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Tacrine-melatonin hybrids were designed and synthesized as new multifunctional drug candidates for Alzheimer's disease. These compounds may simultaneously palliate intellectual deficits and protect the brain against both beta-amyloid (A beta) peptide and oxidative stress. They show improved cholinergic and antioxidant properties, and are more potent and selective inhibitors of human acetylcholinesterase (hAChE) than tacrine. They also capture free radicals better than melatonin. Molecular modeling studies show that these hybrids target both the catalytic active site (CAS) and the peripheral anionic site (PAS) of AChE. At sub-micromolar concentrations they efficiently displace the binding of propidium iodide from the PAS and could thus inhibit A beta peptide aggregation promoted by AChE. Moreover, they also inhibit A beta self-aggregation and display neuroprotective properties in a human neuroblastoma line against cell death induced by various toxic insults, such as A beta(25-35), H(2)O(2), and rotenone. Finally, they exhibit low toxicity and may be able to penetrate the central nervous system according to an in vitro parallel artificial membrane permeability assay for the blood-brain barrier (PAMPA-BBB).

Currently, the potential to interfere with the pathology of beta-amyloid targeting a well-known drugable enzyme, the acetylcholinesterase (AChE), is opened. Peripheral or dual binding site inhibitors of AChE may simultaneously alleviate the cognitive and behavioral deficits in Alzheimer's disease (AD) patients and, more importantly, act as disease-modifying agents delaying amyloid plaque formation. As part of a rational drug design program directed to find dual binding site AChE inhibitors, several families of compounds have been synthesized as potent AChE inhibitors. From these series, several drug candidates were selected based on their potent and selective inhibition of AChE (subnanomolar activity) and their interference with the beta-amyloid aggregation in vitro (IC(50) in the low micromolar range). First in vivo data confirm our initial hypothesis. Oral treatment with NP-61 for 3 months is able to reverse the cognitive impairment (Morris water maze test) and to reduce plaque load in the brains of human amyloid precursor protein transgenic mice (Swedish mutation). These results suggest that NP-61, a potent beta-amyloid modulator, is able to reverse the AD-like neurodegenerative phenotype in transgenic mice, indicating a promising disease-modifying agent for clinical application., (2008 S. Karger AG, Basel)

The therapeutic potential of acetylcholinesterase (AChE) inhibitors has been strengthened recently by evidence showing that besides their role in cognitive function, they might contribute to slow down the neurodegeneration in Alzheimer's disease (AD) patients. It is known that AChE exerts secondary noncholinergic functions, related to its peripheral anionic site, in cell adhesion and differentiation, and recent findings also support its role in mediating the processing and deposition of beta-amyloid (Abeta) peptide. AChE is one of the proteins that colocalizes with Abeta peptide deposits in the brain of AD patients and promotes Abeta fibrillogenesis by forming stable AChEA beta complexes. Additionally, it has also been postulated that AChE binds through its peripheral site to the Abeta nonamyloidogenic form and acts as a pathological chaperone inducing a conformational transition to the amyloidogenic form (Inestrosa et al., 1996; Bartolini et al., 2003). Anew series of dual binding site AChE inhibitors has been designed and synthesized as new potent AChE inhibitors, which might simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting Abeta peptide aggregation through binding to both catalytic and peripheral sites of the enzyme.

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as new potent drugs that may simultaneously alleviate cognitive deficits and behave as disease-modifying agents by inhibiting the beta-amyloid (A beta) peptide aggregation through binding to both catalytic and peripheral sites of the enzyme. Particularly, compounds 5 and 6 emerged as the most potent heterodimers reported so far, displaying IC50 values for AChE inhibition of 20 and 60 pM, respectively. More importantly, these dual AChE inhibitors inhibit the AChE-induced A beta peptide aggregation with IC50 values 1 order of magnitude lower than that of propidium, thus being the most potent derivatives with this activity reported up to date. We therefore conclude that these compounds are very promising disease-modifying agents for the treatment of Alzheimer's disease (AD).

The synthesis of tacrine-thiadiazolidinone hybrids is described. These compounds are designed as dual acetylcholinesterase inhibitors binding simultaneously to the peripheral and catalytic sites of the enzyme. All tested compounds exhibit significant AChE inhibitory activity. Competition assays using propidium as reference of selective ligand for the peripheral anionic site on acetylcholinesterase indicates the influence of the designed compounds over the peripheral site. They can be considered as new leads in the optimization of Alzheimer's disease modifying agents.

