Massively parallel analysis of single-molecule dynamics on next-generation sequencing chips.

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.

Role of P-170 glycoprotein in colchicine brain uptake.

To study the role of P-glycoprotein (P-gp) in the delivery of colchicine from blood to brain, the pharmacokinetics of colchicine in plasma and brain was studied in the rat by an in vivo method and by the in situ brain perfusion technique. Colchicine was administered intravenously at three doses (1, 2.5, and 5 mg/kg) with or without an inhibitor of P-gp, verapamil (0.5 mg/kg i.v.); blood and brain samples were taken at t = 1, 2, and 3 hr. Areas under the colchicine curve at doses from 2.5 to 5 mg/kg were proportional to dose for plasma but not for brain. At a colchicine dose of 5 mg/kg, verapamil co-treated rats showed a 1.65-fold enhancement of the colchicine concentration in plasma but a 4.5-fold enhancement in brain. During short experimental times (in situ brain perfusion technique), a comparable enhancement was found (4.26-fold): mean distribution volumes of colchicine were enhanced from 0.23 +/- 0.17 to 0.98 +/- 0.19 microl/g for the eight gray areas, and no effect was observed in the choroid plexus, which do not express P-gp. These results clearly show that P-gp, present at the luminal surface of the capillary endothelial cells, is responsible for the weak penetration of colchicine into the brain.

Specific modulation of two neuronal digitalis receptors by anaesthesia.

Three isoenzymes of digitalis receptors (alpha 1, alpha 2, alpha 3) in the brain and only one in the kidney (alpha 1) can be distinguished by their ouabain affinities and their responsiveness to sodium. Since we have reported modulations for these digitalis receptors by their fatty acid membrane environment, anaesthesics could bind on and modulate either directly these receptors or indirectly by disturbing membrane lipids. The aim of this study was to evaluate this anaesthetic action on apparent ouabain affinities and sodium dependence of cerebral and renal Na+, K(+)-ATPase isoenzymes activities. Rat brain and kidney membrane fractions with pentobarbital-induced anaesthetized state were compared to an unanaesthetized state for their (1) fatty acid composition of total membrane phospholipids, (2) responsiveness to ouabain and (3) Na+ dependence of digitalis receptors. An anaesthesia period of 10 minutes induced (1) a fatty acid modification of brain membranes and (2) a significant sensibilization to ouabain for the alpha 2 and alpha 3 isoforms of digitalis receptors (alpha 2, IC50; 8.2 +/- 0.5 x 10(-7) mol/l vs 4.5 +/- 0.2 x 10(-7) mol/l; alpha 3, IC50; 6.0 +/- 0.3 x 10(-8) mol/l vs 2.5 +/- 0.1 x 10(-8). In contrast, the ouabain affinity of the alpha 1 subunit expressed in kidney and brain membranes was unaltered. No anaesthetic effect was observed on the Na+ dependence of the alpha 1 isoenzyme in the brain (4 mmol/l) and the kidney (8 mmol/l). Pentobarbital induced a desensibilization for alpha 2-receptors (8.3 +/- 0.5 vs 16.0 +/- 1.4 mmol/l Na+) and a sensibilization for alpha 3-receptors (14.4 +/- 0.8 vs 10 +/- 1.3 mmol/l Na+). These altered properties could be related to a selective modification of the fatty acid composition and/or to the presence of a specific binding site for pentobarbital on these two neuronal digitalis receptors.

Developmental effects of intrauterine growth retardation on cerebral amino acid transport.

Early restriction of nutrients during the perinatal period of life can modify the development of the mammalian fetus and have marked repercussions on the ontogeny of the CNS. The brain is vulnerable to undernutrition, with delayed morphologic and biochemical maturation leading to impaired functions. The aim of the present investigation was to assess whether modified brain neurotransmitter and amino acid concentrations found in an animal model of intrauterine growth retardation were related to modified blood-brain amino acid transport properties. Four amino acids were tested: alanine and taurine, plus two neurotransmitter precursors, tryptophan and tyrosine. Intrauterine growth retardation was induced by restriction of maternal-fetal blood flow from the 17th d of gestation. Blood-brain transport of these amino acids was measured by i.v. injection of radiolabeled amino acids in 7-d-old, 21-d-old, and 60-d-old intrauterine growth-retarded or control rats. No major statistical differences were revealed either for brain regional transport or between intrauterine growth-retarded animals and controls at any age studied. Transfer coefficients and influxes remained statistically similar for almost all brain regions in both groups. A significant decrease and different time course for amino acid transport with age related to the blood-brain barrier maturation are confirmed in this model. Our results are related to a major role of the blood-brain barrier as a part of mechanisms leading to "brain growth sparing."

