Time after seawater transfer influences immune cell abundance and responses to SAV3 infection in Atlantic salmon.

Pancreas disease (PD) caused by salmonid alphavirus (SAV) severely affects salmonid aquaculture during the seawater phase. To characterize immune cells in target tissues for SAV infection, heart, pancreas and pyloric caeca were analysed from two groups of fish adapted to seawater for 2 and 9 weeks. The sections were scored for the relative abundance of cells expressing MHC class II, IgM, CD3, CD8 or neutrophil/granulocyte markers using immuno-histochemical techniques. In general, necrosis of tissue was more severe in fish infected at 2 weeks post-seawater transfer (wpt) compared with those infected at 9 wpt. At 9 wpt, there were higher numbers of MHC II+ cells in heart, pancreas and pyloric caeca, IgM+ cells in heart and pancreas, and CD3+ cells in pancreas compared to those infected at 2 wpt. The majority of the immune cells infiltrating PD-affected tissues were MHC II+ and CD3+ cells suggesting that antigen-presenting cells and T lymphocytes are the main types of immune cells responding to SAV infection. All the investigated cell types were also observed in pyloric caeca of infected fish, suggesting that this tissue may play a role in the immune response to SAV.

Vaccine adjuvant technology: from theoretical mechanisms to practical approaches.

Poorly immunogenic antigens depend on vaccine adjuvants to evoke an immune response. In addition, adjuvants largely determine the magnitude, quality, time of onset and the duration of immune responses to co-administered antigens. As late as 1989, Janeway aptly called adjuvants: "the immunologist's dirty little secret". This statement reflected the ignorance on the mechanisms of action of most known adjuvants. Yet, rational vaccine design involves a logical choice of adjuvant based on a knowledge of their mode of action and their effects on product efficacy and safety. However, even today the key processes critical for immune induction in general and those evoked by vaccine adjuvants in particular are being disputed among immunologists. This paper presents the four most important concepts likely to explain some of the mechanisms of vaccine adjuvants. They include: (i) the geographical concept of immune reactivity; (ii) the depot concept; (iii) the hypothesis of pathogen-structure recognition, and (iv) the damage/endogenous danger theory. These paradigms are based on observations gathered in mammalian species, largely in murine models. In aquatic animals the processes underlying immune induction will at least partly overlap those in mammals. However, due to inherent species differences, certain pathways may be different. Rational vaccine design, a difficult goal in mammals, is further hampered in aquatic animals by the lack of immunological tools in these species. Extensive trial and error-based approaches have yielded adjuvant candidates for various fish species, with acceptable safety and proven efficacy, some of which are presented.

Dopamine oxidation generates an oxidative stress mediated by dopamine semiquinone and unrelated to reactive oxygen species.

Dopamine (100 microM, 10-30 min) inhibits/inactivates the MgATP-dependent generation of a transmembrane proton electrochemical gradient in chromaffin granule ghosts. The dopamine dependent inhibition was enhanced by adding soluble dopamine beta-monooxygenase (DBM, 0.2 U/ml) and completely prevented by ascorbate (1 mM), dithiothreitol (2 mM) and approximately 80% by the DBM inhibitor fusaric acid (10 microM). This indicates that the inhibition is caused by the dopamine semiquinone free radical generated during DBM-dependent dopamine oxidation. Catalase, superoxide dismutase or both did not prevent the inhibition, and DBM-catalysed dopamine oxidation did not change the basal level of lipid peroxidation, excluding the involvement of reactive oxygen species as being responsible for the inhibition. N-ethylmaleimide-sensitive ATPase activity (i.e. the proton translocating ATPase) in the vesicle membranes was inhibited during dopamine incubation, indicating that the toxic metabolite (dopamine semiquinone) inhibits proton pumping by inhibiting the endogenous vacuolar H(+)-ATPase. As this proton pump represents the driving force for the vesicular uptake and storage of catecholamines, the dopamine dependent inhibition, if taking place in vivo, may inhibit dopamine uptake in storage vesicles in sympathetic neurons, e.g. as observed in the myopathic hamster heart.

Determination of manganese superoxide dismutase activity by direct spectrophotometry.

