Efficient generation of transgenic pigs using equine infectious anaemia virus (EIAV) derived vector

Traditional methods of transgene delivery in livestock are inefficient. Recently, human immunodeficiency virus (HIV-1) based lentiviral vectors have been shown to offer an efficient transgene delivery system. We now extend this method by demonstrating efficient generation of transgenic pigs using an equine infectious anaemia virus derived vector. We used this vector to deliver a green fluorescent protein expressing transgene; 31% of injected/transferred eggs resulted in a transgenic founder animal and 95% of founder animals displayed green fluorescence. This compares favourably with results using HIV-1 based vectors, and is substantially more efficient than the standard pronuclear microinjection method, indicating that lentiviral transgene delivery may be a general tool with which to efficiently generate transgenic mammals.     

Growth factor impact on articular cartilage subpopulations

We have examined the effects of growth factor stimulation on superficial and growth zone chondrocyte populations. Zonal articular chondrocytes from 8-month-old Spanish goat distal femurs were plated in monolayer cultures and stimulated by using insulin-like growth factor I (IGF-I), basic fibroblast growth factor (bFGF), and transforming growth factor-β1 (TGF-β1). Gene expression for collagen I and II, aggrecan, and superficial zone protein were evaluated every week for 3 weeks. Finally, proteoglycan and collagen deposition were measured for each experimental group. Major differences existed in the behavior of superficial and growth zone chondrocytes, the most apparent being the higher capacity for protein synthesis by the growth zone population. Variations also existed regarding growth factor treatment. TGF-β1 had the greatest effect on proliferation over 8 days. With respect to differentiation, IGF-I increased average collagen II gene expression in the growth zone populations in comparison with growth zone controls. IGF-I increased aggrecan gene expression for the same groups. Superficial zone populations exhibited lower collagen II, collagen I, and aggrecan gene expression than the growth zone populations under all conditions. However, superficial zone protein expression was dramatically elevated in superficial zone populations by TGF-β1. Collagen I expression showed a general increase under all conditions compared with initial values. Combined biosynthesis results showed that the superficial populations secreted little to no collagen, especially collagen II, in comparison with their growth zone counterparts. Glycosaminoglycan production was also much lower than for the growth zone groups. TGF-β1 and IGF-I increased collagen II production in the growth zone populations. TGF-β1 increased glycosaminoglycan secretions in the superficial zone populations and in the growth zone populations, whereas IGF-I produced an increase in glycosaminoglycan secretion only in the growth zone populations. Thus, growth factors elicit different proliferation, gene expression, and biosynthesis responses from zonal chondrocyte subpopulations.     

Studies of the mechanisms involved in the fate of prostacyclin (PGI2) and 06-keto-PGF1α in the pulmonary circulation

We have investigated the metabolism of [3]H-prostaglandin (PG)I₂ and its non-enzymatic breakdown product [3]H-6-keto-PGF_1α by rat pulmonary tissue and their possible uptake and metabolism upon passage through the isolated perfused rat lung. When incubated with rat lung homogenate in the presence of β-NAD, [3]H-PGI₂ was extensively degraded into at least one metabolite, while [3]H-6-keto-PGF_1α was only minimally metabolized. However, on passage through isolated perfused rat lungs, neither [3]H-PGI₂ nor [3]H-6-keto-PGF_1α were removed from the circulation into the lung or degraded. This demonstration that PGI₂ is not a substrate for the transport system for the removal of PGs from the circulation into the lung further illustrates that this system is a critical determinant for the pulmonary inactivation of circulating prostaglandins. The experimental findings are discussed in reference to the structure-activity requirements necessary for pulmonary transport and subsequent metabolism.     

Polysaccharide deacetylases serve as new targets for the design of inhibitors against Bacillus anthracis and Bacillus cereus

Peptidoglycan N-acetylglucosamine (GlcNAc) deacetylases (PGNGdacs) from bacterial pathogens are validated targets for the development of novel antimicrobial agents. In this study we examined the in vitro inhibition of hydroxamate ligand N-hydroxy-4-(naphthalene-1-yl)benzamide (NHNB), a selective inhibitor of histone deacetylases-8 (HDAC8), against two PGNGdacs namely BC1974 and BC1960 from B. cereus, highly homologous to BA1977 and BA1961 of B. anthracis, respectively. Kinetic analysis showed that this compound functions as a competitive inhibitor of both enzymes with apparent K_i’s of 8.7?μM (for BC1974) and 66?μM (for BC1960), providing thus the most potent CE4 inhibitor reported to date. NHNB was tested in antibacterial assays and showed bactericidal activity against both examined pathogens acting as a multi-target drug. This compound can serve as lead for the development of inhibitors targeting the conserved active sites of the multiple polysaccharide deacetylases (PDAs) of both pathogens.     

