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PubMed2002

Characterization of effector proteins translocated via the SPI1 type III secretion system of Salmonella typhimurium.

Salmonella spp. employ a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63) to translocate effector proteins into the host cytosol. The translocated effector proteins trigger diverse responses including bacterial internalization. In a mutation analysis we have defined the set of effector proteins mediating tissue culture cell invasion. This set includes sopE2 (centisome 40-42), sopB (SPI5, centisome 20) and in the case of S. typhimurium SL1344 also the phage-encoded effector sopE (SopEphi, centisome 59-60). A triple mutant SL1344 derivative deficient of SopE, SopE2 and SopB was more than 100-fold attenuated in tissue culture cell invasion. Phylogenetic analyses indicate that the last common ancestor of all contemporary Salmonella lineages already harbored all genes necessary for host cell invasion, namely the SPI1 type III secretion system, sopE2 and sopB. SopE, which is 70% identical to sopE2 is only present in some Salmonella strains and emerged later well after the divergence of the contemporary Salmonella lineages. Interestingly, S. typhimurium strains that harbor sopE are associated with epidemics, arguing that sopE is one of the factors determining the "fitness" of a strain. We found that SopE can specifically activate a different set of host cellular RhoGTPases than SopE2. This allows the bacteria to fine tune host cellular responses very precisely and may offer an explanation for the improved epidemic fitness of sopE-positive S. typhimurium strains.

Animals↗
PubMed2026

Interspecies Exchange of Mobile Genetic Elements During a Plant Disease Outbreak.

Outbreak sequencing provides insight into the origin and evolutionary processes acting on emerging pathogens. Sequencing a historic multihost outbreak of Ralstonia spp. in Martinique shows the outbreak was caused by two lineages that diverged at separate times from mainland populations. One lineage (Ralstonia pseudosolanacearum I-18) was originally introduced from Asia to South America, where it became well established prior to its dissemination to Martinique, where it retains a signature of specialization on solanaceous hosts. The novel lineage first identified during the outbreak (Ralstonia solanacearum IIB-4NPB) arose from a mainland population endemic to the Americas prior to its arrival in Martinique, where host-range expansion was observed. In contrast to minor changes in secreted effector protein repertoires, the emergent R. solanacearum IIB-4NPB acquired a novel integrative and conjugative element (ICERsoRUN1145). After identifying all Ralstonia spp. ICEs and mapping their spatial and phylogenetic distribution among Ralstonia spp. sampled during the outbreak, we found closely related ICEs circulating in mainland populations of R. pseudosolanacearum, indicating likely exchange between introduced and endemic Ralstonia spp. The family of ICEs in Ralstonia (ICERs) has a conserved bipartite structure and display a striking pattern of functional specialization in each cargo gene insertion hotspot: the first hotspot is a target for metabolic gene acquisition, and the second is a target for defense element acquisition. This work provides unparalleled phylogenetic and spatial resolution of an unusual outbreak and highlights the role of horizontal transfer in shaping the ecological success of an emerging pathogen.

Plant Diseases↗
PubMed2001

Analysis of Fc gammaRIII and IgG Fc polymorphism reveals functional and evolutionary implications of protein-protein interaction.

Fc gamma receptor III (Fc gammaRIII), a low-affinity receptor for the Fc portion of immunoglobulin G (IgG Fc), targets antigen-antibody complexes in a variety of effector cells of the immune system. We have investigated Fc gammaRIII and IgG Fc polymorphism and made comparative analysis of the functional and evolutionary implications of the interaction between these two molecules. Sequence analysis and comparison of the three-dimensional structure suggest that the C-terminal Ig domain of Fc gammaRIII is associated with the binding of IgG. The polymorphic residues of Fc gammaRIII are mainly located in the region of the C-terminal Ig domain that might be involved in IgG binding. Therefore, polymorphism and functional binding affinity seems to be related to each other as has been increasingly implicated in clinical observations. IgG Fcs, the natural ligand of Fc gammaRs, also exhibit significant polymorphism. Three regions have been identified where polymorphism frequently occurs: the putative FcR binding site, the linker region, and the intermolecular domain-domain interface of the second Ig domain. The putative Fc gammaR binding sites where polymorphic, and isotype-specific residues cluster are consistent with the regions that have been identified by mutagenesis and molecular modeling studies. The polymorphic residues of IgG Fc were mainly located in the molecular surface, which could be used in the recognition of other binding molecules. These observations suggest that polymorphic and isotype-specific residues in IgG Fc are closely related to their function and protein-protein interaction. Therefore, the colocalization of the polymorphic residues of Fc gammaRIII and IgG Fcs at their docking sites implies that the polymorphic residues would affect the IgG-Fc gammaRIII binding interactions to optimize their signaling through evolution.

