Enkephalin pseudopeptides: resistance to in vitro proteolytic degradation afforded by amide bond replacements extends to remote sites

Five analogs of leucine enkephalin containing the CH2S group as an amide bond replacement were evaluated with respect to resistance toward degradation by human serum in an HPLC-based assay using both ultraviolet and electrochemical detection. Analogs with the modification at the 1-2, 2-3, 3-4, or 4-5 peptide linkages demonstrated half-lives of 118, 85, 134, and 318 min vs. 12 min for the parent peptide. A pseudopeptide analog with additional D-Ala2 protection had a half-life of greater than 1000 min, while the potent [D-Ala2]-leucine enkephalin analog showed approximately a 10-fold increase in stability. The significant increase in stability for a compound with protection only at the C-terminus suggests that serum enzymes may have greater specificity toward backbone changes than previously realized.     

Site-directed insertion mutagenesis with cloned fragments in Escherichia coli by P1 phage transduction

Summary A cloned gene with an insertion, which was made by introducing cat, was ligated to the cloning site of the phage gt11. P1 phage grown on cells lysogenized with the recombinant phage could transduce the mutant gene into the original site on the Escherichia coli chromosome.     

Lingulid shell mediation in clay formation

Organophosphatic shells of the brachiopod Lingula squarniformis , collected from Scottish Lower Carboniferous shales and mudstones of intertidal to sublittoral provenance, have been studied to ascertain chemico-structural changes resulting from fossilization. Enough original shell has been preserved at ultrastructural and molecular levels to confirm that Carboniferous and Recent integuments are homologous with stratiform successions of apatitic to organic laminae forming rhythmic sets. One of the main organic constituents, the acidic, hydrophilic gel glycosaminoglycans (GAGs), is the dominant component towards the tops of rhythms. During fossilization of the Carboniferous shells, GAGs degraded incrementally without disturbing apatitic ultrastructures, and the spaces so created became partly filled with sheets of recrystal-lized apatite with some kaolinite or with books and plates of kaolinite. The kaolinite in the shells contrasts with the illite of the entombing sediments and suggests that degrading acidic GAGs mediated in clay formation in situ . The sediments also contain framboidal pyrite, which is virtually absent from the shells themselves but is usually even more abundant, with a greater range of trace metals, in the sedimentary fills of complete shells. This imbalance suggests mediation by another gel, the glycocalyx, secreted by the inner epithelium of the brachiopod mantle. The glycocalyx would have lined the shell interior and could have served as a sorption film for dissolved metals precipitated as compounds on decomposition of body tissue.     

Valve movement of the freshwater mussel Anodonta anatina: a reciprocal transplant experiment between lake and river

The effects of source and caging on the valve movements of the freshwater unionid mussel (Anodonta anatina) were studied in a reciprocal transplant experiment between a lake and its outflow. Caged mussels were moved and compared with those remaining in their natural environment on the lake or river bottom. At both sites, the mussels from the study site and the transplanted mussels from the opposite site were monitored simultaneously. In river the averaged weighted valve openness was higher and the number of valve movements was lower than in the lake. The mussels monitored in the lake exhibited a diurnal rhythm of valve movements which differed between the lake-bottom and the caged animals. Caging was found to increase valve openness. On the other hand, little variation appeared in valve openness between caged and bottom animals in the river, where diurnal rhythms were almost nonexistent. In the river the valve movements were more variable in respect to time than in the lake.     

