Structural analysis of nanoscale self-assembled discoidal lipid bilayers by solid-state NMR spectroscopy

Nanodiscs are an example of discoidal nanoscale self-assembled lipid/protein particles similar to nascent high-density lipoproteins, which reduce the risk of coronary artery disease. The major protein component of high-density lipoproteins is human apolipoprotein A-I, and the corresponding protein component of Nanodiscs is membrane scaffold protein 1 (MSP1), a 200-residue lipid-binding domain of human apolipoprotein A-I. Here we present magic-angle spinning (MAS) solid-state NMR studies of uniformly (13)C,(15)N-labeled MSP1 in polyethylene glycol precipitated Nanodiscs. Two-dimensional MAS (13)C-(13)C correlation spectra show excellent microscopic order of MSP1 in precipitated Nanodiscs. Secondary isotropic chemical shifts throughout the protein are consistent with a predominantly helical structure. Moreover, the backbone conformations of prolines derived from their (13)C chemical shifts are consistent with the molecular belt model but not the picket fence model of lipid-bound MSP1. Overall comparison of experimental spectra and (13)C chemical shifts predicted from several structural models also favors the belt model. Our study thus supports the belt model of Nanodisc structure and demonstrates the utility of MAS NMR to study the structure of high molecular weight lipid-protein complexes.     

Free radical equilibrium in interscapular brown adipose tissue: relationship between metabolic profile and antioxidative defense

Interscapular brown adipose tissue (IBAT) hyperplasia involves a new metabolic and structural profile, resulting from acclimation of animals to a cold environment. Cold-induced changes of several antioxidative defense (AD) components in IBAT and their interrelationship with uncoupling protein 1 (UCP1), sympathetic innervation and apoptosis were studied using cold-acclimated adult rat males (4 +/- 1 degrees C, 45 days). Their age-matches were maintained at 22 +/- 1 degrees C serving as the controls. In cold-adapted rats, activities of CuZn- and Mn-superoxide dismutase (SOD) and apoptosis were reduced, while catalase (CAT), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST) activities and glutathione (GSH) content were increased compared to the control. IBAT mass, protein content, plasma free fatty acid (FFA) concentration, sympathetic innervation and UCP1 level were significantly increased in cold-acclimated group compared to the corresponding control. These results suggest that decreased CuZn and MnSOD activities in IBAT represent an adaptive response due to UCP1-induced mitochondrial uncoupling. Additionally, intensive fatty acid oxidation led to an increased H(2)O(2) production which resulted in increased CAT, GSH-Px and GST activities and GSH level. Generally speaking, cold-induced changes of AD in the IBAT are closely connected with newly established metabolic profile in this tissue, thus making an important part of the entire tissue homeostasis including cell survival.     

Genetic transformation of Centaurium erythraea Rafn by Agrobacterium rhizogenes and the production of secoiridoids

Hairy roots of Centaurium erythraea were obtained by infection with Agrobacterium rhizogenes strain LBA 9402. They spontaneously regenerated adventitious shoots in Woody Plant liquid medium without growth regulators. The shoots were grown continuously in Murashige and Skoog (MS) liquid or agar solidified media supplemented with 0.1 mg l⁻¹ indole-3-acetic acid and 1.0 mg l⁻¹ 6-benzylaminopurine. These shoots produced roots 4 weeks after transfer into agar-solidified MS medium without phytohormones. Regenerated plants grown and flowered under greenhouse conditions. The transgenic value of the regenerated plants was confirmed by the polymerase chain reaction amplification. Transformation by Agrobacterium rhizogenes alters plant morphology and production of secoiridoid glucosides. The level of secoiridoids was also modified by development stage of transformed plants. The total content of the compounds (expressed as the sum of gentiopicroside, sweroside and swertiamarin) in 10-week old pRi-transformed regenerants was 280 mg g⁻¹ dry weight and was 8-times the content in the sample of commercially available C. erythraea herb.     

Herpes simplex virus capsids are transported in neuronal axons without an envelope containing the viral glycoproteins

