Beneficial Effects of Snail Helix aspersa Extract in an Experimental Model of Alzheimer's Type Dementia.

BACKGROUND: Alzheimer's disease (AD) is a complex neurodegenerative disease with multifactorial etiology, unsatisfactory treatment, and a necessity for broad-spectrum active substances for cure. The mucus from Helix aspersa snail is a mixture of bioactive molecules with antimicrobial, anti-inflammatory, antioxidant, and anti-apoptotic effects. So far there are no data concerning the capacity of snail extract (SE) to affect neurodegenerative disorders. OBJECTIVE: The effects of SE from Helix aspersa on learning and memory deficits in Alzheimer's type dementia (ATD) induced by scopolamine (Sco) in male Wistar rats were examined and some mechanisms of action underlying these effects were evaluated. METHODS: SE (0.5 mL/100 g) was applied orally through a food tube for 16 consecutive days: 5 days before and 11 days simultaneously with Sco (2 mg/kg, intraperitoneally). At the end of Sco treatment, using behavioral methods, we evaluated memory performance. Additionally, in cortex and hippocampus the acetylcholinesterase (AChE) activity, acetylcholine and monoamines (dopamine, noradrenaline, and serotonin) content, levels of main oxidative stress markers, and expression of brain-derived neurotrophic factor (BDNF) and cAMP response element-binding protein (CREB) were determined. RESULTS: We demonstrated that, according to all behavioral tests used, SE significantly improved the cognitive deficits induced by Sco. Furthermore, SE possessed AChE inhibitory activity, moderate antioxidant properties and the ability to modulate monoamines content in two brain structures. Moreover, multiple SE applications not only restored the depressed by Sco expression of CREB and BDNF, but significantly upregulated it. CONCLUSION: Summarizing results, we conclude that complex mechanisms underlie the beneficial effects of SE on impaired memory in Alzheimer's type dementia.

Antitumor activity of glycosylated molluscan hemocyanins via Guerin ascites tumor.

As observed in most molluscan hemocyanins, high-mannose type glycans were identified in hemocyanins from Rapana venosa (RvH), Helix lucorum (HlH) and keyhole limpet (Megatura crenulata). In addition, a glycan with a branching structure containing xylose, fucose and terminal methyl hexose was identified in ß-HlH. We have examined the immuno-adjuvant properties of hemocyanins, their derivatives and conjugates associated with the cell mediated immunity in experimental tumor-bearing animals with ascites tumor of Guerin. After immunization of the animals with the experimental vaccine preparations, the highest values of splenic lymphocytes were observed in groups immunized with the conjugates RvH-TAg, ß-HlH-TAg and KLH-TAg (42.3%; 40.8% and 40.58%, respectively) than with the native hemocyanins (36.5%; 35.1% and 32.4%, respectively). The immunization of rats with the hemocyanins ß-HlH, RvH and KLH and their conjugates, prolonged the median survival time of tumor-bearing animals compared with non-immunized animals (39, 33, 31 and 7 days, respectively). Both hemocyanins ß-HlH and RvH activate the immune system of the experimental animals and therefore could be a good alternative for KLH. For this reason they could be included into the composition of non-specific anti-tumor vaccines to enhance their effectiveness.

Purification and characterization of L-phenylalanine aminopeptidase from chick-pea cotyledons (Cicer arietinum L.).

Chick-pea (Cicer arietinum L.) cotyledons are unique source of aminopeptidase - 8-9 U/g cotyledons was observed using L-leucine-p-nitroanilide as substrate. The aminopeptidase was purified (65 kDa, pI 4.8 ) reaching a specific activity of 220 U/mg at pH 7.0-7.2 and 35-40 degrees C. The determined constant of specificity k(cat)/K(m) during hydrolysis of N-unsubstituted amino acid-p-nitroanilides showed a decrease order: Phe>Leu>Pro>Ile>Val>Ala. The enzyme was strongly inhibited by p-chloromercuribenzoic acid as well as in a competitive rate by the antihypertensive peptides Ile-Pro-Pro and Val-Pro-Pro.

