Isolation and purification of tyrosine hydroxylase from callus cultures of Portulaca grandiflora

Tyrosine hydroxylase was separated from polyphenol oxidase activity and was highly purified from betacyanin producing callus cultures of Portulaca grandiflora. The purified enzyme catalyzed the formation of DOPA (L-3,4-dihydroxyphenylalanine) from tyrosine and required the pterin compounds (6-methyl-5,6,7,8-tetrahydropterin; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterin) as coenzyme. The K(m) values for tyrosine and 6-methyl-5,6,7,8-tetrahydropterin were 0.5 mM and 0.15 mM, respectively. This enzyme was activated by Fe(2+) and Mn(2+), and inhibited by metal chelating agents.     

The light-harvesting complex of photosystem I in Chlamydomonas reinhardtii: protein composition, gene structures and phylogenic implications

Light-harvesting chlorophyll a/b-binding proteins (LHCI) associated with photosystem I (PSI) and the genes encoding these proteins have been characterized in the unicellular green alga Chlamydomonas reinhardtii, extending previous studies of the PSII-LHCII [Teramoto et al. (2001) Plant Cell Physiol. 42: 849]. In order to assign LHCI proteins in the thylakoid membranes, the PSI-LHCI supercomplex that retains all of the major LHCI proteins was purified. Seven distinct LHCI proteins were resolved from the purified supercomplex by a high-resolution SDS polyacrylamide gel electrophoresis, and their N-terminal amino acid sequences were determined. One LHCI protein (band e) was newly found, although the other six LHCI proteins corresponded to those previously reported. Genomic clones encoding these seven LHCI proteins were newly isolated and the nucleotide sequences were determined. A comprehensive characterization of all members of Lhc gene family in this alga revealed that LHCI proteins are more highly diverged than LHCII, suggesting functional differentiation of the protein components in LHCI. Neighbor joining trees were constructed for LHC proteins from C. reinhardtii and those of Arabidopsis thaliana or Galdieria sulphuraria to assess evolutionary relationships. Phylogenetic analysis revealed that (1). green algal LHCI and LHCII proteins are more closely related to one another than to LHCI proteins in red algae, (2). green algae and higher plants possess seven common lineages of LHC proteins, and (3). Type I and III LHCI proteins are conserved between green algae and higher plants, while Type II and IV are not. These findings are discussed in the context of evolution of multiple diverse antenna complexes.     

Observation of delivery behavior in the rhesus monkey

Seven deliveries of rhesus monkeys maintained in the laboratory were recorded by a video recorder system to determine the signs of impending labor. The characteristic postures; i.e., standing, squatting and crouching, were demonstrated in all cases with cyclic regularity (about 2–4 min) 35–210 min prior to the actual parturition, although prediction of the delivery date several days beforehand failed. The four stages of the delivery process are also presented in this report.     

Elevated Cell Invasion Is Induced by Hypoxia in a Human Pituitary Adenoma Cell Line

Pituitary adenoma tissues are hypovascular, and have a lower partial oxygen pressure compared with neighboring normal organs. In this study, we investigated whether hypoxia influences the cell invasiveness of the human pituitary adenoma cell line, HP-75. HP-75 cells were exposed to hypoxic (1–10% oxygen) or normoxic (21% oxygen) conditions for 24 hours. Gelatin and reverse zymogram assays were used to determine the enzyme activities of matrix metalloproteinases (MMP) and tissue inhibitor of metalloproteinases (TIMP). Cell adhesion and Matrigel cell invasion were examined with a Boiden chamber. Finally, the mRNA gene expression profiles of cells exposed to hypoxia or normoxia were examined by cDNA microarray and confirmed with real-time RT-PCR and flow cytometry. The gelatin and reverse zymograms revealed that the activities of MMP and TIMP were not significantly altered by hypoxia. Matrigel cell invasion and cell adhesion to Matrigel or collagen type IV were increased by hypoxia (3.8- and 4.8-fold, respectively). The cDNA microarray analysis revealed that laminin β2 chain mRNA was specifically up-regulated under hypoxic conditions (4.96-fold). Finally, real-time RT-PCR and flow cytometry verified the elevated expression of laminin β2 chain at the mRNA and protein levels under hypoxic conditions. RNA interference with siRNA targeting laminin β2 inhibited Matrigel invasion and adhesion to collagen type IV in a dose.dependent manner.Collectively, these results suggested that hypoxia (1% oxygen) enhanced the cell invasion properties of a pituitary adenoma cell line in association with elevated expression of laminin β2 and enhanced binding to collagen type IV.Key Words: cell invasion, hypoxia, laminin β2, pituitary adenoma, siRNA     

