bluuebunny/crossref_metadata_2025 huggingface dataset:
Use of Tick Cell Culture‐derived Anaplasma marginale Antigen in a Competitive ELISA for Serodiagnosis of Anaplasmosis
Authors: SALIKI JEREMIAH T. , BLOUIN EDMOUR F. , RODGERS SANDY J. , KOCAN KATHERINE M.
Published: February 2006

ABSTRACT: Anaplasma marginale was propagated in a continuous tick cell line and detergent‐solubilized infected cells were used as antigen in a competitive ELISA (C‐ELISA) for detection of Anaplasma‐specific antibody in bovine sera. Positive control sera competed well (≥35% inhibition) with an A. marginale‐specific monoclonal antibody for binding to this antigen, while negative sera failed to compete (<35% inhibition). The C‐ELISA was compared to the standard complement‐fixation test (CFT) using 2,208 bovine sera. Overall, C‐ELISA was more sensitive than CFT (24.9% versus 9.4%), mainly because CFT yielded “suspicious” or “anti‐complementary” results in 10.5% of the sera and also failed to identify several vaccinated and carrier cattle that were C‐ELISA‐positive. The apparent agreement between CFT and C‐ELISA was 89.6% and the kappa value was 0.6. These results show that this C‐ELISA would be a suitable replacement of the CFT as the standard test for detection of A. marginale antibody.
The Development of a Semi‐automated Latex Agglutination Test for the Detection of Antibodies toAnaplasma marginaleUsing a Cell Culture‐derived Antigen
Authors: RODGERS S. J. , SALIKI J. T. , BLOUIN E. F. , KOCAN K. M.
Published: February 2006

ABSTRACT:Serologic diagnosis of anaplasmosis is currently done by the complement‐fixation, ELISA, and card agglutination tests. These tests have utilizedA. marginaleharvested from bovine erythrocytes as antigen which is often contaminated with erythrocyte stroma. We are currently testingA. marginalepropagated in aIxodes scapulariscell line as antigen for serologic tests. In this study, we report the use of the cell culture‐derivedA. marginaleas antigen for development of a rapid, semi‐automated latex agglutination test. Diluted serum and latex (polystyrene microspheres), sensitized with cell culture‐derivedA. marginaleproteins, were dispensed into 96‐well microtiter plates. An initial reading of light transmission was recorded by a computer‐interfaced scanning autoreader. After 30 minutes, the plates were mixed and read a second time, recording the delta % light transmittance. The sensitized latex microspheres (latex) agglutinated in the presence of A. marginale antibodies, thus producing an increase in light transmittance. In preliminary tests, 724/977 of the sera were positive forA. marginaleantibodies with an apparent agreement of 83.3% when compared with the complement‐fixation test. Sensitization and sera dilution buffers were shown to have a marked effect on the sensitivity and specificity of this assay. Results will be presented on the optimization of buffers and the testing of sera from experimentally and field‐infected cattle.
Global Eradication of Rinderpest: Yea or Nay?
Authors: SCOTT GORDON R.
Published: February 2006

ABSTRACT: Rinderpest is a scourge continuing to change the course of history. The German “Rinderpest” in English fails to convey the spectre of Death, Despair and Desolation long associated with the disease. Although the causal morbillivirus cross‐protects against other morbillivirus, there is only one rinderpest serotype which is both immunosuppressive and immunogenic. Safe vaccines induce lifelong protection in cattle. Transmission follows close contact between sick and healthy artiodactyls. Infectious periods are short and deaths may be curtailed by liberal fluid replacement therapy. Carriers do not exist. In other words rinderpest is a prime candidate for eradication. Why has it not happened? The major obstacle is “man's inhumanity to man”. Rinderpest thrives in a milieu of armed conflict and fleeing refugee masses. Until world peace is secured, the “Nays” win the argument.
Contagious Bovine Pleuropneumonia: The Costs of Control in Central/Southern Africa
Authors: WINDSOR R. S. , WOOD A.
Published: February 2006

ABSTRACT: Contagious bovine pleuropneumonia (CBPP) caused by Mycoplasma mycoides subspecies mycoides small colony (M. mycoides) has been endemic in many parts of Africa for the greater part of this century. Because of an international vaccination campaign the disease was almost eliminated from the continent.

With the deteriorating economic situation of many countries, vaccination programs have been allowed to slip; drought conditions have resulted in great movements of livestock and the disease has spread rapidly to the east and south. For Government Veterinary Departments, the implications of this resurgence of CBPP, are serious. The costs threaten to overwhelm completely under‐funded government services. Where cost‐recovery measures are introduced, the charging for vaccines may damage rapport with communities and erode the credibility of the government services. Farmers may find the loss of cattle and the costs of vaccine severe burdens which threaten their livelihoods and social well‐being (Zambia) and even, in extreme cases, their survival (Botswana). Controlling the disease today presents new possibilities which include increased cross‐border collaboration (Malawi/Tanzania), increased emphasis on monitoring (N. Zambia) and the greater involvement of communities in disease control (W. Zambia).
Serum‐free Media for the in Vitro Cultivation of Cowdria ruminantium
Authors: ZWEYGARTH E. , JOSEMANS A. I. , HORN E.
Published: February 2006

