Expression of mammalian glutathione S-transferase 5-5 in Salmonella typhimurium TA1535 leads to base-pair mutations upon exposure to dihalomethanes

Dihalomethanes can produce liver tumors in mice but not in rats, and concern exists about the risk of these compounds to humans. Glutathione (GSH) conjugation of dihalomethanes has been considered to be a critical event in the bioactivation process, and risk assessment is based upon this premise; however, there is little experimental support for this view or information about the basis of genotoxicity. A plasmid vector containing rat GSH S-transferase 5-5 was transfected into the Salmonella typhimurium tester strain TA1535, which then produced active enzyme. The transfected bacteria produced base-pair revertants in the presence of ethylene dihalides or dihalomethanes, in the order CH2Br2 > CH2BrCl > CH2Cl2. However, revertants were not seen when cells were exposed to GSH, CH2Br2, and an amount of purified GSH S-transferase 5-5 (20-fold excess in amount of that expressed within the cells). HCHO, which is an end product of the reaction of GSH with dihalomethanes, also did not produce mutations. S-(1-Acetoxymethyl)GSH was prepared as an analog of the putative S-(1-halomethyl)GSH reactive intermediates. This analog did not produce revertants, consistent with the view that activation of dihalomethanes must occur within the bacteria to cause genetic damage, presenting a model to be considered in studies with mammalian cells. S-(1-Acetoxymethyl)GSH reacted with 2′-deoxyguanosine to yield a major adduct, identified as S-[1-(N2-deoxyguanosinyl)methyl]GSH. Demonstration of the activation of dihalomethanes by this mammalian GSH S-transferase theta class enzyme should be of use in evaluating the risk of these chemicals, particularly in light of reports of the polymorphic expression of a similar activity in humans.

Differential substrate behaviours of ethylene oxide and propylene oxide towards human glutathione transferase theta hGSTT1-1

The transformation of ethylene oxide (EO), propylene oxide (PO) and 1- butylene oxide (1-BuO) by human glutathione transferase theta (hGSTT1-1) was studied comparatively using 'conjugator' (GSTT1 + individuals) erythrocyte lysates. The relative sequence of velocity of enzymic transformation was PO > EO >> 1-BuO. The faster transformation of PO compared to EO was corroborated in studies with human and rat GSTT1-1 (hGSTT1-1 and rGSTT1-1, respectively) expressed by Salmonella typhimurium TA1535. This sequence of reactivities of homologous epoxides towards GSTT1-1 contrasts to the sequence observed in homologous alkyl halides (methyl bromide, MBr; ethyl bromide, EtBr; n-propyl bromide, PrBr) where the relative sequence MeBr >> EtBr > PrBr is observed. The higher reactivity towards GSTT1-1 of propylene oxide compared to ethylene oxide is consistent with a higher chemical reactivity. This is corroborated by experimental data of acid-catalysed hydrolysis of a number of aliphatic epoxides, including ethylene oxide and propylene oxide and consistent with semi-empirical molecular orbital modelings.

Species differences in the glutathione transferase GSTT1-1 activity towards the model substrates methyl chloride and dichloromethane in liver and kidney

Glutathione transferase (GST) GSTT1-1 is involved in the biotransformation of several chemicals widely used in industry, such as butadiene and dichloro methane DCM. The polymorphic hGSTT1-1 may well play a role in the development of kidney tumours after high and long-term occupational exposure against trichloroethylene. Although several studies have investigated the association of this polymorphism with malignant diseases little is known about its enzyme activity in potential extrahepatic target tissues. The known theta-specific substrates methyl chloride (MC) dichloromethane and 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were used to assay GSTT1-1 activity in liver and kidney of rats, mice, hamsters and humans differentiating the three phenotypes (non-conjugators, low conjugators, high conjugators) seen in humans. In addition GSTT1-1 activity towards MC and DCM was determined in human erythrocytes. No GSTT1-1 activity was found in any tissue of non-conjugators (NC). In all organs high conjugators (HC) showed twofold higher activity towards MC and DCM than low conjugators (LC). The activity in human samples towards EPNP was too close to the detection limit to differentiate between the three conjugator phenotypes. GSTT1-1 activity towards MC was two to seven-times higher in liver cytosol than in kidney cytosol. The relation for MC between species was identical in both organs: mouse > HC > rat > LC > hamster > NC. In rats, mice and hamsters GSTT1-1 activity in liver cytosol towards DCM was also two to seven-times higher than in the kidney cytosol. In humans this activity was twice as high in kidney cytosol than in liver cytosol. The relation between species was mouse > rat > HC > LC > hamster > NC for liver, but mouse > HC > LC/rat > hamster/NC for kidney cytosol. The importance to heed the specific environment at potential target sites in risk assessment is emphasized by these results.

