Detection and biochemical characterization of circulating proteasomes in dog plasma.

A growing body of evidence convincingly indicates that proteasomes are not located exclusively within cells but also in different extracellular compartments. In humans, in fact, this large multimeric protease has been identified in many body fluids and secretions such as blood, urine, tears, sweat, saliva, milk, and cerebrospinal and pericardial fluid. Intriguingly, the exact origins of these extracellular proteasomes as well as the specific biological functions they perform are largely unknown. As no data on this important subject is yet available in domestic animals, the present study was undertaken to investigate the presence of extracellular proteasomes in canine blood. As a result, for the first time, circulating proteasomes could be clearly detected in the plasma of a cohort of 20 healthy dogs. Furthermore, all three main proteasomal peptidase activities were measured and characterized using fluorogenic peptides and highly specific inhibitors. Finally, the effect of ATP and PA28 family activators on this circulating proteasome was investigated. Collectively, our data indicate that at least a part of the proteasome present in dog plasma consists of a particle that in vitro displays the enzymatic properties of the 20S proteasome.

Biological effect of astaxanthin on alcohol-induced gut damage in Carassius auratus used as experimental model.

Alcohol and its metabolites are responsible for damage both within the gastrointestinal tract and other organs. Alcohol abuse promote intestinal inflammation, that may be the cause of multiple organ dysfunctions and chronic disorders. In this research, the effect of astaxanthin, a powerful antioxidant with several biological effects, on alcohol damage-induced in the intestine of Carassius auratus, was investigated. In the fishes exposed to ethanol, an increase of the intestinal epithelium mucous cells and circulating macrophages, with intestinal mucosa disorganization was observed. In contrast, in the fishes treated with astaxanthin intestinal morphology was restored. By immunohistochemical analysis, using α-Smooth Muscle Actin (α-SMA) and Vasoactive intestinal polypeptide (VIP) antibodies, a reduction of inflammatory states alcohol-induced was evident, with more regular muscularis submucosa and more organized intestinal mucosa without inflammatory cells. The results suggest that astaxanthin treatments can be a good candidate for preventing damage within the gastrointestinal associated with excessive alcohol consumption.

Expression of Langerin/CD207 in airways, lung and associated lymph nodes of a stranded striped dolphin (Stenella coeruleoalba).

The airways and lungs of vertebrates are an entrance way for several microbial pathogens. Cetaceans present an upper and lower respiratory anatomy that allows the rapid flow of large air volumes, which may lead to high susceptibility to respiratory infections. Mortality and stranding rate of Cetaceans increased dramatically, so wide the knowledge about the immune system and specific antibodies identifying immune cells populations, is of fundamental importance to monitor and document cetacean health. The aim of this study was to identify the localization of dendritic cells marked by Langerin/CD207 in airways, lungs and associated lymph nodes, of the striped dolphin Stenella coeruleoalba. Samples of trachea, bronchi, lungs and lung-associated lymph nodes were obtained from a stranded adult male of Stenella coeruleoalba. Our results showed abundant lymphoid aggregates (LAs) in the lung of S. ceruleoalba. Langerhans-like dendritic cells were well distributed along the epithelium and interstitium of respiratory tract and in associated lymph nodes. The present study deepens the knowledge about the cetacean's immune system and report about the exploitability of a commercial antibody (Langerin/CD207) for cetacean species.

Experimental exposure of blue mussels (Mytilus galloprovincialis) to high levels of benzo[a]pyrene and possible implications for human health.

