Cytotoxicity of methoctramine and methoctramine-related polyamines

Methoctramine and its analogues are polymethylene tetramines that selectively bind to a variety of receptor sites. Although these compounds are widely used as pharmacological tools for receptor characterization, the toxicological properties of these polyamine-based structures are largely unknown. We have evaluated the cytotoxic effects of methoctramine and related symmetrical analogues differing in polymethylene chain length between the inner nitrogens against a panel of cell lines. Methoctramine caused cell death only at high micromolar concentrations, whereas its pharmacological action is exerted at nanomolar level. Increasing the spacing between the inner nitrogen atoms resulted in a significative increase in cytotoxicity. In particular, an elevated cytotoxicity is associated to a methylene chain length of 12 units dividing the inner amine functions (compound 5). H9c2 cardiomyoblasts were the most sensitive cells, followed by SH-SY5Y neuroblastoma, whereas HL60 leukaemia cells were much more resistant. Methoctramine and related compounds down-regulated ornithine decarboxylase, the first enzyme of polyamine biosynthesis even at non-toxic concentration. Further, methoctramine and compound 5 caused a limited up-regulation of spermine/spermidine N-acetyltransferase, suggesting that interference in polyamine metabolism is not a primary mechanism of toxicity. Methoctramine and its analogues bound to DNA with a higher affinity than spermine, but the correlation with their toxic effect was poor. The highly toxic compound 5 killed the cells in the absence of caspase activation and caused an increase in p53 expression and ERK1/2 phosphorylation. Compound 5 was directly oxidized by cell homogenates producing hydrogen peroxide and its toxic effect was partially subdued by the inhibition of its uptake, by the NMDA ligand MK-801, and by the antioxidant N-acetylcysteine, suggesting that compound 5 can act at different cellular levels and lead to oxidative stress.     

Neuroprotective Effects of Chalcones from <Emphasis Type="Italic">Myracrodruon urundeuva</Emphasis> on 6-Hydroxydopamine-Induced Cytotoxicity in Rat Mesencephalic Cells

In the present work, we showed that a chalcone-enriched fraction (CEF) isolated from the stem bark of a Brazilian medicinal plant, Myracrodruon urundeuva, presents neuroprotective actions on 6-hydroxydopamine (6-OHDA)-induced neuronal cell death, in rat mesencephalic cells. In the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium] assay, which is an index of cell viability, CEF (1–100 μg/ml) reversed in a concentration-dependent manner the 6-OHDA-induced cell death. While cells exposed to 6-OHDA (40 μM) showed an increased concentration of thiobarbituric acid reactive substances (TBARS), the pretreatment with CEF (10–100 μg/ml) significantly decreased the 6-OHDA-induced TBARS formation, indicative of a neuroprotection against lipoperoxidation. Furthermore, the drastic increase of nitrite levels induced by 6-OHDA, indicative of nitric oxide formation and free radicals production, was prevented by CEF. Double staining with acridine orange/ethidium bromide showed that cultures exposed to 6-OHDA (40 and 200 μM) presented an increase of apoptotic and necrotic cell numbers in a concentration-dependent manner. CEF (100 μg/ml) protected cells from apoptosis and necrosis and increased number of cells presenting a normal morphology. The immunohistochemical analysis for tyrosine hydroxylase (TH) positive neurons indicated that 6-OHDA (40 and 200 μM) caused a concentration-dependent loss of TH+ and TH− neurons. CEF protected both cells types from 6-OHDA-induced cell death. All together, our results demonstrated neuroprotective effects of chalcones, which are able to reduce oxidative stress and apoptotic injury caused by 6-OHDA. Our findings suggest that chalcones could provide benefits, along with other therapies, in neurodegenerative injuries, such as Parkinson’s disease.     

Synthesis, characterization of strontium-bile acid salts and their bioactivity vs. the anti-osteoporosis drug strontium ranelate

The strontium salts Sr(cholate)₂, (Compound 1), Sr(dehydrocholate)₂, (Compound 2) and Sr₃(3-dehydrocholanoyliden-l-tartrate)₂, (Compound 3) have been prepared and characterized. The potential anti-osteoporotic activity of these compounds was tested on human primary osteoblasts (hOBs) and human primary osteoclasts (hOCs) in comparison with the bioactivity of strontium ranelate, previously registered as drug in the treatment of post-menopausal osteoporosis. Our results led to the hypothesis that the tested compounds, particularly Compound 2, may have requirements for modulating skeletal tissue regeneration or at least down regulating the loss of bone mass. In fact, all tested compounds have been shown to induce maturation in human primary osteoblasts (hOBs) and apoptosis of human primary osteoclasts (hOCs) at the same time.     

