Switchable bumps of a bead-embedded elastomer surface with variable adhesion.

An extremely simple structural design of a composite material composed of an elastomer sheet and hard beads embedded at the surface is proposed to realize a shape-tunable surface; it reversibly forms bumps/undulations in response to in-plane tensile strain applied to the surface. Tribological properties such as adhesion therefore become switchable.

Observation of Cells on a Simulated Titanium Surface with Transparency.

The main driving force of osseointegration on titanium implants is believed to be the calcification caused by cellular activity. However, owing to the opacity of bulk titanium, live cells on titanium surfaces cannot be observed using an inverted microscope. To overcome this limitation, this study proposes a transparent titanium thin layer as a simulated titanium surface that allows live-cell observation from below. The titanium layer was fabricated on a polystyrene culture dish by magnetron DC sputtering using a pure Ti(JIS1) target. The titanium layer was characterized by transparency, composition, structure, and wettability. Osteoblast-like cells were cultured in the titanium-coated dishes. The cell culture was observed periodically using an inverted microscope, and the images were compiled into time-lapse videos. Cells on the titanium layer were characterized by movement speeds and doubling times. The titanium-coated dish was transparent gray, and its transmittance profile was consistent with that of the polystyrene dish. The titanium layer showed similarities to bulk titanium surfaces in terms of composition and structure; that is, it showed an oxidized titanium outermost layer and titanium metal basal layer. The wettability of the titanium layer was hydrophilic with mean contact angles of 67.52°. Osteoblast-like cells successfully adhered to the titanium layer and proliferated to confluence. The time-lapse videos demonstrated active movement of the cells on the titanium layer, which suggested the involvement of the titanium surface in cellular motility. The cell culture on the titanium layer can be considered cell culture on a titanium surface. In short, the titanium layer enabled the acquisition of information for living cells on titanium that has either been unknown or analogically understood based on cell culture on polystyrene dishes.

Substance P inhibits catecholamine biosynthesis stimulated by carbamylcholine in cultured adrenal chromaffin cells.

The effect of substance P on catecholamine biosynthesis was examined using cultured bovine adrenal chromaffin cells as a model for the sympathoadrenergic system. Substance P markedly inhibited the formation of [14C]catecholamines from L-[14C]tyrosine stimulated by cholinergic agonist, but caused no significant effect on the biosynthesis stimulated by depolarizing agent. In addition, this inhibitory action was completely prevented by the addition of substance P antagonists. Under the conditions in which the inhibition of catecholamine biosynthesis was observed, substance P also inhibited the influx of extracellular 45Ca2+ into these cells, and this inhibitory action on Ca2+ influx was almost identical to that on the biosynthesis. These results provide evidence for a possible role of substance P as a putative neuromodulator in the sympathoadrenergic system.

Dilated cardiomyopathy defines serum autoantibodies against G-protein-coupled cardiovascular receptors.

In order further to identify the prevalence of anti-receptor autoantibodies in the sera of patients with dilated cardiomyopathy (DCM), we attempted to detect autoantibodies against a series of G-protein-coupled cardiovascular receptors in a well-defined population of DCM patients from Japan. Peptides corresponding to the sequences of the second extracellular loops of the human beta 1 and beta 2 adrenoceptors, alpha 1 adrenoceptors, M2 muscarinic acetylcholine receptors and angiotensin II-1 (AT1) receptors were used as antigens in an enzyme immunoassay to screen the sera from patients with DCM (n = 28). Nine sera from patients with DCM (32%) and 2 sera from healthy subjects (9%) recognized the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor peptide. Ten sera from patients (36%) and 3 sera from healthy subjects (13%) recognized the M2 receptor peptide. Thirty-six per cent of the patients with autoantibody against the beta 1 adrenoceptor had autoantibody against the M2 receptor. However, no significantly high frequencies of autoantibodies against the beta 2 adrenoceptor, alpha 1 adrenoceptor and AT1 receptor were found in DCM patients. Our results demonstrate that a subgroup of patients with DCM have a specific spectrum of autoantibodies which are specifically directed against the second extracellular loops of the beta 1 adrenoceptors and M2 muscarinic receptors rather than other cardiovascular receptors.

Progressive cardiac dysfunction in adriamycin-induced cardiomyopathy rats.

