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J W Phillips

Publications and source records attributed to J W Phillips.

At least 37 records · Page 2Linked to original sources
PubMed1995

The contribution of glucose cycling to the maintenance of steady-state levels of lactate by hepatocytes during glycolysis and gluconeogenesis.

When hepatocytes from fasted rats were incubated with 10 mM glucose, there was a linear accumulation of lactate and pyruvate for about 80 min after which steady-state concentrations of these metabolites became established. The rate of glycolysis, determined with [6-3H]glucose, was constant over the entire incubation period and was 50% greater than that calculated from carbon balance studies. This suggests that one-third of the glycolytic products formed were recycled to glucose. To enable study of the factors associated with the generation and maintenance of the lactate steady state and to measure accurately the carbon balance, incubations were performed using supraphysiological concentrations of glucose (20-80 mM). Under these conditions the initial rate of lactate accumulation and its concentration at steady state were shown to be dependent on the concentration of extracellular glucose. Rates of glycolysis were also measured using 40 mM [6-3H]glucose and [U-14C]glucose added alone, or in combination with a steady-state lactate concentration (3 mM). There was no effect on the rate of glycolysis determine with [6-3H]glucose, even when lactate was present in the medium. The difference in rates between measurements with the two isotopes reflect the apparent degree of glucose recycling which in the absence and presence of added lactate increased from 0.26 to 0.54 mumol C3 equivalents min-1.g-1 respectively. Identical studies employing [U-14C]lactate showed that glucose and CO2 were the major products of lactate metabolism under steady-state conditions and that the formation of lactate from [U-14C]glucose exactly balanced the rate of lactate removal as a result of oxidation and gluconeogenesis. These studies provide evidence for the concomitant operation of glycolysis and gluconeogenesis, even in the presence of high glucose concentrations. They also demonstrate that lactate steady states are achieved not by the cessation of glycolysis but rather by the removal of lactate and pyruvate at a rate equal to that of their production.

Animals↗
PubMed1994

The capacity of reducing-equivalent shuttles limits glycolysis during ethanol oxidation.

The inhibition of glycolysis during ethanol oxidation has been examined in isolated hepatocytes from fasted rats. Glycolytic flux was measured by determining the rate of release of tritium from [6-3H]glucose. During ethanol oxidation, the rate of glycolysis was inhibited 80% in freshly prepared hepatocytes, in which shuttle intermediates are depleted, but was depressed only about 20% in the presence of asparagine, a condition under which activity of the malate/aspartate shuttle was restored to normal levels. The inhibition of glycolysis was also partially released by addition of pyruvate and when alcohol dehydrogenase activity was depressed by 4-methylpyrazole. Titrations with this inhibitor revealed inverse linear relationships between the rates of glycolysis and ethanol oxidation. For any given rate of ethanol oxidation, glycolytic flux was lowest and the [lactate]/[pyruvate] ratio highest in the presence of aminooxyacetate, an inhibitor of the malate/aspartate shuttle, whereas flux was highest and the ratio lowest in the presence of asparagine. During these titrations with 4-methylpyrazole the inhibition of ethanol oxidation and concomitant restoration of glycolysis were accompanied by a decline in the [lactate]/[pyruvate] ratio, a substantial fall in the rate of reducing-equivalent transfer from cytoplasm to mitochondria and an increase in lactate accumulation. These findings imply that the reducing equivalents generated during ethanol oxidation compete with those arising in glycolysis for transfer to the mitochondria. This competition leads to an inhibition of aerobic glycolysis, and at the same time contributes to a rise in cytoplasmic NADH and fall in NAD+ that results in depression of anaerobic glycolysis. Allosteric inhibition of 6-phosphofructo-1-kinase due to a decrease in the concentration of fructose 2,6-bisphosphate did not appear to play a primary role in the inhibition of glycolysis by ethanol. Ethanol oxidation had no effect on glucose phosphorylation as measured with [2-3H]glucose, but induced a substantial increase in cycling between glucose and glucose 6-phosphate.

Animals↗
PubMed1994

Effect of citrate infusion on urinary aluminium excretion in the rat.

