Effects of parturition and feed restriction on concentrations and distribution of the insulin-like growth factor-binding proteins in plasma and cerebrospinal fluid of dairy cows.

Hormones and metabolites act as satiety signals in the brain and play an important role in the control of feed intake (FI). These signals can reach the hypothalamus and brainstem, 2 major centers of FI regulation, via the blood stream or the cerebrospinal fluid (CSF). During the early lactation period of high-yielding dairy cows, the increase of FI is often insufficient. Recently, it has been demonstrated that insulin-like growth factors (IGF) may control FI. Thus, we asked in the present study if IGF-binding proteins (IGFBP) are regulated during the periparturient period and in response to feed restriction and therefore might affect FI as well. In addition, we specifically addressed conditional distribution of IGFBP in plasma and CSF. In one experiment, 10 multiparous German Holstein dairy cows were fed ad libitum and samples of CSF and plasma were obtained before morning feeding on d -20, -10, +1, +10, +20, and +40 relative to calving. In a second experiment, 7 cows in second mid-lactation were sampled for CSF and plasma after ad libitum feeding and again after feeding 50% of the previous ad libitum intake for 4 d. Intact IGFBP-2, IGFBP-3, and IGFBP-4 were detected in plasma by quantitative Western ligand blot analysis. In CSF, we were able to predominantly identify intact IGFBP-2 and a specific IGFBP-2 fragment containing detectable binding affinities for biotinylated IGF-II. Whereas plasma concentrations of IGFBP-2 and IGFBP-4 increased during the periparturient period, IGFBP-3 was unaffected over time. In CSF, concentrations of IGFBP-2, both intact and fragmented, were not affected during the periparturient period. Plasma IGF-I continuously decreased until calving but remained at a lower concentration in early lactation than in late pregnancy. Food restriction did not affect concentrations of IGF components present in plasma or CSF. We could show that the IGFBP profiles in plasma and CSF are clearly distinct and that changes in IGFBP in plasma do not simply correspond in the brain. We thus assume independent control of IGFBP distribution between plasma and CSF. Due to the known anorexic effect of IGF-I, elevated plasma concentrations of IGFBP-2 and IGFBP-4 during the postpartum period in conjunction with reduced plasma IGF-I concentrations may be interpreted as an endocrine response against negative energy balance in early lactation in dairy cows.

Hepatic mRNA expression of acid labile subunit and deiodinase 1 differs between cows selected for high versus low concentrations of insulin-like growth factor 1 in late pregnancy.

The somatotropic axis is a key metabolic pathway during transition from late pregnancy to early lactation in dairy cows. The first objective of this study was to determine the feasibility of selecting cows with persistent differences in total insulin-like growth factor 1 (IGF-1) concentration by taking only a single antepartum blood sample. The second objective was to elucidate the underlying causes of differences in peripheral IGF-1 concentrations throughout late pregnancy and whether hormonal axes also differed in dairy cows with low versus high IGF-1. Twenty clinically healthy Holstein Friesian cows were chosen based on their plasma IGF-1 concentration at 244 to 254 d after artificial insemination (AI) and other selection criteria (health status, body condition score, number of lactations). These cows were selected from a large-scale farm, transported to the clinic, and monitored daily from 261 to 275 d after AI. The concentrations of IGF-1, growth hormone, IGF binding proteins 2, 3, and 4, insulin, cortisol, thyroid hormones, progesterone, and estradiol were measured. Ultimately, 7 IGF-1-low and 7 IGF-1-high cows were statistically analyzed. Additionally, a liver biopsy was taken on d 270 ± 1 after AI for analysis of gene expression of somatotropic family members, liver deiodinase 1, and suppressor of cytokine signaling-2. It was possible to select cows with different IGF-1 concentrations based upon only 1 blood sample collected in late pregnancy. Concentrations of IGF-1 in IGF-1-low versus IGF-1-high animals (n=7 each) remained significantly different between groups from the day of selection of the animals until d 275 after AI. Second, the differences in total plasma IGF-1 concentration between experimental groups may be attributed to differences in hepatic production of acid labile subunit. The ability of IGFBP-3 to bind IGF-1 declined before calving in all cows. Furthermore, in addition to decreased mRNA expression of growth hormone receptor 1A and IGF-1 relative to calving, serum binding capacities for IGF-1 also decreased. Insulin-like growth factor binding protein 4 mRNA expression was higher in cows with low IGF-1 concentrations; this binding protein inhibits IGF-1 action at the tissue level and therefore may reduce IGF-1 bioavailability. Finally, other endocrine end points (e.g., insulin and thyroid hormones) differed between the 2 groups.

