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Balancing peptidoglycan (PG) synthesis and degradation with precision is essential for bacterial growth, yet our comprehension of this intricate process remains limited. The NlpI-Prc proteolytic complex plays a crucial but poorly understood role in the regulation of multiple enzymes involved in PG metabolism. In this paper, through fluorescent D-amino acid 7-hydroxycoumarincarbonylamino-D-alanine (HADA) labeling and immunolabeling assays, we have demonstrated that the NlpI-Prc complex regulates the activity of PG transpeptidases and subcellular localization of PBP3 under certain growth conditions. PBP7 (a PG hydrolase) and MltD (a lytic transglycosylase) were confirmed to be negatively regulated by the NlpI-Prc complex by an in vivo degradation assay. The endopeptidases, MepS, MepM, and MepH, have consistently been demonstrated as redundantly essential "space makers" for nascent PG insertion. However, we observed that the absence of NlpI-Prc complex can alleviate the lethality of the mepS mepM mepH mutant. A function of PG lytic transglycosylases MltA and MltD as "space makers" was proposed through multiple gene deletions. These findings unveil novel roles for NlpI-Prc in the regulation of both PG synthesis and degradation, shedding light on the previously undiscovered function of lytic transglycosylases as "space makers" in PG expansion. [ABSTRACT FROM AUTHOR]

The endocannabinoid system (ECS) is considered to be an endogenous protective system in various neurodegenerative diseases. Niemann-Pick type C (NPC) is a neurodegenerative disease in which the role of the ECS has not been studied yet. Most of the endocannabinoid enzymes are serine hydrolases, which can be studied using activity-based protein profiling (ABPP). Here, we report the serine hydrolase activity in brain proteomes of a NPC mouse model as measured by ABPP. Two ABPP methods are used: a gel-based method and a chemical proteomics method. The activities of the following endocannabinoid enzymes were quantified: diacylglycerol lipase (DAGL) α, α/β-hydrolase domain-containing protein 4, α/β-hydrolase domain-containing protein 6, α/β-hydrolase domain-containing protein 12, fatty acid amide hydrolase, and monoacylglycerol lipase. Using the gel-based method, two bands were observed for DAGL α. Only the upper band corresponding to this enzyme was significantly decreased in the NPC mouse model. Chemical proteomics showed that three lysosomal serine hydrolase activities (retinoid-inducible serine carboxypeptidase, cathepsin A, and palmitoyl-protein thioesterase 1) were increased in Niemann-Pick C1 protein knockout mouse brain compared to wild-type brain, whereas no difference in endocannabinoid hydrolase activity was observed. We conclude that these targets might be interesting therapeutic targets for future validation studies. [ABSTRACT FROM AUTHOR]

Background. To date, 4 classes of histone deacetylases (HDACs) have been identified in humans. Class I HDACs are zinc-dependent and NAD+-independent enzymes, and include 4 isoforms closely related to yeast RPD3: HDAC1, 2, 3, and 8. Objectives. The aims of the study were to quantitatively evaluate the expression of HDAC3 in colorectal cancer (CRC) and to correlate its expression levels with clinicopathological parameters. Material and methods. We characterized expression patterns of HDAC3 as class I HDAC isoforms in a cohort of 48 CRC patients by quantitative (real-time) reverse transcription polymerase chain reaction (RT-PCR). In addition, the potential relationship between HDAC3 expression levels and clinicopathological parameters in patients suffering from CRC was explored. Results. We found that HDAC3 was highly expressed in colorectal tumors compared to normal colorectal tissues (p < 0.05). Furthermore, we found significant correlations between HDAC3 expression levels and tumor differentiation grades (p < 0.05). Conclusions. In this prospective study we identified a pronounced HDAC3 expression pattern in CRC. Our findings support an important role of HDAC3 as a complementary molecular marker for existing histopathological diagnostic elements; it might also have applications in prognostic and targeted therapy. Furthermore, HDAC3 can be used as a biomarker to differentiate between tumor borders and margins, and it may also be useful for characterizing field cancerization in CRC. [ABSTRACT FROM AUTHOR]

