Phosphate metabolism during diel vertical migration in the raphidophycean alga, Chattonella antiqua

The ecological advantage of diel vertical migration on the nutrition and accumulation of Chattonella antiqua, which is one of the dominant red-tide forming phytoplankton species in the Seto Inland Sea of Japan, was examined using a large axenic culture tank, in which vertical stratification of salinity, temperature and nutrients was maintained, analogous to natural conditions observed when red tides occur. C. antiqua was capable of migrating through very sharp salinity and temperature gradients. At night the species migrated to the deep nutrient-rich water and assimilated nutrients. During the daytime it migrated to the nutrient-depleted surface water and used the accumulated nutrients for photosynthesis. Nitrogen uptake was synchronized with phosphate uptake. ³¹P-NMR spectroscopy during the migration experiment revealed that C. antiqua has the capability of nocturnal phosphate uptake in the deep nutrient-rich water, but no capability of synthesizing polyphosphate, which was considered to be the intracellular phosphate pool. These findings were compared with those reported for another raphidophycean, Heterosigma akashiwo. Although both species carry out vertical migration and nocturnal nutrient uptake, only H. akashiwo has the capability of making an intracellular polyphosphate pool. This revised version was published online in July 2006 with corrections to the Cover Date.     

Enantioselectivity of bunitrolol 4‐hydroxylation is reversed by the change of an amino acid residue from valine to methionine at position 374 of cytochrome P450‐2D6

The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc.     

Hyperphosphorylated cortactin in cancer cells plays an inhibitory role in cell motility

Cortactin is frequently overexpressed in cancer cells, and changes of the levels of its tyrosine phosphorylation have been observed in several cancer cells. However, how the expression level and phosphorylation state of cortactin would influence the ultimate cellular function of cancer cells is unknown. In this study, we analyzed the role of cortactin in gastric and breast cancer cell lines using RNA interference technique and found that knockdown of cortactin inhibited cell migration in a subset of gastric cancer cells with a lower level of its tyrosine phosphorylation, whereas it greatly enhanced cell migration and increased tyrosine phosphorylation of p130Cas in other subsets of cells with hyperphosphorylated cortactin. Consistent results were obtained when hyperphosphorylation of cortactin was induced in MCF7 breast cancer cells by expressing Fyn tyrosine kinase. Additionally, immunostaining analysis showed that knockdown of hyperphosphorylated cortactin resulted in the recruitment of p130Cas to focal adhesions. These results suggest that cortactin hyperphosphorylation suppresses cell migration possibly through the inhibition of membrane localization and tyrosine phosphorylation of p130Cas.     

Solubility Improvement of Drugs using N-Methyl Pyrrolidone

The solubilization efficiency of N-methyl pyrrolidone (NMP) has been determined and compared to that of ethanol and propylene glycol for 13 poorly soluble drugs. NMP is found to be a more efficient solubilizer for all the drugs studied. The solubility enhancement as high as about 800-fold is obtained in 20% v/v NMP solution as compared to water. The mechanism of drug solubilization by NMP has also been investigated. It is proposed that NMP enhances drug solubility by simultaneously acting as a cosolvent and a complexing agent. A mathematical model is used to estimate the drug solubility in NMP–water mixture, according to which the total solubility enhancement is a sum of the two effects. This model describes the experimental data well and is more accurate than other models. A large and uniform reduction in the surface tension of water as a function of NMP concentration demonstrates its cosolvent effect. The complexation is supported by the fact that it’s strength is affected by the temperature and the polarity of the medium. A strong correlation exists between log K _ow of the drugs and the cosolvency coefficients. The correlation between log K _ow and the complexation coefficients is weak suggesting that factors such as molecular shape and aromaticity of the drug molecule are significant in determining the complexation strength. This has been confirmed by the absence of a significant complexation between NMP and linear drug-like solutes.     

Aerobic exercise training reduces plasma endothelin-1 concentration in older women.

