Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor

Two synthetic analogues of murine epidermal. growth factor, [Abu6, 20] mEGF4-48 (where Abu denotes amino-butyric acid) and [G1, M3, K21, H40] mEGF1-48, have been investigated by NMR spectroscopy. [Abu6, 20] mEGF4-48 was designed to determine the contribution of the 6-20 disulfide bridge to the structure and function of mEGF The overall structure of this analogue was similar to that of native mEGF, indicating that the loss of the 6-20 disulfide bridge did not affect the global fold of the molecule. Significant structural differences were observed near the N-terminus, however, with the direction of the polypeptide chain between residues four and nine being altered such that these residues were now located on the opposite face of the main beta-sheet from their position in native mEGF Thermal denaturation experiments also showed that the structure of [Abu6, 20] mEGF4-48 was less stable than that of mEGF. Removal of this disulfide bridge resulted in a significant loss of both mitogenic activity in Balb/c 3T3 cells and receptor binding on A431 cells compared with native mEGF and mEGF4-48, implying that the structural changes in [Abu6, 20] mEGF4-48, although limited to the N-terminus, were sufficient to interfere with receptor binding. The loss of binding affinity probably arose mainly from steric interactions of the dislocated N-terminal region with part of the receptor binding surface of EGF [G1, M3, K21, H40] mEGF1-48 was also synthesized in order to compare the synthetic polypeptide with the corresponding product of recombinant expression. Its mitogenic activity in Balb/c 3T3 cells was similar to that of native mEGF and analysis of its H-1 chemical shifts suggested that its structure was also very similar to native.

Differentiation of the mononuclear phagocyte system during mouse embryogenesis: The role of transcription factor PU.1

During mouse embryogenesis, macrophage-like cells arise first in the yolk sac and are produced subsequently in the liver. The onset of liver hematopoiesis is associated with the transition from primitive to definitive erythrocyte production. This report addresses the hypothesis that a similar transition in phenotype occurs in myelopoiesis. We have used whole mount in situ hybridization to detect macrophage-specific genes expressed during mouse development. The mouse c-fms mRNA, encoding the receptor for macrophage colony-stimulating factor (CSF-1), was expressed on phagocytic cells in the yolk sac and throughout the embryo before the onset of liver hematopoiesis, Similar cells were detected using the mannose receptor, the complement receptor (CR3), or the Microphthalmia transcription factor (MITF) as mRNA markers. By contrast, other markers including the F4/80 antigen, the macrophage scavenger receptor, the S-100 proteins, S100A8 and S100A9, and the secretory product lysozyme appeared later in development and appeared restricted to only a subset of c-fms-positive cells. Two-color immunolabeling on disaggregated cells confirmed that CR3 and c-fms proteins are expressed on the same cells. Among the genes appearing later in development was the macrophage-restricted transcription factor, PU.1, which has been shown to be required for normal adult myelopoiesis. Mice with null mutations in PU.1 had normal numbers of c-fms-positive phagocytes at 11.5dpc. PU.1(-/-) embryonic stem cells were able to give rise to macrophagelike cells after cultivation in vitro. The results support previous evidence that yolk sac-derived fetal phagocytes are functionally distinct from those arising in the liver and develop via a different pathway. (C) 1999 by The American Society of Hematology.

The plasma membrane calcium pump, its role and regulation: New complexities and possibilities

Significant progress has been achieved in elucidating the role of the plasma membrane Ca2+-ATPase in cellular Ca2+ homeostasis and physiology since the enzyme was first purified and physiology since the enzyme was first purified and cloned a number of years ago. The simple notion that the PM Ca2+-ATPase controls resting levels of [Ca2+](CYT) has been challenged by the complexity arising from the finding of four major isoforms and splice variants of the Ca2+ pump, and the finding that these are differentially localized in various organs and subcellular regions. Furthermore, the isoforms exhibit differential sensitivities to Ca2+, calmodulin, ATP, and kinase-mediated phosphorylation. The latter pathways of regulation can give rise to activation or inhibition of the Ca2+ pump activity, depending on the kinase and the particular Ca2+ pump isoform. Significant progress is being made in elucidating subtle and more profound roles of the PM Ca2+-ATPase in the control of cellular function. Further understanding of these roles awaits new studies in both transfected cells and intact organelles, a process that will be greatly aided by the development of new and selective Ca2+ pump inhibitors. (C) 1999 Elsevier Science Inc.

The moderating role of task characteristics in determining responses to a stressful work simulation

Two experimental studies were conducted to examine whether the stress-buffering effects of behavioral control on work task responses varied as a function of procedural information. Study 1 manipulated low and high levels of task demands, behavioral control, and procedural information for 128 introductory psychology students completing an in-basket activity. ANOVA procedures revealed a significant three-way interaction among these variables in the prediction of subjective task performance and task satisfaction. It was found that procedural information buffered the negative effects of task demands on ratings of performance and satisfaction only under conditions of low behavioral control. This pattern of results suggests that procedural information may have a compensatory effect when the work environment is characterized by a combination of high task demands and low behavioral control. Study 2 (N = 256) utilized simple and complex versions of the in-basket activity to examine the extent to which the interactive relationship among task demands, behavioral control, and procedural information varied as a function of task complexity. There was further support for the stress-buffering role of procedural information on work task responses under conditions of low behavioral control. This effect was, however, only present when the in-basket activity was characterized by high task complexity, suggesting that the interactive relationship among these variables may depend on the type of tasks performed at work. Copyright (C) 1999 John Wiley & Sons, Ltd.

