Search

Purpose: Protective effects of estradiol against H2O2-induced oxidative stress have been demonstrated in lens epithelial cells. The purpose of this study was to investigate the effects of 17β-estradiol (E2) on the different superoxide dismutase (SOD) isoenzymes, SOD-1, SOD-2, and SOD-3, as well as estrogen receptors (ERs), ERα and ERβ, in primary cultured human lens epithelial cells (HLECs). Materials and methods: HLECs were exposed to 0.1 µM or 1 µM E2 for 1.5 h and 24 h after which the effects were studied. Protein expression and immunolocalization of SOD-1, SOD-2, ERα, and ERβ were studied with Western blot and immunocytochemistry. Total SOD activity was measured, and gene expression analyses were performed for SOD1, SOD2, and SOD3. Results: Increased SOD activity was seen after 1.5 h exposure to both 0.1 µM and 1 µM E2. There were no significant changes in protein or gene expression of the different SODs. Immunolabeling of SOD-1 was evident in the cytosol and nucleus; whereas, SOD-2 was localized in the mitochondria. Both ERα and ERβ were immunolocalized to the nucleus, and mitochondrial localization of ERβ was evident by colocalization with MitoTracker. Both ERα and ERβ showed altered protein expression levels after exposure to E2. Conclusions: The observed increase in SOD activity after exposure to E2 without accompanying increase in gene or protein expression supports a role for E2 in protection against oxidative stress mediated through non-genomic mechanisms.

An acquired brain injury often leads to long-lasting mental fatigue, which can have a considerable effect on work and social interactions. Fortunately, the Mindfulness-Based Stress Reduction (MBSR) program has been found to alleviate mental fatigue. The purpose of this feasibility study was to evaluate the success of an interactive MBSR program delivered live online to individuals who have experienced a traumatic brain injury or stroke. We included the following three groups in our study: an Internet group, a face-to-face MBSR group, and an active control group who took weekly walks in natural environments. Thirty-four participants completed the study, and all were suffering from long-lasting mental fatigue after either a traumatic brain injury (16 participants) or a stroke (18 participants). However, seven did not accept to attend an Internet MBSR, and Internet was the only choice for others. We found that, according to the Mental Fatigue Scale (MFS), the program leads to significantly reduced mental fatigue in the Internet group compared with the face-to-face and the control group. Individuals in the MBSR groups also exhibited an improved ability to process two temporally close targets (attentional blink task), while this was not detected in the control group. In conclusion, we believe that it is possible for individuals suffering from mental fatigue after an acquired brain injury to obtain positive results through enrollment in a live, interactive, online MBSR program. This is promising, as the Internet is accessible to many individuals, irrespective of where they live. Further randomized control studies comparing are warranted.

Radiotherapy of thyroid cancer with I-131 is abrogated by inherent loss of radioiodine uptake due to loss of sodium iodide symporter (NIS) expression in poorly differentiated tumor cells. It is also known that ionizing radiation per se down-regulates NIS (the stunning effect), but the mechanism is unknown. Here we investigated whether loss of NIS-mediated iodide transport may be elicited by DNA damage. Calicheamicin, a fungal toxin that specifically cleaves double-stranded DNA, induced a full scale DNA damage response mediated by the ataxia-telangiectasia mutated (ATM) kinase in quiescent normal thyrocytes. At sublethal concentrations (

Samples of the vitreous were analysed in order to identify changes of soluble proteins in vitreo-retinal disease. The soluble proteins of the vitreous were separated on an anion exchange column (Mono-Q). The degree of neutral proteolytic activity in vitreous body was also measured. The vitreous from cataract cases without vitreoretinal disease was characterized by its low content of soluble proteins equivalent to about 1% of that of serum. Albumin and transferrin were the major identified components and their concentrations were approximately 0.85 and 0.03 g/l, respectively. In cases with vitreoretinal disease the vitreous showed changes of total soluble protein and the appearance of additional protein peaks. In patients with PVR the albumin concentration in the vitreous was found to be three times higher as compared to the control group consisting of patients with cataract. Neutral proteolytic activity in the vitreous was relatively low in both normal and pathological vitreous.

OBJECTIVE: Maternal inflammation/infection alone or in combination with birth asphyxia increases the risk for perinatal brain injury. Free radicals are implicated as major mediators of inflammation and hypoxia-ischemia (HI)-induced perinatal brain injury. This study evaluated the neuroprotective efficacy of a scavenging agent, N-acetylcysteine (NAC), in a clinically relevant model. METHODS: Lipopolysaccharide (LPS)-sensitized HI brain injury was induced in 8-day-old neonatal rats. NAC was administered in multiple doses, and brain injury was evaluated at 7 days after HI. RESULTS: NAC (200mg/kg) provided marked neuroprotection with up to 78% reduction of brain injury in the pre+post-HI treatment group and 41% in the early (0 hour) post-HI treatment group, which was much more pronounced protection than another free radical scavenger, melatonin. Protection by NAC was associated with the following factors: (1) reduced isoprostane activation and nitrotyrosine formation; (2) increased levels of the antioxidants glutathione, thioredoxin-2, and (3) inhibition of caspase-3, calpain, and caspase-1 activation. INTERPRETATION: NAC provides substantial neuroprotection against brain injury in a model that combines infection/inflammation and HI. Protection by NAC was associated with improvement of the redox state and inhibition of apoptosis, suggesting that these events play critical roles in the development of lipopolysaccharide-sensitized HI brain injury.

