The tumor suppressor hamartin enhances Dbl protein transforming activity through interaction with ezrin

The Rho guanine nucleotide exchange factor (GEF) Dbl binds to the N-terminal region of ezrin, a member of the ERM (ezrin, radixin, moesin) proteins known to function as linkers between the plasma membrane and the actin cytoskeleton. Here we have characterized the interaction between ezrin and Dbl. We show that binding of Dbl with ezrin involves positively charged amino acids within the region of the pleckstrin homology (PH) domain comprised between β1 and β2 sheets. In addition, we show that Dbl forms a complex with the tuberous sclerosis-1 (TSC-1) gene product hamartin and with ezrin. We demonstrate that hamartin and ezrin are both required for activation of Dbl. In fact, the knock-down of ezrin and hamartin, as well as the expression of a mutant hamartin, unable to bind ezrin, inhibit Dbl transforming and exchange activity. These results suggest that Dbl is regulated by hamartin through association with ezrin.     

Properties of a cobalt-reactivated form of yeast alcohol dehydrogenase

Yeast alcohol dehydrogenase (Y-ADH) is a widely studied metal-enzyme for its well-known biotechnological importance. Although its structure has been extensively investigated, some topics still remain controversial (zinc content and role), and various attempts aiming at modifying its structure to improve its catalytic properties have been made. In this paper, a metal-substituted Y-ADH has been prepared in vitro, in which one Zn atom per molecule (only one of those directly involved in catalysis) has been substituted by one Co atom. The substitution was obtained through zinc removal by a chelating treatment (with Chelex 100) followed by cobalt insertion. The zinc content in the native enzyme was preliminarily evaluated (taking care to avoid contamination) to be 4.1±0.1 g-at./molecule. After cobalt substitution, the ratio Zn:Co in the enzyme results to be 3:1. The active Co-Y-ADH has been compared with the native enzyme: it has lower specific activity (about 50%) and lower substrate affinity but greater thermo-resistance and a pH stability in a wider range than the native Y-ADH. A similar behavior, as far as cobalt content, thermo-resistance and pH stability are concerned, but greater specific activity and substrate affinity, were shown by an in vivo-substituted Co-Y-ADH obtained in a previous study.     

Theranostic Properties of a Survivin-Directed Molecular Beacon in Human Melanoma Cells

Survivin is an inhibitor of apoptosis overexpressed in different types of tumors and undetectable in most terminally differentiated normal tissues. In the current study, we sought to evaluate the in vitro theranostic properties of a molecular beacon-oligodeoxynucleotide (MB) that targets survivin mRNA. We used laser scanning confocal microscopy to study MB delivery in living cells and real-time PCR and western blot to assess selective survivin-targeting in human malignant melanoma cells. We further assess the pro-apoptotic effect of MB by measuring internucleosomal DNA fragmentation, dissipation of mitochondrial membrane potential (MMP) and changes in nuclear morphology. Transfection of MB into A375 and 501 Mel cells generated high signal intensity from the cytoplasm, while no signal was detected in the extracellular environment and in survivin-negative cells (i.e., human melanocytes and monocytes). MB time dependently decreased survivin mRNA and protein expression in melanoma cells with the maximum effect reached at 72 h. Treatment of melanoma cells with MB induced apoptosis by significant changes in MMP, accumulation of histone-complexed DNA fragments in the cytoplasm and nuclear condensation. MB also enhanced the pro-apoptotic effect of standard chemotherapeutic drugs tested at clinically relevant concentrations. The MB tested in the current study conjugates the ability of imaging with the pharmacological silencing activity against survivin mRNA in human melanoma cells and may represent an innovative approach for cancer diagnosis and treatment.     

