A Transmission-Line-Based Cochlear Standing Wave Model To Elucidate Mechanism of Human Auditory System.

How do people hear sounds? As a counterpart of Prof. G. V. Békésy's traveling wave theory, we have proposed resonance theory of outer hair cells and cochlear standing wave theory, respectively. Based on these proposals, this paper develops a transmission-line-based cochlear standing wave model. Since the macroscopic cochlear model is designed as it looks like, various auditory physiology can be explained. Transient analyses with pure-tone excitation and Gaussian pulse excitation are carried out, and Prof. D. Kemp's otoacoustic emission (OAE) is demonstrated successfully.Clinical relevance-Our new model has a great potential to explain auditory physiology including structural inner disorders, hearing loss, and even tinnitus.

Novel pre-therapeutic scoring system using patient and haematological data to predict facial palsy prognosis.

OBJECTIVES: We describe a novel scoring system, the facial Palsy Prognosis Prediction score (PPP score), which we test for reliability in predicting pre-therapeutic prognosis of facial palsy. We aimed to use readily available patient data that all clinicians have access to before starting treatment. DESIGN: Multicenter case series with chart review. SETTING: Three tertiary care hospitals. PARTICIPANTS: We obtained haematological and demographic data from 468 facial palsy patients who were treated between 2010 and 2014 in three tertiary care hospitals. Patients were categorised as having Bell's palsy or Ramsey Hunt's palsy. MAIN OUTCOME MEASURES: We compared the data of recovered and unrecovered patients. PPP scores consisted of combinatorial threshold values of continuous patient data (eg platelet count) and categorical variables (eg gender) that best predicted recovery. We created separate PPP scores for Bell's palsy patients (PPP-B) and for Ramsey Hunt's palsy patients (PPP-H). RESULTS: The PPP-B score included age (≥65 years), gender (male) and neutrophil-to-lymphocyte ratio (≥2.9). The PPP-H score included age (≥50 years), monocyte rate (≥6.0%), mean corpuscular volume (≥95 fl) and platelet count (≤200 000 /µL). Patient recovery rate significantly decreased with increasing PPP scores (both PPP-B and PPP-H) in a step-wise manner. PPP scores (ie PPP-B score and PPP-H score) ≥2 were associated with worse than average prognosis. CONCLUSIONS: Palsy Prognosis Prediction scores are useful for predicting prognosis of facial palsy before beginning treatment.

Galectin fingerprinting by immuno- and lectin histochemistry in cutaneous lymphoma.

Owing to their relevance for growth regulation and cell adhesion monitoring the expression of galectins (endogenous beta-galactoside-binding lectins) and their binding sites has relevance for tumor biology. Using galectin-type-specific reagents (non-crossreactive antibodies for proto-type galectin-1, chimera-type galectin-3 and tandem-repeat-type galectins-4 and -8, and labeled galectins-1, -3, and -4) we determined galectin expression in cutaneous T cell lymphomas (CTCL) and controls. In addition to commonly studied galectins-1 and -3, tandem-repeat-type galectins could be detected, i.e., galectin-8 in six from 15 cases and galectin-4 in one of 15 cases. In view of relevant ligands such as bcl-2 or integrins the presence of galectins-3 and -8 seems to be possibly related to loss of proliferation control and change in cell adhesion properties that are involved in clonal expansion and epidermal spread of malignant T cell clones. Successful chemotherapy of CTCL alters galectin expression selectively as shown for liposomal doxorubicin.

Two domains of rat galectin-4 bind to distinct structures of the intercellular borders of colorectal epithelia.