Cardiotoxicity is a limiting factor in the treatment of cancer with adriamycin. We administered adriamycin by a method which minimizes the risk of peritonitis in an adriamycin-induced cardiomyopathy rat model. Sixty male Wistar rats were given 1 mg/kg of adriamycin intraperitoneally 15 times over a 3-week period (total dose, 15 mg/kg) to induce the cardiomyopathy model. Fifteen control rats received 10 ml/kg body wt. saline 15 times over 3 weeks. The animals were observed for 12 weeks and assessed for mortality, and cardiac volume and function was analyzed by echocardiography at 4, 8, and 12 weeks. In rats treated with adriamycin, the cumulative mortality was 35.8% while in the controls, none of the rats died. Left ventricular diameter of the systole (LVDs) was significantly increased at 4 weeks (4.5 vs. 3.3 mm; P<0.001). Left ventricular diameter of the diastole (LVDd) was significantly increased at 12 weeks (7.9 vs. 7.0 mm; P<0.01) and the % fractional shortening (FS) was significantly decreased at 8 weeks (33.4% vs. 50.0%; P<0.01) in the adriamycin-treated rats. This administration method appears to be useful for investigating the cardiac effect of adriamycin while avoiding the influence of peritonitis typically caused by an intraperitoneal injection of higher single doses of adriamycin.

Influence of cytoplasmic ATP reduction on catecholamine synthesis in cultured bovine adrenal chromaffin cells.

To elucidate a possible role of cytoplasmic ATP in the regulation of catecholamine biosynthesis, cultured bovine adrenal chromaffin cells were pretreated with metabolic inhibitors in the glucose-free medium, and the cytoplasmic concentration of ATP was determined by measuring the amount of ATP released by permeabilization of the plasma membranes with digitonin. Catecholamine biosynthetic activity in these metabolic inhibitor-treated cells was determined by measuring the formation of [(14)C]catecholamines from l-[(14)C]tyrosine. The cytoplasmic concentration of ATP was reduced by pretreatment of the cells with 2,4-dinitrophenol (DNP), oligomycin, carbonyl cyanide m-chlorophenylhydrazone (CCCP) and antimycin. These metabolic inhibitors reduced [(14)C]catecholamine formation without any significant alteration in [(14)C]tyrosine uptake into the cells. In addition, the formation of [(14)C]catecholamines stimulated by high K(+) or cyclic AMP was also reduced by these inhibitors. In contrast, both 2-deoxyglucose (2-DG) and NaN(3) failed to cause any substantial effect on the rate of [(14)C]catecholamine formation as well as the ATP concentration in the cell cytoplasm. While, none of these inhibitors caused a direct inhibitory action on tyrosine hydroxylase prepared from bovine adrenal medulla. The results presented here suggest the possibility that ATP in the cell cytoplasm plays an important role as a factor regulating the catecholamine biosynthetic activity in the adrenal chromaffin cell.

Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells.

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.

Possible involvement of intracellular Ca2+ in hyposmosis-evoked catecholamine release from adrenal chromaffin cells.

The influence of hyposmotic conditions on catecholamine release was studied using cultured adrenal chromaffin cells. Incubation of the cells in hyposmotic solution led to the enhancement of catecholamine release in a manner dependent on the reduction of osmolarity. Hyposmosis-evoked catecholamine release was similarly observed in the presence or absence of extracellular Ca2+, and was not significantly affected by organic and inorganic Ca2+ entry blockers. These results indicated that the hyposmosis-evoked release might be associated with a rise in the intracellular Ca2+ concentration. Further studies showed that neither ryanodine nor thapsigargin caused any significant effect on hyposmosis-evoked catecholamine release, whereas pretreatment of chromaffin cells with carbonyl cyanide m-chlorophenyl hydrazone significantly enhanced the hyposmosis-evoked release. Catecholamine release evoked by exposure to hyposmotic medium is therefore thought to be mediated through intracellular Ca2+, which may be mainly sequestered by the mitochondrial pools. Neither caffeine- nor inositol 1,4,5-trisphosphate-sensitive Ca2+ pools seems likely to be involved in hyposmosis-evoked catecholamine release, although the Ca2+ pools that contribute to the elevation of intracellular Ca2+ observed under hyposmotic conditions are not yet completely identified.

Vinblastine enhancement of hyposmosis-induced catecholamine release in cultured adrenal chromaffin cells: lack of relation to cell swelling and microtubule disruption.