ABSTRACT: The in vitro culture of Cowdria ruminantium, the causative agent of heartwater in domestic ruminants, was first achieved in 1985; since then, most groups working with this culture system have used media which were supplemented with serum and, in addition, most of them contained tryptose phosphate broth. These undefined products vary from batch to batch and often fail to support the growth of C. ruminantium. We are therefore working towards the development of a completely chemically defined medium for Cowdria culture. We attempted the propagation of the Welgevonden stock of C. ruminantium in bovine endothelial cell cultures in a variety of serum‐free culture media. Four synthetic media gave unsatisfactory results, these were: SFRE‐199, Iscove's modified Dulbecco's medium, Dulbecco's modified Eagle's medium, and Leibovitz L‐15. These media were all supplemented with a proprietary solution A (components solution A of the HL‐1 medium kit, containing transferrin, testosterone, sodium selenite, ethanolamine, saturated and unsaturated fatty acids, and stabilizing proteins). Three other serum‐free media did support the growth of C. ruminantium: a modified HL‐1 medium, Dulbecco's modified Eagle's medium nutrient mixture Ham F‐12 (DME/F‐12), and RPMI 1640. The chemical composition of DME/F‐12 and RPMI 1640 are published, but not that of the HL‐1 medium. Each of these media was supplemented with proprietary solution A. Various supplements were investigated as alternatives to the incompletely specified solution A; bovine lipoproteins and bovine transferrin were identified as essential supplements which effectively replaced compound solution A. C. ruminantium was propagated in the three growth‐supportive media for at least 10 passages.
Purification of Cowdria ruminantium Organisms for Use in Genome Analysis by Pulsed‐field Gel Electrophoresis a
Authors: DE VILLIERS E. P. , BRAYTON K. A. , ZWEYGARTH E. , ALLSOPP B. A.
Published: February 2006

ABSTRACT: Cowdria ruminantium is an obligate intracellular rickettsial pathogen which is responsible for a tick‐borne disease of domestic and wild ruminants called heartwater or cowdriosis. Although several genes have been cloned and partially sequenced, the genome size, gross structure, and organization of the C. ruminantium genome is unknown. Genome analysis of the organism has been hindered because it is difficult to obtain C. ruminantium DNA free from contaminating host cell DNA, and this probably accounts for the lack of genome size data for this organism. In this study we investigated several methods for purifying C. ruminantium from bovine cellular contaminants and organisms of a relatively high purity were obtained. These were used to prepare Cowdria DNA which was analyzed by pulsed‐field gel electrophoresis (PFGE) and which revealed a genome approximately 1900 kbp in length plus an additional extra‐chromosomal fragment migrating with an apparent size of 815 kbp. This is the first time that the genome size of C. ruminantium has been determined and the first demonstration of an extrachromosomal element.
Caprine T‐Cell Receptor Variable β‐Chain (TCRVβ) Repertoire Analysis and Potential Applications in Cowdriosis Immune Response Studiesa
Authors: OBEXER‐RUFF G. , FLURI A. , HEIN W. , LAZARY S. , PETERHANS E. , BERTONI G.
Published: February 2006

ABSTRACT: Anchor polymerase chain reaction has been applied to the study of caprine TCR Vβ‐chain repertoire at the mRNA level in peripheral blood of a Saanen goat. Single stranded, g‐tailed cDNA synthesized from total RNA was PCR‐amplified using a sheep Vβ constant region primer (3′) and a poly(dC) anchor primer (5′) at the variable region end of the TCR Vβ‐chain. The obtained amplicon was subsequently cloned into Bluescript plasmid vector. A total of 72 recombinant clones whereof 61 contained an insert of appropriate size were harvested. Up to now, the full length sequences of a total of 55 clones have been obtained. Nine clones were rearranged but not functional due to stop codons. Forty‐five sequences were functionally rearranged and further analyzed. They were classified into 15 different Vβ families on the basis of V‐region sequence homology with their human counterpart. Vβ families corresponding to the 9 published bovine families were represented in our library. This complexity enables us to develop Vβ family‐specific primers in order to study the TCR Vβ repertoire of other goat breeds of interest, i.e., the Creole goat. There the TCR Vβ repertoire analysis and kinetic study of the cowdriosis model will provide insight into the type of the immune response and the status of protection during the immunization process and challenge.
Comparative Ethnoveterinary and Serological Evaluation of the Karimojong Community Animal Health Worker Program in Uganda
Authors: JOST C. , STEM C. , RAMUSHWA M. , TWINAMASIKO E. , MARINER J.
Published: February 2006

ABSTRACT:The Karimojong cahw program, in which members of the pastoral Karimojong community were trained as basic veterinary care providers, was evaluated using ethnoveterinary techniques. A serological study of rinderpest antibody titers was also completed to compare serological results with ethnoveterinary results in the case of one disease, rinderpest. An outbreak of rinderpest‐like disease that occurred during the field phase of the study was also investigated. The results show that rinderpest does exist in Karamoja in an apparently endemic form. Given the significant value placed by Karimojong herders on vaccinated animals, it can be concluded that they would adequately vaccinate their herds if they could trust the vaccine and the vaccinator. Discrepancies between ethnoveterinary and serological results show that ethnoveterinary techniques do not provide numerically accurate data concerning protection from disease, but that they can, nonetheless, provide basic, cost effective indications of disease patterns.
Animal Disease Regionalization and Its Impact on Tropical Countries
Authors: BOKMA BOB H.
Published: February 2006

ABSTRACT: This paper reviews progress that has been made around the world in animal disease regionalization. Signatory countries of the Uruguay Round of the General Agreement on Tariffs and Trade (GATT) and other free‐trade associations are currently implementing regionalization, one of the cornerstone provisions of the Uruguay Round of the GATT and the World Trade Organization's (WTO) Sanitary and Phytosanitary Agreement. Regionalization allows countries to protect the health status of their animal herds and at the same time promote their animal and animal product export markets. The implementation of regionalization by importing countries affects export markets of tropical nations. Veterinary officials of tropical countries must give regionalization due priority such that import and export trade in animals and animal products is not hindered, and the public and animal herd health are protected.
Successes and Failures in the Tropical Bont Tick Eradication Campaigns in the French Antilles
Authors: ROSE‐ROSETTE F. , BARRÉ N. , FOURGEAUD P.
Published: February 2006