This study aimed to investigate redox regulation of the proteasome as well as the effect of proteasome inhibition on intracellular oxidative status and apoptosis.Oxidative stress was induced in cultured human lens epithelial cells (HLECs) and intact mouse lenses by 100 microM H2O2. HLECs were also exposed to the reduced and the oxidized forms of glutathione (GSH/GSSG) and the reducing agent dithiotreitol (DTT). The chymotrypsin-like, the trypsin-like, and the peptidylglutamyl peptidase activities of the proteasome were measured using synthetic fluorogenic substrates. Superoxide as well as peroxide production, mitochondrial membrane potential, and the level of GSH was measured in HLECs after proteasome inhibition by MG-132 or lactacystin. Apoptosis was determined by measuring caspase-3 activation and by studying apoptotic nuclei after staining with Hoechst 33342.All three peptidase activities of the proteasome were inhibited by 100 microM H2O2 and by the oxidized form of glutathione (GSSG), whereas the reduced form (GSH) stimulated chymotrypsin-like and peptidylglutamyl peptidase activities in HLECs lysates. Intact mouse lenses exposed to 100 microM H2O2 exhibited loss of transparency and trends of decreased chymotrypsin-like proteasome activity as well as decreased GSH levels. Inhibition of the proteasome in cultured HLECs caused significant increase in apoptosis and disturbed intracellular redox balance. Simultaneous addition of exogenous GSH completely abolished the increased apoptosis seen after MG-132 treatment.This study supports the hypothesis that intracellular proteolytic and oxidative regulatory systems are tightly coupled. The current data also indicate that apoptosis by proteasome inhibition is mediated through oxidative mechanisms.

Apoptosis has been implied in normal lens development in the embryo as well as in lens fibre differentiation. It has also been suggested to play a role in non-congenital cataract and in the formation of posterior subcapsular opacification, but data on the presence of apoptosis in human lens epithelium from cataractous lenses are scarce and conflicting. The present study aimed to investigate apoptosis in lens epithelium from patients undergoing cataract surgery. The amount of apoptosis detected was correlated to age, gender, type of cataract, medications and disease. Moreover, the ability of human lens epithelial cells in culture to respond to the apoptosis-inducing agent staurosporin by activation of caspase-3 was investigated. Human lens capsulotomy specimens were collected immediately after surgery, frozen and later analysed with respect to caspase-3 activity, using the fluorogenic substrate Ac-DEVD-AMC. Generally, the activity of caspase-3 detected in this manner was very low and in 23% of the specimens it was non-detectable. However, there were differences in caspase activity between lens epithelial cells from different types of cataract, where samples from lenses with posterior subcapsular cataract exhibited significantly lower caspase-3 activity than lenses with a clear subcapsular zone. Age, gender or medications did not show any correlation with caspase activity but human capsulotomy specimens from diabetic patients exhibited significantly lower caspase-3 activity. Staurosporin caused a concentration-dependent increase in caspase activity in cultured human lens epithelial cells and the amount of apoptotic nuclei was also increased as viewed by staining with Hoechst 33342, showing chromatin condensation and nuclear fragmentation. Similar results were obtained when fresh human lens capsulotomy specimens were exposed to 1000 nM staurosporin for 24 hr. To conclude, the present data indicate that human lens epithelial cells have the ability to respond to apoptosis-inducing agents with caspase-3 dependent apoptosis, and that even though the general level of apoptosis in human lens epithelium in vivo is low, there are differences in caspase-3 activity levels in lenses with or without posterior subcapsular cataract. The latter finding supports previous studies indicating that this type of cataract may result from defective differentiation, in which apoptosis may play an important role.

The relative contributions of apoptosis and necrosis in brain injury have been a matter of much debate. Caspase-3 has been identified as a key protease in the execution of apoptosis, whereas calpains have mainly been implicated in excitotoxic neuronal injury. In a model of unilateral hypoxia-ischemia in 7-day-old rats, caspase-3-like activity increased 16-fold 24 h postinsult, coinciding with cleavage of the caspase-3 proenzyme and endogenous caspase-3 substrates. This activation was significantly decreased by pharmacological calpain inhibition, using CX295, a calpain inhibitor that did not inhibit purified caspase-3 in vitro. Activation of caspase-3 by m-calpain, but not μ-calpain, was facilitated in a dose-dependent manner in vitroby incubating cytosolic fractions, containing caspase-3 proform, with calpains. This facilitation required the presence of some active caspase-3 and could be abolished by including the specific calpain inhibitor calpastatin. This indicates that initial cleavage of caspase-3 by m-calpain, producing a 29-kDa fragment, facilitates the subsequent cleavage into active forms. This is the first report to our knowledge suggesting a direct link between the early, excitotoxic, calcium-mediated activation of calpain after cerebral hypoxia-ischemia and the subsequent activation of caspase-3, thus representing a tentative pathway of “pathological apoptosis.”

Caspase-3 is a major effector protease in several apoptotic pathways, but its role in hypoxic-ischemic (HI) brain injury is incompletely understood. Cerebral HI was induced in 7-day-old rats by unilateral carotid artery ligation and exposure to 7.7% oxygen for 55 min. Caspase-3-like activity was significantly increased at 1 h (208%), peaked at 24 h (2,563%) and was still increased 6 days after HI (169%) in the ipsilateral cerebral cortex. Concomitantly, cleavage of the caspase-3 proform (31/33 kD) was detected on immunoblots, producing 29- and 17-kD fragments. Furthermore, significant degradation of the endogenous caspase-3 substrates inhibitor of caspase-activated DNase (DNA fragmentation factor 45), poly(ADP-ribose) polymerase and fodrin occurred. In conclusion, caspase-3 is activated extensively in the immature brain after HI. The subsequent cleavage of proteins involved in cellular homeostasis and repair may contribute to the process of brain injury.

Parkinson's disease is characterized by a selective loss of dopaminergic neurons in the nigrostriatal pathway. However, not all dopaminergic neurons degenerate in this disease, and calcium has been suspected of playing a role in this differential vulnerability. An overexpression of the calcium-dependent protease calpain II has recently been reported in the parkinsonian substantia nigra, suggesting that a rise in intracellular calcium concentrations may be involved in the mechanism leading to cell death. The proteasic activity of calpain is regulated by an endogenous inhibitory protein called calpastatin. Because little is known about the distribution of calpastatin in the primate brain, we first analyzed immunohistochemically the calpastatin expression in normal human and monkey brain. A ubiquitous distribution of calpastatin immunostaining was observed in both cases, but its expression was variable from one region to another. In the basal ganglia, staining was intense in the striatum, in the pallidal complex, and in some nuclei of the thalamus. The cerebellum was stained intensely, particularly in the granular and Purkinje cell layers. A dense, heterogeneous staining was observed in the hippocampal formation, mostly in the pyramidal and granular layers. The distribution of staining was similar in the different cerebral cortices studied, and it was most intense in layer V. In the brainstem, staining was particularly prominent in the substantia nigra pars reticulata and compacta, the central gray substance, the superior colliculus, and the cuneiform nucleus, and staining was moderate in the tegmenti pedonculopontinus nucleus and the griseum pontis. In the second part of the study, the authors compared calpastatin expression in the mesencephalon between patients with Parkinson's disease and control subjects. Sequential double staining revealed that some dopaminergic neurons coexpress calpastatin, the proportion of double-stained neurons ranging between 52% and 76% among the different dopaminergic cell groups. Quantitative analysis of the number of calpastatin-stained neurons evidenced a loss of both calpastatin-positive and calpastatin-negative neurons in the substantia nigra of patients with Parkinson's disease. These data suggest that calpain II overexpression in Parkinson's disease is not compensated for by a concomitant increase in calpastatin expression.