Effect of fish oil diet on fatty acid composition of phospholipids of brain membranes and on kinetic properties of Na+,K(+)-ATPase isoenzymes of weaned and adult rats.

The influence of dietary (n-3) fatty acids (such as eicosapentaenoic and docosahexaenoic acids) as found in fish oil on Na+ sensitivity and ouabain affinity of Na+,K(+)-ATPase isoenzymes (alpha 1, alpha 2, alpha 3) was studied in whole brain membranes from weaned and adult rats fed diets for two generations. The long chain (n-3) fatty acids supplied by fish oil decreased the fatty acids of the (n-6) series compared with the standard diet, resulting in a decrease in the (n-6)/(n-3) molar ratio in both 21- and 60-day-old rats. On the basis of ouabain titration, three inhibitory processes with markedly different affinities were associated with isoenzymes, i.e., low affinity (alpha 1), high affinity (alpha 2), and very high affinity (alpha 3). It appears that the fish oil diet, in part via the modification of membrane fatty acid composition, altered the proportion and ouabain affinity of isoenzymes. Na+ sensitivity is the best criterion of physiologic change induced by fish oil diet. We calculated the Na+ activation for each isoenzyme and found one Na+ sensitivity and two Na+ sensitivities per isoenzyme in weanling and adult rats fed different diets, respectively. In contrast to alpha 2 and alpha 3, alpha 1 appears insensitive to membrane change induced by fish oil diet. Fish oil diet, which is known to confer cardioprotection, induced significant modulation of Na+,K(+)-ATPase isoenzymes at the brain level.

Cerebral amino acid changes in an animal model of intrauterine growth retardation.

As part of a series of experiments to ascertain the effects of prenatal malnutrition on brain development, we measured brain amino acids in an animal model of intrauterine growth retardation (IUGR) obtained by restriction of blood supply to the fetus in utero during the last 5 days of gestation. In the present study, amino acids were measured during development by HPLC as their O-phthaldialdehyde derivatives in cerebral cortex, cerebellum and hippocampus. In rats with IUGR, significant increase of alanine (by 20% to 50%) and taurine (by 20% to 80%) were observed prior to weaning in the cerebellum and the cerebral cortex respectively. Alanine levels were also increased in hippocampus. In control animals, at birth, activities of the GABA nerve terminal marker enzyme glutamic acid decarboxylase (GAD) were found to be 32%, 17%, and 11% of adult values in cerebellum, hippocampus and cerebral cortex respectively. Two-day-old rats with IUGR had significantly lower GAD activities in all brain regions. Thus, impairment of nutrient supply to fetal brain results in selective regional abnormalities of amino acids particularly in the cerebral cortex.

Heterogeneous Na+ sensitivity of Na+,K(+)-ATPase isoenzymes in whole brain membranes.

The Na+ sensitivity of whole brain membrane Na+,K(+)-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (alpha 1, low affinity for ouabain; alpha 2, high affinity; and alpha 3, very high affinity). At 100 mM Na+, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10(-5) and 10(-7) M), we were able to discriminate Na+ sensitivity of Na+,K(+)-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, alpha 1, exhibited high Na+ sensitivity [Ka of 3.88 +/- 0.25 mM Na+ and a Hill coefficient (n) of 1.98 +/- 0.13]; the ouabain high-affinity isoform, alpha 2, had two Na+ sensitivities, a high (Ka of 4.98 +/- 0.2 mM Na+ and n of 1.34 +/- 0.10) and a low (Ka of 28 +/- 0.5 mM Na+ and an n of 1.92 +/- 0.18) Na+ sensitivity activated above a threshold (22 +/- 0.3 mM Na+); and the ouabain very-high-affinity isoform, alpha 3, was resolved by two processes and appears to have two Na+ sensitivities (apparent Ka values of 3.5 and 20 mM Na+). We show that Na+ dependence in the absence of ouabain is the result of at least of five Na+ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.

