EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

Decreased brain weights in rats after long-term barbital treatments

To study dependence on barbiturates, rats were in three experiments given a solution of barbital as their only drinking fluid for periods around 30 weeks. The animals were sacrificed after abstinence periods of up to 30 days and the brains were weighed. Compared with untreated controls the barbital treated animals in these experiments consistently had reduced wet brain weights. This reduction was not restituted after an abstinent period of 30 days. The decrease did not seem to be secondary to a reduced body weight. The decrease in brain weight found throughout the abstinence period was not influenced by changes in water intake.In a 4th experiment 12 weeks of barbital treatment caused a similar slightly smaller reduction in wet brain weight. Since there was no difference in water content the dry weight was also reduced. The possibility that this finding is related to the brain atrophy found in humans after long-term ethanol abuse is pointed out.     

Modulation of dopamine release by the nicotinic agonist epibatidine in the frontal cortex and the nucleus accumbens of naive and chronic nicotine treated rats

The effect of the nicotinic acetylcholine receptors (nAChRs) agonist (+/-)epibatidine on the modulation of dopamine (DA) release was investigated by microdialysis in vivo in the frontal cortex and the nucleus accumbens of naive and chronic nicotine-treated awake rats. (+/-)Epibatidine (2.5 microg/kg, s.c.), contrary to (-)nicotine (0.5 mg/kg, s.c.), decreased the extracellular concentrations of DA in the brain of naive rats. Subchronic nicotine treatment (0.45 mg/kg, s.c., twice daily for 7 days) attenuated the (+/-)epibatidine induced decrease in the DA level. The extracellular concentrations of the DA metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were elevated by (+/-)epibatidine administration in both na?ve and subchronic treated rats. The findings suggest that the decrease in DA extracellular concentrations induced by the high affinity nAChRs agonist (+/-)epibatidine might be due to inactivation of nAChRs, which can be overcome by subchronic treatment with nicotine. Different mechanisms in modulation of DA release appears to be involved in the rat brain by (+/-)epibatidine compare to (-)nicotine.     

Long-lasting acetylcholinesterase splice variations in anticholinesterase-treated Alzheimer's disease patients

Protein levels of different acetylcholinesterase (AChE) splice variants were explored by a combination of immunoblot techniques, using two different antibodies, directed against the C-terminus of the AChE-R splice variant or the core domain common to all variants. Both AChE-R and AChE-S splice variants as well as several heavier AChE complexes were detected in brain homogenates from the parietal cortex of patients with or without Alzheimer's disease (AD) as well as the cerebrospinal fluid (CSF) of AD patients, compatible with the assumption that CSF AChEs might originate from CNS neurons. Long-term changes in the composition of CSF AChE variants were further pursued in AD patients treated with rivastigmine (n = 11) or tacrine (n = 17) in comparison to untreated AD patients (n = 5). In untreated patients, AChE-R was markedly reduced as compared with the baseline level (37%), whereas the medium size AChE-S complex was increased by 32%. Intriguingly, tacrine produced a general and profound up-regulation of all detected AChE variants (up to 117%), whereas rivastigmine treatment caused a mild and selective up-regulation of AChE-R ( approximately 10%, p < 0.05). Moreover, the change in the ratio of AChE-R to AChE-S (R/S-ratio) strongly and positively correlated with sustained cognition at 12 months (p < 0.0001). Thus, evaluation of changes in the composition of CSF AChE variants may yield important information referring to the therapeutic efficacy and/or development of drug tolerance in AD patients treated with anti-cholinesterases.     

Increased levels of tau protein in SH-SY5Y cells after treatment with cholinesterase inhibitors and nicotinic agonists

Several cholinesterase inhibitors used in the treatment of Alzheimer's disease (AD) have been shown to interact with an allosteric site on the nicotinic acetylcholine receptor (nAChR). A possible linkage between the phosphorylation state of tau, the major component of paired helical filaments found in AD brain, and stimulation of nAChRs by cholinesterase inhibitors and nicotinic agonists was investigated. Western blot analysis showed that treatment of SH-SY5Y cells for 72 h with the cholinesterase inhibitors tacrine (10(-5) M), donepezil (10(-5) M), and galanthamine (10(-5) M), nicotine (10(-5) M), and epibatidine (10(-7) M) increased tau levels as detected with Tau-1, AT 8, and AT 270 monoclonal antibodies and binding of [3H]epibatidine. The increase in tau immunoreactivity induced by nicotine, epibatidine, and tacrine, but not the up-regulation of nAChRs, was prevented by the antagonists d-tubocurarine and mecamylamine. Both antagonists were synergistic with the nicotinic agonists in causing up-regulation, but only d-tubocurarine showed a synergistic effect with tacrine. The increased tau immunoreactivity induced by tacrine was not prevented by atropine, indicating that in terms of cholinergic receptors, tacrine modulates tau levels mainly through interactions with nAChRs and not with muscarinic receptors. Additional work is needed to determine the exact mechanism by which cholinesterase inhibitors and nicotinic agonists modulate phosphorylation and levels of tau protein.     

