Isolation and characterization of mammalian D-aspartyl endopeptidase.

The accumulation of D-isomers of aspartic acid (D-Asp) in proteins during aging has been implicated in the pathogenesis of Alzheimer's disease (AD), cataracts and arteriosclerosis. Here, we identified a specific lactacystin-sensitive endopeptidase that cleaves the D-Asp-containing protein and named it D-aspartyl endopeptidase (DAEP). DAEP has a multi-complex structure (MW: 600 kDa) and is localized in the inner mitochondrial membrane. However, DAEP activity was not detected in E. coli, S. cerevisiae, and C. elegans. A specific inhibitor for DAEP, i-DAEP: (benzoyl-L-Arg-L-His-[D-Asp]-CH(2)Cl; MW: 563.01), was newly synthesized and inhibited DAEP activity (IC(50), 3 microM), a factor of ten greater than lactacystin on DAEP. On the other hand, i-DAEP did not inhibit either the 20S or 26S proteasome. And we identified succinate dehydrogenase and glutamate dehydrogenase 1 as components of DAEP by affinity label using biotinylated i-DAEP. In the long life span of mammals, DAEP may serve as a scavenger against accumulation of racemized proteins in aging. Insights into DAEP will provide the foundation for developing treatments of diseases, such as AD, in which accumulation of D-Asp-containing proteins are implicated.

Age-related changes of alpha-crystallin aggregate in human lens.

Lens alpha-crystallin, composed of two subunits alpha A- and alpha B-crystallin, forms large aggregates in the lens of the eye. The present study investigated the aggregate of human lens alpha-crystallin from elderly and young donors. Recombinant alpha A- and alpha B-crystallins in molar ratios of alpha A to alpha B at 1:1, corresponding to the aged sample, were also studied in detail. We found by ultra-centrifugation analysis that the alpha-crystallin aggregate from elderly donors was large and heterogeneous with an average sedimentation coefficient of 30 S and a range of 20-60 S at 37 degrees C. This was higher compared to the young samples that had an average sedimentation coefficient of 17 S. The sedimentation coefficients of recombinant alpha A- and alpha B-crystallins were approximately 12 S and 15 S, respectively. Even when recombinant alpha-crystallins were mixed in molar ratios equivalent to those found in vivo, similar S values as the native aged alpha-crystallin aggregates were not obtained. Changes in the self-association of alpha-crystallin aggregate were correlated to changes in chaperone activity. Alpha-crystallin from young donors, and recombinant alpha A- and alpha B-crystallin and their mixtures showed chaperone activity, which was markedly lost in samples from the aged alpha-crystallin aggregates.

Serum lipid metabolism abnormalities and change in lipoprotein contents in patients with advanced-stage renal disease.

BACKGROUND: Arteriosclerosis is the major cause of death in patients with chronic renal failure. There is much interest in the lipid metabolism of patients treated with hemodialysis. METHODS: We analyzed low-density lipoproteins (LDL) and high-density lipoproteins (HDL) in chronic renal failure (CRF) patients according to patients on hemodialysis (HD), patients with diabetic nephropathy before initiation of dialysis (DN), and patients with chronic glomerulonephritis in the conservative stage (CGN); and compared the lipid metabolic abnormalities in patients on hemodialysis and those not yet on hemodialysis. We also analyzed the qualitative abnormalities of LDL and HDL and their relationship with the pathological stages. RESULTS: Electrophoretic patterns identified small LDL particles and small HDL particles in the three groups, and the degree of denaturation was more enhanced in CRF patients in the conservative stage than in HD patients. For LDL susceptibility to oxidation LDL (oxLDL) by addition of Cu(2+), the lag time was approximately 57 min in healthy controls and CGN patients, but was prolonged to approximately 75 min in HD and DN patients. For HDL susceptibility to oxidation HDL (oxHDL), HD, DN and CGN patients showed lag times shorter than those found in healthy control subjects. These results showed that LDL and HDL in the serum of CRF patients were in a state of enhanced susceptibility to oxidative modification. In Western blot analysis using anti-human-denatured LDL and anti-human-oxidized HDL monoclonal antibodies, bands of low molecular oxLDL at 150-197 kDa were detected in all CRF patients, with marked tailing in CGN patients. Similarly, bands of small oxHDL particles at 110 and 120 kDa were found in HD, DN and CGN patients. CONCLUSIONS: Oxidative modification of both LDL and HDL occurs in patients with advanced CRF resulting in small lipoproteins. Increased production of oxLDL and oxHDL is the main cause of lipid metabolic abnormality in CRF patients.

