Identification of galactofuranose antigens such as galactomannoproteins and fungal-type galactomannan from the yellow koji fungus (Aspergillus oryzae).

Filamentous fungi belonging to the genus Aspergillus are known to possess galactomannan in their cell walls. Galactomannan is highly antigenic to humans and has been reported to be involved in the pathogenicity of pathogenic filamentous fungi, such as A. fumigatus, and in immune responses. In this study, we aimed to confirm the presence of D-galactofuranose-containing glycans and to clarify the biosynthesis of D-galactofuranose-containing glycans in Aspergillus oryzae, a yellow koji fungus. We found that the galactofuranose antigen is also present in A. oryzae. Deletion of ugmA, which encodes UDP-galactopyranose mutase in A. oryzae, suppressed mycelial elongation, suggesting that D-galactofuranose-containing glycans play an important role in cell wall integrity in A. oryzae. Proton nuclear magnetic resonance spectrometry revealed that the galactofuranose-containing sugar chain was deficient and that core mannan backbone structures were present in ΔugmA A. oryzae, indicating the presence of fungal-type galactomannan in the cell wall fraction of A. oryzae. The findings of this study provide new insights into the cell wall structure of A. oryzae, which is essential for the production of fermented foods in Japan.

Alkaloids and Leishmania donovani UDP-Galactopyarnosemutase: A Novel Approach in Drug Designing Against Visceral Leishmaniasis.

BACKGROUND: The unsatisfactory treatment options for Visceral Leishmaniasis (VL), need identification of new drug targets. Among natural products, Alkaloids have been proved to be highly effective against number of diseases. In Leishmania, UDP-galactopyranosemutase (UGM) is a critical enzyme required for cell wall synthesis and thus a drug target for structure based drug designing against L. donovani. OBJECTIVE: The aim was to build the homology model of UDP galactopyransemutase and investigate the interaction of selected alkaloids with this modeled UDP galactopyranosemutase by molecular docking. METHODOLOGY: Since no crystal structure record has been found with this protein, a homology modeling was performed and a three dimensional structure of L. donovani UGM was created using MODELLER v9.9, structure quality was validated using PROCHECK and QMEAN programs which confirms that the structure is reliable. Further Molecular docking was performed with previously reported15 alkaloids. RESULTS: It was found that Protopine with a binding energy of -12.39Kcal/mole, binds at Flavin adenine dinucleotide (FAD) biding site. CONCLUSION: It was concluded that Protopine, an alkaloid could interrupt the functional aspect of L. donovani UGM and thus may be useful for drug designing studies. These finding would contribute to the understanding of the effect of drug on the parasite.

Identification of eukaryotic UDP-galactopyranose mutase inhibitors using the ThermoFAD assay.

Aspergillus fumigatus is a human pathogen responsible for deadly infections in immune-compromised patients. A potential strategy for treating A. fumigatus infections is by targeting the biosynthesis of cell wall components, such as galactofuranase, which is absent in humans. Galactofuranose biosynthesis is initiated by the flavoenzyme UDP-galactopyranose mutase (UGM), which converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). UGM requires the reduced form of the flavin for activity, which is obtained by reacting with NADPH. We aimed to identify inhibitors of UGM by screening a kinase inhibitor library using ThermoFAD, a flavin fluorescence thermal shift assay. The screening assay identified flavopiridol as a compound that increased the melting temperature of A. fumigatus UGM. Further characterization showed that flavopiridol is a non-competitive inhibitor of UGM and docking studies suggest that it binds in the active site. This compound does not inhibit the prokaryotic UGM from Mycobacteria tuberculosis.

Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates HUVEC proteins related to inflammatory and stress responses.

