Development and analysis of various clonal alloantigen-dependent cytotoxic cell lines from channel catfish

To determine the phenotypes of cytotoxic cells in channel catfish, clonal alloantigen-dependent leukocyte lines were established from mixed leukocyte cultures. Each clone was analyzed for expression of TCR alpha and beta genes by RT-PCR and for target cell specificity by 51Cr-release assay. Based on the above criteria, the following five different cell types were identified among the 19 clones analyzed: 1) TCR alphabeta+ allospecific cytotoxic cells, 2) TCR alphabeta+ nonspecific cytotoxic cells, 3) allospecific TCR alphabeta+ noncytotoxic cells, 4) TCR alphabeta- nonspecific cytotoxic cells, and 5) TCR alphabeta- allospecific cytotoxic cells. The demonstration of cloned, TCR alphabeta+, allospecific cytotoxic effectors provides the strongest evidence to date for the existence of cytotoxic T cells in fish.     

Improved aeroponic culture of inocula of arbuscular mycorrhizal fungi

We compared conventional atomizing disc aeroponic technology with the latest ultrasonic nebulizer technology for production of Glomus intraradices inocula. The piezo ceramic element technology used in the ultrasonic nebulizer employs high-frequency sound to nebulize nutrient solution into microdroplets 1 μm in diameter. Growth of pre-colonized arbuscular mycorrhizal (AM) roots of Sudan grass was achieved in both chambers used but both root growth and mycorrhization were significantly faster and more extensive in the ultrasonic nebulizer system than in the atomizing disc system. Shearing of the AM fungi (AMF) infected roots in both the systems did not reduce inoculum viability, as evident from the MPN data. However, sheared roots from the ultrasonic nebulizer system had significantly more infective propagules than those produced in the atomizing disc system. Thus, the latest ultra-sonic nebulizer aeroponic technology appears to be superior and an alternative to conventional atomizing disc or spray nozzle systems for the production of high-quality AMF inocula. These can be used in small doses to produce a large response, which is a prerequisite for commercialization of AMF technology. Accepted: 11 January 2000     

Thioredoxin acts as a B cell growth factor in channel catfish

To identify differentially expressed genes from channel catfish macrophages, a cDNA library from LPS-stimulated catfish macrophages was screened by subtractive hybridization. This screening yielded a 552-bp cDNA coding for catfish thioredoxin (CF-TRX). The deduced amino acid sequence revealed that CF-TRX contains 107 amino acids and is 59% homologous to human adult T cell leukemia-derived factor/TRX, originally described as an IL-2R alpha-inducing factor. Northern blot analyses showed that CF-TRX is expressed in catfish T and macrophage cell lines, but weakly in B cell lines. Similar results were also observed in Western blot analyses using a mAb specific for recombinant CF-TRX (rTRX). The use of rTRX in functional studies demonstrated that rTRX induces in vitro proliferative responses of catfish PBL that were synergistically enhanced by the addition of culture supernatants from catfish T cell lines. In addition, cell separation studies and flow cytometric analyses revealed that the cells proliferating in rTRX-stimulated cultures were mostly B cells. These results suggest that CF-TRX may have an important role in the activation and proliferation of channel catfish B cells.     

Distribution of newly synthesized aggrecan in explant cultures of bovine cartilage treated with retinoic acid.

This paper describes temporal changes in the metabolism and distribution of newly synthesized aggrecan and the organization of the extracellular matrix when explant cultures of articular cartilage maintained in the presence of fetal calf serum were exposed to retinoic acid for varying periods of time. Explant cultures of articular cartilage were incubated with radiolabeled sulfate prior to exposure to retinoic acid. The radiolabeled and chemical aggrecan present in the tissue and appearing in the culture medium was studied kinetically. Changes in the localization of radiolabeled aggrecan within the extracellular matrix were monitored by autoradiography in relation to type VI collagen distribution in the extracellular matrix. In control cultures where tissue levels of aggrecan remain constant the newly synthesized aggrecan remained closely associated with the territorial matrix surrounding the chondrocytes. Exposure of cultures to retinoic acid for the duration of the experiment, resulted in the extensive loss of aggrecan from the tissue and the redistribution of the remaining radiolabeled aggrecan from the chondron and territorial matrix into the inter-territorial matrix. These changes preceded alterations in the organization of type VI collagen in the extracellular matrix that involved the remodeling of the chondron and the appearance of type VI collagen in the inter-territorial matrix; there was also evidence of chondrocyte proliferation and clustering. In cartilage explant cultures exposed to retinoic acid for 24 h there was no loss of aggrecan from the matrix but there was an extensive redistribution of the radiolabeled aggrecan into the inter-territorial matrix. This work shows that maintenance of the structure and organization of the extracellular matrix that comprises the chondron and pericellular microenvironment of chondrocytes in articular cartilage is important for the regulation of the distribution of newly synthesized aggrecan monomers within the tissue.     

