scholarly journals Discovery and functional analysis of a salicylic acid hydroxylase from Aspergillus niger

2021 ◽  
Author(s):  
Ronnie J.M. Lubbers ◽  
Adiphol Dilokpimol ◽  
Jaap Visser ◽  
Kristiina S. Hildén ◽  
Miia R. Mäkelä ◽  
...  
Keyword(s):  
Salicylic Acid ◽  
Aspergillus Niger ◽  
Metabolic Pathways ◽  
Biochemical Characterization ◽  
Plant Biomass ◽  
Pathogenic Fungi ◽  
Alternative Methods ◽  
Muconic Acid

Salicylic acid plays an important role in the plant immune response and its degradation is therefore important for plant pathogenic fungi. However, many non-pathogenic microorganisms can also degrade salicylic acid. In the filamentous fungi Aspergillus niger, two salicylic acid metabolic pathways have been suggested. The first pathway converts salicylic acid to catechol by a salicylate hydroxylase (ShyA). In the second pathway, salicylic acid is 3-hydroxylated to 2,3-dihydroxybenzoic acid followed by the decarboxylation to catechol by 2,3-dihydroxybenzoate decarboxylase (DhbA). A. niger cleaves the aromatic ring of catechol catalyzed by catechol 1,2-dioxygenase (CrcA) to form cis,cis-muconic acid. However, the identification and role of the genes and characterization of the enzymes involved in these pathways are lacking. In this study, we used transcriptome data of A. niger grown on salicylic acid to identify genes (shyA and crcA) involved in salicylic acid metabolism. Heterologous production in Escherichia coli followed by biochemical characterization confirmed the function of ShyA and CrcA. The combination of ShyA and CrcA demonstrated that cis,cis-muconic acid can be produced from salicylic acid. In addition, the in vivo roles of shyA, dhbA and crcA were studied by creating A. niger deletion mutants which revealed the role of these genes in the fungal metabolism of salicylic acid. Importance Nonrenewable petroleum sources are depleting and therefore alternative sources are needed. Plant biomass is one of the most abundant renewable sources on earth and is efficiently degraded by fungi. In order to utilize plant biomass efficiently, knowledge about the fungal metabolic pathways and the genes and enzymes involved is essential to create efficient strategies for producing valuable compounds such as cis,cis-muconic acid. cis,cis-muconic acid is an important platform chemical that is used to synthesize nylon, polyethylene terephthalate (PET), polyurethane, resins and lubricants. Currently, cis,cis-muconic acid is mainly produced through chemical synthesis from petroleum based chemicals. Here, we show that two enzymes from fungi can be used to produce cis,cis-muconic acid from salicylic acid and contributes 40 in creating alternative methods for the production of platform chemicals.

scholarly journals The in vivo ISGylome links ISG15 to metabolic pathways and autophagy upon Listeria monocytogenes infection

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yifeng Zhang ◽  
Fabien Thery ◽  
Nicholas C. Wu ◽  
Emma K. Luhmann ◽  
Olivier Dussurget ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Metabolic Pathways ◽  
Quantitative Proteomics ◽  
Computational Analysis ◽  
Murine Models ◽  
Direct Modification ◽  
Catalytic Sites ◽  
Listeria Monocytogenes Infection

AbstractISG15 is an interferon-stimulated, ubiquitin-like protein, with anti-viral and anti-bacterial activity. Here, we map the endogenous in vivo ISGylome in the liver following Listeria monocytogenes infection by combining murine models of reduced or enhanced ISGylation with quantitative proteomics. Our method identifies 930 ISG15 sites in 434 proteins and also detects changes in the host ubiquitylome. The ISGylated targets are enriched in proteins which alter cellular metabolic processes, including upstream modulators of the catabolic and antibacterial pathway of autophagy. Computational analysis of substrate structures reveals that a number of ISG15 modifications occur at catalytic sites or dimerization interfaces of enzymes. Finally, we demonstrate that animals and cells with enhanced ISGylation have increased basal and infection-induced autophagy through the modification of mTOR, WIPI2, AMBRA1, and RAB7. Taken together, these findings ascribe a role of ISGylation to temporally reprogram organismal metabolism following infection through direct modification of a subset of enzymes in the liver.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Serum interspecies differences in metabolic pathways of bradykinin and [des-Arg9]BK: influence of enalaprilat

