International Commission for Protection against Environmental Mutagens and Carcinogens. ICPEMC Working Paper No. 15/5. Acute aldehyde syndrome and chronic aldehydism

Different types of alcohol dehydrogenase and of aldehyde dehydrogenase lead to different blood acetaldehyde levels. With respect to acetaldehyde levels in human blood 3 types can be distinguished: (1) the normal range, (2) the acute aldehyde syndrome, and (3) the chronic aldehydism. Acetaldehyde is electrophilic and reacts with nucleophilic groups of various macromolecules including DNA. Acetyldehyde inhibits synthetic and metabolic pathways, it interferes with the polymerization of tubulin and stimulates collagen synthesis. By depletion of cellular glutathione levels, acetaldehyde leads to lipid peroxidation and to the formation of malonaldehyde. There are indications that acetaldehyde may play a role in positively reinforcing mood changes induced by alcohol in humans.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

The onset of the respiratory burst in human neutrophils. Real-time studies of H2O2 formation reveal a rapid agonist-induced transduction process

A real-time study of the initiation of the respiratory burst in human neutrophils was made. The cells were stimulated with fMet-Leu-Phe (fMLP) C5a, platelet-activating factor, leukotriene B4, phorbol myristate acetate (PMA), or ionomycin, and H2O2 production was determined by chemiluminescence. Identical average onset times (2.4 s) and closely comparable values for the apparent first-order rate constant (kapp) for the induction of NADPH-oxidase activity (0.21-0.29 s-1) were obtained following stimulation with fMLP, C5a, platelet-activating factor, or leukotriene B4, suggesting that different agonists act through a common transduction sequence. Much longer onset times and lower kapp values were obtained upon stimulation with PMA or ionomycin. Pretreatment with PMA consistently shortened the onset time of the neutrophil's responses to agonists by about 1 s. When H2O2 production was initiated with PMA, a subsequent stimulation with the agonist fMLP elicited an immediate response (onset time less than 0.2 s) which preceded further changes in fura-2-detected [Ca2+]i. The results are consistent with a mechanism in which agonist signals appear to be transduced by two sequences acting in concert--a rate-limiting one liberating Ca2+ and diacylglycerol and turning on the Ca2+/phospholipid-dependent enzyme protein kinase C, and an essentially instantaneous one which does not appear to require further changes in cytosolic Ca2+.     

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic identification of rat liver carboxylesterases isolated in different laboratories

Six carboxylesterases previously isolated from rat liver microsomes, characterized in Brussels and in Kiel, were compared with genetically defined liver esterases of various reference strains using polyacrylamide gel electrophoresis and isoelectric focusing. The six liver carboxylesterases were identified as alloenzymic forms of ES-3, ES-4, ES-8/ES-10 and ES-15 according to the genetic nomenclature recommended by van Zutphen (Van Zutphen, L.F.M. (1983) Transplant. Proceed. 15, 1687-1688). The genetic and biochemical characteristics of the four isoenzymes are summarized, and their identity with several other drug-metabolizing esterases/amidases and lipases of rat liver endoplasmic reticulum is discussed.     

A mutant rat major histocompatibility haplotype showing a large deletion of class I sequences

The LEW.1LM1 inbred rat strain, which has been derived from a (LEW×LEW.1W) F₂ hybrid, carries a major histocompatibility (RT1) haplotype which is distinct from that of the LEW strain (RT1 ¹) in that certainRT1.C region-determined class I antigens are not expressed. Here we show that this phenotypic defect is due to genomic deletion of about 100 kb of theRT1.C region. Certain deleted DNA fragments have been cloned from the wild-type DNA into the EMBL4 vector. Five clones have been characterized and are shown to possess different restriction maps and to each carry a single stretch of class I cross-hybridizing sequences. Probes derived from the non-class I coding part of two clones detect fragments which are present in the wild-type but absent from thelm1 mutant. The type of deletion described here in the rat is discussed in the context ofH-2D/Q deletions in the mouse.     

Early and late effects in the bone marrow of mice following 2 Gy (6 MeV) neutron irradiation

Summary Following 5 Gy gamma irradiation, residual damage in bone marrow persisted up to one year and was ascribed to genetic defects in hemopoietic stem cells (von Wangenheim et al. 1986). To see whether high LET radiation is more efficient in inducing late effects, mice were whole-body irradiated with a single dose of 2 Gy neutrons ( = 6 MeV) and femoral cellularity, CFU-S number, proliferation ability of bone marrow cells (PF) and the compartment ratio (CR), i.e. the splenic 125-iodo-deoxyuridine incorporation per transfused CFU-S were measured up to one year after the radiation insult. Within 12 weeks, femoral cellularity, PF and CR recovered to control or near-control level, whereas CFU-S numbers remained significantly below control. No further recovery was observed. On the contrary, PF and CR deteriorated again after 12 and 26 weeks, respectively. CFU-S per femur tended to decrease as well. Thus it is demonstrated that a single dose of 2 Gy 6 MeV neutrons causes significant injury in function (PF) and structure (CFU-S numbers, CR) of bone marrow which persisted up to one year. While this residual injury can be attributed to genetic defects in hemopoietic stem cells, its increasing expression is probably due to late evolving damage in microenvironmental cells. The RBE of 6 MeV neutrons for the introduction of late effects in the bone marrow is in the range of 3.     

The heat generated by yeast cultures with a mixed metabolism in the transition between respiration and fermentation

The heat generated by both batch and continuous cultures of the yeast K. fragilis was studied using a modified Bench Scale Calorimeter. Batch cultures were used to measure the heat dissipation rates and the heat yields during fully aerobic and completely anaerobic growth, whereas continuous cultures enabled, in addition, a quantitative study of heat dissipation rates during growth on mixed metabolism. In this case, the extent of fermentation versus respiration could be specified and controlled by varying the degree of oxygen limitation. The heat dissipated per unit biomass formed was highest for fully respirative catabolism and fell continuously to a much lower value typical of anaerobic cultures as the catabolism was shifted increasingly to the fermentative mode. The heat generated per mole of oxygen taken up stayed quite close to the fully aerobic value of 506 kJ mol(-1) even when a sizable fraction of the substrate available to catabolism was fermented. If the fraction of respiration in the metabolism is lowered beyond a certain threshold, the ratio of the heat generation to oxygen consumption starts to increase dramatically and finally tends to infinity for fully anaerobic growth. All experimental results were quantitatively analyzed and explained on the basis of a simple model which formally describes the cultures in terms of two parallel "chemical" reactions. In simple cases such as the one presented here, the model enables calculation of the whole stoichiometry of the culture from a single measured heat yield.     

Cleavage-site motifs in mitochondrial targeting peptides

Although mitochondrial targeting peptides lack a common consensus sequence, a certain bias in the positional distribution of amino acids has recently been found. These patterns seem to be associated with cleavage of the precursor proteins by matrix processing proteases. We have extended the previous studies and found new sequence motifs that are conserved within subgroups of mitochondrial targeting peptides. These motifs have certain common themes, indicating that they are associated with cleavage by one single protease. Two of the conserved patterns have a high predictive value, but even for sequences that do not possess these patterns, a fairly accurate prediction of the cleavage site is shown to be possible. We also suggest that a well-conserved RXY decreases (S/A) pattern may be used to engineer efficiently recognized cleavage sites into uncleaved or artificial mitochondrial targeting peptides.