The germination characteristics of phytochrome-deficient aurea mutant tomato seeds

The role of light reactions in anthocyanin synthesis was studied in both attached and detached corollas of Petunia hybrida (cv. Hit Parade Rosa), the latter grown in vitro in media containing 150 m M sucrose and 50 μ M gibberellic acid (GA). Light was essential for the synthesis of anthocyanin in detached corollas, whereas in intact corollas its effect was only to enhance anthocyanin synthesis. Continuous white light at a fluence rate of at least 20 μmol m⁻² s⁻¹ was needed for anthocyanin synthesis in detached corollas. Blue light was more effective than red or green, and far-red was ineffective. Pigmentation of detached corollas exposed to light was inhibited by the photosynthetic inhibitor 3-(4-dichlorophenyl)-1,1-dimethylurea (DCMU). The chloroplast uncoupler NH₄Cl did not affect anthocyanin synthesis, which was, however, inhibited by the blocking of ATP synthesis in both the chloroplast and the mitochondria by dicyclohexylcarbodiimide (DCCD). Sucrose uptake in vitro was inhibited by DCMU and by darkness, and was promoted equally by blue and red light. The activity of phenylalanine ammonialyase (EC 4.3.1.5) was inhibited in detached corollas grown in the dark or in the light in the presence of DCMU. The activity of chalcone isomerase (EC 5.5.1.6) was not affected by light. These findings suggest that at least two different light reactions are involved in the regulation of anthocyanin synthesis in petunia corollas, namely the high irradiance reaction (HIR) and photosynthesis.     

Vanilloid receptor agonists and antagonists are mitochondrial inhibitors: how vanilloids cause non-vanilloid receptor mediated cell death

Time-lapse photomicroscopy of human H460 lung cancer cells demonstrated of the transient receptor potential V1 (TRPV1) channel agonists, (E)-capsaicin and resiniferatoxin, and the TRPV1 antagonists, capsazepine, and SB366791, were able to bring about morphological changes characteristic of apoptosis and/or necrosis. Immunoblot analysis identified immunoreactivity for the transient receptor potential V1 (TRPV1) channel in rat brain samples, but not in rat heart mitochondria or in H460 cells. In isolated rat heart mitochondria, all four ligands caused concentration-dependent decreases in oxygen consumption and mitochondrial membrane potential. (E)-Capsaicin and capsazepine evoked concentration-dependent increases and decreases, respectively, in mitochondrial hydrogen peroxide production, whilst resiniferatoxin and SB366791 were without significant effect. These data support the hypothesis that (E)-capsaicin, resiniferatoxin, capsazepine, and SB366791 are all mitochondrial inhibitors, able to activate apoptosis and/or necrosis via non-receptor mediated mechanisms, and also support the use of TRPV1 ligands as anti-cancer agents.     

The crystal structure of cobalt-substituted pseudoazurin from Alcaligenes faecalis

The Cu(II) center at the active site of the blue copper protein pseudoazurin from Alcaligenes faecalis has been substituted by Co(II) via denaturing of the protein, chelation and removal of copper by EDTA and refolding of the apo‐protein, followed by addition of an aqueous solution of CoCl₂. Sitting drop vapour diffusion experiments produced green hexagonal crystals, which belong to space group P6₅, with unit cell dimensions a = b = 50.03, c = 98.80 Å. Diffraction data, collected at 291 K on a copper rotating anode X‐ray source, were phased by the anomalous signal of the cobalt atom. The structure was built automatically, fitted manually and subsequently refined to 1.86 Å resolution. The Co‐substituted protein exhibits similar overall geometry to the native structure with copper. Cobalt binds more strongly to the axial Met86‐Sδ and retains the tetrahedral arrangement with the four ligand atoms, His40‐Nδ₁, Cys78‐Sγ, His81‐Nδ₁, and 86Met‐Sδ, although the structure is less distorted than the native copper protein. The structure reported herein, is the first crystallographic structure of a Co(II)‐substituted pseudoazurin. © 2010 Wiley Periodicals, Inc. Biopolymers 95: 202–207, 2011.     

On the Active Surface State of Nickel‐Ceria Solid Oxide Fuel Cell Anodes During Methane Electrooxidation

Solid oxide fuel cells (SOFCs) have grown in recognition as a viable technology able to convert chemical energy directly into electricity, with higher efficiencies than conventional thermal engines. Direct feeding of the SOFCs anode with hydrocarbons from fossil or renewable sources, appears more attractive compared to the use of hydrogen as a fuel. The addition of mixed oxide‐ion/electron conductors, like gadolinium‐doped ceria (GDC), to commonly used nickel‐based anodes is a well–known strategy that significantly enhances the performance of the SOFCs. Here we provide in situ experimental evidence of the active surface oxidation state and composition of Ni/GDC anodes during methane electroxidation using realistic solid oxide electrode assemblies. Ambient pressure X‐ray photoelectron and near edge X‐ray absorption fine structure spectroscopies (APPES and NEXAFS respectively) combined with on line electrical and gas phase measurements, were used to directly associate the surface state and the electrocatalytic performance of Ni/GDC anodes working at intermediate temperatures (700°C). A reduced anode surface (Ce³⁺ and Ni), with an optimum Ni to Ce surface composition, were found to be the most favorable configuration for maximum cell currents. Experimental results are rationalized on the basis of first principles calculations, proposing a detailed mechanism of the cell function.