Search PubMed⌕ Search

Biomedical subjects

G K Radda

Publications and source records attributed to G K Radda.

At least 37 records · Page 2Linked to original sources
PubMed1998

Excitatory amino acid synthesis in hypoxic brain slices: does alanine act as a substrate for glutamate production in hypoxia?

Excitatory amino acids are an important cause of cell death in the hypoxic and ischaemic brain. Neuronal glutamate stores are depleted rapidly in hypoxia, but alanine production rises under such conditions and has been suggested to be a potential precursor of glutamate. To test this hypothesis, we have investigated amino acid metabolism using 13C NMR with superfused guinea pig cortical slices subjected to varying degrees of hypoxia. During severe hypoxia, brain slices metabolising 5 mM [2-(13)C]pyruvate exported [2-(13)C]alanine into the superfusion fluid. The metabolic fate of alanine during normoxia and hypoxia was tested by superfusion of brain slices with 10 mM glucose and 2 mM [2-(13)C,15N]alanine. Metabolism of exogenous alanine leads to the release of aspartate into the superfusion fluid. The pattern of labelling of aspartate indicated that it was synthesised via the glial-specific enzyme pyruvate carboxylase. 13C-labelled glutamate was produced with both normoxia and hypoxia, but concentrations were 30-fold lower than for labelled aspartate. Thus, although substantial amounts of glutamate are not synthesised from alanine in hypoxia, there is significant production of aspartate, which also may have deleterious effects as an excitatory amino acid.

Alanine↗
PubMed1998

Brain biochemistry in Williams syndrome: evidence for a role of the cerebellum in cognition?

OBJECTIVE: To determine what biochemical changes may occur in the brain in Williams syndrome (WS) and whether these changes may be related to the cognitive deficits. BACKGROUND: WS is a rare, congenital disorder with a characteristic physical, linguistic, and behavioral phenotype with known cognitive deficits. METHODS: We obtained 31P magnetic resonance spectra (MRS) from a region consisting of mostly frontal and parietal lobe of 14 patients with WS (age, 8 to 37 years) and 48 similarly-aged controls. 1H MRS (27 cm3) localized to the left cerebellum obtained from the WS cohort were compared with those from 16 chronological age- and sex-matched normal controls. A battery of cognitive tests were administered to all subjects undergoing 1H MRS. RESULTS: WS brains exhibited significant biochemical abnormalities. All 31P MRS ratios containing the phosphomonoester (PME) peak were significantly altered in WS, suggesting that PME is significantly decreased. Ratios of choline-containing compounds and creatine-containing compounds to N-acetylaspartate (Cho/NA and Cre/NA) were significantly elevated in the cerebellum in WS cf. controls, whereas the ratio of Cho/Cre was not altered. This suggests a decrease in the neuronal marker N-acetylaspartate in the cerebellum. Significant correlations were found between the cerebellar ratios Cho/NA and Cre/NA and the ability of all subjects at various neuropsychological tests, including Verbal and Performance IQ, British Picture Vocabulary Scale, Ravens Progressive Matrices, and Inspection Time. CONCLUSIONS: The correlations can be interpreted in two ways: 1) Our sampling of cerebellar biochemistry reflects a measure of "global" cerebral biochemistry and is unrelated to cerebellar function, or 2) The relations indicate that cerebellar neuronal integrity is a requirement (on a developmental time scale or in real-time) for ability on a variety of cognitive tests.

Adenosine Triphosphate↗
PubMed1998

Theoretical modelling of some spatial and temporal aspects of the mitochondrion/creatine kinase/myofibril system in muscle.

