Fruit-juice concentrate of Asian plum inhibits growth signals of vascular smooth muscle cells induced by angiotensin II

Bainiku-ekisu, the fruit-juice concentrate of the Oriental plum (Prunus mume) has recently been shown to improve human blood fluidity. We have shown that angiotensin II (AngII) stimulates growth of vascular smooth muscle cells (VSMCs) through epidermal growth factor (EGF) receptor transactivation that involves reactive oxygen species (ROS) production. To better understanding the possible cardiovascular protective effect of Bainiku-ekisu, we have studied whether Bainiku-ekisu inhibits AngII-induced growth promoting signals in VSMCs. Bainiku-ekisu markedly inhibited AngII-induced EGF receptor transactivation. H(2)O(2)-induced EGF receptor transactivation was also inhibited by Bainiku-ekisu. Thus, Bainiku-ekisu markedly inhibited AngII-induced extracellular signal-regulated kinase (ERK) activation. However, EGF-induced ERK activation was not affected by Bainiku-ekisu. AngII stimulated leucine uptake in VSMCs that was significantly inhibited by Bainiku-ekisu. Also, Bainiku-ekisu possesses a potent antioxidant activity. Since the activation of EGF receptor, ERK and the production of ROS play central roles in mediating AngII-induced vascular remodeling, these data suggest that Bainiku-ekisu could exert a powerful cardiovascular protective effect with regard to cardiovascular diseases.     

Changes in the abnormal alpha-subunit upon CO-binding to the normal beta-subunit of Hb M Boston: resonance Raman,EPR and CD study

Heme-heme interaction in Hb M Boston (His alpha 58-->Tyr) was investigated with visible and UV resonance Raman (RR), EPR, and CD spectroscopies. Although Hb M Boston has been believed to be frozen in the T quaternary state, oxygen binding exhibited appreciable co-operativity (n=1.4) and the near-UV CD spectrum indicated weakening of the T marker at pH 9.0. Binding of CO to the normal beta-subunit gave no change in the EPR and visible Raman spectra of the abnormal alpha-subunit at pH 7.5, but it caused an increase of EPR rhombicity and significant changes in the Raman coordination markers as well as the Fe(III)-tyrosine related bands of the alpha-subunit at pH 9.0. The UVRR spectra indicated appreciable changes of Trp but not of Tyr upon CO binding to the alpha-subunit at pH 9.0. Therefore, we conclude that the ligand binding to the beta heme induces quaternary structure change at pH 9.0 and is communicated to the alpha heme, presumably through His beta 92-->Trp beta 37-->His alpha 87.     

Effects of exercise practice on the maintenance of radius bone mineral density in postmenopausal women

The effect of exercise practice on the radius bone mineral density (BMD) was investigated on 480 women at the age of perimenopause and later. The BMD at the 1/3 distal site of the radius on a non-dominant hand was measured using the Dual Energy X-ray Absorptiometry method. The subjects were divided into group E with regular exercising and group C without regular exercising. The difference of mean radius BMD values was not statistically significant between groups E and C. Each of the groups E and C was further classified into a perimenopause group and groups with stratified postmenopausal years (postmenopausal years less than 5, less than 10 and more than 10). Then, the mean radius BMD value of the perimenopause subgroup was taken as 100% for each of the groups E and C, and the relative radius BMD values of subgroups were compared between groups E and C. The relative BMD values for the subgroup of postmenopausal years more than 10 were higher in E group than those in C group, while no difference was observed in below postmenopausal 10 years between the two groups. To clarify the BMD difference among exercise events, group E was further divided into the following three subgroups: the subgroup practising events which give high-impact to arm (HI group), such as tennis, ping-pong, golf and volleyball, the subgroup practising swimming (SW group), and the subgroup practising events which give low-impact to arm (LI group), such as walking, running, aerobic dance and light gymnastics. No significant differences were observed in BMD values among the above sports event-stratified subgroups when postmenopausal years were less than 10 years, but the relative BMD values for the subgroup of postmenopausal years more than 10 were higher in HI and LI group than those in control group. These results suggest the possibility that exercise practice suppresses a postmenopausal decline in bone density for preventing osteoporosis from developing.     

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis

Effects of adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF) were studied in transgenic (Tg) mice model for amyotrophic lateral sclerosis (ALS). Adenoviral vector containing GDNF gene (Ad-GDNF), E. coli lacZ (Ad-LacZ), or vehicle was injected once a week from 35 weeks of age into the right gastrocnemius muscle of Tg mice carrying mutant human Cu/Zn superoxide dismutase (SOD1) gene, and histological analysis was performed at 46 W. Clinical data showed a tendency of improvement, but was not significantly different among the three animal groups. In contrast, total number of and phospho-Akt (p-Akt) positive large motor neurons in the treated side was significantly preserved in Ad-GDNF-treated group than in vehicle- and Ad-LacZ-treated groups (*p < 0.05). Immunoreactivity of phospho-ERK (p-ERK) and active caspases-3 and -9 showed no difference. These results indicate that the Ad-GDNF treatment prevented motor neuron loss with preserving survival p-Akt signal and without affecting caspase activations, suggesting a future possibility for the therapy of the disease.     

