Iron absorption by CaCo 2 cells cultivated in serum-free medium as in vitro model of the human intestinal epithelial barrier

A cell culture system consisting of confluent monolayer of human enterocyte-like CaCo 2 cells, cultivated in a serum-free nutritive medium, on microporous synthetic membranes has been used as an in vitro model of the intestinal epithelial barrier. The uptake of ⁵⁵ferric citrate, as well as the transepithelial passage from the apical to the basolateral pole, have been studied. CaCo 2 cells accumulate iron in a time- and concentration-dependent process, largely specific from the apical pole. When ⁵⁵ferric citrate is added at the apical pole, radioiron appears at the basal pole and the clearance rate is ～four times higher than in the opposite direction; the amounts of ⁵⁵Fe increase with the concentration in iron citrate and the duration of incubation. At least two concurrent mechanisms could be involved in iron absorption across monolayers of CaCo 2 cells. A first route would correspond to a paracellular passage of the metal from the apical to the basal pole. The second route would involve a selective intake of iron at the apical pole and could require a reduction of ferric iron, prior to the entry. Iron accumulated by the cells would, for a minor part, be stored within ferritin, whereas the major part would be excreted at the basolateral pole, either as low molecular weight material of undetermined chemical composition but from which iron is easily mobilized by apotransferrin or associated with neosynthesized apotransferrin. Vesicular transport and protein synthesis seem to be required. © 1994 Wiley-Liss, Inc.     

Immature stages of the bee fly Ligyra satyrus (F.) (Diptera: Bombyliidae): A hyperparasitoid of canegrubs (Coleoptera: Scarabaeidae)

We describe the third- and fourth-instar larva, pupa and biology of the hyperparasitoid bee fly Ligyra satyrus (Fabricius). The larval and pupal morphology of the bee fly is typical for members of the subfamily Anthracinae. The bee fly larvae are found inside cocoons of the scoliid Campsomeris tasmaniensis Saussure, an external parasite of canegrubs (Coleoptera: Scarabaeidae) in sugarcane fields at Ayr and Bundaberg, Queensland. Ligyra satyrus has been recorded attacking only C. tasmaniensis . Ligyra and its relatives are all parasites of predatory and parasitic, ground-nesting aculeate Hymenoptera such as Sphecidae, Pompilidae, Tiphiidae and Scoliidae. Larval morphology of the hyperparasite is similar to other ectoparasitic Bombyliidae and pupal morphology is compared to that of species in the same and related subfamilies. Rates of hyperparasitism at Gordonvale reported in the early 1900s are relatively high, but our results show that currently they are very low at Ayr and Bundaberg. These results suggest that the impact of the bee fly on natural control of the canegrubs by scoliids varies considerably.     

Ethogram of Abdopus aculeatus (d'Orbigny, 1834) (Cephalopoda: Octopodidae): Can behavioural characters inform octopodid taxomony and systematics?

An ethogram is provided for the small, intertidal, diurnal octopusAbdopus aculeatus (d'Orbigny, 1834). Information is based primarilyon in situ observations of adults, and supplemented with photographsof animals in aquaria. Aspects of the dymantic display, matingsystem, activity patterns, and habitat use appear similar tothose expressed by other members of Abdopus, as well as thelarge sister taxon Octopus cyanea, suggesting that these behavioursmay be conserved throughout the evolution of these octopuses.Many skin components are also shared with Octopus bimaculoides,which may reflect either an evolutionary affinity with thisoctopus, or convergence in these characters. If behaviouralunits such as those documented here are compiled in a consistentmanner for other species, then they may facilitate taxonomicidentification, as well as future evolutionary studies of octopodids.     

Song ranging by incubating male Blue-headed Vireos: the importance of song representation in repertoires and implications for song delivery patterns and local/foreign dialect discrimination

ABSTRACT.   Mechanisms used by birds to range their distance from singing conspecifics are being debated. In particular, the idea that an incoming song must be in a bird's repertoire for it to be ranged accurately is controversial, but important to our appreciation of the role ranging plays in song evolution. We tested the relation between ranging accuracy and songs in repertoires in playback experiments to male Blue-headed Vireos ( Vireo solitarius ) whose precise locations were known because they were incubating eggs. Males ranged songs heard while incubating and, when their mates relieved them at the nest, flew directly to the silent playback sites, suggesting that they remembered the locations of simulated intruders. Male vireos approached playback sites of local songs, likely in their own repertoires, more precisely than foreign songs recorded 95–645 km from our study site. Songs included in local and foreign playback tapes differed primarily in frequency modulation, but were similar in other measurements. These results support ranging theory as described by Morton (1986) . If the songs within an individual's repertoire are ranged with greater accuracy, we discuss how the stability of neighborhoods becomes a factor as to whether or not selection will favor repertoire sharing in song evolution. As well, singing style is affected by ranging. Because Blue-headed Vireos present their songs in a stereotyped order, a listener can compare ordered sequential changes in signal degradation. Comparing degradation in a sequence of songs adds a temporal element that should result in more accurate ranging of the singer's location.     

Alpha-proteobacterial symbionts of marine bryozoans in the genus Watersipora

Bacterial symbionts that resembled mollicutes were discovered in the marine bryozoan Watersipora arcuata in the 1980s. In this study, we used PCR and sequencing of 16S rRNA genes, specific fluorescence in situ hybridization, and phylogenetic analysis to determine that the bacterial symbionts of "W. subtorquata" and "W. arcuata" from several locations along the California coast are actually closely related alpha-Proteobacteria, not mollicutes. We propose the names "Candidatus Endowatersipora palomitas" and "Candidatus Endowatersipora rubus" for the symbionts of "W. subtorquata" and "W. arcuata," respectively.     

