Expression of iron-related proteins in feline and canine mammary gland reveals unexpected accumulation of iron.

Dysregulation of cellular iron homeostasis in human breast cancer is reflected by the altered expression of regulatory proteins. The expressions of iron-related proteins in the mammary glands of cats and dogs have not been assessed. We evaluated the expressions of ferritin, ferroportin, hepcidin and transferrin receptor 1 in benign and malignant mammary gland lesions in cats and dogs. Iron deposition was detected using Perls' Prussian blue staining. We found no major differences in the expression of iron-related proteins between benign and malignant mammary gland lesions in either cats or dogs; however, these species exhibited accumulation of iron in benign lesions. Our findings provide an explanation for the absence of higher iron requirements by tumor cells in these animals. Further investigation of local iron homeostasis in cats and dogs and differences in their physiology compared to human breast cancer is required.

Effects of the PPARα agonist WY-14,643 on plasma lipids, enzymatic activities and mRNA expression of lipid metabolism genes in a marine flatfish, Scophthalmus maximus.

Fibrates and other lipid regulator drugs are widespread in the aquatic environment including estuaries and coastal zones, but little is known on their chronic effects on non-target organisms as marine fish. In the present study, turbot juveniles were exposed to the PPARα model agonist WY-14,643 for 21 days by repeated injections at the concentrations of 5mg/kg (lo-WY) and 50mg/kg (hi-WY), and samples taken after 7 and 21 days. Enzyme activity and mRNA expression of palmitoyl-CoA oxidase and catalase in the liver were analyzed as first response, which validated the experiment by demonstrating interactions with the peroxisomal fatty acid oxidation and oxidative stress pathways in the hi-WY treatment. In order to get mechanistic insights, alterations of plasma lipids (free cholesterol, FC; HDL associated cholesterol, C-HDL; triglycerides, TG; non-esterified fatty acids, NEFA) and hepatic mRNA expression of 17 genes involved in fatty acid and lipid metabolism were studied. The exposure to hi-WY reduced the quantity of plasma FC, C-HDL, and NEFA. Microsomal triglyceride transfer protein and apolipoprotein E mRNA expression were higher in hi-WY, and indicated an increased formation of VLDL particles and energy mobilization from liver. It is speculated that energy depletion by PPARα agonists may contribute to a higher susceptibility to environmental stressors.

Morphologic, cytometric and functional characterization of the common octopus (Octopus vulgaris) hemocytes.

The hemocytes of Octopus vulgaris were morphologically and functionally characterized. Light and electron microscopy (TEM and SEM), and flow cytometry analyses revealed the existence of two hemocyte populations. Large granulocytes showed U-shaped nucleus, a mean of 11.6 µm±1.2 in diameter with basophilic granules, polysaccharide and lysosomic deposits in the cytoplasm. Small granulocytes measured a mean of 8.1 µm±0.7 in diameter, and have a round nucleus occupying almost the entire cell and few or not granules in the cytoplasm. Flow cytometry analysis showed that large granulocytes are the principal cells that develop phagocytosis of latex beads (rising up to 56%) and ROS after zymosan stimulation. Zymosan induced the highest production of both ROS and NO. This study is the first tread towards understanding the O. vulgaris immune system by applying new tools to provide a most comprehensive morpho-functional study of their hemocytes.

Activity of purine catabolism enzymes during the reproductive cycle of male and female brown trout (Salmo trutta).

In order to detect predicted changes in purine catabolism during the annual reproductive cycle of brown trout, we measured the activity of xanthine dehydrogenase, urate oxidase, and allantoinase. In kidney, only xanthine dehydrogenase was detected. In female liver and kidney, the activity of these enzymes was higher in May and decreased during vitellogenesis, with urate oxidase being undetectable in this period. In male liver, a similar variation pattern was found; however, in kidney, high activities were found in both May and December. These results suggest an influence of sex hormones in trout purine catabolism, especially in females.

