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.

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.

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.

Magnetic circular dichroism of resonant X-ray emission spectra at L edges of rare-earth compounds.

We study the resonant x-ray emission spectroscopy (RXES) and the magnetic circular dichroism (MCD) at the L2,3 absorption edges of rare-earth elements, especially gadolinium ion. To this end, an atomic model is adopted and the multiplet coupling effect is taken into account. Using the formula of the coherent second order optical process which consists of the excitation process from the 2p core state to the empty 5d state and of the radiative decay from the 3d core state to the 2p core hole, we calculate RXES-MCD spectra. By inspection, we have confirmed that the enhancement of the 2p-5d dipole matrix element proposed for MCD of x-ray absorption spectroscopy. In addition, characteristic features of the spectra observed in gadolinium metallic compounds are reproduced by our calculations.

Multielectron excitations probed by helicity-modulation XMCD at K-edge in 3d transition metal compounds.

Multielectron excitations (MEE) have been investigated using X-ray magnetic circular dichroism (XMCD) at the K-edge in 3d transition metal compounds. In the range of 50-70 eV above the absorption edge, a dichroic signal that is associated with MEE was identified. This phenomenon is thought to occur due to the super Coster-Kronig transition described as the final states (1s)1(3p)5(3d)n+2 resulting from the 3p --> 3d transitions. In order to verify this interpretation, XMCD measurements using the helicity-modulation method and calculations of the dichroic spectrum were performed based on this super Coster-Kronig transition.

A case of XY pure gonadal dysgenesis with 46,XYp-/47,XXYp- karyotype whose gonadoblastoma was removed laparoscopically.

A case of pure gonadal dysgenesis was investigated. The patient was an 18-year-old Japanese woman with a history of primary amenorrhea. She had poorly developed breasts, a hypoplastic uterus, a normal vagina and infantile genitalia. The patient's karyotype was 46,XYp-/ 47,XXYp-. Microsatellite analysis revealed that the X chromosomes of this patient originated from one of the two maternal X chromosomes. DNA analysis of the Y chromosome revealed that she had a deletion of SRY (the sex-determining region on the Y chromosome). She underwent laparoscopic gonadectomies with a final pathology consistent with gonadoblastoma. Laparoscopic surgery is recommended as it is much less invasive and associated with rapid postoperative recovery.

Quantitative assessment of human leukocyte antigen-G protein in amniotic fluid by a double-determinant enzyme-linked immunosorbent assay using anti-human leukocyte antigen-G-specific antibody '87G'.

PROBLEM: The objective of this study was to establish an enzyme-linked immunosorbent assay (ELISA) system, in an attempt to quantify the amount of human leukocyte antigen (HLA)-G protein in amniotic fluid. METHOD OF STUDY: We established a double-determinant ELISA system using the anti-HLA-G specific mouse monoclonal antibody '87G' as a capture antibody and the horseradish-peroxidase labeled rabbit anti-human beta2-microglobulin antibody as a detection antibody. We then measured the concentration of HLA-G protein in amniotic fluid samples from nine normal second-trimester pregnant women and in serum samples from eight normal males. RESULTS AND CONCLUSION: HLA-G protein was detected in amniotic fluid at a concentration of 275 ng/ml (197-343 ng/ml) (median value and 95% confident range), whereas the concentration of HLA-G protein in male serum was below the minimum detection level.

[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.

Prenatal diagnosis of Fukuyama-type congenital muscular dystrophy by microsatellite analysis.

We applied microsatellite analysis to prenatal diagnosis of Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive severe muscular dystrophy associated with brain malformations. Recent identification of the FCMD gene locus at 9q31-q33 provided the basis for prenatal diagnosis and carrier detection. We recently developed new microsatellite markers which are closer to the FCMD gene and improved the phenotype probability. Nine fetuses in eight unrelated FCMD families, including a twin pregnancy, were analysed using the newly developed markers. Four fetuses showed over 99% probability of being healthy either as normal homozygote (n = 1) or heterozygote carrier (n = 3) and were born without signs of FCMD. The other five fetuses were diagnosed with a probability of FCMD of 99% or greater; all of the latter parents decided to terminate the pregnancies. Brain malformations characteristic of FCMD in one of the aborted fetuses confirmed the diagnosis of FCMD at 19 weeks of gestation.

