Driver eye glance behavior and performance with camera-based visibility systems versus mirrors.

OBJECTIVE: Drivers' ability to extract visual information efficiently from mirrors or camera-based visibility systems impacts driving performance when carrying out maneuvers such as lane changes. The objective of the research was to compare drivers' eye gaze behavior and driving performance with mirrors versus camera-based visibility systems (i.e., CMS, or camera monitor system) to identify any differences and possible impacts on safety. METHODS: A test track study was conducted comparing drivers' eye gaze and lane change behavior when driving a vehicle equipped with outside mirrors versus a prototype CMS. Participants' opinions regarding usability and comfort in using mirrors versus the tested CMS were also obtained using a post-drive questionnaire. RESULTS: Study results were somewhat mixed but did demonstrate that with the tested CMS, participants took longer to pass a slower moving vehicle and maintained a greater resultant distance from the passed vehicle. Additionally, participants had a greater number of fixations to the CMS displays compared to the outside rearview mirrors. Results also found slight perceived advantages for the tested CMS in regard to ease of use, comfortability, and visibility. When asked to choose which rear visibility technology they would prefer to use in everyday driving, most participants preferred the outside rearview mirrors over the tested prototype CMS or having both systems. However, not all lane change and gaze metrics followed the same pattern. CONCLUSIONS: In this study, participants' longer time to pass a slower moving vehicle, greater distance when passing a slower moving vehicle, greater number of fixations, and lower subjective ratings with the tested CMS may indicate difficulty in judging distances and focusing on the electronic image. This study provides preliminary findings that suggest differences in driving behavior exist between a single tested prototype CMS and outside rearview mirrors and is a foundational step toward evaluating whether these trends are consistent across different systems and overall implications for safe driving behavior.

CRISPR-based oligo recombineering prioritizes apicomplexan cysteines for drug discovery.

Nucleophilic amino acids are important in covalent drug development yet underutilized as anti-microbial targets. Chemoproteomic technologies have been developed to mine chemically accessible residues via their intrinsic reactivity towards electrophilic probes but cannot discern which chemically reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based oligo recombineering (CORe) platform to support the rapid identification, functional prioritization and rational targeting of chemically reactive sites in haploid systems. Our approach couples protein sequence and function with biological fitness of live cells. Here we profile the electrophile sensitivity of proteinogenic cysteines in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. Electrophile-sensitive cysteines decorating the ribosome were found to be critical for parasite growth, with target-based screening identifying a parasite-selective anti-malarial lead molecule and validating the apicomplexan translation machinery as a target for ongoing covalent ligand development.

A protease-based biosensor for the detection of schistosome cercariae.

Parasitic diseases affect millions of people worldwide, causing debilitating illnesses and death. Rapid and cost-effective approaches to detect parasites are needed, especially in resource-limited settings. A common signature of parasitic diseases is the release of specific proteases by the parasites at multiple stages during their life cycles. To this end, we engineered several modular Escherichia coli and Bacillus subtilis whole-cell-based biosensors which incorporate an interchangeable protease recognition motif into their designs. Herein, we describe how several of our engineered biosensors have been applied to detect the presence and activity of elastase, an enzyme released by the cercarial larvae stage of Schistosoma mansoni. Collectively, S. mansoni and several other schistosomes are responsible for the infection of an estimated 200 million people worldwide. Since our biosensors are maintained in lyophilised cells, they could be applied for the detection of S. mansoni and other parasites in settings without reliable cold chain access.

Indigofera spicata (creeping indigo) poisoning of three ponies.