Absence of contagious yawning in children with autism spectrum disorder

This study is the first to report the disturbance of contagious yawning in individuals with autism spectrum disorder (ASD). Twenty-four children with ASD as well as 25 age-matched typically developing (TD) children observed video clips of either yawning or control mouth movements. Yawning video clips elicited more yawns in TD children than in children with ASD, but the frequency of yawns did not differ between groups when they observed control video clips. Moreover, TD children yawned more during or after the yawn video clips than the control video clips, but the type of video clips did not affect the amount of yawning in children with ASD. Current results suggest that contagious yawning is impaired in ASD, which may relate to their impairment in empathy. It supports the claim that contagious yawning is based on the capacity for empathy.     

SH-containing cellulose acetate derivatives: preparation and characterization as a shape memory-recovery material

Cellulose derivatives having a cross-linkable mercapto group were prepared by esterification of cellulose acetate (CA) with mercaptoacetic acid. The molecular structure of a series of products (CA-MA) was characterized by (1)H and (1)H- (13)C HMQC NMR spectroscopy and gel permeation chromatography. The solubility of CA-MA in water and organic solvents could be controlled by changing the preparation conditions including the degree of acetyl substitution of the starting CA. The CA-MA samples thus synthesized showed a sol-gel transition in solution and a shape memory-recovery behavior in film form through adequate redox treatments due to the reversible, cross-linking association and dissociation between mercapto groups. Dimethyl sulfoxide was usable as the organic solvent and oxidant, while the major reducing reagent was 2-mercaptoethanol or ammonium mercaptoacetic acid. The progress of the redox reactions was followed by using a confocal depth scanning technique in Raman spectroscopy. It was found that the compatibility between the cellulose derivatives and the redox reagents used was an important factor for the successful reactions, especially in the samples of film form. The cross-linking effect on the thermal and viscoelastic properties of the CA-MA films was also estimated by differential scanning calorimetry and dynamic mechanical analysis. Discussion focused on the alternately declining and recovering behavior of a principal loss tan delta peak, observed following the redox treatments repeated for the CA-MA film specimens.     

Pathway engineering by designed divergent evolution

Designed divergent evolution is a proposed protein engineering methodology to redesign enzyme function. The methodology was developed on the basis of the theories of divergent molecular evolution: (i) enzymes with more active and specialized functions have evolved from ones with promiscuous functions; (ii) this process is driven by small numbers of amino acid substitutions (plasticity); and (iii) the effects of double or multiple mutations are often additive (quasi-additive assumption). Thus, in many cases the impact of multiple mutations can be calculated by first determining the effects of a mutation at a single position and subsequently summing these effects using the quasi-additive assumption. In this way, the shape of the fitness landscape of a particular enzyme function can be estimated. The combinations of mutations predicted to yield global optima for desired functions can then be selected and introduced into the enzymes. The methodology has been demonstrated to be very powerful to redesign enzyme function. The use of multiple redesigned enzymes in novel or reconstructed metabolic pathways will enable the production of natural and unnatural products that will find use as pharmaceuticals, agrochemicals and many other applications.