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Fungal infections are a significant global public health challenge because of their widespread occurrence, morbidity, and profound social and economic consequences. Antifungal resistance is also an increasing concern, posing a substantial risk to public health. There is a growing interest in searching for new antifungal drugs isolated from natural sources. This study aimed to evaluate the antifungal activity of novel mollusk fractions against fungal strains resistant to nystatin and amphotericin B. In addition, the role of oxidative stress in the mechanism of damage was determined. The mucus from the garden snail Cornu aspersum (MCa/1-20) and the hemolymph fraction from the marine snail Rapana venosa (HLRv/3-100) were obtained and characterized via 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric -analyses. The results demonstrate that the spores and biomass of both mollusk fractions have a significant fungicidal effect against Penicillium griseofulvum, and Aspergillus niger. Compared to the control group, the release of intracellular proteins and reducing sugars was significantly increased in the treated groups. The data showed increased levels of oxidative stress biomarkers (lipid peroxidation and oxidatively damaged proteins) and a downregulated antioxidant enzyme defense, corresponding to increased antifungal activity. To our knowledge, this is the first study evaluating oxidative stress as a factor in mollusk fractions' antifungal activity. [ABSTRACT FROM AUTHOR]

Many biologically active compounds have been identified in the mucus of the garden snail Cornu aspersum, which are effective in the treatment of several diseases such as cancer, ulcers, wounds, etc. The incorporation of these compounds into the green synthesis of copper nanoparticles (CuONPs-Muc) was demonstrated in our previous study. Based on the synergistic effect of two reducing agents—C. aspersum snail mucus and ascorbic acid (AsA)—on CuSO4.5H2O, which also act as stabilizers of the resulting compound, a new method for the "green" synthesis of CuONPs-Muc is presented. Using two reducing agents has several advantages, such as forming spherical nanoparticles with a diameter of about 150 nm and reducing the formation time of CuONPs-Muc to 3 h. Analyses by ultraviolet–visible spectroscopy (UV-Vis) and Fourier transform infrared spectroscopy (FT-IR) show the formation of CuONPs-Muc, composed of a mixture of copper and copper oxide. This was confirmed by scanning electron microscopy combined with energy-dispersive spectroscopy (SEM/EDS) and X-ray diffraction (XRD). Another important advantage of CuONPs obtained by the new method with two reducing agents is the stronger inhibitory effect on the bacterial growth of some Gram-positive and Gram-negative bacterial strains, compared to CuONPs-Muc prepared with only one reducing agent, i.e., a fraction of mucus with an MW > 20 kDa. [ABSTRACT FROM AUTHOR]

Significant agricultural losses are caused by the phytopathogenic fungi Botrytis cinerea and Phytophthora infestans, as well as bacteria of the Ralstonia solanacearum species. The present work aimed to isolate rhizobacteria for simultaneous biocontrol of these three phytopathogenic species and to suggest the mechanisms of their antagonistic action. Among 120 Bacillus spp. isolated from soils, Bacillus velezensis and Bacillus licheniformis strains displayed the highest activity against all three phytopathogens. A rapid, polymerase chain reaction-based method for detecting nonribosomal peptide synthetase genes was developed to elucidate the genetic basis of these traits. The presence of fenA, srfAA, ppsA, and lchAA genes, encoding fengycin/surfactin/plipastatin synthetases and lichenysin synthase, was revealed in the strains' genomes. The whole genome sequencing (WGS) of B. velezensis R22 showed that it contains 4,081,504 bp (with G + C content 46.35%), 4087 genes for 3935 proteins, 72 tRNAs, 14 rRNAs, and 5 ncRNAs. WGS allowed the prediction of 10 complete clusters for secondary metabolites with putative antimicrobial activity: difficidin, fengycin, bacillaene, butyrosin, bacillibactin, bacilysin, surfactin, macrolactin H, macrolactin R22, and velezensin. LC-MS and high-sensitivity UHPLC-Q-TOF LC-MS/MS analysis were used to search for the predicted metabolites in cell-free supernatants of B. velezensis R22. The compounds with the strongest antifungal activity are surfactin with a C15 β-OH fatty acid chain; two homologous forms of fengycin A; and two fengycin B homologues containing C16 and C17 β-hydroxy fatty acid chains. The broad antimicrobial spectrum of B. velezensis R22 and its molecular characterization provide a good basis for the future development of plant protection preparations. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is the most widespread neurodegenerative disorder. Recently, it was found that mucus extract from Cornu aspersum has beneficial effects on memory and cognitive processes in a rat scopolamine model of AD. The present study elucidated the mechanisms of action of standardized mucus snail extract (SE) enriched with a fraction above 20 kDa on Alzheimer-type dementia in rats. Using proteomic analysis on two-dimensional polyacrylamide gel electrophoresis (2D–PAGE) on rat cortex extracts, we compared protein expression in both groups: the first group was treated intraperitoneally with scopolamine (Sco, 2 mg/kg, 11 days) and the second (Sco + SE) group was treated intraperitoneally with Sco (Sco, 2 mg/kg) and protected by SE (0.5 mL/100 g bw) applied daily orally for 11 days. Brain cortex was separated and the expressions of various proteins related to memory and cognitive functions were identified. We found that the expression of Ubiquitin carboxyl-terminal hydrolase isozyme L1, Calbindin, Vacuolar ATP synthase catalytic subunit A, Tropomyosin beta chain, 14-3-3 zeta/delta, Kinesin-1 heavy chain, and Stathmin-4 significantly differs in SE-protected rats as compared to dement animals treated only by Sco, and these brain proteins might be potential therapeutic targets for Alzheimer's-type dementia treatment. [ABSTRACT FROM AUTHOR]

