Structure and gene cluster of the O-polysaccharide from Pseudomonas veronii A-6-5 and its uranium bonding.

A denitrifying bacterium Pseudomonas veronii A-6-5 was isolated from a deep aquifer contaminated with nitrates and uranium. The O-polysaccharide (OPS) was isolated by mild acid degradation of the lipopolysaccharide of P. veronii A-6-5 and studied using sugar analysis and 1D and 2D 1H and 13C NMR spectroscopy. The trisaccharide O-repeating unit was found to have the following structure: [Formula: see text] [Formula: see text] where Hb is 3-hydroxybutanoyl. The genome of P. veronii A-6-5 was sequenced and a respective OPS gene cluster was identified. Functions of the proteins encoded in the gene cluster, including the enzymes involved in the O-polysaccharide biosynthesis and glycosyl transferases, were putatively assigned by comparison with available database sequences. Formation of a new coordination bond between uranyl and the O-polysaccharide from P. veronii A-6-5 was demonstrated using FTIR spectroscopy; it may affect uranyl migration in the groundwaters due to its immobilization on microbial biofilms. Applied importance of this work is that the structure of the O-polysaccharide of a strain isolated from uranium-contaminated groundwater was determined and the character of interaction between the polysaccharide and the uranyl ion was established. The data obtained are of importance for development of the biotechnologies for treatment of uranium-contaminated groundwater and activated sludge.

Attract and kill: spinosad containing spheres to control onion maggot (Delia antiqua).

BACKGROUND: Onion maggot (Delia antiqua) is a pest of onions worldwide. Current means of managing this pest rely heavily on prophylatic insecticide treatments at planting. These options may not be viable in organic production systems or situations where insecticide-resistant populations occur. Here we explore the efficacy of an attract and kill strategy for control of D. antiqua evaluating the ability of attractive, spinosad containing spheres to kill adult D. antiqua and reduce crop losses. RESULTS: Spinosad containing spheres were able to consistently kill D. antiqua adults over the course of the field season (mortality range: between 49% and 59% on average). Pairing spinosad spheres with Delia Lure increased efficacy by 72% compared with the spheres alone. Performance of this attract and kill strategy also can reduce damage by D. antiqua larvae in the field, but it did not achieve a level of control comparable to the level provided by a conventional insecticide treatment. CONCLUSION: Implementation of this attract and kill strategy could be a valuable tool in situations where conventional pesticides are either not available or desired, where additional control techniques are needed, or to provide a season-long option for control of D. antiqua populations. © 2020 Society of Chemical Industry.

Sting nematodes modify metabolomic profiles of host plants.

Plant-parasitic nematodes are devastating pathogens of many important agricultural crops. They have been successful in large part due to their ability to modify host plant metabolomes to their benefit. Both root-knot and cyst nematodes are endoparasites that have co-evolved to modify host plants to create sophisticated feeding cells and suppress plant defenses. In contrast, the ability of migratory ectoparasitic nematodes to modify host plants is unknown. Based on global metabolomic profiling of sting nematodes in African bermudagrass, ectoparasites can modify the global metabolome of host plants. Specifically, sting nematodes suppress amino acids in susceptible cultivars. Upregulation of compounds linked to plant defense have negative impacts on sting nematode population densities. Pipecolic acid, linked to systemic acquired resistance induction, seems to play a large role in protecting tolerant cultivars from sting nematode feeding and could be targeted in breeding programs.

Antihypoxic and anti-ischemic properties of the North Caucasus flora plant extracts / Propiedades antihipóxicas y antiisquémicas de los extractos de plantas de la flora del norte del Cáucaso

Nowdays it is established that ischemic brain damage like ischemic stroke is one of the leading cause of death and disability in the population that assumes relevance development of anti-ischemic drugs. The work studied the anti-hypoxic and anti-ischemic effect of 7 plant extracts. Antihypoxic activity was assessed on models of hypobaric, hypercapnic, histotoxic, hematotoxic hypoxia. Anti-ischemic activity of test-extracts was studied on the focal cerebral ischemia model. Administration of Tagetes patula, Gaillardia pulchella, Sorbaria sorbifolia, Grossularia reclinata, Ribes nigrum, Rubus caesius and Lysimachia punctata extracts contributed to the necrosis zone reduction by 56.6% (p<0.05); 37.3% (p<0.05); 73.2% (p<0.05); 49.4% (p<0.05); 42.5% (p<0.05); 85.5% (p<0.05); 44.2% (p<0.05) and also restored aerobic metabolism in brain tissue. Test - objects increased of the animal lifespan under hypoxia conditions. Based on the data obtained, it is assumed that further studies of North Caucasus flora plant extracts as cerebro-protective agents are promising.