Effect of a "diluted" diet containing 10% lignocellulose on the gastrointestinal tract, intestinal microbiota, and excreta characteristics of dual purpose laying hens.

Low performing dual purpose hens have different nutritional requirements compared to conventional hybrid hens. Lignocellulose is a low fermentable polymer, acting as a diet diluent and may influence physiological and digestive processes. This study investigated the effect of a 10% dietary lignocellulose dilution on the development of gastrointestinal organs, intestinal morphology, intestinal microbiota, and excreta characteristics of dual purpose hens. One-day-old female Lohmann Dual chicks were allocated to 12 pens and fed two different diets: A standard control diet (CON) and a treatment diet (LC), based on CON but diluted with 10% lignocellulose (ARBOCEL®). At 52 wk of age, gastrointestinal organs were extracted and weights determined. Colorectal tissue samples were chemically fixed and stained for histomorphological examinations. Cecal digesta samples were analyzed for bacterial metabolites and composition using gas chromatography, HPLC, photometry, and PCR. Excreta dry matter and viscosity was consistently assessed during the trial. LC-fed hens showed increased weights of the gizzard (P = 0.003), small (P < 0.001), and large intestine (P = 0.048) compared to hens fed CON. LC-fed hens had a larger colorectal villus area (P = 0.049), a higher mucosal enlargement factor of villi (P = 0.016) and crypts (P = 0.030) than CON-fed hens. The concentration of short-chain fatty acids (SCFAs) (P = 0.017) and ammonia (P = 0.013) was higher in CON-fed hens compared to LC-fed hens. Bacterial composition and activity was generally not affected by feeding the different diets. LC-fed hens had a higher excreta dry matter content than hens fed CON at 10 (P < 0.001), 17 (P < 0.001), and 22 (P = 0.002) wk of age. Correlation analyses revealed a negative relationship between the concentration of SCFAs in the cecum and the colorectal villus surface area (P < 0.01). In conclusion, the feeding of high levels of lignocellulose increased gastrointestinal organ weights and colorectal surface area in dual purpose laying hens. A higher intestinal surface area in combination with lower concentrations of SCFAs might indicate a compensatory reaction of hens fed LC enhancing the absorption of bacterial metabolites by increasing the intestinal mucosal surface.

Effect of feeding different levels of lignocellulose on performance, nutrient digestibility, excreta dry matter, and intestinal microbiota in slow growing broilers.

