Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

Effect of pretreatment on saccharification of sugarcane bagasse by complex and simple enzyme mixtures

Saccharification of sugarcane bagasse pretreated at the pilot-scale with different processes (in combination with steam-explosion) was evaluated. Maximum glucan conversion with Celluclast 1.5 L (15–25 FPU/g glucan) was in the following order: glycerol/HCl > HCl > H2SO4 > NaOH, with the glycerol system achieving ∼100% conversion. Surprisingly, the NaOH substrate achieved optimum saccharification with only 8 FPU/g glucan. Glucan conversions (3.6–6%) obtained with mixtures of endo-1,4-β-glucanase (EG) and β-glucosidase (βG) for the NaOH substrate were 2–6 times that of acid substrates. However, glucan conversions (15–60%) obtained with mixtures of cellobiohydrolase (CBH I) and βG on acidified glycerol substrate were 10–30% higher than those obtained for NaOH and acid substrates. The susceptibility of the substrates to enzymatic saccharification was explained by their physical and chemical attributes. Acidified glycerol pretreatment offers the opportunity to simplify the complexity of enzyme mixtures required for saccharification of lignocellulosics.

Low temperature pretreatment of sugarcane bagasse at atmospheric pressure using mixtures of ethylene carbonate and ethylene glycol

A low temperature lignocellulose pretreatment process was developed using acid-catalysed mixtures of alkylene carbonate and alkylene glycol. Pretreatment of sugarcane bagasse with mixtures of ethylene carbonate (EC) and ethylene glycol (EG) was more effective than that with mixtures of propylene carbonate (PC) and propylene glycol (PG). These mixtures were more effective than the individual components in making bagasse cellulose more amenable to cellulase digestion. Glucan digestibilities of ≥87% could be achieved with a wide range of EC to EG ratios from 9:1 to 1:1 (w/w). Pretreatment of bagasse by the EC/EG mixture with a ratio of 4:1 in the presence of 1.2% H2SO4 at 90 °C for 30 min led to the highest glucan enzymatic digestibility of 93%. The high glucan digestibilities obtained under these acidic conditions were due to (a) the ability of alkylene carbonate to cause significant biomass size reduction, (b) the ability of alkylene glycol to cause biomass defibrillation, (c) the ability of alkylene carbonate and alkylene glycol to remove xylan and lignin, and (d) the magnified above attributes in the mixtures of alkylene carbonate and alkylene glycol.

Oxidative stress induced lung cancer and COPD : opportunities for epigenetic therapy

Reactive oxygen species (ROS) form as a natural by-product of the normal metabolism of oxygen and play important roles within the cell. Under normal circumstances the cell is able to maintain an adequate homeostasis between the formation of ROS and its removal through particular enzymatic pathways or via antioxidants. If however, this balance is disturbed a situation called oxidative stress occurs. Critically, oxidative stress plays important roles in the pathogenesis of many diseases, including cancer. Epigenetics is a process where gene expression is regulated by heritable mechanisms that do not cause any direct changes to the DNA sequence itself, and disruption of epigenetic mechanisms has important implications in disease. Evidence is emerging that histone deacetylases (HDACs) play decisive roles in regulating important cellular oxidative stress pathways including those involved with sensing oxidative stress and those involved with regulating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by HDACs may play critical roles in cancer progression. In this review we discuss the current evidence linking epigenetics and oxidative stress and cancer, using chronic obstructive pulmonary disease and non-small cell lung cancer to illustrate the importance of epigenetics on these pathways within these disease settings. © 2009 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Targeting oxidative stress in cancer

Importance of the field: Reactive oxygen species (ROS) occur as natural by-products of oxygen metabolism and have important cellular functions. Normally, the cell is able to maintain an adequate balance between the formation and removal of ROS either via anti-oxidants or through the use specific enzymatic pathways. However, if this balance is disturbed, oxidative stress may occur in the cell, a situation linked to the pathogenesis of many diseases, including cancer. Areas covered in this review: HDACs are important regulators of many oxidative stress pathways including those involved with both sensing and coordinating the cellular response to oxidative stress. In particular aberrant regulation of these pathways by histone deacetylases may play critical roles in cancer progression. What the reader will gain: In this review we discuss the notion that targeting HDACs may be a useful therapeutic avenue in the treatment of oxidative stress in cancer, using chronic obstructive pulmonary disease (COPD), NSCLC and hepatocellular carcinoma (HCC) as examples to illustrate this possibility. Take home message: Epigenetic mechanisms may be an important new therapeutic avenue for targeting oxidative stress in cancer. © 2010 Informa UK, Ltd.

