Interleukin-18 is elevated in the horny layer in patients with atopic dermatitis and is associated with Staphylococcus aureus colonization.

BACKGROUND: An increase in interleukin (IL)-18 production from epidermal cells has been reported in an atopic dermatitis (AD) mouse model, and subsequent topical application of Staphylococcus aureus results in severe dermatitis. OBJECTIVES: To reveal the relationship between S. aureus colonization of skin lesions and keratinocyte IL-18 production, particularly in AD with relatively low serum IgE levels. We also aimed to establish a simple and noninvasive method of assaying IL-18 produced by epidermal keratinocytes to evaluate local skin inflammation and therapeutic effects in patients with AD. METHODS: IL-18 in the horny layer of the skin was collected via a tape-stripping method and measured in 95 patients with AD and 40 healthy controls by enzyme-linked immunosorbent assay (ELISA). Clinical severity, blood data and S. aureus skin colonization were evaluated before and after treatment. RESULTS: IL-18 levels in the horny layer were significantly higher in the skin lesions of patients with AD than in healthy controls and correlated with SCORAD, levels of serum IL-18, IgE, lactate dehydrogenase, thymus and activation-regulated chemokine, blood eosinophils and transepidermal water loss. In the AD group with serum IgE < 1500 IU mL(-1) , significantly higher IL-18 levels were observed in the horny layer of patients colonized with S. aureus compared with those who were not. CONCLUSIONS: Epidermal IL-18 production was associated with the severity of AD. Staphylococcus aureus colonization seems to contribute to this IL-18 production, especially in the AD group with relatively low IgE production. Tape stripping provides an easy and noninvasive method to assess epidermal IL-18 production by ELISA.

Mechanical regulation of bone homeostasis through p130Cas-mediated alleviation of NF-κB activity.

Mechanical loading plays an important role in bone homeostasis. However, molecular mechanisms behind the mechanical regulation of bone homeostasis are poorly understood. We previously reported p130Cas (Cas) as a key molecule in cellular mechanosensing at focal adhesions. Here, we demonstrate that Cas is distributed in the nucleus and supports mechanical loading-mediated bone homeostasis by alleviating NF-κB activity, which would otherwise prompt inflammatory processes. Mechanical unloading modulates Cas distribution and NF-κB activity in osteocytes, the mechanosensory cells in bones. Cas deficiency in osteocytes increases osteoclastic bone resorption associated with NF-κB-mediated RANKL expression, leading to osteopenia. Upon shear stress application on cultured osteocytes, Cas translocates into the nucleus and down-regulates NF-κB activity. Collectively, fluid shear stress-dependent Cas-mediated alleviation of NF-κB activity supports bone homeostasis. Given the ubiquitous expression of Cas and NF-κB together with systemic distribution of interstitial fluid, the Cas-NF-κB interplay may also underpin regulatory mechanisms in other tissues and organs.

Galactose-carrying polymers as extracellular matrices for liver tissue engineering.

Extracellular matrix (ECM) plays important roles in tissue engineering because cellular growth and differentiation, in the two-dimensional cell culture as well as in the three-dimensional space of the developing organism, require ECM with which the cells can interact. Especially, the bioartificial liver-assist device or regeneration of the liver-tissue substitutes for liver tissue engineering requires a suitable ECM for hepatocyte culture because hepatocytes are anchorage-dependent cells and are highly sensitive to the ECM milieu for the maintenance of their viability and differentiated functions. Galactose-carrying synthetic ECMs derived from synthetic polymers and natural polymers bind hepatocytes through a receptor-mediated mechanism, resulting in enhanced hepatocyte functions. Attachment and functions of hepatocytes were affected by physico-chemical properties including ECM geometry as well as the type, density and orientation of galactose. Also, cellular environment, medium composition and dynamic culture system influenced liver-specific functions of hepatocytes beside ECM.

Raman spectroscopic study on the structure of ribonuclease F1 and the binding mode of inhibitor.

The structure of RNase F1 in aqueous solution has been studied by Raman spectroscopy and compared with that of a homologous enzyme, RNase T1. RNase F1 contains less beta-sheet and alpha-helical structure and more irregular structure than RNase T1. The strength of hydrogen bonding is weak in the beta-sheet and strong in the alpha-helix compared to that of RNase T1. Two disulfide bridges take the gauche-gauche and gauche-trans conformations, respectively. The overall hydrogen bonding of nine Tyr side chains in RNase F1 is very similar to that in RNase T1. Both of two His residues have pKa values around 8.2, which are close to those of the His residues in the active site of RNase T1. Upon binding of 2'-GMP, the hydrogen bonding of some Tyr side chains changes to a more proton-donating state. 2'-GMP is strongly hydrogen bonded with the enzyme at N7 of the guanine ring and takes the C3' endo-syn conformation. The binding mode of the inhibitor is identical to that found for RNase T1. In spite of significant differences in secondary structure, the molecular architecture of the active site seems to be highly conserved.

Raman spectra of some snake venom components.

