Association of MT1A haplotype with cardiovascular disease and antioxidant enzyme defense in elderly Greek population: comparison with an Italian cohort

Metallothioneins (MT), the antioxidant zinc-binding proteins, seem to mediate cardioprotection. It has been postulated that zinc homeostasis and MT function may be altered, as a consequence of oxidative stress, in cardiovascular disease (CVD), with a potential implication of MT genetic polymorphisms. The present study explores the role of +647A/C and +1245A/G MT1A polymorphisms on the susceptibility to CVD, zinc status and enzyme antioxidant activity, in the Greek and Italian populations. The country selection was based on the lower zinc status and the reduced zinc dietary intake in Greece than in Italy despite the similar Mediterranean dietary pattern. A total of 464 old, healthy control subjects and 369 old CVD patients more than 70 years of age were studied. Logistic regression model indicated that +1245 MT1A G+ genotype significantly increased the risk of CVD in Greece (34.4% vs. 23.2%; odds ratio=1.88, 95% confidence interval=1.14–3.08; P=.013) but not in Italy. Haplotype analysis showed an increment of CG haplotype frequency in CVD Greek patients (17.4% vs. 10.6%, P<.05). Differential country-related frequency distribution was also recorded. Applying a multivariate regression model, +647/+1245 MT1A haplotype was associated with a modulation of enzyme antioxidant activities in both countries. Decreased plasma zinc and reduced intracellular Zn release, as well as increased enzyme antioxidant activity, were more apparent in Greek healthy donors than in Italy. In conclusion, +1245 MT1A polymorphism and +647/+1245 MT1A haplotype are implicated in CVD in Greece but not in Italy, suggesting a role of gene–diet interaction in the disease predisposition.     

Changes of the ganglioside pattern and content in human fibroblasts by high density cell population subculture progression

In this study we show that the ganglioside content and pattern of human skin fibroblasts change along the process of cell subculture progression by varying the cell density.GM3, GD3 and GD1a were components of the total cell ganglioside mixtures extracted from cells, but GD1a was in all the extracts a minor component or very scant. Other gangliosides present in traces were not characterised. The fibroblast ganglioside content of 52 pools of cells obtained from 5 different cell lines cultured at variable cell density ranged from 2.0 to 13.1 nmoles per mg of cell protein. The molar ratio between GM3 and GD3 varied from 418 to 0.6 in the ganglioside mixtures, as determined by densitometric quantitative analysis after thin layer chromatographic separation.Both the ganglioside content and the GM3/GD3 molar ratio were constant along several passages of subculture progression performed by plating cells collected at confluence. Instead, when the subculture progression was performed by plating cells collected at a few days after reaching confluence, a progressive increase of the ganglioside content was observed. GD3 increased proportionally more than GM3 so that a progressive decrease of the ratio between GM3 and GD3 was observed. In some experiments, GD3 was very scant at the beginning of the progression, while it was near 30% after 5 passages under these conditions. The progressive increase of GD3 along the high density cell population subculture progression was associated to a moderate increase of the mRNA GD3 synthase. Published in 2003.     

Grafting of "abbreviated" complementarity-determining regions containing specificity-determining residues essential for ligand contact to engineer a less immunogenic humanized monoclonal antibody

Murine mAb COL-1 reacts with carcinoembryonic Ag (CEA), expressed on a wide range of human carcinomas. In preclinical studies in animals and clinical trials in patients, murine COL-1 showed excellent tumor localization. To circumvent the problem of immunogenicity of the murine Ab in patients, a humanized COL-1 (HuCOL-1) was generated by grafting the complementarity-determining regions (CDRs) of COL-1 onto the frameworks of the variable light and variable heavy regions of human mAbs. To minimize anti-V region responses, a variant of HuCOL-1 was generated by grafting onto the human frameworks only the "abbreviated" CDRs, the stretches of CDR residues that contain the specificity-determining residues that are essential for the surface complementarity of the Ab and its ligand. In competition RIAs, the recombinant variant completely inhibited the binding of radiolabeled murine and humanized COL-1 to CEA. The HuCOL-1 and its variant showed no difference in their binding ability to the CEA expressed on the surface of a CEA-transduced tumor cell line. Compared with HuCOL-1, the HuCOL-1 variant showed lower reactivity to patients' sera carrying anti-V region Abs to COL-1. The final variant of the HuCOL-1, which retains its Ag-binding reactivity and shows significantly lower serum reactivity than that of the parental Ab, can serve as a prototype for the development of a potentially useful clinical reagent.     

