Production of lactating dairy cows fed alfalfa or red clover silage at equal dry matter or crude protein contents in the diet.

Two Latin square trials, using 21 or 24 multiparous lactating Holstein cows, compared the feeding value of red clover and alfalfa silages harvested over 2 yr. Red clover silages averaged 2 percentage units lower in crude protein (CP) and more than 2 percentage units lower in neutral detergent fiber and acid detergent fiber than did alfalfa silage. In trial 1, diets were formulated to 60% dry matter (DM) from alfalfa, red clover silage, or alfalfa plus red clover silage (grown together); CP was adjusted to about 16.5% by adding soybean meal, and the balance of dietary DM was from ground high moisture ear corn. Nonprotein N in red clover and alfalfa-red clover silages was 80% of that in alfalfa silage. Although DM intake was 2.5 and 1.3 kg/d lower on red clover and alfalfa plus red clover, yield of milk and milk components was not different among diets. In trial 2, four diets containing rolled high moisture shelled corn were formulated to 60% DM from alfalfa or red clover silage, or 48% DM from alfalfa or red clover silage plus 12% DM from corn silage. The first three diets contained 2.9% soybean meal, and the red clover-corn silage diet contained 5.6% soybean meal; the 60% alfalfa diet contained 18.4% CP, and the other three diets averaged 16.5% CP. Nonprotein N in red clover silage was 62% of that in alfalfa silage. Intake of DM was about 2 (no corn silage) and 1 kg/d (plus corn silage) lower on red clover. Yield of milk and milk components was not different among the first three diets; however, yields of milk, total protein, and true protein were higher on red clover-corn silage with added soybean meal. Replacing alfalfa with red clover improved feed and N efficiency and apparent digestibility of DM, organic matter, neutral detergent fiber, acid detergent fiber, and hemicellulose in both trials. Net energy of lactation computed from animal performance data was 18% greater in red clover than alfalfa. Data on milk and blood urea and N efficiency suggested better N utilization on red clover.

The use of 3D structural data in the design of specific factor Xa inhibitors.

Factor Xa (fXa) is a serine protease that plays a critical role in the blood coagulation process and qualifies as an attractive target for finding new antithrombotics. In the case of fXa several structure based drug design strategies have been followed because of the difficulty in growing fXa co-crystals routinely. This has led to the use of surrogate proteins such as trypsin. Factor Xa inhibitors for which the binding mode has been determined experimentally or modeled are described in this review. The inhibitors are divided into three fragments: a P1 group, a central scaffold and a P4 group. In this review, interactions in each sub-site of fXa with various inhibitor fragments have been examined at the molecular level and were shown to bind, in most cases, independently of the rest of the molecule. Knowledge of the 3D structure of the binding mode of ligands to target proteins has been successfully applied in designing fXa inhibitors with enhanced specificity, affinity and has provided hints to modulate the physico-chemical properties of the small molecule ligand.

Crystal structures of human factor Xa complexed with potent inhibitors.

Involved in the coagulation cascade, factor Xa (FXa) is a serine protease which has received great interest as a potential target for the development of new antithrombotics. Although there is a great wealth of structural data on thrombin complexes, few structures of ligand/FXa complexes have been reported, presumably because of the difficulty in growing crystals. Reproducible crystallization conditions for human des-Gla1-45 coagulation FXa have been found. This has led to an improvement in the diffraction quality of the crystals (about 2.1 A) when compared to the previously reported forms (2.3-2.8 A) thus providing a suitable platform for a structure-based drug design approach. A series of crystal structures of noncovalent inhibitors complexed with FXa have been determined, three of which are presented herein. These include compounds containing the benzamidine moiety and surrogates of the basic group. The benzamidine-containing compound binds in a canonical fashion typical of synthetic serine protease inhibitors. On the contrary, molecules that contain surrogates of the benzamidine group do not make direct hydrogen-bonding interactions with the carboxylate of Asp189 at the bottom of the S1 pocket. The structural data provide a likely explanation for the specificity of these inhibitors and a great aid in the design of bioavailable potent FXa inhibitors.

Design and structure-activity relationships of potent and selective inhibitors of blood coagulation factor Xa.

The discovery of a series of non-peptide factor Xa (FXa) inhibitors incorporating 3-(S)-amino-2-pyrrolidinone as a central template is described. After identifying compound 4, improvements in in vitro potency involved modifications of the liphophilic group and optimizing the angle of presentation of the amidine group to the S1 pocket of FXa. These studies ultimately led to compound RPR120844, a potent inhibitor of FXa (K(i) = 7 nM) which shows selectivity for FXa over trypsin, thrombin, and several fibrinolytic serine proteinases. RPR120844 is an effective anticoagulant in both the rat model of FeCl(2)-induced carotid artery thrombosis and the rabbit model of jugular vein thrombus formation.

Sulfonamidopyrrolidinone factor Xa inhibitors: potency and selectivity enhancements via P-1 and P-4 optimization.