Hoy en día, se ha establecido que el daño cerebral isquémico, como el accidente cerebrovascular isquémico, es una de las principales causas de muerte y discapacidad en la población lo cual hace relevante el desarrollo fármacos antiisquémicos. En este trabajo se estudió el efecto antihipóxico y antiisquémico de siete extractos de plantas. La actividad antihipóxica se evaluó en modelos de hipoxia hipocrática, hipercápnica, histotóxica y hematotóxica. La actividad antiisquémica de los extractos de prueba se estudió en el modelo de isquemia cerebral focal. La administración de los extractos de Tagetes patula; Gaillardia pulchella; Sorbaria sorbifolia; Grossularia reclinata; Ribes nigrum; Rubus caesius y Lysimachia punctata contribuyeron a la reducción de la zona de necrosis en un 56,6% (p<0,05); 37,3% (p<0,05); 73,2% (p<0,05); 49,4% (p<0,05); 42,5% (p<0,05); 85,5% (p<0,05); 44.2% (p<0.05), respectivamente, además, de restaurar el metabolismo aeróbico en el tejido cerebral. Comparado con el control, se observó un aumento en el tiempo de sobrevida del animal en condiciones de hipoxia. Sobre la base de los interesantes datos obtenidos, se sugiere estudios adicionales de extractos de plantas de la flora del Cáucaso Norte como agentes protectores del cerebro.

Alteration of erythrocyte membrane polyunsaturated fatty acids in preterm newborns with retinopathy of prematurity.

Extremely preterm infants are at high risk for retinopathy of prematurity (ROP), a potentially blinding disease characterized by abnormalities in retinal vascularization. Whereas animal studies revealed that n-3 polyunsaturated fatty acids (PUFAs) may be of benefit in preventing ROP, human studies conducted on preterm infants during the 1st weeks of life showed no association between blood n-3 PUFA bioavailability and ROP incidence and/or severity, probably because of the influence of nutrition on the lipid status of infants. In the OmegaROP prospective cohort study, we characterized the erythrocyte concentrations of PUFAs in preterm infants aged less than 29 weeks gestational age (GA) without any nutritional influence. We show that GA is positively associated with the erythrocyte n-6 to n-3 PUFA ratio, and particularly with the ratio of arachidonic acid (AA) to docosahexaenoic acid (DHA), in infants with ROP. A time-dependent accumulation of AA at the expense of DHA seems to occur in utero in erythrocytes of preterm infants who will develop ROP, thus reinforcing previous data on the beneficial properties of DHA on this disease. In addition, preliminary data on maternal erythrocyte membrane lipid concentrations suggest modifications in placental transfer of fatty acids. Documenting the erythrocyte AA to DHA ratio at birth in larger cohorts might be useful to set up new prognostic factors for ROP.

Curare alkaloids from Matis Dart Poison: Comparison with d-tubocurarine in interactions with nicotinic, 5-HT3 serotonin and GABAA receptors.

Several novel bisbenzylisoquinoline alkaloids (BBIQAs) have recently been isolated from a Matis tribe arrow poison and shown by two-electrode voltage-clamp to inhibit mouse muscle nicotinic acetylcholine receptors (nAChR). Here, using radioligand assay with Aplysia californica AChBP and radioiodinated α-bungarotoxin ([125I]-αBgt), we show that BBIQA1, BBIQA2, and d-tubocurarine (d-TC) have similar affinities to nAChR orthosteric site. However, a competition with [125I]-αBgt for binding to the Torpedo californica muscle-type nAChR revealed that BBIQAs1, 2, and 3 are less potent (IC50s = 26.3, 8.75, and 17.0 µM) than d-TC (IC50 = 0.39 µM), while with α7 nAChR in GH4C1 cells, BBIQA1 was less potent that d-TC (IC50s = 162 µM and 7.77 µM, respectively), but BBIQA2 was similar (IC50 = 5.52 µM). In inhibiting the Ca2+ responses induced by acetylcholine in Neuro2a cells expressing the mouse adult α1ß1εδ nAChR or human α7 nAChR, BBIQAs1 and 2 had similar potencies to d-TC (IC50s in the range 0.75-3.08 µM). Our data suggest that BBIQA1 and BBIQA2 can inhibit adult muscle α1ß1εδ nAChR by both competitive and noncompetitive mechanisms. Further experiments on neuronal α3ß2, α4ß2, and α9α10 nAChRs, expressed in Xenopus laevis oocytes, showed that similar potencies for BBIQAs1, 2, and d-TC. With α3ß2γ2 GABAAR currents were almost completely inhibited by d-TC at a high (100 µM) concentration, but BBIQAs1 and 2 were less potent (only 40-50% inhibition), whereas in competition with Alexa Fluor 546-α-cobratoxin for binding to α1ß3γ2 GABAAR in Neuro2a cells, d-TC and these analogs had comparable affinities. Especially interesting effects of BBIQAs1 and 2 in comparison with d-TC were observed for 5-HT3AR: BBIQA1 and BBIQA2 were 5- and 87-fold less potent than d-TC (IC50 = 22.63 nM). Thus, our results reveal that these BBIQAs differ from d-TC in their potencies towards certain Cys-loop receptors, and we suggest that understanding the reasons behind this might be useful for future drug design.