Lignocellulose is a constituent of plant cell walls and might be used as a fiber source in poultry nutrition. The current study investigated the impact of increasing dietary levels of lignocellulose on performance, nutrient digestibility, excreta DM, intestinal microbiota, and bacterial metabolites in slow growing broilers. At an age of 10 wk, 60 male broilers of an intercross line (New Hampshire × White Leghorn) were allocated to cages and fed isoenergetic and isonitrogenous diets containing 0.8% (LC1), 5% (LC2), or 10% (LC3) lignocellulose. After 23 D of feeding, broilers were killed and digesta samples of ileum and excreta analyzed for nutrient digestibility and DM. Cecal contents were analyzed for microbial composition and metabolites. Broiler performance was not affected by feeding dietary lignocellulose. LC3 fed broilers showed reduced ileal digestibility of protein compared to chickens fed LC1 (P = 0.003). Moreover, increasing levels of dietary lignocellulose reduced apparent digestibility of organic matter and gross energy (P < 0.001). Feeding of lignocellulose had no impact on the excreta DM of broilers. Increasing levels of dietary lignocellulose lowered cecal counts of Escherichia/Hafnia/Shigella (P = 0.029) and reduced the total concentration of short-chain fatty acids (P < 0.001), lactate (P < 0.05), and ammonia (P = 0.009). The molar ratio of cecal acetic acid was higher in LC3 fed broilers (P < 0.001), while the proportions of cecal propionic acid and n-butyric acid were higher in LC1 and LC2 fed chickens (P < 0.001). Correlation analyses indicated that dietary lignocellulose was negatively related to the total concentration of cecal bacterial metabolites (P < 0.001). In conclusion, the feeding of lignocellulose did not affect growth performance, but impaired nutrient digestibility of slow growing broilers. While minor changes in cecal microbial composition were detected, cecal bacterial metabolite concentrations were significantly reduced with increasing levels of dietary lignocellulose. These findings suggest that lignocellulose is not extensively degraded by bacteria residing in the large intestine of broilers.

[The contribution of CT-guided transthoracic lung biopsy to the diagnosis of organizing pneumonia]. / Apport de la biopsie pulmonaire transthoracique dans le diagnostic de pneumonie organisée.

BACKGROUND: Organizing pneumonia is a pulmonary disease with variable clinical and radiological features and with many differential diagnoses. Diagnosis is based on histology obtained by either transbronchial or surgical lung biopsy but these techniques have several disadvantages. The aim of this study was to evaluate the diagnostic yield of CT-guided transthoracic lung biopsy in organizing pneumonia and to compare it to the usual diagnostic tools. METHODS: Six cases of organizing pneumonia diagnosed with CT-guided lung biopsy are reported and discussed. A review of literature concerning the role of CT-guided lung biopsy in the diagnosis of organizing pneumonia was performed. RESULTS: CT-guided transthoracic lung biopsies provided a higher rate of adequate samples than transbronchial biopsies (92-100% versus 77-86%). The samples were larger, which reduced the risks of misdiagnosis and increased the diagnostic yield (88-97% versus 26-55% in pulmonary nodules and 42-100% versus 66-75% in diffuse pulmonary disease). Complications were rare and generally not serious. CONCLUSION: CT-guided transthoracic lung biopsy may be considered in place of transbronchial biopsy in the diagnosis of organizing pneumonia. Surgical lung biopsy remains the reference method for diagnosis.

Shifted dynamic interactions between subcortical nuclei and inferior frontal gyri during response preparation in persistent developmental stuttering.

Persistent developmental stuttering is associated with basal ganglia dysfunction or dopamine dysregulation. Here, we studied whole-brain functional connectivity to test how basal ganglia structures coordinate and reorganize sensorimotor brain networks in stuttering. To this end, adults who stutter and fluent speakers (control participants) performed a response anticipation paradigm in the MRI scanner. The preparation of a manual Go/No-Go response reliably produced activity in the basal ganglia and thalamus and particularly in the substantia nigra. Strikingly, in adults who stutter, substantia nigra activity correlated positively with stuttering severity. Furthermore, functional connectivity analyses yielded altered task-related network formations in adults who stutter compared to fluent speakers. Specifically, in adults who stutter, the globus pallidus and the thalamus showed increased network synchronization with the inferior frontal gyrus. This implies dynamic shifts in the response preparation-related network organization through the basal ganglia in the context of a non-speech motor task in stuttering. Here we discuss current findings in the traditional framework of how D1 and D2 receptor activity shapes focused movement selection, thereby suggesting a disproportional involvement of the direct and the indirect pathway in stuttering.

Altered morphology of the nucleus accumbens in persistent developmental stuttering.