[A case of adult Wilms' tumor].

Wilms' tumor is very rarely found in adults and there are no established treatment guidelines for such tumors in adults. A 56-year-old woman was referred to our hospital for further examination of macroscopic hematuria. Computed tomography scan revealed a large right renal mass with enlarged lymph nodes. Angiography showed a hypovascular tumor. She underwent right nephrectomy and resection of lymph node metastasis with a diagnosis of malignant renal tumor. Histopathological examination revealed nephroblastoma with lymph node metastasis. The disease was classified as stage III according to the National Wilms' Tumor Study classification. The patient received adjuvant chemotherapy consisting of ifosfamide, cisplatin, and etoposide. This protocol was selected because of the published poor results with the standard Wilms' tumor chemotherapeutic agents when used in adults. She remained without tumor recurrence as of six months after surgery. Development of better therapeutic approaches to adult Wilms' tumor is awaited.

Atypical alkaline phosphatase isozymes in serum and urine of patients with renal failure.

BACKGROUND: Alkaline phosphatases (ALPs) originating from different organs are frequently detected in the serum and urine of patients with renal failure. METHODS: We investigated the characteristics of ALPs in the serum and urine of 108 patients with chronic renal failure (CRF) and of 106 healthy control subjects. RESULTS: In polyacrylamide gel electrophoresis, three atypical ALP bands in serum of patients were designated as atypical-s1, -s2 and -s3, respectively. In contrast, five atypical bands (u1, u2, u3, u4 and u5) were detected in the urine of patients. The atypical ALPs were electrophoretically isolated and assayed to determine their biochemical properties, i.e., neuraminidase sensitivity, heat stability, reactivity to anti-intestinal or anti-tissue nonspecific ALP antibodies, molecular sizes and sugar chain heterogeneities. From these results, we found that atypical-s1 and -s2 were the intestinal-type ALP, while s3 was the tissue-unspecific type ALP. Atypical-u1, -u2 and -u3 were high-molecular type ALPs, which we suggested as the ones that originated from the intestine. Atypical-u4, a tissue-unspecific type ALP, was detected with considerable frequency in the urine of patients. In patients with CRF, the appearance of these atypical ALPs was accompanied by a deterioration of the creatinine clearance. CONCLUSIONS: The appearance of atypical ALPs in the serum and urine of patients with CRF may be a useful marker for renal disease.

Isolation of an androgen-inducible novel lipocalin gene, Arg1, from androgen-dependent mouse mammary Shionogi carcinoma cells.

Here we report isolation of an androgen-regulated novel gene from an androgen-dependent mouse mammary Shionogi carcinoma SC-3 cell line. Using a polymerase chain reaction-based subtraction method and Northern blotting analysis, we isolated four androgen-inducible genes from SC-3 cells. Nucleotide sequencings identified three of the genes as cyclin D1, beta-catenin, and fatty acid synthase, respectively, but the fourth, a gene tentatively named as Arg1 (androgen-regulated gene 1), remained undefined. The cloned 2.0-kb sized Arg1 cDNA encoded 414 amino acid sequences. The deduced amino acid sequences, sharing about 30% homology with cathepsin family members at a protein level, had relatively conserved residues around the three proteinase active sites reported earlier. In Northern blotting, Arg1 mRNA was found in kidney, heart, lung, and to a lesser degree, in spleen and liver. Its transcripts were also detected in male reproductive organs on RT-PCR. In addition, its expression levels in prostate were markedly reduced after castration. Unexpectedly, Arg1-expressing COS1 cells showed no significant proteinase activity to various synthesized substrates under neutral or acidic conditions in this study. This might have been due to the replacement of the cysteinyl active site for proteinase to serine residue in the Arg1 amino acid sequences. Given that Arg1 also contains a lipocaline signature known as a binding motif for small hydrophobic molecules at the center of its amino acid sequences, Arg1 is a lipocalin family gene regulated by androgens in prostate and Shionogi carcinoma cells.

