Abnormalities in esophageal smooth muscle induced by mutations in collagen XIX.

Collagen XIX is a nonfibrillar collagen that localizes in restricted tissues at very low amounts. A previous study on Col19a1 null mice revealed that collagen XIX is involved in esophageal muscle physiology and morphogenesis. Here, we use histological analysis to show that mice with a Col19a1 mutant lacking the NC3 domain and seven collagen triplets display abnormal transition of smooth to striated muscle in the abdominal segment of esophagus, and a widened esophagus with age. With two newly prepared antibodies, we analyzed the expression of collagen XIX in the mouse esophagus and show that collagen XIX colocalizes with α-smooth muscle actin. By immunoelectron microscopy, we confirmed the localization of collagen XIX in esophageal smooth muscle cells. Col19a1 mutant mice contained reduced levels of mutated Col19a1 mRNA. Interestingly, hepatocyte growth factor, which has an important role in esophageal striated muscle development, was reduced in the esophagus of the Col19a1 mutant mice. These findings suggest that collagen XIX may be critical for the function of esophageal smooth muscle cells as a scaffold for anteroposterior migration of esophagus-striated muscle cells.

Thiazoline-related innate fear stimuli orchestrate hypothermia and anti-hypoxia via sensory TRPA1 activation.

Thiazoline-related innate fear-eliciting compounds (tFOs) orchestrate hypothermia, hypometabolism, and anti-hypoxia, which enable survival in lethal hypoxic conditions. Here, we show that most of these effects are severely attenuated in transient receptor potential ankyrin 1 (Trpa1) knockout mice. TFO-induced hypothermia involves the Trpa1-mediated trigeminal/vagal pathways and non-Trpa1 olfactory pathway. TFOs activate Trpa1-positive sensory pathways projecting from trigeminal and vagal ganglia to the spinal trigeminal nucleus (Sp5) and nucleus of the solitary tract (NTS), and their artificial activation induces hypothermia. TFO presentation activates the NTS-Parabrachial nucleus pathway to induce hypothermia and hypometabolism; this activation was suppressed in Trpa1 knockout mice. TRPA1 activation is insufficient to trigger tFO-mediated anti-hypoxic effects; Sp5/NTS activation is also necessary. Accordingly, we find a novel molecule that enables mice to survive in a lethal hypoxic condition ten times longer than known tFOs. Combinations of appropriate tFOs and TRPA1 command intrinsic physiological responses relevant to survival fate.

Commensal microbiota contributes to chronic endocarditis in TAX1BP1 deficient mice.

Tax1-binding protein 1 (Tax1bp1) negatively regulates NF-κB by editing the ubiquitylation of target molecules by its catalytic partner A20. Genetically engineered TAX1BP1-deficient (KO) mice develop age-dependent inflammatory constitutions in multiple organs manifested as valvulitis or dermatitis and succumb to premature death. Laser capture dissection and gene expression microarray analysis on the mitral valves of TAX1BP1-KO mice (8 and 16 week old) revealed 588 gene transcription alterations from the wild type. SAA3 (serum amyloid A3), CHI3L1, HP, IL1B and SPP1/OPN were induced 1,180-, 361-, 187-, 122- and 101-fold respectively. WIF1 (Wnt inhibitory factor 1) exhibited 11-fold reduction. Intense Saa3 staining and significant I-κBα reduction were reconfirmed and massive infiltration of inflammatory lymphocytes and edema formation were seen in the area. Antibiotics-induced 'germ free' status or the additional MyD88 deficiency significantly ameliorated TAX1BP1-KO mice's inflammatory lesions. These pathological conditions, as we named 'pseudo-infective endocarditis' were boosted by the commensal microbiota who are usually harmless by their nature. This experimental outcome raises a novel mechanistic linkage between endothelial inflammation caused by the ubiquitin remodeling immune regulators and fatal cardiac dysfunction.

Organic chemicals in diesel exhaust particles enhance picryl chloride-induced atopic dermatitis in NC/Nga mice.