Amino Acid Sequence↗
PubMed2002

Connective tissue growth factor gene expression alters tumor progression in esophageal cancer.

The ability of cancer cells to initiate specific fibroblast reactions may subsequently determine tumor evolution. In the present study we examined the coordinated expression of transforming growth factor-beta-1 (TGF-beta1), its signaling receptors, and its downstream mediator-connective tissue growth factor (CTGF)--and their impact on tumor progression and fibrogenesis in esophageal carcinomas. Messenger ribonucleic acid (mRNA) expression of TGF-beta1, CTGF, TGF-beta receptor subtype I ALK5 (TbetaR-IALK5), and TGF-beta receptor type II (TbetaR-II) was studied by Northern blot analysis in esophageal cancer and the normal esophagus. By means of immunohistochemistry and Western blot analysis, the respective proteins were localized in the tissue samples and the protein content was quantitated. Northern blot analysis revealed 3-fold and 4-fold increases (p < 0.05) in TGF-beta1 and CTGF mRNA levels, respectively, in esophageal cancer in comparison with normal controls, whereas TbetaR-I mRNA levels were significantly decreased and TbetaR-II mRNA levels were unchanged in the cancer samples. Immunostaining revealed results similar to those seen on the RNA level. TGF-beta1 and CTGF immunoreactivity were increased, TbetaR-II was unchanged, and TbetaR-IALK5 immunoreactivity was decreased. CTGF immunoreactivity was mainly present in the stroma surrounding the cancer cells but was also present in the cancer cells. The degree of fibrosis was different in squamous and adenocarcinomas and was significantly related to CTGF mRNA expression levels. The presence of CTGF in squamous cell carcinomas was associated with longer survival, whereas in adenocarcinomas it influenced survival negatively. The findings indicate that TGF-beta signaling is disturbed in esophageal cancer. CTGF, a downstream effector of TGF-beta action, differentially influences the composition of tumor microenvironment and distinct cell-matrix interactions in the two histological types of esophageal carcinoma, resulting in differences in tumor progression and patient survival.

Adenocarcinoma↗
PubMed1999

Eosinophils, ribonucleases and host defense: solving the puzzle.

The eosinophil ribonucleases eosinophil-derived neurotoxin (EDN/RNase 2) and eosinophil cationic protein (ECP/RNase 3) are among the major secretory effector proteins of human eosinophilic leukocytes, cells whose role in host defense remains controversial and poorly understood. We have recently described the unusual manner in which this ribonuclease lineage has evolved, with extraordinary diversification observed in primate as well as in rodent EDNs and ECPs. The results of our evolutionary studies suggest that the EDN/ ECP ribonucleases are in the process of being tailored for a specific, ribonuclease-related goal. With this in mind, we have begun to look carefully at some of the intriguing associations that link eosinophils and their ribonucleases to disease caused by the single-stranded RNA viral pathogen, respiratory syncytial virus (RSV). Recent work in our laboratory has demonstrated that eosinophils can mediate a direct, ribonuclease-dependent reduction in infectivity of RSV in vitro, and that EDN can function alone as an independent antiviral agent. The results of this work have led us to consider the possibility that the EDN/ECP ribonucleases represent a heretofore unrecognized element of innate and specific antiviral host defense.

Amino Acid Sequence↗
PubMed1985

Evolution of alloantibodies and suppressor cells in allografted mice treated for passive enhancement.