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The discovery and investigation of new natural compounds with antimicrobial activity are new potential strategies to reduce the spread of antimicrobial resistance. The presented study reveals, for the first time, the promising antibacterial potential of two fractions from Cornu aspersum mucus with an MW < 20 kDa and an MW > 20 kDa against five bacterial pathogens—Bacillus cereus 1085, Propionibacterium acnes 1897, Salmonella enterica 8691, Enterococcus faecalis 3915, and Enterococcus faecium 8754. Using de novo sequencing, 16 novel peptides with potential antibacterial activity were identified in a fraction with an MW < 20 kDa. Some bioactive compounds in a mucus fraction with an MW > 20 kDa were determined via a proteomic analysis on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and bioinformatics. High homology with proteins and glycoproteins was found, with potential antibacterial activity in mucus proteins named aspernin, hemocyanins, H-lectins, and L-amino acid oxidase-like protein, as well as mucins (mucin-5AC, mucin-5B, mucin-2, and mucin-17). We hypothesize that the synergy between the bioactive components determined in the composition of the fraction > 20 kDa are responsible for the high antibacterial activity against the tested pathogens in concentrations between 32 and 128 µg/mL, which is comparable to vancomycin, but without cytotoxic effects on model eukaryotic cells of Saccharomyces cerevisiae. Additionally, a positive effect, by reducing the levels of intracellular oxidative damage and increasing antioxidant capacity, on S. cerevisiae cells was found for both mucus extract fractions of C. aspersum. These findings may serve as a basis for further studies to develop a new antibacterial agent preventing the development of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Several biologically active compounds involved in the green synthesis of silver and gold nanoparticles have been isolated from snail mucus and characterized. This paper presents a successful method for the application of snail mucus from Cornu aspersum as a bioreducing agent of copper sulfate and as a biostabilizer of the copper oxide nanoparticles (CuONPs-Muc) obtained. The synthesis at room temperature and neutral pH yielded nanoparticles with a spherical shape and an average diameter of 150 nm. The structure and properties of CuONPs-Muc were characterized using various methods and techniques, such as ultraviolet–visible spectroscopy (UV–vis), high-performance liquid chromatography (HPLC), one-dimensional polyacrylamide gel electrophoresis (1D-PAGE), up-conversion infrared spectroscopy Fourier transform (FTIR), scanning electron microscopy combined with energy dispersive spectroscopy (SEM/EDS), Raman spectroscopy and imaging, thermogravimetric analysis (TG-DSC), etc. Mucus proteins with molecular weights of 30.691 kDa and 26.549 kDa were identified, which are involved in the biogenic production of CuONPs-Muc. The macromolecular shell of proteins formed around the copper ions contributes to a higher efficiency of the synthesized CuONPs-Muc in inhibiting the bacterial growth of several Gram-positive (Bacillus subtilis NBIMCC2353, Bacillus spizizenii ATCC 6633, Staphylococcus aureus ATCC 6538, Listeria innocua NBIMCC8755) and Gram-negative (Escherichia coli ATCC8739, Salmonella enteitidis NBIMCC8691, Salmonella typhimurium ATCC 14028, Stenotrophomonas maltophilia ATCC 17666) bacteria compared to baseline mucus. The bioorganic synthesis of snail mucus presented here provides CuONPs-Muc with a highly pronounced antimicrobial effect. These results will expand knowledge in the field of natural nanomaterials and their role in emerging dosage forms. [ABSTRACT FROM AUTHOR]