[A case of clinically multifocal renal cell carcinoma].

A 61-year-old woman was diagnosed with a renal tumor of the left kidney by ultrasound sonography during a health check-up. Computerized tomography (CT) and colored Doppler ultrasound sonography demonstrated two hypervascular tumors as typical renal cell carcinomas. A radically nephrectomized specimen was step-sectioned. Four tumor nodules were detected macroscopically, and 47 small nodules were detected microscopically, showing the clear cell type and alveolar growth pattern. Then all nodules including the 47 small nodules were diagnosed renal cell carcinoma.

Cilnidipine more highly attenuates cold pressor stress-induced platelet activation in hypertension than does amlodipine.

The clinical significance of N-type calcium channel blockade has not been fully examined. We here compared the effects of the N-type calcium channel blockers cilnidipine and amlodipine on the sympathetic nervous system and platelet function in hypertension under resting and stressed conditions. Thirty-two patients with hypertension (58+/-9 years) received cilnidipine or amlodipine for 4 weeks in this crossover study. On day 28 of each treatment, plasma levels of epinephrine (EP), norepinephrine (NEP), and beta-thromboglobulin (BTG), and EC50 of ADP-induced platelet aggregation (ADPE50) were determined at rest and after a cold pressor test. On day 29, the group receiving cilnidipine was switched to amlodipine treatment, and vice versa. At rest, the blood pressure, heart rates, EP, NEP, ADPEC50, and BTG, were similar in both treatments. After the cold pressor test, increases in EP (35+/-17 to 44+/-25 pg/ml; p<0.05) and BTG (40+/-13 to 49+/-22 ng/ml; p<0.01) and a decrease in ADPEC50 (32+/-26 to 27+/-24 micromol; p<0.05) were observed in the amlodipine treatment, but not in the cilnidipine treatment. In addition, the increase in NEP was significantly greater (p<0.05) in the amlodipine (276+/-78 to 318+/-87 pg/ml; p<0.01) than in the cilnidipine treatment (273+/-88 to 291+/-100 pg/ml; p<0.05). Cilnidipine more highly attenuates the activation of platelet function in response to cold pressor stress than does amlodipine. Attenuated activation of the sympathetic nervous system via N-type calcium channel blockade may contribute to this phenomenon.

Proteoliposomes colocalized with endogenous mitochondria in mouse fertilized egg.

Colocalization of mitochondria is the first step of intermitochondrial interaction or fusion in a cell. Here, we showed colocalization between exogenous mitochondria and endogenous ones or between exogenous proteoliposomes and endogenous mitochondria in mouse fertilized eggs by confocal laser microscopy. Isolated mitochondria from mouse liver and proteoliposomes containing mitochondrial membrane were directly labeled with red fluorescent aliphatic marker, PKH26, which is incorporated into lipid membrane, and then were microinjected into fertilized mouse eggs. Exogenous mitochondria appeared to be almost colocalized with endogenous mitochondria at the 4- and 8-cell stages, when mitochondria were stained with Rhodamine 123 (green fluorescent marker). On the contrary, when liposomes consisted of soy bean phospholipid were microinjected into the eggs as a control, their localization was different from that of endogenous mitochondria. Next, the submitochondrial particles and proteoliposomes were microinjected. Both the proteoliposomes and the submitochondrial particles appeared to colocalize with endogenous mitochondria at the 4-cell stage. These results suggest the existence of a factor that makes liposomes colocalize with mitochondria. Such a proteoliposome would be useful for the development of mitochondrial gene transfer techniques.

Ganglioside GD3 and its mimetics induce cytochrome c release from mitochondria.

