NTP Levels Research Articles

Quantitative analysis of transcription start site selection reveals control by DNA sequence, RNA polymerase II activity and NTP levels.

Transcription start site (TSS) selection is a key step in gene expression and occurs at many promoter positions over a wide range of efficiencies. Here we develop a massively parallel reporter assay to quantitatively dissect contributions of promoter sequence, nucleoside triphosphate substrate levels and RNA polymerase II (Pol II) activity to TSS selection by 'promoter scanning' in Saccharomyces cerevisiae (Pol II MAssively Systematic Transcript End Readout, 'Pol II MASTER'). Using Pol II MASTER, we measure the efficiency of Pol II initiation at 1,000,000 individual TSS sequences in a defined promoter context. Pol II MASTER confirms proposed critical qualities of S. cerevisiae TSS -8, -1 and +1 positions, quantitatively, in a controlled promoter context. Pol II MASTER extends quantitative analysis to surrounding sequences and determines that they tune initiation over a wide range of efficiencies. These results enabled the development of a predictive model for initiation efficiency based on sequence. We show that genetic perturbation of Pol II catalytic activity alters initiation efficiency mostly independently of TSS sequence, but selectively modulates preference for the initiating nucleotide. Intriguingly, we find that Pol II initiation efficiency is directly sensitive to guanosine-5'-triphosphate levels at the first five transcript positions and to cytosine-5'-triphosphate and uridine-5'-triphosphate levels at the second position genome wide. These results suggest individual nucleoside triphosphate levels can have transcript-specific effects on initiation, representing a cryptic layer of potential regulation at the level of Pol II biochemical properties. The results establish Pol II MASTER as a method for quantitative dissection of transcription initiation in eukaryotes.

Anfractuous assemblies of IMP dehydrogenase and CTP synthase: new twists on regulation?

CTP synthase (CTPS) and IMP dehydrogenase (IMPDH) catalyse the rate-limiting steps of de novo CTP and guanosine nucleotide biosynthesis, respectively, and form filament assemblies in response to inhibitors. A recent study explores the morphology and dynamics of these assemblies using fluorescence and super-resolution confocal microscopy with cell lines expressing CTPS1 and IMPDH2 fusion proteins. The formation and dismantling of mixed assemblies depends on nucleotide levels, suggesting a co-regulation function.

P0683 : Hepatitis C virus NS3-4A protease targets the host factor BNIP1 at a non-canonical cleavage site

oral dosing for the ability to form the active AL-335 NTP in dog liver. Results: In the stable genotype (GT) 1b replicon assay, AL-335 exhibited potent antiviral activity with an EC50 of 75nM. In transient chimeric GT-1b replicons with NS5B regions derived from GT 1–4, AL-335 demonstrated pan-genotypic activity with EC50 values between 40–60nM. AL-335 retained activity vs. replicon mutants resistant to non-nucleoside polymerase, NS3/4A protease and NS5A inhibitors. AL-335 was additive or synergistic in all combinations studies. The AL-335 NTP was a potent inhibitor of the HCV NS5B polymerase with an IC50 of 120nM and a Ki of 24nM, and acted as a chain-terminator of RNA synthesis. The AL-335 NTP was not a substrate for human mitochondrial RNA polymerase and demonstrated no inhibition (IC50 >100mM) of human DNA or RNA polymerases. In vitro, the AL-335 NTP was rapidly formed and demonstrated a long intracellular half-life of >24hrs in primary human hepatocytes. Following oral administration to dog at 5mg/kg parent nucleoside equivalent, the AL-335 NTP was formed at high and sustained levels in liver (AL-335 NTP levels at 24hrs: 2.1mM). Conclusions: AL-335 is a potent uridine based nucleotide analog that demonstrates a desirable preclinical profile. The compound is currently advancing in preclinical studies as a potential treatment for CHC.

Effects of sleep deprivation on brain bioenergetics, sleep, and cognitive performance in cocaine-dependent individuals.

