The gusBC genes of Escherichia coli encode a glucuronide transport system

Two genes, gusB and gusC, from a natural fecal isolate of Escherichia coli are shown to encode proteins responsible for transport of beta-glucuronides with synthetic [(14)C]phenyl-1-thio-beta-d-glucuronide as the substrate. These genes are located in the gus operon downstream of the gusA gene on the E. coli genome, and their expression is induced by a variety of beta-d-glucuronides. Measurements of transport in right-side-out subcellular vesicles show the system has the characteristics of secondary active transport energized by the respiration-generated proton motive force. When the genes were cloned together downstream of the tac operator-promoter in the plasmid pTTQ18 expression vector, transport activity was increased considerably with isopropylthiogalactopyranoside as the inducer. Amplified expression of the GusB and GusC proteins enabled visualization and identification by N-terminal sequencing of both proteins, which migrated at ca. 32 kDa and 44 kDa, respectively. Separate expression of the GusB protein showed that it is essential for glucuronide transport and is located in the inner membrane, while the GusC protein does not catalyze transport but assists in an as yet unknown manner and is located in the outer membrane. The output of glucuronides as waste by mammals and uptake for nutrition by gut bacteria or reabsorption by the mammalian host is discussed.     

Structural reorganization of the copper binding site involving Thr15 of mavicyanin from Cucurbita pepo medullosa (zucchini) upon reduction

Mavicyanin, a glycosylated protein isolated from Cucurbita pepo medullosa (zucchini), is a member of the phytocyanin subfamily containing one polypeptide chain of 109 amino residues and an unusual type-I Cu site in which the copper ligands are His45, Cys86, His91, and Gln96. The crystal structures of oxidized and reduced mavicyanin were determined at 1.6 and 1.9 A resolution, respectively. Mavicyanin has a core structure of seven polypeptide beta-strands arranged as a beta-sandwich organized into two beta-sheets, and the structure considerably resembles that of stellacyanin from cucumber (CST) or cucumber basic protein (CBP). A flexible region was not observed on superimpositioning of the oxidized and reduced mavicyanin structures. However, the Cu(II)-epsilon-O-Gln96 bond length was extended by 0.47 A, and the Thr15 residue was rotated by 60.0 degrees and O-gamma1-Thr15 moved from a distance of 4.78 to 2.58 A from the ligand Gln96 forming a new hydrogen bond between O-gamma1-Thr15 and epsilon-O-Gln96 upon reduction. The reorganization of copper coordination geometry of mavicyanin upon reduction arouses reduction potential decreased above pH 8 [Battistuzzi et al. (2001) J. Inorg. Biochem. 83, 223-227]. The rotation of Thr15 and the hydrogen bonding with the ligand Gln96 may constitute structural evidence of the decrease in the reduction potential at high pH.     

Crystal structure of dynein light chain TcTex-1

TcTex-1, one of three dynein light chains of the dynein motor complex, has been implicated in targeting and binding cargoes to cytoplasmic dynein for retrograde or apical transport. Interactions between TcTex-1 and a diverse set of proteins such as the dynein intermediate chain, Fyn, DOC2, FIP1, the poliovirus receptor, CD155, and the rhodopsin cytoplasmic tail have been reported; yet, despite the broad range of targets, a consensus binding sequence remains uncertain. Consequently, we have solved the crystal structure of the full-length Drosophila homolog of TcTex-1 to 1.7 A resolution using MAD phasing to gain insight into its function and target specificity. The structure is homodimeric with a domain swapping of beta-strand 2 and has a fold similar to the dynein light chain, LC8. Based on structural alignment, the TcTex-1 and LC8 sequences show no identity, although the root mean square deviation between secondary structural elements is less than 1.6 A. Moreover, the N terminus, which is equivalent to beta-strand 1 in LC8, is splayed out and binds to a crystallographic dimer as an anti-parallel beta-strand at the same position as the neuronal nitric-oxide synthase peptide in the LC8 complex. Similarity to LC8 and comparison to the LC8-neuronal nitricoxide synthase complex suggest that TcTex-1 binds its targets in a similar manner as LC8 and provides insight to the lack of strict sequence identity among the targets for TcTex-1.     

