Norepinephrine and dopamine: assay by mass fragmentography in the picomole range.

Gas chromatography-mass spectrometry makes possible the simultaneous measurement of norepinephrine and dopamine in concentrations of 0.1-milligram tissue samples. Specificity of the assay is confirmed both by the retention time of the compound and by the mass to charge ratio of the fragments recorded. The sensitivity is of the order of 0.5 picomole, and linearity of the response is maintained up to at least 200 picomoles.

Gas chromatographic-mass spectrometric assay of four indole alkylamines of rat pineal.

Gas chromatography-mass spectrometry was used to quantitate serotonin, N-acetylserotonin, 5-methoxytryptamine, and melatonin in single rat pineal glands. After gas chromatographic separation, the ion density of specific fragments of each indole was measured with mass spectrometry. Sensitivity of this indole assay is of the order of 10(-12) to 10(-13) mole. Routinely, specificity is based on gas chromatographic retention time and the recording of the ion density generated by specific fragments. Absolute identification of the extracted indoles was based on multiple ion detection.

Trophic actions of extracellular nucleotides and nucleosides on glial and neuronal cells.

In addition to their well-established roles as neurotransmitters and neuromodulators, growing evidence suggests that nucleotides and nucleosides might also act as trophic factors in both the central and peripheral nervous systems. Specific extracellular receptor subtypes for these compounds are expressed on neurons, glial and endothelial cells, where they mediate strikingly different effects. These range from induction of cell differentiation and apoptosis, mitogenesis and morphogenetic changes, to stimulation of synthesis or release, or both, of cytokines and neurotrophic factors, both under physiological and pathological conditions. Nucleotides and nucleosides might be involved in the regulation of development and plasticity of the nervous system, and in the pathophysiology of neurodegenerative disorders. Receptors for nucleotides and nucleosides could represent a novel target for the development of therapeutic strategies to treat incurable diseases of the nervous system, including trauma- and ischemia-associated neurodegeneration, demyelinating and aging-associated cognitive disorders.

Hippocampal synaptic plasticity involves competition between Ca2+/calmodulin-dependent protein kinase II and postsynaptic density 95 for binding to the NR2A subunit of the NMDA receptor.

NMDA receptor, Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII), and postsynaptic density 95 (PSD-95) are three major components of the PSD fraction. Both alphaCaMKII and PSD-95 have been shown previously to bind NR2 subunits of the NMDA receptor complex. The nature and mechanisms of targeting to the NMDA receptor subunits are, however, not completely understood. Here we report that the C-terminal NR2A(S1389-V1464) sequence was sufficient to guarantee the association of both native and recombinant alphaCaMKII and PSD-95. PSD-95(54-256) was able to compete with the binding of both native and recombinant alphaCaMKII to the NR2A C-tail. Accordingly, alphaCaMKII(1-325) competes with both the native PSD-95 and the native kinase itself for the binding to NR2A. In addition, Ser/Ala1289 and Ser/Asp1289 point mutations on the unique CaMKII phosphosite of NR2A did not significantly influence the binding of native alphaCaMKII and PSD-95 to the NR2A C-tail. Finally, the association-dissociation of alphaCaMKII and PSD-95 to and from the NR2A C-tail was significantly modulated by activation of NMDA receptor achieved by either pharmacological tools or long-term potentiation induction, underlining the importance of dynamic and reciprocal interactions of NMDA receptor, alphaCaMKII, and PSD-95 in hippocampal synaptic plasticity.

Evidence for separate mechanisms of cytotoxicity in mammalian cells treated with hydrogen peroxide in the absence or presence of L-histidine.

