Respiratory function impairment in patients with Parkinson's disease--a consideration on the possible pathogenetic relation to autonomic dysfunction]

To investigate the characteristics and clinical significance of respiratory function in patients with Parkinson's disease (PD), we studied 38 patients (male, 19; female, 19: mean age, 65.5 years: mean duration of disease, 6.7 years) who had no history of respiratory disease and smoking. Fifty three non-respiratory disease subjects (male, 26; female, 27: mean age, 67.6 years) were served as age-matched control. We measured spirometry and maximal expiratory flow-volume curve in all patients, and analyzed the relations between respiratory function variables and clinical profiles. The clinical disability of PD was indicated by Hoehn-Yahr (H-Y) scale. The number of PD patients was 15 in H-Y 2, 18 in H-Y 3 and 5 in H-Y 4, respectively. The values of % VC, %FEV 1, FEV 1/FVC, %PEFR, % V50 in H-Y 4 group were significantly smaller than those in H-Y 2 and 3 groups. Small airway dysfunction (SAD) was represented by abnormality of % V25, % V50/V25. The prevalence of impairment in % V25 and % V50/V25 was detected in 13 patients (34.2%) and 15 patients (39.5%), respectively, this was significantly higher than age-matched controls. However, the mean value and prevalence of impairment in % V25, % V50/V25 were not affected by H-Y scale and duration of disease or ideal body weight (%predicted value). Twenty seven patients showed normal ventilatory function based on % VC over 80% and FEV 1/FVC over 70%. The prevalence of impairment in % V25, % V50/V25 was detected in 8 patients (29.6%), 9 patients (33.3%), respectively, among 27 patients with normal ventilatory function. These results suggest that ventilatory dysfunction is concerned with clinical disability but SAD which is independent of clinical disability seen prevalently in patients with PD. It is widely accepted that patients with PD frequently have cardiac or bowel dysfunction based on the visceral autonomic dysfunction. We hypothesize that SAD may also be caused by possible autonomic dysfunction in patients with PD.     

Immunocytochemical studies of aquaporin 4 in the skeletal muscle of mdx mouse.

Immunostainability of anti aquaporin 4 antiserum was investigated in the muscles of dystrophin deficient mdx mice. Western blot analysis showed that the rabbit antiserum against aquaporin 4 reacted with a 28 kDa protein in extracts of normal mouse quadriceps femoris muscles but did not react with the protein in extracts of quadriceps femoris muscles of mdx mice. Immunoperoxidase staining of the muscles from normal and mdx mice revealed the positive immunoreaction at the myofiber surface of normal mice and the negative, or the faint and discontinuous immunostaining at the surface of mdx myofibers. Immunogold electron microscopy disclosed the localization of aquaporin 4 molecules at the myofiber plasma membranes of normal mice and the localization was consistent with that of orthogonal array particles in the protoplasmic face of normal muscle plasma membrane seen in freeze fracture replicas. This study demonstrated that the density of aquaporin 4 molecules was decreased in the muscle plasma membranes of mdx mice, resulting in the faulty function of mdx myofibers.     

Confocal laser microscopy of chondrocytes that received gene transfer using in vitro electroporation

To develop a gene therapy for osteopathies, this study was conducted to establish a method of transferring the BMP gene, a bone formation factor, to cells and administering the cells with BMP expression to patients with osteopathies. Although virus vectors are frequently used for gene transfer, there has been reported a death case of gene therapy using the adenovirus vector. Therefore, various efforts have been made to prevent such complications. In the present study, we used electroporation by which gene transfer can be efficiently performed without inducing severe complications after electric perforation of the cell membrane. Human bone tissues were initially collected intraoperatively, and BMP-2 and Smad4 genes were cloned and integrated into GFP and DsRed plasmid vectors. Using in vitro electroporation, these plasmid vectors were transferred to the cultured chondrocytes (KTN-1) derived from human herniated intervertebral disk. Confocal laser microscopy revealed that the BMP gene was successfully transferred to the nucleus of chondrocytes in the presence of Smad. Since electroporation facilitated human gene transfer to the target cells, gene therapy using electroporation may facilitate individualized treatment for patients.     