BACKGROUND: Diesel exhaust particles (DEP) have been reported to worsen allergic airway inflammation in mice. Recently, the organic chemical components of DEP (DEP-OC) were found to be important contributors to the aggravation of allergic airway inflammation in mice. The purpose of this study was to examine the effects of DEP-OC on atopic dermatitis (AD)-like skin lesions induced by picryl chloride (PiCl) in NC/Nga mice. METHODS: DEP were extracted with benzene/ethanol, and the soluble organic fraction formed the DEP-OC. NC/Nga male mice received simultaneous application of DEP-OC and/or PiCl on their ears once a week for 9 or 3 weeks. We evaluated skin lesions by noting scaling, eruption, excoriation, erosion, hemorrhage, pathologic changes, production of cytokines, and IgE level in the serum. RESULTS: PiCl application alone produced progressively severe AD-like skin lesions. The application of PiCl plus DEP-OC resulted in a marked worsening of skin lesions in the early stages of AD. Moreover, mast cell counts significantly increased in the subcutaneous tissue. Administration of PiCl combined with DEP-OC resulted in a greater increase in the local expression of interleukin-4, keratinocyte chemoattractant, and neutrophils in subcutaneous tissue compared with PiCl treatment alone. In contrast, the combination treatment produced lower levels of IFN-γ compared with PiCl treatment alone. CONCLUSIONS: DEP-OC application to the skin aggravated PiCl-induced AD. This aggravation may be due to activation of the Th2-associated immune responses by the organic chemicals in DEP.

Addition of a single N-glycan to street rabies virus glycoprotein enhances virus production.

Most street rabies virus G proteins have two N-glycosylation sites, i.e. Asn(37) and Asn(319), whereas additional sites are found in fixed (laboratory adapted) viruses. In this study, we performed a pseudotyped virus assay using G-deficient rabies virus and demonstrated that single-N-glycan additions to the G protein of street rabies virus strain 1088, which are found in adapted strains, enhanced virus production in neural and non-neural cell lines, while additions to Asn(194) or Asn(247) enhanced production greatly. Moreover, we found that N-glycan additions at Asn(194) or Asn(247) facilitated the production of cell-associated virus. In contrast, deletion of the sequon at Asn(37) reduced viral production, while a deletion at Asn(319) resulted in extensive loss of production. Furthermore, G proteins lacking an N-glycan at Asn(319) failed to fold into their correct structure and lost their fusion activity, indicating that Asn(319) N-glycosylation is important for the functional expression of street virus G proteins.

Asian sand dust aggravates allergic rhinitis in guinea pigs induced by Japanese cedar pollen.

Asian sand dust (ASD) contains microbial materials, sulfate (SO(4)(2-)), and nitrate (NO(3)(-)), and is derived from air pollutants in East China. ASD reportedly causes adverse respiratory health effects; a case in point is aggravated allergen-associated experimental lung eosinophilia. Guinea pigs were administered normal saline (control), ASD (0.3 mg/animal), ASD (0.6 mg/animal), Japanese cedar pollen (JCP) (0.2 mg/kg body weight), JCP + ASD (0.3 mg/animal), or JCP + ASD (0.6 mg/animal), into their nasal cavities at seven weekly intervals. The number of sneezes, amount of nasal secretions, and nasal obstructing response were measured as indices of nasal responses. Total immunoglobulin E (IgE) antibodies in serum and the number of eosinophils, histamine, and arachidonic acid metabolites in nasal cavity lavage fluids (NCLF) were also measured. ASD enhanced the JCP-associated nasal obstructing response, but not the number of sneezes or amount of nasal secretions. ASD enhanced JCP-associated cysteinyl leukotrienes (C(4), D(4), E(4)) and histamine production in NCLF. ASD augmented the number of eosinophils in NCLF and total IgE in serum induced by JCP. ASD enhanced eosinophil recruitment in the nasal mucosa, and goblet cell proliferation in the nasal epithelium induced by JCP. These results suggest that ASD enhances the nasal allergic reaction induced by repeated JCP administration in guinea pigs.

A novel diagnostic monoclonal antibody specific for Helicobacter pylori CagA of East Asian type.

Molecular biological and epidemiological studies have suggested that Helicobacter pylori producing East Asian CagA protein variant is more virulent than that producing Western CagA. In the present study, we developed and validated an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody specifically recognizing East Asian CagA-positive H. pylori. A total of 32 H. pylori strains were tested and the data were subjected to receiver-operator characteristic (ROC) curve analysis. The accuracy of the test, determined by calculating the area under the curve, was 0.96, which indicated a high level of accuracy. At the ROC optimized cutoff, the sensitivity and specificity of our ELISA method were 88.0% and 100%, respectively. The validated ELISA showed good performance in terms of sensitivity and specificity. These results suggest that this test is suitable for the diagnostic detection of East Asian CagA carrying strains. We also analyzed the localization of the CagA protein in H. pylori-infected gastric mucosa with fluorescence immunohistochemistry, and found that CagA protein expression was up-regulated by adhesion to epithelial cells.