Treating Chinese hamster ovary cells with 1 mM L-histidine markedly increases their susceptibility to killing by H2O2. The mechanism of this effect has not been firmly established, although previous studies have shown that L-histidine in combination with H2O2, in contrast to H2O2 alone, generates DNA double-strand breaks (DSBs), albeit following supralethal concentrations of the oxidant. Using the highly sensitive pulsed field gel electrophoresis technique, we examined the ability of H2O2-L-histidine combinations to induce DSBs in cells over the same oxidant concentration range that causes cytotoxicity. Thus the correlation between DSB induction and cell killing could be investigated directly without the necessity for extrapolating effects across different concentration ranges. We used a number of treatment protocols that allowed the compartmentation of L-histidine inside or outside the cells, or both. Increased cytotoxicity was invariably associated with the appearance of DSBs, and both parameters were dependent on the intracellular fraction of the amino acid. A linear relationship was found between cytotoxicity and DSB formation when the cells were either treated with H2O2 (at > or = 20 microM) and L-histidine concurrently or were exposed to the oxidant following pre-loading with L-histidine. On the other hand, no DSBs were detected in cells treated with: (a) H2O2 alone; (b) L-histidine plus H2O2 at < or = 20 microM; or (c) H2O2 in association with both L-histidine and excess (20 mM) L-glutamine (which prevents L-histidine uptake). Thus, separate mechanisms appear to underlie the cytotoxic response in cells treated with H2O2 in the absence and presence of L-histidine, with the latter process being associated with the induction of DSBs and having a threshold at approximately 20 microM H2O2. The linear correlation between DSBs and cell killing observed in cells treated with H2O2-L-histidine at H2O2 concentrations > or = 20 microM was similar to (but not superimposable on) the correlation curve established for gamma-irradiated cells; DSBs produced by gamma-rays were associated with more cell killing than those generated by the H2O2-L-histidine combination.

Hydrogen peroxide insult in cultured mammalian cells: relationships between DNA single-strand breakage, poly(ADP-ribose) metabolism and cell killing.

We examined the effect of exposure to H2O2 at 37 degrees C on Chinese hamster ovary cell survival, DNA single-strand break (SSB) induction and rejoining, and activation of poly(ADP-ribose) (ADPR) polymerase. The effect of the ADPR polymerase inhibitor 3-aminobenzamide on each of these processes was also determined. SSB induction increased progressively with increasing H2O2 concentration. SSB levels were maximal after approx. 5 min of exposure to H2O2 (100 microM) and then decreased at longer times. This decrease, which paralleled the time-dependent depletion of H2O2, was due to the rejoining of SSBs. 3-Aminobenzamide enhanced the level of SSBs at each time point. H2O2 increased the level of both ADPR synthesis and NAD+ depletion (both measures of ADPR polymerase activity) in a concentration-dependent fashion, with the maximum effect being reached after approx. 20 min. After 100 microM H2O2, the effects on both ADPR and NAD+ were reversible. 3-Aminobenzamide completely blocked the effects of the oxidant on both NAD+ and ADPR levels. Thus, SSB induction by H2O2 at 37 degrees C was accompanied by a marked but reversible stimulation of ADPR polymerase. However, cell killing by H2O2 was only slightly enhanced in the presence of 3-aminobenzamide (5 mM), so the above-mentioned effects do not appear to be relevant to the cytotoxic effect of H2O2 under these conditions. Comparing these results with data obtained previously for cells treated with H2O2 at 4 degrees C suggests that the mechanisms of DNA strand breakage and cell killing may be quite different at the two temperatures, and that DNA damage at 37 degrees C may be indirectly mediated by temperature-dependent metabolic events.

Prenatal methylazoxymethanol treatment in rats produces brain abnormalities with morphological similarities to human developmental brain dysgeneses.

A double methylazoxymethanol (MAM) intraperitoneal injection was prenatally administered to pregnant rats at gestational day 15 to induce developmental brain dysgeneses. Thirty adult rats from 8 different progenies were investigated with a combined electrophysiological and neuroanatomical analysis. The offspring of treated dams was characterized by extensive cortical layering abnormalities, subpial bands of heterotopic neurons in layer I, and subcortical nodules of heterotopic neurons extending from the periventricular region to the hippocampus and neocortex. The phenotype of cell subpopulations within the heterotopic structures was analyzed by means of antibodies raised against glial and neuronal markers, calcium binding proteins, GABA, and AMPA glutamate receptors. Neurons within the subcortical heterotopic nodules were characterized by abnormal firing properties, with sustained repetitive bursts of action potentials. The subcortical nodules were surrounded by cell clusters with ultrastructural features of young migrating neurons. The immunocytochemical data suggested, moreover, that the subcortical heterotopia were formed by neurons originally committed to the neocortex and characterized by morphological features similar to those found in human periventricular nodular heterotopia. The present study demonstrates that double MAM treatment at gestational day 15 induces in rats developmental brain abnormalities whose anatomical and physiological features bear resemblance to those observed in human brain dysgeneses associated with intractable epilepsy. Therefore, MAM treated rats could be considered as useful tools in investigating the pathogenic mechanisms involved in human developmental brain dysgeneses.

Levels of NGF, p75NGFR and ChAT immunoreactivity in brain of adult and aged microencephalic rats.

