Microsomal triglyceride transfer protein -493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.     

GABA(A) receptor function is regulated by lipid bilayer elasticity

Docosahexaenoic acid (DHA) and other polyunsaturated fatty acids (PUFAs) promote GABA(A) receptor [(3)H]-muscimol binding, and DHA increases the rate of GABA(A) receptor desensitization. Triton X-100, a structurally unrelated amphiphile, similarly promotes [(3)H]-muscimol binding. The mechanism(s) underlying these effects are poorly understood. DHA and Triton X-100, at concentrations that affect GABA(A) receptor function, increase the elasticity of lipid bilayers measured as decreased bilayer stiffness using gramicidin channels as molecular force transducers. We have previously shown that membrane protein function can be regulated by amphiphile-induced changes in bilayer elasticity and hypothesized that GABA(A) receptors could be similarly regulated. We therefore studied the effects of four structurally unrelated amphiphiles that decrease bilayer stiffness (Triton X-100, octyl-beta-glucoside, capsaicin, and DHA) on GABA(A) receptor function in mammalian cells. All the compounds promoted GABA(A) receptor [(3)H]-muscimol binding by increasing the binding capacity of high-affinity binding without affecting the associated equilibrium binding constant. A semiquantitative analysis found a similar quantitative relation between the effects on bilayer stiffness and [(3)H]-muscimol binding. Membrane cholesterol depletion, which also decreases bilayer stiffness, similarly promoted [(3)H]-muscimol binding. In whole-cell voltage-clamp experiments, Triton X-100, octyl-beta-glucoside, capsaicin, and DHA all reduced the peak amplitude of the GABA-induced currents and increased the rate of receptor desensitization. The effects of the amphiphiles did not correlate with the expected changes in monolayer spontaneous curvature. We conclude that GABA(A) receptor function is regulated by lipid bilayer elasticity. PUFAs may generally regulate membrane protein function by affecting the elasticity of the host lipid bilayer.     

Nuclear protein kinase C

Protein kinase C (PKC) isozymes constitute a family of ubiquitous phosphotransferases which act as key transducers in many agonist-induced signaling cascades. To date, at least 11 different PKC isotypes have been identified and are believed to play distinct regulatory roles. PKC isoforms are physiologically activated by a number of lipid cofactors. PKC is thought to reside in the cytoplasm in an inactive conformation and to translocate to the plasma membrane or cytoplasmic organelles upon cell activation by different stimuli. However, a sizable body of evidence collected over the last 20 years has shown PKC to be capable of translocating to the nucleus. Furthermore, PKC isoforms are resident within the nucleus. Studies from independent laboratories have to led to the identification of quite a few nuclear proteins which are PKC substrates and to the characterization of nuclear PKC-binding proteins which may be critical for finely tuning PKC function in this cell microenvironment. Several lines of evidence suggest that nuclear PKC isozymes are involved in the regulation of biological processes as important as cell proliferation and differentiation, gene expression, neoplastic transformation, and apoptosis. In this review, we shall highlight the most intriguing and updated findings about the functions of nuclear PKC isozymes.     

Cerato-platanin, the first member of a new fungal protein family

The ascomcete Ceratocystis fimbriata, the causal agent of “canker stain disease,” secretes a protein of 12.4 kDa that elicits phytoalexin synthesis and plant cell death. This protein, named cerato-platanin (CP), is also located in the cell walls of ascospores, hyphae, and conidia; it contains four cysteines (S-S bridged) and is moderately hydrophobic. The cp gene consists of a single exon and has 42 bp codifying for a signal peptide of 14 residues. The recombinant protein was obtained by cloning the cp gene of the mature protein in Escherichia coli (BL21), and a refolding step was needed to achieve the native active form. In the European Molecular Biology data bank, CP is reported as the first member of the CP family; this is the first example of an set of secreted fungal proteins whose primary structure is very similar. Nonetheless, the data also revealed some structural and functional features that make CP simlar to proteins of the hydrophobin family.     

The molecular basis of filamin binding to integrins and competition with talin

The ability of adhesion receptors to transmit biochemical signals and mechanical force across cell membranes depends on interactions with the actin cytoskeleton. Filamins are large, actin-crosslinking proteins that connect multiple transmembrane and signaling proteins to the cytoskeleton. Here, we describe the high-resolution structure of an interface between filamin A and an integrin adhesion receptor. When bound, the integrin beta cytoplasmic tail forms an extended beta strand that interacts with beta strands C and D of the filamin immunoglobulin-like domain (IgFLN) 21. This interface is common to many integrins, and we suggest it is a prototype for other IgFLN domain interactions. Notably, the structurally defined filamin binding site overlaps with that of the integrin-regulator talin, and these proteins compete for binding to integrin tails, allowing integrin-filamin interactions to impact talin-dependent integrin activation. Phosphothreonine-mimicking mutations inhibit filamin, but not talin, binding, indicating that kinases may modulate this competition and provide additional means to control integrin functions.     

