Characterization of phycoerythrin from a Cryptomonas sp.

Summary Two closely similar phycoerythrins were purified from Cryptomonas sp. The two proteins were indistinguishable with respect to native molecular weight, subunit structure, photolability and immunological specificity, and differed only in their isoelectric points (pH 5.74 and 6.35), as determined by isoelectric focussing in polyacrylamide gels. Each protein consisted of two unequal subunits, (mol. wt. 11,800) and (mol. wt. 19,000), and each subunit contained covalently bound chromophore. In contrast to the blue-green and red algal phycoerythrins studied thus far, the Cryptomonas sp. phycoerythrins are extremely photolabile; exposure of the purified proteins to relatively short periods of intense illumination with visible light produces a marked decrease in fluorescence and in absorbance at 567 m.Abbreviation used SDS sodium dodecyl sulfate     

Circadian Rhythms of Plasma Renin Activity and Aldosterone: Changes Related to Age, Sex, Recumbency and Sodium Restriction. Chronobiologic Specification for Reference Values

Plasma renin activity (PRA) and aldosterone (PA) levels are characterized by a circadian rhythmicity (CR). The present study revealed that this rhythmicity is influenced by several factors including posture, sodium intake and age. Time-qualified PRA and PA reference intervals can reduce the incidence of false positives and false negatives in a diagnostic work-up. The circadian rhythmicity of PRA and PA have been quantified in relation to posture, sodium intake and age. The cosinor procedure has been applied to quantify the properties of the circadian rhythmicity under these conditions.

Chronograms and circadian parameters can be used to optimize the use of PRA and PA measurements in clinical practice. The chronobiological specification of reference values for PRA and PA is of valuable importance since the assessment of PRA and PA circadian rhythmicity has a diagnostic interest for a certain type of clinical disorder. It should be noted that several studies have described circannual variations for renin and aldosterone. The next step in the optimation of laboratory time-qualified reference values is the assessment of changes induced by the deterministic factors on a circannual domain.     

THE FINE STRUCTURE OF RHODOSPIRILLUM RUBRUM

The fine structure of Rhodospirillum rubrum grown under a series of defined conditions has been examined in thin sections prepared by the methods of Ryter and Kellenberger. In cells grown anaerobically at different light intensities, the abundance of 500 A membrane-bounded vesicles in the cytoplasm is inversely related to light intensity, and directly related to cellular chlorophyll content. When the chlorophyll content of the cell is low, the vesicles are exclusively peripheral in location; they extend more deeply into the cytoplasm when the chlorophyll content is high. Typical vesicles also occur, though rarely, in cells grown aerobically in the dark, which have a negligible chlorophyll content. When synthesis of the photosynthetic pigment system is induced in a population of aerobically grown cells by incubation under semianaerobic conditions in the dark, the vesicles become increasingly abundant with increasing cellular chlorophyll content, and the cells eventually acquire the cytoplasmic structure that is characteristic of cells growing anaerobically at a high light intensity. Poststaining with lead hydroxide reveals that the membranes surrounding the 500 A vesicles are indistinguishable in structure from the cytoplasmic membrane, and continuous with it in some areas of the sections. The bearing of these observations on current notions concerning the organization of the bacterial photosynthetic apparatus is discussed.     

Abstracts

Abstract Section

Abstracts     

Changes to the geometry and fluid mechanics of the carotid siphon in the pediatric Moyamoya disease

Background: The Moyamoya disease is a cerebrovascular disease that causes occlusion of the distal end of the internal carotid artery, leading to the formation of multiple tiny collateral arteries. To date, the pathogenesis of Moyamoya is unknown. Improved understanding of the changes to vascular geometry and fluid mechanics of the carotid siphon during disease may improve understanding of the pathogenesis, prognosis techniques and disease management. Methods: A retrospective analysis of Magnetic Resonance Angiography (MRA) images was performed for Moyamoya pediatric patients (MMD) (n = 23) and control (Ctrl) pediatric patients (n = 20). The Ctrl group was composed of patients who complained of headache and had normal MRA. We performed segmentation of MRA images to quantify geometric parameters of the artery. Computational fluid dynamics (CFD) was performed to quantify the hemodynamic parameters. Results: MMD internal carotid and carotid siphons were smaller in cross-sectional areas, and shorter in curved vascular length. Vascular curvature remained constant over age and vascular size and did not change between Ctrl and MMD, but MMD carotid siphon had lower tortuosity in the posterior bend, and higher torsion in the anterior bend. Wall shear stress and secondary flows were significantly lower in MMD, but the ratio of secondary flow kinetic energy to primary flow kinetic energy were similar between MMD and Ctrl. Conclusion: There were alterations to both the geometry and the flow mechanics of the carotid siphons of Moyamoya patients but it is unclear whether hemodynamics is the cause or the effect of morphological changes observed.     