NMR structural comparison of the cytoplasmic juxtamembrane domains of G-protein-coupled CB1 and CB2 receptors in membrane mimetic dodecylphosphocholine micelles

The fourth cytoplasmic domain, the so-called C-terminal juxtamembrane segment or helix VIII, has been identified in numerous G-protein-coupled receptors and exhibits unique functional characteristics. Efforts have been devoted to studying the juxtamembrane segment in order to understand the biological importance of the segment in G-protein activation of the cannabinoid CB1 and CB2 receptors. Recent biochemical data revealed that the CB1 C-terminal juxtamembrane peptide fragment CB1-(401-417) can directly activate the G-protein and also showed that the specificity of the signal transduction activation by the C-terminal juxtamembrane region is unique to the CB1 receptor but not to the CB2 receptor (Mukhopadhyay, S., and Howlett, A. C. (2001) Eur. J. Biochem. 268, 499-505). However, there is experimental work, not yet reported, on the conformational analyses and structural comparison between the respective helix VIII segments of the two receptors. In the present study, we have examined the conformational specificities of the cytoplasmic helical domains for both cannabinoid receptors. Three-dimensional structural features of two synthetic CB1 and CB2 peptides, CB1I397-G418 and CB2I298-K319, respectively, in membrane mimetic DPC micelles were studied using a combined high resolution NMR and computer modeling approach. Comparisons of the NMR-determined structures of the two peptides as well as their correspondent mutant peptides revealed their conformational properties and salt bridge dissimilarity, which might help us to understand the different structural roles of the fourth cytoplasmic helices in the function and regulation of CB1 and CB2 receptors.     

Mammalian exocyst complex is required for the docking step of insulin vesicle exocytosis

Glucose stimulates insulin secretion from pancreatic beta cells by inducing the recruitment and fusion of insulin vesicles to the plasma membrane. However, little is currently known about the mechanism of the initial docking or tethering of insulin vesicles prior to fusion. Here, we examined the role of the SEC6-SEC8 (exocyst) complex, implicated in trafficking of secretory vesicles to fusion sites in the plasma membrane in yeast and in regulating glucose-stimulated insulin secretion from pancreatic MIN6 beta cells. We show first that SEC6 is concentrated on insulin-positive vesicles, whereas SEC5 and SEC8 are largely confined to the cytoplasm and the plasma membrane, respectively. Overexpression of truncated, dominant-negative SEC8 or SEC10 mutants decreased the number of vesicles at the plasma membrane, whereas expression of truncated SEC6 or SEC8 inhibited overall insulin secretion. When single exocytotic events were imaged by total internal reflection fluorescence microscopy, the fluorescence of the insulin surrogate, neuropeptide Y-monomeric red fluorescent protein brightened, diffused, and then vanished with kinetics that were unaffected by overexpression of truncated SEC8 or SEC10. Together, these data suggest that the exocyst complex serves to selectively regulate the docking of insulin-containing vesicles at sites of release close to the plasma membrane.     

Residual activity and population effects of noviflumuron for German cockroach (Dictyoptera: Blattellidae) control

A benzoylphenyl urea insect growth regulator with the common name noviflumuron was evaluated for efficacy and residual activity on the German cockroach, Blattella germanica (L.). In laboratory studies evaluating residual activity, 0.05% noviflumuron suspension concentrate produced 100% nymphal mortality 120 d after application to steel and masonite substrates. Residual activity of noviflumuron was more variable on painted plywood substrates compared with stainless steel and masonite. In bioassay arenas, population reductions caused by noviflumuron were significantly greater than Archer and the untreated populations. After 16 wk, populations exposed to 0.05, 0.1, and 0.2% noviflumuron were reduced by 51.9 +/- 19.8, 62.2 +/- 6.5, and 62.6 +/- 18.4%, respectively. Control cockroach populations and populations exposed to 1.3% pyriproxyfen at labeled rate (Archer, 0.61 g/m2) increased by 1286.3 +/- 125.1 and 937.2 +/- 137.1%, respectively, at the end of 16 wk. A field study in multifamily housing complexes showed noviflumuron (0.2 and 0.5%) to provide 73.3 +/- 8.0 and 90.6 +/- 3.6% trap catch reduction at 4 wk posttreatment, respectively. There were no significant differences in the performance of noviflumuron, Maxforce FC Roach Bait Stations (0.05% [AI] fipronil), and Avert dust bait (0.05% [AI] abamectin B1). Noviflumuron shows excellent potential for use in cockroach management programs.     

