Effects of 5-aminolevulinic acid supplementation on milk production, iron status, and immune response of dairy cows.

The objective of this study was to investigate the effect of 5-aminolevulinic acid (5-ALA) as a dietary supplement on milk yield and composition as well as iron status and immune response in lactating dairy cows. In this study 13 lactating Holstein cows were randomly assigned to either a control group or a treatment group supplemented with 10 mg of 5-ALA per kilogram of dry matter. During feeding, 5-ALA was mixed with a small amount of the total mixed ration and top-dressed. The experiments followed a crossover design with 2 periods. Each period consisted of an adaptation period of 12 d and a test period of 2 d. Dairy cows fed the diet supplemented with 5-ALA exhibited increased counts of white blood cells and granulocytes compared with the control group. The rate of phagocytosis and mitogen-induced proliferation of peripheral blood mononuclear cells in cows fed 5-ALA were higher than in cows fed a basal diet. However, 5-ALA did not affect iron status or plasma biochemical composition. Supplementation with 5-ALA improved milk protein and milk casein contents; however, it had no effect on milk production, milk fat, lactose, total solids, or solids-not-fat, compared with the control. We conclude that dietary supplementation of 5-ALA to lactating dairy cows may have a positive effect on milk protein synthesis and the immune response.

Pressure-dependent magnetization and magnetoresistivity studies on tetragonal FeS (mackinawite): revealing its intrinsic metallic character.

The transport and magnetic properties of the tetragonal Fe[Formula: see text]S were investigated using magnetoresistivity and magnetization within [Formula: see text] K, [Formula: see text] 70 kOe and [Formula: see text] 3.0 GPa. In addition, room-temperature x-ray diffraction and photoelectron spectroscopy were also applied. In contrast to previously reported nonmetallic character, Fe[Formula: see text]S is intrinsically metallic but due to a presence of a weak localization such metallic character is not exhibited below room temperature. An applied pressure reduces strongly this additional resistive contribution and as such enhances the temperature range of the metallic character which, for ∼3 GPa, is evident down to 75 K. The absence of superconductivity as well as the mechanism behind the weak localization will be discussed.

Magnetic refrigeration material operating at a full temperature range required for hydrogen liquefaction.

Magnetic refrigeration (MR) is a key technique for hydrogen liquefaction. Although the MR has ideally higher performance than the conventional gas compression technique around the hydrogen liquefaction temperature, the lack of MR materials with high magnetic entropy change in a wide temperature range required for the hydrogen liquefaction is a bottle-neck for practical applications of MR cooling systems. Here, we show a series of materials with a giant magnetocaloric effect (MCE) in magnetic entropy change (-∆Sm > 0.2 J cm-3K-1) in the Er(Ho)Co2-based compounds, suitable for operation in the full temperature range required for hydrogen liquefaction (20-77 K). We also demonstrate that the giant MCE becomes reversible, enabling sustainable use of the MR materials, by eliminating the magneto-structural phase transition that leads to deterioration of the MCE. This discovery can lead to the application of Er(Ho)Co2-based alloys for the hydrogen liquefaction using MR cooling technology for the future green fuel society.

On the ferromagnetic structure of the intermetallic borocarbide TbCo(2)B(2)C.

Based on magnetization, specific heat, magnetostriction and neutron-diffraction studies on single-crystal TbCo(2)B(2)C, it is found out that the paramagnetic properties, down to liquid nitrogen temperatures, are well described by a Curie-Weiss behavior of the Tb(3+) moments. Furthermore, below T(c) = 6.3 K, the Tb sublattice undergoes a ferromagnetic (FM) phase transition with the easy axis being along the (100) direction and, concomitantly, the unit cell undergoes a tetragonal-to-orthorhombic distortion. The manifestation of an FM state in TbCo(2)B(2)C is unique among all other isomorphous borocarbides, in particular TbNi(2)B(2)C (T(N) = 15 K, incommensurate modulated magnetic state) even though the Tb ions in both isomorphs have almost the same crystalline electric field properties. The difference among the magnetic modes of these Tb-based isomorphs is attributed to a difference in their exchange couplings which are in turn caused by a variation in their lattice parameters and in the position of their Fermi levels.

Low temperature specific heat of superconducting ternary intermetallics La(3)Pd(4)Ge(4), La(3)Ni(4)Si(4), and La(3)Ni(4)Ge(4) with U(3)Ni(4)Si(4)-type structure.