1996 ◽  
Vol 271 (4) ◽  
pp. H1340-H1347 ◽  
Author(s):  
A. Decarie ◽  
P. Raymond ◽  
N. Gervais ◽  
R. Couture ◽  
A. Adam
Keyword(s):  
Metabolic Pathways ◽  
Neutral Endopeptidase ◽  
Thrombin Inhibitor ◽  
Rat Serum ◽  
Highly Sensitive ◽  
Significant Difference ◽  
Hydrolysis Of

Among the different enzymes responsible for the metabolism of bradykinin (BK), three peptidases look relevant in vivo: kininase I (KI), which transforms BK into its active metabolite, [des-Arg9]BK; kininase II (KII); and neutral endopeptidase, which inactivate BK and [des-Arg9]BK. The in vitro incubation of BK and [des-Arg9]BK in the serum of four species with or without enalaprilat and the quantification of the immunoreactivity of both peptides at different time intervals allowed the measurement of the kinetic parameters characterizing their metabolic pathways. Highly sensitive chemiluminescent enzyme immunoassays were used to measure the residual concentrations of BK and [des-Arg9]BK. Half-life (t1/2) of BK showed significant difference among species: rats (10 +/- 1 s) = dogs (13 +/- 1 s) < rabbits (31 +/- 1 s) < humans (49 +/- 2 s). t1/2 values of [des-Arg9]BK were also species dependent: rats (96 +/- 6 s) < < rabbits (314 +/- 6 s) = dogs (323 +/- 11 s) = humans (325 +/- 12 s). Enalaprilat significantly prevented the rapid BK and [des-Arg9]BK degradation in all species except that of [des-Arg9]BK in rat serum. Relative amount of BK hydrolyzed by serum KII was given as follows: rabbits (93.7 +/- 14.8%) = rats (83.6 +/- 6.7%) = humans (76.0 +/- 7.5%) > dogs (50.0 +/- 3.9%). Its importance in the hydrolysis of [des-Arg9]BK was 5.2 +/- 0.5% in rats < < 33.9 +/- 1.5% in humans < 52.0 +/- 1.1% in rabbits < 65.1 +/- 3.4% in dogs. The participation of serum KI in the transformation of BK into [des-Arg9]BK was dogs (67.2 +/- 5.3%) > > humans (3.4 +/- 1.2%) = rabbits (1.8 +/- 0.2%) = rats (1.4 +/- 0.3%). Finally, no significant difference on t1/2 values for BK and [des-Arg9]BK could be demonstrated between serum and plasma treated with either sodium citrate or a thrombin inhibitor. These results revealed striking species differences in the serum metabolism of kinins that could address at least partially some of the controversial data related to the cardioprotective role of kinins.

scholarly journals Salicylic Acid Sans Aspirin in Animals and Man

2020 ◽  
Author(s):  
James Ronald Lawrence ◽  
Gwendoline Joan Baxter ◽  
John Robert Paterson
Keyword(s):  
Drug Delivery ◽  
Salicylic Acid ◽  
Drug Delivery Systems ◽  
Protective Action ◽  
Sole Source ◽  
Vegetarian Diet ◽  
Low Dose Aspirin ◽  
Drug Free

