Identification and localization of a Rickettsia sp. in Bemisia tabaci (Homoptera: Aleyrodidae)

Whiteflies (Homoptera: Aleyrodidae) are sap-sucking insects that harbor "Candidatus Portiera aleyrodidarum," an obligatory symbiotic bacterium which is housed in a special organ called the bacteriome. These insects are also home for a diverse facultative microbial community which may include Hamiltonella, Arsenophonus, Fritchea, Wolbachia, and Cardinium spp. In this study, the bacteria associated with a B biotype of the sweet potato whitefly Bemisia tabaci were characterized using molecular fingerprinting techniques, and a Rickettsia sp. was detected for the first time in this insect family. Rickettsia sp. distribution, transmission and localization were studied using PCR and fluorescence in situ hybridizations (FISH). Rickettsia was found in all 20 Israeli B. tabaci populations screened but not in all individuals within each population. A FISH analysis of B. tabaci eggs, nymphs, and adults revealed a unique concentration of Rickettsia around the gut and follicle cells, as well as a random distribution in the hemolymph. We postulate that the Rickettsia enters the oocyte together with the bacteriocytes, leaves these symbiont-housing cells when the egg is laid, multiplies and spreads throughout the egg during embryogenesis and, subsequently, disperses throughout the body of the hatching nymph, excluding the bacteriomes. Although the role Rickettsia plays in the biology of the whitefly is currently unknown, the vertical transmission on the one hand and the partial within-population infection on the other suggest a phenotype that is advantageous under certain conditions but may be deleterious enough to prevent fixation under others.     

Wnt11 controls cell contact persistence by local accumulation of Frizzled 7 at the plasma membrane

Wnt11 is a key signal, determining cell polarization and migration during vertebrate gastrulation. It is known that Wnt11 functionally interacts with several signaling components, the homologues of which control planar cell polarity in Drosophila melanogaster. Although in D. melanogaster these components are thought to polarize cells by asymmetrically localizing at the plasma membrane, it is not yet clear whether their subcellular localization plays a similarly important role in vertebrates. We show that in zebrafish embryonic cells, Wnt11 locally functions at the plasma membrane by accumulating its receptor, Frizzled 7, on adjacent sites of cell contacts. Wnt11-induced Frizzled 7 accumulations recruit the intracellular Wnt signaling mediator Dishevelled, as well as Wnt11 itself, and locally increase cell contact persistence. This increase in cell contact persistence is mediated by the local interaction of Wnt11, Frizzled 7, and the atypical cadherin Flamingo at the plasma membrane, and it does not require the activity of further downstream effectors of Wnt11 signaling, such as RhoA and Rok2. We propose that Wnt11, by interacting with Frizzled 7 and Flamingo, modulates local cell contact persistence to coordinate cell movements during gastrulation.     

A dynamic model for replication protein A (RPA) function in DNA processing pathways

Processing of DNA in replication, repair and recombination pathways in cells of all organisms requires the participation of at least one major single-stranded DNA (ssDNA)-binding protein. This protein protects ssDNA from nucleolytic damage, prevents hairpin formation and blocks DNA reannealing until the processing pathway is successfully completed. Many ssDNA-binding proteins interact physically and functionally with a variety of other DNA processing proteins. These interactions are thought to temporally order and guide the parade of proteins that ‘trade places’ on the ssDNA, a model known as ‘hand-off’, as the processing pathway progresses. How this hand-off mechanism works remains poorly understood. Recent studies of the conserved eukaryotic ssDNA-binding protein replication protein A (RPA) suggest a novel mechanism by which proteins may trade places on ssDNA by binding to RPA and mediating conformation changes that alter the ssDNA-binding properties of RPA. This article reviews the structure and function of RPA, summarizes recent studies of RPA in DNA replication and other DNA processing pathways, and proposes a general model for the role of RPA in protein-mediated hand-off.     

Excitatory and feed-forward inhibitory hippocampal synapses work synergistically as an adaptive filter of natural spike trains

Short-term synaptic plasticity (STP) is an important mechanism for modifying neural circuits during computation. Although STP is much studied, its role in the processing of complex natural spike patterns is unknown. Here we analyze the responses of excitatory and inhibitory hippocampal synapses to natural spike trains at near-physiological temperatures. Our results show that excitatory and inhibitory synapses express complementary sets of STP components that selectively change synaptic strength during epochs of high-frequency discharge associated with hippocampal place fields. In both types of synapses, synaptic strength rapidly alternates between a near-constant level during low activity and another near-constant, but elevated (for excitatory synapses) or reduced (for inhibitory synapses) level during high-frequency epochs. These history-dependent changes in synaptic strength are largely independent of the particular temporal pattern within the discharges, and occur concomitantly in the two types of synapses. When excitatory and feed-forward inhibitory synapses are co-activated within the hippocampal feed-forward circuit unit, the net effect of their complementary STP is an additional increase in the gain of excitatory synapses during high-frequency discharges via selective disinhibition. Thus, excitatory and feed-forward inhibitory hippocampal synapses in vitro act synergistically as an adaptive filter that operates in a switch-like manner and is selective for high-frequency epochs.     

Purification and analysis of prion and amyloid aggregates

Amyloids and prions represent aggregates of misfolded proteins, which consist of protein polymer fibrils with cross-beta sheet structure. Understanding of their occurrence and role is developing rapidly. Initially, they were found associated with mammalian diseases, mainly of neurodegenerative nature. Now they are known to relate to a range of non-disease phenomena in different species from mammals to lower eukaryotes. Uncovering new prion- and amyloid-related processes may be helped greatly by a procedure for purification of amyloid polymers. Studies of growth and propagation of these polymers require methods for determination of their size. Here, we describe such methods. They rely on the treatment with cold SDS or Sarcosyl detergents, which do not dissolve amyloids, but solubilize almost all non-amyloid complexes and associations between amyloid fibers. This allows purifying amyloids by centrifugation in the presence of these detergents. The size of amyloid polymers may be analyzed by electrophoresis in agarose gels containing SDS. Two procedures are described for determining the proportion between polymers and monomers of a particular protein using polyacrylamide gels.     

