Galectin-7 in the stratum corneum: a biomarker of the skin barrier function.

OBJECTIVE: Skin barrier disruption often occurs in diseased and damaged skin conditions such as atopic dermatitis (AD). We focused the galectin-7 protein (Gal-7) as a biomarker of skin condition and assessed whether the content of Gal-7 in stratum corneum (scGal-7) could be used as an indicator of skin barrier disruption and as an index of local skin symptoms in AD patients. METHODS: Alteration of Gal-7 expression levels in keratinocyte and scGal-7 contents after barrier disruption by sodium dodecyl sulphate were evaluated in vitro and in vivo, respectively. Correlation between scGal-7 content and transepidermal water loss (TEWL) was examined in 126 healthy subjects. We performed single measurements of scGal-7 contents in 34 AD patients and serial measurements of 15 inpatients among them. SC samples were collected by the tape-stripping method, and scGal-7 content was determined using enzyme-linked immunosorbent assay. RESULTS: Gal-7 expression in keratinocytes increased after barrier disruption. The scGal-7 content reflected the disruption of the skin barrier. The scGal-7 contents and TEWL values correlated in healthy subjects. The scGal-7 level was higher in AD patients than in healthy subjects. The scGal-7 contents in the cheek and neck of AD patients significantly correlated with the total and local skin lesion severity scores. Serial measurements in the inpatients showed that the scGal-7 contents in the cheek and neck decreased in tandem with local severity scores in response to treatment. CONCLUSION: Measurement of scGal-7 content in tape-stripped samples was useful for the evaluation of the skin barrier function in dry skin conditions such as AD.

Effects of oligomeric proanthocyanidins (OPCs) of red wine to improve skin whitening and moisturizing in healthy women - a placebo-controlled randomized double-blind parallel group comparative study.

OBJECTIVE: This study was undertaken to evaluate the effects of red wine from grapes oligomeric procyanidins (OPCs) intake on skin color and skin moisture in Japanese healthy women. The purpose of this study was to improve skin condition, with the primary endpoint set to improve sunburn by ultraviolet (UV) and the secondary endpoint set to improve dryness. PATIENTS AND METHODS: A randomized, placebo-controlled, double-blind, parallel-group study was conducted on 100 subjects (30 to 59 years of age). They were administered a test beverage, including 200 mg of the red wine OPCs (the test beverage group) or a placebo beverage (the control beverage group) once a day for 12 weeks. The properties of facial skin were measured at 0 (start value), 4th, 8th, and 12th week of the test period. RESULTS: After 12 weeks of administration, the pigmentation scores and melanin index values of the OPC group were significantly reduced from the start value and were lower than the control group (p<0.05). In addition, the OPC group showed a significant increase in water content of the stratum corneum compared to the start value, while that of the control group significantly decreased. CONCLUSIONS: The red wine OPCs showed the effects of skin whitening and moisturizing, and it is suggested that OPCs may improve the skin condition of healthy women.

TNF-alpha increases expression of IL-6 and ICAM-1 genes through activation of NF-kappaB in osteoblast-like ROS17/2.8 cells.

