Mapping of a hapten-binding site: molecular modeling and site-directed mutagenesis study of an anti-atrazine antibody

A three-dimensional model of the variable domain of the atrazine-specific Fab fragment K411B was constructed by molecular modeling using known structures of highly homologous immunoglobulins as templates. Molecular dynamic simulations and cross-reactivity data were used to predict residues responsible for the binding of the hapten 4-chloro-6-(isopropylamino)-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (iPr/Cl/C6) instead of atrazine. Specific binding pockets could be defined for the chlorine, the isopropylamino group and the C6-spacer of the hapten. The influence of various amino acids on hapten binding was investigated by site-directed mutagenesis, and the effect of these mutations was analyzed by capture ELISA using the hapten iPr/Cl/C6 and 4-amino-6-chloro-1,3,5-triazine-2-(6-aminohexanecarboxylic acid) (H/Cl/C6). GlyH100a seems to be important in determining the conformation of the heavy-chain complementarity determining region H3; replacing it with any other residue prevented the binding of the hapten. Altering residues responsible for the binding of the chlorine atom (TrpH33, GluH50 and TyrL96) decreased the affinity significantly. Hapten-spacer recognition can be attributed to the interaction with PheL32; replacing PheL32 by leucine reduced the affinity towards iPr/Cl/C6. A triple mutant Fab fragment (GlnL89Glu, ValH37Ile and GluL3Val) showed an affinity 5-fold greater towards iPr/Cl/C6 compared to the wild-type K411B, as a result of better recognition of the isopropylamino group of iPr/Cl/C6.     

Immunocytochemical localization of a diuretic hormone of the beetle Tenebrio molitor,Tenmo-DH(37), in nervous system and midgut

Although the mealworm Tenebrio molitor inhabits very dry environments, it has at least two diuretic peptides, which increase fluid secretion by the free portions of the Malpighian tubules. Unlike other insect corticotropin-releasing factor (CRF)-related peptides isolated to date, these are non-amidated peptides. The immunocytochemical localization of Tenmo-DH(37) was investigated using antisera raised against this hormone. Immunoreactive neurosecretory cells were found in the brain and abdominal ganglia with immunoreactive processes projecting to the peripheral nervous system. Intense staining of the neurohaemal release site, the corpora cardiaca, was observed. In addition, neurosecretory cells immunoreactive to Tenmo-DH(37) were found in the posterior midgut and a network of immunoreactive nerve processes extended over the surface of the midgut. Tenmo-DH(37) is widely distributed and its staining pattern resembles that found for other, amidated CRF-related diuretic peptides.     

Combining theoretical and experimental approaches to understand the circadian clock

This review is intended as a summary of our work carried out as part of the German Research Association (DFG) Center Program on Circadian Rhythms. Over the last six years, our approach to understanding circadian systems combined theoretical and experimental tools, and Gonyaulax and Neurospora have proven ideal for these efforts. Both of these model organisms demonstrate that even simple circadian systems can have multiple light input pathways and more than one rhythm generator. They have both been used to elaborate basic circadian features in conjunction with formal models. The models introduce the “zeitnehmer,” i.e., a clock-regulated input pathway, to the conceptual framework of circadian systems, and proposes networks of individual feedbacks as the basis for circadian rhythmicity.     

Isolation,identification and localization of a second beetle antidiuretic peptide

We isolated from head extracts of Tenebrio molitor a peptide that inhibits fluid secretion by the Malpighian tubules of this insect. This second antidiuretic factor, ADFb, like the previously published ADFa, works through cyclic GMP as a second messenger. It has primary structure Tyr-Asp-Asp-Gly-Ser-Tyr-Lys-Pro-His-Ile-Tyr-Gly-Phe-OH with an EC(50) of approximately 240 pM in a fluid secretion assay. This peptide is now the second sequenced endogenous insect ADF which inhibits Malpighian tubule fluid secretion. Immunohistochemical techniques show that the peptide is localized in the brain; it appears to be produced mainly in two pairs of bilaterally symmetrical cells in the protocerebrum.     

