Immunolocalization of ubiquitin in degenerating insect flight muscle.

Ubiquitin was localized by immunofluorescence microscopy during post-mating histolysis of fibrillar flight muscle in female fire ants, Solenopsis spp. Normal muscles, as well as histolysing muscles from artificially inseminated and haemolymph-injected females contained ubiquitin in association with nuclei, Z-lines, myofilaments and mitochondria. However, the density of the ubiquitin immunoreaction was markedly increased in the nuclei, Z-lines and mitochondria of degenerating tissues 6, 12 and 24 h posttreatment. At these times the heaviest immunoreactivity for ubiquitin was seen in association with the nuclei, Z-lines and mitochondria. Immuno-controls, incubated in the absence of the primary antibody, showed no similar immunostaining. When insemination was preceded by the injection of actinomycin D, muscle degradation was significantly depressed after a 24-h period. Also, ubiquitin immunofluorescence was markedly reduced in tissues pre-treated with actinomycin D. These observations suggest that insemination increases the ubiquitination of specific myofibrillar proteins destined for degradation.

Superoxide formation preceding flight muscle histolysis in Solenopsis: fine structural cytochemistry and biochemistry.

In Solenopsis spp., muscle histolysis or breakdown is a normal process in females and is initiated in the flight muscles only immediately after a mating flight. Information regarding the presence of the oxyradical scavenging enzyme superoxide dismutase (SOD) and the formation of the radical oxygen intermediate superoxide (SO) during the early stages of flight muscle histolysis in this insect was investigated. In normal fibrillar flight muscles from control animals, SOD was immunolocalized to vesicular and tubular components of the sarcotubular system. Lanthanum tracer studies indicated that some of these SOD-positive structures might be tubulovesicles continuous with the extracellular space. Following the injection of virgin alates with experimental haemolymph obtained from artificially inseminated females, the membrane delimited elements of the sarcotubular system became increasingly swollen and dilated with time (from 60 to 120 minutes postinjection) with a concomitant decrease in SOD activity and an increase in oxyradical formation. Many similar vesicles were lanthanum-positive. SO was not seen in the sarcoplasmic vesicles and tubules of control insects. The biochemical quantification of SO release over a 2-hour period showed a marked increase in oxyradical formation following treatment with the experimental haemolymph in comparison to control insects. Also, the addition of superoxide dismutase depressed SO formation under these conditions. Despite the histochemical and biochemical changes seen in the muscles of experimental insects, by 2 hours post-treatment there was no evidence of muscle necrosis. From these studies on flight muscle histolysis/necrosis in Solenopsis it appears that the formation of oxyradicals might represent an early event in myopathogenesis and subsequent tissue involution. The generation of SO is more than likely to be associated with alterations in the normal structure, biochemistry and permeability of the biomembranes which delimit the sarcotubular system.

The electron microscopic immunolocalization of a copper-zinc superoxide dismutase in association with collagen fibers of periodontal soft tissues.

Periodontal soft tissues were cleaved from freshly extracted human teeth. Tissues were then prepared for subsequent biochemical and morphological studies according to the following plan: 1) immediate immersion in liquid nitrogen for the biochemical assay of superoxide dismutase (SOD); 2) immediate fixation prior to routine preparation for routine transmission electron microscopy; 3) immediate fixation prior to preparation for electron microscopic immunohistochemistry. Biochemical analysis showed that the human periodontal ligament contained about twice as much SOD activity as human skin (dermis), but considerably less enzyme activity than that seen in red blood cells. Interestingly, periodontal SOD activity appeared to decrease with age. Immunohistochemistry localized enzyme activity to the periphery of matrix collagen fibrils and to the glycocalyx of tissue fibroblasts. The pathophysiology of this enzyme regarding inflammatory diseases such as periodontitis is discussed.

Copper-zinc superoxide dismutase activity in normal and inflamed human dental pulp tissue.

