Search

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an arteriopathy related to a genetic defect of the notch 3 gene on chromosome 19. The purpose of this study was to evaluate lesion distribution and volume using MR imaging and to correlate the lesion volume with the neurologic and neuropsychological findings.Twenty members of two families (14 with CADASIL as determined by linkage analysis, six healthy) were studied with MR imaging. Two observers evaluated the MR findings semiquantitatively and quantitatively. MR results were then correlated with neurologic and neuropsychological findings.A typical pattern of lesion distribution in patients with CADASIL was found: the frontal lobe was the site with the highest lesion load, followed by the temporal lobe and the insula. The total lesion volume on T1-weighted MR images correlated significantly with the degree of disability and the degree of impairment in neuropsychological functions (including attention, memory, and conceptual and visuospatial functions).In CADASIL patients, a common pattern of cerebral lesion distribution is found. The total T1 lesion volume is an important parameter to correlate with disability, as it may prove to be helpful in predicting the natural history of the disease.

We have synthesized by solution methods and characterized the lipopeptaibol metabolite LP237-F8 extracted from the fungus Tolypocladium geodes and five selected analogues with the Etn --> Aib or Etn --> Nva replacement at position 8 and/or a triple Gln --> Glu(OMe) replacement at positions 516, and 9 (Etn = Calpha-ethylnorvaline, Aib = alpha-aminoisobutyric acid, Nva = norvaline). Conformation analysis, performed by FT-IR absorption, NMR, and CD techniques, strongly supports the view that the six terminally blocked decapeptides are highly helical in solution. Helix topology and amphiphilic character are responsible for their remarkable membrane activity. At position 8 the combination of high hydrophobicity and Ca tetrasubstitution, as in the Etn-containing LP237-F8 metabolite, has a positive effect on membrane interaction.

Tributyltin (TBT), widely used as an antifouling biocide, is the most abundant pesticide in coastal environments. One of its main toxic effects is immunosuppression in both vertebrates and invertebrates. At sublethal doses of TBT, phagocytes lose their ability to move towards and ingest foreign particles. For short-term cultures of haemocytes (60 min at 25 ° C) of a marine invertebrate, the colonial ascidian Botryllus schlosseri, exposed to 10-5 M TBT, we previously reported dose- and time-dependent impairment of yeast phagocytosis and changes in cell morphology related to cytoskeleton disorganization. These effects are Ca2+-dependent, since inactivation of Ca2+-adenosine triphosphatase and a sustained increase in cytosolic Ca2+ occurred. As TBT can antagonize the effect of chlorpromazine, a specific calmodulin (CaM) inhibitor, and the co- presence of exogenous CaM and TBT in the incubation medium resulted in the absence of effects, we hypothesized an interaction between TBT and CaM. TBT may remove endogenous CaM from cell proteins, thus inactivating them and causing alteration of Ca2+ homeostasis. With the aim of confirming the hypothesis of a direct TBT-CaM interaction, we first studied the effects of co-incubation of TBT with other exogenous proteins on restoring the ability of phagocyte morphology. Although bovine serum albumin was never able to restore cell morphology, the effect of human spectrin was similar to that described for CaM, suggesting a common non-specific mechanism of action based on the interaction of TBT with exposed hydrophobic pouches. We also analysed the conformational changes of pure CaM (10-5 M) in the presence of various concentrations (10-4 to 10-3 M) of TBT and its degradation products, dibutyltin (DBT) and monobutyltin (MBT), by circular dichroism. Results indicate the dose- and time-dependent interaction of TBT with CaM. This interaction is a non-covalent interaction, probably hydrophobic in nature, between the aliphatic chains of TBT and the hydrophobic regions of Ca2+-activated CaM. DBT and MBT turned out to be less active in inducing CaM conformational changes, without any significant differences between the two compounds.

Bombolitins are five natural heptadecapeptides originally isolated from the venom of a bumblebee. They induce lysis of erythrocytes and liposomes and increase the activity of phospholipase A2 (PLA2), that plays an important role in the early steps of the inflammatory process. It has been proposed that PLA2 activation depends on the alteration of the physical state of the membrane. Bombolitin II folds into an alpha-helix in a membrane mimicking environment constituted by SDS micelles [Battistutta et al., Macromol. Chem. Phys., 196 (1995) 2827-2841]. In the present work, the topological orientation of the peptide relative to the micelle was determined, using three spin probes localized in different positions of the water/micelle system. The reduction in intensity of the 1H NMR signals clearly demonstrated that the peptide is located on the surface of the micelle, with its helical axis parallel to the interface. Only a small portion of the helix is exposed to the aqueous environment. Results from NMR experiments were confirmed by molecular dynamics simulations, performed using a two-phase water/decane simulation cell. The timescale for the reorientation of the peptide was between 120 and 450 ps, depending on the starting position of the peptide.

