VP2118 has major roles in Vibrio parahaemolyticus response to oxidative stress

Background

Reactive oxygen species (ROS), including superoxide anion radical, induce chronic risk of oxidative damage to many cellular macromolecules resulting in damage to cells. Superoxide dismutases (SODs) catalyze the dismutation of superoxide to oxygen and hydrogen peroxide and are a primary defense against ROS. Vibrio parahaemolyticus, a marine bacterium that causes acute gastroenteritis following consumption of raw or undercooked seafood, can survive ROS generated by intestinal inflammatory cells. However, there is little information concerning SODs in V. parahaemolyticus. This study aims to clarify the role of V. parahaemolyticus SODs against ROS.

Methods

V. parahaemolyticus SOD gene promoter activities were measured by a GFP reporter assay. Mutants of V. parahaemolyticus SOD genes were constructed and their SOD activity and resistance to oxidative stresses were measured.

Results

Bioinformatic analysis showed that V. parahaemolyticus SODs were distinguished by their metal cofactors, FeSOD (VP2118), MnSOD (VP2860), and CuZnSOD (VPA1514). VP2118 gene promoter activity was significantly higher than the other SOD genes. In a VP2118 gene deletion mutant, SOD activity was significantly decreased and could be recovered by VP2118 gene complementation. The absence of VP2118 resulted in significantly lowered resistance to ROS generated by hydrogen peroxide, hypoxanthine–xanthine oxidase, or Paraquat. Furthermore, both the N- and C-terminal SOD domains of VP2118 were necessary for ROS resistance.

Conclusion

VP2118 is the primary V. parahaemolyticus SOD and is vital for anti-oxidative stress responses.

General significance

The V. parahaemolyticus FeSOD VP2118 may enhance ROS resistance and could promote its survival in the intestinal tract to facilitate host tissue infection.     

Inorganic phosphate uptake in unicellular eukaryotes

Background

Inorganic phosphate (P_i) is an essential nutrient for all organisms. The route of P_i utilization begins with P_i transport across the plasma membrane.

Scope of review

Here, we analyzed the gene sequences and compared the biochemical profiles, including kinetic and modulator parameters, of P_i transporters in unicellular eukaryotes. The objective of this review is to evaluate the recent findings regarding P_i uptake mechanisms in microorganisms, such as the fungi Neurospora crassa and Saccharomyces cerevisiae and the parasite protozoans Trypanosoma cruzi, Trypanosoma rangeli, Leishmania infantum and Plasmodium falciparum.

Major conclusion

P_i uptake is the key step of P_i homeostasis and in the subsequent signaling event in eukaryotic microorganisms.

General significance

Biochemical and structural studies are important for clarifying mechanisms of P_i homeostasis, as well as P_i sensor and downstream pathways, and raise possibilities for future studies in this field.     

Molecular basis of thermal stability in truncated (2/2) hemoglobins

Background

Understanding the molecular mechanism through which proteins are functional at extreme high and low temperatures is one of the key issues in structural biology. To investigate this phenomenon, we have focused on two instructive truncated hemoglobins from Thermobifida fusca (Tf-trHbO) and Mycobacterium tuberculosis (Mt-trHbO); although the two proteins are structurally nearly identical, only the former is stable at high temperatures.

Methods

We used molecular dynamics simulations at different temperatures as well as thermal melting profile measurements of both wild type proteins and two mutants designed to interchange the amino acid residue, either Pro or Gly, at E3 position.

Results

The results show that the presence of a Pro at the E3 position is able to increase (by 8°) or decrease (by 4°) the melting temperature of Mt-trHbO and Tf-trHbO, respectively. We observed that the ProE3 alters the structure of the CD loop, making it more flexible.

Conclusions

This gain in flexibility allows the protein to concentrate its fluctuations in this single loop and avoid unfolding. The alternate conformations of the CD loop also favor the formation of more salt-bridge interactions, together augmenting the protein's thermostability.

General significance

These results indicate a clear structural and dynamical role of a key residue for thermal stability in truncated hemoglobins.     