In vitro assessment of surface congruency and integration of chondrocytes at adjacent edges of micro-fabricated clefts on cartilage

Osteochondral grafts are common treatment options for joint focal defects due to their excellent functionality. However, the difficulty is matching the topography of host and graft(s) surfaces flush to one another. Incongruence could lead to disintegration particularly when the gap reaches subchondoral region. The aim of this study is therefore to investigate cell response to gap geometry when forming cartilage-cartilage bridge at the interface. The question is what would be the characteristics of such a gap if the cells could bridge across to fuse the edges? To answer this, osteochondral plugs devoid of host cells were prepared through enzymatic decellularization and artificial clefts of different sizes were created on the cartilage surface using laser ablation. High density pellets of heterologous chondrocytes were seeded on the defects and cultured with chondrogenic differentiation media for 35 days. The results showed that the behavior of chondrocytes was a function of gap topography. Depending on the distance of the edges two types of responses were generated. Resident cells surrounding distant edges demonstrated superficial attachment to one side whereas clefts of 150 to 250 µm width experienced cell migration and anchorage across the interface. The infiltration of chondrocytes into the gaps provided extra space for their proliferation and laying matrix; as the result faster filling of the initial void space was observed. On the other hand, distant and fit edges created an incomplete healing response due to the limited ability of differentiated chondrocytes to migrate and incorporate within the interface. It seems that the initial condition of the defects and the curvature profile of the adjacent edges were the prime determinants of the quality of repair; however, further studies to reveal the underlying mechanisms of cells adapting to and modifying the new environment would be of particular interest.

Pretreatment of sugarcane bagasse by acidified aqueous polyol solutions

Pretreatments of sugarcane bagasse by three high boiling-point polyol solutions were compared in acid-catalysed processes. Pretreatments by ethylene glycol (EG) and propylene glycol solutions containing 1.2 % H2SO4 and 10 % water at 130 °C for 30 min removed 89 % lignin from bagasse resulting in a glucan digestibility of 95 % with a cellulase loading of ~20 FPU/g glucan. Pretreatment by glycerol solution under the same conditions removed 57 % lignin with a glucan digestibility of 77 %. Further investigations with EG solutions showed that increases in acid content, pretreatment temperature and time, and decrease in water content improved pretreatment effectiveness. A good linear correlation of glucan digestibility with delignification was observed with R2 = 0.984. Bagasse samples pretreated with EG solutions were characterised by scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction, which confirmed that improved glucan enzymatic digestibility is mainly due to delignification and defibrillation of bagasse. Pretreatment by acidified EG solutions likely led to the formation of EG-glycosides. Up to 36 % of the total lignin was recovered from pretreatment hydrolysate, which may improve the pretreatment efficiency of recycled EG solution.

Glycerol carbonate as green solvent for pretreatment of sugarcane bagasse

Background Pretreatment of lignocellulosic biomass is a prerequisite for effective saccharification to produce fermentable sugars. We have previously reported an effective low temperature (90 °C) process at atmospheric pressure for pretreatment of sugarcane bagasse with acidified mixtures of ethylene carbonate (EC) and ethylene glycol (EG). In this study, “greener” solvent systems based on acidified mixtures of glycerol carbonate (GC) and glycerol were used to treat sugarcane bagasse and the roles of each solvent in deconstructing biomass were determined. Results Pretreatment of sugarcane bagasse at 90 °C for only 30 min with acidified GC produced a solid residue having a glucan digestibility of 90% and a glucose yield of 80%, which were significantly higher than a glucan digestibility of 16% and a glucose yield of 15% obtained for bagasse pretreated with acidified EC. Biomass compositional analyses showed that GC pretreatment removed more lignin than EC pretreatment (84% vs 54%). Scanning electron microscopy (SEM) showed that fluffy and size-reduced fibres were produced from GC pretreatment whereas EC pretreatment produced compact particles of reduced size. The maximal glucan digestibility and glucose yield of GC/glycerol systems were about 7% lower than those of EC/ethylene glycol (EG) systems. Replacing up to 50 wt% of GC with glycerol did not negatively affect glucan digestibility and glucose yield. The results from pretreatment of microcrystalline cellulose (MCC) showed that (1) pretreatment with acidified alkylene glycol (AG) alone increased enzymatic digestibility compared to pretreatments with acidified alkylene carbonate (AC) alone and acidified mixtures of AC and AG, (2) pretreatment with acidified GC alone slightly increased, but with acidified EC alone significantly decreased, enzymatic digestibility compared to untreated MCC, and (3) there was a good positive linear correlation of enzymatic digestibility of treated and untreated MCC samples with congo red (CR) adsorption capacity. Conclusions Acidified GC alone was a more effective solvent for pretreatment of sugarcane bagasse than acidified EC alone. The higher glucose yield obtained with GC-pretreated bagasse is possibly due to the presence of one hydroxyl group in the GC molecular structure, resulting in more significant biomass delignification and defibrillation, though both solvent pretreatments reduced bagasse particles to a similar extent. The maximum glucan digestibility of GC/glycerol systems was less than that of EC/EG systems, which is likely attributed to glycerol being less effective than EG in biomass delignification and defibrillation. Acidified AC/AG solvent systems were more effective for pretreatment of lignin-containing biomass than MCC.

