Fusaproliferin production by Fusarium subglutinans and its toxicity to Artemia salina, SF-9 insect cells, and IARC/LCL 171 human B lymphocytes.

Fusarium subglutinans is an important pathogen of maize and other commodities worldwide. We examined MRC-115 and 71 other F. subglutinans strains from various geographic areas for their ability to synthesize fusaproliferin, a novel toxic sesterterpene recently isolated from F. proliferatum. Fusaproliferin production ranged from 30 to 1,500 micrograms/g of dried ground substrate, with 33 strains producing more than 500 micrograms/g. In particular, strain MRC-115 produced as much as 1,100 to 1,300 micrograms/g. In toxicity studies of two invertebrate models, fusaproliferin was toxic to Artemia salina (50% lethal dose, 53.4 microM) and to the lepidopteran cell line SF-9 (50% cytotoxic concentration, approximately 70 microM, after a 48-h exposure). Fusaproliferin was also toxic to the human nonneoplastic B-lymphocyte cell line IARC/LCL 171 (50% cytotoxic concentration, approximately 55 microM in culture in stationary phase after a 48-h exposure). Experiments performed will cells exposed at seeding suggested a possible cytostatic effect at subtoxic concentrations.     

Facile reduction of peptide oxime endothelin antagonist during trialkylsilane/TFA cleavage after solid-phase synthesis

Summary We describe a new solid-phase strategy for the selective reduction of the C=N bond in peptide oximes using a trialkylsilane in trifluoroacetic acid. The reduction is performed directly on the resin-bound peptide, with concomitant cleavage of the peptide from the resin and deblocking of protected side chains.     

Guanylate cyclase in Escherichia coli. I. Purification of the enzyme and evidence for an inhibitor]

Guanylate cyclase activity is present in crude E. coli extract. Guanylate cyclase has been purified 3500 fold from this extract, through ammonium sulfate fractionation, DEAE-cellulose chromatography. Sephadex G-75 gel-filtration and polyacrylamide gel preparative microelectrophoresis. During the purification a guanylate cyclase inhibitor has been separated.     

Predicting potentially pathogenic effects of hRPE65 missense mutations: a computational strategy based on molecular dynamics simulations

The human retinal pigment epithelium-specific 65-kDa protein (hRPE65) plays a crucial role within the retinoid visual cycle and several mutations affecting either its expression level or its enzymatic function are associated with inherited retinal diseases such as Retinitis Pigmentosa. The gene therapy product voretigene neparvovec (Luxturna) has been recently approved for treating hereditary retinal dystrophies; however, the treatment is currently accessible only to patients presenting confirmed biallelic mutations that severely impair hRPE65 function, and many reported hRPE65 missense mutations lack sufficient evidences for proving their pathogenicity. In this context, we developed a computational approach aimed at evaluating the potential pathogenic effect of hRPE65 missense variants located on the dimerisation domain of the protein. The protocol evaluates how mutations may affect folding and conformation stability of this protein region, potentially helping clinicians to evaluate the eligibility for gene therapy of patients diagnosed with this type of hRPE65 variant of uncertain significance.     

A mouse model for Li-Fraumeni-Like Syndrome with cardiac angiosarcomas associated to POT1 mutations

The shelterin protein POT1 has been found mutated in many different familial and sporadic cancers, however, no mouse models to understand the pathobiology of these mutations have been developed so far. To address the molecular mechanisms underlying the tumorigenic effects of POT1 mutant proteins in humans, we have generated a mouse model for the human POT1^R117C mutation found in Li-Fraumeni-Like families with cases of cardiac angiosarcoma by introducing this mutation in the Pot1a endogenous locus, knock-in for Pot1a^R117C. We find here that both mouse embryonic fibroblasts (MEFs) and tissues from Pot1a^+/ki mice show longer telomeres than wild-type controls. Longer telomeres in Pot1a^+/ki MEFs are dependent on telomerase activity as they are not found in double mutant Pot1a^+/ki Tert^-/- telomerase-deficient MEFs. By using complementation assays we further show that POT1a pR117C exerts dominant-negative effects at telomeres. As in human Li-Fraumeni patients, heterozygous Pot1a^+/ki mice spontaneously develop a high incidence of angiosarcomas, including cardiac angiosarcomas, and this is associated to the presence of abnormally long telomeres in endothelial cells as well as in the tumors. The Pot1a^+/R117C mouse model constitutes a useful tool to understand human cancers initiated by POT1 mutations.     