Characterization of an aminopeptidase and a proline iminopeptidase from cabbage leaves.

Aminopeptidase, preferring phenylalanine-p-nitroanilide as substrate, and proline iminopeptidase, highly-specific for proline-p-nitroanilide, were isolated from cabbage leaves (Brassica oleraceae var. capitata). As pH optima, 7.2-7.5 for aminopeptidase activity and 8.0-8.5 for proline iminopeptidase were determined. Both peptidases were strongly inhibited by p-chloromercuribenzoic acid, heavy metal ions and urea. The molecular weights were determined by gel filtration to be 56 and 204 kDa, respectively. The iminopeptidase was decomposed during SDS electrophoresis to four subunits of 50 kDa. Minor impurities of myrosinase-associated protein (approximately 70 kDa) were found in both preparations. Preliminary data of their amino acid sequences showed similarities to those of aminopeptidases N (family M1) and proline iminopeptidases (family S33).

Structure and stability of arthropodan hemocyanin Limulus polyphemus.

In the hemolymph of many arthropodan species, respiratory copper proteins of high molecular weight, termed hemocyanins (Hcs) are dissolved. In this communication, we report on the protein stability of different hemocyanin species (Crustacea and Chelicerata) using fluorescence spectroscopy. Five to seven major electrophoretically separable protein chains (structural subunits) were purified by fast protein liquid chromatography (FPLC) ion exchange chromatography from different hemocyanins with very high sequence homology of the active site regions binding copper ions (CuA and CuB), and especially the relative sequence positions of histidine (His) and tryptophan (Trp) residues of these protein segments are in all cases identical. The conformational stabilities of the native dodecameric aggregates and their isolated structural subunits towards various denaturants (pH and guanidine hydrochloride (Gdn.HCl)) indicate that the quaternary structure is stabilized by hydrophilic and polar forces, whereby both, the oxy- and apo-forms of the protein are considered. These two classes of Crustacea and Chelicerata Hcs have the similar Trp-fluorescence quantum yields, but different values of lambda(max) emission (about 325 and 337 nm, respectively). Differences in the quantum yields are observed of the oxy- and apo-forms, which must be attributed to the fluorescence quenching effect of the two copper ions (CuA and CuB) in the active site. The position of emission maximum indicates tryptophan side chains are situated in a non-polar environment. Denaturation studies of Hcs by Gdn.HCl indicate that the denaturation process consists of two steps: dissociation of the native molecule into its structural subunits and denaturation of the subunits at concentrations >1.5M Gdn.HCl. Two steps of denaturation are also observed after keeping the protein in buffer solutions at different pH values with different pH-stability for holo-oxy and apo-Hc forms.

Unusual location and characterization of Cu/Zn-containing superoxide dismutase from filamentous fungus Humicola lutea.

The present study aims to provide new information about the unusual location of Cu/Zn-superoxide dismutase (Cu/Zn-SOD) in lower eukaryotes such as filamentous fungi. Humicola lutea, a high producer of SOD was used as a model system. Subcellular fractions [cytosol, mitochondrial matrix, and intermembrane space (IMS)] were isolated and tested for purity using activity measurements of typical marker enzymes. Evidence, based on electrophoretic mobility, sensitivity to KCN and H(2)O(2) and immunoblot analysis supports the existence of Cu/Zn-SOD in mitochondrial IMS, and the Mn-SOD in the matrix. Enzyme activity is almost equally partitioned between both the compartments, thus suggesting that the intermembrane space could be one of the major sites of exposure to superoxide anion radicals. The mitochondrial Cu/Zn-SOD was purified and compared with the previously published cytosolic enzyme. They have identical molecular mass, cyanide- and H(2)O(2)-sensitivity, N-terminal amino acid sequence, glycosylation sites and carbohydrate composition. The H. lutea mitochondrial Cu/Zn-SOD is the first identified naturally glycosylated enzyme, isolated from IMS. These findings suggest that the same Cu/Zn-SOD exists in both the mitochondrial IMS and cytosol.