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MLC1 is a membrane protein mainly expressed in astrocytes, and genetic mutations lead to the development of a leukodystrophy, megalencephalic leukoencephalopathy with subcortical cysts disease. Currently, the biochemical properties of the MLC1 protein are largely unknown. In this study, we aimed to characterize the transmembrane (TM) topology and oligomeric nature of the MLC1 protein. Systematic immunofluorescence staining data revealed that the MLC1 protein has eight TM domains and that both the N- and C-terminus face the cytoplasm. We found that MLC1 can be purified as an oligomer and could form a trimeric complex in both detergent micelles and reconstituted proteoliposomes. Additionally, a single-molecule photobleaching experiment showed that MLC1 protein complexes could consist of three MLC1 monomers in the reconstituted proteoliposomes. These results can provide a basis for both the high-resolution structural determination and functional characterization of the MLC1 protein.

The CLC family of proteins are involved in a variety of physiological processes to control cellular chloride concentration. Two distinct classes of CLC proteins, Cl − channels and Cl − /H + antiporters, have been functionally and structurally investigated over the last several decades. Previous studies have suggested that the conformational heterogeneity of the critical glutamate residue, Glu ex , could explain the transport cycle of CLC-type Cl − /H + antiporters. However, the presence of multiple conformations ( Up , Middle , and Down ) of the Glu ex has been suggested from combined structural snapshots of 2 different CLC antiporters: CLC-ec1 from Escherichia coli and cmCLC from a thermophilic red alga, Cyanidioschyzon merolae . Thus, we aimed to investigate further the heterogeneity of Glu ex -conformations in CLC-ec1, the most deeply studied CLC antiporter, at both functional and structural levels. Here, we show that the crystal structures of the Glu ex mutant E148D and wild-type CLC-ec1 with varying anion concentrations suggest a structural intermediate, the “ Midlow ” conformation. We also found that an extra anion can be located above the external Cl − -binding site in the E148D mutant when the anion concentration is high. Moreover, we observed that a carboxylate in solution can occupy either the external or central Cl − -binding site in the ungated E148A mutant using an anomalously detectable short carboxylic acid, bromoacetate. These results lend credibility to the idea that the Glu ex can take at least 3 distinct conformational states during the transport cycle of a single CLC antiporter.

CLC-ec1 is a Cl−/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mol in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4–5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

CLC-ec1 is a Cl-/H+ antiporter that forms stable homodimers in lipid bilayers, with a free energy of −10.9 kcal/mole relative to the 1 subunit/lipid standard state in 2:1 POPE/POPG lipid bilayers. The dimerization interface is formed by four transmembrane helices: H, I, P and Q, that are lined by non-polar side-chains that come in close contact, yet it is unclear as to whether their interactions drive dimerization. To investigate whether non-polar side-chains are required for dimer assembly, we designed a series of constructs where side-chain packing in the dimer state is significantly reduced by making 4-5 alanine substitutions along each helix (H-ala, I-ala, P-ala, Q-ala). All constructs are functional and three purify as stable dimers in detergent micelles despite the removal of significant side-chain interactions. On the other hand, H-ala shows the unique behavior of purifying as a mixture of monomers and dimers, followed by a rapid and complete conversion to monomers. In lipid bilayers, all four constructs are monomeric as examined by single-molecule photobleaching analysis. Further study of the H-helix shows that the single mutation L194A is sufficient to yield monomeric CLC-ec1 in detergent micelles and lipid bilayers. X-ray crystal structures of L194A reveal the protein re-assembles to form dimers, with a structure that is identical to wild-type. Altogether, these results demonstrate that non-polar membrane embedded side-chains play an important role in defining dimer stability, but the stoichiometry is highly contextual to the solvent environment. Furthermore, we discovered that L194 is a molecular hot-spot for defining dimerization of CLC-ec1.

