Homonucleotide tracts, short repeats and CpG/CpNpG motifs are frequent sites for heterogeneous mutations in the neurofibromatosis type 1 (NF1) tumour-suppressor gene

Glutamatergic synaptic potentials induced by micromolar concentrations of the potassium conductance blocker 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in the presence of 10 microM bicuculline (BIC). These synaptic potentials originate from neostriatal cortical and thalamic afferents and were completely blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) plus 100 microM D-2-amino-5-phosphonovaleric acid (2-APV). Their inter-event time intervals could be fitted to exponential distributions, suggesting that they are induced randomly. Their amplitude distributions had most counts around 1 mV and fewer counts with values up to 5 mV. Since input resistance of the recorded neurons is about 40 M omega, the amplitudes agree to quantal size measurements in mammalian central neurons. The action of a D2 agonist, quinpirole, was studied on the frequency of these events. Mean amplitude of synaptic potentials was preserved in the presence of 2-10 microM quinpirole, but the frequency of 4-AP-induced glutamatergic synaptic potentials was reduced in 35% of cases. The effect was blocked by the D2 antagonist sulpiride (10 microM). Input resistance, membrane potential, or firing threshold did not change during quinpirole effect, suggesting a presynaptic site of action for quinpirole in some but not all glutamatergic afferents that make contact on a single cell. The present experiments show that dopaminergic presynaptic modulation of glutamatergic transmission in the neostriatum does not affect all stimulated afferents, suggesting that it is selective towards some of them. This may control the quality and quantity of afferent flow upon neostriatal neurons.     

Rat mature sympathetic neurones derive neurotrophin 3 from peripheral effector tissues

In a previous study we have demonstrated that endogenous neurotrophin 3 (NT3) is required for the survival of most sympathetic neurones in postnatal rats. However, the mechanisms underlying the action of NT3 on sympathetic neurones is not known. Neither is it understood whether NT3 is retrogradely transported from peripheral tissues or acts locally in an autocrine fashion. In the present study, NT3-mRNA was quantified in sympathetic effector tissues and NT3 protein was localized in intact and lesioned sympathetic nerves in rats. NT3-mRNA is expressed and developmentally regulated in many effector tissues including mesenteric arteries, salivary gland, heart and kidney but hardly detectable in the superior cervical ganglia of adult animals. The majority of sympathetic neurones express immunoreactivity for TrkA and TrkC in both neonatal and adult rats. Sympathetic somata are normally immunoreactive for NT3, but the immunoreactivity is abolished by systemic administration of NT3 antibodies or axotomy of postganglionic neurones, suggesting an accumulation of NT3 from extraneuronal sources. Furthermore, the detection of NT3-immunoreactivity in the internal carotid nerve as early as 3 h following a compression and only on the distal side indicates endogenous NT3 is retrogradely transported by sympathetic neurones. These studies provide evidence indicating that NT3, like nerve growth factor, is an effector tissue-derived neurotrophic factor for sympathetic neurones both during development and in the adult animal. Thus, we have provided a clear example that one type of neurone derives, through a retrograde transport mechanism, two neurotrophic factors simultaneously from its peripheral effector tissues.     

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.     

Behavioral and subjective effects of D-amphetamine and modafinil in healthy adults.

Modafinil is indicated for the management of excessive daytime sleepiness; however, recent studies have examined a broad range of potential uses. Given that clinical uses of modafinil may be expanding, this study compared modafinil and d-amphetamine effects on subjective and performance measures. Across 11 sessions, 11 healthy adults were tested after oral doses of placebo (5 sessions), modafinil (1.75 mg/kg, 3.50 mg/kg, or 7.00 mg/kg), and d-amphetamine (0.035 mg/kg, 0.070 mg/kg, 0.140 mg/kg) under double-blind, randomized conditions. Assessments of cognitive performance and subjective effects were completed before drug administration, 30 min after drug administration, and at hourly intervals after drug administration for 5 hr. Modafinil increased ratings on the Amphetamine and Morphine Benzedrine Group scales of the Addiction Research Center Inventory (ARCI) and increased ratings on the Vigor and Total Positive scales of the Profile of Mood States. d-Amphetamine increased visual analog ratings of feeling stimulated and liking the drug and increased ratings on the Morphine Benzedrine Group scale of the ARCI. Both medications significantly reduced visual analog scale ratings of feeling sleepy, and modafinil decreased ratings on the ARCI Pentobarbital-Chlorpromazine-Alcohol Group scale. Both medications sustained performance that deteriorated across time on the Sternberg Number Recognition Test. Modafinil also enhanced performance rate on the Digit-Symbol Substitution Task above baseline levels and increased response rate on the Repeated Acquisition of Response Sequences Task. These results suggest that modafinil engenders alerting effects and increases performance in healthy non-sleep-deprived individuals comparable with that of d-amphetamine. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Inhibition of nitric oxide synthesis extends cerebrovascular autoregulation during hypertension

