Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity

We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.     

Zebrafish β-adrenergic receptor mRNA expression and control of pigmentation

Beta adrenergic receptors (β-ARs) are members of the G-protein-coupled receptor superfamily and mediate various physiological processes in many species. The expression patterns and functions of β-ARs in zebrafish are, however, largely unknown. We have identified zebrafish β-AR orthologs, which we have designated as adrb1, adrb2a, adrb2b, adrb3a and adrb3b. adrb1 was found to be expressed in the heart and brain. Expression of adrb2a predominated in the brain and skin, whereas adrb2b was found to be highly expressed in muscle, pancreas and liver. Both adrb3a and adrb3b were exclusively expressed in blood. Knock-down of these β-ARs by morpholino oligonucleotides revealed a functional importance of adrb2a in pigmentation. Expression of atp5a1 and atp5b, genes that encode subunits of F1F0-ATPase, which is known to be involved in pigmentation, was significantly increased by knock-down of adrb2a. Our data suggest that adrb2a may regulate pigmentation, partly by modulating F1F0-ATPase.     

Modeling daily flowering probabilities: expected impact of climate change on Japanese cherry phenology

Understanding the drivers of phenological events is vital for forecasting species’ responses to climate change. We developed flexible Bayesian survival regression models to assess a 29‐year, individual‐level time series of flowering phenology from four taxa of Japanese cherry trees (Prunus spachiana, Prunus × yedoensis, Prunus jamasakura, and Prunus lannesiana), from the Tama Forest Cherry Preservation Garden in Hachioji, Japan. Our modeling framework used time‐varying (chill and heat units) and time‐invariant (slope, aspect, and elevation) factors. We found limited differences among taxa in sensitivity to chill, but earlier flowering taxa, such as P. spachiana, were more sensitive to heat than later flowering taxa, such as P. lannesiana. Using an ensemble of three downscaled regional climate models under the A1B emissions scenario, we projected shifts in flowering timing by 2100. Projections suggest that each taxa will flower about 30 days earlier on average by 2100 with 2–6 days greater uncertainty around the species mean flowering date. Dramatic shifts in the flowering times of cherry trees may have implications for economically important cultural festivals in Japan and East Asia. The survival models used here provide a mechanistic modeling approach and are broadly applicable to any time‐to‐event phenological data, such as plant leafing, bird arrival time, and insect emergence. The ability to explicitly quantify uncertainty, examine phenological responses on a fine time scale, and incorporate conditions leading up to an event may provide future insight into phenologically driven changes in carbon balance and ecological mismatches of plants and pollinators in natural populations and horticultural crops.     

The levels of main urinary metabolite of prostaglandin F1α and F2α in human subjects measured by radioimmunoassay

Radioimmunoassay technique for measuring 5α,7α-dihydroxy-11-keto-tetranorprosta-1,16-dioic acid, the main urinary metabolite of PGF₁α and PGF₂α (PGF₂α-MUM), was further improved.It was postulated based on some experimental data that the PGF₂α-MUM exists in the urine mostly as dioic acid form, not as δ-lactone formThe daily excretion of PGF₂α-MUM in men ranged from 14.43 μg to 36.14 μg and in women from 5.21 μg to 14.25 μg.     

Crucial Role of Extracellular Polysaccharides in Desiccation and Freezing Tolerance in the Terrestrial Cyanobacterium Nostoc commune

