Interconvertible Kinetic States of t-Butylbicycloorthobenzoate Binding Sites of the γ-Aminobutyric AcidA Ionophores

The kinetics of t-[³H]butylbicycloorthobenzoate (TBOB) binding to the convulsant sites of the γ-aminobutyric acid_A (GABA_A) receptor-ionophore complex were examined in synaptosomal membrane preparations of rat brain. On and off rates of TBOB binding were accelerated by 1 μM GABA and decelerated by 1 μM bicuculline methochloride, a GABA_A antagonist. The presence of GABA and bicuculline methochloride created rapid and slow phases of dissociation, respectively. The three groups of rate constants distinguished for the dissociation of 4 nM and 30 nM [³H]TBOB represent multiaffinity states of the convulsant sites depending on the presence of GABA or bicuculline methochloride. Apparent association rate constants do not obey the equation k_app=k_off±k_on [TBOB] without assuming interconvertibility of the kinetic states during binding. Avermectin B_1a (AVM B_1a), a chloride channel opening agent, accelerated the association and dissociation of TBOB and resulted in a biphasic effect on TBOB binding, i.e., enhancement at low concentrations (EC₅₀, 7.8 nM) followed by displacement at high concentrations (IC₅₀ 6.3 μM) of AVM B_1a. AVM B_1a resulted in similar biphasic effects on t- [³⁵S]butylbicyclophosphorothionate binding. DIDS, an isothiocyanatostilbene derivative with irreversible anion channel blocking effect, selectively inhibited basal [³H]TBOB binding (IC₅₀ 125 μM DIDS) leaving the enhancement by AVM B_1a unaffected.     

Phenotypic and genetic differentiation between diadromous and landlocked puyen Galaxias maculatus

This study reports the phenotypic and genetic differences between individuals of puyen Galaxias maculatus from two sites in the same river basin in Tierra del Fuego National Park, southern South America. Individuals from the two sampling sites presented morphometric and genetic differences. The morphometric differences indicated that individuals from Laguna Negra (LN) were short and more robust and had large eyes, whereas those from Arroyo Negro (AN) were thin and elongated and had small eyes. Genetic differences showed that AN individuals had a greater genetic structuration and an older demographic history than LN individuals. The results of this study affirmed that the individuals from the two sampling sites belong to different populations with a high degree of isolation. The demographic history could indicate that the individuals of G. maculatus which migrated to northern areas during the last glaciation settled in the Beagle Channel after its formation. The LN population could have originated after the retreat of the glaciers, migrating from AN.     

Molecular and iridescent feather reflectance data reveal recent genetic diversification and phenotypic differentiation in a cloud forest hummingbird

The present day distribution and spatial genetic diversity of Mesoamerican biota reflects a long history of responses to habitat change. The hummingbird Lampornis amethystinus is distributed in northern Mesoamerica, with geographically disjunct populations. Based on sampling across the species range using mitochondrial DNA (mtDNA) sequences and nuclear microsatellites jointly analysed with phenotypic and climatic data, we (1) test whether the fragmented distribution is correlated with main evolutionary lineages, (2) assess body size and plumage color differentiation of populations in geographic isolation, and (3) evaluate a set of divergence scenarios and demographic patterns of the hummingbird populations. Analysis of genetic variation revealed four main groups: blue‐throated populations (Sierra Madre del Sur); two groups of amethyst‐throated populations (Trans‐Mexican Volcanic Belt and Sierra Madre Oriental); and populations east of the Isthmus of Tehuantepec (IT) with males showing an amethyst throat. The most basal split is estimated to have originated in the Pleistocene, 2.39–0.57 million years ago (MYA), and corresponded to groups of populations separated by the IT. However, the estimated recent divergence time between blue‐ and amethyst‐throated populations does not correspond to the 2‐MY needed to be in isolation for substantial plumage divergence, likely because structurally iridescent colors are more malleable than others. Results of species distribution modeling and Approximate Bayesian Computation analysis fit a model of lineage divergence west of the Isthmus after the Last Glacial Maximum (LGM), and that the species’ suitable habitat was disjunct during past and current conditions. These results challenge the generality of the contraction/expansion glacial model to cloud forest‐interior species and urges management of cloud forest, a highly vulnerable ecosystem to climate change and currently facing destruction, to prevent further loss of genetic diversity or extinction.     

NMR and CD studies on the conformation of a synthetic peptide containing epitopes of the human immunodeficiency virus 1 transmembrane protein gp41

CD and nmr characterizations are reported for the 23-mer peptide CMC3, corresponding to residues 577–599 of gp41, the transmembrane glycoprotein of the human immunodeficiency virus 1. Concentration, temperature, and pH dependencies of CD and nmr spectra are indicative of self-association with a consequent stabilization of secondary structural elements in water. The addition to the water solution of small amounts of trifluoroethanol induces a secondary structure, mostly due to the presence of helical elements. The amphipathic character of the helix and the presence of three hydrophobic 4/3 heptad repeats suggest that the peptide could be structured in a symmetric association of helices, such as in a coiled-coil structure. This behavior is discussed in terms of a possible role of this segment in the gp41 envelope oligomerization. © 1996 John Wiley & Sons, Inc.     

Celiac disease association with CD8+ T cell responses: identification of a novel gliadin-derived HLA-A2-restricted epitope

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8(+) T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123-132) by CD8(+) T lymphocytes from celiac patients. A-gliadin 123-132-specific IFN-gamma production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123-132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123-132 epitope recognized human APC pulsed with either whole recombinant alpha-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.     

