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Biochemistry Jun 2021is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for...
is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. uses the unusual pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (MTAN) is the direct path for producing downstream metabolites in the pathway. However, genomic analysis indicates is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (AFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and -adenosylhomocysteine. The / value for AFL is 6.8 × 10 M s, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for AFLDA and demonstrated inhibitory effects on growth. Supplementation with futalosine partially restored growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-AFLDA and with MTCF at the catalytic sites show a catalytic site Zn or Fe as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp hydroxyl group of the transition state analogue. Metabolomics analysis revealed that AFLDA has intracellular activity and is inhibited by MTCF. The pathway in bifurcates at aminofutalosine with MTAN producing adenine and depurinated futalosine and AFLDA producing futalosine. Inhibition of cellular MTAN or AFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.
Topics: Catalytic Domain; Crystallography, X-Ray; Deoxyadenosines; Helicobacter pylori; Models, Molecular; N-Glycosyl Hydrolases; Nucleosides; Purine-Nucleoside Phosphorylase; Substrate Specificity; Thionucleosides; Vitamin K 2
PubMed: 34077175
DOI: 10.1021/acs.biochem.1c00215 -
International Journal of Biochemistry... 2021Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5....
Alkaline phosphatase is an enzyme that converts para-nitrophenyl phosphate to para-nitrophenol (yellow coloured) in 2-amino, 2-methyl, 1-propanol buffer at pH 10.5. However, when this protocol is applied to the in vitro cellular model systems to estimate alkaline phosphatase activity, it tends to generate clumps of genomic DNA, leading to inaccurate pipetting for protein estimation. The aim of the study was to introduce minor modifications in the existing protocol to make it simple, cost-effective, with minimal labor-intensive procedures while estimating alkaline phosphatase activity in cellular model systems. The genomic DNA clumps were dissolved by depurination (adding 0.2 N HCl) and fragmentation (adding 0.2 N NaOH) during enzyme estimation. Moreover, these minor modifications have been standardized and optimized extensively by using serum samples (rich source of alkaline phosphatase), hFOB/ER9 (human Fetal osteoblastic cell) and HepG2 cells. Our results suggest that the modification incorporated in previously published method was robust enough to estimate ALP activity and protein concentration accurately. There was no significant variation in ALP activity estimated after modification (P > 0.05). This innovative approach could be beneficial for a researcher by providing an easy, cost effective and less labor-intensive solution for estimation of enzymatic activity in cellular model systems.
PubMed: 33824775
DOI: No ID Found -
Toxins Mar 2021International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and...
International authorities classify ricin toxin present in castor seed as a potential agent for use in bioterrorism. Therefore, the detection, identification, and characterization of ricin in various sample matrices are considered necessary actions for risk assessment during a suspected exposure. This study reports a portable electrochemical assay for detecting active ricin based on the adenine electro-oxidation released from herring sperm DNA substrate by its catalytic action. Also, kinetic parameters were calculated, and the values were of 3.14 µM and 2107 min. A linear response was found in optimized experimental conditions for ricin concentrations ranging from 8 to 120 ng/mL, and with a detection limit of 5.14 ng/mL. This proposed detection strategy emphasizes the possibility of field detection of active ricin in food matrices and can be applied to other endonucleolytic activities.
Topics: Adenine; Animals; DNA; Electrochemical Techniques; Fishes; Kinetics; Male; Reproducibility of Results; Ricin; Spermatozoa; Substrate Specificity
PubMed: 33810228
DOI: 10.3390/toxins13040238 -
International Journal of Molecular... Feb 2021Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a...
Ribosome-inactivating proteins (RIPs) are a class of cytotoxic enzymes that can inhibit protein translation by depurinating rRNA. Most plant RIPs are synthesized with a leader sequence that sequesters the proteins to a cell compartment away from the host ribosomes. However, several rice RIPs lack these signal peptides suggesting they reside in the cytosol in close proximity to the plant ribosomes. This paper aims to elucidate the physiological function of two nucleocytoplasmic RIPs from rice, in particular, the type 1 RIP referred to as OsRIP1 and a presumed type 3 RIP called nuRIP. Transgenic rice lines overexpressing these RIPs were constructed and studied for developmental effects resulting from this overexpression under greenhouse conditions. In addition, the performance of transgenic seedlings in response to drought, salt, abscisic acid and methyl jasmonate treatment was investigated. Results suggest that both RIPs can affect methyl jasmonate mediated stress responses.