Pectin influences the kinetics of in vitro lipid digestion in oil-in-water emulsions.

Oil-in-water emulsions were prepared with 5% (w/v) carrot-enriched olive oil and stabilized with Tween 80 (TW), phosphatidylcholine (PC), citrus pectin (CP) or a combination of these emulsifiers. Additionally, the methylesterification degree (DM) of citrus pectin was modified, resulting in three different studied pectin structures: CP82, CP38 and CP10. All initial emulsions presented small initial oil droplet sizes and were submitted to an in vitro simulated gastric and small intestinal phase. The latter was executed in a kinetic way to determine the time dependency of the lipolysis reaction, micelle formation and carotenoid bioaccessibility. The results showed that the pectin DM mainly influenced the reaction rate constants, while the emulsifier (combination) determined the extent of lipolysis and carotenoid bioaccessibility. Moreover, a direct relation was observed between the lipolysis reaction and bioaccessibility extent. The presented study showed that targeted emulsion design can be used to tailor lipid digestion kinetics.

Thermodynamic analysis questions claims of improved cardiac efficiency by dietary fish oil.

Studies in the literature describe the ability of dietary supplementation by omega-3 fish oil to increase the pumping efficiency of the left ventricle. Here we attempt to reconcile such studies with our own null results. We undertake a quantitative analysis of the improvement that could be expected theoretically, subject to physiological constraints, by posing the following question: By how much could efficiency be expected to increase if inefficiencies could be eliminated? Our approach utilizes thermodynamic analyses to investigate the contributions, both singly and collectively, of the major components of cardiac energetics to total cardiac efficiency. We conclude that it is unlikely that fish oils could achieve the required diminution of inefficiencies without greatly compromising cardiac performance.

Curare Alkaloids: Constituents of a Matis Dart Poison.

A phytochemical study of dart and arrow poison from the Matis tribe led to the identification of D-(-)-quinic acid, L-malic acid, ethyldimethylamine, magnoflorine, and five new bisbenzyltetrahydroisoquinoline alkaloids (BBIQAs), 1-5. D-Tubocurarine could not be identified among these products. BBIQA (3) contains a unique linkage at C-8 and C-11'. All structures were characterized by a combination of NMR and HRESIMS data. The effects of Matis poison and individual BBIQAs (1-3) on rat muscle nAChR expressed in Xenopus oocytes have been investigated using the two-electrode voltage clamp technique.

Dietary supplementation with either saturated or unsaturated fatty acids does not affect the mechanoenergetics of the isolated rat heart.

Abstract It is generally recognized that increased consumption of polyunsaturated fatty acids, fish oil (FO) in particular, is beneficial to cardiac and cardiovascular health, whereas equivalent consumption of saturated fats is deleterious. In this study, we explore this divergence, adopting a limited purview: The effect of dietary fatty acids on the mechanoenergetics of the isolated heart per se. Mechanical indices of interest include left-ventricular (LV) developed pressure, stroke work, cardiac output, coronary perfusion, and LV power. The principal energetic index is whole-heart oxygen consumption, which we subdivide into its active and basal moieties. The primary mechanoenergetic index of interest is cardiac efficiency, the ratio of work performance to metabolic energy expenditure. Wistar rats were divided into three Diet groups and fed, ad libitum, reference (REF), fish oil-supplemented (FO), or saturated fatty acid-supplemented (SFA) food for 6 weeks. At the end of the dietary period, hearts were excised, mounted in a working-heart rig, and their mechanoenergetic performance quantified over a range of preloads and afterloads. Analyses of Variance revealed no difference in any of the individual mechanoenergetic indices among the three Diet groups. In particular, we found no effect of prior dietary supplementation with either saturated or unsaturated fatty acids on the global efficiency of the heart.

