Ca(2+) influx and neurotransmitter release at ribbon synapses

Ca(2+) influx through voltage-gated Ca(2+) channels triggers the release of neurotransmitters at presynaptic terminals. Some sensory receptor cells in the peripheral auditory and visual systems have specialized synapses that express an electron-dense organelle called a synaptic ribbon. Like conventional synapses, ribbon synapses exhibit SNARE-mediated exocytosis, clathrin-mediated endocytosis, and short-term plasticity. However, unlike non-ribbon synapses, voltage-gated L-type Ca(2+) channel opening at ribbon synapses triggers a form of multiquantal release that can be highly synchronous. Furthermore, ribbon synapses appear to be specialized for fast and high throughput exocytosis controlled by graded membrane potential changes. Here we will discuss some of the basic aspects of synaptic transmission at different types of ribbon synapses, and we will emphasize recent evidence that auditory and retinal ribbon synapses have marked differences. This will lead us to suggest that ribbon synapses are specialized for particular operating ranges and frequencies of stimulation. We propose that different types of ribbon synapses transfer diverse rates of sensory information by expressing a particular repertoire of critical components, and by placing them at precise and strategic locations, so that a continuous supply of primed vesicles and Ca(2+) influx leads to fast, accurate, and ongoing exocytosis.     

A family-based approach to preventing excessive weight gain

Objective: Preventing weight gain in adults and excessive weight gain in children is a high priority. We evaluated the ability of a family‐based program aimed at increasing steps and cereal consumption (for breakfast and snacks) to reduce weight gain in children and adults. Research Methods and Procedures: Families (n = 105) with at least one 8‐ to 12‐year‐old child who was at‐risk‐for‐overweight or overweight (designated as the target child) were recruited for the study. Eighty‐two families were randomly assigned to receive the family‐based intervention and 23 families to the control condition. The 13‐week intervention consisted of specific increases in daily steps (an additional 2000 steps/d) and consumption of 2 servings/d of ready‐to‐eat cereal. Results: The intervention was successful in increasing walking (steps) and cereal consumption. The intervention had positive, significant effects on percentage BMI‐for‐age and percentage body fat for target children and weight, BMI, and percentage body fat for parents. On further analysis, the positive effects of the intervention were seen largely in target girls and moms, rather than in target boys and dads. Discussion: This family‐based weight gain prevention program based on small changes holds promise for reducing excessive weight gain in families and especially in growing overweight children.     

MRI Features in a Canine Model of Ischemic Stroke: Correlation between Lesion Volume and Neurobehavioral Status during the Subacute Stage

