The folding of human active and inactive extracellular superoxide dismutases is an intracellular event

Human extracellular superoxide dismutase (EC-SOD) is a tetrameric glycoprotein responsible for the removal of superoxide generated in the extracellular space. Two different folding variants of EC-SOD exist based on the disulfide bridge connectivity, resulting in enzymatically active (aEC-SOD) and inactive (iEC-SOD) subunits. As a consequence of this, the assembly of the EC-SOD tetramers produces molecules with variable activity and may represent a way to regulate the antioxidant level in the extracellular space. To determine whether the formation of these two folding variants is an intra- or extracellular event, we analyzed the biosynthesis in human embryonic kidney 293 cells expressing wild-type EC-SOD. These analyses revealed that both folding variants were present in the intra- and extracellular spaces, suggesting that the formation is an intracellular event. To further analyze the biosynthesis, we constructed mutants with the capacity to generate only aEC-SOD (C195S) or iEC-SOD (C45S). The expression of these suggested that the cellular biosynthetic machinery supported the secretion of aEC-SOD but not iEC-SOD. The coexpression of these two mutants did not affect the expression pattern. This study shows that generation of the EC-SOD folding variants is an intracellular event that depends on a free cysteine residue not involved in disulfide bonding.     

Matrix remodelling enzymes, the protease cascade and glycosylation.

Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1).     

A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

The angiotensin AT₁ receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT₁ receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT₁ receptor and insulin receptor signaling pathways. To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET² technique to monitor the effect of angiotensin II on the interaction between Rluc⁸ tagged insulin receptor and GFP² tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin receptor and IRS4 interactions whereas Gαs- and Gαi-coupled 7TM receptors had no effect. Furthermore, we used a panel of kinase inhibitors to show that angiotensin II engages different pathways when regulating insulin receptor interactions with IRS1 and IRS4. Angiotensin II inhibited the interaction between insulin receptor and IRS1 through activation of ERK1/2, while the interaction between insulin receptor and IRS4 was partially inhibited through protein kinase C dependent mechanisms. We conclude that the crosstalk between angiotensin AT₁ receptor and insulin receptor signaling shows a high degree of specificity, and involves Gαq protein, and activation of distinct kinases. Thus, the BRET² technique can be used as a platform for studying molecular mechanisms of crosstalk between insulin receptor and 7TM receptors.     

Influence of insulin and free fatty acids on contractile function in patients with chronically stunned and hibernating myocardium

It is unknown whether short-term modulation of substrate supply affects cardiac performance in heart failure patients with chronic ischemic myocardium. The aim of this study was to determine whether modulation of myocardial substrate metabolism with insulin and free fatty acids (FFAs) affects contractile function of chronically stunned (CST) and hibernating (HIB) myocardium at rest and after maximal exercise. We studied eight nondiabetic patients with ejection fraction (EF) 30 +/- 4% (SE) and CST/HIB in 49 +/- 6% of the left ventricle: 36 +/- 6% CST and 13 +/- 2% HIB as determined by 99m Technetium-Sestamibi single photon emission computed tomography (SPECT) and [18F]fluorodeoxyglucose (FDG) positron emission tomography (PET). Each patient was subjected to a 3-h infusion of 1) saline, 2) insulin-glucose (i.e., euglycemic insulin clamp; high insulin, suppressed FFA), and 3) somatostatin-heparin (suppressed insulin, high FFA). Echocardiographic endpoints were global EF and regional contractile function [maximum velocity (Vmax) and strain rate (epsilon max)] as determined by tissue Doppler imaging at steady state and after maximal exercise. EF was similar at baseline and steady state and increased after exercise to 36 +/- 5% (P < 0.05). Baseline regional Vmax and epsilon max were highest in control, intermediate in CST and HIB, and lowest in infarct regions (P < 0.05). Steady-state EF, Vmax, and epsilon max were not affected by metabolic modulation in any region. After maximal exercise, contractile function increased in control, CST, and HIB (P < 0.05), but not in infarct, regions. Exercise-induced contractile increments were unaffected by metabolic modulation. Metabolic modulation does not influence contractile function in CST and HIB regions. Chronic ischemic myocardium has preserved ability to adapt to extreme, short-term changes in substrate supply at rest and after maximal exercise.     