A systematic investigation on the thermodynamic properties of La-based ternary intermetallic superconductors crystallizing in a U(3)Ni(4)Si(4)-type structure is presented. The U(3)Ni(4)Si(4)-type structure consists of a characteristic intergrowth of periodic BaAl(4) (ThCr(2)Si(2))- and AlB(2)-type segments. Pristine low temperature specific heat data for recently discovered members La(3)Ni(4)Si(4) and La(3)Ni(4)Ge(4) with T(c)s of 1.0 and 0.7 K, respectively, are presented as well as La(3)Pd(4)Ge(4) with the highest T(c) of 2.5 K in the U(3)Ni(4)Si(4)-type group. Owing to the higher T(c)s of U(3)Ni(4)Si(4)-type superconductors than the related ThCr(2)Si(2)-type compounds, comparisons are drawn in our investigations of the ternary intermetallics of LaPd(2)Ge(2), LaNi(2)Si(2), and LaNi(2)Ge(2) having a ThCr(2)Si(2)-type structure. Our investigations of the thermodynamic properties show that La(3)Ni(4)Si(4) and La(3)Ni(4)Ge(4) have higher values of γ(n), N(E(F)), and Θ(D) than La(3)Pd(4)Ge(4). The same trend was found in ThCr(2)Si(2)-type compounds of LaPd(2)Ge(2), LaNi(2)Si(2), and LaNi(2)Ge(2). It turns out that the difference in T(c) between La(3)Pd(4)Ge(4), La(3)Ni(4)Si(4), and La(3)Ni(4)Ge(4), as well as the relatively higher T(c) of the U(3)Ni(4)Si(4)-type superconductors than of the related ThCr(2)Si(2)-type compounds, are largely due to the strength of electron-phonon coupling.

Quantum conductance-temperature phase diagram of granular superconductor K x Fe2-ySe2.

It is now well established that the microstructure of Fe-based chalcogenide K x Fe2-ySe2 consists of, at least, a minor (~15 percent), nano-sized, superconducting K s Fe2Se2 phase and a major (~85 percent) insulating antiferromagnetic K2Fe4Se5 matrix. Other intercalated A1-xFe2-ySe2 (A = Li, Na, Ba, Sr, Ca, Yb, Eu, ammonia, amide, pyridine, ethylenediamine etc.) manifest a similar microstructure. On subjecting each of these systems to a varying control parameter (e.g. heat treatment, concentration x,y, or pressure p), one obtains an exotic normal-state and superconducting phase diagram. With the objective of rationalizing the properties of such a diagram, we envisage a system consisting of nanosized superconducting granules which are embedded within an insulating continuum. Then, based on the standard granular superconductor model, an induced variation in size, distribution, separation and Fe-content of the superconducting granules can be expressed in terms of model parameters (e.g. tunneling conductance, g, Coulomb charging energy, E c , superconducting gap of single granule, Δ, and Josephson energy J = πΔg/2). We show, with illustration from experiments, that this granular scenario explains satisfactorily the evolution of normal-state and superconducting properties (best visualized on a [Formula: see text] phase diagram) of A x Fe2-ySe2 when any of x, y, p, or heat treatment is varied.

Anti-beta2-glycoprotein I (beta2GPI) monoclonal antibodies with lupus anticoagulant-like activity enhance the beta2GPI binding to phospholipids.

