Helicobacter pylori is a Gram negative bacterium that plays a central role in the etiology of chronic gastritis and peptic ulcer diseases. However, not all H. pylori positive cases develop advanced disease. This discriminatory behavior has been attributed to the difference in virulence of the bacteria. Among all virulence factors, cytotoxin released by H. pylori is the most important factor. In this work, we studied variation in H. pylori isolates from Indian dyspeptic patients on the basis of cytotoxin production and associated changes in K+-dependent ATPase (one of its targets) enzyme activity in HeLa cells.

Sco1, a protein required for the proper assembly of cytochrome c oxidase, has a soluble domain anchored to the cytoplasmic membrane through a single transmembrane segment. The solution structure of the soluble part of apoSco1 from Bacillus subtilis has been solved by NMR and the internal mobility characterized. Its fold places Sco1 in a distinct subgroup of the functionally unrelated thioredoxin proteins. In vitro Sco1 binds copper(I) through a CXXXCP motif and possibly His 135 and copper(II) in two different species, thus suggesting that copper(II) is adventitious more than physiological. The Sco1 structure represents the first structure of this class of proteins, present in a variety of eukaryotic and bacterial organisms, and elucidates a link between copper trafficking proteins and thioredoxins. The availability of the structure has allowed us to model the homologs Sco1 and Sco2 from S. cerevisiae and to discuss the physiological role of the Sco family.

A great challenge in the proteomics and structural genomics era is to predict protein structure and function, including identification of those proteins that are partially or wholly unstructured. Disordered regions in proteins often contain short linear peptide motifs (e.g., SH3 ligands and targeting signals) that are important for protein function. We present here DisEMBL, a computational tool for prediction of disordered/unstructured regions within a protein sequence. As no clear definition of disorder exists, we have developed parameters based on several alternative definitions and introduced a new one based on the concept of "hot loops," i.e., coils with high temperature factors. Avoiding potentially disordered segments in protein expression constructs can increase expression, foldability, and stability of the expressed protein. DisEMBL is thus useful for target selection and the design of constructs as needed for many biochemical studies, particularly structural biology and structural genomics projects. The tool is freely available via a web interface (http://dis.embl.de) and can be downloaded for use in large-scale studies.

Previously, we demonstrated that plasticity of frontal cortex is altered in aging rats: cholinergic lesions of the nucleus basalis magnocellularis (NBM) produce larger declines in dendritic morphology in frontal cortex of middle-aged and aged rats relative to young adults. To more closely examine the interactive effects of age and cholinergic deafferentation on synaptic connectivity in frontal cortex, we assessed the effects of specific cholinergic lesions on spine density of frontal cortical neurons in young adult, middle-aged, and aged rats. Rats received unilateral sham or 192 IgG-saporin lesions of the NBM. Two weeks after surgery, brains were stained using a Golgi-Cox procedure, and spine density was quantified in second-, third-, and fourth-order basilar dendrites of pyramidal neurons in layer II-III of frontal cortex. Spine density was reduced at all branch orders in aged, sham-lesioned rats. In addition, whereas lesions produced a marked increase in spine density on second- and third-order branches in young adult rats, lesions failed to significantly alter spine density in middle-aged and aged rats. Thus, the upregulation of dendritic spines may be a compensatory response to deafferentation, which is lost with advancing age.

The D2 dopamine receptor, short form (D2s) has been shown to stimulate phospholipase D (PLD) activity independent of activation of phospholipase C (PLC) activity in GH4 derived cells stably transfected with the D2s receptor [Mol. Pharm. 58 (2000) 455]. Agonist activation of D2s has been shown to mediate the inhibition of growth in the same cell line [J. Biol. Chem. 276 (1992) 24169; Endocrinology 134 (1994) 783]. In the present study, D2s-HEK 293 cells were generated using Epstein-Barr virus (EBV) based vectors. The stimulation of PLD by D2s can be augmented by the transfection of Rho A, but not Cdc 42 or Rac and nullified by transfection of N19 Rho A, a dominant negative form of Rho A. Addition of ethanol, at 0.5% reduced the ability of dopamine agonists to inhibit growth in D2s-HEK 293 cells, suggesting that PLD is involved in the antiproliferative effects of D2s signaling. In addition, the expression of N19 Rho A ablated the ability of the D2s to inhibit [3H]thymidine incorporation, while the expression of N19 Cdc 42 or N17 Rac had no effect. These results suggest that the D2s stimulation of PLD is Rho A dependent and lies along the signaling pathway which leads to the antiproliferative effects of D2s receptor activation.

Histone deacetylase inhibitors (HDACIs) induce hyperacetylation of core histones modulating chromatin structure and affecting gene expression. These compounds are also able to induce growth arrest, cell differentiation, and apoptotic cell death of tumor cells in vitro as well as in vivo. Even though several genes modulated by HDAC inhibition have been identified, those genes clearly responsible for the biological effects of these drugs have remained elusive. We investigated the pharmacological effect of the HDACI and potential anti-cancer agent Trichostatin A (TSA) on primary T cells.