Three ponies continuously grazed a pasture containing an estimated 24% Indigofera spicata (wet weight basis) for 4-6 weeks in April and May 2004. They developed ataxia, paresis, depression, muscle fasciculations, dysphagia, ptyalism and halitosis. Two also developed corneal opacity. One pony recovered with supportive treatment, but the other two were euthanased and necropsied. Neuropathology was not present in either case, but both livers had periacinar and periportal lymphocytic infiltrations and hydropic degeneration of mid-zonal hepatocytes, with mild to moderate periacinar necrosis also evident in one. The I. spicata contained 2.66 mg 3-nitropropionic acid (3-NPA)/g dry matter and 1.5 mg indospicine/g dry matter. Indospicine, but not 3-NPA, was detected in serum from both of the euthanased ponies and indospicine was detected in heart, liver and muscle from the one pony in which this assay was performed. The clinical syndrome closely resembled 'Birdsville horse disease' caused by I. linnaei and was similar to that reported in horses poisoned by the closely related species I. hendecaphylla and to 3-NPA poisoning of other animals, including humans. 3-NPA is thought to cause this neurological syndrome. To our knowledge, this is the first authenticated report of I. spicata poisoning in grazing animals. We also report here the first published evidence that 3-NPA and indospicine exist in naturalised I. spicata in Australia and of the formation of indospicine residues in tissues of animals grazing paddocks infested with I. spicata.

Standing magnetic resonance imaging detection of bone marrow oedema-type signal pattern associated with subcarpal pain in 8 racehorses: a prospective study.

REASON FOR PERFORMING STUDY: The proximal metacarpal region is a common site of origin of lameness in the performance horse. A number of disease entities are recognised as causes of proximal metacarpal lameness but a definitive diagnosis is often elusive. Magnetic resonance imaging (MRI) is hypothesised to offer advantages over traditional imaging modalities in the investigation of proximal metacarpal pain. OBJECTIVE: To describe clinical and imaging features of cases of lameness in racehorses arising from the proximal metacarpal region in which standing MRI identified 'bone marrow oedema-type' (BMO-type) signal patterns. METHODS: Records for all horses undergoing standing MRI of the proximal metacarpus/distal carpus from September 2006 to December 2008 were reviewed. Cases underwent a standardised protocol for diagnostic analgesia, radiography and ultrasonography of the proximal metacarpus and distal carpus. Cases with proximal metacarpal lameness displaying a characteristic BMO-type signal pattern on MRI were identified and outcomes analysed. RESULTS: Eight cases were identified with characteristic MRI findings of extensive hyperintensity on T2* gradient echo and short tau inversion fast spin echo sequences and corresponding hypointensity on T1 gradient echo images within the palmaroproximal aspect of the third metacarpal bone. Follow-up information was available for all cases; at the time of writing 7/8 had returned to full work and were free from lameness. CONCLUSIONS: The BMO-type signal patterns visible on MR images in these cases may signal the existence of a previously under-diagnosed pathological process associated with proximal metacarpal lameness in racehorses. This finding is postulated to be associated with a stress reaction and possible prodromal stress fracture of the palmaroproximal metacarpus not appreciable radiographically or ultrasonographically. POTENTIAL RELEVANCE: MRI of the proximal metacarpal region permits detection of pathological processes, which may elude conventional imaging and, therefore, has important therapeutic and prognostic implications in these cases.

Equine laminitis: ultrastructural lesions detected 24-30 hours after induction with oligofructose.

REASONS FOR PERFORMING STUDY: The pathology of equine laminitis has been well-documented 48 h after dosing with oligofructose when clinical lameness and lamellar disintegration is well advanced. Further analysis of the earliest lesions, by collecting lamellar samples at the first sign of foot lameness after oligofructose dosing is required in order to increase understanding of the disease. OBJECTIVES: To investigate lamellar epidermal hemidesmosome damage and basement membrane dysadhesion by transmission electron microscopy (TEM). METHODS: Eight clinically normal, mature Standardbred horses were divided randomly into 2 groups of 4. The treatment group were dosed with oligofructose (10 g/kg bwt) and subjected to euthanasia when shifting weight from one foot to other commenced and at the first sign of lameness during walking and turning. This occurred at 24 h in 3 horses and 30 h in one. The sham treatment control group were dosed with water and subjected to euthanasia after 48 h. Lamellar tissues of the front feet were harvested and processed for ultrastructural study using TEM. RESULTS: Examination by TEM showed excessive waviness of the basement membrane zone and pointed tips of some secondary epidermal lamellae, an ultrastructural lesion typical of laminitis. The average number of hemidesmosomes/microm of basement membrane was decreased and their distance from the centre of the lamina densa of the basement membrane was increased. CONCLUSIONS: Laminitis lesions are detectable 24 h after oligofructose administration. POTENTIAL RELEVANCE: Hindgut events occurring in the first 24 h after dosing have begun the destruction of the hoof lamellar interface. Prevention and treatment strategies should precede lameness if they are to be efficacious.

