Biochemical characterization and crystallization of porin from Rhodopseudomonas blastica

Oligomeric porin of the phototrophic bacterium Rhodopseudomonas blastica DSM 2131 was obtained from cell envelopes by differential temperature extraction in the presence of detergent and salt. The isolated porin exhibited strong porin activity after reconstitution into lipid bilayer membranes. The effective channel diameter for the trimer was estimated as 1.5 nm from single channel conductance measurements in the presence of 1 M KCl. Moderate cation-selectivity was observed. Oligomeric porin migrated as a single band (apparent molecular weight 81 kDa) on sodium dodecyl sulfate polyacrylamide gelelectrophoresis when solubilized below 70 °C. The oligomers were converted into monomers on heating to 70 °C or above forming two bands with apparent molecular weight of 36 kDa and 35 kDa. The oligomer was not sensitive to EDTA. Its molecular weight was determined to be 119.3 kDa by analytical ultracentrifugation. The isoelectric point was 5.7. Circular dichroism data indicated a high content of -sheet structure. Gasphase sequencing of the N-terminal residues revealed the sequence: NH₂-Glu-Ile-Ser-Leu-Asn-Gly-Tyr-Gly-Arg-Phe. Crystals with a maximal side length of 300 m and diffracting to 0.32 nm resolution were obtained with the porin oligomer in the presence of C8E4 and 1,2,3-heptanetriol by using the vapor phase equilibration technique.Abbreviations C8E4 n-octyl tetraoxyethylene - M_r apparent molecular weight - Octyl-POE n-octyl polyoxyethylene - LDAO N,N-dimethyl dodecyl aminoxide - LPS lipopolysaccharide - PAGE polyacrylamide gel-electrophoresis - PEG polyethylene glycol     

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon.     

Cellular Concentrations and Uniformity of Cell-Type Accumulation of Two Lea Proteins in Cotton Embryos

The levels and cell-type distribution of late embryogenesis abundant (Lea) proteins D-7 and D-113 have been determined in mature cotton embryos by immunochemical methods. The two proteins were expressed in and purified from Escherichia coli and utilized for antibody production in rabbits. The antiserum to each protein was found to interact with all members of each protein family in cotton extracts by protein gel blotting. Using these antibodies in quantitative "rocket" immunoelectrophoreses, D-7 proteins were found to accumulate to ~8 x 1015 molecules per embryo, which is equivalent to ~109 molecules per "average cell." D-113 proteins accumulate to ~1016 molecules per embryo, which equates to ~1.3 x 109 molecules per average cell. These values calculate to concentrations of about 226 and 283 [mu]M, respectively, in the cell aqueous phase immediately prior to seed desiccation. In immunocytochemical studies using the fluorophor rhodamine linked to the secondary antibody, both proteins appeared to be evenly present in the cytosol of all cell types present in the embryo, including both cotyledon and axis epidermal cells. Thus, their function does not appear related to unique functions of specific cell or tissue types. The very high molar concentrations of the two proteins, coupled with their unusual predicted structure and their cytosol location, would seem to reduce the number of their conceivable functions.     

AFFINITY CONCENTRATION OF LISTERIA MONOCYTOGENES CELLS ON CONCANAVALIN A-COATED POLYESTER CLOTH AND SUBSEQUENT DETECTION BY THE POLYMERASE CHAIN REACTION

The Canavalia ensiformis lectin concanavalin A (con A) was immobilized on macroporous polyester cloth to form an inexpensive high surface area adsorbent (con A-cloth) for the affinity concentration of bacterial cells in aqueous suspensions. When the con A-cloth was packed into a 1 ml pipette tip, the resulting mini-column could be used to concentrate large volumes of dilute L. monocytogenes cell suspensions, followed by the polymerase chain reaction (PCR) amplification of L. monocytogenes-specific hly A sequences from lysates of the captured cells. This improved the effective sensitivity of the PCR as compared to the assay of L. monocytogenes in unconcentrated suspensions. Several enrichment broths (Fraser Broth, Listeria Enrichment Broth and Brain Heart Infusion Broth) were found to be inhibitory to the PCR of L. monocytogenes when introduced directly into the reaction mixture. This inhibitory effect was completely eliminated when the L. monocytogenes cells were captured on the con A-cloth and washed to remove the enrichment broth components prior to performing the PCR. Since the lectin con A is reactive with a broad variety of Gram positive and Gram negative bacteria, this simple and inexpensive affinity concentration method should be applicable to the PCR detection of other pathogens in enrichment cultures.     