Analyses in non-aspirin takers finding salicylic acid (SA) and hydroxylated metabolites in serum also SA and salicyluric acid (SU) in urine led to a re-evaluation of dietary sources of salicylates. Fruit and vegetable sources explained higher levels found in drug-free vegetarians, which overlapped with those from patients on low dose aspirin. That drug’s chemo-protective action in cancer is, at least partially, attributable to its principal metabolite, SA—which we believe contributes to the benefits of a vegetarian diet. However, diet is unlikely to be the sole source of the circulating salicylate found in aspirin-free animals and man. We adduced evidence for its persistence in prolonged fasting and biosynthesis in vivo from labelled benzoic acid. We review the roles, defined and potential, of SA in the biosphere. Emphasis on the antiplatelet effect of aspirin in man has detracted from the likely pivotal role of SA in many potential areas of bioregulation—probably as important in animals as in plants. In this expanding field, some aspirin effects, mediated by apparently conserved receptors responding to SA, are discussed. The perspectives revealed may lead to re-evaluation of the place of salicylates in therapeutics and potentially improve formulations and drug delivery systems.

scholarly journals Some Plants Showing Antagonism to Five Plant Pathogenic Fungi

2020 ◽  
Vol 12 (12) ◽  
pp. 144
Author(s):  
M. E. N. Ferreira ◽  
C. F. Pinto ◽  
R. R. de Sena ◽  
J. G. de Mendoça Neto ◽  
C. R. Lobo ◽  
...  
Keyword(s):  
Plant Extracts ◽  
Petri Dish ◽  
Pathogenic Fungi ◽  
Plant Diseases ◽  
Alternative Methods ◽  
Plant Pathogenic Fungi ◽  
Agar Culture ◽  
Antifungal Potential ◽  
Verticillium Dahlia

Fungi are among the main agents of plant diseases, being responsible for major losses in agriculture. The control of these microorganisms carried out using chemical compounds and numerous cases of resistance have already been observed, which makes it necessary to search for alternative methods of management of these pathogens. Therefore, the aim of this study, to evaluate the antifungal potential in plants. Twenty-four (24) plant extracts were tested for their antifungal potential against five plant pathogenic fungi: Sclerotinia sclerotiorum (Ss), Stromatinia cepivora (Sc), Fusarium oxysporum (Fox), Colletotrichum gloesporioides (Cg) and Verticillium dahlia (Vd). For the evaluation of the fungicidal potential, plant extracts were prepared by liquefying the plants in distilled water. The extracts were incorporated into PDA (Potato-Dextrose-Agar) culture medium to a final concentration of 35% and autoclaved. Then, PDA discs colonized by the aforementioned fungi were added to the center of each plate with the respective treatments (plant extracts). When all control treatments (PDA medium without plant extract) had colonized the entire Petri dish, the diameters of the fungal colonies were measured to calculate the Mycelial Growth Inhibition Index (MGI). Tests showed that all these plant extracts have some antifungal activity, ranging from 0 to 100% inhibition. In general, extracts of basil, lavender, guaco, rue, toxic cassava and black plum were the ones that stood out, with MGIs above 50%. New studies are being conducted to evaluate the activity of plant extracts without autoclaving, inhibition of sclerotia formation, to determine the minimum inhibitory concentration, as well as other parts of plants like roots and seeds, mixtures of plant extracts and in vivo antagonism tests.

scholarly journals Autoantibodies to desmoplakin I and II in patients with erythema multiforme.

1995 ◽  
Vol 181 (1) ◽  
pp. 169-179 ◽  
Author(s):  
D Foedinger ◽  
G J Anhalt ◽  
B Boecskoer ◽  
A Elbe ◽  
K Wolff ◽  
...  
Keyword(s):  
Erythema Multiforme ◽  
Biochemical Characterization ◽  
Newborn Mice ◽  
Humoral Immune ◽  
Intercalated Discs ◽  
Mucosal Involvement ◽  
Microscopic Studies ◽  
Actual Target