The kinetics and quality of the alloimmune reaction were studied in CBA (H-2k) mice treated for passive enhancement of tumor allografts (Sa 1 indigenous of A/J (H-2a or H-2k/d) mice). Serum samples of treated animals were tested for their biological properties relevant to different antibody isotypes in vitro (hemagglutination, complement-dependent cytotoxicity, and anaphylaxis, i.e., mast cell degranulation involving all main Ig isotypes; IgM, IgG2, and IgG1, IgE, respectively) as well as in vivo (allograft enhancement). Spleen cells from these treated animals were examined for their capacity to interfere with the rejection of tumor allografts by adoptive transfers into syngeneic recipients. In vitro, 51Cr release cytolysis assays were performed in order to test their cytolytic and regulatory activities in comparison to rejecting control animals. It has been shown that: grafted mice, pretreated for passive enhancement, kept their grafts longer and synthetized anaphylactic antibodies (mainly IgG1) earlier and at higher titers than normal serum controls, which rejected the same Sa 1 allografts. Mice with enhanced tumors synthetized cytotoxic antibodies (mainly IgG2) later than rejecting controls. Serum samples from treated and control animals, harvested 10 days (early sera) and 30 days (late sera) after grafting, were injected with a "normal dose" (0.2 ml) and a "high" dose (0.4 ml) to new CBA recipients grafted with Sa 1. Early immune sera were only enhancing at high doses when derived from animals previously treated for enhancement (at the low dose both immune sera were enhancing). Late sera, presenting both complement-fixing, cytotoxic (predominantly IgG2), and IgG1 anaphylactic alloantibodies in the two groups, induced enhancement in all cases, but more strongly when derived from the group treated for Sa 1 enhancement. Adoptive transfer of spleen cells from animals treated for passive enhancement were able either to inhibit the accelerated rejection (Day 10) or to promote enhancement of Sa 1 allogeneic cells (Day 30) while similar cells taken (Day 10 and Day 30) from control graft-rejecting mice transferred accelerated rejection. Among the transferred T-cell sub-populations, the suppressive effect was mediated by Lyt 2 T cells. In vitro, these spleen cells showed a weaker cytolytic activity than those of allograft-rejecting mice. Moreover, they were able to regulate the cytolytic activity of cytotoxic effector cells from specifically immunized CBA mice.

Animals↗
PubMed1984

Respiratory tract fluids: analysis of content and contemporary use in understanding lung diseases.

Respiratory tract fluid, produced from an enormous area spanning the mucosa of the nose to the alveolar surface, is a complex mixture of serum transudate and locally secreted proteins and glycomucoproteins and of inflammatory and immune effector cells intermingled. Its analysis is important in understanding the pathogenesis of respiratory diseases and remains essential for the clinical diagnosis of most lung disorders. Many basic facts about the formation and composition of this fluid remain unknown, and little information exists about absorptive mechanisms along the airways. Respiratory fluid is not homogeneous but has unique regional characteristics that are becoming better appreciated as more selective sampling methods are devised. Above all, it is a dynamic substance in healthy airways and diseased ones, and any specimen is just a point-in-time sample that can change in composition, often making serial analysis and comparisons necessary. Nasal fluids currently have limited diagnostic application except in allergic rhinitis. Expectorant (sputum) telescopes fluid and cells from all areas and is not from a specific locale, so the trend is to retrieve more selective and regional specimens of airways fluids. Technology largely does not exist to collect area specimens, except for bronchoalveolar lavage, which generally samples the alveolar surface. Clearly, bronchoalveolar lavage fluid analysis has been the favored way to characterize the peripheral air-spaces for the past 10 years or so, and most of this monograph has been devoted to normal data derived from lavage specimens and to a few examples of lung disease that reflect this burgeoning application. In many respects, results obtained from lavage fluid are virtually in catalogue form at present, and it remains to the future to know how some of the observations will help make diagnosis better or elucidate pathogenic mechanisms. Generally, bronchoalveolar lavage fluid analysis has led to better concepts of immunopathology of many diseases and provided new ways to monitor the evolution of certain diseases, especially the diffuse interstitial lung disorders, but development of specific criteria for diagnosis has been less rewarding. However, certain patterns of lymphocyte-predominant alveolitis, suggesting sarcoidosis or hypersensitivity diseases, recognition of specific T lymphocyte defects and opportunistic microorganisms as in AIDS, and the use of microprobe electron analysis to identify cellular particulates all point to more precision of diagnosis. Alveolar proteinosis and histiocytosis X may be detected from lavage fluid components. The prospects are truly exciting.(ABSTRACT TRUNCATED AT 400 WORDS)

Acquired Immunodeficiency Syndrome↗
PubMed2000

Digression on chemical electromagnetic field effects in membrane signal transduction--cooperativity paradigm of the acetylcholine receptor.

