Yukihide Isogai

Intracellular carboxyl esterase activity is a determinant of cellular sensitivity to the antineoplastic agent KW-2189 in cell lines resistant to cisplatin and CPT-11

KW‐2189, a novel antitumor antibiotic belonging to the duocarmycins, possesses marked DNA‐binding activity upon activation by carboxyl esterase to its active form, DU‐86. Three duocarmycins, KW‐2189, DU‐86 and duocarmycin SA, were active against the cisplatin (CDDP)‐resistant human non‐small cell lung cancer cell lines PC‐9/CDDP and PC‐14/CDDP, and the multidrug‐resistant human small cell lung cancer cell line H69/VP. However, HAC2/0.1, a CDDP‐resistant human ovarian cancer cell line which is also resistant to CPT‐11 because of decreased intracellular activation of CPT‐11, was about 12.8‐fold more resistant to KW‐2189. HAC2/0.1 was not resistant to other duocarmycins as compared to its parental cell line, HAC2. There was no difference between HAC2 and HAC2/0.1 with regard to the intracellular accumulation of KW‐2189. Addition of 130 mU/ml of carboxyl esterase to the culture medium did not influence the sensitivity of HAC2 cells to KW‐2189. However, the sensitivity of HAC2/0.1 cells to KW‐2189 was enhanced to the level of HAC2. These results suggest that HAC2/0.1 is less potent than HAC2 in activating KW‐2189. The carboxyl esterase activity of whole‐cell and microsomal extracts from HAC2/0.1 was approximately 60% of that from HAC2. The cell‐free experiment revealed that KW‐2189 bound to DNA more efficiently in the presence of HAC2 than HAC2/0.1 cell extract. It was concluded that decreased intracellular carboxyl esterase activity in HAC2/0.1 cells caused decreased intracellular conversion of KW‐2189 to its active form, thus producing resistance to KW‐2189. The decreased conversion of CPT‐11 to SN‐38 in HAC2/0.1 cells might be explained by decreased carboxyl esterase activity.

A novel antitumor antibiotic, KW-2189 is activated by carboxyl esterase and induces DNA strand breaks in human small cell lung cancer cells.

KW‐2189 has been selected as a lead compound for clinical trial among duocarmycin derivatives with structural similarity to CC‐1065, a cyclopropylpyrroloindole. The purpose of this study was to examine the DNA‐binding potency and the mechanisms of cytotoxicity of KW‐2189. In order to analyze DNA‐binding activity of KW‐2189, plasmid pBR322 was treated with KW‐2189 with or without pretreatment with carboxyl esterase, which we demonstrated to be an activating enzyme, and the products were examined by agarose gel electrophoresis and restriction enzyme analysis. Cytotoxic activity was examined by exposing a human small cell lung cancer cell line, NCI‐H69 to KW‐2189 with or without carboxyl esterase. Alkaline elution was performed to examine whether KW‐2189 induces DNA strand breaks. DNA treated with KW‐2189 and carboxyl esterase migrated faster than KW‐2189‐treated DNA, which migrated at the same rate as untreated DNA. In addition DNA treated with esterase‐activated KW‐2189 was protected from digestion by some restriction enzymes. KW‐2189 showed concentration‐ and time‐dependent growth inhibitory effect with IC50 values (drug concentration required for 50% growth inhibition) of 58 nM (96 h) to 1900 nM (1 h) in H69 cells. The IC50 values of 4‐h exposure of H69 to KW‐2189 with 0, 26, 130, 650 mU/ml carboxyl esterase were 460, 120, 30, and 7 nM, respectively. Time‐dependent enhancement of cytotoxicity by carboxyl esterase was also observed. KW‐2189 induced DNA strand breaks in H69 cells in a concentration‐dependent manner around the IC50 value. We conclude that 1) KW‐2189 is activated by carboxyl esterase to its active form(s), 2) activated KW‐2189 has a stronger DNA‐binding activity and cytotoxicity than KW‐2189, 3) DNA cleavage is one of the major mechanisms of KW‐2189‐mediated cytotoxicity.

Changes in auditory P300 event‐related potentials and brainstem evoked potentials in diabetes mellitus

To investigate the influence of diabetes mellitus on higher cognitive functions electrophysiologically, we studied auditory P300 event‐related potentials (P300) in 40 NIDDM patients, taking into account wave I‐V latencies (I‐V) in auditory brainstem evoked potentials, clinical parameters and head MRI findings. Compared with 20 controls, diabetics had significantly longer P300 and I‐V latencies. P300 latencies in diabetics correlated with neither I‐V. HbA1, blood glucose levels, nor disease duration. Of the 13 diabetics investigated neuroradiologically, four had lacunar infarcts with prolonged electrophysiological values. The remaining nine had normal MRI scans, but their physiological parameters were still significantly longer than those of controls. These findings suggest that NIDDM can independently alter higher cognitive and the central auditory pathway functions. Our data also suggest that these alterations occur regardless of the recent metabolic derangement and disease duration. Cerebrovascular ischemia, if present, also appears to contribute in part to cognitive alterations.

