Changes in the inducibility of a hepatic aldehyde dehydrogenase by various effectors

A hepatic soluble aldehyde dehydrogenase (ALDH), inducible by polycyclic aromatic hydrocarbons, was studied in Wistar rats in connection with substances known to affect drug metabolism or aldehyde dehydrogenase activity, such as phenobarbital (PB), disulfiram (DS), -diethylaminoethyl diphenylpropylacetate (SKF 525A) and calcium cyanamide (CC). 3-Methylcholanthrene (MC) was given as a model inducer of ALDH (100 mg/kg, i.p., as a single dose) and the animals were killed after 3 days. Pretreatment with PB (1 g/l drinking water, for 2 weeks) enhanced the inducing effect of MC. On the contrary, pretreatment with DS (100 mg/kg, i.p., daily x4) reduced by 70% the expected increase in ALDH activity. Neither SKF 525A (25 mg/kg, i.p., daily x4), nor CC (5 mg/kg, i.p., daily x4) could affect the action of the inducer. At the above doses, basal ALDH activity was inhibited by DS (30%) and CC (70%), but was not affected at all by PB or SKF 525A. The results were somewhat different when the various effectors tested were administered to animals already treated with MC (20 mg/kg, i.p., daily x6). In this case, DS did not affect the already induced ALDH activity. On the contrary, CC was still an effective inhibitor. Unexpectedly, post-treatment with SKF 525A further enhanced the initial induction brought about by MC. Our findings show that substances affecting microsomal drug metabolism can interfere with the process of ALDH induction by MC. The additive result of PB pretreatment is probably due to the enhanced accumulation of an active metabolite of MC. The opposite effect of DS on drug metabolism could explain the decreased ability of MC to induce ALDH activity. The MC-inducible ALDH isozyme can be effectively inhibited with CC, but not with DS.     

The isolation of pure cholinergic nerve terminal sacs (T-sacs) from the electric organ of juvenile Torpedo.

The problem of synaptosome formation in the electric organ of Torpedo has been re-investigated using tissue from juvenile fish. This tissue is softer than adult material and can be easily homogenized in an Aldridge-type homogenizer. Homogenates so prepared contain a significant number of synaptosome-like structures which can be purified by differential and density gradient centrifugation. The purified particles are enriched in acetylcholine and choline acetyltransferase; they also contain lactate dehydrogenase activity, most of which is in an occluded form. The structure of these particles as revealed by electron microscopy is unusual in that they have no post-synaptic adhesions, relatively few synaptic vesicles and no intraterminal mitochondria. Because of their unusual morphology we have named these particles nerve terminal sacs (T-sacs). A high-affinity, hemicholinium-3 sensitive choline uptake system with an apparent K_m of 1–3 μm is associated with the T-sacs.     

Lactic dehydrogenase virus (LDV) impairs the antigen-presenting capacity of macrophages yet fails to affect their phagocytic activity

The effect of acute infection of mice with lactic dehydrogenase virus (LDV) on two major functions of peritoneal macrophages was tested. Using a macrophage-dependent T cell proliferative assay to test the antigen-presenting capacity of LDV-infected macrophages we found that LDV impairs the capacity of antigen-presenting cells to trigger memory T lymphocytes. Endocytosis of antigen by LDV-infected macrophages was similar to that of uninfected cells. In addition, the proportion of intracellular antigen versus membrane-bound antigen in LDV-infected cells were similar to that observed in uninfected mice. It appears therefore, that the impaired immunogenic effect of LDV-infected macrophages results from reduced immunogenicity of the membrane-bound antigen.Testing the phagocytic activity of peritoneal macrophages we found that the uptake of radiolabeled antibody-coated sheep erythrocytes or bacteria (E. coli) by infected cells was similar to that by uninfected macrophages. In addition, LDV failed to affect the ability of peritoneal macrophages in a nitroblue tetrazolium reduction reaction which serves as an alternative parameter for measuring phagocytic activity. Our results support the assumption that LDV, which probably propagates in the cells of the reticuloendothelial system, impairs some of the immunogenic functions of macrophages and thereby affects macrophage-dependent immune responses.     

