Tomoichi Ohkubo

Enhancement of Oxidative Damage to Cultured Cells and Caenorhabditis elegans by Mitochondrial Electron Transport Inhibitors

The mechanisms that lead to mitochondrial damage under oxidative stress conditions were examined in primary and cultured cells as well as in the nematode Caenorhabditis elegans ( C. elegans ) treated simultaneously with electron transport inhibitors and oxygen gas. Oxygen loading enhanced the damage of PC 12 cells by thenoyltrifluoroacetone (TTFA, a complex II inhibitor), but did not by rotenone (a complex I inhibitor), antimycin (a complex III inhibitor), and sodium azide (a complex IV inhibitor). In primary hepatocytes, the enhancement was observed with the addition of sodium azide and rotenone, but not by TTFA or antimycin. In the nematode, only rotenone and TTFA enhanced the sensitivity under hyperoxia. These results demonstrate that highly specific inhibitors of electron transport can induce oxygen hypersensitivity in cell levels such as PC 12 cells and primary hepatocytes, and animal level of C. elegans . In addition the cell damage is different dependent on cell type and organism.

Impaired superoxide production in peripheral blood neutrophils of germ-free rats

A method of separating neutrophils from the peripheral blood of rats with 95% purity is described. To determine the role of antigenic stimulation in neutrophil function, neutrophils front germ‐free (GF) rats were compared with those of conventional (CV) rats. Neutrophil counts were lower in GF rats but the total number of monocytic cells was the same.

Time course of superoxide generation by leukocytes-The MCLA chemiluminescence system

This study was performed to examine the pattern of Superoxide (O2−·) generation from leukocytes using the O2−· specific chemiluminescence (CL) method.Cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-alpha]pyrazin-3-one (MCLA) was used as a CL probe. The appropriate conditions of the MCLA method was first determined for the evaluation of the time course of O2−· generation by leukocytes. The time course of O2−· generation obtained by the MCLA-CL system was compared with that by the luminol-dependent CL, electron spin resonance (ESR)/spin trapping, and cytochromec systems. Following stimulation by three different stimulants (PMA, OZ, FMLP), leukocytes continuously generated O2−· for up to 5 h in the MCLA-CL system, irrespective of the kind of stimulation. The curves obtained by generation ceased more rapidly in the luminol-CL, ESR/spin trapping, and cytochromec systems. A 50% activity of the initial value was observed at 70 min in the MCLA-CL system, but 30, 10 and 35 min in the other systems, respectively. The CL or O2−· generation value decreased to less than 1% (possible termination) at 300, 90, 120 and 180 min, respectively. With the exception of ESR studies with OZ, the cell viability was not significantly affected in any of the trials. These results indicate that leukocytes can generate O2−· much longer than previously estimated and that the MCLA-CL-system is the most suitable system for the measurement of the O2−· generation by leukocytes.

Purification of a serum DNA binding protein (64DP) with a molecular weight of 64,000 and its diagnostic significance in malignant diseases

Abstract A DNA binding protein with a molecular weight of 64,000(64DP) has been purified to homogeneity from human serum, and its quantitative assay has been developed. The average level of serum 64DP in 30 normal controls was 41.4 μg/ml, whereas it was 175 μg/ml in 87 patients with untreated malignant disease. Furthermore it was found to be elevated in all tested patients, 8 cases, with carcinoma in early stages. Serum 64DP has been found to be different from C3DP, CEA # or α-FP # , and it appears that this protein might prove to be a useful tumor marker in malignant diseases.

Peripheral Blood Neutrophils of Germ‐Free Rats Modified by In Vivo Granulocyte–Colony‐Stimulating Factor and Exposure to Natural Environment

Peripheral blood neutrophils from germ‐free (GF), specific‐pathogen‐free (SPF) and conventional (CV) rats were compared. Besides neutropenia and impaired superoxide anion generation as previously reported, it was found that GF rats had lower phagocytic function (70%) and generated less nitric oxide than the other rats. GF and SPF rats were injected with recombinant human granulocyte–colony‐stimulating factor (rhG‐CSF), or were transferred to natural environment. It was found that total number, phagocytosis, intracellular killing, ratio of phagocytosis versus killing (killing efficiency) and nitric oxide production induced by recombinant rat interferon‐γ (rrIFN‐γ) were normalized upon injection of rhG‐CSF. These results indicate that rhG‐CSF may stimulate neutrophil production and induce the expression of neutrophil receptors for phagocytosis and nitric oxide production in GF rats. Although lower than in CV rats, the level of superoxide produced was sufficient for normal neutrophil‐killing efficiency in SPF and GF rats. In SPF rats, this could be amended by exposure to natural environment. However, neither rhG‐CSF injection nor transfer to natural environment could increase the generation of superoxide anion in GF rats.

