Decreased oxidative stress during glycolytic inhibition enables maintenance of ATP production and astrocytic survival

Depressed energy metabolism and oxidative stress are common features in many pathological situations in the brain, including stroke. In order to investigate astrocytic responses to such stress, we induced metabolic depression in cultured rat astrocytes. Iodoacetate (IA), an inhibitor of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used and resulted in a rapid inhibition of GAPDH activity. After 1h of GAPDH inhibition the ATP levels started to decrease and were completely abolished at 4h. In parallel, the activity of reactive oxygen species (ROS) was significantly increased, followed by extensive cell death involving flipping of phosphatidylserine and translocation of apoptosis-inducing factor, but not caspase-3 activation. When IA was combined with azide, a respiratory chain complex IV inhibitor, the ATP levels decreased immediately. Interestingly, with azide present, the ROS activity remained low and the astrocytes remained viable even at very low ATP levels. Addition of exogenous ROS-scavengers prevented the IA-induced ROS activity, the ATP levels were maintained and cell death was prevented. Similar protection could be obtained when astrocytes, prior to addition of IA, were incubated with substances known to activate the nuclear factor erythroid 2-related factor 2 (Nrf2)-regulated endogenous antioxidant system. When IA was washed out, after a relatively moderate ATP depression, massive cell death occurred. This was efficiently prevented by addition of azide or ROS scavengers during the IA treatment or by pre-activation of the Nrf2 system. Our results demonstrate that astrocytes in culture can endure and recover from glycolytic inhibition if the ROS activity remained at a low level and suggest that oxidative stress can be an important component for astrocytic cell death following metabolic stress.     

Lactate dehydrogenase in two digenetic trematodes and their host

Polyacrylamide gel electrophoresis of the two digenetic trematodes, Gigantocotyle explanalum from the liver and Gastrothylax crumenifer from the rumen of the water buffalo, Bubalus bubalis revealed the presence of at least six and seven isoenzymes of lactate dehydrogenase (LDH), respectively in a partially purified enzyme preparation. The respective host tissues showed five isoenzymes of LDH, which are characteristic to the vertebrates. Both parachloromercuribenzoate and iodoacetate affected the LDH activity of the parasites and host tissues differently. Spectrophotometric analysis also showed different specific activity and susceptibility to the action of thiol inhibitors. The host LDH was quite stable at 57°C for 30 min, but that of the parasites was less stable.     

Use of haloacetate dehalogenase genes as selection markers forEscherichia coli andPseudomonas vectors

The haloacetate dehalogenase gene,dehH2, cloned fromMoraxella sp. strain B could be used as a selection marker gene for vectors inEscherichia coli andPseudomonas putida. Haloacetates, especially iodoacetate, inhibit the growth of some microorganisms. ThedehH2 gene introduced into the cells conferred iodoacetate resistance on them. Therefore,E. coli andP. putida transformed with vectors marked withdehH2 could be easily selected on plates containing iodoacetate.     

Development of an in vitro technique for cytological investigations of slices of Fasciola hepatica: Evaluation by physiological criteria

In order to check the via bilityof Fasciola hepatica slices, maintained in M199 under defined physico-chemical conditions, their rates of consumption of oxygen and glucose were determined, throughout a 12-h period, using polarographic and spectrophotometric techniques. The results, when compared with similar determinations on intact animals, indicated that the slices remained as viable as incubated whole flukes, for at least 12 h. This finding supports ultrastructural evidence detailed in a previous publication. The slices were shown to consume more oxygen and less glucose than whole animals, and possible reasons for this are discussed. Studies on the effects of DNP and iodoacetate on the uptake behaviour of slices suggested that in Fasciola hepatica, as in aerobes, most of the oxygen and glucose consumed is involved with energy metabolism. Hence, oxygen and glucose uptake rates are probably valid criteria of tissue viability for the slices.     

Cysteamine restores glutathione redox status in cultured cystinotic proximal tubular epithelial cells

Recent evidence implies that impaired metabolism of glutathione has a role in the pathogenesis of nephropathic cystinosis. This recessive inherited disorder is characterized by lysosomal cystine accumulation and results in renal Fanconi syndrome progressing to end stage renal disease in the majority of patients. The most common treatment involves intracellular cystine depletion by cysteamine, delaying the development of end stage renal disease by a yet elusive mechanism. However, cystine depletion does not arrest the disease nor cures Fanconi syndrome in patients, indicating involvement of other yet unknown pathologic pathways. Using a newly developed proximal tubular epithelial cell model from cystinotic patients, we investigate the effect of cystine accumulation and cysteamine on both glutathione and ATP metabolism. In addition to the expected increase in cystine and defective sodium-dependent phosphate reabsorption, we observed less negative glutathione redox status and decreased intracellular ATP levels. No differences between control and cystinosis cell lines were observed with respect to protein turnover, albumin uptake, cytosolic and mitochondrial ATP production, total glutathione levels, protein oxidation and lipid peroxidation. Cysteamine treatment increased total glutathione in both control and cystinotic cells and normalized cystine levels and glutathione redox status in cystinotic cells. However, cysteamine did not improve decreased sodium-dependent phosphate uptake. Our data implicate that cysteamine increases total glutathione and restores glutathione redox status in cystinosis, which is a positive side-effect of this agent next to cystine depletion. This beneficial effect points to a potential role of cysteamine as anti-oxidant for other renal disorders associated with enhanced oxidative stress.     