Effects of asian sand dust, Arizona sand dust, amorphous silica and aluminum oxide on allergic inflammation in the murine lung.

The aggravating effects of Asian sand dust (SD) and related minerals on the allergic inflammation were examined in the murine lungs. The toxic materials adsorbed onto Asian SD, Arizona SD were inactivated by heat-treatment. ICR mice were administered mineral samples (0.1 mg/mouse) and/or ovalbumin (OVA) (1 microg/mouse) - normal saline (control), Asian SD, Arizona SD, SiO2, Al2O3, OVA, OVA + Asian SD, OVA + Arizona SD, OVA + SiO2, and OVA + Al2O3 - intratracheally four times at two-week intervals. All samples tested enhanced eosinophil recruitment induced by ovalbumin in the submucosa of the airway, which has a goblet cell proliferation in the bronchial epithelium. Arizona SD alone caused a slight increase of neutrophils in bronchoalveolar lavage fluids along with pro-inflammatory mediators, such as keratinocyte chemoattractant, but Asian SD alone or Al2O3 alone showed no effect. The test particles, except Al2O3, synergistically increased the numbers of eosinophils in BALF induced by ovalbumin. In particular, Arizona SD and SiO2 synergistically increased the eosinophil relevant cytokine and chemokine, such as IL-5 and monocyte chemotactic protein (MCP)-3. The aggravating effects of the samples were dependent on the SiO2 content. All samples tested also induced the adjuvant effects to specific IgG1 production by OVA. These results suggest that the aggravated allergic inflammation by mineral dusts may be due to the mineral elements (mainly SiO2). The enhancement by Arizona SD may be mediated, at least partially, by the increased expression of IL-5 and MCP-3 and also by the modulated expression of IL-5 and MCP-3.

Rescue of mutant alpha-galactosidase A in the endoplasmic reticulum by 1-deoxygalactonojirimycin leads to trafficking to lysosomes.

Active-site-specific chaperone therapy for Fabry disease is a genotype-specific therapy using a competitive inhibitor, 1-deoxygalactonojirimycin (DGJ). To elucidate the mechanism of enhancing alpha-galactosidase A (alpha-Gal A) activity by DGJ-treatment, we studied the degradation of a mutant protein and the effect of DGJ in the endoplasmic reticulum (ER). We first established an in vitro translation and translocation system using rabbit reticulocyte lysates and canine pancreas microsomal vesicles for a study on the stability of mutant alpha-Gal A with an amino acid substitution (R301Q) in the ER. R301Q was rapidly degraded, but no degradation of wild-type alpha-Gal A was observed when microsomal vesicles containing wild-type or R301Q alpha-Gal A were isolated and incubated. A pulse-chase experiment on R301Q-expressing TgM/KO mouse fibroblasts showed rapid degradation of R301Q, and its degradation was blocked by the addition of lactacystin, indicating that R301Q was degraded by ER-associated degradation (ERAD). Rapid degradation of R301Q was also observed in TgM/KO mouse fibroblasts treated with brefeldin A, and the amount of R301Q enzyme markedly increased by pretreatment with DGJ starting 12 h prior to addition of brefeldin A. The enhancement of alpha-Gal A activity and its protein level by DGJ-treatment was selectively observed in brefeldin A-treated COS-7 cells expressing R301Q but not in cells expressing the wild-type alpha-Gal A. Observation by immunoelectron microscopy showed that the localization of R301Q in COS-7 cells was in the lysosomes, not the ER. These data suggest that the rescue of R301Q from ERAD is a key step for normalization of intracellular trafficking of R301Q.

Effects of gamma ray irradiation on energy metabolism in the rat brain: a 31P nuclear magnetic resonance spectroscopy study.

