Solid-state 15N NMR of oriented lipid bilayer bound gramicidin A'

Highly oriented samples of lipid and gramicidin A' (8:1 molar ratio) have been prepared with the samples extensively hydrated (approximately 70% water v/w). These preparations have been shown to be completely in a bilayer phase with a transition temperature of 28 degrees C, and evidence is presented indicating that the gramicidin is in the channel conformation. An estimate of the disorder in the alignment of the bilayers parallel with the glass plates used to align the bilayers can be made from the asymmetry of the nuclear magnetic resonances (NMR). Such an analysis indicates a maximal range of disorder of +/- 3 degrees. Uniformly 15N-labeled gramicidin has been biosynthesized by Bacillus brevis grown in a media containing 15N-labeled Escherichia coli cells as the only nitrogen source. When prepared with labeled gramicidin, the oriented samples result in high-resolution 15N NMR spectra showing 12 resonances for the 20 nitrogen sites of the polypeptide. The frequency of the three major multiple resonance peaks has been interpreted to yield the approximate orientation of the N-H bonds in the peptide linkages with respect to the magnetic field. These bond orientations are only partially consistent with the extant structural models of gramicidin.     

3-Azi-1-methoxybutyl D-maltooligosaccharides specifically bind to the maltose/maltooligosaccharide-binding protein of Escherichia coli and can be used as photoaffinity labels

Maltooligosaccharides with two to six (alpha 1-4)-linked glucose residues, carrying at their reducing end a 3-azi-1-methoxybutyl group in either alpha or in beta glycosidic linkage, were synthesized. These maltooligosaccharide analogues inhibit maltose uptake via the maltose-binding-protein-dependent transport system in Escherichia coli. The concentration of half-maximal inhibition of maltose transport, at 15 nM concentration, decreases with increasing chain length of the analogue, levelling off at 40 microM after a chain length of four glucose residues in the alpha series and at 350 microM after a chain length of three glucose residues in the beta series. The inhibition of maltose transport occurs at the level of the periplasmic maltose-binding protein. 3-Azi-1-methoxybutyl alpha-D-[3H]maltotrioside was bound by the maltose-binding protein with a Kd of 0.18 mM. Irradiation at 350 nm of purified maltose-binding protein in the presence of 4 microM of this substrate labeled the protein covalently; labeling was prevented by 1 mM maltose. Using a crude preparation of periplasmic proteins two proteins were labeled, the maltose-binding protein and alpha-amylase. Thus, 3-azi-1-methoxybutyl alpha-D-maltooligosaccharides are potent photoaffinity labels for proteins with maltooligosaccharides-binding sites.     

In Vivo and In Vitro Effects of Some Plant Hormones on Rat Erythrocyte Carbonic Anhydrase and Glucose-6-Phosphate Dehydrogenase Activities

The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects.     

Permanent Cerebral Vessel Occlusion via Double Ligature and Transection

Stroke is a leading cause of death, disability, and socioeconomic loss worldwide. The majority of all strokes result from an interruption in blood flow (ischemia) ¹. Middle cerebral artery (MCA) delivers a great majority of blood to the lateral surface of the cortex ², is the most common site of human stroke ³, and ischemia within its territory can result in extensive dysfunction or death ^1,4,5. Survivors of ischemic stroke often suffer loss or disruption of motor capabilities, sensory deficits, and infarct. In an effort to capture these key characteristics of stroke, and thereby develop effective treatment, a great deal of emphasis is placed upon animal models of ischemia in MCA.Here we present a method of permanently occluding a cortical surface blood vessel. We will present this method using an example of a relevant vessel occlusion that models the most common type, location, and outcome of human stroke, permanent middle cerebral artery occlusion (pMCAO). In this model, we surgically expose MCA in the adult rat and subsequently occlude via double ligature and transection of the vessel. This pMCAO blocks the proximal cortical branch of MCA, causing ischemia in all of MCA cortical territory, a large portion of the cortex. This method of occlusion can also be used to occlude more distal portions of cortical vessels in order to achieve more focal ischemia targeting a smaller region of cortex. The primary disadvantages of pMCAO are that the surgical procedure is somewhat invasive as a small craniotomy is required to access MCA, though this results in minimal tissue damage. The primary advantages of this model, however, are: the site of occlusion is well defined, the degree of blood flow reduction is consistent, functional and neurological impairment occurs rapidly, infarct size is consistent, and the high rate of survival allows for long-term chronic assessment.     

Meander: visually exploring the structural variome using space-filling curves

The introduction of next generation sequencing methods in genome studies has made it possible to shift research from a gene-centric approach to a genome wide view. Although methods and tools to detect single nucleotide polymorphisms are becoming more mature, methods to identify and visualize structural variation (SV) are still in their infancy. Most genome browsers can only compare a given sequence to a reference genome; therefore, direct comparison of multiple individuals still remains a challenge. Therefore, the implementation of efficient approaches to explore and visualize SVs and directly compare two or more individuals is desirable. In this article, we present a visualization approach that uses space-filling Hilbert curves to explore SVs based on both read-depth and pair-end information. An interactive open-source Java application, called Meander, implements the proposed methodology, and its functionality is demonstrated using two cases. With Meander, users can explore variations at different levels of resolution and simultaneously compare up to four different individuals against a common reference. The application was developed using Java version 1.6 and Processing.org and can be run on any platform. It can be found at http://homes.esat.kuleuven.be/~bioiuser/meander.