Exposure of chromaffin cells to hyposmotic solution has been shown to cause catecholamine release through the elevation of intracellular Ca2+ level. While cell volume change observed under hyposmotic conditions has been shown to be accompanied by the movement of various ions and suggested to be associated with the reorganization of cytoskeletons. In the present study, the effects of cytoskeleton-disrupting agents on hyposmosis-induced catecholamine release were examined to investigate a possible relationship between catecholamine release and cell volume change under hyposmotic conditions. Hyposmosis-induced catecholamine release was enhanced by pre-treatment of the cells with a microtubule-disrupting agent vinblastine, but not significantly altered by a microfilament-disrupting agent cytochalasin B. Vinblastine also caused an additional increase in the intracellular Ca2+ but failed to affect the cell volume change under hyposmotic conditions. In contrast, the hyposmosis-induced release was not significantly altered by either colchicine, another microtubule-disrupting agent, or taxol, a microtubule-stabilizing agent. These results indicate that vinblastine enhances hyposmosis-induced catecholamine release through an additional increase in the intracellular Ca2+ and furthermore suggest that this effect of vinblastine on the hyposmosis-induced release is unassociated with the disruption of the microtubule system, providing evidence for a lack of the direct relationship between catecholamine release and the cell volume change observed under hyposmotic conditions.

Effects of bioflavonoids on exocytotic release of catecholamines from digitonin-permeabilized chromaffin cells: A comparison with the effects of other protein kinase C inhibitors.

The effects of bioflavonoids on catecholamine release from permeabilized adrenal chromaffin cells were examined to show their intracellular actions on exocytosis. The release from these permeabilized cells in response to a direct calcium challenge was shown to be markedly inhibited by quercetin in a manner dependent on its concentration. Apigenin was also shown to cause a moderate inhibitory action, but flavone caused no significant effect on the release under the experimental conditions used here. Furthermore, the inhibitory actions of these flavonoids on the phorbol ester-dependent fraction of catecholamine release were shown to be more pronounced than those on the calcium-dependent fraction. The effects of bioflavonoids on the calcium-dependent and the phorbol ester-dependent releases were then compared with those of other protein kinase C inhibitors, and quercetin was shown to cause a potent inhibitory action on the exocytotic secretory process, which was almost equivalent to those caused by polymyxin B and neomycin. Both quercetin and apigenin were clearly shown to inhibit the phorbol ester-dependent as well as the calcium-dependent release of catecholamines from digitonin-permeabilized chromaffin cells. The inhibitory actions of these compounds were therefore thought to be attributed to their inhibitory actions on protein kinase C in the cytoplasmic space of the permeabilized cells. Thus, these results seem to provide further evidence for a possible involvement of protein kinase C as one of the sites for calcium action in the intracellular mechanism of exocytotic secretion.

Effects of bioflavonoids on catecholamine biosynthetic activity in the adrenal gland: In vitro studies using partially purified tyrosine hydroxylase and chromaffin cell cultures.

To elucidate the effects of bioflavonoids on catecholamine biosynthetic activity in the sympathoadrenergic system, the direct actions of bioflavonoids on tyrosine hydroxylase activity were first examined in vitro using the enzyme partially purified from bovine adrenal medulla. The enzyme activity was markedly and rapidly inhibited by quercetin, but not significantly affected by either apigenin or flavone. The inhibitory action of quercetin on tyrosine hydroxylase was shown to be accompanied by an alteration in kinetic properties of the enzyme. Furthermore, the effects of these flavonoid compounds on catecholamine biosynthetic activity were examined using cultured bovine adrenal chromaffin cells, and found that quercetin caused a marked decrease in the formation of [(14)C]catecholamines from [(14)C]tyrosine in the cells. In contrast, neither apigenin nor flavone caused any significant effect on the formation of [(14)C]catecholamines under the same conditions. The findings presented here suggest that quercetin may inhibit catecholamine biosynthesis through its direct action on the rate-limiting enzyme in the adrenal chromaffin cell, thus resulting in modulation of the sympathoadrenergic function.

X-ray photoelectron spectroscopic studies of the adsorption of salivary constituents on enamel.

Time-dependent change of adsorption of salivary components on the outermost surface layer of enamel was studied by x-ray photoelectron spectroscopy. Adsorption of proteinaceous components, as monitored in terms of the relative mass of nitrogen, was detected within 30 min, increased with time, and reached a plateau at 90 min. Thus, the ratio of nitrogen to calcium in the two-hour sample increased to about 240 times that in the control sample. The ratio of carbon to nitrogen on the surface decreased to about one-half of that in the control sample. The data established the time required for equilibrium between the proteinaceous component in saliva and the amount of material adsorbed onto the tooth surface.

Mechanical properties of hydroxyapatite and OH-carbonated hydroxyapatite single crystals.

