A study of non-aromatizing androgen 19-hydroxylase in sheep adrenal

We reported on the unusually high isotope effect of non-aromatizing androgen 19-hydroxylase in sheep and dog adrenals and the validity of the [3H] water method using [19-3H3] androgen. We have extended the study to examine whether this 19-hydroxylation is catalyzed by a cytochrome P-450 dependent enzyme. Sheep adrenal homogenate (1.65 mg prot.) was incubated in the presence of NADPH (5.6mM) with [19-3H3, 4-14C]-androstenedione (A) (3.2 microM, 8.24 x 10(4) dpm 3H/micrograms, 3H/14C = 17.2) in a total of 1.2 ml PO4 buffer under air at pH 7.4 for 2, 5 and 10 min. [19-3H2, 4-14C]-19-hydroxy-A (19-OHA) with added carrier was purified through extraction, TLC, acetylation to form 19-AcOA, and further TLC to give 19-hydroxylase activity as assessed by the product isolation method. Simultaneously, the [3H] water was measured by distillation, and with correction by the apparent kinetic isotope effect (KH/KT = 11.8), used for assessment of 19-hydroxylase activity. The effects on the hydroxylation by cofactor (NADPH, NADH), incubation atmosphere (N2, CO/O2), cytochrome P-450 inhibitors (metyrapone, clotrimazole) and heating were measured by both methods. Compared to the complete system (89.6pmol/min/mg as 100%), carbon monoxide suppressed 15.8, 59.3 and 86.4% of the 19-hydroxylation when a CO/O2 ratio of 0.1, 1 and 9 was used, respectively. Replacement to nitrogen atmosphere decreased the activity by 93.8%. Replacement of NADPH with NADH (7.5mM) caused more than a 92.1% decrease in activity. Metyrapone at 50 and 200 microM and and clotrimazole at 2.5 and 10 microM suppressed the activity by 82.8, 90.4, 85.4 and 94.9%, respectively. A larger scale sheep adrenal incubation of A (250 microM) under 18O2 atmosphere and isolation of 19-AcOA were carried out in a similar manner. The gas chromatography-mass spectrometry analysis of the purified product showed 48.5% of the product to be 18O-labeled as [M+ + 2], m/e 346. Thus, the non-aromatizing androgen 19-hydroxylase requires NADPH and molecular oxygen. It is strongly inhibited by carbon monoxide and cytochrome P-450 inhibitors. These results indicate that the enzyme system responsible for non-aromatizing androgen 19-hydroxylase in adrenal is a cytochrome P-450 dependent monooxygenase.     

Disturbances of cross-localization of fingertips in a callosal patient

If defective cross-localization of fingertips (CLF) in callosal patients is due to a deficit in the interhemispheric transfer of somesthetic information, when the patient's eyes are open, CLF should be affected when the stimulated hand is excluded from vision, not when the responding hand is excluded from vision. In order to investigate this hypothesis, a patient with a callosal lesion was subjected to CLF with eyes closed and open. With eyes closed, the CLF score in the left-to-right direction was significantly lower than that in the right-to-left direction. With eyes open, the CLF performance in the right-to-left direction was impaired when it was the responding hand to be excluded from vision, not when it was the stimulated hand to be excluded from vision. It would, therefore, appear that the patient's CLF disturbance was not due to a somesthetic transfer deficit, but to left unilateral apraxia for the right-to-left direction errors and to left tactile finger anomia for the left-to-right direction errors.     

Reappraisal of projection levels of the corticospinal fibers in the cat, with special reference to the fibers descending through the dorsal funiculus: a WGA-HRP study

The precise course and termination levels of the corticospinal tract (CST) in the cat were studied using the anterograde transport of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP). Following injection of WGA-HRP into the pericruciate (sensorimotor) cortex on one side, we observed the precise caudal termination levels of the CST fibers in the lateral and ventral funiculi. Simultaneously, the bilateral CSTs descending through the dorsal funiculus of the spinal cord were identified. Anterogradely labeled CST fibers within the lateral and ventral funiculi were observed bilaterally to reach the level of the third sacral (S3) spinal segment, which is lower than that ever described. The lowest level of the CST fibers within the dorsal funiculus, however, reached the level of the first sacral (S1) spinal segment. In conclusion, this study demonstrates that, in the cat, there exist 6 different CSTs (crossed and uncrossed lateral, ventral, dorsal) and that the termination levels of the lateral and ventral CSTs are much lower than those described in previous reports.     

Risk factors for invasive aspergillosis in living donor liver transplant recipients.

