A Method for Molecular Analysis of Catalase Gene Diversity in Seawater

Catalase plays an important role in the metabolism of marine bacteria and has potential impact on the marine environment. Four PCR primers were designed to amplify the catalase gene fragments in marine bacteria by applying metagenomic DNA from Yellow Sea surface water as the template. Of the four reproducible target PCR products, the longest one with 900 bp were chosen for catalase gene library construction by the T-vector and the white Escherichia coli colonies in the library was screened through restriction-digesting the reamplified insert fragments by the selected restriction endonuclease MboI, and then the bands of the resulting products were displayed in the agarose gel by electrophoresis. The unique restriction fragment length polymorphism (RFLP) pattern was selected and the corresponding catalase gene fragments were sequenced, which verified that every unique RFLP pattern represented one type of catalase. This PCR–RFLP method above was established to investigate the bacterial catalase diversity in seawater.     

Pseudomonas fluorescens NZI7 repels grazing by C. elegans,a natural predator

The bacteriovorous nematode Caenorhabditis elegans has been used to investigate many aspects of animal biology, including interactions with pathogenic bacteria. However, studies examining C. elegans interactions with bacteria isolated from environments in which it is found naturally are relatively scarce. C. elegans is frequently associated with cultivation of the edible mushroom Agaricus bisporus, and has been reported to increase the severity of bacterial blotch of mushrooms, a disease caused by bacteria from the Pseudomonas fluorescens complex. We observed that pseudomonads isolated from mushroom farms showed differential resistance to nematode predation. Under nutrient poor conditions, in which most pseudomonads were consumed, the mushroom pathogenic isolate P. fluorescens NZI7 was able to repel C. elegans without causing nematode death. A draft genome sequence of NZI7 showed it to be related to the biocontrol strain P. protegens Pf-5. To identify the genetic basis of nematode repellence in NZI7, we developed a grid-based screen for mutants that lacked the ability to repel C. elegans. The mutants isolated in this screen included strains with insertions in the global regulator GacS and in a previously undescribed GacS-regulated gene cluster, ‘EDB'' (‘edible''). Our results suggest that the product of the EDB cluster is a poorly diffusible or cell-associated factor that acts together with other features of NZI7 to provide a novel mechanism to deter nematode grazing. As nematodes interact with NZI7 colonies before being repelled, the EDB factor may enable NZI7 to come into contact with and be disseminated by C. elegans without being subject to intensive predation.     

The Ataxia telangiectasia-mutated and Rad3-related protein kinase regulates cellular hydrogen sulfide concentrations

The ataxia telangiectasia-mutated and Rad3-related (ATR) serine/threonine kinase plays a central role in the repair of replication-associated DNA damage, the maintenance of S and G2/M-phase genomic stability, and the promotion of faithful mitotic chromosomal segregation. A number of stimuli activate ATR, including persistent single-stranded DNA at stalled replication folks, R loop formation, hypoxia, ultraviolet light, and oxidative stress, leading to ATR-mediated protein phosphorylation. Recently, hydrogen sulfide (H₂S), an endogenous gasotransmitter, has been found to regulate multiple cellular processes through complex redox reactions under similar cell stress environments. Three enzymes synthesize H₂S: cystathionine-β-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase. Since H₂S can under some conditions cause DNA damage, we hypothesized that ATR activity may regulate cellular H₂S concentrations and H₂S-syntheszing enzymes. Here we show that human colorectal cancer cells carrying biallelic knock-in hypomorphic ATR mutations have lower cellular H₂S concentrations than do syngeneic ATR wild-type cells, and all three H₂S-synthesizing enzymes show lower protein expression in the ATR hypomorphic mutant cells. Additionally, ATR serine 428 phosphorylation is altered by H₂S donor and H₂S synthesis enzyme inhibition, while the oxidative-stress induced phosphorylation of the ATR-regulated protein CHK1 on serine 345 is increased by H₂S synthesis enzyme inhibition. Lastly, inhibition of H₂S production potentiated oxidative stress-induced double-stranded DNA breaks in the ATR hypomorphic mutant compared to ATR wild-type cells. Our findings demonstrate that the ATR kinase regulates and is regulated by H₂S.     