In cocaine-dependent individuals, sleep is disturbed during cocaine use and abstinence, highlighting the importance of examining the behavioral and homeostatic response to acute sleep loss in these individuals. The current study was designed to identify a differential effect of sleep deprivation on brain bioenergetics, cognitive performance, and sleep between cocaine-dependent and healthy control participants. 14 healthy control and 8 cocaine-dependent participants experienced consecutive nights of baseline, total sleep deprivation, and recovery sleep in the research laboratory. Participants underwent [31]P magnetic resonance spectroscopy (MRS) brain imaging, polysomnography, Continuous Performance Task, and Digit Symbol Substitution Task. Following recovery sleep, [31]P MRS scans revealed that cocaine-dependent participants exhibited elevated global brain β-NTP (direct measure of adenosine triphosphate), α-NTP, and total NTP levels compared to those of healthy controls. Cocaine-dependent participants performed worse on the Continuous Performance Task and Digit Symbol Substitution Task at baseline compared to healthy control participants, but sleep deprivation did not worsen cognitive performance in either group. Enhancements of brain ATP levels in cocaine dependent participants following recovery sleep may reflect a greater impact of sleep deprivation on sleep homeostasis, which may highlight the importance of monitoring sleep during abstinence and the potential influence of sleep loss in drug relapse.

Dissection of Pol II Trigger Loop Function and Pol II Activity–Dependent Control of Start Site Selection In Vivo

Structural and biochemical studies have revealed the importance of a conserved, mobile domain of RNA Polymerase II (Pol II), the Trigger Loop (TL), in substrate selection and catalysis. The relative contributions of different residues within the TL to Pol II function and how Pol II activity defects correlate with gene expression alteration in vivo are unknown. Using Saccharomyces cerevisiae Pol II as a model, we uncover complex genetic relationships between mutated TL residues by combinatorial analysis of multiply substituted TL variants. We show that in vitro biochemical activity is highly predictive of in vivo transcription phenotypes, suggesting direct relationships between phenotypes and Pol II activity. Interestingly, while multiple TL residues function together to promote proper transcription, individual residues can be separated into distinct functional classes likely relevant to the TL mechanism. In vivo, Pol II activity defects disrupt regulation of the GTP-sensitive IMD2 gene, explaining sensitivities to GTP-production inhibitors, but contrasting with commonly cited models for this sensitivity in the literature. Our data provide support for an existing model whereby Pol II transcriptional activity provides a proxy for direct sensing of NTP levels in vivo leading to IMD2 activation. Finally, we connect Pol II activity to transcription start site selection in vivo, implicating the Pol II active site and transcription itself as a driver for start site scanning, contravening current models for this process.

Hepatitis C Virus NS5B Polymerase Exhibits Distinct Nucleotide Requirements for Initiation and Elongation

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase (RdRp) essential for replication of the viral RNA genome. Purified NS5B has been reported to exhibit multiple activities in vitro. Using a synthetic heteropolymeric RNA template with dideoxycytidine at its 3'-end, we examined de novo initiation and primer extension in a system devoid of self-priming and terminal nucleotide transferase activities. Products predominantly of template size and its multiples were detected. High concentrations of nucleoside triphosphates (K(app)(m) approximately 100-400 mum) corresponding to the first three incorporated nucleotides were found to be required for efficient de novo RNA synthesis. In the presence of initiating di- or trinucleotides, however, the amount of NTP needed to achieve maximal activity dropped 10(3)- to 10(4)-fold, revealing a much reduced nucleotide requirement for elongation (K(app)(m) approximately 0.03-0.09 microm). Accordingly, single round extension from an exogenous primer following preincubation of the enzyme with template and primer could also be supported by <0.1 microm levels of NTP. De novo synthesis at high NTP concentrations was shown to be preferred over primer extension. On a dideoxycytidine-blocked synthetic RNA template derived from the 3'-end of the HCV(-)UTR, the addition of the corresponding initiating trinucleotide also dramatically reduced the NTP levels needed to achieve efficient RNA synthesis. Thus, distinct nucleotide requirements exist for initiation and elongation steps catalyzed by the HCV NS5B polymerase.