The purpose of the present investigation was to develop a system for continuous evaluation of extralysosomal proteolytic activity and its regulation in polarized epithelial cells. Filter inserts containing a tight monolayer of primary cultured pig thyrocytes were placed in a thermostated aluminium block. The cell-permeable, fluorogenic calpain and proteasome substrate succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin was added to the apical buffer and fluorescence changes were continuously measured via the fibre optics of a luminometer held at a fixed distance from the cell layer. Basal proteolytic activity was reduced by 60-70% by the proteasome inhibitor lactacystin. Proteolysis was increased within a few minutes after application of Ca(2+)-mobilizing agents (ionomycin, 4-bromo-A23187, thapsigargin and maitotoxin). Forskolin and staurosporine also enhanced the proteolytic activity. We conclude that Ca(2+)mobilization, and possibly also changes of protein kinase activity, rapidly increase non-lysosomal proteolysis in the intact thyroid epithelium.

Retinoic acid is one of the active metabolites of vitamin A and has profound effects on the development of the CNS including retina. Previously, we have shown that rod-specific apoptosis is induced in retinal explants from neonatal mice by exposure to 9-cis-retinoic acid (9CRA) when the retinal pigment epithelium (RPE) is present. In explants lacking RPE, it instead has a differentiation-promoting effect seen as an accelerated opsin expression on postnatal day 3. To investigate the long-term effect of 9CRA exposure, we have explanted retinas from neonatal C3H mice with or without RPE attached and placed in organ culture. After 19 or 48 h in culture or 7, 8 or 13 days in culture, the explants were either fixed for histochemical examination or frozen for assay of DEVDase activity. We found that long-term exposure to 9CRA caused a decrease in the number of cell layers in the outer nuclear layer (ONL) only in explants with the RPE attached. When explants with RPE attached were exposed to 9CRA only during the second postnatal week, neither an increase in DEVDase activity, TUNEL-positive cells, nor a decrease in cell layers of the ONL could be demonstrated, indicating that the retina was insensitive to the apoptosis-inducing effect of 9CRA after the first postnatal week. The absence of RPE in control explants resulted in a higher number of rosettes and the extrusion of cells into the subretinal space.

With ageing, accumulation of modified proteins occur in the lens, forming light scattering aggregates. The multicatalytic proteinase complex, or proteasome, is known to be the major system for removal of damaged proteins in many tissues. In this study we attempted to compare levels of proteasome activity in human lens epithelium from clear vs. cataractous lenses. Normal lenses were obtained from eye donors in a cornea bank and samples from cataractous lenses were obtained from an eye clinic during cataract surgery. Proteolytic activity was quantified using the synthetic peptide substrate N -Succ-Leu-Leu-Val-Tyr-AMC, a substrate often used to measure the chymotrypsin-like activity of the proteasome. Addition of 100 μ m lactacystin, a proteasome specific inhibitor, totally inhibited proteolysis, certifying the specificity of the assay. Hydrolysis was detected over time as the appearance of the flourogenic cleavage product and correlated to the area of the epithelium-capsule specimens. Proteolytic cleavage of N -Succ-Leu-Leu-Val-Tyr-AMC by the proteasome was higher in lens epithelium from clear donor lenses as compared to samples from cataractous lenses. Median activity in the latter was only 19% of that in the former, a highly significant difference. There was no difference in activity of the proteasome when looking at cortical vs. non-cortical cataract, nor was there any difference between genders. Regression analysis did not reveal any age-dependent relationship, either in the clear group or in the cataractous group. This work is the first to show differences in proteasome activity between clear and cataractous lenses.

Proteolysis at neutral pH in the soluble fraction of cultured pig thyroid epithelial cells was examined using a synthetic calpain substrate, succinyl-Leu-Tyr-7-amino-4-methylcoumarin. The Ca2+-independent proteolytic activity was largely inhibited by substances known to affect cysteine- and metalloproteases, whereas no or little effects were obtained with inhibitors affecting serine- and aspartic proteases. Addition of Ca2+ did not significantly alter the rate of substrate degradation. Biochemical separation via hydrophobic interaction chromatography and Western blotting demonstrated the presence of both m-calpain (40% of total calpain) and mu-calpain (60%) in confluent thyrocytes. Determination of calpastatin activity indicated a 30 times higher level of the inhibitor as compared to total calpain activity. Western blotting showed the presence of a 110 kD calpastatin form with additional low mol wt forms possibly representing fragmentation products. In immunofluorescent stainings, m-calpain had a diffuse cytoplasmic distribution whereas mu-calpain was located both in the cytoplasm and at the cell-cell contacts. Calpastatin immunoreactivity was mainly granular and located close to the nucleus, although a fibrillar distribution was also observed. The results show the presence of all components of the calpain/calpastatin system and indicate a strict control of calpain activity in cultured thyrocytes. The different subcellular distributions of calpains and calpastatin suggests that they are compartmentalized and require mobilization to interact.

