Biochemical Studies Research Articles

The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs

MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N"(fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.

One RING to rule them all: Mind bomb-1 ccRING3 controls Notch signaling

In this issue of Structure, Cao etal.1 use X-ray crystallography, biochemical, and genetic studies to define the key role of the Mind bomb-1 ccRING3 domain in triggering Notch signaling, and they demonstrate that ccRING3-mediated dimerization is a key step in ligand activation.

Влияние малых доз ионизирующего излучения при облучении in vitro на показатели крови

Abstract. The purpose is the determination of the degree of influence of small absorbed doses of ionizing radiation on biochemical and hematological parameters during irradiation of peripheral blood samples of cattle in vitro. For the work, blood samples were taken from lactating cows kept under the influence of the background absorbed dose in the Krasnoyarsk Territory, followed by in vitro irradiation at doses of 5, 50, 500 mGy. Methods. Peripheral blood was taken from blood vessels from the tail vein into vacuum tubes with a coagulation activator, in vitro irradiation of samples was carried out on an installation equipped with a Cs-137 source, biochemical studies of blood serum were carried out using a PE-5400UF spectrophotometer. Hematological blood parameters were determined according to generally accepted methods. Results. Ionizing radiation in vitro in doses of 5 mGy, 50 mGy and 500 mGy of peripheral blood samples of cattle affects hematological parameters in different directions, which is characterized by wave-like fluctuations in hemoglobin values, a decrease in the content of erythrocytes and a decrease in the erythrocyte sedimentation rate. It was found that a single irradiation of peripheral blood samples of cows at a dose of 5 mGy reduced the concentration of beta-globulins and creatinine and did not affect the content of total protein and protein fractions. When exposed to ionizing radiation at a dose of 50 mGy, the content of total protein, the level of beta-globulins, creatinine decreased in blood samples, and the content of albumins and alpha-, gamma-globulins did not change. When irradiated at a dose of 500 mGy, the relative content of alpha-globulins, the concentration of ALT and AST decreased, the content of creatinine, total protein, albumins and beta-, gamma-globulins did not change. The stability of alkaline phosphatase, albumins and gamma globulins to ionizing effects at doses of 5 mGy, 50 mGy and 500 mGy has been established. Scientific novelty. The linear dependence of the concentration of alpha-globulins, AST and ALT on the absorbed dose was revealed, the numerical values of the approximation coefficients describing the decrease in concentration and biochemical parameters were determined. The inverse direct linear relationship between creatinine stability and absorbed dose has been established. Practical significance is the established digital values of the approximation coefficients of alpha globulins, AST and ALT can be used in the reconstruction of radiation dose values in the range of 5–50 mGy.

How ATP and dATP Act as Molecular Switches to Regulate Enzymatic Activity in the Prototypical Bacterial Class Ia Ribonucleotide Reductase.

Class Ia ribonucleotide reductases (RNRs) are allosterically regulated by ATP and dATP to maintain the appropriate deoxyribonucleotide levels inside the cell for DNA biosynthesis and repair. RNR activity requires precise positioning of the β2 and α2 subunits for the transfer of a catalytically essential radical species. Excess dATP inhibits RNR through the creation of an α-β interface that restricts the ability of β2 to obtain a position that is capable of radical transfer. ATP breaks the α-β interface, freeing β2 and restoring enzyme activity. Here, we investigate the molecular basis for allosteric activity regulation in the well-studied Escherichia coli class Ia RNR through the determination of six crystal structures and accompanying biochemical and mutagenesis studies. We find that when dATP is bound to the N-terminal regulatory cone domain in α, a helix unwinds, creating a binding surface for β. When ATP displaces dATP, the helix rewinds, dismantling the α-β interface. This reversal of enzyme inhibition requires that two ATP molecules are bound in the cone domain: one in the canonical nucleotide-binding site (site 1) and one in a site (site 2) that is blocked by phenylalanine-87 and tryptophan-28 unless ATP is bound in site 1. When ATP binds to site 1, histidine-59 rearranges, prompting the movement of phenylalanine-87 and trytophan-28, and creating site 2. dATP hydrogen bonds to histidine-59, preventing its movement. The importance of site 2 in the restoration of RNR activity by ATP is confirmed by mutagenesis. These findings have implications for the design of bacterial RNR inhibitors.