Caspase inhibitors of the P35 family are more active when purified from yeast than bacteria

Many insect viruses express caspase inhibitors of the P35 superfamily, which prevent defensive host apoptosis to enable viral propagation. The prototypical P35 family member, AcP35 from Autographa californica M nucleopolyhedrovirus, has been extensively studied. Bacterially purified AcP35 has been previously shown to inhibit caspases from insect, mammalian and nematode species. This inhibition occurs via a pseudosubstrate mechanism involving caspase-mediated cleavage of a "reactive site loop" within the P35 protein, which ultimately leaves cleaved P35 covalently bound to the caspase's active site. We observed that AcP35 purifed from Saccharomyces cerevisae inhibited caspase activity more efficiently than AcP35 purified from Escherichia coli. This differential potency was more dramatic for another P35 family member, MaviP35, which inhibited human caspase 3 almost 300-fold more potently when purified from yeast than bacteria. Biophysical assays revealed that MaviP35 proteins produced in bacteria and yeast had similar primary and secondary structures. However, bacterially produced MaviP35 possessed greater thermal stability and propensity to form higher order oligomers than its counterpart purified from yeast. Caspase 3 could process yeast-purified MaviP35, but failed to detectably cleave bacterially purified MaviP35. These data suggest that bacterially produced P35 proteins adopt subtly different conformations from their yeast-expressed counterparts, which hinder caspase access to the reactive site loop to reduce the potency of caspase inhibition, and promote aggregation. These data highlight the differential caspase inhibition by recombinant P35 proteins purified from different sources, and caution that analyses of bacterially produced P35 family members (and perhaps other types of proteins) may underestimate their activity.     

Screening fungi for improved glucoamylase productivity using buffered Dextran broth cultures

Summary Improved glucoamylase productivity was screened for using surface cultures in small tubes containing Dextran T-10 broth buffered at a pH supra-optimal for activity of the glucoamylase of the parental strain. Visual identification of improved mutations on microtitre plates is based on their relative abilities to produce residual glucose in culture filtrates. A 141% increase in glucoamylase was achieved.     

High Fidelity System Modeling for High Quality Image Reconstruction in Clinical CT

Today, while many researchers focus on the improvement of the regularization term in IR algorithms, they pay less concern to the improvement of the fidelity term. In this paper, we hypothesize that improving the fidelity term will further improve IR image quality in low-dose scanning, which typically causes more noise. The purpose of this paper is to systematically test and examine the role of high-fidelity system models using raw data in the performance of iterative image reconstruction approach minimizing energy functional. We first isolated the fidelity term and analyzed the importance of using focal spot area modeling, flying focal spot location modeling, and active detector area modeling as opposed to just flying focal spot motion. We then compared images using different permutations of all three factors. Next, we tested the ability of the fidelity terms to retain signals upon application of the regularization term with all three factors. We then compared the differences between images generated by the proposed method and Filtered-Back-Projection. Lastly, we compared images of low-dose in vivo data using Filtered-Back-Projection, Iterative Reconstruction in Image Space, and the proposed method using raw data. The initial comparison of difference maps of images constructed showed that the focal spot area model and the active detector area model also have significant impacts on the quality of images produced. Upon application of the regularization term, images generated using all three factors were able to substantially decrease model mismatch error, artifacts, and noise. When the images generated by the proposed method were tested, conspicuity greatly increased, noise standard deviation decreased by 90% in homogeneous regions, and resolution also greatly improved. In conclusion, the improvement of the fidelity term to model clinical scanners is essential to generating higher quality images in low-dose imaging.