1,720,997 research outputs found
New bioactive compounds of vegetable sources in cancer therapy
No full textMany of the current anticancer drugs have their origin from natural sources. Phytochemicals have the ability to suppress cancer cell growth, induce apoptosis, inhibit invasion by targeting multiple signal transduction networks. Research directed toward elucidating underlying molecular mechanisms of chemoprevention led to the identification of several potential cellular targets. These include transcriptional factors and their upstream protein kinases, such as the family of MAPK, protein kinase B/Akt, GSK, etc. The current research in our laboratory concerns evaluation of chemopreventive effects of some anti-cancer phytochemicals and elucidation of their underlying molecular mechanisms using genomic and proteomic techniques. Protein targets of phytochemicals are identified by two-dimensional gel electrophoresis and mass spectroscopy (MS/MS) analysis. The most effective chemopreventive agents including resveratrol, curcumin and carnosol have potential to be used as adjuncts to current cancer therapies in ovarian and breast cancer
Buffer capacity in the blood of the hemoglobinless Antarctic fish Chionodraco hamatus
Full text linkBlood acid-base homeostasis of the hemoglobinless Antarctic teleost Chionodraco hamatus was analyzed by measuring the titratable buffer capacity (β) and carbonic anhydrase (CA) activity in blood samples. Results were compared with those obtained in the red-blooded temperate fish Anguilla anguilla. Data show that the titratable blood buffer capacity of icefish, in the range of red-blooded teleost species, was significantly higher than that of A. anguilla. Furthermore, plasma inorganic phosphate and reactive sulfhydryl content was significantly higher in the Antarctic species. No enzymatic activity of CA was detected in blood samples of Antarctic fish, suggesting the absence of a blood CA-isozyme
Lipid composition of intestinal cell membranes of the Antarctic fish, Pagothenia bernacchii: comparative studies with the temperate fish Anguilla anguilla.
No full textEffect of the prion protein fragment hPrP[173-195] on the proliferation of B104 neuroblastoma cells.
No full textPrion diseases are a group of neurodegenerative pathologies that recognize, as aetiopathologic agent, an aberrant isoform of the prion protein (PrPc), named PrPsc [1]. Recently, a correlation between the structural state of the prion protein and its neurotoxicity has been demonstrated and, by using different prion peptide fragments of the structured portion of the protein, the molecular mechanisms involved in the pathogenesis of prion diseases begin to be elucidated [2]. In this study, we examined the neurotoxicity of the prion protein fragment PrP[173-195] (Ac-NNFVHDCVNITIKQHTV-TTTTKG-NH2) on a neuronal cell line (B104 neuroblastoma cells). PrP[173-195] corresponds to a highly conserved region that could provide a nucleation site for the sequence-dependent unfolding and may be implicated in the conformational transition from PrPC to PrPSc. Stock cultures of B104 cells were maintained in Eagle's minimal essential medium (MEM) as described elsewhere [3]. B104 cells were plated into 96-well trays and maintained in the medium containing 10% fetal bovine serum (FBS) for at least 24 h, then, a cell aliquot was switched to a medium containing 0.1% FBS. Then, all cells were treated with increasing concentrations of PrP[173-195] up to 12 μg/ml and cell viability was assessed by MTT (3,[4,5dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) test after 18, 24 and 48 h of incubation with the peptide fragment. After 18 h incubation, a maximum of 30% reduction of cell viability was observed in all cells at peptide concentrations of 2 μg/ml. After 24 h incubation, the same concentration of peptide induced a maximum of reduction of almost 55% cell viability in 0.1% FBS, while in cell maintained in 10% FBS the toxic effect was maintained at the same level observed after 18 h incubation. After 48 h incubation, a continuous, concentration-dependent reduction of cell viability up to 90% (10% FBS) and 80% (0.1% FBS) was measured up to 12 μg/ml. To assess the specificity of the neurotoxic effects of the fragment, was assayed by performing the same analyses on human MCF7 breast cancer cell line
ANALYSIS OF COPPER TRANSPORT IN B104 NEUROBLASTOMACELLS
No full textCopper is an essential trace metal required as a cofactor in a broad range
of enzymatic functions including cellular antioxidant activity, oxidative
phosphorylation and neurological function. If in excess, it can initiate a
cascade of events leading to cell disruption. Consequently, cells have
developed sophisticated homeostatic mechanisms to maintain adequate
intracellular copper concentrations (Pena et al., J. Nutr. 129(7): 1251-60,
1999). In the attempt to characterise the components of brain copper
homeostasis, we studied the expression and function of copper transport
systems in a rat neuroblastoma cell line (B104). In our cell model copper
fluxes could be related to the functional expression of a member of the
Ctr family named rat Ctr1 and to the primary active copper transport
MNK protein. In both cases we demonstrated their mRNA expression in
B104 cells using specific primers and the RT-PCR amplification method.