α-Amino-β-carboxymuconate-ε-semialdehyde decarboxylase catalyzes Enol/Keto tautomerization of oxaloacetate

ACMSD (α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase) is a key metalloenzyme critical for regulating de novo endogenous NAD+/NADH biosynthesis through the tryptophan-kynurenine pathway. This decarboxylase is a recognized target implicated in mitochondrial diseases and neurodegenerative disorders. However, unraveling its enzyme-substrate complex has been challenging due to its high catalytic efficiency. Here, we present a combined biochemical and structural study wherein we determined the crystal structure of ACMSD in complex with malonate. Our analysis revealed significant rearrangements in the active site, particularly in residues crucial for ACMS decarboxylation, including Arg51, Arg239* (a residue from an adjacent subunit), His228, and Trp194. Docking modeling studies proposed a putative ACMS binding mode. Additionally, we found that ACMSD catalyzes oxaloacetic acid (OAA) tautomerization at a rate of 6.51 ± 0.42 s-1 but not decarboxylation. The isomerase activity of ACMSD on OAA warrants further investigation in future biological studies. Subsequent mutagenesis studies and crystallographic analysis of W194A variant shed light on the roles of specific second-coordination sphere residues. Our findings indicate that Arg51 and Arg239* are crucial for OAA tautomerization. Moreover, our comparative analysis with related isomerase superfamily members underscores a general strategy employing arginine residues to promote OAA isomerization. Given the observed isomerase activity of ACMSD on OAA and its structural similarity to ACMS, we propose that ACMSD may facilitate isomerization to ensure ACMS is in the optimal tautomeric form for subsequent decarboxylation initiated by the zinc-bound hydroxide ion. Overall, these findings deepen the understanding of the structure and function of ACMSD, offering insights into potential therapeutic interventions.

Targeting LDL aggregation decreases atherosclerotic lipid burden in a humanized mouse model of familial hypercholesterolemia. Crucial role of ApoB100 conformational stabilization

Backgroundand aims; Low-density lipoprotein (LDL) aggregation is nowadays considered a therapeutic target in atherosclerosis. DP3, the retro-enantio version of the sequence Gly1127-Cys1140 of LRP1, efficiently inhibits LDL aggregation and foam cell in vitro formation. Here, we investigate whether DP3 modulates atherosclerosis in a humanized ApoB100, LDL receptor (LDLR) knockout mice (Ldlr–/–hApoB100 Tg) and determine the potential LDL-related underlying mechanisms. MethodsTg mice were fed an HFD for 21 days to induce atherosclerosis and then randomized into three groups that received a daily subcutaneous administration (10 mg/kg) of i) vehicle, ii) DP3 peptide, or iii) a non-active peptide (IP321). The in vivo biodistribution of a fluorescent-labeled peptide version (TAMRA-DP3), and its colocalization with ApoB100 in the arterial intima, was analyzed by imaging system (IVIS) and confocal microscopy. Heart aortic roots were used for atherosclerosis detection and quantification. LDL functionality was analyzed by biochemical, biophysical, molecular, and cellular studies. ResultsIntimal neutral lipid accumulation in the aortic root was reduced in the DP3-treated group as compared to control groups. ApoB100 in LDLs from the DP3 group exhibited an increased percentage of α-helix secondary structures and decreased immunoreactivity to anti-ApoB100 antibodies. LDL from DP3-treated mice were protected against passive and sphingomyelinase (SMase)-induced aggregation, although they still experienced SMase-induced sphingomyelin phospholysis. In patients with familial hypercholesterolemia (FH), DP3 efficiently inhibited both SMase-induced phospholysis and aggregation. ConclusionsDP3 peptide administration inhibits atherosclerosis by preserving the α-helix secondary structures of ApoB100 in a humanized ApoB100 murine model that mimicks the hallmark of human hypercholesterolemia.

