Plasmodium falciparum hypoxanthine guanine phosphoribosyltransferase. Stability studies on the product-activated enzyme

Hypoxanthine guanine phosphoribosyltransferases (HGPRTs) catalyze the conversion of 6-oxopurine bases to their respective nucleotides, the phosphoribosyl group being derived from phosphoribosyl pyrophosphate. Recombinant Plasmodium falciparum HGPRT, on purification, has negligible activity, and previous reports have shown that high activities can be achieved upon incubation of recombinant enzyme with the substrates hypoxanthine and phosphoribosyl pyrophosphate [Keough DT, Ng AL, Winzor DJ, Emmerson BT & de Jersey J (1999) Mol Biochem Parasitol98, 29-41; Sujay Subbayya IN & Balaram H (2000) Biochem Biophys Res Commun279, 433-437]. In this report, we show that activation is effected by the product, Inosine monophosphate (IMP), and not by the substrates. Studies carried out on Plasmodium falciparum HGPRT and on a temperature-sensitive mutant, L44F, show that the enzymes are destabilized in the presence of the substrates and the product, IMP. These stability studies suggest that the active, product-bound form of the enzyme is less stable than the ligand-free, unactivated enzyme. Equilibrium isothermal-unfolding studies indicate that the active form is destabilized by 2-3 kcal x mol(-1) compared with the unactivated state. This presents a unique example of an enzyme that attains its active conformation of lower stability by product binding. This property of ligand-mediated activation is not seen with recombinant human HGPRT, which is highly active in the unliganded state. The reversibility between highly active and weakly active states suggests a novel mechanism for the regulation of enzyme activity in P. falciparum.     

Unravelling soil and plant metal relationships in Albanian nickel hyperaccumulators in the genus Odontarrhena (syn. Alyssum sect. Odontarrhena,Brassicaceae)

Plant and Soil - Based on a recent taxonomic revision of the genus Odontarrhena in Albania, our work aimed at comparing the taxa and populations of this genus in terms of nickel accumulation and...     

Functional Analysis of Sesame Diacylglycerol Acyltransferase and Phospholipid: Diacylglycerol Acyltransferase Genes Using In Silico and In Vitro Approaches

Plant Molecular Biology Reporter - Sesamum indicum L. is one of the major oilseed crops of India. Sesame oil is stable with a good source of unsaturated fatty acids. To know the mechanism of oil...     

Evidence for the dispersal of a unique lineage from Asia to America and Africa in the sugarcane fungal pathogen Ustilago scitaminea

The basidiomycete Ustilago scitaminea Sydow, which causes sugarcane smut disease, has been spreading throughout Africa and America since the 1940s. The genetic diversity and structure of different populations of this fungus worldwide was investigated using microsatellites. A total of 142 single-teliospore were isolated from 77 distinct whips (sori) collected in 15 countries worldwide. Mycelium culture derived from on generation of selfing of these single teliospores were analysed for their polymorphisms at 17 microsatellite loci. All these strains but one were homozygous at all loci, indicating that selfing is likely the predominant reproductive mode of U. scitaminea. The genetic diversity of either American or African U. scitaminea populations was found to be extremely low and all strains belong to a single lineage. This lineage was also found in some populations of Asia, where most U. scitaminea genetic diversity was detected, suggesting that this fungal species originated from this region. The strong founder effect observed in U. scitaminea African and American populations suggests that the fungus migrated from Asia to other continents on rare occasions through movement of infected plant material.     

Combination of obesity and co-morbidities leads to unfavorable outcomes in COVID-19 patients

ObjectiveObesity has been described as a significant independent risk factors of COVID-19. We aimed to study the association between obesity, co-morbidities and clinical outcomes of COVID-19.MethodsClinical data from 417 patients were collected retrospectively from the Al Kuwait Hospital, Ministry of Health and Prevention (MOHAP), Dubai, United Arab Emirates, who were admitted between March and June 2020. Patients were divided according to their body mass index (BMI). Various clinical outcomes were examined: presenting symptoms, severity, major co-morbidities, ICU admission, death, ventilation, ARDS, septic shock and laboratory parameters.ResultsThe average BMI was 29 ± 6.2 kg/m². BMI alone was not associated with the outcomes examined. However, class II obese patients had more co-morbidities compared to other groups. Hypertension was the most significant co-morbidity associated with obesity. Patients with BMI above the average BMI (29 kg/m²) and presence of underlying co-morbidities showed significant increase in admission to ICU compared to patients below 29 kg/m² and underlying co-morbidities (21.7% Vs. 9.2%), ARDS development (21.7% Vs. 10.53%), need for ventilation (8.3% Vs. 1.3%), and mortality (10% Vs. 1.3%).ConclusionsOur data suggests that presence of underlying co-morbidities and high BMI work synergistically to affect the clinical outcomes of COVID-19.     

Partial purification of a recombinase activity from human placenta and assay of recombination using rec-blot

We have designed an assay for the detection of recombination intermediates. The assay can also be used for screening for recombinases. In this assay, the nonradioactive plasmid was immobilized on a nylon membrane and the radioactive plasmid counterpart was allowed to recombine in the presence of a DNA binding protein fraction from human placenta. After incubation for 30 min at 30 degrees C, the membrane was washed extensively and dried. The recombination was visualised by autoradiography and quantitated by counting the corresponding areas of the membrane. Recombination was confirmed by transformation using pRDK plasmids and by gel electrophoresis. These results indicate the presence of recombinase activity in the DNA binding protein fraction of human placenta.     

Cloning of the aconitase gene (acn) of Escherichia coli K12

Lambda phages containing the aconitase gene (acn) of Escherichia coli K12 have been isolated by hybridization with an M13 probe containing part of the aconitase gene (citB) of Bacillus subtilis. Aconitase specific activities are amplified 5- to 18-fold in thermally induced lambda acn lysogens and threefold in a strain transformed with a plasmid derivative (pGS181).     

Identification of a Novel Inhibitor of Coactivator-associated Arginine Methyltransferase 1 (CARM1)-mediated Methylation of Histone H3 Arg-17

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, “KAPRK,” where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.     

Latitudinal variation in seeds characteristics of Acer platanoides and A. pseudoplatanus

Climate change will likely affect population dynamics of numerous plant species by modifying several aspects of the life cycle. Because plant regeneration from seeds may be particularly vulnerable, here we assess the possible effects of climate change on seed characteristics and present an integrated analysis of seven seed traits (nutrient concentrations, samara mass, seed mass, wing length, seed viability, germination percentage, and seedling biomass) of Acer platanoides and A. pseudoplatanus seeds collected along a wide latitudinal gradient from Italy to Norway. Seed traits were analyzed in relation to the environmental conditions experienced by the mother trees along the latitudinal gradient. We found that seed traits of A. platanoides were more influenced by the climatic conditions than those of A. pseudoplatanus. Additionally, seed viability, germination percentage, and seedling biomass of A. platanoides were strongly related to the seed mass and nutrient concentration. While A. platanoides seeds were more influenced by the environmental conditions (generally negatively affected by rising temperatures), compared to A. pseudoplatanus, A. platanoides still showed higher germination percentage and seedling biomass than A. pseudoplatanus. Thus, further research on subsequent life-history stages of both species is needed. The variation in seed quality observed along the climatic gradient highlights the importance of studying the possible impact of climate change on seed production and species demography.