Background Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare form of infantile-onset leukodystrophy. The disorder is caused primarily by mutations of MLC1 that leads to a series of phenotypic outcomes including vacuolation of myelin and astrocytes, subcortical cysts, brain edema, and macrocephaly. Recent studies have indicated that functional interactions among MLC1, GlialCAM, and ClC-2 channels play key roles in the regulation of neuronal, glial and vascular homeostasis. However, the physiological role of MLC1 in cellular homeostatic communication remains poorly understood. In the present study, we investigated the cellular function of MLC1 and its effects on cell–cell interactions. Methods MLC1-dependent cellular morphology and motility were analyzed by using confocal and live cell imaging technique. Biochemical approaches such as immunoblotting, co-immunoprecipitation, and surface biotinylation were conducted to support data. Results We found that the altered MLC1 expression and localization led to a great alteration in cellular morphology and motility through actin remodeling. MLC1 overexpression induced filopodia formation and suppressed motility. And, MLC1 proteins expressed in patient-derived MLC1 mutants resulted in trapping in the ER although no changes in morphology or motility were observed. Interestingly knockdown of Mlc1 induced Arp3-Cortactin interaction, lamellipodia formation, and increased the membrane ruffling of the astrocytes. These data indicate that subcellular localization of expressed MLC1 at the plasma membrane is critical for changes in actin dynamics through ARP2/3 complex. Thus, our results suggest that misallocation of pathogenic mutant MLC1 may disturbs the stable cell-cell communication and the homeostatic regulation of astrocytes in patients with MLC.

This article introduces the history and the long-term goals of the Korea Brain Initiative, which is centered on deciphering the brain functions and mechanisms that mediate the integration and control of brain functions that underlie decision-making. The goal of this initiative is the mapping of a functional connectome with searchable, multi-dimensional, and information-integrated features. The project also includes the development of novel technologies and neuro-tools for integrated brain mapping. Beyond the scientific goals this grand endeavor will ultimately have socioeconomic ramifications that not only facilitate global collaboration in the neuroscience community, but also develop various brain science-related industrial and medical innovations.

Additional file 4: Figure S4. Examine the infection efficiency and the effect of MLC1 on the matured primary astrocytes. (A) Knockdown of MLC1 in mouse primary astrocytes was performed by viral infection and was confirmed via Western Blotting with anti-MLC1 antibody. GFP expression levels were analyzed to evaluate the efficiency of AAV infection. Similar GFP expression level in the astrocytes treating with either shMlc1 or the scrambled shMlc1 (shScr) virus indicates that the infection efficiency was similar. Actin was used as loading control. (B and C) To verify the maturation of primary astrocytes, cells were stained with anti-S100β antibody (magenta). Empty and filled arrowheads in the magnified image indicate fibrous actin bundles and branched actin networks, respectively.

The CLC family proteins are involved in a variety of cellular processes, where chloride homeostasis needs to be controlled. Two distinct classes of CLC proteins, Cl- channels and Cl-/H+ antiporters, have been functionally and structurally investigated over the last several decades. Recent studies have revealed that the conformational heterogeneity of the critical glutamate residue, Gluex could explain the transport cycle of CLC-type Cl-/H+ antiporters. However, the presence of multiple conformations of the Gluex has been suggested from combined structural snapshots of two different CLC antiporters. Thus, we aimed to investigate the presence of these three intermediate conformations in CLC-ec1, the most deeply studied CLC at both functional and structural levels. By comparing crystal structures of E148D, E148A mutant and wildtype CLC-ec1 with varying anion concentrations, we suggest that the Gluex indeed take at least three distinct conformational states in a single CLC antiporter, CLC-ec1.

Preclinical Research Emerging evidence suggests that Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has therapeutic potential. This study examined the antiwrinkle effects of ethanol extracts of D. odorifera in UVB-irradiated human skin cells. Ethanol extracts of D. odorifera and thier constituents, dalbergin and sativanone, induced expression of collagen type I and transforming growth factor (TGF)-β1 in human dermal fibroblasts. In HR-1 hairless mice exposed to UVB, the ethanol extract reduced wrinkle formation and skin thickness. This inhibitory effect of ethanol extract was associated with the restoration of collagen type I, TGF-β1, and elastin to levels approaching those in skin tissues not exposed to UVB, which was accompanied by the reduction of matrix metalloproteinase-2 and upregulation of tissue inhibitors of metalloproteinase (TIMP)-2 and TIMP-3 in skin tissue exposed to UVB. These results suggest that the ethanol extracts prevent some effects of photoaging and maintain skin integrity by regulating the degradation of the extracellular matrix proteins. © 2015 Wiley Periodicals, Inc.

