Injury of mitochondrial respiration and membrane potential during iron/ascorbate-induced peroxidation

First functional events during peroxidation in mitochondria consisted in a progressive inhibition of the phosphorylating and uncoupled respiration with succinate and glutamate/malate as substrates, whereas the resting state respiration during the same period was virtually not influenced. The membrane potential registered at a time with the respiration rates was capable of being built up for a relatively long time interval with only minor decreases, and broke down rather promptly when the active respiration was highly diminished. Inhibition of respiration proceeded mainly during the initiation phase of peroxidation. Lag phases of varied length, of malondialdehyde formation which were predominantly attributed to the iron/protein ratios correlated closely with different time intervals needed to attain maximal inhibition of respiration and decrease in glutathione. Hence, the lessening of respiration, drop of membrane potential and loss of the antioxidant, glutathione, represent early stages in the causal chain of events which precede the onset of intensive lipid peroxidation.     

Human hepatic triglyceride lipase: cDNA cloning, amino acid sequence and expression in a cultured cell line

By immunoscreening of a human cDNA expression library and hybridization of colonies, four partially overlapping cDNA clones of human hepatic triglyceride lipase (HTGL) mRNA were isolated. The clones included the complete coding sequence, the 3'- and at least part of the 5'-untranslated region. The length of the composite HTGL cDNA segment (1.7 kb) was consistent with the size of the mRNA identified in an established human hepatoma cell line. DNA-sequence analysis of cDNAs of partially unspliced mRNAs, and of cloned genomic DNA indicated that the HTGL coding sequence comprises at least six exons. As predicted from the cDNA, the unprocessed HTGL protein has a molecular weight of 56, three potential glycosylation sites, and a signal peptide of 23 amino acids. Sequence comparison with cDNA of other lipases, including rat hepatic lipase, revealed 30%-75% protein-sequence homology. The data establish that HTGL is a secretory protein produced in the hepatocyte, and that its synthesis can be continued in permanent cell lines of hepatoma origin. Our studies also showed that HTGL is another member of a lipase gene family which has interfacial binding sites and possibly other functional domains in common.     

Filtration rate capacities in 6 species of European freshwater bivalves

Summary Filtration rate capacities in undisturbed freshwater bivalves were determined by means of two different methods (indirect clearance and suction methods) in Anodonta anatina (L.), Unio tumidus Philipsson, Unio pictorum (L.), Unio crassus Philipsson, Dreissena polymorpha (Pallas) and Sphaerium corneum (L.). In A. anatina, D. polymorpha, and S. corneum the filtration rate (FR, 1 h^-1) at 19–20°C as a function of dry tissue weight (DW, g) or ash-free dry weight (AFDW, g) could be expressed by the equations: 1.10 DW^0.78, 6.82 DW^0.88, and 2.14 AFDW^0.92, respectively. In U. tumidus, U. pictorum, and U. crassus filtration rates were comparable with those of A. anatina. In D. polymorpha the b value of the corresponding regression of gill area on dry weight was 0.87. The rates of water transport in freshwater bivalves are 2–8 times lower than in marine bivalves of comparable size. A corresponding difference in the filtration rate per gill area unit is found. The measured filtration rates in undisturbed bivalves are substantially higher (at least 4 times) than previously reported. This indicates that the impact of bivalve water processing on freshwater ecosystems is greater than hitherto suggested.     

