Distinctive functional features in prokaryotic and eukaryotic Cu,Zn superoxide dismutases

Bacterial and eukaryotic Cu,Zn superoxide dismutases show remarkable differences in the active site region and in their quaternary structure organization. We report here a functional comparison between four Cu,Zn superoxide dismutases from Gram-negative bacteria and the eukaryotic bovine enzyme. Our data indicate that bacterial dimeric variants are characterized by catalytic rates higher than that of the bovine enzyme, probably due to the solvent accessibility of their active site. Prokaryotic Cu,Zn superoxide dismutases also show higher resistance to hydrogen peroxide inactivation and lower HCO3- -dependent peroxidative activity. Moreover, unlike the eukaryotic enzyme, all bacterial variants are susceptible to inactivation by chelating agents and show variable sensitivity to proteolytic attack, with the E. coli monomeric enzyme showing higher rates of inactivation by EDTA and proteinase K. We suggest that differences between individual bacterial variants could be due to the influence of modifications at the dimer interface on the enzyme conformational flexibility.     

Molecular insights into mental retardation: multiple functions for the Fragile X mental retardation protein?

Mental retardation is a frequent cause of intellectual and physical impairment. Several genes associated with mental retardation have been mapped to the X chromosome, among them, there is FMR1. The absence of or mutation in the Fragile Mental Retardation Protein, FMRP, is responsible for the Fragile X syndrome. FMRP is an RNA binding protein that shuttles between the nucleus and the cytoplasm. FMRP binds to several mRNAs including its own mRNA at a sequence region containing a G quartet structure. Some of the candidate downstream genes recently identified encode for synaptic proteins. Neuronal studies indicate that FMRP is located at synapses and loss of FMRP affects synaptic plasticity. At the synapses, FMRP acts as a translational repressor and in particular regulates translation of specific dendritic mRNAs, some of which encode cytoskeletal proteins and signal transduction molecules. This action occurs via a ribonucleoprotein complex that includes a small dendritic non-coding neuronal RNA that determines the specificity of FMRP function via a novel mechanism of translational repression. Since local protein synthesis is required for synaptic development and function, this role of FMRP likely underlies some of the behavioural and developmental symptoms of FRAXA patients. Finally we review recent work on the Drosophila system that connects cytoskeleton remodelling and FMRP function.     

Expression of the Stilbene Synthase (StSy) Gene from Grapevine in Transgenic White Poplar Results in High Accumulation of the Antioxidant Resveratrol Glucosides

When present, stilbene synthase leads to the production of resveratrol compounds, which are major components of the phytoalexin response against fungal pathogens of the plant and are highly bioactive substances of pharmaceutical interest. White poplar (Populus alba L.) was transformed with a construct containing a cDNA insert encoding stilbene synthase from grapevine (Vitis vinifera L.), under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a chimeric kanamycin resistance gene. Southern blot hybridization analysis demonstrated the presence and integration of exogenous DNA sequences in the poplar genome. Expression of the stilbene synthase-encoding gene in different transgenic lines was confirmed by Western blot and Northern analyses. Compared to the controls, in the transgenic plants two new compounds were detected and were identified as the trans- and cis-isomers of resveratrol-3-glucoside (piceid) by high-pressure liquid chromatography (HPLC), UV spectrophotometry, electrospray mass spectrometry (HPLC-ESI-MS) and enzymatic hydrolysis. Since poplar is a good biomass producer and piceids are accumulated in substantial amounts (up to 615.2 microg/g leaf fresh weight), the transgenic plants represent a potential alternative source for the production of these compounds with high pharmacological value. Despite the presence of piceid, in our experimental conditions no increased resistance against the pathogen Melampsora pulcherrima, which causes rust disease, was observed when in vitro bioassays were performed.     

Designed calix[8]arene-based ligands for selective tryptase surface recognition

Basic amino acid calix[8]arene receptors for tryptase surface recognition have been synthesized. The tetrameric arrangement and the negative charge distribution close to the active sites of the enzyme, have suggested the design of complementary multifunctional receptors that might bind to the active region of the protein blocking the approach of the substrate. Kinetic inhibition analysis on recombinant lung tryptase have showed a time-dependent competitive inhibition with both initial and steady-state rate constants in the nanomolar range.     

Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.     

Solution structure of Sco1: a thioredoxin-like protein Involved in cytochrome c oxidase assembly

Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment. The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized. Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins. In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological. The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins. The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S. cerevisiae and to discuss the physiological role of the Sco family.     

A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm

De novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three-color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of approximately 80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, approximately 0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology.     

Immunoglobulins against gp273, the ligand for sperm-egg interaction in the mollusc bivalve Unio elongatulus,are directed against charged O-linked oligosaccharide chains bearing a Lewis-like structure and interact with epitopes of the human zona pellucida

In oocytes of the mollusc bivalve Unio elongatulus, gp273 is the ligand molecule for sperm-egg interaction and binding is mediated by its O-glycans. A serum raised against this protein enabled its localization in the crater region, the area of the vitelline coat where sperm recognition occurs, and showed that after cyanogen bromide fragmentation, the anti-gp273 epitope(s) was retained by a peptide where the O-glycans are localized. In this article, we utilized purified anti-gp273 immunoglobulins to characterize the corresponding epitope by: (i) immunoblotting analysis of the protein after removal of O- and N-glycans; (ii) solid phase binding analysis of anti-gp273 IgG to gp273 N- and O-glycans; and (iii) binding analysis of the same antibody to commercially available oligosaccharides. The results showed that the epitope consists of O-glycans and contains a Lewis-like structure with fucose as determinant. Anti-gp273 IgG were then used to investigate human zona pellucida by immunoelectronmicroscopy and immunoblotting. Epitopes recognized by the antibody were demonstrated on the outer surface of the zona pellucida and shown to belong to a zona pellucida protein having electrophoretic mobility similar to human ZP3. Since human sperm specifically bind to gp273, and anti-gp273 interferes with this binding a functional role for these epitopes is suggested.