A stereochemical and positional isotope exchange study of the mechanism of activation of isoleucine by isoleucyl-tRNA synthetase from Escherichia coli

Isoleucyl-tRNA synthetase from Escherichia coli catalyzes the activation of [18O2]isoleucine by adenosine 5'-[(R)-alpha-17O]triphosphate with inversion of configuration at phosphorus. Moreover, isoleucyl-tRNA synthetase does not catalyze positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of isoleucine or in the presence of the competitive inhibitor isoleucinol, which effectively eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. Together, these observations require that isoleucyl-tRNA synthetase catalyzes the activation of isoleucine by associative "in line" nucleotidyl transfer. The synthesis of adenosine 5'-[(R)-alpha-17O]diphosphate and its conversion to adenosine 5'-[(R)-alpha-17O]triphosphate is described and an explanation provided for the reported differences between the treatment of adenosine 5'-[(S)-alpha-thiodiphosphate] with cyanogen bromide and bromine in [18O]water.     

Carbachol stimulates a different phospholipid metabolism than nerve growth factor and basic fibroblast growth factor in PC12 cells.

We have examined 1,2-diglycerides (DGs) generated in PC12 cells in response to the muscarinic agonist carbachol and compared them with those generated in response to the differentiation factors nerve growth factor and basic fibroblast growth factor. Whereas carbachol stimulates a greater release of inositol phosphates, all three agonists generate similar levels of DGs. In this report, we have analyzed the molecular species of PC12 DGs generated in response to these three agonists. Additionally, we have analyzed the molecular species of PC12 phospholipids. The data indicate that 1) after 1 min of either nerve growth factor or basic fibroblast growth factor stimulation, DGs arise primarily from phosphoinositide hydrolysis; 2) in contrast, after 1 min of carbachol stimulation, DG are generated equally by both phosphoinositide and phosphatidylcholine hydrolysis; and 3) after 15 min of stimulation by any of these agonists, DGs are generated largely by phosphatidylcholine hydrolysis, with a smaller component arising from the phosphoinositides. These results suggest that at least part of the mechanism by which PC12 cells distinguish between different agonists is via alterations in phospholipid sources and kinetics of DG generation.     

Human ventilatory response to acute hyperoxia during and after 8h of both isocapnic and poikilocapnic hypoxia

Tansley, J. G., C. Clar, M. E. F. Pedersen, and P. A. Robbins. Human ventilatory response to acute hyperoxia during andafter 8 h of both isocapnic and poikilocapnic hypoxia.J. Appl. Physiol. 82(2): 513-519, 1997.During 8 h of either isocapnic or poikilocapnic hypoxia,there may be a rise in ventilation(E) thatcannot be rapidly reversed with a return to higherPO₂ (L. S. G. E. Howard and P. A. Robbins. J. Appl. Physiol. 78:1098-1107, 1995). To investigate this further, threeprotocols were compared: 1) 8-hisocapnic hypoxia [end-tidalPCO₂(PET_CO2 ) held atprestudy value, end-tidal PO₂(PET_O2) = 55 Torr],followed by 8-h isocapnic euoxia(PET_O2 = 100 Torr);2) 8-h poikilocapnic hypoxia followed by 8-h poikilocapnic euoxia; and3) 16-h air-breathing control.Before and at intervals throughout each protocol, theE response to eucapnichyperoxia (PET_CO2 held1-2 Torr above prestudy value,PET_O2 = 300 Torr) wasdetermined. There was a significant rise in hyperoxic E over 8 hduring both forms of hypoxia (P < 0.05, analysis of variance) that persisted during the subsequent 8-heuoxic period (P < 0.05, analysis ofvariance). These results support the notion that an 8-h period ofhypoxia increases subsequenthyperoxic E, even if acid-base changes have been minimized through maintenance ofisocapnia during the hypoxic period.

     

A stereochemical and positional isotope-exchange study of the mechanism of activation of methionine by methionyl-tRNA synthetase from Escherichia coli

Methionyl-tRNA synthetase from Escherichia coli catalyses the activation of [18O2]methionine by adenosine 5'-[(R)-alpha 17O]triphosphate with inversion of configuration at P alpha. Furthermore methionyl-tRNA synthetase does not catalyse positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the absence of methionine or in the presence of the competitive inhibitor, methioninol, which eliminates the possibility of either adenylyl-enzyme or adenosine metaphosphate intermediates being involved. These observations require that methionyl-tRNA synthetase catalyses the activation of methionine by an associative 'in-line' nucleotidyl transfer mechanism. A kinetic study of positional isotope exchange in adenosine 5'-[beta-18O2]triphosphate in the presence of methionine, Mg2+ and methionyl-tRNA synthetase showed that torsional equilibration (18O exchange into the P alpha--O--P beta bridge) occurs faster than tumbling (18O exchange into P gamma by rotation about the C2 axis of Mg[18O2]PPi), demonstratings that the positional isotope exchange occurs at least in part in the E X Met-AMP X Mg[18O2]PPi complex.     

A stereochemical study of the mechanism of activation of donor oligonucleotides by RNA ligase from bacteriophage T4 infected Escherichia coli

RNA ligase from bacteriophage T4 infected Escherichia coli catalyzes the activation of adenosine 3',5'-bisphosphate (representing the donor oligonucleotide) by adenosine 5'-[(S)-alpha-17O,alpha,alpha-18O2]triphosphate with retention of configuration at P alpha. Since single-step enzyme-catalyzed nucleotidyl transfer reactions proceed with inversion, this stereochemical result provides support for a double displacement mechanism involving an adenylyl-enzyme intermediate as proposed previously from isotope exchange experiments.     

Spatial Access to Emergency Services in Low- and Middle-Income Countries: A GIS-Based Analysis

Injury is a leading cause of the global disease burden, accounting for 10 percent of all deaths worldwide. Despite 90 percent of these deaths occurring in low and middle-income countries (LMICs), the majority of trauma research and infrastructure development has taken place in high-income settings. Furthermore, although accessible services are of central importance to a mature trauma system, there remains a paucity of literature describing the spatial accessibility of emergency services in LMICs. Using data from the Service Provision Assessment component of the Demographic and Health Surveys of Namibia and Haiti we defined the capabilities of healthcare facilities in each country in terms of their preparedness to provide emergency services. A Geographic Information System-based network analysis method was used to define 5- 10- and 50-kilometer catchment areas for all facilities capable of providing 24-hour care, higher-level resuscitative services or tertiary care. The proportion of a country’s population with access to each level of service was obtained by amalgamating the catchment areas with a population layer. A significant proportion of the population of both countries had poor spatial access to lower level services with 25% of the population of Haiti and 51% of the population of Namibia living further than 50 kilometers from a facility capable of providing 24-hour care. Spatial access to tertiary care was considerably lower with 51% of Haitians and 72% of Namibians having no access to these higher-level services within 50 kilometers. These results demonstrate a significant disparity in potential spatial access to emergency services in two LMICs compared to analogous estimates from high-income settings, and suggest that strengthening the capabilities of existing facilities may improve the equity of emergency services in these countries. Routine collection of georeferenced patient and facility data in LMICs will be important to understanding how spatial access to services influences outcomes.