Yoshikazu Kawakami

Weakened cellular scavenging activity against oxidative stress in diabetes mellitus: regulation of glutathione synthesis and efflux

SummaryGlutathione functions to scavenge oxidants or xenobiotics by covalently binding them and transporting the resulting metabolites through an adenosine 5′-triphosphate-dependent transport system. It has been reported that the intracellular concentration of glutathione decreases in diabetes mellitus. In order to elucidate the physiological significance and the regulation of anti-oxidants in diabetic patients, changes in the activity of the glutathione-synthesizing enzyme, γ-glutamylcysteine synthetase, and transport of thiol [S-(2,4-dinitrophenyl)glutathione] were studied in erythrocytes from patients with non-insulin-dependent diabetes and K562 cells cultured with 27 mmol/l glucose for 7 days. The activity of γ-glutamylcysteine synthetase, the concentration of glutathione, and the thiol transport were 77%, 77% and 69%, respectively in erythrocytes from diabetic patients compared to normal control subjects. Treatment of patients with an antidiabetic agent for 6 months resulted in the restoration of γ-glutamylcysteine synthetase activity, the concentration of glutathione, and the thiol transport. A similar impairment of glutathione metabolism was observed in K562 cells with high glucose levels. The cytotoxicity by a xenobiotic (1-chloro-2,4-dinitrobenzene) was higher in K562 cells with high glucose than in control subjects (50% of inhibitory concentration. 300±24 Μmol/l vs 840±29 Μmol/l, p<0.01). Expression of γ-glutamylcysteine synthetase protein was augmented in K562 cells with high glucose, while enzymatic activity and expression of mRNA were lower than those in the control subjects. These results suggest that inactivation of glutathione synthesis and thiol transport in diabetic patients increases the sensitivity of the cells to oxidative stresses, and these changes may lead to the development of some complications in diabetes mellitus.

Impairment of glutathione metabolism in erythrocytes from patients with diabetes mellitus

The metabolism of glutathione and activities of its related enzymes were studied in erythrocytes from patients with non-insulin-dependent diabetes mellitus (NIDDM). A decrease in the levels of the reduced form of glutathione and an increase in the levels of glutathione disulfide were found in erythrocytes of diabetics. To elucidate these changes in the levels of glutathione, synthetic and degradative processes were studied. The activity of gamma-glutamylcysteine synthetase was significantly lower in diabetics than in normal controls. The activity of glutathione synthetase of each group was the same. The rate of outward transport of glutathione disulfide in diabetics decreased to approximately 70% of that of normal controls. The activity of glutathione reductase decreased in diabetics. These data suggest that the decrease in the levels of reduced form of glutathione in erythrocytes of diabetics is brought about by impaired glutathione synthesis and that the increase in the levels of glutathione disulfide is brought about by the decreased transport activity of glutathione disulfide through the erythrocyte membrane together with a decrease in the activity of glutathione reductase. These data also suggest that the impairment of glutathione metabolism weakens the defense mechanism against oxidative stress in erythrocytes of diabetics.

Relation of Oxygen Delivery, Mixed Venous Oxygenation, and Pulmonary Hemodynamics to Prognosis in Chronic Obstructive Pulmonary Disease

We studied the relation of oxygen delivery, mixed venous oxygenation, and pulmonary hemodynamics to prognosis in 50 randomly selected patients with chronic obstructive pulmonary disease. Cardiac catheterization was performed when the patients were clinically stable. Four years later, 27 patients (54 per cent) had died of respiratory failure. At the time of catheterization, patients who subsequently lived were similar to those who died in age, physical characteristics, and hematocrit. Nonsurvivors had significantly lower arterial and mixed venous oxygen tension and significantly higher arterial and mixed venous carbon dioxide tension. The mean pulmonary arterial pressure, pulmonary arteriolar resistance, right ventricular work, coefficient of oxygen delivery, and cardiac index did not differ between the two groups. After inhalation of 100 per cent oxygen for one hour, the mixed venous oxygen tension of nonsurvivors rose to a level equivalent to that of survivors, and their mean pulmonary arterial pressure fell significantly. These results indicate that, with respect to oxygen supply to the tissues, mixed venous oxygenation is one of the important prognostic factors in chronic obstructive pulmonary disease. Pulmonary and right ventricular hemodynamics measured during periods of clinical stability do not differentiate nonsurvivors from survivors.