Erythema multiforme (EM) represents a syndrome of chronic recurrent inflammatory skin disease. Depending on the severity and extent of skin and mucosal involvement, it is defined either as EM minor or EM major. In this study we demonstrate the presence of autoantibodies (aAbs) against desmoplakin I and II, two major proteins of the desmosomal plaque, in six of six patients with the severe variant of EM, EM major. Light microscopic studies of lesional skin and mucous membranes localized in vivo bound immunoglobulin G (IgG) in a dotted desmosomal pattern along the cytoplasmic membranes of keratinocytes. By immunoelectronmicroscopy, in vivo bound IgG was confined to the desmosomal plaques. These findings were confirmed by indirect immunolocalization studies that demonstrated the presence of IgG aAbs in the serum of patients during active disease. These aAbs did not only bind to desmosomal plaques of epithelial cells where they colocalized with defined murine monoclonal antibodies directed against desmoplakin I and II, but also labeled the intercalated discs of myocardial cells. Biochemical characterization of circulating IgG aAbs revealed desmoplakin I and II as actual target autoantigens. By passive transfer of serum into newborn mice, in vivo binding of serum aAbs to keratinocytes was shown. The findings presented in this study imply a humoral immune response in certain patients with EM major and indicate a potential pathogenetic role of aAbs against desmoplakin I and II in this disease.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

scholarly journals Evaluation of two eco-friendly botanical extracts on fruit rot pathogens of orange (Citrus sinesis (L.) Osbeck)

2018 ◽  
Vol 10 (2) ◽  
pp. 107-112
Author(s):  
Thanh Toan Le ◽  
Trong Ky Vo ◽  
Huy Hoang Nguyen
Keyword(s):  
Aspergillus Niger ◽  
Plant Protection ◽  
Current Control ◽  
Fruit Rot ◽  
Alternative Methods ◽  
Control Treatment ◽  
Lesion Length ◽  
Leaf Extracts ◽  
Botanical Extracts

Fruit rot caused by Aspergillus niger and Colletotrichum sp. could cause rapid and severe damage on orange fruits. Current control method of orange fruits is mainly applied by usage of harmful pesticides, leading to chemical residues on fruits, environmental pollution and human poisoning. One of alternative methods of reducing pesticides is to use botanical extracts. This study was conducted to evaluate the in vivo antifungal efficacy of aqueous extracts from the leaves of neem and basket plants against A. niger and Colletotrichum sp. Orange fruits artificially inoculated by fruit rot pathogens were immersed into leaf extracts of 6% (w/v) neem or basket plants for 30 s, and kept for 11 days to record lesion length at room temperature. Orange fruits immersed into sterile distilled water were used as the control treatment. The results showed that at 11 days after inoculation, extracts of neem and basket plants significantly reduced the Aspergillus rot lesions by 109.08 and 124.00 mm, respectively. In addition, anthracnose lesions on orange fruits were statistically inhibited by treatments of neem and basket plants, with the average lesion diameters approximately 160.00 and 154.75 mm, respectively, at day 11 of the conducting experiment. The results of this study showed that leaf extracts of neem and basket plant at the concentration of 6% could be used as a natural alternative to control the in vivogrowth of rot pathogens of orange fruits. These extracts have a bright future in modern plant protection to replace conventional synthetic pesticides in agro-ecosystem. Thối trái bởi Aspergillus niger và Colletotrichum sp. gây ra các thiệt hại nghiêm trọng trên cam. Biện pháp phòng trừ bệnh trên trái cam hiện nay chủ yếu dựa vào thuốc hóa học, dẫn đến tồn dư thuốc trên trái cây, ô nhiễm môi trường và gây độc cho con người. Một trong các phương pháp thay thế giúp giảm sử dụng thuốc hóa học là sử dụng dịch trích thực vật. Nghiên cứu này đã được thưc hiện để đánh giá hiệu quả in vivo của dịch trích ở nồng độ 6% của neem hoặc lược vàng đối với A. niger và Colletotrichum sp. Các trái cam đã lây nhiễm nhân tạo tác nhân gây thối trái thì được nhúng vào dịch trích ở nồng độ 6% của neem hoặc lược vàng trong 30 giây, và giữ đến 11 ngày để ghi nhận chiều dài vết bệnh ở nhiệt độ phòng. Cái trái cam được nhúng vào nước cất thì dùng như nghiệm thức đối chứng. Kết quả cho thấy ở 11 ngày sau khi chủng bệnh, dịch trích neem và lược vàng làm giảm đáng kể vết thối Aspergillus lần lượt là 109,08 và 124,00 mm. Bên cạnh đó, vết bệnh thán thư trên trái cam đã bị ức chế có ý nghĩa thống kê bởi các dịch trích neem và lược vàng, với đường kính trung bình các vết bệnh lần lượt là 160,00 và 154,75 mm, ở ngày 11 của thí nghiệm. Kết quả của nghiên cứu này đã chỉ ra rằng dịch trích neem và lược vàng ở nồng độ 6% có thể sử dụng như một biện pháp thay thế tự nhiên trong việc phòng trừ sự phát triển của tác nhân gây thối trái cam. Các loại dịch trích này có tương lai trong bảo vệ thực vật hiện đại, thay thế các loại thuốc hóa học tổng hợp truyền thống trong hệ sinh thái nông nghiệp.

