35 research outputs found
Exploiting Structural Analysis, in Silico Screening and functional variants characterization to identify novel inhibitors of cytidine deaminase
No full textThis thesis work, combining a virtual screening study for cytidine deaminase ligands with a study on the effect that genetic polymorphisms have on the enzyme functionality provide a useful base to: a) understand the mechanism of nucleoside recognition by CDA and identify novel effective inhibitors; and b) study the different properties of said inhibitors toward three distinct naturally occurring CDA variants (K27, Q27 and T70). Ultimately, this may assist the future design of novel CDA inhibitors or antitumor drugs not susceptible to deamination, with the aim to get more effective personalized drug therapies
Studies on the antimicrobial activity of Silver Nanoparticles as additive for several kind of materials
No full textStudies on the antimicrobial activity of Silver Nanoparticles as additive for several kind of materials
Rossana, Galassi;a Anna Teresa, Ramadori;a § Alfredo, Burini;a Daniela, Micozzi;b Stefania, Pucciarelli.b
a Scuola di Scienze e Tecnologie, Divisione di Chimica, Università di Camerino, Via Sant’Agostino, 1, Camerino, I-62032, Italy.
b Scuola di Bioscienze e Medicina Veterinaria, Polo di Bioscienze, Università di Camerino, Via Gentile III da Varano, Camerino, I-62032, Italy.
§ fellow granted by project T.R.A.S.P.A.R.E.N.T.E. DGR 1464 del 7/11/2011, Regione Marche.
Silver nanoparticles (AgNPs) have attracted extensive research interest due to their attractive optical, electronic properties and excellent antimicrobial activities. AgNPs exhibit strong cytotoxicity towards a broad range of microorganisms and are widely used as an antibacterial agents.1 The advantage of AgNPs compared to bulk metal or salts is the slow and regulated release of silver from nanoparticles, thereby causing long lasting protection against bacteria. The antimicrobial activity of AgNPs is comparatively better than most prominent antibiotics used worldwide.2 Numerous methods have been developed for the preparation of AgNPs. The most common method is the chemical reduction of silver salt by a reducing agent in the presence of a stabilizing agent. In this work AgNPs have been prepared by reducing silver cations with NaBH4 and using as stabilizer sodium citrate, PVP (polivinylpirrolidone) or polysaccharides. AgNPs so obtained were characterized as average 10 nm particles by DLS and UV-vis spectroscopy. This work has the aim to verify the biocide action of silver nanoparticles mainly in plasters but also in other substrates occurring in a civil environment to reduce exposure to risk of infection by people with weak immune system. The study was focused to develop a method of study for each kind of material both in the AgNPs dispersion’s method and on the antimicrobial activity of the resulting substrate treated with AgNps. The antimicrobial activity has been led on Escherichia coli cells cultured in Luria Broth.3 The minimum concentration needed to have effects has been determined in each case. Moreover a comparative study of the inhibitory effect of AgNPs and silver salts such as AgNO3 and AgCF3SO3 on the regards of DHFR (DeHydroFolateReductase) from E. Coli has been performed.
1) Xihui Zhao; Yanzhi Xia; Qun Li; Xiaomei Ma Fengyu, Quan Cunzhen Geng, Zhenyu Han. Colloids and Surfaces A: Physicochem. Eng. Aspects 444, 2014, 180-188. doi.org/10.1016/j.colsurfa.2013.12.008
2) R. Roy, M. R. Hoover, A. S. Bhalla, T. Slawecki, S. Dey, W. Cao, J. Li, S. Bhaskar. Material Research Innovation, Volume 11, Issue 1 ,2007, 3-18. DOI 10.1179/143307507X196167.
3) Ivan Sondi and Branka Salopek-Sondi. Journal of Colloid and Interface Science 2004, 275, 177-182. doi:10.1016/j.jcis.2004.02.01
Dihydrofolate reductase: a new molecular target for gold(I) drugs?
No full textDehydrofolate reductasi : a new molecular target for gold(I) drugs?