Sulfonamidopyrrolidinones were previously disclosed as a selective class of factor Xa (fXa) inhibitors, culminating in the identification of RPR120844 as a potent member with efficacy in vivo. Recognizing the usefulness of the central pyrrolidinone template for the presentation of ligands to the S-1 and S-4 subsites of fXa, studies to optimize the P-1 and P-4 groups were initiated. Sulfonamidopyrrolidinones containing 4-hydroxy- and 4-aminobenzamidines were discovered to be effective inhibitors of fXa. X-ray crystallographic experiments in trypsin and molecular modeling studies suggest that our inhibitors bind by insertion of the 4-hydroxybenzamidine moiety into the S-1 subsite of the fXa active site. Of the P-4 groups examined, the pyridylthienyl sulfonamides were found to confer excellent potency and selectivity especially in combination with 4-hydroxybenzamidine. Compound 20b (RPR130737) was shown to be a potent fXa inhibitor (K(i) = 2 nM) with selectivity against structurally related serine proteinases (>1000 times). Preliminary biological evaluation demonstrates the effectiveness of this inhibitor in common assays of thrombosis in vitro (e.g. activated partial thromboplastin time) and in vivo (e.g. rat FeCl(2)-induced carotid artery thrombosis model).

Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases.

Human immunodeficiency virus (HIV) integrase is the enzyme responsible for insertion of a DNA copy of the viral genome into host DNA, an essential step in the replication cycle of HIV. HIV-1 integrase comprises three functional and structural domains: an N-terminal zinc-binding domain, a catalytic core domain and a C-terminal DNA-binding domain. The catalytic core domain with the F185H mutation has been crystallized without sodium cacodylate in a new crystal form, free and complexed with the catalytic metal Mg2+. The structures have been determined and refined to about 2.2 A. Unlike the previously reported structures, the three active-site carboxylate residues (D,D-35-E motif) are well ordered and both aspartate residues delineate a proper metal-binding site. Comparison of the active binding site of this domain with that of other members from the polynucleotidyl transferases superfamily shows a high level of similarity, providing a confident template for the design of antiviral agents.

Crystallization of 5-aminolaevulinic acid dehydratase from Escherichia coli and Saccharomyces cerevisiae and preliminary X-ray characterization of the crystals.

5-Aminolaevulinic acid dehydratase (ALAD) catalyzes the formation of porphobilinogen from two molecules of 5-aminolaevulinic acid. Both Escherichia coli and Saccharomyces cerevisiae ALADs are homo-octameric enzymes which depend on Zn2+ for catalytic activity and are potently inhibited by lead ions. The E. coli enzyme crystallized in space group I422 (unit cell dimensions a = b = 130.7 A, c = 142.4 A). The best crystals were obtained in the presence of the covalently bound inhibitor laevulinic acid. The yeast enzyme (expressed in E. coli) crystallized in the same space group (I422) but with a smaller unit cell volume (a = b = 103.7 A, c = 167.7 A). High resolution synchrotron data sets were obtained from both E. coli and yeast ALAD crystals by cryocooling to 100 K.

High-resolution structure (1.33 A) of a HEW lysozyme tetragonal crystal grown in the APCF apparatus. Data and structural comparison with a crystal grown under microgravity from SpaceHab-01 mission.

Crystals of tetragonal hen egg-white lysozyme were grown using Advanced Protein Crystallization Facility (APCF) apparatus under a microgravity environment (SpaceHab-01 mission) and ground control conditions. Crystals were grown from NaCl as a crystallizing agent at pH 4.3. The X-ray diffraction patterns of the best diffracting ground- and space-grown crystals were recorded using synchrotron radiation and an image plate on the W32 beamline at LURE. Both ground- and space-grown crystals showed nearly equivalent maximum resolution of 1.3-1.4 A. Refinements were carried out with the program X-PLOR with final R values of 18.45 and 18.27% for structures from ground- and space- grown crystals, respectively. The two structures are nearly identical with the root-mean-square difference on all protein atoms being 0.13 A. Some residues of the two refined structures show multiple alternative conformations. Two ions were localized into the electron-density maps of the two structures: one chloride ion at the interface between two symmetry-related molecules and one sodium ion stabilizing the loop Ser60-Leu75. The sodium ion is surrounded by six ligands which form a bipyramid around it at distances of 2.2-2.6 A.

The Grb2 adaptor.

Grb2 is an 'adaptor' protein made of one SH2 and two SH3 domains. The SH3 domains bind to prolinerich motifs in the C-terminal part of the ras exchange factor Sos. Binding of the Grb2 SH2 domain to phosphotyrosine motifs on receptors, or other adaptor proteins such as Shc, recruits this Grb2/Sos complex at the plasma membrane where Sos stimulates nucleotide exchange on ras, then ras activates raf and leads to MAP kinase activation. The structure of Grb2, the precise motifs recognised by its SH2 and SH3 domains, the way Grb2 performs its function, a possible regulation of its association with Sos, and its ability to complex with other proteins in vivo, are discussed.

Crystal structure of the mammalian Grb2 adaptor.

The mammalian growth factor receptor-binding protein Grb2 is an adaptor that mediates activation of guanine nucleotide exchange on Ras. Grb2 binds to the receptor through its SH2 domain and to the carboxyl-terminal domain of Son of sevenless through its two SH3 domains. It is thus a key element in the signal transduction pathway. The crystal structure of Grb2 was determined to 3.1 angstrom resolution. The asymmetric unit is composed of an embedded dimer. The interlaced junctions between the SH2 and SH3 domains bring the two adjacent faces of the SH3 domains in van der Waals contact but leave room for the binding of proline-rich peptides.