Detection of viable but nonculturable Vibrio parahaemolyticus induced by prolonged cold-starvation using propidium monoazide real-time polymerase chain reaction.

Viable but nonculturable (VBNC) Vibrio parahaemolyticus cannot be detected by the standard cultivation-based methods. In this study, commonly used viability assessment methods were evaluated for the detection of V. parahaemolyticus in a VBNC state. Vibrio parahaemolyticus cells exposed to nutrient deficiency at cold temperature were used for epifluorescence microscopy with SYTO9 and propidium iodide (PI) staining and real-time polymerase chain reaction (qPCR) with propidium monoazide (PMA), and its resuscitative ability was determined by a temperature upshift in freshly prepared artificial sea water (ASW; pH 7) fluids. Viable cells with intact membranes always exceeded 5·0 log CFU per ml in ASW microcosms at 4°C. After 80 days, cycle thresholds for V. parahaemolyticus ATCC 27969 were 16·15-16·69. During cold-starvation, PMA qPCR selectively excluded DNAs from heat-killed cells. However, there may be some penetration of PMA into undamaged cells that persisted in ASW for 150 days, as evidenced by their ability to resuscitate from a VBNC state after a temperature upshift (25°C); V. parahaemolyticus ATCC 33844 and V. parahaemolyticus ATCC 27969 were successfully reactivated from a VBNC state in ASW microcosms containing <5% NaCl, following enrichment in ASW medium (pH 7). SIGNIFICANCE AND IMPACT OF THE STUDY: Few studies have evaluated the characteristics of and detection methods for viable but nonculturable (VBNC) Vibrio parahaemolyticus induced by cold-starvation. Currently, VBNC cells are routinely detected by SYTO9 and propidium iodide double staining. However, viable cell counts might be overestimated by this approach, suggesting that the fluorescence dyes may be ineffective for accurately determining the viability of bacterial cells. We demonstrated that quantitative real-time polymerase chain reaction with propidium monoazide, which selectively permeates damaged cell membranes, can be used to obtain viable cell counts of V. parahaemolyticus after its evolution to a VBNC state under cold-starvation conditions.

Development of a semi-continuous two-stage simultaneous saccharification and fermentation process for enhanced 2,3-butanediol production by Klebsiella oxytoca.

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising chemical with wide industrial applications, along with various by-products. Previously, we have developed a metabolically engineered K. oxytoca ΔldhA ΔpflB strain to reduce the formation of by-products. To improve 2,3-BD productivity and examine the stability of K. oxytoca ΔldhA ΔpflB strain for industrial application, a semi-continuous two-stage simultaneous saccharification and fermentation (STSSF) process was developed. The STSSF with the K. oxytoca ΔldhA ΔpflB mutant using cassava as a carbon source could produce 108 ± 3·73 g(2,3-BD)  l-1 with a yield of 0·45 g(2,3-BD)  g(glucose)-1 and a productivity of 3·00 g(2,3-BD) l-1  h-1 . No apparent changes in the final titre, yield and productivity of 2,3-BD were observed for up to 20 cycles of STSSF. Also, microbial contamination and spontaneous mutation of the host strain with potential detrimental effects on fermentation efficiency did not occur during the whole fermentation period. These results strongly underpin that the K. oxytoca ΔldhA ΔpflB mutant is stable and that the STSSF process is commercially exploitable. SIGNIFICANCE AND IMPACT OF THE STUDY: There is growing interest in the production of 2,3-butanediol (2,3-BD) from renewable resources by microbial fermentation because of its wide applications to specialty and commodity chemical industries. Klebsiella oxytoca usually produces 2,3-BD as a major end product during the fermentation of carbohydrates. This is the first study to provide a high-efficiency simultaneous saccharification and 2,3-BD fermentation process. Also, this study proves the stability of a metabolically engineered 2,3-BD overproducing K. oxytoca strain for industrial application.

Dual-source, dual-energy multidetector CT for the evaluation of pancreatic tumours.