The Importance of the Prediction Model in the Validation of Alternative Tests

2001 ◽  
Vol 29 (2) ◽  
pp. 135-143 ◽  
Author(s):  
Andrew P. Worth ◽  
Michael Balls
Keyword(s):  
Prediction Model ◽  
Predictive Ability ◽  
Test System ◽  
Alternative Methods ◽  
In Vivo Test ◽  
Testing Strategies ◽  
Adequate Test ◽  
Alternative Test

An overview is presented of the validation process adopted by the European Centre for the Validation of Alternative Methods, with particular emphasis on the central role of the prediction model (PM). The development of an adequate PM is considered to be just as important as the development of an adequate test system, since the validity of an alternative test can only be established when both components (the test system and the PM) have successfully undergone validation. It is argued, however, that alternative tests and their associated PMs do not necessarily need to undergo validation at the same time, and that retrospective validation may be appropriate when a test system is found to be reliable, but the case for its relevance remains to be demonstrated. For an alternative test to be considered “scientifically valid”, it is necessary for three conditions to be fulfilled, referred to here as the criteria for scientific relevance, predictive relevance, and reliability. A minimal set of criteria for the acceptance of any PM is defined, but it should be noted that required levels of predictive ability need to be established on a case-by-case basis, taking into account the inherent variability of the alternative and in vivo test data. Finally, in view of the growing shift in emphasis from the use of standalone alternative tests to alternative testing strategies, the importance of making the PM an integral part of the testing strategy is discussed.

Role of melanotransferrin in iron metabolism: studies using targeted gene disruption in vivo

Blood ◽  
2006 ◽  
Vol 107 (7) ◽  
pp. 2599-2601 ◽  
Author(s):  
Eric O. Sekyere ◽  
Louise L. Dunn ◽  
Yohan Suryo Rahmanto ◽  
Des R. Richardson
Keyword(s):  
Metabolic Pathways ◽  
Gene Disruption ◽  
Vital Role ◽  
Plasminogen Activation ◽  
Serum Chemistry ◽  
Wild Type ◽  
Targeted Disruption ◽  
Targeted Gene Disruption

AbstractMelanotransferrin (MTf) or tumor antigen p97 is a transferrin homolog that binds one iron (Fe) atom and has been suggested to play roles in a variety of processes, including Fe metabolism, eosinophil differentiation, and plasminogen activation. Considering the vital role of Fe in many metabolic pathways, such as DNA and heme synthesis, it is important to understand the function of MTf. To define this, a MTf knockout (MTf–/–) mouse was generated through targeted disruption of the MTf gene. The MTf–/– mice were viable and fertile and developed normally, with no morphologic or histologic abnormalities. Assessment of Fe indices, tissue Fe levels, hematology, and serum chemistry parameters demonstrated no differences between MTf–/– and wild-type (MTf+/+) mice, suggesting MTf was not essential for Fe metabolism.

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