Pulmonary Schizophyllum commune infection developing mucoid impaction of the bronchi

A 54-year-old woman was admitted for cough, sputum, and an abnormal chest X-ray shadow. Bronchoscopy showed mucoid impaction of the bronchi (MIB). Histopathologic evidence of mucous plugs was consistent with one component of allergic bronchopulmonary mycosis. Schizophyllum commune (S. commune) was identified. Two attempts at removal of the mucous plugs were unsuccessful. Itraconazole was then administered, and the mucous plugs disappeared. There are few reports of MIB due to S. commune; we herein report a case of MIB due to S. commune infection.     

Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis

Introduction

Recent studies have revealed that rapamycin activates autophagy in human chondrocytes preventing the development of osteoarthritis (OA) like changes in vitro, while the systemic injection of rapamycin reduces the severity of experimental osteoarthritis in a murine model of OA in vivo. Since the systemic use of rapamycin is associated with numerous side effects, the goal of the current study was to examine the beneficial effect of local intra-articular injection of rapamycin in a murine model of OA and to elucidate the mechanism of action of rapamycin on articular cartilage.

Methods

Destabilization of the medial meniscus (DMM) was performed on 10-week-old male mice to induce OA. Intra-articular injections of 10 μl of rapamycin (10 μM) were administered twice weekly for 8 weeks. Articular cartilage damage was analyzed by histology using a semi-quantitative scoring system at 8 and 12 weeks after surgery. Mammalian target of rapamycin (mTOR), light chain 3 (LC3), vascular endothelial growth factor (VEGF), collagen, type X alpha 1 (COL10A1), and matrix metallopeptidase 13 (MMP13) expressions were analyzed by immunohistochemistry. VEGF, COL10A1, and MMP13 expressions were further examined via quantitative RT-PCR (qPCR).

Results

Intra-articular injection of rapamycin significantly reduced the severity of articular cartilage degradation at 8 and 12 weeks after DMM surgery. A reduction in mTOR expression and the activation of LC3 (an autophagy marker) in the chondrocytes was observed in the rapamycin treated mice. Rapamycin treatment also reduced VEGF, COL10A1, and MMP13 expressions at 8 and 12 weeks after DMM surgery.

Conclusion

These results demonstrate that the intra-articular injection of rapamycin could reduce mTOR expression, leading to a delay in articular cartilage degradation in our OA murine model. Our observations suggest that local intra-articular injection of rapamycin could represent a potential therapeutic approach to prevent OA.     

Disruption of the temporally regulated cloaca endodermal β-catenin signaling causes anorectal malformations

