Molecular Mechanisms behind Circular Dichroism Spectral Variations between Channelrhodopsin and Heliorhodopsin Dimers.

Channelrhodopsin (ChR) and heliorhodopsin (HeR) are microbial rhodopsins with similar structures but different circular dichroism (CD) spectra: ChR shows biphasic negative and positive bands, whereas HeR shows a single positive band. We explored the physicochemical factors underlying these differences through computational methods. Using the exciton model based on first-principles computations, we obtained the CD spectra of ChR and HeR. The obtained spectra indicate that the protein dimer structures and the quantum mechanical treatment of the retinal chromophore and its interacting amino acids are crucial for accurately reproducing the experimental spectra. Further calculations revealed that the sign of the excitonic coupling was opposite between the ChR and HeR dimers, which was attributed to the contrasting second term of the orientation factor between the two retinal chromophores. These findings demonstrate that slight variations in the intermolecular orientation of the two chromophores can result in significant differences in the CD spectral shape.

The Coevolution of Cellularity and Metabolism Following the Origin of Life.

The emergence of cellular organisms occurred sometime between the origin of life and the evolution of the last universal common ancestor and represents one of the major transitions in evolutionary history. Here we describe a series of artificial life simulations that reveal a close relationship between the evolution of cellularity, the evolution of metabolism, and the richness of the environment. When environments are rich in processing energy, a resource that the digital organisms require to both process their genomes and replicate, populations evolve toward a state of non-cellularity. But when processing energy is not readily available in the environment and organisms must produce their own processing energy from food puzzles, populations always evolve both a proficient metabolism and a high level of cellular impermeability. Even between these two environmental extremes, the population-averaged values of cellular impermeability and metabolic proficiency exhibit a very strong correlation with one another. Further investigations show that non-cellularity is selectively advantageous when environmental processing energy is abundant because it allows organisms to access the available energy, while cellularity is selectively advantageous when environmental processing energy is scarce because it affords organisms the genetic fidelity required to incrementally evolve efficient metabolisms. The selection pressures favoring either non-cellularity or cellularity can be reversed when the environment transitions from one of abundant processing energy to one of scarce processing energy. These results have important implications for when and why cellular organisms evolved following the origin of life.

[Clinical efficacy of immunotherapy with house dust in the patients with perennial nasal allergy].

We tried to elucidate the relation of age and severity with the efficacy of immunotherapy. The subjects included 51 patients with perennial allergic rhinitis who had immunotherapy for more than a year. Symptom scores, serum total IgE levels, Dermatophagoides pteronyssinus specific IgE, and peripheral blood eosinophils counts were evaluated before, half a year, one year and two years after the onset of immunotherapy. 64% of the children's group and 69% of the adults' group of patients showed significant improvement in their nasal symptoms, showing that age is not a factor influencing the efficacy of immunotherapy. The severity of the disease influenced the clinical efficacy; the more severe the disease, the poorer the efficacy of the immunotherapy. Periphipheral blood eosinophils counts gradually decreased after the onset of immunotherapy. High eosinophil counts before immunotherapy predicted poor efficacy of the immunotherapy.

CFTR antisense phosphorothioate oligodeoxynucleotides (S-ODns) induce tracheo-bronchial mucin (TBM) mRNA expression in human airway mucosa.

Mucus hypersecretion is a critical component of cystic fibrosis (CF) pathogenesis. The effects of dysfunction of the cystic fibrosis transmembrane regulator (CFTR) on mucin expression were examined using the tracheo-bronchial mucin (TBM) gene as an indicator. TBM mRNA expression was assessed in a human bronchial epithelial cell line (HBE1) and human nasal mucosal explants in vitro. Antisense phosphorothioate oligodeoxynucleotides (S-ODN) to TBM suppressed baseline expression of TBM mRNA in both systems, but had no effect on glyceraldehyde phosphate dehydrogenase mRNA (GAPDH) expression. Sense and missense (multiple scrambled control oligonucleotides) S-ODNs had no effect. 8Br-cAMP and PGE1 significantly elevated TBM mRNA expression. These increases were also specifically inhibited by the antisense S-ODNs. In order to induce a CF-like state, S-ODN to CFTR were added to explants. Antisense CFTR S-ODNs were anticipated to reduce the expression of cellular CFTR protein, and the level of CFTR function. Antisense, but not sense or missense, CFTR S-ODN significantly increased TBM mRNA expression. These data suggest that mucin hypersecretion in CF may be a direct consequence of CFTR dysfunction; the specific mechanism through which this effect is mediated is not known.