The absorption of triclosan on the surface of four different carbon adsorbents, obtained on the base of plant and household waste (RDF), provided by Sofia Waste Plant, was investigated. The obtained results indicate that the most important parameters, which determine the process of triclosan adsorption by adsorbent surface are pore texture, the size of the accessible surface for the molecules of triclosan, and the chemical nature of the adsorbent surface. It was found that the obtained adsorbents can be successfully applied for the extraction of triclosan from waste and drinking water. The possibilities for solid products, obtained by thermal treatment of household and vegetable waste, to be applied successfully in a water purification technique are discussed. [ABSTRACT FROM AUTHOR]

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of some invertebrate species that attracted scientific interest as potential anticancer agents. The present study aims to assess the in vitro and in vivo anticancer activity of hemocyanins isolated from Helix aspersa, Helix lucorum, and Rapana venosa in the Graffi myeloid tumor model. The in vitro antitumor activity of the hemocyanins was determined by a MTT test and cytomorphological analysis by fluorescent and transmission electron microscopy. The in vivo effects of the hemocyanins were examined in hamsters transplanted with Graffi tumor. The serum antibody titers against the tested hemocyanins and tumor antigen were determined by ELISA. Histopathological assessment of the morphological features related to antitumor effect, immune system response, and toxicity in some internal organs was performed. The results of in vitro studies indicated that the tested hemocyanins induced significant antiproliferative and apoptogenic effects. The in vivo investigations demonstrated a protective antitumor effect, expressed in reduced transplantability, suppression of tumor growth and metastasis, reduced mortality, prolonged survival time, and absence of toxic side effects. The present study indicated that the antitumor activity of the studied hemocyanins was due to both immune stimulation and direct effects on the tumor cells, and they displayed their potential as therapeutic agents against hematological malignances. [ABSTRACT FROM AUTHOR]

This study is the first report describing the promising antitumor activity of biologically active compounds isolated from the hemolymph of marine snail Rapana venosa—a fraction with Mw between 50 and 100 kDa and two structural subunits (RvH1 and RvH2), tested on a panel of human breast cell lines—six lines of different molecular subtypes of breast cancer MDA-MB-231, MDA-MB-468, BT-474, BT-549, SK-BR-3, and MCF-7 and the non-cancerous MCF-10A. The fraction with Mw 50–100 kDa (HRv 50–100) showed good antitumor activity manifested by a significant decrease in cell viability, altered morphology, autophagy, and p53 activation in treated cancer cells. An apparent synergistic effect was observed for the combination of HRv 50–100 with cis-platin for all tested cell lines. The combination of HRv 50–100 with cisplatin and/or tamoxifen is three times more effective compared to treatment with classical chemotherapeutics alone. The main proteins in the active fraction, with Mw at ~50 kDa, ~65 kDa, ~100 kDa, were identified by MALDI-MS, MS/MS analyses, and bioinformatics. Homology was established with known proteins with antitumor potential detected in different mollusc species: peroxidase-like protein, glycoproteins Aplysianin A, L-amino acid oxidase (LAAO), and the functional unit with Mw 50 kDa of RvH. Our study reveals new perspectives for application of HRv 50–100 as an antitumor agent used alone or as a booster in combination with different chemotherapies. [ABSTRACT FROM AUTHOR]

