The inhibition of phospholipase A2 by manoalide and manoalide analogues

Manoalide, a natural product from sponge, displays anti-inflammatory activity in vivo. Previous work has shown that manoalide is also a potent covalent inhibitor of the extracellular phospholipase A2 from cobra venom and that the inhibition correlated with a pH-dependent change in manoalide (Lombardo and Dennis (1985) J. Biol. Chem. 260, 7234-7240). Manoalide contains two rings and the opening of either would produce an alpha,beta-unsaturated aldehyde. The cobra venom phospholipase A2 may be able to catalyze the opening or isomerization of one of these rings, raising the possibility that manoalide is acting as a suicide substrate. To ascertain the role of the gamma-lactone ring in the inhibition, we have now investigated a synthetic manoalide analogue, 3(cis,cis-7,10)-hexadecadienyl-4-hydroxy-2-butenolide (HDHB) which contains only the alpha,beta-unsaturated gamma-lactone ring. We have found that the closed and open forms are in rapid equilibrium between pH 4 and 9 with the cyclic form being preferred at acidic pH values and the open cis form preferred at pH 9.5. When the pH is raised above 12, the alpha,beta double bond isomerizes to form trans-HDHB. Once the trans compound is formed, it is stable at all pH values and does not recyclize to the gamma-lactone ring. The observed pKa of 7.7 found for the inhibition of manoalide agrees well with the transition of the closed to the cis form of the gamma-lactone ring. Kinetic experiments with the HDHB compound show that under conditions in which the cis and closed form of the inhibitor are present in equal molar ratios, HDHB is not an irreversible inhibitor, but reversibly competes with substrate. However, the kinetics of this inhibition are complex and do not follow either pure competitive or non-competitive inhibition. The trans-HDHB exhibits similar complex kinetic but is several times more potent. The distinct differences between the behavior of manoalide and HDHB clearly indicate that while the gamma-lactone ring may play an important role in manoalide inhibition, it alone does not produce irreversible inhibition.     

Optimization of 2-piperidin-4-yl-acetamides as melanin-concentrating hormone receptor 1 (MCH-R1) antagonists: Designing out hERG inhibition

Herein, we disclose the discovery and optimization of 2-piperidin-4-yl-acetamide derivatives as MCH-R1 antagonists. Structural investigation of piperidin-4-yl-amide and piperidin-4-yl-ureas identified 2-piperidin-4-yl-acetamide-based MCH-R1 antagonists with outstanding in vivo efficacy but flawed with high affinity towards the hERG potassium channel. While existing hERG SAR information was employed to discover highly potent MCH-R1 antagonists with minimized hERG inhibition, additional hurdles prevented their subsequent clinical exploration.     

Plasma FA composition in familial LCAT deficiency indicates SOAT2-derived cholesteryl ester formation in humans

Mutations in the LCAT gene cause familial LCAT deficiency (Online Mendelian Inheritance in Man ID: #245900), a very rare metabolic disorder. LCAT is the only enzyme able to esterify cholesterol in plasma, whereas sterol O-acyltransferases 1 and 2 are the enzymes esterifying cellular cholesterol in cells. Despite the complete lack of LCAT activity, patients with familial LCAT deficiency exhibit circulating cholesteryl esters (CEs) in apoB-containing lipoproteins. To analyze the origin of these CEs, we investigated 24 carriers of LCAT deficiency in this observational study. We found that CE plasma levels were significantly reduced and highly variable among carriers of two mutant LCAT alleles (22.5 [4.0–37.8] mg/dl) and slightly reduced in heterozygotes (218 [153–234] mg/dl). FA distribution in CE (CEFA) was evaluated in whole plasma and VLDL in a subgroup of the enrolled subjects. We found enrichment of C16:0, C18:0, and C18:1 species and a depletion in C18:2 and C20:4 species in the plasma of carriers of two mutant LCAT alleles. No changes were observed in heterozygotes. Furthermore, plasma triglyceride-FA distribution was remarkably similar between carriers of LCAT deficiency and controls. CEFA distribution in VLDL essentially recapitulated that of plasma, being mainly enriched in C16:0 and C18:1, while depleted in C18:2 and C20:4. Finally, after fat loading, chylomicrons of carriers of two mutant LCAT alleles showed CEs containing mainly saturated FAs. This study of CEFA composition in a large cohort of carriers of LCAT deficiency shows that in the absence of LCAT-derived CEs, CEs present in apoB-containing lipoproteins are derived from hepatic and intestinal sterol O-acyltransferase 2.     