High performance liquid chromatographic enantioseparation of chiral bridged polycyclic compounds on chiralcel OD-H and chiralpak OT(+)

The HPLC enantiomeric separation of 29 racemic bridged polycyclic compounds was examined on commercially available Chiralcel OD-H and Chiralpak OT(+) columns. The separations were evaluated under normal-phase mode (hexane containing mobile phase) for Chiralcel OD-H and under normal-phase as well as under reversed-phase mode (pure MeOH, temperature 5 degrees C) for Chiralpak OT(+). Almost all compounds were resolved either on Chiralcel OD-H or on Chiralpak OT(+), in some cases on both. The use of trifluoroacetic acid (TFA), as modifier of the hexanic mobile phase, had a beneficial effect on the enantioseparation of some polar and acidic compounds on Chiralcel OD-H. The influence of small chemical structural modifications of the analytes on the enantioseparation behavior is discussed. A structure-retention relationship has been observed on both stationary phases. This chromatographic evaluation may provide some information about the chiral recognition mechanism: in the case of Chiralcel OD-H, hydrogen bonding, pi-pi and distereoselective repulsive are supposed to be the major analyte-CSP interactions. In the case of Chiralpak OT(+), a reversed-phase enantioseparation could take place through hydrophobic interactions between the aromatic moiety of the analytes and the chiral propeller structure of the CSP. The synthesis of some unknown racemic bromobenzobicyclo[2.2.1] analytes is also described.     

Amyloidogenic Potential of Transthyretin Variants: INSIGHTS FROM STRUCTURAL AND COMPUTATIONAL ANALYSES*