Generic variation within and among one Finnish and three Swedish populations of Fomitopsis pinicola (Schwarts: Fr.) Karst. were studied by amplifying DNA from hap-loid isolates originating from single spore cultures using two arbitrary primers. Analysis offspring from single fruit bodies revealed only three pairs of codominant alleles among 42 variable genetic markers, the remaining 38 segregated independently. Genetic similarity was measured in terms of Euclidean distance. Individuals in the Finnish population tended to form a distinct cluster in the principal component analysis. Variation within and among populations/regions was partitioned by Analysis of Molecular Variance — AMOVA. Within population variation accounted for 91.6% of the total genetic variation. The remaining 7.68% was accounted for by variation between the Finnish population and each of the three Swedish ones. Variation among the Swedish populations accounted for only 0.72% of the total variation. Wright's Fst was 0.17 for all four populations and 0.13 for the three Swedish populations. These relatively low values indicate that there is gene flow among all populations or that they are derived from a common ancestral population. The observed pattern of genetic variation is probably the result of effective spore dispersal and the continuous distribution of this common early successional species.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min) and allowed to recover for 3 h, 24 h, 2 days or 14 days. Consecutive tissue sections were stained with antibodies against α-fodrin, the 150 kDa breakdown product of α-fodrin (FBDP, marker of calpain proteolysis) or microtubule associated protein 2 (MAP 2, marker of dendrosomatic neuronal injury). Cortical tissue pieces were subjected to Western blotting using the antibody against the FBDP. Areas with brain injury displayed a distinct loss of MAP 2 which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. A reciprocal staining pattern could be seen in the cerebral cortex, i.e. loss of MAP 2 and accumulation of FBDP, most pronounced 14 days after the insult. Fodrin and MAP 2 are known calpain substrates, and degradation of these proteins preceded neuronal degeneration, indicating that these proteases may be involved in the early events triggering the cascades leading to neuronal death.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery +7.70% O 2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62–68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204–292% of controls). No significant changes in FBDP could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.

Background Traditional outcome measures in peripheral arterial disease (PAD) provide insufficient information regarding patient benefit. It has therefore been suggested to add patient-reported outcome measures. The main aim of this study was to validate the Swedish Vascular Quality of Life questionnaire (VascuQoL) version, a patient-reported PAD-specific health-related quality of life (HRQoL) instrument. Methods Two-hundred PAD patients were consecutively recruited from two university hospitals. Out of the 200 subjects, 129 had intermittent claudication and 71 had critical limb ischemia. Mean age was 70 ± 9 y and 57% of the participants were male. All patients completed SF-36 and VascuQoL at the vascular outpatient clinic, when evaluated for invasive treatment. Risk factors and physiological parameters were registered. Construct validity was tested by correlation analysis versus SF-36 and was also assessed with multitrait/multi-item scaling analysis (MTMI). Sensitivity analysis regarding disease severity identification was performed. Reliability was assessed with Cronbach's alpha and responsiveness by standardized response mean (SRM) calculations. Results Significant correlations were demonstrated between relevant subscales of VascuQoL and SF-36. MTMI showed acceptable construct validity, but some scaling-errors. VascuQoL significantly (p Conclusions The Swedish version of VascuQoL is valid and quantifies central aspects of HRQoL in PAD patients. Sensitivity analysis showed high ability to differentiate between disease severity and SRM illustrated excellent responsiveness. The relative abundance of items however makes use in the everyday clinical setting somewhat difficult.

Karlsson, J.-O. 1994. Genetic variation in Heterobasidion annosum detected with M13 fingerprinting and ribosomal DNA probes. Experimental Mycology 18, 48-56. Mitochondrial and nuclear DNA diversity were analyzed in 54 strains of the forest pathogen Heterobasidion annosum (Fr.) Bref. from Northern Europe. Restriction-fragment-length polymorphisms were detected with three different probes on Southern blots of DNA restricted with Hae III. Intersterility groups were clearly differentiated based on the UPGMA clustering of M13 minisatellite bands, and strains with the same geographic origin tended to form clusters. Average band share values were 0.80 and 0.68 within S- and P-groups, respectively, and 0.49 between groups. When the ribosomal mitochondrial ML 5-8 region was used as a probe the two types of patterns observed were in accordance with the results of the mating tests and the UPGMA clustering of fingerprints. No variability was detected in ribosomal nuclear ITS 1-4 region.

DNA from 124 strains of Heterobasidion annosum was amplified using the core sequence of the M13 minisatellite region as primer. The strains were derived from 11 S-, 8 P- and 1 F-intersterility group populations originating in Scandinavia, Germany and Italy. Following electrophoresis the banding patterns of all isolates were compared and 23 fragments were scored. Average band-sharing indices (ABSI) were used to compare genetic diversity within and between populations. Genetic similarity of populations decreased with increasing geographical distance in the S group. In both the S and P intersterility groups, ABSI values were higher within than among populations (70·3 ± 2·3 s.d. and 66·4 ± 2·2, respectively in the S group and 77·0 ± 2·3 and 75·0 ± 2·4, respectively in the P group) indicating local differentiation. The higher values in the P than in the S group indicate that the P group is less variable. ABSI was much lower between S and P group populations (49·2 ± 2·5) than within either group. This result demonstrates the large divergence between these two intersterility groups. In the F population ABSI was higher (68 ± 3·1) than for the comparisons with the S- (50·1 ± 1·6) and P-groups (44·0 ± 2·4). By using discriminant analysis, the agreement between geographical population affiliation and that inferred from banding patterns of amplified DNA was 20·8% within the S group, and 14·9% within the P group. Chi-square test of the discriminant analysis indicated regional differentiation in the S but not in the P intersterility group. The agreement between methods was 98·4% when strains were classified to intersterility group by pairing tests or amplified DNA profiles. Results are consistent with a high degree of exchange between populations of the same intersterility group but low exchange between intersterility groups.

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.

Microtubules isolated from Atlantic cod (Gadus morhua) brains retained assembly competence and ultraculture, although treatment with rabbit calpain resulted in loss of MAPs. In addition, spirals and aberrant structures formed when calpain I was activated post assembly. No such effect was seen with calpain II. Soluble fractions from cod brain were found to contain proteolytic activity that could be blocked by exogenously added calpastatin. Calpain was also isolated from cod muscle tissue with 10 times less yield, compared to rabbit lung. On the basis of Ca(2+)-requirements for activation in the mM range, electrophoretic mobility, antigenicity and hydrophobicity, we conclude that the proteolytic activity was attributable to calpain II. There was no difference in effects of rabbit and cod calpain II on cod microtubule proteins, indicating that calpain is a conserved protein. Our results suggest that calpains might be involved in the Ca(2+)-dependent irreversible regulation of cod brain microtubules.