Transmembrane copper fluxes were measured by loading cells with the
Cu2+-sensitive fluorophore Phen Green SK. Copper addition in the extra
cellular medium induced a rapid fluorescence quenching of the entrapped
probe, probably due to copper entry via Ctr1 protein, followed by a fast
recovery of fluorescence related to a putative MNK-mediated copper
efflux. The initial rate of copper uptake was dependent on external ion
concentrations and saturated at approximately 1 5M Cu2+. Furthermore,
we found that zinc can affect copper transport by partially inhibiting the
uptake. In conclusion, our results support the hypothesis that copper
transport in neuroblastoma cells is a complex phenomenon presumably
mediated by more than a single carrier process
Proteomic map of peripheral blood mononuclear cells
No full textIn the field of proteomics extensive efforts have been focused on the knowledge of proteins
expressed by different cell types. In particular, enormous progress has been done in the
characterization of blood cellular components. In this work, we have established a public
2-DE database for human peripheral blood mononuclear cells (PBMCs) proteins. Two
hundred and forty-six spots corresponding to 174 different proteins have been identified
on 2-DE gels from PBMCs isolated from six healthy individuals. All the identified proteins
have been classified in thirteen categories on the basis of their differential functions or
cellular localization and annotated at the http://physiology.unile.it/proteomics. The role of
several proteins has been discussed in relation to their biological function. We intend to
show the potentiality of PBMCs to investigate the proteomics changes possibly associated
with a large number of pathologies such as autoimmune, neurodegenerative and cancer
diseases
INTESTINAL PEPTIDE TRANSPORTER OF THE ANTARCTIC HAEMOGLOBINLESS TELEOST CHIONODRACO HAMATUS
No full textA member of peptide transporter family, well characterised in higher vertebrates, was found in the intestine of the Antarctic waters haemoglobinless teleost Chionodraco hamatus (PepT-Ice; Maffia et al. J. Exp. Biol. 206:705, 2003). PepT-Ice shares high similarity to the low-affinity mammalian PepT1, but also possesses cold-adapted features. To better clarify the molecular basis of PepT-Ice evolutive adaptation, we studied its structural characteristics and tissue distribution. The full-length nucleotide sequence encoding for PepT-Ice was obtained by RT-PCR and 5’-/3’-RACE. PepT-Ice cDNA was 2509 bp long, with an orf of 2232 bp encoding for a putative protein of 743 amino acids. Hydropathy analysis predicted at least 12 potential transmembrane domains (TMD) with a large extracellular loop between TMD IX and X. Six putative extracellular N-glycosilation sites and nine and three intracellular consensus regions for protein kinase C and protein kinase A were identified. PepT-Ice exhibited higher percentage of identity with mammalian PepT1 (59-61%) than PepT2 (48-50%). Also, highest identity (69%) was found with zebrafish PepT1. Phylogenetic analysis clustered PepT-Ice to the PepT1 branch of the phylogenetic tree, In PepT-Ice, the presence of such hydrophobic amino acids as Gly-599, Leu-160 and Gly-271 in substitution of two Glu and one Asp, respectively, could play a relevant role in adaptive mechanisms to cold, possibly reducing intramolecular interaction and increasing protein flexibility. Using PepT-Ice specific primers, 630 bp RT-PCR amplification products were detected in mRNA extracted from C. hamatus intestine, kidney, liver, gills, brain and heart. The molecular characterization of PepT-Ice has been achieved that might elucidate the low temperature adaptation mechanisms of membrane transporters and functional relationships among vertebrate peptide transporters
Effects of copper availability on Ctr1 expression in B104 rat neuroblastoma cell line
No full textAim: Copper is an essential trace metal required as a cofactor in a broad range of enzymatic functions. Copper transporter-1 (Ctr1), ubiquitously expressed, is believed to play a major role in Cu uptake into the cell. In the present study we used a rat neuroblastoma cell line (B104) as a cell model aiming to examine how extracellular copper availability can modulate the expression of Ctr1 and Cu fluxes.