Nowadays, an increased interest in natural compounds with preventive or therapeutic potential for various diseases has been observed. Given the involvement of oxidative stress in the pathogenesis of gastric ulcer (GU) and the wide range of bioactive compounds isolated from snails, this study aimed to investigate the protective effect of Cornu aspersum (Müller, 1774) mucus on ethanol-induced GUs. Male albino mice were divided into Control, Ethanol, Mucus + Ethanol and Mucus + Omeprazole treated groups. The GUs were induced by administration of 96% ethanol (10 mL/kg, per os). One hour before ulcer induction, the mice of Mucus + Ethanol group were pretreated with mucus (20 mg/kg, per os), and the mice of Mucus + Omeprazole group were pretreated with omeprazole (20 mg/kg, per os). Ethanol administration caused grave lesions of gastric mucosa and a significant decrease of glutathione (GSH) and superoxide dismutase (SOD), catalase, and glutathione reductase (GR) activities. In the animals with mucus or omeprazole pre-administration compared to the Ethanol group, the following were observed: only a small number of hemorrhagic fields, significantly reduced GU index with calculated 73% protection by mucus and 78% protection by omeprazole, and significant recovery of mucosal GSH and SOD and GR activities. In addition, the mucus inhibited Helicobacter pylori growth. Thus, the protective effect of C. aspersum mucus on both gastric mucosa and gastric antioxidant potential in ethanol-induced GU model suggests that it may serve as a good tool for prevention of this disease. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Peptides isolated from the mucus of Cornu aspersum could be prototypes for antibiotics against pathogenic bacteria. Information regarding the mechanisms, effective concentration, and methods of application is an important tool for therapeutic, financial, and ecological regulation and a holistic approach to medical treatment. A peptide fraction with MW < 10 kDa was analyzed by MALDI-TOF-TOF using Autoflex™ III. The strain Escherichia coli NBIMCC 8785 (18 h and 48 h culture) was used. The changes in bacterial structure and metabolic activity were investigated by SEM, fluorescent, and digital image analysis. This peptide fraction had high inhibitory effects in surface and deep inoculations of E. coli of 1990.00 and 136.13 mm2/mgPr/µMol, respectively, in the samples. Thus, it would be effective in the treatment of infections involving bacterial biofilms and homogenous cells. Various deformations of the bacteria and inhibition of its metabolism were discovered and illustrated. The data on the mechanisms of impact of the peptides permitted the formulation of an algorithm for the treatment of infections depending on the phase of their development. The decrease in the therapeutic concentrations will be more sparing to the environment and will lead to a decrease in the cost of the treatment. [ABSTRACT FROM AUTHOR]

Yeasts Saccharomyces cerevisiae, like other microbes in nature, respond to the unavailability of nutrients with entrance in quiescent/G0 state. These cells exist in non-dividing, latent form by maintaining the cellular metabolism at a low level but still able to sense and adapt to environmental stresses. Their quiescent status characteristics are likely close to those of tissues and organs in mammals and humans. This fact makes them an appropriate model system for investigation of the basic mechanisms underlying the toxicity of different chemical compounds. In this study, the toxic effect of H2O2 and menadione on quiescent S. cerevisiae cells was evaluated through the analysis of RNA polymerases transcription profile and ribosomal RNA content. Distinct RNA polymerases subunits were expressed in G0 yeast cells after short exposure to 0.1 mmol/L menadione and 5 mmol/L hydrogen peroxide. Significant transcription repression of RNA polymerases genes was observed as a response to menadione. Both stress agents induced changes in the 25S and 18S rRNA profile in quiescent and proliferating yeast cells. These results strongly suggest that the toxicological response of eukaryotic cells involves rapid alterations in RNA polymerases gene expression and changes in RNA transcriptome profiles, and depends on the specific mechanism of toxic action. [ABSTRACT FROM AUTHOR]

The focus of the present study is to determine proteins responsible for the oxidative and toxic stress response in proliferating and stationary phase (G0) cultures. Therefore, the yeast Saccharomyces cerevisiae was treated with oxidative and drug compounds (H2O2, menadione, zeocin, and ibuprofen) in both phases. These substances were chosen to determine the redox status of the yeast. S. cerevisiae appeared to employ different strategies to ensure their antioxidant defence metabolism. Analysis, including sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry, was used in the search. The proteins were identified by SDS-PAGE, matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry analysis, and Mascot database-fingerprint. The final step was determination of protein profiles of yeast S. cerevisiae in proliferating (M) and stationary phase (G0). Seven bands were determined and the corresponding proteins were proposed: cytochrome c peroxidase, glutathione S-transferase omega-like, NAPDH-dependent diflavin reductase, DNA replication fork-blocking protein, putative aryl alcohol dehydrogenase, AP-1-like transcription factor YAP5, GTP-binding protein. All putative proteins coincide with the literature database. A typical example of such an adaptation mechanism in the defence against oxidative damage is the synthesis of several glutathione and thioredoxin peroxidases in the yeast cell. A deeper investigation of the conserved mechanisms responsible for entry into, survival, and exit from quiescence in higher eukaryotes will help the development of new anticancer therapies, the study in the process of ageing and neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