Cellobiohydrolases hydrolyze cellulose, a linear polymer with glucose monomers linked exclusively by β‐1,4 glycosidic linkages. The widespread hydrogen bonding network tethers individual cellulose polymers forming crystalline cellulose, which prevent the access of hydrolytic enzymes and water molecules. The most abundant enzyme secreted by Myceliophthora thermophila M77 in response to the presence of biomass is the cellobiohydrolase MtCel7A, which is composed by a GH7‐catalytic domain (CD), a linker, and a CBM1‐type carbohydrate‐binding module. GH7 cellobiohydrolases have been studied before, and structural models have been proposed. However, currently available GH7 crystal structures only define separate catalytic domains and/or cellulose‐binding modules and do not include the full‐length structures that are involved in shaping the catalytic mode of operation. In this study, we determined the 3D structure of catalytic domain using X‐ray crystallography and retrieved the full‐length enzyme envelope via small‐angle X‐ray scattering (SAXS) technique. The SAXS data reveal a tadpole‐like molecular shape with a rigid linker connecting the CD and CBM. Our biochemical studies show that MtCel7A has higher catalytic efficiency and thermostability as well as lower processivity when compared to the well‐studied TrCel7A from Trichoderma reesei. Based on a comparison of the crystallographic structures of CDs and their molecular dynamic simulations, we demonstrate that MtCel7A has considerably higher flexibility than TrCel7A. In particular, loops that cover the active site are more flexible and undergo higher conformational fluctuations, which might account for decreased processivity and enhanced enzymatic efficiency. Our statistical coupling analysis suggests co‐evolution of amino acid clusters comprising the catalytic site of MtCel7A, which correlate with the steps in the catalytic cycle of the enzyme. Database: The atomic coordinates and structural factors of MtCel7A have been deposited in the Protein Data Bank with accession number 5W11. [ABSTRACT FROM AUTHOR]

To elucidate molecular mechanisms responsible for the sexually dimorphic phenotype of soluble epoxide hydrolase (sEH) expression, we tested the hypothesis that female-specific down-regulation of sEH expression is driven by estrogen-dependent methylation of the Ephx2 gene. Mesenteric arteries isolated from male, female, ovariec- tomized female (OV), and OV with estrogen replacement (OVE) mice, as well as the human cell line (HEK293T) were used. Methylation-specific PCR and bisulfite genomic sequencing analysis indicate significant increases in DNA/CG methylation in vessels of female and OVE compared with those of male and OV mice. The same increase in CG methylation was also observed in male vessels incubated with a physiological concentration of 17β-estradiol (17β-E2) for 48 hours. All vessels that displayed increases in CG methylation were concomitantly associated with decreases in their Ephx2 mRNA and protein, suggesting a methylation-induced gene silencing. Transient transfection assays indicate that the activity of Ephx2 promoter-coding luciferase was significantly attenuated in HEK293T cells treated with 17β-E2, which was prevented by additional treatment with an estrogen receptor antagonist (ICI). ChIP analysis indicates significantly reduced binding activities of transcription factors (including SP1, AP-1, and NF-κB with their binding elements located in the Ephx2 promoter) in vessels of female mice and human cells treated with 17β-E2, responses that were prevented by ICI and Decitabine (DNA methyltransferase inhibitor), respectively. In conclusion, estrogen/estrogen receptor-dependent methylation of the promoter of Ephx2 gene silences sEH expression, which is involved in specific transcription factor-directed regulatory pathways. [ABSTRACT FROM AUTHOR]

The morphological alteration of lysosomes is a powerful indicator of various pathological disorders. In this regard, we have designed and synthesized a new water soluble fluorescent Schiff-base ligand (L-lyso) containing two hydroxyl groups. L-lyso exhibits excellent two-photon properties with tracking of lysosomes in live cells as well as in 3D tumor spheroids. Furthermore, it can label lysosomes for more than 3 days. Thus, L-lyso has an edge over the commercially available expensive LysoTracker probes and also over other reported probes in terms of its long-term imaging, water solubility and facile synthesis. [ABSTRACT FROM AUTHOR]