Human transthyretin (TTR) is an amyloidogenic protein whose mild amyloidogenicity is enhanced by many point mutations affecting considerably the amyloid disease phenotype. To ascertain whether the high amyloidogenic potential of TTR variants may be explained on the basis of the conformational change hypothesis, an aim of this work was to determine structural alterations for five amyloidogenic TTR variants crystallized under native and/or destabilizing (moderately acidic pH) conditions. While at acidic pH structural changes may be more significant because of a higher local protein flexibility, only limited alterations, possibly representing early events associated with protein destabilization, are generally induced by mutations. This study was also aimed at establishing to what extent wild-type TTR and its amyloidogenic variants are intrinsically prone to β-aggregation. We report the results of a computational analysis predicting that wild-type TTR possesses a very high intrinsic β-aggregation propensity which is on average not enhanced by amyloidogenic mutations. However, when located in β-strands, most of these mutations are predicted to destabilize the native β-structure. The analysis also shows that rat and murine TTR have a lower intrinsic β-aggregation propensity and a similar native β-structure stability compared with human TTR. This result is consistent with the lack of in vitro amyloidogenicity found for both murine and rat TTR. Collectively, the results of this study support the notion that the high amyloidogenic potential of human pathogenic TTR variants is determined by the destabilization of their native structures, rather than by a higher intrinsic β-aggregation propensity.Protein misfolding and aggregation are involved in the pathogenesis of particularly relevant human deposition diseases, known as amyloidoses. In such diseases, normally soluble proteins undergo misfolding and become insoluble, causing the extracellular deposition of fibrillar aggregates (for reviews, see Ref. 1, 2). To date, more than 40 distinct human proteins have been associated with amyloidoses. For some of such proteins, including transthyretin (TTR),⁴ lysozyme, gelsolin, ApoAI, and ApoAII, fibrinogen A α-chain and cystatin C, the amyloidogenic potential is induced, or is enhanced as in the case of TTR (see below), by specific mutations. The most frequent hereditary amyloidoses are caused by the genetic variants of human TTR (2).TTR is a homotetramer of about 55 kDa involved in the transport of thyroxine in the extracellular fluids and in the co-transport of vitamin A, by forming a macromolecular complex with retinol-binding protein, the specific plasma carrier of retinol (3–5). Its three-dimensional structure is known at high resolution (6, 7). The structure is characterized by a large predominance of β-strands, and its four monomers are arranged according to a 222 symmetry, where one of the 2-fold symmetry axes of the molecule coincides with a long channel that transverses the entire tetramer and harbors two symmetrical binding sites for the thyroid hormone thyroxine. Each monomer contains eight β-strands (A-H), arranged in a β-sandwich of two four-stranded β-sheets, with a short α-helix connecting two of the eight β-strands. In the tetramer, the four monomers are organized as a dimer of dimers. Two monomers are held together, forming a stable dimer through a net of H-bond interactions involving the two external β-strands H and F. The two dimers associate back to back and form the tetramer, by interacting mostly through hydrophobic contacts between residues of the AB and GH loops.Normal TTR possesses an inherent potential, albeit low, to generate amyloid fibrils, giving rise to Senile Systemic Amyloidosis (SSA) in ∼25% of the population aged over 80 years (8). More than 100 point mutations are described for human TTR. Most of them are involved in the hereditary amyloidoses known as familial amyloidotic polyneuropathy (FAP) or cardiomyopathy (FAC) (9). Single point mutations enhance the amyloidogenicity of TTR, so that patients show an earlier age of onset and a faster disease progression compared with SSA patients. The observation that single point mutations can drastically influence the disease phenotype is particularly relevant. In fact, the study of pathogenic TTR variants may provide clues to the mechanism of their abnormal behavior leading to amyloid formation. Although amyloidogenic proteins in general may be structurally unrelated to each other, and lead to various pathological phenotypes in humans, the amyloid fibrils originating from different proteins share the common cross-β structure, consisting in continuous β-sheets lying parallel to the longitudinal axis of the fibril, with the constituent β-strands running perpendicular to this axis. Therefore, the amyloidogenic proteins have to undergo structural alterations to be able to generate the cross-β structure, i.e. new β-pairing interactions have to be established on the way to fibril formation. However, the molecular mechanisms underlying protein misfolding and aggregation into highly ordered fibrillar structures are not clarified definitely, although significant progress is recently been made toward their elucidation (1, 10, 11).Based on the seminal observation that the rates of aggregation into amyloid fibrils in vitro correlate with simple physico-chemical amino acid features (12), several algorithms were introduced in recent years to predict, with good success, the intrinsic β-aggregation propensities of protein and peptide sequences (for a review, see Ref. 13). The intrinsic β-aggregation propensity is a measure of the tendency polypeptide chains may have to aggregate into the amyloid structure, provided that aggregation proceeds from unstructured monomers. The prediction of intrinsic propensities to β-aggregation for amyloidogenic or non-amyloidogenic variants of the same sequence was used to explain in several instances their relative ability to speed up/slow down in vitro fibrillogenesis or the enhancement/reduction of their amyloidogenic potential in vivo (14). However, a high intrinsic aggregation propensity may not result in an actual aggregation, due to the protecting role of the ordered native structure (15, 16). Therefore, the amyloidogenic potential in the TTR variants may depend further on the change of stability in the native TTR tetramer induced by mutations. In particular, it remains to be clarified to what extent human TTR possesses an intrinsic propensity to β-aggregation, and whether amyloidogenic mutations enhance such a propensity, or only destabilize the TTR tetramer, thereby facilitating the misfolding and misassembly of a protein which is in itself prone to β-aggregation.With regard to the pathway from native to misfolded TTR and to amyloid aggregation, the results of a number of in vitro studies are consistent with the rate-limiting dissociation of the TTR tetramer, followed by misfolding of TTR monomers and their downhill polymerization to generate pathological aggregates (17–25). The crystal structures of amyloidogenic TTR variants are generally well conserved (26–30). Accordingly, the functional properties of the variants, such as the ability to interact with retinol-binding protein (5), are maintained, being consistent with the fact that large conformational changes are not induced by amyloidogenic mutations, at least under native-like conditions (11). In vitro studies have shown that a moderately acidic medium (pH 4–5) facilitates TTR fibrillogenesis (17) and that the extent of fibril formation is remarkably enhanced for amyloidogenic TTR variants in comparison to wild-type TTR (31). Recently, it has been shown by x-ray analysis that an acidic pH (4.6) causes a large local conformational change in an amyloidogenic TTR variant (I84S) affecting two subunits within the tetramer, which probably destabilizes the TTR tetramer (32). In contrast, no significant structural changes for wild-type TTR at pH 4.6 and for I84S TTR at neutral pH were found, suggesting that conformational changes associated with a destabilization of the TTR native state may be induced or enhanced in amyloidogenic TTR variants by partially denaturing conditions (32). Pursuing these observations, we extend here our investigation to include other amyloidogenic TTR variants in comparison to the wild-type protein, with the aim to unravel structural alterations that are possibly associated with an enhanced amyloidogenic potential, according to the conformational change hypothesis (11). In addition, we report the results of a computational analysis of the mutational effects on both the intrinsic propensity to β-aggregation and the stability of the native β-structure. The same analysis is performed on murine and rat TTRs, whose structural organizations are very similar to that of the human protein (33, 34).     

