Masahiko Taura

Novel Human Homologues of p47phox and p67phox Participate in Activation of Superoxide-producing NADPH Oxidases

Abstract The catalytic core of a superoxide-producing NADPH oxidase (Nox) in phagocytes is gp91phox/Nox2, a membrane-integrated protein that forms a heterodimer with p22phox to constitute flavocytochrome b558. The cytochrome becomes activated by interacting with the adaptor proteins p47phox and p67phox as well as the small GTPase Rac. Here we describe the cloning of human cDNAs for novel proteins homologous to p47phox and p67phox, designated p41nox and p51nox, respectively; the former is encoded by NOXO1 (Nox organizer 1), and the latter is encoded by NOXA1 (Nox activator 1). The novel homologue p41nox interacts with p22phox via the two tandem SH3 domains, as does p47phox. The protein p51nox as well as p67phox can form a complex with p47phox and with p41nox via the C-terminal SH3 domain and binds to GTP-bound Rac via the N-terminal domain containing four tetratricopeptide repeat motifs. These bindings seem to play important roles, since p47phox and p67phox activate the phagocyte oxidase via the same interactions. Indeed, p41nox and p51nox are capable of replacing the corresponding classical homologue in activation of gp91phox. Nox1, a homologue of gp91phox, also can be activated in cells, when it is coexpressed with p41nox and p51nox, with p41nox and p67phox, or with p47phox and p51nox; in the former two cases, Nox1 is partially activated without any stimulants added, suggesting that p41nox is normally in an active state. Thus, the novel homologues p41nox and p51nox likely function together or in combination with a classical one, thereby activating the two Nox family oxidases.

A region N-terminal to the tandem SH3 domain of p47phox plays a crucial role in the activation of the phagocyte NADPH oxidase.

The superoxide-producing NADPH oxidase in phagocytes is crucial for host defence; its catalytic core is the membrane-integrated protein gp91phox [also known as Nox2 (NADPH oxidase 2)], which forms a stable heterodimer with p22phox. Activation of the oxidase requires membrane translocation of the three cytosolic proteins p47phox, p67phox and the small GTPase Rac. At the membrane, these proteins assemble with the gp91phox-p22phox heterodimer and induce a conformational change of gp91phox, leading to superoxide production. p47phox translocates to membranes using its two tandemly arranged SH3 domains, which directly interact with p22phox, whereas p67phox is recruited in a p47phox-dependent manner. In the present study, we show that a short region N-terminal to the bis-SH3 domain is required for activation of the phagocyte NADPH oxidase. Alanine substitution for Ile152 in this region, a residue that is completely conserved during evolution, results in a loss of the ability to activate the oxidase; and the replacement of Thr153 also prevents oxidase activation, but to a lesser extent. In addition, the corresponding isoleucine residue (Ile155) of the p47phox homologue Noxo1 (Nox organizer 1) participates in the activation of non-phagocytic oxidases, such as Nox1 and Nox3. The I152A substitution in p47phox, however, does not affect its interaction with p22phox or with p67phox. Consistent with this, a mutant p47phox (I152A), as well as the wild-type protein, is targeted upon cell stimulation to membranes, and membrane recruitment of p67phox and Rac normally occurs in p47phox (I152A)-expressing cells. Thus the Ile152-containing region of p47phox plays a crucial role in oxidase activation, probably by functioning at a process after oxidase assembly.

Expression and function of Noxo1γ, an alternative splicing form of the NADPH oxidase organizer 1

Activation of the superoxide‐producing NADPH oxidase Nox1 requires both the organizer protein Noxo1 and the activator protein Noxa1. Here we describe an alternative splicing form of Noxo1, Noxo1γ, which is expressed in the testis and fetal brain. The Noxo1γ protein contains an additional five amino acids in the N‐terminal PX domain, a phosphoinositide‐binding module; the domain plays an essential role in supporting superoxide production by NADPH oxidase (Nox) family oxidases including Nox1, gp91phox/Nox2, and Nox3, as shown in this study. The PX domain isolated from Noxo1γ shows a lower affinity for phosphoinositides than that from the classical splicing form Noxo1β. Consistent with this, in resting cells, Noxo1γ is poorly localized to the membrane, and thus less effective in activating Nox1 than Noxo1β, which is constitutively present at the membrane. On the other hand, cell stimulation with phorbol 12‐myristate 13‐acetate (PMA), an activator of Nox1–3, facilitates membrane translocation of Noxo1γ; as a result, Noxo1γ is equivalent to Noxo1β in Nox1 activation in PMA‐stimulated cells. The effect of the five‐amino‐acid insertion in the Noxo1 PX domain appears to depend on the type of Nox; in activation of gp91phox/Nox2, Noxo1γ is less active than Noxo1β even in the presence of PMA, whereas Noxo1γ and Noxo1β support the superoxide‐producing activity of Nox3 to the same extent in a manner independent of cell stimulation.