After discussing approaches to the modelling of mitochondrial regulation in muscle, we describe a model that takes account, in a simplified way, of some aspects of the metabolic and physical structure of the energy production/usage system. In this model, high-energy phosphates (ATP and phosphocreatine) and low energy metabolites (ADP and creatine) diffuse between the mitochondrion and the myofibrillar ATPase, and can be exchanged at any point by creatine kinase. Creatine kinase is not assumed to be at equilibrium, so explicit account can be taken of substantial changes in its activity of the sort that can now be achieved by transgenic technology in vivo. The ATPase rate is the input function. Oxidative ATP synthesis is controlled by juxtamitochondrial ADP concentration. To allow for possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase, we define parameters phi and psi that set the fraction of the total flux carried by ATP rather than phosphocreatine out of the mitochondrial unit and into the ATPase unit, respectively. This simplification is justified by a detailed analysis of the interplay between the mitochondrial outer membrane porin proteins, mitochondrial creatine kinase and the adenine nucleotide translocase. As both processes of possible 'coupling' are incorporated into the model as quantitative parameters, their effect on the energetics of the whole cell model can be explicitly assessed. The main findings are as follows: (1) At high creatine kinase activity, the hyperbolic relationship of oxidative ATP synthesis rate to spatially averaged ADP concentration at steady state implies also a near-linear relationship to creatine concentration, and a sigmoid relation to free energy of ATP hydrolysis. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on these relationships. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, and this is exaggerated when phi or psi is near unity (i.e. little coupling). (2) At high creatine kinase activity, the fraction of flow at steady state carried in the middle of the model by ATP is small, unaffected by the degree of functional coupling, but increases with ADP concentration and rate of ATP turnover. Lowering the creatine kinase activity raises this fraction, and this is exaggerated when psi or psi is near unity. (3) Both creatine and ADP concentrations show small gradients decreasing towards the mitochondrion (in the direction of their net flux), while ATP and phosphocreatine concentration show small gradients decreasing towards the myosin ATPase. Unless phi = psi = 0 (i.e. complete coupling), there is a gradient of net creatine kinase flux that results from the need to transform some of the 'adenine nucleotide flux' at the ends of the model into 'creatine flux' in the middle; the overall net flux is small, but only zero if phi = psi. A reduction in cytosolic creatine kinase activity decreases ADP concentration at the mitochondrial end and increases it at the ATPase end. (4) During work-jump transitions, spatial average responses exhibit exponential kinetics similar to those of models of mitochondrial control that assume equilibrium conditions for creatine kinase. (5) In response to a step increase in ATPase activity, concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. This simplified model embodies many important features of muscle in vivo, and accommodates a range of current theories as special cases. We end by discussing its relationship to other approaches to mitochondrial regulation in muscle, and some possible extensions of the model.

Adenosine Diphosphate↗
PubMed1997

CD40-triggered protein tyrosine phosphorylation on Vav and on phosphatidylinositol 3-kinase correlates with survival of the Ramos-Burkitt lymphoma B cell line.

Signals transduced through CD40 rescue cells of the Ramos-Burkitt lymphoma (Ramos-BL) B cell line from surface immunoglobulin M (sIgM)-triggered growth arrest and apoptosis. This study investigates whether protein tyrosine kinase (PTK) activity and tyrosine phosphorylation on p95(vav) and on the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase) play a role in the regulation of Ramos-BL B cell survival. The PTK inhibitor herbimycin A (HA) triggers significant growth arrest prior to apoptosis from the G1-phase of the cell cycle, indicating that tyrosine phosphorylation of key proteins is critical for Ramos-BL cell cycle progression and survival. Indeed, signals transduced through CD40 fail to rescue Ramos-BL B cells from HA-triggered growth arrest and apoptosis. Since Vav and PI3 kinase are intimately involved in the regulation of cellular growth, their tyrosine phosphorylation status was determined in unstimulated and anti-IgM- and anti-CD40-treated Ramos-BL B cells: Vav and p85 are devoid of tyrosine-phosphorylated epitopes in control cells whereas p85, but not Vav, is significantly phosphorylated following ligation of sIgM and anti-CD40 triggers tyrosine phosphorylation on both proteins. Thus, tyrosine-phosphorylated Vav may be a critical effector of CD40-mediated survival. As tyrosine-phosphorylated PI3 kinase is common to both sIgM-triggered death and CD40-triggered survival pathways, its lipid kinase activity was correlated with tyrosine phosphorylation on p85: Ramos-BL B cells exhibit high basal levels of PI3 kinase activity, determined by immunoprecipitation with anti-p85 and 32P incorporation into phosphatidylinositol, which is not significantly affected by stimulation with anti-IgM but which is elevated by 36 +/- 2.9% following ligation of CD40. Thus, tyrosine phosphorylation on p85 correlates with the CD40-triggered increase in PI3 kinase activity but not with basal levels nor with sIgM-triggered levels of enzymatic activity: these data suggest the presence of different PI3 kinase isoforms or the existence of multiple regulatory pathways for the same PI3 kinase isotype in Ramos-BL B cells.