1. Seven pairs of rats received 1 mmol/l aluminium citrate in their drinking water 5 days before the experiments. Five additional rats were treated identically. Six rats received the same food but drank distilled water. 2. After a 6 h fast, the animal was anaesthetized, the jugular vein and femoral artery were cannulated and the bladder was catheterized, after which an intravenous infusion of Hartmann's solution containing [14C]inulin was begun. The urine was collected at 20 min intervals and 1 ml of arterial blood was obtained before the end of each collection. After at least two basal collections, the infusion was modified to contain, in addition, 5 mmol/l NaHCO3 (control) or 5 mmol/l sodium citrate (experimental). The infusion rate, constant in each pair, differed between pairs across the range 60-125 mumol/min. 3. A total of eight collections was made per animal and urine flow, glomerular filtration rate, plasma and urinary aluminium and citrate were measured. 4. Control and experimental rats had a higher mean basal plasma aluminium level (0.39 +/- 0.21 mumol/l) than the six rats receiving distilled water (0.16 +/- 0.14 mmol/l, P < 0.001). The corresponding urinary aluminium excretion rates were similar (46 +/- 31 and 47 +/- 23 pmol/min, respectively). There was no significant difference between the basal values of any variable in the control and experimental rats. No significant change was observed in any variable during the infusion of NaHCO3 (controls). Among the experimental rats, there was no significant change in urine flow, glomerular filtration rate or plasma aluminium level.(ABSTRACT TRUNCATED AT 250 WORDS)

Aluminum↗
PubMed1994

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.

Adenine Phosphoribosyltransferase↗
PubMed1993

Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes.

We have studied the inhibitory action of long- and short-chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2-3H] and [6-3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the 'cycling' between glucose and glucose-6-phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 mumol/min/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 mumol/min/g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.

Animals↗
PubMed1993

Noncomplementary DNA double-strand-break rejoining in bacterial and human cells.

We examined the rejoining of noncomplementary restriction enzyme-produced DNA double-strand breaks in Escherichia coli and in cultured human cells. The enzymes used in this study, ClaI, BamHI and SalI, produce double-strand breaks with 5 protruding single strands. The joining of a ClaI-produced DNA end to a BamHI-produced end or to a SalI-produced end was examined at the DNA sequence level. End rejoining in E.coli was studied by transforming cultures with linear plasmid DNA that was gel purified from restriction digests, and end rejoining in cultured human cells was studied by introducing enzymes into the cells by electroporation. The human cells used contain an Epstein-Barr virus (EBV)-based shuttle vector, pHAZE, that was recovered and introduced into E.coli for further analysis. The major products of DNA end-joining processes observed in linear plasmid-transformed E.coli and in the human cells exposed to restriction enzymes were identical. Furthermore, the deletions observed in both systems and in the spontaneous mutant plasmids in untreated human cells had a common underlying feature: short stretches of directly repeated DNA at the junction sites.

Base Composition↗
PubMed1993

Cellular resistance to oxidative stress is accompanied by resistance to cisplatin: the significance of increased catalase activity and total glutathione in hydrogen peroxide-resistant fibroblasts.

Studies designed to better understand the involvement of cellular resistance to oxidative stress in mechanisms of cellular resistance to cisplatin were undertaken using H2O2-resistant variants of the HA1 Chinese hamster fibroblast cell line. H2O2-resistant cell lines were resistant to clonogenic inactivation mediated by cisplatin with dose modifying factors at 10% survival of 1.5-3.0, relative to HA1 cells. The most cisplatin resistant of these cell lines (OC5) also demonstrated fewer DNA-DNA crosslinks induced by cisplatin, relative to HA1. Since H2O2-resistant cells contained increased catalase activity as well as total glutathione (GSH) content, the involvement of these cellular antioxidants in the resistance to cisplatin toxicity was evaluated. Treatment of HA1 and H2O2-resistant cell lines (OC5, OC14) with 9 mM aminotriazole reduced catalase activity by 60-65% but had no effect on the cytotoxicity of cisplatin. In contrast, treatment with 5 mM buthionine sulfoximine reduced total GSH by 90% and sensitized the cells to cisplatin cytotoxicity. Furthermore, extracellular reaction of GSH with cisplatin prior to treating HA1 cells reduced the toxicity of the compound, indicating that this reaction is capable of participating in the detoxification of cisplatin. These results indicate that cellular adaptation to oxidative stress renders cells resistant to DNA damage as well as to cytotoxicity associated with cisplatin treatment. Furthermore, increases in total GSH content (but not catalase activity) appear to partially account for cisplatin resistance demonstrated by H2O2-resistant cells.

Animals↗
PubMed1992

Reducing-equivalent transfer to the mitochondria during gluconeogenesis and ureogenesis in hepatocytes from rats of different thyroid status.