There is ongoing public concern on potential hazards and risks of even small electromagnetic fields (EMFs) such as those emanating from electrical appliances. Evolution and persistence of life in the natural geofields and basic scientific experience in using technical EMFs (F) suggest that, in general, living matter is remarkably stable against external field perturbations within the technical safety limits of the EM field strengths. Besides the trivial primary effects of EMF on ionic charges and dipolar matter, it is explicitly elaborated that cellular biochemical reactivity and channel transport processes are field dependent. However, equilibrium (K) and rate (k) constants are only sensitive to F if the reaction moments deltaM are finite, as seen in the general van't Hoff relationship d ln K/dF = deltaM/RT. Indeed, it is the difference (deltaM) in the electric or magnetic moments (M), representing the difference in the field forces on the reaction partners, that determines the extent of the field-induced transitions, say from an inactive conformer of a macromolecule to an active one. If small EM fields, which locally can only cause small shifts in K and k, are to become effective for chemical reactivity, amplification is required. A widely encountered concept of chemical amplification is structural, and thus functional, cooperativity, realized in many biopolymers. The cooperation of n units of such a polymer yields a larger deltaM(n)= ndeltaM and exponentially increases the field sensitivity of the overall equilibrium constant K(n) = Kn. Using the acetylcholine receptor protein as an example for signal amplification by structural cooperativity, explicit proposals are specified for the presumed amplification of small local field effects on proteins of the classical signal transduction cascades. Electric membrane field amplification by interfacial polarization in external fields is discussed in the context of using electric field pulses to transiently permeabilize cells and tissue for the direct transfer of effector substances and genes in cancer and gene therapy.

Cell Membrane↗
PubMed2000

Human immunosenescence: the prevailing of innate immunity, the failing of clonotypic immunity, and the filling of immunological space.

According to the remodeling theory of aging we proposed several years ago, the current data on human immunosenescence depicts a complex scenario where clonotypical immunity deteriorates, while ancestral innate/natural immunity is largely conserved or even up-regulated with age. Under an evolutionary perspective, antigens are the cause of a persistent life-long antigenic stress, responsible for the accumulation of effector CD8+/CD28- T cells, the decrease of naive T cells (CD95-) and the marked shrinkage of T cell repertoire with age. Concomitantly, NK cytotoxicity, chemotaxis, phagocytosis and complement activities remain unaffected or negligibly affected, in comparison to clonotypical immunity. Thus, immunosenescence is not a random deteriorative phenomenon but appears to inversely recapitulate an evolutionary pattern. On the whole, immunosenescence can be envisaged as the result of the continuous challenge of the unavoidable exposure to a variety of potential antigens (viruses, bacteria, but also food and self molecules among others). From this perspective antigens are nothing else than a particular type of stressor and immunosenescence appears to be the price paid to immunological memory, i.e. one of the main characteristics of the most evolutionary recent and sophisticated type of immunity. Together with the age-related thymic involution, and the consequent age-related decrease of thymic output of new T cells, this situation leaves the body practically devoid of virgin T cells, and thus likely more prone to a variety of infectious and non infectious diseases.

Aged↗
PubMed1997

Human natural killer lymphocytes directly recognize evolutionarily conserved oligosaccharide ligands expressed by xenogeneic tissues.

BACKGROUND: In discordant xenogeneic species combinations, vascularized transplants are hyperacutely rejected, due to binding of xenoreactive natural antibodies (XNA) to selected tissues of the graft, followed by activation of the complement and coagulation cascades. A major epitope recognized by human XNA is the terminal disaccharide Gal alpha(1,3)Gal. Poorly defined, early cell-mediated events also contribute to recognition and rejection of discordant xenografts, and we have suggested a role of natural killer (NK) lymphocytes in this process. METHODS: Human NK cells were used as effectors in functional assays of adhesion to and lysis of xenogeneic discordant endothelial cells in vitro. Adhesion and lysis inhibition experiments were performed using a large panel of carbohydrates, as well as F(ab')2 fragments of human XNA. COS cells transduced with the porcine alpha-galactosyltransferase were also used as targets for NK cell adhesion. RESULTS: We demonstrate that XNA-reactive carbohydrate epitopes expressed by xenogeneic cells, including Gal alpha(1,3)Gal, are also directly recognized by human NK cells. First, selected carbohydrates in solution displace with comparable efficiency both XNA and NK cell binding to xenogeneic endothelium; second, XNA F(ab')2 fragments selectively inhibit human NK cell adhesion to porcine endothelium, but not to human endothelium; third, unstimulated NK lymphocytes adhere selectively to COS-7 cells expressing the porcine glycosyltransferase that encodes the Gal alpha(1,3)Gal epitope. CONCLUSIONS: Collectively, our findings suggest that humoral and cellular components of the natural immune response against heterologous species independently evolved recognition patterns directed against overlapping carbohydrate determinants.

