Mutational Studies Research Articles

Evidence for Safety of The Dietary Ingredient Agmatine Sulfate as Assessed by Mutagenicity and Genotoxicity Studies

Agmatine, 1-Amino-4-guanidinobutane, is a ubiquitous naturally occurring molecule present in low amounts in a wide variety of foodstuff. Clinical trials have demonstrated the safety of oral agmatine sulfate and have led to its development as an effective dietary ingredient for promoting resilient nerve functions. Although clearly required, the mutagenic and genotoxic effects of agmatine have not been previously reported. The present study, therefore, undertook to assess the safety profile of agmatine using currently accepted in vitro and in vivo mutagenicity and genotoxicity tests. The test item was G-Agmatine®, a proprietary brand of agmatine sulfate. Using the bacterial reverse mutation assay (Ames test), the study found that G-Agmatine® has no mutagenic effects. It had no clastogenic effects as observed by the in vitro chromosomal aberration test using Chinese Hamster lung cells. And it lacked genotoxic effects as evidenced by the lack of increased frequency of micronucleated polychromatic immature erythrocytes following oral administration in the mouse micronucleus test. Taken together with previously published data, results of the present study further support the safety of agmatine sulfate as a dietary ingredient.

Quantum chemical calculations of nitrosamine activation and deactivation pathways for carcinogenicity risk assessment.

N-nitrosamines and nitrosamine drug substance related impurities (NDSRIs) became a critical topic for the development and safety of small molecule medicines following the withdrawal of various pharmaceutical products from the market. To assess the mutagenic and carcinogenic potential of different N-nitrosamines lacking robust carcinogenicity data, several approaches are in use including the published carcinogenic potency categorization approach (CPCA), the Enhanced Ames Test (EAT), in vivo mutagenicity studies as well as read-across to analogue molecules with robust carcinogenicity data. We employ quantum chemical calculations as a pivotal tool providing insights into the likelihood of reactive ion formation and subsequent DNA alkylation for a selection of molecules including e.g., carcinogenic N-nitrosopiperazine (NPZ), N-nitrosopiperidine (NPIP), together with N-nitrosodimethylamine (NDMA) as well as non-carcinogenic N-nitrosomethyl-tert-butylamine (NTBA) and bis (butan-2-yl) (nitros)amine (BBNA). In addition, a series of nitroso-methylaminopyridines is compared side-by-side. We draw comparisons between calculated reaction profiles for structures representing motifs common to NDSRIs and those of confirmed carcinogenic and non-carcinogenic molecules with in vivo data from cancer bioassays. Furthermore, our approach enables insights into reactivity and relative stability of intermediate species that can be formed upon activation of several nitrosamines. Most notably, we reveal consistent differences between the free energy profiles of carcinogenic and non-carcinogenic molecules. For the former, the intermediate diazonium ions mostly react, kinetically controlled, to the more stable DNA adducts and less to the water adducts via transition-states of similar heights. Non-carcinogenic molecules yield stable carbocations as intermediates that, thermodynamically controlled, more likely form the statistically preferred water adducts. In conclusion, our data confirm that quantum chemical calculations can contribute to a weight of evidence approach for the risk assessment of nitrosamines.

A Nonclinical Safety Evaluation of Cold Atmospheric Plasma for Medical Applications: The Role of Genotoxicity and Mutagenicity Studies.

Atmospheric nonthermal plasma (ANTP) has rapidly evolved as an innovative tool in biomedicine with various applications, especially in treating skin diseases. In particular, the formation of reactive oxygen species (ROS) and nitrogen species (RNS), which are generated by ANTP, plays an important role in the biological signaling pathways of human cells. Unfortunately, excessive amounts of these reactive species significantly result in cellular damage and cell death induction. To ensure the safe application of ANTP, preclinical in vitro studies must be conducted before proceeding to in vivo or clinical trials involving humans. Our study aimed to investigate adverse effects on genetic substances in murine fibroblast cells exposed to ANTP. Cell viability and proliferation were markedly reduced after exposing the cells with plasma. Both extracellular and intracellular reactive species, especially RNS, were significantly increased upon plasma exposure in the culture medium and the cells. Notably, significant DNA damage in the cells was observed in the cells exposed to plasma. However, plasma was not classified as a mutagen in the Ames test. This suggested that plasma led to the generation of both extracellular and intracellular reactive species, particularly nitrogen species, which affect cell proliferation and are also known to induce genetic damage in fibroblast cells. These results highlight the genotoxic and mutagenic effects of ANTP, emphasizing the need for the cautious selection of plasma intensity in specific applications to avoid adverse side effects resulting from reactive species production.