A role for protein kinase B beta/Akt2 in insulin-stimulated GLUT4 translocation in adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKB beta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKB alpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKB beta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKB beta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKB beta in insulin-stimulated glucose transport in adipocytes.

Monooxygenases play only a minor role in resistance to synthetic pyrethroids in the cattle tick, Boophilus microplus

We investigated the role of monooxygenases in resistance to synthetic pyrethroids (SPs) in the cattle tick, Boophilus microplus. We found that monooxygenases play only a minor role in resistance to SPs in both resistant and susceptible strains of B. microplus. We blocked the monooxygenases with piperonyl butoxide (PBO) and simultaneously applied the SPs, flumethrin and cypermethrin to larval B. microplus. PBO increased the effect of flumethrin (synergism ratios 2.7-8.9) more than it increased the effect of cypermethrin (synergism ratios 1.9-3.1). Of the four strains tested, Parkhurst, which is resistant to SPs, was the least affected by the addition of PBO (synergism ratios after cypermethrin was applied 1.9; after flumethrin 2.7) whereas N.R.F.S., the strain susceptible to SPs, was the most affected by synergism between PBO and SPs (synergism ratio after cypermethrin was applied 3.1; after flumethrin 8.9). We hypothesize that B. microplus lacks monooxygenases capable of conferring resistance to SPs because it and its recent ancestors were blood-feeders rather than herbivores.

The role of carbon surface chemistry in N2O conversion to N2 over Ni catalyst supported on activated carbon

The effect of acidic treatments on N2O reduction over Ni catalysts supported on activated carbon was systematically studied. The catalysts were characterized by N-2 adsorption, mass titration, temperature-programmed desorption (TPD), and X-ray photoelectron spectrometry (XPS). It is found that surface chemistry plays an important role in N2O-carbon reaction catalyzed by Ni catalyst. HNO3 treatment produces more active acidic surface groups such as carboxyl and lactone, resulting in a more uniform catalyst dispersion and higher catalytic activity. However, HCl treatment decreases active acidic groups and increases the inactive groups, playing an opposite role in the catalyst dispersion and catalytic activity. A thorough discussion of the mechanism of the N2O catalytic reduction is made based upon results from isothermal reactions, temperature-programmed reactions (TPR) and characterization of catalysts. The effect of acidic treatment on pore structure is also discussed. (C) 1999 Elsevier Science B.V. All rights reserved.

Role of chemical cues from cotton in mediating host selection and oviposition behaviour in Helicoverpa armigera (Hubner) (Lepidoptera : Noctuidae)

We investigated the role of chemoreception in the host selection and oviposition behaviour of Helicoverpa armigera in the laboratory using five cotton genotypes and synthetic volatile terpenes. Female moths oviposited on substrates treated with methanol, ethanol, acetone and pentane extracts of leaves, squares and flowers of the cotton genotypes. Phytochemicals soluble in pentane were the most efficient in eliciting oviposition behaviour. In a two-way bioassay, pentane extracts of leaves or squares of a Multiple Host-plant Resistance genotype (MHR11), Deltapine commercial (DP90), and Smith Red Leaf (SRL) received significantly more eggs than solvent-treated controls. Extracts of squares of the native genotype Gossypium nelsonii did not receive more eggs. Females preferred DP90 and MHR11 to SRL and G. nelsonii. Female moths also laid more eggs on pentane extracts of MHR11 flowers than MHR11 leaves from preflowering, early flowering and peak-flowering plants. In a flight chamber, female moths used olfactory cues at short range to mediate oviposition and discrimination between host plants. Egg-laying, mated females were attracted at a distance (1.5 m) to volatile compounds released by whole plants and odours emanating from filter papers treated with synthetic volatile terpenes. Individually, the terpenes did not stimulate any significant oviposition response. However, there was a significant oviposition response to a mixture of equal volumes of the terpenes (trans-beta-caryophyllene, alpha-pinene, beta-pinene, myrcene, beta-bisabolol, and alpha-humulene). Conversely, antennectomised (moths with transected antennae), egg-laying, mated females did not stimulate any significant oviposition response. The significance of these findings in relation to H. armigera hostplant selection are discussed.

Is there a role for transversus abdominis in lumbo-pelvic stability?