Hypertonic saline nasal provocation stimulates nociceptive nerves, substance P release, and glandular mucous exocytosis in normal humans.

Hypertonic saline (HTS) induces bronchoconstriction. Potential mechanisms were evaluated in a human nasal provocation model. Aliquots of normal saline (1 x NS, 100 microliters) and higher concentrations (3 x NS, 6 x NS, 12 x NS, 24 x NS) were sprayed into one nostril at 5-min intervals. Lavage fluids were collected from the ipsilateral and contralateral sides to determine the concentrations of specific mucus constituents. Nasal cavity air-space volume was assessed by acoustic rhinometry (AcRh). The distribution of substance-P-preferring neurokinin-1 (NK-1) receptor mRNA was assessed by in situ reverse transcriptase-polymerase chain reaction. Unilateral HTS induced unilateral dose-dependent increases in sensations of pain, blockage, and rhinorrhea, the weights of recovered lavage fluids, and concentrations of total protein, lactoferrin, mucoglycoprotein markers, and substance P. Contralateral, reflex-mediated effects were minor. There were no changes in IgG or AcRh measurements. NK-1 receptor mRNA was localized to submucosal glands. HTS caused pain with unilateral substance P release. The presumed nociceptive nerve efferent axon response led to glandular exocytosis, presumably through actions on submucosal gland NK-1 receptors. Vascular processes, including plasma exudation, filling of venous sinusoids, and mucosal edema were not induced in these normal subjects.

Rhinovirus infection induces mucus hypersecretion.

Rhinorrhea is a prominent symptom of the common cold. Although increases in vascular permeability and serous cell secretion have been demonstrated in human nasal mucus during active rhinovirus infections, changes in mucin constituents have not been quantified. Nonallergic (n = 48) and asymptomatic allergic rhinitis (n = 32) subjects were inoculated with rhinovirus type hanks before the spring allergy season. Nasal lavages were performed before inoculation (day 0), then daily for 5 days afterward. The subjects were divided into infected and noninfected groups on the basis of evidence of successful rhinovirus infection (nasal shedding of virus or fourfold increases in specific serum antibodies). Concentrations of interleukin (IL)-8, markers of vascular leak (IgG), seromucous cells (lysozyme), and mucoglycoprotein exocytosis [7F10-immunoreactive mucin (7F10-irm) and Alcian blue staining of acidic mucoglycoproteins] were measured in lavage fluids. The infected subgroup had maximal increases in nasal lavage fluid concentrations of IL-8 (sevenfold), IgG (fourfold), total protein (twofold), and gel-phase 7F10-irm (twofold) on day 3. There were no differences between infected allergic and nonallergic subjects. IL-8 and gel-phase 7F10-irm were significantly higher in infected than in noninfected subjects. In addition to promoting plasma exudation, rhinovirus hanks infection increases IL-8 and gel-phase mucin secretion. These processes may contribute to a progression from watery rhinorrhea to mucoid discharge, with mild neutrophilic infiltration during the common cold.

[Japanese cedar pollinosis in children in our allergy clinic].