R. Galassi,a A. Burini,a D. Micozzi,a A. Dolmella,b S. Pucciarellic
a School of Science and Technology, Chemistry Division, University of Camerino, 62032 Camerino (Italy);
b Department of Pharmaceutical Sciences, University of Padova, 35131 Padova (Italy);
c School of Biosciences and Biotechnology, University of Camerino, 62032 Camerino (Italy).
[email protected]
DHFR is an important enzyme mainly involved in the synthesis of pyrimidine and thymine basilar for the DNA replication and hence the cell proliferation. To perform its functions the DHFR enzyme needs the presence of a substrate and a cofactor such as NADPH and H2F.[1] Despite its fundamental importance in the cell’s metabolism this enzyme has never been tested as molecular target for gold(I) compounds which anti-proliferative action was ascertained. In fact, it is generally accepted that gold compounds act as multi-target drug and the proposed mechanisms mainly involve enzymes. Moreover, their action is function of their overall structure and of the nature of the ligands around the central metal atom.
Some pyrazolyl and imidazolyl gold(I) phosphane complexes synthesized by us have shown anticancer properties and they successfully inhibit some seleno-dependent enzymes such as thioredoxina reductase and glutathione peroxidase.[2] To investigate in depth their action on the regards of enzyme, inhibitory studies on DHFR have been carried out. Moreover, appropriate modifications in their structure have been performed in order to get information about the structure/properties relationship.
DTNB assays, stability studies in solution and affinity constant determinations have been performed too. Some conclusions about the possible mechanism of action may be taken accordingly.
Acknowledgements
Authors are grateful to CIRCSMB for Daniela Micozzi fellowship.
References
[1] M. Brandsch, I. Knütter, E. Bosse-Doenecke, J. Pharm. Pharmacol. 2008, 60, 543.
[2] R. Galassi, A. Burini, S. Ricci, M. Pellei, M. P. Rigobello, A. Citta, A. Dolmella, V. Gandin, C Marzano. Dalton Trans., 2012, 41, 530
A proteomic study on the effect of polyphenolic extract from extra-virgin olive oil on a yeast model of aging
No full textSaccharomyces cerevisiae can serve as a model to study the molecular mechanism underlying human neurodegenerative diseases and aging [1, 2]. In this study, a S. cerevisiae strain that express a glutamine repeat domain (103Q) of Huntingtin, the causing agent of protein misfolding leading to Huntington disease (HD), has been used for monitoring the effect of a polyphenol extracts from extra-virgin olive oil (EVOO) on neurodegeneration. In fact, it is well known that olive polyphenols such as tyrosol, hydroxytyrosol, and their complex derivatives exert a protective effect against oxidative damage, one of the main factors associated with age-related pathologies.
At this purpose, the chronological aging of yeast strain 103Q, has been monitored in comparison with the wild type strain (GFP) by assessing the cell viability and the proteomic pattern. Polyphenols, after their extraction from EVOOs, have been introduced in the culture broth of both 103Q and GFP strains. Cells from both strains were harvested 6 (young), 15 (adult) and 26 (old cells) days after inoculation and the protein expression was evaluated in both strains by two-dimensional electrophoresis (2DE), consisting of a first dimension on an immobilized pH gradient 3-10 gel (Immobiline DryStrip gel, 18cm, GE Healthcare) and a second dimension through a 13% SDS-PAGE [3]. After electrophoresis, the silver stained gel was subjected to image analysis (by the PDquest software) and to mass spectrometry for protein identification.
As a result, we found that polyphenols induce the expression of several proteins both in the neurodegeneration model (103Q) and in the controls (GFP). Until now, two spots were identified, one as "hypothetical protein AWRI1631_45230" [S. cerevisiae AWRI1631], and the other one as "YOR012W-like protein" [S. cerevisiae AWRI796], whose function is not known. Interestingly, in the system where huntingtin is expressed (103Q), we found a protein region, with a broad molecular weight (MW) around 22.2 kDa, and an isoelectric point (pI) of 3.8 which decreases after the administration of polyphenols. Ocampo and co-workers [2], during immunoblotting experiments performed to follow the induced 103Q expression and oligomerization, found the presence of degradation products at low molecular weight (about 35 kDa). With this in mind, we could speculate that the protein region showing the broad molecular weigh observed in our experiments may be a degradation product of the 103Q protein. Obviously, further studies should be carried out in order to validate this hypothesis.
