Characterisation and migration properties of silicone materials during typical long-term commercial and household use applications: a combined case study.

In consequent continuation of previous described studies, pre-characterised silicone materials were assessed for chemical and physical parameters during long-term usage. In a particular case study silicone moulds were used in a commercial pizza bakery on a daily basis up to 1700 times. Migration behaviour, uptake of fat, the amount of volatiles and extractables, as well as physical properties (elongation, tensile strength) were monitored for the whole period. The main question was whether a significant degradation or even breakdown of the silicone elastomer could take place yielding enhanced migration of dimethyl siloxanes. Oligomeric dimethyl siloxanes are reaction side-products of the polymerisation process and despite their origin as so-called non-intentionally added substances (NIAS) were found to be the by far most dominating constituents of the overall migration. Furthermore, the influence of long-term thermal stress on the functionality of the elastomer was proven. Migration into food was determined by (1)H-NMR and was found to decrease during the experiment from values between 11 and 18 mg kg(-1) to levels below the limit of detection (LOD < 1 mg kg(-1)). No formation of migrating siloxanes beside the initial amount in the new, unused moulds could be observed. The loss of extractable siloxanes of the used compared with the new moulds was compensated by an uptake of fat and other lipophilic food constituents. The release of volatile organic compounds (VOC) decreased from 0.44% for the new moulds to 0.14% for the longest used ones (about 1700 individual uses; the corresponding summarised baking time was approximately 400 h at 180°C). GC-MS analysis of evaporating volatile compounds showed only cyclic oligomers for the new moulds but exclusively incorporated food components for the heavily used moulds. The physical properties of the silicone moulds remained almost constant during the experiment; no limitations in function due to the repeated thermal stress were observed. Similar results were obtained for baby teats under household conditions of use: a 100 times repeated simulated use in contact with milk followed by subsequent microwave sterilisation did not influence the function or mechanical properties. Because milk is only a weak extracting agent no significant changes in the amount of extractable siloxanes between new and used teats could was seen. Again an uptake of fat was seen and the amount of VOC decreased from 0.26% to 0.17%.

Comparison of the migration of melamine from melamine-formaldehyde plastics ('melaware') into various food simulants and foods themselves.

A variety of melaware articles were tested for the migration of melamine into the food simulant 3% w/v acetic acid as a benchmark, and into other food simulants, beverages and foods for comparison. The results indicate that the acidity of the food simulant plays a role in promoting migration, but not by as much as might have been anticipated, since 3% acetic acid gave migration values about double those obtained using water under the same time and temperature test conditions. In contrast, migration into the fatty food simulant olive oil was not detectable and at least 20-fold lower than with the aqueous food simulants. This was expected given the solubility properties of melamine and the characteristics of the melaware plastic. Migration levels into hot acidic beverages (apple juice, tomato juice, red-fruit tea and black coffee) were rather similar to the acetic acid simulant when the same time and temperature test conditions are used, e.g. 2 h at 70°C. However, migration levels into foods that were placed hot into melaware articles and then allowed to cool on standing were much lower (6-14 times lower) than if pre-heated food was placed into the articles and then maintained (artificially) at that high temperature in the same way that a controlled time-temperature test using simulants would be conducted. This very strong influence of time and especially temperature was manifest in the effects seen of microwave heating of food or beverage in the melaware articles. Here, despite the short duration of hot contact, migration levels were similar to simulants used for longer periods, e.g. 70°C for 2 h. This is rationalized in terms of the peak temperature achieved on microwave heating, which may exceed 70°C, counterbalancing the shorter time period held hot. There was also evidence that when using melaware utensils in boiling liquids, as for stovetop use of spatulas, the boiling action of circulating food/simulant can have an additional effect in promoting surface erosion, increasing the plastic decomposition and so elevating the melamine release.

Radio fluorination and positron emission tomography (PET) as a new approach to study the in vivo distribution and elimination of the advanced glycation endproducts N epsilon-carboxymethyllysine (CML) and N epsilon-carboxyethyllysine (CEL).