Phenytoin metabolism by human cytochrome P450 : involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Matrix metalloproteinases during wound healing - a double edged sword

The extracellular matrix (ECM) provides a framework for cells and gives skin its tensile strength and elasticity. Loss of its integrity necessitates the clearing of damaged components and the deposition of firstly a provisional matrix and later remodelling of the ECM to support a functionally intact tissue. Matrix metalloproteinases (MMPs) are an important family of enzymes that function in the breakdown of the ECM and modulate the function of many biologically active molecules housed in the ECM. Through their enzymatic actions MMPs play a role in fundamental processes such as immune cell infiltration and ECM remodelling during wound repair. Their tight control is necessary for timely wound healing and excessive MMP activity participates in the development and persistence of chronic wounds, while reduced activity contributes to fibrosis. A number of inhibitors have been designed to target this activity and improve wound healing with limited success. Novel strategies are currently being investigated to improve wound healing by targeting MMP modulating molecules.

Identification, functional expression and kinetic analysis of two primary amine oxidases from Rhodococcus opacus

Two native copper-containing amine oxidases (EC 1.4.3.21) have been isolated from Rhodococcus opacus and reveal phenotypic plasticity and catalytic activity with respect to structurally diverse natural and synthetic amines. Altering the amine growth substrate has enabled tailored and targeted oxidase upreg-ulation, which with subsequent treatment by precipitation, ion exchange and gel filtration, achieved a 90–150 fold purification. MALDI-TOF mass spectrometric and genomic analysis has indicated multiple gene activation with complex biodegradation pathways and regulatory mechanisms. Additional post-purification characterisation has drawn on the use of carbonyl reagent and chelating agent inhibitors. Michaelis–Menten kinetics for common aliphatic and aromatic amine substrates and several structural analogues demonstrated a broad specificity and high affinity with Michaelis constants (K M) ranging from 0.1 to 0.9 mM for C 1 –C 5 aliphatic mono-amines and <0.2 mM for a range of aromatic amines. Potential exploitation of the enzymatic versatility of the two isolated oxidases in biosensing and bioprocessing is discussed.

Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida

L-Amino acid oxidases (LAAOs) are useful catalysts for the deracemisation of racemic amino acid sub-strates when combined with abiotic reductants. The gene nadB encoding the L-aspartate amino acid oxidase from Pseudomonas putida (PpLASPO) has been cloned and expressed in E. coli. The purified PpLASPO enzyme displayed a K M for l-aspartic acid of 2.26 mM and a k cat = 10.6 s −1 , with lower activity also displayed towards L-asparagine, for which pronounced substrate inhibition was also observed. The pH optimum of the enzyme was recorded at pH 7.4. The enzyme was stable for 60 min at up to 40 • C, but rapid losses in activity were observed at 50 • C. A mutational analysis of the enzyme, based on its sequence homology with the LASPO from E. coli of known structure, appeared to confirm roles in substrate binding or catalysis for residues His244, His351, Arg386 and Arg290 and also for Thr259 and Gln242. The high activity of the enzyme, and its promiscuous acceptance of both L-asparagine and L-glutamate as substrates, if with low activity, suggests that PpLASPO may provide a good model enzyme for evolution studies towards AAOs of altered or improved properties in the future.