SLC45A2 mutation frequency in Oculocutaneous Albinism Italian patients doesn't differ from other European studies

Background

Oculocutaneous Albinism (OCA) is a heterogeneous group of inherited diseases involving hair, skin and eyes. To date, six forms are recognized on the effects of different melanogenesis genes.OCA4 is caused by mutations in SLC45A2 showing a heterogeneous phenotype ranging from white hair, blue irides and nystagmus to brown/black hair, brown irides and no nystagmus. The high clinic variety often leads to misdiagnosis.Our aim is to contribute to OCA4 diagnosis defining SLC45A2 genetic variants in Italian patients with OCA without any TYR, OCA2 and TYRP1 gene defects.

Materials and methods

After the clinical diagnosis of OCA, all patients received genetic counseling and genetic test. Automatic sequencing of TYR, OCA2, and TYRP1 genes was performed on DNA of 117 albino patients. Multiplex Ligation-dependent Probe Amplification (MLPA) was carried out on TYR and OCA2 genes to increase the mutation rate. SLC45A2 gene sequencing was then executed in the patients with a single mutation in one of the TYR, OCA2, TYRP1 genes and in the patients, which resulted negative at the screening of these genes.

Results

SLC45A2 gene analysis was performed in 41 patients and gene alterations were found in 5 patients. Four previously reported SLC45A2 mutations were found: p.G100S, p.W202C, p.A511E and c.986delC, and three novel variants were identified: p.M265L, p.H94D, and c.1156+1G>A. All the alterations have been detected in the group of patients without mutations in the other OCA genes.

Conclusions

Three new variants were identified in OCA4 gene; the analysis allowed the classification of a patient previously misdiagnosed as OA1 because of skin and hair pigmentation presence. The molecular defects in SLC45A2 gene represent the 3.4% in this cohort of Italian patients, similar to other Caucasian populations; our data differ from those previously published by an Italian researcher group, obtained on a smaller cohort of patients.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.     

Reduced glycerol incorporation into phospholipids contributes to impaired intra-erythrocytic growth of glycerol kinase knockout Plasmodium falciparum parasites

Background

Malaria is a devastating disease and Plasmodium falciparum is the most lethal parasite infecting humans. Understanding the biology of this parasite is vital in identifying potential novel drug targets. During every 48-hour intra-erythrocytic asexual replication cycle, a single parasite can produce up to 32 progeny. This extensive proliferation implies that parasites require substantial amounts of lipid precursors for membrane biogenesis. Glycerol kinase is a highly conserved enzyme that functions at the interface of lipid synthesis and carbohydrate metabolism. P. falciparum glycerol kinase catalyzes the ATP-dependent phosphorylation of glycerol to glycerol-3-phosphate, a major phospholipid precursor.

Methods

The P. falciparum glycerol kinase gene was disrupted using double crossover homologous DNA recombination to generate a knockout parasite line. Southern hybridization and mRNA analysis were used to verify gene disruption. Parasite growth rates were monitored by flow cytometry. Radiolabelling studies were used to assess incorporation of glycerol into parasite phospholipids.

Results

Disruption of the P. falciparum glycerol kinase gene produced viable parasites, but their growth was significantly reduced to 56.5 ± 1.8% when compared to wild type parasites. ¹⁴C-glycerol incorporation into the major phospholipids of the parasite membrane, phosphatidylcholine and phosphatidylethanolamine, was 48.4 ± 10.8% and 53.1 ± 5.7% relative to an equivalent number of wild type parasites.

Conclusions

P. falciparum glycerol kinase is required for optimal intra-erythrocytic asexual parasite development. Exogenous glycerol may be used as an alternative carbon source for P. falciparum phospholipid biogenesis, despite the lack of glycerol kinase to generate glycerol-3-phosphate.

General significance

These studies provide new insight into glycerolipid metabolism in P. falciparum.     

Polymorphisms of the TLR4 gene and risk of gastric cancer

Objective

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene–environmental interactions on the risk of GC in Northeastern China.

Methods

We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models.

Results

Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR = 0.33, 95% CI 0.18–0.60, P < 0.001 and adjusted OR = 0.37, 95% CI 0.21–0.67, P = 0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC.

Conclusion

Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC.     

miRNA–miRNA interaction implicates for potential mutual regulatory pattern

Background

Natural or endogenous sense/antisense miRNAs, located on sense and antisense strands in the same genomic region, respectively, are detected recently. However, little is known about these miRNA pairs, especially for their distributions in different animal species. We herein present systematic analysis of them in human, mouse and rat miRNAs, and their expression patterns based on deep sequencing datasets.