The chloride channel (CLC) family of proteins consists of channels and transporters that share similarities in architecture and play essential roles in physiological functions. Among the CLC family, CLC-1 channels have the representative homodimeric double-barreled structure carrying two gating processes. One is protopore gating that acts on each pore independently by glutamate residue (Eext). The other is common gating that closes both pores simultaneously in association with large conformational changes across each subunit. In skeletal muscle, CLC-1 is associated with maintaining normal sarcolemmal excitability, and a number of myotonic mutants were reported to modify the channel gating of CLC-1. In this study, we characterized highly conserved helix O as a key determinant of structural stability in CLC-1. Supporting this hypothesis, myotonic mutant (G523D) at N-terminal of helix O showed the activation at hyperpolarizing membrane potentials with a reversed voltage dependency. However, introducing glutamate at serine residue (S537) at the C-terminal of the helix O on G523D restored WT-like voltage dependency of the common gate and showed proton insensitive voltage dependency. To further validate this significant site, site-specific mutagenesis experiments was performed on V292 that is highly conserved as glutamate in antiporter and closely located to S537 and showed that this area is essential for channel function. Taken together, the results of our study suggest the importance of helix O as the main contributor for stable structure of evolutionary conserved CLC proteins and its key role in voltage dependency of the CLC-1. Furthermore, the C-terminal of the helix O can offer a clue for possible proton involvement in CLC-1 channel.

A subclass of bacterial CLC anion-transporting proteins, phylogenetically distant from long-studied CLCs, was recently shown to be specifically up-regulated by F - . We establish here that a set of randomly selected representatives from this “CLC F ” clade protect Escherichia coli from F - toxicity, and that the purified proteins catalyze transport of F - in liposomes. Sequence alignments and membrane transport experiments using 19 F NMR, osmotic response assays, and planar lipid bilayer recordings reveal four mechanistic traits that set CLC F proteins apart from all other known CLCs. First, CLC F s lack conserved residues that form the anion binding site in canonical CLCs. Second, CLC F s exhibit high anion selectivity for F - over Cl - . Third, at a residue thought to distinguish CLC channels and transporters, CLC F s bear a channel-like valine rather than a transporter-like glutamate, and yet are F - /H + antiporters. Finally, F - /H + exchange occurs with 1∶1 stoichiometry, in contrast to the usual value of 2∶1.

As previously reported, the activity of the large-conductance calcium (Ca(2+))-activated potassium (K(+)) (BK(Ca)) channel is strongly potentiated from the extracellular side of the cell membrane by certain benzofuroindole derivatives. Here, the mechanism of action of one of the most potent activators, 4-chloro-7-(trifluoromethyl)-10H-benzofuro[3,2-b]indole-1-carboxylic acid (CTBIC), is characterized. This compound, Compound 22 in the previous report (Chembiochem 6:1745-1748, 2005), potentiated the activity of the channel by shifting its conductance-voltage relationship toward the more negative direction. Cotreatment with CTBIC reduced the affinity of charybdotoxin, a peptide pore-blocker, whereas that of tetraethylammonium, a small pore-blocking quaternary ammonium, was not significantly altered. Guided by these results, scanning mutagenesis of the outer vestibule of the BK(Ca) channel was launched to uncover the molecular determinants that affect CTBIC binding. Alanine substitution of several amino acid residues in the turret region and the S6 helix of the channel decreased potentiation by CTBIC. Homology modeling and molecular dynamics simulation showed that some of these residues formed a CTBIC binding pocket between two adjacent α-subunits in the outer vestibule of the channel. Thus, it can be envisioned that benzofuroindole derivatives stabilize the open conformation of the channel by binding to the residues clustered across the extracellular part of the subunit interface. The present results indicate that the interface between different α-subunits of the BK(Ca) channel may play a critical role in the modulation of channel activity. Therefore, this interface represents a potential therapeutic target site for the regulation of K(+) channels.