After synthesis of fluorine-18 labelled analogues by [18F]fluorobenzoylation at the alpha-amino group, biodistribution and elimination of individual advanced glycation endproducts, namely N epsilon-carboxymethyllysine and N epsilon-carboxyethyllysine, were studied in comparison to lysine in rats after intravenous injection using positron emission tomography (PET). The [18F]radiofluorinated amino acids were fast distributed via the blood, followed by a rapid excretion through the kidneys. Elimination kinetics were similar for both AGEs and lysine. For CML and CEL, but not for lysine, a temporary liver accumulation could be observed, which was not connected with any metabolisation or enterohepatic circulation. No further accumulation in any tissues was observable, indicating that increased tissue levels of CML or CEL, which have been described for certain disorders, are exclusively derived from endogenous origin and should not depend on a dietary intake. However, under uremic conditions, an impaired kidney function might result in a significant increase of the AGE-load of blood and tissues. PET based on 18F-labelled AGEs proved to be a promising tool to elucidate the physiological fate of post-translationally modified amino acids and to clarify the role of AGEs as possible "glycotoxins".

The designability of protein structures.

It has been noted that natural proteins adapt only a limited number of folds. Several researchers have investigated why and how nature has selected this small number of folds. Using simple models of protein folding, we demonstrate systematically that there is a "designability principle" behind nature's selection of protein folds. The designability of a structure (fold) is measured by the number of sequences that can design the structure--that is, sequences that possess the structure as their unique ground state. Structures differ drastically in terms of their designability. A small number of highly designable structures emerge with a number of associated sequences much larger than the average. These highly designable structures possess proteinlike secondary structures, motifs, and even tertiary symmetries. In addition, they are thermodynamically more stable and fold faster than other structures. These results suggest that protein structures are selected in nature because they are readily designed and stable against mutations, and that such a selection simultaneously leads to thermodynamic stability.

Pathway choice in glutamate synthesis in Escherichia coli.

Escherichia coli has two primary pathways for glutamate synthesis. The glutamine synthetase-glutamate synthase (GOGAT) pathway is essential for synthesis at low ammonium concentration and for regulation of the glutamine pool. The glutamate dehydrogenase (GDH) pathway is important during glucose-limited growth. It has been hypothesized that GDH is favored when the organism is stressed for energy, because the enzyme does not use ATP as does the GOGAT pathway. The results of competition experiments between the wild-type and a GDH-deficient mutant during glucose-limited growth in the presence of the nonmetabolizable glucose analog alpha-methylglucoside were consistent with the hypothesis. Enzyme measurements showed that levels of the enzymes of the glutamate pathways dropped as the organism passed from unrestricted to glucose-restricted growth. However, other conditions influencing pathway choice had no substantial effect on enzyme levels. Therefore, substrate availability and/or modulation of enzyme activity are likely to be major determinants of pathway choice in glutamate synthesis.

Emergence of preferred structures in a simple model of protein folding.

Protein structures in nature often exhibit a high degree of regularity (for example, secondary structure and tertiary symmetries) that is absent from random compact conformations. With the use of a simple lattice model of protein folding, it was demonstrated that structural regularities are related to high "designability" and evolutionary stability. The designability of each compact structure is measured by the number of sequences that can design the structure-that is, sequences that possess the structure as their nondegenerate ground state. Compact structures differ markedly in terms of their designability; highly designable structures emerge with a number of associated sequences much larger than the average. These highly designable structures possess "proteinlike" secondary structure and even tertiary symmetries. In addition, they are thermodynamically more stable than other structures. These results suggest that protein structures are selected in nature because they are readily designed and stable against mutations, and that such a selection simultaneously leads to thermodynamic stability.

icdB mutants of Escherichia coli.

icdB mutations map at 16 min, lead to the specific loss of citrate synthase, and are complemented by a prophage containing a gltA+ gene. Thus, they are allelic with gltA.

Why does Escherichia coli have two primary pathways for synthesis of glutamate?

Escherichia coli has two primary pathways for glutamate synthetase-glutamate synthase pathway is known to be essential for synthesis at low ammonium concentrations and for regulation of the glutamine pool, but the necessity for glutamate dehydrogenase (GDH) has been uncertain. The results of competition experiments between the wild type and a GDH-deficient mutant during nutrient-limited growth and of direct enzyme measurements suggest that GDH is used in glutamate synthesis when the cell is limited for energy (and carbon) but ammonium and phosphate are present in excess, while the glutamine synthetase-glutamate synthase pathway is used when the cell is not under energy limitation. The use of alternative routes for glutamate synthesis implies that the energy cost of biosynthesis may be less when energy is limited than when energy is unlimited.