Patients with colorectal tumors with microsatellite instability and large deletions in HSP110 T17 have improved response to 5-fluorouracil­based chemotherapy.

BACKGROUND & AIMS: Patients with colorectal tumors with microsatellite instability (MSI) have better prognoses than patients with tumors without MSI, but have a poor response to 5-fluorouracil­based chemotherapy. A dominant-negative form of heat shock protein (HSP)110 (HSP110DE9) expressed by cancer cells with MSI, via exon skipping caused by somatic deletions in the T(17) intron repeat, sensitizes the cells to 5-fluorouracil and oxaliplatin.We investigated whether HSP110 T(17) could be used to identify patients with colorectal cancer who would benefit from adjuvant chemotherapy with 5-fluorouracil and oxaliplatin. METHODS: We characterized the interaction between HSP110 and HSP110DE9 using surface plasmon resonance. By using polymerase chain reaction and fragment analysis, we examined how the size of somatic allelic deletions in HSP110 T(17) affected the HSP110 protein expressed by tumor cells. We screened 329 consecutive patients with stage II­III colorectal tumors with MSI who underwent surgical resection at tertiary medical centers for HSP110 T(17). RESULTS: HSP110 and HSP110DE9 interacted in a1:1 ratio. Tumor cells with large deletions in T(17) had increased ratios of HSP110DE9:HSP110, owing to the loss of expression of full-length HSP110. Deletions in HSP110 T(17) were mostly biallelic in primary tumor samples with MSI. Patients with stage II­III cancer who received chemotherapy and had large HSP110 T(17) deletions (≥5 bp; 18 of 77 patients, 23.4%) had longer times of relapse-free survival than patients with small or no deletions (≤4 bp; 59 of 77 patients, 76.6%) in multivariate analysis (hazard ratio, 0.16; 95% confidence interval, 0.012­0.8; P = .03). We found a significant interaction between chemotherapy and T17 deletion (P =.009). CONCLUSIONS: About 25% of patients with stages II­III colorectal tumors with MSI have an excellent response to chemotherapy, due to large, biallelic deletions in the T(17) intron repeat of HSP110 in tumor DNA.

Dietary pre-exposure of rats to fish oil does not enhance myocardial efficiency of isolated working hearts or their left ventricular trabeculae.

Numerous epidemiological studies, supported by clinical and experimental findings, have suggested beneficial effects of dietary fish or fish oil supplementation on cardiovascular health. One such experimental study showed a profound (100%) increase in myocardial efficiency (i.e. the ratio of work output to metabolic energy input) of the isolated whole heart, achieved by a corresponding decrease in the rate of myocardial oxygen consumption. However, a number of other investigations have returned null results on the latter energetic index. Such conflicting findings have motivated us to undertake a re-examination. To that effect, we investigated the effects of dietary fatty acid supplementation on myocardial mechano-energetics, with our primary focus on cardiac efficiency. We used both isolated hearts and isolated left ventricular trabeculae of rats fed with one of three distinct diets: reference (REF), fish oil-supplemented (FO) or saturated fat-supplemented (SFA). For all three groups, and at both spatial levels, we supplied 10 mm glucose as the exogenous metabolic substrate. In the working heart experiments, we found no difference in the average mechanical efficiency among the three dietary groups: 14.8 ± 1.1% (REF), 13.9 ± 0.6% (FO) and 13.6 ± 0.7% (SFA). Likewise, we observed no difference in peak mechanical efficiency of left ventricular trabeculae among the REF, FO and SFA groups: 13.3 ± 1.4, 11.2 ± 2.2 and 12.5 ± 1.5%, respectively. We conclude that there is no effect of a period of pre-exposure to a diet supplemented with either fish oil or saturated fatty acids on the efficiency of the myocardium at either spatial level: tissue or whole heart.

Erythrocyte selenium concentration as a marker of selenium status.