There has been considerable interest in the literature regarding the function of transversus abdominis, the deepest of the abdominal muscles, and the clinical approach to training this muscle. With the development of techniques for the investigation of this muscle involving the insertion of fine-wire electromyographic electrodes under the guidance of ultrasound imaging it has been possible to test the hypotheses related to its normal function and function in people with low back pain. The purpose of this review is to provide an appraisal of the current evidence for the role of transversus abdominis in spinal stability, to develop a model of how the contribution of this muscle differs from the other abdominal muscles and to interpret these findings in terms of the consequences of changes in this function.

DCC plays a role in navigation of forebrain axons across the ventral midbrain commissure in embryonic Xenopus

In the developing vertebrate brain, growing axons establish a scaffold of axon tracts connected across the midline via commissures. We have previously identified a population of telencephalic neurons that express NOC-2, a novel glycoform of the neural cell adhesion molecule N-CAM that is involved in axon guidance in the forebrain. These axons arise from the presumptive telencephalic nucleus, course caudally along the principal longitudinal tract of the forebrain, cross the ventral midline in the midbrain, and then project to the contralateral side of the brain. In the present study we have investigated mechanisms controlling the growth of these axons across the ventral midline of the midbrain. The axon guidance receptor DCC is expressed by the NOC-2 population of axons both within the longitudinal tract and within the ventral midbrain commissure. Disruption of DCC-dependent interactions, both in vitro and in vivo, inhibited the NOC-2 axons from crossing the ventral midbrain. Instead, these axons grew along aberrant trajectories away from the midline, suggesting that DCC-dependent interactions are important for overcoming inhibitory mechanisms within the midbrain of the embryonic vertebrate brain. Thus, coordinated responsiveness of forebrain axons to both chemostimulatory and chemorepulsive cues appears to determine whether they cross the ventral midline in the midbrain, (C) 2000 Academic Press.

The role of disulfide-linked dimerization in interleukin-3 receptor signaling and biological activity

Cysteine residues 86 and 91 of the beta subunit of the human interleukin (hIL)-3 receptor (h beta c) participate in disulfide-linked receptor subunit heterodimerization. This linkage is essential for receptor tyrosine phosphorylation, since the Cys-86 --> Ala (Mc4) and Cys-91 --> Ala (Mc5) mutations abolished both events. Here, we used these mutants to examine whether disulfide-linked receptor dimerization affects the biological and biochemical activities of the IL-3 receptor. Murine T cells expressing hIL-3R alpha and Mc4 or Mc5 did not proliferate in hIL-3, whereas cells expressing wild-type h beta c exhibited rapid proliferation. However, a small subpopulation of cells expressing each mutant could be selected for growth in IL-3, and these proliferated similarly to cells expressing wild-type h beta c, despite failing to undergo IL-3-stimulated h beta e tyrosine phosphorylation. The Mc4 and Mc5 mutations substantially reduced, but did not abrogate, IL-3-mediated anti-apoptotic activity in the unselected populations. Moreover, the mutations abolished IL-3-induced JAK2, STAT, and AKT activation in the unselected cells, whereas activation of these molecules in IL-3-selected cells was normal. In contrast, Mc4 and Mc5 showed a limited effect on activation of Erk1 and -2 in unselected cells. These data suggest that whereas disulfide-mediated cross-linking and h beta c tyrosine phosphorylation are normally important for receptor activation, alternative mechanisms can bypass these requirements.

The role of colour in signalling and male choice in the agamid lizard Ctenophorus ornatus

Bright coloration and complex visual displays are frequent and well described in many lizard families. Reflectance spectrometry which extends into the ultraviolet (UV) allows measurement of such coloration independent of our visual system. We examined the role of colour in signalling and mate choice in the agamid lizard Ctenophorus ornatus. We found that throat reflectance strongly contrasted against the granite background of the lizards' habitat. The throat may act as a signal via the head-bobbing and push-up displays of C. ornatus. Dorsal coloration provided camouflage against the granite background, particularly in females. C. ornatus was sexually dichromatic for all traits examined including throat UV reflectance which is beyond human visual perception. Female throats were highly variable in spectral reflectance and males preferred females with higher throat chroma between 370 and 400 nm. However, female throat UV chroma is strongly correlated to both throat brightness and chest UV chroma and males may choose females on a combination of these colour variables. There was no evidence that female throat or chest coloration was an indicator of female quality. However, female brightness significantly predicted a female's laying date and, thus, may signal receptivity. One function of visual display in this species appears to be intersexual signalling, resulting in male choice of females.

The role of mobile belts for the longevity of deep cratonic lithosphere: The crumple zone model

Many Archean cratons are surrounded by Proterozoic mobile belts that have experienced episodes of tectonic re-activation over their lifetimes. This suggests that mobile belt lithosphere may be associated with long lived, inherited weakness. It is proposed that the proximity of this weakness can increase the longevity of deep Archean lithosphere by buffering Archean cratons from mantle derived stresses. The physical plausibility of this idea is explored through numerical simulations of mantle convection that include continents and allow for material rheologies that model the combined brittle and ductile behavior of the lithosphere. Within the simulations, the longevity of deep cratonic lithosphere does increase if it is buffered by mobile belts that can fail at relatively low stress levels.