Antimicrobial peptides from chilli pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells

During the last few years, a growing number of cysteine-rich antimicrobial peptides has been isolated from plants and particularly from seeds. It has become increasingly clear that these peptides play an important role in the protection of plants against microbial infection. In this work, proteins from chilli pepper (Capsicum annuum L.) seeds were extracted in phosphate buffer, pH 5.4 and peptides purification were performed by employing ion-exchange chromatographies on DEAE, CM-Sepharose, Sephacryl S-100 and reverse phase in HPLC. Three peptide enriched fractions, namely F1, F2 and F3, were obtained after the CM-Sepharose chromatography. The F1 fraction, mainly composed of three peptides ranging from 6 to 10 kDa, was submitted to N-terminal amino acid sequencing. The closer to 10 kDa peptide showed high sequence homology to lipid transfer proteins (LTPs) previously isolated from others seeds. F1 fraction exhibited strong fungicidal activity against Candida albicans, Saccharomyces cerevisiae and Schizosaccharomyces pombe and also promoted several morphological changes to C. albicans, including the formation of pseudohyphae, as revealed by scanning electron micrography. F1 fraction also reduced the glucose stimulated acidification of the medium mediated by H⁺-ATPase of S. cerevisiae cells in a dose-dependent manner and caused the permeabilization of yeast plasma membrane to the dye SYTOX Green, as verified by confocal laser microscopy.     

Giant noctule bats face conflicting constraints between roosting and foraging in a fragmented and heterogeneous landscape

Noting that some ground-dwelling passerine birds have remarkably long claws, reflected in such names as longclaw (Motacillidae) and longspur (Emberizidae), this comparative study assessed whether these features might aid movement over grassy ground vegetation. Using measurements of museum specimens, we found that, corrected for body size, larks (Alaudidae) living in grassy habitats ( n =31) had significantly longer toes and claws and hence larger footspans than species dwelling on bare substrates ( n =25). Using phylogenetically corrected contrasts, the claw result remained strongly significant, and the footspan result marginally significant, while there was no significant difference between the toes of the two groups. Among 15 matched pairs of other ground-dwelling passerines, where one member of the pair inhabits vegetated ground and the other inhabits bare ground, the former had significantly longer toes and claws and hence larger footspans. We conclude that ground-dwelling passerines living on vegetated, typically grassy ground, have larger feet, due to both longer toes and claws. This probably facilitates movement over uneven and unstable vegetation.     

Activation of phospholipase A2 by temporin B: Formation of antimicrobial peptide-enzyme amyloid-type cofibrils

Phospholipases A2 have been shown to be activated in a concentration dependent manner by a number of antimicrobial peptides, including melittin, magainin 2, indolicidin, and temporins B and L. Here we used fluorescently labelled bee venom PLA2 (PLA2D) and the saturated phospholipid substrate 1,2-dipalmitoyl-glycero-sn-3-phosphocholine (L-DPPC), exhibiting a lag-burst behaviour upon the initiation of the hydrolytic reaction by PLA2. Increasing concentrations of Cys-temporin B and its fluorescent Texas red derivative (TRC-temB) caused progressive shortening of the lag period. TRC-temB/PLA2D interaction was observed by Förster resonance energy transfer (FRET), with maximum efficiency coinciding with the burst in hydrolysis. Subsequently, supramolecular structures became visible by microscopy, revealing amyloid-like fibrils composed of both the activating peptide and PLA2. Reaction products, palmitic acid and 1-palmitoyl-2-lyso-glycero-sn-3-phosphocholine (lysoPC, both at > 8 mol%) were required for FRET when using the non-hydrolysable substrate enantiomer 2,3-dipalmitoyl-glycero-sn-1-phosphocholine (D-DPPC). A novel mechanism of PLA2 activation by co-fibril formation and associated conformational changes is suggested.     

Cryoelectron Microscopy Structure of Purified γ-Secretase at 12 Å Resolution

γ-Secretase, an integral membrane protein complex, catalyzes the intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal production of the amyloid β-peptide. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer's disease. Existing biochemical studies conflict on the oligomeric assembly state of the protease complex, and its detailed structure is not known. Here, we report that purified active human γ-secretase in digitonin has a total molecular mass of ∼ 230 kDa when measured by scanning transmission electron microscopy. This result supports a complex that is monomeric for each of the four component proteins. We further report the three-dimensional structure of the γ-secretase complex at 12 Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction. The structure reveals several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove in the transmembrane region of the complex.     

Adaptive dose-finding: Proof of concept with type I error control

We consider an adaptive dose‐finding study with two stages. The doses for the second stage will be chosen based on the first stage results. Instead of considering pairwise comparisons with placebo, we apply one test to show an upward trend across doses. This is a possibility according to the ICH‐guideline for dose‐finding studies (ICH‐E4). In this article, we are interested in trend tests based on a single contrast or on the maximum of multiple contrasts. We are interested in flexibly choosing the Stage 2 doses including the possibility to add doses. If certain requirements for the interim decision rules are fulfilled, the final trend test that ignores the adaptive nature of the trial (naïve test) can control the type I error. However, for the more common case that these requirements are not fulfilled, we need to take the adaptivity into account and discuss a method for type I error control. We apply the general conditional error approach to adaptive dose‐finding and discuss special issues appearing in this application. We call the test based on this approach Adaptive Multiple Contrast Test. For an example, we illustrate the theory discussed before and compare the performance of several tests for the adaptive design in a simulation study.     