The structural stability of the hеmocyanin purified from the hеmolymph of gаrden snаils Helix lucorum (HlH) was investigated by means of far-UV circular dichroism (CD), differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). For the first time, TEM analyses showed the presence of tubular polymers in hemocyanins after three-day dialysis against a stabilizing buffer containing high concentrations of Ca2+ and Mg2+ ions (100 mmol L−1). The conformational stability study of native HlH by means of CD in a wide pH range (2.5-11.5) defined the pH stability region of HlH at pH 6.5 − 8.0. DSC analyses demonstrated the thermal stability of this hemocyanin. One transition, with an apparent transition tеmperature (Tm) at 82.3 °C, was detected in the heаt capacity curve of HlH in 50 mmol L−1 Tris-HCl buffer, pH 7.2, at a heating rate of 1.0 °C min−1. The calorimetrically observed thermal transition correlates well with the unfolding transition monitored by CD measurements. The two-state kinetic model was used to analyse the process of irreversible thermal denaturation of HlH; Еa of 451 ± 4 kJ mol−1 was calculated. The obtained results on the conformational stability of HlH will facilitate the further investigation of the properties and potential biomedical applications of this respiratory protein. [ABSTRACT FROM AUTHOR]

Hemocyanins are oxygen-transporting glycoproteins in molluscs and arthropods. In this study, we assayed the biomacromolecules from the molluscs Helix lucorum (HlH), Rapana venosa (RvH) and Megatura crenulata (KLH) including their functional units (FUs), for therapy of bladder cancer permanent cells. In vitro studies antitumour activities of these proteins were performed with bladder cancer permanent cell line CAL-29 and the normal urothelial cell line HL 10/29 in comparison to doxorubicin. The obtained results showed that the human tumour CAL-29 cell line is sensitive to the action of the tested hemocyanins and their isoforms. We observed a dose- and time-dependent inhibition of tumour cell growth after incubation with native HlH and two FUs (βc-HlH-a and FU βc-HlH-h); and of particular significance, FU βc-HlH-h showed a surprisingly stronger effect than that the doxorubicin-treated cells. Cells treated with βc-HlH-h, showed both, apoptotic and necrotic cells. In addition, two-dimensional polyacrylamide gel electrophoresis (PAGE) found for differential up-regulation of several proteins after hemocyanin treatment. Eight different down-regulated and two up-regulated proteins were identified, which may be associated with the apoptosis pathway. No inhibition of the normal urothelial cell line HL 10/29 was observed after treatment with HlH and its isoforms. The most effective inhibition of CAL-29 tumour cells was observed after treatment with βc-HlH-h, probably caused by a specific oligosaccharide structure of HlH with methylated hexoses. These results suggest that hemocyanin glycosylation plays an important role in its anticancer activity. [ABSTRACT FROM AUTHOR]

The increasing antibiotic resistance among pathogenic bacteria is a challenge that drives the development of new antibacterial substances. Marine inhabitants are excellent sources of antimicrobial proteins and considered as promising candidates for the treatment of microbial infections. In the present study, we obtained haemocyanin from Eriphia verrucosa and studied its potential to suppress the growth of some pathogenic bacteria and yeasts. The putative antibacterial molecules were isolated from the haemolymph by chromatography appropriate for producing the native haemocyanin (EvH) and its five structural units (SUs). The results showed that EvH had no antimicrobial activity unlike its glycosylated SUs. All haemocyanin SUs exhibited differential antibacterial activity depending on their grade of glycosylation. The strongest antimicrobial activity of SU1 (with highest carbohydrate content) was against Escherichia coli and Bacillus subtilis. The least glycosylated SU3 and SU4 exhibited the lowest antimicrobial activity against all strains. The fraction SU1 has the potential to be applied as a substitute for some commonly used antibiotics. It was demonstrated that the grade of haemocyanin glycosylation plays an important role in its functional antibacterial properties. [ABSTRACT FROM AUTHOR]

