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The intercalation of the μ-oxo Fe(III)-phenanthroline 1:1 complex [(OH2)3(Phen)FeOFe(Phen)(OH2)3]+4inside montmorillonite yielded a nanostructured material with strong and selective entrapping ability toward thiol molecules and hydrogen sulfide. In this work, experiments and computational molecular modeling by means of quantum mechanical calculations has been applied to study the molecular structure and interactions between this complex and the interlayer of montmorillonite. This approach allowed the identification of the geometrical disposition of the complexes inside the interlayer, the characterization of the hydration and coordination water molecules, and the explanation of the physico-chemical properties of these functionalized materials. The antiferromagnetic spin configuration of the Fe(III) ions results in the most stable state. Two conformers of the complex have been considered, having the phenanthroline rings in twisted or in parallel planes, respectively, and the transition of one conformer into the other has been explored by molecular dynamics simulations. The conformer with phenanthroline rings in parallel planes is found to be the favored species for intercalation in montmorillonite. Both experimental nuclear magnetic resonance analysis and adsorption isotherms are consistent with the modeling results. Different complex amount, equal and double of the cation exchange capacity (CEC) of montmorillonite, and hydration states inside the interlayer have been investigated reproducing faithfully the experimental d(001) spacing of the montmorillonite in the different conditions. The complex molecules intercalated over the CEC of montmorillonite adopt a disposition of the phenanthroline rings perpendicular to that of the complex already introduced by cation exchange.

Genetic studies on the populations of Austropotamobius pallipes complex from Alessandria province (N-Italy), conducted on the basis of variations recorded in the sequences of mitochondrial DNA in the gene 16S RNA, have allowed to single out an overlapping zone of the distribution areas of the two taxa pallipes and italicus. In the crayfishes from a creek where both species have been ascertained to be present, the authors have applied the same molecular marker in order to investigate the distribution of the two taxa, stressing the syntopy of A. pallipes and A. italicus. Multivariate statistical techniques on 21 morphological parameters have been used in order to single out possible distinctive features of 31 crayfishes collected in this site, and of 225 crayfishes obtained from four sites in neighbouring areas. The results confirm the presence of a high morphological variability, not only at interspecific, but also at intraspecific level, which does not allow to distinguish the two species on the basis of the features measured in the present study.

Information was gathered on the distribution of white-clawed crayfish Austropotamobius pallipes complex in the watercourses of the Alessandria province (NW Italy), on the biological and ecological preferences of the species, and on the features of the biotopes in which it is found. A total of 409 sites on 361 watercourses connected to the main sub-basins of the Po River were analysed, with data gathered during the field research phase, conducted for three consecutive summers from 2002 through 2004. Thirteen percent of the sites investigated were found to currently house crayfish populations. The persistence in time of superficial water and natural morphology of the watercourse were found to be the chief requirements for the presence of crayfish. Data on land use, human density and other factors which potentially limit the survival of crayfish populations were also analysed.

High LDL-cholesterol (LDL-C) characterizes familial hypercholesterolemia (FH) and familial combined hyperlipidemia (FCH). LDL-apheresis, used in these patients to reduce LDL-C levels, has been shown to also affect HDL levels and composition. We studied LDL-apheresis effects on six FH and nine FCH subjects’ serum capacity to modulate cellular cholesterol efflux, an index of HDL functionality, and to load macrophages with cholesterol. Serum cholesterol efflux capacity (CEC) and macrophage cholesterol loading capacity (CLC) were measured before, immediately after, and two days after LDL-apheresis. The procedure reduced total cholesterol (TC), LDL-C, and apoB plasma levels (–69%, –80% and –74%, respectively), parameters only partially restored two days later. HDL-C and apoA-I plasma levels, reduced after LDL-apheresis (–27% and –16%, respectively), were restored to almost normal levels two days later. LDL-apheresis reduced serum aqueous diffusion (AD) CEC, SR-BI-CEC, and ABCA1-CEC. AD and SR-BI were fully restored whereas ABCA1-CEC remained low two days later. Sera immediately and two days after LDL-apheresis had a lower CLC than pre-LDL-apheresis sera. In conclusion, LDL-apheresis transiently reduces HDL-C levels and serum CEC, but it also reduces also serum capacity to deliver cholesterol to macrophages. Despite a potentially negative effect on HDL levels and composition, LDL-apheresis may counteract foam cells formation.