Increased lipoperoxide value and glutathione peroxidase activity in blood plasma of Type 2 (non-insulin-dependent) diabetic women

SummaryThe lipoperoxide values and glutathione peroxidase activity in blood plasma, along with the glutathione peroxidase, catalase and cupro-zinc superoxide dismutase activities in erythrocytes were investigated in 60 women with Type 2 (non-insulin-dependent) diabetes mellitus and in 71 healthy women. The mean lipoperoxide value and the mean plasma glutathione peroxidase activity in the diabetic patients were significantly higher than those in the control subjects (lipoperoxidep<0.001, plasma glutathione peroxidase activityp<0.01). The plasma glutathione peroxidase activities did not, however, correlate with the plasma lipoperoxide values. The erythrocyte glutathione peroxidase activity was approximately ten times higher than that of the plasma glutathione peroxidase activity, nor did they correlate with each other. In contrast to the findings of other authors on the activities of the protective enzymes in erythrocytes against oxidative damage, there were no significant differences of erythrocytes glutathione peroxidase, catalase and superoxide dismutase activities between diabetic and control women.

Prognostic significance of the expression of ras oncogene product in non-small cell lung cancer

The clinical significance of ras oncogene expression in non‐small cell lung cancer was evaluated in 116 surgically treated patients. Archival paraffin sections of the tumors were analyzed immunohistochemically using anti‐ras p21 monoclonal antibody (MoAb) rp‐35, and p21 staining was correlated with clinicopathologic parameters and survival. Positive reactions (+ and ++) were observed in 72.5% of the adenocarcinomas and 55.6% of the squamous cell carcinomas studied. The T1 tumors showed a ++ reaction less frequently than T2 and T3 tumors (P < 0.05). Stage I tumors also were less reactive with MoAb rp‐35 than tumors in more advanced stages (P < 0.05). Survival analysis showed that patients with p21‐negative tumors had significantly longer survival times (a 5‐year survival rate of 64.1%) than those with p21 + tumors (38.0%, P < 0.05) or those with p21 ++ tumors (11.5%, P < 0.005). The significant correlation between p21 staining and patient survival was independent of histologic type, stage of disease, tumor or node status, and the resectability of tumors. On Coxs multivariate analysis, p21 staining was a major and independent prognostic determinant of survival. These results suggest that enhanced ras p21 expression may be one of the important biologic and clinical markers indicating the malignant potential of non‐small cell lung cancer.

Effect of insulin on impaired antioxidant activities in aortic endothelial cells from diabetic rabbits

The defense system of aortic endothelial cells against oxidative stress was studied in alloxan-induced diabetic rabbits, and the effect of insulin on the antioxidant activities was estimated. Endothelial cells were prepared from 10 diabetic rabbits, 18 diabetic rabbits treated with insulin, and 10 age-matched controls after 17 days of diabetes. These cells were used for the estimation of glutathione (GSH) levels and its related enzyme activities. The antioxidant activities in these endothelial cells from diabetic rabbits were compared with those from control subjects. The concentration of GSH decreased in diabetic rabbits (1.6 +/- 0.2 nmol/mg protein [mean +/- SD] v 3.7 +/- 0.6 nmol/mg protein). Decreases in the activities of Cu, Zn-superoxide dismutase (Cu,Zn-SOD) (62.7 +/- 11.0 U/mg protein v 172.9 +/- 20.2 U/mg protein), catalase (7.6 +/- 2.1 U/mg protein v 12.3 +/- 3.2 U/mg protein), and GSH peroxidase (134.0 +/- 27.0 mU/mg protein v 179.1 +/- 26.2 mU/mg protein) were observed. The activities of other GSH-related enzymes such as GSH S-transferase or GSH reductase did not change in endothelial cells from diabetic rabbits. Most of these antioxidant activities were prevented when diabetic rabbits were treated with insulin (1 to 2 U/kg/d). These antioxidant activities were also determined in the diabetic liver and kidney. Similar decreases in the cellular defense activities and prevention of the decrease in activities by insulin were observed in the diabetic liver, while these antioxidant enzyme activities in the kidney were resistant to diabetic conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Prognostic and therapeutic significance of the flow cytometric nuclear DNA content in non-small cell lung cancer