A new series of 3,6-disubstituted 2-(methylthio)-4-(trifluoromethyl)-3,4-dihydropyrimidin-4-ols displaying methyl, phenyl, aryl, and heteroaryl groups at the 6-position; and methyl, ethyl, allyl, and phenyl groups at the 3-position of the dihydropyrimidine ring, were synthesized and evaluated in vitro for acetylcholinesterase inhibitory activity. Seven compounds showed activity with IC50 values in the lower micromolar range. The compound 4-trifluoromethyl-6-(4-fluorophenyl)-3-methyl-2-methylthio-3,4-dihydropyrimidin-4-ol ( 6e) had the best inhibitory activity ( IC50 2.2 ± 0.9 μ m) and this inhibition was characterized as competitive. The molecular docking study showed that the acetylcholinesterase enzyme accommodates compound 6e in its catalytic site. The enantiomers of compound 6e, present similar interactions: π-π stacking interactions between the aromatic ring of the ligand's 4-fluorophenyl moiety and the aromatic rings of the electron-rich Trp84; and H-bonds between the hydroxyl group of Tyr121 and the hydroxyl moiety from 6e. The antioxidant effect of the dihydropyrimidin-4-ols was also investigated. [ABSTRACT FROM AUTHOR]

Background: There is increasing evidence in support of the role of lncRNAs in tumor cell proliferation, differentiation and apoptosis. Methods: We examined the expression of the lncRNA ABHD11-AS in epithelial ovarian cancer (EOC) tissues and normal ovarian tissues by real-time quantitative PCR (qRT-PCR). After inducing ABHD11-AS downregulation by small interfering RNA (siRNA) or ABHD11-AS overexpression by plasmid transfection, we examined the EOC cell phenotypes and expression of related molecules. Results: Expression of the lncRNA ABHD11-AS in EOC tissues was higher than that in normal ovarian tissue. It was positively associated with the tumor stage (stage I/II vs. stage III/IV), and it was lower in the well-differentiated group than in the poorly/moderately differentiated group. Overexpression of ABHD11-AS in the ovarian cancer cell lines A2780 and OVCAR3 promoted ovarian cancer cell proliferation, invasion and migration, and inhibited apoptosis. Silencing of ABHD11-AS had the opposite effect. Subcutaneous injection of tumor cells in nude mice showed that ABHD11-AS could significantly promote tumor growth. In addition, intraperitoneal injection of tumor cells in the nude mice resulted in an increase in the metastatic ability of the tumor. Further, overexpression of ABHD11-AS upregulated the expression of RhoC and its downstream molecules P70s6k, MMP2 and BCL-xL. Silencing of ABHD11-AS had the opposite effect. The RNA pull-down assay showed that ABHD11-AS can combine directly with RhoC. Silencing of RhoC was found to inhibit the cancer-promoting effects of lncRNA ABHD11-AS. Thus, it seems that RhoC is a major target of the lncRNA ABHD11-AS. Conclusions: This is the first study to demonstrate the role of RhoC in the tumor-promoting effects of the lncRNA ABHD11-AS. The present findings shed light on new therapeutic targets for ovarian cancer treatment. [ABSTRACT FROM AUTHOR]