Galectin-4 (G4) is a member of a family of soluble galactoside-binding lectins found in various mammalian tissues. To determine the function of this protein in colorectal tissue, we separately produced the N- and C-terminal carbohydrate binding domains (CBD) of rat G4 as a recombinant glutathione S-transferase (GST) fusion protein (G4-N and G4-C) and examined the tissue binding site(s) of each CBD by light and electron microscopy (LM and EM). At the LM level, both fusion proteins stained the intercellular borders of the surface-lining epithelial cells of colorectal mucosa. At the EM level, two proteins recognized spatially close but distinct subcellular structures. G4-N stained electron-lucent flocculent substances freely located in the intercellular spaces, whereas G4-C bound to the lateral cell membranes demarcating the intercellular spaces. These findings suggest that colorectal G4 may be involved in crosslinking the lateral cell membranes of the surface-lining epithelial cells, thereby reinforcing epithelial integrity against mechanical stress exerted by the bowel lumen. (J Histochem Cytochem 47:75-82, 1999)

Recombinant galectin-1 recognizes mucin and epithelial cell surface glycocalyces of gastrointestinal tract.

Rat gastrointestinal (GI) tract is rich source of galectins, a family of mammalian galactoside-binding lectins. To determine which tissue component is the relevant glycoconjugate ligand for the galectins, we produced recombinant galectin-1 and surveyed its binding sites on tissue sections of rat GI tract. Mucin and epithelial surface glycocalyces of both gastric and intestinal mucosa were intensely stained. This finding raises the possibility that some GI tract galectins known to be secreted by the epithelia may recognize these glycoconjugates and crosslink them into a macromolecular mass. This galectin-ligand complex may play a role in protecting the epithelial surface against luminal contents such as gastric acid, digestive enzymes, and foreign organisms.

Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.

Colocalization of connexin 43 and connexin 45 but absence of connexin 40 in granulosa cell gap junctions of rat ovary.

The expression and localization of gap junction family proteins (connexins) were examined in nonstimulated and gonadotrophin-stimulated ovarian follicles of immature rats. Immunoblot and RNA blot analysis showed the presence of connexin (Cx) 43, Cx40 and Cx45 in ovarian tissue. Of these connexin proteins, Cx43 and Cx45 were identified by immunofluorescent microscopy between granulosa cells in characteristic expression patterns related to follicular developmental stages, while Cx40 was not expressed in granulosa cells but was detected in blood vessels in ovarian stroma. In some plaques of gap junction between granulosa cells, Cx45 was found to be colocalized with Cx43. In immunofluorescent microscopy, the expression of Cx43 was increased with follicular growth, but decreased after induction of ovulation by injection of human chorionic gonadotrophin. In contrast, the Cx45 protein was constantly expressed through follicular development; however, after ovulation, no staining of Cx45 was detected in the corpus luteum. Dual expression and the functional role of Cx43 and Cx45 in cell-to-cell communication in ovarian granulosa cells at various developmental stages were discussed.

Rat intestinal galactoside-binding lectin L-36 functions as a structural protein in the superficial squamous cells of the esophageal epithelium.

Using an affinity purified antibody raised against the RI-H fragment of rat intestinal lectin L-36, the latter protein has been identified within the esophageal epithelium by means of ultracryotomy followed by immunogold labeling. The epithelium consists of 4 morphologically distinct cell-types, namely, the basal, spiny, granular and squamous cells, and each of these exhibits a different immunolabeling pattern. The basal cells form a layer on the basal lamina, and in these a diffuse cytoplasmic staining is observed. This basal cell layer is overlaid by spiny cells that extend many cell processes into wide intercellular spaces. In these cells, immunogold particles are found only on small granular inclusions consisting of an electron-lucent homogeneous substance. The granular cells form a third layer over the spiny cells, and are characterized by a number of large granular inclusions with an electron-dense core rimmed by a less electron-dense substance. Immunogold labeling is found on these granules, both on the core and peripheral region. Squamous cell-types constitute the most superficial layer of the epithelium. They are without granular inclusions, and immunogold labeling is confined to the cytoplasmic surface of the thickened plasma membrane. These findings suggest that L-36 is produced in the basal cells as free cytosolic protein, then becomes progressively aggregated into the granular inclusions of the spiny and granular cells, and is eventually transferred onto the cytoplasmic surface of the squamous cell plasma membrane where it may interact with complementary glycoconjugate(s) located at this site.(ABSTRACT TRUNCATED AT 250 WORDS)

Lamellar bodies of rat alveolar type 2 cells have late endosomal marker proteins on their limiting membranes.