Extender Supplementation with Antioxidants Selected after the Evaluation of Sperm Susceptibility to Oxidative Challenges in Goats

This study aimed to detect the most deleterious ROS for goat sperm and then supplemented the extender with a proper antioxidant. For this, 12 adult goats (aged 1–7) were used. Fresh samples were submitted to challenge with different ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and malondialdehyde (MDA—toxic product of lipid peroxidation). After experiment 1, sperms were cryopreserved in extenders supplemented to glutathione peroxidase (Control: 0?UI/mL; GPx1: 1?UI/mL; GPx5: 5?UI/mL, and GPx10: 10?UI/mL) and catalase (Control: 0?UI/mL; CAT60: 60?UI/mL; CAT120: 120?UI/mL, and CAT240: 240?UI/mL). Each sample was evaluated by motility, plasma membrane integrity (eosin/nigrosin), acrosome integrity (fast green/rose bengal), sperm morphology, assay of the sperm chromatin structure, mitochondrial activity (3,3-diaminobenzidine), and measurement of lipid peroxidation (thiobarbituric acid reactive substances [TBARS]). It was possible to observe a mitochondrial dysfunction (DAB—Class IV) and low membrane integrity after hydrogen peroxide action. However, the high rates of TBARS were observed on hydroxyl radical. CAT240 presents the lower percentage of plasma membrane integrity. It was possible to attest that hydrogen peroxide and hydroxyl radical are the more harmful for goat sperm. Antioxidant therapy must be improving perhaps using combination between antioxidants.     

Using neutron crystallography to elucidate the basis of selective inhibition of carbonic anhydrase by saccharin and a derivative

Up-regulation of carbonic anhydrase IX (CA IX) expression is an indicator of metastasis and associated with poor cancer patient prognosis. CA IX has emerged as a cancer drug target but development of isoform-specific inhibitors is challenging due to other highly conserved CA isoforms. In this study, a CA IX_mimic construct was used (CA II with seven point mutations introduced, to mimic CA IX active site) while maintaining CA II solubility that make it amenable to crystallography. The structures of CA IX_mimic unbound and in complex with saccharin (SAC) and a saccharin-glucose conjugate (SGC) were determined using joint X-ray and neutron protein crystallography. Previously, SAC and SGC have been shown to display CA isoform inhibitor selectivity in assays and X-ray crystal structures failed to reveal the basis of this selectivity. Joint X-ray and neutron crystallographic studies have shown active site residues, solvent, and H-bonding re-organization upon SAC and SGC binding. These observations highlighted the importance of residues 67 (Asn in CA II, Gln in CA IX) and 130 (Asp in CA II, Arg in CA IX) in selective CA inhibitor targeting.     

Sequestration and biosynthesis of cyanogenic glucosides in passion vine butterflies and consequences for the diversification of their host plants

The colorful heliconiine butterflies are distasteful to predators due to their content of defense compounds called cyanogenic glucosides (CNglcs), which they biosynthesize from aliphatic amino acids. Heliconiine larvae feed exclusively on Passiflora plants where ~30 kinds of CNglcs have been reported. Among them, some CNglcs derived from cyclopentenyl glycine can be sequestered by some Heliconius species. In order to understand the evolution of biosynthesis and sequestration of CNglcs in these butterflies and its consequences for their arms race with Passiflora plants, we analyzed the CNglc distribution in selected heliconiine and Passiflora species. Sequestration of cyclopentenyl CNglcs is not an exclusive trait of Heliconius, since these compounds were present in other heliconiines such as Philaethria, Dryas and Agraulis, and in more distantly related genera Cethosia and Euptoieta. Thus, it is likely that the ability to sequester cyclopentenyl CNglcs arose in an ancestor of the Heliconiinae subfamily. Biosynthesis of aliphatic CNglcs is widespread in these butterflies, although some species from the sara‐sapho group seem to have lost this ability. The CNglc distribution within Passiflora suggests that they might have diversified their cyanogenic profile to escape heliconiine herbivory. This systematic analysis improves our understanding on the evolution of cyanogenesis in the heliconiine–Passiflora system.     