Spectral analyses of R-R interval and systolic blood pressure in diabetic autonomic neuropathy

We studied autonomic nervous system function using the principle of maximum entropy (ME) to perform spectral analyses of the R-R interval and systolic blood pressure in 32 diabetic patients and 40 healthy controls. The R-R interval and systolic blood pressure were measured using a continuous, noninvasive monitoring system. The power spectra of both the R-R interval (RR) and systolic blood pressure (SYS) were obtained using ME and the areas of two frequency components were measured: a low- (LFC) and a high-frequency component (HFC). The RR-LFC, RR-HFC and SYS-LFC of diabetic patients were significantly smaller than those of healthy controls. The results of the spectral analyses in diabetic patients correlated with neither disease duration nor nephropathy, while the SYS-LFC showed significant correlations with both retinopathy and the delay in median motor nerve conduction velocity. In the mild autonomic neuropathy group, the RR-LFC and SYS-LFC were not differ from those of healthy controls or patients without autonomic neuropathy. However, the RR-HFC was significantly smaller than that of healthy controls or patients without atonomic neuropathy. In the setting of mild diabetic autonomic neuropathy, it was suggested that cardiac parasympathetic dysfunction preceded both alpha and beta sympathetic dysfunction.

Social and Economic Impact on Youth-Onset Diabetes in Japan

OBJECTIVE To investigate the social and economic backgrounds of youth-onset insulin-treated diabetes mellitus in Japan. RESEARCH DESIGN AND METHODS We conducted a case-control study on 35 diabetic patients with age at onset of 19.5 ±; 5.1 yr and duration of diabetes 14.9 ±; 6.7 yr. Sex- and age-matched (within 5 yr) siblings were selected as control subjects. Thirty-five matched pairs were asked to complete a questionnaire, including employment status and educational achievement. RESULTS Overall, diabetic patients were more likely to encounter job refusal in their lives than sibling control subjects (20 vs. 0%), and most patients (6/7) who had an experience of job refusal told job interviewers about their diabetes. Although the full-time employment rate and unemployment rate did not differ significantly between patients and control subjects, income levels were lower among patients than in the sibling (1600 vs. 2500 thousand yen). A multivariate analysis indicated that patients had lower incomes than control subjects after adjusting for the effect of physical disability. Educational achievements in the patients were similar to those in the siblings. CONCLUSIONS These results suggested that diabetic patients had several social and economic problems in Japan. Further studies in more subjects are required to grasp the social and economic impact on diabetes precisely, and minimize the social handicaps on diabetic patients.

Viscosity of Blood and Plasma in Various Diseases

It is presumed that increased blood viscosity gives rise to disturbances in the microcirculation, resulting in disorder of tissue metabolism, changes in the wall of microvascularure and thrombus formation. Thus, investigation of the influence of blood high viscosity on the cause and development of a disease seems of great importance from a clinical standpoint. The present paper deals with whole blood and plasma viscosity in various diseases.

Red cell filterability in diabetes

A negative pressure red cell filteration technique using polycarbonate sieve (Nuclepore) was devised for the observation of red cell filterability (RCF) in diabetes. A significant increase of HbA1 was suggested to be closely correlated to the decrease of RCF. A greater decrease of RCF was observed in diabetics with advanced retinopathy. The increased 2,3-DPG of red cells has been observed in diabetes, but P50 changed much less than might be expected. Improved RCF was observed when Pentoxifylline was added into red cell suspensions. This suggests that Pentoxifylline will have a good effect on microcirculation where impared circulation is observed.

The effect of macrophage colony-stimulating factor (M-CSF) on the progression of lipid-induced nephrotoxicity in diabetic nephropathy

In order to elucidate the role of macrophage in lipid-induced nephrotoxicity in diabetic nephropathy, we examined the effect of macrophage colony-stimulating factor (M-CSF) on the progression of renal lesions in hypercholesterolemic steptozotocin (STZ)-diabetic rats fed with high cholesterol chow. Hypercholesterolemia aggravated albuminuria in diabetic rats accompanied by infiltration of macrophages in glomeruli. Treatment with M-CSF suppressed simultaneously infiltration of glomerular macrophages and urinary albumin excretion in hypercholesterolemic diabetic rats. These results suggest that infiltration of glomerular macrophage has a primary role in lipid-induced nephrotoxicity in diabetic nephropathy, and M-CSF is involved in this process as a preventive factor.

Correlation of Altered Q-T Interval and Sympathetic Nervous System Dysfunction in Diabetic Autonomic Neuropathy

We examined the relationship between the Q-T interval and autonomic dysfunction in 74 diabetic patients and age-matched controls. The expected Q-T interval was standardized as a function of the R-R interval in 646 healthy controls, and the delta Q-T defined as the difference from the expected value. Propranolol increased delta Q-T, confirming this as a parameter of autonomic function. No relationship between delta Q-T and duration of disease, glucose control, retinopathy or proteinuria was observed. However, significant correlations were found between the delta Q-T and delay of nerve conduction velocity, orthostatic hypotension, altered Valsalva ratio, abnormal Valsalva overshoot, cold pressor response, decreased norepinephrine concentration and sympathetic function score in diabetic patients.

Comparison of Color Flow and 3D Image By Computer Graphics for the Evaluation of Carotid Disease

Morphologic characteristics and hemodynamics in the carotid artery were investigated in patients with stroke by use of Doppler color-flow analysis and three-dimensional (3D) analysis by computer graphics. The 3D imaging of the carotid bifurcation was reconstructed from a series of transverse B-mode images. Comparison of color-flow and 3D images demonstrated that wall configuration in vivo, dilation, and curvature play an important role in reversal of flow and stagnation at the carotid bifurcation. Furthermore, 3D analysis is very useful in mapping the distribution of the atheromatous plaques. The analyses suggest that it is important to investigate the wall configuration of the bifurcation in vivo on flow patterns, especially flow separation and stagnation, in relation to the development of atheromatous plaques.