[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.

Comparative concentrations of growth hormone-binding protein in maternal circulation, fetal circulation, and amniotic fluid.

Fetal growth is thought to be independent of the concentration of GH, although circulating levels of GH are high in the human fetus. To elucidate the role of GH in fetal development, levels of GH-binding protein (GHBP) were measured in the serum of nonpregnant and pregnant women and neonates as well as in amniotic fluid obtained at various stages of gestation. Total GHBP (the sum of free GHBP and GHBP bound to GH) is measured by a ligand-mediated immunofunctional assay. GHBP concentrations in adult serum were not changed by pregnancy or the stage of gestation. A significant correlation was observed between the concentration of GHBP in the umbilical artery and vein. No correlations were observed between the GHBP concentration and such measures of fetal growth as fetal weight and fetal age. Although the neonatal concentrations of GHBP were significantly lower than those of pregnant women, no correlation was observed between them. GHBP was also present in the amniotic fluid from early to late gestation at concentrations higher than in the cord serum of the neonate. The amniotic GHBP concentration in late gestation was significantly higher than in early gestation. GHBP appears to be derived from GH receptors of fetal organs (most probably fetal liver). The low level of GHBP in fetal serum may be the result of a decrease in GH receptors caused by high levels of circulating GH. GHBP levels in amniotic fluids may be related to the development or maturation of the fetus.

DNA polymorphism analysis of a case of complete hydatidiform mole coexisting with a fetus.

A complete hydatidiform mole coexisting with a fetus is a rare condition. The diagnosis is often difficult because of the morphological similarity to a partial mole, but is crucial to management in the postmolar course. We present a case of molar pregnancy coexisting with a fetus in which DNA polymorphism analysis revealed a different genetic origin for the fetal and molar parts. This is the only known case of a complete mole in a twin pregnancy complicated by pre-eclampsia followed by maternal pulmonary oedema. During follow-up, the patient developed a clinically invasive mole which was successfully treated with chemotherapy. In this case, genetic analysis unequivocally diagnosed a twin pregnancy consisting of a complete hydatidiform mole and a fetus.

Treatment of cystic lesions of soft tissue using fibrin sealant.

A new method was devised for treating 29 patients with cystic lesions of the soft tissue (popliteal cysts, bursitis, and ganglia) by injecting a fibrin sealant into the cyst after the aspiration of the contents. During the followup period (mean, 2.2 years), cysts did not recur in 22 (76%) patients. Of the 7 patients with recurrences, 4 were treated again in the same manner and 3 of these cases have not recurred. Additionally, to investigate the healing process in cysts, fibrin sealant was injected into the space between the abdominal muscle and the peritoneum of rats. Specimens were obtained after the rats were sacrificed at 1, 2, 4, and 8 weeks after injection, and were examined histopathologically. The fibrin sealant was found to be completely resorbed and replaced with organized granulation tissue. In case of multiple failed aspirations to the cystic lesions, this new method is recommended before surgery.

[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.

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.

[The effects of glucocorticoids on angiogenesis in vitro].

Glucocorticoids administration presumably affects the vascular system and causes avascular necrosis of the femoral head. However, the mechanism by which this occurs is still unknown. In order to clarify the action of glucocorticoids on the vascular system, we investigated the effects of dexamethasone on an angiogenesis model in vitro. The angiogenesis model was obtained by co-culturing vessel fragments and myofibroblastic cells from rat epididymal fat pads. In this model, myofibroblastic cells induce capillary formation by producing an endothelial cell growth factor and collagen. Dexamethasone at physiological doses inhibited significantly capillary growth by suppressing the collagen synthesis by myofibroblastic cells. However, dexamethasone had no effect on endothelial cells. These results indicate that glucocorticoids are related to the pathogenesis of avascular necrosis of the femoral head by inhibiting the repair of the vascular system in vivo.

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.

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 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.