Characterization and partial purification of a chloride- and calcium-dependent glutamate-binding protein from rat brain

A glutamate-binding protein was solubilized from rat brain synaptic plasma membranes using sodium cholate. Its properties were characterized after addition of exogenous phospholipids and formation of proteoliposomes. Glutamate binding was dependent on calcium and chloride ions with maximal binding at concentrations of 10(-5) M calcium and 10 mM chloride ions. The effects of the two ions were synergistic rather than additive. In addition, glutamate binding was not affected by inhibitors specific for N-methyl-D-aspartate and kainate receptor subtypes, but was inhibited by quisqualate (Ki = 50 microM) and DL-2-amino-4-phosphonobutyrate (Ki = 1.3 mM). Furthermore, binding was abolished by 100 microM 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid and 1 mM dithiothreitol. These properties resemble those of the chloride- and calcium-dependent binding site. Starting from the detergent extract, the glutamate-binding protein was purified 123-fold using fractionated ammonium sulfate precipitation, chromatography on hydroxyapatite and on DEAE-Sephacel as sequential purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified protein fraction showed two major bands migrating with Mr values of 51,000 and 105,000. The properties of the partially purified binding protein were similar to those of the detergent extract. Glutamate binding to the partially purified protein is not due to a sequestration process or product binding to N-acetylated alpha-linked dipeptidase. Thus, the functional role of the binding protein remains to be established.     

Synthesis and evaluation of unsaturated caprolactams as interleukin-1beta converting enzyme (ICE) inhibitors

Peptidomimetic compounds possessing a caprolactam ring constraint were prepared and evaluated as interleukin-1beta converting enzyme (ICE) inhibitors. The caprolactam ring was used to constrain the P3 region of our inhibitors. This strategy proved to be effective for the synthesis of ICE inhibitors, maintaining key hydrogen bond interactions with the enzyme and invoking a preferred conformation for binding. Several compounds exhibited IC(50) values less than 10nM in a caspase-1 enzyme assay and less than 100nM in a THP-1 whole cell assay measuring IL-1beta production. Two compounds, 13c and 13j, were found to have good oral bioavailability (>50%) in rats when administered as prodrugs.     

The ecology of Mytilus edulis L. in exposed rocky intertidal communities

Information is given to show that Mytilus edulis L. is an important component of exposed rocky coast communities of the Western Hemisphere. It is abundant on the exposed coast of Washington State with a potential distribution extending from + 10.6 ft ( + 3.2 m) down to at least 10 m depth: it is not continuous along this entire gradient. It dominates a band from +10.6 ft down to +9.6 ft ( + 3.2 to + 2.9 m) tidal height where it usually intergrades with its congener M. californianus Conrad. Its upper limit appears to be determined by desiccation stress and the lower probably by competition and or predation.In the mid-intertidal zone, from + 5.6 to + 9.6 ft ( + 1.7 to 2.9 m), where M. californianus is dominant, M. edulis occurs in patches of cleared space which usually form in the M. californianus bed matrix in winter months. Here, it acts as an opportunistic species by colonizing quickly and growing rapidly to reproductive size. It is preyed upon by Thais spp. which prefer it to Mytilus californianus and eventually is eliminated from these patches. It also occurs, sometimes abundantly, in tufts of the red alga Endocladia muricata (Postels & Ruprect) J. Agardh at high and mid-intertidal heights.In low intertidal and subtidal regions, to ≈ 10 m depth, Mytulis edulis can be found in refuges of filamentous or intricately arranged substrata which generally offer protection from predation. These are represented by a wide variety of forms such as hydroids. bryozoans, filamentous algae, coralline algae, and kelp with convoluted holdfasts or complex stipes.     

The source of spontaneous activity in the main olfactory bulb of the rat

Introduction

In vivo, most neurons in the main olfactory bulb exhibit robust spontaneous activity. This paper tests the hypothesis that spontaneous activity in olfactory receptor neurons drives much of the spontaneous activity in mitral and tufted cells via excitatory synapses.

Methods

Single units were recorded in vivo from the main olfactory bulb of a rat before, during, and after application of lidocaine to the olfactory nerve. The effect of lidocaine on the conduction of action potentials from the olfactory epithelium to the olfactory bulb was assessed by electrically stimulating the olfactory nerve rostral to the application site and monitoring the field potential evoked in the bulb.

Results

Lidocaine caused a significant decrease in the amplitude of the olfactory nerve evoked field potential that was recorded in the olfactory bulb. By contrast, the lidocaine block did not significantly alter the spontaneous activity of single units in the bulb, nor did it alter the field potential evoked by electrical stimulation of the lateral olfactory tract. Lidocaine block also did not change the temporal patters of action potential or their synchronization with respiration.

Conclusions

Spontaneous activity in neurons of the main olfactory bulb is not driven mainly by activity in olfactory receptor neurons despite the extensive convergence onto mitral and tufted cells. These results suggest that spontaneous activity of mitral and tufted is either an inherent property of these cells or is driven by centrifugal inputs to the bulb.     

The first isolation of <Emphasis Type="Italic">Westerdykella dispersa</Emphasis> in a critically burned patient

Patients with critical thermal trauma belong to one of the most high-risk groups for development of infectious complications. Fungal infections are not among frequent complications during therapy of patients with thermal trauma, yet their incidence dramatically aggravates the prognosis for patients with this disorder. In the case report, we present the case of a young man with a critical burn, where Westerdykella dispersa was isolated. Identification of the pathogen was provided with a combination of cultivation and molecular biological confirmation. In this case, the distinction between infection and colonization was very complicated. Histopathological examination for definitive diagnosis of infection was not performed because the material from unburned soft tissue sampling could further compromise the function of the hand. Repeated cultivation and molecular identification W. dispersa before and after the necrectomy is indicative of infection rather than colonization. It is the second documented case of positive cultivation with this pathogen in humans and the first such case in a non-neutropenic host.     

Introduction of intersubunit disulfide bonds in the membrane-distal region of the influenza hemagglutinin abolishes membrane fusion activity.

Influenza virus hemagglutinin (HA) mediates viral entry into cells by a low pH-induced membrane fusion event in endosomes. A number of structural changes occur throughout the length of HA at the pH of fusion. To probe their significance and their necessity for fusion activity, we have prepared a site-directed mutant HA containing novel intersubunit disulfide bonds designed to cross-link covalently the membrane-distal domains of the trimer. These mutations inhibited the low pH-induced conformational changes and prevented HA-mediated membrane fusion; conditions that reduced the novel disulfide bonds restored membrane fusion activity. We conclude that structural rearrangements in the membrane distal region of the HA are required for membrane fusion activity.