BACKGROUND & AIMS: Plasma selenium concentration and glutathione peroxidase (GPx) activity are commonly used as markers of selenium nutritional status. However, plasma selenium concentrations fall independently of selenium status during the acute phase response and GPx is analytically problematic. The assay for erythrocyte selenium is robust and concentrations are unaffected by the systemic inflammatory response. This study was performed to investigate the validity of erythrocyte selenium measurement in assessing selenium status. METHODS: C-reactive protein (CRP), plasma and erythrocyte selenium concentrations and GPx activity were measured in 96 women from two regions of Malawi with low and high selenium dietary intakes. CRP and plasma and erythrocyte selenium was measured in 91 critically ill patients with a systemic inflammatory response. RESULTS & CONCLUSIONS: The median CRP value of all subjects from Malawi was 4.2 mg/L indicating no inflammation. The median CRP value for the critically ill patients was 126 mg/L indicating this group was inflamed. In the non-inflamed population there was a strong positive correlation (r = 0.95) between erythrocyte and plasma selenium and a strong positive correlation (r = 0.77) between erythrocyte selenium and erythrocyte GPx up to 6.10 nmol/g Hb after which maximal activity was reached. In the inflamed population, plasma selenium was low, erythrocyte selenium was normal and there was a weak correlation (r = 0.30) between selenium concentrations in plasma and erythrocytes. This demonstrates that plasma selenium is affected by the inflammatory response while erythrocyte selenium concentration is unaffected and can be used to reliably assess selenium status across a wide range of selenium intakes.

Energetics of stress production in isolated cardiac trabeculae from the rat.

The heat liberated upon stress production in isolated cardiac muscle provides insights into the complex thermodynamic processes underlying mechanical contraction. To that end, we simultaneously measured the heat and stress (force per cross-sectional area) production of cardiac trabeculae from rats using a flow-through micromechanocalorimeter. In a flowing stream of O(2)-equilibrated Tyrode solution (∼22°C), the stress and heat production of actively contracting trabeculae were varied by 1) altering stimulus frequency (0.2-4 Hz) at optimal muscle length (L(o)), 2) reducing muscle length below L(o) at 0.2 and 2 Hz, and 3) changing extracellular Ca(2+) concentrations ([Ca(2+)](o); 1 and 2 mM). Linear regression lines were adequate to fit the active heat-stress data. The active heat-stress relationships were independent of stimulus frequency and muscle length but were dependent on [Ca(2+)](o), having greater intercepts at 2 mM [Ca(2+)](o) than at 1 mM [Ca(2+)](o) (3.5 and 2.0 kJ·m(-3)·twitch(-1), respectively). The slopes among the heat-stress relationships did not differ. At the highest experimental stimulus frequency, pronounced elevation of diastolic Ca(2+) resulted in incomplete twitch relaxation. The resulting increase of diastolic stress, which occurred with negligible metabolic energy expenditure, subsequently diminished due to the time-dependent loss of myofilament Ca(2+)-sensitivity.

Synergistic effects of stress and omega-3 fatty acid deprivation on emotional response and brain lipid composition in adult rats.

The aim was to determine the consequences of multi-generational n-3 polyunsaturated fatty acids (PUFA) deficiency on emotional response in rats subjected to maternal separation (MS) as chronic early life stress. Pups fed a control or an n-3 PUFA deficient diet were daily separated for 2 weeks before weaning. In adult rats, reward response was assessed by sucrose consumption and reactivity to novelty using openfield test. Both n-3 PUFA deficiency and MS increased reward response and impulsivity. Moreover, nutritional deficiency and stress acted in synergy to elevate sucrose intake by 80%, compared to control conditions. n-3 PUFA deprivation induced a depletion of docosahexanoeic acid of brain membranes by 70% compensated by increase in 22:5 n-6 and arachidonic acid (AA) levels. The diet-induced AA increase was, however, significantly higher in MS rats. This suggests that n-3 PUFA deficit could be an environmental risk increasing vulnerability to depressive-like response induced by chronic stress.

Evaluation of a dynamic in vitro model to simulate the porcine ileal digestion of diets differing in carbohydrate composition.

