Timothy P. Higgins

Biochemical and molecular characterisation of the 2,3-dichloro-1-propanol dehalogenase and stereospecific haloalkanoic dehalogenases from a versatile Agrobacterium sp.

AbstractWe previously reported the presence of both haloalcohol and haloalkanoate dehalogenase activity in the Agrobacterium sp. strain NHG3. The versatile nature of the organism led us to further characterise the genetic basis of these dehalogenation activities. Cloning and sequencing of the haloalcohol dehalogenase and subsequent analysis suggested that it was part of a highly conserved catabolic gene cluster. Characterisation of the haloalkanoate dehalogenase enzyme revealed the presence of two stereospecific enzymes with a narrow substrate range which acted on d -2-chloropropionic and I-2-chloropropionoic acid, respectively. Cloning and sequencing indicated that the two genes were separated by 87 bp of non-coding DNA and were preceded by a putative transporter gene 66 bp upstream of the d-specific enzyme.

Metabolism of methanesulfonic acid involves a multicomponent monooxygenase enzyme

A novel methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was the subject of these investigations. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Partial purification of MSAMO from cell-free extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by the mixing of three distinct protein fractions designated A, B and C. The reconstituted enzyme had a narrow substrate specificity relative to crude cell-free extracts. Addition of FAD and ferrous ions to the reconstituted enzyme complex resulted in a fivefold increase in enzyme activity, suggesting the loss of FAD and ferrous ion from the multicomponent enzyme on purification. Analysis of mutants of strain M2 defective in the metabolism of C1 compounds indicated that methanol was not an intermediate in the degradative pathway of MSA and also confirmed the involvement of a multicomponent enzyme in the degradation of MSA by methylotroph strain M2.

Heterogeneous responses of personalised high intensity interval training on type 2 diabetes mellitus and cardiovascular disease risk in young healthy adults

Hypertension, decreased glucose tolerance, adverse lipid profiles and low physical activity levels are associated with increased type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD) risk. High intensity interval training (HIIT), a low volume, reduced time, high intensity programme, may be a useful alternative to current government guidelines which specify a minimum of 150 minutes of physical activity per week. We describe a personalised programme of high intensity exercise which provides significant improvements in CVD risk markers. Healthy volunteers undertook 6 weeks of HIIT. T2DM and CVD risk predictors including glucose tolerance, VO2max, blood pressure (BP), and lipids were measured before and after HIIT. HIIT training was associated with beneficial changes in a range of predictors of blood flow and cardiovascular risk. There was a heterogeneous response to HIIT, with some subjects responding with favourable changes and others being non-responders to HIIT. In responders, HIIT was associated with a statistically significant (p = 0.023) increase in VO2max, from 45.4 (38.4,52.5) to 56.9 (51.2,65.7) (median (interquartile range)(ml/min/kg)). In responders HIIT resulted in a decrease in systolic BP from 127 (126,129) to 116 (106,122) (mmHg) with p = 0.026 and a decrease is diastolic blood pressure from 72 (69,74) to 57 (56,66) with p = 0.026. There was also some evidence of a beneficial change in blood lipid and glucose concentrations with HIIT. In conclusion, personalised HIIT has potential as an intervention to improve blood flow and cardiovascular health.

Comparison of pathways for biodegradation of monomethyl sulphate in Agrobacterium and Hyphomicrobium species

Different mechanisms have been proposed previously for the biodegradation of monomethyl sulphate (MMS) in Agrobacterium sp. and Hyphomicrobium sp. Sulphate liberation from MMS in Agrobacterium sp. M3C was previously shown to be O2-dependent, whereas in several Hyphomicrobium spp. the initiating step has been considered hitherto to be hydrolytic and catalysed by methyl sulphatase. In the present study, Agrobacterium and Hyphomicrobium strains were compared for their ability to oxidize MMS and its potential metabolites in the oxygen electrode. MMS-grown Agrobacterium sp. M3C and Hyphomicrobium sp. MS223 oxidized MMS with consumption of 0.5 mol O2 per mol of substrate, but they were unable to oxidize methanol. By repeatedly challenging MMS-grown Hypomicrobium with MMS in the electrode chamber, all the O2 in the electrode became exhausted, at which point SO4(2-) liberation stopped although excess MMS was available. SO4(2-) release resumed immediately when O2 was re-admitted to the electrode chamber. Thus liberation of SO4(2-) from MMS in the oxygen electrode was dependent on the continuing availability of O2. Hyphomicrobium sp. MS223 therefore closely resembled Agrobacterium sp. M3C in its obligatory requirement for O2 in MMS degradation. Unlike Agrobacterium sp. M3C, Hyphomicrobium sp. MS223 was able to grow on methanol and methanol-grown cells oxidized methanol (0.5 mol O2 per mol of substrate) but not MMS. Cyclopropanol, an inhibitor of methanol dehydrogenase, abolished oxidation of methanol by methanol-grown Hyphomicrobium sp. MS223 but did not affect oxidation of MMS by MMS-grown cells. Thus Hyphomicrobium sp. MS223 expresses enzymes for oxidation of methanol when needed for growth on this compound, but not when grown on MMS.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of physical activity on haematological predictors of cardiovascular risk – evidence of a dose response