AbstractThe term ‘dyslipidemia’ includes a wide family of lipoprotein metabolic defects often related to cardiovascular diseases. Despite the complexity of lipid disorders, current guidelines indicate LDL-C as the single most important therapeutic target; however, other lipido–proteic parameters, such as the concentration of lipoprotein subfractions (e.g., small-dense LDL) or their functional capacity (e.g., HDL reverse cholesterol transport), may display direct pro- or anti-atherogenic properties. Traditional lipid profiles may therefore be inadequate in representing dyslipidemia complexity and may prove to be ineffective in estimating the overall patient’s risk in this context. We designed a research project attempting to provide a coherent framework integrating traditional lipid profiling and advanced diagnostics, to assess qualitative and functional lipoprotein features in relevant clinical conditions and to correlate them with preclinical atherosclerosis.

Alkyl-5,6-diphenylpyridazine derivatives combining several main features of ACAT inhibitors, such as a long alkyl side chain linked to a heterocycle and the o-diphenyl system, were synthesized and tested. Moreover, modeling studies on representative terms were performed. Some compounds displayed ACAT inhibition in the micromolar range, both on the enzyme isolated from rat liver microsomes and in cell-free homogenate of murine macrophages.

1Cholesterol esterification and accumulation in the arterial wall is a hallmark of atherogenesis. Several preclinical studies suggest that calcium antagonists may exert antiatherosclerotic activity by directly affecting atherogenesis in the arterial wall. We investigated the effect of the second generation dihydropyridine calcium antagonist lacidipine on cholesterol metabolism in vivoin the aortic arch of cholesterol fed rabbits, and in vitroin mouse cultured peritoneal macrophages.2Treatment of cholesterol‐fed rabbits with 1, 3 and 10 mg kg−1day−1of lacidipine for two months reduced, in a dose‐dependent manner, cholesterol esterification in the aortic arch: 24±6, 30±12, and 41±8% inhibition, respectively (P<0.001 vs HC control). Concomitantly, drug treatment reduced total cholesterol content of the vessel wall. Lacidipine 3 and 10 mg kg−1day−1reduced cholesterolaemia (∼20%); no effect was observed at the lowest dose used (1 mg kg−1day−1). These results suggest that the action of lacidipine on cholesterol esterification in the arterial wall involves, at least in part, a direct effect on cellular cholesterol metabolism. Inhibition of cholesterol esterification in the arterial wall was observed also in a reference group of animals treated with the specific ACAT inhibitor CI‐976.3To evaluate the action of lacidipine on intracellular cholesterol metabolism we performed in vitroexperiments with murine macrophages, the main cell type that accumulates cholesterol in the arterial wall. Lacidipine almost completely inhibited cholesterol esterification in cholesterol loaded macrophages in culture. The effect was observed independently of esterification stimuli and in cell free homogenates. The drug modified intracellular cholesterol distribution, doubling the free‐ to esterified sterol ratio, but did not influence the cellular rate of cholesteryl ester hydrolysis in the cell. All together these results indicate an inhibitory effect of lacidipine on cholesterol esterification catalyzed by the enzyme ACAT in murine macrophages.4We concluded that lacidipine influences cellular cholesterol metabolism. This effect may contribute to the potential antiatherosclerotic activity of this drug.

Summary The regulation of cellular LDL metabolism by hypertriglyceridemic LDL was tested before and after treatment with acipimox, a nicotinic acid derivative, in 11 type IV hyperlipidemic patients. Large, less dense LDL particles were found in plasma after acipimox treatment. HDL subfractions were only slightly modified, with an increase of dense, cholesteryl ester-enriched and triglyceride-poor HDL3 particles. The LDL (B, E) receptor activity in human skin fibroblasts of LDL isolated before and after treatment was also evaluated. Hypertriglyceridemic LDL proved rather inefficient in regulating receptor activity, with a close to 30% lower capacity than normal LDL to inhibit receptor-mediated uptake and degradation of125I-LDL. This abnormality was fully corrected after acipimox. The reported findings indicate that acipimox treatment in type IV patients can normalize the defective interaction of hypertriglyceridemic LDL with the LDL (B, E) receptor.