Sheep grazing and rodent populations: evidence of negative interactions from a landscape scale experiment

Inter-specific competition, facilitation and predation influence herbivore assemblages, but no study has experimentally explored the interactions between large ungulates and small rodents. In a fully replicated, landscape scale experiment, we manipulated densities of domestic sheep in mountain pastures in Norway. We then determined population growth and densities of rodents by live trapping in each of the areas with different sheep densities. We found that the (summer) population growth rate and autumn density of the field vole (Microtus agrestis) was lower at high sheep density. This provides the first experimental evidence of negative interactions between an ungulate and small rodent species. There was no effect on the bank vole (Clethrionomys glareolus), whose diet differs from sheep. Sheep density, therefore, potentially alters the pattern of inter-specific population synchrony amongst voles. Our study shows that negative interactions between large ungulates and small rodents may be species-specific and negative population consequences for the rodent population appear above threshold ungulate densities.Electronic supplementary material is available for this article at     

The human SH-SY5Y neuroblastoma cell-line expresses a functional P2X7 purinoceptor that modulates voltage-dependent Ca2+ channel function

Fura-2 imaging of purinergic stimulation of non-differentiated neuronal human SH-SY5Y cells resulted in a rapid elevation in intracellular Ca2+ ([Ca2+]i) that was dependent on extracellular Ca2+. The rank order of agonists (200 micro m) was as follows: 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP) > ATP4- > ATP; whereas 2-(methylthio)-ATP, ADP, UTP and alpha,beta-methylene-ATP and beta,gamma-methylene-ATP were ineffective. The response to BzATP was inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic-acid (PPADS, 1 micro m), 1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine (KN-62, 100 nm) and 8-(3-benzamido-4-4-methylbenzamido)-naphthalene-1,3,5-trisulfonic-acid (suramin, 200 micro m). The presence of a P2X7 receptor was confirmed by western blot studies using anti-P2X7. EC50 for BzATP was 212 +/- 6 micro m. BzATP > 30 micro m induced an initial, transient increase in [Ca2+]i before a plateau level was reached. BzATP < 30 micro m only produced a monophasic increase to the plateau level. The transient phase was reduced by the introduction of nimodipine (3 micro m) and to a smaller degree by omega-conotoxin GVIA (1 micro m) despite an almost equal presence of L and N-type Ca2+-channels. In whole-cell voltage-clamp studies at - 90 mV, BzATP (300 micro m) produced a fast activating inward current with a similar pharmacology as observed with Fura-2 imaging. Current clamp studies showed a dose-dependent depolarization to BzATP and ATP4-. BzATP also triggered transmitter release. Thus, the human neuronal SH-SY5Y cell line expresses a functional P2X7 receptor coupled to activation of Ca2+-channels.     

CNTF inhibits high voltage activated Ca2+ currents in fetal mouse cortical neurones

Neurotrophic factors yield neuroprotection by mechanisms that may be related to their effects as inhibitors of apoptosis as well as their effects on ion channels. The effect of ciliary neurotrophic factor (CNTF) on high-threshold voltage-activated Ca channels in cultured fetal mouse brain cortical neurones was investigated. Addition of CNTF into serum-free growth medium resulted in delayed reduction of the Ca2+ currents. The currents decreased to 50% after 4 h and stabilized at this level during incubation with CNTF for 48 h. Following removal of CNTF the inhibition was completely reversed after 18 h. CNTF reduced the current of all pharmacological subtypes of Ca channels as shown by use of selective blockers of L, N, and P/Q type Ca channels (nifedipine, omega-conotoxin MVIIA, omega-agatoxin IVA). The Ca channel depression was mediated via the CNTF receptor, because enzymatic cleavage of the alpha-subunit glycerophosphatidylinositol anchor of the receptor eliminated the response. The CNTF effect was not elicited through pertussis toxin-sensitive G proteins. Other neurotrophic factors like neurotrophin-3 and insulin-like growth factor-I had no effect on the Ca2+ currents. These results may have important implications for the possible functions of CNTF in the nervous system, such as altered synaptic activity, neuronal excitability and susceptibility to brain ischaemia.