Tumor necrosis factor-alpha (TNF-alpha) plays a key role in inflammatory diseases such as rheumatoid arthritis and in postmenopausal osteoporosis. In various tissues, TNF-alpha action is mediated by a transcription factor, nuclear factor-kappa B (NF-kappaB). However, little is known about how TNF-alpha exerts its action in osteoblasts. We thus examined the effect of TNF-alpha on the activation of NF-kappaB in rat osteoblast-like osteosarcoma cells (ROS17/2.8). Electrophoretic mobility shift assay revealed that the activation of the p50-p65 heterodimer NF-kappaB was induced by TNF-alpha as early as 15 minutes followed by a persistent activation for 48 h. When the binding activity of NF-kappaB in cytosol was examined using detergents that dissociate NF-kappaB from an inhibitory protein IkappaB, it decreased during the initial 30 minutes and then increased to the unstimulated level. Northern blot analysis revealed a marked increase in the mRNA levels of p105, a precursor of p50, 6 h after TNF-alpha and a gradual increase in p65 mRNA levels during the initial 1 h. Significant increase in both mRNA levels continued until 24 h after TNF-alpha. These results suggest that the rapid activation of NF-kappaB by TNF-alpha is mainly due to the nuclear translocation of NF-kappaB pre-existing in cytosol, and that the subsequent increase in the expression of p50 and p65 may result in the persistent activation of NF-kappaB during TNF-alpha stimulation. TNF-alpha also increased the mRNA levels of interleukin-6 (IL-6) and intercellular adhesion molecule-1 (ICAM-1). An antioxidant, N-acetyl-L-cysteine, significantly attenuated the TNF-alpha-dependent increase in these mRNAs, and simultaneously reduced the activation of NF-kappaB by TNF-alpha, indicating that NF-kappaB mediates the TNF-alpha-dependent expression of IL-6 and ICAM-1 in ROS17/2.8 cells. These results suggest that the activation of NF-kappaB by TNF-alpha may play an important role in the production of cytokines and cell adhesion molecules from osteoblasts, leading to the promotion of bone resorption and inflammation.

Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse.

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.

Production of polyclonal antibody specific for human natriuretic peptide receptor B.

Polyclonal antibody against human natriuretic peptide receptor B (NPR-B) was produced using as immunogen a soluble chimeric protein consisting of the extracellular domain of the receptor fused with Fc portion of human IgG. The antibody was purified with protein A column, and then subjected to an adsorption of anti-Fc antibody using IgG column. The purified antibody recognized human NPR-B but not the related receptor NPR-A. The antibody inhibited C-type natriuretic peptide (CNP)-mediated intracellular cGMP accumulation in a dose-dependent manner. With regard to specific activity for the neutralization, the antibody purified with IgG column was significantly stronger than that before the adsorption step, indicating that the purification of the antibody with IgG column was extremely effective to remove the contaminating anti-Fc antibody from anti-NPR-B antibody. Western blot analysis using the purified antibody revealed that while the native NPR-B exists as an oligomer, the truncated NPR-B lacking most of its cytoplasmic domain is a monomer. This finding suggests that the cytoplasmic region may be involved in the oligomerization of the receptor. The results in this study demonstrate that soluble IgG fusion protein is very effective and useful for generating specific antibodies to the proteins expressed on cell surface.

The immunosuppressive effect of 5-lipoxygenase inhibitor on liver allotransplantation in rats.

This study was performed to examine the immunosuppressive effect of a 5-lipoxygenase inhibitor, AA-861, on liver transplantation in rodents, and also to examine the production of eicosanoids during rejection of liver allograft in these animals. Rats were divided into three groups: group I (syngenic orthotopic liver transplantation from LEW to LEW), group II (allogenic OLT from ACI to LEW with dimethyl sulfoxide), and group III (allogenic OLT from ACI to LEW with AA-861 [20 mg/kg/day] s.c. dissolved in DMSO). Histological examinations were performed, survival time was monitored, and eicosanoid levels at 3, 5, and 7 days after transplantation were measured. Mean survival time in group III was significantly longer than that in group II (36.0 +/- 6.8 vs. 11.1 +/- 0.7 days, mean +/- SEM; P < 0.01). Histologically, the degree of rejection in group III was moderate compared with that in group II. On day 3, the LTB4 level in group II was significantly higher than that in group I (3361 +/- 985 vs. 407 +/- 70 pg/ml, P < 0.05), and the PGE2 level in group III was significantly higher than that in group 1 (50.3 +/- 4.8 vs. 23.5 +/- 4.7 pg/ml, P < 0.01) and in group II (32.9 +/- 4.2 pg/ml, P < 0.05). These findings suggest that AA-861 reduced liver allograft rejection by suppressing the elevation of 5-lipoxygenase products and increasing PGE2 production in the early stage of rejection.

Nucleolin stimulates viral internal ribosome entry site-mediated translation.