The purpose of this study was to evaluate the diagnostic value of magnetic resonance imaging (MRI) and assess the correlation between the volume of the ischemic lesion and neurobehavioral status during the subacute stage of ischemic stroke. Ischemic stroke was induced in 6 healthy laboratory beagles through permanent occlusion of the middle cerebral artery (MCAO). T2-weighted and fluid-attenuated inversion recovery (FLAIR) imaging, diffusion-weighted imaging (DWI), measurement of the apparent diffusion coefficient (ADC) ratio, and neurobehavioral evaluation were performed 3 times serially by using a 1.5-T MR system: before and 3 and 10 d after MCAO. Ischemic lesions demonstrated T2 hyperintensity, FLAIR hyperintensity, and DWI hyperintensity. The ADC ratio was decreased initially but then was increased at 10 d after MCAO. Ischemic lesion volumes on T2-weighted and FLAIR imaging were not significantly different from those on DWI. The lesion volume and neurobehavioral score showed strong correlation. Our results suggest that conventional MRI may be a reliable diagnostic tool during the subacute stage of canine ischemic stroke.Abbreviations: ADC, apparent diffusion coefficient; DWI, diffusion-weighted imaging; FLAIR, fluid-attenuated inversion recovery; MCAO, middle cerebral artery occlusion; MRI, magnetic resonance imaging; PWI, perfusion-weighted imagingIn human medicine, stroke is a leading cause of adult mortality and neurologic disability worldwide.¹ Strokes previously were thought to be uncommon in small animals, but the true prevalence is unknown.⁴ These events are now recognized more frequently in dogs because of increased use of magnetic resonance imaging (MRI).^5,14,17Because the infusion of thrombolytic agents, such as urokinase or tissue plasminogen activator, within 3 to 6 h of the onset symptoms is effective in restoring blood flow and improving stroke outcome in humans,¹⁹ the detection of early ischemic changes is now thought to be necessary to improve patient outcome. Computed tomography and conventional MRI are not sufficiently sensitive to predict the presence and extent of ischemic damage during the acute stage after a stroke.^12,20 Therefore several MRI sequences, such as fluid-attenuated inversion recovery (FLAIR), diffusion-weighted imaging (DWI), perfusion-weighted imaging (PWI), and MR angiography, have been developed for early diagnosis and subsequent follow-up of ischemic stroke.³ High-field magnetic strengths (at least 1.5 T) are necessary to perform these sequences.In contrast to the situation in humans, ischemic stroke in many dogs is diagnosed during the subacute stage—24 h to 6 wk after the vascular insult—due to the time lag between the onset of clinical signs to referral and to the lack of standard diagnostic protocols for ischemic stroke in dogs. In most reports of strokes in dogs, the median interval between the onset of neurologic dysfunction and performance of an MRI was more than 2 d.^5,14,17 Whereas DWI has marked sensitivity to very early ischemic changes in the brain, T2-weighted and FLAIR images gradually become more hyperintense later (that is, during the first 24 h after the insult).³ Therefore, hyperintensity on T2-weighted and FLAIR images is believed to be representative of mature lesions.¹⁵ In light of these findings, we hypothesized that conventional MR sequences, such as T2-weighted and FLAIR imaging as well as DWI would be used for the diagnosis of the subacute stage of ischemic stroke in dogs.The purpose of this study was to evaluate the diagnostic value of MRI and assess the correlation between the volume of ischemic lesions and neurobehavioral status during the subacute stage of ischemic stroke in dogs. We therefore investigated the lesion volume of T2-weighted and FLAIR images compared with that on DWI images. Furthermore, we assessed the relationship between the apparent diffusion coefficient (ADC) of the ischemic lesions and the neurobehavioral status of the dogs.     

Draft Genome Sequencing of Bacillus sp. Strain M2-6, Isolated from the Roots of Korean Ginseng,Panax ginseng C. A. Meyer,after High-Hydrostatic-Pressure Processing

A bacterium, designated M2-6, was isolated from Korean ginseng, Panax ginseng C. A. Meyer, roots after high-hydrostatic-pressure processing. On the basis of 16 rRNA gene phylogeny, the isolate was presumptively identified as a Bacillus sp. Here we report the draft genome sequence of Bacillus sp. strain M2-6 (= KACC 16563).     

Characterisation and transferability of apple SSRs to two European pear F<Subscript>1</Subscript> populations

European pear (Pyrus communis L.) is among the important fruit species for which only few genetic studies have been carried out. Available evidence indicates that simple sequence repeats (SSR) are very useful as molecular markers because they are codominant, highly polymorphic, abundant and reproducible. The present paper reports more than 100 apple SSR markers in two populations of European pear; a total of 41 SSR markers were then positioned on a genetic linkage map of the cross Passe Crassane × Harrow Sweet and 31 in the map Abbè Fétel × Max Red Bartlett. Syntenic relationships between pear and apple maps have been considered for the chromosomes carrying two or more SSR markers. The alignment among the two maps supports the colinearity of the two genomes with respect both to identification and to orientation of the linkage groups.     