Aspergillus fumigatus, the main etiologic agent causing invasive aspergillosis, can induce an inflammatory response and a prothrombotic phenotype upon contact with human umbilical vein endothelial cells (HUVECs). However, the fungal molecules involved in this endothelial response remain unknown. A. fumigatus hyphae produce an extracellular matrix composed of galactomannan, galactosaminogalactan and α-(1,3)-glucan. In this study, we investigated the consequences of UGM1 gene deletion in A. fumigatus, which produces a mutant with increased galactosaminogalactan production. The ∆ugm1 mutant exhibited an HUVEC-hyperadhesive phenotype and induced increased endothelial TNF-α secretion and tissue factor mRNA overexpression in this "semi-professional" immune host cell. Using a shotgun proteomics approach, we show that the A. fumigatus ∆ugm1 strain can modulate the levels of proteins in important endothelial pathways related to the inflammatory response mediated by TNF-α and to stress response pathways. Furthermore, a purified galactosaminogalactan fraction was also able to induce TNF-α secretion and the coincident HUVEC pathways regulated by the ∆ugm1 mutant, which overexpresses this component, as demonstrated by fluorescence microscopy. This work contributes new data regarding endothelial mechanisms in response to A. fumigatus infection. SIGNIFICANCE: Invasive aspergillosis is the main opportunistic fungal infection described in neutropenic hematologic patients. One important clinical aspect of this invasive fungal infection is vascular thrombosis, which could be related, at least in part, to the activation of endothelial cells, as shown in previous reports from our group. It is known that direct contact between the A. fumigatus hyphal cell wall and the HUVEC cell surface is necessary to induce an endothelial prothrombotic phenotype and secretion of pro-inflammatory cytokines, though the cell surface components of this angioinvasive fungus that trigger this endothelial response are unknown. The present work employs a discovery-driven proteomics approach to reveal the role of one important cell wall polysaccharide of A. fumigatus, galactosaminogalactan, in the HUVEC interaction and the consequent mechanisms of endothelial activation. This is the first report of the overall panel of proteins related to the HUVEC response to a specific and purified cell wall component of the angioinvasive fungus A. fumigatus.

Dataset of differentially regulated proteins in HUVECs challenged with wild type and UGM1 mutant Aspergillus fumigatus strains.

Invasive aspergillosis is the primary opportunistic invasive fungal infection described in neutropenic hematologic patients, caused by the angioinvasive pathogen Aspergillus fumigatus. The molecular mechanisms associated with A. fumigatus infection in the vascular endothelium are poorly understood. In this context, we used a high-throughput proteomic approach to unveil the proteins modulated in HUVECs after interaction with a wild type strain and the UGM1 mutant (Δugm1) of A. fumigatus. The proteomic analysis was also performed in HUVECs challenged with a galactosaminogalactan (GAG) purified from A. fumigatus cell wall. The dataset presented here correspond to all proteins identified that fit a 2-fold change criteria (log 2 ratio ≥ 1 or ≤ -1), disregarding the statistical validation cut off, in order to supplement the research article entitled "Modifications to the composition of the hyphal outer layer of Aspergillus fumigatus modulates the HUVEC proteins associated with inflammatory and stress responses" (G.W.P. Neves, N.A. Curty, P.H. Kubitschek-Barreira, T. Fontaine, G.H.M.F. Souza, M. Lyra Cunha, G.H. Goldman, A. Beauvais, J.P. Latgé, L.M. Lopes-Bezerra, 2016) [1]. The mass spectrometry proteomic data have been deposited in the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PRIDE: PXD002823.

Mechanism-based candidate inhibitors of uridine diphosphate galactopyranose mutase (UGM).

Uridine diphosphate-galactopyranose mutase (UGM), an enzyme found in many eukaryotic and prokaryotic human pathogens, catalyzes the interconversion of UDP-galactopyranose (UDP-Galp) and UDP-galactofuranose (UDP-Galf), the latter being used as the biosynthetic precursor of the galactofuranose polymer portion of the mycobacterium cell wall. We report here the synthesis of a sulfonium and selenonium ion with an appended polyhydroxylated side chain. These compounds were designed as transition state mimics of the UGM-catalyzed reaction, where the head groups carrying a permanent positive charge were designed to mimic both the shape and positive charge of the proposed galactopyranosyl cation-like transition state. An HPLC-based UGM inhibition assay indicated that the compounds inhibited about 25% of UGM activity at 500 µM concentration.

Synthesis and biological evaluation of nonionic substrate mimics of UDP-Galp as candidate inhibitors of UDP galactopyranose mutase (UGM).

The synthesis of 1-[5-O-(α-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione and 1-[(5-O-(ß-D-galactopyranosyl)-D-glucityl]pyrimidine-2,4(3H)-dione as non-ionic substrate mimics of UDP-Galp are described. UDP-Galp is a precursor of Galf, which is a primary component of the cell-wall glycans of several microorganisms. The interconversion of UDP-Galp and UDP-Galf is catalyzed by UDP galactopyranose mutase (UGM); its inhibition comprises a mode of compromising the microorganisms. The nonionic polyhydroxylated chain was intended to mimic the ionic pyrophosphate group and the ribose moiety in UDP-Galp and increase the bioavailabilities of the candidate inhibitors. Inhibition assays with UGM of Mycobacterium tuberculosis showed only weak inhibition of the enzyme by these compounds.

Combined molecular dynamics, STD-NMR, and CORCEMA protocol yields structural model for a UDP-galactopyranose mutase-inhibitor complex.