The Raman spectra of aqueous solutions of cobrotoxin, erabutoxin b (short-chain neurotoxins), alpha-bungarotoxin (a long-chain neurotoxin), cardiotoxin, cytotoxin I (cardiotoxins), 6-carboxyl-modified cobrotoxin and cobrotoxin treated with 5 M guanidine-HCl (denatured neurotoxins) were investigated. The main-chain structure of the toxins was found to consist predominantly of beta-pleated sheets and random-coils. The relative amounts of the beta-conformations were estimated to be in order of: cardiotoxins approximately short-chain neurotoxins greater than long-chain neurotoxins greater than Laticauda semifasciata III (exceptionally weak neurotoxin). Most of the disulfide-bridges of every toxin take the gauche-gauche structure about the CC-SS-CC linkage. However, the number of gauche-gauche structures is not the same among the neurotoxins. Tyr-25 of cobrotoxin is found to form a strong hydrogen bond similar to that of other short-chain neurotoxins investigated earlier.

Characteristics of substrates and inhibitors in binding to rat liver L-tryptophan 2,3-dioxygenase: a Fourier transform infrared and kinetic study.

Infrared spectroscopy and steady-state kinetics were applied to rat liver L-tryptophan 2,3-dioxygenase, in order to find relations between the structure and binding characteristics of its substrates and inhibitors. The binding characteristics were reflected by changes in the infrared CO stretch band(s) of an Fe(II)-CO complex of the enzyme upon addition of L-tryptophan and 12 analogs. The CO stretch band around 1961 cm-1 of the complex was not much affected by 1-methyl-D,L-tryptophan, a noncompetitive inhibitor, implying a binding at a site distant from the Fe(II)-CO vicinity. The spectral pattern was significantly changed by any of the other compounds which conserved an indole NH, indicative of its binding to the catalytic site. All substrates, which contained a complete CH(NH2)COOH group in addition to the NH, gave spectra similar to that of an L-tryptophan-bound complex. Spectral changes caused by six inhibitors, which lacked the complete CH(NH2)COOH, were different from one another and from those by the substrates. Hence, for an analog, the indole NH is indispensable to bind to the catalytic site, and the CH(NH2)COOH is important to take a correct configuration appropriate to the catalytic reaction. The reason why L- and D-isomers of 5-hydroxytryptohan are not substrates, in spite of their conservation of the required functional groups and correct binding to the catalytic site, has been ascribed to a possible distortion of the protein structure in the heme pocket due to a strong hydrogen bond from the hydroxyl group to an amino acid side chain.

Structural specificity of peptides in Z-DNA formation and energetics of the peptide-induced B-Z transition of poly(dG-m5dC).

The effects of oligopeptide binding in the conformation of double-stranded poly(dG-m5dC) have been examined by circular dichroism spectroscopy. Conversion from the right-handed B-form to the left-handed Z-form occurs very efficiently in the presence of peptides having amino acid sequences of the (Lys-X)n-Lys type, where X is a non-bulky amino acid residue such as Gly, Ala and Lys. The ability to induce the B-Z transition increases rapidly with increasing repeat number n, and the peptides with n = 2-3 readily convert the B-form DNA to the Z-form at a peptide/nucleotide molar ratio of 0.06 to 0.08 (one peptide per 13 to 17 nucleotides). The peptide-induced B-Z transition of poly(dG-m5dC) duplexes takes place even at a physiological salt concentration (150 mM NaCl). The activation energy and van't Hoff enthalpy of the B-Z conversion have been determined to be 124 (+/- 7) and 138 (+/- 12) kJ/mol, respectively, for the (Lys-Gly)2-Lys-induced transition. The van 't Hoff enthalpy of the peptide-induced B-Z transition is particularly small compared to those of the transitions caused by other inducers. The large enthalpic contribution to the Z-DNA formation indicates that the peptide binds preferentially to the Z-arranged DNA phosphate groups and stabilizes the Z-stretch significantly. It is pointed out that some DNA-binding proteins have amino acid sequences that favor Z-DNA.

Raman spectroscopic study on the conformation of a peptide fragment representing the DNA-binding domain of filamentous virus Pf3 coat protein.

Raman spectra have been measured of a nonapeptide which has an amino acid sequence identical to that of the C-terminal region of the major coat protein subunit of filamentous bacteriophage Pf3. The peptide shows a strong tendency to form a beta-sheet structure in aqueous solution. The beta-sheet formation is significantly promoted by complexation with single-stranded DNA but not with double-stranded DNA. It is suggested that the C-terminal region of the Pf3 coat protein binds to the single-stranded DNA genome in the virion with a beta-sheet conformation, in sharp contrast with the alpha-helical binding in other filamentous bacteriophages.

Structural properties of connectin studied by ultraviolet resonance Raman spectroscopy and infrared dichroism.

