Peroxisomes in digestive gland cells of the mussel Mytilus galloprovincialis Lmk. Biochemical, ultrastructural and immunocytochemical characterization.

The present study was undertaken because of the paucity of information on peroxisomes in molluscs and the increasing importance of these organisms as sensitive indicators of environmental pollution. Peroxisomes were identified by electron microscopy in all three main cell types of the digestive gland of the bivalve mollusc Mytilus galloprovincialis Lmk. They stained weakly with the alkaline diaminobenzidine reaction but showed distinct immunolabeling with an antibody against mammalian catalase by the postembedding protein A-gold procedure. In addition, mussel digestive gland peroxisomes were isolated by differential and metrizamide-density gradient centrifugation, and a 30-fold enrichment of catalase and a 20-fold enrichment of palmitoyl-CoA oxidase was obtained over the initial homogenate. By Western blotting, isolated peroxisomes crossreacted with antibodies to catalase and, furthermore, specific and prominent labeling of isolated peroxisomes was also demonstrated in thin sections incubated with anti-catalase antibodies. These observations establish that peroxisomes in molluscan digestive gland contain the peroxisomal marker enzymes catalase and acyl-CoA oxidase and that they can be labeled by cytochemical and immunocytochemical techniques. Further studies of alterations of molluscan peroxisomes by environmentally relevant xenobiotics are warranted.     

Optimized labeling of bone marrow mesenchymal cells with superparamagnetic iron oxide nanoparticles and <Emphasis Type="Italic">in vivo</Emphasis> visualization by magnetic resonance imaging

Background  

Stem cell therapy has emerged as a promising addition to traditional treatments for a number of diseases. However, harnessing the therapeutic potential of stem cells requires an understanding of their fate in vivo. Non-invasive cell tracking can provide knowledge about mechanisms responsible for functional improvement of host tissue. Superparamagnetic iron oxide nanoparticles (SPIONs) have been used to label and visualize various cell types with magnetic resonance imaging (MRI). In this study we performed experiments designed to investigate the biological properties, including proliferation, viability and differentiation capacity of mesenchymal cells (MSCs) labeled with clinically approved SPIONs.     

Chlamydia-like organisms in digestive and duct cells of mussels from the Basque coast.

Chlamydia-like organisms have been detected in digestive cells and duct cells of the digestive gland of mussels, Mytilus galloprovincialis, collected from the Basque coast. The organisms appeared as basophilic inclusion bodies within digestive cells and consisted of elongate initial reticulate bodies and previously undescribed condensed forms, interpreted as intermediate bodies. Of the 414 mussels examined by light microscopy, 5.31% showed this type of infection. A second type of chlamydia-like organism was found in nonciliated duct cells. The microorganisms were found mostly free in duct cells and large elongate reticulate bodies, intermediate condensing forms, and fully condensed elementary bodies were clearly distinguished. No serious histopathological or ultrastructural changes were observed in host cells but evidences of a possible localized metabolic damage within infected digestive cells is presented.     

Sublethal damage during cryopreservation of rainbow trout sperm

Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at -20 degreesC/min in a programmable cooler and thawed in a 25 degreesC water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. Copyright 1998 Academic Press.     

Consequences of keepingMytilus in the laboratory as assessed by different cellular condition indices

Mytilus galloprovincialis Lmk. were maintained in the laboratory for three months in a semicontinuous water flow system. Animals were fed a commercial filter-feeder food and sampled after 0, 21, 35, 49, 77, and 91 days. In order to establish whether laboratory conditions and the food used were deleterious to mussels, their health status was assessed by quantifying different histological parameters of the digestive gland tissue. It was concluded that mussels kept for more than 35 days under the described laboratory conditions showed signs of stress presumably caused by the reproductive state of the mussels investigated. The food used and the nutrition-related health status of the animals were adequate, as shown by transmission electron microscopical studies after the 91-day maintenance period. A stress response was also evoked by a 10-day starvation period, which was reflected by an increased proportion of type I and type IV digestive tubules, and a reduced “Mean Epithelial Thickness” (MET). Finally, the results demonstrate the sensitivity of quantitative histological diagnosis in comparison to subjective tubule grading procedures in the assessment of the degree of stress experienced by mussels.     

Analysis of silver binding nucleolar organizer regions in exfoliative cytology smears of potentially malignant and malignant oral lesions

Nucleolar organizer regions are nucleolar components that contain proteins that are stained selectively by silver methods; they can be identified as black dots throughout the nucleolus and are known as silver binding nucleolar organizer regions (AgNOR). The number of AgNOR is related to the cell cycle and the proliferative activity of the cells. We investigated AgNOR using exfoliative cytology smears of potentially malignant oral lesions. Eighty individuals were divided into four equal groups: healthy controls, oral leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma. The mean number of AgNOR in each study group gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. The proliferative index was increased in the oral premalignant and malignant patients compared to normal subjects. The mean AgNOR size gradually increased from control to oral leukoplakia to oral submucous fibrosis to oral squamous cell carcinoma. Spherical shaped AgNOR were most common in controls, whereas large, clustered and kidney shapes were most common in oral squamous cell carcinoma. Multiparameter analysis of AgNOR in oral exfoliative smears is a simple, sensitive and cost-effective method for differentiating premalignant from malignant lesions and can be used in conjunction with routine cytomorphological evaluation.     

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: a biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.