DNA polymerase mu (Pol mu) is a DNA-dependent DNA polymerase closely related to terminal deoxynucleotidyl transferase (TdT), and prone to induce template/primer misalignments and misincorporation. In addition to a proposed general role in non-homologous end joining of double-strand breaks, its mutagenic potential and preferential expression in secondary lymphoid tissues support a role in somatic hypermutation (SHM) of immunoglobulin genes. Here, we show that human Pol mu protein is expressed in the nucleus of centroblasts obtained from human tonsils, forming a characteristic foci pattern resembling that of other DNA repair proteins in response to DNA damage. Overexpression of human Pol mu in Ramos cells, in which the SHM process is constitutive, augmented the somatic mutations specifically at the variable (V) region of the immunoglobulin genes. The nature of the mutations introduced, mostly base substitutions, supports the contribution of Pol mu to mutation of G and C residues during SHM. In vitro analysis of Pol mu misincorporation on specific templates, that mimic DNA repair intermediates and correspond to mutational hotspots, indicated that many of the mutations observed in vivo can be explained by the capacity of Pol mu to induce transient template/primer misalignments.

DNA polymerase lambda (pol lambda) is a novel family X DNA polymerase that has been suggested to play a role in meiotic recombination and DNA repair. The recent demonstration of an intrinsic 5'-deoxyribose-5-phosphate lyase activity in pol lambda supports a function of this enzyme in base excision repair. However, the biochemical properties of the polymerization activity of this enzyme are still largely unknown. We have cloned and purified human pol lambda to homogeneity in a soluble and active form, and we present here a biochemical description of its polymerization features. In support of a role in DNA repair, pol lambda inserts nucleotides in a DNA template-dependent manner and is processive in small gaps containing a 5'-phosphate group. These properties, together with its nucleotide insertion fidelity parameters and lack of proofreading activity, indicate that pol lambda is a novel beta-like DNA polymerase. However, the high affinity of pol lambda for dNTPs (37-fold over pol beta) is consistent with its possible involvement in DNA transactions occurring under low cellular levels of dNTPs. This suggests that, despite their similarities, pol beta and pol lambda have nonredundant in vivo functions.

The catestatin fragment of chromogranin A is the first known endogenous compound able to inhibit catecholamine release elicited by the activation of neuronal nicotinic acetylcholine receptors (nAChRs) of different animal species and catecholaminergic cell types. However, how catestatin regulates the receptor activity, which subunit combination of the heteropentameric forms of receptor is better blocked by the peptide, or how it affects the different stages of the exocytotic process have not yet been evaluated. To address these questions, we have assayed the effects of catestatin: (first) on the inward currents elicited by ACh (I(ACh)) in voltage-clamped oocytes expressing different combinations of nAChR subunits; and (second) on the cytosolic Ca2+ concentration, [Ca2+]c, and quantal release of catecholamines simultaneously monitored in single adrenal chromaffin cells stimulated with ACh. Catestatin potently blocks all the subtypes of nAChRs studied. Furthermore, it inhibits the alpha3beta4 current in a reversible, noncompetitive, voltage-, and use-dependent manner, a behavior compatible with open-channel blockade. In fura-2-loaded single chromaffin cells, the peptide reduced the [Ca2+]c signal and the total release of catecholamines elicited by ACh; however, catestatin did not modify the kinetics or the last step of the exocytotic process. Our results suggest that catestatin might play an autocrine regulatory role in neuroendocrine secretion through its interaction with different native nAChR subtypes; the extent of receptor blockade by the peptide could be acutely regulated by the intensity and duration of the presynaptic stimulus.

This study aimed at determining the distribution and expression levels of different subtypes of Ca(2+) channels in the bovine adrenal medulla, and whether individual subtypes were more abundant in chromaffin cells exhibiting an adrenergic or a noradrenergic phenotype. In situ hybridization using riboprobes specific for the pore-forming Ca(2+) channel alpha(1D) (L-type channel), alpha(1B) (N-type channel), and alpha(1A) (P/Q-type channel) subunits of bovine chromaffin cells showed a broad distribution of the three transcripts in adrenal medulla tissue. However, a tissue-specific expression pattern of individual subunits was found; whereas alpha(1B) mRNA was homogeneously distributed throughout the medulla, alpha(1D) and alpha(1A) transcripts were present at higher densities in the internal medullary area, far away from the adrenal cortex. These results were corroborated by comparative analysis of the alpha(1B), alpha(1D), and alpha(1A) products amplified by RT-PCR from total RNA extracted from small pieces of tissue dissected out from external or internal medullary areas. Interestingly, immunohistochemical experiments performed in adrenal gland sections, using antidopamine-beta-hydroxylase and anti-phenylethanolamine-N-methyltransferase antibodies, indicated a higher density of noradrenergic over adrenergic chromaffin cells in the internal medullary region. These results provide direct evidence in favor of a heterogeneous distribution of Ca(2+) channel subtypes in the adrenal medulla, in agreement with previous functional data showing that blockade of the high K+ -elicited responses by dihydropyridines was greater in noradrenergic than in adrenergic chromaffin cells. These differences may be relevant for the differential release regulation of each catecholamine under physiological and pathophysiological conditions., (Copyright 2001 S. Karger AG, Basel)

