaction between crop, soil and climate. Besides, there are substantial differences between the spatial patterns of carbon fluxes that result from different measures.     

Selective alkylation and acylation of alpha and epsilon amino groups with PEG in a somatostatin analogue: tailored chemistry for optimized bioconjugates

The effects of the type and location of polymer grafting on the biological activity of different mono-PEG derivatives of the somatostatin analogue RC160 were evaluated. A chemical strategy to obtain mono-PEG alkylation or acylation of the peptide's alpha-terminal or lysil-epsilon primary amines was devised. Selective BOC protection of the two available primary amines, followed by reaction with two different PEG reagents and removal of the protecting group, was carried out. Chemical characterization, structural studies, and the evaluation of the biological activity of the bioconjugates synthesized allowed the identification of the one having characteristics more suitable for therapeutic application. This corresponds to the mono-epsilon-lysil-pegylated form, obtained by reductive alkylation, where the amine's positive charge is preserved. The results obtained suggest the importance of preliminary studies in the development of new polymer-peptide conjugates with improved pharmacological properties.     

The reproduction and development of sharks,skates, rays and ratfishes: introduction,history, overview,and future prospects

References This volume had its origin in aSymposium on the Reproduction and Development of Cartilaginous Fishes that was held at the annual meetings of the American Elasmobranch Society and the American Society of Ichthyologists and Herpetologists in Charleston, South Carolina in June 1990. The aim of this symposium was to bring together many of those scientists interested in chondrichthyan reproduction and development in order to assess the current state of knowledge in these fields.The chondrichthyan fishes occupy a pivotal position in comparative and evolutionary studies of vertebrate reproduction and development. They are the oldest surviving group of jawed vertebrates and they possess both the adult vertebrate Bauplan and the vertebrate program of embryonic development. The major features of the female reproductive system, including its embryonic origin, structure, physiological function, and biochemistry, apparently were established early in vertebrate evolution and are fully developed in chondrichthyan fishes. These features of the female reproductive system have been retained during the evolution of the other classes of vertebrates. Much the same can be said for the male reproductive system. Moreover, viviparity, placental nourishment of developing embryos, and the hormonal regulation of these events made an initial appearance in this group.The twenty-two articles contained in this volume bring together a wide variety of complementary research by investigators from seven countries. It is hoped that presentation of this disparate body of research and thought in one place will provide perspective on current research activity, call attention to those areas in which the research endeavour is deficient, and identify opportunities for future study. The appearance at this time of a volume on the reproduction and development of cartilaginous fishes is quite opportune. The continued existence of these fishes, which survived the great extinction events of Earth"s history, is now threatened by over-exploitation unless immediate steps for their conservation are undertaken. Knowledge of their reproduction and development not only is an end in itself, but is of critical importance in devising successful conservation and resource management strategies.