Production of a fusion protein consisting of the enterotoxigenic<Emphasis Type="Italic"> Escherichia coli</Emphasis> heat-labile toxin B subunit and a tuberculosis antigen in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Transgenic plants are potentially safe and inexpensive vehicles to produce and mucosally deliver protective antigens. However, the application of this technology is limited by the poor response of the immune system to non-particulate, subunit vaccines. Co-delivery of therapeutic proteins with carrier proteins could increase the effectiveness of the antigen. This paper reports the ability of transgenic Arabidopsis thaliana plants to produce a fusion protein consisting of the B subunit of the Escherichia coli heat-labile enterotoxin and a 6 kDa tuberculosis antigen, the early secretory antigenic target ESAT-6. Both components of the fusion protein were detected using GM1-ganglioside-dependent enzyme-linked immunosorbant assay. This suggested the fusion protein retained both its native antigenicity and the ability to form pentamers.Abbreviations ELISA Enzyme linked immunosorbant assay - ESAT-6 Early secretory antigenic target (6 kDa) - ETEC Enterotoxigenic Escherichia coli - LTB B subunit of E. coli heat-labile enterotoxin Communicated by W.A. Parrott     

Disabilities caused by unstable mutations in Costa Rica

Motonic dystrophy and fragile X syndrome are two genetically determined relatively common disabilities. Both are examples of a new type of mutation mechanism called unstable or dynamic mutations, triple repeats expansions or DNA amplification. Fragile X syndrome is recognized as the main cause of hereditary mental retardation and myotonic dystrophy is considered the most common muscular dystrophy of adults. This is a prospective non randomized study of clinically affected people, in order to confirm the diagnosis with molecular techniques (Southern blot and PCR) and to perform cascade screening of the rest of the family to offer them adequate genetic counseling. We were able to corroborate the initial diagnosis in most clinical cases of myotonic dystrophy, but in the cases of mental retardation more than half studies were negative for fragile X syndrome, stressing the difficulties encountered by medical practitioners to diagnose this syndrome. The reasons for this are several; probable the main culprit is the subtle and unspecific clinical picture affected individuals exhibit, particularly children before puberty. Cascade screening, genetic counseling and selective abortion are the only tools available to prevent these disabling diseases for the moment.     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

Validation of the Diagnostic Interview for Genetic Studies (DIGS) in Colombia

An interview tool, Diagnostic Interview for Genetic Studies (DIGS 3.0), was translated into Spanish for application in studies of psychiatric disorders in Colombia. Two Spanish translations of the original English version of DIGS were prepared and back-translated into English. A review committee verified the linguistic and cultural equivalence of the translations. The evaluator and test-retest reliability were assessed calculating Cohen's kappa for samples of 65 and 91 patients respectively. DIGS proved valid in both appearance and content. The confidence interval (C.I.) was excellent for schizophrenia (kappa = 0.81, C.I. 95% = 0.68-0.93), bipolar disorder (kappa = 0.87, C.I. 95% = 0.75-0.99), major depressive disorder (kappa = 0.86, C.I. 95% = 0.70-1.00), and for a normal diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89); it was good for other psychiatric diagnosis (kappa = 0.65, C.I. 95% = 0.41-0.89) and poor for schizoaffective disorder (kappa = 0.37, C.I. 95% = -0.02-0.76). Test-retest reliability was excellent for all diagnoses (kappa > 0.8), except for "other psychiatric diagnoses" (kappa = 0.64, C.I. 95% = 0.31-0.96). The Spanish translation of the DIGS was comprehensible, with face and content validity, and good test-retest and evaluator reliability. This translation will be a useful tool for genetic studies of psychiatric disorders in Latin America, particularly where schizophrenia and affective disorders are involved.     

Aza-bicyclic amino acid sulfonamides as alpha(4)beta(1)/alpha(4)beta(7) integrin antagonists

The design, synthesis, and biological activity of novel alpha(4)beta(1) and alpha(4)beta(7) integrin antagonists, containing a bridged azabicyclic nucleus, are reported. Conformational analysis of targets containing an azabicyclo[2.2.2]octane carboxylic acid and known integrin antagonists indicated that this azabicycle would be a suitable molecular scaffold. Variation of substituents on the pendant arylsulfonamide and phenylalanine groups resulted in potent alpha(4)beta(1)-selective and dual alpha(4)beta(1)/alpha(4)beta(7) antagonists. Potent compounds 11i, 11h, and 14 were effective in the antigen-sensitized sheep model of asthma.     

Southern Sea Otter as a Sentinel of Marine Ecosystem Health

The southern sea otter (Enhydra lutris nereis) is listed as threatened under the Endangered Species Act (ESA) and is a keystone species, strongly influencing the abundance and diversity of the other species within its kelp forest ecosystem. This is accomplished primarily by preying upon urchins that eat the kelp stipe and holdfast, which can reduce a kelp forest to an urchin barren. Sea otters are very susceptible to marine pollutants such as petroleum, which may be directly toxic and/or alter their furs insulating properties. Sea otters are an excellent sentinel species. They eat approximately 25% of their body weight per day in shellfish and other invertebrates, and can concentrate and integrate chemical contaminants. In addition, they appear to be susceptible to a number of diseases and parasites that may have anthropogenic origins, and shellfish may serve as an intermediary for some of these infections. Many of the shellfish the otters eat are also harvested for human food. In their role as sentinels, sea otter health has implications for human health, economic sustainability of shellfisheries, as well as overall marine ecosystem health. The recent southern sea otter decline has been viewed with some alarm by conservationists and, indeed, recovery seems a long way off. High mortality rather than depressed recruitment appears to underlie the decline. A good deal of debate has centered on the role of infectious diseases and parasites, exposure to contaminants, nutrition and prey availability, net and pot fishery interactions, and other sources of mortality. Current research is being done related to major classes of mortality, various types of pollutants and some specific organisms causing southern sea otter mortality, and their implications for marine ecosystem health and sustainability.     

Over-expression of Rififylin, a new RING finger and FYVE-like domain-containing protein, inhibits recycling from the endocytic recycling compartment

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate-binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.     

Ontogeny has a phylogeny: background to adjunctive behaviors in pigeons and budgerigars

Animals coping with operant conditioning tasks often show behaviors that are not recorded by keys, levers and similar response transducers. Nevertheless, these adjunctive behaviors should not be disposed of by classifying them as incidental. Often they are found to be at least partially influenced by the experimentally programmed contingencies, and under certain conditions they can in turn influence conditioned behaviors. Here we describe the occurrence and characteristics of two such behaviors, stimulus grasping in operantly key-pecking pigeons and intra-delay stereotypies in a delayed matching-to-sample task with budgerigars. It is argued that for a proper account of these behaviors it is necessary to refer to a behavioral systems approach that appeals to longer ranging ontogenetic and phylogenetic histories than is usually considered in the psychological literature. The gaping towards on-key stimuli by pigeons is attributed to the hypothesis that operantly conditioned key-pecks probably relate to a grasp-pecking response that is normally executed towards non-edible items covering food. The intra-delay behaviors shown by the budgerigars are assumed to have originated from stress-induced displacement responses that adventitiously came under the influence of differential reinforcement contingencies. Finally, we discuss what kinds of evidence are needed to put these hypothetical explanations on a more certain footing.