To evaluate prognostic and therapeutic significance, tumor DNA content was determined by flow cytometry in 310 paraffin‐embedded tissue samples obtained surgically from 130 patients with non‐small cell lung cancer. Ninety‐six (76.8%) patients had DNA aneuploid patterns that were statistically higher in adenocarcinoma than in squamous cell carcinoma. A better 5‐year survival rate was observed in Group A (DNA diploidy, 69.6%) than in Group B (DNA aneuploidy and DNA peridiploidy, 33.2%; P < 0.001). The survival curves of the patients in Group B continued to decrease during the next 2.5 years. Coxs model analysis showed that both the pathologic stage and the DNA content were the significant prognostic factors for survival. However, the DNA content was an independent prognostic factor in squamous cell carcinoma, but not in adenocarcinoma. These results indicate that DNA content analysis is useful for the evaluation of clinical behavior and prognosis, and that the clinical value of the DNA content must be differentiated between squamous cell carcinoma and adenocarcinoma.

Deletion polymorphism in the angiotensin I converting enzyme (ACE) gene as a genetic risk factor for sarcoidosis.

BACKGROUND: Genetic control of serum angiotensin I converting enzyme (SACE) levels has been suggested. A study was undertaken to elucidate the role of this polymorphism in sarcoidosis. METHODS: Three hundred and forty one unrelated healthy controls and 103 consecutive patients with sarcoidosis participated in the study. SACE levels and an insertion/deletion (I/D) polymorphism in intron 16 of the ACE gene were studied in each subject and new reference intervals for SACE activity for each genotype were determined. The difference in genotype and allele frequencies between controls and patients was analysed and odds ratios were calculated to estimate the relative risk. RESULTS: A significant association was seen between ACE gene polymorphism and SACE levels in both patients and controls. The new reference intervals for each genotype discriminated abnormal SACE levels in patients more accurately, especially those with genotype II. In women the frequencies of allele I were 0.68 (allele D 0.32) in controls and 0.58 (allele D 0.42) in patients, and the difference between the two female groups was significant (p < 0.05). Thus, an excess of genotype ID or DD was observed in female patients (odds ratio 2.18; 95% confidence interval 1.18 to 4.01; p = 0.01). CONCLUSIONS: These findings suggest that ACE gene polymorphism is associated with SACE levels in both patients with sarcoidosis and controls. ACE gene polymorphism should be further evaluated as a candidate marker for an increased risk of sarcoidosis.

Cysteine proteinases and cystatin C in bronchoalveolar lavage fluid from subjects with subclinical emphysema

This study examined the role of cysteine proteinases and their inhibitor in the development of emphysema in comparison with neutrophil elastase (NE) complexed with alpha1-protease inhibitor (NE-alpha1-PI), which was previously demonstrated to be increased in bronchoalveolar lavage (BAL) fluid from subjects with subclinical emphysema. Eight nonsmokers and 31 current smokers with (n=17) and without (n=14) emphysema, as evidenced by lung computed tomographic scans, were studied. The concentrations of immunologically detected cathepsin L and cystatin C, but not cathepsin B, were significantly increased in BAL fluid from the smokers with emphysema compared with those without emphysema, although the activity of cathepsin L, measured using a synthetic substrate and cathepsin L, released from cultured alveolar macrophages at 24 h, did not show any significant difference between the two groups. When comparison was made only for the subjects aged <60 yrs, the difference between the two groups disappeared for cathepsin L, but remained for NE-alpha1-PI. There was no significant correlation between the level of cathepsin L and that of NE-alpha1-PI in BAL fluid from the subjects with emphysema. In conclusion, increased levels of cathepsin L and cystatin C were demonstrated in bronchoalveolar lavage fluid from subjects with subclinical emphysema. However, the roles of cathepsin L and neutrophil elastase in the development of emphysema may vary between subjects and between the young and the old.

Lack of linkage between atopy and locus 11q13

Atopy as defined in terms of IgE responsiveness was reported to be controlled by a single gene in British families, and this concept was further supported by a significant linkage between atopy and restriction fragment length polymorphism (RFLP) detected by a DNA probe specific to chromosome 11q13. To confirm this observation in a Japanese population, segregation and linkage analyses were done in four large families. Although segregation patterns of atopy were in agreement with the pattern of autosomal dominant inheritance, there was no significant linkage between atopy and locus 11q13. Alterations in the definitions of atopy did not affect the results. These findings suggested the presence of heterogeneity in genetic elements of atopy, even though atopy may be determined mainly by a single dominant gene.