OBJECTIVE: To investigate the potential diagnostic value of dual-energy CT (DECT) with virtual non-enhanced (VNE) and iodine-only images, and to determine the optimal mixed ratio of blended images for evaluation of pancreatic diseases. METHODS: Multiphasic DECT was performed in 44 patients with focal pancreatic disease. DECT was used during the pancreatic and hepatic venous phases, and a peak kilovoltage of 120 kVp was used for both non-contrast phases. For qualitative analysis of the CT images, two radiologists assessed three image sets (VNE, iodine-only and blended images) in order to determine the acceptability of VNE in replacing true non-enhanced (TNE) images, the added value of iodine-only images and the preferred blending ratio. For quantitative analyses, the CT numbers and image noise of the pancreatic parenchyma, lesions, aorta and psoas muscle were measured. The contrast-to-noise ratio of the lesion was calculated on the pancreatic phase images. The effective radiation dose for DECT and TNE images was calculated. Statistical comparisons were made using the Friedman test, the Wilcoxon test, the paired t-test and repeated measures of analysis of variation with Bonferroni correction for multiple comparisons. RESULTS: The level of acceptance of the VNE images in replacing TNE images was 90.9%. Regarding the iodine-only images, 50% of the cases were found to have an added value. The linear-blended images with a weighting factor of 0.5 were preferred. CONCLUSIONS: DECT was able to provide high-quality VNE images that could replace TNE images and iodine-only images showing an added value. Blended images with a weighting factor of 0.5 were preferred by the reviewers.

Transcriptional repression of RUNX2 is associated with aggressive clinicopathological outcomes, whereas nuclear location of the protein is related to metastasis in prostate cancer.

BACKGROUND: Runt-related transcription factor 2 (RUNX2) is a transcription factor that is closely related to bone formation, and prostate cancer (CaP) is the most common cancer to metastasize to bone. The present study investigated the expression levels of RUNX2 in human prostate tissue, and the correlation between RUNX2 levels and the clinicopathological characteristics of CaP. METHODS: A case-control study was conducted including 114 cases of newly diagnosed CaP and 114 age-matched BPH patients as controls. RUNX2 expression was estimated using real-time PCR and immunohistochemical staining. RESULTS: The mRNA expression of RUNX2 did not differ between CaP tissues and non-cancer BPH controls (P=0.825). However, RUNX2 expression was significantly decreased in patients with elevated PSA levels (≥20 ng ml(-1)), a Gleason score ≥8 and metastatic disease compared to those with low PSA, low Gleason score and non-metastatic disease (P=0.023, 0.005 and 0.014, respectively). Immunohistochemical analysis showed that 65.2% of the patients with positive RUNX2 nuclear staining had metastatic disease, which was present in only 25.9% of those with negative staining (P=0.010). CONCLUSIONS: RUNX2 mRNA expression was negatively correlated with CaP aggressiveness. Moreover, the nuclear location of RUNX2 may be a prognostic marker of metastasis in CaP.

Acute but not chronic differences in skilled reaching for food following motor cortex devascularization vs. photothrombotic stroke in the rat.

The variability in the behavioral outcome of human and nonhuman animals after stroke raises the question whether the way that a stroke occurs is a contributing factor. Photothrombotic stroke in rats has been reported to produce especially variable results, with some animals showing either slight to no impairment to other animals displaying severe impairments. The present study investigated this variability. Rats received three different-sized photothrombotic treatments and were contrasted to rats receiving a "standard" motor cortex stroke produced by pial stripping. Rats were assessed acutely and chronically on a skilled reaching for food task using end-point measures and movement assessment in a constraint-induced rehabilitation paradigm. The results indicated that as the size of the photothrombotic infarct approached the size of the pial strip infarct so did chronic behavioral deficits. Nevertheless there were differences in the time course of recovery. Rats with photothrombotic lesions of all sizes were less impaired in the acute period of recovery both on measures of learned nonuse and constrained-induced recovery. The findings are discussed in relation to the idea that whereas the course of recovery might be altered as a function of the type of stroke, chronic deficits are more closely related to the ensuing damage.

Combination drug therapy using edaravone and Daio-Orengedoku-to after transient focal ischemia in rats.