Methylazoxymethanol (MAM)-induced microencephalic aged animals with reduced cortical mass and unmodified basal nucleus were used to study the relationship between cells that produce and cells that utilize NGF. Total cortical ChAT activity of MAM 2, 19 and 27 month old animals was reduced compared to their age-matched controls. To verify whether the reduction of enzyme activity can be ascribed to changes in or ablation of projecting neurons, we carried out immunohistochemical analysis of ChAT and low affinity NGF receptor (p75NGFR) in the basal nucleus of control and MAM-treated animals. ChAT and p75NGFR immunostaining of basal forebrain cholinergic neurons showed morphological changes in MAM animals, as revealed by cellular atrophy, reduced dendritic arborization and decreased staining intensity. In the cerebral cortex of microencephalic animals, reduced levels of NGF compared to controls were observed at all examined ages. These results suggest that MAM treatment induces long-lasting ablation of cortical NGF-synthesizing cells leading to reduced trophic support to basal forebrain cholinergic neurons, which might be responsible for the cellular atrophy observed in the basal nucleus.

Direct excision of 50 kb pair DNA fragments from megabase-sized fragments produced during apoptotic cleavage of genomic DNA.

The DNA of U937 cells exposed to two different apoptotic stimuli, namely the cocktail H2O2/3-aminobenzamide (3AB) and etoposide, was analyzed using field inversion gel electrophoresis (FIGE) as well as programmable, autonomously controlled electrode electrophoresis (PACE). The results obtained indicate that FIGE is not appropriate for sizing apoptotic DNA fragments. PACE appears to be more accurate and reliable and the results obtained with this technique strongly suggest that the 50 kb DNA fragments are directly excised from Mb-sized DNA fragments without the intermediate cleavage of 200-300 kb products.

The L-histidine-mediated enhancement of hydrogen peroxide-induced cytotoxicity is a general response in cultured mammalian cell lines and is always associated with the formation of DNA double strand breaks.

Micromolar concentrations of L-histidine increase the cytotoxicity of hydrogen peroxide in a number of cell lines including CHO (hamster), EAHY, McCoy's, U937 and CCRF-CEM (human), Vero (monkey) and SC-1 (mouse). Importantly, these cell lines displayed different degrees of sensitivity to H2O2 alone and the extent of enhancement elicited by the amino acid was more pronounced in resistant cell lines. The increased cytotoxicity was invariably associated with the formation of DNA DSBs and a remarkable correlation was found by plotting the level of DNA DSBs against the cytotoxic response. These results strongly support the hypothesis that the mechanism whereby L-histidine increases the toxicity elicited by H2O2 involves the formation of DNA DSBs and are consistent with the possibility that the amino acid might participate in the regulation of the physio-pathological response to oxidative stress in mammals.

Chilling followed by incubation at 37 degrees C causes a reduction in NAD+ levels which can be prevented by the poly(ADP-ribose)transferase inhibitor 3-aminobenzamide.

The exposure of cells for 60 min to a serum free medium at ice temperature followed by a return to normal culture conditions (30 min at 37 degrees C) caused a dramatic decrease in NAD+ levels. This decrease in NAD+ was prevented by 3-aminobenzamide. Alkaline elution analysis of DNA from cultures that were sisters to the ones utilized for measuring cellular NAD+ content revealed an absence of DNA breakage. These data suggest that poly(ADP-ribose)transferase may be induced in conditions not involving DNA fragmentation. The induction of this enzyme could therefore represent a cellular emergency reaction and not just a response to DNA damage.

Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells.

A 3 h exposure to 1 mM H2O2 followed by 6 h post-challenge growth in peroxide-free medium induces necrosis in U937 cells. Addition of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide during recovery prevents necrosis and triggers apoptosis, as shown by the appearance of apoptotic bodies, extensive blebbing and formation of multimeric DNA fragments as well as 50 kb double stranded DNA fragments. Thus, the same initial damage can be a triggering event for both apoptotic and necrotic cell death. Furthermore, necrosis does not appear to be a passive response to overwhelming damage.

Measurement of relative amounts of phospho- and dephospho-B-50(GAP-43) peptides by fast atom bombardment-mass spectrometry.