Cardiovascular disease is a major cause of morbidity and mortality in the developed world. Large epidemiological studies have reported a strong association between increases in haematological factors and increased cardiovascular risk. Haematological risk factors predicted cardiovascular disease at least as strongly as traditional risk factors such as blood lipid concentrations. Lifestyle factors such as physical activity level could significantly reduce risk. The aim of this study was to determine the effect of physical activity level on haematological predictors of cardiovascular risk. Healthy subjects (156) were recruited. Physical activity in subjects was assessed by IPAQ physical activity questionnaire. Blood was collected and blood cell counts were determined by automated cell counter; neutrophil elastase was determined by ELISA. Increased levels of physical activity were associated with reduced red cell (p = 0.001), white cell (p = 0.002) and platelet counts (p = 0.001) and with reduced plasma neutrophil elastase concentration (p = 0.001). There was a continuous linear relationship between increase in physical activity and decrease in haematological risk factors. Hence, the authors conclude that increased levels of physical activity improve the flow properties of blood and thus reduce the risk of developing cardiovascular disease. Even small increases in activity result in some reduction in cardiovascular risk.

Bacterial Metabolism of Methanesulfonic Acid

The principal organic sulfur compound entering the atmosphere from aquatic and terrestrial environments is dimethyl sulfide (DMS). The majority of this is released into the oceans from the cleavage of dimethyl sulfoniopropionate, an osmoregulator found in phytoplankton such as Emiliana and Phaeocystis and the marsh grass Spartina (see chapters by Kiene and Visscher, this volume). Global estimates of DMS release vary, although it has been estimated that between 40-70 Tg is lost to the atmosphere annually representing a significant fraction of the global biogeochemical sulfur cycle. DMS in the atmosphere is photochemically oxidized to methanesulfonic acid (MSA) and sulfur dioxide. DMS is also oxidized to MSA by atmospheric NO3 radicals in the dark (see Kelly et al. 1993), resulting in an annual global rate of MSA formation of 20-50 Tg MSA. MSA is known to be important in cloud condensation nuclei (i.e. it may contribute to cloud albedo) and therefore it may play an important role in global temperature control (Charlson et al. 1987). MSA is a very stable strong acid which is not further oxidized in the atmosphere but falls on the Earth’s surface in rain and snow, thus re-entering the terrestrial and marine biosphere.

Increased monocyte actin polymerization in rat blood after intratracheal instillation of air pollution particles.

BACKGROUND Exposure to particulate air pollution is associated with an increased risk of cardiovascular disease. The mechanism by which exposure increases risk is poorly understood but could involve changes in the flow properties of blood. OBJECTIVE The aim of this investigation was to assess the effect, in rats, of intratracheal instillation of particulate air pollution on leukocyte flow properties by measurement of polymorphonucleocyte (PMN) and monocyte actin polymerisation. METHODS Rats were exposed to particulate air pollution by intratracheal instillation of PM10. Blood was collected from test and control animals at 3 days (n=10) and 6 weeks (n=10) after dust instillation. Partial differential leukocyte counts were performed. The intracellular F-actin content of blood PMNs and monocytes was determined by staining with FITC-phalloidin and flow cytometric determination of mean florescence intensity (MFI). RESULTS There were no significant changes in PMN MFI (p=0.369, ANOVA) or cell counts (p=0.753, ANOVA). There was a significant increase in monocyte MFI (p=0.004, ANOVA) and a decrease in monocyte cell count (p=0.003, ANOVA) in instilled rats. CONCLUSIONS Intratracheal instillation of air pollution particles resulted in an increase in blood monocyte actin polymerisation, which may cause trapping of monocytes. This could be a mechanism by which exposure to air pollution increases the risk of cardiovascular disease.