B-Lymphocytes↗
PubMed1997

Wild-type but not mutant p53 activates the hepatocyte growth factor/scatter factor promoter.

p53 transactivates the expression of a variety of genes by binding to specific DNA sequences within the promoter. We have investigated the ability of wild-type p53 and a non-DNA binding p53 mutant to activate the hepatocyte growth factor/scatter factor (HGF/SF) promoter using chloramphenicol acetyltransferase reporter constructs. We also used deletion sequences of the HGF/SF promoter to identify which regions, if any, were responsible for p53 binding. Our results show that wild-type but not mutant p53 activates the HGF/SF promoter when using -3000 and -755 bp upstream of the HGF/SF gene. This activation is lost when promoter sequences covering -365 and -239 bp are used. Analysis of the DNA sequence between -365 and -755 bp shows one putative p53 half-site with 80% homology to the consensus sequence and another half-site 3 bases downstream of this with 100% homology to the consensus sequence. In contrast to previously identified p53 binding DNA sequences, the downstream half-site is inverted. We propose that the HGF/SF promoter can be activated by wild-type p53 in vivo and that this could be as a result of a novel form of sequence-specific DNA binding.

3T3 Cells↗
PubMed1997

Quantitative analysis of skeletal muscle bioenergetics and proton efflux in migraine and cluster headache.

Phosphorus MR spectroscopy (31P-MRS) was used to quantify skeletal muscle bioenergetics and proton efflux in 63 patients with migraine (23 with migraine without aura, MwoA, 22 with migraine with aura, MwA, and 18 with prolonged aura or stroke, CM) and in 14 patients with cluster headache (CH), all in an attack-free period. At rest mitochondrial function was abnormal only in CM, as shown by a low phosphocreatine (PCr) concentration. At the end of a mixed glycolytic/aerobic exercise all three migraine groups showed a significantly smaller decrease of cytosolic pH compared to controls with a similar end-exercise PCr breakdown, while end-exercise pH was normal in cluster headache patients. The normal rate of proton efflux in all headache groups suggests that the reduced end-exercise acidification was due to a reduction of glycolytic flux in migraine patients. The maximum rate of mitochondrial ATP production (Qmax), calculated from the rate of post-exercise PCr recovery and the end-exercise [ADP], was low in cluster headache patients as well as in migraine patients except MwoA. In migraine the degree of the mitochondrial impairment, that apparently is associated with a reduced glycolytic flux, is related to the severity of the clinical phenotype.

Adolescent↗
PubMed1997

Nitric oxide stimulates glucose transport and metabolism in rat skeletal muscle in vitro.