Previous results from our laboratory have identified a small (60 nt) RNA from the yeast S. cerevisiae that specifically inhibits internal ribosome entry site (IRES)-mediated translation programmed by poliovirus (PV) and hepatitis C virus (HCV) 5'-untranslated region (5'UTR). The yeast inhibitor RNA (called IRNA) was found to efficiently compete with viral 5'UTR for binding of several cellular polypeptides that presumably play important roles in IRES-mediated translation. One such IRNA (and 5'UTR)-binding protein has previously been identified as the La autoantigen. In this report, we have identified a 110-kDa IRNA-binding protein (which also interacts with viral 5'UTR) as nucleolin, a nucleolar RNA binding protein that was previously shown to translocate into the cytoplasm following infection of cells with poliovirus. We demonstrate that nucleolin (called C23) stimulates viral IRES-mediated translation both in vitro and in vivo. We also show that nucleolin mutants containing the carboxy-terminal RNA binding domains but lacking the amino terminal domain inhibit IRES-mediated translation in vitro. The translation inhibitory activity of these mutants correlates with their ability to bind the 5'UTR sequence. These results suggest a role of nucleolin/C23 in viral IRES-mediated translation.

Production and characterization of monoclonal antibodies against human natriuretic peptide receptor-A or -B.

Monoclonal antibodies (mAbs) against human natriuretic peptide receptor-A (NPR-A) or NPR-B were produced using NPR-expressing Chinese hamster ovary (CHO) cells and soluble chimeric NPRs consisting of the extracellular domain of each receptor fused to Fc region of human IgG. Three anti-NPR-A mAbs, designated as A144, A397 and A416, bound to human NPR-A but not to NPR-B, while an anti-NPR-B mAb B136 reacted with human NPR-B but not with NPR-A. Competition analysis with the anti-NPR-A mAbs revealed that two mAbs, A144 and A416, recognize an identical or the adjacent site of the receptor and that A397 is directed against another epitope. No anti-NPR-A mAb affected binding of atrial natriuretic peptide (ANP) to NPR-A, while the anti-NPR-B mAb B136 inhibited binding of C-type natriuretic peptide (CNP) to NPR-B. Inhibition of the ligand-binding by B136 is specific in that the mAb showed no effect on the binding of ANP to NPR-A. B136 also blocked CNP-mediated intracellular cGMP accumulation in NPR-B-expressing cells. These results suggest that the region recognized by B136 may be related to the ligand-binding region of NPR-B. NPR-A- and NPR-B-expressing cells were selectively detected by immunostaining using the mAbs. These findings demonstrate that the mAbs will be useful to elucidate the role of the natriuretic peptides and their receptors in normal and disease states in humans [correction of human].

Experimental pancreatolithiasis in the dog.

Little is known about the pathogenesis of pancreatolithiasis, and although several theories have been proposed, no experimental models of pancreatic calculi production are documented in the literature. This study examines such a model in which partial or complete pancreatic duct obstruction was surgically created in dogs. We found that (1) pancreatic calculi were demonstrated in 46.7% of the dogs (14 of 30) with partial outflow obstruction for 4 months or longer; (2) no pancreatic calculi were found in any of the dogs with complete pancreatic duct obstruction; (3) all calculi produced were localized in the ductal system; (4) the organic and inorganic composition of canine and human pancreatic calculi were quite similar; and (5) connective tissue proliferation and mucous cell metaplasia of the ductal epithelium, often seen in association with clinical pancreatolithiasis, are also detected in the pancreata of dogs that have had partial obstruction of pancreatic excretion. These findings suggest that an experimental model of pancreatolithiasis can be accomplished in dogs by the partial obstruction of pancreatic excretion.

Hepatic resection guided by needles inserted under ultrasonographic guidance.