Alveolar type 2 cells are known to take up surfactant phospholipids and proteins from the alveolar space and recycle them into secretory organelles via a receptor-mediated endocytic pathway. To clarify the intracellular route(s) through which materials ingested by the cells are processed, we examined the immunocytochemical localization of late endosomal and lysosomal membrane markers, rab 7 and lamp 1 proteins, within rat alveolar type 2 cells. The limiting membranes of lamellar bodies (LBs) showed positive immunoreactivity for both proteins, whereas multivesicular bodies (MVBs) exhibited positive immunoreactivity only for lamp 1 protein on free vesicles in the MVB lumen. From these findings, it is suggested that LBs are not only secretory granules, but also constitute one of the late endosomal compartments of the cells and that MVBs of this cell type may be targeted to cell organelle(s) other than lysosomes.

Morphological heterogeneity of secretory granules of rat Clara cells: an immunocytochemical study.

The secretory granules of rat bronchiolar Clara cells were classified into different types by their ultrastructural appearances followed by immunocytochemistry using anti-rat 10 kDa Clara cell-specific protein (10 kDa CCSP) antibody. One predominant type was the oval to round granule (type A granule), of which the matrix was composed of a map-like mixture of electron-dense and less electron-dense material. Another predominant type was the rod-shaped granule (type B granule). The content of type B granules varied from a finely fibrillar (type B1 granule) to an electron-dense, rod-like (type B3 granule) structure. Various intermediate types (type B2 granule) between type B1 and B3 granules were also found. Small cytoplasmic vesicles were found occasionally in close proximity to type B2 or B3 granule. Another type of granule (type C granule) was large, up to 8 microns in diameter, and contained a moderately electron-dense amorphous matrix. Both type A and C granules stained at a similar density with the antibody. The nascent form of type A granules, which was found in the vicinity to the trans face of the Golgi apparatus, was also labeled. On the other hand, the labeling density of type B granules varied: type B1 granules were almost devoid of immunolabeling, whereas type B3 granules were intensely labeled. Type B2 granules stained with the antibody; however, the labeling density was less than that of type B3 granules. The small cytoplasmic vesicles of type B2 granules were labeled.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs.

67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and -4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.

Immunoelectron microscopic analysis of Ia antigen expression in rat skin during limb allograft rejection.

Using a whole-limb graft model in rats, morphologic changes and variations in the expression of Ia antigen on epidermal cells were investigated in the allografted skin during acute rejection. BN right limbs were transplanted to F344 recipients. Skin tissues were excised during acute rejection on days 1, 3, 5, 7, and 9 after the transplantation. Sections were examined for Ia antigen expression using immunohistologic techniques, and in situ quantification of Ia antigen was made using an immunogold method. Epidermal keratinocytes expressed Ia antigen before the grafts were rejected and the amount of Ia antigen expression increased and exceeded the amount of Ia antigen of Langerhans cells during the course of rejection. The progressive increase in class II antigen expression on EKs correlated with the appearance and relative accumulation of dermal lymphocytic cells. On the other hand, Ia antigen was not expressed on vascular endothelial cells during rejection. Our results suggest that the Ia-positive keratinocytes can serve as target cells in skin rejection of limb allografts. The immunogold technique we used seems most pertinent for a quantitative examination of cell-surface antigens in situ.

Immunohistochemical localization of 14 kDa beta-galactoside-binding lectin in various organs of rat.