The cloaca is temporally formed and eventually divided by the urorectal septum (URS) during urogenital and anorectal organ development. Although congenital malformations, such as anorectal malformations (ARMs), are frequently observed during this process, the underlying pathogenic mechanisms remain unclear. β-Catenin is a critical component of canonical Wnt signaling and is essential for the regulation of cell differentiation and morphogenesis during embryogenesis. The expression of β-catenin is observed in endodermal epithelia, including URS epithelia. We modulated the β-catenin gene conditionally in endodermal epithelia by utilizing tamoxifen-inducible Cre driver line (Shh^CreERT2). Both β-catenin loss- and gain-of-function (LOF and GOF) mutants displayed abnormal clefts in the perineal region and hypoplastic elongation of the URS. The mutants also displayed reduced cell proliferation in the URS mesenchyme. In addition, the β-catenin GOF mutants displayed reduced apoptosis and subsequently increased apoptosis in the URS epithelium. This instability possibly resulted in reduced expression levels of differentiation markers, such as keratin 1 and filaggrin, in the perineal epithelia. The expression of bone morphogenetic protein (Bmp) genes, such as Bmp4 and Bmp7, was also ectopically induced in the epithelia of the URS in the β-catenin GOF mutants. The expression of the Msx2 gene and phosphorylated-Smad1/5/8, possible readouts of Bmp signaling, was also increased in the mutants. Moreover, we introduced an additional mutation for a Bmp receptor gene: BmprIA. The Shh^CreERT2/+; β-catenin^flox(ex3)/+; BmprIA^flox/− mutants displayed partial restoration of URS elongation compared with the β-catenin GOF mutants. These results indicate that some ARM phenotypes in the β-catenin GOF mutants were caused by abnormal Bmp signaling. The current analysis revealed the close relation of endodermal β-catenin signaling to the ARM phenotypes. These results are considered to shed light on the pathogenic mechanisms of human ARMs.During embryonic development, the cloaca is a temporal structure that is subsequently divided into the urogenital sinus and rectum by the urorectal septum (hereafter referred to as the URS).^{1, 2, 3} The URS develops from the proximal umbilical mesenchyme (the rostral portion of the cloaca) around embryonic day 10.5 (E10.5) (Figure 1a, asterisk)^{3, 4, 5} and subsequently extends caudally along the cloaca reaching the cloacal membrane. Part of the cloacal membrane degrades with the approximation of the URS tip. As a result, the cloaca is divided into the urogenital sinus and rectum, and the tip of the URS (endodermal epithelia) contributes to forming the ectodermal epithelia of the perineum and external genitalia.⁶ The contribution of these cells and growth factors expressed in the endodermal epithelia are essential for the proper morphogenesis of the perineal region, based on the phenotypes of several mutants. For instance, Sonic hedgehog (Shh) is expressed in the endodermal epithelia and has an essential role in both URS formation and GT protrusion by affecting neighboring mesenchymal cells.^{7, 8, 9, 10, 11} Apoptotic cells are observed in the urogenital tract during URS formation, being distributed primarily in the epithelial layers of the URS, cloacal membrane and mesenchyme of the dorsal surface of the caudal hindgut at E11.5 and E11.75.^{5, 12} From E12.5, apoptotic cells are also observed in the URS mesenchyme and are thought to be involved in the transformation of the URS and disintegration of the cloacal membrane.⁵Open in a separate windowFigure 1Temporally labeled Shh-expressing URS endodermal cells contribute to the ectodermal epithelia. The expression of the Shh gene in sagittal sections of wild-type embryos at E10.5, E11.5, E12.5 and E13.5 (a–d). The asterisk in (a) indicates future URS derived from the proximal umbilical mesenchyme. Ventral view of the genital tubercle (e–j). Tissue labeling experiments of Shh-expressing cells were performed at E15.5. Shh^CreERT2/+; R26^LacZ/+ embryos subsequent to TM administration at E8.5, E9.5, E10.5, E11.5, E12.5 or E13.5 (e–j). The red arrows indicate the LacZ-positive ectodermal cells. β-Catenin was expressed in the endoderm, including the URS epithelia, at E11.5 in the wild-type embryos (k). b, Bladder; c, cloaca; gt, genital tubercle; hl, hindlimb; r, rectum; t, tail; uc, umbilical cord; urs, urorectal septumSeveral congenital anomalies are frequently observed during urogenital organ development. These abnormalities are usually accompanied by deficient excretory and copulatory functions, influencing the quality of life of the patient. In particular, the incidence of anorectal malformations (ARMs) is approximately 1 in 5000 human births;¹³ however, the underlying pathogenic mechanisms of this condition are currently unknown. ARM phenotypes are observed in several diseases, including Currarino syndrome, Townes Brocks syndrome and VACTERL complex.^{14, 15, 16, 17} Affected patients often display other malformations, such as anal fistulas, sacral malformations and renal malformations. Human and mouse genetic analyses have shed light on the possible genetic causes of some of these abnormalities.Several mouse mutants for hedgehog signaling genes, ephrin-Eph signaling genes, fibroblast growth factor (Fgf) signaling genes and Wnt signaling genes are reported to display ARM phenotypes.^{18, 19, 20, 21, 22} Another causative factor for the development of ARM is all-trans retinoic acid (RA), a teratogen and active form of vitamin A. RA treatment in pregnant mice results in imperforate anus in embryos.^{23, 24} Although these reports have identified causative factors for ARM phenotypes, the pathogenic mechanisms underlying the development of ARM and URS remain elusive.The Wnt signaling pathway is essential for embryonic development, and its dysregulation has been implicated in developmental disorders and human diseases. Wnt signaling is transmitted primarily via three divergent pathways: the canonical Wnt/β-catenin pathway, the planner cell polarity pathway and the Wnt/Ca²⁺ pathway.^{25, 26} β-Catenin is a key factor for the canonical Wnt pathway and also acts as a subunit of the cadherin protein complex, which controls cell–cell adhesion. Owing to the early lethality of β-catenin-deficient mice,²⁷ analyses of the function of β-catenin in organogenesis have been performed using conditional mutants. Previous reports have revealed that β-catenin induces the differentiation of hair follicles during hair/skin development according to loss- and gain-of-function (LOF and GOF) approaches.^{28, 29} The ectopic activation of canonical Wnt signaling results in the ectopic induction of bone morphogenetic protein (Bmp) signaling, which is essential for hair follicle formation.^{30, 31} With respect to genital tubercle (primordia of external genitalia: GT) development, region-specific (ectodermal, endodermal and mesenchymal) modulation of the β-catenin gene has revealed essential functions, such as regulation of cell proliferation, epithelial integrity and protrusion/elongation of GT.^{8, 32, 33} However, the regulatory functions of the β-catenin gene during the development of the URS have not been investigated.The current study aimed to investigate the function of the β-catenin gene in URS development. We modulated the β-catenin activity in the endodermal epithelia and studied the phenotypic consequences of dysregulated endodermal β-catenin signaling for urorectal development. Both β-catenin LOF and GOF mutations resulted in ARM phenotypes. The β-catenin GOF mutation led to the ectopic induction of Bmp signaling. Moreover, the ARM phenotypes in the β-catenin GOF mutants were restored by additionally introducing the BmprIA gene mutation. These results suggest that adequately controlled β-catenin signaling and its downstream growth factor signaling are essential for proper URS formation.     

