Influence of high temperature and humidity on rumen bacterial diversity in Holstein heifers

The effect of heat and humidity stresses on the rumen bacterial molecular diversity of heifers was studied. No statistically significant changes in the rumen microbiota composition were found in the first experiment (average body mass 250kg) while in the second and third experiments (additional variables included the relative humidity and body weight), the microbiota composition was significantly different at elevated environmental temperatures and humidity. These shifts were accompanied by the decrease in concentration of short-chain fatty acids in the rumen.     

Hopping and climbing gait of Japanese Pygmy Woodpeckers (Picoides kizuki)

Single cycles of hopping and climbing were investigated in Japanese Pygmy Woodpeckers Picoides kizuki using motion analyses on video. Body movements on substrate angled from 0–90° were compared for every 10°. The body was inclined forward during stance phase for both small and large substrate angles, and the inclination amplitude increased when the substrate angle increased. The tail was bent ventrally almost simultaneously to this body inclination, and its amplitude was apparently high at large substrate angles. Most of the gait parameters changed when the stride length increased. The minimum body–tail angle and most of the parameters representing body movements during stance phase changed when the substrate angle increased, probably because gravity pulled the birds further backward when they were moving on a steeper slope. These parameters showed a clear difference between the data on substrate steeper than 40° and lower than 30°. The abrupt changes in these parameters most likely mean that the motor pattern changed from hopping to climbing between these angles.     

Binding of Bacillus thuringiensis Cry1A toxins to brush border membrane vesicles of midgut from Cry1Ac susceptible and resistant Plutella xylostella

Plutella xylostella strain resistant (PXR) to Bacillus thuringiensis Cry1Ac toxin was not killed at even more than 1000 μg Cry1Ac/g diet but killed by Cry1Ab at 0.5 μg/g diet. In contrast, susceptible strain (PXS) was killed by Cry1Ac at 1 μg/g diet. Cy3-labeld Cry1A(s) binding to brush border membrane vesicles (BBMV) prepared from both strains were analyzed with direct binding assay. The Kd value of Cry1Aa to both BBMV was almost identical: 213.2 and 205.8 nM, and 263.5 and 265.0 nM for Cry1Ac. The highest Kd values were in Cry1Ab which showed most effective insecticidal activity in PXS and PXR, 2126 and 2463 nM, respectively. These results clearly showed that the BBMV from PXR and PXS could equally bind to Cry1Ac. The binding between BBMV and Cy3-labeled Cry1Ac was inhibited only by anti-175 kDa cadherin-like protein (CadLP) and -252 kDa protein antisera, but not by anti-120 kDa aminopeptidase. This supports that resistance in PXR resulted from the abortion of pore formation after the binding of Cry1Ac to the BBMV. And furthermore, the importance of 175K CadLP and P252 proteins in those bindings was suggested. We briefly discuss possible mechanisms of the resistance.     

Determination of the role of the Carboxyl-terminal leucine-122 in FMN-binding protein by mutational and structural analysis

Mutants of flavin mononucleotide-binding protein (FMN-bp) were made by site-directed mutagenesis to investigate the role of carboxyl-terminal Leu122 of the pairing subunit in controlling redox potentials, binding the prosthetic group, and forming the tertiary and quaternary structure. We compared the oxidation-reduction potentials, FMN-binding properties, and higher structures of wild-type FMN-bp and four mutant proteins (L122Y, L122E, L122K and L122-deleted). We found that the redox potentials were affected by mutations. Also, the affinities of L122E, L122K and L122 deletion mutant apoproteins for FMN were lower than for the wild-type apoprotein, whereas the affinity of L122Y for FMN was increased. Analytical ultracentrifugation showed that the dissociation constants for dimerization of L122E and L122K were larger than for wild-type FMN-bp, whereas the dissociation constants for L122Y and the deletion mutant were lower than for the wild type. Finally, we determined the higher structures of L122Y, L122E and L122K mutants by X-ray crystallography. Our results show that the mutation of Leu122 in FMN-bp changes midpoint potentials, dissociation constants for FMN, and dimer formation, indicating that this residue is important in the pairing subunit.     

Characterization of two brassinosteroid C-6 oxidase genes in pea

C-6 oxidation genes play a key role in the regulation of biologically active brassinosteroid (BR) levels in the plant. They control BR activation, which involves the C-6 oxidation of 6-deoxocastasterone (6-DeoxoCS) to castasterone (CS) and in some cases the further conversion of CS to brassinolide (BL). C-6 oxidation is controlled by the CYP85A family of cytochrome P450s, and to date, two CYP85As have been isolated in tomato (Solanum lycopersicum), two in Arabidopsis (Arabidopsis thaliana), one in rice (Oryza sativa), and one in grape (Vitis vinifera). We have now isolated two CYP85As (CYP85A1 and CYP85A6) from pea (Pisum sativum). However, unlike Arabidopsis and tomato, which both contain one BR C-6 oxidase that converts 6-DeoxoCS to CS and one BR C-6 Baeyer-Villiger oxidase that converts 6-DeoxoCS right through to BL, the two BR C-6 oxidases in pea both act principally to convert 6-DeoxoCS to CS. The isolation of these two BR C-6 oxidation genes in pea highlights the species-specific differences associated with C-6 oxidation. In addition, we have isolated a novel BR-deficient mutant, lke, which blocks the function of one of these two BR C-6 oxidases (CYP85A6). The lke mutant exhibits a phenotype intermediate between wild-type plants and previously characterized pea BR mutants (lk, lka, and lkb) and contains reduced levels of CS and increased levels of 6-DeoxoCS. To date, lke is the only mutant identified in pea that blocks the latter steps of BR biosynthesis and it will therefore provide an excellent tool to further examine the regulation of BR biosynthesis and the relative biological activities of CS and BL in pea.     