Immunohistochemical localization of 14 kDa beta-galactoside-binding lectin in various organs of adult rat was achieved using a monospecific antibody raised against lectin purified from rat lung. The antibody-stained cells were formed into small aggregates, thin fascicles, or thick bundles in the walls of blood vessels, gastrointestinal tracts and urogenital organs. From the patterns of distribution, as well as their organization, these immunoreactive cells were regarded as smooth muscle cells. This was confirmed by a double immunofluorescence study using a mixture of anti 14 kDa lectin and anti alpha-smooth muscle-specific actin antibodies. Strong 14 kDa lectin immunoreactivity was seen in the pericellular matrix of smooth muscle cells in intact organs as well as in detergent-treated organs from which all cellular components were extracted. From these findings, it is suggested that the 14 kDa lectin may be externalized by smooth muscle cells into their pericellular matrix and participate in the crosslinking of the complementary glycoconjugate(s) localized at that site. The macromolecular xomplex of glycoconjugates thus formed around smooth muscle cells may play a role in anchoring smooth muscle cells to the pericellular connective tissue thereby permitting the force of muscle contraction to be efficiently transmitted to the surrounding connective tissue proper.

Rat lung 29 kD beta-galactoside-binding lectin is secreted by bronchiolar Clara cells into airways.

We isolated a mixture of beta-galactoside-binding lectins from rat lung and raised polyclonal antibody against 14 kD lectin purified from the mixture of lectins. Immunoblotting of the mixture of lectins, which was separated with SDS-PAGE under reducing condition and transferred onto a NC paper, showed that the antibody reacted with two bands at 14 and 29 kD, indicating that these two lectins have common antigenic determinants(s). Immunohistochemically, the antibody recognized only bronchiolar Clara cells with intense immunofluorescence in their apical cytoplasmic protrusions where the secretory granules of the cells are known to be stored. Thus, to determine if the lectin(s) might be secreted into airways, we next raised antibody against airway secretions free from serum as well as surfactant proteins. By immunoblot analysis, the resulting antibody stained 29,45 and 55 kD bands, but not 14 kD band, on a NC paper transferred with the mixture of lectins. These findings suggest that at least 29 kD lung lectin is located in bronchiolar Clara cells and secreted by these cells into airways.

Lectin-gold cytochemistry of mucin oligosaccharide biosynthesis in Golgi apparatus of airway secretory cells of the hamster.

To elucidate the mechanism for the biosynthesis of O-linked mucin oligosaccharides, airway secretory cells of the hamster trachea were embedded in Lowicryl K4M resin, and sections were examined by lectin-gold cytochemistry with special attention focused on the Golgi apparatus. The interrelations between the Golgi cisternae stained with five different lectins were determined by double-staining procedures using various combinations of lectins conjugated with 14-nm and 8-nm colloidal gold. Several cis cisternae were stained only with HPA (Helix pomatia agglutinin specific for terminal alpha-N-acetylgalactosamine). The next medial cisternae were not stained with HPA, but reacted positively with two lectins, GSII (Griffonia simplicifolia agglutinin II specific for terminal alpha- or beta-N-acetylglucosamine) and RCAI (Ricinus communis agglutinin I specific for beta-galactose). The trans cisternae as well as condensing and mature secretory granules were labeled with four lectins, UEAI (Ulex europaeus agglutinin I specific for terminal alpha-L-fucose) and LFA (Limax flavus agglutinin specific for terminal N-acetyl or N-glycolyl neuraminic acid) in addition to HPA and RCAI. The same number of trans cisternae were positive to HPA and UEAI, whereas LFA bound to a few transmost cisternae but fewer than were stained with HPA or UEAI. The observed sequential appearance of different sugar residues in different levels of Golgi cisternae (from cis to trans cisternae) coincides quite well with the sugar sequence of airway mucin oligosaccharide (from reducing to nonreducing ends) proposed by biochemical analysis. It is suggested that airway mucin oligosaccharides elongate during a vectorial movement through the Golgi stack from cis toward trans and that the stack consists of at least three functionally distinct segments, cis, medial, and trans; in these three segments there take place, respectively, the initial O-glycosylation of mucin core peptide, the formation of a core region of oligosaccharide chain, and the completion of chain growth by addition of terminal sugar moieties.

Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells.