Summary After reviewing the literature, a personal series of 10 adult patients with cerebellar infarction diagnosed by CT scan is described. The clinical picture in young adult men is characterized by rapid onset of headache, vomiting, vertigo, ataxia and blurred vision. After this sudden onset the patients may present a stable course or a rapid or delayed onset of brain stem compression, revealed by impairment of consciousness.

Summary A case of congenital absence of the right internal carotid and of the left vertebral arteries, associated with an aneurysm on the anterior communicating artery, is reported.

Ten cases of primary empty sella with clinical findings of a secreting pituitary adenoma were studied using computed tomography (CT). Scans performed before contrast medium infusion confirmed the diagnosis of empty sella. After infusion, eight cases were found to have a nodular area of increased density within the sella turcica. In two patients, the enhancement probably corresponded to the pituitary stalk: the other six patients demonstrated CT evidence of pituitary microadenoma. These six patients had surgical confirmation of the existence of neoplasm. Tomodensitometric diagnosis of empty sella combined with microadenoma allows transsphenoidal microsurgery for complete tumor removal with preservation of the residual pituitary gland. For comparison, in a series of 21 patients suspected of having microadenomas, but without evidence of empty or enlarged sella, enhancing microadenomas were shown in 13 (62N)

Proteins conjugated with lactose residues exhibit enhanced hepatic uptake mediated by the galactose receptor. In this study, we demonstrate that lactosaminated Fab fragments (lac-Fab) of IgG can induce hepatic catabolism of specific antigens, especially low density lipoproteins (LDL). lac-Fab and human LDL-lac-Fab complex exhibited specific uptake in isolated rat hepatocytes. In vivo in the rat, lactosamination enhanced plasma clearance of Fab fragments 2-fold and hepatic localization 20-fold. Fab fragments retained their affinity after lactosamination. Hepatic uptake of rat 125I-IgG complexed in vitro with anti-rat lac-Fab was increased almost 5-fold, compared to rat 125I-IgG alone. Injection of rats with anti-LDL lac-Fab induced plasma clearance and hepatic uptake of tracer amounts of previously injected human 125I-LDL, which decreased 50% 10 min after injection of lac-Fab, with 30% present in the liver. Asialofetuin completely inhibited these processes. After a bolus of 6 mg of human LDL, administration of anti-LDL lac-Fab reduced the serum cholesterol of rats to basal values within 2.5 h. These findings suggest that lactosaminated Fab fragments of specific IgGs are effective reagents for inducing hepatic uptake of macromolecules through the galactose receptor. lac-Fab specific for LDL may be an effective hypocholesterolemic agent in vivo.

In J774 murine macrophages, chemically oxidized LDL (OxLDL) and biologically oxidized LDL (BioOxLDL) have similar metabolic fates, characterized by a relatively poor degradation when compared with acetylated LDL (AcLDL), and a modest ability to activate acyl-CoA:cholesterol acyltransferase (ACAT) (850 and 754 pmol [14C]oleate/mg cell protein in OxLDL- and BioOxLDL-incubated cells, versus 425 and 7070 pmol [14C]cholesteryl oleate/mg cell protein in control and AcLDL-incubated cells) with a massive increase of cellular free cholesterol. Therefore, OxLDL were used to investigate the cellular processing of oxidatively modified LDL. Binding and fluorescence microscopy studies demonstrated that OxLDL are effectively bound and internalized by macrophages and accumulate in organelles with density properties similar to those of endo/lysosomes. Although the overall metabolism of OxLDL is modestly affected by 100 microM chloroquine, owing to the poor cellular degradation of the substrate, the drug can further depress OxLDL degradation, indicating that this process takes place in an acidic compartment. Failure to detect products of extensive degradation of OxLDL in the medium is due to their relative resistance to enzymatic hydrolysis, as demonstrated also by in vitro experiments with partially purified lysosomal enzymes, rather than to the intracellular accumulation of degradation products (degraded intracellular protein is, at most, 8.5% of total). This sluggish degradation process is not due to a cytotoxic effect since OxLDL do not affect the intracellular processing of other ligands like AcLDL or IgG. The accumulation of OxLDL-derived products within macrophages may elicit cellular responses, the relevance of which in the atherosclerotic process remains to be addressed.