PURPOSE: Neuroimaging studies in persistent developmental stuttering repeatedly report altered basal ganglia functions. Together with thalamus and cerebellum, these structures mediate sensorimotor functions and thus represent a plausible link between stuttering and neuroanatomy. However, stuttering is a complex, multifactorial disorder. Besides sensorimotor functions, emotional and social-motivational factors constitute major aspects of the disorder. Here, we investigated cortical and subcortical gray matter regions to study whether persistent developmental stuttering is also linked to alterations of limbic structures. METHODS: The study included 33 right-handed participants who stutter and 34 right-handed control participants matched for sex, age, and education. Structural images were acquired using magnetic resonance imaging to estimate volumetric characteristics of the nucleus accumbens, hippocampus, amygdala, pallidum, putamen, caudate nucleus, and thalamus. RESULTS: Volumetric comparisons and vertex-based shape comparisons revealed structural differences. The right nucleus accumbens was larger in participants who stutter compared to controls. CONCLUSION: Recent theories of basal ganglia functions suggest that the nucleus accumbens is a motivation-to-movement interface. A speaker intends to reach communicative goals, but stuttering can derail these efforts. It is therefore highly plausible to find alterations in the motivation-to-movement interface in stuttering. While behavioral studies of stuttering sought to find links between the limbic and sensorimotor system, we provide the first neuroimaging evidence of alterations in the limbic system. Thus, our findings might initialize a unified neurobiological framework of persistent developmental stuttering that integrates sensorimotor and social-motivational neuroanatomical circuitries.

First-in-man study with a novel PEGylated recombinant human insulin-like growth factor-I.

OBJECTIVE: This study is a first time assessment of safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 in human, in comparison with unmodified rhIGF-I. DESIGN: The study was conducted as a single-center, randomized, double-blinded, placebo-controlled, single ascending dose, parallel group study in a clinical research unit in France. A total of 62 healthy volunteers participated in this clinical trial. RO5046013 was given as single subcutaneous injection, or as intravenous infusion over 48h, at ascending dose levels. The active comparator rhIGF-I was administered at 50µg/kg subcutaneously twice daily for 4days. Safety and tolerability, pharmacokinetics, and pharmacodynamics of RO5046013 were evaluated. RESULTS: PEGylation resulted in long exposure to RO5046013 with a half-life of 140-200h. Exposure to RO5046013 increased approximately dose proportionally. RO5046013 was safe and well tolerated at all doses, injection site erythema after SC administration was the most frequent observed AE. No hypoglycemia occurred. Growth hormone (GH) secretion was almost completely suppressed with rhIGF-I administration, whereas RO5046013 caused only a modest decrease in GH at the highest dose given IV. CONCLUSIONS: PEGylation of IGF-I strongly enhances half-life, reduces the negative GH feedback and hypoglycemia potential, and therefore offers a valuable alternative to rhIGF-I in treatment of relevant diseases.

Phenotype analysis of male transgenic mice overexpressing mutant IGFBP-2 lacking the Cardin-Weintraub sequence motif: Reduced expression of synaptic markers and myelin basic protein in the brain and a lower degree of anxiety-like behaviour.