Effect of dietary alpha-linolenic acid on functional characteristic of Na+/K(+)-ATPase isoenzymes in whole brain membranes of weaned rats.

The influence of dietary fatty acids on Na+ sensitivity and ouabain affinity of Na+/K(+)-ATPase isoenzymes of whole brain membranes were studied in weaned rats fed for two generations with diets either devoid of alpha-linolenic acid (sunflower oil diet) or rich in alpha-linolenic acid (soya oil diet). The (n--3) deficiency induced by the sunflower oil diet led to an increase in the (n--6)/(n--3) molar ratio in whole brain membranes. Na+/K(+)-ATPase isoenzymes were discriminated on the basis of their differential affinities for ouabain. In rats fed sunflower oil diet, the ouabain titration displayed three inhibitory processes with markedly different affinities: low affinity (alpha 1); high affinity (alpha 2); and very high affinity (alpha 3). Membranes of rats fed soya oil diet exhibited only two inhibitory processes, i.e., low affinity (likely alpha 1+ alpha 2) and high affinity (likely alpha 2+ alpha 3) with the low affinity form intermediate between the sunflower alpha 1 and alpha 2 forms, and the high affinity form intermediate between the sunflower alpha 2 and alpha 3 forms. In fact, the Na+ response shows that the three isoenzymes have different Na+ sensitivities. Regardless of the diet, alpha 1 has a similar Na+ sensitivity (less than 1 mM), whilst alpha 2 and alpha 3 are more sensitive in soya oil membranes compared to sunflower oil membranes (5.1 vs. 7.2 mM and about 11 vs. 22.5 mM, respectively). Thus, sodium appears to be a better criterion of heterogeneity than ouabain.

Temperature-dependent immunoreactive assay to screen for digoxin-like immunoreactive factor(s).

Endogenous circulating digoxin-like immunoreactive factors (DLIF) are known to cross-react with antibodies to digoxin and to inhibit Na+/K(+)-transporting ATPase (Na+K+ATPase; EC 3.6.1.37). Moreover, increasing the immunoassay temperature from 4 to 37 degrees C markedly decreases DLIF from human cord serum. We tested several compounds, including hormonal steroids, bile salts, lipids, and methionine-enkephalin, for their ability to cross-react with two commercially available 125I digoxin RIAs, to inhibit porcine Na+K+ATPase, and to see whether they present the same incubation temperature dependence as human cord serum. Except for methionine-enkephalin, all compounds were inhibitors of Na+K+ATPase in the range of 1-10 mmol/L. Progesterone exhibited the highest cross-reactivity in the two RIAs. The apparent digoxin immunoreactivity for the majority of the cross-reacting steroids, bile salts, and linoleic acid was markedly decreased by increasing the incubation temperature from 4 to 37 degrees C, whereas estriol, pregnanediol, and nonspecific compounds (e.g., ethanol, human serum albumin) did not appear to be temperature-sensitive. Both lysophosphatidyl lipids gave an increased apparent digoxin concentration with increasing incubation temperature. Our data suggest that numerous weakly cross-reactive compounds can parallel the response of human cord serum. However, the temperature-dependent effect could be an additional criterion for identifying DLIF.

Existence of a digitalis-like compound in the human fetus.

Digoxin-like inhibitors of Na+,K(+)-ATPase have been implicated in several pathophysiological problems in the perinatal period. Aqueous endogenous digoxin-like immunoreactive substance (DLIS) was extracted from 9 different organs of a 24-week-old human fetus whose mother died after paraquat poisoning. The results indicate that this endogenous DLIS has a wide distribution in fetal tissues. The highest levels were found in gut and adrenals, and there was a correlation between these high levels and the inhibition of Na+,K(+)-ATPase. The hypothesis that DLIS originated in the fetus is of particular relevance.

Development of the cholinergic system in control and intra-uterine growth retarded rat brain.