Tetraiodobenzimidazoles are potent inhibitors of protein kinase CK2

A series of novel iodinated benzimidazoles have been prepared by iodination of respective benzimidazole with iodine and periodic acid in sulfuric acid solution. Additionally several 2-substituted- and N-1-carboxymethyl-substituted derivatives of 4,5,6,7-tetraiodobenzimidazole (TIBI) were obtained. For sake of comparison, some new 4,5,6,7-tetrabromobenzimidazoles were also synthesized. The ability of the new compounds to inhibit protein kinase CK2 has been evaluated. The results show that 4,5,6,7-tetraiodobenzimidazoles are more powerful inhibitors of CK2 than their tetrabrominated analogs. Molecular modeling supports the experimental data showing that tetraiodobenzimidazole moiety fills better the binding pocket than respective tetrabromo and tetrachlorocompounds. To note that 4,5,6,7-tetraiodobenzimidazole (TIBI) is one of the most efficient CK2 inhibitors (K_i = 23 nM) described to date.     

Poly(ADP-ribose) Catabolism Triggers AMP-dependent Mitochondrial Energy Failure

Upon massive DNA damage, hyperactivation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP)-1 causes severe depletion of intracellular NAD and ATP pools as well as mitochondrial dysfunction. Thus far, the molecular mechanisms contributing to PARP-1-dependent impairment of mitochondrial functioning have not been identified. We found that degradation of the PARP-1 product poly(ADP-ribose) through the concerted actions of poly(ADP-ribose) glycohydrolase and NUDIX (nucleoside diphosphate-X) hydrolases leads to accumulation of AMP. The latter, in turn, inhibits the ADP/ATP translocator, prompting mitochondrial energy failure. For the first time, our findings identify NUDIX hydrolases as key enzymes involved in energy derangement during PARP-1 hyperactivity. Also, these data disclose unanticipated AMP-dependent impairment of mitochondrial exchange of adenine nucleotides, which can be of relevance to organelle functioning and disease pathogenesis.     

A retrospective analysis of human cystic echinococcosis in Sardinia (Italy), an endemic Mediterranean region,from 2001 to 2005

To assess the current impact of human CE in Sardinia (Italy) and to monitor the changes over time, a survey has been carried out for the period 2001–2005 using hospital inpatient discharge reports (HDR) as information source, supplementing data wherever possible with additional information retrieved directly from medical records. The total of 726 admissions with “Echinococcosis” as primary diagnosis (annual rate of 8.9 per 100,000 inhabitants) concerned 540 CE cases with an annual mean incidence rate of 6.62 per 100,000 inhabitants. Male-to-female ratio was 1.36, suggesting a marked risk associated with traditional male occupations. Age-specific incidence showed increasing rates of clinical CE with age for both genders. The liver was found to be the most common localization, affecting 72% of patients, while pulmonary CE was more frequent in males than in females. CE risk was unevenly distributed in the island. The more pastoral areas had the highest probability of humans becoming infected, with an incidence rate of clinical cases of ~ 14.0 per 100,000 for areas with sheep/inhabitants index of > 6. Compared to the past, incidence rates appear to be decreasing both for pulmonary and hepatic localizations, while there is a reversal of the CE “urbanization” trend resulting in “ruralization”, accompanied by a greater degree of parasite ecological “isolation” and focus-points of infection risk. In spite of this decrease, the cost of hospital care alone (~4 million euros) suggests that the monetary plus non-monetary costs of CE are still very high but not fully recognised.