Modulation of the serotonin-activated K+ channel by G protein subunits and nucleotides in rat hippocampal neurons

In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K⁺ current via G protein. We identified the K⁺ channel activated by 5-HT (K_5-HT channel) and studied the effects of G protein subunits and nucleotides on the K⁺ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 m 5-HT in the pipette, application of GTP (100 m) to the cytoplasmic side of the membrane activated an inwardly rectifying K⁺ channel with a slope conductance of 36±1 pS (symmetrical 140 mm K⁺) at –60 mV and a mean open time of 1.1±0.1 msec (n=5). Transducin activated the (K_5-HT) channels and this was reversed by -GDP. Whether the K_5-HT channel was activated endogenously (GTP, GTPS) or exogenously (), the presence of 1 mm ATP resulted in a 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G activates the G protein-gated K⁺ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.     

Modelling human power and endurance

A generalised three component hydraulic model has been proposed to represent the human bioenergetic processes relating internal energy stores to performance during exercise, and into recovery. Further development of the model allows testable predictions to be made. In particular in this paper I examine certain hypotheses of chemical fuel shortage as a subgroup of the potential causes of fatigue, and their implications for maximal power and for endurance. The assumption that the limitation to sustainable power is direct proportionality to the glycogen store remaining, appears the most feasible. Based on this assumption, equations for the decline in maximum attainable power over time, the endurance at fixed workrates and the endurance at incremental tests (as a function of the increment slope) are obtained. Using published data for fit males, the maximum exertable power declines after about 6 s at 972 W to very low levels after about 2 min. For constant powers selected between 208 and 927 W, endurance declines from ad infinitum to only 6 s. Endurance at VO2max is predicted to be about 9 min. For incremental exercise tests of slope ranging from 30 W/min to 60 W/min, endurance lessens from 14 to 9 min. In these tests the anaerobic threshold is reached in times between 6 and 3 min. Although the power at termination of a test increases with incremental slope, terminal oxygen consumption is effectively constant. Almost all these model predictions are observed to correspond well with published experimental findings. These results suggest that the model can be used to represent an adequate overview of the operation of the human bioenergetic system.     

Effect of high-intensity endurance training on isokinetic muscle power

The purpose of this study was to determine the effects of high-intensity endurance training on isokinetic muscle power. Six male students majoring in physical-education participated in high intensity endurance training on a cycle ergometer at 90% of maximal oxygen uptake (VO2max) for 7 weeks. The duration of the daily exercise session was set so that the energy expenditure equalled 42 kJ.kg-1 of lean body mass. Peak knee extension power was measured at six different speeds (30 degrees, 60 degrees, 120 degrees, 180 degrees, 240 degrees, and 300 degrees.s-1) with an isokinetic dynamometer. After training, VO2max increased significantly from mean values of 51.2 ml.kg-1.min-1, SD 6.5 to 56.3 ml.kg-1.min-1, SD 5.3 (P less than 0.05). Isokinetic peak power at the lower test speeds (30 degrees, 60 degrees and 120 degrees.s-1) increased significantly (P less than 0.05). However, no significant differences in muscle peak power were found at the faster velocities of 180 degrees, 240 degrees, and 300 degrees.s-1. The percentage improvement was dependent on the initial muscle peak power of each subject and the training stimulus (intensity of cycle ergometer exercise).     

Use of isotopic signatures to assess the food web in a tropical shallow marine ecosystem of Southeastern Brazil

A dual isotope approach was used to assess the relative importance of terrestrial vegetation detritus and other primary producers in the trophic web of Flamengo Sound (Ubatuba, SP), SE Brazil, surrounded by the Atlantic Rain Forest. Primary producers showed distinct C signatures and the observed values suggest that little terrestrial or bulk sediment organic matter enter the food web of the sound. Suspended particulate organic matter (POM, supports the bulk of the consumers, with some contribution by macroalgae . Consumers C values ranged from −17.4 to . At least three trophic levels were detectable in the food web. The N value of POM was , while that of sediment and detritus was . The N values of suspension feeding benthic invertebrates were 8.2–, deposit feeders 8.3–, and carnivores 10.7–. Values for fishes were for detritivore, 11.4– for benthic feeders, 12.4– for zooplanktivores, and for piscivores/benthic invertebrate feeders. Squid mean value was . There is a reasonable agreement between feeding habits information from the literature and N values from this study. In the sound, the first and second trophic steps seem to be about 1– higher than those of similar organisms studied in temperate waters and this may reflect an input of allochtonous anthropogenic nitrogen enriched in ¹⁵N from human activities.     