3T3 Cells↗
PubMed1990

Cytokines of the lung.

Communication between cells determines the steady-state composition of the lung in health and becomes a critical determinant of outcome in pathologic processes resulting in anatomic remodeling. This review presents the evolving concepts of the biology of cytokines (also known as peptide growth factors or biological response modifiers) in maintaining normal tissue growth and homeostasis. How these extracellular signaling proteins are involved in such pathologic disorders as spontaneous pulmonary fibrosis, sarcoidosis, pneumoconiosis, and the evolution and recovery from acute lung injury is also discussed. During the past decade the cytokines have come to the fore as important multifunctional mediators of cell behavior and cell-cell communication. A wide range of cellular responses are influenced or triggered when cytokines interact with cells. These include mitosis, chemotaxis, angiogenesis, cytoskeleton arrangement, immunomodulation, and extracellular matrix production. Cytokines influence cell behavior by binding to specific high affinity surface receptors on target cells. These receptors are linked in turn at the cell membrane to a complex array of intracellular signaling pathways. Individual cytokines may inhibit as well as promote cellular functions such as mitosis and thereby play a critical role in homeostasis of normal tissue elements. Hence, cytokines are intimately involved in normal tissue homeostasis as well as in processes eventuating in growth and remodeling. All cells produce and secrete cytokines at some time during their life. Each cytokine is capable of modulating more than one cellular function. Although produced by a variety of cell types, the triggers that induce a specific cytokine to be produced differ between cells. Many of the cytokines share regions of homologous nucleic acid sequences, suggesting that they are members of larger gene families. Given that tissues and cells are exposed to complex cytokine mixtures rather than to individual cytokines, recent attention has turned to understanding how cytokines interact. The combined effects of cytokine mixtures have proved to be both complex and unpredictable based on knowledge of the separate actions of the individual cytokines involved. In studies of the role of cytokines in lung disease, early research attention has focused on those cytokines released by alveolar macrophages (the so-called macrophage-derived growth factors). However, structural cells as well as immune effector cells of the lung are capable of cytokine production and release. The cytokines receiving the most attention to date in relation to pulmonary diseases include platelet-derived growth factor (PDGF), interleukin-1 (IL-1), transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), insulinlike growth factor I (IGF-I), and, most recently, interleukin-6 (IL-6).(ABSTRACT TRUNCATED AT 400 WORDS)

Biological Factors↗
PubMed2002

Immune pathophysiology of aplastic anemia.

Aplastic anemia (AA) remains an elusive disease. Its pathophysiology is not only fascinating by the seemingly simple findings of cytopenia and marrow hypoplasia, but may also contain key information to the understanding of other fundamental processes such as stem cell regeneration, evolution, and immune control of clonal diseases. Although measurements of blood counts provide an objective tool to assess the disease activity and response to the therapy, immune pathophysiology of AA, as inferred from the successes of immunosuppression, provides only few other clinical clues. Similarly, the current laboratory evidence remains mostly indirect. In spite of the recognition of immune pathways of hematopoietic inhibition and apoptosis in AA, the fundamental question about the nature of the antigen(s) inciting or maintaining the pathologic immune response that ultimately leads to bone marrow failure, remains open. However, recognition of the immune targets may aid in understanding not only the pathogenesis but also many of clinical associations and the late squelae of AA. For example, abnormal cells in AA and myelodysplastic syndrome (MDS) MDS may harbor inciting antigens but the immune response lacks selectivity. Clonal selection pressure may be a result of this process or alternatively, emergence of tolerance could lead to the establishment of abnormal hematopoiesis. Clonal proliferation of large granular lymphocytosis could represent an example of an exaggerated response to an immunodominant hematopoietic antigen. In addition to the traditional functional or phenotypic analysis, pathologic immune response in AA can be studied on molecular level by identifying and quantitating T cell clones based on the presence of unique variable B-chain CDR3 sequences. Detection of clonal expansion is based on the observation that in infections and autoimmune conditions, the presence of antigenic drive will lead to the expansion and overrepresentation of T cell clones recognizing this antigen. However, simple analysis of clonal representation is not sufficient to resolve the complex nature of the immune repertoire in the context of genetic and clinical heterogeneity. Therefore, we analyzed VB and CDR3 repertoire in CD4 and CD8 cells, activated or effector cell subsets. To distinguish truly expanded and likely immunodominant clones, we first studied VB distribution and cloned CDR3 sequences from expanded VB families. Identified clonotypic sequences can be used to design molecular tests to quantitate the strength of pathologic immune response. Clonotype sharing has been confirmed in patients with similar clinical features indicating presence of common antigens. In addition, quantitative analysis showed correlation with the therapy response. Persistence and patterns of clonotypes may be helpful in the classification of immune-mediated marrow failure based on the immune characteristics and will allow inferences into the inciting pathways.

