永井 良三

The clinical utility of doxorubicin, an antineoplastic agent, is limited by its cardiotoxicity. Our objective was to determine whether expression of genes encoding proteins that affect Ca2+ homeostasis were altered in the hearts of rabbits chronically treated with doxorubicin. Twelve male New Zealand white rabbits received an injection of doxorubicin (2.5 mg/kg i.v.) once a week for 8 weeks. Eight rabbits were similarly injected with saline as controls. The cardiac function of both groups was evaluated 8 weeks after the final injection, as were the levels of expression of mRNA for Ca2+ transport proteins in the sarcoplasmic reticulum and plasma membrane. The amount of the sarcoplasmic reticulum Ca2+-ATPase and the Ca2+ uptake capacity of the protein were also quantitated. Cardiac output was significantly decreased in the doxorubicin-treated group (71+/-21 ml/min, P<0.05) compared with the control group (118+/-15 ml/min). The mRNA levels for the sarcoplasmic reticulum proteins were significantly diminished in the doxorubicin-treated hearts: ryanodine receptor-2 (relative expression level compared with controls, 0.35+/-0.13, P<0.01), sarcoplasmic reticulum Ca2+-ATPase (0.56+/-0.13, P<0.01), phospholamban (0.62+/-0.20, P<0.01) and cardiac calsequestrin (0. 57+/-0.26, P<0.01). In addition, both relative amount of sarcoplasmic reticulum Ca2+-ATPase protein (doxorubicin-treated group, 69+/-17% of control, P<0.01) and the Ca2+ uptake capacity (46. 9+/-9.8 nmol Ca2+/mg protein-5 min in doxorubicin group v 63.2+/-10. 4 in the control group, P<0.01) were concomitantly decreased with its mRNA expression level. Conversely, the mRNA levels for the plasma membrane proteins did not differ from those of control rabbits: the dihydropyridine receptor (relative expression level, 1. 03+/-0.30, N.S.), plasma membrane Ca2+-ATPase (0.93+/-0.33, N.S.) and the Na+/Ca2+ exchanger (0.87+/-0.34, N.S.). These findings suggest that a selective decrease in mRNA expression for sarcoplasmic reticulum Ca2+ transport proteins is responsible for the impaired Ca2+ handling, and thus, for the reduced cardiac function seen in the cardiomyopathy induced in rabbits by the long-term treatment with doxorubicin.

STUDY OBJECTIVES: The pathophysiologic role of nitric oxide (NO) released in the lung is not well understood. To determine whether the production of endogenous NO is correlated with any hemodynamic parameters, we measured the amount of NO released from the lung tissue of patients with heart disease. METHODS: Twenty patients (14 with ischemic heart disease, 4 with dilated cardiomyopathy, and 2 with mitral stenosis) and 16 normal control subjects were enrolled in the study. We measured exhaled air samples by using a method developed in our laboratory. The NO release rate from the lungs was calculated from the amount of exhaled NO and the duration of the exhalation. RESULTS: The rate of NO release was significantly lower in the patients with moderate-to-severe heart failure (New York Heart Association [NYHA] II or III) than in those with mild heart failure (NYHA I) or in normal control subjects. The rate of NO release was positively correlated with the cardiac index (r=0.50, p<0.05), and was negatively correlated with either the systemic (r= -0.58, p<0.01) or pulmonary vascular resistance (r=-0.45, p<0.05). In the patients with moderate-to-severe heart failure, the amount of NO released and the oxygen tension in the pulmonary artery were significantly lower compared with those parameters in patients with mild heart failure. CONCLUSIONS: Results suggest that the basal production of endogenous NO in the lung tissue of patients with heart failure is impaired, perhaps leading to the elevated pulmonary vascular tone seen in patients with moderate-to-severe heart failure.