Molecular characterization of bla(IMP-5), a new integron-borne metallo-beta-lactamase gene from an Acinetobacter baumannii nosocomial isolate in Portugal

Postbloom fruit drop (PFD) of citrus is caused by Colletotrichum acutatum. PFD isolates infect flower petals, induce abscission of small fruit and can cause severe yield loss on most citrus cultivars. Isolates from Key lime anthracnose (KLA) cause that disease on the Mexican lime, but also cause PFD on sweet orange. Both PFD and KLA isolates exhibited resistance to the common selection agents including hygromycin, bialaphos, benomyl and geneticin/G418. A genetic transformation system was developed for C. acutatum to confer resistance to sulfonylurea (chlorimuron ethyl) by expressing an acetolactate synthase gene (sur) cassette from Magnaporthe grisea. The protocol was tested on 11 different KLA and PFD isolates. The transformation frequencies were highly variable among isolates and among experiments (0-17.9 per microg circular DNA using 10(7) protoplasts). Southern blot analysis of transformants indicated that the plasmid vector was randomly integrated in multiple copies into the genome of C. acutatum. Addition of restriction enzymes or use of a vector with homologous sequences did not change the transformation frequencies, but tended to reduce the number integrated. Over 97% of the transformants retained the sulfonylurea resistance phenotype under non-selective conditions. Of 300 transformants tested, three were unable to cause necrotic lesions on detached Key lime leaves. The transformation method opens up opportunities for the genetic manipulation of C. acutatum.     

2-styrylchromones as novel inhibitors of xanthine oxidase. A structure-activity study

The purpose of this study was the evaluation of the xanthine oxidase (XO) inhibition produced by some synthetic 2-styrylchromones. Ten polyhydroxylated derivatives with several substitution patterns were synthesised, and these and a positive control, allopurinol, were tested for their effects on XO activity by measuring the formation of uric acid from xanthine. The synthesised 2-styrylchromones inhibited xanthine oxidase in a concentration-dependent and non-competitive manner. Some IC50 values found were as low as 0.55 microM, which, by comparison with the IC50 found for allopurinol (5.43 microM), indicates promising new inhibitors. Those 2-styrylchromones found to be potent XO inhibitors should be further evaluated as potential agents for the treatment of pathologies related to the enzyme's activity, as is the case of gout, ischaemia/reperfusion damage, hypertension, hepatitis and cancer.     

High-performance thin-layer chromatography-bioautography for multiple antibiotic residues in cow's milk

An analytical method to identify and quantify multiple antibiotic residues (chloramphenicol, ampicillin, benzylpenicillin, dicloxacillin and erythromycin) in cow's milk by high-performance thin-layer chromatography (HPTLC) combined with bioautography was developed. The test microorganism used for bioautography was Bacillus subtilis ATCC 6633. Antibiotic residues were extracted with acetonitrile, fat eliminated with petroleum ether and residues isolated with dichloromethane The sensitivity of the method guarantees the detection of the above-mentioned antibiotics at levels below maximum residue limits (MRL) allowed for milk. Percentage recoveries ranged between 90 and 100%, with coefficients of variation between 7.2 and 21.3%. Some advantages of this methodology over thin-layer chromatography (TLC)/bioautography are also discussed.     

Lernanthropus kroyeri infections in farmed sea bass Dicentrarchus labrax: pathological features

Twenty sea bass Dicentrarchus labrax L. from a fish farm (floating cage) in Greece were examined for the presence of parasites. The gills of 7 (35%) fish were infected with adult female specimens of the parasitic copepod Lernanthropus kroyeri van Beneden, 1851, and the intensity of infection ranged from 1 to 24 parasites per host. The most infected portion of the gills appeared to be the primary lamellae. Erosion, desquamation and necrosis of the secondary lamellae were noticed near the site of copepod attachment; furthermore, the terminal claw of the second antennae lacerated tissue and vessels of infected gill. Parasitism by L. kroyeri affected the host's condition factor (mean +/- SE in uninfected vs parasitized; 1.88 +/- 0.04 vs 1.66 +/- 0.12; p < 0.05).     

Immunohistochemistry,ultrastructure and pathology of gills of Abramis brama from Lake Mondsee,Austria, infected with Ergasilus sieboldi (Copepoda)

Immunohistochemical, ultrastructural and pathological studies were carried out on the gills of bream Abramis brama (L.) from Lake Mondsee, Austria, that were naturally infected with Ergasilus sieboldi Nordmann, 1832. Of a total of 14 specimens of bream examined, the gills of 7 (50%) were parasitized with this copepod and the intensity of infection ranged from 1 to 23 crustaceans per host. Histopathological investigations of infected gill showed extensive tissue damage due to attachment and feeding of the crustacean. Parasites attached close to the base of filaments near the gill arch. Pressure exerted by the ectoparasite attached to the lateral margin of the gill filaments induced atrophy of the secondary lamellae. Tissue reactions included hyperplasia and mucous cell proliferation of the respiratory epithelium. Mucous cells displayed an intense immunohistochemical reactivity with the anti-nitric oxide synthase antibody. In parasitized primary and secondary lamellae, a high number of eosinophilic granular cells and rodlet cells were noticed. Rodlet cells represent an inflammatory cell type closely linked to other piscine inflammatory cells. Presence of a high number of inflammatory cells at the site of E. sieboldi attachment is related to intense host cellular reaction.