Gain of function of mutant p53 by coaggregation with multiple tumor suppressors

Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.     

Melatonin Prevents Oxidative Stress and Hepatocyte Cell Death Induced by Experimental Cholestasis

The induction of oxidative stress precedes liver injury during experimental obstructive jaundice (OJ). In this sense, different evidences suggest that melatonin (MEL), as antioxidant, may be useful in the protection against apoptosis and necrosis during experimental cholestasis. In addition, we will also assess if MEL-dependent protection is related to a recovery of antioxidant status disturbances induced by OJ. Cholestasis was achieved by double ligature and sectioning of the principal bile duct. MEL was injected intraperitoneally (500?μg/kg/day). Lipid peroxidation was evaluated by the measurement of malondialdehyde (MDA) content in liver. Different parameters related to antioxidant status, such as reduced glutathione (GSH), glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD) were determined in liver. Liver injury was assessed by alanine aminotransferase (ALT) in serum, histological examination, DNA fragmentation and TUNEL assay. The activation of perisinusoidal stellate cells was evaluated by immunohistochemical measurement of α-smooth muscle actin in liver sections. The induction of OJ increased all the parameters related to apoptosis and necrosis in liver. The induction of liver injury was associated with stellate cell activation, as well as an increase in MDA (p<0.0001) and a reduction in GSH, GPx, catalase and SOD content (p<0.0001) in liver. MEL reduced hepatic apoptosis and necrosis (p<0.004) with a significant improvement in all oxidative stress markers. In conclusion, our results showed that MEL recovered the antioxidant status and reduced apoptosis and necrosis induced by experimental cholestasis.     

MTPA: a crustacean metallothionein that affects hepatopancreatic mitochondrial functions

Metallothioneins are cysteine-rich proteins, with a high capacity to bind metallic ions, and for which a precise biological role has not been established. Here we investigated the effects of MTPA, a metallothionein from the lobster Panulirus argus, on mitochondrial oxygen consumption and ROS production. An HPLC-RP-ESI-MS analysis of recombinant MTPA showed that despite its extra Cys, MTPA binds 6 Zn²⁺ per molecule akin to other crustacean metallothioneins with 18 Cys. The extra Cys is not involved in zinc binding, since its side-chain would be oriented to the outside of the molecule according to a preliminary model of the tridimensional structure of MTPA. MTPA-Zn²⁺₆ is imported into the hepatopancreatic mitochondria intermembrane space and inhibits mitochondrial oxygen consumption, increasing thereby ROS production. Nevertheless, the stimulation of ROS production by MT-bound Zn²⁺ is weaker compared to equivalent amounts of free Zn²⁺, suggesting that MTPA protects against oxidative stress. This constitutes the first report on metallothioneins effects on mitochondrial function in invertebrates and agrees with the results described for mammals, suggesting a connection between metallothioneins and energy metabolism.     

Cloning and production of Xylanase B from Paenibacillus barcinonensis in Bacillus subtilis hosts

Xylanase B from Paenibacillus barcinonensis was cloned in shuttle vectors for Escherichia coli and Bacillus subtilis, and expressed in Bacillus hosts. Several recombinant strains were constructed, among which B. subtilis MW15/pRBSPOX20 showed the highest production. This recombinant strain consists of a protease double mutant host containing P. barcinonensis xynB gene under the control of a phage SPO2 strong promoter. Maximum production was found when the strain was cultured in nutrient broth supplemented with xylans. Analysis of xylanase B location in B. subtilis MW15/pRBSPOX20 showed that the enzyme remained cell-associated in young cultures, consistent with its intracellular location in its original host, P. barcinonensis, and the lack of a signal peptide. However, when cultures reached the stationary phase, xylanase B was released to the external medium as a result of cell lysis. The amount of enzyme located in the supernatants of old cultures could account for 50% of total xylanase activity. Analysis by SDS-PAGE showed that xylanase B is an abundant protein found in the culture medium in late stationary phase cultures.     