It was recently shown that the covalent tethering of the N-terminus of parathyroid hormone (PTH )to the seventh helical bundle of the G-protein coupled PTH-receptor (PTH1R)leads to autoactivation [Shimizu et al., J.Biol.Chem. 275 (2000) 19456–19460]. Here, we have developed molecular models for the interaction of PTH(1–11)tethered to PTH1R and refined them with molecular dynamics simulations. The starting structure of the ligand/receptor complex is based on experimental data from a series of spectroscopic structural studies of PTH(1–34)and the extracellular domains of PTH1R and intermolecular contact points derived from photoaffinity labeling. The resulting PTH1R/[Arg11]PTH(1–11)complex has the N-terminus of PTH interacting with residues of the third extracellular loop of PTH1R, as a possible mode for receptor activation. The hydrophobic residues leucine-5 and methionine-8,centrally located in the N-terminal alpha-helix of PTH(1–11), are located in deep, well-defined hydrophobic pockets in the central core of the seventh helical bundle, consistent with the requirement of these amino acids for autoactivation. We postulate that the improved signaling properties of [Arg]PTH(1–11)over wild type PTH(1–11)is due to a stable hydrogen bond between Arg11 and E444, at the beginning of TM7. The model provides atomic insight into currently available biochemical data as well as numerous putative ligand/receptor interactions, and thereby may further the rational design of reduced-size PTH agonists at the PTH1 receptor.

Bovine beta-lactoglobulin represents an interesting example of context-dependent secondary structure induction. In fact, secondary structure predictions indicated that this beta-barrel protein has a surprisingly high alpha-helical preference, which was retained for short fragments. Cooperative transitions from the native beta-sheet to alpha-helical structures were additionally induced by organic solvents, in particular trifluoroethanol. As a result of this high alpha-helical preference, it has been proposed that non-native alpha-helical intermediates could be formed in the unfolding pathway of this protein. In order to provide a better understanding of the processes that underlie conformational plasticity in this protein, CD measurements in the presence of increasing amounts of urea and in the presence of organic solvents were performed. Urea unfolding studies, performed at pH 2.1 and 37 degrees C, revealed an apparent two-state transition, and afforded no evidence of non native alpha-helical intermediates. The protein treated with up to 6M urea, refolded to the native structure, while treatment with higher molar concentration urea, lead to partial misfolding. A 29-mer peptide covering the region of strands a and b of the intact protein, characterized by the presence of 4/3 heptad repeats, was synthesized and studied by CD in the presence of different solvents. On the basis of the obtained results, a mechanism was proposed to explain the structural transition from the beta to alpha structure, provoked by organic solvents in the intact protein.

Multifarious applications of NMR (high-resolution NMR in liquid-state and in semi-solid matrices, low-field NMR relaxometry, and NMR-imaging) in the analysis of food components and entire food samples are described using examples of different food matrices and different problems related to food safety, traceability, geographical and botanical origin, farming methods, food processing, maturation and ageing, etc. Althoug NMR has not yet been recognized as an official methodology for the food control the numerous applications of NMR reported in the literature show the potenziality of this methodology also as an approach complementary to the other recognized conventional methodologies.

Multifarious applications of NMR (high-resolution NMR in liquid-state and in semi-solid matrices, low-field NMR relaxometry, and NMR-imaging) in the analysis of food components and entire food samples are described using examples of different food matrices and different problems related to food safety, traceability, geographical and botanical origin, farming methods, food processing, maturation and ageing, etc. Althoug NMR has not yet been recognized as an official methodology for the food control the numerous applications of NMR reported in the literature show the potenziality of this methodology also as an approach complementary to the other recognized conventional methodologies.