Epstein-Barr virus BNRF1 protein allows efficient transfer from the endosomal compartment to the nucleus of primary B lymphocytes.

Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.

Resting B cells as a transfer vehicle for Epstein-Barr virus infection of epithelial cells.

Epstein-Barr virus (EBV), an orally transmitted herpesvirus, efficiently targets B lymphocytes through binding of the viral envelope glycoprotein gp350 to the complement receptor CD21. How the virus accesses epithelial cells is less well understood, because such cells are largely resistant to infection with cell-free virus in vitro. Here, we show that, after binding to primary B cells, most Epstein-Barr virions are not internalized but remain on the B cell surface and from there can transfer efficiently to CD21-negative epithelial cells, increasing epithelial infection by 10(3)- to 10(4)-fold compared with cell-free virus. Transfer infection is associated with the formation of B cell-epithelial conjugates with gp350/CD21 complexes focused at the intercellular synapse; transfer involves the gp85 and gp110 viral glycoproteins but is independent of gp42, the HLA class II ligand that is essential for B cell entry. Therefore, through efficient binding to the B cell surface, EBV has developed a means of simultaneously accessing both lymphoid and epithelial compartments; in particular, infection of pharyngeal epithelium by orally transmitted virus becomes independent of initial virus replication in the B cell system.

Defective infectious particles and rare packaged genomes produced by cells carrying terminal-repeat-negative epstein-barr virus.

The Epstein-Barr virus (EBV) lytic program includes lytic viral DNA replication and the production of a viral particle into which the replicated viral DNA is packaged. The terminal repeats (TRs) located at the end of the linear viral DNA have been identified as the packaging signals. A TR-negative (TR(-)) mutant therefore provides an appropriate tool to analyze the relationships between EBV DNA packaging and virus production. Here, we show that supernatants from lytically induced 293 cells carrying TR mutant EBV genomes (293/TR(-)) contain large amounts of viral particles devoid of viral DNA which are nevertheless able to bind to EBV target cells. This shows that viral DNA packaging is not a prerequisite for virion formation and egress. Rather surprisingly, supernatants from lytically induced 293/TR(-) cells also contained rare infectious viruses carrying the viral mutant DNA. This observation indicates that the TRs are important but not absolutely essential for virus encapsidation.

Divergent roles for ferric ions in the biological activity of amidated and non-amidated gastrins.

Amidated forms of the peptide hormone gastrin act via the cholecystokinin-2 receptor to stimulate gastric acid secretion, whereas non-amidated forms stimulate colonic mucosal proliferation via a novel, as yet uncharacterised, receptor. Nuclear magnetic resonance (NMR) and fluorescence spectroscopic studies have revealed that glycine-extended gastrin17 bound two ferric ions, and that ferric ion binding was essential for biological activity. We have therefore investigated the role of ferric ions in the biological activity of amidated gastrin17. As with glycine-extended gastrin17, fluorescence quenching experiments indicated that Glu7 Ala and Glu8,9 Ala mutants of amidated gastrin17 each bound only one ferric ion. The affinity of the mutant peptides for the cholecystokinin-2 receptor on transfected COS-7 cells or on Tlymphoblastoid Jurkat cells, and their potency in stimulation of proliferation in Jurkat cells and inositol phosphate production in transfected COS-7 cells, were similar to the values obtained for amidated gastrin17. In addition, the iron chelator desferrioxamine did not significantly inhibit either binding of amidated gastrin17 to the cholecystokinin-2 receptor, or stimulation of inositol phosphate production by amidated gastrin17 in transfected COS-7 cells. We conclude that, in contrast to glycine-extended gastrin17, binding of ferric ions is not essential for the biological activity of amidated gastrin17. Our results support the concept of distinct modes of action for amidated and non-amidated gastrins, and raise the possibility of developing selective antagonists of the actions of non-amidated and amidated gastrins.

Mutation of lysine residues of the 78-kDa gastrin-binding protein reduces gastrin binding.

