Membrane particle changes attending the acrosome reaction in guinea pig spermatozoa

To examine the freeze-fracture appearance of membrane alterations accompanying the preparation of sperm membranes for fusions-the first preparatory stage occurring before physiological release of the acrosomal content, the second afterward-we induced the acrosome reaction in capacitated guinea pig spermatozoa by adding calcium to the mixture. The most common features observed before fusion of the acrosomal and plasma membranes were the deletion of fibrillar intramembranous particles from the E-fracture faces of both membranes, and the clearance of globular particles from the P face of the plasma membrane-events taking place near the terminus of the equatorial segment. Large particles, >12nm, remained not far from the cleared E-face patches. The P face of the outer acrosomal membrane is virtually clear from the outset. In addition, when fusion was completed, occasional double lines of large particles transiently embossed the P face of the plasma membrane (postacrosomal) side of the fusion zone. Behind the line of fusion, another series of particle-cleared foci emerged. We interpreted these postfusion membrane clearances as a second adaptation for sperm-egg interaction. Induction of the acrosome reaction in media containing phosphatidylcholine liposomes resulted in their apparent attachment, incorporation, or exchange in both the originally and secondarily cleared regions. Our observations support the concepts that membranes become receptive to union at particle- deficient interfaces, and that the physiologically created barren areas in freeze-fracture replicas may herald incipient membrane fusion.     

Modifications of anionic-lipid domains preceding membrane fusion in guinea pig sperm

The relationship between anionic-lipid concentration and the functional properties of plasma-membrane domains was explored using the guinea-pig sperm membrane as a model, with polymyxin B (PXB) as a probe. Areas of plasmalemma specialized for fusion during the acrosome reaction had a higher affinity for the probe than adjacent nonfusigenic regions. In addition, capacitation--a process preceding acrosome:plasma-membrane fusion--markedly enlarged the area susceptible to PXB binding over the acrosomal cap. Protease treatment mimicked capacitation by increasing the acrosome-reaction incidence as well as PXB binding, at enzyme concentrations not affecting the surface coat nor altering filipin/sterol localization. Both proteolytic digestion and capacitation failed to augment PXB- or filipin-affinity in nonfusigenic zones, such as the post-acrosomal segment, including its particle-free maculae. Incubation of sperm in capacitating medium supplemented with 32P-labeled phosphate, followed by lipid extraction, thin-layer chromatography, and autoradiography, revealed a radioactive band comigrating with cardiolipin and phosphatidic acid. Vermiform protrusions elicited by PXB in the outer lamellae of cardiolipin- phosphatidylcholine liposomes resembled those seen in fusional regions of sperm membrane. We conclude that (a) differing concentrations of anionic lipids are found in adjacent domains of the sperm plasma membrane; (b) these domains mirror the functional regions of the membrane, with higher anionic-lipid concentrations localized over fusional zones; (c) the surface coat does not participate in the maintenance of such domains; (d) anionic-lipid synthesis may contribute to their formation; and (e) anionic-lipid concentrations increase as the membrane becomes fusionally competent, indicating that cellular modulation of lipid domains accompanies regulation of membrane function.     

Monoclonal antibodies to rabbit liver cytochrome P-448

Cytochrome P-448 (mol wt 55,000 Daltons) from rabbit liver was purified to a specific content of 16.6 nmol/mg. Mice were immunised with this preparation, their spleens removed and dissociated lymphocytes hybridised with myeloma cells. Four monoclonal antibodies against cytochrome P-448 were raised and partially characterised. All four antibodies interacted with cytochrome P-448 in intact microsomal fractions and selectively immunoadsorbed cytochrome P-448 from solubilised microsomal preparations. One of the antibodies inhibited benzo[a] pyrene hydroxylase activity in a reconstituted system, one had no effect on activity and two increased activity. The possible applications of such antibodies are discussed.     

Improving Education in Medical Statistics: Implementing a Blended Learning Model in the Existing Curriculum

Background

Although recent studies report on the benefits of blended learning in improving medical student education, there is still no empirical evidence on the relative effectiveness of blended over traditional learning approaches in medical statistics. We implemented blended along with on-site (i.e. face-to-face) learning to further assess the potential value of web-based learning in medical statistics.

Methods

This was a prospective study conducted with third year medical undergraduate students attending the Faculty of Medicine, University of Belgrade, who passed (440 of 545) the final exam of the obligatory introductory statistics course during 2013–14. Student statistics achievements were stratified based on the two methods of education delivery: blended learning and on-site learning. Blended learning included a combination of face-to-face and distance learning methodologies integrated into a single course.

Results

Mean exam scores for the blended learning student group were higher than for the on-site student group for both final statistics score (89.36±6.60 vs. 86.06±8.48; p = 0.001) and knowledge test score (7.88±1.30 vs. 7.51±1.36; p = 0.023) with a medium effect size. There were no differences in sex or study duration between the groups. Current grade point average (GPA) was higher in the blended group. In a multivariable regression model, current GPA and knowledge test scores were associated with the final statistics score after adjusting for study duration and learning modality (p<0.001).