Charge contrast imaging of biomaterials in a variable pressure scanning electron microscope

Charge contrast imaging (CCI) is a dynamic phenomenon recently reported in insulating and semiconducting materials imaged with low vacuum or variable pressure scanning electron microscopes (SEM). Data presented in this paper illustrates that CCI can also be applied to biominerals and biological soft-tissues and that useful and unique structural information can be obtained from routine samples. Various resin-embedded samples were considered and example images from several different biomaterials are presented. Due to the diverse nature of samples that appear to exhibit charge contrast, this imaging technique has prospective application in a wide range of biological and biomedical research. This work represents the first application of CCI to biomaterials and in particular, highlights a new method for investigating the formation, structure and growth of biominerals.     

Spatial and acoustic pressure dependence of microbubble-mediated gene delivery targeted using focused ultrasound

BACKGROUND: Ultrasound/microbubble-mediated gene delivery has the potential to be targeted to tissue deep in the body by directing the ultrasound beam following vector administration. Application of this technology would be minimally invasive and benefit from the widespread clinical experience of using ultrasound and microbubble contrast agents. In this study we evaluate the targeting ability and spatial distribution of gene delivery using focused ultrasound. METHODS: Using a custom-built exposure tank, Chinese hamster ovary cells in the presence of SonoVue microbubbles and plasmid encoding beta-galactosidase were exposed to ultrasound in the focal plane of a 1 MHz transducer. Gene delivery and cell viability were subsequently assessed. Characterisation of the acoustic field and high-resolution spatial analysis of transfection were used to examine the relationship between gene delivery efficiency and acoustic pressure. RESULTS: In contrast to that seen in the homogeneous field close to the transducer face, gene delivery in the focal plane was concentrated on the ultrasound beam axis. Above a minimum peak-to-peak value of 0.1 MPa, transfection efficiency increased as acoustic pressure increased towards the focus, reaching a maximum above 1 MPa. Delivery was microbubble-dependent and cell viability was maintained. CONCLUSIONS: Gene delivery can be targeted using focused ultrasound and microbubbles. Since delivery is dependent on acoustic pressure, the degree of targeting can be determined by appropriate transducer design to modify the ultrasound field. In contrast to other physical gene delivery approaches, the non-invasive targeting ability of ultrasound makes this technology an attractive option for clinical gene therapy.     

Polydisulfide Gd(III) chelates as biodegradable macromolecular magnetic resonance imaging contrast agents

Macromolecular gadolinium (Gd)(III) complexes have a prolonged blood circulation time and can preferentially accumulate in solid tumors, depending on the tumor blood vessel hyperpermeability, resulting in superior contrast enhancement in magnetic resonance (MR) cardiovascular imaging and cancer imaging as shown in animal models. Unfortunately, safety concerns related to these agents' slow elimination from the body impede their clinical development. Polydisulfide Gd(III) complexes have been designed and developed as biodegradable macromolecular magnetic resonance imaging (MRI) contrast agents to facilitate the clearance of Gd(III) complexes from the body after MRI examinations. These novel agents can act as macromolecular contrast agents for in vivo imaging and excrete rapidly as low-molecular-weight agents. The rationale and recent development of the novel biodegradable contrast agents are reviewed here. Polydisulfide Gd(III) complexes have relatively long blood circulation time and gradually degrade into small Gd(III) complexes, which are rapidly excreted via renal filtration. These agents result in effective and prolonged in vivo contrast enhancement in the blood pool and tumor tissue in animal models, yet demonstrate minimal Gd(III) tissue retention as the clinically used low-molecular-weight agents. Structural modification of the agents can readily alter the contrast-enhancement kinetics. Polydisulfide Gd(III) complexes are promising for further clinical development as safe, effective, biodegradable macromolecular MRI contrast agents for cardiovascular and cancer imaging, and for evaluation of therapeutic response.     

Role of calcium in apoptosis of HL-60 cells induced by harringtonine

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Measurement of bioreactor perfusion using dynamic contrast agent-enhanced magnetic resonance imaging.

Dynamic magnetic resonance imaging was used to monitor solute diffusion through aggregates of Chinese hamster ovary cells growing on macroporous carriers in a fixed-bed bioreactor. Diffusion-weighted (1)H magnetic resonance imaging (MRI) and scanning electron microscopy demonstrated that cell growth in the bioreactor was heterogeneous, with the highest cell densities being found at the periphery of the carriers. T(1)-weighted magnetic resonance imaging measurements of the inflow of a commonly used magnetic resonance contrast agent, gadolinium-diethylenetriaminopentaacetic acid (Gd-DTPA), showed that migration of the agent through the peripheral cell masses could be explained by diffusion. However, appearance of the contrast agent in the center of the carriers was too fast to be explained by simple diffusion and indicated that these regions were perfused by convective flow. The average diffusivity of Gd-DTPA through the cell mass was found to be (2.4 +/- 0.2) x 10(-10) m(2) sec(-) (mean +/- SEM). This technique will be useful in the characterization and development of high-cell-density bioreactor systems, in which solute transport plays a critical role in cell growth and physiology.