Monoclonal Antibodies Allow Precipitation of Esterasic but Not Peptidasic Activities Associated with Butyrylcholinesterase

Commercially available and affinity-purified butyrylcholinesterases isolated from human serum were examined for their esterasic activity and their ability to hydrolyze various neuropeptides, including neurotensin, substance P, and leucine-enkephalin. The three pools that displayed the lowest esterasic activities were shown to hydrolyze neurotensin with the same HPLC degradative pattern. By contrast, noticeable qualitative and quantitative discrepancies were observed when hydrolyses of substance P and leucine-enkephalin by these three butyrylcholinesterase pools were studied. The pool that exhibited the highest esterasic activity appeared to be homogeneously constituted by 90- and 180-kDa protein bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and was totally unable to hydrolyze these three neuropeptides. This suggested that the three other butyrylcholinesterase preparations could be contaminated by exogenous peptidases. This was confirmed by means of three distinct monoclonal antibodies directed toward human serum butyrylcholinesterase. The three IgG-purified fractions precipitated the esterasic activity, whereas they failed to precipitate the neuropeptide-hydrolyzing activities whatever the substrate examined. Altogether, these results demonstrate that peptidases associated with butyrylcholinesterase are contaminating enzymes that cannot be considered as intrinsic activities of this enzyme.     

Aspects of neural plasticity in the central nervous system—III. Methodological studies on the microdialysis technique

Some methodological aspects of the intracerebral microdialysis technique have been investigated: the existence of a pressure gradient at the level of the dialyzing membrane, the substance diffusion from the microdialysis probe and the extent of tissue damage induced by the implantation of the microdialysis probe. At the level of the dialyzing membrane a rough balance between the pressure inside the probe and the one present in the extracellular fluid compartment has been observed. The pattern of substance diffusion in the tissue showed a large variability depending on the substance used and the experimental conditions. Relevant deductions can be made by the use of labeled markers. By means of this approach, the diffusion pattern of tritiated ganglioside GM1 in the tissue around the probe could be shown to follow a biexponential pattern, suggesting a two-step process of diffusion. The degree of tissue damage induced by the microdialysis probe was assessed by analyzing the glial reaction, and was measured by means of semiquantitative immunocytochemistry of glial fibrillary acidic protein immunoreactivity. Only a limited area of neuronal damage was observed in the region surrounding the microdialysis probe. The amount of glial reaction after probe implantation was shown to be comparable with that induced by the implantation of a microinjection cannula.     

Biologically active interleukin 1 in human eccrine sweat: site-dependent variations in alpha/beta ratios and stress-induced increased excretion

Human eccrine sweat devoid of epidermal protein contamination was collected from palms, soles, and different sites on the trunk. Interleukin 1 alpha (IL 1 alpha) and interleukin 1 beta (IL 1 beta) content were analyzed for immunoreactivity by enzymo-immunoassay and immunoblotting and for bioactivity by the stimulation of prostaglandin E2 (PGE2) production in human dermal fibroblasts. The bioassay was validated by using blocking antibodies against IL 1 alpha and beta. All sweat samples were found to contain significant amounts of immunoreactive and biologically active IL 1. The immunoreactive forms were at 17 kDa as shown by immunoblotting analysis, indicating that they were mature (secreted), undegraded IL 1 peptides. Whereas IL 1 alpha was detectable in sweat samples obtained from both truncal and palmo-plantar regions, IL 1 beta was only detectable in the sweat of palms and soles (IL 1 alpha/beta ratio greater than 700 in trunk and 5.4 in palms and soles) indicating a site-dependent difference in the excretion of the two IL 1 molecules. IL 1 concentration was high in spontaneous (IL 1 alpha, 3.7; IL 1 beta, 0.3 ng/mL) and pilocarpine induced sweat (IL 1 alpha, 3.9; IL 1 beta, 1.2 ng/mL), and it was much increased during jogging and sauna (IL 1 alpha, 22.6; IL 1 beta, 3.3 ng/mL). This does not appear to represent an excretory process aimed at clearing blood IL 1, but rather a stress-induced increased production of IL 1 by sweat gland cells.     

Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores ofPhytophthora cinnamomi

Summary The role of tubular mastigonemes in the reversal of thrust of the anterior flagellum ofPhytophthora cinnamomi was analysed using mastigoneme-specific monoclonal antibodies and immunoflu-orescence and video microscopy. Exposure of live zoospores ofP. cinnamomi to the mastigoneme-specific Zg antibodies caused alterations in the arrangement of mastigonemes on the flagellar surface and at Zg concentrations above 0.3 /ml, mastigonemes became detached from the flagellum. As a consequence of antibody binding to the mastigonemes there were concentration-dependent perturbations in zoospore swimming behaviour and anterior flagellum beat pattern. With increasing antibody concentration zoospores swam more slowly and other parameters of their swimming pattern, such as the wavelength of the swimming helix and the frequency of rotation, were also reduced. The effects of Zg antibodies were specific at two levels: control immunoglobulins or antibodies that bound to other flagellar surface components did not have an effect on motility, and Zg antibodies did not interfere with the motility of zoospores of oomycete species to which they did not bind. The effects of antibody-induced disruption of mastigoneme arrangement strongly support previous hypotheses that tubular mastigonemes are responsible for thrust reversal by the anterior flagellum, enabling it to pull the cell through the surrounding medium.     

Unusual conformational preferences of β-alanine containing cyclic peptides. VII

In the present paper we describe the synthesis, purification, and single crystal x-ray analysis of the cyclic pentapeptide cyclo-(Pro-Phe-Phe-β-Ala-β-Ala). This compound crystallizes in the orthorhombic space group P2₁2₁2₁ from methanol and adopts in the solid state an unusual conformation characterized by a cis β-Ala⁵-Pro¹ peptide bond and by an intramolecular hydrogen bond stabilizing a C₁₁- and a C₁₂-ring structure. The C₁₁, structure contains the Phe³ and the β-Ala⁴ at the corner position of the turn; it is the first observation of a type II β-turn enlargement due to the insertion of an extra methylene group of the β-alanine residue. The rest of the molecule participates in a newly characterized C₁₂-ring structure, which incorporates a β-Ala residue at position i of the turn. © 1996 John Wiley & Sons, Inc.     

Preliminary evidence of endotoxic activity of Bilophila wadsworthia

In this study we describe two properties of the Gram-negative bacterium Bilophila wadsworthia, namely the ability to clot Limulus lysate and the capacity to induce the production of tissue factor-like procoagulant activity by human mononuclear cells in vitro. Although exhibited at a lower degree when compared with those of typical Gram-negative bacteria or Gram-negative endotoxin those activities may account in part for Bilophila's pathogenicity. The capacity indeed to induce fibrin formation through the interaction with mononuclear cells suggests one mechanism by which the microorganism might cause abscess formation in the host. Moreover, since this activity is dependent on the number of Bilophila interacting with mononuclear cells, we hypothesize that this biological activity is closely influenced by growth environment.     

A model and lexicon for pollen fate

As pollination biology undergoes unprecedented growth as a discipline, confusion in the use of terms has become increasingly common. The need for a flexible yet unambiguous terminology has become urgent. As an example we discuss how the term “pollination efficiency” is used differently by 18 studies, and “pollinator effectiveness” by seven others. Here we present flowcharts of two general models of pollination systems (biotic and abiotic) that trace all the events from pollen production to development of seed or fruit, and we develop a lexicon for the quantities of pollen, processes of transfers (to a vector, to a stigma), and ratios of quantities that are of interest in studies of pollination and mating systems. An appendix includes a glossary of the definitions we suggest.     

Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny

The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.     

Intracellular aminopeptidases inStreptomyces lividans 66

Summary We have investigated the aminopeptidase activities present inStreptomyces lividans strains. The majority of these activities proved to be intracellular with multiple active species. Two aminopeptidase P genes were identified to be responsible for the ability to hydrolyze amino terminal peptide bonds adjacent to proline residues. Two other broad spectrum aminopeptidases were found to display homology at both the DNA and protein levels. One showed significant homology to PepN proteins, particularly around the putative zinc-binding residues which are important for catalysis. The second broad spectrum activity was not analyzed in detail but showed a different spectrum of substrate specificity to that of PepN.