N-Acetylaspartylglutamate (NAAG) is a peptide neurotransmitter present in the brain and spinal cord. It is hydrolysed by glutamate carboxypeptidase II (GCPII); thus, the GCP-II inhibitor 2-[phosphono-methyl]-pentanedioic acid (2-PMPA) protects endogenous NAAG from degradation, allowing its effects to be studied in vivo. We recorded the effect of spinal 2-PMPA (50-1000 microg) on the electrical-evoked activity of dorsal horn neurones in normal and carrageenan-inflamed animals, and in the spinal nerve ligation (SNL) model of neuropathy and sham-operated animals. In normal animals, 1000 microg 2-PMPA selectively inhibited noxious-evoked activity (input, post-discharge and C- and Adelta-fibre-evoked responses), and not low threshold Abeta-fibre-evoked responses. After carrageenan inflammation, the lower dose of 100 microg 2-PMPA inhibited input, post-discharge, C- and Adelta-fibre-evoked responses by a significantly greater amount than the same dose in normal animals. 2-PMPA inhibited neuronal responses less consistently in sham-operated and SNL animals, and effects were not significantly different from those seen in normal animals. NAAG is an agonist at the inhibitory metabotropic glutamate receptor mGluR3, and 2-PMPA may inhibit nociceptive transmission in normal animals by elevating synaptic NAAG levels, allowing it to activate mGluR3 and thus reducing transmitter release from afferent nerve terminals. mGluR3 expression in the superficial dorsal horn is upregulated after peripheral inflammation, perhaps explaining the greater inhibition of neuronal responses we observed after carrageenan inflammation. These results support an important role of endogenous NAAG in the spinal processing of noxious information.

Burkholderia cepacia (formerly Pseudomonas cepacia) grows in media containing acetamide or propionamide as C and N sources. Chromosomal DNA from a hospital isolate of B. cepacia served as a template in PCRs using primers designed for the amplification of the P. aeruginosa amiE gene that encodes an aliphatic amidase. Partial sequencing of the PCR products gave a translated sequence 100% identical with the amino acid sequence of P. aeruginosa amidase. A search of Burkholderia genomes detected a putative amidase in B. cepacia J2315 with high identity to the P. aeruginosa amidase and predicted that other Burkholderia species also possessed CN_hydrolases that use the same catalytic triad (Glu-Lys-Cys) as amidase. Superimposition of theoretical three-dimensional models suggested that differences in the amino acid sequences between amidases from B. cepacia (hospital isolate) and B. cepacia J2315 do not affect their three-dimensional structure.

Mushroom bodies (MB) are substructures in the Drosophila brain that are essential for memory. At present, MB anatomy is rather well described when compared to other brain areas, and elucidation of the genetic control of the development and projection patterns of MB neurons will be important to the understanding of their functions. We have performed a gain-of-function screen in order to identify genes that are involved in MB development. We drove expression of genes in MB neurons by crossing 2407 GAL4-driven UY element lines to lines containing an MB GAL4 source and UAS-GFP elements, and looked for defects in the MB structure. We have molecularly identified the genomic regions adjacent to the 26 positive UY insertions and found 18 potential genes that exhibit adult MB gain-of-function phenotypes. The proteins encoded by these candidate genes include, as well as genes with yet unknown function, transcription factors (e.g., tramtrack), nanos RNA-binding protein, microtubule-severing protein, vesicle trafficking proteins, axon guidance receptor, and the Src64 cytoplasmic protein tyrosine kinase. These genes are involved in key features of neuron cell biology. In three cases, tramtrack, nanos, and Src64, we show that the open reading frame located directly downstream of the UY P element is indeed the expressed target gene. Loss-of-function mutations of both ttk and Src64 lead to MB phenotypes proving that these genes are involved in the genetic control of MB development. Moreover, Src64 is shown here to act in a cell-autonomous fashion and is likely to interact with the previously-identified linotte/derailed receptor tyrosine kinase in MB development.

To facilitate rapid detection of a future bioterrorist attack, an increasing number of public health departments are investing in new surveillance systems that target the early manifestations of bioterrorism-related disease. Whether this approach is likely to detect an epidemic sooner than reporting by alert clinicians remains unknown. The detection of a bioterrorism-related epidemic will depend on population characteristics, availability and use of health services, the nature of an attack, epidemiologic features of individual diseases, surveillance methods, and the capacity of health departments to respond to alerts. Predicting how these factors will combine in a bioterrorism attack may be impossible. Nevertheless, understanding their likely effect on epidemic detection should help define the usefulness of syndromic surveillance and identify approaches to increasing the likelihood that clinicians recognize and report an epidemic.