Animal locomotion is mediated by a sensory system referred to as proprioception. Defects in the proprioceptive coordination of locomotion result in uncontrolled and inefficient movements. However, the molecular mechanisms underlying proprioception are not fully understood. Here, we identify two transient receptor potential cation (TRPC) channels, trp-1 and trp-2, as necessary and sufficient for proprioceptive responses in C. elegans head steering locomotion. Both channels are expressed in the SMDD neurons, which are required and sufficient for head bending, and mediate coordinated head steering by sensing mechanical stretches due to the contraction of head muscle and orchestrating dorsal head muscle contractions. Moreover, the SMDD neurons play dual roles to sense muscle stretch as well as to control muscle contractions. These results demonstrate that distinct locomotion patterns require dynamic and homeostatic modulation of feedback signals between neurons and muscles., Author summary Proprioception provides the nervous system with feedback about body posture in animals and is essential for the generation of coherent locomotive behaviors, such as walking, running, or crawling. However, little is known about the identity of proprioceptive receptors that sense body movement to regulate locomotion and the extent to which proprioception modulates sensorimotor coordination. Here, we analyze the molecular mechanisms that control head steering locomotion of Caenorhabditis elegans. We show that this movement is regulated by the transient receptor potential cation (TRPC) channels TRP-1 and TRP-2 and the SMDD proprioceptive neurons. We observe that mutant animals for both channels are defective in head steering locomotion and that ectopic expression of TRP-1 or TPR-2 in a C. elegans chemosensory neuron confers head bending–dependent responses, suggesting roles for these channels in proprioception. We also find that SMDD neurons are both necessary and sufficient to generate head steering locomotion via the two channels. Moreover, we demonstrate that the proprioceptive system mediates locomotion coordination by desynchronizing activities in motor systems. We conclude that two TRPC channels in collaboration with the proprioceptive receptor SMDD neurons control head steering in worms during forward locomotion.

CLC-ec1, a bacterial homologue of the CLC family's transporter subclass, catalyzes transmembrane exchange of Cl(-) and H(+). Mutational analysis based on the known structure reveals several key residues required for coupling H(+) to the stoichiometric countermovement of Cl(-). E148 (Glu(ex)) transfers protons between extracellular water and the protein interior, and E203 (Glu(in)) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H(+) transport while preserving Cl(-) transport. We tested the role of Glu(in) by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H(+)/Cl(-) exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H(+) coupling and severely inhibit Cl(-) flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H(+) transport with substantial Cl(-) transport. Influence on H(+) transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Glu(in) is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl(-) movements to bring about Cl(-)/H(+) exchange.

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor (iGluR). We have solved the crystal structure of the ligand-binding core of NpGluR0 in complex with l -glutamate at a resolution of 2.1 A. The structure exhibits a noncanonical ligand interaction and two distinct subunit interfaces. The side-chain guanidium group of Arg80 forms a salt bridge with the γ-carboxyl group of bound l -glutamate: in GluR0 from Synechocystis (SGluR0) and other iGluRs, the equivalent residues are Asn or Thr, which cannot form a similar interaction. We suggest that the local positively charged environment and the steric constraint created by Arg80 mediate the selectivity of l -glutamate binding by preventing the binding of positively charged and hydrophobic amino acids. In addition, the NpGluR0 ligand-binding core forms a new subunit interface in which the two protomers are arranged differently than the known iGluR and SGluR0 dimer interfaces. The significance of there being two different dimer interfaces was investigated using analytical ultracentrifugation analysis.

Spinorphin, an endogenous antinociceptive peptide (LVVYPWT), showed potent and non-competitive antagonism at the ATP-activated human P2X3 receptor (IC50 = 8.3 pM) in a two-electrode voltage clamp assay with recombinant human P2X3 receptors expressed in Xenopus oocytes. Single alanine substitutions from 1st to 4th amino acids and the cyclic form of LVVYPWT sustained antagonistic properties at the human P2X3 receptors, whereas the threonine to alanine substitution resulted in an enhancing effect of the agonistic activity.

Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has been widely used in northern and eastern Asia to treat diverse diseases. Here, we investigated the anti-senescent effects of ethanolic extracts of Dalbergia odorifera (EEDO) in ultraviolet (UV) B-irradiated skin cells. EEDO significantly inhibited UVB-induced senescence of human keratinocytes in a concentration-dependent manner, concomitant with inhibition of reactive oxygen species (ROS) generation. UVB-induced increases in the levels of p53 and p21, biomarkers of cellular senescence, were almost completely abolished in the presence of EEDO. Sativanone, a major constituent of EEDO, also attenuated UVB-induced senescence and ROS generation in keratinocytes, indicating that sativanone is an indexing (marker) molecule for the anti-senescence properties of EEDO. Finally, treatment of EEDO to mice exposed to UVB significantly reduced ROS levels and the number of senescent cells in the skin. Thus, EEDO confers resistance to UVB-induced cellular senescence by inhibiting ROS generation in skin cells.

Preclinical Research Emerging evidence suggests that Dalbergia odorifera T. Chen (Leguminosae), an indigenous medicinal herb, has therapeutic potential. This study examined the antiwrinkle effects of ethanol extracts of D. odorifera in UVB-irradiated human skin cells. Ethanol extracts of D. odorifera and thier constituents, dalbergin and sativanone, induced expression of collagen type I and transforming growth factor (TGF)-β1 in human dermal fibroblasts. In HR-1 hairless mice exposed to UVB, the ethanol extract reduced wrinkle formation and skin thickness. This inhibitory effect of ethanol extract was associated with the restoration of collagen type I, TGF-β1, and elastin to levels approaching those in skin tissues not exposed to UVB, which was accompanied by the reduction of matrix metalloproteinase-2 and upregulation of tissue inhibitors of metalloproteinase (TIMP)-2 and TIMP-3 in skin tissue exposed to UVB. These results suggest that the ethanol extracts prevent some effects of photoaging and maintain skin integrity by regulating the degradation of the extracellular matrix proteins. © 2015 Wiley Periodicals, Inc.

Large-conductance Ca2+-activated K+ (BK(Ca)) channels are widely distributed and play key roles in various cell functions. We previously reported the chemical synthesis of several benzofuroindole compounds that act as potent openers of BK(Ca) channels. In this study, we investigated the mechanism of channel potentiation by one of the compounds, 7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC), using electrophysiological means. This chemical highly activated cloned BK(Ca) channels from extracellular side independent of beta subunits and regardless of the presence of intracellular Ca2+. The EC50 and Hill coefficient for rat BK(Ca) channel alpha subunit, rSlo, were estimated as 8.9 +/- 1.5 microM and 0.9, respectively. TBIC shifted the conductance-voltage curve of rSlo channels to more hyperpolarized potentials without altering its voltage dependence. Single-channel recording revealed that TBIC increased the open probability of the channel in a dose-dependent manner without any changes in single-channel conductance. Strong potentiation by TBIC was also observed for native BK(Ca) channels from rat hippocampus pyramidal neurons. Thus, TBIC and the related benzofuroindole compounds can be useful tools to unravel the mechanism of this novel allosteric activation of BK(Ca) channels.

Background & Aims: Interstitial cells of Cajal are pacemakers in the gastrointestinal tract, regulating rhythmicity by activating nonselective cation channels. In Caenorhabditis elegans, the melastatin-type transient receptor potential (TRPM) channel, especially TRPM7, was suggested as being involved in defecation rhythm. The aim here was to show that the nonselective cation channel in interstitial cells of Cajal in mouse small intestine has properties essentially identical to those of murine TRPM7, heterologously expressed in human embryonic kidney cells. Methods: The patch-clamp technique for whole-cell recording was used in cultured or single interstitial cells of Cajal. TRPM7-specific small interfering RNAs were used for specific inhibition of TRPM7. Results: Electrophysiological and pharmacological properties of the nonselective cation channel in interstitial cells of Cajal were the same as those of TRPM7. Reverse-transcription polymerase chain reaction, Western blotting, and immunohistochemistry all showed abundant and localized expression of TRPM7 messenger RNA and protein in mouse small intestine. Treatment of primary cultured interstitial cells of Cajal with TRPM7-specific small interfering RNA resulted in inhibition of pacemaking activity. Conclusions: TRPM7 is required for intestinal pacemaking. The protein is a likely potential target for pharmacological treatment of motor disorders of the gut.