First Comprehensive Evaluation of Genetic Variability for Bacterial Blight Resistance in Wild Punica granatum L. Populations from the North-Western Himalayas

Identifying and selecting disease-resistant sources from wild germplasm pools is pivotal due to their ecological benefits and cost-effectiveness. This study encompassed the survey and collection of wild pomegranate genotypes alongside commercial cultivars, followed by screening against native pathogens. Additionally, biochemical analyses were conducted to elucidate defense mechanisms in both resistant and susceptible genotypes. Cuttings from selected germplasm across four districts were obtained, grown for one year, and assessed for pathogenicity and biochemical responses. Microscopic and molecular analyses confirmed the pathogen as Xanthomonas axonopodis pv. punicae (Xap), identified by its distinctive morphology and a 1500 bp amplicon along with phylogeny analysis. Germplasm screening via detached and attached leaf assays identified genotypes with minimal disease severity, highlighting SH-14 and SH-16 as highly resistant in the attached leaf assay. Principal component analysis categorized genotypes based on disease parameters, with PC1 explaining approximately 75% of the variability. Agglomerative hierarchical clustering further grouped genotypes into ten clusters, emphasizing clusters IX and X for their significant resistance traits. Biochemical studies revealed that the resistant genotype SH-16 exhibited higher levels of constitutive defense components like chlorophyll and vitamin C, as well as induced defense components such as phenols, flavonoids, and antioxidants, compared to the susceptible cultivar Bhagwa. These findings underscore the critical roles of both constitutive and induced defense mechanisms in conferring pomegranate resistance to Xap. The study's comprehensive approach in evaluating genetic and biochemical resistance provides valuable insights for breeding disease-resistant pomegranate varieties, marking a significant advancement in exploring pomegranate wild relatives from the Himalayan region for their resistance to bacterial blight.

Shedding light on cancer immunology at the molecular level: A quantum biochemistry study of representative PD-1/PD-L1 conformations

BackgroundProgrammed death 1 (PD-1) binding to PD-L1 is a potent mechanism used by immunogenic tumors to evade the immune system and the immune checkpoint PD-1PD-L1 has emerged as a promising target in the search for new drugs to improve cancer treatment. The crystallographic structure of humanPD-1humanPD-L1 shed light on the molecular characterization of this system and allowed computational studies to be carried out to characterize structural behaviors. MethodsThis study demonstrated the importance of analyzing the flexibility of protein systems through molecular dynamics simulations (MDS) and its impacts on the interaction energy obtained through quantum biochemistry. ResultsThe computational results obtained provide a description of the flexibility and energetic profile of the PD-1PD-L1 contact surface using representative conformations from MDS. Variations of up to 50 % in the total interaction energy values were detected depending on the scrutinized conformation, which can be mainly attributed to the flexibility of the CC' loop, FG loop and ASP85-GLN91 of PD-1 and the MET58-LYS62 segment of PD-L1. Quantum biochemistry revealed the three hot spots in PD-L1: ARG113L-ARG125L > ILE54L-VAL76L > ALA18L-ASP26L; and two energetic hot spots in PD-1: ALA125-ARG139 > VAL63-GLN88. Nonetheless, VAL63-GLN88 and GLY124-ARG139 exhibit significant variation in interaction energy between different conformations, while ARG113L-ARG125L is the only hot spot with high energetic fluctuation on the PD-L1 surface. ConclusionThis is the first application of MDS coupled to dimensionality reduction and density functional theory (DFT) demonstrating new structural and energetic features that might be useful in discovering/designing more potent PD-1PD-L1 inhibitors.