NM23-H2 Is a Tumor Associated Antigen (TAA) in Chronic Myeloid Leukemia (CML).

Introduction: The human Nm23 family consists of 8 genes, termed Nm23-H1 through -H8. H1 and H2 are 88% identical and localized to chromosome 17. Both Nm23-H1 and -H2 display nucleotide-diphosphate kinase (NDPk) activity responsible for balancing NTP levels by transferring gamma-phosphates between nucleotides, and individually demonstrate a variety of other activities including DNA binding, transcriptional regulation and apoptotic cleavage of DNA. A variety of studies imply a role for Nm23 proteins in development and in a range of cancers, in which level of expression rather than mutation correlates to prognosis. Specifically, a mutual transcriptional activation between the nm23-H2 and c-myc genes has been implicated in transformation. Our group described the identification of Nm23-H2 as a candidate tumor-associated antigen in a case of CML and demonstrated the presence of Nm23-H2 reactive T cells 5 years after transplantation. Here, we compare the expression of Nm23-H2 and H1 proteins in leukaemic patients and healthy donors.Materials and Methods: PBMC were collected from 10 CML patients at initial diagnosis, from 4 Glivec-resistant CML patients, from 12 AML patients at diagnosis and from 5 healthy donors. PBMC, CD3+, CD14+, CD33+ and CD34+ populations were stained, fixed and sorted by FACS onto microscope slides. Immunocytochemical analysis was performed with Nm23-H1 (Novacastra), Nm23-H2 (Santa Cruz) and β-Actin (Sigma) specific antibodies. Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss).Results: PBMC from 9 out of 10 untreated CML patients (90%) showed a strong Nm23-H2 staining. Fractionation of the PBMC population revealed that CD14+, CD33+ and CD34+ but not CD3+ cells are Nm23-H2 positive in the majority of these patients. Nm23-H2 was also detected in the PBMC of 3 out of 4 Glivec-resistant CML patients. In most cases, Nm23-H2 co-localized with β-actin. Under the same staining conditions, PBMC and sorted subpopulations from 11 of 12 AML patients (92%) and from all healthy donors were Nm23-H2 negative, while very weak staining was detected in PBMC of one AML patient. Nm23-H1 protein was clearly detectable in a number of cell lines (HL-60, U-937, K562 and T47D), but was very weak or absent in PBMC from CML patients and healthy donors.Conclusion: Taken together, our results show that those myeloid populations functionally affected by the bcr/abl mutation specifically over express Nm23-H2 but not Nm23-H1 protein in 9 out of 10 cases of CML. While the molecular basis of this expression pattern and the possible involvement of c-Myc remains to be resolved, these results support the proposal that immune responses to Nm23-H2 may mediate GvL effects and that peptides derived from Nm23-H2 might therefore be attractive targets for specific immunotherapy in CML patients.

Energizing an Invertebrate Embryo: Bafilomycin‐Dependent Respiration and the Metabolic Cost of Proton Pumping by the V‐ATPase

We examine herein the contribution of V-ATPase activity to the energy budget of aerobically developing embryos of Artemia franciscana and discuss the results in the context of quiescence under anoxia. (31)P-NMR analysis indicates that intracellular pH and NTP levels are unaffected by acute incubation of dechorionated embryos with the V-ATPase inhibitor, bafilomycin A(1). Bafilomycin A(1) also has no significant effect on oxygen consumption by isolated mitochondria. Taken together, these data indicate that bafilomycin does not affect energy-producing pathways in the developing embryo. However, the V-ATPase inhibitor exhibits a concentration-dependent inhibition of oxygen consumption in aerobic embryos. A conservative analysis of respirometric data indicates that proton pumping by the V-ATPase, and processes immediately dependent on this activity, constitutes approximately 31% of the aerobic energy budget of the preemergent embryo. Given the complete absence of detectable Na(+)K(+)-ATPase activity during the first hours of aerobic development, it is plausible that the V-ATPase is performing a role in both the acidification of intracellular compartments and the energization of plasma membranes. Importantly, the high metabolic cost associated with maintaining these diverse proton gradients requires that V-ATPase activity be downregulated under anoxia in order to attain the almost complete metabolic depression observed in the quiescent embryo.

