Plasmodium knowlesi is the leading cause of malaria in Malaysia. Serine Repeat Antigens (SERAs) have an essential role in the parasite life cycle. However, genetic characterization on P. knowlesi SERA3 Ag2 (PkSERA3 Ag2) is lacking. In the present study, nucleotide diversity, natural selection, and haplotypes of PkSERA3 Ag2 in clinical samples from Peninsular Malaysia and Malaysian Borneo were investigated. A total of 50 P. knowlesi clinical samples were collected from Peninsular Malaysia and Malaysian Borneo. The PkSERA3 Ag2 gene was amplified using PCR, and subsequently cloned and sequenced. Genetic diversity, haplotype, natural selection as well as genetic structure and differentiation of PkSERA3 Ag2 were analysed. In addition, in silico analyses were performed to identify repeat motifs, B-cell epitopes, and antigenicity indices of the protein. Analysis of 114 PkSERA3 Ag2 sequences revealed high nucleotide diversity of the gene in Malaysia. A codon-based Z-test indicated that the gene underwent purifying selection. Haplotype and population structure analyses identified two distinct PkSERA3 Ag2 clusters (K = 2, ΔK = 721.14) but no clear genetic distinction between PkSERA3 Ag2 from Peninsular Malaysia and Malaysian Borneo. FST index indicated moderate differentiation of the gene. In silico analyses revealed unique repeat motifs among PkSERA3 Ag2 isolates. Moreover, the amino acid sequence of PkSERA3 Ag2 exhibited potential B-cell epitopes and possessed high antigenicity indices. These findings enhance the understanding of PkSERA3 Ag2 gene as well as its antigenic properties. Further validation is necessary to ascertain the utility of PkSERA3 Ag2 as a serological marker for P. knowlesi infection.
Zidovudine (ZDV) is converted to its active triphosphate (ZDVTP) by intracellular kinases. The intermediate ZDV monophosphate (ZDVMP) is believed to play a major role in ZDV toxicity. Manipulation of ZDV phosphorylation is a possible therapeutic strategy for altering the risk-benefit ratio. Here we investigate whether combining RBV with ZDV is able to modulate efficacy and toxicity of ZDV. We have measured the intracellular activation of ZDV (0.3 microM) in the absence and presence of ribavirin (RBV; 2 and 20 microM) in Molt 4 and U937 cells. MTT cytotoxicity of ZDV (10-1000 microM) was also measured with and without RBV (2 microM) in Molt 4 and U937 cells. Measurement of endogenous deoxythymidine triphosphate (dTTP) allowed investigation of the dTTP/ZDVTP ratio. The antiviral efficacy of ZDV in combination with RBV (2 microM) was assessed by HIV p24 antigen measurements. In the presence of RBV (2 and 20 microM) a decrease in total ZDV phosphates was observed, owing mainly to an effect primarily on ZDVMP rather than the active ZDVTP. RBV also increased endogenous dTTP pools in both cell types, resulting in an increase in the dTTP/ZDVTP ratio. ZDV alone significantly reduced p24 antigen production, with an IC50 of 0.34 microM. Addition of RBV increased the IC50 approximately fivefold (1.52 microM). However, at higher concentrations of ZDV (10 and 100 microM) the antagonistic effect of RBV (2 microM) on ZDV was lost. The RBV-mediated decrease in ZDVMP may explain the reduction in ZDV toxicity when combined with RBV (2 microM). Cytotoxicity of ZDV was reduced in the presence of RBV (2 microM) at all concentrations in both cell lines, probably owing to saturation of ZDVTP formation. The interaction of ZDV and RBV is concentration dependent.
The biochemical events associated with the onset of lipid accumulation in Mucor circinelloides and Mortierella alpina, under conditions of nitrogen-limited growth, have been elucidated; they differ in key aspects from those described in oleaginous yeasts. The NAD+:isocitrate dehydrogenases of Mc. circinelloides and Mort. alpina were not absolutely dependent on AMP for activity. Furthermore, changes in the cellular adenine nucleotide pools and energy charge were different from those reported for oleaginous yeasts. In Mc. circinelloides ATP, ADP and AMP concentrations all decreased by 50% after nitrogen limitation, leading to a constant energy charge at the expense of the size of the total adenylate pool. Pyruvate carboxylase in Mc. circinelloides was cytosolic, having implications for the organization of lipid synthesis in filamentous fungi. As a result of the data obtained, a revised and more concerted mechanism for the initiation of storage lipid accumulation is put forward for filamentous fungi.
