Three-dimensional crystal structure of human eosinophil cationic protein (RNase 3) at 1.75 A resolution

Eosinophil cationic protein (ECP; RNase 3) is a human ribonuclease found only in eosinophil leukocytes that belongs to the RNase A superfamily. This enzyme is bactericidal, helminthotoxic and cytotoxic to mammalian cells and tissues. The protein has been cloned, heterologously overexpressed, purified and crystallized. Its crystal structure has been determined and refined using data up to 1. 75 A resolution. The molecule displays the alpha+beta folding topology typical for members of the ribonuclease A superfamily. The catalytic active site residues are conserved with respect to other ribonucleases of the superfamily but some differences appear at substrate recognition subsites, which may account, in part, for the low catalytic activity. Most strikingly, 19 surface-located arginine residues confer a strong basic character to the protein. The high concentration of positive charges and the particular orientation of the side-chains of these residues may also be related to the low activity of ECP as a ribonuclease and provides an explanation for its unique cytotoxic role through cell membrane disruption.     

Isolation of a Tobacco cDNA Encoding Sar1 GTPase and Analysis of Its Dominant Mutations in Vesicular Traffic Using a Yeast Complementation System

The cDNA clone of NtSARl, a gene encoding the small GTPase Sar1pwhich is essential for vesicle formation from the endoplasmicreticulum (ER) membrane in yeast, has been isolated from Nicotianatabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated fromArabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205]could complement the lethality of the disruption of SARI inyeast cells in a temperature-sensitive fashion. They also suppressedyeast sec12 and sec16 temperature-sensitive mutations as yeastSARI does. Using this complementation system, we analyzed thephenotypes of several mutations in plant SAR1 cDNAs in yeastcells. The expression of NtSAR1 H74L and AtSAR1 N129I showeddominant negative effect in growth over the wild-type SARI,which was accompanied by the arrest of ER-to-Golgi transport.Such dominant mutations will be useful to analyze the role ofmembrane trafficking in plant cells, if their expression canbe regulated conditionally. (Received October 29, 1997; Accepted March 17, 1998)     

Fear Conditioning Induced by Interpersonal Conflicts in Healthy Individuals

Psychophysiological markers have been focused to investigate the psychopathology of psychiatric disorders and personality subtypes. In order to understand neurobiological mechanisms underlying these conditions, fear-conditioning model has been widely used. However, simple aversive stimuli are too simplistic to understand mechanisms because most patients with psychiatric disorders are affected by social stressors. The objective of this study was to test the feasibility of a newly-designed conditioning experiment using a stimulus to cause interpersonal conflicts and examine associations between personality traits and response to that stimulus. Twenty-nine healthy individuals underwent the fear conditioning and extinction experiments in response to three types of stimuli: a simple aversive sound, disgusting pictures, and pictures of an actors’ face with unpleasant verbal messages that were designed to cause interpersonal conflicts. Conditioned response was quantified by the skin conductance response (SCR). Correlations between the SCR changes, and personality traits measured by the Zanarini Rating Scale for Borderline Personality Disorder (ZAN-BPD) and Revised NEO Personality Inventory were explored. The interpersonal conflict stimulus resulted in successful conditioning, which was subsequently extinguished, in a similar manner as the other two stimuli. Moreover, a greater degree of conditioned response to the interpersonal conflict stimulus correlated with a higher ZAN-BPD total score. Fear conditioning and extinction can be successfully achieved, using interpersonal conflicts as a stimulus. Given that conditioned fear caused by the interpersonal conflicts is likely associated with borderline personality traits, this paradigm could contribute to further understanding of underlying mechanisms of interpersonal fear implicated in borderline personality disorder.     

Variation in photoinhibition among Sasa senanensis, Quercus mongolica, and Acer mono in the understory of a deciduous broad-leaved forest exposed to canopy gaps caused by typhoons

To elucidate mechanisms for tolerating sudden increases in light intensity following canopy gap formation, we investigated susceptibility to photoinhibition in the evergreen clonal plant bamboo, Sasa senanensis, and two deciduous broadleaf woody plants, Quercus mongolica, and Acer mono. We measured pre-dawn photochemical efficiency of photosystem II (F _v /F _m) in plants exposed to canopy gaps and in shade-grown plants through the month following gap formation. Photoinhibition (indicated by decreased F _v /F _m) was smallest in S. senanensis and largest in A. mono. S. senanensis had the highest area-based net CO₂ assimilation rate (A _area) and electron transport rate (ETR) under high light conditions. This species also had the highest leaf mass per area (LMA) and leaf nitrogen content per area (N _area). Higher values of LMA and N _area under shade conditions probably contribute to circumvent photoinhibition through maintenance of a higher ETR capacity. Q. mongolica, a gap-dependent species, had properties intermediate between S. senanensis and A. mono; it appeared less susceptible to photoinhibition than the shade-tolerant A. mono. None of the species examined had increased photosynthetic capacity 1 month after gap formation, indicating that shade-grown leaves were unable to fully acclimate to increased light.     

