Yoshio Nomura

Culture in Vector-Averaged Gravity Under Clinostat Rotation Results in Apoptosis of Osteoblastic ROS 17/2.8 Cells

Space flight experiments and studies carried out in altered gravity environments have revealed that exposure to altered gravity conditions results in (mal)adaptation of cellular function. In the present study, we used a clinostat to generate a vector‐averaged gravity environment. We then evaluated the responses of osteoblast‐like ROS 17/2.8 cells subsequent to rotation at 50 revolutions per minute (rpm) for 6–24 h. We found that the cells started to detach from the substrate between 12 h and 24 h of rotation in clinostat but not in stationary cultures or after horizontal rotation (the latter serving as a motion control for turbulence, shear forces, and vibrations). At 24 h, 35% of clinorotated cells had detached and the cells underwent apoptotic death as evidenced by DNA fragmentation analysis, terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate‐biotin nick end labeling (TUNEL) staining, and flow cytometry with Annexin V staining. The apoptotic death was associated with perinuclear distribution of cell‐surface integrin β1 and disorganization of actin cytoskeleton. These results suggest that vector‐averaged gravity causes apoptosis of osteoblasts by altering the organization of the cytoskeleton. We hypothesize that apoptotic death of osteoblasts might play an important role in the pathogenesis of osteoporotic bone loss as observed in actual space flights.

4-(O-Benzylphenoxy)-N-Methylbutylamine (Bifemelane) and Other 4-(O-Benzylphenoxy)-N-Methylalkylamines as New Inhibitors of Type A and B Monoamine Oxidase

Abstract: 4‐(O‐Benzylphenoxy)‐N‐methylbutylamine (Bifemelane, BP‐N‐methylbutylamine), a new psychotropic drug, was found to inhibit monoamine oxidase (MAO) in human brain synaptosomes. It inhibited type A MAO (MAO‐A) competitively and type B (MAO‐B) noncompetitively. BP‐N‐methylbutylamine had a much higher affinity to MAO‐A than an amine substrate, kynuramine, and it was a more potent inhibitor of MAO‐A than of MAO‐B. The Ki values of MAO‐A and ‐B were determined to be 4.20 and 46.0 μM, respectively, while the Km values of MAO‐A and ‐B with kynuramine were 44.1 and 90.0 μM, respectively. The inhibition of MAO‐A and ‐B by BP‐N‐methylbutylamine was found to be reversible by dialysis of the incubation mixture. MAO‐A in human placental and liver mitochondria and in a rat clonal pheochromocytoma cell line, PC12h, was inhibited competitively by BP‐N‐methylbutylamine, while MAO‐B in human liver mitochondria was inhibited noncompetitively, as in human brain synaptosomes. BP‐N‐methylbutylamine was not oxidized by MAO‐A and ‐B. The effects of other BP‐N‐methylalkylamines, such as BP‐N‐methylethylamine, ‐propylamine, and ‐pentanylamine, on MAO activity were examined. BP‐N‐methylbutylamine was the most potent inhibitor of MAO‐A, and BP‐N‐methylethylamine and ‐propylamine inhibited MAO‐B competitively, whereas BP‐N‐methylbutylamine and ‐pentanylamine inhibited it noncompetitively. Inhibition of these BP‐N‐methylalkylamines on MAO‐A and ‐B is discussed in relation to their chemical structure.

Quinolinic acid: an endogenous inhibitor specific for type B monoamine oxidase in human brain synaptosomes.

Quinolinic acid (QUIN), a well-known excitotoxin, was found to inhibit type B monoamine oxidase (MAO-B) in human brain synaptosomal mitochondria. By kinetic analysis, the inhibition of MAO-B activity by QUIN was competitive with the substrate, kynuramine. On the other hand, type A MAO (MAO-A) activity in human brain synaptosomal mitochondria and human placental mitochondria was not affected by QUIN. The selective inhibition of MAO-B by QUIN was confirmed using human liver mitochondria; only MAO-B was inhibited by QUIN and MAO-A was not inhibited. The inhibition was completely reversible. Among compounds structurally related to QUIN, 4-pyrimidine carboxaldehyde was the most potent substrate-competitive inhibitor of MAO-B, while 3-hydroxyanthranilic acid and xanthrenic acid, other metabolites of tryptophan, inhibited MAO non-competitively with the substrate. The inhibition of MAO-B by QUIN may be related to the causes of the neurotoxicity of QUIN.

1,4-benzoquinone as a new inhibitor of monoamine oxidase

The effect of 1,4-benzoquinone (BQ) on type A and B monoamine oxidase (MAO-A and -B) in human brain synaptosomes was examined. MAO-A was found to be markedly inhibited by BQ competitively with the substrate, kynuramine, while MAO-B was less sensitive to this inhibitor and the inhibition was non-competitive with the substrate. The Ki value of MAO-A (9.62 +/- 0.35 microM) was much smaller than the Km value of the enzyme with the substrate (56.3 +/- 3.5 microM) or the Ki value of MAO-B with BQ (20.3 +/- 0.9 microM). The inhibition of MAO-A by BQ was also confirmed by the use of platelet mitochondria and clonal rat pheochromocytoma PC12h cells as enzyme sources. The inhibition of MAO activity by BQ proved to be reversible: the inhibited enzyme activity could be recovered by column chromatography on Sephadex G-25.