Iron deficiency caused by 7 weeks of intensive physical exercise

The present study was designed to evaluate the effect of an intensive physical training program involving both isometric and isotonic activities on the body iron status of 8 females and 11 males (age 20 +/- 1 year). The training was carried out over a 7 week period and included 8 h of varying physical activities each day. Venous blood samples were obtained from the subjects prior to the beginning of the training, on day 2 and in weeks 2, 4, 6 and 7 of the program. Blood samples were analyzed for iron, ferritin and hemoglobin (Hb) concentrations, total iron binding capacity (TIBC) and red blood cell count (RBC). Iron levels of males and females decreased 65% after 2 weeks of training (p less than 0.001). At the end of the training program 5 males and 6 females had lower than normal iron values (less than 13.4 mumol.l-1). TIBC increased 25% in women and 18% in men following 2 and 4 weeks of training (p less than 0.001) and remained at this elevated level throughout the training period. Ferritin levels decreased 50% in both sexes after 4 weeks of exercise (p less than 0.05) and remained at this level until the end of the training. Hb and RBC decreased 8-10% in both sexes during the training period. In two of the women anemia occurred after 4 weeks of training. The development of latent iron deficiency in a substantial number of participants after a relatively short period of training is uncommon and may reflect the high intensity of exercise required in this program.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen delivery does not limit peak running speed during incremental downhill running to exhaustion

Oxygen consumption (VO2), ventilation (VI), respiratory exchange ratio (R), stride frequency and blood lactate concentrations were measured continuously in nine trained athletes during two continuous incremental treadmill runs to exhaustion on gradients of either 0 degree or -3 degrees. Compared to the run at 0 degree gradient, the athletes reached significantly higher maximal treadmill velocities but significantly lower VO2, VI, R and peak blood lactate concentrations (P less than 0.001) during downhill running. These lower VO2 and blood lactate concentrations at exhaustion indicated that factors other than oxygen delivery limited maximal performance during the downhill run. In contrast, stride frequencies were similar at each treadmill velocity; the higher maximal speed during the downhill run was achieved with a significantly longer stride length (P less than 0.001); maximal stride frequency was the same between tests. Equivalent maximal stride frequencies suggested that factors determining the rate of lower limb stride recovery may have limited maximal running speed during downhill running and, possibly, also during horizontal running.     

GTP-binding protein-independent potentiation by mastoparan of IL-1β-induced nitric oxide release from insulin-secreting HIT-T15 cells

Our recent data implicated small molecular weight G-proteins (e.g., H-Ras) in interleukin 1beta (IL 1beta)-induced metabolic dysfunction and apoptotic demise of the islet beta cell (Tannous et al., Biochem Pharmacol 2001; 62:1459-1468, Kowluru and Morgan, Biochem Pharmacol, 2002; 63:1027-1035, Chen et al. Biochem Pharmacol, 2003; 66:1681-1694). Recently, we have shown that mastoparan, a tetradecapeptide from wasp venom, has been shown to directly activate islet endogenous G-proteins and regulate islet function (Amin et al., Endocrinology 2003; 144: 4508-4518). Herein, we investigated potential contributory roles, if any, of mastoparan (Mas)-sensitive G-proteins in IL-induced nitric oxide (NO) release from insulin-secreting HIT-T15 cells. While, ineffective by itself, Mas significantly potentiated IL-induced NO release from HIT-T15 cells. Interestingly, Mas-17, an inactive analog of Mas, also potentiated IL-induced NO release, suggesting that the potentiating effect of Mas may not involve activation of specific G-proteins. Such potentiating effects on IL-induced NO release were also demonstrable in the presence of another polycationic compound, melittin. Together, these findings suggest that Mas-induced potentiation of IL-induced NO release may in part be due to its amphiphilic and polycationic nature. These data also warrant caution in the use of Mas to study its regulation of cellular function without the use of an appropriate negative control, such as Mas-17.     

Neuroprotection by α-Lipoic Acid in Streptozotocin-Induced Diabetes

Glial cells provide structural and metabolic support for neurons, and these cells become reactive to any insult to the central nervous system. The streptozotocin (STZ) rat model was used to study glial reactivity and the prevention of gliosis by alpha-lipoic acid (alpha-LA) administration. The expression of glial fibrillary acidic protein (GFAP), S100B protein, and neuron specific enolase (NSE) was determined as well as lipid peroxidation (LPO) and glutathione (GSH) levels in some brain tissues. Western blot analyses showed GFAP, S100B, and NSE levels significantly increased under STZ-induced diabetes in brain, and LPO level increased as well. Administration of alpha-LA reduced the expression both of glial and neuronal markers. In addition, alpha-LA significantly prevented the increase in LPO levels found in diabetic rats. GSH levels were increased by the administration of alpha-LA. This study suggests that alpha-LA prevents neural injury by inhibiting oxidative stress and suppressing reactive gliosis.     