Anemia, Aplastic↗
PubMed1994

Evolutionary conservation of a human function-associated molecule on murine natural killer cells: expression and function.

Using a novel anti-natural killer (NK) cell monoclonal antibody (MoAb), we have recently identified an evolutionary conserved function-associated molecule (FAM) present on fish, rat and human NK cells. This molecule is involved in NK cell function as anti-FAM MoAbs inhibit cytotoxicity, stimulate lymphokine secretion and inhibit conjugate formation between effector cells and target cells. We now have examined murine NK cells for the presence of this structure. It was observed by two-colour flow cytometric analysis that the anti-FAM MoAb 5C6 specifically bound to a subpopulation of nylon wool non-adherent splenic lymphocytes (19-20%). The expression of the FAM molecule was restricted to NK cells that expressed the NK1.1 antigen. Neither T cells, B cells, nor macrophages reacted with the anti-FAM MoAb. Analysis of FAM expression in various lymphoid tissues revealed that splenocytes expressed the greatest numbers of MoAb(+) cells. Generation of lymphokine-activated killer (LAK) cells and adherent lymphokine-activated killer (ALAK) cells resulted in higher levels of FAM expression. The anti-FAM MoAb 5C6 also detected the presence of FAM on fresh SCID NK cells. It was demonstrated that the anti-FAM MoAb 5C6 inhibited the lysis of target cells by endogenous NK cells, activated NK cells, 5d LAK cells, ALAK cells and SCID NK cells. Moreover, conjugate assays demonstrated involvement of this molecule in recognition between NK cells and target cells.

Animals↗
PubMed1998

Evolutionary analysis of G-proteins in early metazoans: cloning of alpha- and beta-subunits from the sponge Geodia cydonium.

G-protein-coupled (seven-transmembrane segment)-receptors represent a major group of metazoan receptors, involved in transduction of extracellular signals. The G-proteins, which are made up of Galpha/beta/gamma-subunits, link the receptors to the effector system(s). To analyze the phylogenetic relationships among the metazoan alpha-subunits of G-proteins, cDNAs of alpha-subunits were isolated from Geodia cydonium, a marine sponge belonging to the lowest metazoan phylum, Porifera. One encodes a putative isotype of a stimulator of the adenylyl cyclase (Galpha s), another one a putative inhibitor of the adenylyl cyclase (Galpha i/o) and the third one a putative activator of phospholipase C (Galpha q). In addition one putative beta-subunit was cloned from the same species. The deduced amino acid sequences of the sponge Galpha s -(putative Mr 44749), the Galpha i/o -(Mr 41064) and the Galpha q subunits (Mr 41363) were found to display high similarity with the corresponding sequences from higher Metazoa, and are only distantly related to those of slime mold, yeast or plants. Of lower similarity are the sequences of the beta-subunits among animals and plants, thus not allowing robust grouping. These data demonstrate that the phylogenetic relationships, obtained from analyses of the alpha subunits from metazoan G-proteins, support the conclusion that all metazoan phyla, including the Porifera are of monophyletic origin.

Amino Acid Sequence↗
PubMedDate not supplied

Complexity of human T-cell antigen receptor beta-chain constant- and variable-region genes.