Recently, it is pointed out that the incidence of Japanese cedar pollinosis has increased in children. We studied on the rate of sensitization and the onset of the disease in children under sixteen who visited allergy clinic in the Department of Otorhinolaryngology in Mie University Hospital. The percentage of positive rate of skin test and IgE antibodies of house dust mite was about 80 to 90% in 1981, 1991, and 1996. However, the percentage of positive rate of skin test and IgE antibodies of Japanese cedar pollen was 43% and 26%, respectively in 1981, but both increased to 58% in 1996. Most of Japanese cedar pollinosis patients also had house dust mite allergy. Among 115 pediatric patients who visited our allergy clinics in the past seven years, 32.2% were allergic to house dust mite alone, 8.6% were allergic to Japanese cedar pollen alone, 40.9% were allergic to both, and 6.1% were allergic to house dust mite, Japanese cedar pollen, and orchard grass pollen. 68% of the total 115 patients were boys, but significantly more girls had the pollinosis. 17.4% of those who are sensitized to the pollen were asymptomatic during the pollen season. Thus, it was confirmed that the rate of children sensitized to the pollen has apparently increased for the past twenty years. We should take care of those children who are sensitized to the pollen but asymptomatic during the pollen season.

Detection of thromboxane A2 receptor mRNA in the nasal mucosa of patients with allergic rhinitis.

Previous reports suggest that thromboxane (TX) A2 plays an important role in bronchial asthma, especially in the development of airway hypersensitivity. Similarly to asthmatic subjects, the nasal mucosa in patients with allergic rhinitis is hypersensitive to non-specific stimuli. Allergic rhinitis is mediated by chemical mediators released from inflammatory cells. However, the involvement of TXA2 in allergic rhinitis has been studied very little. Recently, TXA2 receptors from humans and mice have been characterized at the molecular level. We performed RT-PCR to examine whether TXA2 receptor mRNA expression is elevated higher in allergic rhinitis than in chronic sinusitis. We found that TXA2 receptor mRNA was expressed both in the nasal inferior turbinates with allergic rhinitis and in the maxillary sinus mucosa with chronic inflammation at a similar level. This result suggests that TXA2 receptor mRNA expression is not enhanced in allergic rhinitis.

Nasal secretion analysis in allergic rhinitis, cystic fibrosis, and nonallergic fibromyalgia/chronic fatigue syndrome subjects.

Rhinitis symptoms are present in approximately 70% of subjects with fibromyalgia and chronic fatigue syndrome (FM/CFS). Because only 35% to 50% have positive allergy skin tests, nonallergic mechanisms may also play a role. To better understand the mechanisms of nonallergic rhinitis in FM/CFS, nasal lavages were performed, and markers of vascular permeability, glandular secretion, and neutrophil and eosinophil infiltration measured in 27 nonallergic FM/CFS, 7 allergic rhinitis, 7 cystic fibrosis, and 9 normal subjects. Allergic rhinitis subjects had significantly increased vascular permeability (IgG) and ECP levels. Cystic fibrosis subjects had significantly higher elastase and total protein levels. There were no significant differences between FM/CFS and normal lavage fluids. Analysis of the constituents of nasal mucus provides information about ongoing secretory processes in rhinitis. There were no differences in the basal secretion of these markers of vascular permeability, submucosal gland serous cell secretion, eosinophil and neutrophil degranulation in nonallergic FM/CFS subjects. This suggests that constitutively active secretory processes that regulate continuous production of nasal secretions are not altered in FM/CFS. Future studies should examine alternative mechanisms such as inducible, irritant-activated, or reflex-mediated effects.

Glucocorticoids induce beta2-adrenergic receptor function in human nasal mucosa.

Glucocorticoids are hypothesized to induce beta2-adrenergic receptors (beta2-R) and their functions. The ability of dexamethasone (DEX) in vitro and beclomethasone dipropionate (BDP) in vivo to induce beta2-R messenger RNA (mRNA) and function was investigated in human nasal mucosa. In this tissue, albuterol does not stimulate exocytosis either in vivo or in vitro (Mullol and coworkers, 1992). Therefore, induction of beta2-R-mediated glandular exocytosis by glucocorticoids was proposed as an unambiguous outcome measure. Human nasal mucosa was cultured for 3 d with and without 1 microM DEX, then challenged with media or 100 microM albuterol. Culture supernatants were collected for measurement of exocytosed glandular products. Explant mRNA was extracted for reverse transcriptase-polymerase chain reaction (RT-PCR), and in situ hybridization of beta2-R mRNA performed. In vivo, normal subjects received saline or BDP for 3 d before albuterol nasal provocation. Concentrations of exocytosed products were measured in nasal secretions. RNA was extracted from nasal epithelial scrapings for RT-PCR. In vitro, DEX treatment induced albuterol-mediated glandular exocytosis (p < 0.04), and increased the steady-state beta2-R/beta-actin mRNA ratio (p < 0.05), and expression of beta2-R mRNA in glands. In vivo, BDP increased the beta2-R/beta-actin mRNA ratio in epithelial scrapings (p < 0.04), but did not induce albuterol-mediated glandular secretion. We conclude that glucocorticoids increase steady-state beta2-R mRNA levels in vivo and in vitro, and can induce beta2-R function as assessed by submucosal gland exocytosis in vitro. While topical BDP induced epithelial beta2-R mRNA, it did not modulate exocytosis from the deeper submucosal glands.