Anti-diabetic effect of ginsenoside Re in ob/ob mice

We evaluated the anti-diabetic effects of ginsenoside Re in adult male C57BL/6J ob/ob mice. Diabetic ob/ob mice with fasting blood glucose levels of approximately 230 mg/dl received daily intraperitoneal injections of 7, 20 and 60 mg/kg ginsenoside Re for 12 consecutive days. Dose-related effects of ginsenoside Re on fasting blood glucose levels were observed. After the 20 mg/kg treatment, fasting blood glucose levels were reduced to 188+/-9.2 and 180+/-10.8 mg/dl on Day 5 and Day 12, respectively (both P<0.01 compared to vehicle group, 229+/-9.5 and 235+/-13.4 mg/dl, respectively). The EC(70) of ginsenoside Re was calculated to be 10.3 mg/kg and was used for subsequent studies. Consistent with the reduction in blood glucose, there were significant decreases in both fed and fasting serum insulin levels in mice treated with ginsenoside Re. With 12 days of ginsenoside treatment, glucose tolerance of ob/ob mice increased significantly, and the area under the curve for glucose decreased by 17.8% (P<0.05 compared to vehicle treatment). The hypoglycemic effect of the ginsenoside persisted even at 3 days of treatment cessation (blood glucose levels: 198+/-13.1 with ginsenoside treatment vs. 253+/-20.3 mg/dl with vehicle, P<0.01). There were no significant changes in body weight or body temperature. Preliminary microarray analysis revealed differential expression of skeletal muscle genes associated with lipid metabolism and muscle function. The results suggest that ginsenoside Re may prove to be useful in treating type 2 diabetes.     

Neutrophil-derived heparin-binding protein (HBP/CAP37) deposited on endothelium enhances monocyte arrest under flow conditions

In acute inflammation, infiltration of neutrophils often precedes a second phase of monocyte invasion, and data in the literature suggest that neutrophils may directly stimulate mobilization of monocytes via neutrophil granule proteins. In this study, we present a role for neutrophil-derived heparin-binding protein (HBP) in monocyte arrest on endothelium. Adhesion of neutrophils to bovine aorta endothelial cells (ECs) or HUVEC-triggered secretion of HBP and binding of the protein to the EC surface. Blockade of neutrophil adhesion by treatment with a mAb to CD18 greatly reduced accumulation of HBP. In a flow chamber model, immobilized recombinant HBP induced arrest of human monocytes or monocytic Mono Mac 6 (MM6) cells to activated EC or plates coated with recombinant adhesion molecules (E-selectin, P-selectin, VCAM-1). However, immobilized recombinant HBP did not influence arrest of neutrophils or lymphocytes. Treatment of MM6 cells with recombinant HBP evoked a rapid and clear-cut increase in cytosolic free Ca(2+) that was found to be critical for the HBP-induced monocyte arrest inasmuch as pretreatment with the intracellular calcium chelating agent BAPTA-AM abolished the evoked increase in adhesion. Thus, secretion of a neutrophil granule protein, accumulating on the EC surface and promoting arrest of monocytes, could contribute to the recruitment of monocytes at inflammatory loci.     

Synthesis, characterization and supramolecular structures of two Ni(II) complexes with potentially heptadentate ligand N,N-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol

A potentially heptadentate ligand H₃L (N,N^′-bis(2-hydroxybenzyl)-1,3-bis[(2-aminoethyl)amino]-2-propanol) and its two Ni(II) complexes, [Ni(H₂L)H₂O](H₂O)₃ClO₄ (1) and [Ni(H₂L)(H₂O)](H₂O)Cl (2) were prepared and characterized. X-ray structural analyses indicate that complex 1 has a distorted octahedral coordination geometry, with four amine N atoms of H₂L⁻ defining the equatorial plane, one aqua O atom and one phenoxo O atom of the ligand occupying two axial positions, respectively. The Ni(II) center of 2 has coordination geometry similar to that of 1. IR and electronic spectra of 1 and 2 are in agreement with their crystal structural features. Approximately along the ab plane, 2D supramolecular structure of 1 is assembled through multiple hydrogen bonds between hydroxy groups of the ligands, coordinated and crystal lattice H₂O and π-π stacking interactions between adjacent phenyl rings of the ligands, while for that of 2, probably along the a axis, 1D chain structure is also formed by multiple hydrogen bonds, but lack of π-π stacking interactions.