γ-glutamyl-hydrolase (GGH) is a ubiquitously-expressed enzyme that regulates intracellular folate metabolism for cell proliferation, DNA synthesis, and repair. Employing GGH immunohistochemistry on a tissue microarray with 12,427 prostate cancers, we found that GGH expression was negative to low in normal prostate epithelium, whereas 88.3% of our 10,562 interpretable cancers showed GGH expression. GGH staining was considered as low intensity in 49.6% and as high intensity in 38.6% of cancers. High GGH expression was linked to the TMPRSS2:ERG-fusion positive subset of cancers (p < 0.0001), advanced pathological tumor stage, and high Gleason grade (p < 0.0001 each). Further analysis revealed that these associations were merely driven by the subset of ERG-negative cancers, High GGH expression was weakly linked to early biochemical recurrence in ERG negative cancers (p < 0.0001) and independent from established histo-pathological parameters. Moreover, GGH expression was linked to features of genetic instability, including presence of recurrent deletions at 3p, 5q, 6q, and 10q (PTEN, p ≥ 0.01 each), as well as to accelerated cell proliferation as measured by Ki67 immunohistochemistry (p < 0.0001). In conclusion, the results of our study identify GGH as an ERG subtype specific molecular marker with modest prognostic relevance, which may have clinical relevance if analyzed in combination with other molecular markers. [ABSTRACT FROM AUTHOR]

Dendrogenin A (DDA) is the first steroidal alkaloid (SA) to be identified in human tissues to date and arises from the stereoselective enzymatic conjugation of 5,6α-epoxycholesterol (5,6α-EC) with histamine (HA). DDA induces the re-differentiation of cancer cells in vitro and in vivo and prevents breast cancer (BC) and melanoma development in mice, evidencing its protective role against oncogenesis. In addition, DDA production is lower in BCs compared with normal tissues, suggesting a deregulation of its biosynthesis during carcinogenesis. The discovery of DDA reveals the existence of a new metabolic pathway in mammals which lies at the crossroads of cholesterol and HA metabolism and which leads to the production of this metabolic tumour suppressor. [ABSTRACT FROM AUTHOR]

BACKGROUND This study analyzed the effects of chronic administration of N[omega]-hydroxy-nor-l-arginine (nor-NOHA), an inhibitor of arginase, on the hemodynamic, oxidative stress, morphologic, metabolic, and renal manifestations of hyperthyroidism in rats. METHODS Four groups of male Wistar rats were used: control, nor-NOHA-treated (10 mg/kg/day), thyroxine (T4)-treated (75 μg/rat/day), and thyroxine-plus nor-NOHA-treated rats. All treatments were maintained for 4 weeks. Body weight, tail systolic blood pressure (SBP), and heart rate (HR) were recorded weekly. Finally, morphologic, metabolic, plasma, and renal variables were measured. Arginase I and II protein abundance and arginase activity were measured in aorta, heart, and kidney. RESULTS The T4 group showed increased arginase I and II protein abundance, arginase activity, SBP, HR, plasma nitrates/nitrites (NOx), brainstem and urinary isoprostanes, proteinuria and cardiac and renal hypertrophy in comparison to control rats. In hyperthyroid rats, chronic nor-NOHA prevented the increase in SBP and HR and decreased proteinuria in association with an increase in plasma NOx and a decrease in brainstem and urinary isoprostanes. In normal rats, nor-NOHA treatment did not significantly change any hemodynamic, morphologic, or renal variables. Acute nor-NOHA administration did not affect renal or systemic hemodynamic variables in normal or T4-treated rats. CONCLUSION Hyperthyroidism in rats is associated with the increased expression and activity of arginase in aorta, heart, and kidney. Chronic arginase inhibition with nor-NOHA suppresses the characteristic hemodynamic manifestations of hyperthyroidism in association with a reduced oxidative stress. These results indicate an important role for arginase pathway alterations in the cardiovascular and renal abnormalities of hyperthyroidism. [ABSTRACT FROM AUTHOR]

Coprinopsis cinerea is a model species for molecular genetics studies of sexual development in agaricomycetes or homobasidiomycetes. Recently, efficient gene targeting was established in this fungus by generating Cc.ku70 or Cc.lig4 disruptants. To determine the molecular mechanisms underlying sexual development, which involves many genes, generating multiple gene disruptants is required. However, the number of transformation markers available for C. cinerea is limited. This problem would be solved by establishing marker recycling. In this study, we found that C. cinerea lacks a gene encoding a homolog of Saccharomyces cerevisiae cytosine deaminase (Fcy1p) in its genome, which is present in many other fungi. We also observed that C. cinerea is resistant to 5-fluorocytosine. Based on these findings, we established a simple marker recycling method in this fungus using 5-fluorocytosine counterselection after heterologous expression of FCY1 derived from Pleurotus ostreatus, together with the hygromycin resistance gene. This study proposes a simple genetic manipulation system that can be performed using wild-type strains of several fungi that lack a gene homologous to S. cerevisiae FCY1 in their genomes. [ABSTRACT FROM AUTHOR]