Lithium-induced alterations in nucleoside triphosphate levels in human brain: a proton-decoupled 31P magnetic resonance spectroscopy study

We examined how lithium's demonstrated effects on various cellular processes in human brain would be reflected in the 31P magnetic resonance spectra of living human beings with respect to brain high-energy phosphate metabolites. Eight healthy volunteers received a baseline 31P magnetic resonance spectroscopy (MRS) scan, after which they received lithium carbonate, 900 mg/day, for 14 days. Follow-up MRS scans were obtained on day 7 and on day 14. We detected a lithium-induced decrease in alpha-, beta-, gamma- and total nucleoside triphosphate NTP levels with chronic administration of lithium. On day 7, significant decreases were noted in gamma-NTP (14%) and total NTP (11%) levels. There was a trend for a decrease in beta-NTP (11%) levels. On day 14, significant decreases were noted in alpha-NTP (7%) and total NTP (8%) levels. There was a trend for a decrease in beta-NTP (16%) levels. Lithium caused a 25% reduction in inorganic phosphate (P i) levels on day 14. The theoretical relevance of the lithium-induced alterations on brain high-energy phosphates to the lithium-induced modifications of neuroplasticity is discussed.

The Escherichia coli fis Promoter Is Regulated by Changes in the Levels of Its Transcription Initiation Nucleotide CTP

Expression of the Escherichia coli nucleoid-associated protein Fis (factor for inversion stimulation) is controlled at the transcriptional level in accordance with the nutritional availability. It is highly expressed during early logarithmic growth phase in cells growing in rich medium but poorly expressed in late logarithmic and stationary phase. However, fis mRNA expression is prolonged at high levels throughout the logarithmic and early stationary phase when the preferred transcription initiation site (+1C) is replaced with A or G, indicating that initiation with CTP is a required component of the regulation pattern. We show that RNA polymerase-fis promoter complexes are short lived and that transcription is stimulated over 20-fold from linear or supercoiled DNA if CTP is present during formation of initiation complexes, which serves to stabilize these complexes. Use of fis promoter fusions to lacZ indicated that fis promoter transcription is sensitive to the intracellular pool of the predominant initiating NTP. Growth conditions resulting in increases in CTP pools also result in corresponding increases in fis mRNA levels. Measurements of NTP pools performed throughout the growth of the bacterial culture in rich medium revealed a dramatic increase in all four NTP levels during the transition from stationary to logarithmic growth phase, followed by reproducible oscillations in their levels during logarithmic growth, which later decrease during the transition from logarithmic to stationary phase. In particular, CTP pools fluctuate in a manner consistent with a role in regulating fis expression. These observations support a model whereby fis expression is subject to regulation by the availability of its initiating NTP.

P.1.043 Lithium induced alterations in NTP levels in human brain: A proton decoupled 31P MRS study

P.1.043 Lithium induced alterations in NTP levels in human brain: A proton decoupled 31P MRS study

HTERT associates with human telomeres and enhances genomic stability and DNA repair.