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and has a cosmopolitan distribution. In this study, the role of extracellular polysaccharides (EPS) in the desiccation tolerance of photosynthesis in N. commune was examined. Although photosynthetic O₂ evolution was not detected in desiccated colonies, the ability of the cells to evolve O₂ rapidly recovered after rehydration. The air-dried colonies contained approximately 10% (wt/wt) water, and field-isolated, natural colonies with EPS were highly water absorbent and were rapidly hydrated by atmospheric moisture. The cells embedded in EPS in Nostoc colonies were highly desiccation tolerant, and O₂ evolution was not damaged by air drying. Although N. commune was determined to be a mesophilic cyanobacterium, the cells with EPS were heat tolerant in a desiccated state. EPS could be removed from cells by homogenizing colonies with a blender and filtering with coarse filter paper. This treatment to remove EPS did not damage Nostoc cells or their ability to evolve O₂, but O₂ evolution was significantly damaged by desiccation treatment of the EPS-depleted cells. Similar to the EPS-depleted cells, the laboratory culture strain KU002 had only small amount of EPS and was highly sensitive to desiccation. In the EPS-depleted cells, O₂ evolution was also sensitive to freeze-thaw treatment. These results strongly suggest that EPS of N. commune is crucial for the stress tolerance of photosynthesis during desiccation and during freezing and thawing.     

A nationwide attempt to standardize growth hormone assays

The Growth Hormone (GH) and Its Related Factors Study Committee of the Foundation for Growth Science, Japan, has been conducting a quality control study for 15 years to improve the equality of diagnosis of GH deficiency. It found that the greatest differences in measured GH values were due to the different potencies of the kit standards, which were primarily adjusted to WHO standards for human GH of pituitary origin. With the collaboration of kit makers and the Study Group of Hypothalamo-Pituitary Disorders of the Ministry of Health, Labor and Welfare, all GH kits in Japan have begun using the same recombinant human GH standard since April 2005. As a result the diagnostic cut-off peak GH has changed from 10 to 6 ng/ml.     

The pretreatment of corn stover with Gloeophyllum trabeum KU-41 for enzymatic hydrolysis

Background

Pretreatment is an essential step in the enzymatic hydrolysis of biomass for bio-ethanol production. The dominant concern in this step is how to decrease the high cost of pretreatment while achieving a high sugar yield. Fungal pretreatment of biomass was previously reported to be effective, with the advantage of having a low energy requirement and requiring no application of additional chemicals. In this work, Gloeophyllum trabeum KU-41 was chosen for corn stover pretreatment through screening with 40 strains of wood-rot fungi. The objective of the current work is to find out which characteristics of corn stover pretreated with G. trabeum KU-41 determine the pretreatment method to be successful and worthwhile to apply. This will be done by determining the lignin content, structural carbohydrate, cellulose crystallinity, initial adsorption capacity of cellulase and specific surface area of pretreated corn stover.

Results

The content of xylan in pretreated corn stover was decreased by 43% in comparison to the untreated corn stover. The initial cellulase adsorption capacity and the specific surface area of corn stover pretreated with G. trabeum were increased by 7.0- and 2.5-fold, respectively. Also there was little increase in the cellulose crystallinity of pretreated corn stover.

Conclusion

G. trabeum has an efficient degradation system, and the results indicated that the conversion of cellulose to glucose increases as the accessibility of cellulose increases due to the partial removal of xylan and the structure breakage of the cell wall. This pretreatment method can be further explored as an alternative to the thermochemical pretreatment method.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) (1) were designed to examine the importance of each residue on mu-opioid receptor interaction. Replacement of Tyr(1) by 2',6'-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt(1)]EM-2 (9) elevated mu-opioid affinity 4.6-fold (K(i mu=0.15 nM) yet selectivity fell 330-fold as delta-affinity rose (K(i)delta=28.2 nM). This simultaneous increased mu- and delta-receptor bioactivities resulted in dual agonism (IC(50)=0.07 and 1.87 nM, respectively). While substitution of Phe(4) by a phenethyl group (4) decreased mu affinity (K(i)mu=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak delta antagonism (pA(2)=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.     

An arylbenzofuran and four isoflavonoids from the roots of Erythrina poeppigiana

An arylbenzofuran, erypoegin F and four isoflavonoids, erypoegins G-J, together with six known compounds were isolated from the roots of Erythrina poeppigiana, and their structures were elucidated on the basis of spectroscopic evidence. Erypoegin F is a rare 2-arylbenzofuran possessing a formyl group from a natural source, and erypoegin I is the first naturally occurring isoflavonoid with a 2-oxo-3-methylbutyl group.