The 78-kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin/cholecystokinin receptor antagonists on colorectal carcinoma cell lines. Both the N- and C-terminal halves of the GBP bind gastrin, but the affinity of the N-terminal half for gastrin is 7.2-fold higher than the affinity of the C-terminal half. In order to define the gastrin-binding sites of the GBP in greater detail, we have constructed a truncation mutant lacking residues 221-318 of the N-terminal domain and a series of point mutants in which the lysine residues in the first 220 residues of the N-terminal domain were mutated to arginine residues. The effect of these mutations on both the extent of covalent cross-linking of iodinated gastrin2,17 and on the affinity for gastrin17 was investigated. Deletion of residues 221-318 of the GBP decreased the affinity 5.5-fold and reduced, but did not abolish, the extent of covalent cross-linking. Mutation of the 17 lysines in residues 1-220 of the GBP decreased the affinity for gastrin between 1.7- and 3.5-fold and in some cases reduced, but did not abolish, the extent of covalent cross-linking. We conclude that one or more lysine residues are involved in binding of gastrin to the GBP, but that no single lysine residue is the preferred target for covalent cross-linking of iodinated gastrin2,17 to the GBP.

Short term infusion of glycine-extended gastrin(17) stimulates both proliferation and formation of aberrant crypt foci in rat colonic mucosa.

Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo and for colorectal carcinoma cell lines in vitro. The effect of short term administration of synthetic gastrins on the colonic mucosa in vivo, however, has not been reported. The aim of our study was to determine whether continuous systemic infusion of glycine-extended gastrin(17) stimulated proliferation and accelerated carcinogenesis in the colorectal mucosa. A significant increase in colonic mucosal proliferation as assessed by metaphase index was seen in the caecum (23%, p < 0.02) and distal colon (27%, p < 0.001), but not the rectum, after treatment of intact rats with glycine-extended gastrin(17) for 1 week using implanted miniosmotic pumps. Defunctioning of the rectum reduced both the proliferative index and crypt height of the rectal mucosa of untreated rats. Treatment of rectally defunctioned animals with glycine-extended gastrin(17) for either 1 or 4 weeks resulted in a significant increase in both the proliferative index (40% and 93%, respectively) and crypt height (11% and 19%, respectively) of the rectal mucosa. The total number of aberrant crypt foci in intact rats treated with the procarcinogen azoxymethane plus glycine-extended gastrin(17) was increased by 48% compared to the value in controls treated with azoxymethane only (p = 0.01). We conclude that short term administration of glycine-extended gastrin(17) to mature rats not only has a proliferative effect upon colonic mucosa, but also increases the number of aberrant crypt foci formed in the colorectal mucosa after treatment with azoxymethane. Glycine-extended gastrin(17) could thus potentially act as a promoter of carcinogenesis.

HIV type 1 Gag and nucleocapsid proteins: cytoskeletal localization and effects on cell motility.

Cell motility is likely to play a pivotal role in HIV infection by promoting the dissemination of infected cells. On the basis of observations indicating an interaction between HIV-1 Gag and target cell filamentous actin, we hypothesized that these interactions would promote cell motility of HIV-infected cells. Indeed, we have found that HIV-1 infection enhances the chemotactic response of macrophages. To specifically investigate the significance of the interactions between Gag and cellular actin, we transfected NIH 3T3 fibroblasts and HeLa cells with a construct that permits the expression of HIV-1 Gag in the absence of any other viral protein. Fractionation experiments showed that Gag was present in cytoskeletal fraction containing long actin filaments and in a high-speed postcytoskeletal fraction with short actin filaments. We have also localized HIV-1 Gag to the lamellipodia of chemoattractant-stimulated cells. Significantly, the motility of Gag-expressing cells was enhanced in chemotaxis assays. In vitro mutagenesis experiments showed that HIV-1 Gag binds filamentous actin through the nucleocapsid domain (NC). An NC-green fluorescent protein fusion had the same cellular distribution as the complete protein, and its expression increased cell motility. These data suggest that interactions between HIV-1 Gag and actin in infected cells enhance cell motility. Ultimately this enhanced motility of infected cells could promote the dissemination of virus into the brain and other tissues.

Biology and pathology of non-amidated gastrins.