High-density lipoprotein and apolipoprotein A-I deficiency induced by combination therapy with probucol and bezafibrate

The effects of the administration of slow-release bezafibrate to hypercholesterolaemic patients who were already receiving long-term probucol treatment (mean 865 days, 500–1000 mg·day^–1) were investigated. Bezafibrate was administered at either 200 mg·day^–1 (13 males, 13 females, mean age 55.2 years) or 400 mg·day^–1 (11 males, 14 females, mean age 57.2 years), and blood was taken at 0, 3, 6 and 12 months after the beginning of combination therapy. Overall, serum total cholesterol (TC), triglyceride (TG), very low density lipoprotein (VLDL)-TC, high-density lipoprotein (HDL)-TG, VLDL-TG, VLDL-phospholipid (PL), lipoprotein (a) [Lp(a)], apolipoprotein (apo) C-III, apo E levels and LCAT activity decreased significantly with this combination therapy, while HDL cholesterol (C), HDL3-C, HDL-PL, apo A-I and apo A-II levels significantly increased, as assessed by analysis of variance (ANOVA). Five patients (one receiving 200 mg·day^–1, four receiving 400 mg·day^–1 bezafibrate) showed drastic reductions in HDL-C (HDL-C levels were reduced by a mean of 46.2%, 59.3% and 61.6% at 3, 6 and 12 months, respectively) after beginning combination therapy. These HDL-C reductions were maintained for the 1 year of combination therapy, but then returned to pre-combination treatment levels 1 month after discontinuation of bezafibrate. Serum probucol concentrations and cholesteryl ester transfer protein (CETP) mass were assayed at 6 months, and the probucol concentration was higher in the HDL-deficient group (56.2 vs 26.5 g/ml). In contrast, CETP mass was significantly lower in HDL-deficient patients than in non-HDL-deficient patients (2.08 vs 2.87 mg·l^–1). When the patients in the non-HDL-deficient group were divided into two groups, receiving low (200 mg·day^–1, n–25) and high (400 mg·day^–1, n–21) doses of bezafibrate, the former group showed a significant increase in probucol-lowered HDL-C and apo A-I, although these levels did not return to pre-probucol treatment levels, while the latter group showed no changes in HDL. These data suggest that the addition of a low dose of bezafibrate to probucol tended to reverse probucol-induced HDL lowering, while 9.8% (5 of 51 patients) of the patients exhibited a severe HDL deficiency. Since it is unclear whether or not such an extreme HDL reduction is harmful, HDL deficiency should be carefully monitored with this combination therapy.Part of this work was presented at the XIth International Symposium on Drugs Affecting Lipid Metabolism (DALM), 15 May 1992, Florence, Italy.     

Efficient nuclear delivery of antisense oligodeoxynucleotides and selective inhibition of CETP expression by apo E peptide in a human CETP-stably transfected CHO cell line.

N,N-Dipalmitylglycyl-apolipoprotein E (129-169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP-stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.     

Long-term cadmium exposure accelerates age-related mitochondrial changes in renal epithelial cells

Long-term cadmium exposure leads to mitochondrial dysfunction in the proximal tubular epithelial cells. Mitochondrial DNA deletion may contribute to the pathogenesis of cadmium-induced nephropathy. The aim of our study is to clarify the accumulation of mitochondrial DNA deletion and mitochondrial dysfunction in the renal cortex of rats injected three times/week with 1 ml of 1 mM CdCl2 or saline for 80 weeks. After 40-week cadmium injection, mitochondrial number diminished, and cadmium in the renal cortex reached a saturation level. At this time interval, nearly 30% of cadmium in the whole cell fraction was found in the mitochondria. Cytochrome c oxidase (COX) activity in the proximal tubular epithelial cells decreased after 40-week exposure of cadmium. Oxidized phosphatidylcholine (oxPC) started to accumulate in the cytochrome c-positive mitochondria in some tubular epithelial cells after 80-week exposure. After 40 weeks, accumulation of the 4834-bp deletion in mitochondrial DNA was evident in both control and cadmium-treated groups. However, the amount of accumulated mitochondrial DNA deletion tended to increase after 40-week exposure, and was significantly greater after 80 weeks of exposure, compared to the control. Our results indicate that long-term cadmium exposure in rats accelerates accumulation of 4834-bp mitochondrial DNA deletions and impairment of mitochondrial function associated with accumulation of oxidized product.     

Effect of AGEs on human disc herniation: intervertebral disc hernia is also effected by AGEs

Currently, extracellular matrix MMP has been discussed in relation to the extrusion and spontaneous regression of the herniated mass observed in lumbar disc herniation. However, the question remains as to whether degenerated protein is really the cause of this condition's pathogenesis. We confirmed immunologically by means of electron microscopy that extrusion is caused by the AGEs (advanced glycation end products)-induced cross-linking of collagen, and that spontaneous regression is due to AGE receptors on macrophages. Further, AGEs were found to be already exposed during histogenesis, suggesting a relation to apoptosis. In lumbar disc herniation and aging, glucose-derived AGEs cross-link proteins and cause vascular tissue damage.