Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.     

New amphiphilic lactic acid oligomer-hyaluronan conjugates: synthesis and physicochemical characterization

The "grafting onto" strategy was used to conjugate DL-lactic acid oligomers (OLA) to hyaluronan (HA) for the sake of developing novel degradable HA-based self-assembling polymeric systems. Grafting was achieved by reacting COCl-terminated OLA with cetyltrimethylammonium hyaluronate (CTA-HA) in dimethyl sulfoxide (DMSO). The resulting CTA-HAOLA conjugates were purified and turned to sodium form (Na-HAOLA) by dissolution in a phosphate buffer-DMSO mixture and successive dialyses against DMSO, ethanol, and water. In contrast, when the same protocol was applied to CTA-HAOLA, phase separation with gel formation was observed. The solution phase was composed of Na-HAOLA whereas the gel phase was made of mixed CTA-Na-HAOLA salt with ca. 25% of the carboxyl groups neutralized by CTA. Gelation was assigned to intramolecular hydrophobic associations between OLA and cetyl alkyl chains that complemented electrostatic interactions between CTA and HA COO- groups synergistically. Therefore, the corresponding stabilized CTA ions required more drastic conditions to be released. Under the selected dialysis conditions, the CTA-Na-HAOLA gels formed tiny tubes. Na-HAOLA and CTA-Na-HAOLA were characterized by FTIR, one-dimensional 1H and two-dimensional 1H NMR. The extent of grafting was ca. 5% per disaccharidic repeating unit, regardless of the molecular weight, as determined by NMR and capillary zone electrophoresis. Amphiphilic Na-HAOLA molecules were aggregated and formed spherical species in water according to size exclusion chromatography combined with multiangle laser light scattering detection. The critical aggregation concentration ranged between 0.2 and 0.35% (w/v), depending of the molecular weight of the parent hyaluronan.     

Insecticidal effects of Buthus occitanus tunetanus BotIT6 toxin expressed in Escherichia coli and baculovirus/insect cells

BotIT6 is a neurotoxin polypeptide derived from the venom of the scorpion Buthus occitanus tunetanus (Bot). Its mature form is composed of 62 amino acids. BotIT6 has been reported to be the most potent toxin from Bot venom that has a strict selectivity for insects. Such toxin may have potential as a potent animal-harmless tool against insects. Using RT-PCR, we isolated and sequenced a cDNA encoding 62 amino acid residues corresponding to the known amino acid sequence of BotIT6. We have expressed a recombinant active form of BotIT6 in significantly high amounts in Escherichia coli. We have also engineered the cDNA into the Autographa californica Nuclear Polyhedrosis Virus (AcMNPV) genome and expressed the protein under control of the polyhedrin promoter. Supernatants of AcIT6-virus infected Sf9 insect cells exhibit a typical intoxication effect when injected to Spodoptera littoralis larvae. Moreover, injection of the recombinant virus showed enhanced insecticidal potency against S. littoralis larvae compared with wild type AcMNPV.     

In vitro effect of metal ions on the activity of two amphibian glyceraldehyde-3-phosphate dehydrogenases: potential metal binding sites

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.     

A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.     

Selective uptake of high density lipoproteins cholesteryl ester in the dog, a species lacking in cholesteryl ester transfer protein activity; An in vivo approach using stable isotopes