UDP-galactopyranose mutase (UGM) is an enzyme involved in the biosynthesis of the Mycobacterium tuberculosis cell wall, and is essential for the growth and survival of the organism. A micromolar inhibitor developed by tetrafluorination of the UGM substrate has been previously studied by saturation transfer difference (STD) NMR spectroscopy. To elucidate the bioactive conformation of the inhibitor bound to UGM, we employ molecular dynamics (MD) simulations to construct a structural model. The MD model is subsequently validated by a good fit between experimental and theoretical STD effects, the latter calculated by a complete relaxation and conformational exchange matrix (CORCEMA) analysis. This structural model is used to explain the relative binding affinities of the inhibitor and the parent substrate.

Unconventional P-35S sequence identified in genetically modified maize.

The Cauliflower Mosaic Virus 35S promoter sequence, CaMV P-35S, is one of several commonly used genetic targets to detect genetically modified maize and is found in most GMOs. In this research we report the finding of an alternative P-35S sequence and its incidence in GM maize marketed in Jordan. The primer pair normally used to amplify a 123 bp DNA fragment of the CaMV P-35S promoter in GMOs also amplified a previously undetected alternative sequence of CaMV P-35S in GM maize samples which we term V3. The amplified V3 sequence comprises 386 base pairs and was not found in the standard wild-type maize, MON810 and MON 863 GM maize. The identified GM maize samples carrying the V3 sequence were found free of CaMV when compared with CaMV infected brown mustard sample. The data of sequence alignment analysis of the V3 genetic element showed 90% similarity with the matching P-35S sequence of the cauliflower mosaic virus isolate CabbB-JI and 99% similarity with matching P-35S sequences found in several binary plant vectors, of which the binary vector locus JQ693018 is one example. The current study showed an increase of 44% in the incidence of the identified 386 bp sequence in GM maize sold in Jordan's markets during the period 2009 and 2012.

Substrate-dependent dynamics of UDP-galactopyranose mutase: Implications for drug design.

Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that represents one of the major health challenges of the Latin American countries. Successful efforts were made during the last few decades to control the transmission of this disease, but there is still no treatment for the 10 million adults in the chronic phase of the disease. In T. cruzi, as well as in other pathogens, the flavoenzyme UDP-galactopyranose mutase (UGM) catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, a precursor of the cell surface ß-galactofuranose that is involved in the virulence of the pathogen. The fact that UGM is not present in humans makes inhibition of this enzyme a good approach in the design of new Chagas therapeutics. By performing a series of computer simulations of T. cruzi UGM in the presence or absence of an active site ligand, we address the molecular details of the mechanism that controls the uptake and retention of the substrate. The simulations suggest a modular mechanism in which each moiety of the substrate controls the flexibility of a different protein loop. Furthermore, the calculations indicate that interactions with the substrate diphosphate moiety are especially important for stabilizing the closed active site. This hypothesis is supported with kinetics measurements of site-directed mutants of T. cruzi UGM. Our results extend our knowledge of UGM dynamics and offer new alternatives for the prospective design of drugs.

Androgen receptors in hormone-dependent and castration-resistant prostate cancer.

In the United States, prostate cancer (PCa) is the most commonly diagnosed non-cutaneous cancer in males and the second leading cause of cancer-related death for men. The prostate is an androgen-dependent organ and PCa is an androgen-dependent disease. Androgen action is mediated by the androgen receptor (AR), a hormone activated transcription factor. The primary treatment for metastatic PCa is androgen deprivation therapy (ADT). For the most part, tumors respond to ADT, but most become resistant to therapy within two years. There is persuasive evidence that castration resistant (also termed castration recurrent) PCa (CRPC) remains AR dependent. Recent studies have shown that there are numerous factors that contribute to AR reactivation despite castrate serum levels of androgens. These include changes in AR expression and structure through gene amplification, mutation, and alternative splicing. Changes in steroid metabolism, cell signaling, and coregulator proteins are also important contributors to AR reactivation in CRPC. Most AR targeted therapies have been directed at the hormone binding domain. The finding that constitutively active AR splice variants that lack the hormone binding domain are frequently expressed in CRPC highlights the need to develop therapies that target other portions of AR. In this review, the role of AR in normal prostate, in PCa, and particularly the mechanisms for its reactivation subsequent to ADT are summarized. In addition, recent clinical trials and novel approaches to target AR are discussed.