A comparison of the high-affinity peripheral benzodiazepine receptor ligands DAA1106 and (R)-PK11195 in rat models of neuroinflammation: implications for PET imaging of microglial activation

Activated microglia are an important feature of many neurological diseases and can be imaged in vivo using 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195), a ligand that binds the peripheral benzodiazepine receptor (PBR). N-(2,5-dimethoxybenzyl)-N-(5-fluoro-2-phenoxyphenyl) acetamide (DAA1106) is a new PBR-specific ligand that has been reported to bind to PBR with higher affinity compared with PK11195. We hypothesized that this high-affinity binding of DAA1106 to PBR will enable better delineation of microglia in vivo using positron emission tomography. [(3)H]DAA1106 showed higher binding affinity compared with [(3)H](R)-PK11195 in brain tissue derived from normal rats and the rats injected intrastriatally with 6-hydroxydopamine or lipopolysaccharide at the site of the lesion. Immunohistochemistry combined with autoradiography in brain tissues as well as correlation analyses showed that increased [(3)H]DAA1106 binding corresponded mainly to activated microglia. Finally, ex vivo autoradiography and positron emission tomography imaging in vivo showed greater retention of [(11)C]DAA1106 compared with [(11)C](R)-PK11195 in animals injected with either lipopolysaccaride or 6-hydroxydopamine at the site of lesion. These results indicate that DAA1106 binds with higher affinity to microglia in rat models of neuroinflammation when compared with PK11195, suggesting that [(11)C]DAA1106 may represent a significant improvement over [(11)C](R)-PK11195 for in vivo imaging of activated microglia in human neuroinflammatory disorders.     

Novel Ca2+‐independent carbohydrate recognition of the C‐type lectins,SPL‐1 and SPL‐2, from the bivalve Saxidomus purpuratus

Novel Ca²⁺‐independent C‐type lectins, SPL‐1 and SPL‐2, were purified from the bivalve Saxidomus purpuratus. They are composed of dimers with either identical (SPL‐2 composed of two B‐chains) or distinct (SPL‐1 composed of A‐ and B‐chains) polypeptide chains, and show affinity for N‐acetylglucosamine (GlcNAc)‐ and N‐acetylgalactosamine (GalNAc)‐containing carbohydrates, but not for glucose or galactose. A database search for sequence similarity suggested that they belong to the C‐type lectin family. X‐ray crystallographic analysis revealed definite structural similarities between their subunits and the carbohydrate‐recognition domain (CRD) of the C‐type lectin family. Nevertheless, these lectins (especially SPL‐2) showed Ca²⁺‐independent binding affinity for GlcNAc and GalNAc. The crystal structure of SPL‐2/GalNAc complex revealed that bound GalNAc was mainly recognized via its acetamido group through stacking interactions with Tyr and His residues and hydrogen bonds with Asp and Asn residues, while widely known carbohydrate‐recognition motifs among the C‐type CRD (the QPD [Gln‐Pro‐Asp] and EPN [Glu‐Pro‐Asn] sequences) are not involved in the binding of the carbohydrate. Carbohydrate‐binding specificities of individual A‐ and B‐chains were examined by glycan array analysis using recombinant lectins produced from Escherichia coli cells, where both subunits preferably bound oligosaccharides having terminal GlcNAc or GalNAc with α‐glycosidic linkages with slightly different specificities.     

A Stable Chimeric Fibroblast Growth Factor (FGF) Can Successfully Replace Basic FGF in Human Pluripotent Stem Cell Culture

Fibroblast growth factors (FGFs) are essential for maintaining self-renewal in human embryonic stem cells and induced pluripotent stem cells. Recombinant basic FGF (bFGF or FGF2) is conventionally used to culture pluripotent stem cells; however, because of the instability of bFGF, repeated addition of fresh bFGF into the culture medium is required in order to maintain its concentration. In this study, we demonstrate that a heat-stable chimeric variant of FGF, termed FGFC, can be successfully used for maintaining human pluripotent stem cells. FGFC is a chimeric protein composed of human FGF1 and FGF2 domains that exhibits higher thermal stability and protease resistance than do both FGF1 and FGF2. Both human embryonic stem cells and induced pluripotent stem cells were maintained in ordinary culture medium containing FGFC instead of FGF2. Comparison of cells grown in FGFC with those grown in conventional FGF2 media showed no significant differences in terms of the expression of pluripotency markers, global gene expression, karyotype, or differentiation potential in the three germ lineages. We therefore propose that FGFC may be an effective alternative to FGF2, for maintenance of human pluripotent stem cells.