A method to determine Mn-superoxide dismutase activity by measuring directly the rate of decay of O2- in a spectrophotometer, is described. Decay of O2- generated by KO2 at pH 9.5, was monitored as the fall in absorbance (A250nm-A360nm). Mn-superoxide dismutase was determined as the activity of cyanide-resistant superoxide dismutase, calculated from the rate of O2- dismutation. Mn-superoxide dismutase could be determined in the presence of a 700 times higher Cu,Zn-superoxide dismutase activity. The alkaline pH did not cause analytical problems. The assay was used to measure both Mn- and Cu,Zn-superoxide dismutase activity in mitochondrial preparations. The assay had a detection limit of 2.8 ng/ml when Mn-superoxide dismutase from E. coli was used, and the between-day CV was 5.8%. The assay is an alternative to indirect methods for detecting superoxide dismutase activity.

Effect of decreased ferrochelatase activity on iron and porphyrin content in mitochondria of mice with porphyria induced by griseofulvin.

The content of iron and protoporphyrin in liver mitochondria from mice with porphyria induced by griseofulvin was measured. The amount of porphyrin was 0.0076 +/- 0.0043, 4.11 +/- 0.58 and 22.2 +/- 6.8 nmol/mg protein (n = 5) in mitochondria from control animals and animals treated with griseofulvin for 3 days and 4-5 weeks, respectively. The energy coupling of the mitochondria was greatly diminished after 4-5 weeks of treatment, and the ferrochelatase activity was inhibited 80-90%, compared to that of control animals. Mitochondrial preparations isolated by differential centrifugation were contaminated with iron-containing lysosomes which could be removed by Percoll density-gradient centrifugation. In purified mitochondrial preparations no change in the amount of non-heme iron was found after griseofulvin feeding, representing 3.36 +/- 0.15, 3.97 +/- 0.40 and 3.59 +/- 0.23 nmol/mg protein for control animals, 3 days- and 4-5 weeks-treated animals, respectively (n = 4). A mitochondrial iron pool previously identified in rat liver mitochondria and shown to be available for heme synthesis in vitro (Tangerås, A. (1985) Biochim. Biophys. Acta 843, 199-207) was also present in mitochondria from mice. The magnitude of this iron pool, as well as its availability for heme synthesis, was not changed after treatment of the animals with griseofulvin. The fact that porphyrin, but not iron, accumulated in the mitochondria when ferrochelatase was inhibited is discussed with regard to our understanding of the process of heme synthesis and its regulation.

Lysosomes, but not mitochondria, accumulate iron and porphyrins in porphyria induced by hexachlorobenzene.

In female rats with porphyria induced by hexachlorobenzene, the amounts of non-haem iron and porphyrins in liver mitochondrial fractions were increased almost 3-fold and greater than 500-fold respectively compared with that of untreated animals. A considerable fraction of both iron and porphyrins in this fraction was shown to be located in lysosomes. Thus mitochondrial preparations, which were further depleted of lysosomes by Percoll-density-gradient centrifugation, contained 2.78 +/- 0.75 and 2.99 +/- 0.49 nmol of non-haem iron/mg of protein when isolated from the liver of control rats and hexachlorobenzene-treated rats respectively. Mitochondria isolated from the liver of hexachlorobenzene-treated animals contained a pool of iron (about 1 nmol/mg of protein) that was available for haem synthesis in vitro. This pool is similar to that previously reported for mitochondria isolated from the liver of rats with normal haem synthesis. Hexachlorobenzene treatment, therefore, does not affect the iron status of the mitochondria.

Effects of essential fatty acid deficiency on mitochondria and peroxisomes in rat hepatocytes with special reference to a partially hydrogenated fish oil diet.

Feeding male rats a high cal% partially hydrogenated fish oil diet induced morphological and biochemical changes in hepatocytes at the mitochondrial and peroxisomal level. At the mitochondrial level, formation of megamitochondria was related to the development of an essential fatty acid deficiency, as measured by a high 20:3/20:4 fatty acid ratio. These mitochondrial changes were fully prevented by adding linoleic acid to the partially hydrogenated fish oil diet. The megamitochondria revealed a normal specific content of respiratory chain pigments, normal specific respiratory rates and a normal energy coupling. At the peroxisomal level, feeding of the partially hydrogenated fish oil diet caused a considerable proliferation, which was unrelated to essential fatty acid deficiency. The total number of peroxisomes increased 1.9-fold, and 2.6-fold in the presence of added linoleic acid. Essential fatty acid deficiency seemed to result in an inhibition of peroxisomal biogenesis. It was concluded that the induction of megamitochondria by partially hydrogenated fish oil was fully attributable to essential fatty acid deficiency, whereas peroxisomal proliferation must be attributed to other factors in the diet.

Mitochondrial iron not bound in heme and iron-sulfur centers and its availability for heme synthesis in vitro.

