A novel class of protease targets of phosphatidylethanolamine-binding proteins (PEBP): a study of the acylpeptide hydrolase and the PEBP inhibitor from the archaeon Sulfolobus solfataricus

This work describes the identification and characterization of a Sulfolobus solfataricus acylpeptide hydrolase, named APEH(Ss), recognised as a new protease target of the endogenous PEBP inhibitor, SsCEI. APEH is one of the four members of the prolyl oligopeptidase (POP) family, which removes acylated amino acid residues from the N terminus of oligopeptides. APEH(Ss) is a cytosolic homodimeric protein with a molecular mass of 125 kDa. It displays a similar exopeptidase and endopeptidase activity to the homologous enzymes from Aeropyrum pernix and Pyrococcus horikoshii. Herein we demonstrate that SsCEI is the first PEBP protein found to efficiently inhibit APEH from both S. solfataricus and mammalian sources with IC(50) values in the nanomolar range. The 3D model of APEH(Ss) shows the typical structural features of the POP family including an N-terminal β-propeller and a C-terminal α/β hydrolase domain. Moreover, to gain insights into the binding mode of SsCEI toward APEH(Ss), a structural model of the inhibition complex is proposed, suggesting a mechanism of steric blockage on substrate access to the active site or on product release. Like other POP enzymes, APEH may constitute a new therapeutic target for the treatment of a number of pathologies and this study may represent a starting point for further medical research.     

β‐Cell Function Improvement After Biliopancreatic Diversion in Subjects With Type 2 Diabetes and Morbid Obesity

In subjects with obesity and type 2 diabetes mellitus (T2DM), biliopancreatic diversion (BPD) improves glucose stimulated insulin secretion, whereas the effects on other secretion mechanisms are still unknown. Our objective was to evaluate the early effects of BPD on nonglucose‐stimulated insulin secretion. In 16 morbid obese subjects (9 with T2DM and 7 with normal fasting glucose (NFG)), we measured insulin secretion after glucose‐dependent arginine stimulation test and after intravenous glucose tolerance test (IVGTT) before and 1 month after BPD. After surgery the mean weight lost was 13% in both groups. The acute insulin response during IVGTT was improved in T2DM after BDP (from 55 ± 10 to 277 ± 91 pmol/l, P = 0.03). A reduction of insulin response to arginine was observed in NFG, whereas opposite was found in T2DM. In particular, acute insulin response to arginine at basal glucose concentrations (AIR_basal) was reduced but insulin response at 14 mmol/l of plasma glucose (AIR₁₄) was increased. Therefore, after BPD any statistical difference in AIR₁₄ between NFG and T2DM disappeared (1,032 ± 123 for NFG and 665 ± 236 pmol/l for T2DM, P = ns). The same was observed for Slope_AIR, a measure of glucose potentiation, reduced in T2DM before BPD but increased after surgery, when no statistically significant difference resulted compared with NFG (Slope_AIR after BPD: 78 ± 11 in NFG and 56 ± 18 pmol/l in T2DM, P = ns). In conclusion, in obese T2DM subjects 1 month after BPD we observed a great improvement of both glucose‐ and nonglucose‐stimulated insulin secretions. The mechanisms by which BDP improve insulin secretion are still unknown.     

Parasites of European hedgehogs (<Emphasis Type="BoldItalic">Erinaceus europaeus</Emphasis>) in Britain: epidemiological study and coprological test evaluation

Seventy-four European hedgehogs (Erinaceus europaeus) that had died in wildlife rehabilitation centres were dissected and their parasite burdens documented. Overall parasite prevalence was 91%, and a total of six helminth species were isolated: five nematodes (Crenosoma striatum, Eucoleus aerophilus, Capillaria erinacei, Capillaria ovoreticulata and Capillaria spp.), one trematode (Brachylaemus erinacei) and one acanthocephalan (Oliganthorhynchus erinacei). The tick Ixodes hexagonus and flea Archeopsylla erinacei were also collected. The effect of parasite infection on body condition was assessed by correlation of burdens with the residuals of weight–skeletal length regression. Tick presence was positively related to body condition; for other parasites, no significant relationship was found. Faecal egg or larval count was closely correlated with adult parasite burden for C. striatum and Capillaria/Eucoleus spp., but not for other species. Coprological analysis should therefore be useful for in vivo studies of nematode parasite infection in hedgehogs. The epidemiology of parasites in hedgehogs and their possible role in recent population declines are discussed.     

