Influence of ethnic background on left atrial markers of inflammation,endothelial function and tissue remodelling

Background

It has been suggested that ethnicity can make a significant difference to the likelihood of thromboembolic stroke related to atrial fibrillation. Ethnic differences have been shown to alter inflammatory and haemostatic factors; however, this may all be confounded by differences in cardiovascular risk factors between different ethnicity. The impact of different ethnicities on the thrombogenic profile is not known. The aim of this study was to investigate differences in markers of inflammation, endothelial function and tissue remodelling between Caucasian and Indian populations with supraventricular tachycardia (SVT).

Mutation detection using immobilized mismatch binding protein (MutS).

An accurate and highly sensitive mutation detection assay has been developed. The assay is based on the detection of mispaired and unpaired bases by immobilized mismatch binding protein (Escherichia coli MutS). The assay can detect most mismatches and all single base substitution mutations, as well as small addition or deletion mutations. The assay is simple to use and does not require the use of either radioactivity or gel electrophoresis.     

Vanadium chemistry and biochemistry of relevance for use of vanadium compounds as antidiabetic agents

The stability of 11 vanadium compounds is tested under physiological conditions and in administration fluids. Several compounds including those currently used as insulin-mimetic agents in animal and human studies are stable upon dissolution in distilled water but lack such stability in distilled water at pH7. Complex lability may result in decomposition at neutral pH and thus may compromise the effectiveness of these compounds as therapeutic agents; Even well characterized vanadium compounds are surprisingly labile. Sufficiently stable complexes such as the VEDTA complex will only slowly reduce, however, none of the vanadium compounds currently used as insulin-mimetic agents show the high stability of the VEDTA complex. Both the bis(maltolato)oxovanadium(IV) and peroxovanadium complexes extend the insulin-mimetic action of vanadate in reducing cellular environments probably by increased lifetimes under physiological conditions and/or by decomposing to other insulin mimetic compounds. For example, treatment with two equivalents of glutathione or other thiols the (dipicolinato)peroxovanadate(V) forms 9dipicolinato)oxovanadate(V) and vanadate, which are both insulin-mimetic vanadium(V) compounds and can continue to act. The reactivity of vanadate under physiological conditions effects a multitude of biological responses. Other vanadium complexes may mimic insulin but not induce similar responses if the vanadate formation is blocked or reduced. We conclude that three properties, stability, lability and redox chemistry are critical to prolong the half-life of the insulin-mimetic form of vanadium compounds under physiological conditions and should all be considered in development of vanadium-based oral insulin-mimetic agents.Abbreviations ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - ADP-V adenosine 5-diphosphate-vanadate - bpV bis(peroxo)oxovanadium(V) - (bpV)2 bis(peroxo)oxovanadium(V) dimer - bpVpic bis(peroxo)picolinatooxovanadate(V) - ¹³C carbon-13 - EDTA ethylenediaminetetraacetic acid - EPR electron paramagnetic resonance - EXSY exchange spectroscopy - ¹H proton - HSG glutathione - NAD -nicotinamide adenine dinucleotide - NADP -nicotinamide adenine dinucleotide phosphate - NADV -nicotinamide adenine dinucleotide vanadate - NMR nuclear magnetic resonance (also referred to as magnetic resonance imaging) - pVdipic (dipicolinato)peroxovanadate(V) - Vcit (citrato)dioxovanadate(V) - VEDTA (ethylenediaminetetraacetato)dioxovanadate(V) - Vmalto bis(maltolato)-oxovanadium(IV) - Voxal bis(oxalato)dioxovanadate(V) - ⁵¹V vanadium-51 - V₁ vanadate monomer - V₂ vanadate dimer - V₄ vanadate tetramer - V₅ vanadate pentamer - UV-vis spectroscopy ultraviolet-visible spectroscopy     

Expression of the inducible nitric oxide synthase gene in diaphragm and skeletal muscle

Thompson, Marita, Lisa Becker, Debbie Bryant, Gary Williams,Daniel Levin, Linda Margraf, and Brett P. Giroir. Expression ofthe inducible nitric oxide synthase gene in diaphragm and skeletal muscle. J. Appl. Physiol. 81(6):2415-2420, 1996.Nitric oxide (NO) is a pluripotent molecule thatcan be secreted by skeletal muscle through the activity of the neuronalconstitutive isoform of NO synthase. To determine whether skeletalmuscle and diaphragm might also express the macrophage-inducible formof NO synthase (iNOS) during provocative states, we examined tissuefrom mice at serial times after intravenous administration ofEscherichia coli endotoxin. In thesestudies, iNOS mRNA was strongly expressed in the diaphragm and skeletalmuscle of mice 4 h after intravenous endotoxin and was significantlydiminished by 8 h after challenge. Induction of iNOS mRNA was followedby expression of iNOS immunoreactive protein on Western immunoblots.Increased iNOS activity was demonstrated by conversion of arginine tocitrulline. Immunochemical analysis of diaphragmatic explants exposedto endotoxin in vitro revealed specific iNOS staining in myocytes, inaddition to macrophages and endothelium. These results may be importantin understanding the pathogenesis of respiratory pump failure duringseptic shock, as well as skeletal muscle injury during inflammation ormetabolic stress.