Discerning the stability behaviour of mavacamten availing liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy: In silico toxicity and mutagenicity prediction of degradation products.

The present study aimed to separate, identify, and characterise the degradation products formed when mavacamten is exposed to stress degradation as well as the stability of the drug in various environments and also to understand its degradation chemistry. Prediction of in silico toxicity and mutagenicity was aimed at the observed degradation products. Stress degradation along with stability studies and degradation kinetics were performed on mavacamten, and separation of degradation products was carried out by high-performance liquid chromatography. Tandem mass spectrometry studies were executed to characterise the structures of degradation products using product ion fragments. Orthogonally, nuclear magnetic resonance experiments were conducted to elucidate the structures having ambiguity in characterising them. Deductive Estimation of Risk from Existing Knowledge and Structure Activity Relationship Analysis using Hypotheses software were used to establish in silico toxicity and mutagenic profiles of mavacamten and its degradation products. Two degradation products of mavacamten found in acidic hydrolytic stress conditions were separated, identified, characterised, and proposed as 1-isopropylpyrimidine-2,4,6(1H,3H,5H)-trione and 1-phenylethanamine. Mavacamten was found to be stable under different pH and gastrointestinal conditions. The degradation kinetics of mavacamten under 1 N acidic condition followed zero-order kinetics, and it was degraded completely within 6 h. In silico toxicity and mutagenicity studies revealed that 1-phenylethanamine can be a skin sensitiser. A high-performance liquid chromatography method was developed for the separation of degradation products of mavacamten and characterised by liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance. During the manufacturing and storage of drug product, precautions need to be taken when dealing with acidic solutions as the drug is prone to hydrolysis in acidic conditions. The formation of 1-phenylethanamine under these conditions is to be monitored as it is a skin sensitiser.

Single Ion Pair Is Essential for Stabilizing SHP2's Open Conformation.

Src-homology-2-domain-containing PTP-2 (SHP2) is a widely expressed signaling enzyme whose misregulation is associated with multiple human pathologies. SHP2's enzymatic activity is controlled by a conformational equilibrium between its autoinhibited ("closed") state and its activated ("open") state. Although SHP2's closed state has been extensively characterized, the putative structure of its open form has only been revealed in the context of a highly activated mutant (E76K), and no systematic studies of the biochemical determinants of SHP2's open-state stabilization have been reported. To identify amino-acid interactions that are critical for stabilizing SHP2's active state, we carried out a mutagenic study of residues that lie at potentially important interdomain interfaces of the open conformation. The open/closed equilibria of the mutants were evaluated, and we identified several interactions that contribute to the stabilization of SHP2's open state. In particular, our findings establish that an ion pair between glutamate 249 on SHP2's PTP domain and arginine 111 on an interdomain loop is the key determinant of SHP2's open-state stabilization. Mutations that disrupt the R111/E249 ion pair substantially shift SHP2's open/closed equilibrium to the closed state, even compared to wild-type SHP2's basal-state equilibrium, which strongly favors the closed state. To the best of our knowledge, the ion-pair variants uncovered in this study are the first known SHP2 mutants in which autoinhibition is augmented with respect to the wild-type protein. Such "hyperinhibited" mutants may provide useful tools for signaling studies that investigate the connections between SHP2 inhibition and the suppression of human disease progression.

Biochemical and biophysical characterization of natural polyreactivity in antibodies

To become specialized binders, antibodies undergo a process called affinity maturation to maximize their binding affinity. Despite this process, some antibodies retain low-affinity binding to diverse epitopes in a phenomenon called polyreactivity. Here we seek to understand the molecular basis of this polyreactivity in antibodies. Our results highlight that polyreactive antigen-binding fragments (Fabs) bind their targets with low affinities, comparable to Tcell receptor recognition of autologous classical major histocompatibility complex. Extensive mutagenic studies find no singular amino acid residue or biochemical property responsible for polyreactive interaction, suggesting that polyreactive antibodies use multiple strategies for engagement. Finally, our crystal structures and all-atom molecular dynamics simulations of polyreactive Fabs show increased rigidity compared to their monoreactive relatives, forming a neutral and accessible platform for diverse antigens to bind. Together, these data support a cooperative strategy of rigid neutrality in establishing the polyreactive status of an antibody molecule.

Toxicity and mutagenicity studies of 6PPD-quinone in a marine invertebrate species and bacteria.