ABSTRACT: The Amblyomma variegatum eradication campaigns, based on three phases and a 5‐year program, as in other islands of the Caribbean, started in the French Antilles in April 1994 with various financial inputs. The progress of the campaigns in the two islands of Guadeloupe and Martinique contrast sharply. Owing to a shortage and inadequacy of results with regard to the initial objectives, particularly in organization, communication efforts, cattle identification, and application of acaricides on livestock, the European Community which was the most important donor, did not support the Guadeloupean program in 1996 and 1997. This induced a deep crisis in the local sanitary organization, which has the responsibility for the execution of the project, and the almost total interruption of acaricide treatments in Guadeloupe and its dependencies (Marie‐Galante, St Martin, Désirade). In Martinique on the contrary, a well‐defined organization, communication strategy, and personnel training effort resulted in a generalized census of livestock owners. Identification of cattle increased dramatically in number and the systematic treatment of livestock started in satisfying conditions. It is too early to anticipate on the final result of the campaign, but it is clear that a longer period of time than previously planned, will be necessary to achieve the program. If this prolongation is accepted, it may allow Guadeloupe to take part again in the campaign, a condition absolutely necessary for its regional success.
Entomopathogenic Nematodes as a Potential Biological Control Method for Ticksa
Authors: KOCAN KATHERINE M. , BLOUIN EDMOUR F. , PIDHERNEY MARSHA S. , CLAYPOOL P. L. , SAMISH MICHAEL , GLAZER ITAMAR
Published: February 2006

ABSTRACT: Entomopathogenic nematodes have been used for biological control of certain insect pests. In these studies the nematodes were tested as a possible biological control agent for engorged female ticks. Five species of infective juveniles (IJs) were tested initially for their ability to penetrate and kill ticks, including Steinernema glaseri (SG), S. riobravus (SR), S. carpocapsae (DT), S. feltiae (SF) and Heterorhabiditis bacteriophora (HP88). Infective juveniles (IJs) of SRs and SFs appeared to be the most effective in killing ticks and invaded and killed 30 to 100% of replete females. These two nematode species were tested on several tick species including Amblyomma americanum, A. cajennense, A. maculatum, Dermacentor variabilis and Rhipicephalus sanguineus. Although the killing rate of each tick species varied, the nematodes did not appear to be host specific and were able to kill ticks of all species tested. Egg mass weights of exposed ticks of each species were significantly lower than those of the controls. Ticks were examined with microscopy to determine whether nematodes entered and multiplied inside ticks. Partially fed female Amblyomma americanum and Dermacentor variabilis exposed to 5000 IJs in petri dishes were collected at 8, 24, 48 and 96 hrs (Trial 1) and 1,2,3,4, 7 and 9 days (Trial 2) post‐exposure, and fixed, processed and embedded in resin for microscopy studies. Only a few nematodes were seen in the hemocoel and tissues and they were surrounded by a clear space. Bacteria, released from the nematodes, were present in the exposed ticks and appeared to increase daily causing a generalized infection. Degeneration of tick tissues and death of the ticks appeared to result from bacterial proliferation. Nematodes did not multiply within ticks as they do in insect larvae. In these controlled laboratory studies, exposure of ticks to nematodes resulted in tick mortality and reduced egg production. Entomopathogenic nematodes appear to have potential as a biological control agent of ticks, but future studies will be required to determine whether nematode/tick interactions will occur in the field.
Intestinal Epithelial‐Mesenchymal Cell Interactions
Authors: KEDINGER M. , DULUC I. , FRITSCH C. , LORENTZ O. , PLATEROTI M. , FREUND J. N.
Published: February 2006

ABSTRACT: Intestinal morphogenesis, as well as maintenance of the stem cell population and of the steady state between cell proliferation and differentiation, results from controlled cell interactions. There is growing evidence that the mesenchymal cells control epithelial cell behavior via their own expression and induction in the epithelial cells of key regulatory genes. This heterologous cross talk involves basement membrane molecules and paracrine factors. New in vitro/in vivo cellular models allowed us to analyze various mesenchymal cell phenotypes and to show that they exhibit different inductive properties on epithelial cells and that their proliferation and metabolic properties are differentially modulated by cytokines. Finally the epithelial‐mesenchymal unit is controlled by hormonal and exogenous factors.
Interactions between Epithelial Cells and Immune Cells in the Intestine
Authors: STROBER WARREN
Published: February 2006

ABSTRACT: Not merely a passive barrier as previously assumed, epithelial cells act as sensitive indicators of infection that initiate defense responses. Noninvasive as well as invasive organisms have been demonstrated to elicit production of chemoattractants. That noninvasive organisms have this capacity suggests that receptor‐mediated signaling pathways may be involved. Epithelial cells have been found to release both chemokines and cytokines in a precise mixture that varies according to the origin or maturity of the cell. Different microorganisms also elicit different response patterns. The information presented provides a basis for a new view of epithelial cell function in relation to host defense.
The Laminins: Role in Intestinal Morphogenesis and Differentiation
Authors: SIMON‐ASSMANN P. , LEFEBVRE O. , BELLISSENT‐WAYDELICH A. , OLSEN J. , ORIAN‐ROUSSEAU V. , DE ARCANGELIS A.
Published: February 2006