Rat liver mitochondrial fractions have previously been shown to contain a pool of iron which was bound neither in cytochromes nor in iron-sulfur centers (Tangerås, A., Flatmark, T., Bãckstrõm, D. and Ehrenberg, A. (1980) Biochim. Biophys. Acta 589, 162-175), and in the present study the availability of this iron pool for heme synthesis has been studied in isolated mitochondria. A minor fraction of this iron is here shown to originate from iron-rich lysosomes present as a contaminant in mitochondrial fractions isolated by differential centrifugation, and a method for the selective quantitation of this iron pool was developed. The availability of the mitochondrial iron pool for heme synthesis by mitochondria in vitro was studied using a recently developed HPLC method for the assay of ferrochelatase activity. When deuteroporphyrin was used as the substrate, 1.04 +/- 0.13 nmol/mg protein of deuteroheme was formed after 6 h incubation at 37 degrees C when a plateau was approached, and the initial rate of heme synthesis was 0.3 nmol/h per mg protein. Heme formation from the physiological substrate protoporphyrin was also seen. The heme synthesis increased with the amount of mitochondria used and was blocked by both Fe(II) and Fe(III) chelators. The heme synthesis was independent of mitochondrial oxidizable substrates and no difference was observed between pH 7.4 and 6.5. FMN slightly stimulated the formation of heme from endogenous iron, probably by mobilization of a small amount of contaminating lysosomal iron present in the preparations. The possibility that the mitochondrial iron pool functions as the proximate iron donor for heme synthesis by ferrochelatase in vivo is discussed.

Separation of haem compounds by reversed-phase ion-pair high-performance liquid chromatography and its application in the assay of ferrochelatase activity.

The separation of haems and porphyrins was achieved in a reversed-phase ion-pair high-performance liquid chromatography system using tetrabutylammonium hydrogen sulphate as the pairing ion. The concentration of methanol and pH in the mobile phase were determinative parameters for the elution pattern of the compounds. Two isocratic systems--one for the assay of protohaem IX and one for deuterohaem IX--were developed. The chromatographic systems were applied to the assay of ferrochelatase activity in mitochondria using either protoporphyrin or deuteroporphyrin as the substrate. The ferrochelatase activity was also measured in reticulocytes, which contain high levels of endogenous haem.

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats.

1. The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats, as revealed by analytical differential centrifugation, indicated single population of mitochondria with -S4,B values of 13 200 +/- 560 S and 14 200 +/- 590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction obtained by differential centrifugation decreased from 69 +/- 28 nmol/mg protein to 5.6 +/- 1.1 nmol/mg protein and from 19.3 +/- 5.6 nmol/mg protein to 3.3 +/- 0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminating the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, W.G., et al. (1981) Biochim. Biophys. Acta 678, 293-299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.

Oxygen uptake, ATPase activity, and superoxide dismutase activity in isolated rat liver mitochondria are not influenced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate.

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) influences neither the State 3 nor the State 4 respiration in rat liver mitochondria. The respiratory control and ADP/O ratio were also unaffected by TPA. The oligomycin-sensitive ATPase activity in submitochondrial particles remained unaltered upon TPA addition, whereas the NADH oxidase activity was slightly inhibited at a very high concentration of TPA (15% decrease at 17 microM TPA). The activity of the superoxide dismutase located to the mitochondria was insensitive to the tumor promoter, and no change in the rate of H2O2 production was found on TPA treatment in vitro. Thus, the mitochondrion is not a likely candidate for the site of action of the tumor promoter.

Inhibition of mitochondrial protein synthesis and energy coupling by fragment A of diphtheria toxin.

The effect of intact diphtheria toxin and its fragment A on energy-dependent functions in mouse liver mitochondria/mitoplasts has been studied. Fragment A was found to inhibit protein synthesis in mitoplasts to the same extent (approximately 80%) as the uncoupler carbonylcyanide p-trifluoromethoxyphenylhydrazone, but had no similar effect in lysed mitoplasts. Intact diphtheria toxin had no effect in either case. Fragment A was found to function as a potent uncoupler in isolated mitochondria and mitoplasts, inhibiting oxidative phosphorylation by approximately 80% at a concentration (2 micrograms fragment A/mg of protein) that did not inhibit protein synthesis. In contrast, intact toxin slightly increased the tightness of energy coupling in isolated mitoplasts. 125I-labelled intact diphtheria toxin was bound to mitoplasts to about the same extent as labelled fragment A. At concentrations which efficiently inhibited mitochondrial protein synthesis, fragment A had no effect on the intramitochondrial concentration of nicotinamide adenine dinucleotides, nor was it capable of ADP-ribosylating mitochondrial proteins, indicating that the well known enzymatic activity of fragment A is not involved in the observed effect in mitochondria. The results indicate that fragment A of diphtheria toxin inhibits protein synthesis in mitochondria and mitoplasts by inhibiting mitochondrial energy transduction. The detailed mechanism of the uncoupling effect and its possible significance in intact cells remain to be elucidated.