Uptake kinetics of 99Tc in common duckweed

The uptake of the nuclear waste product technetium-99 was studied in common duckweed (Lemna minor). In addition to measurements, a model involving two compartments in duckweed with different chemical forms of technetium was derived. The model was tested by chemical speciation, i.e. differentiating between reduced Tc-compounds and Tc(VII)O(4)(-). The TcO(4)(-) concentrations measured were in good agreement with those predicted by the model. Two processes determine technetium uptake: (1) transport of Tc(VII)O(4)(-) across the cell membrane, and (2) reduction of Tc(VII). The TcO(4)(-) concentration in duckweed reaches a steady state within 2 h while reduced Tc-compounds are stored, as a result of absence of release or re-oxidation processes. Bioaccumulation kinetic properties were derived by varying 99Tc concentration, temperature, nutrient concentrations, and light intensity. The reduction of technetium in duckweed was highly correlated with light intensity and temperature. At 25 degrees C the maximum reduction rate was observed at light intensities above 200 μmol m(-2) s(-1) while half of the maximum transformation rate was reached at 41 μmol m(-2) s(-1). Transport of TcO(4)(-) over the cell membrane requires about 9.4 kJ mol(-1), indicating an active transport mechanism. However, this mechanism behaved as first-order kinetics instead of Michaelis-Menten kinetics between 1x10(-14) and 2.5x10(-5) mol l(-1) TcO(4)(-). Tc uptake could not be inhibited by 10(-3) mol l(-1) nitrate, phosphate, sulphate or chloride.     

YODA: selecting signature oligonucleotides

MOTIVATION: Selecting oligonucleotide probes for use in microarray design, and other applications requiring signature sequences, involves identifying sequences which will bind strongly to their intended target, while binding only weakly (or preferably, not at all) to non-target sequences which may be present in the hybridization reaction. While many tools to assist in selection of such sequences exist, all the ones we examined lack important oligo design and software features. RESULTS: YODA is an application for assisting biological researchers in selecting signature sequences. It incorporates a custom sequence similarity search to find potential cross-hybridizing non-target sequences. For this task, most oligo design tools rely on BLAST, which is ill suited for it due to an unacceptable risk of false negatives. YODA supports multiple probe design goals including single-genome, multiple-genome, pathogen-host and species/strain-identification. A graphical interface is provided as well as a command-line interface, both of which support many user-controlled parameters. YODA is easy to install and use and runs on Windows, Mac OS X and Linux platforms. AVAILABILITY: Freely available (LGLP) along with source code and additional documentation at http://pathport.vbi.vt.edu/YODA CONTACT: enordber@vbi.vt.edu.     

Optimized expression of soluble cyclomaltodextrinase of thermophilic origin in Escherichia coli by using a soluble fusion-tag and by tuning of inducer concentration

Cyclomaltodextrinases are multidomain and often dimeric proteins from the alpha-amylase family (glycoside hydrolase family 13) which frequently have been very difficult to express in active form in Escherichia coli. To express the soluble form of this type of proteins in larger quantities the expression has to be optimized. We have used and combined two strategies to increase the yield of soluble recombinant cyclomaltodextrinase expressed from a gene originating from the thermophilic Gram-positive bacterium Anoxybacillus flavithermus. One strategy involved tuning of the inducer concentration while the other involved fusion of the gene encoding the target protein to the gene encoding the solubility-enhancing protein NusA. The enzyme activity could be increased 6-7 times solely by finely tuning the IPTG concentration, but the activity level was very sensitive to the amount of inducer applied. Hence, the IPTG concentration may have to be optimized for every protein under the conditions used. The fusion protein-strategy gave a slightly lower total activity but the level of soluble recombinant protein obtained was in this case significantly less sensitive to the inducer concentration applied. Moreover, the activity could be increased about 2-fold by cleaving off the solubility-tag (NusA) by enterokinase.