ABSTRACT: Dynamic and reciprocal heterotypic cell interactions are crucial for intestinal morphogenesis and differentiation. This paper emphasizes the role of basement membrane molecules and in particular of laminins as potent mediators in this intercellular cross talk. Changes in the expression or localization of laminin isoforms or of integrins during development and cell migration strengthen the concept that heterogeneity in cell‐matrix interactions could mediate distinct cell responses. A combination of genetic or biochemical approaches associated with in vitro models allows us to study the potential role of each laminin isoform in basement membrane assembly, cell migration, or cell differentiation.
From Cytoskeleton to Polarity and Chemoreception in the Gut Epithelium
Authors: HÖFER DIRK , JÖNS THOMAS , KRAEMER JUTTA , DRENCKHAHN DETLEV
Published: February 2006

ABSTRACT: The membrane surface of polarized epithelial cells can be separated in apical and basolateral domains that differ in molecular composition and function. Components of the cytoskeleton obviously play an important role in both generation and maintenance of epithelial polarity. The microtubular system is uniformly aligned in enterocytes and pancreatic acinar cells with the minus ends of the microtubules located apically and the plus ends basally. Drug‐induced disassembly of microtubules results in mistargeting of pancreatic zymogen granules and of apical but not basolateral membrane proteins of enterocytes. Association of zymogen granules with the minus end motor protein, cytoplasmic dynein, and components of its receptor, dynactin, indicates that microtubules are important for vectorial delivery of apical but not basolateral post‐Golgi vesicles. The lateral membrane domain of the gut epithelium is scaffolded by a spectrin‐based membrane cytoskeleton consisting basically of actin, spectrin, and ankyrin. Ankyrin binds to different integral membrane proteins, such as the sodium pump in glandular and kidney epithelial cells or to the anion exchanger (AE2) of gastric parietal cells, thereby probably playing a critical role in maintenance and/or generation of the polarized distribution of these basolateral membrane proteins. Scattered epithelial cells sharing apical cytoskeletal features of gustatory receptor cells were identified as brush cells (tufted cells). These cells are rich in nitric oxide synthase and contain in their apical brush border the gustatory trimeric G‐protein, α‐gustducin, indicating that brush cells are involved in chemoreceptive signaling.
Experimental Models to Study Molecular Mechanisms Underlying Intestinal Inflammation
Authors: ELSON C. O. , CONG Y. , BRANDWEIN S. , WEAVER C. T. , McCABE R. P. , MÄHLER M. , SUNDBERG J. P. , LEITER E. H.
Published: February 2006

ABSTRACT: Experimental animal models, particularly the newer mouse models, have convincingly demonstrated that CD+ T cells play a central role in chronic intestinal inflammation. Such CD4+ effector T cells are induced by the bacterial flora. In at least one model, it is conventional protein antigens that are stimulating these pathogenic T cells. The antigens driving disease seem to be a selective subset of immunodominant proteins, likely derived from a subset of organisms. Multiple genes contribute to colitis susceptibility and a number of these genes are being localized.
Helicobacter pylori Infection and the Pathogenesis of Duodenal Ulceration
Authors: WALKER M. M. , CRABTREE J. E.
Published: February 2006

ABSTRACT: Helicobacter pylori is a gram‐negative spiral bacterium confined to the habitat of gastric‐type epithelium. H. pylori causes duodenal ulceration by a cumulative effect of antral predominant gastritis with increased acid secretion, consequent gastric metaplasia in the duodenum (a site of further colonization by H. pylori), duodenitis, reduced duodenal bicarbonate secretion, and mucosal damage. Bacterial factors influence outcome. Major determinants are the production of a vacuolating toxin and the presence of CagA, an immunodominant product of a nonconserved gene cagA, a marker for the cag pathogenicity island that encodes virulence genes involved in induction of epithelial chemokine responses. In ulcer patients the mucosal immune response is polarized to a T‐helper‐1 (Th1) cell‐mediated response, which may contribute to mucosal damage. Eradication of H. pylori restores acid output to normal. Loss of both acid and bacteria halts gastroduodenitis and allows ulcer healing. Gastric metaplasia does not regress in the short term.
Heterogeneity of Celiac Disease: Clinical, Pathological, Immunological, and Genetic
Authors: FERGUSON ANNE , GILLETT HELEN , HUMPHREYS KENNETH , KINGSTONE KATHLEEN
Published: February 2006

ABSTRACT: In this paper we consider recent new data on the pathological features of gluten sensitivity and on the disease‐associated antigens, in the context of a multistage hypothesis that we have been developing for the last five years. This incorporates concepts of oral tolerance induction, mucosal T‐cell and antibody‐mediated injury, and genetic contributions. Until now, there has been complete agreement that the diagnosis of celiac disease must be based on small bowel histology. There are patients with low‐grade gluten‐sensitive enteropathy, in whom the only morphological abnormality is a high count of intraepithelial lymphocytes (IEL). Some, but not all, also have positive serum IgA anti‐endomysium antibody (AEA). With good techniques, in a properly accredited laboratory, in a patient suspected on clinical grounds to have celiac disease, a positive serum IgA AEA test (perhaps, alternatively, high‐titer anti‐transglutaminase by ELISA), is virtually diagnostic of the condition. Our hypothesis of a stepwise pathogenesis of severe gluten‐sensitive enteropathy is re‐examined in the light of these new data. It is evident that there are at least five different levels at which genetic influences may operate.
Identification of the Autoantigen of Celiac Disease
Authors: SCHUPPAN DETLEF , DIETERICH WALBURGA , EHNIS TOBIAS , BAUER MICHAEL , DONNER PETER , VOLTA UMBERTO , RIECKEN ERNST OTTO
Published: February 2006

