Functional Expression of GFP-linked Human Heart Sodium Channel (hH1) and Subcellular Localization of the a Subunit in HEK293 Cells and Dog Cardiac Myocytes

Recent evidence suggests that biosynthesis of the human heart Na+ channel (hH1) protein is rapidly modulated by sympathetic interventions. However, data regarding the intracellular processing of hH1 in vivo are lacking. In this study we sought to establish a model that would allow us to study the subcellular localization of hH1 protein. Such a model could eventually help us to better understand the trafficking of hH1 in vivo and its potential role in cardiac conduction. We labeled the C-terminus of hH1 with the green fluorescent protein (GFP) and compared the expression of this construct (hH1-GFP) and hH1 in transfected HEK293 cells. Fusion of GFP to hH1 did not alter its electrophysiological properties. Confocal microscopy revealed that hH1-GFP was highly expressed in intracellular membrane structures. Immuno-electronmicrographs showed that transfection of hH1-GFP and hH1 induced proliferation of three types of endoplasmic reticulum (ER) membranes to accommodate the heterologously expressed proteins. Labeling with specific markers for the ER and the Golgi apparatus indicated that the intracellular channels are almost exclusively retained within the ER. Immunocytochemical labeling of the Na+ channel in dog cardiomyocytes showed strong fluorescence in the perinuclear region of the cells, a result consistent with our findings in HEK293 cells. We propose that the ER may serve as a reservoir for the cardiac Na+ channels and that the transport from the ER to the Golgi apparatus is among the rate-limiting steps for sarcolemmal expression of Na+ channels.     

Evaluation of the involvement of nitric oxide and substance P in reducing baroreflex gain in the genetically hypertensive (GH) rat

The attenuation of baroreflex gain associated with hereditary hypertension could involve abnormal signalling by nitric oxide or substance P. Baroreflex gain was measured in age-matched male genetically hypertensive (GH) and nonnotensive (N) anaesthetised rats from heart rate changes in response to i.v. phenylephrine or sodium nitroprusside. In subgroups of these animals, nitric oxide synthesis was inhibited using NG-nitro-L-arginine methyl ester (L-NAME, 30 mg x kg(-1) i.v.), substance P transmission was blocked using the antagonist SR 140333 (360 nmoles x kg(-1) i.v.) or substance P release was inhibited with resiniferatoxin (4 doses of 0.3 microg x kg(-1) i.v. at 4 min intervals). Baroreflex gain was markedly reduced in GH compared to N animals (N -0.37 +/- 0.04 beat x min(-1) x mm Hg(-1), GH -0.17 +/- 0.02 beat x min(-1) x mm Hg(-1), p < 0.0001). Inhibition of nitric oxide synthase increased baroreflex gain in each strain, but the inter-strain difference in gain persisted (post-treatment N -0.57 +/- 0.07 beat x min(-1) x mm Hg(-1), GH -0.24 +/- 0.05 beat x min(-1) x mm Hg(-1) (p < 0.001). Blockade of receptors or inhibition of substance P release did not affect gain in either strain. Nitric oxide, but not substance P, appears to play an inhibitory role in the rat arterial baroreflex. Impairment of baroreflex gain in GH rats is not secondary to altered nitric oxide signaling.     

Searching for preventive measures of cardiovascular events in aged Japanese taxi drivers--the daily rhythm of cardiovascular risk factors during a night duty day

Previous studies have shown that Japanese taxi drivers are exposed to more risk factors and have a higher mortality rate due to cardiovascular disease than other occupational groups. We investigated the effect of night taxi driving with a view to preventing acute events of cardiovascular disease among aged taxi drivers. Twenty-nine taxi drivers (41-67 years old) were examined for urine normetanephrine/creatinine, von Willebrand factor, anti-thrombin III, t-plasminogen activator-plasminogen activator inhibitor 1-complex, hematocrit, blood glucose and blood pressure in the morning and at midnight during a duty day and in the following morning. At the same time, the blood pressure and blood glucose of 46 taxi drivers (43-67 years old) in the morning after a night duty with little sleep and in the morning after daytime work and subsequent night sleep were compared. The results obtained indicate that the aggravation of sympathetic nervous system functions with disturbed circadian rhythms, increased blood coagulation and blood concentration, endothelial injury and the elevation of blood glucose at midnight or the next morning were induced by their night work. These conditions are supposed to favour acute vascular events in aged taxi drivers. Preventive measures considered include social support for anticoagulant food and water intake, short exercise and walking as well as taking a rest and a nap during night work.     