Tyrosinase (Ty) is a copper-containing enzyme ubiquitously distributed in nature. In recent years, Ty has attracted interest as a potential detoxifying agent for xenobiotic compounds with phenolic structure. Among these, chlorophenols are particularly relevant pollutants, commonly found in waste waters. The activity of Streptomyces antibioticus tyrosinase toward isomeric monochlorophenols was studied. Tyrosinase oxidizes both 3- and 4-chlorophenol to the same product, 4-chloro-1,2-ortho-quinone, which subsequently undergoes a nucleophilic substitution reaction at the chlorine atom by excess phenol to give the corresponding phenol-quinone adduct. By contrast, 2-chlorophenol is not reactive and acts as a competitive inhibitor. Docking calculations suggest that the substrates point to one of the copper atoms of the dinuclear center (copper B) and appear to interact preferentially with one of the two coordinated oxygen atoms. The approach of the substrate toward the active site is favored by a π-stacking interaction with one of the copper-coordinated histidines (His194) and by a hydrogen bonding interaction with the O1 oxygen. With this study, we provide the first characterization of the early intermediates in the biotechnologically relevant reaction of Ty with chlorophenols. Additionally, combining experimental evidences with molecular modeling simulations, we propose a detailed reaction scheme for Ty-mediated oxidation of monochlorophenols. © 2010 Elsevier Inc. All rights reserved.

The two peptides, rich in C(alpha)-tetrasubstituted amino acids, Ac-[Aib-L-(alphaMe)Val-Aib](2)-L-His-NH(2) (1) and Ac-[Aib-L-(alphaMe)Val-Aib](2)-O-tBu (2 a) are prevalently helical. They present the unique property of changing their conformation from the alpha- to the 3(10)-helix as a function of the polarity of the solvent: alpha in more polar solvents, 3(10) in less polar ones. Conclusive evidence of this reversible change of conformation is reported on the basis of the circular dichroism (CD) spectra and a detailed two-dimensional NMR analysis in two solvents (trifluoroethanol and methanol) refined with molecular dynamics calculations. The X-ray diffractometric analysis of the crystals of both peptides reveals that they assume a prevalent 3(10)-helix conformation in the solid state. This conformation is practically superimposable on that obtained from the NMR analysis of 1 in methanol. The NMR results further validate the reported CD signature of the 3(10)-helix and the use of the CD technique for its assessment.

In this study, we compared a fast fluid-attenuated inversion recovery (fast-FLAIR) sequence to conventional spin-echo (CSE) in the evaluation of brain MRI lesion loads of seven patients with clinically definite multiple sclerosis. Interleaved CSE (3000/20, 5 mm contiguous axial slices) and fast-FLAIR (9000/150/2200, 5 mm contiguous axial slices) sequences were performed on a 1.0 T machine. Lesions were counted consensually by two observers and then segmented independently by two other observers using a local thresholding technique, with subsequent manual editing in the case of poorly defined lesions. Four hundred and two lesions were detected in at least one of the two sequences: 128 were seen only on fast-FLAIR, 17 only on CSE. Forty-one lesions were larger on fast-FLAIR, while no lesion was considered larger on CSE. The numbers of periventricular (P = 0.05), cortical/subcortical (P = 0.02) and discrete (P = 0.05) lesions detected using fast-FLAIR were higher than those detected using CSE. The median lesion load was 7185 mm3 on CSE and 8418 mm3 on the fast-FLAIR, the average being 18% (range = 11.6-29%) higher on the fast-FLAIR images. Lesion contrast ratio was higher for lesions on the fast-FLAIR than on the CSE sequence (P < 0.0001). The percentages of poorly defined lesions which needed manual editing after the local thresholding technique was applied and the total time needed for the measurements were lower (P < 0.001) when fast-FLAIR images were used compared with CSE. This resulted in a reduced inter-rater coefficient of variation in measuring lesion volumes. Our data indicate that fast-FLAIR sequences are more sensitive than CSE in detecting multiple sclerosis lesion burden and that fast-FLAIR is a promising technique for natural history studies and clinical trials in multiple sclerosis.

We evaluated the correlations between cognitive impairment, clinical and brain magnetic resonance imaging (MRI) findings in 100 patients with multiple sclerosis (MS). The performance on one or more neuropsychological tests was abnormal in 47% of the 64 patients who completed the entire neuropsychological battery; the cognitive impairment was mild in 14 (22%) and severe in 16 (25%). Performance on any single neuropsychological test was unrelated to clinical parameters (age, duration of the disease, disability). The neuropsychological performance of relapsing-remitting patients was better than in patients with a chronic-progressive disease. The mean scores for almost all the neuropsychological tests were significantly lower in patients with severe ventricular dilatation and corpus callosum atrophy than in patients in whom these structures were little affected. Mean scores for WMS, performance Intelligence Quotient (IQ), total IQ and Token Test (TT) were also significantly correlated with the widening of cortical sulci and total lesional scores. Our data support the contention that the involvement of pathways that are critical for a given cognitive process as well as the progression of the axonal degeneration and sclerosis seem to play important roles in the pathophysiology of cognitive dysfunction in MS.