ABSTRACT: Tissue transglutaminase is demonstrated to be the unknown endomysial autoantigen by means of immunoprecipitations from a fibrosarcoma cell culture. A novel hypothesis for the pathogenesis of celiac disease is formulated: The mainly intracellular tissue transglutaminase is released from cells during wound healing where it aids in stabilizing the wound area by cross‐linking a small set of extracellular matrix components.
Clinical Models of Intestinal Adaptation
Authors: SCHULZKE JÖRG‐DIETER , SCHMITZ HEINS , FROMM MICHAEL , BENTZEL CARL J. , RIECKEN ERNST OTTO
Published: February 2006

ABSTRACT: Mucosal adaptation of the small intestine is morphologically restricted to only three different patterns, namely, atrophy, hyperplasia, and hyperregeneration. The hyperplastic mucosa in the experimental short bowel syndrome exhibits unchanged epithelial barrier properties and a differential functional adaptation with a 150% increase in Na‐glucose cotransport but no change in electroneutral NaCl cotransport. In the hyperregeneratively transformed mucosa of the self‐filling blind loop of rat jejunum, absorption is seriously impaired, as indicated by the 80% decrease in Na‐glucose cotransport. To compensate for this, epithelial barrier function is upregulated by an increase in tight junction complexity to prevent leak flux of ions and substrates. In contrast, the hyperregeneratively transformed mucosa in celiac sprue shows reduced tight junction complexity. Possible candidates responsible for the heterogeneity of tight junction adaptation in these conditions could be cytokines, because tumor necrosis factor‐a can specifically downregulate the tight junction, as indicated in the intestinal HT‐29/B6 cell model.
Molecular Mechanisms of Enteroendocrine Differentiaton
Authors: HÖCKER MICHAEL , WIEDENMANN BERTRAM
Published: February 2006

ABSTRACT: Passing through a complex series of developmental steps, the visceral endoderm differentiates into four intestinal epithelial lineages comprising enterocytes, goblet cells, paneth cells, and enteroendocrine cells. The intestinal enteroendocrine system consists of at least 15 different cell types, which can be classified on the basis of morphological criteria, expression of secretory products, and abundance of specific marker molecules. During intestinal development and in the adult gut, neuroendocrine subpopulations display strictly controlled differences in their geographical distribution that go along with dramatic differences in cell type‐specific gene expression. Identification to transcription factors and regulatory DNA elements responsible for cell‐specific gene expression in different neuroendocrine cell types as well as various transgenic and “knock‐out” mouse models have largely added to our understanding of mechanisms controlling appropriate spacial and temporal activation of enteroendocrine differentiation programs. This article reviews current in vitro and in vivo studies analyzing different molecular aspects of enteroendocrine differentiation. In addition, the influence of intestinal diseases including malignant transformation on enteroendocrine differentiation and the underlying mechanisms will be discussed.
Signaling by the Cytokine Receptor Superfamilya
Authors: IHLE JAMES N. , THIERFELDER WILLIAM , TEGLUND STEPHAN , STRAVAPODIS DIMITRIOS , WANG DEMIN , FENG JIAN , PARGANAS EVAN
Published: February 2006

Abstract: A variety of cytokines that regulate functions of multiple lineages share the utilization of receptors that are structurally and functionally related and are referred to as the cytokine receptor superfamily. These receptors associate with one or more of the four mammalian Janus kinases (Jaks) and ligand‐induced receptor aggregation results in their activation. Critical roles for Jak3 and Jak2 are demonstrated by the phenotypes of mice that lack each gene. Among the substrates of the Jaks are one or more of the seven members of the signal transducers and activators of transcription (Stats). Each Stat family member plays a critical role in the biological functions of specific cytokines as demonstrated by the phenotype of mice lacking one or more of these genes.
Cis‐Regulatory Elements Controlling Basal and Inducible VIP Gene Transcription
Authors: HAHM SUNG HO , EIDEN LEE E.
Published: February 2006

Abstract: The cis‐acting elements of the VIP gene important for basal and stimulated transcription have been studied by transfection of VIP‐reporter gene constructs into distinct human neuroblastoma cell lines in which VIP transcription is constitutively high, or can be induced to high levels by protein kinase stimulation. The 5.2 kb flanking sequence of the VIP gene conferring correct basal and inducible VIP gene expression onto a reporter gene in these cell lines was systematically deleted to define its minimal components. A 425‐bp fragment (−4656 to −4231) fused to the proximal 1.55 kb of the VIP promoterenhancer was absolutely required for cell‐specific basal and inducible transcription. Four additional components of the VIP gene were required for full cell‐specific expression driven by the 425 bp TSE (region A). Sequences from −1.55 to −1.37 (region B), −1.37 to −1.28 (region C), −1.28 to −.094 (region D), and the CRE‐containing proximal 94 bp (region E) were deleted in various combinations to demonstrate the specific contributions of each region to correct basal and inducible VIP gene expression. Deletion of region B, or mutational inactivation of the CRE in region E, resulted in constructs with low transcriptional activity in VIP‐expressing cell lines. Deletion of regions B and C together resulted in a gain of transcriptional activity, but without cell specificity. All five domains of the VIP gene were also required for cell‐specific induction of VIP gene expression with phorbol ester. Gelshift analysis of putative regulatory sequences in regions A‐D suggests that both ubiquitous and neuron‐specific trans‐acting proteins participate in VIP gene regulation.
p22/PACAP Response Gene 1 (PRG1): A Putative Target Gene for the Tumor Suppressor p53
Authors: SCHÄFER HEINER , TRAUZOLD ANNA , SEBENS THORSTEN , DEPPERT WOLFGANG , FÖLSCH ULRICH R. , SCHMIDT WOLFGANG E.
Published: February 2006