Behavioral compensations in a positional learning and memory task by aged monkeys

The present experiment assessed learning and memory of a positional task by evaluating behavioral strategies as well as accuracy of a task in four young and four aged monkeys. They were tested in a delayed response (DR) task that has been widely used to study animal models of aging. The task consisted of two phases; an acquisition of the task and a positional memory test with five delay times (1-30 s). There was no clear difference between age groups in the number of trials needed for acquisition of the task. However, an analysis of behavior revealed differences in behavioral characteristics displayed during testing. The young monkeys showed various irrelevant behaviors during the execution of the task. In contrast, the aged monkeys consistently concentrated on the task exhibiting no behaviors irrelevant to the task. These results showed than the aged monkeys' performance was supported by a different behavioral strategy from the young monkeys. The results of the memory test were similar to those of the acquisition on the accuracy and the behavior. The aged monkeys depended on behavioral cues to preserve their positional memory, especially during the task. The present study suggests that cognitive impairments in aged monkeys can be compensated for by employing behavioral strategies.     

Tolerance to high osmolality of Lactococcus lactis subsp. lactis and cremoris is related to the activity of a betaine transport system

Lactococcus lactis strains from the subsp. cremoris are described as more sensitive to osmotic stress than subsp. lactis strains. We examined the relation between osmotic tolerance and the activity of the betaine transporter BusA among 34 strains of L. lactis. The cremoris strains that showed reduced growth at high osmolality failed to accumulate betaine. The nature of the defect was found to vary among cremoris strains: lack of the busA encoding region, absence of synthesis or synthesis of an inactive form of BusA. The results suggest that the selection of strains well fitted to the dairy production lead to the loss of an otherwise efficient adaptation mechanism.     

Effects of double mutation at two distant IgE-binding sites in the three-dimensional structure of the major house dust mite allergen Der f 2 on IgE-binding and histamine-releasing activity

Recently, we reported that introduction of mutations that induced conformational changes of the major mite allergen Der f 2 was an efficient strategy to reduce the allergenicity for safer allergen-specific immunotherapy. In this study, we evaluated another strategy, disruption of two independent IgE epitopes without inducing conformational change. We analyzed allergenicities of the wild-type Der f 2, two single mutants with a mutation at either of the two IgE-binding sites (K15A and K77A), and a double mutant with mutations at both of the sites (K15/77A). Purified recombinant forms of Der f 2 expressed in Escherichia coli had correct disulfide bonds, equivalent apparent molecular masses of approximately 15 kDa, and similar secondary structures. The mutants of Der f 2 had less IgE reactivities than the wild-type Der f 2 and reduced inhibitory activities for IgE-binding to the wild-type Der f 2. However, the mutations did not significantly reduce histamine-releasing activity.     

Physiology of vertebrates under micro-gravity with special reference to the Ca metabolism]

On April 12, 1961, Major Yurii A. Gagarin of the former-U.S.S.R. Air Force circled the Earth in a spacecraft named "Vostok", a word which means "east". He spent 1 hour and 48 minutes in space. Since then, the U.S.S.R. and the U.S.A. have sent many astronauts into space. In one case, the stay in space exceeded a year in length, reaching 438 days. Through these experiences, it became clear that micro-gravity caused various problems in human physiology. One of the most serious problems was the loss of Ca from bones, as a result of the negative expenditure of Ca. Under 1G on the ground, bone absorption and bone formation proceed in accordance. Under micro-gravity, however, this balance is broken. Although this phenomenon has been widely analyzed from the viewpoint of molecular biology as well, studies to clarify the mechanism that causes the disorder of Ca metabolism in bones have just started. At present, no perfect treatment to prevent the loss of Ca from bones is available.     

Neodictyoprolenol and dictyoprolenol, the possible biosynthetic intermediates of dictyopterenes, in the Japanese brown algae Dictyopteris

Neodictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecatrien-3-ol] and dictyoprolenol [(-)-(3S)-(1,5Z,8Z)-undecadien-3-ol]), which had been proposed as possible biosynthetic intermediates of the sex pheromones of marine brown algae such as dictyopterene B [(-)-trans-1-((1'E,3'Z)-hexadienyl)-2-vinylcyclopropane], D' [(+)-6-((1'Z)-butenyl)-1,4-cycloheptadiene] and C' [(+)-6-butyl-1,4-cycloheptadiene], were again identified in the essential oils from Dictyopteris prolifera, D. latiscula, and in D. undulata, together with the C11-related volatile compounds such as neodictyoprolene, dictyoprolene and dictyopterenes. Incubation of D. prolifela preparation with racemic neodictyoprolenol and dictyoprolenol as substrates showed (S)-enantioselective decreases of the added substrates and increases in dictyopterenes. From these results, a possible pathway to form dictyopterenes is discussed.