Differences in changes of the alpha1-beta2 subunit contacts between ligand binding to the alpha and beta subunits of hemoglobin A: UV resonance raman analysis using Ni-Fe hybrid hemoglobin

The alpha1-beta2 subunit contacts in the half-ligated hemoglobin A (Hb A) have been explored with ultraviolet resonance Raman (UVRR) spectroscopy using the Ni-Fe hybrid Hb under various solution conditions. Our previous studies demonstrated that Trpbeta37, Tyralpha42, and Tyralpha140 are mainly responsible for UVRR spectral differences between the complete T (deoxyHb A) and R (COHb A) structures [Nagai, M., Wajcman, H., Lahary, A., Nakatsukasa, T., Nagatomo, S., and Kitagawa, T. (1999) Biochemistry, 38, 1243-1251]. On the basis of it, the UVRR spectra observed for the half-ligated alpha(Ni)beta(CO) and alpha(CO)beta(Ni) at pH 6.7 in the presence of IHP indicated the adoption of the complete T structure similar to alpha(Ni)beta(deoxy) and alpha(deoxy)beta(Ni). The extent of the quaternary structural changes upon ligand binding depends on pH and IHP, but their characters are qualitatively the same. For alpha(Ni)beta(Fe), it is not until pH 8.7 in the absence of IHP that the Tyr bands are changed by ligand binding. The change of Tyr residues is induced by binding of CO, but not of NO, to the alpha heme, while it was similarly induced by binding of CO and NO to the beta heme. The Trp bands are changed toward R-like similarly for alpha(Ni)beta(CO) and alpha(CO)beta(Ni), indicating that the structural changes of Trp residues are scarcely different between CO binding to either the alpha or beta heme. The ligand induced quaternary structural changes of Tyr and Trp residues did not take place in a concerted way and were different between alpha(Ni)beta(CO) and alpha(CO)beta(Ni). These observations directly indicate that the phenomenon occurring at the alpha1-beta2 interface is different between the ligand binding to the alpha and beta hemes and is greatly influenced by IHP. A plausible mechanism of the intersubunit communication upon binding of a ligand to the alpha or beta subunit to the other subunit and its difference between NO and CO as a ligand are discussed.     

Time-resolved UV resonance Raman investigation of protein folding using a rapid mixer: characterization of kinetic folding intermediates of apomyoglobin

The 244-nm excited transient UV resonance Raman spectra are observed for the refolding intermediates of horse apomyoglobin (h-apoMb) with a newly constructed mixed flow cell system, and the results are interpreted on the basis of the spectra observed for the equilibrium acid unfolding of the same protein. The dead time of mixing, which was determined with the appearance of UV Raman bands of imidazolium upon mixing of imidazole with acid, was 150 micros under the flow rate that was adopted. The pH-jump experiments of h-apoMb from pH 2.2 to 5.6 conducted with this device demonstrated the presence of three folding intermediates. On the basis of the analysis of W3 and W7 bands of Trp7 and Trp14, the first intermediate, formed before 250 micros, involved incorporation of Trp14 into the alpha-helix from a random coil. The frequency shift of the W3 band of Trp14 observed for this process was reproduced with a model peptide of the A helix when it forms the alpha-helix. In the second intermediate, formed around 1 ms after the start of refolding, the surroundings of both Trp7 and Trp14 were significantly hydrophobic, suggesting the formation of the hydrophobic core. In the third intermediate appearing around 3 ms, the hydrophobicity was relaxed to the same level as that of the pH 4 equilibrium intermediate, which was investigated in detail with the stationary state technique. The change from the third intermediate to the native state needs more time than 40 ms, while the appearance of the native spectrum after the mixing of the same solutions was confirmed separately.     

Studies on the antimicrobial mechanisms of capsaicin using yeast DNA microarray

Capsaicin is a pungent element in a variety of red peppers that are widely used as food additives and considered to be an antimicrobial factor. For our tests, we used yeast DNA micro-array methods to understand the mechanisms of inhibitory effects of capsaicin. The capsaicin treatment significantly induced 39 genes from approximately 6,000 genes. These induced genes were classified as multi-drug resistance transporter genes, membrane biosynthesis genes, genes encoding stress proteins, and uncharacterized genes. The growth abilities of the strains with the deletion of the induced genes suggest that capsaicin is pumped out of the yeast cells by the PDR5 transporter.     

Formation of a persistent inhibitory state of brain adenylate cyclase by GTP analog: Regional differences

Inhibition of adenylate cyclase by pretreatment with guanyl-5′-ylimidodiphosphate (10 μM) at 0°C was examined in particulates obtained from 8 non-neural tissues and from various areas of the CNS of the guinea pig. The excess of the GTP analog used for the pretreatment was removed by washing prior to the enzyme assay. Under these conditions, adenylate cyclase from all the non-neural tissues was activated or not altered while that from the brain was persistently inhibited with kinetics properties similar to those reported previously (Yamamoto and Shimizu, 1983). In the CNS, in general, the inhibition was more marked in those areas containing high densities of neuronal plasma membrane (cerebral cortex, cerebellum, hippocampus) than in areas consisting predominantly of myelin and glial cells (pons, medulla, spinal cord). The formation of a persistently inhibitory state presented herein represents one of the properties specific to brain adenylate cyclase.