Rac and protein kinase C-delta regulate ERKs and cytosolic phospholipase A2 in FcepsilonRI signaling to cysteinyl leukotriene synthesis in mast cells

Although cysteinyl leukotrienes (cysLTs) are known to be principal inflammatory lipid mediators released from IgE-stimulated mast cells, the signaling mechanisms involved in the synthesis of cysLTs remain largely unknown. In the present study, therefore, we investigated the signaling pathway by which IgE induces cysLTs synthesis after binding to its high affinity receptor (FcepsilonRI) in RBL-2H3 mast cells. We found that IgE-induced cysLT synthesis is completely abolished in RBL-2H3(Rac-N17) cells, a stable cell line expressing Rac(N17), a dominant negative Rac1 mutant; conversely, synthesis was enhanced in cells expressing Rac(V12), a constitutively active Rac1 mutant, suggesting that Rac1 is a key mediator of IgE signaling to cysLT synthesis. Further analysis aimed at identifying mediators downstream of Rac1 revealed that pretreating cells with a protein kinase C-delta (PKC-delta) inhibitor or infection with an adenoviral vector harboring a dominant negative PKC-delta mutant significantly attenuates IgE-induced ERKs phosphorylation, cytosolic phospholipase A(2) phosphorylation/translocation, and cysLT synthesis. In addition, the expression of Rac(N17) blocked PKC-delta translocation and impaired the phosphorylation of ERKs and cytosolic phospholipase A(2) otherwise elicited by IgE stimulation. Taken together these results suggest that PKC-delta also plays a critical mediatory role in the IgE signaling pathway leading to cysLT synthesis, acting downstream of Rac1. Finally, the physiological significance of PKC-delta in the IgE signaling pathway was demonstrated in an Ag (OVA)-challenged in vivo mouse model, in which induced levels of cysLTs and airway responsiveness in lung airways were significantly diminished by prior i.p. injection of a PKC-delta inhibitor.     

Intestinal Helminthic Infections Diagnosed by Colonoscopy in a Regional Hospital during 2001-2008

The present study investigated characteristics of 24 parasite infection cases detected during colonoscopy in a regional hospital from January 2001 to December 2008. Sixteen patients were confirmed with Trichuris trichiura infection, 6 patients were with Ascaris lumbricoides infection, 1 patient with Enterobius vermicularis infection, and 1 patient with Anisakis infection. Among them, 7 patients (43.8%) were asymptomatic. Colonoscopy findings were normal in 18 patients (75.0%). Among the patients with T. trichiura infection, colonoscopy showed several erosions in 2 patients (8.3%) and non-specific inflammation of the affected segment of the colon in 3 patients (12.5%). In 1 patient with anisakiasis, colonoscopy revealed a markedly swollen colonic wall. Stool examinations were performed before treatment in 7 patients (29.2%) and were all negative for parasite eggs or worms. These results suggest that colonoscopy is a useful diagnostic approach for parasitic infections even for asymptomatic patients and for patients with negative stool examinations.     

Discovery and optimization of 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazines as novel,CNS penetrant pan-muscarinic antagonists

This letter describes the synthesis and structure activity relationship (SAR) studies of structurally novel M₄ antagonists, based on a 3-(4-aryl/heteroarylsulfonyl)piperazin-1-yl)-6-(piperidin-1-yl)pyridazine core, identified from a high-throughput screening campaign. A multi-dimensional optimization effort enhanced potency at human M₄ (hM₄ IC₅₀s < 200 nM), with only moderate species differences noted, and with enantioselective inhibition. Moreover, CNS penetration proved attractive for this series (rat brain:plasma K_p = 2.1, K_p,uu = 1.1). Despite the absence of the prototypical mAChR antagonist basic or quaternary amine moiety, this series displayed pan-muscarinic antagonist activity across M_1-5 (with 9- to 16-fold functional selectivity at best). This series further expands the chemical diversity of mAChR antagonists.     

Multiplex gene removal by two-step polymerase chain reactions

Precise DNA manipulation is critical for molecular biotechnology. Restriction enzyme-based approaches are limited by their requirement of specific enzyme sites. Restriction-free cloning has greatly improved the flexibility and speed of precise DNA assembly. Most of these approaches focus on DNA assembly rather than gene removal. Here we present a polymerase chain reaction (PCR)-based cloning method that allows removal of multiple gene segments from plasmids without using restriction enzymes and thermostable ligase. We demonstrate simultaneous removal of three gene segments from a plasmid. This approach could be beneficial to DNA library construction, genetic and protein engineering, and synthetic biology.