The activity of leucine aminopeptidase in serum was determined by measuring the increase over a given time period in current due to the oxidation of 5-aminosalicylic acid as enzymatically released from leucine-3-carboxy-4-hydroxyanilide using a dialysis membrane-covered glassy-carbon electrode. The activities determined amperometrically using this method for eighteen serum samples were in excellent agreement with those determined using a colorimetric method with leucine-p-nitroanilide as a substrate (correlation coefficient between the two sets of results, 0.998).

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We have developed an HPLC system for the simultaneous determination of neurotransmitters such as catecholamines, serotonin, and their precursors and metabolites. This system consists of two reversed-phase columns, a column switching device, a pair of electrodes for elimination of interferents and two sets of new electrochemical detectors with four electrodes. Using this system, adequate separation of peaks for 17 kinds of neurotransmitters in a standard solution required only 17 min. Norepinephrine (NE), 4-hydroxy-3-methoxyphenylethyleneglycol (MHPG), 3, 4-dihydroxyphenylacetic acid (DOPAC), 5-hydroxyindoleacetic acid (5-HIAA), homovanillic acid (HVA) and tryptophan (TRP) levels in cerebrospinal fluid (CSF) were determined without any pretreatment of CSF samples. The between-day CVs were less than 5% for all analytes in CSF. The analytical rates of recoveries were between 91 and 101%. CSF samples from patients with Alzheimer's disease, vascular dementia and Parkinson's disease were analyzed, and it was found that the concentrations of DOPAC, 5-HIAA and HVA were significantly lower in CSF of patients with these diseases than in CSF of the age-matched controls.

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Atrial natriuretic peptide (ANP)(10-8mol/l), brain natriuretic peptide (BNP)(10-8mol/l), the protein kinase C (PKC) inhibitors H-7 (10-5mol/l) and staurosporin (10-8mol/l) suppressed aldosterone secretion in cultured bovine adrenal glomerulosa cells by 40 to 50 % compared with untreated control cells. When cells incubated with 10-8mol/l angiotensin II (All) were also treated with ANP, BNP, or PKC inhibitors, aldosterone secretion was 50 to 60 % of that seen in untreated cells. ANP, BNP and PKC inhibitors suppressing the expression of PKC, suggested that PKC plays a role in aldosterone secretion. Our results suggest that ANP and BNP may have an unknown second messenger pathway in which the suppression of PKC is essential for the expression of their physiological actions.

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The infectivity of human papillomavirus (HPV) is closely linked to characteristics of epithelial lesions in the uterine cervix. In our previous study, we reported amplification of c-myc gene, and pointed out the possibility that tumorigenesis might be enhanced by the amplification of this oncogene. Furthermore, we found that HPV exists in the host cell in various forms, such as the Episome or Integration form. Based on these results, we proposed the hypothesis that the chromosomal integration of HPV-DNA (deoxyribonucleic acid) might play a significant role in tumorigenesis of cervical cancer. In order to substantiate this hypothesis, we studied the state of HPV-DNA in tissue samples of patients with condyloma acuminatum, mild dysplasia or invasive carcinoma using a two-dimensional Southen hybridization technique. The HPV-DNA types found were: HPV6 in one patient with condyloma acuminatum, HPV16 in thirteen patients with mild dysplasia or invasive carcinoma, and HPV18 in two patients with invasive carcinoma. As for the form of HPV in host cells, it was notewothy that the DNA in all cases of benign lesions such as condyloma acuminatum and mild dysplasia existed as Episomes, whereas the DNA in 11 out of 12 cases of invasive carcinoma was Integrations.

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The stimulated productions of superoxide anion in polymorphonuclear leukocytes obtained from diabetic patients and control subjects were measured. Superoxide production was estimated with a flowcytometer by measuring oxidized fluorescent products of 2, 7-dichlorofluorescin diacetate incorporated in leukocytes.
The superoxide production stimulated by phorbol myristate acetate (PMA) was significantly less in diabetic patient's leukocytes than in control subjects' leukocytes (mean±SE 12900±257 versus 11400±463 counts; p<0.01), and that stimulated by opsonized zymozan also tended to be less in diabetic patients' leukocytes than in those of control subjects. The PMA stimulated polymorphonuclear leukocytes was negatively correlated with fasted blood glucose levels in the diabetic patients (r=-0.500, p<0.05), but not with glycated protein marker levels.
Stimulated superoxide production in polymorphonuclear leukocytes decreased after incubation with glucose at concentrations between 20 to 50mmo1/1 (p<0.05).
We conclude that stimulated superoxide anion production in polymorphonuclear leukocytes was impaired by hyperglycemia. This impairment may be the consequence of a shrunken NADPH pool depleted via the sorbitol pathway in conditions of hyperglycemia and thought to be one of the reasons for the vulnerability to infection of diabetic patients.