We investigated the therapeutic effects of OK432 (picibanil; CAS39325-1-4), an immunomodulator that is derived from the Su strain of Streptococcus pyogenes. This agent was administered alone or combined with human interferon-alpha in a murine model of insulin-dependent diabetes mellitus. Interferon-alpha inhibits viral replication, reducing the incidence of virus-induced IDDM. Groups of DBA/2 mice (N = 25 per group) received an intraperitoneal injection of OK432 and interferon-alpha daily for 16 d beginning 1 d after inoculation with 500 plaque-forming units of encephalomyocarditis virus (EMCV). The dose of OK432 was one clinical unit (corresponding to 0.1 mg dried cells) per mouse, and that of interferon-alpha was 1 x 10(4) u/g. The animals were killed at random at 3 or 7 d after inoculation with EMCV. The survival rate of mice treated with the combination of OK432 and with interferon-alpha was significantly greater than that of the non-treated infected control animals (P < 0.01). Fasting levels of blood glucose were significantly lower in the mice administered the combination, than in the controls, both on day 3 (68 +/- 21 mg/dl vs. 270 +/- 135 mg/dl, P < 0.01) and on day 7 (101 +/- 29 mg/dl vs. 219 +/- 112 mg/dl, P < 0.01). Serum levels of insulin were significantly higher in the treated mice than in the controls (65 +/- 5 vs. 55 +/- 1 microU/ml, P < 0.05). However, in the mice treated with OK432 or interferon-alpha alone, the survival rate and the blood level of glucose and insulin did not differ from those of infected controls. Natural killer (NK) cell activity was significantly higher in the mice treated with the drug combination than in the controls on both days evaluated: day 3, 65 +/- 5 vs. 55 +/- 1%, n = 3, P < 0.05; day 7, 44 +/- 3 vs. 22 +/- 8%, n = 3, P < 0.05). Serum levels of murine interferon in the treated mice exceeded those in controls on both days evaluated (day 3, 671 U/ml vs. 442 U/ml; day 7, 57 U/ml vs. 43 U/ml). There were no significant differences in NK cell activity or in the interferon level in mice treated with either OK432 or interferon-alpha alone as compared with the infected, non-treated controls. Results suggest that the combination of OK432 and interferon-alpha protects against virally induced IDDM by increasing the activity of NK cells as well as the plasma level of interferon.

Accordingly, we induced streptozotocin diabetes in rats and evaluated the effects of ligating the coronary artery to produce myocardial infarct by analyzing hemodynamics and the expression of brain natriuretic peptide (BNP) messenger (m) RNA. Eight-week diabetic rats and age-matched nondiabetic rats underwent ligation of the coronary artery for 1 week. Left ventricular end-diastolic pressure (LVEDP) was not statistically different between diabetic rats (15+/-6 mmHg) and nondiabetic rats (13+/-9 mmHg) 1 week after coronary ligation, size of infarct, systolic blood pressure were also similar in both groups after coronary ligation. The BNP mRNA/beta-actin mRNA ratio in right ventricle of nondiabetic rats with MI was increased to 350+/-60%, however, in diabetic rats with MI, that was slightly increased to 200+/-50% (P < 0.01). The level of BNP mRNA in the left ventricle of diabetic rats with MI was not increased significantly (120+/-30% versus that in diabetic rats without MI), although that in left ventricle of nondiabetic rats with MI was increased to 280+/-40% versus nondiabetic rats without MI (P < 0.01). Cardiac BNP synthesis in diabetic rats completely reverted to control levels after insulin therapy.

Cardiomegaly is one of the commonest findings encountered in daily clinical practice, and its differential diagnosis is a common clinical problem. There are many electrocardiological (ECG) criteria known for left ventricular hypertrophy (LVH), but its limitations have also been suggested. We evaluated 102 patients fulfilling the ECG criteria of precordial and limb lead for LVH with echocardiographic findings as a gold standard. Among these 102 patients, the echocardiogram revealed 38 subjects with LVH, 26 subjects with left ventricular dilatation (LVD), 7 subjects with both findings, and 31 subjects with neither findings. Precordial criteria such as SV1+RV5 or RV6 > 30 mm, SV1 or SV2+RV5 > 35 mm, R+S > 40 mm, SV1 or SV2+RV5 or RV6 > 35 mm, SV2+RV4 or RV5 > 35 mm, high in sensitivity and low in specificity for LVD and LVH, are appropriate for screening LVD and LVH. Cornell limb lead criterion, SV3+RaVL > 28 mm (male), SV3+RaVL > 20 mm (female), high in sensitivity and specificity only for LVH, is the best elecrocardiographic criterion to evaluate LVH. Precordial and limb lead criteria such as R> 13 mm, RaVL > 12 mm, RaVF > 20 mm, onset of intrinsicoid deflection in V5 or V6> 0.05 sec, left axis deviation -30 degrees to -90 degrees, low in sensitivity, and high in specificity, are useful to rule out LVH and/or LVD. Our findings suggest LVD and LVH can be evaluated by ECG, but similar sensitivity and specificity for both LVH and LVD makes separation of LVH from LVD unattainable.