Experimental Induction of Localized Reproduction in a Marine Bryozoan.

The control of reproduction and growth rate within colonies of marine invertebrates is often conditional and can be very localized. We demonstrate experimentally large and localized shifts in the timing and pattern of reproduction within colonies of a temperate bryozoan (Membranipora membranacea) in response to simulated damage by predators and crowding by conspecifics. In these protandrously hermaphrodite colonies, zooids on the damaged side of a colony reproduced sooner than in unmanipulated regions of the same colony. To examine the influence of the pattern of edge damage on localized reproduction, we damaged the perimeter of circular colonies in two patterns: (1) a continuous half of the edge was trimmed (1/2-Damage) and (2) the edge was trimmed in four alternating one-eighth sections (4/8-Damage). The 1/2-damage treatment triggered localized reproduction, and the more localized four-eighths-damage did not. These experiments demonstrate that the configuration rather than the total amount of edge damage affects the localization of reproduction. In parallel experiments, conspecifics were allowed to crowd half the perimeter of experimental colonies. This treatment also resulted in localized and accelerated reproduction near the contact zone adjacent to a conspecific. Not only do patterns of reproduction change in crowded or damaged colonies, but obstructed colonies also compensate for reduced growth at an obstructed edge by extending the adjacent unobstructed perimeter edge at a greater rate. One model to explain the sort of local cues governing the observed shifts in reproduction and growth rate is a source-sink model. A similar mechanism is proposed to underly growth and reproductive allocation in plants. We suggest that the balance between growth and onset of reproduction in zooids is determined by the rate of translocate moving through each zooid. The rate of translocate movement through zooids is, in turn, affected by the strength and proximity of sinks for that translocate, such as the growing edge of the colony. We propose a simple source-sink model of carbon flow to explain our experimental results. This model would account for the induction of localized reproduction in 1/2 damaged colonies and the lack of localization in 4/8 damaged colonies.

Genetic changes accompanying increased fitness in evolving populations of Escherichia coli.

Two populations of Escherichia coli, each initiated with a single clone containing a derivative of the plasmid pBR322, were maintained for long periods in glucose-limited continuous culture. In both populations, after an extensive number of generations had elapsed, clones were isolated in which the transposon Tn3 from the plasmid had integrated into the bacterial chromosome. In both cases examined, the transpositions were shown to increase relative fitness approximately 6-7%, in the environment in which the populations were maintained. The loci of integration were mapped to approximately 13.2 min (population 1) and approximately 32.8 min (population 2).

The glutamate dehydrogenase structural gene of Escherichia coli.

The glutamate dehydrogenase structural gene, gdhA, was mapped at 38.6 min on the genetic map and at 1860 kb on the physical map. A detailed map of this region is presented.

Processing of chloroplast ribosomal RNA transcripts in Euglena gracilis bacillaris.

The ribosomal RNA operons (rrn operons) of Euglena gracilis chloroplasts contain genes for (in order) 16S rRNA, tRNA(Ile), tRNA(Ala),23S rRNA and 5S rRNA. Major sites of cleavage of the primary rrn transcript were identified by Northern blot hybridization and S1-mapping. The presumptive termini of all of the mature products have now been identified. During initial processing in the chloroplast, the primary transcript is cleaved between the two tRNAs and between the 23S and 5S rRNAs so as to separate the sequences found in the different mature rRNAs. Subsequently the tRNAs are separated from the rRNAs, further trimming provides the remaining proper ends, and the 3'-ends of the tRNAs are added.

Initiation of rrn transcription in chloroplasts of Euglena gracilis bacillaris.

The site of initiation of chloroplast rRNA synthesis was determined by S1-mapping and by sequencing primary rRNA transcripts specifically labeled at their 5'-end. Transcription initiates at a single site 53 nucleotides upstream of the 5'-end of the mature 16S rRNA under all growth conditions examined. The initiation site is within a DNA sequence that is highly homologous to and probably derived from a tRNA gene-region located elsewhere in the chloroplast genome. A nearly identical sequence (102 of 103 nucleotides) is present near the replication origin. The near identity of the two sequences suggests a common mode for control of transcription of the rRNA genes and initiation of chloroplast DNA replication. The related sequence in the tRNA gene-region does not appear to serve as a transcript initiation site.