A genetic component in human lysosomal enzyme excretion

Acid hydrolases are present in normal human urine in appreciable amounts. Their source appears to be lysosomes released by kidney proximal tubule epithelial cells. For a given lysosomal enzyme the total amount excreted is the product of two parameters, a general one describing the rate of lysosome secretion and a specific one describing the relative concentration of that enzyme in lysosomes. There is considerable population variation in both parameters. Studies of -glucuronidase, -galactosidase, -hexosaminidase, and -galactosidase in monozygotic and dizygotic twins show that an appreciable part of this variation is genetic in origin. This appears to be true for both total enzyme excretion and lysosome composition. Although it was not possible to test directly whether this is also true for the rate of lysosome secretion, the fact that the two former parameters are both heritable strongly suggests that the rate of lysosome excretion is also a heritable trait. Taken together with previous data, the results suggest polygenic control of these biochemical traits. It is particularly significant that -glucuronidase excretion in normal individuals is a heritable trait since the excretion of this enzyme has frequently been used as a measure of normal and pathological physiological changes.This study was supported by grants from the National Institutes of Health (GM-19521) and the Council for Tobacco Research—U.S.A., Inc. (1080). The work was done while the authors were in the Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York 14263.     

Posttranslational Arginylation of Brain Proteins

The knowledge of brain protein metabolism is important in understanding nervous system brain function. Protein synthesis rates are high in young brain, decline rapidly at adult stages, and thereafter continue falling slowly with age. The breakdown of protein appears to follow a similar rate (1). Protein synthesis and degradation however, are only the two extremes of a complex phenomena which includes a variety of other protein modifications. Proteolytic cleavage is the most common covalent modification of proteins; probably all proteins that have been isolated were modified by proteolysis, since only few are found with the starting amino acid (methionine) attached. This suggests that most proteins were subject to one or more co- and/or posttranslational modifications (2). One of these posttranslational modifications is the arginylation of proteins, described 30 years ago, which now is being recognized as a widespread modification of proteins. In this review, the current status of posttranslational arginylation of brain proteins is discussed.     

Identification of very small embryonic like (VSEL) stem cells in bone marrow

Bone marrow (BM) develops in mammals by the end of the second/beginning of the third trimester of gestation and becomes a major hematopoietic organ in postnatal life. The alpha-chemokine stromal derived factor-1 (SDF-1) to CXCR4 ([Formula: see text]-protein-coupled seven transmembrane-spanning chemokine receptor) axis plays a major role in BM colonization by stem cells. By the end of the second trimester of gestation, BM becomes colonized by hematopoietic stem cells (HSC), which are chemoattracted from the fetal liver in a CXCR4-SDF-1-dependent manner. Whereas CXCR4 is expressed on HSC, SDF-1 is secreted by BM stroma and osteoblasts that line BM cavities. Mounting evidence indicates that BM also contains rare CXCR4(+) pluripotent stem cells (PSC). Recently, our group has identified a population of CXCR4(+) very small embryonic like stem cells in murine BM and human cord blood. We hypothesize that these cells are deposited during development in BM as a mobile pool of circulating PSC that play a pivotal role in postnatal tissue turnover, both of non-hematopoietic and hematopoietic tissues.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

The production of a new extracellular putative long-chain saturated polyester by smooth variants of Mycobacterium vaccae interferes with Th1-cytokine production

Mycobacterium vaccae is of major pharmaceutical interest as an immunotherapeutic agent. Although M. vaccae 15483 ATCC(T) strain displays smooth and rough colonial morphologies on solid culture media, it is not known in which conditions M. vaccae switches from one colonial morphotype to the other or whether there are biological differences, especially immunological, between them. We have found that the change from a smooth to rough stable variant occurs spontaneously at 30 degrees C. The analysis of the composition of the cell wall in both variants showed that the smooth morphotype presents an extracellular material that has never previously been described and was identified as a long-chain saturated polyester that, interestingly, is not produced by the rough morphotype. Our results also indicate that this compound could be implicated in the spreading ability of smooth colonies. Proliferation, IFN-gamma and IL-12(p40) production by splenocyte cultures was significantly higher in mice immunised with the rough variant compared with those immunised with the smooth one. This latter finding suggests that the different colonial morphology of M. vaccae may affect the immunomodulatory effects induced from M. vaccae preparations.     