Mucoglycoprotein hypersecretion in allergic rhinitis and cystic fibrosis.

There is little information about specific changes in submucosal gland exocytosis in diseases such as allergic rhinitis (AR), nonallergic rhinitis (NAR), and cystic fibrosis (CF). Nasal lavage fluids were collected from normal, AR, NAR, and CF subjects. Concentrations of lysozyme, Alcian blue-staining mucoglycoconjugate material (AB + m), and human high-molecular-weight mucoglycoconjugates recognized by the 7F10 murine monoclonal antibody [7F10-immunoreactive mucoglycoconjugates (7F10-irm)] were measured. AB + m and 7F10-irm were characterized by Sepharose-2B column chromatography and glycosidase digestion. 7F10-irm was increased in CF (2.4-fold; P = 0.001) and AR (12.7-fold; P = 0.00007) subjects. AB + m was increased in CF (1.8-fold; P = 0.049) and AR (1.2-fold; P = 0.07) subjects. There were no changes in NAR subjects. On Sepharose-2B columns, AB + m peaks were at 1.3-3.0 x 10(6) and 0.36-0.65 x 10(6) Da. 7F10-irm showed four distinct peaks at 1.5, 1.2, 0.85, and 0.53 x 10(6) Da that were nearly identical in both normal and CF samples. Sialic acid was present in both 7F10-irm and AB + m. 7F10-irm and AB + m are mutually exclusive sialylated mucoglycoproteins that are significantly induced in AR and CF but not in NAR.

Increased expression of histamine H1 receptor mRNA in allergic rhinitis.

BACKGROUND: Histamine plays an important role in producing nasal symptoms via histamine H1 receptor (H1R) in allergic rhinitis. It is reported that the minimum histamine concentration that induces sneezing is lower in allergic patients than in normal control subjects. Previous studies by binding assay on H1R gave divided results on whether the number of H1Rs is increased in allergic rhinitis or not. OBJECTIVE: The objective of this study was to examine if H1R mRNA expression is increased in patients with allergic rhinitis compared with normal healthy volunteers. METHODS: We extracted RNA from scrapings of inferior turbinate mucosa of 10 patients suffering from allergic rhinitis and 10 control subjects. As the H1R gene lacks introns, we treated RNA pellets by DNase to distinguish RNA from contaminating genomic DNA. Since amplification of H1R and beta-actin mRNA remained in an exponential phase at 35 cycles, H1R and beta-actin mRNAs were amplified for 35 cycles by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were hybridized with internal probes and band intensities were quantitated by a densitometer. RESULTS: The mean +/- SD of H1R/beta-actin ratio was 0.88 +/- 0.62 for the patients with allergic rhinitis and 0.29 +/- 0.17 for the normal subjects; the difference was statistically significant (P < 0.01). CONCLUSIONS: These data suggest that expression of H1R mRNA is increased in the nasal mucosa of the patients with allergic rhinitis.

Analysis of T cell receptor beta chain repertoire in middle ear effusions.