Oxidative stress is a major contributor to slowly developing diseases like Parkinson's disease, Alzheimer's disease and cancer and one of the main causes of tissue damage following ischemic insults in the brain. Nrf2 is a transcription factor responsible for much of the inducible cellular defense against oxidative stress. Nrf2 can also be activated by xenobiotics like sulforaphane, a component highly enriched in cruciferous vegetables such as broccoli. Ingestion of broccoli or sulforaphane results in long-term protection against radical damage, although absorbed sulforaphane is cleared from the body within a few hours. Here we have examined whether the prolonged protection induced by sulforaphane is explained by a slow down regulation of the Nrf2 response. Furthermore, to simulate daily ingestion of sulforaphane, we examined the hypothesis that repeated transient sulforaphane stimulation results in an accumulation of Nrf2-mediated gene expression and an increased protection against oxidative damage. The kinetics of sulforaphane-induced Nrf2 response was studied in astrocytes, a cell type known to be highly involved in the defense against oxidative stress in the brain. Sulforaphane stimulation for 4 h induced an Nrf2-dependent increase of Nqo1 and Hmox1 mRNA that remained elevated for 24 h, and the corresponding proteins remained elevated for over 48 h. In addition, peroxide-clearing activity and the levels of glutathione were elevated for more than 20 h after stimulation for 4 h with sulforaphane, resulting in an increased resistance to superoxide-induced cell damage. Repeated sulforaphane stimulation resulted in an accumulation of mRNA and protein levels of Nqo1 and a persistent cell protection against oxidative damage. These findings indicate that brief stimulation of the Nrf2 pathway by sulforaphane results in long-lasting elevation of endogenous antioxidants in astrocytes. The findings also demonstrate that part of this response can be built up by repeated transient stimulation, possibly explaining how intermittent intake of sulforaphane can result in long-term protection from radical-induced disease.

Ca-independent and Ca-dependent proteolytic activity were significantly higher in erythrocytes from male patients with Alzheimer''s disease compared to control subjects. The level of calpastatin, an endogenous inhibitor of the proteolytic enzyme calpain, was lower in male patients with Alzheimer''s disease. The small changes observed in the female group were not statistically significant. Control male subjects had significantly higher levels of calpastatin compared to female controls. The observed changes in the calpain/calpastatin system of erythrocytes may be caused by a general dysregulation of Ca homeostasis in cells of Alzheimer patients and may lead to calpain-induced pathological changes.

Apoptosis-related mechanisms are important in the pathophysiology of hypoxic–ischemic injury in the neonatal brain. Caspases are the major executioners of apoptosis, but there are a number of upstream players that influence the cell death pathways. The Bcl-2 family proteins are important modulators of mitochondrial permeability, working either to promote or prevent apoptosis. In this study we focused on the anti-apoptotic Bcl-2 protein after neonatal cerebral hypoxia–ischemia (HI) in 8-day-old rats. Bcl-2 translocated to nuclei and accumulated there over the first 24 h of reperfusion after HI, as judged by immunohistochemistry and immuno-electron microscopy. We also found that the total level of Bcl-2 decreased after HI in vivo and after ionophore challenge in cultured human neuroblastoma (IMR-32) cells in vitro. Furthermore, the Bcl-2 reduction was calpain-dependent, because it could be prevented by the calpain inhibitor CX295 both in vivo and in vitro, suggesting cross-talk between excitotoxic and apoptotic mechanisms.

Pectinolytic activities in Armillaria ostoyae, Armillaria mellea, Armillaria gallica, Armillaria borealis, and Armillaria cepistipes were assessed by measuring the ability of fungal strains to reduce the viscosity of their pectic growth media. Isozyme patterns of pectin esterases and polygalacturonases were determined directly from the culture filtrate. A total of 94 strains, representing isolations from various parts of Europe, were analyzed for their isozyme patterns. Armillaria mellea and A. borealis caused a 50% reduction in viscosity within 7 and 9 days, respectively. Growth medium from the other species were slower to reach the 50% level, i.e., means were 13 days for A. ostoyae and 17 days for A. cepistipes and A. gallica. All species produced more isozymes on spruce wood than on citrus pectin medium, and pectic isozyme patterns differed between media. The pectic isozyme pattern for A. mellea differed distinctly from those of the other four species by having two bands of polygalacturonase not found in the others. The pectic isozyme patterns of the other four species were separated using multivariate analysis. The value of such analyses for use in distinguishing between European Armillaria species is discussed, as is the relation between enzyme activity and fungal pathogenicity. Key words: root rot, diagnostic tests, Agaricales, polygalacturonase, pectin esterase.

Interfertility among European and North American strains of Heterobasidion annosum was examined in hetero- x homokaryon pairings with testers of known intersterility group affiliation. In a set of 164 heterokaryotic strains, approximately 10% interfertility was found between the Scandinavian S and P groups. Twelve percent of European S group strains were partially interfertile with North American P group tester strains and 16% of European P group strains were interfertile with North American S group tester strains. Homokaryotic European F group strains were highly interfertile with the North American S group.

Pectic isozymes produced in liquid culture by strains of Heterobasidion annosum were separated by gel electrophoresis. Activity staining of the gel revealed six distinct zymogram groups which corresponded with three different intersterility (IS) groups in Europe, two IS groups in North America and one group in Australasia. Banding patterns were consistent and may be used as biochemical markers of affiliation to intersterility group.

The Ca2(+)-dependent neutral proteases calpain I and II as well as their specific inhibitor, calpastatin, were isolated from normal and Alzheimer-degenerated frozen human brain tissue. In the Alzheimer group calpain I activity was higher in cortex than in mesencephalon. The calpastatin activity was lower in cortex in both groups. This may implicate a higher Ca2(+)-dependent proteolysis in cortex compared to mesencephalon. In the Alzheimer group the cortical calpain II level decreased with an increasing degree of neuropathological changes. In the control group, the level of calpastatin decreased as the number of plaques and tangles increased. Evidence was obtained for a correlation of net calpain activity and the extent of neuropathological changes in cortex.