Liver and Heart Mitochondrial Succinate Dehydrogenase Activity of Newborn Rats in Anoxic Hypoxia and Starvation

Succinate dehydrogenase activity was determined in the liver and heart of newborn rats after 3 and 48 hours' exposure to anoxic hypoxia (10% O₂) and after 48 hours' starvation. Control determinations were made on newborn animals of corresponding ages, full term foetuses (21 days), infantile (1 and 2 weeks) and full grown animals. Hypoxia for 3 h had no influence on succinate dehydrogenase activity at all in either the heart or liver mitochondria of the newborn animals. After 48 h no difference was observed in the liver between the hypoxic animals and the starved controls of the same age, though starvation itself had resulted in a significant increase in activity, as much as 42%. When liver mitochondrial succinate dehydrogenase in normal mitochondria was activated by preincubation mitochondria with the substrate, the activity increase obtained was greater than that resulting from starvation. The increase in activity in the heart of the hypoxic or starved animals was not significant (< 10%).     

Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the glyceraldehyde 3-phosphate dehydrogenases from different species

It was previously proposed that an immunological cross-reaction between two denatured proteins is evidence for an homology betweeen their amino sequence (Arnon &; Maron, 1971; Arnheim et al., 1971) and that detection of such a cross-reaction could then be a rapid method to detect sequence homologies (Zakin et al., 1978). In order to test the possibilities of such a methodology, using proteins of known structure, glyceraldehyde 3-phosphate dehydrogenases from different sources are compared by immunochemical techniques. The antibodies raised against the native enzyme from E. coli K 12 can only recognize the homologous antigen, the glyceraldehyde 3-phosphate dehydrogenase from B. stearothermophilus and to a lesser extent that from halibut. In contrast, the antibodies raised against the denatured enzyme from E. coli K 12 can recognize the glyceraldehyde 3-phosphate dehydrogenases from man, ostrich, chicken, sturgeon, halibut, lobster and yeast, when in their denatured state. The present results show unambiguously that through exposure of buried sequences, the immunochemical detection of sequence homologies among proteins is more discriminating when unfolded proteins are used, rather than native ones. It is also proposed that the use of denatured proteins both as immunogens and antigens would be a useful tool in studying biochemical evolution.     

Changes in blood lactate and respiratory gas exchange measures in sports with discontinuous load profiles

This study compares two different sport events (orienteering = OTC; tennis = TEC) with discontinuous load profiles and different activity/recovery patterns by means of blood lactate (LA), heart rate (HR), and respiratory gas exchange measures (RGME) determined via a portable respiratory system. During the TEC, 20 tennis-ranked male subjects [age: 26.0 (3.7) years; height: 181.0 (5.7) cm; weight: 73.2 (6.8) kg; maximal oxygen consumption (V˙O₂max): 57.3 (5.1) ml·kg⁻¹·min⁻¹] played ten matches of 50 min. During the OTC, 11 male members of the Austrian National Team [age: 23.5 (3.9) years; height: 183.6 (6.8) cm; weight: 72.4 (3.9) kg; V˙O₂max: 67.9 (3.8) ml·kg⁻¹·min⁻¹] performed a simulated OTC (six sections; average length: 10.090 m). In both studies data from the maximal treadmill tests (TT) were used as reference values for the comparison of energy expenditure of OTC and TEC. During TEC, the average V˙O₂ was considerably lower [29.1 (5.6) ml^·kg^−1·min⁻¹] or 51.1 (10.9)% of VO₂max and 64.8.0 (13.3)% of V˙O₂ determined at the individual anaerobic threshold (IAT) on the TT. The short high-intensity periods (activity/recovery = 1/6) did not result in higher LA levels [average LA of games: 2.07 (0.9) mmol·l⁻¹]. The highest average V˙O₂ value for a whole game was 47.8 ml·kg^−1·min⁻¹ and may provide a reference for energy demands required to sustain high-intensity periods of tennis predominately via aerobic mechanism of energy delivery. During OTC, we found an average V˙O₂ of 56.4 (4.5) ml·kg⁻¹·min⁻¹ or 83.0 (3.8)% of V˙O₂max and 94.6 (5.2)% of V˙O₂ at IAT. In contrast to TEC, LA were relatively high [5.16 (1.5) mmol·l⁻¹) although the average V˙O₂ was significantly lower than V˙O₂ at IAT. Our data suggest that portable RGEM provides valuable information concerning the energy expenditure in sports that cannot be interpreted from LA or HR measures alone. Portable RGEM systems provide valuable assessment of under- or over-estimation of requirements of sports and assist in the optimization and interpretation of training in athletes. Electronic Publication     