The filamentous bacteria Streptomyces spp. produces diverse extracellular enzymes and other secondary metabolites. Proteomic analysis of the secretome of holocellulolytic Streptomyces sp. ssr-198 was done by tandem mass spectrometry using an Orbitrap Velos hybrid mass spectrometer. A wide range of hydrolytic enzymes, including glycoside hydrolases (17), proteases (17), polysaccharide lyases (3), esterases (2), and hypothetical proteins (14) were detected in the secretome analyzed. Overall, the secretome composition constituted of 12.50% cellulases, 17.50% hemicellulases, 21.25% proteases, 17.50% hypothetical proteins, and 31.25% other proteins. Comprehensive analysis of secretome will be useful in gaining better understanding of the unique role of hydrolytic enzymes in lignocellulose hydrolysis and helps in determining the industrial applications of these potent enzymes. [ABSTRACT FROM AUTHOR]

Background In susceptible individuals, exposure to Aspergillus fumigatus can lead to the development of atopic lung diseases such as allergic bronchopulmonary aspergillosis ( ABPA) and severe asthma with fungal sensitization ( SAFS). Protease allergens including Asp f 5 and Asp f 13 from Aspergillus fumigatus are thought to be important for initiation and progression of allergic asthma. Objective To assess the importance of secreted protease allergens Asp f 5 (matrix metalloprotease) and Asp f 13 (serine protease) in Aspergillus fumigatus-induced inflammation, airway hyperactivity, atopy and airway wall remodelling in a murine model following chronic exposure to secreted allergens. Methods BALB/c mice were repeatedly intranasally dosed over the course of 5 weeks with culture filtrate from wild-type ( WT), Asp f 5 null (∆5) or Asp f 13 null (∆13) strains of Aspergillus fumigatus. Airway hyper-reactivity was measured by non-invasive whole-body plethysmography, Th2 response and airway inflammation by ELISA and cell counts, whilst airway remodelling was assessed by histological analysis. Results Parent WT and ∆5 culture filtrates showed high protease activity, whilst protease activity in ∆13 culture filtrate was low. Chronic intranasal exposure to the three different filtrates led to comparable airway hyper-reactivity and Th2 response. However, protease allergen deleted strains, in particular ∆13 culture filtrate, induced significantly less airway inflammation and remodelling compared to WT culture filtrate. Conclusion Aspergillus fumigatus-secreted allergen proteases, Asp f 5 and Asp f 13, are important for recruitment of inflammatory cells and remodelling of the airways in this murine model. However, deletion of a single allergen protease fails to alleviate airway hyper-reactivity and allergic immune response. Targeting protease activity of Aspergillus fumigatus in conditions such as SAFS or ABPA may have beneficial effects in preventing key aspects of airway pathology. [ABSTRACT FROM AUTHOR]

