The XcpR protein of Pseudomonas aeruginosa dimerizes via its N-terminus

Extracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild-type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino-terminal portion of the cI repressor protein of phage λ was used as a reporter of dimerization in Escherichia coli following fusion to full-length as well as a truncated form of XcpR. The cI–XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by λ phage. The full-length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cI–XcpR protein. The disruption of cI–XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N-terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N-terminus and that this dimerization is essential for extracellular protein secretion.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

Reintroducing the European Beaver to Britain: nostalgic meddling or restoring biodiversity?

The European Beaver Castor fiber once occurred throughout Europe, but in many countries was exterminated or greatly reduced by over-hunting. In the UK, Beavers were last recorded in Scotland in the sixteenth century. Thirteen countries have carried out reintroduction programmes to restore the range of the Beaver in Europe, We provide a basis for discussing the feasibility and desirability of reintroducing the Beaver to Britain. The basic biology of the Beaver is described, followed by summaries of reintroductions in Europe and an evaluation of their successes and failures. We address the fundamental questions of propagule size, habitat requirements, habitat size and provenance of Beavers to be released, before examining in theory whether reintroducing Beavers to Britain is likely to fulfil UKCINC and IUCN criteria relating to environmental impact, socioeconomics and conservation. We then make suggestions as to how a reintroduction to Britain could proceed. Essential work outstanding includes site assessment and public consultation. The restoration of the Beaver to Britain could be a fitting start to the millennium.     

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.     

The effect of lipopolysaccharide desensitization on the regulation of in vivo induction of immunologic tolerance and antibody production and in vitro release of IL-1

As previously reported, LPS and 8-derivatized guanosine (both generators of IL-1 release), as well as IL-1 itself interfere with the in vivo induction of tolerance to DHGG in A/J mice. In the present studies it was demonstrated that desensitization of either A/J or CBA/CaJ mice with LPS aborts the ability of LPS to interfere with the induction of tolerance to DHGG. The abrogation of the ability of LPS to interfere with tolerance by LPS desensitization is not the result of neutralization of LPS by antibody produced to LPS during desensitization. Desensitization with LPS also aborts the interference with tolerance induction by 7-methyl-8-oxoguanosine. LPS desensitization inhibits the ability of LPS and/or 7-methyl-8-oxoguanosine to both convert a tolerogenic signal to an immunogenic signal and interfere with the induction of a tolerant state to a subsequent injection of Ag. The effects resulting from desensitization may be in part attributed to the depletion of IL-1. LPS desensitization also modulates the antibody response to injection of the AG, AHGG. Desensitization with LPS markedly suppresses the antibody response to a subsequent injection of AHGG in CBA/CaJ mice. Desensitization with LPS also inhibits the anti-HGG antibody response in A/J mice, but in this strain its effect is dependent on the route of injection of AHGG. In an experiment directly comparing the responses of normal and desensitized A/J mice to either intravenous or intraperitoneal injection of AHGG, desensitization only suppressed the response in mice injected with AHGG i.p.. Desensitization with LPS also inhibits the ability of LPS to act as an adjuvant in a subsequent antibody response to AHGG. Not only does desensitization interfere with the primary antibody response to AHGG, but it also interferes with the secondary response, suggesting that the primary injection after desensitization induces a state of immunologic tolerance.     

A stereotaxic atlas and implantation technique for nuclei of the diencephalon of Atlantic salmon (Salmo salar) parr.

A stereotaxic apparatus and technique for its implantation in diencephalic nuclei of Atlantic salmon parr of 20 to 30 g body weight is described. An atlas of nuclei in the diencephalon is also presented.     

Inhibitory effect of aspirin on cholera toxin-induced phospholipase and cyclo-oxygenase activity

Cholera toxin (CT) stimulated phospholipase activity and caused [3H]arachidonic acid (3H-AA) release in a murine macrophage/monocyte cell line. Pretreatment of cells with dexamethasone, a phospholipase A2 (PLA2) inhibitor, did not affect CT-induced 3H-AA release. In contrast, aspirin, which is an inhibitor of phospholipase C (PLC), blocked CT-induced 3H-AA release and subsequent prostaglandin (PC) synthesis. The inhibitory effect of aspirin was dose dependent, with 4 mM reducing the CT response by approximately 50%. Similarly, inhibition was time dependent, occurring when the drug was added to the culture medium as late as 30 min after CT. Brief exposure (30 min) of the cells to aspirin did not alter their subsequent response to CT, but 3H-AA release from cells exposed to aspirin for 2.5 h was irreversibly inhibited. The data suggested that CT stimulation of AA metabolism may involve increased PLC activity.