Invasive aspergillosis (IA) is a severe complication of liver transplantation. Risk factors for IA after deceased donor liver transplantation (DDLT) have been presented in several reports, but are not well established for living donor liver transplant recipients. Here, a retrospective case-control study was performed. Five cases with IA were investigated after living donor liver transplantation (LDLT) between January 1999 and December 2002 at Kyoto University Hospital. For comparison, living donor liver transplant recipients without IA were taken as controls. These patients had undergone LDLT 1 month before or after each IA case and had the same survival times as the latter. We evaluated the clinical and laboratory findings for both groups up until their demise. Patients with IA after LDLT had a very poor prognosis. By univariate analysis, risk factors for IA were preoperative intensive care unit stay (P = 0.02) and preoperative steroid administration (P = 0.02). Preoperative steroid administration for fulminant hepatitis possibly predisposed to the development of IA after LDLT.     

DIFFERENCES IN ENDOTHELIUM-DEPENDENT RELAXATION IN VARIOUS ARTERIES FROM WATANABE HERITABLE HYPERLIPIDAEMIC RABBITS WITH INCREASING AGE

1. Endothelium-dependent relaxation in response to acetylcholine (ACh) and the calcium ionophore A 23187 was examined in aorta, coronary, basilar and renal arteries isolated from Watanabe heritable hyperlipidaemic (WHHL) rabbits of 2, 6 and 12 months of age, with normolipidaemic heterozygous WHHL rabbits as controls. 2. In the rings of WHHL rabbit aortae and coronary arteries preconstricted with vasoconstrictors, endothelium-dependent relaxation in response to ACh was attenuated with age compared to the heterozygous WHHL rabbits. A significant negative correlation was found between the total cholesterol content and the relaxation response to ACh in the aortae or coronary arteries from 6 and 12 month old WHHL rabbits. 3. In the rings of basilar arteries, endothelium-dependent relaxations to ACh were not modified with age. Similarly, in the rings of renal arteries, the relaxation response to ACh was not changed with age, but in the 6 and 12 month preparations, after the age of 6 months, a contraction following the relaxation appeared at higher concentrations of ACh (10^?7 to 10^?6 mol/L). The contraction was endothelium-dependent and inhibited by indomethacin. 4. A 23187-induced endothelium-dependent relaxations were also markedly attenuated in the aorta and significantly in the coronary artery with age. 5. Endothelium-independent relaxation to sodium nitroprusside was not changed in all arteries from WHHL rabbits of different ages. 6. These findings indicate that in the aorta and coronary artery of the WHHL rabbit, the endothelium-dependent relaxation to ACh and A 23187 becomes impaired with increasing age (i.e., with the progression of cholesterol deposition in the arterial wall) but is preserved in the basilar and renal artery.     

Treatment of a patient with end-stage renal disease, severe iron overload and ascites by weekly phlebotomy combined with recombinant human erythropoietin

A 41-year-old hemodialyzed woman developed ascites and was found to have secondary iron overload. The dose of administered iron was approximately 11-12 g, and her serum ferritin level was 15,000 ng/ml (15,000 micrograms/l). There were no signs of congestive heart failure, fluid overload, or liver cirrhosis. A program of weekly phlebotomy combined with recombinant human erythropoietin (rhEPO) therapy was tried to eliminate the iron congestion. After 9 months of this therapy, about 5 g of iron had been removed. The ascites completely disappeared, and her serum ferritin level fell to 5,800 ng/ml (5,800 micrograms/l). This suggests that such combined therapy would be useful when iron overload must be corrected rapidly. Before therapy, the sterile ascitic fluid showed exudative characteristics with 3.7 g/dl (37 g/l) of total protein. The serum-ascites albumin difference was 0.6 g/dl (6 g/l), and the fluid contained 1,400 inflammatory cells/mm3 (1.4 X 10(9)/l). Notably, the serum-ascites albumin difference increased in parallel with iron elimination. These findings suggested that iron deposition may have played a role in changing the permeability of the peritoneum, or in impairing lymphatic drainage, both of which are presumed to be pathogenetic factors of nephrogenic ascites.     