Ectopic expression of telomerase in telomerase-silent cells is sufficient to overcome senescence and to extend cellular lifespan. We show here that the catalytic subunit of human telomerase (hTERT) crosslinks telomeres. This interaction is blocked by the telomere repeat binding factor 1, but not by a dominant negative form of this protein. It is also abolished by destruction of the RNA component of telomerase as well as by mutations in the hTERT protein. Ectopic expression of hTERT leads to transcriptional alterations of a subset of genes and changes in the interaction of the telomeres with the nuclear matrix. This is associated with reduction of spontaneous chromosome damage in G(1) cells, enhancement of the kinetics of DNA repair and an increase in NTP levels. The effect on DNA repair is likely indirect as TERT does not directly affect DNA end rejoining in vitro or meiotic recombination in vivo. The observed effects of hTERT occurred rapidly before any significant lengthening of telomeres was observed. Our findings establish an intimate relationship between hTERT-telomere interactions and alteration in transcription of a subset of genes that may lead to increased genomic stability and enhanced repair of genetic damage. These novel functions of telomerase are distinct from its known effect on telomere length and have potentially important biological consequences.

Effect of gemcitabine and cis-platinum combinations on ribonucleotide and deoxyribonucleotide pools in ovarian cancer cell lines.

Gemcitabine (dFdC) and cisplatin (CDDP) act synergistically by an increase in platinum-DNA adduct formation. Since ribonucleotide (NTP) and deoxyribonucleotide (dNTP) levels are essential for DNA-synthesis and repair of DNA damage, we investigated whether disturbances might account for differences in effects between sensitive and resistant cell lines. The human ovarian cancer cell line A2780, its CDDP-resistant variant ADDP and its dFdC-resistant variant AG6000 were exposed for 24 h to dFdC or CDDP alone, or a combination causing moderate to strong growth inhibition. In AG6000 cells UTP levels were 2-fold lower and in ADDP cells almost 2-fold higher than in A2780 cells. Levels of dTTP, dATP and dCTP were 2-5-fold lower in the resistant cell lines. Drug treatment affected NTP and dNTP levels most pronounced in A2780 cells. dFdC alone, at 1.5 nM to 1 micro M increased ATP, GTP and CTP pools 1.2 to 2.0-fold, while 10 micro M dFdC increased UTP 2.5-fold. Combination of dFdC and CDDP increased all NTP levels at low dFdC and CDDP concentrations more than 1.2-fold, but at 20 micro M CDDP only CTP increased 2.4-fold. Only 1.5 nM dFdC increased all dNTP pools more than 1.6-fold, but at 0.1 and 1 micro M dFdC, dATP and dGTP decreased down to 10-fold, while dTTP increased 3-5-fold. CDDP and the combination increased all dNTP pools over 1.4 and 1.9-fold, respectively. In AG6000 cells dFdC and CDDP hardly affected the NTP and dNTP status, except at the high concentrations, which decreased ATP, GTP and UTP levels 1.2-1.8-fold. Both CDDP alone and the combination increased dTTP, dCTP and dATP pools up to 1.6-fold. In ADDP cells NTP and most dNTP levels were hardly affected, except dGTP levels which decreased to non-detectable levels. In conclusion, both dFdC and CDDP induce concentration and combination dependent changes in NTP and dNTP pools.

In vivo nuclear magnetic resonance studies of glycolytic kinetics in Lactococcus lactis.

The metabolism of glucose by nongrowing cells of L. lactis strain MG5267 was studied under controlled conditions of pH, temperature, and gas atmosphere (anaerobic and aerobic) using a circulating system coupled to nuclear magnetic resonance (NMR) detection that allowed a noninvasive determination of intracellular pools of intermediate metabolites by 13C-NMR with a time resolution of 30 seconds. In addition, intracellular parameters, such as pH, NTP levels, and concentration of inorganic phosphate in the cytoplasm, could be monitored on-line by 31P-NMR with a time resolution of approx. 3 min. The time course for the concentrations of intracellular fructose 1,6-bisphosphate (FBP), 3-phosphoglycerate (3-PGA), and phosphoenolpyruvate (PEP), together with kinetic measurements of substrate consumption and endproducts formation, were used as a basis for the construction of a mechanistic model for glycolysis. In vivo measurements were complemented with determinations of phosphorylated metabolites in perchloric acid extracts. A top-down model was developed by simplifying the metabolism to the resolution allowed by the experimental data collected by in vivo NMR (grouped in seven metabolic steps). This simplified mechanistic model was adjusted to the metabolite concentrations determined by in vivo NMR. The results obtained led to the rationalization of the dynamics of glucose metabolism as being driven largely by ATP surplus. This excess causes accumulation of FBP due to NAD+ limitation, whose regeneration is dependent on downstream pyruvate reduction. The model was capable of predicting qualitative shifts in the metabolism of glucose when changing from anaerobic to aerobic conditions.