Amongst the processes involved in the reverse cholesterol transport (RCT) from organs to liver, including high density lipoproteins-apolipoprotein AI (HDL-apoAI) dependent tissue uptake and cholesteryl ester transfer protein (CETP)-mediated transfers, the selective uptake of cholesteryl ester (CE) is of increasing interest through its antiatherogenic role. The purpose of this report is to develop a simple protocol allowing study of this process in an animal model with easier quantification of CE selective uptake. The dog was chosen essentially because this animal has a low CETP activity and an appropriate size to conduce a kinetic study. Tracer kinetics were performed to estimate in vivo the contributions of the pathways involved in HDL-CE turnover in dogs. Stable isotopes, ¹³C-acetate and D₃-leucine as labeled precursors of CE and apoAI, were infused to fasting dogs. Isotopic enrichments were monitored in plasma unesterified cholesterol and in HDL-CE and apoAI by mass spectrometry. Kinetics were analyzed using compartmental modeling. Results concerned the measurement of the activity of cholesterol esterification (0.13±0.032 h⁻¹), rate of HDL-apoAI catabolism (0.024±0.012 h⁻¹), HDL-CE turnover (0.062±0.010 h⁻¹) and CE selective uptake (0.038±0.014 h⁻¹). Our results show that CE in dogs is mainly eliminated by selective uptake of HDL-CE (60% of HDL-CE turnover), unlike in other species studied by similar methods in our laboratory. This study shows that among species used to analyze cholesterol metabolism, the dog appears to be the animal in whom HDL-CE selective uptake represents the largest part of HDL-CE turnover.     

NrdH-redoxin of Mycobacterium tuberculosis and Corynebacterium glutamicum Dimerizes at High Protein Concentration and Exclusively Receives Electrons from Thioredoxin Reductase

NrdH-redoxins are small reductases with a high amino acid sequence similarity with glutaredoxins and mycoredoxins but with a thioredoxin-like activity. They function as the electron donor for class Ib ribonucleotide reductases, which convert ribonucleotides into deoxyribonucleotides. We solved the x-ray structure of oxidized NrdH-redoxin from Corynebacterium glutamicum (Cg) at 1.5 Å resolution. Based on this monomeric structure, we built a homology model of NrdH-redoxin from Mycobacterium tuberculosis (Mt). Both NrdH-redoxins have a typical thioredoxin fold with the active site CXXC motif located at the N terminus of the first α-helix. With size exclusion chromatography and small angle x-ray scattering, we show that Mt_NrdH-redoxin is a monomer in solution that has the tendency to form a non-swapped dimer at high protein concentration. Further, Cg_NrdH-redoxin and Mt_NrdH-redoxin catalytically reduce a disulfide with a specificity constant 1.9 × 10⁶ and 5.6 × 10⁶ m⁻¹ min⁻¹, respectively. They use a thiol-disulfide exchange mechanism with an N-terminal cysteine pK_a lower than 6.5 for nucleophilic attack, whereas the pK_a of the C-terminal cysteine is ∼10. They exclusively receive electrons from thioredoxin reductase (TrxR) and not from mycothiol, the low molecular weight thiol of actinomycetes. This specificity is shown in the structural model of the complex between NrdH-redoxin and TrxR, where the two surface-exposed phenylalanines of TrxR perfectly fit into the conserved hydrophobic pocket of the NrdH-redoxin. Moreover, nrdh gene deletion and disruption experiments seem to indicate that NrdH-redoxin is essential in C. glutamicum.     

A new Palaeocene Megalopidae (Teleostei,Elopomorpha) from the phosphate basins of Morocco

Abstract

A new Megalopidae (Teleostei, Elopomorpha, Elopiformes), Protarpon boualii sp. nov., is described on the basis of two neurocrania gathered from the Lower Palaeocene (Danian) beds of the Oulad Abdoun basin, in Morocco. Its inclusion in the family Megalopidae and the genus Protarpon is supported by the L-shaped pterotics, the flat skull roof, the well developed epiotic processes and the roofed dilatator fossae. It differs from Protarpon priscus and P. oblongus from the Ypresian of the London Clay Formation (England) mainly by the proportions of the subtemporal and the post-temporal fossa openings, the proportions of skull roof bones and its larger size. Protarpon boualii sp. nov. represents the first fossil occurrence of a megalopid in North Africa. Its close phylogenetic relationships with forms from the London Clay Formation highlight the strong biogeographical affinities between the faunas of these two localities during the Palaeocene-Eocene period.

http://www.zoobank.org/urn:lsid:zoobank.org:pub:D5FEE8B3-B220-461F-B635-31DD7F2CF921     