BACKGROUND: Operative ultrasonography for orienting the direction of transection of the liver is often useful in obtaining an adequate disease-free surgical margin. We have devised a new technique for hepatectomy guided by needles inserted under ultrasonographic guidance. METHODS: One hundred two hepatectomies were performed between January 1987 and September 1991, and the hepatectomy with this technique was begun in January 1989. RESULTS: In 10 of 29 limited hepatectomies performed in the first phase of the period in which this technique was not available, disease-free surgical margin of less than 1 cm was left because of inadequately directed division. Disease-free surgical margin of more than 1 cm was left in 18 of 23 limited hepatectomies in the second phase of the period in which this technique was available. In the other five operations where disease-free surgical margin of less than 1 cm was left, carcinomas were located too close to the major hepatic vessels. The average blood loss during the limited hepatectomies was reduced by this technique. Two-year and 3-year survival of patients undergoing hepatectomy for hepatocellular carcinoma were more favorable in the second phase than in the first phase. CONCLUSIONS: Although the difference between the two groups was not significant, this technique is useful in performing adequate transection of the liver.

Detection of human parvovirus B19 DNA by nested polymerase chain reaction.

We describe the development of a nested polymerase chain reaction (PCR) technique used to detect human parvovirus B19 DNA. It was performed in a two-step reaction, first with a pair of outer primers, then with a pair of inner (ie, nested) primers. Primer sequences were selected in the VP1 gene, corresponding to the capsid protein, of human parvovirus B19. To establish the nested PCR assay, the plasmid pGEM-1 containing almost the entire coding sequence of a human parvovirus B19 isolate was used. The nested PCR could detect up to 1.8 x 10(-3) ag of B19 DNA, equivalent to 10(-4) genomes, by electrophoresis. No amplification product was detected by gel electrophoresis when the reaction mixture contained human placental DNA, cytomegalovirus DNA, and sterile distilled water as templates. We used this assay to evaluate four cases of maternal B19 infection that were diagnosed by determination of the presence of anti-B19 immunoglobulin-M in maternal serum. The advantages of our nested PCR for detecting B19 DNA are plating simplicity, safety, sensitivity, and specificity. Our results suggest that this method may have general applicability in the evaluation of nonimmune hydrops fetalis and in the documentation of the natural history of fetal infection with B19 when applied to specimens of amniotic fluid or fetal blood.

Cholinergic and glutamatergic transmission in medial vestibular nucleus neurons responding to lateral roll tilt in rats.

The responses of the medial vestibular nucleus (MVN) neurons to lateral tilt and the neurotransmitters mediating otolith information to MVN neurons were investigated using rats. A computer-operated goniometer was tilted 20 degrees clockwise and counterclockwise at an angular speed of 5 degrees /s and paused in the inclined positions for 10 s to record neuronal responses in the static phase. The 185 MVN neurons recorded were classified into eight types according to their responses to tilt (alpha, beta, gamma, delta, epsilon, zeta, eta and theta). A majority showed increased firing in response to ipsilateral tilting and decreased firing in response to contralateral tilting (alpha type: 31.4%) or exhibited the reverse pattern (beta type: 36.8%). Further, other groups of neurons increased (gamma type) or decreased (delta type) firing rates to either side tilting and increased (epsilon and zeta type) or decreased (eta and theta type) firing only on one side. Atropine or L-glutamic acid diethyl ester hydrochloride (GDEE) applied microiontophoretically antagonized tilt-induced firing of alpha type neurons in 58.8% or 60.0%, respectively, and of beta type neurons in 66.7% or 58.3%, respectively. When the effects of atropine and GDEE were examined in the same neurons, antagonizing effects of both drugs on tilt-induced firing were obtained in 28.6% and 40.0% of alpha and beta type neurons, respectively. These results suggest that both acetylcholine and glutamate act as neurotransmitters in the transmission of otolith information to most MVN neurons.

Morphology of single afferents of the saccular macula in cats.

The morphology of single saccular afferents was studied by the intracellular horseradish peroxidase (HRP) method. Four neurons were sufficiently stained to allow reconstruction of their axonal arborizations. The main axon of these neurons bifurcated into an ascending and a descending branch at the level of the lateral nucleus. The ascending branches of two axons gave off collaterals with boutons in the caudal part of the superior nucleus, while the other two ascending branches lacked such terminations. By contrast, characteristics of the descending axonal arborization patterns of all the four neurons were substantially the same. The descending branches coursed caudally through the lateral part of the descending nucleus, and gave off up to 14 collaterals with boutons that extended throughout this nucleus. These collaterals also reached the ventral part of the lateral nucleus, the lateral border of the medial nucleus, and group f. A few axon collaterals ramified even outside the border of the vestibular nuclei into the spinal trigeminal nucleus and the reticular formation surrounding it. Axon collaterals from the stem axon also terminated in the interstitial nucleus of the vestibular nerve. There was a noticeable absence of any projection to the y group.