Retrotransposon-based molecular markers for grapevine species and cultivars identification

Insertional polymorphisms of two copia-like (Vine-1, Tvv1) and one gypsy-like (Gret1) retrotransposon found in the grapevine genome were studied in 29 Vitis genotypes (Vitis arizonica, Vitis cinerea, Vitis labrusca, Vitis rupestis, Vitis rotundifolia, Vitis vinifera subsp. sylvestris and 23 V. vinifera subsp. sativa) using inter-retrotransposon amplified polymorphism (IRAP), retrotransposon-microsatellite amplified polymorphism (REMAP) and sequence-specific amplified polymorphism (SSAP) techniques. IRAP, REMAP and SSAP polymorphisms were compared with amplified fragment length polymorphism (AFLP), Inter-single sequence repeats (ISSR) and SSR polymorphisms by evaluating the information content, the number of loci simultaneously analysed per experiment, the effectiveness of the analyses in assessing the relationship between accessions and the number of loci needed to obtain a coefficient of variation of 10%. The UPGMA dendrograms of each molecular marker system were compared and the Mantel matrix correspondence test was applied. Furthermore, the corresponding insertion ages of the transposable elements were estimated for each retrotransposon subfamily analysed. The presence of Gret1, Tvv1 and Vine-1 retrotransposons in all analysed genotypes suggests that copia-like and gypsy-like retrotransposons are widespread in Vitis genus. The results indicate that these retrotransposons were active before Vitis speciation and contributed to Vitis genus evolution. IRAP, REMAP and SSAP markers allow the discrimination of Vitis species and V. vinifera subsp. sativa cultivars with certainty as has been shown with AFLP, ISSR and SSR analyses, but phylogenetic trees obtained by retrotransposon-based molecular markers polymorphisms show some significant differences in the allocation of the analysed accessions compare to those obtained by ISSR, AFLP and SSR molecular markers. The phylogenetic tree resulting from REMAP polymorphism appeared the most representative of the effective relationship between all analysed accessions.     

Inhibitory Activity by Barley Coffee Components Towards Streptococcus Mutans Biofilm

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (<1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml⁻¹ that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.     

Multiple tumor marker elevation in androgen ablation-refractory prostate cancer with long-term response to metronomic chemotherapy: a case report

Outcomes in hormone-refractory prostate cancer are very poor. The time from progression to death is only 12-19 months. We present the case of a 69-year-old man with hormone-refractory prostate cancer and bone metastases treated with metronomic chemotherapy (cyclophosphamide based). He had had a colon adenocarcinoma ten years before. The atypical features of this case were an unusually long-lasting response to metronomic chemotherapy and an increase in serum levels of some non-prostate-specific tumor markers (CEA and CA 19-9) that was not related to a relapse of colon cancer. We hypothesize a potential role of hypoxia inducing CA 19-9 and CEA expression in tumor cells, which may predict the development of progressive resistance to antiangiogenic therapies.     

d-Aspartate binding sites in rat Harderian gland

Radioligand binding of d-[³H]aspartic and l-[³H]glutamic acids to plasma membranes from rat Harderian gland was evaluated. Binding was optimal under physiological conditions of pH and temperature, and equilibrium was reached within 50 min. Specific binding for d-Asp and l-Glu was saturable, and Eadie–Hofstee analysis revealed interaction with a single population of binding sites (for d-Asp K _d = 860 ± 28 nM, B _max = 27.2 ± 0.5 pmol/mg protein; for l-Glu, K _d = 580 ± 15 nM and B _max = 51.3 ± 0.8 pmol/mg protein). l-[³H]glutamate had higher affinity and a greater percentage of specific binding than did d-[³H]aspartate. The pharmacological binding specificity of l-[³H]glutamate indicated an interaction with NMDA-type receptors. Specifically, the order of potency of the displacing compound tested was l-Glu > d-Asp > NMDA > MK801 > d-AP5 > glycine. For d-[³H]aspartate, the data revealed an interaction of d-Asp with either NMDA-type receptors or putative specific binding sites.