The genetic control of chemically and radiation-induced skin tumorigenesis: a study with carcinogenesis-susceptible and carcinogenesis-resistant mice

Outbred carcinogenesis-resistant (Car-R) and carcinogenesis-susceptible (Car-S) mouse lines were generated by phenotypic selection for resistance or susceptibility to two-stage skin carcinogenesis. These two Car mouse lines differ by >100-fold in susceptibility. In the present study, we tested the hypothesis that a subset of genetic loci responsible for susceptibility or resistance to chemical skin tumorigenesis may also be involved in radiation-induced skin tumorigenesis. Skin tumorigenesis was tested in groups of Car-S/R mice after X-ray initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA) promotion. We found that ionizing radiation can initiate skin tumors in Car-S mice but not in Car-R mice. In Car-S mice, the most effective radiation doses (6 and 10 Gy given in four fractions) gave a threefold increase in tumor multiplicity and a twofold increase in tumor incidence compared to a TPA-only control group. We performed a molecular analysis of Hras gene mutations in skin tumors of Car-S mice induced by X-ray initiation/TPA promotion or by TPA promotion alone. The most notable difference emerging from the comparison of these mutation patterns is the high incidence ( approximately 50%) of papillomas lacking Hras gene mutations in X-ray-initiated/TPA-promoted papillomas compared to 13% in papillomas induced by TPA alone, suggesting that lack of Hras gene mutations is a consistent feature of radiation-induced papillomas.     

A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions

Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N-terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid-long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.     

The b Subunit of Escherichia coli ATP Synthase

The b subunit of ATP synthase is a major component of the second stalk connecting the F₁and F₀ sectors of the enzyme and is essential for normal assembly and function. The156-residue b subunit of the Escherichia coli ATP synthase has been investigated extensivelythrough mutagenesis, deletion analysis, and biophysical characterization. The two copies ofb exist as a highly extended, helical dimer extending from the membrane to near the top ofF₁, where they interact with the subunit. The sequence has been divided into four domains:the N-terminal membrane-spanning domain, the tether domain, the dimerization domain, andthe C-terminal -binding domain. The dimerization domain, contained within residues 60–122,has many properties of a coiled-coil, while the -binding domain is more globular. Sites ofcrosslinking between b and the a, , , and subunits of ATP synthase have been identified,and the functional significance of these interactions is under investigation. The b dimer mayserve as an elastic element during rotational catalysis in the enzyme, but also directly influencesthe catalytic sites, suggesting a more active role in coupling.     

Genetic fusions of globular proteins to the epsilon subunit of the Escherichia coli ATP synthase: Implications for in vivo rotational catalysis and epsilon subunit function