N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-quinone), an oxidation product of the tire additive, 6PPD, has been associated with high mortality of salmonids (0.1 μg/L). The objective of this study was to determine the acute toxicity using neonates and mutagenicity (micronuclei in hemolymph of exposed adults) of 6PPD-quinone in the marine amphipod Parhyale hawaiensis. Also, we studied its mutagenicity in the Salmonella/microsome assay using five strains of Salmonella with and without metabolic system (rat liver S9, 5%). 6PPD-quinone did not present acute toxicity to P. hawaiensis from 31.25 to 500 μg/L. Micronuclei frequency increased after 96 h-exposure to 6PPD-quinone (250 and 500 μg/L) when compared to the negative control. 6PPD-quinone also showed a weak mutagenic effect for TA100 only in the presence of S9. We conclude that 6PPD-quinone is mutagenic to P. hawaiensis and weakly mutagenic to bacteria. Our work provides information for future risk assessment of the presence of 6PPD-quinone in the aquatic environment.

Current situation and genomic characterization of fowlpox virus in lower Egypt during 2022

A total of 45 samples of vaccinated and non-vaccinated layer chickens were collected from farms in the Egyptian governorates of Sharqia, Ismailia, Menofia, Gharbia, Kafr El Sheikh, Qalyubia, and Dakahlia in the year 2022. They exhibited nodular lesions on their combs, mouth corners, and eyelids, suggesting they were infected with pox disease, which was associated with a 3 to 5% mortality rate. The samples were grown on the chorioallantoic-membrane of embryonated chicken eggs to ensure their viability. In both vaccinated and non-vaccinated farms, 35 of 45 virus isolates were confirmed positive via polymerase chain reaction (PCR) of fpv167 (P4b), based on the amplicon length of the fpv167 gene locus. The 6 strains from various Egyptian governorates were chosen for sequencing and genetic characterization. Phylogenetic investigation of the fpv167 (P4b) gene of sequenced strains clustered within sub clade A1 showed 100% correlation between FWPVD, TKPV13401 and fowlpox-AN2, fowlpox-AN3, and fowlpox-AN6, but only a 98.6% correlation between fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5. Comparing the fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 strains with commercial vaccine strains (HP1-444-(FP9), vaccine-VSVRI), they had 98.6% identity, while other strains had 100% identity. The results of this study's mutation research showed that fowlpox-AN1, fowlpox-AN4, and fowlpox-AN5 had acquired novel mutations; fowlpox-AN1 had R201G and T204A; fowlpox-AN4 and fowlpox-AN5 had L141F and H157P. Further research is required to determine the effectiveness of the current vaccine in order to develop a new vaccine.

A [4Fe-4S] cluster resides at the active center of phosphomevalonate dehydratase, a key enzyme in the archaeal modified mevalonate pathway.

The recent discovery of the archaeal modified mevalonate pathway revealed that the fundamental units for isoprenoid biosynthesis (isopentenyl diphosphate and dimethylallyl diphosphate) are biosynthesized via a specific intermediate, trans-anhydromevalonate phosphate. In this biosynthetic pathway, which is unique to archaea, the formation of trans-anhydromevalonate phosphate from (R)-mevalonate 5-phosphate is catalyzed by a key enzyme, phosphomevalonate dehydratase. This archaea-specific enzyme belongs to the aconitase X family within the aconitase superfamily, along with bacterial homologs involved in hydroxyproline metabolism. Although an iron-sulfur cluster is thought to exist in phosphomevalonate dehydratase and is believed to be responsible for the catalytic mechanism of the enzyme, the structure and role of this cluster have not been well characterized. Here, we reconstructed the iron-sulfur cluster of phosphomevalonate dehydratase from the hyperthermophilic archaeon Aeropyrum pernix to perform biochemical characterization and kinetic analysis of the enzyme. Electron paramagnetic resonance, iron quantification, and mutagenic studies of the enzyme demonstrated that three conserved cysteine residues coordinate a [4Fe-4S] cluster-as is typical in aconitase superfamily hydratases/dehydratases, in contrast to bacterial aconitase X-family enzymes, which have been reported to harbor a [2Fe-2S] cluster.