The activity of choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and muscarinic receptors was studied in control rats and in rats growth-retarded in utero because of reduction of the blood supply 5 days before birth. The different markers of the cholinergic system were estimated at P (postnatal day) 6, 9, 12, 15, 22 and 60 in cerebellum, hypothalamus, septum, striatum and CA1, CA3 and fascia dentata of the hippocampus. In control rats, there was a transient increase in ChAT activity in the septum during the second week of postnatal development. In the intrauterine growth retarded rats there was a marked delay in this developmental rise in CA1, CA3 at P6 and P9 and in the fascia dentata at P14 respectively. This delayed rise enzyme activity was associated with a significant reduction of muscarinic binding sites [( 3H]QNB) in the hippocampus. AChE staining showed a similar development in both groups. Therefore, the undernutrition produced by a reduction of the blood supply 5 days before birth is associated with a delayed maturation of cholinergic functions.

Effect of organic and inorganic mercuric salts on Na+K+ATPase in different cerebral fractions in control and intrauterine growth-retarded rats: alterations induced by serotonin.

An intrauterine growth-retarded (IUGR) model based on restriction of blood supply to the rat fetus at the 17th day of pregnancy was studied. We investigated in vitro the effects of thimerosal and mercuric chloride on Na+K+ATPase activity in total brain homogenate, synaptosomes and myelin at weaning. In addition, we evaluated the reversal effect of serotonin on mercury-inhibited Na+K+ATPase activity. The toxicity, in terms of inhibition of Na+K+ATPase activity was greater with mercuric chloride than with thimerosal. Synaptosomes and principally myelin were more sensitive to the metal salts than total homogenate. Serotonin stimulated the Na+K+ATPase activity in total brain homogenate and synaptosomes but inhibited the enzyme in the myelin fraction. This effect was more marked in the IUGR group than in the control group. Serotonin (1 mM) added to total homogenate pretreated with the mercury salts produced variable reversal effects. In the synaptosomal fraction reverse effect was noted with serotonin. In myelin fraction, added serotonin increased inhibition caused by thimerosal.

Na(+) K(+) ATPase activity in synaptosomes and myelin of developing control and intra-uterine growth retarded rats: effects of lead and serotonin.

An intrauterine growth retarded (IUGR) model based on restriction of blood supply to fetuses at 17 days of pregnancy in rats was studied. We investigated in vitro the effects of lead on Na(+)K(+) ATPase activity in synaptosomes and myelin of IUGR and control rats from 6 to 60 days after birth. In both groups an age-dependent effects existed in synaptosomes for the lowest doses of lead. The experimental group tended to be more sensitive to the metal than the control group and the Na(+)K(+)ATPase activity was less inhibited in the younger rats as compared to mature rats. Serotonin (5-HT) added to the subcellular preparations produced different changes in Na(+)K(+)ATPase activity. In synaptosomes, 5-HT stimulated the enzyme activity in a dose-related manner and apparently reversed the inhibiton induced by lead up to 22 days after birth in the control group. This action was less marked in the IUGR group. In myelin fractions, the Na(+)K(+)ATPase activity was inhibited by lead in both groups but the "protective effect of monoamines" was never observed. The Na(+)K(+) ATPase activity was modulated by monoamines in synaptosomes and not in myelin, perhaps through a mechanism involving soluble factor(s).

Effect of lead on Na+,K+ATPase activity in the developing brain of intra-uterine growth-retarded rats.

Lead (Pb) intoxication in developing mammals, including humans, produces serious brain damage. In addition, it is known that nutritional status influences the susceptibility to Pb toxicity. We developed an in utero undernutrition model based on restriction of blood supply to fetuses on d 17 of pregnancy (IUGR rats). The aim of this study was to investigate in vitro the possible effect of Pb on Na+, K+ATPase activity in the brain of developing IUGR and control rats from 6 to 60 d after birth. In addition, we measured the stimulation of Na+, K+ATPase by the monoamines noradrenaline and serotonin. Our results show that: The neurotoxic effect of Pb is an age-related phenomenon. Both IUGR and control rats were more sensitive to Pb in the first week of life. In adults, Pb had a weak inhibitory potency; the delayed matured brain in IUGR animals seemed less sensitive to Pb when compared to age-paired control rats; in the IUGR group, at 15 and 22 d, low doses of Pb had a stimulatory effect on Na+, K+ATPase instead of an inhibitory effect; noradrenaline and serotonin stimulated Na+, K+ATPase activity to an equivalent extent, but this was greater in IUGR than control rats; and at low Pb concentrations, the studied monoamines reversed Pb-induced inhibition.