Various aspects of biomedical applications of molluscan hemocyanins, associated with their immunogenic properties and antitumor activity, promoted us to perform structural studies on these glycoproteins. The stability and reassociation behavior of native Cornu aspersum hemocyanin (CaH) are studied in the presence of different concentrations of Ca2+ and Mg2+ ions and pH values using electron microscopy. Higher concentrations of those ions led to a more rapid reassociation of CaH, resulting in stable multidecamers with different lengths. The conformational changes of native CaH are investigated within a wide pH-temperature range by UV circular dichroism. The relatively small changes of initial [θ]λ indicated that many secondary structural elements are preserved, even at high temperatures above 80°C, especially at neutral pH. The mechanism of thermal unfolding of CaH has a complicated character, and the process is irreversible. The conformational stability of the native didecameric aggregates of CaH toward various denaturants indicates that hydrophilic and polar forces stabilize the quaternary structure. For the first time, the unfolding of native CaH in water solutions in the presence of four different denaturants is investigated. The free energy of stabilization in water, ∆GDH2O, was calculated in the range of 15.48–16.95 kJ mol−1. The presented results will facilitate the further investigation of the properties and potential applications of CaH. [ABSTRACT FROM AUTHOR]

The recent appearance of a growing number of resistant to conventional antibiotics, has become a serious medical problem. To overcome this resistance, the development of new compounds is encouraged. Hemolymph and mucus of Helix lucorum and Helix aspersa garden snails and Rapana venosa marine snail are a complex mixture of biochemically and pharmacologically active components. Glycoprotein 'hemocyanin' and antimicrobial peptides from the hemolymph and mucus are important components of the innate immunity. Some isoforms and peptides serve as effector molecules of the defense system, providing an efficient initial effect against infectious pathogens. The in vitro antitumor activity of Helix and Rapana hemocyanins and their isoforms with different oligosaccharide structures was established on the bladder carcinoma permanent cell lines T-24. This is probably due to the specific oligosaccharide structures of hemocyanins which are exposed on the surface of the molecule. [ABSTRACT FROM AUTHOR]

Molluscan hemocyanins (Hcs) have recently particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. The oligosaccharide structures of the structural subunits RvH2 from Rapana venosa hemocyanin (RvH) and βc-Helix lucorum hemocyanin (βc-HlH) were comparative investigated by mass spectrometry. Our study revealed a highly heterogeneous mixture of N-glycans with compositions Hex3-7HexNAc2-5MeHex0-4Pent0-1Fuc0-1 and Hex0-9HexNAc2-4MeHex0-1HexA0-1Pent0-1Fuc0-3, isolation from βc-HlH and RvH2, respectively. N-glycans from βc-HlH contain mainly a terminal methyl-hexose (MeHex) residue, in some cases even more than one. Several carbohydrate moieties from βc-HlH are core-fucosylated, and also possess on methylation. In contrast with RvH2, N-glycans with an internal fucose residue, substituted at two positions with N-acetylhexosamine and hexuronic acid, as and glycans containing internal fucose were not found in βc-HlH. The glycosylation sites occupancy was subsequently elucidated by precursor ion scanning of the intact glycopeptides from RvH and βc-HlH using a nano-ESI mass spectrometry and Q-trap-LC/MS system. The oligosaccharide moieties found in HlH and RvH reveal a complex N-glycan pattern combining typical structural features of different higher organisms. Moreover, they are a potential source of novel N-glycans that are important for the stimulation of the immune response and/or for the production of antibodies used in diagnosis and therapy. [ABSTRACT FROM AUTHOR]

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 β-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, β-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 β-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 β-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. [ABSTRACT FROM AUTHOR]

Hemocyanins are copper-binding proteins that play a crucial role in the physiological processes in crustaceans. In this study, the cDNA encoding hemocyanin subunit 5 from the Black sea crab Eriphia verrucosa (EvHc5) was cloned using EST analysis, RT-PCR and rapid amplification of the cDNA ends (RACE) approach. The full-length cDNA of EvHc5 was 2254 bp, consisting of a 5′ and 3′ untranslated regions and an open reading frame of 2022 bp, encoding a protein consisting of 674 amino acid residues. The protein has an N-terminal signal peptide of 14 amino acids as is expected for proteins synthesized in hepatopancreas tubule cells and secreted into the hemolymph. The 3D model showed the presence of three functional domains and six conserved histidine residues that participate in the formation of the copper active site in Domain 2. The EvHc5 is O-glycosylated and the glycan is exposed on the surface of the subunit similar to Panulirus interruptus. The phylogenetic analysis has shown its close grouping with γ-type of hemocyanins of other crustacean species belonging to order Decapoda, infraorder Brachyura. [ABSTRACT FROM AUTHOR]