Cys34-Cysteinylated Human Serum Albumin Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.     

Method of enzymatic determination of pyrroloquinoline quinone

An improved enzymatic method for the determination of pyrroloquinoline quinone, a novel prosthetic group of some important oxidoreductases, has been developed with cytoplasmic membrane of Escherichia coli K-12, in which D-glucose dehydrogenase (EC 1.1.99.17) was completely resolved to apo-enzyme by EDTA treatment. Incubation of the EDTA-treated membrane with exogenous pyrroloquinoline quinone in the presence of magnesium ions gave a quantitative determination of pyrroloquinoline quinone by assaying the restored D-glucose dehydrogenase activity. This novel enzymatic method was confirmed to be highly reproducible up to 10 ng of pyrroloquinoline quinone and could be applied to a routine assay of pyrroloquinoline quinone.     

Crystallization and Properties of Amine Dehydrogenase from Pseudomonas sp.

An amine dehydrogenase was purified and crystallized from the cell free extract of a Pseudomonas sp., isolated from soil by means of the enrichment technique. The crystalline enzyme gave a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was estimated to be 100,000 by gel filtration on a Sephadex column. Upon SDS-gel electrophoresis, the enzyme was dissociated into two nonidentical subunits having molecular weights of 60,000 (dehydrogenase) and 39,000 (cytochrome c). The absorption spectrum of the enzyme showed absorption maxima at 550 nm, 524 nm, 411 nm and 280 nm, and a broad shoulder at around 350 nm, indicating that the enzyme was purified as a dehydrogenase-cytochrome c complex. The prosthetic group of the dehydrogenase was identified as covalently bound pyrroloquinoline quinone. The enzyme showed a broad substrate specificity toward various amines including aliphatic monoamines, aliphatic diamines, aromatic amines and polyamines.     

