Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Reactive oxygen species as essential mediators of cell adhesion: the oxidative inhibition of a FAK tyrosine phosphatase is required for cell adhesion

Signal transduction by reactive oxygen species (ROS; "redox signaling") has recently come into focus in cellular biology studies. The signaling properties of ROS are largely due to the reversible oxidation of redox-sensitive target proteins, and especially of protein tyrosine phosphatases, whose activity is dependent on the redox state of a low pKa active site cysteine. A variety of mitogenic signals, including those released by receptor tyrosine kinase (RTKs) ligands and oncogenic H-Ras, involve as a critical downstream event the intracellular generation of ROS. Signaling by integrins is also essential for the growth of most cell types and is constantly integrated with growth factor signaling. We provide here evidence that intracellular ROS are generated after integrin engagement and that these oxidant intermediates are necessary for integrin signaling during fibroblast adhesion and spreading. Moreover, we propose a synergistic action of integrins and RTKs for redox signaling. Integrin-induced ROS are required to oxidize/inhibit the low molecular weight phosphotyrosine phosphatase, thereby preventing the enzyme from dephosphorylating and inactivating FAK. Accordingly, FAK phosphorylation and other downstream events, including MAPK phosphorylation, Src phosphorylation, focal adhesion formation, and cell spreading, are all significantly attenuated by inhibition of redox signaling. Hence, we have outlined a redox circuitry whereby, upon cell adhesion, oxidative inhibition of a protein tyrosine phosphatase promotes the phosphorylation/activation and the downstream signaling of FAK and, as a final event, cell adhesion and spreading onto fibronectin.     

DISCO--a general computer program for the computation of acid dissociation constants of polyprotic molecules in water and biological fluids,from nuclear magnetic resonance data: application to polyamines

A new computer program, DISCO, running under Windows, has been developed under the project CSA98P22 falling within the Competitive Support Activities initiative launched within the EU 4th Framework Programme. DISCO allows the calculation of the stepwise acid dissociation constants of polyprotic molecules in water and in complex media (i.e., biofluids, etc.) from nuclear magnetic resonance (NMR) data (chemical shifts) by means of two derivative-free methods: Pit-mapping and Simplex. DISCO performances were tested using simulated-unaffected by experimental error-data sets, for systems having up to seven equilibrium constants and experimental NMR data of spermine, 6-monofluorospermine, and 6,6-difluorospermine, dissolved in D(2)O and in physiological solution (D(2)O/NaCl). Results demonstrated that (i) DISCO enables the determination of pK(A) values with high precision even when small-sized raw data sets are employed, when chemical shifts are measured with low precision (the usual condition in biofluids due to the impossibility to obtain narrow line shape), and when the guess solution, necessary as an initial step of the mathematical iterative process, is fixed within a large interval of variation; (ii) DISCO always converges to the root; (iii) DISCO permits the calculation of pK(A) values which lie within the observed pH range, independent of the narrowness of the pH range.     

Determination of dissociation constants of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] in water and biological fluids by means of nuclear magnetic resonance spectroscopy and the DISCO software package

The pK(A) values of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] (BOPTA), a polyprotic molecule whose gadolinium complex is an important magnetic resonance imaging contrast agent for clinical use, have been determined in water, in physiologic solution (PS), in serum (S), and in cerebrospinal fluid (CSF), by means of 13C nuclear magnetic resonance spectroscopy data processed by a dedicated software package called DISCO. The aim of this study was to supply the BOPTA pK(A) values in media very similar to the in vivo environment and, consequently, to get a picture of the in vivo behavior of its Gd complex, whose thermodynamic stability is directly linked to the pK(A) values. The pK(A) values appeared to be almost equal both in D(2)O and in PS, while pK(1) and pK(5) values in CSF differ a little. In S, only pK(2) and pK(3) were calculated due to the narrow pH range used for data collection. However, these pK(A) values were found equal to those in the other media. These results represent the first direct spectroscopic evidence of a substantial invariability of BOPTA behavior in different media and they justify the extrapolation to biological fluids of the data obtained in water. The values also confirmed the high-quality performance of DISCO in calculating pK(A) values of polyprotic molecules in complex media.     

Small-Scale Distribution of Bacteria,Enzymatic Activities,and Organic Matter in Coastal Sediments

apd: 9 February 2001     

Primate Population Densities and Sizes in Atlantic Forest Remnants of Northern Espírito Santo,Brazil

We surveyed primates in seven fragments of Atlantic forest ranging from 210 to 24,250 ha in Espírito Santo, southeastern Brazil. The objective was to assess their population densities and fragment-specific population sizes in order to discuss the survival of primates in this highly fragmented landscape. We used line-transect sampling for censusing primates and estimated densities via DISTANCE program. Alouatta fusca, Cebus apella, Callicebus personatus, and Callithrix geoffroyi live in the study area. Although variable, primate numbers were higher in larger than in smaller fragments, the likely reasons being the lack of space and food resources (fruits) and higher impact of mesopredators (small cats) in small fragments. The estimated densities are within the range reported for other Atlantic forest sites, except for brown howlers (Alouatta fusca), which were extremely rare. Estimates of population sizes were on the order of several hundreds to thousands of individuals/species in the large-sized fragments (ca. 20,000 ha), while comparable estimates for the other areas indicate <500 individuals/species in the medium-sized reserves (ca. 2,000 ha), and <50 individuals/species in the small fragments (ca. 200 ha). These estimates suggest that only Atlantic forest fragments 20,000 ha can harbor primate populations large enough to escape extinction in the long term. Given that <20% of all protected areas remaining in the Atlantic forest are 20,000 ha, management measures are urgently needed to improve primate conservation in this biome.     

Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.     

Sperm quality and reproductive traits in male offspring of female rabbits exposed to lindane (gamma-HCH) during pregnancy and lactation

Fifteen Grimaud female hybrid rabbits, 135 days old and weighting an average of 3.74+/-0.01 kg each, were administered an oral dose of 1 mg x kg(-1) body weight of Lindane during gestation and lactation period. Fertility rate, libido, volume of ejaculate, concentration and morphology of spermatozoa were investigated to test the effects of the treatment on reproductive traits of first generation male rabbits. Ultrastructure of abnormal spermatozoa was described by Transmission Electron Microscopy and the different abnormalities were quantified. The results obtained indicate that low dose exposure of Lindane has effects on spermatozoa ultrastructure that proved to be susceptible to the treatment with the pesticide (cytoplasmic droplets: 5.3% in control group and 10.3% in Lindane group, P < or = 0.05; coiled tails: 1.3% in control group and 4.3% in Lindane group, P < or = 0.05) and could be utilised as a good marker of toxicity.     

Ectoenzymatic ratios in relation to particulate organic matter distribution (Ross Sea,Antarctica)

The results of a study on ectoenzymatic activity (the enzyme activity bound to particles larger than 0.2 micro m) and its relation to organic particle concentration are reported here. The sampling was carried out during the 1994 Antarctic spring, at a fixed station (Station 11) in the polynya of the Ross Sea, an area characterized by quick changes in sea ice cover. The sampling was repeated 4 times over a 20-day time period. The particulate organic matter distribution followed the physical structure of the water column, which depends on ice dynamics and is mainly determined by salinity. In the mixed-water surface layer (0-50 m) the concentrations were higher (on average 65.6 micro gC/L) than in the deeper water layer (50 m-bottom) (on average 19.1 micro gC/L). This distribution and quality, expressed by the protein:carbohydrate ratio, linked the particulate organic matter to the phytoplanktonic bloom which was in progress in the area. We determined the kinetic parameters of the glycolytic and proteolytic ectoenzymes and also the total activity for the proteolytic enzyme, in order to evaluate the contribution of the particle-bound activity. We observed higher values in the surface layer than in the deeper layer. b-Glucosidase activity ranged between 0.03 and 0.92 nmol L(-1) h(-1); b-N-acetylglucosaminidase activity was in the range of 0.04-0.58 nmol (L-1) (h-1). The total proteolytic activity (leucine aminopeptidase) ranged between 0.85 and 33.71 nmol L(-1) (h-1). The ectoproteolytic activity was about 35-60% of the total. The Km values were slightly higher for the proteolytic activity (on average 0.43 micro M for ectoproteolytic activity and 0.58 micro M for total proteolytic activity) than for the b-glucosidase (on average 0.36 micro M) and b-N-acetylglucosaminidase (on average 0.17 micro M), showing no remarkable variations in the water column. The ectoenzymatic ratios and their relationship with particulate organic substrates confirm the close link between organic substrate availability and degradation system response. The significant and positive correlations are not specific and suggest a prompt and efficient systemic response to the input of trophic resources. Nevertheless, changes in ectoenzyme activity and synthesis may act as adaptive responses to changing features of the ecosystem. In particular, variations in the proteolysis:glycolysis ratio depend on the functional features of the ecological system. In our study area this ratio is higher (about 10 or more) during production (particularly autotrophic) and lower (about 5 or less) during degradation/consumption events. The analysis of previous data, collected over a larger area characterized by different environmental conditions due to the changes of the pack ice cover, during the same cruise, confirms the existence of a significant relationship. Furthermore, the analysis of enzyme-uptake systems, expressed as Vmax:Km ratio, suggests that glycolytic ectoenzymes, although poorly expressed, may encourage microconsumers to grow rapidly on a wide range of organic substrates, including the refractory ones such as cellulose and chitin. However, low ectoenzyme potential exploitation rates of available organic substrates (on average about 5% for glycolytic and 12% for proteolytic ectoenzymes) would suggest that, during spring, zooplankton grazing or vertical and lateral transport are likely to play an important role in the removal of organic materials from the system.     

Characterization of dioxygenated cobalt(II)-carnosine complexes by Raman and IR spectroscopy

Raman and IR studies are carried out on carnosine (beta-alanyl-L-histidine, Carnos) and its complexes with cobalt(II) at different metal/ligand ratios and basic pH. Binuclear complexes that bind molecular oxygen are formed and information regarding the O-O bridge is obtained from the Raman spectra. When the Co(II)/Carnos ratio is 相似文献