The regulation and extent of progastrin processing has assumed greater importance with the realisation that progastrin and its processing intermediates have functions quite separate from the biosynthetic end product gastrin-amide. The pattern of processing products generated are organ and disease specific with amidated forms predominating in the stomach and non-amidated forms being more important in colorectal carcinoma. In the stomach, non amidated gastrins sustains the acid stimulatory effect of gastrin amide on gastric acidity. The proliferation of colorectal carcinomas and cell lines are stimulated by nonamidated gastrins presumably by an autocrine regulatory loop acting through distinct receptors. The potential role of non-amidated gastrins as therapeutic targets will remain uncertain until the nature of the receptors are determined and specific antagonists developed.

Selective, high-affinity binding of ferric ions by glycine-extended gastrin(17).

Uptake of dietary iron is essential for replenishment of body stores. A role for the hormone gastrin in iron uptake as a chelator of ferric ions in the gastric lumen has been proposed previously [Baldwin, G. S. (1992) Med. Hypotheses 38, 70-74]. Here, spectroscopic evidence of selective, high-affinity binding of ferric ions to progastrin-derived peptides in aqueous solution at low pH is provided. The maximum at 281 nm in the absorption spectrum of glycine-extended gastrin(17) at pH 4.0 increased (2.07 +/- 0.30)-fold in the presence of > or =2 equiv of ferric ions. Titration of glycine-extended gastrin(17) with ferric ions under stoichiometric conditions indicated that the stoichiometry of binding was 2.00 +/- 0.28 mol of Fe(3+)/mol of peptide. Fluorescence quenching experiments yielded values for the stoichiometry and apparent dissociation constant of the ferric ion-glycine-extended gastrin(17) complex at pH 4.0 of 2.39 +/- 0.17 mol of Fe(3+)/mol and 0.62 +/- 0.19 microM, respectively. No interaction was detected with Co(2+), Cu(2+), Mn(2+), or Cr(3+). Electron paramagnetic resonance spectroscopy suggested that the iron ligands were either oxygen or sulfur atoms. Fluorescence quenching experiments with peptides derived from the glycine-extended gastrin(17) sequence indicated that one or more of the five glutamic acid residues were necessary for iron binding. The binding of ferric ions by glycine-extended gastrin(17) at low pH is consistent with a role for progastrin-derived peptides in iron uptake from the lumen of the gastrointestinal tract.

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells.

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.

A kinetic analysis of substrate recognition by uracil-DNA glycosylase from herpes simplex virus type 1.

Uracil-DNA glycosylase (UDG) is responsible for the removal of uracil from DNA. It has previously been demonstrated that UDG exhibits some sequence dependence in its activity, although this has not been well characterised. This study has investigated the sequence-dependent activity of UDG from herpes simplex virus type-1 (HSV-1). A more detailed analysis has been possible by using both kinetic and binding assays with a variety of different oligonucleotide substrates. The target uracil has been placed in substrates with either A-T-rich or G-C-rich flanking sequences and analyses have been performed on both the single- and double-stranded forms of each substrate. In the latter the uracil has been placed in both a U.A base pair and a U.G mismatch. It is observed that the sequences flanking the target uracil have a greater effect on UDG activity than the partner base of the uracil. Furthermore, the sequence context effects extend to single-stranded DNA. Systematic examination of the kinetics and binding of UDG with these different substrates has enabled us to examine the origin of the sequence preferences. We conclude that the damage recognition step in the HSV-1 UDG reaction pathway is modulated by local DNA sequence.

Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinases in glycine-extended gastrin-induced dissociation and migration of gastric epithelial cells.