Using reverse transcription followed by PCR amplification (RT-PCR), we have identified multiple messenger RNAs encoding for the neuronal pore-forming Ca(2+) channel subunits alpha(1A) (P/Q channel), alpha(1B) (N channel), alpha(1D) (neuronal/endocrine L channel), alpha(1E) (R channel), alpha(1G-H) (T channel) and alpha(1S) (skeletal muscle L channel) in bovine chromaffin cells. mRNAs for the auxiliary beta(2), beta(3), beta(4), alpha(2)/delta and gamma(2) subunits were also identified. In agreement with these molecular data, perforated patch-clamp recordings of whole-cell Ca(2+) currents reveal the existence of functional R-type Ca(2+) channels in these cells that were previously undetected with other techniques. Our results provide a molecular frame for a much wider functional diversity of Ca(2+) channels in chromaffin cells than that previously established using pharmacological and electrophysiological approaches.

Incubation of bovine adrenal chromaffin cells in high K+ (38 mM) during 24-48 h enhanced 2.5 to five times the expression of SNAP-25 protein and mRNA, respectively. This increase was reduced 86% by furnidipine (an L-type Ca2+ channel blocker) but was unaffected by either omega-conotoxin GVIA (an N-type Ca2+ channel blocker) or -agatoxin IVA (a P/Q-type Ca2+ channel blocker). Combined blockade of N and P/Q channels with omega-conotoxin MVIIC did, however, block by 76% the protein expression. The inhibitory effects of fumidipine were partially reversed when the external Ca2+ concentration was raised from 1.6 to 5 mM. These findings, together with the fact that nicotinic receptor activation or Ca2+ release from internal stores also enhanced SNAP-25 protein expression, suggest that an increment of cytosolic Ca2+ concentration ([Ca2+]), rather than its source or Ca2+ entry pathway, is the critical signal to induce the protein expression. The greater coupling between L-type Ca2+ channels and protein expression might be due to two facts: (a) L channels contributed 50% to the global [Ca2+]i rise induced by 38 mM K+ in indo-1-loaded chromaffin cells and (b) L channels undergo less inactivation than N or P/Q channels on sustained stimulation of these cells.

Rat alpha3beta4 or alpha7 neuronal nicotinic acetylcholine receptors (AChRs) were expressed in Xenopus laevis oocytes, and the effects of various toxins and non-toxin Ca2+ channel blockers studied. Nicotinic AChR currents were elicited by 1 s pulses of dimethylphenylpiperazinium (DMPP, 100 microM) applied at regular intervals. The N/P/Q-type Ca2+ channel blocker omega-conotoxin MVIIC inhibited alpha3beta4 currents with an IC50 of 1.3 microM; the blockade was non-competitive and reversible. The alpha7 currents were unaffected. At 1 microM, omega-conotoxin GVIA (N-type Ca2+ channel blocker) inhibited by 24 and 20% alpha3beta4 and alpha7 currents, respectively. At 1 microM, omega-agatoxin IVA (a P/Q-type Ca2+ channel blocker) did not affect alpha7 currents and inhibited alpha3beta4 currents by only 15%. L-type Ca2+ channel blockers furnidipine, verapamil and, particularly, diltiazem exhibited a preferential blocking activity on alpha3beta4 nicotinic AChRs. The mechanism of alpha3beta4 currents blockade by omega-conotoxins and diltiazem differed in the following aspects: (i) the onset and reversal of the blockade was faster for toxins; (ii) the blockade by the peptides was voltage-dependent, while that exerted by diltiazem was not; (iii) diltiazem promoted the inactivation of the current while omega-toxins did not. These data show that, at concentrations currently employed as Ca2+ channel blockers, some of these compounds also inhibit certain subtypes of nicotinic AChR currents. Our data calls for caution when interpreting many of the results obtained in neurons and other cell types, where nicotinic receptor and Ca2+ channels coexist.