Genetic and Infectious Profiles of Japanese Multiple Sclerosis Patients

Background

Nationwide surveys conducted in Japan over the past thirty years have revealed a four-fold increase in the estimated number of multiple sclerosis (MS) patients, a decrease in the age at onset, and successive increases in patients with conventional MS, which shows an involvement of multiple sites in the central nervous system, including the cerebrum and cerebellum. We aimed to clarify whether genetic and infectious backgrounds correlate to distinct disease phenotypes of MS in Japanese patients.

Methodology/Principal Findings

We analyzed HLA-DRB1 and -DPB1 alleles, and IgG antibodies specific for Helicobacter pylori, Chlamydia pneumoniae, varicella zoster virus, and Epstein-Barr virus nuclear antigen (EBNA) in 145 MS patients and 367 healthy controls (HCs). Frequencies of DRB1*0405 and DPB1*0301 were significantly higher, and DRB1*0901 and DPB1*0401 significantly lower, in MS patients as compared with HCs. MS patients with DRB1*0405 had a significantly earlier age of onset and lower Progression Index than patients without this allele. The proportion and absolute number of patients with DRB1*0405 successively increased with advancing year of birth. In MS patients without DRB1*0405, the frequency of the DRB1*1501 allele was significantly higher, while the DRB1*0901 allele was significantly lower, compared with HCs. Furthermore, DRB1*0405-negative MS patients were significantly more likely to be positive for EBNA antibodies compared with HCs.

Conclusions

Our study suggests that MS patients harboring DRB1*0405, a genetic risk factor for MS in the Japanese population, have a younger age at onset and a relatively benign disease course, while DRB1*0405-negative MS patients have features similar to Western-type MS in terms of association with Epstein-Barr virus infection and DRB1*1501. The recent increase of MS in young Japanese people may be caused, in part, by an increase in DRB1*0405-positive MS patients.     

Characterization of a Ca2+ uniporter from Bacillus subtilis by partial purification and reconstitution into phospholipid vesicles

Ca2+ was accumulated by right-side-out membrane vesicles of Bacillus subtilis following imposition of a diffusion potential, inside-negative, owing to K+-efflux via valinomycin. Uptake was dependent on the magnitude of the membrane potential. This voltage-dependent Ca2+ uptake was inhibited by Ca2+ channel blockers such as nitrendipine, verapamil and LaCl3, and was competitively inhibited by Ba2+ and Sr2+. The system showed saturation kinetics with an apparent Km for Ca2+ of about 250 microM. Proteins responsible for the voltage-dependent Ca2+ uptake were partially purified by preparative isoelectric focusing in a Sepharose bed. A fraction at pH 5.28-5.33 contained the activity. The characteristics of Ca2+ uptake in reconstituted proteoliposomes were the same as those in membrane vesicles (sensitive to Ca2+ channel blockers; inhibited by Ba2+ and Sr2+). In addition, uptake was not influenced by a pH gradient imposed on the vesicles. The apparent Km for Ca2+ in the reconstituted system was about 260 microM. The specific activity was increased about 50-fold by purification with isoelectric focusing.     

Regulated expression of endothelial cell-derived lipase

A lipoprotein lipase-like gene was recently cloned from endothelial cells. In vitro functional experiments have suggested that this endothelial-derived lipase (EDL) has phospholipase activity, and preliminary in vivo studies have suggested a role in the regulation of high-density lipoprotein metabolism. To investigate local control of lipase activity and lipid metabolism in the blood vessel wall, we have examined the regulation of EDL expression in cultured human umbilical vein and coronary artery endothelial cells. EDL mRNA levels were upregulated in both cell types by inflammatory cytokines implicated in vascular disease etiology, including TNF-alpha and IL-1beta. In addition, both fluid shear stress and cyclic stretch were found to increase the EDL mRNA levels in these cultured cells. This highly regulated expression of EDL in vascular endothelial cells suggests that this recently identified lipase is intricately involved in modulating vessel wall lipid metabolism and may play a role in vascular diseases such as atherosclerosis.