T cell recognition of melanoma antigens in association with HLA-A1 on allogeneic melanoma cells

Previous studies have shown that recognition of melanoma by cytotoxic T lymphocytes may be restricted by HLA-A1, A2 and other HLA antigens. The present study examined the cytotoxic specificity and major histocompatibility complex restriction of cloned cytotoxic T lymphocytes (CTL) isolated from a patient with the HLA phenotype A3,31 who had been immunized with a vaccine prepared from HLA-A1,3 melanoma cells. Cytotoxic assays against HLA-typed allogeneic melanoma cells indicated that cloned CTL from the patient were able to kill allogeneic melanoma cells expressing HLA-A1 but not other HLA-A1-positive cells. Studies on a representative clone indicated that proliferation and cytokine (tumour necrosis factor ) production in response to melanoma cells was also associated with HLA-A1 on melanoma cells. Response to the melanoma cells was associated with interleukin-4 (IL-4) rather than IL-2 production. The antigen recognized in the context of HLA-A1 on allogeneic melanoma cells was detected in cytotoxic assays on cells from 9 of 12 HLA-A1⁺ melanoma cell lines and did not appear to be the product of the MAGE-1 or-3 genes. These findings suggest that T cells can recognize melanoma antigens in the context of alloantigens and that allogeneic vaccines containing immunodominant alloantigens may generate CTL that are ineffective against autologous melanoma. The study does not, however, exclude the possibility that CTL with specificity to the latter may be activated by allogeneic vaccines, and further studies are needed to answer this question.     

IRE-Bubble PCR: A Rapid Method for Efficient and Representative Amplification of Human Genomic DNA Sequences from Complex Sources

A significant issue in the analysis of any genomic DNA segment is the generation of a unique set of short single-copy sequences that are representative of that region. In this report we describe a novel technique, IRE-bubble PCR, which was designed to amplify the human DNA content of somatic cell hybrids, YACs, cosmids, and λ phage and result in greater complexity and representation than standard inter-IRE, PCR. Here we demonstrate that IRE-bubble PCR is species specific and that it results in the generation of a product that is at least 10-fold more complex and representative than that produced by standard inter-IRE PCR. In addition, we have addressed the factors that contribute to the representation of the IRE-bubble PCR product and show how they may be used to further increase the complexity of this reaction. Finally, we have illustrated how the complexity and distribution of products generated by IRE-bubble PCR can be exploited and applied to FISH mapping and "chromosome painting" as well as to the generation of STSs targeted to specific chromosomal or subchromosomal regions.     

A lack of locomotor activity rhythms inDrosophila melanogaster larvae (Diptera: Drosophilidae)

We examined the locomotor activity ofDrosophila melanogaster for the existence of circadian rhythms, using the wild type and two mutants of theperiod (per) gene,per ^o andper ^s. This was accomplished using a newly described apparatus for the recording and measurement of larval path lengths over a 96-h test period. None of the larvae examined exhibited appreciable diel rhythms under cycling conditions of light or temperature. Larvae were also not rhythmic under free-running conditions. Our results suggest that theper gene does not influence an observable locomotor behavioral phenotype in the larval stage of development.     

Kanamycin resistance as a selectable marker for plastid transformation in tobacco

We report on a novel chimeric gene that confers kanamycin resistance on tobacco plastids. The kan gene from the bacterial transposon Tn5, encoding neomycin phosphotransferase (NPTII), was placed under control of plastid expression signals and cloned between rbcL and ORF512 plastid gene sequences to target the insertion of the chimeric gene into the plastid genome. Transforming plasmid pTNH32 DNA was introduced into tobacco leaves by the biolistic procedure, and plastid transformants were selected by their resistance to 50 g/ml of kanamycin monosulfate. The regenerated plants uniformly transmitted the transplastome to the maternal progeny. Resistant clones resulting from incorporation of the chimeric gene into the nuclear genome were also obtained. However, most of these could be eliminated by screening for resistance to high levels of kanamycin (500 g/ml). Incorporation of kan into the plastid genome led to its amplification to a high copy number, about 10000 per leaf cell, and accumulation of NPTII to about 1% of total cellular protein.     