Ultraviolet resonance Raman spectra of solubilized connectin indicated the presence of beta-sheets and hydrogen-bonded irregular structures. Some Trp and Tyr sidechains are located in hydrophobic environments and some NHs of mainchain amides and Trp indoles are not easily reached by solvent water, suggesting the presence of folded structures constructed of the beta- and irregular parts. Infrared spectra showed an abundance of beta-sheets in a connectin fiber, some of which were aligned with their mainchain axes parallel to the fiber axis. Thus, the beta-spiral structure proposed for elastin is improbable in connectin. This conclusion is also supported by their different amide III frequencies in the visible Raman spectra. A possible filamentous structure of repeated domains, consisting of beta-sheets and irregular parts, is discussed.

B-Z transition of poly(dG-m5dC) induced by binding of Lys-containing peptides.

Effects of oligopeptides containing Lys residues on the conformation of poly(dG-m5dC) have been investigated by circular dichroism spectroscopy. Lys-Ala-Lys (KAK) and its longer analogs with Lys-Ala repeats are found to convert the B-form polynucleotide to the Z form very efficiently. The ability to induce the B-Z transition is characteristic of alternating Lys-Ala sequences and increases exponentially with increasing number of the repeats. The heptapeptide KAKAKAK has an ability comparable with that of spermine, one of the most effective inducers hitherto known. The present results provide the first example of the B-Z transition of poly(dG-m5dC) induced by peptide binding.

4-vinyl and 2,4-divinyl deuteration effects on the low frequency resonance Raman bands of myoglobin: correlation with the structure of vinyl group.

Resonance Raman spectra of myoglobin (Mb) reconstituted with 4-vinyl and 2,4-divinyl deuterated protoheme IX were studied in several oxidation, spin, and ligation states (deoxyMb, MbO2, MbCO, metMbH2O, and metMbCN-) with special attention to the low frequency vibrations. Frequency shifts observed on deuteration enabled us to assign some Raman bands to vibrations specifically involving the 2- or 4-vinyl group. The most significant deuteration effect was found for a band around 410 cm-1 in the ferrous state, which loses intensity on 4-vinyl deuteration and is ascribed to a porphyrin in-plane vibration strongly coupled with the skeletal bend of the vinyl group at position 4. Such strong coupling implies that the vinyl group lies in the same plane as the pyrrole II ring, as in the crystalline state. Thus, frequency shifts on vinyl deuteration may be useful as a probe of the orientation of the vinyl group. Resonance Raman spectra of Mb coordinated with various sizes of isocyanides are interpreted in terms of vinyl orientational changes induced by ligation.

[In vitro antifungal activity of rilopirox, a new hydroxypyridone antimycotic agent].

In vitro antifungal activities of rilopirox (RIL), a new topical hydroxypyridone antifungal agent, were studied against 7 species (31 strains) of yeast-like fungi and 15 species (17 strains) of mycerial fungi from stock cultures of a wide range of medically important fungi. Tests were carried out by the liquid dilution method using Neopeptone dextrose broth with ciclopirox olamine (CPO) and oxiconazole nitrate (OCZ) as reference drugs. RIL exhibited a broad spectrum of antifungal activities; the MIC of RIL against yeasts were about 1 microgram/ml, those against other fungi were 0.5-4 micrograms/ml. Antifungal activities were similar to CPO, and compare to OCZ, RIL showed characteristically little differences in its activities against different species or strains of target organisms.

[The in vitro properties of a new hydroxypyridone antimycotic rilopirox, with special reference to its anti-Candida activity].

In vitro properties of a new hydroxypyridone antimycotic rilopirox (RIL) with special reference to its anti-Candida activities were studied in comparison with the three reference drugs, ciclopirox olamine (CPO), oxiconazole nitrate (OCZ) and isoconazole nitrate (ICZ), using several strains of Candida albicans and Candida glabrata as the test organisms. RIL was potently fungicidal for growing cultures of these Candida strains, whereas all the three reference drugs were slightly fungicidal or fungistatic. Unlike OCZ and ICZ whose anti-Candida activity was decreased by lowering pH or adding serum to culture media, the activity of RIL was scarecely affected by change in pH or serum addition. However, RIL became less potent in the presence of Fe3+ at concentrations of 10(-5) mmol/ml or above. These findings suggest that RIL will be useful as a topical anti-Candida agent.

[An operative case of the dumbbell type retromediastinal schwannoma].

Neurogenic tumors of the mediastinum with an intraspinal component connected by a narrowed segment in the intervertebral one are generally described as dumbbell or hour-glass tumors, which need cautious and precise diagnoses and remedies, compared with other neurological tumors. A 62-year-old male was admitted to our hospital for abnormal tumor shadow in the chest X-ray film. We diagnosed this case as dumbbell type neurogenic tumor by MRI and CT. An operation was performed by modified Grillo's method: One-stage operation. With patient in prone position, L-shaped skin incision was made. Through total laminectomy of T-5 and T-6 and resection of the 6th rib, paravertebral portion of the tumor was removed, and thoracotomy in the same position under the same view enabled us to remove the residual tumor. Histopathological diagnosis was schwannoma. After the operation, no neurological complications were detected.