The hepatic response to Ca2+ is inhibited by Mg2+ and enhanced by phenylephrine or ouabain

Net hepatic Ca2+ efflux, K+ uptake and glycogen breakdown in response to the alpha 1-adrenergic agonist phenylephrine were studied. Rat livers were perfused with CO2/bicarbonate-buffered solutions containing 10 microM Ca2+ and different amounts of Mg2+. K+-free medium and/or ouabain were used to block (Na+ + K+)-ATPase-dependent K+ uptake. In some experiments a sharp increase in extracellular Ca2+ concentrations was produced by infusing CaCl2 into the medium entering the liver. Perfusion with K+-free medium and ouabain enhanced the phenylephrine-induced Ca2+ efflux and diminished the glycogenolytic response, indicating a dissociation of Ca2+ release and glycogenolysis. Exogenous Ca2+ had practically no effect if livers were perfused with regular medium containing 1.2 mM Mg2+. In the presence of phenylephrine and if extracellular Mg2+ concentrations were lowered by omitting Mg2+ from the medium or by preperfusion with EGTA, exogenous Ca2+ was glycogenolytically effective and also produced a transient K+ uptake. Increased extracellular concentrations of Mg2+ inhibited the effects of exogenous Ca2+. In the presence of phenylephrine, higher concentrations of Mg2+ were needed than in the absence of alpha 1-adrenergic agonist to achieve a similar degree of inhibition. In one respect ouabain effects were comparable to those of phenylephrine: the glycoside also increased the metabolic response to exogenous Ca2+ and diminished the sensitivity towards Mg2+. Phenylephrine and ouabain may both enhance the permeability of plasma membranes for Ca2+.     

Characterization of very low density lipoproteins and intermediate density lipoproteins of normo- and hyperlipidemic apolipoprotein E-2 homozygotes

Triglyceride-rich lipoproteins derived from ten normo- and hyperlipidemic apoE-2 homozygotes were analyzed for their composition, beta-VLDL content, and their ability to induce cholesteryl ester storage in macrophages. In six of these probands apoE sequence analysis revealed that the cysteine residues were at positions 112 and 158 of the amino acid sequence (Rall et al. 1983. J. Clin. Invest. 71: 1023-1031). ApoE-2 of these six and the other four patients was further analyzed by SDS electrophoresis to exclude the presence of apoE-2* (Rall et al. 1982. Proc. Natl. Acad. Sci. USA. 79: 4696-4700). The relative serum concentrations of free and esterified cholesterol transported in the d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins of the apoE-2 homozygotes was significantly higher as compared to controls. Compositional analysis of these lipoproteins revealed a relative reduction of triglycerides and a relative increase of cholesteryl esters as compared to controls. In most patients, with increasing serum triglyceride levels the cholesteryl ester concentration increased in d less than 1.006 g/ml and d 1.006-1.019 g/ml lipoproteins. However, in three patients with a low content of beta-VLDL, the increase in the d less than 1.006 g/ml fraction cholesterol was mostly due to free cholesterol and not due to cholesteryl esters. The degree of the macrophage cholesteryl ester accumulation induced by d less than 1.006 g/ml lipoproteins was mostly dependent on the concentration of the beta-migrating fraction (beta-VLDL). The amount of beta-VLDL and pre-beta-VLDL contained in the d less than 1.006 g/ml fraction was determined densitometrically after electrophoretic separation. It could be demonstrated that the beta-VLDL content in the d less than 1.006 g/ml fraction of the apoE-2 homozygous patients was largely independent of serum triglyceride and serum cholesterol levels. When macrophages were incubated with the IDL fraction (d 1.006-1.019 g/ml) from the apoE-2 patients, no significant increase in cellular cholesteryl esters above control levels was observed. Studies with purified lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) clearly revealed that both enzymes interacted with apoE-2 VLDL (binding, hydrolysis) to a lesser degree compared to control preparations. However, the apoE-2 VLDL preparations containing a low content of beta-VLDL were better substrates for LPL and HTGL than those containing a high beta-VLDL content. It is concluded from our studies that the plasma beta-VLDL content in apoE-2 homozygotes is a major determinant for cholesteryl ester accumulation in macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)     

Diadenosine 5′,5‴-P1,P3-triphosphate in eukaryotic cells: Identification and quantitation

