Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.     

Chromosomal localization of ARSB,the gene for human N-acetylgalactosamine-4-sulphatase

Summary A deficiency of N-acetylgalactosamine-4-sulphatase (G4S, gene symbol ARSB), results in the accumulation of undegraded substrate and the lysosomal storage disorder, Maroteaux-Lamy syndrome (mucopolysaccharidosis type VI). In situ hybridization using an ³H-labelled human G4S genomic DNA fragment to human metaphase chromosomes localized ARSB to chromosome 5q13–5q14. This location is consistent with, an refines, previous chromosomal assignments based on the expression of human G4S in somatic cell hybrids.     

Morphological alterations and cytoskeletal reorganization in opossum kidney (OK) cells during osmotic swelling and volume regulation

Cells from a variety of tissues regulate their volume when exposed to anisotonic conditions. After exposure of cells to hypotonic conditions, the rapid phase of cell swelling is followed by a slower phase of cell shrinkage towards the initial volume. The present study investigates morphological alterations of adherent and fully spread cells after exposure to hypotonic conditions and the reorganization of cytoskeletal components such as F-actin, actin-binding proteins, microtubules and intermediate-sized filaments. We used cells of a continuous epithelial cell line from the opossum kidney (OK cells), which were exposed to hypotonic conditions for a period of 60 min at 25° C. The osmolarity was reduced by 40% from 320 mosmol/l (isotonic conditions) to 192 mosmol/l (hypotonic conditions). The initial swelling after exposure of OK cells to hypotonic conditions caused enhanced ruffling membrane activity, formation of lamellipodia and an extended space between adjacent cells which was caused by a more rounded cell shape. Moreover, the height of cells located in the centre of cell clusters increased by 32±8% (mean value±SEM) as checked by morphometric analysis of the vertical distance between the apical and basolateral F-actin domain. Although the fluorescence intensity and organization of F-actin in a horizontal direction remained unaltered during cell swelling, we observed a loss of periodicity and irregular distribution of myosin aggregates and a partial rear-rangement of vimentin filaments in the form of short fragments. In all experiments the organization of microtubules was observed to be unaltered. The alterations described above were reversible during cell shrinkage towards the initial volume, i.e. at 60 min after exposure to hypotonic conditions cell morphology and cytoskeletal organization no longer differed from the corresponding controls which were kept under isotonic conditions for the whole experimental period. The results demonstrate that only certain intracellular cytoskeletal components are actively involved in cell swelling and regulatory volume decrease.     

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.     

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.