Summary A new case of an unusual anomalous anastomotic vessel between the carotid syphon and the pericallosal artery is reported. The description of their most characteristic angiographic and anatomical features is completed with a survey of analogous anomalies found in the literature. The incidence of associated other congenital vascular anomalies and berry aneurysms is high. An explanation of the embryonic development of this vessel is hypothetical. Its angiographic recognition may be useful.

Summary The authors describe two cases in which computerized tomography (CT) revealed the presence of cervical intramedullary metastases in the form of increased density areas.

The classical taxon, Steganacarus (Steganacarus) magnus (Nicolet, 1855) is redescribed on the basis of topotypic material. The study and the comparison of many European and North African populations reveals a variation in the size of the anterior notogastral tectum and consequently enables another classic entity, S. (Steganacarus) anomalus (Berlese, 1883), to be put into synonymy. These two different ecophenotypes, in the authors' opinion, seem to be linked to the presence of xeric low-altitude southern environments (S. (S.) magnus f. anomala) or cool higher-altitude mid-northern environments (S. (S.) magnus f. magna).

SIM 6080 is a new calcium antagonist, structurally related to diphenylalkylamines, which combines transmembrane and intracellular calcium antagonist activities. In the present study we investigated the effect of SIM 6080 on atherogenesis in cholesterol-fed rabbits. Subcutaneous administration of the compound at 0.33, 1, and 3 mg kg^-1/bid for 60 days neither affected plasma lipids nor blood pressure. However at 1 and 3 mg kg^-1/bid SIM 6080 reduced in a dosedependent manner both the area of the aorta covered by plaques and aortic cholesterol content. Determination of SIM 6080 plasma and aortic content indicated that the compound could concentrate up to 10 times in the arterial tissue. In vitro studies demonstrated that at concentrations similar to those observed in the aorta this compound may stimulate rabbit βVLDL catabolism by smooth muscle cells in an homologous system suggesting that the up-regulation of LDL-receptors in the aorta may contribute to the antiatherosclerotic properties of SIM 6080. Copyright 1993, 1999 Academic Press

Atherosclerosis results from multiple factors and involves several mechanisms, including endothelial monocyte and smooth muscle cell (SMC) changes, cholesterol accumulation, plaque rupture and thromboembolism. Calcium ions play a role in the initial and chronic development of atherosclerotic lesions. Several studies in experimental animal models have demonstrated the potential direct antiatherosclerotic effects of calcium antagonists. In this study the antiatherogenic activity of lercanidipine, a new lipophilic, second-generation calcium antagonist, was investigated. Lercanidipine and its enantiomers inhibited the replication and migration of arterial myocytes in concentrations ranging from 10 to 50 µM. The antiproliferative effect of lercanidipine was dose dependent, with a potency similar to that of lacidipine and nifedipine, and was unrelated to the stereoselectivity of enantiomers to bind L-type calcium channels. Lercanidipine and its enantiomers (25 µM) decreased the serum-induced elevation of [Ca2+]i in SMC, with the (S)-enantiomer (69% inhibition) being 2.4-fold more active than the (R)- counterpart (29% inhibition). The studies performed with enantiomers of lercanidipine suggest that the observed effects are not related to the blockade of voltage-dependent Ca2+channels and confirm, at least in vitro, the pharmacological potential of the compound to influence negatively the process of atherogenesis.

The first European specimens belonging to the Eohypochthonius genus are reported and described. Some diagnostically important characteristics, such as genital plate morphology and body microsculpture, are considered and clarified by SEM analysis. In addition some suggestions are given to explain the presence of this genus in Italy.