A highly sensitive enzymatic assay for diadenosine 5′,5?-P¹,P³-triphosphate (Ap₃A) has been established on the basis of the coupled luminescence assay for diadenosine 5′,5?-P¹,P³-tetraphosphate (A. Ogilvie (1981)Anal. Biochem.115, 302–307). Snake venom phosphodiesterase splits Ap₃A into AMP plus ADP which can be measured in a luminescence reaction containing pyruvate kinase, phosphoenolpyruvate and luciferin-luciferase. The procedure is linear with Ap₃A levels ranging from 0.1 to 2 pmol. The assay has been used to measure Ap₃A in various eukaryotic cells after ion-exchange chromatography and high-performance liquid chromatography of acidic extracts of the cells. The level of diadenosine triphosphate was higher in all instances than the level of diadenosine tetraphosphate. When growing in the abdominal cavity of mice, Ehrlich ascites tumor cells contained high amounts of Ap₃A ($\text{0.1}\text{nmol}\text{10}{}^6\text{cells}$), allowing direct optical determination in the HPLC chromatography. The quantitative measurement of Ap₃A with the luminescence assay gave identical results. Ap₃A extracted from Ehrlich cells was also chromatographed with authentic nucleotide in two thin-layer systems providing additional proof for the existence of Ap₃A in biological material.     

Desmethylimipramine Enhances the Release of Endogenous GABA and Other Neuro trans mitter Amino Acids from the Rat Thalamus

The influence of desmethylimipramine (DMI) on the release of endogenous gamma-aminobutyric acid (GABA) and some other amino acids from the rat thalamus was studied with a push-pull perfusion technique. Following HPLC the amino acids were fluorimetrically estimated. Added to the perfusion medium at a concentration of 10 mumol L-1, DMI caused a 5- to 10-fold increase in the release of GABA. Similar effects were found with imipramine, trimeprimine, haloperidol, and propranolol. The elevation of GABA release induced by DMI was Ca dependent. The release of aspartate and glutamate was also increased by DMI, but in contrast to K ions, DMI did not reduce the thalamic output of glutamine.     

The complete heart-lead relationship in the einthoven triangle

The relationship between the source strength and the “manifest vector” in the Einthoven Triangle is derived for a line and a point dipole source and confirmed experimentally. The result permits the interpretation of the standard ECG leads in absolute terms and corrected for body size. The manifest vector is shown to be approximately times what it would be in an otherwise similar circular slab which circumscribes the triangle.     

Recovery Patterns of Spores of Putrefactive Anaerobe 3679 in Various Subculture Media after Heat Treatment

A comparative study was made of the heat resistance of spores of putrefactive anaerobe 3679 grown in two different sporulation media and of the recovery pattern of these spores in several subculturing media after treatment with moist and dry heat. The heat resistance of the spores was characterized in the form of D and z values. The D values were determined by the modified Schmidt method. The z values were established by the graphic method. The results revealed significant differences in D and z values, depending on the type of heat and sporulation and subculture media. Spores grown in beef heart infusion showed higher heat resistance than those grown in Trypticase. Among the seven subculture media used, the largest number of spores was recovered in beef infusion. The magnitude of the D values at 121.1 C obtained with spores heated in moist heat decreased, depending on the subculture medium used, in the following order: beef infusion, pea infusion, yeast extract, liver infusion, Eugonbroth, Trypticase, synthetic medium. With spores subjected to dry heat, D values at 148.9 C decreased with the subculture medium in the following order: beef infusion, yeast extract, pea infusion and liver infusion, Trypticase, Eugonbroth, synthetic medium. The z values obtained with spores subjected to dry heat were approximately double those obtained with moist heat. Their relative magnitude varied slightly, depending on the type of subculture medium used. However, the relative magnitudes of the D values and z values with reference to the subculture media used were different with moist heat from those obtained with dry heat. Two theories are discussed as possible explanations for the logarithmic order of death of bacterial spores. The results obtained in these experiments, together with the findings of other workers, are most compatible with the theory that heat treatment of spores results in an increased rate of random injury to the genetic material of the spores.