Salicylaldoxime derivatives as new leads for the development of carbonic anhydrase inhibitors

New compounds containing a novel zinc binding group (salicylaldoxime system) were identified as effective inhibitors of carbonic anhydrases (CAs). This structural motif seems to bind the catalytic zinc ion of CAs, revealing itself as a new valid alternative to the sulfonamide group. Computational procedures were used to investigate the binding mode of this class of compounds, within the active site of CAII. This study suggests that the salicylaldoxime moiety binds the zinc ion through the oxime oxygen atom that also forms an H-bond with T199. The results herein obtained will allow the development of new CA-inhibitors bearing the salicylaldoxime moiety.     

Marked Increase in PROP Taste Responsiveness Following Oral Supplementation with Selected Salivary Proteins or Their Related Free Amino Acids

The genetic predisposition to taste 6-n-propylthiouracil (PROP) varies among individuals and is associated with salivary levels of Ps-1 and II-2 peptides, belonging to the basic proline-rich protein family (bPRP). We evaluated the role of these proteins and free amino acids that selectively interact with the PROP molecule, in modulating bitter taste responsiveness. Subjects were classified by their PROP taster status based on ratings of perceived taste intensity for PROP and NaCl solutions. Quantitative and qualitative determinations of Ps-1 and II-2 proteins in unstimulated saliva were performed by HPLC-ESI-MS analysis. Subjects rated PROP bitterness after supplementation with Ps-1 and II-2, and two amino acids (L-Arg and L-Lys) whose interaction with PROP was demonstrated by ¹H-NMR spectroscopy. ANOVA showed that salivary levels of II-2 and Ps-1 proteins were higher in unstimulated saliva of PROP super-tasters and medium tasters than in non-tasters. Supplementation of Ps-1 protein in individuals lacking it in saliva enhanced their PROP bitter taste responsiveness, and this effect was specific to the non-taster group.¹H-NMR results showed that the interaction between PROP and L-Arg is stronger than that involving L-Lys, and taste experiments confirmed that oral supplementation with these two amino acids increased PROP bitterness intensity, more for L-Arg than for L-Lys. These data suggest that Ps-1 protein facilitates PROP bitter taste perception and identifies a role for free L-Arg and L-Lys in PROP tasting.     

Coupling Demographic and Genetic Variability from Archived Collections of European Anchovy (Engraulis encrasicolus)

It is well known that temporal fluctuations in small populations deeply influence evolutionary potential. Less well known is whether fluctuations can influence the evolutionary potentials of species with large census sizes. Here, we estimated genetic population parameters from as survey of polymorphic microsatellite DNA loci in archived otoliths from Adriatic European anchovy (Engraulis encrasicolus), a fish with large census sizes that supports numerous local fisheries. Stocks have fluctuated greatly over the past few decades, and the Adriatic fishery collapsed in 1987. Our results show a significant reduction of mean genetic parameters as a consequence of the population collapse. In addition, estimates of effective population size (N_e) are much smaller than those expected in a fishes with large population census sizes (N_c). Estimates of N_e indicate low effective population sizes, even before the population collapse. The ratio N_e/N_e ranged between 10⁻⁶ and 10⁻⁸, indicating a large discrepancy between the anchovy gene pool and population census size. Therefore, anchovy populations may be more vulnerable to fishery effort and environmental change than previously thought.