One-Step Multiplex RT-qPCR Assay for the Detection of Peste des petits ruminants virus,Capripoxvirus, Pasteurella multocida and Mycoplasma capricolum subspecies (ssp.) capripneumoniae

Respiratory infections, although showing common clinical symptoms like pneumonia, are caused by bacterial, viral or parasitic agents. These are often reported in sheep and goats populations and cause huge economic losses to the animal owners in developing countries. Detection of these diseases is routinely done using ELISA or microbiological methods which are being reinforced or replaced by molecular based detection methods including multiplex assays, where detection of different pathogens is carried out in a single reaction. In the present study, a one-step multiplex RT-qPCR assay was developed for simultaneous detection of Capripoxvirus (CaPV), Peste de petits ruminants virus (PPRV), Pasteurella multocida (PM) and Mycoplasma capricolum ssp. capripneumonia (Mccp) in pathological samples collected from small ruminants with respiratory disease symptoms. The test performed efficiently without any cross-amplification. The multiplex PCR efficiency was 98.31%, 95.48%, 102.77% and 91.46% whereas the singleplex efficiency was 93.43%, 98.82%, 102.55% and 92.0% for CaPV, PPRV, PM and Mccp, respectively. The correlation coefficient was greater than 0.99 for all the targets in both multiplex and singleplex. Based on cycle threshold values, intra and inter assay variability, ranged between the limits of 2%–4%, except for lower concentrations of Mccp. The detection limits at 95% confidence interval (CI) were 12, 163, 13 and 23 copies/reaction for CaPV, PPRV, PM and Mccp, respectively. The multiplex assay was able to detect CaPVs from all genotypes, PPRV from the four lineages, PM and Mccp without amplifying the other subspecies of mycoplasmas. The discriminating power of the assay was proven by accurate detection of the targeted pathogen (s) by screening 58 viral and bacterial isolates representing all four targeted pathogens. Furthermore, by screening 81 pathological samples collected from small ruminants showing respiratory disease symptoms, CaPV was detected in 17 samples, PPRV in 45, and PM in six samples. In addition, three samples showed a co-infection of PPRV and PM. Overall, the one-step multiplex RT-qPCR assay developed will be a valuable tool for rapid detection of individual and co-infections of the targeted pathogens with high specificity and sensitivity.     

The Ubiquitin Ligase CBLC Maintains the Network Organization of the Golgi Apparatus

The Golgi apparatus plays a pivotal role in the sorting and post-translational modifications of secreted and membrane proteins. In mammalian cells, the Golgi is organized in stacks of cisternae linked together to form a network with a ribbon shape. Regulation of Golgi ribbon formation is poorly understood. Here we find in an image-based RNAi screen that depletion of the ubiquitin-ligase CBLC induces Golgi fragmentation. Depletions of the close homologues CBL and CBLB do not induce any visible defects. In CBLC-depleted cells, Golgi stacks appear relatively unperturbed at both the light and electron microscopy levels, suggesting that CBLC controls mostly network organization. CBLC partially localizes on Golgi membranes and this localization is enhanced after activation of the SRC kinase. Inhibition of SRC reverts CBLC depletion effects, suggesting interplay between the two. CBLC’s regulation of Golgi network requires its ubiquitin ligase activity. However, SRC levels are not significantly affected by CBLC, and CBLC knockdown does not phenocopy SRC activation, suggesting that CBLC’s action at the Golgi is not direct downregulation of SRC. Altogether, our results demonstrate a role of CBLC in regulating Golgi ribbon by antagonizing the SRC tyrosine kinase.     

In vitro transformation of human amniotic fluid cells in the presence of fecalase. Method for detecting environmental chemicals with oncogenic potential

A short-term method for detecting potential environmental carcinogens is described using in vitro transformation assay of human epithelial-like amniotic fluid cells. According to Styles, after a short exposition to the carcinogen, only transformed cells grow and form large colonies when cultured in semi-solid agar. In our assay addition of liver homogenate was not necessary to activate the carcinogens. Nevertheless adjunction to the exposure medium of human fecalase (after Ames) is advised for studying carcinogenicity of food glycosides. Fecalase efficiency in metabolic activation of glycosidic compounds has been demonstrated in the use of 8-hydroxyquinoline-beta-D-glycopyranoside.     

Bacterial endophyte-mediated naphthalene phytoprotection and phytoremediation

Polyaromatic hydrocarbons (PAHs) are major and recalcitrant pollutants of the environment and their removal presents a significant problem. Phytoremediation has shown much promise in PAH removal from contaminated soil, but may be inhibited because the plant experiences phytotoxic effects from low-molecular-weight PAHs such as naphthalene. This paper describes the construction of a naphthalene-degrading endophytic strain designated Pseudomonas putida VM1441(pNAH7). This strain was found to be an efficient colonizer of plants, colonizing both the rhizosphere and interior root tissues. The inoculation of plants with P. putida VM1441(pNAH7) resulted in the protection of the host plant from the phytotoxic effects of naphthalene. When inoculated plants were exposed to naphthalene, both seed germination and plant transpiration rates were higher than those of the uninoculated controls. The inoculation of plants with this strain also facilitated higher (40%) naphthalene degradation rates compared with uninoculated plants in artificially contaminated soil.     

Structure-function studies of two synthetic anti-vascular endothelial growth factor Fabs and comparison with the Avastin Fab

In the quest to discover new research tools and to develop better agents in the fight against cancer, two antibodies, G6 and B20-4, were isolated from synthetic antibody phage libraries. Unlike the AVASTINtrade mark antibody, a recently approved agent for the treatment of patients with colorectal cancer, B20-4 and G6 bind and block both human and murine vascular endothelial growth factor (VEGF). Here we have analyzed and compared the binding epitopes on VEGF for these three antibodies using alanine-scanning mutagenesis and structural analyses. The epitopes recognized by both synthetic antibodies are conserved between human and mouse VEGF, and they match closely to the receptor epitopes both structurally and functionally. In contrast, the Avastin epitope overlaps minimally with the receptor binding surface and centers around a residue that is not conserved in mouse. Our structural and functional analyses elucidate the cross-species reactivity of all three antibodies and emphasize the potential advantages of antibody generation using phage display as the resulting antibodies do not depend on sequence differences across species and preferentially target natural protein-protein interaction surfaces.