Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.     

Response of avian embryonic brain to spatially segmented x-ray microbeams.

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam irradiation as compared with broad beam irradiation.     

Salmonella enterica Serotype Bredeney: Antimicrobial Susceptibility and Molecular Diversity of Isolates from Ireland and Northern Ireland

Salmonella enterica serotype Bredeney has emerged as the third most commonly identified serotype among human clinical isolates referred to the Irish National Salmonella Reference Laboratory in the years 1998 to 2000. A collection of 112 isolates of S. enterica serotype Bredeney collected during the period 1995 to 1999 from animal, food, and human sources from both Ireland and Northern Ireland were studied. Antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and DNA amplification fingerprinting (DAF) were performed on all isolates. Plasmid profiles were examined on a subset of 33 isolates. A high proportion (74%) of isolates were susceptible to all antimicrobial agents tested. Resistance to both sulfonamide and trimethoprim was observed in 21% of isolates, and resistance to multiple (five) antimicrobial agents was observed in a single isolate (0.9%). Eight different PFGE patterns were obtained, with 87% of isolates grouping as PFGE type A. PFGE type A was predominant in animals, food, and humans. There was good overall concordance between the groups identified by PFGE and DAF. Overall results indicate that most S. enterica serotype Bredeney isolates in Ireland and Northern Ireland from animal and human sources are clonally related.     

An aromatic schiff base stabilised as a coordinated enol iminium zwitterion by complex formation with a series of divalent metal ions

The Schiff base N-(2-hydroxy-3-carboxy-1-naphthylidine)-4-methyl-2-sulphonic acid aniline (bonsaH₃) has been found to react with a range of divalent metal ions (Mg²⁺, Mn²⁺, Co²⁺, Ni²⁺ and Zn²⁺ and UO₂²⁺ to give red-yellow insoluble complexes (bonsaH)_m(H₂O)_n. The solid state diffuse reflectance spectra of all the complexes have an intense visible band at ca. 470 nm. This fact, together with evidence from infrared spectra and room-temperature magnetic-moment measurements, suggests that in all cases the ligand is coordinated to the metal ion in the solid state in the enol-iminium zwitterionic form. The ¹H NMR spectra of the Mg²⁺ and Zn²⁺ complexes in DMSO-d₆ indicate that a different structure is adopted in this solvent. Comparisons with the spectra of bonsa-H₃ and (bonsa-H₂)K·H₂O suggest that the solution structure is that of an enol-imine.     

ESTIMATING POPULATION SIZE IN ASYNCHRONOUS AGGREGATIONS: A BAYESIAN APPROACH AND TEST WITH ELEPHANT SEAL CENSUSES

Many organisms reproduce in temporary aggregations where estimates of colony size can be made by direct counts. When individuals are not synchronous, however, early breeders depart before the last arrive, so counts underestimate the total breeding population. We present a model describing a colony's census as a function of arrival, breeding tenure, and the correlation between them, and we use it to illustrate how variance in arrival and tenure affect the census. Counts of breeding female northern elephant seals ( Mirounga angustirostris ) from 1975 to 2007 were used to test the model. Four of the model's parameters—population size, mean and variance of arrival date, and the correlation between arrival date and breeding tenure—could be estimated from census data using a Bayesian approach; prior estimates of two other parameters—mean tenure and its variance—had to be used to avoid overparameterization. The model's predictions fit observed censuses well and produced reliable estimates of population size and arrival behavior, showing that the maximum census was 8%–16% below the total number of breeding females. This method could be used for estimating abundance in any asynchronous aggregation, given independent information on one of the defining distributions: arrival, tenure, or departure.     

Comparative Proteomics of Dehalococcoides spp. Reveals Strain-Specific Peptides Associated with Activity

Anaerobic reductive dehalogenation by Dehalococcoides spp. is an ideal system for studying functional diversity of closely related strains of bacteria. In Dehalococcoides spp., reductive dehalogenases (RDases) are key respiratory enzymes involved in the anaerobic detoxification of halogenated compounds at contaminated sites globally. Although housekeeping genes sequenced from Dehalococcoides spp. are >85% identical at the amino acid level, different strains are capable of dehalogenating diverse ranges of compounds, depending largely on the suite of RDase genes that each strain harbors and expresses. We identified RDase proteins that corresponded to known functions in four characterized cultures and predicted functions in an uncharacterized Dehalococcoides-containing mixed culture. Homologues within RDase subclusters containing PceA, TceA, and VcrA were among the most frequently identified proteins. Several additional proteins, including a formate dehydrogenase-like protein (Fdh), had high coverage in all strains and under all growth conditions.     

Interactions of amphipathic carrier peptides with membrane components in relation with their ability to deliver therapeutics.

To identify rules for the design of efficient CPPs that can deliver therapeutic agents such as nucleic acids (DNAs, siRNAs) or proteins and PNAs into subcellular compartments, we compared the properties of several primary and secondary amphipathic CPPs. Studies performed with lipid monolayers at the air-water interface have enabled identification of the nature of the lipid-peptide interactions and characterization of the influence of phospholipids on the ability of these peptides to penetrate into lipidic media. Penetration and compression experiments reveal that both peptides interact strongly with phospholipids, and observations on Langmuir-Blodgett transfers indicate that they can modify the lipid organization. Conformational investigations indicate that the lipid-peptide interactions govern the conformational state(s) of the peptides. On the basis of the ability of both peptides to promote ion permeation through both natural and artificial membranes, models illustrating the translocation processes have been proposed. One is based on the formation of a beta-barrel pore-like structure while another is based on the association of helices.