Estudo proteômico da interação do Aspergillus fumigatus com células endoteliais da veia umbilical humana (HUVECs) / Proteomic study of the interaction of Aspergillus fumigatus with human umbilical vein endothelial cells (HUVECs)

O Aspergillus fumigatus é o principal agente etiológico da aspergilose invasiva, uma infecção fúngica oportunista que acomete, principalmente, pacientes de Unidades Hematológicas, como aqueles com neutropenia profunda e prolongada. Após a filamentação este fungo angioinvasivo é capaz de ativar e causar danos em células endoteliais de veia umbilical humana (HUVEC) que passam a expressar um fenótipo pró-trombótico. A ativação destas células, dependente de contato célulacélula, é mediada por TNF-α e caracterizada pela expressão de moléculas próinflamatórias, como citocinas, quimiocinas e moléculas de adesão. Recentemente, nosso grupo comparou a ativação endotelial de HUVECs desafiadas com cepas selvagens e uma cepa mutante para o gene UGM1. Nestes experimentos a cepa mutante Δugm1, que apresenta um fenótipo de maior produção de galactosaminogalactana (GAG) na parede celular, mostrou um fenótipo hiperadesivo e uma capacidade maior de ativar células endoteliais. Entretanto, os receptores e as vias de sinalização envolvidos nesta ativação permanecem desconhecidos. Assim, o objetivo deste trabalho foi verificar as proteínas envolvidas nestes processos através do estudo das proteínas diferencialmente expressas nas HUVECs após a interação com A. fumigatus, usando a técnica proteômica 2D-DIGE. Brevemente, as HUVECs foram infectadas com tubos germinativos da cepa selvagem (AF293) e da cepa Δugm1 de A. fumigatus. Em seguida, as proteínas foram marcadas com diferentes fluorocromos e separadas por eletroforese bidimensional. A análise quantitativa foi realizada utilizando o software DeCyder...

Aspergillus fumigatus is the main etiological agent of invasive aspergillosis, the main opportunistic fungal infection of Hematologial Unity’s patients, especially those with long-term neutropenia. Upon filamentation, this angioinvasive fungus can activate and damage the human umbilical vein endothelial cells (HUVEC), which in response switch to a pro-thrombotic phenotype. HUVEC activation is mediated by TNF-α once cell-cell contact occurs. This activation is characterized by the expression of pro-inflammatory molecules such cytokines, chemokines and adhesion molecules. Recently, our group performed the comparison of HUVEC activation upon interaction with a wild type and the UGM1 mutant strains of A. fumigatus. The Δugm1 strain, which presents an increased production of the cell wall galactosaminogalactan, showed a hyper adherent phenotype and an increased capability to cause endothelial cell stimulation and activation, when compared with the wild type strain. The receptors involved in the pathogen-host interaction or the signaling pathways after endothelial activation by A. fumigatus remain unknown. Thus, the aim of this study was to investigate the differentially expressed proteins in HUVECs upon interaction with A. fumigatus, using the 2D-DIGE proteomic approach. Briefly, HUVECs were challenged with germlings of A. fumigatus wild type Af293 and Δugm1 strains and then submitted to protein extraction. The total HUVEC protein extracts were labeled with different CyDyes and fractionated by 2D electrophoresis. Quantitative analysis to determine the differences in protein abundance amongst interacted cells vs. control endothelial cells was performed using the software DeCyder. Five differentially expressed proteins were identified by MS/MS including galectin-1 and annexin A2, both overexpressed after the interaction. These two proteins are described elsewhere to be associated with host-pathogen interaction...

Establishment and charaterization of normal and transformed fetal rat urogenital sinus mesenchymal cells: a comparative study / 대한비뇨기과학회지

Experiment on fetal urogenital sinus mesenchyme is invaluable to investigate the role of which stromal-epithelial interaction plays in the proliferation, differentiation and hormonal dependence of normal epithelial tissue as well as epithelial tumors. Normal and transformed embryonic fibroblast may have different effect on epithelial tumors. We have recently established normal (NUGM) and spontaneously transformed fetal rat urogenital sinus mesenchymal cell (TUGM) lines and herein present comparative characteristics of these cell lines including cytokinetic study, chromosomal study, effect of sex hormones on the growth, immunohistochemical study for intermediate filaments and in vivo tumorigenicity. TUGM has cellular characteristics of transformed fibroblast and shows increased expression of cell surface fibronectin. Addition of different concentration of serum and various sex hormones did not affect the growth of TUGM significantly, whereas in TUGM, The growth was significantly enhanced by increase of serum concentration in medium and dihyrotestosterone at the concentration of 100 ng/ml of medium. NUGM does not show any tumorigenicity in vivo. TUGM shows strong tumorigenicity (19/21) and forms fibrosarcoma when inoculated into nude mouse. The size of tumor was significantly smaller when inoculated into castrated male and female mice compared to non castrated males. In conclusion, NUGM and TUGM have different cellular characteristics and hormonal dependence. NUGM and TUGM may be help to elucidate the relative role of normal and transformed embryonic fibroblast in the mechanism of evolution, invasion and metastasis of epithelial cancer.