Isolated hepatocytes from hypothyroid, euthyroid and hyperthyroid rats have been employed to investigate the relative importance of reducing-equivalent shuttles for the transfer of hydrogen between cytoplasm and mitochondria during simultaneous ureogenesis and gluconeogenesis. In cells from hypothyroid animals, a 58% depression of glucose formation and 68% reduction in ureogenesis were induced by n-butylmalonate, an inhibitor of the malate shuttle. A more reduced state of the cytoplasmic compartment and a substantial fall in the concentrations of pyruvate, aspartate, alanine and glutamate accompanied this inhibition. Preincubation of cells with n-butylmalonate yielded greater inhibitory effects than observed in the absence of preincubation. The inhibitory effects on gluconeogenesis and ureogenesis were less in cells from euthyroid rats and were very much reduced in the case of glucose synthesis and absent in the case of ureogenesis, in cells from hyperthyroid rats. It is inferred that both the malate-aspartate and alpha-glycerophosphate shuttles may function in the transfer of reducing equivalents from cytoplasm to mitochondria during ureogenesis in hepatocytes. The major inhibition by n-butylmalonate of glucose and urea synthesis in hepatocytes from hypothyroid rats is due to the diminished activity of the alpha-glycerophosphate shuttle in these cells. Moreover, it follows that the NADH arising from the cytoplasmic malate dehydrogenase-catalysed reaction is accessible to both the malate-aspartate shuttle and the alpha-glycerophosphate shuttle.

Alanine↗
PubMed1992

Operation and energy dependence of the reducing-equivalent shuttles during lactate metabolism by isolated hepatocytes.

The participation and energy dependence of the malate-aspartate shuttle in transporting reducing equivalents generated from cytoplasmic lactate oxidation was studied in isolated hepatocytes of fasted rats. Both lactate removal and glucose synthesis were inhibited by butylmalonate, aminooxyacetate or cycloserine confirming the involvement of malate and aspartate in the transfer of reducing equivalents from the cytoplasm to mitochondria. In the presence of ammonium ions the inhibition of lactate utilization by butylmalonate was considerably reduced, yet the transfer of reducing equivalents into the mitochondria was unaffected, indicating a substantially lesser role for butylmalonate-sensitive malate transport in reducing-equivalent transfer when ammonium ions were present. Ammonium ions had no stimulatory effect on uptake of sorbitol, a substrate whose oxidation principally involves the alpha-glycerophosphate shuttle. The role of cellular energy status (reflected in the mitochondrial membrane electrical potential (delta psi) and redox state), in lactate oxidation and operation of the malate-aspartate shuttle, was studied using a graded concentration range of valinomycin (0-100 nM). Lactate oxidation was strongly inhibited when delta psi fell from 130 to 105 mV whereas O2 consumption and pyruvate removal were only minimally affected over the valinomycin range, suggesting that the oxidation of lactate to pyruvate is an energy-dependent step of lactate metabolism. Our results confirm that the operation of the malate-aspartate shuttle is energy-dependent, driven by delta psi. In the presence of added ammonium ions the removal of lactate was much less impaired by valinomycin, suggesting an energy-independent utilization of lactate under these conditions. The oxidizing effect of ammonium ions on the mitochondrial matrix apparently alleviates the need for energy input for the transfer of reducing equivalents between the cytoplasm and mitochondria. It is concluded that, in the presence of ammonium ions, the transport of lactate hydrogen to the mitochondria is accomplished by malate transfer that is not linked to the electrogenic transport of glutamate across the inner membrane, and, hence, is clearly distinct from the butylmalonate-sensitive, energy-dependent, malate-aspartate shuttle.

Ammonia↗
PubMed1992

Differential growth of brain and retinal bovine pericytes.

Within the central nervous system, pericyte degeneration in diabetes mellitus occurs only in the retinal microcirculation and is not seen in the brain. This study sought to elucidate differences between bovine retinal and brain pericytes. When pairs of retinal and brain pericytes from individual calves were cultured in vitro, the morphological organisation of early post-confluent retinal pericyte cultures was consistently different from that of brain pericyte cultures. When retinal and brain pericyte cultures were grown to second passage in high or normal glucose medium supplemented with fetal calf serum, brain pericyte cultures grew significantly faster than retinal pericytes in either medium (p less than 0.0001). Brain pericytes thus appeared to grow intrinsically faster than retinal pericytes and this effect was largely independent of glucose concentration. Brain pericytes also grew faster than retinal pericytes in high glucose medium containing human diabetic or control serum (p less than 0.002). The proliferative effect of serum from diabetic patients with non-proliferative diabetic retinopathy on pericytes grown in high glucose medium was not significantly different from that of control serum. Both brain and retinal pericytes showed variation in their ability to replicate in high concentrations of glucose. The selectivity of pericyte degeneration to the retinal circulation does not appear to be due to changes in the mitogenic activity of diabetic serum for retinal pericytes, but may relate to the intrinsic relative inability of the retinal pericyte to reproliferate in response to the metabolic injury of diabetes mellitus.