Abstract: In this study we describe a novel putative p53‐responsive gene, designated p22/PACAP response gene 1 (PRG1), recently identified as a proliferation‐associated early‐response gene in rats. By means of electrophoretic mobility shift assay and CAT‐reporter gene assay, we could demonstrate that the p53 binding site residing in the promoter of p22/PRG1 is functional in vitro. Furthermore, in clone 6 cells expression of p22/PRG1 is induced in parallel to p21/Waf1 under conditions permitting mutant p53 to adopt wild‐type configuration. An increase of p22/PRG1 transcription was also observed in γirradiated rat splenocytes, which undergo p53‐dependent apoptosis. Our findings demonstrate that p22/PRG1 fulfills all essential criteria as a p53 target gene and might be implicated in p53‐dependent apoptosis.
Cloning and Functional Characterization of PACAP‐Specific Receptors in Zebrafish
Authors: WEI Y. , MARTIN S. C. , HEINRICH G. , MOJSOV S.
Published: February 2006

Abstract: Two PACAP receptors were isolated from total zebrafish cDNA library prepared from 6‐day old fish by a homology‐based cloning strategy. The two zebrafish PACAP receptors have the same topology as the one found in other members of this class of seven membrane‐spanning G‐protein‐coupled receptors. Each of the two zebrafish PACAP receptors shares about 70% sequence identity at the amino acid level with the human PACAP‐type 1 receptor, and about 50% amino acid identity with PACAP/VIP R‐1 and PACAP/VIP R‐2 receptors. One of these zebrafish receptors contains the hop2 configuration found in the human and rat PACAP‐type 1 receptors. On the basis of these structural characteristics the zebrafish PACAP receptors were classified as PACAP‐type 1 and PACAP‐type 2 receptors. In competitive binding experiments zebrafish PACAP‐type 1 and PACAP‐type 2 receptors showed similar binding specificity for zebrafish and human PACAP‐38 and PACAP‐27. Furthermore, the specificity of PACAP‐type 1 and PACAP‐type 2 receptors for zebrafish and human PACAPs is about 1,000‐fold higher than for human VIP. These results demonstrate that zebrafish PACAP‐type 1 receptor is a structural and pharmacological homolog of the mammalian PACAP‐type 1 receptor. Additional pharmacological characterization is needed in order to classify the zebrafish PACAP‐type 2 receptor.
Cloning and Functional Characterization of the Human VIP1/PACAP Receptor Promoter
Authors: COUVINEAU ALAIN , MAORET JEAN JOSE , ROUYER‐FESSARD CHRISTIANE , CARRERO ISABEL , LABURTHE MARC
Published: February 2006

Abstract: The 5′‐flanking region (1.5 kb) of the gene coding for the human VIP1/PACAP receptor was isolated, sequenced, and characterized. Transient expression of constructs containing sequentially deleted 5′‐flanking sequences of the VIP1/PACAP receptor fused to a luciferase reporter gene showed that this sequence was active as a promoter in the intestinal cancer cell line, HT‐29, expressing endogenous VIP1/PACAP receptor. The shortest DNA fragment with significant promoter activity encompassed the region from −205 to +76 bp. Deletion of a CCAAT‐box sequence in the construction corresponding to −173 to +76 bp dramatically reduced the promoter activity. The promoter −205 to +76 bp has a housekeeping gene structure without TATA‐box. It contains GC‐rich regions characterized by potential Sp1 and AP2 sites and some potential regulatory elements, such as CRE and ATF, and a CCAAT‐box sequence (−182 to −178) crucial for gene transcription.
Desensitization of the Human Vasoactive Intestinal Peptide Receptor (hVIP2/PACAP R): Evidence for Agonist‐Induced Receptor Phosphorylation and Internalizationa
Authors: MCDONALD TERENCE P. , DINNIS DIANE M. , MORRISON CHRISTINE F. , HARMAR ANTHONY J.
Published: February 2006

Abstract: To investigate the role of phosphorylation and internalization in the desensitization of the hVIP2/PACAP receptor, we expressed a C‐terminal epitope‐tagged (hemagglutinin; YPYDVPDYASL) receptor in COS7 and HEK293 cell lines. Radiolabeling experiments demonstrated that exposure to agonist induced receptor phosphorylation significantly above basal levels. This receptor phosphorylation was greater than that induced by receptor‐independent activation of PKA with forskolin and that induced by co‐application of forskolin and agonist. This suggests that receptor occupancy promotes phosphorylation and also that receptor phosphorylation may involve a specific G protein‐coupled receptor kinase in addition to PKA. Immunocytochemical analysis showed that the receptor was internalized in response to agonist to a single site of accumulation within the cell and this was dependent on temperature, agonist concentration, and time. Further studies will focus on identifying phosphorylation sites and endocytic signals within the hVIP2/PACAP R.
Rat Aortic Smooth‐Muscle Cell Proliferation Is Bidirectionally Regulated in a Cell Cycle‐Dependent Manner via PACAP/VIP Type 2 Receptora
Authors: MIYATA ATSURO , SATO KUMI , HINO JUN , TAMAKAWA HIROKI , MATSUO HISAYUKI , KANGAWA KENJI
Published: February 2006