In this study, we investigated the effect of Daio-Orengedoku-to (DOT) on ischemic brain damage in a rat model of focal ischemia-reperfusion and attempted to identify synergistic effects for the combination of edaravone and DOT against ischemic insult. Ischemia was induced by intraluminal occlusion of the right middle cerebral artery for 2 h and reperfusion followed for 22 h. To determine the neuroprotective effect of DOT, it was administered orally just before reperfusion and then 2 h after reperfusion. To examine the effects of combination therapy on survival, rats were divided into groups treated with edaravone, DOT, and edaravone and DOT. Microglial activation, neutrophil infiltration and brain-derived neurotrophic factor (BDNF) expression were examined in surviving animals. Infarct volume was significantly reduced by DOT (100, 200 and 400 mg/kg; P < 0.05), and edaravone plus DOT markedly improved the survival rate after transient ischemia (P = 0.0133). Microglial activation was reduced by edaravone and DOT and their combination (P < 0.05), and neutrophil infiltration was lowered in these groups (P < 0.05). BDNF-positive cells were increased in the combination edaravone and DOT group (P < 0.05). It appears that the neuroprotective mechanisms of combined therapy involve inhibition of microglial activation, reduction of invading neutrophils and enhancement of BDNF expression.

Flunarizine induces Nrf2-mediated transcriptional activation of heme oxygenase-1 in protection of auditory cells from cisplatin.

We investigated the cytoprotective mechanisms of flunarizine in cisplatin-induced death of auditory cells. Concomitant with an increase in viability, treatment with flunarizine resulted in a marked dissociation of Nrf2/Keap1 and subsequent intranuclear translocation of Nrf2, which was mediated by PI3K-Akt signaling. Overexpression of Nrf2 protected cells from cisplatin along with transcriptional activation of ARE to generate heme oxygenase-1 (HO-1). Pretreatment with flunarizine predominantly increased the transcriptional activity of HO-1 among Nrf2-driven transcripts, including HO-1, NQO1, GCLC, GCLM, GST micro-1, and GSTA4. Furthermore, both pharmacological inhibition and siRNA transfection of HO-1 completely abolished the flunarizine-mediated protection of HEI-OC1 cells and the primary rat (P2) organ of Corti explants from cisplatin. These results suggest that Nrf2-driven transcriptional activation of ARE through PI3K-Akt signaling augments the generation of HO-1, which may be a critically important determinant in cellular response toward cisplatin and the cytoprotective effect of flunarizine against cisplatin.

Genetic basis of hearing loss associated with enlarged vestibular aqueducts in Koreans.

Sensorineural hearing loss associated with enlargement of the vestibular aqueduct (EVA) can be associated with mutations of the SLC26A4 gene. In western populations, less than one-half of the affected individuals with EVA have two mutant SLC26A4 alleles, and EVA is frequently caused by unknown genetic or environmental factors alone or in combination with a single SLC26A4 mutation as part of a complex trait. In this study, we ascertained 26 Korean probands with EVA and performed nucleotide sequence analysis to detect SLC26A4 mutations. All subjects had bilateral EVA, and 20 of 26 were sporadic (simplex) cases. Fourteen different mutations were identified, including nine novel mutations. Five mutations were recurrent and accounted for 80% of all mutant alleles, providing a basis for the design and interpretation of cost-efficient mutation detection algorithms. Two mutant alleles were identified in 21 (81%), one mutant allele was detected in three (11%), and zero mutant allele was detected in two (8%) of 26 probands. The high proportion of Korean probands with two SLC26A4 mutations may reflect a reduced frequency of other genetic or environmental factors causing EVA in comparison to western populations.

Origins and frequencies of SLC26A4 (PDS) mutations in east and south Asians: global implications for the epidemiology of deafness.

Recessive mutations of SLC26A4 (PDS) are a common cause of Pendred syndrome and non-syndromic deafness in western populations. Although south and east Asia contain nearly one half of the global population, the origins and frequencies of SLC26A4 mutations in these regions are unknown. We PCR amplified and sequenced seven exons of SLC26A4 to detect selected mutations in 274 deaf probands from Korea, China, and Mongolia. A total of nine different mutations of SLC26A4 were detected among 15 (5.5%) of the 274 probands. Five mutations were novel and the other four had seldom, if ever, been identified outside east Asia. To identify mutations in south Asians, 212 Pakistani and 106 Indian families with three or more affected offspring of consanguineous matings were analysed for cosegregation of recessive deafness with short tandem repeat markers linked to SLC26A4. All 21 SLC26A4 exons were PCR amplified and sequenced in families segregating SLC26A4 linked deafness. Eleven mutant alleles of SLC26A4 were identified among 17 (5.4%) of the 318 families, and all 11 alleles were novel. SLC26A4 linked haplotypes on chromosomes with recurrent mutations were consistent with founder effects. Our observation of a diverse allelic series unique to each ethnic group indicates that mutational events at SLC26A4 are common and account for approximately 5% of recessive deafness in south Asians and other populations.