Identification of novel antigens in blood vessels in rectovaginal endometriosis

To identify specific markers of rectovaginal endometriotic nodule vasculature, highly enriched preparations of vascular endothelial cells and pericytes were obtained from endometriotic nodules and control endometrial and myometrial tissue by laser capture microdissection (LCM), and gene expression profiles were screened by microarray analysis. Of the 18 400 transcripts on the arrays, 734 were significantly overexpressed in vessels from fibromuscular tissue and 923 in vessels from stromal tissue of endometriotic nodules, compared with vessels dissected from control tissues. The most frequently expressed transcripts included known endothelial cell-associated genes, as well as transcripts with little or no previous association with vascular cells. The higher expression in blood vessels was further corroborated by immunohistochemical staining of six potential markers, five of which showed strong expression in pericytes. The most promising marker was matrix Gla protein, which was found to be present in both glandular epithelial cells and vascular endothelial cells of endometriotic lesions, although it was barely expressed at all in normal endometrium. LCM, combined with microarray analysis, constitutes a powerful tool for mapping the transcriptome of vascular cells. After immunohistochemical validation, markers of vascular endothelial and perivascular cells from endometriotic nodules could be identified, which may provide targets to improve early diagnosis or to selectively deliver therapeutic agents.

Methods for extracting genomic DNA from whole blood samples : current perspectives

Deoxyribonucleic acid (DNA) extraction has considerably evolved since it was initially performed back in 1869. It is the first step required for many of the available downstream applications used in the field of molecular biology. Whole blood samples are one of the main sources used to obtain DNA, and there are many different protocols available to perform nucleic acid extraction on such samples. These methods vary from very basic manual protocols to more sophisticated methods included in automated DNA extraction protocols. Based on the wide range of available options, it would be ideal to determine the ones that perform best in terms of cost-effectiveness and time efficiency. We have reviewed DNA extraction history and the most commonly used methods for DNA extraction from whole blood samples, highlighting their individual advantages and disadvantages. We also searched current scientific literature to find studies comparing different nucleic acid extraction methods, to determine the best available choice. Based on our research, we have determined that there is not enough scientific evidence to support one particular DNA extraction method from whole blood samples. Choosing a suitable method is still a process that requires consideration of many different factors, and more research is needed to validate choices made at facilities around the world.

Nanostructured silver-gold bimetallic SERS substrates for selective identification of bacteria in human blood

Surface-enhanced Raman spectroscopy (SERS) is a potentially important tool in the rapid and accurate detection of pathogenic bacteria in biological fluids. However, for diagnostic application of this technique, it is necessary to develop a highly sensitive, stable, biocompatible and reproducible SERS-active substrate. In this work, we have developed a silver–gold bimetallic SERS surface by a simple potentiostatic electrodeposition of a thin gold layer on an electrochemically roughened nanoscopic silver substrate. The resultant substrate was very stable under atmospheric conditions and exhibited the strong Raman enhancement with the high reproducibility of the recorded SERS spectra of bacteria (E. coli, S. enterica, S. epidermidis, and B. megaterium). The coating of the antibiotic over the SERS substrate selectively captured bacteria from blood samples and also increased the Raman signal in contrast to the bare surface. Finally, we have utilized the antibiotic-coated hybrid surface to selectively identify different pathogenic bacteria, namely E. coli, S. enterica and S. epidermidis from blood samples.

The role of other people and emotion for blood donation : an altruistic social service

Blood donation is a critical part of health services with a viable blood supply underpinning an effective health program in any country. Typically blood is provided by voluntary donations from citizens and is therefore reliant on the goodwill and altruistic commitment of donors. In Australia, like many other developed nations, there are many challenges in maintaining a sufficient and sustainable blood supply. The Australian Red Cross Blood Service Donor and Community research group aim is to understand the barriers, motivations and perceptions of donors. Blood donation is a ‘people-processing’ service (Lovelock 1983, Russell-Bennett et al 2013) with the marketing exchange relating to bodily fluid rather than money and is an altruistic social service that has no direct benefit for the customer donor rather the benefit is for other people and society (Kotler and Zaltman 1971). Emotion has been shown to be a motivator and a barrier in a variety of Blood Service studies, this is a key insight that is further explored in the current study. Other key social factors that impact blood donor behavior are classified as social because they involve perceptions of other people’s beliefs and responses (such as moral or subjective norms), peer pressure, other people’s expectations and other people as a form of support. Given that emotions are social phenomena (Parkinson 1996), this study focuses on the role of other people in the blood donation process and how other people relates to the emotional experience of blood donors. We argue in this paper that overcoming emotional barriers to blood donation by leveraging the role of other people will influence low donation rates in Australia. To date, there has been little evidence in service research that identifies. In this paper we explore how other people influence the emotional experience of donors and how, donor emotions create the need for other people as a coping resource.