Diversification of cardiac insulin signaling involves the p85 alpha/beta subunits of phosphatidylinositol 3-kinase

Ventricular cardiomyocytes and cardiac tissue of lean and genetically obese (fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 alpha and p85 beta for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these mechanisms for cardiac insulin resistance. Western blot analysis of cardiomyocyte lysates revealed expression of p85 alpha and p85 beta but no detectable amounts of the splice variants of p85 alpha. Essentially no p85 alpha subunit of PI 3-kinase was found to be associated with insulin receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in p85 beta associated with IRS-1, leading to a three- to fourfold increase in p85 beta-associated PI 3-kinase activity. This response was significantly reduced in obese animals. Comparable results were obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 +/- 0.7-fold over basal) of p85 alpha was observed after insulin stimulation of lean animals, with no significant effect in the obese group. No p85 beta could be detected in GLUT-4-containing vesicles. Recruitment of the p110 catalytic subunit of PI 3-kinase and a twofold increase in enzyme activity in GLUT-4-containing vesicles by insulin was observed only in lean rats. We conclude that, in the heart, p85 alpha recruits PI 3-kinase activity to GLUT-4 vesicles, whereas p85 beta represents the main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation. Furthermore, multiple defects of PI 3-kinase activation, involving both the p85 alpha and the p85 beta adaptor subunits, may contribute to cardiac insulin resistance.     

Flow cytometric determination of E-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers

BACKGROUND: Endothelial cell adhesion molecules are involved in initiation and progression of vascular diseases. The purpose of this study was to determine conditions of fixation and dissociation of human umbilical vein endothelial cell (HUVEC) monolayers that permit a reliable flow cytometric determination of intracellular and surface content of E-selectin, vascular cell adhesion molecule-1 (VCAM-1), and intercellular adhesion molecule-1 (ICAM-1). METHODS: TNFalpha-treated HUVEC monolayers were fixed with 0.5% formaldehyde at the end of the experimental incubation. Subsequently, either the monolayer was trypsinized and thereafter the cells were subjected to indirect fluorescence labeling or the monolayer was first labeled and then dissociated by trypsinization. Cell integrity was assessed by vimentin staining. Total adhesion molecule content was detected in saponin-permeabilized cells. RESULTS: HUVEC integrity was maintained when the fixation time of the monolayer did not exceed 5 min and trypsin/EDTA was used for dissociation. Surface adhesion molecules were partially hydrolyzed by trypsin when trypsinization preceded labeling but antibody binding protected adhesion molecules from degradation. VCAM-1 and E-selectin exhibited substantial trypsin-sensitive surface fractions but surface ICAM-1 was mainly trypsin resistant. Permeabilization with 0.06% saponin allowed the detection of considerable intracellular pools of the investigated adhesion molecules. CONCLUSIONS: The described method permits the reliable determination of surface and intracellular fractions of adhesion molecules in formaldehyde-fixed HUVEC monolayers and may be used for studies on the regulation of adhesion molecule expression.     

Insect larvae contain substances toxic to adults: the discovery of paralysins

Acidic methanolic extracts of larvae of nine different insect species were found to contain substances that cause a lethal effect in the adult stage of the same species and of other species. These endogenous toxic substances, apparently being widely spread over the class of insects, were designated as paralysins, because of their immediate and observable paralytic effect upon injection. The developmental concentration curves of five different species of insects (Galleria mellonella (Lepidoptera), Neobellieria bullata (Diptera), Spodoptera frugiperda (Lepidoptera), Tenebrio molitor (Coleoptera) and Schistocerca gregaria (Orthoptera) indicate that the toxins are not present throughout all the developmental stages in the same concentration. The strongest paralytic activity was found in late instar larvae or in the early pupal stage. The temporal distribution of paralysins during development suggests that they might be involved in metamorphosis.     

Bioactive peptides,networks and systems biology

Bioactive peptides are a group of diverse intercellular signalling molecules. Almost half a century of research on this topic has resulted in an enormous amount of data. In this essay, a general perspective to interpret all these data will be given. In classical endocrinology, neuropeptides were thought of as simple signalling molecules that each elicit one response. However, the fact that the total bioactive peptide signal is far from simple puts this view under pressure. Cells and tissues express many different bioactive peptides and they are also able to respond to many different bioactive peptides, indicating that multiple receptors and signal transduction pathways are present in a single cell. Therefore, the authors suggest that the bioactive peptide signalling system should be regarded in the context of network and systems biology. Bioactive peptides can best be viewed as an extension of the protein interaction network that allows regulating and fine‐tuning the metabolism of the different cells and tissues in the body. The cell thus responds to the ‘peptidome’ instead of to a single peptide. The intracellular part of this signalling network consists of the various signalling transduction cascades. Recently, new systems biology approaches have emerged for the modelling of cell signalling. The network and systems biology approach is also able to shed new light on the evolution of intercellular signalling.