Methods and results

The phenomenon of miRNA–miRNA interaction could be detected in different animal species. The common miRNAs pairs were found across species. These miRNA pairs could form miRNA:miRNA duplex with complete complementary structure, and were prone to be located on specific chromosomes. They might be homologous miRNA genes (especially in human), or clustered in a gene cluster (especially in rat), or simultaneously detected in different genomic regions due to multicopy pre-miRNAs. Remarkably, some miRNA pairs, located in different genomic regions, also showed complementarity as well as endogenous sense/antisense miRNAs. Based on published deep sequencing datasets, one member of miRNA pairs always was abundantly expressed, whereas another was quite rare. Rare common target mRNAs of these miRNA pairs were predicted.

Conclusions

Interaction between miRNAs and significant expression divergence implied complex potential mutual regulatory pattern in the miRNA world. The study would enrich miRNA regulatory network.     

Importance of cytochrome c redox state for ceramide-induced apoptosis of human mammary adenocarcinoma cells

Background

Ceramides are intracellular lipid mediator implicated in various cellular responses, including oxidative stress and programmed cell death. Studies demonstrated strong links between ceramide and the mitochondria in the regulation of apoptosis. However, the mechanism of apoptosis induced by ceramides is not fully understood. The present study delineates importance of the redox state of cytochrome c for release of cytochrome c and apoptosis of human mammary adenocarcinoma MCF-7 and MDA-MB-231 cells induced by ceramides.

Methods

The study uses MCF-7 and MDA-MB-231 cells, isolated mitochondria, submitochondrial particles, and oxidized and reduced cytochrome c. Methods used include flow cytometry, immunoblotting, spectroscopy, and respirometry.

Results

We show that ceramides induce mitochondrial oxidative stress and release of cytochrome c from the mitochondria of these cells. Our findings show that ceramides react with oxidized cytochrome c whereas reduced cytochrome c does not react with ceramides. We also show that oxidized cytochrome c reacted with ceramides exerts lower reducibility and function to support mitochondrial respiration. Furthermore, our data show that glutathione protects cytochrome c of reacting with ceramides by increasing the reduced state of cytochrome c.

Conclusions

Ceramides induce oxidative stress and apoptosis in human mammary adenocarcinoma cells by interacting with oxidized cytochrome c leading to the release of cytochrome c from the mitochondria. Our findings suggest a novel mechanism for protective role of glutathione.

General significance

Our study suggests that the redox state of cytochrome c is important in oxidative stress and apoptosis induced by ceramides.     

Polyunsaturated fatty acids cause apoptosis in C. albicans and C. dubliniensis biofilms

Background

Polyunsaturated fatty acids (PUFAs) have antifungal properties, but the mode by which they induce their action is not always clear. The aim of the study was to investigate apoptosis as a mode of action of antifungal PUFAs (stearidonic acid, eicosapentaenoic acid and docosapentaenoic acid) which are inhibitory towards biofilm formation of C. albicans and C. dubliniensis.

Methods

Candida biofilms were grown in the absence or presence of 1 mM PUFAs (linoleic acid, stearidonic acid, eicosapentaenoic acid, docosapentaenoic acid) for 48 h at 37 °C. The effect of these PUFAs on the membrane fatty acid profile and unsaturation index, oxidative stress, mitochondrial transmembrane potential and apoptosis was evaluated.

Results

When biofilms of C. albicans and C. dubliniensis were exposed to certain PUFAs there was an increase in unsaturation index of the cellular membranes and accumulation of intracellular reactive oxygen species (ROS). This resulted in apoptosis, evidenced by reduced mitochondrial membrane potential and nuclear condensation and fragmentation. The most effective PUFA was stearidonic acid.

Conclusions

The resultant cell death of both C. albicans and C. dubliniensis is due to apoptosis.

General significance

Due to the increase in drug resistance, alternative antifungal drugs are needed. A group of natural antifungal compounds is PUFAs. However, understanding their mechanisms of action is important for further use and development of these compounds as antifungal drugs. This paper provides insight into a possible mode of action of antifungal PUFAs.