Synthesis and activity of a novel class of tribasic macrocyclic antibiotics: the triamilides

The stereoselective synthesis of two novel series of tribasic macrocyclic antibiotics with potent in vitro activity against Pasteurella multocida and Escherichia coli strains of bacteria is described. The in vitro activity can be significantly influenced by the nature of the substituents on the C-4" aminoalcohol, with the stereochemistry of the C-4" alcohol playing a less critical role. The effect of substitution and stereochemistry on the in vivo activity in a murine model of respiratory infection is also described.     

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.     

Human complement C3 is a substrate for transglutaminases. A functional link between non-protease-based members of the coagulation and complement cascades

In this study, we report the finding of functional cross-talk between two non-protease components of the complement and coagulation cascades. We show that complement C3, a central component of the complement system, is associated with the fibrin clot and that C3 becomes covalently cross-linked to other proteins during coagulation. Enzymatic incorporation of dansylcadaverine and dansyl-PGGQQIV into C3 by coagulation factor XIIIa and tissue transglutaminase demonstrated that C3 is a transglutaminase substrate. This suggested that coagulation factor XIIIa covalently cross-links C3 to clot components during coagulation. Using mass spectrometry, we verified that C3 indeed is covalently associated with the fibrin clot in a ratio of 0.05:1 relative to the known coagulation factor XIIIa substrate α2-antiplasmin.     

PHYLOGENETIC CONSTRAINTS IN KEY FUNCTIONAL TRAITS BEHIND SPECIES’ CLIMATE NICHES: PATTERNS OF DESICCATION AND COLD RESISTANCE ACROSS 95 DROSOPHILA SPECIES

Species distributions are often constrained by climatic tolerances that are ultimately determined by evolutionary history and/or adaptive capacity, but these factors have rarely been partitioned. Here, we experimentally determined two key climatic niche traits (desiccation and cold resistance) for 92–95 Drosophila species and assessed their importance for geographic distributions, while controlling for acclimation, phylogeny, and spatial autocorrelation. Employing an array of phylogenetic analyses, we documented moderate‐to‐strong phylogenetic signal in both desiccation and cold resistance. Desiccation and cold resistance were clearly linked to species distributions because significant associations between traits and climatic variables persisted even after controlling for phylogeny. We used different methods to untangle whether phylogenetic signal reflected phylogenetically related species adapted to similar environments or alternatively phylogenetic inertia. For desiccation resistance, weak phylogenetic inertia was detected; ancestral trait reconstruction, however, revealed a deep divergence that could be traced back to the genus level. Despite drosophilids’ high evolutionary potential related to short generation times and high population sizes, cold resistance was found to have a moderate‐to‐high level of phylogenetic inertia, suggesting that evolutionary responses are likely to be slow. Together these findings suggest species distributions are governed by evolutionarily conservative climate responses, with limited scope for rapid adaptive responses to future climate change.     

STIM1 is necessary for store-operated calcium entry in turning growth cones

J. Neurochem. (2012) 122, 1155-1166. ABSTRACT: Coordinated calcium signalling is vital for neuronal growth cone function and axon pathfinding. Although store-operated calcium entry (SOCE) has been suggested to be an important source of calcium in growth cone navigation, the mechanisms that regulate calcium signalling, particularly the regulation of internal calcium stores within growth cones, are yet to be fully determined. Stromal Interaction Molecule 1 (STIM1) is a calcium-sensing protein localized in the endoplasmic reticulum membrane that interacts with Orai proteins in the plasma membrane to initiate SOCE and refilling of intracellular calcium stores. We hypothesize that STIM1- and Orai1/2-mediated SOCE are necessary for growth cone turning responses to extracellular guidance cues. We show that STIM1 and Orai reorganize into puncta upon store depletion and during growth cone turning with STIM1 localization biased towards the turning side (high calcium side) of the growth cone. Importantly, STIM1 knock-down perturbed growth cone turning responses to the guidance cues brain-derived neurotrophic factor and semaphorin-3a (Sema-3a), as well as abolishing Sema-3a-induced growth cone collapse. Furthermore, STIM1 knock-down abolished SOCE induced by brain-derived neurotrophic factor, but not Sema-3a. Our data suggest that STIM1 is essential for correct growth cone navigation, playing multiple roles in growth cone motility, including the activation of SOCE.