δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo

Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ¹-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.     

Differentiation under isolation and genetic structure of Sardinian hares as revealed by craniometric analysis,mitochondrial DNA and microsatellites

Hares (Lepus capensis Linnaeus 1758) were probably introduced into Sardinia in historical times. Previous studies indicated North Africa as the most likely source area but did not exclude the occurrence of hybridization events with continental brown hares (L. europaeus Pallas 1778) perhaps introduced for hunting purposes. We implemented both morphometric and genetic approaches to verify the genetic isolation of the Sardinian population. Specifically, we conducted a multivariate analysis of craniometric data and analysed 461 bp of the mitochondrial control region and 12 autosomal microsatellites in Sardinian hares, using North African cape hares and European brown hares as reference populations. Sardinian hares displayed a peculiar skull shape. In agreement, both nuclear and mitochondrial markers remarked the distinctiveness of this population. Observed and expected heterozygosity were 0.52 and 0.61, while haplotype and nucleotide diversity were 0.822 and 0.0129. Self‐assignment based on Bayesian cluster analysis was high (average membership 0.98), and no evident signs of introgression from continental brown hares were found. Our results support the hypothesis that the Sardinian hares have been introduced from North Africa, remained genetically isolated since the founding event and evolved independently from the source population. This long‐lasting isolation and the consequent genetic drift resulted in a differentiation, perhaps accompanied by an adaptation to local environmental conditions.     

Giant dedifferentiated liposarcoma of the right hemifacial area involving the oral cavity

Although liposarcoma is a reasonably common soft tissue sarcoma in adults, its occurrence within the head and neck region is very rare. The following report presents the case of a giant dedifferentiated liposarcoma initially located in the temporal region and then extending to the entire right maxillofacial region. Clinical as well as histopathological features and therapeutic approaches of dedifferentiated liposarcoma are discussed, and a literature review is presented.     

GSAFold: a new application of GSA to protein structure prediction

The folding process defines three‐dimensional protein structures from their amino acid chains. A protein's structure determines its activity and properties; thus knowing such conformation on an atomic level is essential for both basic and applied studies of protein function and dynamics. However, the acquisition of such structures by experimental methods is slow and expensive, and current computational methods mostly depend on previously known structures to determine new ones. Here we present a new software called GSAFold that applies the generalized simulated annealing (GSA) algorithm on ab initio protein structure prediction. The GSA is a stochastic search algorithm employed in energy minimization and used in global optimization problems, especially those that depend on long‐range interactions, such as gravity models and conformation optimization of small molecules. This new implementation applies, for the first time in ab initio protein structure prediction, an analytical inverse for the Visitation function of GSA. It also employs the broadly used NAMD Molecular Dynamics package to carry out energy calculations, allowing the user to select different force fields and parameterizations. Moreover, the software also allows the execution of several simulations simultaneously. Applications that depend on protein structures include rational drug design and structure‐based protein function prediction. Applying GSAFold in a test peptide, it was possible to predict the structure of mastoparan‐X to a root mean square deviation of 3.00 Å. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

The mitochondrial effects of small organic ligands of BCL-2: sensitization of BCL-2-overexpressing cells to apoptosis by a pyrimidine-2,4,6-trione derivative

We have investigated the mitochondrial effects of BH3I-2', Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2' (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 microm) > HA14-1 (half-maximal uncoupling at about 20 microm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.     

Dysfunctions of cellular oxidative metabolism in patients with mutations in the NDUFS1 and NDUFS4 genes of complex I

The pathogenic mechanism of a G44A nonsense mutation in the NDUFS4 gene and a C1564A mutation in the NDUFS1 gene of respiratory chain complex I was investigated in fibroblasts from human patients. As previously observed the NDUFS4 mutation prevented complete assembly of the complex and caused full suppression of the activity. The mutation (Q522K replacement) in NDUFS1 gene, coding for the 75-kDa Fe-S subunit of the complex, was associated with (a) reduced level of the mature complex, (b) marked, albeit not complete, inhibition of the activity, (c) accumulation of H(2)O(2) and O(2)(.-) in mitochondria, (d) decreased cellular content of glutathione, (e) enhanced expression and activity of glutathione peroxidase, and (f) decrease of the mitochondrial potential and enhanced mitochondrial susceptibility to reactive oxygen species (ROS) damage. No ROS increase was observed in the NDUFS4 mutation. Exposure of the NDUFS1 mutant fibroblasts to dibutyryl-cAMP stimulated the residual NADH-ubiquinone oxidoreductase activity, induced disappearance of ROS, and restored the mitochondrial potential. These are relevant observations for a possible therapeutical strategy in NDUFS1 mutant patients.     

A scoring system based on the expression of six surface molecules allows the identification of three prognostic risk groups in B-cell chronic lymphocytic leukemia

We have previously identified 12 surface antigens whose differential expression represented the signature of B-cell chronic lymphocytic leukemia (B-CLL) subsets with different prognosis. In the present study, expression data for these antigens, as determined in 137 B-CLL cases, all with survivals, were utilized to devise a comprehensive immunophenotypic scoring system of prognostic relevance for B-CLL patients. In particular, univariate z score was employed to identify the markers with greater prognostic impact, while maximally selected log-rank statistics were chosen to define the optimal cut-off points capable to split patients into two groups with different survivals. A weighted immunophenotypic scoring system was developed by integrating results from these analyses. Six antigens were selected: three positive prognosticators (CD62L, CD54, CD49c) and three negative prognosticators (CD49d, CD38, CD79b), with cut-off values ranging from 30% to 50% of positive cells. By weighing the expression of each marker according to its statistical power, a complete scoring system, with point values comprised between 0 (complete absence of phenotypic conditions associated with good prognosis) and 9 (all the phenotypic conditions associated with good prognosis fulfilled), allowed to split the whole set of B-CLL patients, into three distinctive prognostic groups (P = 4.78 x 10(-11)) with high- (score 0-3), intermediate- (score 4-6), and low- (score 7-9) risk of death. The three risk groups showed different distribution of cases as for Rai's stages, IgVH mutations, and ZAP-70 expression. The proposed immunophenotypic scoring system may be an additional useful tool in routine diagnostic/prognostic procedures for B-CLL.