Cassava brown streak disease (CBSD) is a leading cause of cassava losses in East and Central Africa, and is currently having a severe impact on food security. The disease is caused by two viruses within the Potyviridae family: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV), which both encode atypical Ham1 proteins with highly conserved inosine triphosphate (ITP) pyrophosphohydrolase (ITPase) domains. ITPase proteins are widely encoded by plant, animal, and archaea. They selectively hydrolyse mutagenic nucleotide triphosphates to prevent their incorporation into nucleic acid and thereby function to reduce mutation rates. It has previously been hypothesized that U/CBSVs encode Ham1 proteins with ITPase activity to reduce viral mutation rates during infection. In this study, we investigate the potential roles of U/CBSV Ham1 proteins. We show that both CBSV and UCBSV Ham1 proteins have ITPase activities through in vitro enzyme assays. Deep-sequencing experiments found no evidence of the U/CBSV Ham1 proteins providing mutagenic protection during infections of Nicotiana hosts. Manipulations of the CBSV_Tanza infectious clone were performed, including a Ham1 deletion, ITPase point mutations, and UCBSV Ham1 chimera. Unlike severely necrotic wild-type CBSV_Tanza infections, infections of Nicotiana benthamiana with the manipulated CBSV infectious clones do not develop necrosis, indicating that that the CBSV Ham1 is a necrosis determinant. We propose that the presence of U/CBSV Ham1 proteins with highly conserved ITPase motifs indicates that they serve highly selectable functions during infections of cassava and may represent a euphorbia host adaptation that could be targeted in antiviral strategies.
The effects of sulfide on the energy metabolism of Boleophthalmus boddaerti in normoxia and hypoxia were examined. The 24-, 48-, and 96-h LC50 values of sulfide for B. boddaerti with body weight ranging from 11.6 to 14.2 g were 0.786, 0.567, and 0.467 mM, respectively. The tolerance of B. boddaerti to sulfide was not due to the presence of a sulfide-insensitive cytochrome c oxidase. There was no accumulation of lactate in the muscle and liver of specimens exposed to sulfide in normoxia. In addition, the levels of ATP, AMP, and energy charge in both the muscle and the liver were unaffected. These results indicate that B. boddaerti was able to sustain the energy supply required for its metabolic needs via mainly aerobic respiration when exposed to sulfide (up to 0.4 mM) in normoxia. Exposure of B. boddaerti simultaneously to hypoxia and 0.2 mM sulfide for 48 h resulted in decreases in the ATP levels in the muscle and liver. However, the energy charge in both tissues remained unchanged, and the level of lactate accumulated in the muscle was too low to have any major contribution to the energy budget of the fish. Our results reveal that B. boddaerti possesses inducible mechanisms to detoxify sulfide in an ample supply or a lack of O2. In normoxia, it detoxified sulfide to sulfate, sulfite, and thiosulfate. There were significant increases in the activities of sulfide oxidase in the muscle and liver of specimens exposed to sulfide, with that in the liver being >13-fold higher than that in the muscle. However, in hypoxia, sulfide oxidase activity in the liver was suppressed in response to environmental sulfide. In such conditions, there were significant increases in the activities of sulfane sulfur-forming enzyme(s) in the muscle and liver that were not observed in specimens exposed to sulfide in normoxia. Correspondingly, there were no changes in the levels of sulfate or sulfite in the muscle or liver. Instead, B. boddaerti detoxified sulfide mainly to sulfane sulfur in hypoxia. In conclusion, B. boddaerti was able to activate different mechanisms to detoxify sulfide, producing different types of detoxification products in normoxia and hypoxia.
MTH1 is an enzyme that hydrolyzes 8-oxo-dGTP, which is an oxidatively damaged nucleobase, into 8-oxo-dGMP in nucleotide pools to prevent its mis-incorporation into genomic DNA. Selective and potent MTH1-binding molecules have potential as biological tools and drug candidates. We recently developed 8-halogenated 7-deaza-dGTP as an 8-oxo-dGTP mimic and found that it was not hydrolyzed, but inhibited enzyme activity. To further increase MTH1 binding, we herein designed and synthesized 7,8-dihalogenated 7-deaza-dG derivatives. We successfully synthesized multiple derivatives, including substituted nucleosides and nucleotides, using 7-deaza-dG as a starting material. Evaluations of the inhibition of MTH1 activity revealed the strong inhibitory effects on enzyme activity of the 7,8-dihalogenated 7-deaza-dG derivatives, particularly 7,8-dibromo 7-daza-dGTP. Based on the results obtained on kinetic parameters and from computational docking simulating studies, these nucleotide analogs interacted with the active site of MTH1 and competitively inhibited the substrate 8-oxodGTP. Therefore, novel properties of repair enzymes in cells may be elucidated using new compounds.