Methyllycaconitine (MLA), alpha-conotoxin ImI, and alpha-bungarotoxin inhibited the release of catecholamines triggered by brief pulses of acetylcholine (ACh) (100 microM, 5 s) applied to fast-superfused bovine adrenal chromaffin cells, with IC50s of 100 nM for MLA and 300 nM for alpha-conotoxin ImI and alpha-bungarotoxin. MLA (100 nM), alpha-conotoxin ImI (1 microM), and alpha-bungarotoxin (1 microM) halved the entry of 45Ca2+ stimulated by 5-s pulses of 300 microM ACh applied to incubated cells. These supramaximal concentrations of alpha7 nicotinic receptor blockers depressed by 30% (MLA), 25% (alpha-bungarotoxin), and 50% (alpha-conotoxin ImI) the inward current generated by 1-s pulses of 100 microM ACh, applied to voltage-clamped chromaffin cells. In Xenopus oocytes expressing rat brain alpha7 neuronal nicotinic receptor for acetylcholine nAChR, the current generated by 1-s pulses of ACh was blocked by MLA, alpha-conotoxin ImI, and alpha-bungarotoxin with IC50s of 0.1 nM, 100 nM, and 1.6 nM, respectively; the current through alpha3 beta4 nAChR was unaffected by alpha-conotoxin ImI and alpha-bungarotoxin, and weakly blocked by MLA (IC50 = 1 microM). The functions of controlling the electrical activity, the entry of Ca2+, and the ensuing exocytotic response of chromaffin cells were until now exclusively attributed to alpha3 beta4 nAChR; the present results constitute the first evidence to support a prominent role of alpha7 nAChR in controlling such functions, specially under the more physiological conditions used here to stimulate chromaffin cells with brief pulses of ACh.

We have studied capacitative Ca2+ entry into Xenopus oocytes by depleting intracellular Ca2+ stores with inositol 1,4,5-trisphosphate or thapsigargin. Capacitative Ca2+ entry was evoked by hyperpolarisation and monitored via the Ca(2+)-activated Cl- current. Hyperpolarisation-evoked currents increased with extracellular [Ca2+] in the range 0.9-5 mM, and were reversibly inhibited by extracellular Mg2+ (0.1-10 mM) by up to 60%. Currents were decreased by the voltage-gated Ca2+ channel antagonists omega-conotoxin GVIA, MVIIA and MVIIC (0.3-10 microM) and the inhibition of Ca2+ entry in individual oocytes by omega-conotoxins GVIA and MVIIA was highly heterogeneous, but not additive. Flunarizine (10 microM) and the imidazoles SK&F 96365 (10 microM), miconazole (40 microM) and econazole (40 microM) partly blocked Ca2+ entry. Ca2+ entry was unaffected by calciseptine (300 nM) or alpha-bungarotoxin (1 microM). The possibility that these compounds might inhibit the Ca(2+)-activated Cl- current rather than capacitative Ca2+ entry itself was examined by recording the Cl- current activated by the increase in [Ca2+]i activated by the flash photolysis of caged Ca2+. Eicosatetraynoic acid (2-10 microM) markedly inhibited, and La3+ (1 mM but not 100 microM) potentiated the increase in Ca(2+)-activated Cl- current. In contrast, omega-conotoxins and Mg2+ had no effect on the Ca(2+)-activated Cl- current itself. These findings support the hypothesis that capacitative Ca2+ entry into Xenopus oocytes occurs through channels with a pharmacology similar to that of neuronal non-L type voltage-gated Ca2+ channels.

In strips of pig coronary arteries incubated in oxygenated Krebs-bicarbonate solution at 37 degrees C, dotarizine blocked the phasic contractions evoked by 5-HT (0.5 microM) or K+ depolarization (35 mM K+) with an IC50 of 0.22 and 3.7 microM, respectively. Flunarizine inhibited both types of contractions with IC50 values of 1.7 microM for 5-HT and 2.4 microM for K+ responses. In Xenopus oocytes injected with in vitro transcribed RNA encoding for 5-HT2A or 5-HT2C receptors, 5-HT (100 nM for 20 s) applied every 10 min caused, in both cases, a reproducible inward current through Ca2(+)-activated Cl- channels (ICl). Dotarizine inhibited the 5-HT2A response in a concentration-dependent manner, with an IC50 of 2.2 nM. In contrast, the 5-HT2C response was unaffected by 1 microM dotarizine and blocked around 62% by 10 microM of this drug. The ICl activated either by intracellular injection of inositol 1,4,5-trisphosphate (IP3) in oocytes or by direct photorelease of Ca2+ in DM-nitrophen-injected oocytes was unaffected by 10 microM dotarizine. It is concluded that dotarizine blocks 5-HT2A receptors with a high affinity; the compound is devoid of intracellular effects on any further steps of the transduction pathway (i.e., IP3 receptor). Contrary to flunarizine that blocks equally well the serotonergic and the K+ vascular responses, dotarizine exhibits 17-fold higher affinity for vascular 5-HT receptors. These findings might be relevant to an understanding of the mechanism involved in the use of dotarizine and flunarizine as prophylactic agents in migraine.