Identification of dual specificity phosphatases induced by olfactory bulbectomy in rat olfactory neuroepithelium.

Dual specificity protein tyrosine phosphatases (dsPTPs) are a subfamily of protein tyrosine phosphatases implicated in the regulation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 mitogen-activated protein kinases (MAPKs) which are target enzymes activated by a wide range of cell-surface stimuli. Like these kinases, a class of dsPTP has been implicated in cell differentiation, regeneration, and apoptosis. In order to isolate dsPTPs which might play an important role in neuronal regeneration and apoptosis in olfactory neuroepithelium, we subcloned DNA fragments amplified by reverse transcription-polymerase chain reaction (RT-PCR), using degenerate oligonucleotide primers based on the conserved amino acid regions within the catalytic domain of dsPTPs, from rat olfactory epithelial RNA 1 and 4 h after an olfactory bulbectomy. The PCR products were subcloned into the pCRII vector, and 23 clones were chosen for further characterization. The sequence of these 23 individual clones revealed that two clones were identical to the rat dsPTP, MKP-3, and the other 21 clones were identical to the rat dsPTP, MKP-1. By Northern analysis, the MKP-1 transcript was induced and peaked 4 h following a bulbectomy. Similar results were obtained with the MKP-3 transcript. These results suggest that MKP-1 and MKP-3 may be involved in the early steps of apoptosis in vivo in rat olfactory neuroepithelium.

Mitogen-activated protein kinase phosphatase-1 in olfactory neural regeneration.

Uniquely, olfactory neurons continuously replace themselves. Olfactory bulb ablation induces coordinated degeneration and regeneration in olfactory neuroepithelium; up-regulated growth factors bind to their receptors, initiating a phosphorylation cascade activating mitogen-activated protein kinases (MAPK). MAPK then relay proliferation signals to the nucleus. Mitogen-activated protein kinase phosphatase-1 (MKP-1) inactivates MAPK. We examined MKP-1 expression in adult mouse olfactory epithelium following bulbectomy using a reverse transcription-polymerase chain reaction (RT-PCR) as well as Western analysis. While MKP-1 expression was high in olfactory epithelium from control mice, it decreased greatly 2-3 weeks after bulbectomy in temporal association with increased tyrosine phosphorylation of MAPK. Thus, reduced MKP-1 expression may contribute to regeneration of olfactory neuroepithelium after bulbectomy via decreased dephosphorylation of MAPK.

Induction of KGF, basic FGF, and TGFalpha mRNA expression during healing of experimental TM perforations.

KGF (KGF), synthesized and secreted exclusively by stromal cells in epithelialized organs, specifically promotes proliferation of cells of epithelial origin, including keratinocytes. A related peptide, basic fibroblast growth factor (bFGF), has mitogenic properties for fibroblasts and endothelial cells. KGF expression is stimulated markedly in the skin during wound healing. To investigate the physiologic action of KGF in the healing of TM (TM) perforations, we examined KGF and KGF receptor (KGFR) mRNA transcript levels as well as those of bFGF and transforming growth factor-alpha (TGFalpha) in normal and wounded rat TM at varying intervals, using a semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). We found KGF and TGFalpha mRNA expression to be induced rapidly, peaking 3 days after wounding and then declining. Expression of bFGF was induced gradually and remained increased until 7 days. In contrast, we found KGFR to be expressed in normal TM, remaining unchanged during TM repair. These results indicate that KGF and TGFalpha may mediate migration and proliferation of epithelial cells of the outer layer in the early stage of TM repair while bFGF may mediate the connective tissue reaction in the middle layer in a subsequent stage.

Keratinocyte growth factor and receptor mRNA expression in cholesteatoma of the middle ear.

Keratinocyte growth factor (KGF) is synthesized and secreted exclusively by stromal cells in epithelialized organs, and specifically promotes proliferation of cells of epithelial origin, including keratinocytes. Cholesteatoma is a proliferative form of keratinocyte dysregulation with aggressive growth that often leads to destruction of adjacent bone. We examined the expression of KGF, closely related peptides and their receptors in the development of cholesteatoma by comparing cholesteatoma with normal ear skin using the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis. All surgical specimens of cholesteatoma and most normal ear skin samples expressed detectable levels of KGF and KGF receptor (KGFR). Semiquantitative RT-PCR using beta-actin mRNA as an internal standard revealed significantly higher expression of KGF mRNA in cholesteatoma than in normal skin, while no significant difference in KGFR mRNA expression was noted between cholesteatoma tissue and normal skin. These results suggest that excessive KGF synthesis may contribute to the hyperproliferative state in cholesteatoma.

Stroboscopic observation of the larynx after radiation in patients with T1 glottic carcinoma.

We studied laryngeal video stroboscopy (LVS) system for evaluation of patients with glottic carcinoma (T1N0M0) before and after radiotherapy. There were 10 patients with T1 glottic squamous cell carcinoma (9 men and 1 woman) who received radiotherapy at the Hitachi General Hospital. We performed LVS before and after radiotherapy. The presence or absence of mucosal waves (MW) was particularly noted. No MW were present before radiotherapy but at 1-6 months after, MW gradually appeared. One year after radiotherapy all patients showed MW on LVS. In patients with glottic carcinoma MW recovered after radiation therapy. LVS may be useful for the clinical follow-up of post-radiation patients for early detection of recurrence of glottic carcinoma.

Auditory nerve disease of both ears revealed by auditory brainstem responses, electrocochleography and otoacoustic emissions.

We report on two patients who showed absence of auditory brainstem response (ABR) but broad compound action potentials on electrocochleograms and almost normal otoacoustic emissions, together with absence of caloric response and preservation of per rotatory nystagmus for both ears. Patient 1, a 53-year-old woman, had noted auditory and vestibular problems since the age of 15 years, and Patient 2, a 68-year-old woman, had noted problems of the same age of 30 years. They could hear words and understand sentences if spoken slowly, but they could not discriminate monosyllables very well. Their auditory examinations disclosed mild threshold elevation in pure-tone audiometry and markedly poor scores in speech audiometry and good scores in auditory comprehension test. They were diagnosed as having auditory nerve disease of unknown cause.