The rotational mechanism of ATP synthase was investigated by fusing three proteins from Escherichia coli, the 12-kDa soluble cytochrome b(562), the 20-kDa flavodoxin, and the 28-kDa flavodoxin reductase, to the C terminus of the epsilon subunit of the enzyme. According to the concept of rotational catalysis, because epsilon is part of the rotor a large domain added at this site should sterically clash with the second stalk, blocking rotation and fully inhibiting the enzyme. E. coli cells expressing the cytochrome b(562) fusion in place of wild-type epsilon grew using acetate as the energy source, indicating their capacity for oxidative phosphorylation. Cells expressing the larger flavodoxin or flavodoxin reductase fusions failed to grow on acetate. Immunoblot analysis showed that the fusion proteins were stable in the cells and that they had no effect on enzyme assembly. These results provide initial evidence supporting rotational catalysis in vivo. In membrane vesicles, the cytochrome b(562) fusion caused an increase in the apparent ATPase activity but a minor decrease in proton pumping. Vesicles bearing ATP synthase containing the larger fusion proteins showed reduced but significant levels of ATPase activity that was sensitive to inhibition by dicyclohexylcarbodiimide (DCCD) but no proton pumping. Thus, all fusions to epsilon generated an uncoupled component of ATPase activity. These results imply that a function of the C terminus of epsilon in F(1)F(0) is to increase the efficiency of the enzyme by specifically preventing the uncoupled hydrolysis of ATP. Given the sensitivity to DCCD, this uncoupled ATP hydrolysis may arise from rotational steps of gammaepsilon in the inappropriate direction after ATP is bound at the catalytic site. It is proposed that the C-terminal domain of epsilon functions to ensure that rotation occurs only in the direction of ATP synthesis when ADP is bound and only in the direction of hydrolysis when ATP is bound.     

ATP-citrate lyase activity and carotenoid production in batch cultures of Phaffia rhodozyma under nitrogen-limited and nonlimited conditions

ATP-citrate lyase (ACL) is the key cytoplasmic enzyme which supplies acetyl-CoA for fatty acids in oleaginous yeast. Although it has been suggested that fatty acid and carotenoid biosynthesis may have a common source of acetyl-CoA in Phaffia rhodozyma, the source for carotenoids is currently unknown. The purpose of this work was to analyze the development of ACL activity during batch cultures of P. rhodozyma under ammonium-limited and nonammonium-limited conditions and study its possible relationship with carotenoid synthesis. Every experiment showed carotenoid accumulation linked to an increasing ACL activity. Moreover, the ACL activity increased with dissolved oxygen (DO), i.e., ACL responded to DO in a similar way as carotenoid synthesis. Additionally, in the ammonium-limited culture, ACL activity increased upon ammonium depletion. However, the contribution to carotenoid accumulation in that case was negligible. This suggests that P. rhodozyma has developed two components of ACL, each one responsive to a different environmental stimulus, i.e., DO and ammonium depletion. The role of each component is still unknown; however, considering that the former responds to DO and the known role of carotenoids as antioxidants, it may be a provider of acetyl-CoA for carotenoid synthesis.     

Interfacing 3D Engineered Neuronal Cultures to Micro-Electrode Arrays: An Innovative In Vitro Experimental Model

Currently, large-scale networks derived from dissociated neurons growing and developing in vitro on extracellular micro-transducer devices are the gold-standard experimental model to study basic neurophysiological mechanisms involved in the formation and maintenance of neuronal cell assemblies. However, in vitro studies have been limited to the recording of the electrophysiological activity generated by bi-dimensional (2D) neural networks. Nonetheless, given the intricate relationship between structure and dynamics, a significant improvement is necessary to investigate the formation and the developing dynamics of three-dimensional (3D) networks. In this work, a novel experimental platform in which 3D hippocampal or cortical networks are coupled to planar Micro-Electrode Arrays (MEAs) is presented. 3D networks are realized by seeding neurons in a scaffold constituted of glass microbeads (30-40 µm in diameter) on which neurons are able to grow and form complex interconnected 3D assemblies. In this way, it is possible to design engineered 3D networks made up of 5-8 layers with an expected final cell density. The increasing complexity in the morphological organization of the 3D assembly induces an enhancement of the electrophysiological patterns displayed by this type of networks. Compared with the standard 2D networks, where highly stereotyped bursting activity emerges, the 3D structure alters the bursting activity in terms of duration and frequency, as well as it allows observation of more random spiking activity. In this sense, the developed 3D model more closely resembles in vivo neural networks.