Bovine lactoferrin in involuting mammary tissue

Bovine lactoferrin in involuting mammary tissue was identified by immunohistochemistry and tissue explant culture. Immunoreactive lactoferrin was associated with mammary epithelial cells. Immunostaining for lactoferrin increased during involution, in contrast to declining immunostaining of epithelia for the milk-specific protein β-lactoglobulin. Immunostaining for lactoferrin also was observed at the basal region of alveolar epithelia, perhaps in association with basement membrane components. Lactoferrin was preferentially synthesized in involuting mammary tissue compared with lactating tissue. Synthesis of lactoferrin in the involuting mammary gland occurs despite the apparent decline in synthesis of milk-specific proteins.     

Phosphorylation of the GABAA receptor by cAMP-dependent protein kinase and by protein kinase C: Analysis of the substrate domain

Previous work has shown that the GABA_A-receptor (GABA_A-R) could be phosphorylated by cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and a receptor associated kinase. However, no clear picture has yet emerged concerning the particular subunit subtypes of the GABA_A-R that were phosphorylated by PKA and PKC. In the present report we show that an antibody raised against a 23 amino acid polypeptide corresponding to a sequence in the putative intracellular loop of the 1 subunit of the receptor blocks the in vitro phosphorylation of the purified receptor by PKA and PKC. Moreover, N-terminal sequence analysis of the principal phosphopeptide fragment obtained after proteolysis of the receptor yielded a sequence that corresponds to the 3 subunit of the receptor. Such data provide additional support for our hypothesis (Browning et al., 1990, Proc. Natl. Acad. Sci. USA 87:1315–1317) that both PKA and PKC phosphorylate the -subunit of the GABA_A-R.Special issue dedicated to Dr. Paul Greengard.     

Leaf nutrients in relation to stature and life form in tropical rain forest

Abstract. To document the relationship between a plant's position in the canopy and its leaf nutrient content, leaf nitrogen and phosphorus were determined for 30 species growing in mature evergreen lowland rain forest at La Selva Biological Station, Costa Rica. Species that grow either in the understory, midstory, or the canopy were selected. Species were further separated into three life forms: self-supporting monocots, self-supporting dicots, and climbers. Mass-based nutrient concentrations were expected to decrease with stature, as has been reported in studies of other forests. In fact, mass-based nitrogen and phosphorus did not vary significantly among the three adult-stature classes, although area-based values differed greatly: canopy plants averaged 60 % more nitrogen and 90 % more phosphorus per unit leaf area than understory plants. Differences in leaf characteristics were evident among the three life forms. Most notably, area-based phosphorus and leaf specific mass were lowest in climbers, intermediate in self-supporting dicots, and highest in self-supporting monocots. These results support the characterization of climbers as investing in inexpensive structures, perhaps in order to gain competitive advantage in light capture by allocating resources to maximize elongation rates.     

Deep-water macroalgae from the Canary Islands: new records and biogeographical relationships

Due to the geographical location and paleobiogeography of the Canary Islands, the seaweed flora contains macroalgae with different distributional patterns. In this contribution, the biogeographical relations of several new records of deep-water macroalgae recently collected around the Canarian archipelago are discussed. These areBryopsidella neglecta (Berthold) Rietema,Discosporangium mesarthrocarpum (Meneghini) Hauck,Hincksia onslowensis (Amsler et Kapraun) P. C. Silva,Syringoderma floridana Henry,Peyssonnelia harveyana J. Agardh,Cryptonemia seminervis (C. Agardh) J. Agardh,Botryocladia wynnei Ballantine,Gloiocladia blomquistii (Searles) R. E. Norris,Halichrysis peltata (W. R. Taylor) P. Huvé et H. Huvé,Leptofauchea brasiliensis Joly, andSarcodiotheca divaricata W. R. Taylor. These new records, especially those in the Florideophyceae, support the strong affinity of the Canary Islands seaweed flora with the warm-temperate Mediterranean-Atlantic region. Some species are recorded for the first time from the east coast of the Atlantic Ocean, enhancing the biogeographic relations of the Canarian marine flora with that of the western Atlantic regions.