In anesthetized intact rats, cerebral blood flow is autoregulated until mean arterial blood pressure (MAP) exceeds 150 mmHg. At higher pressures cerebral blood flow breaks through autoregulation and rapidly increases. However, interruption of the arterial baroreceptor reflex eliminates breakthrough of autoregulation. Thus, breakthrough may reflect active rather than passive vasodilatation. We, therefore, sought to determine if breakthrough depends upon synthesis of the vasodilator nitric oxide. Thirty-eight anesthetized adult male Sprague-Dawley rats were studied. In all, MAP was raised by slow i.v. infusion of phenylephrine. In rats pretreated with the nitric oxide synthase inhibitor L-nitroarginine (L-NA; 22 mg/kg i.v.) or with a combination of L-NA plus D-arginine (D-Arg; 240 mg/kg i.v.), breakthrough did not occur even when MAP exceeded 185 mmHg (L-NA) and 165 mmHg (D-Arg). In contrast, breakthrough occurred in rats treated with L-NA plus L-arginine (L-Arg; 240 mg/kg i.v.) and in rats whose basal vascular tone had been increased by pretreatment with arginine vasopressin prior to infusion of phenylephrine. Removal of sympathetic innervation to cerebral vessels attenuated, but did not eliminate, effects of L-NA on breakthrough. Thus, vasodilatation seen with breakthrough of autoregulation depends upon release of nitric oxide or a nitric oxide donor.     

Simple protocols to determine dust potentials from cattle feedlot soil and surface samples

Cattle feedlot dust is an annoyance and may be a route for nutrient transport, odor emission, and pathogen dispersion, but important environmental factors that contribute to dust emissions are poorly characterized. A general protocol was devised to test feedlot samples for their ability to produce dust under a variety of environmental conditions. A blender was modified to produce dust from a variety of dried feedlot surface and soil samples and collect airborne particles on glass fiber filters by vacuum collection. A general blending protocol optimized for sample volume (150-175 cm3), blending time (5 min of pre-blending), and dust collection time (15 s) provided consistent dust measurements for all samples tested. The procedure performed well on samples that varied in organic matter content, but was restricted to samples containing less than 200 to 700 g H2O kg(-1) dry matter (DM). When applied to field samples, the technique demonstrated considerable spatial variability between feedlot pen sites. Mechanistically, dust potential was related to moisture and organic matter content. An alternative protocol also demonstrated differences within pen sites in maximum dust potential and dust airborne residence time. The two protocols were not intended, nor are they suitable, for predicting actual particulate matter emissions from agricultural sources. Rather, the protocols rapidly and inexpensively compared the potential for dust emission from samples of differing composition under a variety of environmental conditions.     

Mannolipid donor specificity of glycosylphosphatidylinositol mannosyltransferase-I (GPIMT-I) determined with an assay system utilizing mutant CHO-K1 cells

The microsomal enzyme glycosylphosphatidylinositol mannosyltransferase I (GPIMT-I) catalyses the transfer of a mannosyl residue from beta-mannosylphosphoryldolichol (beta-Man-P-Dol) to glucosamine-alpha(1,6)(acyl)phosphatidylinositol (GlcN-aPI) to form Man alpha(1,4)GlcN-aPI (ManGlcN-aPI), an intermediate in glycosylphosphatidylinositol (GPI) synthesis. While the transfer of [3H]mannosyl units to endogenous GlcN-aPI was not seen when membrane fractions from normal Chinese hamster ovary (CHO) K1 cells were incubated with exogenous [3H]Man-P-Dol, GPIMT-I activity could be characterized with an in vitro enzyme assay system employing membrane fractions from Lec15 or Lec35 cells. These CHO cell mutants apparently contain elevated levels of endogenous GlcN-aPI due to the inability to synthesize (Lec15) or utilize (Lec35) beta-Man-P-Dol in vivo. The presence of a saturated alpha-isoprene unit in the dolichyl moiety is required for optimal GPIMT-I activity since beta-mannosylphosphorylpolyprenol (beta-Man-P-Poly), which contains a fully unsaturated polyisoprenyl chain, was only 50% as effective as beta-[3H]Man-P-Dol as a mannosyl donor. When beta-[3H]-Man-P-Dol and alpha-[3H]Man-P-Dol were compared as substrates, GPIMT-I exhibited a strict stereospecificity for the mannolipid containing the beta-mannosyl-phosphoryl linkage. beta-[3H]Man-P-dolichols containing 11 or 19 isoprenyl units were equally effective substrates for GPIMT-I. Membrane fractions from Lec 9, a CHO mutant that apparently lacks polyprenol reductase activity and synthesizes very little beta-Man-P-Dol, but accumulates beta-Man-P-Poly, synthesized no detectable Man-GlcN-aPI when incubated with beta-[3H]Man-P-Dol in vitro. This indirect assay suggests that GlcN-aPI does not accumulate in Lec 9 cells, possibly because it is mannosylated via beta-Man-P-Poly, or perhaps the small amount of Man-P-Dol formed by the mutant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)