OBJECT: Gamma Knife surgery (GKS) is performed to treat patients with functional neurological diseases, but the neurophysiological mechanisms of GKS's biological effects with subnecrotic doses remain largely undefined. The purpose of the present study was to investigate the effects of gamma irradiation on energy metabolism in the rat brain by using 31P nuclear magnetic resonance spectroscopy (31P-NMRS). METHODS: The whole brains of Wistar rats were irradiated with a subnecrotic (60-Gy) dose of radiation. One week after the irradiation, brain slices (400 microm thick) were incubated in standard artificial cerebrospinal fluid to undergo 31P-NMRS investigation. Changes in high-energy phosphate, phosphocreatine (PCr), and gamma-ATP, as well as inorganic phosphate levels before, during, and after ischemic stress for 64 minutes were measured. Histological findings were also evaluated using light and electron microscopy. The decrease in the PCr level was significantly slower during ischemia and recovery after reperfusion was significantly faster and greater in the gamma-irradiated rats than in the control animals. The gamma-ATP level after ischemia was also higher in the gamma-irradiated rats than in the controls. Neither neuronal damage nor astrocytosis was observed in the irradiated cerebral cortices. CONCLUSIONS: Gamma irradiation with a subnecrotic dose may have neuroprotective effects that maintain a more stable cellular phosphorylation potential after ischemic stress. Such effects of GKS on energy metabolism coupled with neurotransmission (glutamate-glutamine cycling between neurons and astrocytes) may play a role in the treatment of neurological disease.

Enhancement of mite allergen-induced eosinophil infiltration in the murine airway and local cytokine/chemokine expression by Asian sand dust.

Data on the effects of sand dust toward allergic asthma produced by indoor allergens, such as house dust mites, are not currently available. This study was undertaken to clarify the role of Asian sand dust on mite allergen, Dermatophagoides farinae (D. farinae)-induced eosinophilic inflammation in the murine lung, using sand dusts from the Maowusu Desert (Inner Mongolia) (SD-1) and the Tengger Desert (China) (SD-2). ICR mice were intratracheally administered saline; SD-1 alone; SD-2 alone; D. farinae alone; D. farinae + SD-1; and D. farinae + SD-2, 4 times at 2-wk intervals. The two sand dusts enhanced infiltration of eosinophil in the airway, along with goblet-cell proliferation related to D. farinae. The degree of eosinophil infiltration induced with SD-2 was greater than with SD-1. The SD-1, which contained higher amounts of beta-glucan, increased the expression of interferon (IFN)-gamma in bronchoalveolar lavage fluids (BALF) with or without D. farinae, but SD-2 did not. Synergistically or cumulatively elevated levels of interleukin (IL)-5, eotaxin, and monocyte chemotactic protein in BALF related to D. farinae were higher with D. farinae + SD-2 than with D. farinae + SD-1. These results suggest that increased cytokine and chemokines in BALF play an important role in the enhancement of eosinophil infiltration in the airway induced by D. farinae + sand dusts. The reduced eosinophil infiltration in the SD-1-treated mice could be due to suppression of Th-2 cytokine and eotaxin via interferon-gamma induced by microbial materials, such as beta-glucan.

Cytoarchitecture and intercalated disks of the working myocardium and the conduction system in the mammalian heart.

Working and specialized cardiac myocytes and their intercalated disks (ID) in the mammalian heart were examined by transmission and scanning electron microscopy. The NaOH/ultrasonication treatment of cardiac tissues resulted in the digestion of collagen fibers and separation of intercellular junctions. Auricular and ventricular myocytes were cylindrical in shape, bifurcated, and connected end-to-end at the ID. The ID in the working myocardium showed a stair-like profile, consisting of steps (plicate segments) and corresponding risers (interplicate segments). The ventricular myocytes had many steps and risers. The steps were filled with numerous finger-like microprojections, including desmosomes, fasciae adherentes, and small gap junctions. The risers showed the smooth surface, including desmosomes and large gap junctions. The cell strands of the sinoatrial node were oriented linearly, while those of the atrioventricular node formed a reticular network. The ID in both nodal cells was underdeveloped, having few microprojections. Myocytes in the His bundle and its branches were arranged in parallel, and Purkinje cell strands formed reticular networks. The ID in the His-Purkinje system was irregular in appearance, and the microprojections were larger in size and smaller in number than those of working myocytes. There were few microprojections in the sheep Purkinje cells. The gap junctions in the conduction system were few or small in size in the nodal tissue, but large in the His-Purkinje system.