A surface-focused biotinylation procedure identifies the Yersinia pestis catalase KatY as a membrane-associated but non-surface-located protein

This study identified major surface proteins of the plague bacterium Yersinia pestis. We applied a novel surface biotinylation method, followed by NeutrAvidin (NA) bead capture, on-bead digestion, and identification by liquid chromatography-tandem mass spectrometry (LC-MS-MS). The use of stachyose during biotinylation focused the reaction to the surface. Coupled with NA pulldown and immunoblot analysis, this method determined whether a protein was accessible to the surface. We applied the method to test the hypothesis that the catalase KatY is a surface protein of the plague bacterium Y. pestis. A rabbit serum recognized the catalase KatY as a major putative outer membrane-associated antigen expressed by Y. pestis cells grown at 37 degrees C. Similar findings by other groups had led to speculations that this protein might be exposed to the surface and might be a candidate for evaluation as a protective antigen for an improved plague vaccine. KatY was obtained only in the total membrane fraction, and stachyose greatly reduced its biotinylation as well as that of the periplasmic maltose binding protein, indicating that KatY is not on the bacterial surface. LC-MS-MS analysis of on-bead digests representing ca. 10(9) cells identified highly abundant species, including KatY, Pal, and OmpA, as well as the lipoprotein Pcp, all of which bound in a biotin-specific manner. Pla, Lpp, and OmpX (Ail) bound to the NA beads in a non-biotin-specific manner. There was no contamination from abundant cytoplasmic proteins. We hypothesize that OmpX and Pcp are highly abundant and likely to be important for the Y. pestis pathogenic process. We speculate that a portion of KatY associates with the outer membrane in intact cells but that it is located on the periplasmic side. Consistent with this idea, it did not protect C57BL/6 mice against bubonic plague.     

Computational definition of a mixed valent Fe(II)Fe(I) model of the [FeFe]hydrogenase active site resting state

Density-functional calculations have been used to examine the electronic structure and bonding in the recently reported complex [(PMe(3))(CO)(2)Fe(mu-pdt)(mu-CO)Fe(CO)(IMes)](+) (1(+), IMes=1,3-bis(2,4,6-trimethylphenyl)-imidazol-2-ylidene). This mixed valent Fe(II)Fe(I) complex features a rotated geometry that places a carbonyl ligand in a semi-bridging position, which makes it an accurate model of the S =(1/2) resting state of the [FeFe]-hydrogenase active site. Calculations indicate that the unpaired electron in this complex lies almost entirely on the rotated iron center, implying that this iron remains in the Fe(I) oxidation state, while the unrotated iron has been oxidized to Fe(II). The frontier molecular orbitals in 1(+) are compared with those in the neutral Fe(I)Fe(I) precursor (PMe(3))(CO)(2)Fe(mu-pdt)(mu-CO)Fe(CO)(IMes) at both its optimized geometry (1) and constrained to a rotated geometry (1(rot)). These theoretical results are used to address the role of the bridging CO ligand in 1(+) and to predict reactivity patterns; they are related back to the intricate biological mechanism of [FeFe]-hydrogenase.     

Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA

Plasmid DNA for biopharmaceutical applications is mainly produced in E. coli cells. The first and most crucial step for recovering the plasmid is the cell lysis. Governed by the physico-chemical properties of the polynucleotide, alkaline lysis has been the lysis-method of choice. This chemical disintegration technique was initially developed for the lab scale and non-pharmaceutical applications. A continuous, fully automated and closed system combining alkaline lysis, neutralization and clarification in one gentle and generic operation was developed. This system consists of a three units. One unit controls mixing and contact time during the alkaline treatment, another one controls the neutralization and the concurrent formation of flocs and a third one the separation of flocs and pDNA containing lysate. Based on optimization experiments the selected process parameters resulted in yields up to 100% and homogeneities comparable to that obtained by gentle manual lysis. The process does not need enzymes and it is scalable and routinely used for cGMP-production of pharmaceutical grade plasmid DNA from 200 L fermentations.     

Redox regulation of chloroplast enzymes in Galdieria sulphuraria in view of eukaryotic evolution

Redox modulation is a general mechanism for enzyme regulation, particularly for the post-translational regulation of the Calvin cycle in chloroplasts of green plants. Although red algae and photosynthetic protists that harbor plastids of red algal origin contribute greatly to global carbon fixation, relatively little is known about post-translational regulation of chloroplast enzymes in this important group of photosynthetic eukaryotes. To address this question, we used biochemistry, phylogenetics and analysis of recently completed genome sequences. We studied the functionality of the chloroplast enzymes phosphoribulokinase (PRK, EC 2.7.1.19), NADP-dependent glyceraldehyde 3-phosphate dehydrogenase (NADP-GAPDH, GapA, EC 1.2.1.13), fructose 1,6-bisphosphatase (FBPase, EC 3.1.3.11) and glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49), as well as NADP-malate dehydrogenase (NADP-MDH, EC 1.1.1.37) in the unicellular red alga Galdieria sulphuraria (Galdieri) Merola. Despite high sequence similarity of G. sulphuraria proteins to those of other photosynthetic organisms, we found a number of distinct differences. Both PRK and GAPDH co-eluted with CP12 in a high molecular weight complex in the presence of oxidized glutathione, although Galdieria CP12 lacks the two cysteines essential for the formation of the N-terminal peptide loop present in higher plants. However, PRK inactivation upon complex formation turned out to be incomplete. G6PDH was redox modulated, but remained in its tetrameric form; FBPase was poorly redox regulated, despite conservation of the two redox-active cysteines. No indication for the presence of plastidic NADP-MDH (and other components of the malate valve) was found.