Hirofumi Katayama

A Negative Feedback Loop Controlling bHLH Complexes Is Involved in Vascular Cell Division and Differentiation in the Root Apical Meristem.

Controlling cell division and differentiation in meristems is essential for proper plant growth. Two bHLH heterodimers consisting of LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5)/TMO5-LIKE1 (T5L1) regulate periclinal cell division in vascular cells in the root apical meristem (RAM). In this study, we further investigated the functions of LHW-T5L1, finding that in addition to controlling cell division, this complex regulates xylem differentiation in the RAM via a novel negative regulatory system. LHW-T5L1 upregulated the thermospermine synthase gene ACAULIS5 (ACL5), as well as SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3), which encodes a bHLH protein, in the RAM. The SACL3 promoter sequence contains a conserved upstream open reading frame (uORF), which blocked translation of the main SACL3 ORF in the absence of thermospermine. Thermospermine eliminated the negative effect of uORF and enhanced SACL3 production. Further genetic and molecular biological analyses indicated that ACL5 and SACL3 suppress the function of LHW-T5L1 through a protein-protein interaction between LHW and SACL3. Finally, we showed that a negative feedback loop consisting of LHW-T5L1, ACL5, SACL3, and LHW-SACL3 contributes to maintain RAM size and proper root growth. These findings suggest that a negative feedback loop regulates the LHW-T5L1 output level to coordinate cell division and differentiation in a cell-autonomous manner.

Inhibitory Effect of Ambroxol on Superoxide Anion Production and Generation by Murine Lung Alveolar Macrophages

We examined the effect of ambroxol on superoxide anion production before and generation after phorbol-myristate acetate (PMA) stimulation of lung alveolar macrophages. Lung free cells including lung alveolar macrophages were obtained from Fischer 344 rats and guinea pigs using bronchoalveolar lavage. The superoxide anion produced by lung alveolar macrophages with or without stimulation of PMA was measured by lucigenin-dependent chemiluminescence method using a photon counter. Ambroxol inhibited the superoxide anion production and generation by lung alveolar macrophages harvested from both F344 rats and guinea pigs in a dose-dependent fashion. Approximately 16 mumol/L of ambroxol inhibited 50% of superoxide production of lung alveolar macrophages in rats and guinea pigs, whereas a slightly greater dose of ambroxol, i.e., 18-26 mumol/L, was necessary to inhibit 50% of PMA-enhanced superoxide generation by lung alveolar macrophages. These results suggest that ambroxol acts as an antioxidant in murine lungs and may be a potential therapeutic option for reactive oxygen species-associated lung disorders including bronchial asthma.

Effects of Angiotensin-Converting Enzyme (ACE) Inhibitors on Oxygen Radical Production and Generation by Murine Lung Alveolar Macrophages

We examined the effect of angiotensin-converting enzyme (ACE) inhibitors on oxygen radical production before and generation after phorbol-myristate acetate (PMA) stimulation of lung alveolar macrophages. Lung free cells, predominantly pulmonary alveolar macrophages, were obtained from Fischer 344 rats and guinea pigs using bronchoalveolar lavage. The oxygen radicals produced by pulmonary alveolar macrophages with or without stimulation of PMA were measured by lucigenin-dependent chemiluminescence method using a photon counter, Lumat 9501 (Berthold, Germany). Alacepril, an ACE inhibitor with SH-group, inhibited the oxygen radical production and generation by lung alveolar macrophages harvested from both rats and guinea pigs in a dose-dependent fashion. Approximately 0.3 mM of alacepril inhibited 50% of oxygen radical production of lung alveolar macrophages in both rats and guinea pigs, whereas a higher concentration (1-5 mM) of lisinopril, an ACE inhibitor without SH-group, was necessary to inhibit 50% of oxygen radical production of lung alveolar macrophages in the animals. These results suggest that an ACE inhibitor with SH-group acts as an antioxidant in murine lungs and the treatment with the ACE inhibitor may reduce oxidant stress in hypertensive patients with asthma.