     

Receptor Activity-modifying Proteins 2 and 3 Generate Adrenomedullin Receptor Subtypes with Distinct Molecular Properties

Adrenomedullin (AM) is a peptide hormone with numerous effects in the vascular systems. AM signals through the AM₁ and AM₂ receptors formed by the obligate heterodimerization of a G protein-coupled receptor, the calcitonin receptor-like receptor (CLR), and receptor activity-modifying proteins 2 and 3 (RAMP2 and RAMP3), respectively. These different CLR-RAMP interactions yield discrete receptor pharmacology and physiological effects. The effective design of therapeutics that target the individual AM receptors is dependent on understanding the molecular details of the effects of RAMPs on CLR. To understand the role of RAMP2 and -3 on the activation and conformation of the CLR subunit of AM receptors, we mutated 68 individual amino acids in the juxtamembrane region of CLR, a key region for activation of AM receptors, and determined the effects on cAMP signaling. Sixteen CLR mutations had differential effects between the AM₁ and AM₂ receptors. Accompanying this, independent molecular modeling of the full-length AM-bound AM₁ and AM₂ receptors predicted differences in the binding pocket and differences in the electrostatic potential of the two AM receptors. Druggability analysis indicated unique features that could be used to develop selective small molecule ligands for each receptor. The interaction of RAMP2 or RAMP3 with CLR induces conformational variation in the juxtamembrane region, yielding distinct binding pockets, probably via an allosteric mechanism. These subtype-specific differences have implications for the design of therapeutics aimed at specific AM receptors and for understanding the mechanisms by which accessory proteins affect G protein-coupled receptor function.     

Co‐expression of the protease furin in Nicotiana benthamiana leads to efficient processing of latent transforming growth factor‐β1 into a biologically active protein

Transforming growth factor beta (TGF‐β) is a signalling molecule that plays a key role in developmental and immunological processes in mammals. Three TGF‐β isoforms exist in humans, and each isoform has unique therapeutic potential. Plants offer a platform for the production of recombinant proteins, which is cheap and easy to scale up and has a low risk of contamination with human pathogens. TGF‐β3 has been produced in plants before using a chloroplast expression system. However, this strategy requires chemical refolding to obtain a biologically active protein. In this study, we investigated the possibility to transiently express active human TGF‐β1 in Nicotiana benthamiana plants. We successfully expressed mature TGF‐β1 in the absence of the latency‐associated peptide (LAP) using different strategies, but the obtained proteins were inactive. Upon expression of LAP‐TGF‐β1, we were able to show that processing of the latent complex by a furin‐like protease does not occur in planta. The use of a chitinase signal peptide enhanced the expression and secretion of LAP‐TGF‐β1, and co‐expression of human furin enabled the proteolytic processing of latent TGF‐β1. Engineering the plant post‐translational machinery by co‐expressing human furin also enhanced the accumulation of biologically active TGF‐β1. This engineering step is quite remarkable, as furin requires multiple processing steps and correct localization within the secretory pathway to become active. Our data demonstrate that plants can be a suitable platform for the production of complex proteins that rely on specific proteolytic processing.     

Unique Characteristics of Recombinant Hybrid Manganese Superoxide Dismutase from <Emphasis Type="Italic">Staphylococcus equorum and S. saprophyticus</Emphasis>

A recombinant hybrid of manganese dependent-superoxide dismutase of Staphylococcus equorum and S. saprophyticus has successfully been overexpressed in Escherichia coli BL21(DE3), purified, and characterized. The recombinant enzyme suffered from degradation and aggregation upon storage at ?20 °C, but not at room temperature nor in cold. Chromatographic analysis in a size exclusion column suggested the occurrence of dimeric form, which has been reported to contribute in maintaining the stability of the enzyme. Effect of monovalent (Na⁺, K⁺), divalent (Ca²⁺, Mg²⁺), multivalent (Mn^2+/4+, Zn^2+/4+) cations and anions (Cl^?, SO₄ ^2?) to the enzyme stability or dimeric state depended on type of cation or anion, its concentration, and pH. However, tremendous effect was observed with 50 mM ZnSO₄, in which thermostability of both the dimer and monomer was increased. Similar situation was not observed with MnSO₄, and its presence was detrimental at 200 mM. Finally, chelating agent appeared to destabilize the dimer around neutral pH and dissociate it at basic pH. The monomer remained stable upon addition of ethylene diamine tetraacetic acid. Here we reported unique characteristics and stability of manganese dependent-superoxide dismutase from S. equorum/saprophyticus.     

Some effects of humic acid on two different biological systems

Summary Humic acid, and the water-and acid-extracted residue fractions obtained from it, stimulated the development of invertase activity in beet disks during aging. Those fractions which showed most effect on invertase development also caused the greatest numbers of sporangia to be formed on several species of the Actinoplanaceae (Actinomycetales) cultured on agar with incorporated humic acid. The results are discussed in relation to an aromatic core present in the humic acid.