An arachidonic acid generation/export system involved in the regulation of cholesterol transport in mitochondria of steroidogenic cells

Recent studies demonstrated the importance of the mitochondrial ATP in the regulation of a novel long-chain fatty acid generation/export system in mitochondria of diabetic rat heart. In steroidogenic systems, mitochondrial ATP and intramitochondrial arachidonic acid (AA) generation are important for steroidogenesis. Here, we report that mitochondrial ATP is necessary for the generation and export of AA, steroid production and steroidogenic acute regulatory protein induction supported by cyclic 3'-5'-adenosine monophosphate in steroidogenic cells. These results demonstrate that ATP depletion affects AA export and provide new evidence of the existence of the fatty acid generation and export system involved in mitochondrial cholesterol transport.     

Study of the inhibition of cyclin-dependent kinases with roscovitine and indirubin-3′-oxime from molecular dynamics simulations

Molecular dynamics simulations were performed to elucidate the interactions of CDK2 and CDK5 complexes with three inhibitors: R-roscovitine, S-roscovitine, and indirubin-3′-oxime. The preference of the two complexes for R-roscovitine over the S enantiomer, as reported by the experiment, was also found by the simulations. More importantly, the simulations showed that the cause of the stronger affinity for the R enantiomer is the presence of an important hydrogen bond between R-roscovitine and the kinases not found with S-roscovitine. The simulations also showed two amino acid mutations in the active site of CDK5/R-roscovitine that favor binding-enhanced electrostatic contributions, making the inhibitor more effective for CDK5 than for CDK2. This suggests that the effectiveness of roscovitine-like inhibitors can be improved by enhancing their electrostatic interaction with the kinases. Finally, molecular mechanics–Possion–Boltzmann/surface area calculations of the CDK5/indirubin-3′-oxime system in both water-excluded and water-included environments gave significantly different electrostatic contributions to the binding. The simulations detected the displacement of a water molecule in the active site of the water-included CDK/indirubin-3′-oxime system. This resulted in a more conserved binding pattern than the water-excluded structure. Hence, in the design of new indirubin-like inhibitors, it is important to include the water molecule in the analysis. Figure Hydrogen bonding networks at the active sites of both CDK5/R-roscotivine (light grey) and CDK2/R-roscovitine (black).     

Resistance modulatory and efflux-inhibitory activities of capsaicinoids and capsinoids

Capsaicinoids are reported to have a bunch of promising pharmacological activities, among them antibacterial effects against various strains of bacteria. In this study the effect on efflux pumps of mycobacteria was investigated. The importance of efflux pumps, and the inhibition of these, is rising due to their involvement in antibiotic resistance development. In order to draw structure and activity relationships we tested natural and synthetical capsaicinoids as well as synthetical capsinoids. In an accumulation assay these compounds were evaluated for their ability to accumulate ethidium bromide into mycobacterial cells, a well-known substrate for efflux pumps. Capsaicin and dihydrocapsaicin, the two most abundant capsaicinoids in Capsicum species, proved to be superior efflux pump inhibitors compared to the standard verapamil. A dilution series showed dose dependency of both compounds. The compound class of less pungent capsinoids qualified for further investigation as antibacterials against Mycobacterium smegmatis.     

Metabolic engineering for the optimization of hydrogen production in Escherichia coli: A review

Hydrogen is a potential sustainable energy source and it could become an alternative to fossil fuel combustion, thus helping to reduce greenhouse gas emissions. The biological production of hydrogen, instead of its chemical synthesis, is a promising possibility since this process requires less energy and is more sustainable and eco-friendly. Several microorganisms have been used for this purpose, but Escherichia coli is one of the most widely used in this field. The literature in this area has increased exponentially in the last 10 years and several strategies have been reported in an effort to improve hydrogen production. In this work, the stay of the art of hydrogen biosynthesis by E. coli and metabolic engineering strategies to enhance hydrogen production are reviewed. This work includes a discussion about the hydrogenase complexes responsible for the hydrogen synthesis in this microorganism and the central carbon metabolism pathways connected to this process. The main metabolic engineering strategies applied are discussed, including heterologous gene expression, adaptive evolution and metabolic and protein engineering. On the other hand, culture conditions, including the use of carbon sources such as glycerol, glucose or organic wastes, have also been considered. Yields and productivities of the most relevant engineered strains reported using several carbon sources are also compared.