Topics: Abscisic Acid; Acetates; Cyclopentanes; Cytosol; Gene Expression Regulation, Plant; Green Fluorescent Proteins; Oryza; Oxylipins; Phenotype; Plant Proteins; Plants, Genetically Modified; Protein Biosynthesis; Ribosomes; Salts; Saporins; Seedlings; Stress, Physiological
PubMed: 33535383
DOI: 10.3390/ijms22031434 -
Toxins Jan 2021Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1...
Ribosome-inactivating proteins (RIPs) are plant toxins that irreversibly damage ribosomes and other substrates, thus causing cell death. RIPs are classified in type 1 RIPs, single-chain enzymatic proteins, and type 2 RIPs, consisting of active A chains, similar to type 1 RIPs, linked to lectin B chains, which enable the rapid internalization of the toxin into the cell. For this reason, many type 2 RIPs are very cytotoxic, ricin, volkensin and stenodactylin being the most toxic ones. From the caudex of (Mast.) Engl., a new type 2 RIP, named kirkiin, was purified by affinity chromatography on acid-treated Sepharose CL-6B and gel filtration. The lectin, with molecular weight of about 58 kDa, agglutinated erythrocytes and inhibited protein synthesis in a cell-free system at very low concentrations. Moreover, kirkiin was able to depurinate mammalian and yeast ribosomes, but it showed little or no activity on other nucleotide substrates. In neuroblastoma cells, kirkiin inhibited protein synthesis and induced apoptosis at doses in the pM range. The biological characteristics of kirkiin make this protein a potential candidate for several experimental pharmacological applications both alone for local treatments and as component of immunoconjugates for systemic targeting in neurodegenerative studies and cancer therapy.
Topics: Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cell Survival; Erythrocyte Aggregation; Humans; Molecular Weight; Neuroblastoma; Passifloraceae; Protein Biosynthesis; Protein Synthesis Inhibitors; Ribosome Inactivating Proteins, Type 2; Ribosomes
PubMed: 33499082
DOI: 10.3390/toxins13020081 -
Ecology and Evolution Jan 2021Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still...
Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next-generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole-genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.
PubMed: 33437437
DOI: 10.1002/ece3.7056 -
Chemosphere Jan 2021Naphthalene is the simplest representative of polycyclic aromatic hydrocarbons (PAHs). It is detected as major pollutant in the different compartments of the...
Naphthalene is the simplest representative of polycyclic aromatic hydrocarbons (PAHs). It is detected as major pollutant in the different compartments of the environment. This compound is considered by the international agency for research on cancer (IARC), the specialized cancer agency of the World Health Organisation (WHO), as a possible carcinogenic (group 2B) since 2002, mainly based on studies on chronic inhalation in rodent by the national toxicology program of the U.S. department of health and human services. In humans, its main metabolites correspond to derivatives substituted in position and 1 and 2 as 1,2-naphthoquinone (1,2-NphQ). Based on previous studies, 1,2-NphQ is supposed to react with DNA to form mostly depurinating adducts, a possible initiating step of carcinogenicity. To confirm this potentiality, adducts were synthetized by the reaction of 1,2-NphQ with 2'-deoxyguanosine (2'-dG) in N,N-dimethylformamide (DMF), water and calf thymus DNA. 2'-dG adducts were analyzed by P post-labelling, HPLC with ultra-violet detection and ultra-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). We found stable DNA adducts detected in DNA. We proposed a formation mechanism by a 1,4-Michael addition with 2'-dG. Adducts with 2'-deoxyxanthosine are formed after a spontaneous deamination of 2'-dG. These adducts are good candidates as biomarkers allowing evaluation of exposure to naphthalene and its derivatives in the development of pathologies such as cancer.
Topics: Chromatography, High Pressure Liquid; DNA Adducts; Naphthalenes; Naphthoquinones; Tandem Mass Spectrometry
PubMed: 33297078
DOI: 10.1016/j.chemosphere.2020.128079 -
SLAS Discovery : Advancing Life... Mar 2021Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic -glycosidase,...