The aim of the study was to assess the ability of a dynamic in vitro model to determine the digestibility of OM, CP, and starch compared with a validated, static, in vitro method and in vivo ileal digestibility obtained from growing pigs fitted with a T-cannula. Five experimental diets with different carbohydrate types and level were assessed: a standard corn-based diet (ST) or the same diet with coarse ground corn (CC), 8% sugar beet pulp (BP), 10% wheat bran (WB), or 8% sugar beet pulp and 10% wheat bran (HF). In the in vivo experiment, diets CC and HF reduced (P = 0.015) ileal digestibility of OM compared with the ST diet. The inclusion of sugar beet pulp reduced (P = 0.049) ileal CP digestibility of the BP diet. This reduction was not statistically significant when sugar beet pulp was combined with the wheat bran in the HF diet. No differences were shown for in vivo starch digestibility among diets. With the static in vitro method, the OM disappearance was greater than that observed in the in vivo experiment. In this static method, the BP and HF diets reduced (P = 0.004 and < 0.001, respectively) the disappearance of the OM compared with the ST diet. The coarse grinding of corn did not alter OM digestibility but decreased (P = 0.005) the starch digestibility. The R(2) between the in vivo results and the static in vitro methods for OM and starch digestibility was 0.99 when the CC diet was not considered. The dynamic in vitro model yielded OM and CP digestibility coefficients comparable with those obtained in vivo for the ST and CC diets. However, the values were considerably affected by the incorporation of the fibrous ingredients. Diets BP, WB, and HF had decreased (P = 0.009, 0.058, and 0.004, respectively) OM digestibility compared with the ST diet. Protein digestibility was also decreased (P < 0.001, P = 0.019, and P = 0.003, respectively) with the BP, WB, and HF diets compared with the ST diet. However, digestibility was decreased to a greater extent in the BP diet than in the WB and HF diets, both of which contained wheat bran. The R(2) between the dynamic in vitro model and the in vivo results for CP digestibility was 0.99 when the CC diet was not considered. No differences were detected for starch digestibility among the diets with the dynamic in vitro model. This dynamic in vitro model yielded ileal digestibility results comparable with those obtained in vivo for CP and OM with a corn-soybean diet, or with a diet including coarse corn, but it underestimated digestibility when fibrous ingredients were included in the diet.

n-3 polyunsaturated fatty acid supplementation reverses stress-induced modifications on brain monoamine levels in mice.

The aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors.

Mutations in the gene encoding peroxisomal sterol carrier protein X (SCPx) cause leukencephalopathy with dystonia and motor neuropathy.

In this report, we describe the first known patient with a deficiency of sterol carrier protein X (SCPx), a peroxisomal enzyme with thiolase activity, which is required for the breakdown of branched-chain fatty acids. The patient presented with torticollis and dystonic head tremor as well as slight cerebellar signs with intention tremor, nystagmus, hyposmia, and azoospermia. Magnetic resonance imaging showed leukencephalopathy and involvement of the thalamus and pons. Metabolite analyses of plasma revealed an accumulation of the branched-chain fatty acid pristanic acid, and abnormal bile alcohol glucuronides were excreted in urine. In cultured skin fibroblasts, the thiolytic activity of SCPx was deficient, and no SCPx protein could be detected by western blotting. Mutation analysis revealed a homozygous 1-nucleotide insertion, 545_546insA, leading to a frameshift and premature stop codon (I184fsX7).

Inhibition of mitochondrial bioenergetics: the effects on structure of mitochondria in the cell and on apoptosis.

The effects of specific inhibitors of respiratory chain, F(o)F(1)ATP synthase and uncouplers of oxidative phosphorylation on survival of carcinoma HeLa cells and on the structure of mitochondria in the cells were studied. The inhibitors of respiration (piericidin, antimycin, myxothiazol), the F(1)-component of ATP synthase (aurovertin) and uncouplers (DNP, FCCP) did not affect viability of HeLa cells, apoptosis induced by TNF or staurosporin and the anti-apoptotic action of Bcl-2. Apoptosis was induced by combined action of respiratory inhibitors and uncouplers indicating possible pro-apoptotic action of reactive oxygen species (ROS) generated by mitochondria. Short-term incubation of HeLa cells with the mitochondrial inhibitors and 2-deoxyglucose followed by 24-48 h recovery resulted in massive apoptosis. Apoptosis correlated to transient (3-4 h) and limited (60-70%) depletion of ATP. More prolonged or more complete transient ATP depletion induced pronounced necrosis. The inhibitors of respiration and uncouplers caused fragmentation of tubular mitochondria and formation of small round bodies followed by swelling. These transitions were not accompanied with release of cytochrome c into the cytosol and were fully reversible. The combined effect of respiratory inhibitors and uncouplers developed more rapidly indicating possible involvement of ROS generated by mitochondria. More prolonged (48-72 h) incubation with this combination of inhibitors caused clustering and degradation of mitochondria.

Phytanoyl-CoA hydroxylase from rat liver. Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation.

Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway. Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes. In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial. In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein. We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE. Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38. 6 kDa protein. The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2). Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa. This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2. This type of processing has been reported in several other peroxisomal proteins that contain a PTS2. Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat. The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.