Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary A modified method for the histochemical demonstration of the activity of -glucan phosphorylase is described. In the histochemical system the enzyme catalyses the synthesis of glycogen by transglucosylation from -d-glucosylphosphate. The incubation medium contains dextran as glucosyl acceptor. Therefore, in contrast with the unmodified method, the new technique is able to demonstrate the activity of phosphorylase in ischaemic glycogen-depleted tissues.     

Integrated mechanism for the generation of the 5′ junctions of LINE inserts

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5′ ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5′ junctions revealed that the 5′-end-joining pathways of LINEs can be divided into two fundamental types—annealing or direct. We also found that the generation of 5′ inversions depends on host and LINE species. These results led us to propose a new model for 5′-end joining, the type of which is determined by the extent of exposure of 3′ overhangs generated after the second-strand cleavage and by the involvement of host factors.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary The activity and localization of -glucan phosphorylase in experimental canine glycogen-depleted heart tissue has been investigated with biochemical and histochemical methods using dextran as enzyme acceptor. Only linear, essentially unbranched, dextrans exhibit acceptor properties; highly branched dextrans are not suitable acceptors for the enzyme. Results of Michaelis-Menten constant measurements for the linear essentially unbranched dextran fractions used, indicate that the affinity of the enzyme for the non-reducing end group of the dextran molecule increases with increasing molecular weight of the acceptor.In the glycogen-depleted tissue of anoxic and ischaemic cardiac musculature there is a gradual inactivation of the enzyme during the ischaemic period. Shortly before total inactivation the affinity of the enzyme, especially for the lower molecular dextran fractions, is greatly reduced. Therefore, for the histochemical demonstration of phosphorylase activity in infarcted areas of the heart it is essential to use as acceptor an unbranched dextran fraction with a high average molecular weight.This investigation was partially supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Extension of the Applicability of δJ-Values for the Configurational Assignment of Diastereomeric Phosphate-Modified Dideoxynucleotides

Abstract

The stereochemical assignment of dinucleoside-S-p-nitrobenzyl-phosphorothioates by NMR spectroscopy is reported. It was found that the method based on the difference of the vicinal phosphorus-carbon coupling constants (δJ =³J_C4′,p-³J_C2′,p) can widely be applied for the determination of the configuration at the pohosphorus atom in phosphate-modified dideoxynucleotides.     

Importance of the Sphingoid Base Length for the Membrane Properties of?Ceramides

The sphingoid bases of sphingolipids, including ceramides, can vary in length from 12 to >20 carbons. To study how such length variation affects the bilayer properties of ceramides, we synthesized ceramides consisting of a C12-, C14-, C16-, C18-, or C20-sphing-4-enin derivative coupled to palmitic acid. The ceramides were studied in mixtures with palmitoyloleoylphosphocholine (POPC) and/or palmitoylsphingomyelin (PSM), and in more complex bilayers also containing cholesterol. The trans-parinaric acid lifetimes showed that 12:1- and 14:1-PCer failed to increase the order of POPC bilayers, whereas 16:1-, 18:1-, and 20:1-PCer induced ordered- or gel-phase formation. Nevertheless, all of the analogs were able to thermally stabilize PSM, and a chain-length-dependent increase in the main phase transition temperature of equimolar PSM/Cer bilayers was revealed by differential scanning calorimetry. Similar thermal stabilization of PSM-rich domains by the ceramides was observed in POPC bilayers with a trans-parinaric acid-quenching assay. A cholestatrienol-quenching assay and sterol partitioning experiments showed that 18:1- and 20:1-PCer formed sterol-excluding gel phases with PSM, reducing the overall bilayer affinity of sterol. The effect of 16:1-PCer on sterol distribution was less dramatic, and no displacement of sterol from the PSM environment was observed with 12:1- and 14:1-PCer. The results are discussed in relation to other structural features that affect the bilayer properties of ceramides.     

Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

A Nomograph for Calculating the Optimal Frequency for Dielectrophoresis and the Characteristic Frequency of the β-Dispersion of Cell Membrane Vesicles

The first stage of the two-stage cell electrofusion technique involves the dielectrophoretic apposition, in an AC field, of protoplasts suspended in a medium of relatively low specific conductivity. A frequency at which the maximum dielectrophoretic force is exerted is given by the characteristic frequency for the dielectric relaxation by a Maxwell-Wagner type of mechanism. We provide a nomograph for the rapid calculation of this frequency.     

Neurofeedback—The Significance of Reinforcement and the Search for an Appropriate Strategy for the Success of Self-regulation

Nine healthy children took part in five sessions of feedback and instrumental conditioning of slow cortical potentials (SCPs). The feedback conditions (the relation between the feedback signal and amplitude of SCP) were inverted after two sessions. Neither the children nor the therapists were aware of this change. The adjustment of the children to the new feedback setting and the self-regulation strategies employed were investigated. The results were as follows: (a) Healthy children achieved control over cortical negativity within two sessions. (b) The change of feedback conditions worsened the regulation abilities, which then improved again within the following three sessions. (c) After the first two sessions, the participants were able to describe strategies that were successful during different phases of self-regulation. (d) Following the change in the feedback conditions, the children re-evaluated the way they influenced their SCPs. However, they did not alter the cognitive or behavioral strategies. The study demonstrated that positive and negative reinforcement and the knowledge of results are more important for successful self-regulation than the search for effective strategies. The relevance of these findings is discussed.     

What kind of lamp for the cultivation of algae?

A method for quantitative evaluation of light sources from the point of their suitability for algal cultivation is described. Two parameters are used for the evaluation:

What is the evidence for the clinical value of SBRT in cancer of the cervix?

AimThe aim of this review is to describe and analyze indications and results of the use of SBRT in uterine cervix cancer, reviewing articles published from January 2010 up to August 2017, for any one of the four indications listed:

• 1Patient refusal or anatomic impediments to interstitial or intracavitary brachytherapy (BCT), i.e. SBRT as an “alternative” for BCT;
• 2Patients with voluminous tumors, or asymmetric tumors where BCT alone would not achieve curative doses, i.e. SBRT as a primary adjunct to BCT;
• 3Pelvic and para aortic adenopathy where SBRT could be used as a boost, i.e. SBRT as a primary adjunct to external beam pelvic radiotherapy;
• 4Small volume recurrences (postoperative or post radiotherapy), i.e. SBRT for salvage.

BackgroundCervix cancer standard treatment involves pelvic irradiation and chemotherapy, recent advances in irradiation techniques might offer new possible approaches.Material and methodsSystematic review of the English language literature about Cervix cancer, SBRT, published from January 2010 to January 2018 identified through a database search of PubMed, and Ovid MEDLINE, using pre-defined search phrases.ResultsThe results in the literature, in general, demonstrate rather weak efficacy of SBRT. In this review, we did not find strong evidence to recommend routine SBRT as a primary treatment for cervico-uterine cancers, i.e. as a replacement for BCT; in highly selected cases it might be considered useful as salvage therapy for relapsed cervix cancer.ConclusionThe existing data to not warrant recommending SBRT for the definitive treatment of cervix cancer, but may have some value in the recurrent/relapsed setting.     

VGLUTs--potential targets for the treatment of seizures?

Vesicular glutamate transporters (VGLUTs) load glutamate into synaptic vesicles. In this issue of Neuron, Juge et?al. report that ketone bodies compete with chloride-dependent activation of VGLUTs, leading to suppression of glutamate release and seizures. These findings provide a surprising explanation for the efficacy of the ketogenic diet in controlling epilepsy.