Effect of intrauterine growth retardation on developmental changes in DNA and [14C]thymidine metabolism in different regions of rat brain: histological and biochemical correlations.

Intrauterine growth retardation was induced in the rat by clamping the uterine artery on day 17 of gestation. The effect of hypotrophy on DNA synthesis was studied in two different cerebral structures: hippocampus and cerebellum. Accumulation of DNA in these structures was biochemically measured in parallel to the incorporation of methyl-[14C]thymidine into nucleic acid at different ages and correlated with autoradiography. The various metabolites of thymidine in acid-soluble fraction were determined by using chromatographic procedures. Phosphorylation defects or reduced utilization of thymidine were found in hypotrophic rats and may delay the DNA synthesis. An essay of catch-up occurred with a different timing according to the cerebral region studied. A morphological and DNA synthesis. An essay of catch-up occurred with a different timing according to the cerebral region studied. A morphological and autoradiographic study after incorporation of [3H]thymidine was carried out in parallel. The neuronal and glial components of cytogenesis were analyzed separately and a good correlation was observed between histological and biochemical data in both groups of animals.

Carnitine and carnitine transferases in the intestinal mucosa of suckling rats.

Carnitine acetyltransferase and palmitoyltransferase activity in the mucosa of the small intestine of rats rises after birth and falls at the time of weaning. The carnitine contents of the mucosa (free, acetyl-, palmitoyl- and total) decrease postnatally, reaching adult levels at the time of weaning. Orally administered 14C-carnitine is only slowly absorbed so that radioactivity is still high in plasma and organs 6 h later, whereas label given subcutaneously disappears from the plasma and tissues more rapidly. The intestinal mucosa also takes up carnitine from 14C-carnitine administered subcutaneously. It is concluded that carnitine plays an important role in the gut of suckling rats.

[Specific contribution of dietary polyunsaturated fatty acids of the n-3 series to the development of nervous system membranes]. / Importance spécifique des acides gras polyinsaturés alimentaires de la série n-3 dans l'élaboration des membranes du système nerveux.

Polyunsaturated fatty acids, especially omega 3 fatty acids, made a specific contribution to the nervous tissue. Retarded intrauterine growth caused a twofold reduction in the oligodendrocyte ratio of omega 3/omega 6 while the same ratio increased by more than twofold in the neurons. A fatty acid-poor diet composed of arachid or sunflower oil dramatically altered the composition of cells and subcellular particles when compared with a soya or rapeseed oil diet containing omega 3 fatty acids; phosphatidylethanolamine was particularly affected. Nerve cell culture showed that brain essential fatty acids are probably docosahexaenoic acid and arachidonic acid.

Long lasting effects of intrauterine growth retardation on basal and 5-HT-stimulated Na(+)/K(+)-ATPase in the brain of developing rats.

Intrauterine growth retardation induced by ligation of the uterine vessels in pregnant rats on the 5th day before delivery was associated with brain and body weights of hypotrophic offspring significantly lower than those of pair-aged control rats, even after 6 weeks of postnatal rearing under normal conditions. In vitro measurements in homogenates indicated that Na(+)/K(+)-ATPase in the forebrain, cerebellum and hippocampus was less active in hypotrophic rats than in pair-aged controls for at least the first month after birth. However, 5-HT and related agonists (RU-24969, bufotenine, and to a lower extent, tryptamine) stimulated Na(+)/K(+)-ATPase activity more efficiently in tissues from hypotrophic rats than in those from control animals. Opposite changes were noted in the brain stem: basal Na(+)/K(+)-ATPase activity was higher in hypotrophic rats during the second half of the first postnatal month but the stimulatory effect of 5-HT was lower than in pair-aged control animals. Since potent 5-HT antagonists such as cinanserin, methiothepin and methysergide, prevented the 5-HT induced-activation of Na(+)/K(+)-ATPase in brain homogenates, these results are discussed in relation with the possible existence of a specific 5-HT receptor controlling Na(+)/K(+)-ATPase activity in the rat brain.