Induction of megamitochondria by cuprizone(biscyclohexanone oxaldihydrazone). Evidence for an inhibition of the mitochondrial division process.

1. Feeding weanling mice a diet containing 0.5% (w/w) cuprizone (biscyclohexanone oxaldihydrazone) induced megamitochondria of a different type of that previously described (Suzuki, K., Science 163, 81-82 (1969). 2. As in the normal controls, a single population of mitochondria with a narrow size distribution, was detected by analytical differential centrifugation of liver homogenates from mice fed a cuprizone-diet. However, their average S-value increased 2.4fold, i.e. from 11 500 S to 27 500 S. The marked increase in the mitochondrial size (megamitochondria) were also detected by morphometric analysis of hepatocytes and isolated mitochondrial fractions, and the 1.6fold increase in the average mitochondrial diameter (as determined by scanning electron microscopy) was as expected from the increase in the S-value. On the other hand, the relative volume density or volume fraction of the mitochondria was unchanged. 3. The megamitochondria revealed a normal content of respiratory chain pigments, normal specific respiratory rates with succinate and NADH-linked substrates as well as normal energy coupling. 4. Removal of cuprizone from the diet normalized the size distribution of mitochondria in hepatocytes. The transition followed first-order kinetics with a half-life of about 30 h. During the period dividing mitochondria were frequently observed, and the division process appeared to follow both a transverse-partition and an elongation-constriction mechanism.

Mitochondrial iron not bound in heme and iron-sulfur centers. Estimation, compartmentation and redox state.

A method is described for the assay of total mitochondrial non-heme iron and a fraction which does not belong to the iron-sulfur proteins (FeS centers) of the outer and inner membrane. The assay of the latter fraction, which is termed 'non-heme non-FeS iron', is based on the formation of a chelate of Fe(II) with bathophenanthroline sulfonate in osmotically swollen mitochondria under conditions where the FeS centers are quite stable as determined by EPR spectroscopy at 20.4 K, 93 K and 123 K. The 'non-heme non-FeS iron', which in normal rat liver mitochondria amounts to approx. one third of the total mitochondrial iron (i.e. 1.7 +/- 0.3 nmol . mg-1 protein), does not represent a homogeneous pool of iron. Based on studies of its reaction with bathophenanthroline sulfonate and the dependency of this reaction on reducing agents in mitochondria and mitoplasts, evidence is presented that this non-heme iron is present in two major pools in which the inner membrane constitutes the barrier. A minor fraction (i.e. 0.4 +/- 0.2 nmol . mg-1 protein) is localized to the 'outer' compartment and a major fraction (i.e. 1.1 +/- 0.1 nmol . mg-1 protein) is localized to the 'inner' compartment and is equally distributed between the inner membrane and the matrix. The experiments described in this study also indicate that approximately half of the 'non-heme non-FeS iron' of the 'inner' pool is in the ferrous form in mitochondria as isolated, and this was not increased when oxidizable substrates were added to the mitochondria. Although the biological significance of this iron pool is not yet clear, it is likely that it represents a transit iron pool being the proximate iron donor for heme synthesis catalyzed by the enzyme ferrochelatase.

In vitro binding of protoheme IX and protoporphyrin IX to components in the matrix of rat liver mitochondria.

The matrix fraction of rat liver mitochondria contains several components capable of binding protoheme IX and protoporphyrin IX in vitro. When binding is analyzed by sucrose density gradient centrifugation, two main groups of components can be separated; i.e. one of high and one of low molecular weight. Experimental evidence is presented indicating a protein nature of these components, and at least six species were detected by gel electrophoresis. The binding of protoheme IX and protoporphyrin IX to matrix components is also evident from a marked change in the heme and porphyrin absorbance spectra in the presence of added matrix. The data obtained indicate that the matrix fraction has a high total binding capacity for heme, but the affinity for heme of the various components seems to be different. Based on the separation of bound and free heme by gel electrophoresis it is concluded that the heme-binding capacity of the high affinity components does not exceed 14 nmol heme . mg-1 protein.