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Calbindin D-28k EF-hand ligand binding and oligomerization: Four high-affinity sites-three modes of action

Calbindin D-28k, a highly conserved protein with Ca2+-sensing and Ca2+-buffering capabilities, is abundant in brain and sensory neurons. This protein contains six EF-hand subdomains, four of which bind Call with high affinity. Calbindin D28k can be reconstituted from six synthetic peptides corresponding to the six EF-hands, indicating a single-domain structure with multiple interactions between th

Localization of thrombin cleavage sites in the amino-terminal region of bovine protein S

Protein S is a vitamin K-dependent plasma protein. It functions as a cofactor to activated protein C in the inactivation of factors Va and VIIIa by limited proteolysis. Protein S is very sensitive to proteolysis by thrombin which reduces its calcium ion binding and leads to a loss of its cofactor activity. We have now determined the sequence of the 100 amino-terminal amino acid residues and locali

Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails

Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the

Site-directed primary in vitro immunization : Production of HIV-1 neutralizing human monoclonal antibodies from lymphocytes obtained from seronegative donors

The design of an in vitro immunization system based on a synthetic heterotope immunogen, which was a peptide containing both T- and B-cell epitopes, that elicited a neutralizing, primary human humoral immune response against the human immunodeficiency virus (HIV-1) is reported here. This heterotope construct contained the major neutralizing B-cell epitope, within the V3 region of glycoprotein 120

Dimerisation and an increase in active site aromatic groups as adaptations to high temperatures: X-ray solution scattering and substrate-bound crystal structures of Rhodothermus marinus endoglucanase Cel12A

Cellulose, a polysaccharide consisting of beta-1,4-linked glucose, is the major component of plant cell walls and consequently one of the most abundant biopolymers on earth. Carbohydrate polymers such as cellulose are molecules with vast diversity in structure and function, and a multiplicity of hydrolases operating in concert are required for depolymerisation. The bacterium Rhodothermus marinus,

Three-dimensional outgrowth of a wood-rotting fungus added to a contaminated soil from a former gasworks site

The capability of wood-rotting fungi (WRF) to colonise contaminated soil is an important fungal characteristic in the development of WRF-based soil bioremediation, it is also important to have methods that monitor the presence of the WRF in the soil. In this lab-scale study, it was shown that it was possible to re-capture, localise and identify a brown-rot fungus, Antrodia vaillantii, after it has

The cooling capacity of mosses : controls on water and energy fluxes in a siberian tundra site

Arctic tundra vegetation composition is expected to undergo rapid changes during the coming decades because of changes in climate. Higher air temperatures generally favor growth of deciduous shrubs, often at the cost of moss growth. Mosses are considered to be very important to critical tundra ecosystem processes involved in water and energy exchange, but very little empirical data are available.

Inferring the migratory status of woodland birds usisng ringing data: the case of a constant-effort site located in the Iberian highlands.

Understanding the spatiotemporal distribution of birds is crucial for effective management and conservation of their populations. However, we still have only limited knowledge not only of the wintering destinations of many Iberian breeding migrants but also of aspects as general as the migratory behaviour of the populations of many common avian species that breed in the Iberian highlands. We used

Human vascular smooth muscle cells from restenosis or in-stent stenosis sites demonstrate enhanced responses to p53: implications for brachytherapy and drug treatment for restenosis

The p53 tumor suppressor gene regulates growth arrest and apoptosis after DNA damage. Recent studies suggest that p53 is inactive in vascular smooth muscle cells (VSMCs) in human angioplasty restenosis, promoting VSMC accumulation and vessel stenosis. In contrast, the success of irradiation (brachytherapy) for in-stent restenosis argues that DNA-damage p53 responses are intact. We examined p53 exp

Factor Va-factor Xa interactions: molecular sites involved in enzyme:cofactor assembly.

The generation of thrombin by the prothrombinase complex is a key event in coagulation. In this complex, the activated form of coagulation factor V (FVa) serves as an essential cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. The enzyme FXa is virtually ineffective in the absence of its cofactor. The assembly of FXa with its cofactor FVa on negatively charged phospholipid

Antigen-binding sites dominate the surface properties of antibodies

We have found a remarkable relationship between the specificity of antibodies and their chromatographic behavious upon liquid-liquid partition chromatography (LLPC). Well characterized human and murine monoclonal antibodies and Fab/Fv fragments thereof as well as mouse/human chimeric antibodies were employed. While, lgG 1, 2 and 4 antibodies with identical specificities (affinity constants) have i

Modulation of GABA(A) Receptors by Natural Products and the Development of Novel Synthetic Ligands for the Benzodiazepine Binding Site.

Nature provides science and society with a virtually unlimited supply of structurally diverse and biologically active molecules; the natural products. While some are directly useful in commercial applications, others are valuable for studying and understanding biological phenomena at the molecular level. An example is the signaling of nerve cells, which has been explored in considerable detail usi

Pedon-scale silicate weathering : comparison of the PROFILE model and the depletion method at 16 forest sites in Sweden

Weathering of soil minerals is important for the recovery from acidification and for the sustainability of forestry. However, there is still substantial uncertainty about its absolute rate. This study presents a harmonized comparison of field weathering rates estimated with the mechanistic model PROFILE and the depletion method for 16 intensively sampled soil profiles across Sweden representing di