Brain growth and function are regulated by insulin-like growth factors I and II (IGF-I and IGF-II) but also by IGF-binding proteins (IGFBPs), including IGFBP-2. In addition to modulating IGF activities, IGFBP-2 interacts with a number of components of the extracellular matrix and cell membrane via a Cardin-Weintraub sequence or heparin binding domain (HBD1). The nature and the signalling elicited by these interactions are not fully understood. Here, we examined transgenic mice (H1d-hBP2) overexpressing a mutant human IGFBP-2 that lacks a specific heparin binding domain (HBD1) known as the Cardin-Weintraub sequence. H1d-hBP2 transgenic mice have the genetic background of FVB mice and are characterized by severe deficits in brain growth throughout their lifetime (p<0.05). In tissue lysates from brain hemispheres of 12-21day old male mice, protein levels of the GTPase dynamin-I were significantly reduced (p<0.01). Weight reductions were also found in distinct brain regions in two different age groups (12 and 80weeks). In the younger group, impaired weights were observed in the hippocampus (-34%; p<0.001), cerebellum (-25%; p<0.0001), olfactory bulb (-31%; p<0.05) and prefrontal cortex (-29%; p<0.05). At an age of 12weeks expression of myelin basic protein was reduced (p<0.01) in H1d-BP-2 mice in the cerebellum but not in the hippocampus. At 80weeks of age, weight reductions were similarly present in the cerebellum (-28%; p<0.001) and hippocampus (-31; p<0.05). When mice were challenged in the elevated plus maze, aged but not younger H1d-hBP2 mice displayed significantly less anxiety-like behaviour, which was also observed in a second transgenic mouse model overexpressing mouse IGFBP-2 lacking HBD1 (H1d-mBP2). These in vivo studies provide, for the first time, evidence for a specific role of IGFBP-2 in brain functions associated with anxiety and risk behaviour. These activities of IGFBP-2 could be mediated by the Cardin-Weintraub/HBD1 sequence and are altered in mice expressing IGFBP-2 lacking the HBD1.

Altered dendritic development of cerebellar Purkinje cells in slice cultures from protein kinase Cgamma-deficient mice.

Protein kinase C (PKC) is a key molecule for the expression of long-term depression at the parallel fiber-Purkinje cell synapse in the cerebellum, a well known model for synaptic plasticity. We have recently shown that activity of PKC also profoundly affects the dendritic morphology of Purkinje cells in rat cerebellar slice cultures suggesting that synaptic efficacy and dendritic development may be controlled by similar intracellular signalling pathways. Here we have analyzed the role of the gamma-isoform of protein kinase C (PKCgamma), which is strongly and specifically expressed in Purkinje cells, during dendritic development. After pharmacological treatment with PKC modulators, phosphorylation of PKCgamma at serine 660 was altered in cerebellar slices suggesting that a change of PKCgamma activity by these treatments was taking place within the Purkinje cells. In PKCgamma-deficient mice, Purkinje cell dendritic trees were enlarged and had an increased number of branching points compared to wild-type mice indicating a role for the PKCgamma isoform as a negative regulator of dendritic growth and branching. Furthermore, the branching-stimulating effects of the PKC inhibitors 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl)maleimide and Gö6976 found in wild-type cultures were abolished in the absence of PKCgamma. In contrast, the strong inhibitory effect on dendritic growth by the PKC activator phorbol-12-myristate-13-acetate (PMA) did not require the presence of the PKCgamma isoform since it was still present in the cultures of PKCgamma-deficient mice. Our results clearly demonstrate an involvement of PKCgamma in Purkinje cell dendritic differentiation in cerebellar slice cultures.

Purkinje cell dendritic tree development in the absence of excitatory neurotransmission and of brain-derived neurotrophic factor in organotypic slice cultures.

The development of the dendritic tree of a neuron is a complex process which is thought to be regulated strongly by signals from afferent fibers. In particular the synaptic activity of afferent fibers and activity-dependent signaling by neurotrophic factors are thought to affect dendritic growth. We have studied Purkinje cell dendritic arbor development in organotypic cultures under suppression of glutamate-mediated excitatory neurotransmission, achieved with multiple combinations of blockers of glutamate receptors. Despite the presence of either single receptor blockers or combinations of blockers predicted to fully suppress glutamate-mediated excitatory neurotransmission Purkinje cell dendritic arbors developed similar to those of control cultures. Furthermore, Purkinje cell dendritic arbors in organotypic cultures from brain-derived neurotrophic factor (BDNF) knockout mice or after pharmacological blockade of trk-receptors also developed in a way similar to control cultures. Our results demonstrate that during the stage of rapid dendritic arbor growth signals from afferent fibers are of minor importance for Purkinje cell dendritic development because a seemingly normal Purkinje cell dendritic tree developed in the absence of excitatory neurotransmission and BDNF signaling. Our results suggest that many aspects of Purkinje cell dendritic development can be achieved by an intrinsic growth program.

Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10.

1. The ATP-sensitive K+ channel (KATP channel) in A10 cells, a cell line derived from rat thoracic aorta, was characterized by binding studies with the tritiated KATP channel opener, [3H]-P1075, and by electrophysiological techniques. 2. Saturation binding experiments gave a KD value of 9.2 +/- 5.2 nM and a binding capacity (BMax) of 140 +/- 40 fmol mg-1 protein for [3H]-P1075 binding to A10 cells; from the BMax value a density of binding sites of 5-10 per microns2 plasmalemma was estimated. 3. KATP channel modulators such as the openers P1075, pinacidil, levcromakalim and minoxidil sulphate and the blocker glibenclamide inhibited [3H]-P1075 binding. The extent of inhibition at saturation depended on the compound, levcromakalim inhibiting specific [3H]-P1075 binding by 85%, minoxidil sulphate and glibenclamide by 70%. The inhibition constants were similar to those determined in strips of rat aorta. 4. Resting membrane potential, recorded with microelectrodes, was -51 +/- 1 mV. P1075 and levcromakalim produced a concentration-dependent hyperpolarization by up to -25 mV with EC50 values of 170 +/- 40 nM and 870 +/- 190 nM, respectively. The hyperpolarization induced by levcromakalim (3 microM) was completely reversed by glibenclamide with an IC50 value of 86 +/- 17 nM. 5. Voltage clamp experiments were performed in the whole cell configuration under a physiological K+ gradient. Levcromakalim (10 microM) induced a current which reversed around -80 mV; the current-voltage relationship showed considerable outward rectification. Glibenclamide (3 microM) abolished the effect of levcromakalim. 6. Analysis of the noise of the levcromakalim (10 microM)-induced current at -40 and -20 mV yielded estimates of the channel density, the single channel conductance and the probability of the channel to be open of 0.14 micron-2, 8.8 pS and 0.39, respectively. 7. The experiments showed that A10 cells are endowed with functional KATP channels which resemble those in vascular tissue; hence, these cells provide an easily accessible source of channels for biochemical and pharmacological studies. The density of binding sites for [3H]-P1075 was estimated to be one order of magnitude higher than the density of functional KATP channels; assuming a plasmalemmal localization of the binding sites this suggests a large receptor reserve for the openers in A10 cells.

Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes.

1. Tedisamil is a new antiarrhythmic drug with predominant class III action. The aim of the present study was to investigate the blocking pattern of the compound on the transient outward current (I(to)) in human subepicardial myocytes isolated from explanted left ventricles. Using the single electrode whole cell voltage clamp technique, I(to) was analysed after appropriate voltage inactivation of sodium current and block of calcium current. 2. Tedisamil reduced the amplitude of peak I(to), but did not affect the amplitude of non-inactivating outward current. The drug accelerated the apparent rate of I(to) inactivation. The reduction in time constant of I(to) inactivation depended on drug concentration, the apparent IC50 value was 4.4 microM. 3. Tedisamil affected I(to) amplitude in a use-dependent manner. After 2 min at -80 mV, maximum block of I(to) was reached after 4-5 clamp steps either at the frequency of 0.2 or 2 Hz, indicating that the block was not frequency-dependent in an experimentally relevant range. Recovery from block was very slow and proceeded with a time constant of 12.1+/-1.8 s. Also in the presence of drug, a fraction of channels recovered from inactivation with a similar time constant as in control myocytes (i.e. 81+/-40 ms and 51+/-8 ms, respectively, n.s.). 4. From the onset of fractional block of I(to) by tedisamil during the initial 60 ms of a clamp step, we calculated k1 = 9 x 10(6) mol(-1) s(-1) for the association rate constant, and k2 = 23 s(-1) for the dissociation rate constant. The resulting apparent KD was 2.6 microM and is similar to the IC50 value. 5. The effects of tedisamil on I(to) could be simulated by assuming a four state channel model where the drug binds to the channel in an open (activated) conformation. It is concluded that in human subepicardial myocytes tedisamil is an open channel blocker of I(to) and that this effect probably contributes to the antiarrhythmic potential of this drug.