Single-crystal hydroxyapatite and OH-carbonated hydroxyapatite have bending strength much higher than that of dense hydroxyapatite ceramic, indicating potential applicability to a load-bearing biomaterial. However, the effects of carbonate on the strength are less clear. The objective of the present study was to determine the bending strength and Young's modulus of these single crystals with carbonate contents from 0 to 0.62 in CO2 wt%. Three-point bending tests were performed by means of a modified ultra-microhardness tester with a span of 380 microm and a bending direction <210> of the single crystals. The crystals were broken in air, water, and air after immersion in a cell culture medium for 3 wks. The average Young's modulus of the single crystals was from 54 to 79 GPa. The average bending strength of the single crystals in air was 500+/-184, 468+/-205, 513+/-151, and 450+/-162 MPa for those with 0, 0.09, 0.37, and 0.62 wt% carbonate, respectively. No significant decrease in strength was found for hydroxyapatite single crystals both in water and after the immersion in the medium. However, the strength of OH-carbonated hydroxyapatite single crystals decreased significantly by 23 to 43% in water in proportion to the carbonate content. The strength of single crystals with 0.37 and 0.62 wt% carbonate decreased significantly, even after the immersion in the medium. Therefore, hydroxyapatite single crystals are superior to OH-carbonated hydroxyapatite single crystals as a biomaterial for a load-bearing purpose.

Sex differences in appetitive learning of mice.

Learning abilities of male and female ddY mice were compared in two appetitive tasks (a lever-press task and an 8-arm radial maze task), and two avoidance tasks (a shuttle box task and a light-dark discrimination T-maze task). In the two types of appetitive learning, male mice were significantly superior to female mice. Sex differences were particularly apparent in the acquisition process. In contrast, there was no significant sex difference in learning of the two avoidance tasks. A sex difference in appetitive learning was not found in juvenile mice prior to sexual maturation, and the mice which had established a lever-press response as juveniles did not show any significant difference in the performance level when tested as adults. Thus, a sex difference appeared only in the acquisition stage of adult mice. These results suggest that there exists a sex difference in motivation level for hunting food but not for feeding, and that it causes a sex difference in appetitive learning.

Sudden cardiac death from hypertrophic cardiomyopathy and acute idiopathic (Fiedler's) myocarditis: autopsy report.

A 45-year-old man with a rapidly deteriorating heart condition died suddenly, two hours after admission, after resistance to attempts of cardiac pacing. At autopsy, the heart weighed 600g with asymmetric septal hypertrophy. Histological examination revealed an extensive and diffuse disarray of myocardial fibers in the ventricular septum and in the free wall of both ventricles. Pronounced mononuclear cell infiltrations and interstitial edema were distributed widely in both ventricles. There were few abnormal findings in the other organs. The diagnosis was hypertrophic cardiomyopathy accompanied by Fiedler's myocarditis. Such a case appears to be rare.

[A flow cytometric study of the effects of benzalkonium chloride on the cell cycle].

The effects of benzalkonium chloride (BAK) on the cell cycle of Chang's cultured human conjunctival cells were investigated by a flow cytometer (FCM). The cells were exposed to BAK solutions for 60 sec and after 12-48 hrs were fixed in 20% ethanol, treated by 0.25% RNase and stained by 0.005% propidium iodide. DNA histograms were analyzed by the FCM. As a result, although many cells were damaged by exposure to solutions of 0.0025% or 0.005% BAK, they began to grow again 48 hrs later. BAK decreased red fluorescence intensity in DNA histograms. The histogram shifted to the left 12 hrs after the cells were exposed to 0.0025% or 0.005% BAK solutions and recovered 48 hrs later. The DNA synthetic phase of the cell cycle was inhibited by exposure to solutions of 0.0025% BAK and then recovered 48 hrs later.

[An intervention trial to postpone aging in competent elderly. Trial of nutritional improvement in the retirement home].