In conclusion, the results of the present study are very encouraging, and we can conclude that the yeast model of neurodegeneration proved to be a very promising system for the evaluation of antioxidant/nutraceutical substances efficacy towards neurodegenerative diseases.
References:
[1] Mortimer R.K., Johnston J.R. Life span of individual yeast cells. Nature. 1959; 183:1751-1752.
[2] Ocampo A., Liu J. and Barrientos A. NAD salvage pathway proteins suppress proteotoxicity in yeast models of neurodegeneration by promoting the clearance of misfolded/oligomerized proteins. Hum Mol Genet. 2013;22:1699-1708.
[3] S. Vincenzetti, A. Amici, S. Pucciarelli, A. Vita, D. Micozzi, F. M. Carpi, V. Polzonetti, P.
Natalini, P. Polidori. A Proteomic Study on Donkey Milk. Biochem Anal Biochem. 2012; 1, 109
Enzymology of Pyrimidine Metabolism and Neurodegeneration
No full textIt is well known that disorders of pyrimidine pathways may lead to neurological, hematological, immunological diseases, renal impairments, and association with malignancies. Nucleotides homeostasis depends on the three portion of pyrimidine metabolism: de novo synthesis, catabolism and recycling of these metabolites. Cytidine and uridine, in addition to be used as substrates for pyrimidine nucleotide salvaging, also act as the precursors of cytidine triphosphate used in the biosynthetic pathway of both brain phosphatidylcholine and phosphatidylethanolamine via the Kennedy cycle. The synthesis in brain of phosphatidylcholine and other membrane phosphatides can utilize, in addition to glucose, three compounds present in the blood stream: choline, uridine, and a polyunsaturated fatty acis like docosahexaenoic acid. Some authors, using rat models, found that oral administration of two phospholipid precursors such as uridine and omega-3 fatty acids, along with choline from the diet, can increase the amount of synaptic membrane generated by surviving striatal neurons in rat models with induced Parkinson's disease. Other authors found that in hypertensive rat fed with uridine and choline, cognitive deficit resulted improved. Uridine has also been recently considered as a neuroactive molecule, because of its involvement in important neurological functions by improving memory, sleep disorders, anti-epileptic effects, as well as neuronal plasticity. Cytidine and uridine are uptaken by the brain via specific receptors and successively salvaged to the corresponding nucleotides. The present review is devoted to the enzymology of pyrimidine pathways whose importance has attracted the attention of several researchers investigating on the mechanisms underlying the physiopathology of brain
Antioxidant Properties of Donkey Milk
No full textIn the present work the well-known beneficial donkey’s milk (DM)
characteristics to human health have been investigated, focusing
the attention on anti-oxidant properties. For this purpose, vitamin
C, lactoferrin, lactoperoxidase activity and total antioxidant power
(PAO) were determined on milk whereas the reactive oxygen
metabolites (ROM) and PAO concentrations were evaluated on
blood. The trial was carried out on 10 pluriparous jennets. Sterile
blood and milk samples were collected during three different periods
of lactation. Microbiological tests, SCC (by a fluoro-optoelettronic
method), PAO and ROM determinations were performed
by commercial kits. Quantitative determination of lactoferrin from
DM was determined by an HPLC method, lactoperoxidase activity
was evaluated by a continuous spectrophotometric method, vitamin
C content determination was performed using a commercial
kit. The obtained data were subjected to the analysis of variance
(ANOVA, SPSS). Mean values of the investigated param -
eters±standard errors were calculated. No significant differences
were observed for ROM, SCC and neither blood or milk PAO
(respectively, 4.45±0.1 mmol/L; 50.9±19.9¥103/mL; 166.2±4.1 and
55.5±3.2 μmol HClO/mL). The fresh DM contains 57 mg/L of vitamin
C, similar to that of human milk but higher compared with
that of cow’s milk. The lactoferrin concentration in DM is