Fovea-Periphery Axis Symmetry of Surround Modulation in the Human Visual System

A visual stimulus activates different sized cortical area depending on eccentricity of the stimulus. Here, our aim is to understand whether the visual field size of a stimulus or cortical size of the corresponding representation determines how strongly it interacts with other stimuli. We measured surround modulation of blood-oxygenation-level-dependent signal and perceived contrast with surrounds that extended either towards the periphery or the fovea from a center stimulus, centered at 6° eccentricity. This design compares the effects of two surrounds which are identical in visual field size, but differ in the sizes of their cortical representations. The surrounds produced equally strong suppression, which suggests that visual field size of the surround determines suppression strength. A modeled population of neuronal responses, in which all the parameters were experimentally fixed, captured the pattern of results both in psychophysics and functional magnetic resonance imaging. Although the fovea-periphery anisotropy affects nearly all aspects of spatial vision, our results suggest that in surround modulation the visual system compensates for it.     

Extraction, purification and characterization of ADH1 from the budding yeast Kluyveromyces marxianus

The enzyme ADH1 has been extracted and purified from the budding yeast Kluyveromyces marxianus, and its enzymatic activity has been compared, with the ADH1 extracted and purified in the same way from the well known yeast Saccharomyces cerevisiae. K. marxianus ADH1 has an optimal temperature higher than the S. cerevisiae enzyme (45-50 degrees vs 35 degrees C), a better stability to pH variations in the oxidative reaction (pH optimum 7.5), a lower Michaelis constant for acetaldehyde, and a good catalytic activity both for fermentative and oxidative reactions. In fact, while in Saccharomyces the constants ratio (velocity constant fermentation/velocity constant oxidation) is about 20,000, in Kluyveromyces the same ratio is only 15. Even if these two Genera are quite related (they belong to the same subfamily) it seems that their ADH1s possess different catalytic properties.     

Carnivore identity mediates the effects of light and nutrients on aquatic food‐chain efficiency

相似文献   

Inactivation and degradation of yeast glucose-6-phosphate dehydrogenase selectively modified by pyridoxal-5-phosphate

Modification by pyridoxal-5-phosphate of glucose-6-phosphate dehydrogenase (EC 1.1.1.49) purified from Saccharomyces cerevisiae produces an inactivation effect, partially reversible by dilution in the presence of substrates. Spectroscopic analysis of the enzyme pyridoxal-5-phosphate complex reduced with NaBH4 provides the values expected for the binding of the aldehydic group to Lys residue. One Lys residue appears to be responsible for the observed enzyme inactivation, and the presence of the phosphate group is required for the effect. Besides the change of activity, the binding of pyridoxal-5-phosphate to the enzyme causes an increase in susceptibility to degradation by the intracellular yeast proteinase A at pH 7.6.     

Neutral maltase of human granulocytes: localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.     

Correlation between presence of estradiol receptors and epidemiological and clinical data on breast cancer

The aim of the study is to find an eventual correlation between the presence of Er receptors and epidemiologic (age, domestic anamnesis, weight, menarca) and clinicist factors (cancerous diameter, linfonodes, first symptoms). The presence of receptors is very important for the start of endocrine therapy. In conclusion we can affirm the absence of correlation between the presence of receptors and the factors we considered. The only exception is about the age of patients; very probably because too little number of patients was considered. On the contrary a correlation was observed between receptors and severity of cancerous receptors in neoplastic tissues was obtained with Poffanelli method using an Ultra-Turrax homogenization followed by a centrifugation at 3800 rpm; the separation was achieved with carbon-dextran.     

Effect of natural or synthetic detergents on the transport of D-glucose in the membranes of vesicles of the brush border of the intestine of the rabbit

We describe here the effects of natural and synthetic detergents on the D-glucose transport into brush-border membranes of vesicles of rabbit's intestine. Two synthetic detergents: Triton X-100 and dodecyltrimethylammonium bromide have been found very strong inhibitors (more than 50 p. 100 of inhibition of maximal D-glucose uptake). Kinetic studies showed that these detergents behaved as mixed type inhibitors. The Na+-dependent transport of amino acids (aspartic acid, lysine, phenylalanine) is only poorly affected by dodecyltrimethylammonium bromide, while Triton X-100 inhibits unspecifically all the transport studied.