Analysis of Variance↗
PubMed1992

The role of lipid peroxidation in McArdle's disease: applications for treatment of other myopathies.

Experimental therapies for McArdle's disease have been directed toward increasing substrate availability to exercising muscle. Such therapies to date have proven largely unsuccessful. These include administration of isoproterenol to increase blood flow, glucagon treatment to elevate serum glucose and increased dietary fat intake. Each of these therapies also results in greater levels of unesterified fatty acids in blood. More recently, a high protein diet is suggested to provide increased amounts of amino acids which would be available as fuel sources. We hypothesize that the absence of myophosphorylase in McArdle's disease creates an imbalance between the enzymes of the redox systems that control the generation, propagation and inactivation of free radicals. This occurs because muscle cells are forced to rely more heavily on fatty acid oxidation. The resulting free radical damage to cellular components disrupts metabolic control and increases the permeability of membranes. Elevated levels of Ca2+ in the sarcoplasm activate proteases, phospholipases and other catabolic enzymes initiating muscle fatigue and cramping. Lipid peroxidation is a consequence of normal muscle activity and may occur unchecked in individuals with McArdle's disease. Continued muscle activity in the absence of a favorable nutritional environment may promote the progression of the disease by increasing susceptibility to oxidative stress.

Antioxidants↗
PubMed1992

Interactions between mitochondria and cytoplasm in isolated hepatocytes.

Results from a wide variety of metabolic studies have provided indirect support for conclusions derived from enzymological approaches that the enzymes of the so-called soluble cytoplasm (and the mitochondrial matrix) exist within the cell and function in the form of multienzyme complexes and that metabolite channeling takes place between the enzymes of each complex. Our studies support the possibility that the enzymes of glycolysis in liver are segregated from those of gluconeogenesis. Thus, the segregation and aggregation of Krebs cycle enzymes in the mitochondrial matrix, elucidated by Paul Srere, may be an example of a general pattern of enzyme organization pertaining to all metabolic pathways.

Animals↗
PubMed1992

Evaluation of support bandaging during measurement of proximal sesamoidean ligament strain in horses by use of a mercury strain gauge.

Liquid mercury strain gauges were implanted in the forelimb proximal sesamoidean ligaments (PSL) of 8 adult horses. The gauges measured PSL strain while horses were standing with or without external support. In 6 of the horses, the gauges also measured PSL strain in horses at a walk, with or without external support. Gauges were enclosed within sliding polypropylene tubes to prevent nonaxial deformation. Each gauge was placed in 1 arm of a low-resistance half-bridge circuit. To provide temperature compensation, a dummy gauge was placed in the adjacent arm of the bridge circuit and was implanted next to the active gauge in the surrounding fascial tissue. External support included fiberglass cast support (CAST), dorsal fetlock splint support (DFS), support wraps of 3 bandage materials (SW1, SW2, and SW3), and support wrap with caudal splint (SW4). The cast was applied, with the fetlock and foot in weightbearing position, from the proximal portion of the metacarpus distal to and including the foot. The DFS was applied by placing the cranial half of the fiberglass cast on the dorsal aspect of the instrumented limb. The SW1, SW2, and SW3 were applied in a figure-8 pattern around the fetlock, using 50% of the linear stretch capacity of the bandage material, with the horse standing squarely on all 4 limbs. The SW4 was applied identically to the other support wraps, with the exception of addition of a flexible caudal splint incorporated in the support wrap. Mean maximal strain while standing (epsilon S) without external support for 8 horses was 6.0% (range, 3.8 to 7.5%).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Chromosomal aberration induction in CHO cells by combined exposure to restriction enzymes and X-rays.