Congenital antithrombin III deficient neonate treated with antithrombin III concentrates.

A patient with antithrombin III deficiency developed deep vein thrombosis during her first pregnancy. Her pregnancy and delivery were successfully treated with simultaneous administration of antithrombin III concentrates and low molecular weight heparin. She delivered a 2,412g girl at 39 weeks' gestation. The baby was administered with antithrombin III concentrates as prophylaxis for neonatal arterial and venous thrombosis because the antithrombin III level was extremely low to be 2%. Her second pregnancy was uneventful at 38 weeks' gestation, and she was treated with administration of antithrombin III concentrates prophylactically. She delivered a 3,256g boy at 42 weeks' gestation without any complications. The antithrombin III level of the second baby was normal. These results showed that in a neonate with congenital antithrombin III deficiency the antithrombin III concentrates would be administered to prevent neonatal arterial and venous thrombosis.

Ellagic acid/phospholipid-induced coagulation and dextran sulfate-induced fibrinolytic activities in beta 2-glycoprotein I-depleted plasma.

Beta 2-glycoprotein I (beta 2-GPI) binds negatively charged substances and inhibits intrinsic blood coagulation in the presence of ellagic acid-phospholipid suspension. Beta 2-GPI is thought to be an important protein in the reaction between negatively charged phospholipids and anti-phospholipid antibodies which appear in patients with lupus anticoagulant/antiphospholipid antibody syndrome. We prepared a monoclonal antibody against beta 2-GPI purified from human plasma and obtained beta 2-GPI-depleted plasma using a monoclonal antibody-coupled column. Either partial thromboplastin time or the activation of prekallikrein induced by diluted ellagic acid-phospholipid suspension in beta 2-GPI-depleted plasma was not different from that in control plasma. Beta 2-GPI inhibited the intrinsic blood coagulation only when added to control or beta 2-GPI-depleted plasma in excess (more than physiological concentrations). The intrinsic fibrinolysis in beta 2-GPI-depleted plasma induced by dextran sulfate was not impaired and, again, beta 2-GPI inhibited the intrinsic fibrinolysis only when added to control or beta 2-GPI-depleted plasma in excess. These results indicate that both in vitro Actin-induced intrinsic coagulation and dextran sulfate-induced fibrinolytic activities are significantly inhibited by more than physiological concentrations of beta 2-GPI.

Space environment, electromagnetic fields, and circadian rhythm.

Human space activity began in 1961. About 400 persons have gone to space since then, and about 70 of them have stayed more than 1 month. Circadian rhythm and sleep in space have been investigated several times, though the effect of longer stays in space has not been adequately clarified. Electromagnetic fields are different in the space environment, especially in deeper space missions, such as the Moon or Mars, but their effects on human health have rarely been studied. In this article, we summarize the current status of the International Space Station project, study circadian rhythm and sleep in space, investigate electromagnetic fields, and state the necessity for investigating this research field.

Odontogenic myxofibroma arising from the periodontal ligament in the maxillary molar region.

A rare case of odontogenic myxofibroma, which arose from the periodontal ligament and expanded into the oral cavity resulting in an epulis-like lesion in a 52-year-old man, is reported including details of studies using lectin histochemistry, transmission electron microscopy (TEM), and immunohistochemistry. Most of the tumor cells, which appeared spindle-like with abundant rough endoplasmic reticulum and some microfilaments by TEM, showed immunoreactivity for mesenchymal markers. Some tumor cells, which were polygonal and contained many microfilaments and some filament bundles, were immunoreactive for muscle markers. The present case was considered to consist of many fibroblasts and some myofibroblasts.