Cytochemical localisation of lysosomal enzymes and acidic mucopolysaccharides in the salivary glands of Aplysia depilans (Opisthobranchia).

Three types of secretory cells were reported in the salivary glands of Aplysia depilans: granular cells, vacuolated cells and mucocytes. To improve the characterisation of these cells, cytochemical methods for the detection of lysosomal enzymes and acidic mucopolysaccharides were applied. In granular cells, acid phosphatase and arylsulphatase were present in small lysosomes and in some secretory granules. The secretory granules could have received these enzymes after fusion with the small lysosomes that were frequently found very close to them. These cells were not stained with colloidal iron because they do not contain acidic mucopolysaccharides. In vacuolated cells, acid phosphatase and arylsulphatase were detected in lysosomes but not in the secretory vacuoles. Colloidal iron staining revealed the presence of acidic mucopolysaccharides in the vacuoles and in the Golgi apparatus of these cells. In mucocytes, lysosomes were very rare, but the secretion of these cells was very rich in acidic mucopolysaccharides. The filamentous network within the secretory vesicles was completely covered with iron particles, but practically no particles were observed over the granular masses attached to the membrane of the vesicles. Iron particles were also found in the trans-face cisternae of the U-shaped Golgi stacks, but were not seen in the cis-face cisternae or in the rough endoplasmic reticulum.

The digestive cells of the hepatopancreas in Aplysia depilans (Mollusca, Opisthobranchia): ultrastructural and cytochemical study.

Digestive cells are the most abundant cell type in the digestive diverticula of Aplysia depilans. These are tall columnar or club shaped cells, covered with microvilli on their apical surface. A large number of endocytic vesicles containing electron-dense substances can be found in the apical zone, but the presence of many heterolysosomes of large diameter is the main feature of these cells. Glycogen particles and some lipid droplets were also observed. Peroxisomes with a circular or oval profile were common, but crystalline nucleoids were not detected in them, although a dense spot in the matrix was observed in a few cases. These organelles were strongly stained after cytochemical detection of catalase activity. The Golgi stacks are formed by 4 or 5 cisternae, with dilated zones containing electron dense material. Arylsulphatase activity was detected in the Golgi stacks and also in lysosomes. Cells almost entirely occupied by a very large vacuole containing a residual dense mass seem to be digestive cells in advanced stages of maturation. The observation of semithin and ultrathin sections indicates that these very large vacuoles are the result of a fusion among the smaller lysosomes. Some images suggest that the content of these large vacuoles is extruded into the lumen of the digestive diverticula.

Ultrastructural and cytochemical aspects of the basophilic cells in the hepatopancreas ofAplysia depilans(Mollusca, Opisthobranchia).

The basophilic cells ofAplysia depilanshave a pyramidal shape and a large nucleus usually located near the center or in the basal half of the cell. The nucleus possesses several clumps of condensed chromatin and a prominent nucleolus. The great profusion of rough endoplasmic reticulum cisterns in a major feature of these cells. Secretion granules are accumulated in the apical zone, and arylsulphatase was detected in some of them. In some basophilic cells a very substantial part of the cell volume was occupied by clear vacuoles, some of them reaching 9 mum. However, in other cells only a few vacuoles were observed. Probably the cells with just a few vacuoles are still young, and after a progressive accumulation, the vacuoles become abundant in old cells. The presence of a dark nucleus in the cells with a large number of vacuoles suggests that they are in a final stage of their life. Arylsulphatase was detected in the vacuoles and also in small secondary lysosomes containing substances in digestion. Bundles of tubules with 50 nm in diameter were found within some cisterns of rough endoplasmic reticulum. A cell fraction enriched in mannitol oxidase, extracted from the hepatopancreas of a terrestrial slug, consisted in very similar tubular structures. Using a histochemical method, mannitol oxidase was detected in the basophilic cells ofA. depilans, and it may be associated with the tubular structures of the endoplasmic reticulum. This is the first report of mannitol oxidase in opisthobranch molluscs. Almost spherical peroxisomes with a small nucleoid were abundant in these cells. The nucleoids presented a rectangular section, but a crystalline structure was not evident. The peroxisomes were stained after the cytochemical detection of catalase activity.