Isolation and Molecular Characterization of Five Marine Cyanophages Propagated on Synechococcus sp. Strain WH7803

Five marine cyanophages propagated on Synechococcus sp. strain WH7803 were isolated from three different oceanographic provinces during the months of August and September 1992: coastal water from the Sargasso Sea, Bermuda; Woods Hole harbor, Woods Hole, Mass.; and coastal water from the English Channel, off Plymouth Sound, United Kingdom. The five cyanophage isolates were found to belong to two families, Myoviridae and Styloviridae, on the basis of their morphology observed in the transmission electron microscope. DNA purified from each of the cyanophage isolates was restricted with a selection of restriction endonucleases, and three distinguishably different patterns were observed. DNA isolated from Myoviridae isolates from Bermuda and the English Channel had highly related restriction patterns, as did DNA isolated from Styloviridae isolates from Bermuda and the English Channel. DNA isolated from the Myoviridae isolate from Woods Hole had a unique restriction pattern. The genome size for each of the Myoviridae isolates was ca. 80 to 85 kb, and it was ca. 90 to 100 kb for each of the Styloviridae isolates. Southern blotting analysis revealed that there was a limited degree of homology among all cyanophage DNAs probed, but clear differences were observed between cyanophage DNA from the Myoviridae and that from the Styloviridae isolates. Polypeptide analysis revealed a clear difference between Myoviridae and Styloviridae polypeptide profiles, although the major, presumably structural, protein in each case was ca. 53 to 54 kDa.     

The evolution and ecology of multiple antipredator defences

Prey seldom rely on a single type of antipredator defence, often using multiple defences to avoid predation. In many cases, selection in different contexts may favour the evolution of multiple defences in a prey. However, a prey may use multiple defences to protect itself during a single predator encounter. Such “defence portfolios” that defend prey against a single instance of predation are distributed across and within successive stages of the predation sequence (encounter, detection, identification, approach (attack), subjugation and consumption). We contend that at present, our understanding of defence portfolio evolution is incomplete, and seen from the fragmentary perspective of specific sensory systems (e.g., visual) or specific types of defences (especially aposematism). In this review, we aim to build a comprehensive framework for conceptualizing the evolution of multiple prey defences, beginning with hypotheses for the evolution of multiple defences in general, and defence portfolios in particular. We then examine idealized models of resource trade-offs and functional interactions between traits, along with evidence supporting them. We find that defence portfolios are constrained by resource allocation to other aspects of life history, as well as functional incompatibilities between different defences. We also find that selection is likely to favour combinations of defences that have synergistic effects on predator behaviour and prey survival. Next, we examine specific aspects of prey ecology, genetics and development, and predator cognition that modify the predictions of current hypotheses or introduce competing hypotheses. We outline schema for gathering data on the distribution of prey defences across species and geography, determining how multiple defences are produced, and testing the proximate mechanisms by which multiple prey defences impact predator behaviour. Adopting these approaches will strengthen our understanding of multiple defensive strategies.     

Combining industrial ecology tools to assess potential greenhouse gas reductions of a circular economy: Method development and application to Switzerland

Industrial ecology (IE) methodologies, such as input/output or material flow analysis and life cycle assessment (LCA), are often used for the environmental evaluation of circular economy strategies. Up to now, an approach that utilizes these methods in a systematic, integrated framework for a holistic assessment of a geographic region's sustainable circular economy potential has been lacking. The approach developed in this study (IE4CE approach) combines IE methodologies to determine the environmental impact mitigation potential of circular economy strategies for a defined geographic region. The approach foresees five steps. First, input/output analysis helps identify sectors with high environmental impacts. Second, a refined analysis is conducted using material flow and LCA. In step 3, circular strategies are used for scenario design and evaluated in step 4. In step 5, the assessment results are compiled and compared across sectors. The approach was applied to a case study of Switzerland, analyzing 8 sectors and more than 30 scenarios in depth. Carbon capture and storage (CCS) from waste incineration, biogas and cement production, food waste prevention in households, hospitality and production, and the increased recycling of plastics had the highest mitigation potential. Most of the scenarios do not influence each other. One exception is the CCS scenarios: waste avoidance scenarios decrease the reduction potential of CCS. A combination of scenarios from different sectors, including their impact on the CCS scenario potential, led to an environmental impact mitigation potential of 11.9 Mt CO₂-eq for 2050, which equals 14% of Switzerland's current consumption-based impacts.     

Development and use of probability models: The industry perspective

Summary In the processed meat industry, food safety and microbiological shelf life issues lend themselves to the use of probability modeling. Our research concentrated on predicting the effectiveness of sodium lactate as an antibotulinal agent in vacuum packaged, uncured and cured turkey breast model systems. In uncured turkey breast containing 1.4% NaCl, 0.3% Na phosphate, and 0–3% Na lactate, the antibotulinal effect of sodium lactate can be predicted using the following model: Days to toxicity = 3.13+0.39(Na lactate)². Using cured turkey breast with 0.3% Na phosphate, 0.2% sucrose, 0–3% Na lactate, the time to toxicity can be predicted from the following model: Days to toxicity = 1.69+4.88(NaCl)–11.16(Na lactate)+7.23(Na lactate)². Probability models have also been developed to predict the refrigerated shelf life of specific processed meat products. The usefulness of the predictive modeling for food safety and quality in the food industry will also be discussed.This paper was presented at The International Conference on the Application of Predictive Microbiology and Computer Modeling Techniques to the Food Industry, April 12–15 1992, Hyatt Regency Hotel, Tampa, FL, USA.