In order to elucidate the immune response in otitis media with effusion (OME), the polymerase chain reaction was employed to examine T cells in middle ear effusions in patients with OME for utilization of T cell receptor (TCR) variable region genes. Specimens of RNA were extracted from 13 ears of 12 patients (9 children and 3 adults). Oligonucleotide primers specific for individual TCR Vbeta gene families were used to amplify TCR gene products in each sample. Although the number of Vbeta families utilized by each sample varied from 1 family to 21, a few significant trends emerged. Eleven ears out of 13 expressed Vbeta7, which was the most frequently utilized (84.6%) Vbeta family among the 24 Vbeta families. In 5 of the 13 samples, the number of Vbeta families utilized was restricted to 1, which was Vbeta7 in all 5 samples. This result indicates the possibility that Vbeta7-bearing T cells in the middle ear are responding to a certain common antigen in some cases of OME.

MUC2 mucin gene expression in the nose and maxillary sinus.

PURPOSE: The MUC2 gene encodes the core protein of a mucin expressed in the intestine and lower airway. The purpose of this study is to examine if the MUC2 gene is expressed in the nose and maxillary sinus. MATERIALS AND METHODS: A Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR) were performed. For the Northern blot analysis, RNAs were extracted from maxillary mucosae and nasal polyps of patients with chronic sinusitis and from the inferior turbinates of a nasal allergy patient. For RT-PCR, RNAs were extracted from 10 patients with chronic sinusitis, 4 patients with allergic rhinitis, 2 patients with hypertrophic rhinitis, and 6 volunteers with normal nasal mucosa. RESULTS: Hybridization of the Northern blot with SMUC41 (a part of MUC2) cDNA probe showed clear bands in 2 of the 3 samples. In RT-PCR, the first round of amplification (35 cycles) failed to give any bands, but the additional 30 cycles with internal primers gave bands in 6 of 22 samples. CONCLUSIONS: These results indicate that MUC2 mucin gene is expressed in the nose and paranasal sinus. This two-round RT-PCR method will be useful for analysis of MUC2 mucin gene expression using relatively small amount of RNA.

Interleukin-8 gene expression in chronic sinusitis.

PURPOSE: Interleukin-8 (IL-8), a monocyte-derived and macrophage-derived cytokine, displays potent chemotactic activating properties toward neutrophils and thus may contribute to the pathogenesis of chronic sinusitis. The object of this investigation was to show the expression of the IL-8 gene in chronic sinusitis by Northern blot analysis and a reverse transcription-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: For Northern blot analysis, RNAs were extracted from maxillary mucosa and nasal polyps from two patients with chronic sinusitis, respectively, and from the inferior turbinate of a nasal allergy patient. For RT-PCR, RNAs were extracted from 11 patients with chronic sinusitis, 8 patients with allergic rhinitis, and 4 patients with hypertrophic rhinitis. RESULTS: Whereas IL-8 mRNA was expressed in the maxillary mucosa, IL-8 transcript was not detected in the inferior turbinate by Northern blot analysis. IL-8 transcripts were detected in 45% of chronic sinusitis RNAs (5/11) and in 50% of allergic rhinitis RNAs (5/10) by RT-PCR. CONCLUSION: These data suggest IL-8 may contribute to neutrophil involvement in chronic sinusitis.

Interleukin-8 gene expression in middle ear effusions.

Interleukin-8 (IL-8), a monocyte- and macrophage-derived cytokine, displays potent chemotactic-activating properties toward neutrophils, and thus may contribute to the pathogenesis of otitis media with effusion (OME). The objective of this investigation was to demonstrate the expression of the IL-8 gene in middle ear effusion (MEEs) of children and adults with OME. Ribonucleic acids (RNAs) were extracted from MEEs from 16 ears of 13 pediatric patients and 12 ears of 12 adult patients with OME. The RNAs were reverse-transcribed and amplified by the polymerase chain reaction. Interleukin-8 transcripts were detected in 75% of both pediatric (12/16) and adult MEEs (9/12). The levels of expression of IL-8 and beta-actin messenger RNAs were quantitated. No significant difference was observed in IL-8/beta-actin ratios between pediatric MEEs and adult MEEs. These data suggest that IL-8 may contribute to neutrophil involvement in both pediatric and adult OME.

[Effects of frequent antral lavage on chronic sinusitis in children].