Coronary ostial stenosis in otherwise normal coronary vessels, is a rare complication of syphilitic aortitis, and most of the cases are found at autopsy. We report here a case in which bilateral coronary ostial stenosis and aortic regurgitation due to syphilitic aortitis was diagnosed; coronary artery bypass graft and aortic valve replacement were then performed. The macroscopic finding and the histopathological examination of the ascending aorta revealed the presence of syphilitic aortitis. It is important to note that syphilis is one of the causes of coronary ostial stenosis in young adults associated with aortic regurgitation.

The role of lymphocytes in the pathogenesis of viral-induced insulin dependent diabetes mellitus (IDDM) is controversial. To better understand how a virus-induced IDDM depends on the infiltrating lymphocytes, encephalomyocarditis virus (EMCV) was inoculated intraperitoneally into three kinds of mice; virus-susceptible C57BL/6, virus-resistant 129/SV and recombination activity gene-2 (Rag2) knockout 129/SV mice. Pancreatic inflammation and beta cell necrosis were evaluated after EMCV, D variant (10(3) pfu/mouse) inoculation. On post-inoculation day 14, the lethal rates of C57BL/6, 129/SV and Rag2 knockout mice were 52, 10 and 100%, respectively. The blood glucose in Rag2KO mice on day 8 was significantly elevated as compared with 129SV mice (231 +/- 49 vs 169 +/- 32 mg/dl, P<0.05). In situ hybridization demonstrated the EMCV genome in the pancreas of Rag2 knockout and C57BL/6 mice, but not in 129/SV mice. Beta cell necrosis were more severe in Rag-2 knockout mice than in wild type 129/SV mice, but lymphocyte infiltration was less severe than C57BL/6. Pancreas in Rag2 knockout mice infected with virus were affected more severely than the virus-resistant strain of mice. Diabetogenic virus induced IDDM in virus-resistant mice without mature lymphocytes.

We evaluated the effects of FR901533, endothelin converting enzyme inhibitor, on development of right ventricular overload and medial thickening of pulmonary arteries in rats with monocrotaline-induced pulmonary hypertension. Pulmonary hypertension was induced by a single injection of monocrotaline (80 mg/kg). Twenty-four hours later (day 1), continuous subcutaneous injection of FR901533 (100 mg/kg/day) was started. Right ventricular systolic pressure, mass ratio of right ventricle to left ventricle, right ventricular wall thickness, right ventricular myocardial fiber diameter, percent medial thickness, and percent smooth muscle area in pulmonary arteries were significantly less in rats that received FR901533 than in the control with monocrotaline on day 28. Both immunoreactivities of endothelin-1 in pulmonary arteries and plasma endothelin-1 levels were observed significantly less in rats treated with FR901533 than in the control with monocrotaline. There were significant increased immunoreactivities of endothelin-B receptor in pulmonary arteries in rats that received FR901533 as compared with those in the control with monocrotaline. FR901533 (100 mg/kg/day), protected the development of right ventricular overload and medial thickening of pulmonary arteries in a rat model of pulmonary hypertension.