Evolution of aromatic amino acid metabolism in plants: a key driving force behind plant chemical diversity in aromatic natural products.

A diverse array of plant aromatic compounds contributes to the tremendous chemical diversity in the plant kingdom that cannot be seen in microbes or animals. Such chemodiversity of aromatic natural products has emerged, occasionally in a lineage-specific manner, to adopt to challenging environmental niches, as various aromatic specialized metabolites play indispensable roles in plant development and stress responses (e.g. lignin, phytohormones, pigments and defence compounds). These aromatic natural products are synthesized from aromatic amino acids (AAAs), l-tyrosine, l-phenylalanine and l-tryptophan. While amino acid metabolism is generally assumed to be conserved between animals, microbes and plants, recent phylogenomic, biochemical and metabolomic studies have revealed the diversity of the AAA metabolism that supports efficient carbon allocation to downstream biosynthetic pathways of AAA-derived metabolites in plants. This review showcases the intra- and inter-kingdom diversification and origin of committed enzymes involved in plant AAA biosynthesis and catabolism and their potential application as genetic tools for plant metabolic engineering. I also discuss evolutionary trends in the diversification of plant AAA metabolism that expands the chemical diversity of AAA-derived aromatic natural products in plants. This article is part of the theme issue 'The evolution of plant metabolism'.

Native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in mitochondria of etiolated pea sprouts

Numerous biochemical and structural studies into the native organization of oxidative phosphorylation in the mitochondria of various eukaryotic organisms have convincingly shown that respiratory complexes can associate with one another to form higher-order structures referred to as supercomplexes. Plant mitochondria are distinguished by a more complicated organization of the respiratory chain due to the presence of a number of alternative oxidoreductases. It is considered that these enzymes do not physically interact with those of the cytochrome pathway. However, the available literature data obtained on yeast mitochondria suggests the possibility of such an association. In this regard, we aimed to study the native organization of alternative NAD(P)H-dehydrogenases NDA and NDB in plant mitochondria. The work was performed on six-day etiolated pea seedlings. The 2D BN/SDS-PAGE in combination with immunochemistry found that, in pea organelles, the main part of the populations of NDA and NDB alternative NAD(P)H dehydrogenases were included in superstructures with masses of 700, 780, and 900 kDa. Additionally, NDA was detected in the region of 1480 and 1600 kDa, and NDB was registered at values of 1330, 340, and 100–120 kDa. An analysis of subunit profiles of the observed associations and a colorimetric detection of ATPase activity in 1D BN-gel suggested that the major part of the NDA and NDB populations identified by the available antibodies was associated with ATP synthase and represented a heterogeneous population of ATP-synthasomes, assumably, with a NDA2/NDB2Va/b1-2 composition. The rest of the enzymes were likely to be part of the NDA2/NDB2III2IV and NDA2IV1Va2 supercomplexes. The physiological significance of the association of alternative NAD(P)H dehydrogenases with ATP synthase requires further study.

DNA-Based Molecular Clamp for Probing Protein Interactions and Structure under Force.

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology, and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA-based device in which the transition between single- and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension, while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Review of p.(Val429Met), a Variant of LDLR That Is Associated with Familial Hypercholesterolemia

Patients affected by familial hypercholesterolemia possess elevated low-density lipoprotein cholesterol and therefore have greater risk for cardiovascular disease. About 90% of familial hypercholesterolemia cases are associated with aberrant LDLR. Over 3500 LDLR variants have been identified, 15% of which are considered “pathogenic.” Given the genetic diversity of LDLR variants, specific variants rarely receive attention. However, investigators have proposed the critical evaluation of individual variants as a method to clarify knowledge and to resolve discrepancies in the literature. This article reviews p.(Val429Met) (rs28942078) in the areas of pathology, epidemiology, lipid-lowering therapy, and genetic testing. The p.(Val429Met) variant is associated with a missense point substitution in exon 9 of chromosome 19. Biochemical studies have found severely reduced low-density lipoprotein receptor protein in autologous and heterologous expression systems. Additionally, there are inconsistencies regarding the functional classification of p.(Val429Met). Considered to be of European origin, p.(Val429Met) is found in extant populations due to founder effects. Evidence from clinical trials have also demonstrated variable responses to newer lipid-lowering therapies in patients with a p.(Val429Met) variant. Proper clinical detection and adequate genetic testing have been shown to greatly improve outcomes. Future research may be aimed at resolving discrepancies to better comprehend the implications of familial hypercholesterolemia.