0.080±0.0035 g/L, very close to that obtained for mare, bovine but
lower if compared with human milk. Mature DM contains low but
detectable LPO activity (0.11±0.027 mg/L) similarly to value
obtained from human milk. Concluding, for the first time were
determined the PAO and ROM reference range in DM, furthermore
the high content of vitamin C in DM confirms the important
nutritional characteristics of this product
The effects of low voltage electrical stimulation on donkey meat
No full textThe effects of early post-mortem low voltage electrical stimulation (28 V, 60 Hz) on biochemical changes and on final tenderness in Longissimus Thoracis et Lumborum (LTL) muscle from donkey carcasses was studied. ES significantly accelerated the glycolytic process only during the first hours post-mortem, with a faster pH fall at 1, 3 and 6 h post-mortem, and a reduction in ATP content at 3 and 6 h post-mortem. The activities of μ-calpain and calpastatin were reduced (P < 0.05) by the treatment in the first 6 h post-mortem. Tenderness was improved (P < 0.05) at 4 and 7 days post-mortem by the application of ES. Muscle colour (L*) was lighter (P < 0.05) in stimulated carcasses
Role of polyamines in aging: impact of spermidine consumption on neurodegeneration.
No full textAge-dependent neurodegeneration is associated with both environmental factors and genetic mutations, determining a complex range of common denominators like genomic instability, loss of proteostasis, mitochondrial dysfunction, epigenetic alterations, altered cell signaling and impaired nutrient sensing [1]. Deregulation of the cellular mechanisms of defense uncharged of controlling and antagonizing the time-dependent accumulation of cellular damage can lead to accelerated aging and neurodegenerative disease. Age dependent memory impairment starts in midlife and worsens with advancing age, suggesting that the greatest driving factor is age itself [2]. The senescence-accelerated mouse prone 8 (SAMP8) strain is a spontaneous animal model of accelerated aging, and age dependent cognitive impairment. Polyamines have been found to be associated with aging: in several organisms, including yeast and humans, the levels of polyamines decrease with age [3]. Remarkably, restoring their levels have resulted to be beneficial.
In this study we have simulated a spermidine-rich diet in SAMP8 and its control strain SAMR1, by supplementing 2mM spermidine ad libitum in drinking water, starting from 2 months of age. We analyzed the levels of different metabolites in blood, brain and small intestine of mice both in treated and control groups of both mice strains. In particular, we have focused on polyamines metabolism (putrescine, spermidine, spermine), NAD metabolism (nicotinate mononucleotide, NaMN, nicotinamide mononucleotide, NMN, nicotinate adenine dinucleotide, NaAD, nicotinamide adenine dinucleotide, NAD, nicotinamide, Nm) and ATP metabolism (ATP, ADP, AMP), in order to assess differences between treated and control groups and among the strains, and also to infer a possible interplay between spermidine administration and energetic metabolism. We also tested cognitive performance of SAMP8 and SAMR1 mice, by using Novel Object Recognition (NOR), a short-term memory test. The obtained results shown that spermidine treatment has the following effects on SAMP8 mice:
it favours putrescine increase in blood, brain and small intestine. The increase of putrescine is a mechanism normally adopted to counteract the effects of aging;
it decreases NaMN levels in blood and favours NAD synthesis in small intestine;
it determines the increase of ATP in blood and brain;
spermidine consumption has an impact in ameliorating cognitive performance of ten months old SAMP8 mice.
References:
[1] López-Otín, C., Blasco, M. A., Partridge, L., Serrano, M., & Kroemer, G. (2013). The hallmarks of aging. Cell, 153(6), 1194-1217.
[2] Gupta,V.K., Pech, U., Bhukel, A., Fulterer, A., Ender, A., Mauermann, S.F., et al. (2016) Spermidine suppresses age-associated memory impairment by preventing adverse increase of presynaptic active zone size and release. PLoSBiol 14(9):e1002563.