Aging, oxidative responses, and proliferative capacity in cultured mouse aortic smooth muscle cells.

The cellular mechanisms that contribute to the acceleration of atherosclerosis in aging populations are poorly understood, although it is hypothesized that changes in the proliferative capacity of vascular smooth muscle cells is contributory. We addressed the relationship among aging, generation of reactive oxygen species (ROS), and proliferation in primary culture smooth muscle cells (SMC) derived from the aortas of young (4 mo old) and aged (16 mo old) mice to understand the phenotypic modulation of these cells as aging occurs. SMC from aged mice had decreased proliferative capacity in response to alpha-thrombin stimulation, yet generated higher levels of ROS and had constitutively increased mitogen-activated protein kinase activity, in comparison with cells from younger mice. These effects may be explained by dysregulation of cell cycle-associated proteins such as cyclin D1 and p27Kip1 in SMC from aged mice. Increased ROS generation was associated with decreased endogenous antioxidant activity, increased lipid peroxidation, and mitochondrial DNA damage. Accrual of oxidant-induced damage and decreased proliferative capacity in SMC may explain, in part, the age-associated transition to plaque instability in humans with atherosclerosis.

The angiopoietin-tie2 system in coronary artery endothelium prevents oxidized low-density lipoprotein-induced apoptosis.

OBJECTIVES: A healthy, intact coronary artery endothelium is important because most common coronary artery diseases result from loss of endothelial integrity. In this study, we explored the biological significance of the angiopoietin-Tie2 system in porcine coronary artery. METHODS: Cultured porcine coronary artery endothelial cells and explanted coronary arteries were used. RESULTS: Immunohistochemical analyses indicated that Ang1 is selectively expressed in vascular muscular cells, whereas angiopoietin-2 (Ang2) and Tie2 are selectively expressed in endothelial cells. Accordingly, Ang1 mRNA is mainly expressed in cultured porcine coronary artery vascular smooth muscle cells, whereas Ang2 and Tie2 mRNAs are mainly expressed in cultured porcine coronary artery endothelial cells (PCAECs). Ang1 (200 ng/ml) induced Tie2 phosphorylation, while Ang2 (200 ng/ml) did not produce Tie2 phosphorylation. Ang1 increased the survival of cultured PCAECs during apoptosis induced by oxidized low-density lipoprotein (OxLDL). This survival effect was does-dependent and PI. Furthermore, Ang1 also protected endothelial cells of explanted coronary artery against OxLDL-induced apoptosis artery. CONCLUSION: These results suggest that adult coronary artery contains Ang1-Tie2 components that enhance endothelial cell survival to help maintain the normal integrity of the coronary artery endothelium.

Effects of uwhangchungsimwon on cell viability, proliferation, and gene expression of human neuronal cell line IMR32.

Uwhangchungsimwon (pill, UC) is one of the traditional Korean medical prescriptions that has been most frequently used for stroke. To characterize the effects of UC on human neuronal cells, the human neuroblastoma cell line IMR32 was treated with UC, and cell viability, cell proliferation, apoptosis, and gene expression were analyzed. The effect of UC on recovery of cell viability was analyzed following stress induction by nutrient depletion or cold shock. Flow cytometric analysis of the cell cycle showed that UC inhibits cell cycle progression of IMR32 in a dose- and time-dependent manner. UC was also identified to increase cell viability and suppress apoptosis induction by a DNA-damaging agent, etoposide. Quantitative RT-PCR analysis revealed that expressions of the p53 tumor suppressor gene and its downstream effect, Waf1, are stimulated whereas expressions of positive cell cycle regulators, c-Myc, c-Fos, and Cyclin D1 were repressed by UC treatment. Moreover, while expression levels of apoptosis inhibitors, Bcl-2 and Bcl-XL were increased following UC treatment, that of an apoptosis promoter, Bax, was decreased. In addition, expression of BMP-7, which has been recently demonstrated to improve the motor neuron recovery from stroke, was induced by UC while it was not detected in untreated cells. Taken together, our data suggest that the pharmacoclinical effects of UC might be derived in part from its negative regulation of cell proliferation and apoptosis through the transcriptional control of related genes.

Incidence of presbycusis of Korean populations in Seoul, Kyunggi and Kangwon provinces.