A number of studies have shown that glycosylation of proteins plays diverse functions in the lives of organisms, has crucial biological and physiological roles in pathogen–host interactions, and is involved in a large number of biological events in the immune system, and in virus and bacteria recognition. The large amount of scientific interest in glycoproteins of molluscan hemocyanins is due not only to their complex quaternary structures, but also to the great diversity of their oligosaccharide structures with a high carbohydrate content (2–9%). This great variety is due to their specific monosaccharide composition and different side chain composition. The determination of glycans and glycopeptides was performed with the most commonly used methods for the analysis of biomolecules, including peptides and proteins, including Matrix Assisted Laser Desorption/Ionisation–Time of Flight (MALDI-TOF-TOF), Liquid Chromatography - Electrospray Ionization-Mass Spectrometry (LC/ESI-MS), Liquid Chromatography (LC-Q-trap-MS/MS) or Nano- Electrospray Ionization-Mass Spectrometry (nano-ESI-MS) and others. The molluscan hemocyanins have complex carbohydrate structures with predominant N-linked glycans. Of interest are identified structures with methylated hexoses and xyloses arranged at different positions in the carbohydrate moieties of molluscan hemocyanins. Novel acidic glycan structures with specific glycosylation positions, e.g., hemocyanins that enable a deeper insight into the glycosylation process, were observed in Rapana venosa, Helix lucorum, and Haliotis tuberculata. Recent studies demonstrate that glycosylation plays a crucial physiological role in the immunostimulatory and therapeutic effect of glycoproteins. The remarkable diversity of hemocyanin glycan content is an important feature of their immune function and provides a new concept in the antibody–antigen interaction through clustered carbohydrate epitopes. [ABSTRACT FROM AUTHOR]

Natural products have long played a major role in medicine and science. The garden snail Cornu aspersum is a rich source of biologically active natural substances that might be an important source for new drugs to treat human disease. Based on our previous studies, nine fractions containing compounds with Mw <3 kDa; <10 kDa; <20 kDa; >20 kDa; >30 kDa>50 kDa and between 3 and 5 kDa; 5 and 10 kDa; and 10 and 30 kDa were purified from the mucus of C. aspersum and analyzed by tandem mass spectrometry (MALDI-TOF/TOF). Seventeen novel peptides with potential antibacterial activity were identified by de novo MS/MS sequencing using tandem mass spectrometry. The different fractions were tested for antibacterial activity against Gram─ (Pseudomonas aureofaciens and Escherichia coli) and Gram+ (Brevibacillus laterosporus) bacterial strains as well the anaerobic bacterium Clostridium perfringens. These results revealed that the peptide fractions exhibit a predominant antibacterial activity against B. laterosporus; the fraction with Mw 10–30 kDa against E. coli; another peptide fraction <20 kDa against P. aureofaciens; and the protein fraction >20 kDa against the bacterial strain C. perfringens. The discovery of new antimicrobial peptides (AMPs) from natural sources is of great importance for public health due to the AMPs' effective antimicrobial activities and low resistance rates. [ABSTRACT FROM AUTHOR]

Hemocyanins are oxygen-transporting glycoproteins in the hemolymph of arthropods and mollusks that attract scientific interest with their diverse biological activities and potential applications in pharmacy and medicine. The aim of the present study was to assess the in vitro antitumor activity of hemocyanins isolated from marine snail Rapana venosa (RvH) and garden snails Helix lucorum (HlH) and Helix aspersa (HaH), as well the mucus of H. aspersa snails, in the HT-29 human colorectal carcinoma cell line. The effects of the hemocyanins on the cell viability and proliferation were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the alterations in the tumor cell morphology were examined by fluorescent and transmission electron microscopy. The results of the MTT assay showed that the mucus and α-subunit of hemocyanin from the snail H. aspersa had the most significant antiproliferative activity of the tested samples. Cytomorphological analysis revealed that the observed antitumor effects were associated with induction of apoptosis in the tumor cells. The presented data indicate that hemocyanins and mucus from H. aspersa have an antineoplastic activity and potential for development of novel therapeutics for treatment of colorectal carcinoma. [ABSTRACT FROM AUTHOR]