Abstract: In the cardiovascular system, vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) have been well characterized as potent vasodepressors or vasodilators. However, their pathophysiological implication in proliferation of vascular smooth muscle cells has not yet been elucidated. In the present study, we have first identified PACAP/VIP type 2 receptor as a dominant type in rat vascular smooth muscle cell (VSMC) by RT‐PCR. PACAP and VIP increased cyclic AMP accumulation with similar potency. In 24‐h [3H]thymidine incorporation assay, PACAP or VIP exhibited a suppressive effect on the DNA synthesis of rat VSMC stimulated by serum when added at the late G1 phase. In contrast, when added at G0/G1 phase of the cell cycle, PACAP or VIP enhanced the serum‐induced DNA synthesis. In 24‐h incubation, PACAP alone has little mitogenic activity. However, when incubated up to 48 h, PACAP stimulated significantly the DNA synthesis and the cell proliferation of rat VSMC. These results suggest that PACAP and VIP regulate the proliferation of rat VSMC by enhancing or suppressing in a cell cycle‐dependent manner and induce delayed mitogenesis and cell proliferation.
Identification of Binding Domains of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) for its Type 1 Receptor by Photoaffinity Labelinga
Authors: CAO YONG‐JIANG , KOJRO ELZBIETA , JASIONOWSKI MAREK , LANKIEWICZ LESZEK , GRZONKA ZBIGNIEW , FAHRENHOLZ FALK
Published: February 2006

Abstract: Structure‐function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1‐27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1‐27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N‐terminal or the helical C‐terminal domain or a short loop region within the C‐terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C‐terminal domain of PACAP(1‐27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125I‐labeled photoreactive analogues specifically label a protein band of Mr66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr22 and Lys15 in PACAP(1‐27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the α‐helical C‐terminal region of PACAP is directly involved in receptor binding.
The Neurotrophic Activity of PACAP on Rat Cerebellar Granule Cells Is Associated with Activation of the Protein Kinase A Pathway and c‐fos Gene Expressiona,
Authors: VAUDRY D. , BASILLE M. , ANOUAR Y. , FOURNIER A. , VAUDRY H. , GONZALEZ B. J.
Published: February 2006

Abstract: In vitro studies have shown that PACAP promotes cell survival and neurite outgrowth in immature cerebellar granule cells. In the present study, we have examined the transduction pathways involved in the neurotrophic activity of PACAP. Incubation of cultured granule cells with graded concentrations of PACAP produced a dose‐dependent increase in c‐fos mRNA level. The effects of PACAP on c‐fos gene expression and granule cell survival were both mimicked by dbcAMP but not by PMA. The maximum effect of PACAP on c‐fos gene expression was observed after 1 h of treatment. Similar effects of the peptide on granule cell survival were observed whether the cells were continuously incubated with PACAP for 48 h or only exposed to PACAP during 1 h. The PKA inhibitor H89 significantly reduced the effect of PACAP on c‐fos mRNA level, whereas the specific PKC inhibitor chelerytrine had no effect. These data indicate that the action of PACAP on cerebellar granule cell survival and c‐fos gene expression are both mediated through the adenylyl cyclase/PKA pathway.
PACAP‐38 Protects Cerebellar Granule Cells from Apoptosis
Authors: JOURNOT LAURENT , VILLALBA MARTIN , BOCKAERT JOËL
Published: February 2006

Abstract: Pituitary adenylate cyclase‐activating polypeptides (PACAP‐27 and ‐38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions including the cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP‐38 on apoptosis induced by potassium deprivation. We demonstrated that serum and potassium withdrawal induces a mixture of apoptosis and necrosis rather than apoptosis only. We showed that PACAP‐38 increased survival of cerebellar neurons in a dose‐dependent manner by specifically decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP‐38 induced activation of the extracellular signal‐regulated kinase (ERK)‐type of MAP kinase through a cAMP‐dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the anti‐apoptotic effect of PACAP‐38, suggesting that MAP kinase pathway activation is necessary for PACAP‐38 effect.
Miniglucagon: A Local Regulator of Islet Physiology
Authors: DALLE STÉPHANE , BLACHE PHILIPPE , LE‐NGUYEN DUNG , LE BRIGAND LAURENCE , BATAILLE DOMINIQUE
Published: February 2006

Abstract: Miniglucagon, or glucagon‐[19–29], is partially processed from glucagon in its target tissues where it modulates the glucagon action. In the islets of Langerhans, the glucagon‐producing A cells contain miniglucagon at a significant level (2–5% of the glucagon content). We studied a possible control of insulin release by miniglucagon using as a model the MIN6 cell line. Miniglucagon, in the 10−14 to 10−9 M range, inhibited insulin release induced by glucose, glucagon, tGLP‐1, or glibenclamide by 85–100% with an IC50 close to 1 pM. While no change in the cyclic AMP content was noted, Ca2+ influx was reduced in parallel with the inhibition of insulin release. Use of pharmacological modulators of L‐type voltage‐sensitive Ca2+ channels and bacterial toxins indicates that miniglucagon blocks insulin release by closing this type of channel via a pertussis toxin‐sensitive G protein. Miniglucagon is a novel, possibly physiologically relevant, local regulator of islet function.
Pituitary Adenylate Cyclase Activating Polypeptide Induces Degranulation of Rat Peritoneal Mast Cells via High‐Affinity PACAP Receptor‐Independent Activation of G Proteins
Authors: SEEBECK JÖRG , KRUSE MARIE‐LUISE , SCHMIDT‐CHOUDHURY ANJONA , SCHMIDT WOLFGANG E.
Published: February 2006