A cross-linked complex between horse pancreatic lipase and colipase

The water soluble carbodiimide N-cyclohexyl-N'-2-morpholinoethyl-carbodiimide-methyl-p-toluolsulfona te was found to effectively covalently cross-link pancreatic colipase to lipase as evidenced by Western blotting experiments using antibodies directed either against lipase or colipase. Moreover the resulting covalent complex has a Mr consistent with a stoichiometry of 1 mol colipase per mol lipase. Cross-linked lipase and colipase retain their activity implying a correct covalent binding between the two proteins. The specificity of the lipase-colipase binding was further supported by the very low amount of cross-linked products when lipase or colipase alone were incubated in the presence of carbodiimide. The formation of a covalent lipase-colipase complex in the presence of carbodiimide clearly demonstrates that the binding between both proteins involves ion pairing. Furthermore, the formation of an active covalent complex strongly suggests that the lipase-colipase binding site is distinct from the colipase interfacial recognition site as well as from the lipase catalytic site.     

Modulation of the cell-mediated immune function by interferon α, β or γ can partially reverse the immunosuppression induced by human T-cell leukemia virus I in human cord blood cultures

Summary Infection with human T-cell leukemia virus type I (HTLV-I) is associated in vitro and in vivo with a remarkable depression of cell-mediated immune functions. In the present report it is shown that early events following virus-induced suppression of the cell-mediated immune response of freshly isolated cord blood mononuclear cells (CBL) infected with HTLV-I can be partially counteracted by treatment with interferons , or (IFN). All three types of IFN exerted a protective effect on CBL cultures exposed to the virus. This resulted in: (a) a reduced number of virus-positive cells until 4 weeks of culture; (b) delay in the clonal expansion of infected cells (IFN and ); (c) increased natural killer cell activity of CBL, 1 week post-infection (p.i.), mediated by IFN; (d) increase of allospecific recognition of infecting and priming HTLV-I donor MT-2 cells by CBL in a cytotoxic-T-lymphocyte-like response, mediated by IFN and particularly by IFN; (e) phenotype distribution of CBL subpopulations, tested 4 days p.i., more similar to that of non-infected CBL cultures.In contrast, the overall CBL proliferation, that is profoundly depressed during the first week p.i., was not restored by IFN treatments, suggesting that boosting of the cell-mediated killing induced by IFN might involve the maturation of undifferentiated precursor cells rather than stimulation of their proliferation. The improvement of the efficiency of the antiviral immune response induced by treatment with IFN is likely to contribute to the clearance of virus-positive cells during the early phase of infection. This would provide experimental evidence to support an immunopharmacological approach contributing to the conversion of HTLV-I carriers from positive to negative.     

A mite survey in the dumpsters for urban solid rubbish

Summary The presence of house dust mites and storage mites in dumpsters was investigated in 3 different areas of Venice hinterland. The aim of this study was to find a relationship between some reported respiratory symptoms among 6 workers who were responsible for moving the dumpsters to the truck and the bio-aerosol released from such operation. These symptoms were closely related to the work and after allergological evaluation we found sensitisation to house dust mites in all 6 employees. The mine exposure in the workplace was assessed by a standardized sampling and analysis of dust obtained after brushing the inside surfaces of dumpsters. In 50% of the specimens (10/20) we found mites belonging to the following families: Pyroglyphidae (Dermatophagoides spp.), Acaridae (Acarus siro) and a lower amount of Cheyletidae (Cheyletus spp.). An indirect test (guanine test) was also performed and gave positivity in 16 dust samples (80%). The insides of dumpsters showed to be an optimal environment for mites survival and reproduction because of cracks and grooves on the walls, the high humidity level and the presence of mould. These mites probably come from dwelling places and other environments (markets, slaughters-houses, farms, etc.). This suggests that mites allergens can be released during rubbish discharging from the dumpsters and represent a possible risk for the employees.     

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW.

Summary The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the -subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif^– phenotype to bacteria grown in the free-living state and a Fix^– phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.     

Specificity and mechanism of influence of amino acid residues on hepatic clearance of oligopeptides

We have investigated the influence of amino acid residues on hepatic clearance of oligopeptides by determining the rate of disappearance (nmol.(min.g liver)-1) of selective oligopeptides from the medium during isolated rat liver perfusion. (a) N terminus: the rate of disappearance of Ala-Leu was greater (p less than 0.01) than those of Gly-Leu, Phe-Leu, and Arg-Leu (208 +/- 13, 135 +/- 13, 116 +/- 12, and 127 +/- 12, respectively). (b) C terminus: the rate of disappearance of Leu-Ala (244 +/- 18) was significantly greater (p less than 0.01) than that of Leu-Gly (145 +/- 16). (c) Number of residues: with each increase in the number of alanine residues (2-4) there was a significant increase in the rate of peptide disappearance, and conversely, with each increase in the number of glycine residues (2-6) there was a significant decrease in the rate of peptide disappearance. Further studies showed no peptide transport by isolated liver plasma membrane vesicles and no significant correlation between the rates of peptide disappearance and hydrolase activities of the perfusion medium but highly significant correlation with hydrolase activity of plasma membrane. We conclude that certain amino acid residues, such as alanine, enhance hepatic clearance of oligopeptides by increasing their affinity as substrates for plasma membrane peptide hydrolases.