Lack of competition of substrates for P-glycoprotein in MCF-7 breast cancer cells overexpressing MDR1.

The MCF-7/Adr cells overexpress MDR-1 which contributes to the drug-resistant phenotype. Our studies show: 1. The retention of daunomycin in the MCF-7/Adr cells relates to a temperature-dependent and energy-dependent process. 2. The MCF-7/Adr cells retain less rhodamine-123 than the parental MCF-7 cells. 3. The MCF-7/Adr cells retain less daunomycin than the parental MCF-7 cells as measured by mean fluorescence or radioactive daunomycin. 4. Cyclosporin A and verapamil effectively block the effluxes of rhodamine-123 and daunomycin from the MCF-7/Adr cells. 5. On short-term incubation, 2-deoxyglucose lowers the NTP levels to a greater extent than sodium azide, showing the importance of glycolysis in the MCF-7 cell lines. 6. Although the MCF-7/Adr cells show cross-resistance to VP-16, actinomycin D and vinblastine, these drugs do not compete with daunomycin for the efflux pump. 7. These data suggest that either there must be multiple MDR-1 pumps that differ in substrate specificity or that there are distinct substrate sites on MDR-1.

The immunosuppressive metabolite of leflunomide, A77 1726, affects murine T cells through two biochemical mechanisms.

The immunosuppressive metabolite of leflunomide, A77 1726, inhibits the enzymatic activity of protein tyrosine kinases and of dihydro-orotic acid dehydrogenase, an enzyme involved in pyrimidine biosynthesis. Here murine CTLL cell lines were studied to determine which of the biochemical targets of A77 1726 was responsible for the observed inhibition of proliferation and cytotoxic activity. At low concentrations of A77 1726, pyrimidine biosynthesis is the target, since inhibition of proliferation correlates with a reduction in pyrimidine NTP levels and is reversed by uridine. At higher concentrations of A77 1726, uridine no longer reverses the inhibition of proliferation even though pyrimidine NTP levels are restored. This second mechanism for inhibiting proliferation is probably inhibition of protein tyrosine kinases, since these higher concentrations of A77 1726 inhibit IL-2-induced tyrosine phosphorylation of Jak1 and Jak3, the protein tyrosine kinases initiating signaling by the IL-2R. Tyrosine phosphorylation of the beta-chain of the IL-2R, which is required for IL-2-driven proliferation, is also inhibited by A77 1726. Cytotoxicity of a CTLL line that overexpresses the Lck protein tyrosine kinase is inhibited by A77 1726; this inhibition is not affected by uridine, but does correlate with inhibition of an Lck in vitro kinase reaction. These studies establish that inhibition of pyrimidine biosynthesis and that of protein tyrosine kinase both contribute to the effects of A77 1726 on CTLL cell lines.

Effects of experimental anaemia on intra-erythrocytic phosphate levels in rainbow trout, Oncorhynchus mykiss

The concentrations of intra-erythrocytic adenylates (ATP, ADP and AMP) and guanylates (GTP, GDP and GMP) were determined in rainbow trout subjected to 10% blood removal every 12 h for 96 h. Haemoglobin concentration, [Hb], decreased from 6·043±0·617 to 0·957 ± 0·195 g dl−1. This decrease in [Hb] was followed by a continuous increase in total organic phosphates, e.g. adenylates plus guanylates. Intra-erythrocytic NTP (ATP plus GTP) levels increased significantly after 48 h when haemoglobin concentration was 2·427 ± 0·256 g dl−1. Although a significant increase in GDP levels in animals with [Hb] less than 1·677 ± 0·235 g dl−1 was observed, the general increase in guanylate level was mainly due to the GMP which increased about 85-fold during the experimental period. It is suggested that the erythrocytes of anaemic rainbow trout have the capacity to increase NTP/Hb4 ratios which may represent an advantage for anaemic fish.