Mandible preserving pull-through oropharyngectomy for advanced oropharyngeal cancer: A pilot study

OBJECTIVE Through our experiences in the parapharyngeal space (PPS) surgery, we have learned that it is possible to gain wide exposure of the PPS near to the skull base with a transcervical approach alone. Thus, we presumed that if this type of transcervical approach would be combined with a transoral approach, a less invasive oropharyngectomy without mandibulotomy and lip-splitting might be feasible for the resection of advanced oropharyngeal cancer, sparing the morbidities associated with conventional mandibular swing approach or its modified procedures. We termed this method as a mandible preserving pull-through oropharyngectomy (MPPO) and evaluated its feasibility and efficacy in this pilot study. MATERIALS AND METHODS MPPO was applied for a series of 7 patients with advanced lateral and/or upper oropharyngeal cancer including 2 patients with T4 stage. Our current application of MPPO excludes tumors, which involves mandibular bone, the higher part of the medial pterygoid muscle, and the lateral pterygoid muscle. RESULTS Safe and sufficient excision of tumors was feasible by MPPO avoiding morbidities associated with mandibulotomy or lip-splitting without compromising oncological outcomes. CONCLUSIONS Although preliminary, our favorable outcomes indicate that MPPO might be a useful alternative to conventional mandibular swing approach or its modified procedures for selected cases with advanced oropharyngeal cancer. Further accumulation of data is encouraged.

Salvage of recurrent hypopharyngeal carcinoma after primary curative treatment.

Abstract Conclusions: There was a moderate chance of cure after surgical salvage of recurrent hypopharyngeal squamous cell carcinoma (SCC). However, surgical salvage was only feasible for early recurrent tumor. Close follow-up surveillance to detect early recurrence is essential after primary treatment of patients. Objectives: Despite improvements in surgery, radiotherapy, and chemotherapy, hypopharyngeal SCC has one of the worst prognoses among head and neck malignant diseases. To improve the overall survival and cure rates in patients with hypopharyngeal SCC, the management of recurrent disease as well as initial treatment is important. In this study, the efficacy and results of salvage treatment of recurrent hypopharyngeal SCC after primary curative treatment were evaluated. Methods: The management outcomes of 49 patients who were treated for recurrent hypopharyngeal SCC between January 2002 and December 2010 at Kyushu University Hospital were reviewed. Results: The median time for detection of recurrence from the initial curative treatment was 10.3 months (range 2.1–61.1 months). The total salvage rates of recurrence were 45% (local, 85%; locoregional, 100%; regional, 23%; distant, 19%). The 1- and 3-year tumor-free actuarial survival rates of those patients who received salvage surgery followed by chemotherapy and/or radiotherapy were 96% and 79%, respectively. There was no 3-year survivor among the patients who received only chemotherapy and/or radiotherapy.

The treatment and outcome analysis of primary squamous cell carcinoma of the thyroid

OBJECTIVES Primary squamous cell carcinoma (SCC) of the thyroid is a rare disease. It usually presents with locally advanced disease and has an overall poor prognosis. In this study, we investigated the characteristics and outcomes of patients with SCC of the thyroid, and reported our experience with chemotherapy with lenvatinib in the treatment of SCC of the thyroid. METHODS The management outcome of 10 patients who had SCC of the thyroid between January 2000 and 2015 at Kyushu University Hospital or associated facilities was reviewed. RESULTS There were 3 males and 7 females, ranging in age from 53 to 77 years. Extent of disease was staged as follows: stage IVA, 3 cases; stage IVB, 3 cases; stage IVC, 4 cases. Only tracheostomy was applied for 2 cases, surgical resection, such as total thyroidectomy and neck dissection, for the other 8 cases. Radiotherapy following surgical treatment was applied for 9 cases. Four patients started on oral lenvatinib due to recurrent or progressive SCC of the thyroid. The one year actuarial survival rate of patients was 22.7%. There was no 2-year survivor of all patients. CONCLUSIONS Treatment should primarily be targeted at surgical resection with negative margins in patients with resectable disease. Lenvatinib may show promise to potentially extend survival.

Morphological reconstruction of the neoepiglottis after hyo-sub-glosso-epiglottectomy (anteriorly extended supraglottic laryngectomy)

INTRODUCTION Primary closure (PC) has been used as the standard surgical technique for defects after supraglottic laryngectomy (SL). In the head and neck cancer surgery textbooks and literature, this relatively simple method has also been recommended for the closure of surgical defects after hyo-sub-glosso-epiglottectomy (HSE) or anteriorly extended supraglottic laryngectomy, in which the base of the tongue is removed with supraglottic structures during SL. However, this closing method ignores the original three-dimensional morphology of the supraglottic space that plays a critical functional role in phonation, respiration, and swallowing. Consequently, even after simple SL, patients who undergo PC suffer from frequent postoperative complications, including unclear voice, insufficient airway, and aspiration pneumonia, which often require placement of permanent tracheal stoma, gastric tube, and gastrostomy. These unfavorable outcomes become more prominent in patients with HSE due to the reduced volume of the base of the tongue. In particular, the enhanced risks of acute and persistent postoperative aspiration pneumonia occasionally require the sacrifice of the preserved larynx to save the life of the patient. In addition, this reconstruction method imposes excessive tension between sutured tissues, causing frequent postoperative leakage, infection, and fistula formation, especially in cases of salvage surgery. To overcome these critical problems, we reconstructed the supraglottic space after HSE with a rectus abdominis myocutaneous (RAMC) flap in a series of three patients including one salvage case. The purpose of this method was to create a sufficient convex shape that hangs over the remaining larynx expecting that it might function as a neoepiglottis. This method worked well, and all three patients demonstrated quite satisfactory functional results compared to the PC method. Surgical techniques of this novel reconstruction method are described herein.