1. The regulation of pigment migration in erythrophores and melanophores of Labrus ossifagus was compared. 2. Both types of chromatophores are regulated by α 2 -adrenoceptors, but the receptors of the erythrophores were more sensitive to adrenoceptor agonists than were those of melanophores. 3. The α 2 -adrenoceptor of the erythrophores were prazosin-sensitive. 4. Forskolin (10μM) completely blocked pigment aggregation in both types of chromatophores. 5. Li + at a concentration of 100 μM interfered with pigment aggregation in erythrophores but not in melanophores. 6. The Na + /H + blocker amiloride inhibited pigment migration induced by the α 2 -selective agonist B-HT 920 in both types of chromatophores. 7. There are differences in the α 2 -adrenoceptor systems regulating pigment migration in erythrophores and melanophores of Labrus ossifagus .

Purpose: Experiments were carried out to study the effects of siramesine on markers for apoptosis, oxidative damage and mitochondrial function in primary cultures of human lens epithelial cells (HLEC). Methods: HLEC were incubated with 25 μM siramesine for 1, 2, 3, 4, 6 and 8 hours. Caspase-3 was assayed in cell extracts with DEVD-AMC. Mitochondrial depolarization was assayed with JC-1. Peroxide production was studied with DCF-DA and superoxide levels with hydroethidium. Glutathione levels were assayed with monochlorobimane. Results: Siramesine induced a significant increase of caspase-3 activity after 6h exposure to 25 μM siramesine. Nuclear morphology examined with Hoechst 33342 showed signs of apoptosis after the same time intervals. A significant increase in the production of peroxide and superoxide were found up to 4 -8 hours after administration of siramesine. Conclusions: Siramesine, a piperidine analogue, was developed for the treatment of psychiatric disorders and is considered to be relatively nontoxic. This study, and others, indicate effects on cell growth, apoptosis and production of ROS. The sigma-2 receptor may be a regulator of HLEC growth and apoptosis.

Hyperhomocysteinemia is commonly associated with polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene. The level of homocysteine can be lowered by dietary intake of folate. A protective effect of folate supplementation has been reported against cataract. Here we investigate MTHFR polymorphisms in human cataract.Retrospective case-control association study.Patients with nuclear (n = 77), cortical (n = 155), posterior subcapsular (n = 119), and mixed (n = 151) cataract, and 187 controls were analyzed for the MTHFR 677C--T and 1298A--C polymorphisms using minisequencing technique.The wild-type MTHFR 677CC/1298AA genotype was strongly overrepresented among cataract cases (P = .003). This effect was most pronounced in the mixed cataract group (P.001). Hyperhomocysteinemia-associated genotypes had similar frequencies in cataract and control groups.The previously reported protective effect of folate against cataract is not due to overrepresentation of hyperhomocysteinemia-associated MTHFR genotypes. Instead, the strong predominance of wild-type MTHFR in cataract may suggest impaired DNA synthesis as a cataractogenic factor.

Purpose: To study possible toxic effects of indomethacin, diclofenac, and celecoxib (NSAIDs) and acetylsalicylic acid (ASA) as well as potentially protective effects of these substances in oxidatively stressed human lens epithelial cells (HLEC) and in intact mouse lenses in culture. Methods: HLEC and mouse lenses were incubated with NSAIDs or ASA alone or in the presence of H2O2. To study apoptosis the cells were then either stained with Hoechst 33342 or assayed for caspase-3 activity. Mouse lenses were studied with respect to lens transparency. Results: Low concentrations of NSAIDs/ASA caused a significant protection against H2O2-induced apoptosis in HLEC whereas higher concentrations were toxic. Conclusion: The protective effects of NSAIDs/ASA against oxidative damage are confined to a relatively small therapeutic window.

OBJECTIVE: Hydrogen peroxide (H2O2), necessary for thyroid hormonogenesis, is produced at the apical surface of the thyroid follicular epithelium. Excess H2O2 is potentially cytotoxic and may contribute to the development of hypothyroidism, e.g. in severe selenium deficiency. Yet it is unclear how H2O2 contributes to thyroid cell death. DESIGN AND METHODS: H2O2-induced apoptosis and necrosis were studied in primary cultured pig thyroid cells. Glutathione peroxidase (GPx) activity was altered by culture in low serum with or without selenite substitution. Apoptosis was evaluated by spectrofluorometric measurement of caspase-3-specific substrate cleavage, and by analysis of DNA fragmentation by agarose gel electrophoresis. Necrosis was detected by 51Cr release from prelabeled cells. RESULTS: Exogenous H2O2 dose-dependently (100-400 micromol/l) activated caspase-3 within 3-12 h, and DNA degradation was observed after 24 h. The potency of H2O2 to induce apoptosis was low compared with that of staurosporine, a strong proapoptotic agent. H2O2-treated cells with reduced GPx activity showed increased caspase-3 activation. Incubation of serum-starved cells with selenite (10-100 nmol/l) normalized the GPx activity and reduced the activation of caspase-3 by H2O2. High H2O2 concentrations (400-800 micromol/l) were required to obtain necrosis. The H2O2-induced necrosis was exaggerated by both low GPx activity and catalase inhibition. CONCLUSIONS: Cytotoxic effects of H2O2 on thyroid cells include caspase-3-dependent apoptosis that occurs at H2O2 concentrations insufficient to induce necrosis. Selenium deficiency aggravates the apoptotic response, probably due to impaired capacity of GPx to degrade H2O2.