Studies on the regulation of differentiation in airway epithelial cells have been hampered by the lack of cell culture systems that differentiate in vitro. One such system that does exhibit differentiation is hamster tracheal epithelial cells (HTE). A major problem with this system, however, is that at the time cells differentiate, they lyze the collagen gel upon which they grow, resulting in termination of the culture. Here we report that by growing the HTE cells at 32 degrees instead of 37 degrees C we can totally prevent lysis of the collagen gel. Cells grown at this lower temperature maintain their differentiated phenotype as evidenced by abundant mucus granules and the secretion of authentic mucus glycoproteins into the culture media. We have also developed a method for subculturing the primary cells which allows growth and differentiation in secondary culture. The HTE cells were capable of being passaged at least three times and did not become transformed as judged by their inability to grow in soft agar and to produce tumors in syngeneic animals. This improved HTE cell culture system will allow detailed studies on the mechanisms which regulate growth, differentiation, and mucus secretion in surface airway epithelial cells.

Membrane differentiation markers of airway epithelial secretory cells.

We describe here a system for culturing epithelial cells isolated from hamster trachea, which results in a highly enriched population of mucus-secreting cells. The culture system has enabled us to study the process of secretory cell differentiation in vitro. We found that epithelial secretory cells, in vivo and after 5 days in vitro, selectively bind the lectin Helix pomatia agglutinin (HPA) to apical and, to a lesser extent, basolateral surfaces as well as to mucin granules and intracellular secretory organelles. SDS-PAGE gels of detergent extracts of secretory cells cultured for 5 days reveal three HPA-binding glycoproteins with MW of 120 KD, 220 KD, and greater than 400 KD. The high-MW glycoprotein appears identical to mucin, since it is found in secretions from intact trachea and in spent media from 5-day cultures. It does not appear in spent media from 3-day cultures when cells contain few mucous granules and secrete little mucin. The 220 KD HPA-binding glycoprotein is also present in 5-day but not in 3-day cultures. In contrast, the 120 KD glycoprotein is present at both times. HPA-gp120 is a hydrophobic integral membrane protein, whereas HPA-gp220 and mucin are hydrophilic and are membrane associated. These studies define three membrane glycoproteins, one of which is specific for the tracheal epithelial secretory cell regardless of its mucous content, whereas the other two glycoproteins correlate with mucin secretion. They also demonstrate that, in the fully differentiated state, mucin is bound in a non-covalent fashion to the apical plasma membrane of the tracheal epithelial secretory cell.

Human neutrophil elastase releases cell surface mucins from primary cultures of hamster tracheal epithelial cells.

Primary hamster tracheal epithelial cells growing on a collagen gel matrix produce high molecular weight mucins indistinguishable from mucins produced in vivo. Using a modified version of these confluent cultures, we have demonstrated here that (i) release of mucins can be stimulated by human neutrophil elastase (HNE; EC 3.4.21.37); (ii) HNE can degrade mucins, and both mucin release and degradation by HNE require an active catalytic site; and (iii) there are at least two pools of mucins in these cells: one is a rapidly turning-over spontaneously releasable constitutive pool, the other is a slowly turning-over HNE-releasable pool. We provide evidence that the HNE-releasable mucins are membrane bound and associated with the secretory cell apical surface.

Three-dimensional architecture of elastic tissue in early atherosclerotic lesions of the rat aorta.

The overall three-dimensional architecture of elastic tissue in early atherosclerotic lesions of the rat aorta was studied using scanning electron microscopy (SEM) after hot-formic acid extraction followed by a freeze-drying method. These lesions were induced by feeding the rats a diet containing 2% cholesterol, 0.5% cholic acid and 0.2% methylthiouracil. SEM revealed two types of alterations in the elastic tissue; one was an increase in the dome-like elastic lamina with few fenestrations that might be due to the reduplication of the internal elastic lamina (IEL), and the other was an increase in fibrous elastin, generally oriented longitudinally in the intima. The former was discussed with respect to its barrier function to such macromolecules as fibrinogen and low density lipoprotein (LDL), and was assumed to be a structure related to prevention of early atherosclerotic lesions.