Polycyclic aromatic hydrocarbons (PAHs) are lipophilic compounds able to accumulate in the food chain. Mussels showed to bioaccumulate contaminants, such as PAHs, so that recurrent consumption of such contaminated food represents a risk for human health. This study was aimed to elucidate if acute exposure of Mediterranean blue mussel (Mytilus galloprovincialis), a bivalve of great economic importance in several countries, to a PAH, benzo[a]pyrene (B[a]P), at doses able to induce cytochrome P450 1A (CYP1A) and pathological changes in mussel gills, can produce accumulation in soft tissue. We explored the cytotoxic effects (cell viability, DNA laddering, and glutathione levels) of in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to organic extracts obtained from blue mussels previously exposed for 12 and 72h via water to B[a]P (0.5-1mg/L). In our experimental conditions, B[a]P induced CYP1A induction and morphological changes in mussel gills and a significant B[a]P accumulation in soft tissue. Conversely, exposing PBMCs to organic extracts obtained from contaminated mussels, resulted in a significant reduction of cell viability and cell glutathione content, and in an increase in DNA laddering. This confirms that consumption of mussels from B[a]P polluted waters might affect human health. Our data lead us to suggest that CYP1A activity in mussel gills may be useful (more than the amount of detected PAHs in the mussel edible tissue) as a marker in assessment of risk for health of consumers exposed to PAHs through ingestion of shellfish.

Effects of PCB-126 on aryl hydrocarbon receptor, ubiquitin and p53 expression levels in Sparus aurata.

The aim of the present study is to determine if Ahr ligands as PCB-126, a dioxin-like, might contribute to inhibition of the tumor suppressor p53 by promoting its degradation through proteasome-ubiquitin system (UPS). The findings show, in the presence of PCB-126, a significant increase of p53 immunoreactivity in fish compared to the control. Subsequently, there is a decrease of p53 immunoreactivity at 24h which is maintained even at 72h. At the same time there is a slight decrease of ubiquitin immunoreactivity to 12h compared to the control and a marked decrease to 24 and 72h. The induction of ubiquitin expression is resulted very marked in the control and preserved at 12h. It's very important to underline as in our study we demonstrate a marked decrease of ubiquitin and p53 immunoreactivity at 24h and 72h. AHR activation, by ligands as PCB-126, increases p53 ubiquitation inhibiting its expression, in addition it decreases the free ubiquitin promoting disruption of Ub homeostasis; this is the first report that establishes a relationship between AhR, increases p53 ubiquitation, and reduction of free ubiquitin. Our result emphasize the need to deeply the role of this receptor in UPS regulation as potential therapeutic target for cancer treatment.

Exposure of sea bream (Sparus aurata) to toxic concentrations of benzo[a]pyrene: possible human health effect.

Polycyclic aromatic hydrocarbons (PAHs) can accumulate in the food chain, due to their lipophilic properties. Fish can accumulate contaminants including PAHs and frequent consumption of such contaminated fish can pose risk to human health. The aim of this study was to clarify if acute exposure of sea bream (Sparus aurata, a fish species of great economic importance in the Atlantic and Mediterranean areas) to a PAH, benzo[a]pyrene (B[a]P), at a dose that can induce CYP1A and pathological changes in fish gills, liver and muscle, can induce accumulation in muscle. We investigated the cytotoxic effects (as changes in cell viability, DNA laddering and glutathione content) of in vitro exposure of human peripheral blood mononuclear cells (PBMCs) to organic extracts obtained from muscle of sea breams previously exposed via water to B[a]P (2mg/l, for 12, 24 and 72 h). At this level of exposure, B[a]P caused morphological changes, inflammatory response and CYP1A induction not only in sea bream gills and liver but also in muscle; furthermore, in fish muscle we observed a substantial B[a]P accumulation, which may be associated with the increased CYP1A activity in liver and especially in muscle. However, when PBMCs were exposed to organic extracts obtained from sea bream muscle contaminated with B[a]P, a toxic, although modest effect was revealed, consisting in a significant decrease in cell glutathione levels without alterations in cell viability and DNA laddering. This suggests that consumption of sea breams from B[a]P contaminated waters might represent a risk for human health.

Estrogenic followed by anti-estrogenic effects of PCBs exposure in juvenil fish (Spaurus aurata).