Abstract: In this study, the secretory effects of PACAP and PACAP analogues on [3H]serotonin‐loaded purified rat peritoneal mast cells (RPMCs) were investigated. PACAP(1‐27) and PACAP(6‐27) stimulated [3H]serotonin release with low potency (ED50: 2 × 10−6 M) but high efficacy. The N‐terminally truncated PACAP form, PACAP(6‐27), stimulated tracer release with an ED50 of 0.2 × 10−6 M, indicating a high‐affinity PACAP receptor‐independent mechanism of action. The secretory response to PACAP(1‐27) could be inhibited by 60‐min preincubation with pertussis toxin (ptx), which inhibits G proteins. U73122, a cell‐permeable phospholipase C inhibitor, dose‐dependently inhibited the secretory effect of 5 μM PACAP(1‐27) with an IC50 value of 4 μM (N= 4; p > 0.006). We conclude that PACAP exerts a secretory effect in RPMCs by high‐affinity PACAP receptor‐independent direct activation of one or more G proteins, which may then activate the PLC‐dependent signal‐transduction pathway.
The Pituitary Adenylate Cyclase Activating Polypeptide Type 1 Receptor (PAC1‐R) Is Expressed on Gastric ECL Cells: Evidence by Immunocytochemistry and RT‐PCR
Authors: ZENG NINGXIN , KANG TAO , LYU RONG‐MING , WONG HELEN , WEN YI , WALSH JOHN H. , SACHS GEORGE , PISEGNA JOSEPH R.
Published: February 2006

Abstract: The current study was undertaken to determine the presence and distribution of PAC1‐Rs within the gastric mucosa. Polyclonal antibodies to the carboxyl terminus of the rat PAC1‐R were generated and shown to be specific against the PAC1‐R expressed in NIH 3T3 cells. Western blot analysis using isolated (∼85% pure) ECL cell membranes identified a 48 kD protein consistent with the calculated molecular mass of the cloned PAC1‐R. RT/PCR performed using specific primers for the PAC1‐R confirmed the presence of splice variants of the rat PAC1‐R, but not VPAC1‐R or VPAC2‐R. These data provide the first direct evidence for the existence of functional PACAP Type I receptors on ECL cells of the gastric mucosa and suggest a potential role for PACAP in the stimulation of gastric acid secretion and in the regulation of the growth of ECL cells.
Differential Display PCR Reveals Induction of Immediate Early Genes by Vasoactive Intestinal Peptide in PC12 Cellsa
Authors: ESCHELBACH ANDREAS , HUNZIKER ANDREAS , KLIMASCHEWSKI LARS
Published: February 2006

Abstract: In order to identify genes regulated by vasoactive intestinal peptide, we performed differential display PCR as originally described by Liang and Pardee. 1 Messenger RNA of PC12 cells treated with vasoactive intestinal peptide or nerve growth factor for one hour was reverse transcribed and amplified using different sets of oligo‐dT and random primers. Radioactively labeled PCR products were displayed on polyacrylamide gels and candidate cDNAs extracted from the gel, re‐amplified by PCR, cloned, and sequenced. Differential expression was verified by RT‐PCR applying sets of specific primers obtained from the sequence. The specificity of the PCR product was confirmed by Southern blotting using a radioactively labeled internal primer and semi‐quantitative densitometric analysis. This rapid and sensitive protocol led to the isolation of two immediate early genes, pip92 2 and PC4, 3 known to be increased on mRNA level by nerve growth factor in PC 12 cells.
Developmental Regulation of Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) and Its Receptor 1 in Rat Brain: Function of PACAP as a Neurotrophic Factora
Authors: LINDHOLM DAN , SKOGLÖSA YLVA , TAKEI NOBUYUKI
Published: February 2006

Abstract: To function as a trophic factor PACAP and PACAP‐R must be expressed in the nervous system during early development. We report here on the distribution of PACAP mRNA in the developing nervous system of the rat and compare its expression with that of PACAP‐R. We discuss primary neuron culture experiments that study the neurotrophic activity of PACAP. Experimental results that indicate the presence of PACAP and its receptor in the developing nervous system, together with the observed neuropeptide activity on various populations of neurons, support the view that PACAP exhibits important neurotrophic activities comparable to those of the classical neurotrophic factors.
Pituitary Adenylate Cyclase Activating Peptide (PACAP) in the Retinohypothalamic Tract: A Daytime Regulator of the Biological Clocka
Authors: HANNIBAL JENS , DING JIAN M. , CHEN DONG , FAHRENKRUG JAN , LARSEN PHILIP J. , GILLETTE MARTHA U. , MIKKELSEN JENS D.
Published: February 2006

Abstract: The retinohypothalamic tract (RHT) relays photic information from the eyes to the brain biological clock in the suprachiasmatic nucleus (SCN). Activation of this pathway by light plays a role in adjusting circadian timing to light exposure at night. Here we report a new signaling pathway by which the RHT regulates circadian timing in the daytime as well. Using dual‐immunocytochemistry for PACAP and the in vivo tracer Cholera toxin subunit B (ChB), intense PACAP immunoreactivity (PACAP‐IR) was observed in retinal afferents at the rat SCN as well as in the intergeniculate leaflet (IGL) of the thalamus. This PACAP‐IR was nearly lost upon bilateral eye enucleation. PACAP afferents originated from ganglion cells distributed throughout the retina. The phase of circadian rhythm measured as SCN neuronal activity in vitro was significantly advanced by application of PACAP‐38 during the subjective day, but not at night. The effect is channelled to the clock via a PACAP 1 receptor‐cAMP signaling mechanism. Thus, in addition to its role in nocturnal regulation by glutamatergic neurotransmission, the RHT can adjust the biological clock by a PACAP‐cAMP‐dependent mechanism during the daytime.