Mutational analysis of cysteine 328 and cysteine 368 at the interface of Plasmodium falciparum adenylosuccinate synthetase

Plasmodium falciparum adenylosuccinate synthetase, a homodimeric enzyme, contains 10 cysteine residues per subunit. Among these, Cys250, Cys328 and Cys368 lie at the dimer interface and are not conserved across organisms. PfAdSS has a positively charged interface with the crystal structure showing additional electron density around Cys328 and Cys368. Biochemical characterization of site directed mutants followed by equilibrium unfolding studies permits elucidation of the role of interface cysteines and positively charged interface in dimer stability. Mutation of interface cysteines, Cys328 and Cys368 to serine, perturbed the monomer-dimer equilibrium in the protein with a small population of monomer being evident in the double mutant. Introduction of negative charge in the form of C328D mutation resulted in stabilization of protein dimer as evident by size exclusion chromatography at high ionic strength buffer and equilibrium unfolding in the presence of urea. These observations suggest that cysteines at the dimer interface of PfAdSS may indeed be charged and exist as thiolate anion.     

Glycolysis–respiration relationships in a neuroblastoma cell line

Background

Although some reciprocal glycolysis–respiration relationships are well recognized, the relationship between reduced glycolysis flux and mitochondrial respiration has not been critically characterized.

Methods

We concomitantly measured the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of SH-SY5Y neuroblastoma cells under free and restricted glycolysis flux conditions.

Results

Under conditions of fixed energy demand ECAR and OCR values showed a reciprocal relationship. In addition to observing an expected Crabtree effect in which increasing glucose availability raised the ECAR and reduced the OCR, a novel reciprocal relationship was documented in which reducing the ECAR via glucose deprivation or glycolysis inhibition increased the OCR. Substituting galactose for glucose, which reduces net glycolysis ATP yield without blocking glycolysis flux, similarly reduced the ECAR and increased the OCR. We further determined how reduced ECAR conditions affect proteins that associate with energy sensing and energy response pathways. ERK phosphorylation, SIRT1, and HIF1a decreased while AKT, p38, and AMPK phosphorylation increased.

Conclusions

These data document a novel intracellular glycolysis–respiration effect in which restricting glycolysis flux increases mitochondrial respiration.

General significance

Since this effect can be used to manipulate cell bioenergetic infrastructures, this particular glycolysis–respiration effect can practically inform the development of new mitochondrial medicine approaches.     

Peptide mimic of the HIV envelope gp120-gp41 interface

The human immunodeficiency virus envelope glycoprotein (Env) is composed of surface (gp120) and transmembrane (gp41) subunits, which are noncovalently associated on the viral surface. Human immunodeficiency virus Env mediates viral entry after undergoing a complex series of conformational changes induced by interaction with cellular CD4 and a chemokine coreceptor. These changes propagate from gp120 to gp41 via the gp120-gp41 interface, ultimately exposing gp41 and allowing it to form the trimer-of-hairpins structure that provides the driving force for membrane fusion. Key unresolved questions about the gp120-gp41 interface include the specific regions of gp41 and gp120 involved, the mechanism by which receptor and coreceptor-binding-induced conformational changes in gp120 are communicated to gp41, how trimer-of-hairpins formation is prevented in the prefusogenic gp120-gp41 complex, and, ultimately, the structure of the prefusion gp120-gp41 complex. Here, we develop a biochemical model system that mimics a key portion of the gp120-gp41 interface in the prefusogenic state. We find that a gp41 fragment containing the disulfide bond loop and C-peptide region binds primarily to the gp120 C5 region and that this interaction is incompatible with trimer-of-hairpins formation. Based on these data, we propose that in prefusogenic Env, gp120 sequesters the gp41 C-peptide region away from the N-trimer region, preventing trimer-of-hairpins formation until coreceptor binding disrupts this interface. This model system is a valuable tool for studying the gp120-gp41 complex, conformational changes induced by CD4 and coreceptor binding, and the mechanism of membrane fusion.     

The Rapid L- and D-Aspartate Uptake in Cultured Astrocytes

Astrocytes in primary culture possess a rapid L-aspartate saturable transport system (K_m = 93 M; V_max = 81 nmol/min/mg protein), which shows certain stereospecificity since V_max was 36% lower for D-aspartate uptake. These are values obtained at short incubation time (15 seconds), to obtain approximate initial rate conditions. Metabolic energy inhibitors, rotenone and iodoacetate very potently inhibited the L- and D-aspartate uptake processes, indicating that the transport process is an active one. However, the accumulation of L-aspartate was "enhanced by inhibitors of L-aspartate metabolism, such as the aspartate aminotransferase inhibitor, aminooxyacetate and L-methionine sulfoximine, an inhibitor of glutamine synthetase, whereas D-aspartate (a non-metabolizable analog of L-aspartate) uptake was not affected. The accumulated levels of L-aspartate in the presence of aminooxyacetate were similar to the levels of D-aspartate. These effects of L-aspartate metabolic inhibitors, suggest that due to metabolism of the the L-aspartate, short incubation time (eg., 15 seconds) is necessary to measure the initial rate of L-aspartate uptake, in order to obtain the "true kinetic parameters.     

Transfer of kanamycin resistance fromNicotiana tabacum toNicotiana plumbaginifolia by fusion of x-irradiated protoplasts

Summary Nuclear hybrids have been obtained by fusion of mesophyll protoplasts ofNicotiana plumbaginifolia and x-irradiated or iodoacetate-treated mesophyll protoplasts of a kanamycin-resistant line ofN. tabacum. The effect of irradiation on the recovery of asymmetric hybrids was evaluated by analysis of their morphology, fertility, chromosome number, isozyme patterns, restriction patterns in their organelle DNAs, and presence of the kanamycin-resistance gene. The results presented in this paper show that x-ray irradiation leads to a significant reduction in the amount ofN. tabacum genome present in the hybrids and demonstrates, once more, the power of this technique to induce directional loss of genomic traits of the irradiated parent.