Ganglioside GD3 induced the release of cytochrome c from isolated rat liver mitochondria. This process was completely prevented by cyclosporin A and partially prevented by a cysteine protease inhibitor, n-acetyl-leu-leu-norleucinal. Cyclosporin A is a potent inhibitor of the permeability transition pore, whereas n-acetyl-leu-leu-norleucinal has no effect on this pore. These results indicate that the release of cytochrome c from mitochondria requires both the opening of the permeability transition pore and a cysteine protease inhibitor-sensitive mechanism. Gangliosides GD1a, GD1b, GT1b, and GQ1b along with the synthetic GD3 mimetics TMS-42 and CI-22, which are glycerophospholipids carrying a disialo residue, also induced cytochrome c release. In contrast, gangliosides GM1, GM2, and GM3 did not induce cytochrome c release. These results indicate that two sialo residues must play an important role in the induction of cytochrome c release by gangliosides.

Close relationship of abnormal glucose tolerance with endothelial dysfunction in hypertension.

Hypertension is frequently accompanied by left ventricular hypertrophy, endothelial dysfunction, and abnormal glucose metabolism. However, no study has examined the relative pathological significance of left ventricular hypertrophy and abnormal glucose metabolism on endothelial dysfunction in hypertension. This study was conducted to evaluate whether abnormal glucose tolerance assessed by 75-g oral glucose tolerance test or left ventricular hypertrophy is more closely associated with endothelial dysfunction in never-treated hypertensive patients without elevated fasting blood glucose. We studied 107 unmedicated hypertensive patients (mean age, 54+/-10 years) whose fasting blood glucose was <7.0 mmol/L. Endothelial function was assessed by change in brachial artery diameter in response to reactive hyperemia, and left ventricular mass index was determined by ultrasonography. Simple linear regression analysis demonstrated that endothelial function significantly correlated with left ventricular mass index and 2-hour blood glucose in 75-g oral glucose tolerance test, but not with fasting blood glucose. Multiple linear regression analysis revealed that endothelial function significantly correlated with 2-hour blood glucose (beta=-2.68, P<0.05) after we controlled for other clinical variables. Patients were divided into 3 groups according to 2-hour blood glucose levels. Endothelial function was more impaired in patients with diabetes (n=12; 4.7+/-1.8%) and in those with impaired glucose tolerance (n=31; 6.3+/-2.9%) than in those with normal glucose tolerance (n=64; 8.4+/-4.5%) (P<0.05), but left ventricular mass index was similar in these 3 groups. Abnormal glucose tolerance assessed by 75-g oral glucose tolerance test, rather than left ventricular hypertrophy, may have direct pathophysiological relevance to endothelial dysfunction in borderline to moderate hypertensive patients.

Thrombin stimulates pertussis toxin-sensitive and -insensitive GTPase activities and ADP-ribosylation of G(i) in human neuroblastoma SH-EP.

Kinetic interaction between thrombin receptor and G proteins was investigated in human epithelial neuroblastoma cell line, SH-EP. In these cells, both alpha-thrombin and SFLLRNP (one-letter amino-acid code) stimulated GTPase activity and enhanced cholera toxin-catalyzed ADP-ribosylation of G(i2) in a concentration-dependent manner. Basal GTPase activity was attenuated by pertussis toxin treatment by 35%, however, agonist stimulation was preserved significantly. These results together indicated that thrombin receptor simultaneously activates G(i2) and PTX-insensitive G protein(s).

A dose-escalation and pharmacokinetic study of subcutaneously administered recombinant human interleukin 12 and its biological effects in Japanese patients with advanced malignancies.

A pilot dose-escalation study of recombinant human interleukin 12 (rhIL-12) was conducted in Japanese patients with advanced malignancies. Cohorts of three patients received escalating doses of rhIL-12 that increased from 50 to 300 ng/kg/day s.c. three times a week for 2 weeks followed by 1-week rest. The same dosage and schedule was repeated for two additional courses. Sixteen previously treated patients were registered, and 15 were evaluated. Common toxicities were fever and leukopenia; the abnormality of laboratory tests included elevations in aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, C-reactive protein, and beta2-microglobin. Dose-limiting toxicity was the grade 3 elevation of aminotransferases, and was observed in two of six patients at the 300-ng/kg dose level after the first course in one patient and after the third course in the other. Leukopenia was observed at all of the dose levels; two of six patients at 300 ng/kg experienced grade 3 leukopenia. Thus, 300 ng/kg was determined to be the maximum acceptable dose. Peak plasma levels of rhIL-12 decreased in the second courses, but the areas under the curve were almost the same in the first and second courses. Biological effects included increases of plasma levels of IFN-gamma, tumor necrosis factor-alpha, IL-6, IL-10, and neopterin. In two patients with renal cell carcinoma, complete response and partial response of metastatic tumors were observed with 50 and 300 ng/kg; the responses lasted for 5 and 3.5 months, respectively. Although immunological response to rhIL-12 varies depending on administration route and schedule and on patients' physiological conditions, the recommended dose for Phase II studies is 300 ng/kg s.c. three times a week for 2 weeks followed by 1-week rest.