The peroxisomes of the hepatopancreas in two species of chitons

. This paper presents the first description of peroxisomes in polyplacophorans. As in other molluscs, the hepatopancreas of chitons is composed of basophilic and digestive cells. In the basophilic cells, the endoplasmic reticulum is abundant and several Golgi stacks can be observed. These cells also possess secretion granules and vacuoles with spherites. The digestive cells are mainly characterized by the presence of many food vacuoles. Several peroxisomes were observed in the basophilic cells of Acanthochiton crinita, most of them almost spherical. The matrix is filled with tubular structures and a crystalline nucleoid is also present in these organelles. In the digestive cells of A. crinita, peroxisomes are also almost spherical and possess two kinds of nucleoids. One of them presents a diamond shape and a bundle of tubular structures forms a second kind of nucleoid, which shows an elongated form. In Lepidochitona cinerea, the peroxisomes of basophilic cells are spherical or oval. Within the matrix, a cluster of dense rods and a prismatic nucleoid were observed. In the digestive cells of this species, almost spherical or oval peroxisomes are common, but they are smaller than the peroxisomes of the preceding cells. Nucleoids were not detected, but a few dense rods could be observed in the matrix. In both cell types of the two species, catalase activity was detected in the peroxisomal matrix. In addition, the elongated nucleoid of A. crinita digestive-cell peroxisomes and the nucleoid of L. cinerea basophilic-cell peroxisomes also present catalase activity.

Disruption of the nuclear gene encoding the 20.8-kDa subunit of NADH: ubiquinone reductase of Neurospora mitochondria.

The nuclear gene coding for the 20.8-kDa subunit of the membrane arm of respiratory chain NADH: ubiquinone reductase (Complex I) from Neurospora crassa, nuo-20.8, was localized on linkage group I of the fungal genome. A genomic DNA fragment containing this gene was cloned and a duplication was created in a strain of N. crassa by transformation. To generate RIP (repeat-induced point) mutations in the duplicated sequence, the transformant was crossed with another strain carrying an auxotrophic marker on chromosome I. To increase the chance of finding an isolate with a non-functional nuo-20.8 gene, random progeny from the cross were selected against this auxotrophy since RIP of the target gene will only occur in the nucleus carrying the duplication. Among these, we isolated and characterised a mutant strain that lacks the 20.8 kDa mitochondrial protein, indicating that this cysteine-rich polypeptide is not essential. Nevertheless, the absence of the 20.8-kDa subunit prevents the full assembly of complex I. It appears that the peripheral arm and two intermediates of the membrane arm of the enzyme are still formed in the mutant mitochondria. The NADH: ubiquinone reductase activity of sonicated mitochondria from the mutant is rotenone insensitive. Electron microscopy of mutant mitochondria does not reveal any alteration in the structure or numbers of the organelles.

Flow Cytometric Analysis of Molt-Related Changes in Hemocyte Type in Male and Female Penaeus japonicus.

Hemocyte cell suspensions obtained from male and female Penaeus japonicus were individually analyzed by flow cytometry through forward and side light-scatter parameters. The hemocyte cell suspensions were further characterized after cell sorting. This type of cell analysis has several advantages over microscopy techniques. After staining with phenoloxidase and peroxidase, the hemocytes were classified into the three classic categories of hyaline, semigranular, and granular cells. Significant cyclic differences were detected among the molting stages in both sexes. The hyaline cell population was predominant before and soon after the molt, decreasing over the intermolt. This decrease was, however, more prolonged in females. Thus, the hyaline cell population was dominant in stages B, D0, and D1 in males and only in stages B and D1 in females. Semigranular cells became predominant in females during the D0 stage.