Evolution of Escherichia coli during growth in a constant environment.

Populations of Escherichia coli, initiated with a single clone and maintained for long periods in glucose-limited continuous culture, developed extensive polymorphisms. In one population, examined after 765 generations, two majority and two minority types were identified. Stable mixed populations were reestablished from the isolated strains. Factors involved in the development of this polymorphism included differences in the maximum specific growth rate and in the transport of glucose, and excretion of metabolites by some clones which were utilized by minority clones.

Sequence and evolution of the regions between thr rrn operons in the chloroplast genome of Euglena gracilis bacillaris.

The rRNA genes are arranged in three sequential operons preceded by a fourth partial operon. Part or all of a 1462 nucleotide sequence extending from within the 3'-end of the 23S rRNA gene, across the 5S rRNA gene and a presumptive transcription terminator, to within the first structural gene (for 16S rRNA) of the rrn operon was determined for each region between operons. Homologies of the 3'-end of the 23S rRNA gene with the 4.5S rRNA genes of higher plant chloroplasts, and of the 5S rRNA gene with other 5S rRNA genes were examined. The region preceding the 16S rRNA gene, which is expected to contain sites for initiation and regulation of rrn transcription, includes a 305 base-pair sequence with substantial homology with structural genes elsewhere in the chloroplast genome. The homologies suggest that this portion of the leader evolved from copies of parts of the structural genes which had been inserted before the 16S rRNA genes. Thus the chloroplast rrn leader may provide a unique opportunity to study how a regulatory sequence evolved from well-defined structural genes.

Structure and rearrangements of rRNA genes in chloroplast DNA in two strains of Euglena gracilis.

The organisation of the rRNA genes in the chloroplast genomes of two strains of Euglena gracilis were analyzed and compared. It was previously shown that the bacillaris strain contains three complete rrn (rRNA) operons (7) and that the Z-S strain contains one operon (21). Using heteroduplex analysis it was found that the bacillaris strain contains, apart from the three complete rrn operons, an extra 16S rRNA gene, an extra partial 23S rRNA gene sequence and an inverted duplication of a stretch within the 5S-16S spacer. In addition a short (<100 bp) inverted repeat sequence (13) which forms a stem/loop structure in single-stranded cpDNA was located between the 3'-end of the extra 16S rRNA gene and the partial 23 S rRNA sequence.The Z-S strain differs from the bacillaris strain by a deletion of two units of the complete rrn operons. The region upstream of the single complete rrn operon, including the inverted repeats, the partial 23S and the extra 16S rRNA sequences is identical with the bacillaris strain.The only non-homology found in heteroduplexes between the SalI fragments of B of the two strains is the deletion-insertion loop which represents the two rrn operons. A small deletion loop was found occasionally in hetero-and in homoduplexes of both strands in the region of variable size. Apart from the deletion/insertion of two rrn operons the two genomes appear to be colinear as can be seen from partial denaturation mapping. The organisation of the rRNA genes of the two strains is compared with those of the Z strain and the bacillaris-ATCC strain.

Cloning of mouse beta-casein gene sequences.

Casein messenger RNAs (mRNAcsn) were purified from lactating mammary glands of BALB/c mice and used as a starting material for cloning of casein gene sequences. Double-stranded casein cDNA (ds-cDNAcsn) was prepared and blunt-end ligated to HindIII-specific DNA linker molecules. After digestion with HindIII, the dsDNAcsn was inserted into the HindIII site of plasmid pBR322, using T4 DNA ligase. Escherichia coli strain RH202 was transformed with the hybrid plasmids, and transformants were selected for resistance to ampicillin. Electrophoresis of HindIII-digested hybrid plasmid DNAs, followed by Southern transfer and hybridization to [32P]cDNAcsn, revealed that one of the hybrid-plasmid-containing colonies, designated pCas51, contained a 400-bp insert which hybridized to the [32P]cDNAcsn. Purification of the individual casein mRNAs (mRNAcsn alpha, beta, and gamma) and solution hybridization of nick-translated insert DNA to each of these revealed that pCas51 contained sequences complementary primarily to mRNAcsn beta.