Vitellogenin (Vtg) is a phospho-lipo-glycoprotein produced by oviparous animals in response to estrogen receptor (ER) binding. The presence of Vtg in juvenile and male fish liver and plasma has been used as biomarker to evaluate levels of environmental contaminants as dioxin and PCBs. Interaction of dioxins and PCBs with aryl hydrocarbon receptor (AhR) may affect reproduction by recruitment of estrogen receptor alpha (ERalpha). The aim of this study was to investigate the effects of PCB-126, a co-planar PCB prototypical AhR agonist, and of PCB-153, a non-coplanar PCB lacking dioxine-like activity, on Vtg expression in young fish (Spaurus aurata) after a 12 or 24h exposure to PCBs as well as 48h following PCBs removal. Vtg expression was evaluated by immunohistochemistry and by Western-blot analysis. Our results showed an increased Vtg expression following PCBs administration, with a maximum level after 12h of exposure to either PCB-126, PCB-153 or a mixture of both PCBs. Following this estrogenic activity, an anti-estrogenic activity was detected after 24h of incubation with PCB-126 (alone or mixed with PCB-153), suggested by a decrease in Vtg expression likely through AhR, as a consequence of a hypothetic defence mechanism to endogenous or exogenous ligands.

Proteasomes are not a target for doxorubicin in feline injection-site sarcoma.

The potent anti-cancer agent doxorubicin (DOX) induces apoptosis of rapidly proliferating cells by inhibiting cellular proteasomes. The aim of the present study was to determine whether DOX modulates the level of expression and function of proteasomes in feline injection-site sarcoma (FISS). Tissue extracts from primary sarcoma lesions and the related healthy subcutis of 18 cats affected by FISS were investigated. Nine of these cats had received neoadjuvant DOX treatment and nine cats did not receive this therapy. There was enhanced proteasome expression in FISS, but this was not affected by administration of DOX. This finding may account for the low clinical effectiveness of DOX therapy in FISS and provides the rationale for developing new therapeutic protocols aimed at achieving better proteasomal inhibition in FISS and other tumours that respond poorly to DOX therapy.

Protective effects of IRFI-042 in monensin induced neurotoxicity in chicks.

Monensin, a well known ionophore antibiotic, may cause severe damage in neuronal cells by altering Na+/K+-ATPase and Ca2+-ATPase. We investigated whether IRFI-042, a synthetic analogue of vitamin E, may block lipid peroxidation in neuronal cells and protect against monensin neurotoxicity in chicks. Monensin toxicity was induced in chicks by once-daily administration (150 mg/kg by oral gavages), for 8 days. Sham animals received a saline solution and were used as controls. All animals were randomized to receive either IRFI-042 (20 mg/kg) or its vehicle. Survival rate, brain lipid peroxidation, mRNA for neuronal and inducible nitric oxide synthases (nNOS and iNOS) and brain histological evaluations, including immunohistochemical expression of nNOS and iNOS were performed. Monensin administration decreased survival rate, induced behavioural changes, increased brain lipid peroxidation, reduced brain nNOS mRNA and immunostaining and enhanced iNOS mRNA and immunostaining in the brain in chicks. IRFI-042 significantly improved the survival rate and counteracted monensin-induced changes in chick brains. Our data suggest that monensin is responsible of neurotoxicity in chicks by inducing oxidative stress/lipid peroxidation and that IRFI-042 might represent a useful pharmacological approach to protect against the neuronal damage induced by this monovalent carboxylic ionophorous polyether antibiotic.

Immunohistochemical study of STAT3 expression in feline injection-site fibrosarcomas.

STAT3 (signal transducer and activator of transcription 3) is a cytoplasmic transcription factor that plays a role in the G1 to S phase cell-cycle transition and is induced by cytokines and growth factors. In the present study, the relation between histological grade and anti-STAT3 immunoreactivity was evaluated in 39 feline injection-site sarcomas treated surgically, 24 of the cats having received preoperative treatment with doxorubicin. Anti-STAT3 immunoreactivity was significantly lower in cases receiving preoperative doxorubicin, specifically with regard to nuclear localization. Moreover, STAT3 expression (intranuclear) was significantly correlated with mitotic activity in the animals that did not receive doxorubicin (P=0.019), and with differentiation score (P=0.009). STAT3 expression was correlated with the histological grade in both doxorubicin-treated and -untreated cats; in the treated cats, however, this correlation applied only to cytoplasmic STAT3 (P=0.018). Doxorubicin treatment induced a significant decrease in STAT3 expression (nuclear, P<0.0001; cytoplasmic, P=0.033) as compared with cases treated by surgery alone. These findings support existing evidence from human and experimental pathology on the potential role of STAT3 in oncogenesis and tumour progression.

Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of proteasome.

Mutations in SOD1 cause selective motor neuron degeneration in familial amyotrophic lateral sclerosis patients and transgenic mice overexpressing the mutant enzyme. Formation and accumulation of ubiquitinated aggregates in motor neurons are thought to be involved in the toxic gain of function of mutant SOD1. The present study shows that the accumulation of soluble and detergent-insoluble mutant SOD1 in spinal cord of symptomatic SOD1G93A transgenic mice is due to impaired degradation of mutant SOD1 rather than to increased transcript levels. This effect was accompanied by a decrease of constitutive proteasome levels and a concomitant increase of immunoproteasome in the spinal cord homogenate which resulted in overall unchanged proteasome activity. A decrease of constitutive proteasome occurred in the motor neurons of SOD1G93A mice at the presymptomatic stage and became remarkable with the progression of the disease. This provides further evidence for an involvement of proteasome impairment in the toxicity of mutant SOD1.

Immunohistochemical investigation of the nitrergic system in the taste organ of the frog, Rana esculenta.

We have studied by immunocytochemistry, the taste discs of the frog, Rana esculenta, with the aim of providing morphological and neurochemical data on the nitrergic system and of assessing the eventual presence of intrinsic neurons associated with the gustatory organs. In taste discs, antibodies against neuronal nitric oxide synthase (nNOS) revealed a positive immunoreaction in the taste receptor cell bodies and processes. The basal cells were also stained. All the fungiform papillae contained intragemmal nerve fibers showing nNOS immunoreactivity; these fiber were mainly located in the basal plexus. Immunoreactive nerve fibers were also visible at the periphery of the papilla-contacting ciliate cells, which form a ring around the taste disc. In conclusion, the findings obtained in this study suggest that the occurrence of nNOS-immunoreactivity in basal cells, taste cells and nerves might reflect a role for nitric oxide in taste mechanisms of Amphibia. The results may also sustain the physiological implication of NO as a molecule involved in the local target function of maintaining the taste bud mucosal integrity and in regulating the blood flow to the epithelium.

Effects of monensin on Na+/K(+)-ATPase and Ca(++)-AtPase activities in chick skeletal muscle and myocardium after subacute treatment.

Chicks were treated at 2 weeks of age with 4,15, 40, 100 and 150 mg/kg of monensin, an ionophore used for its anticoccidial and growth-promoting properties. In the present immunohistochemical study, the expressions and distribution of Na+/K(+)-ATPase and Ca(++)-ATPase were studied in myocardium and skeletal muscles (pectoral and quadriceps femoris). We detected an increase of Na+/K(+)-ATPase immunostaining with prominent staining of the sarcolemma and a slight increase of Ca(+)-ATPase with prominent staining of the sarcoplasma.

The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.

26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide.

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).

The unfolding of substrates and ubiquitin-independent protein degradation by proteasomes.

26S proteasomes are composed of a 20S proteolytic core and two ATPase-containing 19S regulatory particles. To clarify the role of these ATPases in proteolysis, we studied the PAN complex, the archaeal homolog of the 19S ATPases. When ATP is present, PAN stimulates protein degradation by archaeal 20S proteasomes. PAN is a molecular chaperone that catalyzes the ATP-dependent unfolding of globular proteins. If 20S proteasomes are present, this unfoldase activity is linked to degradation. Thus PAN, and presumably the 26S ATPases, unfold substrates and facilitate their entry into the 20S particle. 26S proteasomes preferentially degrade ubiquitinated proteins. However, we found that calmodulin (CaM) and troponin C are degraded by 26S proteasomes without ubiquitination. Ca(2+)-free native CaM and in vitro 'aged' CaM are degraded faster than the Ca(2+)-bound form. Ubiquitination of CaM does not enhance its degradation. Degradation of ovalbumin normally requires ubiquitination, but can occur without ubiquitination if ovalbumin is denatured. The degradation of these proteins still requires ATP and the 19S particle. Thus, ubiquitin-independent degradation by 26S proteasomes may be more important than generally assumed.