beta2-Glycoprotein I (beta2GPI), a plasma glycoprotein with phospholipid-binding property, is known to be the actual target antigen for autoimmune type anticardiolipin antibodies (aCLs). Certain groups of aCLs (anti-beta2GPI antibodies) exert lupus anticoagulant (LA) activity and perturb the function of vascular endothelial cells. This investigation aimed at highlighting some insights into the molecular basis by which aCLs exert their biological effects by using anti-beta2GPI mAbs with well-characterized epitopes from mice and from patients with antiphospholipid syndrome. Anti-beta2GPI mAbs directed against the third domain (Cof-20 and Cof-22) and fourth domain (Cof-21, EY1C8, and EY2C9) of beta2GPI inhibited the thrombin generation induced by Russell's viper venom in diluted plasma and that induced by the prothrombinase complex reconstituted with purified clotting factors. This anticoagulant activity was abrogated in the presence of an excess amount of phospholipids, thus resembling the LA activity. In stark contrast, anti-beta2GPI mAbs directed against the fifth domain and the carboxy-terminal region of the fourth domain showed no LA-like activity. These findings suggest that the LA activity of anti-beta2GPI antibodies depends on their epitope specificity. Experiments carried out to clarify the mechanism of the LA activity showed that anti-beta2GPI mAbs with LA-like activity, but not those without this effect, enhance the beta2GPI binding to phospholipids. In addition, the F(ab')2 fragment, but not the Fab' fragment, of the anti-beta2GPI mAbs was found to enhance the LA activity and the beta2GPI binding to phospholipids, suggesting that anti-beta2GPI antibodies induce formation of multiple complexes of beta2GPI on the surface of phospholipids because of their bivalent property. This clustering of beta2GPI molecules induced by anti-beta2GPI antibodies, probably because of their multivalent property and epitope specificity, might hinder the lateral mobility and activation of clotting factors on the surface of phospholipids and thus exert LA activity. Clustering of beta2GPI molecules may also explain the molecular mechanism by which anti-beta2GPI antibodies alter the function of leukocytes and endothelial cells. The well-documented heterogeneous LA activity of aCLs (anti-beta2GPI antibodies) may also be explained by their epitope specificity.

Reentrant behaviour in Y-doped Ho(0.75)Y(0.25)Ni(2)B(2)C single crystal.

The transport and superconducting properties of Ho(0.75)Y(0.25)Ni(2)B(2)C single crystals were investigated to study the competing effects between superconductivity and magnetism. The superconducting transition temperature T(c) is 7.6 K, determined from the resistivity transition; meanwhile, the commensurate antiferromagnetic (AFM) transition occurs at T(N) of 3.9 K, which is lower than that of pure HoNi(2)B(2)C (T(N)≈5 K). Ho(0.75)Y(0.25)Ni(2)B(2)C reentered into the normal state at T(m) (T(N)

Note: Novel diamond anvil cell for electrical measurements using boron-doped metallic diamond electrodes.

A novel diamond anvil cell suitable for electrical transport measurements under high pressure has been developed. A boron-doped metallic diamond film was deposited as an electrode on a nano-polycrystalline diamond anvil using a microwave plasma-assisted chemical vapor deposition technique combined with electron beam lithography. The maximum pressure that can be achieved by this assembly is above 30 GPa. We report electrical transport measurements of Pb up to 8 GPa. The boron-doped metallic diamond electrodes showed no signs of degradation after repeated compression.

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.

beta 2-Glycoprotein I modulates the anticoagulant activity of activated protein C on the phospholipid surface.

The objective of this study was to determine whether beta 2-glycoprotein I (beta 2GPI) has procoagulant activity by inhibiting the anticoagulant activity of activated protein C (APC). beta 2GPI inhibited significantly the APC-catalyzed inactivation of factor Va in an assay using factor V-deficient plasma and physiological levels of protein S and factor Va. This inhibitory effect was diminished by the addition of increasing concentrations of phospholipids, suggesting that beta 2GPI competitively inhibits the binding of APC to the phospholipid surface. beta 2GPI inhibited weakly factor Va- and phospholipid-dependent prothrombinase activity at concentrations similar to those to inhibit APC activity. The depletion of beta 2GPI from plasma led to only a slight shortening of the diluted Russell's viper venom-dependent clotting time, but to a strong and significant potentiation of the anticoagulant activity of APC. These results suggest that under certain physiological conditions beta 2GPI has procoagulant property by inhibiting the phospholipid-dependent APC anticoagulant activity.

Prothrombin and its derivatives stimulate motility of melanoma cells.

Several studies indicated that activation of the clotting system may promote the growth and the invasive behavior of tumor cells. In the present study, we evaluated the migratory response of various melanoma cell lines to several clotting factors and prothrombin derivatives (thrombin, fragment 1, fragment 2 and kringle 1 fragment). Prothrombin, thrombin and fragment 1 stimulated chemotaxis of the murine (K-1735 M2, X21) and human A375 (SM) melanoma cell lines. Prothrombin and prothrombin fragment 1 showed their maximal chemotactic activity at 0.5 approximately 1 microM. Chemotaxis induced by thrombin was inhibited by hirudin, but not that induced by prothrombin or fragment 1. Other clotting proteins and the fragment 2 and kringle 1 fragment of prothrombin did not elicit chemotactic activity. Checkerboard analysis indicated that motility was directional with a significant chemokinetic component. The K-1735 M2 cells also migrated in a concentration-dependent manner to substratum-bound insoluble prothrombin, thrombin or fragment 1. Ligand binding assays showed that both prothrombin and fragment 1 bound to K-1735 M2 cells with apparent Kds of 0.5 microM. This binding was inhibited by an excess concentration of unlabeled prothrombin and fragment 1 but not by similar concentrations of other prothrombin fragments. These findings suggest that prothrombin and its fragment 1 exert chemotactic activity on melanoma cells by different mechanisms and different binding sites from that induced by thrombin.

