Down-regulation of caveolin-1, an inhibitor of transforming growth factor-beta signaling, in acute allergen-induced airway remodeling

Asthma can progress to subepithelial airway fibrosis, mediated in large part by transforming growth factor-beta (TGF-beta). The scaffolding protein caveolin-1 (cav1) can inhibit the activity of TGF-beta, perhaps by forming membrane invaginations that enfold TGF-beta receptors. The study goals were 1) to evaluate how allergen challenge affects lung expression of cav1 and the density of caveolae in vivo 2) to determine whether reduced cav1 expression is mediated by interleukin (IL)-4 and 3) to measure the effects of decreased expression of cav1 on TGF-beta signaling. C57BL/6J, IL-4-deficient mice, and cav1-deficient mice, sensitized by intraperitoneal injections of phosphate-buffered saline or ovalbumin (OVA) at days 0 and 12, received intranasal phosphate-buffered saline or OVA challenges at days 24, 26, and 28. Additionally, another group of C57BL/6J mice received IL-4 by intratracheal instillation for 7 days. We confirmed that the OVA-allergen challenge increased eosinophilia and T-helper type 2-related cytokine levels (IL-4, IL-5, and IL-13) in bronchoalveolar lavage. Allergen challenge reduced lung cav1 mRNA abundance by 40%, cav1 protein by 30%, and the number of lung fibroblast caveolae by 50%. Administration of IL-4 in vivo also substantially decreased cav1 expression. In contrast, the allergen challenge did not decrease cav1 expression in IL-4-deficient mice. The reduced expression of cav1 was associated with activation of TGF-beta signaling that was further enhanced in OVA-sensitized and challenged cav1-deficient mice. This study demonstrates a previously unknown modulation of TGF-beta signaling by IL-4, via cav1, suggesting novel therapeutic targets for controlling the effects of TGF-beta and thereby ameliorating pathological airway remodeling.     

Identification of the Schistosoma mansoni TNF-Alpha Receptor Gene and the Effect of Human TNF-Alpha on the Parasite Gene Expression Profile

Background

Schistosoma mansoni is the major causative agent of schistosomiasis. The parasite takes advantage of host signals to complete its development in the human body. Tumor necrosis factor-alpha (TNF-α) is a human cytokine involved in skin inflammatory responses, and although its effect on the adult parasite''s metabolism and egg-laying process has been previously described, a comprehensive assessment of the TNF-α pathway and its downstream molecular effects is lacking.

Methodology/Principal Findings

In the present work we describe a possible TNF-α receptor (TNFR) homolog gene in S. mansoni (SmTNFR). SmTNFR encodes a complete receptor sequence composed of 599 amino acids, and contains four cysteine-rich domains as described for TNFR members. Real-time RT-PCR experiments revealed that SmTNFR highest expression level is in cercariae, 3.5 (±0.7) times higher than in adult worms. Downstream members of the known human TNF-α pathway were identified by an in silico analysis, revealing a possible TNF-α signaling pathway in the parasite. In order to simulate parasite''s exposure to human cytokine during penetration of the skin, schistosomula were exposed to human TNF-α just 3 h after cercariae-to-schistosomula in vitro transformation, and large-scale gene expression measurements were performed with microarrays. A total of 548 genes with significantly altered expression were detected, when compared to control parasites. In addition, treatment of adult worms with TNF-α caused a significantly altered expression of 1857 genes. Interestingly, the set of genes altered in adults is different from that of schistosomula, with 58 genes in common, representing 3% of altered genes in adults and 11% in 3 h-old early schistosomula.

Conclusions/Significance

We describe the possible molecular elements and targets involved in human TNF-α effect on S. mansoni, highlighting the mechanism by which recently transformed schistosomula may sense and respond to this host mediator at the site of cercarial penetration into the skin.     