Drugs Targeting Sirtuin 2 Exhibit Broad-Spectrum Anti-Infective Activity.

Direct-acting anti-infective drugs target pathogen-coded gene products and are a highly successful therapeutic paradigm. However, they generally target a single pathogen or family of pathogens, and the targeted organisms can readily evolve resistance. Host-targeted agents can overcome these limitations. One family of host-targeted, anti-infective agents modulate human sirtuin 2 (SIRT2) enzyme activity. SIRT2 is one of seven human sirtuins, a family of NAD+-dependent protein deacylases. It is the only sirtuin that is found predominantly in the cytoplasm. Multiple, structurally distinct SIRT2-targeted, small molecules have been shown to inhibit the replication of both RNA and DNA viruses, as well as intracellular bacterial pathogens, in cell culture and in animal models of disease. Biochemical and X-ray structural studies indicate that most, and probably all, of these compounds act as allosteric modulators. These compounds appear to impact the replication cycles of intracellular pathogens at multiple levels to antagonize their replication and spread. Here, we review SIRT2 modulators reported to exhibit anti-infective activity, exploring their pharmacological action as anti-infectives and identifying questions in need of additional study as this family of anti-infective agents advances to the clinic.

The possible effects of chronic administration of amiodarone hydrochloride on the seminiferous tubules of adult male albino rats: histological and biochemical study

ABSTRACT Amiodarone hydrochloride is an antiarrhythmic agent that is widely prescribed. However, it has serious side effects that approximately affect the whole body organs. In our study, we aimed to assess the possible effects of chronic administration of two different doses of amiodarone hydrochloride on the oxidative and inflammatory parameters as well as the histological morphology and ultrastructure of the seminiferous tubules of adult male albino rats. Forty rats were divided into four groups; Control group 1: each rat did not receive any drugs at all. Control group 2: each rat received 3 ml of 0.16% methylcellulose, orally and daily for 4 weeks. Low dose amiodarone group: each rat received 3 ml of 0.16% methylcellulose contained 3.6 mg amiodarone, orally and daily for 4 weeks. High dose amiodarone group: each rat received 3 ml of 0.16% methylcellulose contained 7.2 mg amiodarone, orally and daily for 4 weeks. Blood samples were collected for measuring serum levels of malondialdehyde, superoxide dismutase, interleukin-6 and tumor necrosis factor-alpha. Testes specimens were examined to assess the morphological changes and the level of expression of caspase-3 apoptotic marker. The results indicated that; amiodarone hydrochloride could induce a dose-dependent toxicity, causing oxidative stress, inflammation, cellular degeneration, deposition of collagen and enhanced apoptosis in the seminiferous tubules.

The role(s) of NF-Y in development and differentiation.

NF-Y is a conserved sequence-specific trimeric Transcription Factor -TF- binding to the CCAAT element. We review here the role(s) in development, from pre-implantation embryo to terminally differentiated tissues, by rationalizing and commenting on genetic, genomic, epigenetic and biochemical studies. This effort brings to light the impact of NF-YA isoforms on stemness and differentiation, as well as binding to distal vs promoter proximal sites and connections with selected TFs.

Evolutionary dynamics of polyadenylation signals and their recognition strategies in protists.