OBJECTIVE: Proinflammatory cytokines are known to affect the follicular epithelium in autoimmune thyroid disease. Here we investigated the effect of interferon-gamma (IFN-gamma) on the barrier function of primary cultured human thyrocytes. DESIGN: Graves' thyroid follicle segments were cultured as a tight and polarised monolayer on the filter of a bicameral chamber, thereby allowing the in vivo epithelial characteristics to be maintained. METHODS: Transepithelial electrical resistance was measured with a Millicell ERS ohmmeter. The tight junction proteins claudin-1 and occludin were analysed by immunofluorescence and Western blotting. Cell morphology was studied by transmission electron microscopy. RESULTS: Thyrotrophin (TSH; 1 mU/ml) promoted the development of a tight epithelium monitored as a persistent increase in the transepithelial resistance to about 800 omega x cm2. IFN-gamma (100 U/ml), on the other hand, decreased the resistance to 60-150 omega x cm2 after 48 h. In IFN-gamma-treated cells the expression of claudin-1, but not that of occludin, was decreased along with a diminished intracellular and cell surface immunostaining. In addition, claudin-1 was disrupted at cell-cell contacts. IFN-gamma also caused profound cell shape changes and a multilayered cellular organisation, without ultrastructural or biochemical (caspase-3 activity) signs of cytotoxicity. TSH was unable to counteract the effects of IFN-gamma. CONCLUSIONS: IFN-gamma destroys the barrier function of filter-cultured human thyroid epithelial cells. The loss of barrier involves down-regulation and an altered distribution of claudin-1. This novel effect of IFN-gamma on target cells in thyroid autoimmunity might be of pathophysiological relevance to the exposure of hidden autoantigens.

Purpose. The aim of this study was to measure proteasome activity in human lens nuclei resulting from cataract surgery and in different regions of donor lenses. Methods. The chymotrypsin-like, the trypsin-like and the peptidylglutamyl-peptide hydrolysing activities of the proteasome were studied using synthetic flourogenic substrates. Results. Proteasome activity did not show any correlation with age of the patients or with gender. Increased opacification of the lens nucleus, as estimated prior to surgery using a 4-grade scale, was significantly correlated with decreased activity of all peptidase activities in the insoluble fraction. In the donor lenses, all peptidase activities were highest in the epithelium and decreased rapidly towards the nucleus. Conclusions. The present study demonstrates that proteasome activity is preserved in the nucleus of lenses from elderly individuals, although a decrease can be seen with cataract formation. This finding may be of importance for elucidating the mechanism behind the formation of nuclear cataract.

Blockade of mitochondrial permeability transition protects against hypoglycemic brain damage. To study the mechanisms downstream from mitochondria that may cause neuronal death, we investigated the effects of cyclosporin A on subcellular localization of apoptosis-inducing factor and cytochrome c, activation of the cysteine proteases calpain and caspase-3, as well as its effect on brain extracellular calcium concentrations. Redistribution of cytochrome c occurred at 30 min of iso-electricity, whereas translocation of apoptosis-inducing factor to nuclei occurred at 30 min of recovery following 30 min of iso-electricity. Active caspase-3 and calpain-induced fodrin breakdown products were barely detectable in the dentate gyrus and CA1 region of the hippocampus of rat brain exposed to 30 or 60 min of insulin-induced hypoglycemia. However, 30 min or 3 h after recovery of blood glucose levels, fodrin breakdown products and active caspase-3 markedly increased, concomitant with a twofold increase in caspase-3-like enzymatic activity. When rats were treated with neuroprotective doses of cyclosporin A, but not with FK 506, the redistribution of apoptosis-inducing factor and cytochrome c was reduced and fodrin breakdown products and active caspase-3 immuno-reactivity was diminished whereas the extracellular calcium concentration was unaffected. We conclude that hypoglycemia leads to mitochondrial permeability transition which, upon recovery of energy metabolism, mediates the activation of caspase-3 and calpains, promoting cell death.

The aim was to study the effects of an NMDA receptor antagonist on caspase-3 activation and DNA fragmentation after hypoxia-ischemia (HI) in 7-day-old rats. Animals were treated with vehicle or MK-801 (0.5 mg/kg) directly after HI and sacrificed 8, 24 or 72 h later. MK-801 reduced injury (by 53%), cells positive for active caspase-3 (by 39%) and DNA fragmentation (by 79%) in the cerebral cortex. Furthermore, MK-801 significantly decreased caspase-3 activity, and Western blots revealed a tendency towards decreased proteolytic cleavage of the caspase-3 proform. The data imply that NMDA receptors are involved in the activation of apoptotic processes in the immature brain after HI.

The proteasome is a large protease complex that is thought to be responsible for proteolytic removal of damaged proteins. We have previously shown that the level of proteolytic activity due to the proteasome is lower in lens epithelium from human cataractous lenses compared to the activity in epithelium from clear donor lenses. This study aimed to characterize the three main peptidase activities of the proteasome in human lens epithelium with respect to kinetic properties and sensitivity to heat and oxidation. Human lens epithelia were obtained from cataract surgery and analysis performed on pools of epithelial cell cytoplasm. Using the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (LLVY), Boc-Val-Gly-Arg-AMC (VGR) and Z-Leu-Leu-Glu-betaNA (LLE), Km-values of 56, 678 and 108 micrometers were obtained. All peptidase activities were inhibited by lactacystin, a specific proteasome inhibitor, but at very different rates; with LLVY-hydrolysing activity being the most sensitive (Ki50%=0.15 micrometers). Thermostability was investigated by performing the proteolytic assay at 20 degrees, 37 degrees and 53 degrees C. The trypsin-like activity, as measured by VGR, was completely stable at 53 degrees C for at least 24 hr whereas hydrolysis of LLVY and LLE declined after a few hours at 37 degrees C. Oxidative inhibition was induced by incubation of the samples in 0.5 m m H2O2for 1 or 24 hr. One hour exposure to H2O2caused moderate inhibition of all peptidase activities. The activity could be partially restored by adding 1 m m dithiotreitol, indicating the dependency on intact SH-groups. After 24 hr, peptidase activities were decreased to 25% (LLVY), 73% (VGR) and 44% (LLE) of corresponding control. This inhibition was irreversible for VGR and LLE, but could be partly prevented by the presence of heat shock protein 90 (LLVY and VGR) or alpha-crystallin (LLVY). These data show that the peptidase activities of the human lens proteasome can be modulated by metabolites, such as reactive oxygen species, and by endogenous proteins such as alpha-crystallin and heat shock protein 90.