The biological role of phosphoproteins depends upon their degree of phosphorylation in vivo. Methods currently available to measure the degree of phosphorylation of a protein involve indirect procedures to detect the 32P-phosphate incorporation. We report here a direct method to measure relative amounts of phospho- and dephospho-forms of peptides based upon a mass spectrometric technique. The intensities of the molecular ions corresponding to the two forms of the peptides are proportional to their relative amounts. This is demonstrated for a peptide fragment of the protein B-50(GAP-43) and for kemptide, respectively substrates for protein kinases C and A, and demonstrates the applicability of fast atom bombardment-mass spectrometry to quantitate peptides bearing post-translational modifications.

Differential translocation of protein kinase C isozymes in rats characterized by a chronic lack of LTP induction and cognitive impairment.

The translocation of protein kinase C isozymes was investigated in an animal model of cognitive deficit and lack of induction of long-term potentiation (LTP). In MAM rats, presynaptic alpha, beta, epsilon PKC showed enhanced translocation, while postsynaptic gamma PKC displayed decreased translocation when compared to control levels. This imbalance of PKC isozyme translocation between the pre- and post-synaptic compartment might therefore represent a possible molecular cause for the lack of synaptic plasticity observed in these animals.

AlphaCaMKII binding to the C-terminal tail of NMDA receptor subunit NR2A and its modulation by autophosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), a multifunctional, widely distributed enzyme, is enriched in post-synaptic densities (PSDs). Here, we demonstrate that CaMKII binds to a discrete C-terminal region of the NR2A subunit of NMDA receptors and promotes the phosphorylation of a Ser residue of this NMDA receptor subunit. Glutathione S-transferase (GST)-NR2A(1349-1464) binds native CaMKII from solubilised hippocampal PSDs in 'pull-out' and overlay experiments and this binding is competed by recombinant alphaCaMKII(1-315). The longer GST-NR2A(1244-1464), although containing the CaMKII phosphosite Ser-1289, binds the kinase with a lower efficacy. CaMKII association to NR2A(1349-1464) is positively modulated by kinase autophosphorylation in the presence of Ca2+/calmodulin. These data provide direct evidence for a mechanism modulating the synaptic strength.

Determination of the endogenous phosphorylation state of B-50/GAP-43 and neurogranin in different brain regions by electrospray mass spectrometry.

Electrospray mass spectrometry coupled to liquid chromatography was utilized to measure two PKC neuronal substrates, B-50/GAP-43 and neurogranin, in single rat brain areas. Aliquots of perchloric acid extracts were directly injected and mass spectra recorded. At elution times of 14.2 and 27.0 min two molecular species of MW 7450 and 23 602 Da were observed. These values are in excellent agreement for the expected MW for rat neurogranin and B-50/GAP-43. The presence of molecular species shifted by 80 mass units in both cases indicates that these proteins are present in phosphorylated forms in cortical and hippocampal extracts.

Abnormal pattern of platelet APP isoforms in Alzheimer disease and Down syndrome.

OBJECTIVE: To determine if changes in levels of amyloid precursor protein (APP) isoforms in periphery are associated with Alzheimer disease and Down syndrome. DESIGN: After subjects were grouped according to diagnosis, APP isoform levels in platelets were compared. SETTING: University medical center. SUBJECTS: Ten patients who fulfilled diagnostic criteria for probable Alzheimer disease, 22 healthy volunteers, and 7 elderly (mean age, 42.7 years) and 7 young (mean age, 19.0 years) patients with Down syndrome. MAIN OUTCOME MEASURES: The levels of APP isoforms were evaluated by means of Western blot analysis and immunostaining of whole platelets. RESULTS: The ratio between the 130- and the 106- to 110-kd APP isoforms was markedly lower in patients with Alzheimer disease and in elderly patients with Down syndrome than in control subjects. In young patients with Down syndrome, the ratio did not significantly differ from that in control subjects. CONCLUSIONS: A consistent alteration in platelet APP isoforms has been found in Alzheimer disease and Down syndrome. Further studies will determine whether this alteration could provide a peripheral biochemical marker of the disorder and whether it could intervene in the pathogenesis of Alzheimer disease.

Amyloid precursor protein in platelets of patients with Alzheimer disease: effect of acetylcholinesterase inhibitor treatment.

