Sohtaro Komiyama

Molecular cloning of a human cDNA encoding a novel protein, DAD1, whose defect causes apoptotic cell death in hamster BHK21 cells.

The tsBN7 cell line, one of the mutant lines temperature sensitive for growth which have been isolated from the BHK21 cell line, was found to die by apoptosis following a shift to the nonpermissive temperature. The induced apoptosis was inhibited by a protein synthesis inhibitor, cycloheximide, but not by the bcl-2-encoded protein. By DNA-mediated gene transfer, we cloned a cDNA that complements the tsBN7 mutation. It encodes a novel hydrophobic protein, designated DAD1, which is well conserved (100% identical amino acids between humans and hamsters). By comparing the base sequences of the parental BHK21 and tsBN7 DAD1 cDNAs, we found that the DAD1-encoding gene is mutated in tsBN7 cells. The DAD1 protein disappeared in tsBN7 cells following a shift to the nonpermissive temperature, suggesting that loss of the DAD1 protein triggers apoptosis.

Prevalence of mitochondrial gene mutations among hearing impaired patients

The frequency of three mitochondrial point mutations, 1555A→G, 3243A→G, and 7445A→G, known to be associated with hearing impairment, was examined using restriction fragment length polymorphism (RFLP) analysis in two Japanese groups: (1) 319 unrelated SNHL outpatients (including 21 with aminoglycoside antibiotic injection history), and (2) 140 cochlear implantation patients (including 22 with aminoglycoside induced hearing loss). Approximately 3% of the outpatients and 10% of the cochlear implantation patients had the 1555A→G mutation. The frequency was higher in the patients with a history of aminoglycoside injection (outpatient group 33%, cochlear implantation group 59%). One outpatient (0.314%) had the 3243A→G mutation, but no outpatients had the 7445A→G mutation and neither were found in the cochlear implantation group. The significance of the 1555A→G mutation, the most prevalent mitochondrial mutation found in this study of a hearing impaired population in Japan, among subjects with specific backgrounds, such as aminoglycoside induced hearing loss, is evident.

Analysis of Vocal Abuse: Fluctuations in Phonation Time and Intensity in 4 Groups of Speakers

We have seen many patients with a voice disorder due to vocal abuse. However, there is little information about the speaking behaviour of such patients. The object of this study was to analyze speaking behaviour and to evaluate the relationship between the cause of voice disorders and its effects on speech. We had previously measured phonation time with a speech time accumulator. Recently, we have developed a speech intensity/speech time accumulator. We obtained data by accumulating the phonation time at 4 degrees of vocal intensity, ranging from weak to strong. By using this instrument, we measured the speaking habits of 29 subjects for 131 days and collected data about the criteria for vocal abuse. Our results showed that the office workers exhibited a phonation time (33.6 +/- 13.6 min for 8 h) three times shorter than that of teachers and patients with vocal fold nodules (102.1 +/- 22.9 min for 8 h). For the teachers and patients with a long phonation time, half of the total phonation time was at high intensity.

Cisplatin blocks mechano-electric transducer current in chick cochlear hair cells

The effects of cisplatin (cis-dichlorodiammine platinum II, CDDP) on the mechano-electrical transduction (MET) current were investigated with a whole-cell patch-electrode voltage clamp technique in dissociated cochlear hair cells of chicks. CDDP blocked the MET channel in a dose- and voltage-dependent manner. At -50 mV, CDDP blocked the MET channel with a Hill coefficient of approximately 2 and a dissociation constant (KD) of 1.5 x 10(-3) M. The kinetics of CDDP blockade consist of a voltage-independent and a voltage-dependent component.

The highly conserved DAD1 protein involved in apoptosis is required for N-linked glycosylation

Background: The tsBN7 cell line is one of the temperature‐sensitive mutants for cell proliferation which have been isolated from the BHK21 cell line derived from the golden hamster. It has a mutation in the DAD1 gene encoding a 12.5 kDa highly conserved protein through evolution, and enters apoptosis at the restrictive temperature due to this mutation.