Immunological study on chyluria

Immunological studies were performed in 87 patients with chyluria referred to our clinic from January 1982 to December 1988. White blood cell count in 78 patients was 5210.3 +/- 1440.9/mm3. The fraction and the number of lymphocyte were 18.7 +/- 9.5% and 934.1 +/- 521.6/mm3, respectively: they were lower than normal limit. The percentages of T and B lymphocytes studied in 46 patients were 79.3% +/- 11.2% (normal range: 76-86) and 10.4 +/- 7.9% (normal range: 8-16), respectively: both lymphocytes tended to decrease in percentage. Lymphocyte blast formation stimulated with phytohemagglutinin (PHA) was carried out in 20 patients. The mean of the blast formation was 17410.0 +/- 10275.1 c.p.m. (normal range: 377700-62400), and much lower than normal value. Of 22 patients who had PPD skin test, only 9 (40.9%) were positive. These findings signified that cellular immunity was suppressed in patients with chyluria. On the other hand, the value of immunoglobulin was within normal range (IgG: 1325.3 +/- 475.6 mg/dl, IgA: 242.0 +/- 98.3 mg/dl, IgM 130.4 +/- 95.9 mg/dl). Study on the values of laboratory examinations showed statistically positive correlation between total lymphocyte population and T cell number, and between total lymphocyte population and lymphocyte blast formation. In patients with chyluria, serious sequelae have not been reported. However, care should be taken for possible opportunistic infection and, particularly, malignant tumors because suppression of cellular immunity may be one of the promoting factors of malignant tumors.     

Pulmonary sequestration associated with asymptomatic aspergillosis.

Intralobar pulmonary sequestration associated with asymptomatic aspergillosis is a rare case. We describe the case of a 65-year-old woman with intrapulmonary sequestration, anomalous systemic arterial supply to the left lower lobe and aspergillosis who underwent left lower lobectomy and ligation of an anomalous artery by Video-Assisted Thoracoscopic surgery (VATS). Pathological examination showed the parenchymal distortion and chronic inflammation. Aspergillus were found in the cyst. VATS lobectomy for intralobar pulmonary sequestration is a safe and valid procedure.     

Characteristics of young-onset hypertension identified by targeted screening performed at a university health check-up.

Since the prevalence and clinical characteristics of young-onset hypertension are still to be elucidated, we performed targeted-screening at an annual university health check-up for two consecutive years. Out of 16,464 subjects in 2003 and 17,032 in 2004 that were aged less than 30 years, 22 and 26 students (all males) exhibited high blood pressure (BP), respectively, on three occasions during casual BP measurements at the Tohoku University Health Center (systolic and diastolic BP of 140 and/or 90 mmHg or greater, respectively). These students were asked to measure their BP at home, and 9 subjects in total were diagnosed as having essential hypertension (EH). The remaining students were diagnosed as having white coat hypertension (WCH). In 8 out of 9 EH students, their father and/or mother had also been treated with antihypertensive medication. Adjustment by attendance ratio for each BP measurement suggested that the incidence of EH was around 0.1% and that of hypertension (EH and WCH) was around 0.5% in university students aged less than 25 years, since most of the subjects and hypertensive students were between 18 and 24 years old. Body mass index of the EH, which was more than 25 kg/m2 (overweight), was significantly higher than that with WCH. In conclusion, the combination of repeated casual BP measurements and home BP effectively identified young-onset EH. The clinical parameters indicated that male gender, genetic background, and excessive weight were risk factors for young-onset hypertension.     

Synthetic/secreting and apoptotic phenotypes in renal biopsy tissues from hypertensive nephrosclerosis patients.

The major glomerular abnormalities in hypertensive nephrosclerosis are described as glomerular obsolescence (GO), glomerulosclerosis (GS), and glomerular collapse (GC). However, glomerular cellular changes caused by hypertensive insults have not been well analyzed. Using an immunoenzyme method, we examined eleven biopsy samples from patients with hypertensive nephrosclerosis for two synthetic and secreting phenotypes, a-smooth muscle actin (alpha-SMA) and collagen type III (Col. III), and two apoptotic phenotypes, pro-apoptotic molecule Bax and anti-apoptotic molecule BcI-2. Together with the glomerular and vascular changes and interstitial fibrosis (IF) area, the results were scored quantitatively and semi-quantitatively and compared to the clinical findings, which included systolic blood pressure (SBP), mean arterial pressure (MAP), serum creatinine levels (sCr) and creatinine clearance (Ccr), using univariate and multivariate analyses. As a result, GS was frequently observed in the mild-to-moderate hypertensive group (140 < or = SBP<180 mmHg), whereas GC was positively correlated with SBP. Furthermore, there was a positive correlation of GS with mesangial alpha-SMA and Col. III, suggesting that GS was the reflection of these synthetic and secreting phenotypic changes in mesangial cells. Endothelial Bax was positively correlated with Ccr (p<0.01); in contrast, podocytic Bax was positively correlated with sCr (p<0.05) and showed a tendency to correlate with MAP (p=0.054). In conclusion, these findings support the view that mesangial synthetic and secreting phenotypic changes may be a reflection of cellular activation caused by mild-to-moderate hypertension and that apoptotic phenotypic expression in podocytes, rather than endothelial cells, may be related to the development of a severe form of hypertensive nephrosclerosis.