Demographics and survivial of a cohort of blood and breast cancer patients who developed heart failure following cancer treatment

Background/Aim: Cardiotoxicity resulting in heart failure is a devastating complication of cancer therapy. It is possible that a patient may survive cancer only to develop heart failure (HF), which is more deadly than cancer. The aim of this project was to profile the characteristics of patients at risk of cancer treatment induced heart failure. Methods: Linked Health Data Analysis of Queensland Cancer Registry (QCR) from 1996-2009, Death Registry and Hospital Administration records for HF and chemotherapy admissions were reviewed. Index heart failure admission must have occurred after the date of cancer registry entry. Results: A total of 15,987 patients were included in this analysis; 1,062 (6.6%) had chemotherapy+HF admission (51.4% Female) and 14,925 (93.4%) chemotherapy_no HF admission. Median age of chemotherapy+HF patients was 67 years (IQR 58 to 75) vs. 54 years (IQR 44 to 64) for chemotherapy_no HF admission. Chemotherapy+HF patients had increased risk of all cause mortality (HR 2.79 [95% CI 2.58-3.02] and 1.67 [95% CI, 1.54 to 1.81] after adjusting for age, sex, marital status, country of birth, cancer site and chemotherapy dose). Index HF admission occurred within one year of cancer diagnosis in 47% of HF patients with 80% of patinets having there index admission with 3 years. The number of chemotherapy cycles was not associated with significant reduction in survival time in chemotherapy+HF patients. Mean survival for heart failure patients was 5.3 years (95% CI, 4.99 - 5.62) vs.9.57 years (95% CI, 9.47-9.68) for chemotherapy_no HF admission patients. Conclusion: All-cause mortality was 67% higher in patients diagnosed with HF following chemotherapy in adjusted analysis for covariates. Methods to improve and better coordinate of the interdisciplinary care for cancer patients with HF involving cardiologists and oncologists are required, including evidence-based guidelines for the comprehensive assessment, monitoring and management of this cohort.

Altering the redox state of skeletal muscle by glutathione depletion increases the exercise-activation of PGC-1α

We investigated the relationship between mitochondrial biogenesis, cell signalling and antioxidant enzymes by depleting skeletal muscle glutathione with diethyl maleate (DEM) which resulted in a demonstrable increase in oxidative stress during exercise. Animals were divided into six groups: (1) sedentary control rats; (2) sedentary rats treated with DEM; (3) exercise control rats euthanized immediately after exercise; (4) exercise rats + DEM; (5) exercise control rats euthanized 4 h after exercise, and; (6) exercise rats + DEM euthanized 4 h after exercise. Exercising animals ran on the treadmill at a 10% gradient at 20 m/min for the first 30 min. The speed was then increased every 10 min by 1.6 m/min until exhaustion. There was a reduction in total glutathione in the skeletal muscle of DEM treated animals compared to the control animals (P<0.05). Within the control group, total glutathione was higher in the sedentary group compared to after exercise (P<0.05). DEM treatment also significantly increased oxidative stress, as measured by increased plasma F2-isoprostanes (P<0.05). Exercising animals given DEM showed a significantly greater increase in peroxisome proliferator activated receptor γ coactivator-1α(PGC-1α) mRNA compared to the control animals that were exercised (P<0.05). This study provides novel evidence that by reducing the endogenous antioxidant glutathione in skeletal muscle and inducing oxidative stress through exercise, PGC-1α gene expression was augmented. These findings further highlight the important role of exercise induced oxidative stress in the regulation of mitochondrial biogenesis.