Competition between electron acceptors in photosynthesis: Regulation of the malate valve during CO2 fixation and nitrite reduction

For maximal rates of CO₂ assimilation in isolated intact spinach chloroplasts the generation of the adequate NADPH/ATP ratio is achieved either by cyclic electron flow around photosystem I or by linear electron transport to oxaloacetate, nitrite or oxygen (Mehler-reaction). The interrelationships between these poising mechanisms turn out to be strictly hierarchical. In the presence of antimycin A, an inhibitor of ferredoxin-dependent cyclic electron transport, the reduction of both, oxaloacetate and nitrite, but not that of oxygen restores CO₂ fixation. When oxaloacetate and nitrite are added at low concentrations simultaneously during steady-state CO₂ fixation, the reduction of nitrite is clearly preferred over the reduction of oxaloacetate, but CO₂ fixation is not influenced. Nitrite reduction is not decreased upon addition of oxaloacetate, but vice versa. This is due to the regulation of NADP-malate dehydrogenase activation by electron pressure via the ferredoxin/thioredoxin system on the one hand, and by the NADPH/(NADP+NADPH) ratio (anabolic reduction charge, ARC) on the other hand. Thus the closing of the malate valve prevents drainage of reducing equivalents from the chloroplast (1) when a low ARC indicates a high demand for NADPH in the stroma and (2) when nitrite reduction reduces the electron pressure at ferredoxin. The malate valve is opened when cyclic electron transport is inhibited by antimycin A. Under these conditions the rate of malate formation is higher than in the absence of the inhibitor even in the presence of oxaloacetate, thus indicating that the regulation of the malate valve functions at various redox states of the acceptor side of Photosystem I.Abbreviations ARC anabolic reduction charge (NADPH/(NADP+NADPH)) - Chl chlorophyll - DTT dithiothreitol; Fd-ferredoxin - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetate - PS photosystem - qN non-photochemical quenching - qP photochemical quenching - _E quantum efficiency of PS II Dedicated to Prof. Dr. Hans Walter Heldt on the occasion of his 60th birthday.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes

Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides. Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin. The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase. Subsequently, it was unambiguously demonstrated by overexpression studies that theenvM gene encodes the bacterial enoyl-ACP reductase. An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic. The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of anenvM mutant ofEscherichia coli. Ultimately,envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex. Finally, lipid analysis of recombinantE. coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids.     

Crystallization and preliminary crystallographic studies of the precursor and mature forms of a neutral lipase from the fungus Rhizopus delemar

A neutral lipase from the filamentous fungus Rhizopus delemar has been crystallized in both its proenzyme and mature forms. Although the latter crystallizes readily and produces a variety of crystal forms, only one was found to be suitable for X-ray studies. It is monoclinic (C2, a = 92.8 Å, b = 128.9 Å, c = 78.3 Å, β = 135.8) with two molecules in the asymmetric unit related by a noncrystallographic diad. The prolipase crystals are orthorhombic (P2₁2₁2₁, with a = 79.8 Å, b = 115.2 Å, c = 73.0 Å) and also contain a pair of molecules in the asymmetric unit. Initial results of molecular replacement calculations using the refined coordinates of the related lipase from Rhizomucor miehei identified the correct orientations and positions of the protein molecules in the unit cells of crystals of both proenzyme and the mature form. © 1994 John Wiley & Sons, Inc.     

Until death do us part? In-depth insights into Dutch consumers’ considerations about product lifetimes and lifetime extension

Long-lasting electronic products contribute to a sustainable society; however, both expected and actual lifetimes are in decline. This research provides in-depth insights into consumers’ considerations about product lifetimes, barriers to extending lifetimes, and responses to a product lifetime label. Results of interviews (n = 22) with Dutch consumers suggest a positive view on long-lasting products. Nevertheless, their products’ value depreciated during their lifetimes. Consumers consider themselves unable to estimate how long products should last, which can be detrimental as low expectations tend to negatively influence actual lifetimes. Also, use intensity and consumers’ care(less) behavior influence the lifetime. To extend product lifetimes, consumers often disregard the option of repairing malfunctioning products. They have limited knowledge and ability, and believe repair provides poor value for money. Lifetime extension can also be hindered by market-related factors, such as convenient replacement services, new technological developments, and (attractive) deals. We suggest a product lifetime label should contain relevant and reliable information; furthermore, we recommend including (extended) warranty information. When information about repairability is included, potential negative responses should be considered. Finally, raising awareness about the environmental impact of short-lived products via a label may have a positive effect but requires more research attention.