Fine structure and fluorescence morphology of adrenergic nerves after 6-hydroxydopamine in vivo and in vitro

Summary In parallel fine structural, fluorescence histochemical and biochemical experiments the effect of 6-OH-DA administered in vivo and in vitro on the adrenergic nerves in the mouse iris was studied. As seen in the electron microscope, in vivo administration of 6-OH-DA causes a selective, rapid degeneration of the adrenergic axon terminals similar to that found after axotomy, whereas the cholinergic nerves are unaffected at all time intervals studied. Already 1 hr after the injection of 6-OH-DA the axonal enlargements swell and the size of the dense core of the granular vesicles is strongly reduced. Since the NA stores are almost completely depleted at this time interval, the small core present may be due to a reaction between 6-OH-DA and the fixative. From 2–4 hr after the injection increasing numbers of axonal enlargements with a high electron density are observed in the Schwann cell cytoplasm, which later are digested and completely absent about 48–72 hr after the 6-OH-DA injection. During the following weeks adrenergic axons reappear. This time course of degeneration obtained is considerably faster than that seen after axotomy in other studies. After incubation in 6-OH-DA containing media similar changes were observed in the axonal enlargements, starting already after 30 min of incubation. At this time-point there is a considerable reduction of endogenous NA and a severe damage of the membrane pump uptake mechanism. Incubation with 6-OH-DA and subsequent rinsing for 2 hr caused marked changes, including partly swelling of axons and partly shrinking of the axons into electron dense bodies.The fluorescence histochemical and biochemical results are in good agreement with the ultrastructural studies demonstrating a rapid loss of NA from the adrenergic nerve terminals and main axons and a long lasting depletion of the NA, with a gradual recovery to 75% 6 weeks after the injection.The investigation has been supported by research grants from the Swedish Medical Research Council (14X-2295, 14X-2887 and 04X-3881) Karolinska Institutet, Magnus Bergvalls and Carl-Berthel Nathorst Stiftelser. For generous supplies of drugs we are indebted to the following companies: AB Hässle (6-OH-DA, through Dr H. Corrodi), Pfizer (Niamid^®), Ciba (Serpasil^®). The skilful technical assistance of Miss Bodil Flock, Mrs Waltraut Hiort and Mrs Eva Lindqvist is gratefully acknowledged.     

Enzyme-linked immunosorbent assay. II. Quantitative assay of protein antigen, immunoglobulin G, by means of enzyme-labelled antigen and antibody-coated tubes

Alkaline phosphatase from calf intestinal mucosa has been conjugated to a protein antigen, rabbit IgG. Such conjugates, prepared by glutardialdehyde, have been used in a competitive solid phase immunoassay. In this test native antigen inhibits the binding of the conjugate to homologous antibodies adsorbed to plastic tubes. Using this assay 1-100 ng/ml of the antigen could be determined.     

Studies on reaction and binding of monoamines after fixation and processing for electron microscopy with special reference to fixation with potassium permanganate

Summary In the present paper certain properties of potassium permanganate (KMnO₄), a fixative used for electron microscopical investigations, have been studied in model test tube experiments and on tissues. Evidence was obtained that KMnO₄ reacts with different types of biogenic monoamines resulting in a formation of a precipitate. In addition, also various monoamine analogues, precursors and metabolites reacts with KMnO₄. The reaction taking place may be an oxidation-reduction-reaction in which KMnO₄ is reduced, probably mainly to manganese dioxide by hydroxyl groups of the amines and related compounds. This is corroborated by the fact that no reaction takes place between KMnO₄ and -phenylethylamine or amphetamine, two substances, which lack hydroxyl groups.Using labelled monoamines evidence was obtained that the amine partly is retained within the precipitate formed after the reaction with KMnO₄ and also in tissues fixed with KMnO₄, indicating a possibility to perform autoradiographic studies on KMnO₄ fixed tissue.Electron microscopic studies on tissues fixed under various conditions revealed that fixation with low concentrations (0.6 and 1.0%) of KMnO₄ and at high temperatures (about 20° C) leads to inferior results as to general morphology and as to the visualization of intraneuronal amine stores.Different types of permanganates were tested as fixatives. These results show that fixation with permanganates with monovalent metallic ions (K⁺, Li⁺ and Na⁺) give good results of comparable quality, whereas fixation with zinc permanganate results in seriously destroyed tissues. However, tissue fixed with calcium permanganate reveals very distinct membranes. Furthermore, evidence was obtained that fixation with high concentrations of LiMnO₄ (6 and 9%) and NaMnO₄ (6 and 9%) was more sensitive as to the demonstration of monoamines at the ultrastructural level as compared to 3% KMnO₄. Thus, with e.g. 6 and 9% LiMnO₄ small granular vesicles could be seen in slices from the caudate nucleus after incubation with -methyl-dopamine. This was not possible when using 3% KMnO₄ as a fixative.     