Binding of [3H]-P1075, an opener of ATP-sensitive K+ channels, to rat glomerular preparations.

ATP-sensitive K+ channels (KATP channels) in the kidney have been found in the tubular system and in the afferent arteriole. In this study we have examined the binding of [3H]-P1075 ([3H]-N-cyano-N'-(1, 1-dimethylpropyl)-N"-3-pyridylguanidine), a selective opener of KATP channels, in rat glomerular preparations. Equilibrium (saturation, competition) and kinetic experiments indicated that [3H]-P1075 binds to a single class of sites with a dissociation constant of about 3 nM and a maximum binding capacity of 10 fmol mg-1 glomerular protein. The association rate constant of the complex was 6,5 x 10(7) M-1 min-1; dissociation occurred with a half-time of 6.2 min. Specific [3H]-P1075 binding was strongly reduced when the metabolic state of the glomerular preparation was impaired during the preparation procedure or the binding assay or when the preparation was subjected to mild collagenase treatment. In different metabolically competent preparations, the amount of specific [3H]-P1075 binding correlated well with the number of vascular endings adherent to the glomeruli; no specific binding was found in mesangial cells in culture. Specific [3H]-P1075 binding was inhibited by representatives of the different classes of KATP channel openers and by sulphonylurea-type blockers with inhibition constants similar to those obtained in rat aortic rings. It is concluded that rat glomerular preparations possess specific binding sites for KATP channel openers with vascular characteristics. The sensitivity of binding to mild collagenase treatment suggests that these sites are located on a membrane protein; in addition, the data suggest that these sites are localized on smooth muscle and/or renin secreting cells of the afferent vascular endings attached to some of the glomeruli. Their estimated density (1,500 microns-2) is much higher than that of KATP channels in smooth muscle.

Sulphonylurea binding in rat isolated glomeruli: pharmacological characterization and dependence on cell metabolism and cytoskeleton.

The kidney is endowed with ATP-sensitive K+ channels (KATP channels) both at the vascular and at the epithelial level. In this study we have characterized the binding of the sulphonylurea glibenclamide, the most widely used blocker of KATP channels, in rat isolated glomeruli. In metabolically intact glomeruli, 3H-glibenclamide labelled two different binding components with affinities of 47 +/- 12 nM and 10 +/- 1 microM and estimated binding capacities of 1.2 +/- 0.1 and 501 +/- 11 pmol/mg protein, respectively. 3H-glibenclamide binding was inhibited differentially by other sulphonylureas (tolbutamide, glibornuride, gliquidone and glipizide) and benzoic acid analogues such as meglitinide, AZ-DF 265 and UL-DF 9. Sulphonylureas interacted with the high affinity component and, in some cases, also with the low affinity component whereas the benzoic acid derivatives inhibited exclusively low affinity glibenclamide binding. Severe metabolic stress affected both components of glibenclamide binding by shifting high affinity binding to the right and reducing the capacity of the low affinity component. Disruption of the cytoskeletal actin filaments by cytochalasin B and D mimicked the effect of metabolic stress on the high affinity component but left the low affinity component unchanged. In crude membranes, the affinity of the first component was again reduced and a major loss of the low affinity sites was observed. The data show that the two binding components of glibenclamide binding in rat isolated glomeruli have very different properties. The high affinity component is not recognized by the benzoic acid derivatives; its affinity is modulated by cell metabolism and the actin component of the cytoskeleton. The low affinity sites are, in their majority, cytosolic. The function and cellular localization of the high affinity sites are under further study.

Mechanical and excitotoxic lesion of motoneurons: effects of neurotrophins and ciliary neurotrophic factor on survival and regeneration.