The purpose of this study was to evaluate the effectiveness of a comprehensive intervention program in changing dietary habits and nutritional status, for postponing aging in competent elderly. The intervention group (n = 44, 11 men, 33 women, mean age: 74.3 +/- 5.4 years) comprised residents of a retirement home in a suburb of Tokyo. The intervention program, which consisted of 82 lectures and practice sessions focusing mainly on improvement of life styles, was performed between May 1993 and May 1995. In addition, individualized dietary consultations were provided every week during the intervention period. The age and sex matched control group (n = 133, 33 men, 100 women) was randomly selected from participants of the community-based health examination in Tokyo. Changes in dietary habits were measured by a 15-food frequency questionnaire method. As parameters of nutritional status, serum albumin (ALB), total cholesterol (TC), HDL cholesterol (HDL-C) and body mass index (BMI) were simultaneously examined at pre- and post-intervention. The results obtained were as follows: 1) The mean score of Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG Index of Competence) was 11.3 +/- 1.6 in the intervention group, 12.0 +/- 1.6 in the control group, the competent level of functional capacity was equally high in both groups. 2) Food frequencies of meat, fruit, and fat and oil significantly increased in the intervention group. A significant increase in the food frequency of meat was seen in the control group. 3) Significant increases in ALB, HDL-C and BMI were consequently observed in the intervention group, whereas levels of ALB and BMI significantly decreased in the control group. 4) The number of times of participation in the comprehensive intervention program was significantly positively related to the change in ALB between pre- and post-intervention, after adjusted for age, sex, education, TMIG Index of Competence score and ALB level at baseline. These results indicate that the comprehensive intervention program is effective for postponing aging in the competent elderly.

[Alcohol-related high blood pressure, and erythrocyte Na+/K(+)-ATPase activity, sodium and potassium concentrations].

Erythrocyte Na+/K(+)-ATPase activity (Ery-ATPase) and intra-erythrocyte sodium and potassium concentrations (Ery-Na, Ery-K) were determined in 83 men aged between 36 and 60 years. Volumes of alcohol consumed during the preceding one week (Alc) correlated significantly with blood pressure (BP), but not correlated with Ery-ATPase and Ery-K. Ery-Na showed a week inverse correlation with Alc, but the partial correlation after adjusting Ery-ATPase was not significant. Therefore, elevations of BP found in alcohol consumers are not related to changes in the cell-membrane Na+/K(+)-ATPase activity and intracellular Na and K concentrations. Ery-ATPase showed a borderline significant positive correlation with diastolic BP (0.05 < or = p < 0.10), independently of age, body mass index and Alc. The significance of the weak association in the pathogenesis of hypertension remains unclear.

[Determination of human erythrocyte membrane Na+/K(+)-ATPase activity in small volume of blood sample].

Na+/K(+)-ATPase of the cell membrane is considered to be closely related to the pathology of various diseases including hypertension and heart failure. The activity of this enzyme in the erythrocyte membrane has been determined in earlier reports by the assay of inorganic phosphate generated from the substrate ATP or radioimmunoassay after binding 3H ouabain to the erythrocyte membrane, using a large volume of blood samples. However, as neither method was appropriate for wide routine use, we developed a method to assay this enzyme in a small volume (10 ml) of fresh human blood samples with re-evaluation of conditions for the inorganic phosphate assay. In this method, the coefficient value (CV) of membrane protein amount and the NA+/K(+)-ATPase activity were 2.2% and 2.5% respectively, indicating sufficient precision of the assay. Moreover, in 97 subjects without abnormalities in blood biochemical tests (77 males and 20 females) aged 35-59 years, the enzyme activity showed no differences according to age or sex, ranging from 0.217 to 0.071 mumols Pi/mg/hr with a mean of 0.130.

[Study on health awareness of home helper for dependent elderly].

The purpose of this study was to investigate the level of oral health awareness and oral health care activities of home helper for dependent elderly. A questionnaire was mailed to persons who took the profession course of care worker training and education (second and third grade) for home helper and short term course (special subject) at care worker support center foundation (33 branches). Responses were obtained from 220 persons qualified as home helpers. Of 220 home helpers, 104 (47.3%) had worked as a home helper for fewer than six years. One hundred forty two (64.5%) were trained and educated for a home helper as a second grade. The results were as follows: 1. One hundred eleven (50.5%) had used a dental floss and an interdental brush. 2. Two hundred eighteen (99.1%) thought it was necessary to perform oral cleaning of dependent elderly. 3. Two hundred fifteen (97.7%) hoped to perform the oral cleaning of dependent elderly. 4. Two hundred nine (95.0%) considered oral cleaning distasteful. 5. Two hundred three (92.3%) recognized food debris between natural teeth and the denture of dependent elderly who could perform oral cleaning themselves. 6. Two hundred three (92.3%) recognized food debris between natural teeth and the denture of dependent elderly who could not perform oral cleaning themselves. 7. One hundred twenty (54.5%) had been trained in oral cleaning for dependent elderly. 8. Two hundred twelve (96.4%) hoped to receive training in oral cleaning for dependent elderly.