1. The effects of the nitric oxide (NO) donor sodium nitroprusside (SNP) on the rates of glucose transport and utilization and its interaction with insulin were investigated in rat soleus muscle in vitro. SNP stimulated the rate of 2-deoxyglucose transport and insulin-mediated (100 mu-units/ml) rates of both net and [14C]lactate release and the rate of glucose oxidation. The effects of SNP were independent of the concentration-dependent effects of insulin on glucose metabolism. 2. SNP stimulated the insulin-stimulated rates of net and [14C]lactate release and glucose oxidation in a concentration-dependent manner. The rate of [14C]lactate release was also stimulated by another NO donor, (Z)-1-(N-[aminopropyl]-N-[4-(3-aminopropylammonio) butyl]-amino)-diazen-l-ium-1,2-diolate (spermine NONOate). 3. SNP at 5, 10 and 15 mM inhibited the insulin-stimulated rate of glycogen synthesis and this rate was further decreased at 20 and 25 mM SNP. SNP did not affect the rate of glycogen synthesis in the absence of insulin. 4. Haemoglobin, which is a NO scavenger, prevented the stimulation of the rates of [14C]lactate release by SNP or spermine NONOate. 5. The cGMP content was increased maximally (by approx. 80-fold) within 15 min by SNP (15 mM). The cGMP content, raised maximally by SNP, was significantly decreased by the guanylate cyclase inhibitor LY-83583 (10 microM). The cGMP analogue 8-bromo-cGMP (100 microM) significantly increased the rate of net lactate release. 6. LY-83583 significantly inhibited SNP-stimulated rates of 2-deoxyglucose transport, [4C]lactate release and glucose oxidation. Methylene Blue (another guanylate cyclase inhibitor) also inhibited SNP-stimulated rates of [14C]lactate release. 7. The results suggest that in rat skeletal muscle: (a) nitric oxide (from SNP or spermine NONOate) increases the rate of glucose transport and metabolism, an effect independent of insulin; (b) SNP inhibits insulin-mediated rates of glycogen synthesis; (c) SNP stimulates cGMP formation, which mediates, at least partly, the effects on glucose metabolism; (d) nitric oxide-mediated stimulation of glucose utilization might occur in fibre contraction. The implications of the effects of NO on glucose metabolism are discussed.

Animals↗
PubMed1997

A 31P-magnetic resonance spectroscopy and biochemical study of the mo(vbr) mouse: potential model for the mitochondrial encephalomyopathies.

31P-magnetic resonance spectroscopy (31P-MRS) provides new biochemical information on mitochondrial disorders affecting brain and muscle. To elucidate the mechanisms of mitochondrial abnormalities, however, animal models are needed. We assessed the mo(vbr) (mottled viable brindled) mouse for its value in studying (1) energetics of a mitochondrial disorder and (2) 31P-MRS changes associated with mitochondrial abnormalities in vivo. The maximal activity of succinate-cytochrome c reductase was significantly reduced in mo(vbr) muscle compared to controls, whereas cytochrome oxidase activity was only reduced in mo(vbr) brain. 31P-MRS of mo(vbr) brain showed an increased pH, but no changes in any metabolite ratios. The phosphocreatine (PCr) recovery rate after exercise was reduced in muscles from mo(vbr) mice, indicating impairment of oxidative metabolism. We conclude that mo(vbr) brain and muscle tissue have biochemical abnormalities consistent with mitochondrial impairment. The PCr recovery rate, measured by 31P-MRS, was sensitive to the muscle abnormality. This strain is best described as having chronic mitochondrial dysfunction.

Animals↗
PubMed1997

In vivo skeletal muscle mitochondrial function in Leber's hereditary optic neuropathy assessed by 31P magnetic resonance spectroscopy.

We used 31P magnetic resonance spectroscopy (31P-MRS) to assess in vivo skeletal muscle mitochondrial function in 10 Leber's hereditary optic neuropathy patients/carriers with a mitochondrial DNA (mtDNA) mutation at one of three nucleotide positions, 11,778, 14,484, and 3,460. We studied one affected patient for each mutation and two unaffected carriers with the 11,778 or 3,460 mutation and three carriers with 14,484. All subjects were homoplasmic except the two 3,460 carriers, who showed 80% and 15% of mutated mtDNA. 31P-MRS at rest disclosed some abnormalities in all subjects. In particular, the phosphorylation potential was below the normal range in all cases. During recovery from exercise, the maximum rate of mitochondrial ATP production (Vmax) was reduced to 27% of normal in the 11,778 mutation and to 53% in the 14,484 mutation patient/carrier groups. Mitochondrial Vmax was within the normal range in all subjects with the 3,460 mutation but correlated inversely with the percentage of mutated mtDNA. This in vivo study shows that the 11,778 mutation causes a mitochondrial impairment more severe than the 14,484 and that the 3,460 mutation results in only a mild depression of muscle mitochondrial function.