Epidemics of meningococcal meningitis occur in areas with particular environmental characteristics. We present evidence that the relationship between the environment and the location of these epidemics is quantifiable and propose a model based on environmental variables to identify regions at risk for meningitis epidemics. These findings, which have substantial implications for directing surveillance activities and health policy, provide a basis for monitoring the impact of climate variability and environmental change on epidemic occurrence in Africa.

An outbreak of severe acute respiratory syndrome (SARS) occurred in Singapore in March 2003. To illustrate the problems in diagnosing and containing SARS in the hospital, we describe a case series and highlight changes in triage and infection control practices that resulted. By implementing these changes, we have stopped the nosocomial transmission of the virus.

The host range of swinepox virus (SPV) is restricted to swine, although SPV has been shown to infect mammalian, non-swine cells, without recovery of infectious virus. SPV is a reasonable candidate for development as a non-productively replicating viral vector for use in non-swine, mammalian species, such as the cat. A novel SPV gene deletion (SPV 043) was created and found to be non-attenuating. This deletion was utilized to generate a stable recombinant virus expressing the Gag-Pro and Env proteins of feline leukemia virus (FeLV). Expression and replication of this vector was studied in embryonic swine kidney cells (ESK-4), and two feline cell lines, Crandell feline kidney cells (CRFK) and feline skin fibroblasts (FSF). Our results showed that feline cells were susceptible to infection by SPV and supported expression of foreign genes driven by synthetic poxvirus promoters, however, SPV viral DNA was not replicated in feline cells and infectious virus was not recovered. In addition, FeLV Gag virus-like particles were produced from both ESK-4 and CRFK cells and foreign antigens were incorporated into infectious SPV intracellular mature virions (IMV). These results suggest that SPV may have potential as a safe vaccine delivery vector for cats.

Integrin-mediated adhesion and B cell antigen receptor (BCR) signaling play a critical role in B cell development and function, including antigen-specific B cell differentiation. Here we show that the BCR controls integrin alpha4beta1 (VLA-4)-mediated adhesion of B cells to vascular cell adhesion molecule-1 and fibronectin. Molecular dissection of the underlying signaling mechanism by a combined biochemical, pharmacological, and genetic approach demonstrates that this BCR-controlled integrin-mediated adhesion requires the (consecutive) activation of Lyn, Syk, phosphatidylinositol 3-kinase, Bruton's tyrosine kinase (Btk), phospholipase C (PLC)gamma2, IP3R-mediated Ca2+ release, and PKC. In contrast, activation of mitogen-activated protein kinase kinase (MEK) or extracellular signal-regulated kinase (ERK) is not required, and simultaneous activation of MEK, ERK, and PKB is not sufficient either. Furthermore, Btk is also involved in the control of integrin-mediated adhesion of preB cells. The control of integrin alpha4beta1-mediated B cell adhesion by the BCR involves cytoskeletal reorganization and integrin clustering. These results reveal a novel function for the BCR and Btk, i.e., regulation of integrin alpha4beta1 activity, thereby providing new insights into the control of B cell development and differentiation, as well as into the pathogenesis of the immunodeficiency disease X-linked agammaglobulineamia (XLA).

In this study, we identify and characterize a novel transmembrane adaptor protein, designated Lck-interacting membrane protein (LIME), as a binding partner of the Lck Src homology (SH)2 domain. LIME possesses a short extracellular domain, a transmembrane domain, and a cytoplasmic tail containing five tyrosine-based motifs. The protein is primarily expressed in hematopoietic cells and lung. Interestingly, LIME expression is up-regulated by TCR stimulation and sustained up to 24 h, suggesting that LIME acts throughout the early to late stages of T cell activation. LIME is localized to membrane rafts and distributed within the T cell-APC contact site. Upon TCR stimulation of Jurkat T cells, LIME associates with Lck as a tyrosine-phosphorylated protein. Experiments using Jurkat T cells expressing CD8-LIME chimera reveal that the protein associates with phosphatidylinositol 3-kinase, Grb2, Gads, and SHP2, and activates ERK1/2 and JNK but not p38. Moreover, overexpression of LIME in Jurkat T cells induces transcriptional activation of the IL-2 promoter. Our data collectively show that LIME is a raft-associated transmembrane adaptor protein linking TCR stimuli to downstream signaling pathways via associations with Lck.