The poly(A) signal, together with auxiliary elements, directs cleavage of a pre-mRNA and thus determines the 3' end of the mature transcript. In many species, including humans, the poly(A) signal is an AAUAAA hexamer, but we recently found that the deeply branching eukaryote Giardia lamblia uses a distinct hexamer (AGURAA) and lacks any known auxiliary elements. Our discovery prompted us to explore the evolutionary dynamics of poly(A) signals and auxiliary elements in the eukaryotic kingdom. We use direct RNA sequencing to determine poly(A) signals for four protists within the Metamonada clade (which also contains G. lamblia) and two outgroup protists. These experiments reveal that the AAUAAA hexamer serves as the poly(A) signal in at least four different eukaryotic clades, indicating that it is likely the ancestral signal, whereas the unusual Giardia version is derived. We find that the use and relative strengths of auxiliary elements are also plastic; in fact, within Metamonada, species like G. lamblia make use of a previously unrecognized auxiliary element where nucleotides flanking the poly(A) signal itself specify genuine cleavage sites. Thus, despite the fundamental nature of pre-mRNA cleavage for the expression of all protein-coding genes, the motifs controlling this process are dynamic on evolutionary timescales, providing motivation for future biochemical and structural studies as well as new therapeutic angles to target eukaryotic pathogens.

A binding site for the antibiotic GE81112 in the ribosomal mRNA channel.

The initiation phase is the rate-limiting step of protein synthesis (translation) and is finely regulated, making it an important drug target. In bacteria, initiation is guided by three initiation factors and involves positioning the start site on the messenger RNA within the P-site on the small ribosomal subunit (30S), where it is decoded by the initiator tRNA. This process can be efficiently inhibited by GE81112, a natural hydrophilic, noncyclic, nonribosomal tetrapeptide. It is found in nature in three structural variants (A, B and B1 with molecular masses of 643-658 Da). Previous biochemical and structural characterisation of GE81112 indicates that the primary mechanism of action of this antibiotic is to (1) prevent the initiator tRNA from binding correctly to the P-site and (2) block conformational rearrangements in initiation factor IF3, resulting in an unlocked 30S pre IC state. In this study, using cryoEM, we have determined the binding site of GE81112 in initiation complexes (3.2-3.7Å) and on empty ribosomes (2.09 Å). This binding site is within the mRNA channel (E-site) but remote from the binding site of the initiation factors and initiator tRNA. This suggests that it acts allosterically to prevent the initiator tRNA from being locked into place. The binding mode is consistent with previous biochemical studies and recent work identifying the key pharmacophores of GE81112.

Biochemical Studies on Efficiency of Natural Gum in Chronic Kidney Failure and Liver Cirrhosis in Rats

It is well-established that apoptosis, oxidative stress, and inflammation are associated with several disorders, including chronic renal disease and hepatic disease. Oxidative stress (OS) is a major cause of death from end-stage renal disease which also contributes to atherosclerosis and cardiac issues. The present study aimed to assess the efficacy of Gum Arabic (GA) in mitigating renal damage and hepatotoxicity in rats induced by Chloropyrifos-methyl (CPM). A total of 42 male Wistar rats were divided into seven groups, with four groups (group 2 [IC], group 5 [GA1+IC]a, group 6 [GA2+IC], and group 7 [GA1+IC]b treated with CPM for eight weeks to induce hepatic and renal damage. Two models of GA administration, including the standard oral model in drinking water (15% w/v) and the oral model by gavage at a dose of 1 g/kg body weight were administered. Physiological parameters of kidney and liver functions, including urea, creatinine, AST, and ALT along with anti-oxidant factors (Melaodialdehyde, superoxide dismutase, reduced glutathione, and catalase) were measured in plasma, and homogenates of renal and hepatic tissues on day 57 of the experiment. In addition, histopathological examination was conducted on liver and kidney tissues using hematoxylin and eosin stain to evaluate the efficacy of GA on damaged tissues. Gum Arabic was found to significantly reduce CPM toxic effects in the liver and kidney in groups treated with CPM as liver and kidney parameters were reduced to normal levels. Furthermore, GA reduced histological indicators of inflammation, fibrosis, and apoptosis, as well as renal morphological damage. Additionally, it reduced OS in liver and kidney homogenates. In conclusion, GA effectively reduced the damage that CPM inflicted on liver and kidney tissue by stabilizing physiological parameters to normal levels and repairing cellular structures damaged by OS. Keywords: Antioxidant, Anti-inflammatory, Gum Arabic, Kidney, Liver, Oxidative stress