The various molecular forms of gastrin can act as promoters of proliferation and differentiation in different regions of the gastrointestinal tract. We report a novel stimulatory effect of glycine-extended gastrin(17) only on cell/cell dissociation and cell migration in a non-tumorigenic mouse gastric epithelial cell line (IMGE-5). In contrast, both amidated and glycine-extended gastrin(17) stimulated proliferation of IMGE-5 cells via distinct receptors. Glycine-extended gastrin(17)-induced dissociation preceded migration and was blocked by selective inhibitors of phosphatidylinositol 3-kinase (PI3-kinase) but did not require mitogen-activated protein (MAP) kinase activation. Furthermore, glycine-extended gastrin(17) induced a PI3-kinase-mediated tyrosine phosphorylation of the adherens junction protein beta-catenin, partial dissociation of the complex between beta-catenin and the transmembrane protein E-cadherin, and delocalization of beta-catenin into the cytoplasm. Long lasting activation of MAP kinases by glycine-extended gastrin(17) was specifically required for the migratory response, in contrast to the involvement of a rapid and transient MAP kinase activation in the proliferative response to both amidated and glycine-extended gastrin(17). Therefore, the time course of MAP kinase activation appears to be a critical determinant of the biological effects mediated by this pathway. Together with the involvement of PI3-kinase in the dissociation of adherens junctions, long term activation of MAP kinases seems responsible for the selectivity of this novel effect of G(17)-Gly on the adhesion and migration of gastric epithelial cells.

HGF regulates tight junctions in new nontumorigenic gastric epithelial cell line.

The regulation of intercellular adhesion by hepatocyte growth factor (HGF) was examined on a novel nontumorigenic gastric epithelial cell line (IMGE-5) derived from H-2Kb-tsA58 transgenic mice. IMGE-5 cells constitutively expressed cytokeratin 18 and HGF receptors. Under permissive conditions (33 degrees C + interferon-gamma), IMGE-5 cells proliferated rapidly but did not display membrane expression of adherens and tight junction proteins. Under nonpermissive conditions, their proliferation was decreased and they displayed a strong, localized membrane expression of E-cadherin/beta-catenin and occludin/ZO-1. HGF treatment largely prevented the targeting of ZO-1 to the tight junction and induced a significant decrease of the transepithelial resistance measured across a confluent IMGE-5 cell monolayer. HGF rapidly increased the tyrosine phosphorylation of ZO-1 and decreased its association with occludin in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent manner. PI 3-kinase was also involved in HGF-induced migration of IMGE-5 cells. Our results demonstrate that 1) HGF prevents the appearance of ZO-1 in the membrane during epithelial cell differentiation; 2) HGF causes partial relocalization of ZO-1 to the cytoplasm and nucleus and concomitantly stimulates cell dissociation and migration; and 3) IMGE-5 cells offer a useful model for the study of gastric epithelial cell differentiation.

Inhibition of the preferential binding of actin to the N-terminal hydratase domain of the 78-kDa gastrin-binding protein by non-steroidal anti-inflammatory drugs and gastrin receptor antagonists.

The 78 kDa gastrin-binding protein (GBP) is a likely target for the antiproliferative effects of gastrin receptor antagonists and non-steroidal anti-inflammatory drugs (NSAIDs) on colorectal carcinoma cells (Baldwin GS, Murphy VJ, Yang Z, and Hashimoto T. J Pharmacol Exp Ther 1998;286:1110-14). This study tested the hypotheses that the GBP bound actin, and that the interaction could be disrupted by gastrin receptor antagonists and NSAIDs. Binding of actin to the GBP was assessed by competition with (125)I-[Nle(15)]-gastrin(2,17) in a covalent cross-linking assay, and by comparison of (125)I-actin binding to the N- and C-terminal GBP domains, which had been expressed independently in E. coli as glutathione-S-transferase (GST) fusion proteins. The ability of gastrin receptor antagonists and NSAIDs to interfere with the actin-GBP interaction was measured by release of (125)I-actin from preformed complexes with the N- and C-terminal domain-GST fusion proteins. Actin purified from skeletal muscle or from gastric mucosal cytosol competed with (125)I-[Nle(15)]-gastrin(2,17) for binding to the GBP with IC(50) values of 2.6 +/- 0.7 microM, and 2.1 +/- 0.7 microM, respectively. The amount of (125)I-actin from either source bound to the N-terminal GBP domain was 8.2 times greater than the amount bound to the C-terminal domain. Binding of actin to both domains was inhibited by the gastrin receptor antagonists proglumide and benzotript, and by NSAIDs. We conclude that the GBP may associate with the cytoskeleton via an interaction between its N-terminal domain and actin, and that the association may be disrupted either by gastrin receptor antagonists or by NSAIDs.