Background Nudix hydrolases (Nd) is a widespread superfamily, which is found in all classes of organism, hydrolyse a wide range of organic pyrophosphates and has a „housecleaning‟ function. The previous study showed that Trichinella spiralis Nd (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with T7 phage-displayed TsNd polypeptides produced protective immunity. The aim of this study was to clone, express and identify the full-length TsNd and to investigate its immune protection against T. spiralis infection. Methods he full-length cDNA sequence of TsNd gene encoding a 46 kDa protein from T. spiralis intestinal infective larvae (IIL) was cloned and identified. The antigenicity of recombinant TsNd protein (rTsNd) was analyzed by Western blot. Transcription and expression of TsNd at T. spiralis different developmental stages were observed by RT-PCR and Immunofluorescence test (IFT). The levels of the specific total IgG, IgG1 and IgG2a antibodies to rTsNd were determined by ELISA. The immune protection of rTsNd against T. spiralis infection in BALB/c mice was investigated. Results Sequence and phylogenetic analysis revealed that TsNd had a nudix motif located at 226- 244aa, which had high homology and the closest evolutionary status with T. pseudospiralis. An 89 kDa fusion protein, the cDNA clone (46 kDa) and MBP tag (43 kDa), was obtained after expression and purification. Western blot analysis showed that anti-rTsNd serum recognized the native TsNd protein with 46 kDa in crude antigens of muscle larvae (ML), IIL, adult worms (AW) and newborn larvae (NBL), and ES antigens of ML. Transcription and expression of TsNd gene was observed in all different developmental stages of T. spiralis (ML, IIL, AW and NBL), with high level expression in ILL. An immunolocalization analysis identified TsNd in the cuticle, stichocytes and reproductive organs of the parasite. Following immunization, anti-rTsNd IgG levels were increased, and the levels of IgG1 were more significantly higher than that of IgG2a. After a challenge infection with T. spiralis, mice immunized with the rTsNd displayed a 57.7% reduction in adult worms and a 56.9% reduction in muscle larval burden. Conclusions TsNd induced a partial protective immunity in mice and could be considered as a novel candidate vaccine antigen against trichinellosis. [ABSTRACT FROM AUTHOR]

The pathophysiological events that lead to renal interstitial fibrogenesis are incompletely understood. Epoxyeicosatrienoic acid (EET), an arachidonic acid metabolite, has anti-inflammatory and profibrinolytic functions. Soluble epoxide hydrolase (sEH) converts EET to less active dihydroxyeicosatrienoic acid. Here, we tested the hypothesis that sEH deficiency would prevent tubulointerstitial fibrosis and inflammation induced by unilateral ureteral obstruction (UUO) in mouse kidneys. The loss of sEH enhanced levels of EET regioisomers and abolished tubulointerstitial fibrosis as demonstrated by reduced collagen deposition and myofibroblast formation at 3 and 10 days after UUO. The inflammatory response was prevented as demonstrated by decreased influx of neutrophil and macrophage, expression of inflammatory cytokines, and chemotactic factors in sEH-deficient UUO kidneys. Pharmacological inhibition of sEH also prevented inflammation and fibrosis after UUO. Next, we delved into the molecular mechanisms piloting the beneficial effects of sEH deficiency in renal fibrosis. UUO upregulated profibrotic factors associated with transforming growth factor (TGF)-1/Smad3 signaling, oxidative stress, and NF-B activation, and downregulated antifibrotic factors including peroxisome proliferator-activated receptor (PPAR) isoforms, especially PPAR, but the loss of sEH prevented these adverse effects in UUO kidneys. Furthermore, administration of PPAR antagonists enhanced myofibroblast formation and activation of Smad3 and NF-B p65, effects that were prevented by sEH deficiency in UUO kidneys. These data demonstrate that loss of sEH promotes anti-inflammatory and fibroprotective effects in UUO kidneys via activation of PPAR isoforms and downregulation of NF-B, TGF-1/Smad3, and inflammatory signaling pathways. Our data suggest the potential use of sEH inhibitors in treating fibrotic diseases. [ABSTRACT FROM AUTHOR]