The relative contribution of glucose and fatty acids to ATP production in hearts reperfused following ischemia

High levels of fatty acids decrease the extent of mechanical recovery of hearts reperfused following a transient period of severe ischemia. Glucose oxidation rates during reperfusion are low under these conditions, which can result in a decreased recovery of mechanical function. Stimulation of glucose oxidation with the carnitine palmitoyl transferase I inhibitor, Etomoxir, or by directly stimulating pyruvate dehydrogenase activity with dichloroacetate (DCA) results in an improvement in mechanical function during reperfusion of previously ischemic hearts. Addition of DCA (1 mM) to hearts perfused with 11 mM glucose and 1.2 mM palmitate results in an increase in contribution of glucose oxidation to overall ATP production from 6 to 23%, with a parallel decrease in that of fatty acid oxidation from 90 to 69%. In aerobic hearts, endogenous myocardial triglycerides are an important source of fatty acids for -oxidation. Using hearts in which the myocardial triglycerides were pre-labeled, the contribution of both endogenous and exogenous fatty acid oxidation to myocardial ATP production was determined in hearts perfused with 11 mM glucose, 1.2 mM palmitate and 500 µU/ml insulin. In hearts reperfused following a 30 min period of global no flow ischemia, 91.9% of ATP production was derived from endogenous and exogenous fatty acid oxidation, compared to 87.7% in aerobic hearts. This demonstrates that fatty acid oxidation quickly recovers following a transient period of severe ischemia. Furthermore, therapy aimed at overcoming fatty acid inhibition of glucose oxidation during reperfusion of ischemic hearts appears to be beneficial to recovery of mechanical function.     

Histochemical and chemical studies on pre- and postnatal development of the different systems of “short” and “long” adrenergic neurons in peripheral organs of the rat

Summary The adrenergic innervation in the submaxillary gland, heart, kidney, small intestine, and accessory male genital organs and the development of the adrenal chromaffin cells and the sympathetic ganglia were studied in the rat from 15 days post coitum to 16 days post partum using the fluorescence histochemical method of Falck and Hillarp. The postnatal development of the noradrenaline concentrations in the heart and vas deferens was followed by fluorometric determinations.At about 15 days post coitum, the anlagen of the sympathetic chains were well visible in the form of two dorsal segmented columns of small branching sympathicoblasts exhibiting an intense catecholamine fluorescence. In the midline, ventrally to these two anlagen, another column of sympathicoblasts developed; this seemed to give rise to the prevertebral ganglia and to the short adrenergic neurons supplying the internal genital organs. At the level of the adrenal anlagen, small intensely fluorescent chromaffin cells were collected in two bilateral groups which became enclosed by adreno-cortical cells. This enclosure was, however, not complete even at two weeks post partum.Bundles of growing sympathetic nerves were visible in the periphery of the various organs studied at 19–21 days post coitum. A terminal innervation of the organs suggestive of a functional transmitter mechanism did not start to establish until at or immediately after birth. The final pattern of innervation was usually reached at about one week post partum, and the following development proceeded largely in the form of a quantitative increase in the number of nerves participating in the innervation apparatus. The adult level of noradrenaline in the heart and vas deferens was reached three to five weeks after birth. The small intestine was an exception in that the final pattern of innervation in the wall was attained immediately after birth.There was no overt difference in the rate of development of the terminal sympathetic innervation in organs supplied by short adrenergic neurons (accessory male genital organs) compared to the innervation of the submaxillary gland, heart and kidney, which receive classical long adrenergic neurons.The work was supported by a grant from the Association for the Aid of Crippled Children, New York, and was carried out within a research organization sponsored by the Swedish Medical Research Council (grants No. B71-14X-56-07A and B71-14X-712-06A).     

GM2 Promotes Ciliary Neurotrophic Factor-Dependent Rescue of Immortalized Motor Neuron-Like Cell (NSC-34)

We have examined whether ciliary neurotrophic factor (CNTF) can alter serum-free cell survival of immortalized motor neuron-like cells, which were established by fusing mouse neuroblasoma N18TG2 with mouse motor neurons. One of the cell lines, NSC-34 exhibited cell survival in the presence of CNTF. NSC-34 preserves the most characteristics of motor neurons, such as the formation of neuromuscular junctions on co-cultured myotube. GM2 ganglioside is characteristic of motor neurons, and expressed highly in NSC-34. When NSC-34 was cultured with exogenous GM2 ganglioside and CNTF, GM2 facilitated the cell survival effect of CNTF. In the addition, 1,4 N-acetylgalactosaminyltransferase (GM2 synthase) activity was enhanced up to 3.9-fold by culture in the presence of CNTF. GM2 might be a functional modulator of CNTF in motor neurons. It might be presented to cell surface by its enzyme activation, and become a signal of early stage, when CNTF rescues motor neurons.     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.