Adult↗
PubMed1997

Influence of cytosolic pH on in vivo assessment of human muscle mitochondrial respiration by phosphorus magnetic resonance spectroscopy.

The authors present an in vivo phosphorus magnetic resonance spectroscopy systematic study on the effects of cytosolic pH on skeletal muscle mitochondrial respiration in human calf muscle. In 49 normal subjects, the effect of cytosolic pH on kinetics of phosphocreatine and adenosine diphosphate recovery and on maximum rate of mitochondrial adenosine triphosphate production (Q(MAX)) was evaluated. The results show a strong relationship between the rate of postexercise phosphocreatine recovery and the lowest value of cytosolic pH reached during recovery from exercise (termed minimum pH; r = 0.89); in contrast, both adenosine diphosphate recovery halftime and Q(MAX) were independent of cytosolic pH at the end of exercise.

Adenosine Diphosphate↗
PubMed1997

Proton efflux in human skeletal muscle during recovery from exercise.

In recovery from exercise, phosphocreatine resynthesis results in the net generation of protons, while the net efflux of protons restores pH to resting values. Because proton efflux rate declines as pH increases, it appears to have an approximately linear pH-dependence. We set out to examine this in detail using recovery data from human calf muscle. Proton efflux rates were calculated from changes in pH and phosphocreatine concentration, measured by 31P magnetic resonance spectroscopy, after incremental dynamic exercise to exhaustion. Results were collected post hoc into five groups on the basis of end-exercise pH. Proton efflux rates declined approximately exponentially with time. These were rather similar in all groups, even when pH changes were small, so that the apparent rate constant (the ratio of efflux rate to pH change) varied widely. However, all groups showed a consistent pattern of decrease with time; the halftimes of both proton efflux rate and the apparent rate constant were longer at lower pH. At each time-point, proton efflux rates showed a significant pH-dependence [slope 17 (3) mmol x l(-1) x min(-1) x pH unit(-1) at the start of recovery, mean (SEM)], but also a significant intercept at resting pH [16 (3) mmol x l(-1) x min(-1) at the start of recovery]. The intercept and the slope both decreased with time, with halftimes of 0.37 (0.06) and 1.4 (0.4) min, respectively. We conclude that over a wide range of end-exercise pH, net proton efflux during recovery comprises pH-dependent and pH-independent components, both of which decline with time. Comparison with other data in the literature suggests that lactate/proton cotransport can be only a small component of this initial recovery proton efflux.

Adult↗
PubMed1997

Fetal growth and insulin resistance in adult life: role of skeletal muscle morphology.

1. Thinness at birth is associated with insulin resistance in adult life and an apparent delay in activation of glycolysis/glycogenolysis in exercising skeletal muscle. As developmental abnormalities of skeletal muscle histology or metabolism may explain this association we examined muscle histology, biochemistry and blood flow in a group of 27 adult women whose birth details were known. 2. Subjects were examined by near-infrared spectroscopy to determine forearm muscle oxygen supply, and by muscle biopsy and forearm plethysmography. Those with a ponderal index at birth < 23 kg/m3 were insulin resistant (assessed by the short insulin-tolerance test-mean rate constants for glucose disappearance = 4.14 compared with 4.83%/min, P = 0.045) and had significantly more rapid muscle reoxygenation than the remainder of the subjects (13 compared with 22 s, P = 0.004). 3. Thinness at birth did not influence muscle capillary density, muscle glycogen content, glycogen synthase activity, citrate synthase activity or resting forearm blood flow. 4. Insulin resistance seen after fetal malnutrition was not associated with abnormal muscle histology, resting muscle blood flow, mitochondrial volume or glycogen content. 5. The increase in muscle reoxygenation rate in adult subjects who were thin at birth could occur to promote oxidative ATP synthesis in compensation for the delay in activation of glycolysis/glycogenolysis. It suggests altered regulation rather than structure of the muscle microcirculation. These changes appear to antedate the structural and biochemical changes seen in muscle from patients with established diabetes.

Embryonic and Fetal Development↗