[3] Minois, N., Carmona-Gutierrez, D., & Madeo, F. (2011). Polyamines in aging and disease. Aging (Albany NY), 3(8), 716-732
VITELLOGENIN IN GRAY MULLET (MUGIL CEPHALUS): ONE-STEP PURIFICATION, CHARACTERIZATION, AND ELISA DEVELOPMENT
No full textGray mullet, Mugil cephalus L., is a cosmopolitan fish species, widely distributed in coastal waters, lagoons, and estuaries between latitudes 42° N and 42° S. This mullet is an economically important species for both aquaculture and commercial fisheries around the world. A new single-step purification procedure was developed to purify gray mullet vitellogenin (VTG), from 17β-estradiol (E2)-treated gray mullet plasma. This method was performed by high performance liquid chromatography (HPLC) technique, using weak anion-exchange chromatography with a discontinuous gradient of NaCl (buffer B was 25 mM Tris-HCl, pH 8.5 + 0.416 M NaCl) with steps of 16.6 mM for 5.50 min from 0 to 0.416 M NaCl. SDS-PAGE electrophoresis revealed the presence of an intense protein band in E2-treated fish with a MW of 190 KDa. The 190 kDa is also the major protein band in the eluted fractions. Lower molecular weight proteins, corresponding to breakdown products of VTG, were also visible. However, by performing native-PAGE electrophoresis followed by Coomassie blue stain, two pure and non-degraded protein bands were detected. The two bands can be attributed to a monomeric and a dimeric form of VTG, as it has been already observed for VTG purified from other fish plasma samples. A polyclonal antibody against gray mullet VTG was raised in rabbits and found to be specific for gray mullet VTG through western blot analysis. Additionally, an enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of VTG in plasma samples of gray mullet
Effect of polyphenolic extract from extra-virgin olive oil on a yeast model of aging and proteotoxicity.
No full textIn 1959, Mortimer and Johnson suggested that the budding yeast Saccharomyces cerevisiae might serve as a model for aging. Although the causes of aging are multifactorial, all eukaryotes present conserved longevity pathways and molecular mechanisms involved in this process [1].
Yeast cells are able to divide in a nutrient rich medium until an hypometabolic state, where can remain for weeks, characterized by nutrients exhaustion. Yeast chronological aging is also characterized by a shift in energy utilization, which is derived mainly from mitochondrial respiration; this leads to an increase in mitochondrial-derived ROS which contribute to the aging of post-mitotic cells like observed in neurons. Saccharomyces cerevisiae can serve as a model to study the molecular mechanism underlying human neurodegenerative disease, including Huntington’s disease [2]. For this reason we have created a yeast strain that can express a glutamine repeat domain (103Q) of Huntingtin (HD) which represents the causing agent of protein misfolding leading to Huntington disease. In our study we have monitored the chronological aging of yeast strains containing the mutated glutamine repeat domain of Huntingtin, in comparison with the wild type strain, by assessing the cell viability, the NAD related metabolic profile and the proteomic pattern. NAD biosynthesis and utilization pathways play a major role in cell protection against proteotoxicity and in increasing yeast lifespan. In both yeast strains we have also tested the effect of an extra virgin olive oil polyphenolic extract added to the culture medium. Tyrosol, hydroxytyrosol, and their complex derivatives (e.g oleuropein and other secoiridoids), the most abundant olive polyphenols, are known to exert a protective effect against oxidative damage, one of the main factors associated with age-related pathologies.
In our study we have observed that the yeast strain expressing the 103Q mutant HD protein decreases cell viability more rapidly than the wild type, due to the neurotoxicity of the mutant protein. Regarding nucleotide analysis, we have observed an increase in NaMN levels in the 103Q HD containing strain could be in agreement with the increase of NAD+, which exerts a protective effect against neurodegeneration; the increase of NaM levels in the strain expressing the mutant protein and treated with the polyphenolic extract could suggest the role of these compounds on sirtuins activation