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We have described the diazo coupling reactions of the photoproducts of bilirubin separated by thin-layer chromatography using silicagel FM258 plate-Wako (Wako Pure Chemical Industries, Ltd.) and a mixture of chloroform: methanol: acetic acid (97: 2: 1, v/v) as a developing solvent. In the bilirubin solution irradiated with a fluorescent lamp (visible light), eleven substaces including biliverdin were detected. The ten substances separated other than biliverdin were classified into three groups, with absorption maxima of about 450 nm (six substances), 420 nm (three substances) and 388 nm (one substance). All these photoproducts reacted with the diazonium salt of sulfanilic acid to form a purple product with absorption maximum of about 550 nm. However, upon oxidation with a nitrous acid solution, the six photoproducts with absorption maxima of about 450 nm each yielded a green product with absorption maxima at about 700 and 370 nm, and the four photoproducts with absorption maxima of about 420 or 388 nm yielded a colorless substance with an absorption peak of about 360 nm. In addition, it was confirmed that bilirubin IIIα and XIIIα were also produced from bilirubin IXα by irradiation with a fluorescent lamp.

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An immunoradiometric assay for group II phospholipase A2 (PLA2) in human serum was developed using a combination of two monoclonal antibodies.
Polystyrene beads were coated with monoclonal antibody (No.PL-71), using glutaraldehyde as the coupling reagent, and 125 I-labeled monoclonal antibody (No.PL-78) was prepared using the chloramine T method.
The working range of this assay was 0.5 to 200ng/ml, and the assay was unaffected by the serum matrix. The within-and between-assay coefficients of variation were 1.3-2.9% and 2.6-9.3%, respectively. The group II PLA2 level in sera of healthy individuals was 2.13±0.49ng/ml, while that of patients with rheum atoid arthritis varied between 4.9-111ng/ml. The method described here was highly reliable and required very short incubation time. It appears to be useful in clinical testing for rheumatoid arthritis.

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A newly developed plastic formed carbon electrode (PFC electrode) produced a reproducible anodic current due to the oxidation of NADH at potentials less positive than those required by ordinary electrodes such as glassy-carbon electrodes. A dialysis membrane-covered PFC electrode produced steady-state currents linearly proportional to concentrations of NADH ranging from 5 to 1500μmol/l. The within-day variation for measurements was 3.1% for five runs at an NADH concentration of 100μmol/l. Activities of lactate dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (HBD) in serum were determined by measuring the decrease in current due to oxidation of NADH for 3 min at 400mV and 37°C in Tris buffer of pH 7.5 containing 350μmol/l NADH and 2 mmol/l pyrurate, or 4 mmol/l α-ketobutyrate. Serum was diluted to yield a concentration one-fifteenth that of the sample. Measurements of activities obtained using this method agreed well with those obtained using UV method (r=0.993, of LDH and HBD n=15 for LDH; r=0.999, n=15 for HBD).

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We developed a high-performance liquid chromatographic method with fluorometric detection using a column-switching system for the determination of retinol levels in human serum. The lower limit of detection of retinol in serum was 30 ng/ml. Retinol in sample solutions was stable at room temperature in room lighting for at least 30 h. The recovery of retinol in serum was 99% over the range 19.6 to 196 ng/0.2ml serum. Withinand between-assay reproducibilities (CV) for samples containing three concentrations of retinol (low, medium and high) were 0.8, 1.4 and 1.1% (n=10 each), and 5.0, 2.3 and 1.8% (n=5 each), respectively. A high degree of correlation was present between serum levels of retinol as determined using the present method and those determined using the conventional method (r=0.95, Syx=61, n=35).

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