The potential interaction between restriction enzyme-induced double-strand breaks (dsb) and X-ray-induced lesions in the formation of chromosomal aberrations was investigated in Chinese hamster ovary cells. Either Alu I, which induces blunt-end dsb, or Sau 3AI, which induces cohesive-end dsb, was electroporated into cells, which were irradiated with 2 Gy of X-rays immediately or 15, 30, 60, 120, or 180 min after electroporation. A significant increase in Alu I-induced chromosomal aberrations was observed when cells were irradiated with 0, 15, 30, or 60 min after enzyme exposure, but only additive effects were found when cells were irradiated 120 or 180 min after enzyme exposure. In one of three experiments, cells exposed to Sau 3AI showed a large increase in aberrations when X-irradiated 0 or 15 min after Sau 3AI exposure, and no increase at any time-points thereafter. These results indicate that restriction enzyme-induced dsb can interact with X-ray-induced lesions, resulting in a synergistic increase in chromosomal aberration formation. Furthermore, this interaction depends on both the type of dsb and the time between enzyme and X-ray exposure.

Animals↗
PubMed1991

Analysis of restriction enzyme-induced DNA double-strand breaks in Chinese hamster ovary cells by pulsed-field gel electrophoresis: implications for chromosome damage.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.

Animals↗
PubMed1991

Modulation of restriction enzyme-induced damage by chemicals that interfere with cellular responses to DNA damage: a cytogenetic and pulsed-field gel analysis.

The electroporation of restriction enzymes into mammalian cells results in DNA double-strand breaks that can lead to chromosome aberrations. Four chemicals known to interfere with cellular responses to DNA damage were investigated for their effects on chromosome aberrations induced by AluI and Sau3AI; in addition, the number of DNA double-strand breaks at various times after enzyme treatment was determined by pulsed-field gel electrophoresis (PFGE). The poly(ADP-ribose) polymerase inhibitor 3-aminobenzamide (3AB) dramatically increased the yield of exchanges and deletions and caused a small but transitory increase in the yield of double-strand breaks induced by the enzymes. 1-beta-D-Arabinofuranosylcytosine, which can inhibit DNA repair either by direct action on DNA polymerases alpha and delta or by incorporation into DNA, potentiated aberration induction but to a lesser extent than 3AB and did not affect the amount of DNA double-strand breakage. Aphidicolin, which inhibits polymerases alpha and delta, had no effect on AluI-induced aberrations but did increase the aberration yield induced by Sau3AI. The postreplication repair inhibitor caffeine had no effect on aberration yields induced by either enzyme. Neither aphidicolin nor caffeine modulated the amount of DNA double-strand breakage as measured by PFGE. These data implicate poly(ADP-ribosyl)ation and polymerases alpha and delta as important components of the cellular processes required for the normal repair of DNA double-strand breaks with blunt or cohesive ends. Comparison of these data with the effect of inhibitors on the frequency of X-ray-induced aberrations leads us to the conclusion that X-ray-induced aberrations can result from the misjoining or nonrejoining of double-strand breaks, particularly breaks with cohesive ends, but that this process accounts for only a portion of the induced aberrations.

Animals↗
PubMed1990

Chromosome aberration induction in Chinese hamster ovary cells by restriction enzymes with different methylation sensitivity.

The isoschizomer pair MspI and HpaII were used to investigate whether the putative specificity of restriction endonucleases would be maintained when they were introduced into mammalian cells. Although both enzymes recognize the sequence CCGG, HpaII will cut only if the internal cytosine is unmethylated, whereas MspI will cut regardless of the methylation status. Cleavage results in a cohesive-end DNA double-strand break, which can lead to the formation of chromosome aberrations. Since mammalian DNA is heavily methylated, one would expect MspI to be much more effective than HpaII at inducing chromosome aberrations in Chinese hamster ovary cells. In fact, during G1, MspI induced a greater than 90-fold higher number of aberrations than did HpaII. Cell cycle studies indicated that during early S there was a 30-fold increase in HpaII-induced aberrations. This increase may be due to increased accessibility of replicating hypomethylated DNA. Cells that were treated with the demethylating agent 5-aza-2'-deoxycytidine (AzdC) displayed only a moderate increase in HpaII-induced aberrations during G1. This observation, together with the results of restriction enzyme analysis of genomic DNA, indicated that demethylation was incomplete. The effects of AzdC on the induction of aberrations by MspI suggested that AzdC increases chromatin accessibility. Our results were consistent with the expected specificity of MspI and HpaII. Thus, it appears that restriction endonucleases can play a useful role in determining the biological consequences of DNA double-strand breaks.

Animals↗