Effects of experimental anaemia on intra‐erythrocytic phosphate levels in rainbow trout, Oncorhynchus mykiss

The concentrations of intra‐erythrocytic adenylates (ATP, ADP and AMP) and guanylates (GTP, GDP and GMP) were determined in rainbow trout subjected to 10% blood removal every 12 h for 96 h. Haemoglobin concentration, [Hb], decreased from 6·043±0·617 to 0·957 ± 0·195 g dl−1. This decrease in [Hb] was followed by a continuous increase in total organic phosphates, e.g. adenylates plus guanylates. Intra‐erythrocytic NTP (ATP plus GTP) levels increased significantly after 48 h when haemoglobin concentration was 2·427 ± 0·256 g dl−1. Although a significant increase in GDP levels in animals with [Hb] less than 1·677 ± 0·235 g dl−1 was observed, the general increase in guanylate level was mainly due to the GMP which increased about 85‐fold during the experimental period. It is suggested that the erythrocytes of anaemic rainbow trout have the capacity to increase NTP/Hb4 ratios which may represent an advantage for anaemic fish.

42 HIGH SPIN-SPIN RELAXATION TIMES SEEN BY PROTON MAGNETIC RESONANCE SPECTROSCOPY IN PIGLET BRAINS CORRELATE WITH ATP DEPLETION AFTER ACUTE HYPOXIA-ISCHAEMIA

Using 1H and 31P magnetic resonance spectroscopy (MRS) we aimed to investigate relationships between 1H spin-spin relaxation times (T2s) and nucleotide triphosphate (NTP; largely ATP) levels in newborn piglet brains after acute hypoxia-ischaemia. Six newborn piglets had bilateral common-carotid occlusion and hypoxaemia for ≤ 1.5 hr followed by reperfusion and normal oxygenation. NTP levels were defined relative to the exchangeable 31P pool (EPP = inorganic phosphate + phosphocreatine (PCr) + (γ + α + β)-NTP). Control T2s (mean (SD)) of choline (Cho), creatine + PCr (Cr), and N-acetylaspartate (Naa) were 255 (50), 160 (23), and 215 (34) ms respectively; NTP/EPP was 0.21 (.01). During the insult, Cr T2 rose to 263 (51) ms (p<.0002; paired t-test) as NTP/EPP fell to 0.05 (.05) (p<.0005). An hour after resuscitation, both NTP/EPP and Cr T2 reverted to control values. In 3 piglets, studies continued for up to 50 hr. NTP/EPP fell as secondary energy failure (SEF) developed and the Cho, Cr, and Naa T2s rose linearly to 520 (p<.02), 620 (p<.001), and 590 (p<.001) ms respectively. The increased T2s during the development of SEF were probably due to failure of the ATP-dependcnt Na+/K+ pump indicating their value as indices of cellular oedema following birth asphyxia.

Aerobic Metabolism of Carbon Reserves by the "Obligate Anaerobe" Desulfovibrio gigas

The sulfate-reducing bacterium, Desulfovibrio gigas, is shown by in vivo 31P-NMR to be capable of generating NTP from the utilization of internal carbon reserves both in anaerobic and in aerobic conditions. Acetate, glycerol and ethanol are the major end-products, but the production of alcohols decreases strongly when oxygen is present. When the glycolylic pathway is inhibited with fluoride, NTP levels decrease drastically but can be remarkably restored when an electron acceptor, such as oxygen, is provided. Our data are in favour of a NADH-linked electron transfer chain enabling transfer of reducing power derived from polyglucose to oxygen which provides this so-called "strict anaerobe" with the capability of surviving to oxic environments.