Structure and Dynamics of the CCCH-Type Tandem Zinc Finger Domain of POS-1 and Implications for RNA Binding Specificity.

CCCH-type tandem zinc finger (TZF) motifs are found in many RNA-binding proteins involved in regulating mRNA stability, translation, and splicing. In Caenorhabditis elegans, several RNA-binding proteins that regulate embryonic development and cell fate determination contain CCCH TZF domains, including POS-1. Previous biochemical studies have shown that despite high levels of sequence conservation, POS-1 recognizes a broader set of RNA sequences compared to the human homologue tristetraprolin. However, the molecular basis of these differences remains unknown. In this study, we refined the consensus RNA sequence and determined the differing binding specificities of the two zinc fingers of POS-1. We also determined the solution structure and characterized the internal dynamics of the TZF domain of POS-1. From the structure, we identified unique features that define the RNA binding specificity of POS-1. We also observed that the TZF domain of POS-1 is in equilibrium between interconverting conformations. Transitions between these conformations require internal motions involving many residues with correlated dynamics in each ZF. We propose that the correlated dynamics are necessary to allow allosteric communication between the nucleotide-binding pockets observed in the N-terminal ZF. Our study shows that both the structure and conformational plasticity of POS-1 are important in ensuring recognition of its RNA binding targets.

Nigella sativa oil mitigate paraquat-induced neurobehavioural, oxidative and histological alterations in the cerebellum of adult male wistar rat

Over the past few decades, several cases of poisoning and death resulting from environmental exposure to the herbicide, paraquat (PQ), has been reported. However, nigella sativa oil, with several antioxidants and anti-inflammatory properties present a significant tool against PQ-induced toxicity. Thus, the study aim was to assess the possible mitigative effect of nigella sativa oil administration on paraquat-induced neurotoxicity in the cerebellum. Twenty-eight (28) adult Wistar rats were randomly divided into four groups of seven (7) rats each. Group I (control) received 0.3ml of normal saline daily; Group II received 0.1ml of paraquat daily; Group III received 0.1ml of paraquat and 0.3ml of nigella sativa daily; and Group IV received 0.3ml of nigella sativa daily. Administrations were done via oral gavage and experimentation lasted for 14 days. On day 13 of the experiment, animals from all groups were subjected to neurobehavioral tests using hanging wire test for grip period. Afterwards, the rats were sacrificed using anesthesia. Blood samples was collected and the brains were harvested for biochemical and histological studies. The result from this study revealed a decrease in grip time in the hanging wire test, in the group administered PQ only when compared to the control. However, the groups administered nigella sativa oil (NSO) showed an increase in grip time compared to the PQ only treated group, these differences are however insignificant. Also, a marked decrease in the GST and TAC levels were observed in the group exposed to PQ only when compared to the control group. However, the groups treated with NSO showed marked increase in these antioxidants level compared to the PQ group. Also, however insignificant, PQ exposure caused a slight increase in ROS levels compared to the control, and slight decrease in ROS levels in the PQ+NSO and NSO respectively was recorded. Distortion in the histoarchitecture of the cerebellum, were also recorded in the PQ-treated group when compared to the control. However, preservation of general histoarchitecture was observed in these brain regions in the treatment groups. Keywords: Sativia Oil, Experiment, Wire Test.