Methods: B104 cells were cultured under Cu depletion by exposure to cuprizone, a specific Cu chelating agent, and Cu supplementation for rising time periods (48, 96 hours). Ctr1 protein levels were assessed by Western Blotting analysis. Changes in transmembrane copper fluxes were measured by a fluorimetric method employing the Cu+-sensitive fluorophore Phen Green SK.
Results: Treatment of cells with 50 μM CuCl2 for 48 h determined a significant increase in Ctr1 expression, while Cu supplementation prolonged for 96 h was found to restore basal protein levels. Exposure to over-physiological Cu level didn’t alter ion transmembrane fluxes as measured by the fluorescence method, indicating that excess protein was unfunctional. Analogously to Cu supplementation, exposure of cells to ion depletion for 48 h induced an increase in protein synthesis, probably as an immediate adaptive response to inadequate copper levels in basal culture medium. No change in protein expression was detected after 96 h treatment.
Conclusion: In summary, our results suggest that short-term adapting strategies of B104 cells to varying Cu availability in culture medium involve directly Ctr1, even if we are not able to exclude other possibilities. For a longer time period treatment, presumably other mechanisms can be involved, such as Ctr1 co-regulation with other ion transporters (i.e. DMT1, Divalent Metal Transporter 1)
Analysis of copper transport in B104 neuroblastoma cell model
No full textThe arising of a number of neurodegenerative disorders in mammals has been correlated
to the impairment of copper homeostasis in the CNS that is far from being completely
understood. In the attempt to give a contribution to clarify some molecular aspects of
this relationship, we used a rat neuroblastoma cell line (B104) as a cell model aiming to
examine how extracellular copper availability can affect the protein expression pattern
and Cu fluxes. Briefly, B104 cells were cultured under Cu depletion for rising time
periods (48, 96 hours). To reveal possible changes in the protein expression pattern
(CTR1, DCT1, PRPc, ATP7A) we conducted Real Time PCR and Immunoblotting
analysis, that enlightened an expected complexity of cuproprotein genes expression and
regulation. Surprisingly the activation of a transcriptional adaptive response involving
the cellular prion protein and the high affinity copper transporter CTR1 was evidenced,
that hasn’t been reported before. We tried to estimate possible alterations of
transmembrane copper fluxes in ion deprived cells by the employment of a fluorimetric
method based on the use of the Cu-sensitive probe Phen Green SK. To verify the
suitability of such a method with respect to our intent a preliminary study was carried
out to verify the dependence of the rate of copper influx on external potassium and the
presence of metal ions (Ag, Cd, Mn, Zn), typically associated to CTR1-mediated Cu
entry. A confocal microscopy approach was additionally developed to confirm the
specificity of the response of the intracellular Phen Green SK probe to copper intake.
In conclusion, our results support the hypothesis that the adapting strategies developed
by B104 neuroblastoma cell model in response to reduced copper availability pass
through the activation of a wide transcriptional adaptive response with suspected
implications in the pathophysiology of neurodegenerative disorders
Molecular aspects of the adaptation to low temperature of functional protein in Antarctic poikilotherms
No full textAim: Physiological processes are impaired by low temperature. The aim of our research was: i) to understand the molecular aspects of the physiologic and phylogenetic adaptation of functional mechanisms of life to the cold, which allow poikilotherms to survive well in thermodynamically disadvantageous conditions; ii) to provide the scientific background for the biotechnological exploitation of cold-adapted functional mechanisms.
Methods: Functional protein (such as carbonic anhydrase and the dipeptide carrier) were isolated from Antarctic teleosts, purified, sequenced and processed for the biochemical and the physiological characterisation. The analysis of the protein lipid microenvironment was performed to assess the role of the lipid-protein interactions on the overall functionality of the protein. FPLC and HPLC were used for protein isolation and purification. Sequencing was carried out by direct protein and cDNA sequencing and mass spectrometry. Isoelectric focusing and Western blot analysis were used to show post-translational modifications. Fluorimetry, spectrophotometry, isotope labelled substrates were used for the biochemical/physiological characterisation of enzymes and transporters. Gas-chromatography, TLC, fluorimetry and spectrophotometry allowed the analysis of the lipid microenvironment. Bioinformatics was applied to perform the phylogenetic analyses.
Results: Some molecular aspects of the capability of protein to perform well at low temperature have been identified. The phylogenetic relationships with analogues from higher temperature have been assessed. The lipid analysis showed the adaptive contribution of the membrane lipid microenvironment to the protein adaptation to cold.
Conclusions: The results obtained extend our knowledge about the key molecular characteristics and mechanisms which contrast the thermodynamic disadvantages of low temperature toward any physiological process
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