Functional characterization of CmCCD1, a carotenoid cleavage dioxygenase from melon

Carotenoids are nutritionally important tetraterpenoid pigments that upon oxidative cleavage give rise to apocarotenoid (norisoprene) aroma volatiles. beta-Carotene is the predominant pigment in orange-fleshed melon (Cucumis melo L.) varieties, reaching levels of up to 50 microg/gFW. Pale green and white cultivars have much lower levels (0-10 microg/gFW). In parallel, beta-ionone, the 9,10 cleavage product of beta-carotene, is present (12-33ng/gFW) in orange-fleshed melon varieties that accumulate beta-carotene, and in much lower levels (0-5 ng/gFW) in pale green and white fleshed varieties. A search for a gene putatively responsible for the cleavage of beta-carotene into beta-ionone was carried out in annotated melon fruit EST databases yielding a sequence (CmCCD1) highly similar (84%) to other plant carotenoid cleavage dioxygenase genes. To test its function, the clone was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. We show here that the CmCCD1 gene product cleaves carotenoids at positions 9,10 and 9',10', generating geranylacetone from phytoene; pseudoionone from lycopene; beta-ionone from beta-carotene, as well as alpha-ionone and pseudoionone from delta-carotene. CmCCD1 gene expression is upregulated upon fruit development both in orange, pale-green and white melon varieties, despite the lack of apocarotenoid volatiles in the later. Thus, the accumulation of beta-ionone in melon fruit is probably limited by the availability of carotenoid substrate.     

Cloning and expression analysis of a UDP-galactose/glucose pyrophosphorylase from melon fruit provides evidence for the major metabolic pathway of galactose metabolism in raffinose oligosaccharide metabolizing plants

The Cucurbitaceae translocate a significant portion of their photosynthate as raffinose and stachyose, which are galactosyl derivatives of sucrose. These are initially hydrolyzed by alpha-galactosidase to yield free galactose (Gal) and, accordingly, Gal metabolism is an important pathway in Cucurbitaceae sink tissue. We report here on a novel plant-specific enzyme responsible for the nucleotide activation of phosphorylated Gal and the subsequent entry of Gal into sink metabolism. The enzyme was antibody purified, sequenced, and the gene cloned and functionally expressed in Escherichia coli. The heterologous protein showed the characteristics of a dual substrate UDP-hexose pyrophosphorylase (PPase) with activity toward both Gal-1-P and glucose (Glc)-1-P in the uridinylation direction and their respective UDP-sugars in the reverse direction. The two other enzymes involved in Glc-P and Gal-P uridinylation are UDP-Glc PPase and uridyltransferase, and these were also cloned, heterologously expressed, and characterized. The gene expression and enzyme activities of all three enzymes in melon (Cucumis melo) fruit were measured. The UDP-Glc PPase was expressed in melon fruit to a similar extent as the novel enzyme, but the expressed protein was specific for Glc-1-P in the UDP-Glc synthesis direction and did not catalyze the nucleotide activation of Gal-1-P. The uridyltransferase gene was only weakly expressed in melon fruit, and activity was not observed in crude extracts. The results indicate that this novel enzyme carries out both the synthesis of UDP-Gal from Gal-1-P as well as the subsequent synthesis of Glc-1-P from the epimerase product, UDP-Glc, and thus plays a key role in melon fruit sink metabolism.     

Spectroscopic detection and identification of infected cells with herpes viruses

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and Fourier transform infrared (FTIR) microspectroscopy were previously applied for the identification of various biological samples. In the present study, normal cells in culture and cells infected with herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV) were analyzed by MALDI-TOF and FTIR microscopy. Specific spectral biomarkers for rapid and reliable monitoring and identification of infected cells and probably for the discrimination between these viruses were searched. The results show consistent spectral peaks in all examined normal uninfected human fibroblast cells both in MALDI-T0F and FTIR microscopy. In HSV-2- or VZV-infected cells, two unique peaks for each appeared at m/z 5397 and 5813 or at m/z 3501 and 4951, respectively, in MALDI-TOF spectra. In addition, several peaks that appeared in control uninfected cells at the region m/z 13,000-20,000 disappeared completely in all examined infected samples. When these infected cells were examined by FTIR microscopy, a band at 859 cm(-1) in control uninfected cells was significantly shifted to 854 cm(-1) in both HSV2- and VZV-infected cells. In addition, phosphate levels were considerably increased in all infected cells compared to normal uninfected cells. These parameters could be used as a basis for developing a spectral method for the detection and identification of cells infected with herpes viruses.     

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice. HYPERStack vessels allow closed system scale up from the current stacked plate products and delivers >2.5X more cells in the same volumetric footprint. The HYPERStack vessels function via gas permeable material which allows gas exchange to occur, therefore eliminating the need for internal headspace within a vessel. The elimination of headspace allows the compartment where cell growth occurs to be minimized to reduce space, allowing more layers of cell growth surface area within the same volumetric footprint.For many applications such as cell therapy or vaccine production, a closed system is required for cell growth and harvesting. The HYPERStack vessel allows cell and reagent addition and removal via tubing from media bags or other methods.This protocol will explain the technology behind the gas permeable material used in the HYPERStack vessels, gas diffusion results to meet the metabolic needs of cells, closed system cell growth protocols, and various harvesting methods.Download video file.^{(70M, mov)}