Endothelial function deteriorates with aging. On the other hand, exercise training improves the function of vascular endothelial cells. Endothelin-1 (ET-1), which is produced by vascular endothelial cells, has potent constrictor and proliferative activity in vascular smooth muscle cells and, therefore, has been implicated in regulation of vascular tonus and progression of atherosclerosis. We previously reported significantly higher plasma ET-1 concentration in middle-aged than in young humans, and recently we showed that plasma ET-1 concentration was significantly decreased by aerobic exercise training in healthy young humans. We hypothesized that plasma ET-1 concentration increases with age, even in healthy adults, and that lifestyle modification (i.e., exercise) can reduce plasma ET-1 concentration in previously sedentary older adults. We measured plasma ET-1 concentration in healthy young women (21-28 yr old), healthy middle-aged women (31-47 yr old), and healthy older women (61-69 yr old). The plasma level of ET-1 significantly increased with aging (1.02 +/- 0.08, 1.33 +/- 0.11, and 2.90 +/- 0.20 pg/ml in young, middle-aged, and older women, respectively). Thus plasma ET-1 concentration was markedly higher in healthy older women than in healthy young or middle-aged women (by approximately 3- and 2-fold, respectively). In healthy older women, we also measured plasma ET-1 concentration after 3 mo of aerobic exercise (cycling on a leg ergometer at 80% of ventilatory threshold for 30 min, 5 days/wk). Regular exercise significantly decreased plasma ET-1 concentration in the healthy older women (2.22 +/- 0.16 pg/ml, P < 0.01) and also significantly reduced their blood pressure. The present study suggests that regular aerobic-endurance exercise reduces plasma ET-1 concentration in older humans, and this reduction in plasma ET-1 concentration may have beneficial effects on the cardiovascular system (i.e., prevention of progression of hypertension and/or atherosclerosis by endogenous ET-1).     

Structure,interaction and real‐time monitoring of the enzymatic reaction of wild‐type APOBEC3G

Human APOBEC3G exhibits anti‐human immunodeficiency virus‐1 (HIV‐1) activity by deaminating cytidines of the minus strand of HIV‐1. Here, we report a solution structure of the C‐terminal deaminase domain of wild‐type APOBEC3G. The interaction with DNA was examined. Many differences in the interaction were found between the wild type and recently studied mutant APOBEC3Gs. The position of the substrate cytidine, together with that of a DNA chain, in the complex, was deduced. Interestingly, the deamination reaction of APOBEC3G was successfully monitored using NMR signals in real time. Real‐time monitoring has revealed that the third cytidine of the d(CCCA) segment is deaminated at an early stage and that then the second one is deaminated at a late stage, the first one not being deaminated at all. This indicates that the deamination is carried out in a strict 3′ → 5′ order. Virus infectivity factor (Vif) of HIV‐1 counteracts the anti‐HIV‐1 activity of APOBEC3G. The structure of the N‐terminal domain of APOBEC3G, with which Vif interacts, was constructed with homology modelling. The structure implies the mechanism of species‐specific sensitivity of APOBEC3G to Vif action.     

Tuning of the Zernike phase-plate for visualization of detailed ultrastructure in complex biological specimens

In order to acquire phase-contrast images with adequate contrast, conventional TEM requires large amount of defocus. Increasing the defocus improves the low-frequency components but attenuates the high-frequency ones. On the other hand, Zernike phase-contrast TEM (ZPC-TEM) can recover low-frequency components without losing the high-frequency ones under in-focus conditions. ZPC-TEM however, has another problem, especially in imaging of complex biological specimens such as cells and tissues; strong halos appear around specimen structures, and these halos hinder the interpretation of images. Due to this problem, the application of ZPC-TEM has been restricted to imaging of smaller particles. In order to improve the halo appearance, we fabricated a new quarter-wave thin film phase-plate with a smaller central hole and tested it on vitreous biological specimens. ZPC-TEM with the new plate could successfully visualize, in in-focus images, the intracellular fine features of cultured cells and brain tissues. This result indicates that reduction of the central hole diameter makes ZPC-TEM applicable on size scales ranging from protein particles to tissue sections. The application of ZPC-TEM to vitreous biological specimens will be a powerful method to advance the new field of imaging science for ultrastructures in close-to-physiological state.