Saporin, a type I ribosome-inactivating protein from soapwort plant, is a potent protein synthesis inhibitor. Catalytically, saporin is a characteristic -glycosidase, and it depurinates a specific adenine residue from a universally conserved loop of the major ribosomal RNA (rRNA) of eukaryotic cells. It is well-known that saporin induces apoptosis through different pathways, including ribotoxic stress response, cell signal transduction, genomic DNA fragmentation and RNA abasic lyase (RAlyase) activity, and NAD depletion by poly-(ADP)-ribose polymerase hyperactivation. Saporin's high enzymatic activity, high stability, and resistance to conjugation procedures make it a well-suited tool for immunotherapy approaches.In the present study, we focus on saporin-based targeted toxins that may be efficacious therapeutic agents for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Our discussed points suggest that saporin may be a strategic molecule for therapeutic knockout treatments and a powerful candidate for novel drugs in the struggle against coronavirus 2019 (COVID-19).
Topics: Antiviral Agents; Apoptosis; Humans; Immunotoxins; NAD; Poly(ADP-ribose) Polymerases; Saporins; Signal Transduction; COVID-19 Drug Treatment
PubMed: 33155515
DOI: 10.1177/2472555220970911 -
Scientific Reports Sep 2020Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes...
Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and bacteria. Previously, we have described for the first time RIP genes in mosquitoes belonging to the Culicidae family. We showed that these genes are derived from a single event of horizontal gene transfer (HGT) from a prokaryotic donor. Mosquito RIP genes are evolving under purifying selection, strongly suggesting that these toxins have acquired a functional role. In this work, we show the existence of two RIP encoding genes in the genome of the whitefly Bemisia tabaci, a hemiptera species belonging to the Aleyrodidae family distantly related to mosquitoes. Contamination artifacts were ruled out analyzing three independent B. tabaci genome databases. In contrast to mosquito RIPs, whitefly genes harbor introns and according to transcriptomic evidence are transcribed and spliced. Phylogeny and the taxonomic distribution strongly support that whitefly RIP genes are derived from an independent HGT event from a plant source. These results, along with our previous description of RIPs in Diptera, suggest that the acquired genes are functional in these insects and confer some fitness advantage.
Topics: Animals; Gene Expression Profiling; Gene Transfer, Horizontal; Genes, Insect; Genes, Plant; Genome, Insect; Hemiptera; Phylogeny; RNA, Ribosomal, 28S; Ribosome Inactivating Proteins; Selection, Genetic; Sequence Alignment; Sequence Analysis, DNA
PubMed: 32968092
DOI: 10.1038/s41598-020-72267-1 -
Journal of the American Chemical Society Sep 2020Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis but suffers from...
Solid-phase oligonucleotide synthesis (SPOS) based on phosphoramidite chemistry is currently the most widespread technique for DNA and RNA synthesis but suffers from scalability limitations and high reagent consumption. Liquid-phase oligonucleotide synthesis (LPOS) uses soluble polymer supports and has the potential of being scalable. However, at present, LPOS requires 3 separate reaction steps and 4-5 precipitation steps per nucleotide addition. Moreover, long acid exposure times during the deprotection step degrade sequences with high A content (adenine) due to depurination and chain cleavage. In this work, we present the first one-pot liquid-phase DNA synthesis technique which allows the addition of one nucleotide in a one-pot reaction of sequential coupling, oxidation, and deprotection followed by a single precipitation step. Furthermore, we demonstrate how to suppress depurination during the addition of adenine nucleotides. We showcase the potential of this technique to prepare high-purity 4-arm PEG-T (T = thymine) and 4-arm PEG-A building blocks in multigram scale. Such complementary 4-arm PEG-DNA building blocks reversibly self-assemble into supramolecular model network hydrogels and facilitate the elucidation of bond lifetimes. These model network hydrogels exhibit new levels of mechanical properties (storage modulus, bond lifetimes) in DNA bonds at room temperature (melting at 44 °C) and thus open up pathways to next-generation DNA materials programmable through sequence recognition and available for macroscale applications.
Topics: DNA; Hydrogels; Models, Molecular; Molecular Structure; Polyethylene Glycols
PubMed: 32902960
DOI: 10.1021/jacs.0c05488