Purification, characterization and cDNA cloning of a novel anticoagulant of the intrinsic pathway, (prolixin-S) from salivary glands of the blood sucking bug, Rhodnius prolixus.

The salivary glands of the blood sucking insect, Rhodnius prolixus, have an anticoagulant, prolixin-S, which was reported as a specific inhibitor of intrinsic coagulant pathway. Prolixin-S was purified from the salivary glands extract of Rhodnius prolixus by gel filtration and anion exchange HPLC by assaying prolongation of activated partial thromboplastin time (APTT). The isolated protein specifically inhibited factor IXa-catalyzed activation of factor X in the presence of Ca2+ and phospholipids irrespective of the presence or absence of factor VIIIa. The anticoagulant factor had red color and a specific absorbance peak at 402 nm and thus it was identified as a heme protein. A Rhodnius prolixus salivary gland cDNA library was prepared, screened with an antibody against prolixin-S and its complete cDNA sequence was determined. cDNA and deduced amino acid sequences showed that prolixin-S is a novel anticoagulant of 19,922 Da, which has no sequence homology with any other anticoagulant reported so far.

The zymogen prothrombin stimulates cell locomotion and calcium influx in murine osteosarcoma cells by different mechanism from thrombin.

The coagulation system plays a role in tumor invasion and metastasis. Prothrombin was previously found to stimulate the motility of melanoma cells. We compared the effect of prothrombin and thrombin on cell motility, calcium influx and actin stress filament contraction in highly metastatic osteosarcoma LM8 cells. Prothrombin and thrombin stimulated chemotaxis and chemokinesis of LM8 cells in a dose-dependent manner. Calcium influx was significantly increased after stimulation with thrombin and prothrombin. Thrombin and prothrombin markedly stimulated actin contraction in LM8 cells. Hirudin inhibited the effects of thrombin but not those of prothrombin. Prothrombin appears to stimulate cell locomotion, actin contraction, calcium influx in LM8 cells by different mechanism from thrombin.

Imaging of a vortex lattice transition in YNi2B2C by scanning tunneling spectroscopy

The vortex lattices in YNi2B2C under the magnetic fields H up to 3 T applied along both the a and the c axes have been studied by scanning tunneling spectroscopy at 4.2 K. The vortex lattice transition has been found to occur in different manners for H parallela and H parallelc; in H parallela a slightly distorted hexagonal vortex lattice has been found to transform to a nearly square one above 1.0 T with increasing H, while in H parallelc the transition occurs at a much lower field around 0.1 T. The unconventional steep increase of the quasiparticle density of states outside the vortex core has also been found well below H(c2).

Blood coagulation factor Xa interacts with a linear sequence of the kringle 2 domain of prothrombin.

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.

Primary structure of a hemorrhagic metalloproteinase, HT-2, isolated from the venom of Crotalus ruber ruber.

Crotalidae and Viperidae snake venoms contains several kinds of metalloproteinases which cause localized hemorrhage by direct action on blood vessel walls. We report here the entire amino acid sequence and the disulfide bridge locations of HT-2, one of the hemorrhagic toxins isolated from the venom of Crotalus ruber ruber (red rattlesnake). The non-reduced protein was first cleaved at methionine residues to provide a set of 8 fragments, which covered the entire sequence of HT-2. The disulfide bridge locations of HT-2 were also determined by using these primary fragments. The unambiguous sequence for the whole protein was then established by conventional methods using lysyl endopeptidase and thermolysin digests. HT-2 consisted of 202 amino acid residues with two disulfide bridges, which were assigned to Cys-117-Cys-197 and Cys-157-Cys-164. HT-2 had a typical zinc-chelating sequence His-Glu-X-X-His (residues 142-146) found in thermolysin, and its overall sequence showed, respectively, 50, 52, and 53% identities to those of HR2a, H2-proteinase, and the metalloproteinase domain of HR1B. However, the disulfide bridge locations of HT-2 were different from those in the other metalloproteinases. The primary structure of HT-2 was more closely related to that of Ht-d from Crotalus atrox recently determined (81% identity). From the structural comparison with five metalloproteinases so far elucidated, six conservative amino acid residues, which may possibly be related to the induction of the hemorrhagic activity, were suggested to be present in these toxins.