Mechanical lesion of peripheral nerves leads to extensive death of corresponding motoneurons in newborn rodents. The extent of cell death can be significantly reduced by neurotrophic factors. These molecules are produced by glial and neuronal cells and play an important role in supporting survival and regeneration of various neuronal populations in the central nervous system, in particular after mechanical, excitotoxic and ischemic insults. In addition, factors such as ciliary neurotrophic factor and neurotrophin-3 influence glial cell proliferation and survival. We have investigated the role of neurotrophic factors on motoneurons, both in cell culture and after axotomy in vivo. Moreover, the role of excitatory neurotransmission in modulating dendritic architecture of these cells was analyzed. Our data suggest that motoneurons are a suitable model for investigating the complex functional and morphological changes after brain lesion and for the identification of new therapeutic strategies to influence survival and functional recovery under such circumstances.

Morphological study of organotypic cerebellar cultures.

Organotypic cerebellar cultures from 8-days-old (P8) mouse pups were studied following 11 days of in vitro (I IDIV) culturing. The cerebellar cytoarchitectonic structure was maintained in most parasagittal cerebellar cortical slice cultures (also containing the deep cerebellar nuclei). The two main extrinsic excitatory inputs (the climbing and the mossy fibers) seem to be replaced by other axonal types: in the molecular layer mostly by parallel fibers (for climbing fibers) and in the granular layer by intrinsic mossy fiber collaterals of local excitatory interneurons, the unipolar brush cells. However, in a few organotypic cultures, which (although preserving the trilaminar cerebellar cortical structure) were "granuloprival" but also contained some of the deep cerebellar nuclei, the participation of extracortical axons from the deep cerebellar nuclei in the replacement of the missing afferents is suggested.

Calorie restriction and resveratrol supplementation prevent age-related DNA and RNA oxidative damage in a non-human primate.

Oxidative stress is a key factor in the aging process and in the development of age-related diseases. Because nutritional interventions such as caloric restriction (CR) delay the onset of age-related diseases and increase the lifespan of many species, the impact of a moderate CR was tested on male grey mouse lemur (Microcebus murinus), which have a median survival time of 5.7 years in captivity. The effects of CR on these lemurs were compared with a potential mimetic, resveratrol (RSV), a polyphenol naturally found in grapes. We hypothesized that both CR and RSV impact oxidative DNA and RNA damage compared to standard-fed control (CTL) animals. Adult (3-4 years old) male mouse lemurs were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200 mg·day(-1)·kg(-1)). Oxidative stress was estimated after 3, 9, 15 and 21 months of treatment using the measurement of oxidized nucleosides in urine samples by mass spectrometry. The resting metabolic rate, adjusted for changes in body composition, was also measured to assess the potential relationship between oxygen consumption and oxidative damage markers. This study provides evidence for oxidative stress accumulation with age in grey mouse lemur. Dietary interventions resulted in a short-term increase in oxidative stress levels followed by reduced levels with increasing age. Moreover, in this photoperiod-dependent heterotherm primate, seasonal variations in oxidative stress were observed, which was likely due to a season-dependent, cost-benefit trade-off between torpor use and oxidative stress.

Early brain amyloidosis in APP/PS1 mice with serum insulin-like growth factor-I deficiency.

The influence of insulin-like growth factor I (IGF-I) on the progression of Alzheimer's disease (AD) is discussed controversially. To help clarify the role of this circulating neurotrophic factor in brain amyloidosis, the major pathological trait in AD, we analyzed plaque formation in a mouse model of AD transgenic for human APP and PS1 mutations with reduced serum IGF-I levels (LIDAD mice). We found that brain amyloidosis in LIDAD mice appeared earlier than in AD mice, at 2 months of age, while attained comparable levels at 6 months. In parallel, early microgliosis was observed in LIDAD mice also at 2 months and remained exacerbated at 6 months. Collectively, these observations suggest a role of serum IGF-I in delaying early brain amyloidosis.