Eighty cases of pediatric sinusitis (4 to 15 years old, averaged 8 years old) were treated with frequent antral irrigations. Antral puncture was made through the inferior meatus with a Kyoto University needle under topical or general anesthesia. Then a plastic tube of 14G Medicut intravenous cannula was left in place to serve as an indwelling catheter. Antral lavages were performed through this tube 3 times daily for 7 to 10 days. The opacity of the maxillary sinus at post-treatment was improved significantly compared with that at pre-treatment in Waters view. This improvement was continued even at about 5.7 months after treatment. The opacity of the ethmoid sinus was also improved significantly after the treatment in occipito-frontal view. Our results suggest that the frequent antral lavage is an useful therapeutic modality for pediatric sinusitis.

Clinical records: a case report of Wegener's granulomatosis limited to the ear.

A 12-year-old girl had a 1-week history of left otalgia with hearing loss in May, 1989. Audiometry showed a conductive hearing loss of 60 dB. The conventional medical treatment for acute otitis media was initiated, but the symptoms and signs failed to respond this treatment. Four weeks after initial presentation, a diagnostic mastoidectomy was performed. The histological findings of the granulomatous tissue in the mastoid cavity showed nonspecific granulomatous inflammation, infiltrated with multinucleated giant cells. Initial laboratory studies revealed elevated ESR, positive CRP and RA, inverted A/G ratio, and increased Ig level. She had no other sites of involvement, such as the nose, paranasal sinus, pharynx, and larynx, and also no pulmonary or renal involvement was indicated. The diagnosis of a limited form of Wegener's granulomatosis was made on the above clinical and histological findings and laboratory examinations. The patient was then treated with cyclophosphamide, 80 mg/day (2 mg/kg) and prednisolone, 60 mg/day. After 1 week her symptoms and general condition were dramatically improved. Out-patient follow-up 1 year later showed her hearing level was improved to 28 dB, and she continues to do well without evidence of recurrence of the disease.

[A study of microdensitometric evaluation of opacity of maxillary sinuses in patients with nasal allergy].

The opacity of the maxillary sinuses on X-ray film in Waters' view in patients with nasal allergy was evaluated by a microdensitometer. The patients with nasal allergy were diagnosed with the positive results of the following; 1) eosinophilia in nasal secretion, 2) nasal provocation test, 3) intradermal skin test or radioallergosorbent test (RAST). The ratio of density of the maxillary sinuses and orbits was determined by a microdensitometer, and the ratio of the average degree of the opacity of the maxillary sinus to that of the ipsilateral orbit was designated as the M/O ratio. The normal range of the M/O ratio was set over the mean value -2 standard deviations in the normal group. The results are followings. 1) The abnormal shadows in the maxillary sinuses were found out to be 36.9% in patients with nasal allergy. 2) Patients under 15 years old had significantly higher incidence of abnormal shadows, especially in the bilateral sinuses compared with patients over 15 years old. 3) Polyps in the maxillary sinus were recognized in 5.0% of all the sinuses. 4) There were no significant differences for the incidence of abnormal shadow among the patients with nasal allergy caused by different antigens.

[Management of chronic sinusitis in children].

The managements and the results of therapy for children with chronic sinusitis were reported. During the period of 1980 to 1988, 190 patients, aged 3 to 15, were examined at our outpatient clinic. The pathologic conditions of the maxillary sinus were examined by the contrast x-ray study (X-MFT). The mild, moderate and severe lesions in the maxillary sinus evaluated by X-MFT were 22%, 44; and 34%, respectively. Sixty-two per cent of ethmoid sinus was opacified in occipito-frontal view. The degree of patency of the maxillary ostium was evaluated by an application of the constant pressure to the maxillary sinus. Almost all maxillary ostia of these patients were patent. Nasal mucociliary clearance was reduced in 37% of the patients examined after the saccharin method. Antral lavages were performed once in a week for the therapy of 72% of children with chronic sinusitis. The efficacy of this modality was evaluated by the above mentioned items. The maxillary and ethmoid lesions, and nasal mucociliary transit time were significantly improved by the modality after 25 months in average. These results indicate that the antral lavage is an useful therapy for chronic sinusitis in children.