Possibilities of using micronucleus analysis to detect gene mutations in animals

Relevance. The main purpose of micronuclear analysis is to identify substances that cause cytotoxic damage and lead to the formation of micronuclei containing lagging fragments of chromosomes or whole chromosomes. For the study of chromosomal disorders, micronuclear analysis is now very often used, since it is quite easy to perform and does not require large costs.Methods and results. Micronuclei are cytoplasmic chromatin-containing bodies formed when delayed in anaphase or telophase during cell division or fragmentation of the nucleus during apoptosis. The method of micronuclear analysis is used as a biomarker of chromosomal aberrations in the study of mutagenicity, as well as a marker of the development of oncological diseases. With quantitative changes in DNA in the cell, the formation of micronuclei occurs. It is their presence that is an indicator that the body was exposed to environmental pollution (pesticides, pesticides, radiation, heavy metals, etc.), leading to inflammatory and pathological processes. Recently, many studies have been conducted aimed at studying the influence of environmental factors on the genetic apparatus of the body. Under the influence of mutagens, micronuclei are formed in proliferating cells, the counting of which allows for the diagnosis of both genotoxic stress and genetic instability of the organism. To determine early genetic changes in humans and animals, sensitive and labor-intensive methods are needed, which can be attributed to micronuclear analysis. Recently, micronuclear analysis has become relevant for its use in the livestock sector, mainly in research aimed at quality control of livestock products used in the future for human nutrition.

Mapping allosteric pathway in NIa‐Pro using computational approach

BackgroundComputer simulation studies complement in vitro experiments and provide avenue to understand allosteric regulation in the absence of other molecular viewing techniques. Molecular dynamics captures internal motion within the protein and enables tracing the communication path between a catalytic site and a distal allosteric site. In this article, we have identified the communication pathway between the viral protein genome linked (VPg) binding region and catalytic active site in nuclear inclusion protein‐a protease (NIa‐Pro).MethodsMolecular dynamics followed by in silico analyses have been used to map the allosteric pathway.ResultsThis study delineates the residue interaction network involved in allosteric regulation of NIa‐Pro activity by VPg. Simulation studies indicate that point mutations in the VPg interaction interface of NIa‐Pro lead to disruption in these networks and change the orientation of catalytic residues. His142Ala and His167Ala mutations do not show a substantial change in the overall protease structure, but rather in the residue interaction network and catalytic site geometry.ConclusionOur mutagenic study delineates the allosteric pathway and facilitates the understanding of the modulation of NIa‐Pro activity on a molecular level in the absence of the structure of its complex with the known regulator VPg. Additionally, our in silico analysis explains the molecular concepts and highlights the dynamics behind the previously reported wet lab study findings.

Mutagenicity Studies of Potable Water Sources in Okerenkoko community, Gbaramatu, Delta State, Nigeria

Mutagenic testing of portable water is fast becoming an important protocol for assessing water quality. Two potable water samples were selected from both ground and surface water sources within the Okerenkoko community, Gbaramatu Kingdom, Delta State Nigeria. Ame’s test was employed in the assessment of potential carcinogens in the potable water available to the community. The study was carried out in triplicate to broaden the reproducibility of the study. Sensitivity of the test organism was evaluated using the Salmonella TA98 and Salmonella TA100 which was reconstituted for the study. The average revertant colonies from the study was enumerated to indicate that the test conducted on S3OK (Surface water) had 428.1 and 637.84 revertant colonies using Salmonella TA 100 for both +S9 mix and -S9 mix-conditions respectively while similar replica result was for Salmonella TA 98 was 629.7 and 66.12 revertant colonies. The samples obtained from S1OK had slightly lower number of revertant colonies of 209.16 and 115.94 colonies for Salmonella T.A 100 while for Salmonella T.A 98 had 116.82 and 108.90 colonies for treatments containing S9 mix preparations. The study identified the test organism was Salmonella T.A 98 was more sensitive to the carcinogen present in the S3OK sample being used by a wide population of indigenes of the lower economic class. This study further buttresses the need to develop a more robust monitoring protocol for packaged water sources in Nigeria. Regulatory agencies such as NAFDAC must enforce strict intervention limits for potable water sources.

Comparative investigation of ligand binding of heme-containing dioxygenases by computer modeling.