The liver haemopoietic activity of three species of Urodeles (Triturus carnifex, Triturus alpestris and Speleomantes ambrosii) was examined by morphocytochemical approaches (light and electron microscopy, anti-BrdU immunocytochemistry, flow cytometry). The proliferation of haemopoietic cells, detected by the anti-BrdU labelling index, was accompanied by absence of mitotic cell division and the appearance of cells showing features of amitosis (e.g. nuclear constrictions with bundles of electron-dense chromatin) sometime positive to the anti-BrdU immuno-gold reaction. The possible unbalanced segregation of chromatin during the direct division of the nucleus was detected by flow cytometric measurement in terms of heterogeneous relative DNA content in peripheral blood cells. The presence in the bloodstream samples of cells (erythrocytes) with replicating DNA, nuclear constrictions and binucleations is also consistent with a situation of direct nuclear division.

Artificial hyperventilation was used during fractional encephalography. In 9 patients who underwent conventional fractional encephalography, the hyperventilation was started at the end of the investigation. In the remaining cases, in which fractional encephalography by the conventional technique gave unsatisfactory results, the examination was repeated 5 days later after mechanical hyperventilation for 30 min under intermittent positive pressure. In 3 patients with expanding processes of the posterior cranial fossa, hyperventilation encephalography confirmed the failure of passage of air into the ventricular system, and in 12 patients either visualisation or improved filling of the ventricular system and subarachnoid spaces was obtained. The great incidence of good filling of both the ventricular system and the subarachnoid spaces reduces the number of patients requiring ventriculography. Fractional encephalography following hyperventilation was well tolerated in all the patients.

The authors have made a full anatomical description and roentgenological study of the internal auditory or labyrinthine artery. The internal auditory artery seldom arises from the basilar trunk and reaches the inner ear without giving off collateral branches to the cerebellum; more often it originates as a branch of the cerebello-labyrinthine artery near to the internal auditory meatus. The first segment of the internal auditory artery goes through the pontocerebellar cistern (precanalicular or cisternal segment) ending with a slight angle close to the internal auditory meatus; at this level the intracanalicular segment arises and follows the inferior wall of the internal auditory canal with a fluctuating path.

Calcium antagonists (CA) exert an anti-atherosclerotic effect in cholesterol-fed rabbits through reduction of cholesterol accumulation in the arterial wall. Further studies in our Institute indicate that verapamil-like compounds and diltiazem stimulate receptor-mediated LDL uptake by human fibroblasts in culture, while nifedipine-like compounds and flunarizine are inactive. Verapamil and diltiazem stimulated LDL-receptor activity also in cells from a heterozygous FH patient, while they were inactive in a receptor defective homozygous FH patient. A basic group needs to be present on the CA molecule to modulate the LDL receptor expression. Preliminary data in our laboratory suggest that some CA can achieve concentrations in the aortic wall likely to exert effects on LDL receptors. This stimulatory activity may improve lipid metabolism in the arterial wall.

Cultured human umbilical vein endothelial cells (EC) exposed to native and acetylated low density lipoproteins (LDL and acetyl-LDL) show an increased synthesis of PAI-1. Confluent EC monolayers were incubated for 16–18 hours in medium 199 with or without different concentrations of LDL and acetyl-LDL and PAI-1 antigen levels were measured in conditioned medium. LDL and acetyl-LDL increased the release of PAI-1 by EC in a concentration-dependent manner. The effect was specific for PAI-1 because tissue type plasminogen activator (t-PA) and expression of procoagulant activity were not affected by either lipoprotein. The observation that native and acetyl-LDL, which are known to interact with different receptors on EC, exert the same stimulatory effect on PAI-1 release rules out the possibility of an involvement of the LDL receptor in mediating this effect. Experiments carried out incubating native LDL in the presence of a monoclonal antibody against LDL receptor and using binding-defective LDL with a reduced affinity for the LDL receptor (~50% with respect to normal LDL) further excluded an involvement of the classical LDL receptor in mediating the effect of the lipoproteins on PAI-1 synthesis by EC.