Ankyrin-repeat family A protein (ANKRA) was originally cloned in mouse as an interacting protein to megalin, a member of low-density lipoprotein receptor superfamily. Here, we report that the isolation of rat ANKRA as a new binding partner for the α-subunit of rat large-conductance Ca2+-activated K+channel (rSlo). We mapped the binding region of each protein by using yeast two-hybrid and in vitro binding assays. ANKRA expressed together with rSlo channels were colocalized near the plasma membrane and coimmunoprecipitated in transfected cells. We also showed that BKCachannel in rat cerebral cortex coprecipitated with rANKRA and colocalized in cultured rat hippocampal neuron. Although the coexpression of ANKRA did not affect the surface expression of rSlo, the gating kinetics of rSlo channel was significantly altered and the effects were highly dependent on the intracellular calcium. These results indicate that ANKRA could modulate the excitability of neurons by binding directly to endogenous BKCachannel and altering its gating kinetics in a calcium-dependent manner.

Defensins, a prominent group of antimicrobial peptides, are an important component of the innate immune response, particularly at mucosal surfaces that are vulnerable to colonization by potential pathogens. The present study was undertaken to investigate the expression of defensins in inferior turbinate mucosa of normal subjects and inferior turbinate mucosa and nasal polyps of patients with chronic sinusitis. Expression of beta-defensin 1 and 2 and alpha-defensin 5 and 6 messenger RNAs (mRNAs) was investigated by reverse transcriptase-polymerase chain reaction, and their expression level was semiquantitatively evaluated by dot blot hybridization. Immunohistochemical analysis was used for detection of alpha-defensins 1, 2, and 3 in tissue sections. Beta-defensin 1 mRNA was expressed in all tissue samples, at levels that did not differ significantly. Beta-defensin 2 mRNA was detected in the turbinate mucosa and nasal polyps of patients with chronic sinusitis, but not in normal mucosa. Its expression level was significantly higher in nasal polyps than in turbinate mucosa. Alpha-defensin 5 and 6 mRNAs were not expressed in any tissues, but alpha-defensins 1, 2, and 3 were detected in all tissue samples obtained from patients with chronic sinusitis. These results suggest that beta-defensin 1 may play a constitutive role in nasal defenses, whereas alpha-defensins 1, 2, and 3 and beta-defensin 2 may be induced in response to local infection or inflammation.

It has been suggested that the formation and growth of nasal polyp require the remodeling of extracellular matrix. Proteoglycans (PGs) are major components of the extracellular matrix that maintain the integrity of structural tissue. The leucine-rich repeat PGs include lumican, decorin and biglycan and have many important biologic activities in various pathologic conditions, including the remodeling of the extracellular matrix. Therefore, these small-PG families may be involved in the formation and growth of nasal polyp. In the present study, surgical specimens of nasal polyps and nasal mucosa were assessed for expression of mRNA coding for lumican, decorin and biglycan using reverse transcriptase-polymerase chain reaction followed by dot blot hybridization. Lumican, decorin and biglycan mRNA were expressed in all tissue samples examined. Semiquantitative dot blot hybridization revealed that the levels of the lumican and biglycan messages are lower in nasal polyp tissues than in nasal mucosa. The decorin messages in nasal polyp were expressed at levels similar to those in nasal mucosa. These results suggest that lumican, decorin and biglycan may be important components of the extracellular matrix in nasal mucosa. Considering the function of these PGs, normal levels of decorin associated with low levels of biglycan and lumican may play a role in the pathogenesis of nasal polyposis.

The incidence of acute mastoiditis has declined dramatically during the postantibiotic era. Even so, antibiotic-resistant or unusual pathogens can still cause this disease entity. At our hospital, we documented an increase in antibiotic-resistant and atypical pathogens such as Actinomyces spp. and Mycobacterium tuberculosis. In this paper, we discuss the optimal diagnosis and treatment strategy for acute mastoiditis, and we describe our retrospective review of 13 patients with mastoiditis who were treated at our hospital from 1988 through 1998. Eight of these patients recovered following treatment with intravenous antibiotics, with or without myringotomy, and five who had complications of disease were managed surgically. Among these five, one developed chronic otitis media and one developed cholesteatoma 3 years later. For patients with acute mastoiditis, we emphasize the need to be aware of any unusual pathogens that do not respond to empiric antibiotic therapy.

