A new synthetic class A amphipathic peptide analogue protects mice from diet-induced atherosclerosis

Several synthetic class A peptide analogues have been shown to mimic many of the properties of human apo A-I in vitro. A new peptide [acetyl-(AspTrpLeuLysAlaPheTyrAspLysValPheGluLysPheLysGluPhePhe)-NH2; 5F], with increased amphipathicity, was administered by intraperitoneal injection, 20 microg/day for 16 weeks, to C57BL/6J mice fed an atherogenic diet. Mouse apo A-I (MoA-I) (50 microg/day) or phosphate-buffered saline (PBS) injections were given to other mice as controls. Total plasma cholesterol levels and lipoprotein profiles were not significantly different between the treated and control groups, except that the mice receiving 5F or MoA-I had lower high density lipoprotein (HDL) cholesterol when calculated as a percentage of total cholesterol. No toxicity or production of antibodies to the injected materials was observed. When HDL was isolated from high fat diet-administered mice injected with 5F and presented to human artery wall cells in vitro together with human low density lipoprotein (LDL), there were substantially fewer lipid hydroperoxides formed and substantially less LDL-induced monocyte chemotactic activity than with HDL from PBS-injected animals. Injection of human apo A-I produced effects similar to 5F on lipid peroxide formation and LDL-induced monocyte chemotactic activity, but injection of MoA-I was significantly less effective in reducing lipid hydroperoxide formation or lowering LDL-induced monocyte chemotactic activity. Mice receiving peptide 5F had significantly less aortic atherosclerotic lesion area compared with mice receiving PBS, whereas lesion area in mice receiving MoA-I was similar to that of the PBS-injected animals. This is the first in vivo demonstration that a model class A amphipathic helical peptide has antiatherosclerotic properties. We conclude that 5F inhibits lesion formation in high fat diet-administered mice by a mechanism that does not involve changes in the lipoprotein profile, and may have potential in the prevention and treatment of atherosclerosis.     

Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3). The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway.     

PKC signaling mediates global enhancement of excitatory synaptogenesis in neurons triggered by local contact with astrocytes

Here we provide evidence that astrocytes affect neuronal synaptogenesis by the process of adhesion. Local contact with astrocytes via integrin receptors elicited protein kinase C (PKC) activation in individual dissociated neurons cultured in astrocyte-conditioned medium. This activation, initially focal, soon spread throughout the entire neuron. We then demonstrated pharmacologically that the arachidonic acid cascade, triggered by the integrin reception, is responsible for the global activation of PKC. Local astrocytic contact also facilitated excitatory synaptogenesis throughout the neuron, a process which could be blocked by inhibitors of both integrins and PKC. Thus, propagation of PKC signaling represents an underlying mechanism for global neuronal maturation following local astrocyte adhesion.     

Enantioselective transesterification using immobilized <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> overexpressing lipase

In the present study, we used gene manipulation to construct a recombinant Aspergillus oryzae strain overexpressing lipase and investigated its application to the optical resolution of chiral compounds. A. oryzae niaD300, which was derived from the wild-type strain RIB40, was used as the host strain. The tglA gene, which encodes a triacylglycerol lipase, was cloned from the A. oryzae niaD300 chromosomal genome, then reintroduced, with and without a secretion-signal sequence, into the genome and expressed under the control of the improved glaA promoter of plasmid pNGA142. The resulting recombinant strain overexpressing A. oryzae lipase was immobilized within biomass-support particles and used as a whole-cell biocatalyst. The immobilized lipase-overexpressing strain with secretion-signal sequence showed high activity and was used to selectively synthesize (R)-1-phenylethyl acetate from (RS)-1-phenylethanol and vinyl acetate. After 48 h reaction at 30°C with molecular sieve 4A, the yield and enantiomeric excess (%ee) of (R)-1-phenylethyl acetate reached approximately 90 and 95%ee, respectively. The whole-cell biocatalyst for optical resolution of chiral compounds produced in this study maintained its activity over 25 batch-reaction cycles.     

PtdIns(3,5)P2 is required for delivery of endocytic cargo into the multivesicular body

The endocytic pathway transports cargo from the plasma membrane to early endosomes, where certain cargoes are sorted to the late endosome/multivesicular body. Biosynthetic cargo destined for the lysosome is also trafficked through the multivesicular body. Once delivered to the multivesicular body, cargo destined for the interior of the lysosome is selectively sorted into vesicles that bud into the lumen of the multivesicular body. These vesicles are released into the lumen of the lysosome upon the fusion of the multivesicular body and lysosomal limiting membranes. The yeast protein Fab1, which catalyzes the production of phosphatidylinositol (3,5) bisphosphate [PtdIns(3,5)P₂], is necessary for proper sorting of biosynthetic cargo in the multivesicular body. Utilizing an endocytosis screen, we isolated a novel allele of FAB1 that contains a point mutation in the lipid kinase domain. Characterization of this allele revealed reduced PtdIns(3,5)P₂ production, altered vacuole morphology, and biosynthetic protein sorting defects. We also found that endocytosis of the plasma membrane protein Ste3 is partially blocked downstream of the internalization step, and that delivery of the dye FM4-64 to the vacuole is delayed in fab1 mutants. Additionally, Ste3 is not efficiently sorted into multivesicular body vesicles in fab1 mutants and instead localizes to the vacuolar limiting membrane. These data show that PtdIns(3,5)P₂ is necessary for proper trafficking and sorting of endocytic cargo through the late endosome/multivesicular body.     