Colonization of the land by plants required major modifications in cellular structural composition and metabolism. Intercellular communication through plasmodesmata (PD) plays a critical role in the coordination of growth and cell activities. Changes in the form, regulation or function of these channels are likely linked to plant adaptation to the terrestrial environments. Constriction of PD aperture by deposition of callose is the best-studied mechanism in PD regulation. Glycosyl hydrolases family 17 (GHL17) are callose degrading enzymes. In Arabidopsis this is a large protein family, few of which have been PD-localized. The objective here is to identify correlations between evolution of this protein family and their role at PD and to use this information as a tool to predict the localization of candidates isolated in a proteomic screen. With this aim, we studied phylogenetic relationship between Arabidopsis GHL17 sequences and those isolated from fungi, green algae, mosses and monocot representatives. Three distinct phylogenetic clades were identified. Clade alpha contained only embryophytes sequences suggesting that this subgroup appeared during land colonization in organisms with functional PD. Accordingly, all PD-associated GHL17 proteins identified so far in Arabidopsis thaliana and Populus are grouped in this 'embryophytes only' phylogenetic clade. Next, we tested the use of this knowledge to discriminate between candidates isolated in the PD proteome. Transient and stable expression of GFP protein fusions confirmed PD localization for candidates contained in clade alpha but not for candidates contained in clade beta. Our results suggest that GHL17 membrane proteins contained in the alpha clade evolved and expanded during land colonization to play new roles, among others, in PD regulation. [ABSTRACT FROM AUTHOR]

Background Zearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus. Results We screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro). In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models. Conclusions Our research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. 'multigermtubi'). [ABSTRACT FROM AUTHOR]

The allophanate hydrolase from Pseudomonas sp. strain ADP was expressed and purified, and a tryptic digest fragment was subsequently identified, expressed and purified. This 50 kDa construct retained amidase activity and was crystallized. The crystals diffracted to 2.5 Å resolution and adopted space group P21, with unit-cell parameters a = 82.4, b = 179.2, c = 112.6 Å, β = 106.6°. [ABSTRACT FROM AUTHOR]

Adipogenesis is a tightly regulated cellular process that involves the action of multiple signaling pathways. Characterization of regulators that are associated with adipose development is crucial to understanding the mechanisms underlying obesity and other metabolic disorders. Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein that was first described as a neurotrophic factor. The role of PEDF in lipid metabolism was established when adipose triglyceride lipase (ATGL), a major triglyceride hydrolase, was characterized as its binding partner. In this study, we investigated the downstream effects of PEDF on adipogenic differentiation using rat adipose-derived stem cells (AdSCs) and the mouse pre-adipocyte cell line 3T3-L1. Knocking down PEDF in differentiating cells resulted in elevated levels of ATGL and CD36, as well as other adipogenic markers, with a concomitant increase in adipocyte number. CD36, a scavenger receptor for a variety of ligands, regulated proliferation and lipogenic gene expression during adipogenesis. The CD36 increase due to PEDF down-regulation might be a result of elevated PPARγ. We further demonstrated that PEDF expression was regulated by dexamethasone, a synthetic glucocorticoid that is widely used for adipogenesis at the transcriptional level. Taken together, our findings highlight that PEDF negatively regulates adipogenesis through the regulation of various signaling intermediates, and it may play a crucial role in lipid metabolic disorders. [ABSTRACT FROM AUTHOR]

Background: Deep eutectic solvents have attracted considerable attention in numerous fields. There is little information on mung bean epoxide hydrolase-catalyzed epoxides in deep eutectic solvent-containing system.Results: Adding deep eutectic solvents with hydrogen bond donor of acids to phosphate buffer resulted in an obvious decrease in the optical purity of product; nevertheless, the relative slight change was observed by the addition of the deep eutectic solvents with hydrogen bond donor of alcohols and urea. Of the tested deep eutectic solvents, 10% additional amount of choline chloride/trithylene glycol can cause a significant improvement in the enantiopurity of product, from 83.2 ± 1.3% to 87.9 ± 0.3%. Moreover, with the increase in the addition amount of choline chloride/triethylene glycol from 10 to 30%, the enantiomeric excess of product enhanced from 87.9 to 94%, but a decline of product yield was observed. Finally, the evaluation of enzyme stability showed that the additional amount from 10% to 30% was not beneficial to the activity recovery.Conclusion: In short, adding choline chloride/triethylene glycol contributes to the improvement in the optical purity of (R)-1-phenyl-1, 2-ethanediol in the catalysis of styrene oxide by mung bean epoxide hydrolases; meanwhile, the enzyme immobilization could be essential. [ABSTRACT FROM AUTHOR]