Changes in Ca homeostasis in non-neuronal cells have been found both in aging and in Alzheimer’s disease (1). Similar changes have also been described in the brain, including hippocampal neurons (2–5). A comprehensive review of Ca changes in aging cells and during Alzheimer’s disease has been published (6). Dysregulation of free Ca in the cytosol may induce changes in Ca-dependent proteolytic systems in the cell. Calpains (EC 3.4.22.17) are Ca-activated, neutral cysteine proteases found in all cells. Two types of calpains have been identified: μ-calpain and m-calpain, requiring micro- and millimolar concentrations of Ca, respectively for, in vitro, initiation of activity (7,8). A specific inhibitory protein, calpas-tatin, is also part of this highly regulated proteolytic system. Two lines of evidence suggest that calpains have a regulatory rather than a degradative function in the cell. First, Ca is a well established regulator of many coordinated cellular functions. Second, calpains appear to cleave substrates only to a limited extent, preferentially between rather than within the domains of the substrate proteins (8). Limited proteolysis by calpains may be an important regulatory mechanism mediating biological responses elicited by extracellular stimuli. In an earlier study on post mortem brain material (9) we obtained evidence for an increased calpain activity in the brain during an active stage of Alzheimers disease characterized by a high amount of neuropathological changes. We have also obtained evidence for an increased proteolytic activity and a decreased calpastatin activity in erythrocytes from patients with Alzheimer’s disease (10). A reduced calpastatin activity in erythrocytes from patients with Alzheimer’s disease has also been observed by Soldati et al. (11).

Lens epithelium from patients with cataract was obtained during surgery and frozen. The samples were subjected to SDS-electrophoresis and Western blotting. Calpains were quantified using polyclonal antibodies against m- and μ-calpain. m-Calpain could be detected but not the isoenzyme μ-calpain, indicating that m-calpain is the significant most important calpain in human lens epithelium. Quantification of m-calpain showed no relationship to age or gender, but there were significant differences between different types of cataract.

The localization of the two Ca-activated extralysosomal proteases m-calpain and μ-calpain in the eye of the adult rabbit was examined by immunohistochemistry, using poly- and monoclonal antibodies against the corresponding rabbit antigens. Immunoreactivity against the two forms of calpains was observed in the epithelial cells on the external and internal surfaces of the cornea as well as in the epithelial cells covering the iris and ciliary body. The sclera and choroid layers showed a relatively weak immunoreactivity. Using anti m-calpain antibodies, the pigment epithelium in the retina was heavily labelled as well as the outer and inner plaxiform layers. The outer and inner borders of the Muller cells were clearly labelled. The outer segments on the receptor cells showed a strong immunorectivity for both μ-calpain and m-calpain. Lebelling was also observed in the retinal ganglion cells in tin the nerve fiber layer. The immunohistochemical localization of calpastatin, an endogenous inhibitor of both m- and μ-calpain was also examined. A high level of calpains, especially immunoreactivity was observed in the outer segments of the cells. The results may be compatible with a role for calpains, especially m-calpain, in the secretory/phagocytic process and as modulators of the cytoskeleton in cell processes.

Marchi-positive bodies are structures present paranodally in large myelinated nerve fibers. They have morphological and biochemical characteristics closely resembling the partially degraded myelin fragments formed during the early phases of Wallerian degeneration. Levels of calcium-activated neutral proteases (calpains) and their endogenous specific inhibitor calpastatin were measured in highly purified rabbit myelin and a spinal cord subcellular light ('floating') fraction heavily enriched in Marchi-positive bodies. Calpain levels were found to be significantly higher in the floating fraction as compared to myelin. No calpastatin was detectable in either fraction.

Calpains and calpastatin in the brain of the rabbit were examined in experimental situations that could mimic some features of brain ischemia. Incubations of bisected brains in saline at 39 degrees C for 0.5, 1, or 1.5 h resulted in a decreased calpain I activity in the cytosol and in an increased hydrophobicity of cytosolic calpain II activity. Incubation of brain homogenates at different pH levels demonstrated an almost-complete transfer of calpains from the cytoplasmic compartment to the membranes when pH was lowered from 6 to 5. At pH values lower than 5, the total calpain activity (soluble plus membrane-bound) markedly decreased. No significant changes of calpastatin activity or its subcellular distribution was found following incubation of the homogenates at different pH levels.

Following intravitreal injections of 35 S-amino acids to label axonally transported proteins, calpain I and II from the superior colliculi and the lateral geniculate bodies were isolated via hydrophobic interaction chromatography, anion exchange HPLC and sodium-dodecyl sulphate electrophoresis. Among the slowly transported proteins radioactive label was associated with molecular weight components corresponding to both the light (approx. 30 kDa) and the heavy (approx. 80 kDa) subunits of calpain I as well as with the corresponding subunits of calpain II. Among the fast transported material no radioactive protein bands were detected in these fractions.

Summary This study was undertaken to elucidate the physiological significance of material involved in the rapid axonal transport. The effects of colchicine-induced inhibition of axonal transport in the retinal ganglion cells on the electrophysiological properties of the retrobulbar visual pathways were investigated in Albino rabbits. An impaired signal transmission to the contralateral visual cortex, superior colliculus and lateral geniculate body following flash light stimulation as well as direct optic nerve stimulation appeared 4–6 days after an intravitreous injection of 10–25 μg colchicine. It was concluded that inhibition of the fast axonal transport within the retinal ganglion cells interferes with transsynaptic signal transmission from optic nerve terminals in the subcortical nuclei. This indicates a functional relationship between material supplied via the rapid phase of axonal transport and an unimpaired transsynaptic signal transmission, previously not revealed in the central nervous system of mammals.

1. Innervation and adrenoceptor-mediated pigment granule (i.e. melanosome) aggregation were investigated in melanophores of isolated scales from Labrus berggylta . 2. Tetrodotoxin (10 −6 M) and guanethidine (10 −4 M) blocked the electrically induced melanosome aggregation. 3. Electrically induced melanosome aggregation was inhibited by yohimbine (10 −6 M) but not by prazosin (10 −6 M). 4. The adrenoceptor-agonist order of potency was: medetomidine (alpha 2 -adrenoceptor-selective) > noradrenaline ≥ B-HT 920 > clonidine > dopamine > isoprenaline. 5. Forskolin (10 −5 M) blocked the medetomidine-induced melanosome aggregation. 6. There were no signs of beta-adrenoceptor-mediated melanosome dispersion. 7. These results indicate that the melanophores are innervated by adrenergic nerve fibres, and the aggregation of the melansomes is mediated by an alpha 2 -adrenoceptor, which probably acts via inhibition of adenylate cyclase.