Hybrid graphite film–carbon nanotube platform for enzyme immobilization and protection

A novel platform consisting of a multilayered substrate, activated graphite-like carbon film, and dense forest of long, vertically-aligned multiwall carbon nanotubes grown by the chemical vapor deposition is designed, fabricated, and tested for covalent immobilization of enzymatic biocatalysts with the aim of protecting them from shear forces and microbial attacks present in bioreactors. The covalent bonding ensures enzyme retention in a flow, while the dense nanotube forest may serve as a protection of the enzymes from microbial attack without impeding the flow of reactants and products. This platform was demonstrated for the two reference enzymes, horseradish peroxidase and catalase, which were immobilized without degrading their biological activity. This combination of an activated carbon layer for an efficient immobilization of biocatalysts with a protective layer of inert carbon nanotubes could dramatically improve the efficiency and longevity of enzymatic bio-catalysis employed in a large variety of advanced biotechnological processes.

The crude skin secretion of the pepper frog leptodactylus labyrinthicus is rich in metallo and serine peptidases

Peptidases are ubiquitous enzymes involved in diverse biological processes. Fragments from bioactive peptides have been found in skin secretions from frogs, and their presence suggests processing by peptidases. Thus, the aim of this work was to characterize the peptidase activity present in the skin secretion of Leptodactylus labyrinthicus. Zymography revealed the presence of three bands of gelatinase activity of approximately 60 kDa, 66 kDa, and 80 kDa, which the first two were calcium-dependent. These three bands were inhibited either by ethylenediaminetetraacetic acid (EDTA) and phenathroline; thus, they were characterized as metallopeptidases. Furthermore, the proteolytic enzymes identified were active only at pH 6.0–10.0, and their activity increased in the presence of CHAPS or NaCl. Experiments with fluorogenic substrates incubated with skin secretions identified aminopeptidase activity, with cleavage after leucine, proline, and alanine residues. This activity was directly proportional to the protein concentration, and it was inhibited in the presence of metallo and serine peptidase inhibitors. Besides, the optimal pH for substrate cleavage was determined to be 7.0–8.0. The results of the in gel activity assay showed that all substrates were hydrolyzed by a 45 kDa peptidase. Gly-Pro-AMC was also cleaved by a peptidase greater than 97 kDa. The data suggest the presence of dipeptidyl peptidases (DPPs) and metallopeptidases; however, further research is necessary. In conclusion, our work will help to elucidate the implication of these enzymatic activities in the processing of the bioactive peptides present in frog venom, expanding the knowledge of amphibian biology.

Identification of salivary N-glycoproteins and measurement of glycosylation site occupancy by boronate glycoprotein enrichment and liquid chromatography/electrospray ionization tandem mass spectrometry

RATIONALE Diseases including cancer and congenital disorders of glycosylation have been associated with changes in the site-specific extent of protein glycosylation. Saliva can be non-invasively sampled and is rich in glycoproteins, giving it the potential to be a useful biofluid for the discovery and detection of disease biomarkers associated with changes in glycosylation. METHODS Saliva was collected from healthy individuals and glycoproteins were enriched using phenylboronic acid based glycoprotein enrichment resin. Proteins were deglycosylated with peptide-N-glycosidase F and digested with AspN or trypsin. Desalted peptides and deglycosylated peptides were separated by reversed-phase liquid chromatography and detected with on-line electrospray ionization quadrupole-time-of-flight mass spectrometry using a 5600 TripleTof instrument. Site-specific glycosylation occupancy was semi-quantitatively determined from the abundance of deglycosylated and nonglycosylated versions of each given peptide. RESULTS Glycoprotein enrichment identified 67 independent glycosylation sites from 24 unique proteins, a 3.9-fold increase in the number of glycosylation sites identified. Enrichment of glycoproteins rather than glycopeptides allowed detection of both deglycosylated and nonglycosylated versions of each peptide, and thereby robust measurement of site-specific occupancy at 21 asparagines. Healthy individuals showed limited biological variability in occupancy, with partially modified sites having characteristics consistent with inefficient glycosylation by oligosaccharyltransferase. Inclusion of negative controls without enzymatic deglycosylation controlled for spontaneous chemical deamidation, and identified asparagines previously incorrectly annotated as glycosylated. CONCLUSIONS We developed a sample preparation and mass spectrometry detection strategy for rapid and efficient measurement of site-specific glycosylation occupancy on diverse salivary glycoproteins suitable for biomarker discovery and detection of changes in glycosylation occupancy in human disease.