Heme-containing enzymes are typically used as catalysts of oxidation-reduction reactions. These enzymes range from monooxygenases and dioxygenases to peroxidases and catalases to oxidases and dehydrogenases. Mechanistic aspects of these enzymes, including heme chemistry, have been extensively studied. Cyclooxygenase (COX), a heme-containing dioxygenase, has a cyclooxygenase activity and an associated peroxidase activity. COX shows similarity in the organization of the peroxidase active site to the other peroxidases. Cyclooxygenase active site, to which arachidonic acid binds, is on the proximal side of the heme and is the main target of explosive research interest, which results in great success. In plants, pathogen-inducible oxygenase (PIOX), another heme-containing enzyme, generates a potent lipid mediator by oxygenating 14-20 fatty acid substrates. COX and PIOX exhibit catalytic and structural similarities. The ligand-protein interactions of COX and PIOX have been characterized by crystallographic and mutagenic studies. Interestingly, the orientation of the fatty acid substrate inside the PIOX active site is different from the case of COX because of the α-dioxygenation of a cognate substrate by PIOX. This paper analyzes the characteristics of the substrate binding of mammalian and plant heme-containing dioxygenases using computer-aided modeling.

Preclinical study of mutagenicity and safety of a vaccine against pneumococcal infection based on a recombinant hybrid protein

Pneumococcal infection is the cause of pneumo-nia. Vaccination can prevent the severe course of this disease. The pneumococcal vaccine considered in the work was created by a genetically modified method based on a hybrid recombinant protein. The aim of the work was preclinical investigation of the mutagenic, genetic safety of the new pneumococcal vaccine, Irivax, against pneumococcal infection in rats. Materials and methods. Studies of the Irivax vaccine were carried out in male Wistar rats’ cell populations of the bone marrow and peripheral blood mononuclear cells. The karyoty-pic stability of cells and myelogram of the bone mar-row were studied. Results. After the introduction of the vaccine, the content of the lymphoid germ cells in the blood of rats was increased to 74.2% (p <0.05), which proves the body’s immune response. The frequency of cells with an unstable genome, as an indicator of the genetic safety of the AL-vaccine, was rare both in expe-rimental and control animals, and did not exceed 1.5%. Conclusion. Thus, the new pneumococcal vaccine Irivax and its components have mutagenic and toxic safety.

Cloned antibodies from patients with HIT provide new clues to HIT pathogenesis

AbstractHeparin-induced thrombocytopenia (HIT) is a serious adverse drug reaction characterized by antibodies that recognize platelet factor 4/heparin complexes (PF4/H) and activate platelets to create a prothrombotic state. Although a high percentage of heparin-treated patients produce antibodies to PF4/H, only a subset also makes antibodies that are platelet activating (PA). A close correlation between PA antibodies and the likelihood of experiencing HIT has been demonstrated in clinical studies, but how PA (presumptively pathogenic) and nonactivating (NA) (presumptively benign) antibodies differ from each other at the molecular level is unknown. To address this issue, we cloned 7 PA and 47 NA PF4/H-binding antibodies from 6 patients with HIT and characterized their structural and functional properties. Findings showed that PA clones differed significantly from NA clones in possessing 1 of 2 heavy chain complementarity-determining region 3 (HCDR3) motifs, RX1-2R/KX1-2R/H (RKH) and YYYYY (Y5), in an unusually long complementarity-determining region 3 (≥20 residues). Mutagenic studies showed that modification of either motif in PA clones reduced or abolished their PA activity and that appropriate amino acid substitutions in HCDR3 of NA clones can cause them to become PA. Repertoire sequencing showed that the frequency of peripheral blood IgG+ B cells possessing RKH or Y5 was significantly higher in patients with HIT than in patients without HIT given heparin, indicating expansion of B cells possessing RKH or Y5 in HIT. These findings imply that antibodies possessing RKH or Y5 are relevant to HIT pathogenesis and suggest new approaches to diagnosis and treatment of this condition.

A manually curated annotation characterises genomic features of P. falciparum lncRNAs

BackgroundImportant regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum. However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum.ResultsWe generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes.ConclusionsThe insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

Cytotoxicity, oral toxicity, genotoxicity, and mutagenicity evaluation of essential oil from Psidium glaziovianum Kiaersk leaves