Milk-borne insulin with trypsin inhibitor in milk induces pancreatic amylase development at the onset of weaning in rats.

BACKGROUND: The physiologic significance of milk-borne hormones and growth factors for internal organs of suckling animals is poorly understood. In this study the significance of milk-borne insulin was evaluated, as well as its combination with trypsin inhibitor, and its role in the development of pancreatic digestive capacity at the time of weaning was investigated. METHODS: Experiments were performed using insulin-deficient milk formula (standard formula), insulin (20 ng/ml) formula, or insulin with trypsin inhibitor (1 U/ml) formula by a rat artificial-rearing technique. RESULTS: In 17-day-old rats administered standard formula, the plasma insulin level was as low as that in 14-day-old rats. When insulin-trypsin inhibitor formula was administered to rat pups, the plasma insulin level was significantly higher than those in rats given standard or insulin formula. In rats artificially reared on standard formula, the usual developmental increases in pancreatic amylase activity and plasma insulin concentration at the beginning of weaning did not occur. Insulin formula elevated the pups' plasma insulin concentration and amylase activity at the onset of weaning but not to the levels observed in mother-reared rats. In rats reared on insulin-trypsin inhibitor formula, the developmental increases in the plasma insulin concentration and amylase activity observed in mother-reared rats were induced. CONCLUSIONS: The present study demonstrates the necessity of milk-borne insulin for the development of pancreatic amylase during the weaning period.

Low susceptibility of Long-Evans Cinnamon rats to N-butyl-N-(4-hydroxybutyl)-nitrosamine-induced urinary bladder carcinogenesis and inhibitory effect of urinary copper.

We studied the susceptibilities to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis of male Long-Evans Cinnamon (LEC), F344 and Long-Evans Agouti (LEA) rats. Male rats (n=21) were given 0.1% BBN in their drinking water from week 6, 8 and 10 for one week, and killed in week 56. The incidences of transitional cell tumors (papillomas plus carcinomas) in BBN-treated LEC and F344 rats were 12% and 76%, respectively (P<0.001, experiment 1), and those in LEC and LEA rats were 11% and 95%, respectively (P<0.001, experiment 2). When male LEC and F344 rats were given 0.1% BBN in their drinking water for 7 days, the intake of BBN and the urinary concentration of its active metabolite, N-butyl-N-(3-carboxypropyl)nitrosamine (BCPN), were higher in the LEC rats (P<0.01). The urinary pHs of untreated LEC and F344 rats were similar between week 6 and 30. The urinary copper concentration was lower in LEC rats before jaundice than in F344 rats, but its concentrations in 28- and 50-week-old LEC rats were 1.7 and 2.3 times those in F344 rats. In a two-stage carcinogenesis study using F344 rats, i.p. injections of cupric nitrilotriacetate increased urinary copper excretion, and inhibited BBN-induced bladder carcinogenesis. In a two-stage carcinogenesis study using LEC rats, oral administration of D-penicillamine decreased urinary copper excretion, and increased BBN-induced bladder cancer, although the difference was not significant. These data show that LEC rats are resistant to bladder carcinogenesis and suggest that urinary copper has a significant role in their resistance.

Inhibitory effects of tea extracts on the mutagenicity of 1-methyl-1, 2,3,4-tetrahydro-beta-carboline-3-carboxylic acid on treatment with nitrite in the presence of ethanol.