Purification and properties of recombinant β-galactosidase from Bacillus circulans

A gene encoding β-galactosidase from Bacillus circulans which had hydrolysis specificity for the β1-3 linkage was expressed in Escherichia coli. The β-galactosidase was purified from crude cell lysates of E. coli by column chromatographies on Resource Q and Sephacryl S-200 HR. The enzyme released galactose with high selectivity from oligosaccharides which had terminal β1-3 linked galactose residues. However it did not hydrolyse β1-4 linked galactooligosaccharides. Moreover, Galβ1-3GlcNAc, Galβ1-3GalNAc, and their p-nitrophenyl glycosides were regioselectively synthesized in 10–46% yield by the transglycosylation reaction using this enzyme.     

The Candida Genome Database: facilitating research on Candida albicans molecular biology

The Candida Genome Database (CGD; http://www.candidagenome.org) is a resource for information about the Candida albicans genomic sequence and the molecular biology of its encoded gene products. CGD collects and organizes data from the biological literature concerning C. albicans, and provides tools for viewing, searching, analysing, and downloading these data. CGD also serves as an organizing centre for the C. albicans research community, providing a gene-name registry, contact information, and research community news. This article describes the information contained in CGD and how to access it, either from the perspective of a bench scientist interested in the function of one or a few genes, or from the perspective of a biologist or bioinformatician interpreting large-scale functional genomic datasets.     

Occurrence of Taurine-Pyruvate Aminotransferase in Bacterial Extract

Natural ( + )-(1R,2S,3S)-methyl cucurbate (1b) and the ( – )-δ-lactone of 3-epi-cucurbic acid (16) were synthesized from (+)-(1R,6S,7R)-bicyclo [4.3.0] non-3-en-7-ol (5). Asymmetric hydrolysis of the acetate (8) of ( ± )-5 with pancreatin gave optically pure the ( + )-(7R)-alcohol (5) and (–)-(7S)-acetate (8). An ozonolysis product of ( + )-5 was transformed to ( – )-16 and ( + )-(3S)-1b with inversion of the (7R)-hydroxyl group. Similarly, unnatural (–)-1b and (+)-16 were prepared from optically pure ( — )-5. The growth inhibitory activities of these synthesized chiral compounds toward lettuce seedlings were examined.     

Refolding of the recombinant protein Sm29, a step toward the production of the vaccine candidate against schistosomiasis

Schistosomiasis is an important parasitic disease, with about 240 million people infected worldwide. Humans and animals can be infected, imposing an enormous social and economic burden. The only drug available for chemotherapy, praziquantel, does not control reinfections, and an efficient vaccine for prophylaxis is still missing. However, the tegumental protein Sm29 of Schistosoma mansoni was shown to be a promising antigen to compose an anti-schistosomiasis vaccine. Though, recombinant Sm29 is expressed in Escherichia coli as insoluble inclusion bodies requiring an efficient process of refolding, thus, hampering its production in large scale. We present in this work studies to refold the recombinant Sm29 using high hydrostatic pressure, a mild condition to dissociate aggregated proteins, leading to refolding on a soluble conformation. Our studies resulted in high yield of rSm29 (73%) as a stably soluble and structured protein. The refolded antigen presented protective effect against S. mansoni development in immunized mice. We concluded that the refolding process by application of high hydrostatic pressure succeeded, and the procedure can be scaled-up, allowing industrial production of Sm29.     

Binuclear schiff base complexes of nickel(II) or copper(II) linked by dithiols as a bridging group

Binuclear Schiff base complexes of nickel(II) or copper(II) were synthesized by bridging unsymmetrical Schiff base complexes of nickel(II) and copper(II) with 2,5-dimercapto-1,3,4-thiadiazole or 2-mercaptoethyl ether.Although the magnetic susceptibility and ESR measurements showed that copper(II)copper(II) interaction of the binuclear copper(II) complexes is very weak in the solid state, they catalyzed chemiluminescence of luminol by hydrogen peroxide in DMF solution, suggesting that small amounts of the complexes might be present in the form of the appropriate copper(II)copper(II) distance to activate hydrogen peroxide in solution.     