Crystal structures of MKK4 kinase domain reveal that substrate peptide binds to an allosteric site and induces an auto-inhibition state

MKK4 activates both JNKs and p38s. We determined the crystal structures of human non-phosphorylated MKK4 kinase domain (npMKK4) complexed with AMP-PNP (npMKK4/AMP) and a ternary complex of npMKK4, AMP-PNP and p38α peptide (npMKK4/AMP/p38). These crystal structures revealed that the p38α peptide-bound npMKK4 at the allosteric site rather than at the putative substrate binding site and induced an auto-inhibition state. While the activation loop of the npMKK4/AMP complex was disordered, in the npMKK4/AMP/p38 complex it configured a long α-helix, which prevented substrate access to the active site and αC-helix movement to the active configuration of MKK4.     

Protein kinase Cdelta plays a non-redundant role in insulin secretion in pancreatic beta cells

Protein kinase C (PKC) is considered to modulate glucose-stimulated insulin secretion. Pancreatic beta cells express multiple isoforms of PKCs; however, the role of each isoform in glucose-stimulated insulin secretion remains controversial. In this study we investigated the role of PKCdelta, a major isoform expressed in pancreatic beta cells on beta cell function. Here, we showed that PKCdelta null mice manifested glucose intolerance with impaired insulin secretion. Insulin tolerance test showed no decrease in insulin sensitivity in PKCdelta null mice. Studies using islets isolated from these mice demonstrated decreased glucose- and KCl-stimulated insulin secretion. Perifusion studies indicated that mainly the second phase of insulin secretion was decreased. On the other hand, glucose-induced influx of Ca2+ into beta cells was not altered. Immunohistochemistry using total internal reflection fluorescence microscopy and electron microscopic analysis showed an increased number of insulin granules close to the plasma membrane in beta cells of PKCdelta null mice. Although PKC is thought to phosphorylate Munc18-1 and facilitate soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors complex formation, the phosphorylation of Munc18-1 by glucose stimulation was decreased in islets of PKCdelta null mice. We conclude that PKCdelta plays a non-redundant role in glucose-stimulated insulin secretion. The impaired insulin secretion in PKCdelta null mice is associated with reduced phosphorylation of Munc18-1.     

Role of Septin cytoskeleton in spine morphogenesis and dendrite development in neurons

Septins are GTP-binding proteins that polymerize into heteromeric filaments and form microscopic bundles or ring structures in vitro and in vivo. Because of these properties and their ability to associate with membrane, F-actin, and microtubules, septins have been generally regarded as cytoskeletal components [1, 2]. Septins are known to play roles in cytokinesis, in membrane trafficking, and as structural scaffolds; however, their function in neurons is poorly understood. Many members of the septin family, including Septin 7 (Sept7), were found by mass-spectrometry analysis of postsynaptic density (PSD) fractions of the brain [3, 4], suggesting a possible postsynaptic function of septins in neurons. We report that Sept7 is localized at the base of dendritic protrusions and at dendritic branch points in cultured hippocampal neurons--a distribution reminiscent of septin localization in the bud neck of budding yeast. Overexpression of Sept7 increased dendrite branching and the density of dendritic protrusions, whereas RNA interference (RNAi)-mediated knockdown of Sept7 led to reduced dendrite arborization and a greater proportion of immature protrusions. These data suggest that Sept7 is critical for spine morphogenesis and dendrite development during neuronal maturation.     

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper-like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild-type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L-RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X-ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L-RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L- and 2L-RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L-RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L-RNase 1) was also evaluated. N4L-RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper-like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.     

Spirodiketopiperazine-based CCR5 antagonists: Lead optimization from biologically active metabolite

Hydroxylated derivatives were designed and synthesized based on the information of oxidative metabolites. Compounds derived from beta-substituted (2R,3R)-2-amino-3-hydroxypropionic acid showed improved inhibitory activities against the binding of MIP-1alpha to human CCR5, compared with the non-hydroxylated derivatives and the other isomers.