Immunoregulatory Role of Ocular Macrophages: The Macrophages Produce RANTES to Suppress Experimental Autoimmune Uveitis

Murine experimental autoimmune uveitis (EAU) is a model of human uveitis. Ocular-infiltrating macrophages play a crucial role in the generation of tissue damage in EAU. In fact, several chemokines are actually produced in the inflamed eye. The aim of this study was to elucidate the role of ocular macrophage-derived chemokines in EAU. C57BL/6 mice were immunized with human interphotoreceptor retinoid binding protein peptide 1–20, and the EAU severity was scored at multiple time points based on microscopic fundus observations (retinal vascular dilatation and exudates) and histological examinations. The peak inflammatory response was observed 1 wk (day 16) after the beginning of macrophage infiltration to the eye (day 9). Ocular-infiltrating cells were enriched or depleted of macrophages by magnetic beads and analyzed by real-time RT-PCR for chemokine mRNA production. We found that only the macrophage-enriched cells from the eye produced RANTES, and thus proposed that macrophage-derived RANTES facilitated the ocular inflammations. In contrast to our postulate, neutralization of RANTES by specific Ab in vivo on days 9 and 13 exacerbated EAU. We also found that the ratio of ocular CD4/CD8 T cells was markedly increased after treatment. As a result, RANTES neutralization might exacerbate EAU by modulating the type of T cell subsets recruited to the eye. In conclusion, our data provide insight into the immunoregulatory role of macrophages and RANTES in the pathogenesis of ocular inflammation. Not all macrophage-derived chemokines cause local inflammation, since RANTES produced by ocular macrophages appears to suppress EAU.

Pre- and postsynaptic ATP-sensitive potassium channels during metabolic inhibition of rat hippocampal CA1 neurons

Presynaptic and postsynaptic membrane activities during experimental metabolic inhibition were analysed in mechanically dissociated rat hippocampal neurons using nystatin‐perforated and conventional whole‐cell patch clamp recordings. NaCN, an inhibitor of mitochondrial ATP synthesis, induced an outward current across the postsynaptic soma membrane. This current was blocked by tolbutamide, a sulfonylurea, which blocks ATP‐sensitive K+ (KATP) channels. The presynaptic effect of metabolic inhibitors such as NaCN, NaN3, or glucose‐free solution was to increase the frequency of GABAergic miniature inhibitory postsynaptic currents (mIPSCs). Tolbutamide had no effect on this increase in mIPSC frequency induced by metabolic inhibition. Diazoxide, a KATP channel opener, evoked a similar somatic outward current in a dose‐dependent manner. In addition, diazoxide decreased the frequency of mIPSCs in a dose‐dependent fashion. Both these pre‐ and postsynaptic effects of diazoxide were reversed by tolbutamide, suggesting the existence of KATP channels on both pre‐ and postsynaptic membranes. These results confirm the presence of KATP channels on both the pre‐ and postsynaptic membranes but indicate that the channels have significantly different sensitivities to metabolic inhibition.

The Roles of JNK1 and Stat3 in the Response of Head and Neck Cancer Cell Lines to Combined Treatment with All trans‐retinoic Acid and 5‐Fluorouracil