Conclusion

This study provides empirical evidence to support educator decisions to implement different learning environments for teaching medical statistics to undergraduate medical students. Blended and on-site training formats led to similar knowledge acquisition; however, students with higher GPA preferred the technology assisted learning format. Implementation of blended learning approaches can be considered an attractive, cost-effective, and efficient alternative to traditional classroom training in medical statistics.     

Nanocrystalline SrS:Ce3+ system for the generation of white light‐emitting diodes

Nanocrystalline SrS phosphors doped with Ce³⁺ ions at different concentrations (0.5, 1, 1.5 and 2 mol%) are synthesized via the solid‐state diffusion method (SSDM), which is suitable for the large‐scale production of phosphors in industrial applications. The as‐prepared samples are characterized using an X‐ray diffraction (XRD) technique, field emission scanning electron microscopy (FESEM), high‐resolution transmission electron microscopy (HRTEM) and energy‐dispersive X‐ray (EDX) analysis. The optical properties of these phosphors are analyzed using reflectance spectra, photoluminescence spectra and afterglow decay curves. The cubic structure of the SrS phosphor is confirmed by XRD analysis and the crystallite size calculated by Scherer's formula using XRD data shows the nanocrystalline nature of the phosphors. No phase change is observed with increasing concentrations of Ce³⁺ ions. The surface morphology of the prepared phosphors is determined by FESEM, which shows a sphere‐like structure and good connectivity of the grains. The authenticity of the formation of nanocrystalline phosphors is examined by HRTEM analysis. Elemental compositional information for the prepared phosphors is gathered by EDX analysis. Photoluminescence studies reveal that the emission spectra of the prepared phosphor shows broad band emission centered at 458 and 550 nm due to the transition of electrons from the 5d → 4f energy levels. The afterglow decay characteristics of different as‐synthesized SrS:Ce³⁺ nanophosphors are conceptually described. Copyright © 2016 John Wiley & Sons, Ltd.     

The impact of aerobic and anaerobic training regimes on blood pressure in normotensive and hypertensive rats: focus on redox changes

Molecular and Cellular Biochemistry - Melatonin is a crucial neurohormone synthesized in the pineal gland that influences the physiology of animals. The molecular mechanism of norepinephrine...     

Mapping of the KHSRP gene to a region of conserved synteny on human chromosome 19p13.3 and mouse chromosome 17

The K homology-type splicing regulatory protein, KSRP, activates splicing through intronic splicing enhancer sequences. It is highly expressed in neural cells and is required for the neural-specific splicing of the c-src N1 exon. In this study, we mapped the gene (gene symbols KHSRP and Khsrp) to human chromosome 19 by using radiation hybrid panels and to mouse chromosome 17 by studying an interspecific backcross panel. Human KHSRP is a positional candidate gene for familial febrile convulsion and Cayman type cerebellar ataxia. Comparative analysis of the human and mouse genomes indicates that the KHSRP gene is located in regions of conserved synteny between the two species.     

A mechanism for substrate-Induced formation of 6-hydroxyflavin mononucleotide catalyzed by C30A trimethylamine dehydrogenase

Experiments are described to determine the origin of the 6-hydroxyl group of 6-hydroxyFMN produced by the substrate-induced transformation of FMN in the C30A mutant of trimethylamine dehydrogenase. The conversion of FMN to 6-hydroxyFMN is carried out in the presence of H(2)(18)O and 18O(2), and the results clearly show that the 6-hydroxyl group is derived from molecular oxygen and not from water.     

Cooperative assembly of an hnRNP complex induced by a tissue-specific homolog of polypyrimidine tract binding protein

Splicing of the c-src N1 exon in neuronal cells depends in part on an intronic cluster of RNA regulatory elements called the downstream control sequence (DCS). Using site-specific cross-linking, RNA gel shift, and DCS RNA affinity chromatography assays, we characterized the binding of several proteins to specific sites along the DCS RNA. Heterogeneous nuclear ribonucleoprotein (hnRNP) H, polypyrimidine tract binding protein (PTB), and KH-type splicing-regulatory protein (KSRP) each bind to distinct elements within this sequence. We also identified a new 60-kDa tissue-specific protein that binds to the CUCUCU splicing repressor element of the DCS RNA. This protein was purified, partially sequenced, and cloned. The new protein (neurally enriched homolog of PTB [nPTB]) is highly homologous to PTB. Unlike PTB, nPTB is enriched in the brain and in some neural cell lines. Although similar in sequence, nPTB and PTB show significant differences in their properties. nPTB binds more stably to the DCS RNA than PTB does but is a weaker repressor of splicing in vitro. nPTB also greatly enhances the binding of two other proteins, hnRNP H and KSRP, to the DCS RNA. These experiments identify specific cooperative interactions between the proteins that assemble onto an intricate splicing-regulatory sequence and show how this hnRNP assembly is altered in different cell types by incorporating different but highly related proteins.