The Alzheimer Disease Protective Mutation A2T Modulates Kinetic and Thermodynamic Properties of Amyloid-β (Aβ) Aggregation

Missense mutations in alanine 673 of the amyloid precursor protein (APP), which corresponds to the second alanine of the amyloid β (Aβ) sequence, have dramatic impact on the risk for Alzheimer disease; A2V is causative, and A2T is protective. Assuming a crucial role of amyloid-Aβ in neurodegeneration, we hypothesized that both A2V and A2T mutations cause distinct changes in Aβ properties that may at least partially explain these completely different phenotypes. Using human APP-overexpressing primary neurons, we observed significantly decreased Aβ production in the A2T mutant along with an enhanced Aβ generation in the A2V mutant confirming earlier data from non-neuronal cell lines. More importantly, thioflavin T fluorescence assays revealed that the mutations, while having little effect on Aβ42 peptide aggregation, dramatically change the properties of the Aβ40 pool with A2V accelerating and A2T delaying aggregation of the Aβ peptides. In line with the kinetic data, Aβ A2T demonstrated an increase in the solubility at equilibrium, an effect that was also observed in all mixtures of the A2T mutant with the wild type Aβ40. We propose that in addition to the reduced β-secretase cleavage of APP, the impaired propensity to aggregate may be part of the protective effect conferred by A2T substitution. The interpretation of the protective effect of this mutation is thus much more complicated than proposed previously.     

The linkage between inflammation and fibrosis in muscular dystrophies: The axis autotaxin–lysophosphatidic acid as a new therapeutic target?

Muscular dystrophies (MDs) are a diverse group of severe disorders characterized by increased skeletal muscle feebleness. In many cases, respiratory and cardiac muscles are also compromised. Skeletal muscle inflammation and fibrosis are hallmarks of several skeletal muscle diseases, including MDs. Until now, several keys signaling pathways and factors that regulate inflammation and fibrosis have been identified. However, no curative treatments are available. Therefore, it is necessary to find new therapeutic targets to fight these diseases and improve muscle performance. Lysophosphatidic acid (LPA) is an active glycerophospholipid mainly synthesized by the secreted enzyme autotaxin (ATX), which activates six different G protein-coupled receptors named LPA₁ to LPA₆ (LPARs). In conjunction, they are part of the ATX/LPA/LPARs axis, involved in the inflammatory and fibrotic response in several organs-tissues. This review recapitulates the most relevant aspects of inflammation and fibrosis in MDs. It analyzes experimental evidence of the effects of the ATX/LPA/LPARs axis on inflammatory and fibrotic responses. Finally, we speculate about its potential role as a new therapeutic pharmacological target to treat these diseases.     

Gene flow and allozymic population structure in the clam Venus antiqua (King of Broderip), (Bivalvia, Veneriidae) from Southern Chile

Using horizontal electrophoresis, 13 allozyme loci were screened in 144 adult clams (Venus antiqua) (King of Broderip) from 5 geographic samples in southern Chile. Polymorphism was detected at 11 loci across the sampling localities. Gene frequency differences rather than the alternative fixation of alleles characterize these collecting sites. Direct-count heterozygosity fluctuated between 6.8% and 10.3%, with a generalized and significant heterozygote deficiency detected in every locality. Similar levels of genetic diversity were detected in both exploited banks and one presumed unmanaged population. Substantial estimates of demic structuration were found at the total population (F_ST=0.107) and regional levels (F_ST=0.143), with coinciding high inbreeding estimates (F_IS=0.57). Although no statistically significant evidence of genetic substructuring was found at the within-region comparison (F_ST=0.007), a generalized heterozygote deficiency (F_IS=0.57) also characterized that hierarchical level. Spatial correlation of gene frequencies indicated a nearly random distribution of genotypes along the latitudinal distribution of samples. The high rates of global, and the within-region rates of gene flow suggest substantial larval interchange via long-distance dispersal that prevent differentiation by drift alone. Accordingly, no discrete genetic units can be recognized for the species at any spatial scale. Apparently, high recruitment rates help in replenishing genetic variants lost by continuous overfishing, thus explaining why human extraction has not resulted in major losses of genetic variation for individual populations. The slow-growing rate and delayed sexual maturation of this clam species are apparently stronger limiting factors than human-induced genetic erosion for the conservation of natural beds and viable captive management of V. antiqua.