The purpose of the present study was to examine the effects of a long-acting calcium antagonist, amlodipine, on the development of cardiac remodeling. Dihydropyridine calcium antagonists have been used widely for many years in the treatment of hypertension and angina pectoris. It has been reported, however, that a prototype of dihydropyridines, nifedipine, does not reduce mortality of patients with ischemic heart disease, possibly because of reflex stimulation of the sympathetic nervous system. A calcium antagonist, amlodipine, has been reported to have potential benefits by virtue of a gradual onset of action and a long duration of effects. Amlodipine (8 mg/kg per day, once a day) or nifedipine (24 mg/kg per day, three times a day) was administered to spontaneously hypertensive 12-week-old rats for 12 weeks. Left ventricular wall thickness was measured by echocardiography, and relative amounts of myosin heavy chain isoforms were assessed by pyrophosphate gels. Expressions of "fetal type" genes and type 1 collagen gene were examined by Northern blot analysis. Amlodipine and nifedipine both markedly reduced systolic blood pressure. However, the decrease in systolic blood pressure caused by nifedipine continued for no more than 8 hours, whereas the blood pressure-lowering effect of amlodipine continued for more than 16 hours post dose. Amlodipine markedly reduced left ventricular wall thickness, whereas nifedipine only weakly attenuated an increase in the wall thickness. Amlodipine, but not nifedipine, prevented an increase in the relative amount of V3 myosin heavy chain isoform and suppressed an increase in mRNA levels of beta-myosin heavy chain, skeletal alpha-actin, and type 1 collagen. Unlike nifedipine, amlodipine effectively prevented cardiac remodeling secondary to high blood pressure at biochemical levels and morphological levels. These results suggest that a long-acting calcium antagonist is more effective than a short-acting one in preventing organ injury in hypertensive subjects.

The purpose of this study was to examine the role of endothelium-derived nitric oxide in modulating the effect of renal perfusion pressure (RPP) on renal interstitial hydrostatic pressure (RIHP) and urinary Na+ excretion (UNaV). The effects of RPP on renal hemodynamics, RIHP, and Na+ and Li+ excretions were determined in control Sprague-Dawley rats, in Sprague-Dawley rats pretreated with intravenous infusion of N-G-nitro-L-arginine methyl ester (L-NAME) at doses of 1, 5, and 50 mu g/kg/min, and in rats pretreated with L-NAME (5 mu g/kg/min) plus L-arginine (10 mg/kg/min). The RPP was changed from 95 to 135 mm Hg by an electronically servo-controlled aortic occluder above the renal arteries in all groups. Increasing RPP in control rats from 95 to 135 mm Hg increased RIHP (from 4.4 +/- 0.5 to 8.7 +/- 1.2 mm Hg), UNaV (from 2.37 +/- 0.61 to 8.29 +/- 1.59 mu Eq/min), and fractional excretion of Li+ (from 38.0 +/- 2.5 to 51.4 +/- 6.0%). In rats pretreated with L-NAME (5 mu g/kg/min), increases in RPP from 95 to 135 mm Hg had no effect on RIHP (from 1.6 +/- 0.4 to 2.2 +/- 0.6 mm Hg) or fractional excretion of Li+ and markedly attenuated pressure-natriuresis relationship (from 1.84 +/- 0.50 to 2.88 +/- 0.65 mu Eq/min). Although L-NAME did reduce renal plasma flow and glomerular filtration rate, the autoregulatory responses to RPP were maintained. In rats pretreated with L-NAME plus L-arginine, RIHP, UNaV, and fractional excretion of Li+ responses to RPP were similar to the control rats. The results of this study indicate that endothelium-derived nitric oxide plays an important role in modulating the effect of RPP on Na+ excretion by enhancing the transmission of RPP into the renal interstitium.

Inactivation of the klotho (kl) gene in mice results in multiple disorders that resemble human aging. The mouse hi gene encodes a novel single-pass membrane protein with homology to beta-glucosidases. In this study, we have isolated a human homologue of the kl gene and determined its gene structure. The human kl gene is composed of 5 exons and ranges over 50 kb on chromosome 13q12. We have further identified two transcripts that encode a membrane or secreted protein. These transcripts arise from a single kl gene through alternative RNA splicing. Expression of the putative secreted form predominates over that of the membrane form. The present study provides fundamental information necessary for further analyses of the human kl gene and its functions. (C) 1998 Academic Press.

A new gene, termed klotho, has been identified that is involved in the suppression of several ageing phenotypes. A defect in klotho gene expression in the mouse results in a syndrome that resembles human ageing, including a short lifespan, infertility, arteriosclerosis, skin atrophy, osteoporosis and emphysema. The gene encodes a membrane protein that shares sequence similarity with the beta-glucosidase enzymes. The klotho gene product may function as part of a signalling pathway that regulates ageing in vivo and morbidity in age-related diseases.