Primary structure of H2-proteinase, a non-hemorrhagic metalloproteinase, isolated from the venom of the habu snake, Trimeresurus flavoviridis.

The complete amino acid sequence of and the locations of disulfide bridges in H2-proteinase, a major non-hemorrhagic proteinase isolated from the venom of the habu Trimeresurus flavoviridis, have been determined and compared with those of HR2a, one of the hemorrhagic metalloproteinases in this venom. The strategy involved consisted of structural analysis of peptides in digests with cyanogen bromide, lysyl endopeptidase, trypsin, Staphylococcus aureus V8 protease and thermolysin. Peptides were purified by gel filtration followed by reversed-phase HPLC. H2-proteinase is a non-glycosylated single chain polypeptide consisting of 201 amino acids with an amino-terminal pyroglutamic acid, a calculated molecular weight of 22,991 and a net charge of +14 at neutral pH. There was no evidence of heterogeneity of the sequence. H2-proteinase has a typical zinc-chelating sequence and its overall sequence identity with HR2a is 73.6%. The 3 disulfide bridges in H2-proteinase link Cys-117 to Cys-196, Cys-158 to Cys-180, and Cys-160 to Cys-163, in the same manner as in the case of HR2a. In striking contrast to HR2a, it contains en extra free cysteine residue at position 94 which becomes reactive to a sulfhydryl reagent in the presence of a denaturant.

The role of human factor X activation peptide in activation of factor X by factor IXa.

We studied the interaction of factor X activation peptide (XAP) with factor IXa and factor Xa and the effect of XAP on factor IXa-catalyzed activation of factor X. XAP associated with factor Xa in the presence of 5 mM Ca2+ was dissociated from factor Xa by gel chromatography using Ultrogel AcA54 in 5 mM EDTA, or in 8 M urea-0.1% SDS. An exogenous isolated XAP inhibited the factor IXa-catalyzed factor X activation both in the presence and absence of factor VIIIa. 4-Amidinophenylmethylsulfonyl (aPMS)-factor Xa independent of XAP also inhibited the factor X activation more effectively than XAP alone in the presence of factor VIIIa. However, aPMS-factor Xa independent of XAP hardly inhibited the factor X activation in the absence of factor VIIIa. The binding of 125I-labeled factor X to the aPMS-factor IXa fixed to a microwell plate was inhibited by unlabeled factor X or XAP, but not by aPMS-factor Xa with or without XAP. Factor IXa directly bound to XAP and aPMS-factor Xa with XAP, but did not bind to aPMS-factor Xa without XAP. These findings suggest that the region of XAP in factor X directly interacts with factor IXa, and factor Xa region other than XAP interacts with factor VIIIa. Desialation or deletion of N-linked carbohydrates of XAP reduced the inhibitory activity of XAP for the factor X activation by factor IXa to approximately 50% of that of the intact XAP. This suggests that the sialic acids in the carbohydrate chains of the XAP region partly contribute to the interaction with factor IXa during its activation.

Primary structure of hemorrhagic protein, HR2a, isolated from the venom of Trimeresurus flavoviridis.

The complete amino acid sequence and disulfide bridge location of HR2a, one of the hemorrhagic proteins isolated from the snake venom of Trimeresurus flavoviridis, have been determined by analysis of peptides derived from digests with cyanogen bromide, lysyl endopeptidase, trypsin, and Staphylococcus aureus V8 protease. Peptides were purified by gel filtration followed by reversed-phase HPLC. HR2a has the amino-terminal sequence of less than Glu-Gln-Arg- and consists of a total of 202 residues with a calculated molecular weight of 23,015. Sequence analysis indicates the presence of another isoform which lacks the amino-terminal residue, making 201 amino acid residues with a molecular weight of 22,887. Three disulfide bridges of HR2a link Cys-118 to Cys-197, Cys-159 to Cys-181, and Cys-161 to Cys-164. HR2a contains a segment which is similar to the zinc-chelating sequences found in thermolysin and several mammalian metalloproteinases, suggesting that HR2a is a metalloproteinase with limited substrate specificity. However, there is no other significant sequence homology with thermolysin except for the zinc-ligand region.