Deformation of a three-dimensional red blood cell in a stenosed micro-capillary

Red blood cells (RBCs) exhibit different types of motions and deformations when the blood flows through capillaries. Interestingly, due to the complex three-dimensional structure of the RBC membrane, RBCs show three-dimensional motions and deformations in the blood flow. These motions and deformations of the RBCs highly depend on the stiffness of the RBC membrane and on the geometrical parameters of the capillary through which blood flows. However, capillaries always do not have uniform cross sections and some capillaries have stenosed segments, where cross sectional area suddenly reduces. Further, some diseases can alter the stiffness of the RBC membrane drastically. In this study, the deformation behaviour of a single three-dimensional RBC is examined, when it moves through a stenosed capillary. A three-dimensional spring network is used to model the RBC membrane. The RBC’s inside and outside fluids are discretized into a finite number of mass points and treated by smoothed particle hydrodynamics (SPH) method. The capillary is considered as a rigid tube with a stenosed section. The deformation index, mean velocity and total energy of the RBC are analysed when it flows through the stenosed capillary. Further, motion and deformation of the RBCs with different membrane stiffness (KB) are compared when they flow through the stenosed segment of the capillary. The simulation results demonstrate the RBCs are subjected to a larger deformation when they move through the stenosed part of the capillary and the RBCs with lower KBvalues easily pass through the stenosed segment of the capillary. Further, RBCs having higher KBvalues have a lower mean velocity and it leads to slow down the overall blood flow rate

Deformation of a single red blood cell in a microvessel

Red blood cells (RBCs) are the most common type of cells in human blood and they exhibit different types of motions and deformed shapes in capillary flows. The behaviour of the RBCs should be studied in order to explain the RBC motion and deformation mechanism. This article presents a numerical simulation method for RBC deformation in microvessels. A two dimensional spring network model is used to represent the RBC membrane, where the elastic stretch/compression energy and the bending energy are considered with the constraint of constant RBC surface area. The forces acting on the RBC membrane are obtained from the principle of virtual work. The whole fluid domain is discretized into a finite number of particles using smoothed particle hydrodynamics concepts and the motions of all the particles are solved using Navier--Stokes equations. Minimum energy concepts are used to simulate the deformed shape of the RBC model. To verify the model, the motion of a single RBC is simulated in a Poiseuille flow and the characteristic parachute shape of the RBC is observed. Further simulations reveal that the RBC shows a tank treading motion when it flows in a linear shear flow.

Formation of the three-dimensional geometry of the red blood cell membrane

Red blood cells (RBCs) are nonnucleated liquid capsules, enclosed in deformable viscoelastic membranes with complex three dimensional geometrical structures. Generally, RBC membranes are highly incompressible and resistant to areal changes. However, RBC membranes show a planar shear deformation and out of plane bending deformation. The behaviour of RBCs in blood vessels is investigated using numerical models. All the characteristics of RBC membranes should be addressed to develop a more accurate and stable model. This article presents an effective methodology to model the three dimensional geometry of the RBC membrane with the aid of commercial software COMSOL Multiphysics 4.2a and Fortran programming. Initially, a mesh is generated for a sphere using the COMSOL Multiphysics software to represent the RBC membrane. The elastic energy of the membrane is considered to determine a stable membrane shape. Then, the actual biconcave shape of the membrane is obtained based on the principle of virtual work, when the total energy is minimised. The geometry of the RBC membrane could be used with meshfree particle methods to simulate motion and deformation of RBCs in micro-capillaries

Estimating the burden of disease attributable to high blood pressure in South Africa in 2000

Objectives To estimate the burden of disease attributable to high blood pressure (BP) in adults aged 30 years and older in South Africa in 2000. Design World Health Organization comparative risk assessment (CRA) methodology was followed. Mean systolic BP (SBP) estimates by age and sex were obtained from the 1998 South African Demographic and Health Survey adult data. Population-attributable fractions were calculated and applied to revised burden of disease estimates for the relevant disease categories for South Africa in 2000. Monte Carlo simulation modelling techniques were used for uncertainty analysis. Setting South Africa Subjects Adults aged 30 years and older. Outcome measures Mortality and disability-adjusted life years (DALYs) from ischaemic heart disease (IHD), stroke, hypertensive disease and other cardiovascular disease (CVD). Results High BP was estimated to have caused 46 888 deaths (95% uncertainty interval 44 878 - 48 566) or 9% (95% uncertainty interval 8.6 - 9.3%) of all deaths in South Africa in 2000, and 390 860 DALYs (95% uncertainty interval 377 955 - 402 256) or 2.4% of all DALYs (95% uncertainty interval 2.3 - 2.5%) in South Africa in 2000. Overall, 50% of stroke, 42% of IHD, 72% of hypertensive disease and 22% of other CVD burden in adult males and females (30+ years) were attributable to high BP (systolic BP ≥ 115 mmHg). Conclusions High BP contributes to a considerable burden of CVD in South Africa and results indicate that there is considerable potential for health gain from implementing BP-lowering interventions that are known to be highly costeffective.