Impact of the Distance from the Stent Edge to the Residual Plaque on Edge Restenosis following Everolimus-Eluting Stent Implantation

Objectives

This study aimed to assess the relation between stent edge restenosis (SER) and the distance from the stent edge to the residual plaque using quantitative intravascular ultrasound.

Background

Although percutaneous coronary intervention with drug-eluting stents has improved SER rates, determining an appropriate stent edge landing zone can be challenging in cases of diffuse plaque lesions. It is known that edge vascular response can occur within 2 mm from the edge of a bare metal stent, but the distance to the adjacent plaque has not been evaluated for drug-eluting stents.

Methods

A total of 97 proximal residual plaque lesions (plaque burden [PB] >40%) treated with everolimus-eluting stents were retrospectively evaluated to determine the distance from the stent edge to the residual plaque.

Results

The SER group had significantly higher PB (59.1 ± 6.1% vs. 51.9 ± 9.1% for non-SER; P = 0.04). Higher PB was associated with SER, with the cutoff value of 54.74% determined using receiver operating characteristic (ROC) curve analysis. At this cutoff value of PB, the distance from the stent edge to the lesion was significantly associated with SER (odds ratio = 2.05, P = 0.035). The corresponding area under the ROC curve was 0.725, and the cutoff distance value for predicting SER was 1.0 mm.

Conclusion

An interval less than 1 mm from the proximal stent edge to the nearest point with the determined PB cutoff value of 54.74% was significantly associated with SER in patients with residual plaque lesions.     

Eyelid Opening with Trigeminal Proprioceptive Activation Regulates a Brainstem Arousal Mechanism

Eyelid opening stretches mechanoreceptors in the supratarsal Müller muscle to activate the proprioceptive fiber supplied by the trigeminal mesencephalic nucleus. This proprioception induces reflex contractions of the slow-twitch fibers in the levator palpebrae superioris and frontalis muscles to sustain eyelid and eyebrow positions against gravity. The cell bodies of the trigeminal proprioceptive neurons in the mesencephalon potentially make gap-junctional connections with the locus coeruleus neurons. The locus coeruleus is implicated in arousal and autonomic function. Due to the relationship between arousal, ventromedial prefrontal cortex, and skin conductance, we assessed whether upgaze with trigeminal proprioceptive evocation activates sympathetically innervated sweat glands and the ventromedial prefrontal cortex. Specifically, we examined whether 60° upgaze induces palmar sweating and hemodynamic changes in the prefrontal cortex in 16 subjects. Sweating was monitored using a thumb-mounted perspiration meter, and prefrontal cortex activity was measured with 45-channel, functional near-infrared spectroscopy (fNIRS) and 2-channel NIRS at Fp1 and Fp2. In 16 subjects, palmar sweating was induced by upgaze and decreased in response to downgaze. Upgaze activated the ventromedial prefrontal cortex with an accumulation of integrated concentration changes in deoxyhemoglobin, oxyhemoglobin, and total hemoglobin levels in 12 subjects. Upgaze phasically and degree-dependently increased deoxyhemoglobin level at Fp1 and Fp2, whereas downgaze phasically decreased it in 16 subjects. Unilateral anesthetization of mechanoreceptors in the supratarsal Müller muscle used to significantly reduce trigeminal proprioceptive evocation ipsilaterally impaired the increased deoxyhemoglobin level by 60° upgaze at Fp1 or Fp2 in 6 subjects. We concluded that upgaze with strong trigeminal proprioceptive evocation was sufficient to phasically activate sympathetically innervated sweat glands and appeared to induce rapid oxygen consumption in the ventromedial prefrontal cortex and to rapidly produce deoxyhemoglobin to regulate physiological arousal. Thus, eyelid opening with trigeminal proprioceptive evocation may activate the ventromedial prefrontal cortex via the mesencephalic trigeminal nucleus and locus coeruleus.     

Development of recombinant Aspergillus oryzae whole-cell biocatalyst expressing lipase-encoding gene from Candida antarctica

To expand the industrial applications of Candida antarctica lipase B (CALB), we developed Aspergillus oryzae whole-cell biocatalyst expressing the lipase-encoding gene from C. antarctica. A. oryzae niaD300, which was derived from the wild type strain RIB40, was used as the host strain. The CALB gene was isolated from C. antarctica CBS6678 and expression plasmids were constructed with and without secretion signal peptide. The lipase gene was expressed under the control of improved glaA and pNo-8142 promoters of plasmids pNGA142 and pNAN8142, respectively. The Southern blot analysis demonstrated the successful integration of the CALB gene in the genome of A. oryzae. To determine the role of signal peptide, the expression plasmids were constructed with homologous and heterologous secretion signal sequences of triacylglycerol lipase gene (tglA) from A. oryzae and lipase B (CALB) from C. antarctica, respectively. The C-terminal FLAG tag does not alter the catalytic properties of the lipase enzyme and Western blotting analysis using anti-FLAG antibodies demonstrated the presence of cell wall and membrane bound lipase responsible for the biocatalytic activity of the whole-cell biocatalyst. The resultant recombinant A. oryzae was immobilized within biomass support particles (BSPs) made of polyurethane foam (PUF) and the BSPs were successfully used for the hydrolysis of para-nitrophenol butyrate (p-NPB) and for the optical resolution of (RS)-1-phenyl ethanol by enantioselective transesterification with vinyl acetate as acyl donor.