Presbycusis, a bilateral sensorineural hearing loss caused by changes in the inner ear, is related to multiple factors such as noise exposure and otologic disease. In institute-based studies, we tried to determine the incidence of presbycusis in Korean populations living in Seoul, Kyunggi and Kangwon provinces by gender and age groups. The subjects were people who had visited health promotion centers. Pure tone audiometry was done over 20 years on 6,028 subjects. In a community-based study, the subjects were elderly residents of Kanghwa-do area. There were no obvious factors that could cause hearing impairment in the subjects. For the pure tone audiometry, hearing threshold was obtained by using the six-dimension method. The incidence of presbycusis for subjects aged 65 years and older was 37.8% and 8.3% for > or = 27 dB HL criterion and > or = 41 dB HL criterion, respectively. The incidence increased with age. A statistically significant difference in the hearing threshold was found between men and women aged 65 years or older. No differences were found between the community-based study and the institute- based studies. There was a high incidence (about 40%) of presbycusis among Koreans aged 65 years or older (for > or = 27 dB HL criterion). With an aging population, we anticipate that this report could be used to provide a basic data for the study of presbycusis.

Inhibition of human smooth muscle cell proliferation by gamigeonsim-tang through the transcriptional regulation of cell cycle-controlling genes.

The effects of gamigeonsim-tang (GGT) on cellular proliferation and expression of cell cycle-related genes were investigated in human smooth muscle cell HISM. HISM cells were treated with an aqueous extract of GGT. Cellular proliferation was investigated by an immunocytometric analysis of PCNA expression and a flow cytometric analysis of the cell cycle progression. Reduced expression of PCNA and a significant accumulation of G1 phase cells were observed following treatment, indicating that GGT inhibits cellular proliferation of human smooth muscle cells. To explore whether GGT affects the transcription of cell cycle-regulating genes, we evaluated mRNA expression of p53, p21Waf1 PCNA, Cyclin D1, Cdc2, Histone H3, c-Myc, and c-Fos using a quantitative RT-PCR analysis. While increased expressions of two negative cell cycle regulators, p53 and p21Waf1 were found, reduced expressions of cell cycle stimulators, PCNA, c-Fos, and c-Myc, were identified following treatment. Taken together, our study demonstrates that GGT inhibits cellular proliferation of human smooth muscle cell through the up- and down-regulation of growth-inhibiting and growth-promoting genes, respectively.

Design and synthesis of highly potent fumagillin analogues from homology modeling for a human MetAP-2.

New fumagillin analogues were designed through structure-based molecular modeling with a human methionine aminopeptidase-2. Among the fumagillin analogues, cinnamic acid ester derivative CKD-731 showed 1000-fold more potent proliferation inhibitory activity on endothelial cell than TNP-470.

Hwansodan protects PC12 cells against serum-deprivation-induced apoptosis via a mechanism involving Ras and mitogen-activated protein (MAP) kinase pathway.

Hwansodan has been used as a prescription for senile and vascular dementia in Oriental medicine. We investigated the neuroprotective effects of Hwansodan water extract on the apoptotic death of PC12 cells by serum deprivation. Hwansodan significantly rescued PC12 cells from apoptotic death by serum deprivation in a dose-dependent manner. The nuclear staining of PC12 cells clearly showed that Hwansodan attenuated nuclear condensation and fragmentation, which represents typical neuronal apoptotic characteristics. Hwansodan also prevents DNA fragmentation and caspase-3-like protease activation in serum-deprived PC12 cells and induces the tyrosine phosphorylation of proteins around 44 kDa, which was identified as ERK1 with electrophoretic gel mobility shift by Western blot. In addition, MEK inhibitor PD98059 and Ras inactivator, alpha-hydroxyfarnesylphosphonic acid and mevastatin, attenuated the neuroprotective effects of Hwansodan in serum-deprived PC12 cells. These results indicate that Ras/MEK/ERK signaling pathway plays a role in neuroprotective effects of Hwansodan in serum-deprived PC12 cells.

Activation of caspase cascades in Korean mistletoe (Viscum album var. coloratum) lectin-II-induced apoptosis of human myeloleukemic U937 cells.

