Induction of DNA breakage and suppression of DNA synthesis by the OH radical generated in a Fenton-like reaction

The effect of the OH radical, generated in a Fenton-like reaction, on DNA structure and function was studied in a monkey kidney cell line (Vero). DNA single strand breaks were detected following exposure to 10- 100 microM concentrations of H2O2 on ice. These breaks were repaired very rapidly, and addition of the poly (ADP-ribose) transferase inhibitor, 3-aminobenzamide, resulted in an accumulation of breaks. DNA synthesis was inhibited at concentrations as high as 1- 30 mM, this effect also being reversible in approximately 60 min. 3-aminobenzamide did not affect the rate of DNA synthesis inhibition by H2O2.     

A MASS FRAGMENTOGRAPHIC ASSAY OF 3-METHOXYTYRAMINE IN RAT BRAIN

Abstract— A mass fragmentographic method for the measurement of 3-methoxytyramine, a metabolite of dopamine formed after its neuronal release, is described. This method allows the assay of this dopamine metabolite in the picogram range, thus utilizing only few milligrams of brain areas containing dopaminergic neurons. Moreover, the method is simple and possesses the high specificity intrinsic to mass fragmentography.     

Measurement of 3,4-dihydroxyphenylacetic acid and 3-methoxytyramine specific activity rat striatum.

A method is described for the simultaneous determination in rat striatum of the specific activities of tyrosine, dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC), and 3-methoxytyramine (3-MT) after administration of [³H]tyrosine. [³H]Tyrosine was given intraventricularly to nonanesthetized rats, and the animals were killed by exposure to microwave radiations. Combined chromatographic elutions on Dowex 50W-X4 columns and alumina or solvent extractions were devised to separate the compounds. Fluorimetric, mass-fragmentographic, and radiometric techniques were used for their detection. Recovery was 94% for tyrosine, 72% for dopamine, 63% for DOPAC, and 50% for 3-MT. Concentrations of the labeled compounds in rat striatum 15 min after the [³H]tyrosine injection were at least five to eight times higher than background. Identity of the final fractions containing 3-MT and DOPAC tissue extracts was verified by thin-layer chromatography. α-Methyltyrosine pretreatment of rats markedly reduced the formation of labeled dopamine. DOPAC, and 3-MT from [³H]tyrosine.     

Induction of DNA double strand breaks in cultured mammalian cells exposed to hydrogen peroxide and histidine

The amino acid histidine was found to increase the toxicity of H₂O₂ in cultured mammalian cells. Histidine also augmented the level of DNA single strand breaks (SSB) detectable in cells exposed to the oxidant and, in addition, resulted in the appearance of DNA double strand breaks (DSB), a lesion which is not produced by H₂O₂ alone.     

CHANGES IN SPECIFIC ACTIVITY OF DOPAMINE METABOLITES AS EVIDENCE OF A MULTIPLE COMPARTMENTATION OF DOPAMINE IN STRIATAL NEURONS

The functional status of dopaminergic nerve terminals has been studied with a method that allows the simultaneous determination of the specific activities of dopamine (DM), tyrosine (Tyr), 3-methoxytyramine (3-MT) and 3,4-dihydroxyphenylacetic acid (DOPAC), after administration of [³H]tyrosine ([³H]Tyr). Combined fluorimetric, mass fragmentographic and radiometric techniques have been used. [³H]Tyrosine was given intraventricularly to unanaesthetized rats and the animals were killed by exposure for 4 s to high energy microwave radiations. The specific activities of 3-MT and DOPAC measured 5 and 20 min after administration of [³H]Tyr, i. e. at time intervals in which the specific activity of DM is rising, are much higher than those their physiological precursor, suggesting that they are generated by more than one DM compartment. This hypothesis seems to be supported by the finding that in animals killed by decapitation instead of microwave radiations the post mortem accumulation of 3-MT occurs to a much smaller extent for the radioactive fraction than for the endogenous one, indicating that 3-MT formed after death may be mainly derived from DM coming from a compartment where this monoamine has been poorly labeled by the radioactive precursor.     

Effects of streptozotocin-diabetes on the hippocampal NMDA receptor complex in rats

In animal models of diabetes mellitus, such as the streptozotocin-diabetic rat (STZ-rat), spatial learning impairments develop in parallel with a reduced expression of long-term potentiation (LTP) and enhanced expression of long-term depression (LTD) in the hippocampus. This study examined the time course of the effects of STZ-diabetes and insulin treatment on the hippocampal post-synaptic glutamate N-methyl-D-aspartate (NMDA) receptor complex and other key proteins regulating hippocampal synaptic transmission in the post-synaptic density (PSD) fraction. In addition, the functional properties of the NMDA-receptor complex were examined. One month of STZ-diabetes did not affect the NMDA receptor complex. In contrast, 4 months after induction of diabetes NR2B subunit immunoreactivity, CaMKII and Tyr-dependent phosphorylation of the NR2A/B subunits of the NMDA receptor were reduced and alphaCaMKII autophosphorylation and its association to the NMDA receptor complex were impaired in STZ-rats compared with age-matched controls. Likewise, NMDA currents in hippocampal pyramidal neurones measured by intracellular recording were reduced in STZ-rats. Insulin treatment prevented the reduction in kinase activities, NR2B expression levels, CaMKII-NMDA receptor association and NMDA currents. These findings strengthen the hypothesis that altered post-synaptic glutamatergic transmission is related to deficits in learning and plasticity in this animal model.     

alpha]-Secretase ADAM10 as well as [alpha]APPs is reduced in platelets and CSF of Alzheimer disease patients

BACKGROUND: Members of membrane-bound disintegrin metalloproteinases (ADAMs) were shown to be capable of cleaving amyloid precursor protein (APP) at the alpha-cleavage site in different cell systems. One of the candidate alpha-secretases identified in this family is ADAM10. The present study addresses the following major questions: 1) Are the levels of an alpha-secretase candidate (i.e., ADAM10) reduced in accessible cells of Alzheimer Disease (AD) patients? 2) Are ADAM10 levels in the peripheral cells of AD patients related to a concomitant decrease in alpha APPs? MATERIALS AND METHODS: Western Blot analysis of ADAM10 is performed on platelet homogenates from 33 sporadic AD patients and on 26 age-matched control subjects. Moreover, the levels of alpha-secretase metabolite (alpha APPs) are tested both in platelets and cerebrospinal fluid (CSF) of the same pool of subjects by means of Western blot with a specific antibody. RESULTS: A significant decrease of platelet ADAM10 levels is observed in patients affected by probable AD when compared to control subjects and this is paralleled by a reduced level of alpha APPs released from platelets. Moreover, in the same pool of AD patients, alpha APPs levels were reduced concomitantly in CSF. CONCLUSIONS: ADAM10 is expressed in platelets. A reduced level of ADAM10 is observed in platelets obtained from AD patients compared to age-matched controls. Further, in the same pool of AD patients, a qualitatively and quantitatively similar decrease in alpha APPs is present both in thrombin-activated platelets and CSF, thus suggesting that alterations of APP processing might occur both in the neuronal compartment and peripheral cells.