To examine the molecular mechanisms that regulate the expression of the SMemb/NMHC-B gene, a nonmuscle myosin heavy chain isoform predominantly expressed in fetal aorta, we have isolated and characterized the 5'-flanking region of the rabbit SMemb/NMHC-B gene. Transient transfection experiments demonstrated that 105 base pairs of 5'-flanking sequence was necessary to direct high level transcription in C2/2 cells, vascular smooth muscle cells derived from rabbit aorta. An essential cis-regulatory element was localized between -100 and -91 base pairs from the transcription start site based on the results that replacement mutagenesis within this region significantly reduced promoter activity. Sequence of this region is completely conserved between mouse and rabbit and fits no known DNA binding consensus. Gel mobility shift assays revealed that a specific DNA-protein complex was formed at this site with nuclear extracts from C2/2 cells, which can be competed by H-2Kb CCAAT box but not by Hsp70 CCAAT box or other CCAAT-containing sequences. We conclude that expression of the SMemb/NMHC-B gene is regulated through an interaction between a sequence element located at -100 and a distinct member of CCAAT-binding proteins.

To date, there has been neither a good method to clarify the three-dimensional distribution of pulmonary ventilation/perfusion ((V) over dot(A)/(Q) over dot) ratios, nor a convenient way to assess (V) over dot(A)/(Q) over dot inequality, The purpose of this study was to develop a functional image of pulmonary (V) over dot(A)/(Q) over dot over dot ratios based on data acquired with simultaneous dual-radionuclide single-photon emission tomography (SPET) and to assess (V) over dot(A)/(Q) over dot unevenness through the (V) over dot(A)/(Q) over dot histogram in patients with various pulmonary diseases. Dual-radionuclide SPET was performed with technetium-99m macroaggregated albumin (MAA) and krypton-81m, with the patient in the supine position, Af ter correction for linear cross-talk, the total acquisition counts of both radionuclides were equalized, The (V) over dot(A)/(Q) over dot ratio, which was calculated in each pixel by dividing the Kr-81m count by the Tc-99m-MAA count, was expressed as a (V) over dot(A)/(Q) over dot image. A histogram of the pixel number plotted against the (V) over dot(A)/(Q) over dot ratios was then produced and its centre of weight (CW) and standard deviation (SD) determined, Ten healthy volunteers and 46 patients [seven with pulmonary vascular disease (PVD), nine with pulmonary emphysema (PE), 18 with bronchogenic carcinoma and 12 with miscellaneous diseases] participated in this study, In normal volunteers, (V) over dot(A)/(Q) over dot ratios were generally even, but were slightly lower in dorsal regions, Patients with PVD had lobar and/or segmental areas with a high (V) over dot(A)(Q) over dot ratio, Low (V) over dot(A)(Q) over dot areas extended widely in patients with PE, Bronchogenic carcinoma exceeding 3 cm in diameter was detected as a very low (V) over dot(A)/(Q) over dot area, The SD of (V) over dot(A)/(Q) over dot ratios had a significant positive correlation both with A-aDO(2) (r=0.64, P&lt;0.001) and with cigarette smoking history (r=0.72, P&lt;0.001), It is concluded that the (V) over dot(A)/(Q) over dot ratio image produced with simultaneous dual-radionuclide SPET using Tc-99m-MAA and Kr-81m is a unique and simple method for demonstrating the three-dimensional distribution of (V) over dot(A)/(Q) over dot ratios, The unevenness of (V) over dot(A)/(Q) over dot distribution can be assessed through the SD of the (V) over dot(A)/(Q) over dot histogram.

We describe the novel karyotype of a 33-year-old woman with severe mental retardation and multiple cardiac anomalies, including patent ductus arteriosus, a ventricular septal defect, pulmonary atresia, and an overriding aorta. Her karyotype was 46, XX, add(17)(p13). The short arm of chromosome 17 was slightly elongated owing to the deletion of the distal portion of that chromosome and the addition of extra material from another chromosome. Miller-Dieker syndrome is characterized by a patent ductus arteriosus, lissencephaly, and the deletion of chromosome 17p13.3; however, as the patient's brain surface appeared normal on computed tomography, Miller-Dieker syndrome was excluded. The breakpoint in her chromosome 17 was probably located distal to band 17p13.3. In fact, fluorescence in situ hybridization analysis demonstrated that band 17p13.3 was intact. To date, genes distal to 17p13.3 have not been implicated in cardiac anomalies. This patient probably carries a novel deletion on the short arm of chromosome 17.