Electron micrographic studies of neuronal axons have produced contradictory conclusions on how alphaherpesviruses are transported from neuron cell bodies to axon termini. Some reports have described unenveloped capsids transported on axonal microtubules with separate transport of viral glycoproteins within membrane vesicles. Others have observed enveloped virions in proximal and distal axons. We characterized transport of herpes simplex virus (HSV) in human and rat neurons by staining permeabilized neurons with capsid- and glycoprotein-specific antibodies. Deconvolution microscopy was used to view 200-nm sections of axons. HSV glycoproteins were very rarely associated with capsids (3 to 5%) and vice versa. Instances of glycoprotein/capsid overlap frequently involved nonconcentric puncta and regions of axons with dense viral protein concentrations. Similarly, HSV capsids expressing a VP26-green fluorescent protein fusion protein (VP26/GFP) did not stain with antiglycoprotein antibodies. Live-cell imaging experiments with VP26/GFP-labeled capsids demonstrated that capsids moved in a saltatory fashion, and very few stalled for more than 1 to 2 min. To determine if capsids could be transported down axons without glycoproteins, neurons were treated with brefeldin A (BFA). However, BFA blocked both capsid and glycoprotein transport. Glycoproteins were transported into and down axons normally when neurons were infected with an HSV mutant that produces immature capsids that are retained in the nucleus. We concluded that HSV capsids are transported in axons without an envelope containing viral glycoproteins, with glycoproteins transported separately and assembling with capsids at axon termini.     

Characterization of a novel 5' subgenomic RNA3a derived from RNA3 of Brome mosaic bromovirus

The synthesis of 3' subgenomic RNA4 (sgRNA4) by initiation from an internal sg promoter in the RNA3 segment was first described for Brome mosaic bromovirus (BMV), a model tripartite positive-sense RNA virus (W. A. Miller, T. W. Dreher, and T. C. Hall, Nature 313:68-70, 1985). In this work, we describe a novel 5' sgRNA of BMV (sgRNA3a) that we propose arises by premature internal termination and that encapsidates in BMV virions. Cloning and sequencing revealed that, unlike any other BMV RNA segment, sgRNA3a carries a 3' oligo(A) tail, in which respect it resembles cellular mRNAs. Indeed, both the accumulation of sgRNA3a in polysomes and the synthesis of movement protein 3a in in vitro systems suggest active functions of sgRNA3a during protein synthesis. Moreover, when copied in the BMV replicase in vitro reaction, the minus-strand RNA3 template generated the sgRNA3a product, likely by premature termination at the minus-strand oligo(U) tract. Deletion of the oligo(A) tract in BMV RNA3 inhibited synthesis of sgRNA3a during infection. We propose a model in which the synthesis of RNA3 is terminated prematurely near the sg promoter. The discovery of 5' sgRNA3a sheds new light on strategies viruses can use to separate replication from the translation functions of their genomic RNAs.     

Role of hydroxyprolines in the in vitro oxidative folding and biological activity of conotoxins

Hydroxylation of proline residue occurs in specific peptides and proteins derived from plants and animals, but the functional role of this modification has been characterized primarily in collagen. Marine cone snails produce disulfide-rich peptides that have undergone a plethora of posttranslational modifications, including proline hydroxylation. Although Conus snails extensively utilize proline hydroxylation, the consequences of this modification remain largely unexplored. In this work, we investigated the function of 4-hydroxyproline (Hyp) in conotoxins from three distinct gene families: mu-, omega-, and alpha-conotoxins. Analogues of mu-GIIIA, omega-MVIIC, alpha-GI, and alpha-ImI were synthesized with either Pro or Hyp, and their in vitro oxidative folding and biological activity were characterized. For GIIIA, which naturally contains three Hyp residues, the modifications improved the ability to block NaV1.4 sodium channels but did not affect folding. In contrast, the presence of Hyp in MVIIC had a significant impact on the oxidative folding but not on the biological activity. The folding yields for the MVIIC[Pro7Hyp] analogue were approximately 2-fold higher than for MVIIC under a variety of optimized oxidation conditions. For alpha-conotoxins ImI and GI, the hydroxylation of the conserved Pro residue improved their folding but impaired their activities against target receptors. Since prolyl-4-hydroxylase and protein disulfide isomerase coexist as a heterotetramer in the ER, we discuss the effects of Hyp on the folding of conotoxins in the context of cis-trans isomerization of Pro and Hyp. Taken together, our data suggest that proline hydroxylation is important for both in vitro oxidative folding and the bioactivity of conotoxins.     

Seidel–Herzel model of human baroreflex in cardiorespiratory system with stochastic delays

The stochastic versus deterministic solution of the Seidel–Herzel model describing the baroreceptor control loop (which regulates the short-time heart rate) are compared with the aim of exploring the heart rate variability. The deterministic model solutions are known to bifurcate from the stable to sustained oscillatory solutions if time delays in transfer of signals by sympathetic nervous system to the heart and vasculature are changed. Oscillations in the heart rate and blood pressure are physiologically crucial since they are recognized as Mayer waves. We test the role of delays of the sympathetic stimulation in reconstruction of the known features of the heart rate. It appears that realistic histograms and return plots are attainable if sympathetic time delays are stochastically perturbed, namely, we consider a perturbation by a white noise. Moreover, in the case of stochastic model the bifurcation points vanish and Mayer oscillations in heart period and blood pressure are observed for whole considered space of sympathetic time delays.