We have used a combination of vitamin A (all‐trans‐retinyl palmitate), 5‐fluorouracil (5‐FU) and radiation to treat human head and neck squamous cell carcinoma (HNSCC). This chemoradiother‐ apy is called “FAR therapy” In this study we examined the effects of all‐trans‐retinoic acid (ATRA), the active metabolite of vitamin A, and ATRA plus 5‐FU on two HNSCC cell lines (YCU‐ N861 and YCU‐H891) to gain insight into the molecular mechanisms of FAR therapy. ATRA at 1 μM (the order of concentration found in HNSCC tumors treated with FAR therapy) inhibited cell proliferation and caused Gl cell cycle arrest in both cell lines. This was associated with a decrease in cyclin D1, an increase in p27Kipl and a reduction in the hyperphosphorylated form of retinoblastoma protein (pRB). With YCU‐N861 cells, ATRA also caused a decrease in Bcl‐2 and Bcl‐XL and an increase in Bax. Both ATRA and 5‐FU activated c‐Jun N‐terminal kinase (JNK) 1 and the combination of both agents resulted in additive or synergistic activation of JNK1, and also enhanced the induction of apoptosis. The YCU‐H891 cells, in which the epidermal growth factor receptor (EGFR)‐signal transducer and activator of transcription 3 (Stat3) pathway is constitutively activated, were more resistant to treatments with ATRA, 5‐FU and the combination of both agents than YCU‐N861 cells. A dominant negative Stat3 construct strongly enhanced the cellular sensitivity of this cell line to 5‐FU but not to ATRA. In addition there is evidence that activation of Stat3 is associated with cellular resistance to radiation in HNSCC. Therefore, the addition to FAR therapy of agents that inhibit activation of the Stat3 pathway may enhance the clinical response of patients with HNSCC to FAR therapy.

A Newly Devised Speech Accumulator

Voice therapy is often most effective for treating patients with vocal cord polyp, polypoid degeneration and singers nodule. However, little is known about the total speaking times in 1 day, the ratio of speech per hour and the sound level during speech, in individual patients. If these parameters can be readily detected, it could be clarified as to how speaking times or patterns are related to a particular voice disorder and/or what instruction a doctor had given a patient. We devised a speech accumulator which records the vibration time of the vocal cords by a small contact microphone attached to the neck, but does not record the actual speech, thus monitoring the privacy of the individual. The time of speech, at any sound level can be read digitally, at any time. This system was clinically used for 11 patients and proved to be most useful. The longest speaking time in 1 day was 182 min, for a bus guide, and the shortest time was 33 min for an office clerk.

Establishment of tumor cell lines from a patient with head and neck cancer and their different sensitivities to anti-cancer agents

The authors established five cell lines from a human head and neck tumor. The five cell lines (HC‐2, HC‐3, HC‐4, HC‐7, and HC‐9) exhibited different sensitivities to Adriamycin, cisplatin, bleomycin, 5‐fluorouracil, vincristine, and daunomycin. The D50 was 200 ng/ml Adriamycin (doxorubicin) for HC‐7 and 45 ng/ml for HC‐2. At the inception of long‐term culture (11 months) in the absence of any drug, the sensitivity to Adriamycin of HC‐7‐5 (subcloned from HC‐7) was 3.4 times greater than that of HC‐2‐6 (subcloned from HC‐2); by 11 months, it decreased to 1.6 times that of HC‐2‐6. The cytocidal action of Adriamycin on HC‐2‐6 and HC‐7‐5 was potentiated when Adriamycin was combined with verapamil or cepharanthine. Cepharanthine also potentiated daunomycin and vincristine (VCR) against HC‐2‐6 and HC‐7‐5 cells, and it almost completely overcame drug‐resistance to daunomycin and vincristine in HC‐7‐5/VCR, a multidrug‐resistant variant isolated after long exposure to vincristine of HC‐7‐5 cells in culture. The cellular accumulation of [3H]‐daunomycin by HC‐7‐5 cells was about 70% that of HC‐2‐6 cells. By Northern blot analysis, using a multidrug‐resistance gene (mdr‐1) probe, neither HC‐2‐6 nor HC‐7‐5 expressed the mdr‐1 gene, but HC‐7‐5/VCR or other multidrug‐resistant variants showed active expression of the mdr‐1 gene. Differential sensitivities among the five cell lines to 5‐fluorouracil, cisplatinum, and bleomycin appear to be mediated through other mechanism beside the mdr‐1 gene.