Diacylglycerol (DAG) lipase activity is required for axonal growth during development and for retrograde synaptic signaling at mature synapses. This enzyme synthesizes the endocannabinoid 2-arachidonoyl-glycerol (2-AG), and the CB1 cannabinoid receptor is also required for the above responses. We now report on the cloning and enzymatic characterization of the first specific sn-1 DAG lipases. Two closely related genes have been identified and their expression in cells correlated with 2-AG biosynthesis and release. The expression of both enzymes changes from axonal tracts in the embryo to dendritic fields in the adult, and this correlates with the developmental change in requirement for 2-AG synthesis from the pre- to the postsynaptic compartment. This switch provides a possible explanation for a fundamental change in endocannabinoid function during brain development. Identification of these enzymes may offer new therapeutic opportunities for a wide range of disorders.

p120-catenin stabilizes epithelial cadherin (E-cadherin) in SW48 cells, but the mechanism has not been established. Here, we show that p120 acts at the cell surface to control cadherin turnover, thereby regulating cadherin levels. p120 knockdown by siRNA expression resulted in dose-dependent elimination of epithelial, placental, neuronal, and vascular endothelial cadherins, and complete loss of cell-cell adhesion. ARVCF and delta-catenin were functionally redundant, suggesting that proper cadherin-dependent adhesion requires the presence of at least one p120 family member. The data reveal a core function of p120 in cadherin complexes, and strongly predict a dose-dependent loss of E-cadherin in tumors that partially or completely down-regulate p120.

The NC1 domains of human type IV collagen, in particular alpha3NC1, are inhibitors of angiogenesis and tumor growth (Petitclerc, E., Boutaud, A., Prestayko, A., Xu, J., Sado, Y., Ninomiya, Y., Sarras, M. P., Jr., Hudson, B. G., and Brooks, P. C. (2000) J. Biol. Chem. 275, 8051-8061). The recombinant alpha3NC1 domain contained a RGD site as part of a short collagenous sequence at the N terminus, designated herein as RGD-alpha3NC1. Others, using synthetic peptides, have concluded that this RGD site is nonfunctional in cell adhesion, and therefore, the anti-angiogenic activity is attributed exclusively to alpha(v)beta(3) integrin interactions with non-RGD motifs of the RGD-alpha3NC1 domain (Maeshima, Y., Colorado, P. C., and Kalluri, R. (2000) J. Biol. Chem. 275, 23745-23750). This nonfunctionality is surprising given that RGD is a binding site for alpha(v)beta(3) integrin in several proteins. In the present study, we used the alpha3NC1 domain with or without the RGD site, expressed in HEK 293 cells for native conformation, as an alternative approach to synthetic peptides to assess the functionality of the RGD site and non-RGD motifs. Our results demonstrate a predominant role of the RGD site for endothelial adhesion and for binding of alpha(v)beta(3) and alpha(v)beta(5) integrins. Moreover, we demonstrate that the two non-RGD peptides, previously identified as the alpha(v)beta(3) integrin-binding sites of the alpha3NC1 domain, are 10-fold less potent in competing for integrin binding than the native protein, indicating the importance of additional structural and/or conformational features of the alpha3NC1 domain for integrin binding. Therefore, the RGD site, in addition to non-RGD motifs, may contribute to the mechanisms of endothelial cell adhesion in the human vasculature and the anti-angiogenic activity of the RGD-alpha3NC1 domain.

This chapter describes three assays to monitor receptor-induced association of the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus (ASLV) with target bilayers: (1) the original assay for monitoring binding of the EnvA ectodomain (EnvA-PI) to target membranes (liposomes), (2) a modified and miniaturized EnvA-PI-liposome binding assay, and (3) an assay to measure binding of intact sarcoma/leukosis virus subtype A (ASLV-A) virus particles to target membranes. These assays are also useful for studying other receptor-activated viral fusion proteins. When one viral glycoprotein and one “simple” host cell receptor are involved, it should be possible to develop assays directly analogous to those described above for studying Tva-induced binding of the EnvA ectodomain (EnvA-PI) to target membranes. A general prerequisite for a fusion protein/target membrane binding assay is a soluble and correctly oligomeric form of the viral fusion protein ectodomain. The simplest host cell receptors that would be amenable to this type of analysis are type I or type II integral membrane proteins. The soluble versions of the ectodomains of these receptors, produced by genetic engineering or proteolytic release, could then be used to trigger the cognate fusion protein. The methodology could, similarly, be applicable to multimembrane-spanning host cell receptors when the functional part of the receptor is tethered at only one end or where an ectodomain loop preserves enough structure to function as a soluble analog, perhaps by generating a cyclic peptide analog of the loop. The same “receptor reagents” could be employed for intact virus particle/target membrane binding assays.

Nonsynonymous mutations in the coding regions of human genes are responsible for phenotypic differences between humans and for susceptibility to genetic disease. Computational methods were recently used to predict deleterious effects of nonsynonymous human mutations and polymorphisms. Here we focus on understanding the amino-acid mutation spectrum of human genetic disease. We compare the disease spectrum to the spectra of mutual amino-acid mutation frequencies, non-disease polymorphisms in human genes, and substitutions fixed between species.