BACKGROUND: Amyloid precursor protein (APP) forms with apparent molecular weights of 130, 110, and 106 kd are present in human platelets. It has been demonstrated that Alzheimer disease (AD) is specifically associated with a decreased APP forms ratio in platelets. OBJECTIVE: To investigate whether acetylcholinesterase (AChE) inhibitor treatment modifies the ratio of platelet APP forms in patients with AD. PATIENTS AND METHODS: From a large sample of patients with probable AD, 30 with mild to moderate AD were selected. Each patient underwent a clinical evaluation including the Mini-Mental State Examination (MMSE) and platelet APP forms analysis at baseline and after 30 days. During this interval, 20 of 30 patients with AD were treated with donepezil hydrochloride (5 mg/d), a piperidine phosphate-based cholinesterase inhibitor. Platelets were subjected to Western blot analysis using monoclonal antibody (22C11). The ratio between the immunoreactivity of the higher-molecular-weight APP form (130 kd) and the lower forms (106 and 110 kd) was measured. RESULTS: All patients taking donepezil completed the 30 days of treatment without adverse effects. The platelet APP forms ratio at baseline did not differ between the 2 AD groups (mean +/- SD optical density ratio: untreated AD, 0.47 +/- 0.12; treated AD, 0.38 +/- 0.18), whereas a significant difference was found at follow-up (mean +/- SD optical density ratio: untreated AD, 0.45 +/- 0.17; treated AD, 0.77 +/- 0.29; P<.001). A significant improvement in MMSE scores in treated AD patients was observed from baseline (16.9 +/- 3.8) to 30 days (18.9 +/- 4.42) (P<.009, 30 days vs baseline), but no significant correlation was found in treated AD patients between MMSE score improvement and APP forms/ratio increase (P =.09). CONCLUSIONS: Administration of AChE inhibitors increases the ratio of APP forms in platelets of patients with AD, suggesting a potential effect of AChE inhibitors on APP trafficking or processing in a peripheral cell.

Differential level of platelet amyloid beta precursor protein isoforms: an early marker for Alzheimer disease.

OBJECTIVE: To determine whether a differential level of platelet amyloid beta precursor protein (APP) isoforms is specifically related to Alzheimer disease (AD) and whether it shows a correlation with the progression of clinical symptoms. DESIGN: After subjects were grouped according to diagnosis and severity of dementia, APP isoform levels in platelets were compared. SETTING: University medical centers. PATIENTS: Thirty-two patients who fulfilled diagnostic criteria for probable AD, 25 age-matched control subjects, and 16 patients with non-AD dementia. MAIN OUTCOME MEASURE: The levels of APP isoforms were evaluated by means of Western blot analysis and immunostaining of whole platelets. Messenger RNAs for APP transcripts were also evaluated by means of reverse transcriptase polymerase chain reaction. RESULTS: The ratio between the intensity of the 130-kd and 106- to 110-kd APP isoforms was significantly lower in the AD group (0.31 +/- 0.15, mean +/- SD) compared with both controls (0.84 +/- 0.2) and non-AD subjects (0.97 +/- 0.4). The ratio of platelet APP isoforms in patients with AD grouped by Clinical Diagnostic Rating score significantly correlated with the severity of the disease (Pearson correlation coefficient, followed by Bonferroni correction, P = .01). Reverse transcriptase polymerase chain reaction experiments showed that APP transcripts in all experimental groups were equally expressed. CONCLUSIONS: The pattern of platelet APP isoforms is specifically altered in patients with AD. In addition, the alteration of platelet APP isoforms shows a positive correlation with the progression of clinical symptoms, supporting the possibility to consider this peripheral parameter as a marker of progression of the disease. These alterations are not related to abnormalities of APP isoforms messenger RNAs in platelets.

Platelet amyloid precursor protein forms in AD: a peripheral diagnostic tool and a pharmacological target.

Alzheimer Disease (AD) is characterized by the progressive deposition of beta-amyloid in the parenchyma and cerebral microvasculature. The beta-amyloid peptide derives from the metabolism of a larger precursor, Amyloid Precursor Protein (APP). This protein is present in central nervous system, but it is also expressed in peripheral tissues such as circulating cells. An alteration of the APP forms pattern in platelets has been recently reported in AD patients when compared to platelets both of control subjects or non AD patients (NADD). The accuracy of the assay to identify AD is high and decreased levels are found throughout the course of AD with a significant association with severity of symptoms. Moreover, a recent study has demonstrated that AD patients on donepezil (5 mg daily) for 4 weeks displayed two-fold increase in their APPr baseline levels up to normal range. Thus, platelet APP ratio (APPr) holds the potential to be a clinical marker, which might be of helpful and adjunctive value in the diagnosis of AD and in tracking the course of illness, also in the early stages when pharmacological treatment has the greatest potential of being effective.