Mistletoe lectins are of high biological activity and exert cytotoxic effects. We have previously shown that Korean mistletoe, Viscum album var. coloratum, lectin-II specifically induces apoptotic cell death in cancer cells, not normal lymphocytes. The destructive mechanism by mistletoe lectins on tumor cells was mediated by activation of c-JUN N-terminal kinase (JNK)/stress-activated protein kinase. Herein, we investigated the involvement of caspase cascade and its proteolytic cleavage effects on biosubstrates of human myeloleukemic U937 cells by D-galactoside and N-acetyl-galactosamine-specific Korean mistletoe lectin-II. Mistletoe lectin-II induced ladder pattern DNA fragmentation and activation of caspase-3, -8, and -9 of U937 cells, but not caspase-1 protease, in a time- and dose-dependent manner. Consistent with catalytic activation of protease, both poly(ADP-ribose) polymerase (PARP) and protein kinase C-delta (PKC-delta) are also cleaved in mistletoe lectin-II-treated U937 cells. An inhibitor of caspase-3-like protease, DEVD-CHO peptide, significantly inhibited mistletoe lectin-II-induced apoptosis, PARP cleavage, and fragmentation of DNA. These results provide the evidence that Korean mistletoe lectin-II induces apoptotic death of U937 cells via activation of caspase cascades.

Cell biology of tubotympanum in relation to pathogenesis of otitis media - a review.

The sterility of the eustachian tube and tympanic cavity of normal individuals is maintained not only by the adaptive immune system, but also by the mucociliary system and the antimicrobial molecules of innate immunity. Mucin production and periciliary fluid homeostasis are essential for normal mucociliary function and dysfunction of this system is an important risk factor for otitis media. The secreted antimicrobial molecules of the tubotympanum include lysozyme, lactoferrin, beta defensins, and the surfactant proteins A and D (SP-A, SP-D). Defects in the expression or regulation of these molecules may also be the major risk factor for otitis media.

A new biological strategy for high productivity of recombinant proteins in animal cells by the use of hypoxia-response enhancer.

Oxygen supply is one of the major problems in the production of useful proteins by cultured animal cells and therefore it is of importance to devise a system by which a high productivity of human therapeutic recombinant proteins can be maintained or enhanced under low oxygen concentrations. A number of hypoxia-inducible genes have been found in animal cells and the induction in most cases is due to hypoxic activation of the gene transcription. A consensus sequence (HRE = hypoxia-response enhancer) responsible for the hypoxic activation exists in these genes and the binding of a protein, which is widely distributed in animal cells, to this sequence responding to hypoxia activates the promoter activity. The promoter of lactate dehydrogenase A gene is active in Chinese hamster ovary (CHO) cells and the vicinal HRE stimulates the promoter activity efficiently in hypoxia. We have prepared a number of permanent CHO cell lines producing recombinant human erythropoietin (Epo) under control of this promoter/HRE. Epo production was highly hypoxia-inducible when the wild-type of HRE was used but uninducible when the mutant HRE was used. There was little difference in the in vitro and in vivo activities, and glycosylation between Epo produced by the cells cultured in 21% and 2% oxygen. Furthermore, forced expression of hypoxia-inducible factor-1alpha (HIF-1alpha) enhanced Epo production in all oxygen concentrations. These results indicate that a biological strategy based on the hypoxic induction of gene transcription provides a novel system which guarantees a high productivity even uner low oxygen concentrations.

Mucin gene expression in cultured human middle ear epithelial cells.

For the advanced study of the cell and molecular biology of middle ear mucosa, an in vitro cell culture system is required. Although middle ear epithelial cells have been cultured from various species of laboratory animal, there have been no reports concerning a serial subculture system of human middle ear epithelial cells. In this paper, we describe the establishment of a primary culture system of human middle ear epithelial cells using a serum-free conditioned medium and the characterization of these cells by the expression of phenotypic characteristics of epithelial cells and mucin genes. Cultured cells were anchorage-dependent in terms of growth and showed a polygonal cobblestone-like appearance: desmosomes in the cell junction were observed by electron microscopy. In the immunocytochemical study, cytokeratin (epithelial cell marker) was expressed in all cultured cells. but von Willebrand factor (endothelial cell marker) was not. Unexpectedly, vimentin (fibroblast marker) was locally expressed, and a double stain showed the co-expression of both cytokeratin and vimentin in the same cell. The products of reverse transcriptase polymerase chain reaction from cultured cells yielded distinct bands compatible with the expected sizes of the MUC1, MUC2, MUC5AC and MUC5B genes. This culture system will allow us to prepare the cell line and to perform advanced studies of human middle ear mucosal biology.