Immune systems of vertebrates function via two types of effector cells, B and T cells, which are capable of antigen-specific recognition. The immunoglobulins, which serve as antigen receptors on B cells, have been well characterized with respect to gene structure, unlike the T-cell receptors. Recently, cDNA clones thought to correspond to the beta-chain locus of the human and mouse T-cell receptor have been described. The presumptive beta-chain clones detect gene rearrangement specifically in T-cell DNA and show homology with immunoglobulin light chains. The similarity of the T-cell beta-chain gene system to the immunoglobulin genes has been further demonstrated by the recent observation of variable- and constant-region gene segments as well as joining segments and putative diversity segments. We report here the characterization of cDNA and genomic clones encoding human T-cell receptor beta-chain genes. There are two constant-region genes (C beta 1 and C beta 2), each capable of rearrangement and expression as RNA. The gene arrangement, analogous to that of mouse beta-chain genes, shows strong evolutionary conservation of the dual C beta gene system in these two species.

Base Sequence↗
PubMed2001

Microbial origin of plant-type 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate synthases, exemplified by the chorismate- and tryptophan-regulated enzyme from Xanthomonas campestris.

Enzymes performing the initial reaction of aromatic amino acid biosynthesis, 2-keto-3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthases, exist as two distinct homology classes. The three classic Escherichia coli paralogs are AroA(I) proteins, but many members of the Bacteria possess the AroA(II) class of enzyme, sometimes in combination with AroA(I) proteins. AroA(II) DAHP synthases until now have been shown to be specifically dedicated to secondary metabolism (e.g., formation of ansamycin antibiotics or phenazine pigment). In contrast, here we show that the Xanthomonas campestris AroA(II) protein functions as the sole DAHP synthase supporting aromatic amino acid biosynthesis. X. campestris AroA(II) was cloned in E. coli by functional complementation, and genes corresponding to two possible translation starts were expressed. We developed a 1-day partial purification method (>99%) for the unstable protein. The recombinant AroA(II) protein was found to be subject to an allosteric pattern of sequential feedback inhibition in which chorismate is the prime allosteric effector. L-Tryptophan was found to be a minor feedback inhibitor. An N-terminal region of 111 amino acids may be located in the periplasm since a probable inner membrane-spanning region is predicted. Unlike chloroplast-localized AroA(II) of higher plants, X. campestris AroA(II) was not hysteretically activated by dithiols. Compared to plant AroA(II) proteins, differences in divalent metal activation were also observed. Phylogenetic tree analysis shows that AroA(II) originated within the Bacteria domain, and it seems probable that higher-plant plastids acquired AroA(II) from a gram-negative bacterium via endosymbiosis. The X. campestris AroA(II) protein is suggested to exemplify a case of analog displacement whereby an ancestral aroA(I) species was discarded, with the aroA(II) replacement providing an alternative pattern of allosteric control. Three subgroups of AroA(II) proteins can be recognized: a large, central group containing the plant enzymes and that from X. campestris, one defined by a three-residue deletion near the conserved KPRS motif, and one possessing a larger deletion further downstream.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed2000

RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis.

RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

ATP-Binding Cassette Transporters↗
PubMed2000

Design of large metabolic responses. Constraints and sensitivity analysis.

Metabolic control analysis (Kacser & Burns (1973). Symp. Soc. Exp. Biol.27, 65-104; Heinrich & Rapoport (1974). Eur. J. Biochem.42, 89-95) has been extensively used to describe the response of metabolic concentrations and fluxes to small (infinitesimal) changes in enzyme concentrations and effectors. Similarly, metabolic control design (Acerenza (1993). J. theor. Biol.165, 63-85) has been proposed to design small metabolic responses. These approaches have the limitation that they were not devised to deal with large (non-infinitesimal) responses. Here we develop a strategy to design large changes in the metabolic variables. The only assumption made is that, for all the parameter values under consideration, the system has a unique stable steady state. The procedure renders the kinetic parameters of the rate equations that when embedded in the metabolic network produce the pattern of large changes in the steady-state variables that we aim to design. Structural and kinetic constraints impose restrictions on the type of responses that could be designed. We show that these conditions can be transformed into the language of mean-sensitivity coefficients and, as a consequence, a sensitivity analysis of large metabolic responses can be performed after the system has been designed. The mean-sensitivity coefficients fulfil conservation and summation relationships that in the limit reduce to the well-known theorems for infinitesimal changes. Finally, it is shown that the same procedure that was used to design metabolic responses and analyse their sensitivity properties can also be used to determine the values of kinetic parameters of the rate laws operating "in situ".

Animals↗