Rapid activation of glycogen synthase and protein phosphatase in human skeletal muscle after isometric contraction requires an intact circulation

The effects of isometric contraction (66% of maximal force) and recovery on glycogen synthase fractional activity (GSF) in human skeletal muscle have been studied. Biopsies were taken from the quadriceps femoris muscle at rest, at fatigue and 5 min postexercise on two occasions: after one of the contractions, the circulation to the thigh was occluded during the 5 min recovery (OCC), and after the other contraction, the circulation was intact (control, CON). During CON, GSF decreased from (mean ± SE) 0.34±0.05 at rest to 0.24±0.02 at fatigue and then increased to 0.74±0.04 at 5 min postexercise; corresponding values for OCC were 0.37±0.04, 0.25±0.04 and 0.48±0.05 (P<0.001 vs. CON for 5 min postexercise only). Compared with the value at fatigue, protein phosphatase activity (PP) increased by 79±16% during CON recovery (P<0.01), whereas no change was observed during OCC recovery. Uridine diphosphate glucose increased by approximately 2.5-fold at fatigue, remained elevated during OCC recovery, but reverted to the preexercise level during CON recovery (P<0.001 vs. OCC recovery). Glucose 6-P increased approximately 5-fold at fatigue and was higher at 5 min postexercise in OCC vs. CON recovery (8.6±1.5 vs. 4.1±0.9 mmol/kg dry wt; P<0.01). It is concluded that the rapid increase in GSF after intense exercise with an intact circulation may be at least partly attributed to an increase in the specific activity of PP. The increase in GSF during recovery in OCC may be at least partly attributed to the high glucose 6-P content in vivo, which enhances the substrate suitability of GS for PP. Thus, separate mechanisms exist for the activation of PP and GS during recovery from intense short term exercise.     

Sequence analysis of the gene coding for glyceraldehyde-3-phosphate dehydrogenase (gpd) of Podospora anserina: use of homologous regulatory sequences to improve transformation efficiency

Summary The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene of Podospora anserina has been isolated from a genomic library by heterologous hybridization with the corresponding gene of Curvularia lunata. The coding region consists of 1014 nucleotides and is interrupted by a single intron. The amino-acid sequence encoded by the gpd gene shows a high degree of sequence identity with the corresponding gene products of various fungi. Multiple alignments of all fungal GPD sequences so far available resulted in the construction of a phylogenetic tree. The evolutionary relationships of the various fungi belonging to different taxa will be discussed on the basis of these data. Sequence analysis of 1.9 kbp of the 5 non-coding region revealed the presence of typical fungal promoter elements. Utilizing different parts of the 5 regulatory sequence of the Podospora gpd gene, expression vectors containing a dominant selectable marker gene (hygromycin B phosphotransferase) have been constructed for the transformation of P. anserina protoplasts. The use of these homologous gpd regulatory sequences resulted in a significant increase in transformation efficiencies compared to those obtained with vectors in which the selectable marker gene is under the control of the corresponding heterologous promoter of Aspergillus nidulans.     

Mitochondrial antigens as targets of cellular and humoral auto-immunity in primary biliary cirrhosis

Several factors point toward an auto-immune pathogenesis for primary biliary cirrhosis (PBC), mostly based on the presence of serum auto-antibodies to mitochondrial antigens (AMAs) and autoreactive T cells (both helper and cytotoxic). Interestingly, epitopes recognized by AMA and T-cell clones are located within overlapping areas of the antigens. Moreover, a role for an imbalance in cytokine pattern and for natural-killer lymphocytes has also been proposed. Despite several experimental reports, no clear evidence is available regarding the interaction of these factors leading to bile duct destruction. This article reviews the current reports regarding the auto-immune reaction against mitochondrial auto-antigens in PBC.