It has been shown that the mutagenicity of 1-methyl-1,2,3, 4-tetrahydro-beta-carboline-3-carboxylic acid (MTCCA), a major mutagen precursor in soy sauce on treatment with nitrite and ethanol, was strongly decreased by the addition of hot water extracts of green, black and oolong teas in the reaction mixture when it was treated with 50mM nitrite at pH3.0, 37 degrees C for 60min in the presence of 7.5% ethanol. The mutagenicity-decreasing activity of the teas was scarcely decreased by washing the teas with chloroform and benzene and was partly decreased by butanol and ethyl acetate. Typical polyphenols such as catechins were shown to have the antimutagenicity dose dependently. The antimutagenicity and the reducing power of tea extracts gave a positive good correlation. The results suggest that the mutagenicity of MTCCA on treatment with nitrite in the presence of ethanol may be decreased by the mixed fractions of lyophilic components such as polyphenols, which have high reducing power such as catechins and the other compounds which have little reducing power including the derivatives of the catechins and so on. Although the antimutagenicity of teas and catechins was also considerably effective when they were added after the nitrosation, that of black tea and some catechins was less effective.

Metabolism of amyloid precursor protein in COS cells transfected with a beta-secretase candidate.

Thimet oligopeptidase (TOP) is a thiol- andmetallo-dependent peptidase and has been shown to beone of the beta-secretase candidates. TOPexpressed in COS cells cleaved amyloid precursorprotein (APP) at the beta-secretase site, and wefound a proteolytic product of APP called secretedform of APP by beta-secretase (sAPPbeta) in theconditioned media. Here we demonstrate thatsAPPbeta was increased in conditioned media whenTOP was coexpressed in COS cells with APP and treatedwith an ADAM inhibitor SI-27. In addition, althoughTOP expressed in COS cell was localized at nuclei orGolgi apparatus, it exclusively colocalized at Golgiapparatus when APP was coexpressed with TOP.

Incidence rate of satellite tumors in renal cell carcinoma.

BACKGROUND: Nephron-sparing surgery for incidentally detected small renal tumors has been performed. The main objection to such surgery concerns the incidence rate of satellite renal tumors. In this study, the authors analyzed the rate of incidence and proliferative potential of satellite renal tumors. METHODS: The tumors of 124 renal cell carcinoma patients with a clinically identified unilateral and single tumor measuring

Characteristics of cardiovascular morphology and function in the high-normal subset of hypertension defined by JNC-VI recommendations.

A cross-sectional study was conducted to compare the morphological and functional characteristics of the cardiovascular system among subgroups of hypertension defined by the JNC-VI recommendations. One hundred and sixteen subjects (normotensives and unmedicated hypertensives: 49+/-10 yr) were classified into 4 groups based on the criteria of JNC-VI: normotensive (NOR: n = 38), high-normal blood pressure (HN: n = 16), stage 1 hypertensive (SI: n = 28), and stage 2 to 3 hypertensive (SII-III: n = 34). Ultrasonographic examinations of the heart and carotid artery were performed in all subjects, and the following parameters were obtained: left ventricular mass index (LVMI), relative wall thickness at end-diastole (RWTd), cardiac diastolic function (A/E), common carotid artery diameter (CAD), intimal media thickness of the common carotid artery (IMT), and distensibility of the common carotid artery (Distens). RWTd, A/E, and IMT in SI (RWTd, 0.41+/-0.07; A/E, 1.21+/-0.41; IMT, 0.69+/-0.17 mm) and SII-III patients (0.40+/-0.08, 1.38+/-0.33, 0.80+/-0.21 mm) were larger than those in NOR patients (0.33+/-0.03, 0.86+/-0.21, 0.56+/-0.10 mm) (p < .01). Furthermore, LVMI in SII-III (135.5+/-35.5 g/m2) patients was larger than that in NOR patients (99.4+/-17.5 g/m2) (p < .05). RWTd in HN patients (0.37+/-0.06) was significantly higher than that in NOR patients (p < .05). A/E tended to be larger in HN than in NOR patients (p < 0.1). In the normotensives, no significant difference in any of the parameters was detected between those with optimal (n = 19) and normal (n = 19) blood pressure. Thus, both morphological and functional changes were associated with elevation of blood pressure. Cardiac morphological adaptation and functional impairment were present even in subjects with high-normal blood pressure level, while there were no significant differences between the normal and optimal subsets.