Cofilin1 Controls Transcolumnar Plasticity in Dendritic Spines in Adult Barrel Cortex

During sensory deprivation, the barrel cortex undergoes expansion of a functional column representing spared inputs (spared column), into the neighboring deprived columns (representing deprived inputs) which are in turn shrunk. As a result, the neurons in a deprived column simultaneously increase and decrease their responses to spared and deprived inputs, respectively. Previous studies revealed that dendritic spines are remodeled during this barrel map plasticity. Because cofilin1, a predominant regulator of actin filament turnover, governs both the expansion and shrinkage of the dendritic spine structure in vitro, it hypothetically regulates both responses in barrel map plasticity. However, this hypothesis remains untested. Using lentiviral vectors, we knocked down cofilin1 locally within layer 2/3 neurons in a deprived column. Cofilin1-knocked-down neurons were optogenetically labeled using channelrhodopsin-2, and electrophysiological recordings were targeted to these knocked-down neurons. We showed that cofilin1 knockdown impaired response increases to spared inputs but preserved response decreases to deprived inputs, indicating that cofilin1 dependency is dissociated in these two types of barrel map plasticity. To explore the structural basis of this dissociation, we then analyzed spine densities on deprived column dendritic branches, which were supposed to receive dense horizontal transcolumnar projections from the spared column. We found that spine number increased in a cofilin1-dependent manner selectively in the distal part of the supragranular layer, where most of the transcolumnar projections existed. Our findings suggest that cofilin1-mediated actin dynamics regulate functional map plasticity in an input-specific manner through the dendritic spine remodeling that occurs in the horizontal transcolumnar circuits. These new mechanistic insights into transcolumnar plasticity in adult rats may have a general significance for understanding reorganization of neocortical circuits that have more sophisticated columnar organization than the rodent neocortex, such as the primate neocortex.     

Regioselectivity in beta-galactosidase-catalyzed transglycosylation for the enzymatic assembly of D-galactosyl-D-mannose

The regioselectivity of beta-galactosidase derived from Bacillus circulans ATCC 31382 (beta-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for beta-1,3-galactosidase but also for beta-galactosidases of different origins. The relative hydrolysis rate of Gal beta-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of beta-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of beta-1,3-, beta-1,4- and beta-1,6-linked D-galactosyl-D-mannose was subsequently established.     

A superagonistic monoclonal antibody for CD28 ameliorates crescentic glomerulonephritis in wistar-kyoto rats

Regulatory T (Treg) cells play an important role in the resolution of crescentic glomerulonephritis, where a T helper 1 (Th1)-predominant immune response promotes crescent formation. Therefore, agents that increase Treg cells appear to be ideal for suppressing T-cell-mediated renal pathology. We hypothesized that a superagonistic monoclonal antibody for CD28 (JJ316), which has been known to preferentially expand Treg cells in vivo, could prevent nephrotoxic serum-induced nephritis in Wistar-Kyoto rats, one of the experimental models of crescentic glomerulonephritis. Administration of JJ316 attenuated crescent formation, proteinuria and glomerular accumulation of macrophages and CD8(+) T cells. These changes were accompanied by increased infiltration of Treg cells. Among glomerular macrophages, the CD163(+) subset was significantly increased after treatment, suggesting that Treg cells may modulate the phenotype of macrophages leading to resolution of glomerulonephritis. In an adoptive transfer experiment, two T-cell subsets (CD4(+)CD25(+) and CD4(+)CD25(-) T cells) purified from spleens and lymph nodes of donor rats primed with JJ316 3 d before were inoculated into nephritic recipient rats, which recapitulated the beneficial effects of in vivo administration of JJ316. Furthermore, a single injection of JJ316 administered 3 d after disease induction completely protected nephritic rats from death for 2 months. In conclusion, we demonstrated that treatment with JJ316 has a dramatic therapeutic effect on an experimental crescentic glomerulonephritis, possibly due to expansion and activation of Treg cells.