Sixty-three biopsy specimens were obtained from the olfactory region of 15 patients with persistent anosmia and 6 patients with normosmia after sinus surgery. Immunohistochemical examination of all specimens with microtubule-associated protein 5 (MAP5) antisera demonstrated olfactory epithelium in 11 of 18 specimens from normosmic patients and in 12 of 45 samples from anosmic patients. There was a significant difference in the proportion of specimens containing olfactory epithelium between the two groups of patients. In normosmic patients, most of the biopsy samples contained normal-appearing olfactory tissue. However, 2 main patterns of histologic findings were noted in the olfactory mucosa of anosmic patients. First, the olfactory receptor cells were remarkably decreased in number. Second, the orderly arrangement of cells characteristic of normal olfactory epithelium was lost, demonstrating a degenerative appearance. These data suggest that olfactory epithelium can be degenerated even in chronic sinusitis and thereafter extensively replaced with respiratory epithelium, resulting in increased sampling error. Moreover, an unimproved olfactory deficit after sinus surgery may be due to the abnormalities observed at the olfactory epithelium level.

Antimicrobial peptides are cationic proteins that are found in a wide range of organisms. Recent reports suggested that human beta-defensin 1 (hBD-1), a prominent group of antimicrobial peptides, is an important component of the innate immune response, particularly at mucosal surfaces that are vulnerable to colonization by potential pathogens. Therefore, hBD-1 may participate in providing intrinsic nasal mucosal defence against microbial infections. The present study aimed to look for hBD-1 mRNA in human nasal mucosa without obvious signs of inflammation. Total RNA was isolated from human inferior turbinate mucosa and hBD-1 mRNA was detected in these tissues by using reverse transcription and polymerase chain reaction (RT-PCR). By in situ hybridization, hBD-1 mRNA was predominantly localized in superficial epithelial cells and submucosal glandular epithelium of human inferior turbinate mucosa. These data suggest that nasal epithelia and submucosal glands may secrete hBD-1, contributing to the mucosal defences of the nose.

Increased vascular permeability and endothelial cell growth are important in the pathogenesis of otitis media with effusion (OME) and the vascular endothelial growth factor (VEGF) is known to play an important role in the increased vascular permeability and angiogenesis. To date, at least five isoforms of the VEGF family have been identified as VEGF transcripts, encoding polypeptides of 206, 189, 165, 145 and 121, but their physiological roles are unclear. The purpose of this study was to investigate the expression of VEGF, in both endotoxin-induced OME of the rat and human otitis media. We instilled endotoxin and saline as a control into the middle ear cavity of the rat. Middle ear mucosa were taken at 0 h, 1 h, 3 h, 6 h, 12 h, 1 day, 3 days, 7 days and 14 days and the expression of VEGF mRNA and VEGF protein was evaluated using semi-quantitative RT-PCR and immunohistochemistry. Expression of VEGF164 mRNA and VEGF120 mRNA was first identified 1 h after endotoxin instillation and was dramatically increased over the period 6 h-1 day and then progressively decreased by day 7. The level of expression of VEGF120 mRNA was slightly higher than that of VEGF164 mRNA and that of VEGF164 mRNA was much higher than that of VEGF188 mRNA. Immunostaining revealed expression of VEGF during 6 h to day 3 and its expression was localized to ciliated cells and some inflammatory cells. We also performed RT-PCRs of cDNA from middle ear fluids of 8 human OME patients and middle ear mucosa of 4 chronic otitis media patients for the identification of VEGF mRNA expression. VEGF121 mRNA was highly expressed in all samples compared with VEGF165 mRNA. These results suggest that VEGF may be primarily responsible for increased vascular permeability and endothelial cell growth in OME and that VEGF seems to play a significant role in the pathogenesis of OME.