Glucotoxic and diabetic conditions induce caspase 6-mediated degradation of nuclear lamin A in human islets,rodent islets and INS-1 832/13 cells

Nuclear lamins form the lamina on the interior surface of the nuclear envelope, and regulate nuclear metabolic events, including DNA replication and organization of chromatin. The current study is aimed at understanding the role of executioner caspase 6 on lamin A integrity in islet β-cells under duress of glucotoxic (20 mM glucose; 24 h) and diabetic conditions. Under glucotoxic conditions, glucose-stimulated insulin secretion and metabolic cell viability were significantly attenuated in INS-1 832/13 cells. Further, exposure of normal human islets, rat islets and INS-1 832/13 cells to glucotoxic conditions leads to caspase 6 activation and lamin A degradation, which is also observed in islets from the Zucker diabetic fatty rat, a model for type 2 diabetes (T2D), and in islets from a human donor with T2D. Z-Val-Glu-Ile-Asp-fluoromethylketone, a specific inhibitor of caspase 6, markedly attenuated high glucose-induced caspase 6 activation and lamin A degradation, confirming that caspase 6 mediates lamin A degradation under high glucose exposure conditions. Moreover, Z-Asp-Glu-Val-Asp-fluoromethylketone, a known caspase 3 inhibitor, significantly inhibited high glucose-induced caspase 6 activation and lamin A degradation, suggesting that activation of caspase 3 might be upstream to caspase 6 activation in the islet β-cell under glucotoxic conditions. Lastly, we report expression of ZMPSTE24, a zinc metallopeptidase involved in the processing of prelamin A to mature lamin A, in INS-1 832/13 cells and human islets; was unaffected by high glucose. We conclude that caspases 3 and 6 could contribute to alterations in the integrity of nuclear lamins leading to metabolic dysregulation and failure of the islet β-cell.     

Safety and Immunogenicity of H1/IC31?, an Adjuvanted TB Subunit Vaccine,in HIV-Infected Adults with CD4+ Lymphocyte Counts Greater than 350 cells/mm3: A Phase II,Multi-Centre,Double-Blind,Randomized, Placebo-Controlled Trial

Background

Novel tuberculosis vaccines should be safe, immunogenic, and effective in various population groups, including HIV-infected individuals. In this phase II multi-centre, double-blind, placebo-controlled trial, the safety and immunogenicity of the novel H1/IC31 vaccine, a fusion protein of Ag85B-ESAT-6 (H1) formulated with the adjuvant IC31, was evaluated in HIV-infected adults.

Methods

HIV-infected adults with CD4+ T cell counts >350/mm³ and without evidence of active tuberculosis were enrolled and followed until day 182. H1/IC31 vaccine or placebo was randomly allocated in a 5∶1 ratio. The vaccine was administered intramuscularly at day 0 and 56. Safety assessment was based on medical history, clinical examinations, and blood and urine testing. Immunogenicity was determined by a short-term whole blood intracellular cytokine staining assay.

Results

47 of the 48 randomised participants completed both vaccinations. In total, 459 mild or moderate and 2 severe adverse events were reported. There were three serious adverse events in two vaccinees classified as not related to the investigational product. Local injection site reactions were more common in H1/IC31 versus placebo recipients (65.0% vs. 12.5%, p = 0.015). Solicited systemic and unsolicited adverse events were similar by study arm. The baseline CD4+ T cell count and HIV viral load were similar by study arm and remained constant over time. The H1/IC31 vaccine induced a persistent Th1-immune response with predominately TNF-α and IL-2 co-expressing CD4+ T cells, as well as polyfunctional IFN-γ, TNF-α and IL-2 expressing CD4+ T cells.

Conclusion

H1/IC31 was well tolerated and safe in HIV-infected adults with a CD4+ Lymphocyte count greater than 350 cells/mm³. The vaccine did not have an effect on CD4+ T cell count or HIV-1 viral load. H1/IC31 induced a specific and durable Th1 immune response.

Trial registration

Pan African Clinical Trials Registry (PACTR) PACTR201105000289276