Ethnopharmacological relevanceMembers of the Psidium genus have been suggested in ethnobotanical research for the treatment of various human diseases, and some studies have already proven their popular uses through research, such as Psidium glaziovianum, which is found in Brazil's northeast and southeast regions and has antinociceptive and anti-inflammatory properties; however, the safety of use has not yet been evaluated. Aim of the studyThis study investigated the safety of using essential oil obtained from P. glaziovianum leaves (PgEO) in vitro and in vivo models. Materials and methodsCytotoxicity was evaluated in murine erythrocytes, while acute toxicity, genotoxicity (comet assay) and mutagenicity (micronucleus test) studies were performed using Swiss albino mice. ResultsIn the cytotoxicity assay, the hemolysis rate indicated a low capacity of PgEO to cause cell lysis (0.33–1.78%). In the acute oral toxicity study, animals treated with up to up to 5000 mg/kg body weight did not observe mortality or physiological changes. Neither dosage caused behavioral problems or death in mice over 14 days. The control and 2,000 mg/kg groups had higher feed intake and body weight than the 5,000 mg/kg PgEO group. Erythrocyte count, hemoglobin level, mean corpuscular volume, and MCV decreased, but serum alanine and aspartate aminotransferases increased. In the genotoxic evaluation, 5000 mg/kg PgEO enhanced nucleated blood cell DI and DF. ConclusionsThe present study describes that PgEO can be considered well tolerated in acute exposure at doses up to 2000 mg/kg, however the dose of 5000 mg/kg of PgEO should be used with caution.

Characterization of Lantana Camara Roots (Pentacyclic Triterpenoid) and Mutagenicity Testing of Extracted Oleanolic Acid Using Salmonella Typhimurium

The objective of the present study was to isolate oleanolic acid from the roots of Lantana camara, and characterize it chemically using DSC, HPLC, and FTIR methods, and additionally carrying out a short term assay for assessment of its mutagenic potential by conducting bacterial reverse mutation test to evaluate the ability of the “Oleanolic acid (Pentacyclic Triterpenoid)” to induce point mutations in tester strains of Salmonella typhimurium in both presence and absence of exogenous metabolic activation system (S9) containing microsomal enzymes. Differential scanning calorimetry (DSC), High Performance Liquid chromatography (HPLC), and Fourier transform infrared (FTIR) spectrometers were used to chemically analyze the isolated molecule. Oleanolic acid was utilized to carry out Preliminary Cytotoxicity and mutagenicity study. According to the results of spectrophotometric research, oleanolic acid extracted from Lantana camara roots exhibits identical spectrum characteristics to standard oleanolic acid also the mutagenic potential of Oleanolic acid (Pentacyclic Triterpenoid). Oleanolic acid was found to be non-mutagenic in all five test strains of Salmonella typhimurium—TA98, TA100, TA102, TA1535, and TA1537 employing plate incorporation assays. It may be determined that oleanolic acid isolated from Lantana camara roots gives identical, identifiable signals and absorbance like previously reported reference standard based on the results of DLC, HPLC, and FTIR spectra and their interpretation and was determined that oleanolic acid purified from Lantana camara roots is non-mutagenic in Salmonella typhimurium.

Uncovering lipopolysaccharide regulation in bacteria via the critical lipid binding tunnel of YciS/YciM

SummaryGram-negative bacteria contain an asymmetric outer membrane, in which the outer leaflet is composed of lipopolysaccharide (LPS). LPS, a drug target of polymyxin, plays an essential role in drug resistance, biofilm formation, and pathogenesis. An important inner membrane protein, YciM, may be responsible for the regulation of LPS biosynthesis and transport. Here, we report the crystal structure of YciM from Salmonella typhimurium in a complex with a non-specifically bond molecule, an ethylene glycol, which identified a tunnel that could bind lipids. Our in vitro assays showed that YciM could bind lipid molecules with affinity in the micromolar range, while mutagenic and functional studies confirmed that lipid-binding residues are critical for the function of YciM. Additionally, our data also showed that YciM accurately regulates LPS biosynthesis and transport with YciS, which could help to better understand the regulation mechanism of LPS.

Evidence for Plant-Conserved Region Mediated Trimeric CESAs in Plant Cellulose Synthase Complexes.

Higher plants synthesize cellulose using membrane-bound, six-lobed cellulose synthase complexes, each lobe containing trimeric cellulose synthases (CESAs). Although molecular biology reports support heteromeric trimers composed of different isoforms, a homomeric trimer was reported for in vitro studies of the catalytic domain of CESA1 of Arabidopsis (AtCESA1CatD) and confirmed in cryoEM structures of full-length CESA8 and CESA7 of poplar and cotton, respectively. In both structures, a small portion of the plant-conserved region (P-CR) forms the only contacts between catalytic domains of the monomers. We report inter-subunit lysine-crosslinks that localize to the small P-CR, negative-stain EM structure, and modeling data for homotrimers of AtCESA1CatD. Molecular dynamics simulations for AtCESA1CatD trimers based on the CESA8 cryoEM structure were stable and dependent upon a small set of residue contacts. The results suggest that homomeric CESA trimers may be important for the synthesis of primary and secondary cell walls and identify key residues for future mutagenic studies.