Environmental change influences the life history of salmon Salmo salar in the North Atlantic Ocean

Annual mean total length (L_T) of wild one‐sea‐winter (1SW) Atlantic salmon Salmo salar of the Norwegian River Imsa decreased from 63 to 54 cm with a corresponding decrease in condition factor (K) for cohorts migrating to sea from 1976 to 2010. The reduction in L_T is associated with a 40% decline in mean individual mass, from 2 to 1·2 kg. Hatchery fish reared from parental fish of the same population exhibited similar changes from 1981 onwards. The decrease in L_T correlated negatively with near‐surface temperatures in the eastern Norwegian Sea, thought to be the main feeding area of the present stock. Furthermore, S. salar exhibited significant variations in the proportion of cohorts attaining maturity after only one winter in the ocean. The proportion of S. salar spawning as 1SW fish was lower both in the 1970s and after 2000 than in the 1980s and 1990s associated with a gradual decline in post‐smolt growth and smaller amounts of reserve energy in the fish. In wild S. salar, there was a positive association between post‐smolt growth and the sea survival back to the River Imsa for spawning. In addition, among smolt year‐classes, there were significant positive correlations between wild and hatchery S. salar in L_T, K and age at maturity. The present changes may be caused by ecosystem changes following the collapse and rebuilding of the pelagic fish abundance in the North Atlantic Ocean, a gradual decrease in zooplankton abundance and climate change with increasing surface temperature in the Norwegian Sea. Thus, the observed variation in the life‐history traits of S. salar appears primarily associated with major changes in the pelagic food web in the ocean.     

Trans‐generational maternal effect: temperature influences egg size of the offspring in Atlantic salmon Salmo salar

Effect of increased temperature during egg maturation on the mass of single eggs produced by the offspring was investigated experimentally in Atlantic salmon Salmo salar. Mass of eggs produced by next‐generation females was larger when their mothers experienced warmer water during the last two months of egg maturation, relative to those that experienced unheated river water. There was no similar trans‐generational paternal effect on offspring egg mass.     

Conformational rearrangements of tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC)-bound actin

The mechanism of chaperonins is still under intense investigation. Earlier studies by others and us on the bacterial chaperonin GroEL points to an active role of chaperonins in unfolding the target protein during initial binding. Here, a natural eukaryotic chaperonin system [tail-less complex polypeptide 1 (TCP-1) ring complex (TRiC) and its target protein actin] was investigated to determine if the active participation of the chaperonin in the folding process is evolutionary-conserved. Using fluorescence resonance energy transfer (FRET) measurements on four distinct doubly fluorescein-labeled variants of actin, we have obtained a fairly detailed map of the structural rearrangements that occur during the TRiC-actin interaction. The results clearly show that TRiC has an active role in rearranging the bound actin molecule. The target is stretched as a consequence of binding to TRiC and further rearranged in a second step as a consequence of ATP binding; i.e., the mechanism of chaperonins is conserved during evolution.     

In Swedish families with hereditary prostate cancer, linkage to the HPC1 locus on chromosome 1q24-25 is restricted to families with early-onset prostate cancer.

Prostate cancer clusters in some families, and an estimated 5%-10% of all cases are estimated to result from inheritance of prostate cancer-susceptibility genes. We previously reported evidence of linkage to the 1q24-25 region (HPC1) in 91 North American and Swedish families each with multiple cases of prostate cancer (Smith et al. 1996). In the present report we analyze 40 (12 original and 28 newly identified) Swedish families with hereditary prostate cancer (HPC) that, on the basis of 40 markers spanning a 25-cM interval within 1q24-25, have evidence of linkage. In the complete set of families, a maximum two-point LOD score of 1.10 was observed at D1S413 (at a recombination fraction [theta] of.1), with a maximum NPL (nonparametric linkage) Z score of 1.64 at D1S202 (P=.05). The evidence of linkage to this region originated almost exclusively from the subset of 12 early-onset (age <65 years) families, which yielded a maximum LOD score of 2.38 at D1S413 (straight theta=0) and an NPL Z score of 1.95 at D1S422 (P=.03). Estimates from heterogeneity tests suggest that, within Sweden, as many as 50% of early-onset families had evidence of linkage to the HPC1 region. These results are consistent with the hypothesis of linkage to HPC1 in a subset of families with prostate cancer, particularly those with an early age at diagnosis.     

Mitigation of acidified salmon rivers – effects of liming on young brown trout Salmo trutta

In southern Norway, 22 acidified rivers supporting anadromous salmonids were mitigated with lime to improve water quality and restore fish populations. In 13 of these rivers, effects on Salmo trutta and Salmo salar densities were monitored over 10–12 years, grouped into age 0 and age ≥ 1 year fish. These rivers had a mean annual discharge of between 4·9 and 85·5 m³ s^?1, and six of them were regulated for hydro‐power production. Salmo salar were lost in six of these rivers prior to liming, and highly reduced in the remaining seven rivers. Post‐liming, S. salar became re‐established in all six rivers with lost populations, and recovered in the seven other rivers. Salmo trutta occurred in all 13 study rivers prior to liming. Despite the improved water quality, both age 0 and age ≥ 1 year S. trutta densities decreased as S. salar density increased, with an average reduction of >50% after 10 years of liming. For age 0 year S. trutta this effect was less strong in rivers where S. salar were present prior to liming. In contrast, densities of S. trutta increased in unlimed streams above the anadromous stretches in two of the rivers following improved water quality due to natural recovery. Density increases of both age 0 and age ≥ 1 year S. salar showed a positive effect of river discharge. The results suggest that the decline in S. trutta density after liming is related to interspecific resource competition due to the recovery of S. salar. Thus, improved water quality through liming may not only sustain susceptible species, but can have a negative effect on species that are more tolerant prior to the treatment, such as S. trutta.