Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.     

A multiscale computational fluid dynamics approach to simulate the micro-fluidic environment within a tissue engineering scaffold with highly irregular pore geometry

Mechanical stimulation can regulate cellular behavior, e.g., differentiation, proliferation, matrix production and mineralization. To apply fluid-induced wall shear stress (WSS) on cells, perfusion bioreactors have been commonly used in tissue engineering experiments. The WSS on cells depends on the nature of the micro-fluidic environment within scaffolds under medium perfusion. Simulating the fluidic environment within scaffolds will be important for gaining a better insight into the actual mechanical stimulation on cells in a tissue engineering experiment. However, biomaterial scaffolds used in tissue engineering experiments typically have highly irregular pore geometries. This complexity in scaffold geometry implies high computational costs for simulating the precise fluidic environment within the scaffolds. In this study, we propose a low-computational cost and feasible technique for quantifying the micro-fluidic environment within the scaffolds, which have highly irregular pore geometries. This technique is based on a multiscale computational fluid dynamics approach. It is demonstrated that this approach can capture the WSS distribution in most regions within the scaffold. Importantly, the central process unit time needed to run the model is considerably low.

     

Temporal changes of genetic population structure and diversity in the endangered Blakiston's fish owl (Bubo blakistoni) on Hokkaido Island, Japan, revealed by microsatellite analysis

The Blakiston's fish owl (Bubo blakistoni) population on Hokkaido Island, Japan, decreased to less than one hundred individuals over the last century due to habitat disruption by human activity. Although the ongoing conservation management has slightly restored the population, it remains endangered. In order to assess the genetic variation and population structure of the Blakiston's fish owl in Hokkaido, we genotyped eight microsatellite loci on 120 individuals sampled over the past three decades. The genotype data set showed low levels of genetic variation and gene flow among the geographically isolated five subpopulations. Comparative analysis of past and current populations indicated that some alleles shared by past individuals had been lost, and that genetic variation had declined over the last three decades. The result suggests that the genetic decline may have resulted from inbreeding and/or genetic drift due to bottlenecks in the Hokkaido population. The present study provides invaluable genetic information for the conservation and management of the endangered Blakiston's fish owl in Hokkaido.     

Wider Retinal Artery Trajectories in Eyes with Macular Hole Than in Fellow Eyes of Patients with Unilateral Idiopathic Macular Hole

Purpose

To determine whether the width of the retinal artery (RA) trajectory was associated with the presence of a macular hole (MH).

Methods

A retrospective cross sectional case-control study was performed. The fundus photographs were rotated 90 degrees, and the coordinates of the best fit curve of the RA trajectory were determined automatically based on these plots using the ImageJ program. The converted coordinates were fit to a second degree polynomial (ax²/100 + bx + c) equation. The width and steepness of the RA trajectory, “a”, of the eyes with a MH eye were compared to that of the fellow eyes.

Results

One hundred and ten eyes of 55 consecutive patients (30 women) with a unilateral MH and healthy fellow eyes were analyzed. The mean age was 64.9 years (range 47-81 years). The constant ‘a’ was significantly smaller in eyes with a MH than that of the fellow eyes (0.379 ± 0.094 vs 0.416 ± 0.121, P = 0.001, paired t test), indicating that the RA trajectory was wider in the MH eyes than in the fellow eyes. There was a significant correlation between the axial length and ‘a’ of the RA trajectory in the MH eyes (R = 0.273, P = 0.044) and in the fellow eyes (R = 0.356, P = 0.008; Spearman’s rank correlation coefficient).

Conclusions

Because eyes with a MH have a significantly wider and flatter RA trajectory, there may be greater traction on the fovea which is located between the RA arches. The causative role of this finding is still unclear.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Genetic Diversity Between Japanese and Chinese Threeline Grunt (<Emphasis Type="Italic">Parapristipoma trilineatum</Emphasis>) Examined by Microsatellite DNA Markers

Threeline grunt (Parapristipoma trilineatum) distributes around the southwestern coast of Japan and the east coast of China. The Chinese P. trilineatum was imported by Japan as an aquacultural seed because of its rapid growth compared with that of the Japanese P. trilineatum. The Japanese P. trilineatum differs from the Chinese P. trilineatum in some quantitative traits, and it has been suggested that these two P. trilineatum populations are genetically different. In order to identify the population structures around Japan and China, 5 local populations of the Japanese P. trilineatum and 2 local populations of the Chinese P. trilineatum were analyzed using 4 microsatellite DNA markers. Significant differences were detected between Japanese and Chinese P. trilineatum and among samples of Chinese P. trilineatum; however, among the samples of Japanese P. trilineatum, no significant differences were detected. These results suggest that care must be taken to prevent the escape of the Chinese P. trilineatum from culture cages around the Japanese coast, in order to preserve the genetically different population structures of Japanese and Chinese P. trilineatum.     

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.     

winged eye Induces Transdetermination of Drosophila Imaginal Disc by Acting in Concert with a Histone Methyltransferase,Su(var)3-9

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A sclerostin-based theory for strain-induced bone formation

Bone formation responds to mechanical loading, which is believed to be mediated by osteocytes. Previous theories assumed that loading stimulates osteocytes to secrete signals that stimulate bone formation. In computer simulations this 'stimulatory' theory successfully produced load-aligned trabecular structures. In recent years, however, it was discovered that osteocytes inhibit bone formation via the protein sclerostin. To reconcile this with strain-induced bone formation, one must assume that sclerostin secretion decreases with mechanical loading. This leads to a new 'inhibitory' theory in which loading inhibits osteocytes from inhibiting bone formation. Here we used computer simulations to show that a sclerostin-based model is able to produce a load-aligned trabecular architecture. An important difference appeared when we compared the response of the stimulatory and inhibitory models to loss of osteocytes, and found that the inhibitory pathway prevents the loss of trabeculae that is seen with the stimulatory model. Further, we demonstrated with combined stimulatory/inhibitory models that the two pathways can work side-by-side to achieve a load-adapted bone architecture.