Immunohistochemical localization of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta).

The present immunohistochemical study provides the first evidence of the presence of calcium-binding proteins (CaBPs) in the epidermis of the earthworm Lumbricus terrestris (Annelida, Oligochaeta) a lower invertebrate. The entire epidermis was labelled for calmodulin which is in agreement with its ubiquitous occurrence. Immunopositivity for calbindin D28K was limited to mucous cells, while that for S-100 protein was present only in neuroendocrine-like small granular cells. Finally, labelling for parvalbumin was specifically present in the subcutaneous nerve plexus. S-100 protein is considered to be a marker of neuroendocrine cells, at least in lower invertebrates such as Annelida. Although calbindin D28K is considered to be a marker of these cells in vertebrates, the same function cannot be attributed in Lumbricus terrestris. However, we can conclude that S-100 protein, as a regulatory protein, is phylogenetically older than calbindin D28K. We assume that the latter has an autoregulatory function in secretory processes. In agreement with previous data, we suggest that small granular cells exert a paracrine action in osmoregulatory and secretory processes.

Distinct proteolytic processes generate the C and N termini of MHC class I-binding peptides.

Most of the MHC class I peptides presented to the immune system are generated during the course of protein breakdown by the proteasome. However, the precise role of the proteasome, e.g., whether this particle or some other protease generates the carboxyl (C) and amino (N) termini of the presented 8- to 10-residue peptides, is not clear. Here, we show that presentation on Db of ASNENMETM, a peptide from influenza nucleoprotein, and on Kb of FAPGNYPAL, a peptide from Sendai virus nucleoprotein, was blocked by the proteasome inhibitor, lactacystin. Using plasmid minigene constructs encoding oligopeptides of various lengths, we found that presentation of ASNENMETM from C-terminally extended peptides that contain this antigenic peptide plus three or five additional amino acids and presentation of FAPGNYPAL from a peptide containing FAPGNYPAL plus one additional C-terminal residue required the proteasome. In contrast, the proteasome inhibitor did not reduce presentation of cytosolically expressed ASNENMETM or FAPGNYPAL or N-terminally extended versions of these peptides, suggesting involvement of aminopeptidase(s) in trimming these N-extended variants. Accordingly, when the N termini of these 3N-extended peptides were blocked by acetylation, they were resistant to hydrolysis by cellular aminopeptidases and pure leucine aminopeptidase. Moreover, if introduced into the cytosol, Ag presentation of these peptides occurred to a much lesser extent than from their nonacetylated counterparts. Thus, the proteasome is essential for the generation of ASNENMETM and FAPGNYPAL peptides from the full-length nucleoproteins. Although it generates the C termini of these presented peptides, distinct aminopeptidase(s) can trim the N termini of these presented peptides to their proper size.

Affinity purification and characterization of protein gene product 9.5 (PGP9.5) from retina.

Protein gene product 9.5 (PGP9.5) is a cytosolic protein that is highly expressed in vertebrate neurons, which is now included in the ubiquitin C-terminal hydrolase subclass (UCH) on the basis of primary-structure homology and hydrolytic activity on the synthetic substrate ubiquitin ethyl ester (UbOEt). Some UCHs show affinity for immobilized ubiquitin, a property exploited to purify them. In this study we show that this property can also be applied to PGP9.5, since a protein has been purified to homogeneity from bovine retina by affinity chromatography on a ubiquitin-Sepharose column that can be identified with: (a) PGP9.5 with respect to molecular mass, primary structure and immunological reactivity; (b) the known UCHs with respect to some catalytic properties, such as hydrolytic activity on UbOEt, (which also characterizes PGP9.5), Km value and reactivity with cysteine and histidine-specific reagents. However, it differs with respect to other properties, e.g. inhibition by UbOEt and a wider pH range of activity.