Ultrastructural and cytochemical identification of peroxisomes in the ciliate Ichthyophthirius multifiliis.

The peroxisomes of the parasitic ciliate Ichthyophthirius multifiliis were studied, using ultrastructural and cytochemical techniques. In this ciliate most peroxisomes possess a circular or oval section less than 0.6 micron in diameter. However, some dumbbell-shaped and elongated peroxisomes could also be observed. These organelles were frequently associated with the mitochondria and were more abundant in the cell cortex than in the center of the ciliate. Small vesicles and dense nucleoids were usually present in the ultrathin sections of these peroxisomes. Peroxisomal vesicles and tubular structures were selectively impregnated with osmium tetroxide. Catalase was detected by cytochemical techniques in I. multifiliis peroxisomes.

Processing of food vacuoles in the parasitic ciliate Ichthyophthirius multifiliis after exit from the host.

The digestive cycle of the fish parasite Ichthyophthirius multifiliis (Ciliophora) can be divided into three main stages. During stage A the vacuoles are not yet condensed. This stage can be subdivided into an early phase in which food vacuoles contain almost intact fish cells and a later phase in which dense material accumulates at the periphery of the vacuoles. At stage B, food vacuoles attain a very high density, and at stage C the vacuole expands when the membrane pulls away from a condensed mass of substances in digestion. After its exit from the host the parasite encysts and divides, but new food vacuoles are not formed during this phase of the life cycle. Type A vacuoles are the first to disappear after exit from the host. The percentage of type B vacuoles increases during the first few hours of free life, decreasing later when the percentage of type C vacuoles starts to increase. At the end of the division phase, type C vacuoles are the most common. Food-vacuole egestion was observed only 20 h after exit from the host. At the theront stage, food vacuoles were not evident, but small vacuoles with acid phosphatase activity were observed.

Enzyme cytochemistry of the alveolar sacs and golgian-like cisternae in the ciliate Ichthyophthirius multifiliis.

In the cell cortex of the parasitic ciliate Ichthyophthirius multifiliis different kinds of cisternae were observed: the alveolar sacs, thick membrane cisternae and the endoplasmic reticulum. The thick membrane cisternae possess coated dilated rims and sometimes could be observed close to the endoplasmic reticulum. Using cytochemical techniques acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in the thick membrane cisternae and in the alveolar sacs of trophozoites. In the endoplasmic reticulum acid phosphatase activity was not detected and only very small amounts of thiamine pyrophosphatase and nucleoside diphosphatase reaction product were observed. After exit from the host, a reduction in acid phosphatase activity was evident in the alveolar sacs. At theront stage acid phosphatase activity is absent from these structures. However, high thiamine pyrophosphatase and nucleoside diphosphatase activities remain in the alveolar sacs during the whole life cycle. On the other hand, acid phosphatase, thiamine pyrophosphatase and nucleoside diphosphatase activities were detected in thick membrane cisternae of theronts. Based on the morphological aspects and enzymatic content the thick membrane cisternae of the cell cortex are designated as golgian-like cisternae. The cytochemical results point out a relationship between the alveolar sacs and the Golgi complex.

Ultrastructural and cytochemical study of food vacuoles and primary lysosomes in Ichthyophthirius multifiliis (Ciliophora).

In the trophozoites of the fish parasite Ichthyophthirius multifiliis many food vacuoles can be found in different stages of the digestive cycle. Newly formed food vacuoles are large and contain remains of the fish cells that have been phagocyted by the ciliate. In some food vacuoles it was possible to observe evaginations in the membrane. Primary lysosomes of I. multifiliis are small dense bodies with about 0.2 µn in diameter. Enzyme cytochemistry revealed the presence of acid phosphatase and aryl sulphatase in food vacuoles and primary lysosomes, but trimetaphosphatase was not detected. Since aryl sulphatase activity was observed at pH 4.5 and 6.0 it is possible that two isoenzymes, A and B, are present.