Heat production is a quantitative parameter for intracellular cell function

Abstract Heat measurements were found to provide a quantitative assay of phagocytosis in which there is a dramatic increase in heat production. Glycyrrhizin, an extract from Glycyrrhizaglabra, has a beneficial effect on patients with viral infections including hepatitis and AIDS. Therefore the in vivo effect of glycyrrhizin on the phagocytic function of blood neutrophils in rats was investigated. Glycyrrhizin was injected in vivo into rats twice a day for 2 days and the blood was taken from the descending aorta. Neutrophils were isolated and the phagocytic function was determined both by heat production using a thermoactive cell analyser (ESCO 3000) and by the conventional assay system using Saccharomyces cerevisiae. In vivo treatment of glycyrrhizin seemed to enhance heat production by 40% during phagocytosis, as compared with neutrophils from non-treated rats. However, it was not possible to detect this difference using the conventional assay system in which small functional changes cannot be determined. Therefore microcalorimetry can provide clear evidence of a small change in cell function and may lead to a new field in cell biology.

Association of sick building syndrome with neuropathy target esterase (NTE) activity in Japanese

Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP‐induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty‐eight SNP markers within the PNPLA6 gene were tested for association in a case–control study of 188 affected individuals and 401 age‐matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS.

Senescent case of cholesterol ester storage disease that progressed to liver cirrhosis with a novel mutation (N250H) of lysosomal acid lipase gene

The patient, a 69‐year‐old man, had a chief complaint of hepatomegaly. The liver was palpated four fingerbreadths below the costal margin, and the spleen was three fingerbreadths below the costal margin. There were no other abnormal findings. Laparoscopy showed that the liver resembled an orange‐yellow crayon in appearance and was nodular. The pathological findings of the liver biopsy tissue were consistent with liver cirrhosis. Inside the fibrous septum was an apparent aggregation of enlarged macrophages that phagocytosed lipid components, as well as enlarged Kupffer cells that phagocytosed lipid droplets. Electron microscopy showed the lipid droplets to have a moth‐eaten appearance. Using monocytes extracted from the peripheral blood, acid lipase activity was measured by fluorescence spectrometry using 4‐methylumbelliferone palmitate as a substrate. This patients human lysosomal acid lipase activity was 0.020 nM/min per 106 cells, corresponding to 5.9% of that in healthy subjects (0.332 ± 0.066 nM/min per 106 cells). Cholesterol ester storage disease was therefore diagnosed. The acid lipase A base sequence obtained from leukocytes by direct sequencing was compared with a library. This patient had a point mutation of N250H/N250H in exon 7, a novel gene abnormality that has not previously been reported.

A novel in vitro system for studying cardiomyocyte differentiation with medaka embryonic cells.

Our studies revealed that dissociated cells from medaka (the fresh-water fish, Oryzias latipes ) blastula-stage embryos differentiate into many rhythmically contracting cells when incubated with a conditioned medium from a cell line. Analyses of these cells by immunostaining, electron microscopy, expression of marker genes, action potential recordings and pharmacological responses all showed the characteristics of myocardial cells. We succeeded in purifying the cardiac cell-inducing factor from the conditioned medium by 523,100-fold with 8% recovery of the protein. Analysis of its amino-acid sequence by mass spectrometry revealed the factor to be medaka activin A2 (homodimer of inhibin betaA2 subunit). Recombinant human activin A showed the same cardiac cell-inducing activity toward medaka embryonic cells. Our results demonstrate that activin A is a potent factor for medaka myocardial cell differentiation. This culture method should provide a novel and simple experimental system to study cardiomyocyte differentiation in vitro.

Illustrations of the value of calorimetry in biology

Calorimetry was used to measure heat generated by both living microorganisms and macroorganisms. This seemingly simple measurement can give an evaluation of the state of an organism and can differentiate between rest, sleep, under anaesthesia, activity and inactivity. The great advantage of calorimetry is that the measurement is not harmful and does not cause any damaging effect on the samples.