Information regarding the presence of the free radical scavenging (inactivating, dismutating) enzyme superoxide dismutase in human dental pulp was sought. Free radicals, such as the superoxide anion radical (O2-) and the hydroxyl anion radical (OH.), are powerful biological oxidants produced by phagocytes during the normal tissue response to injury and infection. Also produced is hydrogen peroxide (H2O2), an aggressive oxygen species formed by the reaction of superoxide with itself, i.e., a dismutation in which one molecule of O2- is oxidized by the other. These three reactive oxygen intermediates serve as part of the normal host biological defense mechanism for the inactivation of microorganisms and the breakdown of their toxic products. Both normal and inflamed dental pulps were assayed for the presence of this enzyme. Superoxide dismutase activity was identified in the normal pulpal tissues. There was a slight decrease in activity with age. In the inflamed pulpal tissues, enzyme activity was markedly and significantly increased in comparison to that in the normal tissues. These observations indicate that human dental pulp possesses an endogenous defense mechanism designed to protect the tissue components (cells and matrix) from the toxic effects of the reactive oxygen intermediates. In this regard, the inflammatory response of this specialized and somewhat isolated (compartmentalized) tissue is not unlike that seen in other connective tissues.

An ultrastructural and immunohistochemical study of human dental pulp: identification of Weibel-Palade bodies and von Willebrand factor in pulp endothelial cells.

Special and specific immunohistochemical techniques as well as routine transmission electron microscopy were used to identify the presence of von Willebrand factor (vWF), a blood clotting factor essential to normal hemostasis, and Weibel-Palade bodies (WPB's), respectively, in the endothelial cells lining the blood vessels from both normal and inflamed human pulpal tissues. In human endothelial cells, WPB's are peculiar and specialized organelles which store vWF. All classes of blood vessels (capillaries, arterioles, arteries, venules, and veins) were vWF positive. The fine structural studies showed similar results with regard to the presence of WPB's. Interestingly, morphometric analyses conducted on the same tissues using either light or transmission electron microscopy showed that significantly more vWF-positive blood vessels were seen in the inflamed tissues. In agreement with the latter observation, transmission electron microscopy showed that more vascular endothelial cells contained WPB's in the inflamed tissues when compared with the normal tissues. From this it appears that during pulpal inflammation, the cascade of events associated with hemostasis may be activated with the increased synthesis and release of vWF by endothelial cells.

Changes in cytosolic calcium, bleb formation, and cell death in neural crest cells treated with isotretinoin and 4-oxo-isotretinoin.

Information regarding the cytopathologic mechanism of action of the retinoids [isotretinoin (IR) and 4-oxo-isotretinoin (4-OIR)] on neural crest cells (NCCs) in culture was sought. Those pathophysiologic alterations in cell metabolism studied were: cell blebbing (xieosis), free radical formation, cell viability, and cellular calcium homeostasis. Cells were treated with IR or 4-OIR in the presence of high (1.4 mM) and low (5.0 microM) levels of extracellular calcium ions. Recently developed techniques utilizing fluorescent molecular probes for calcium analyses, i.e., Fura 2AM, were used to study the effects of these drugs on the cytosolic calcium concentration of NCCs. The effects of IR and 4-OIR on NCC viability, [Ca++]int, were contrasted with the effects of certain sulfhydryl drugs (HgCl2, NEM, PCMBS) and calcium ionophores (ionomycin, A23187), agents known to perturb cell membranes, increase cytosolic calcium loads, and induce cell injury and subsequent cell death. Both retinoids were shown to induce an increase in the generation of superoxide radicals (SO) and increase the influx of calcium ions by the NCCs, thus increasing [Ca++]int by several hundred percent within 5 to 10 min. The liberation of SO was calcium dependent. These early effects were accompanied by an increase in cell blebbing activity. Also, a significant decrease in NCC viability was seen as early as 10 min after the addition of IR or 4-OIR to the incubation medium. 4-OIR proved to be the more potent of the two retinoids tested. The severity of these effects on NCC metabolism was dependent on medium calcium concentration with all changes being increased in the presence of the higher extracellular calcium levels. From the data presented it appears as though the retinoids cause a rapid elevation in cytosolic [Ca++]int possibly by purturbing the integrity of the cell membrane, denaturing membrane Ca-ATPase activity, or both. Retinoid-induced changes in membrane activity are evidenced by increased surface blebbing and superoxide formation. The prolonged elevation of intracellular [Ca++] may be directly related to depressed NCC viability and thus explain the known teratogenic effects of these drugs and their relationship to ectomesenchymal cell hypoplasia and craniofacial dysmorphogenesis.

Immunolocalization of secretory protein-I or chromogranin A in amphibian urinary bladder granular cell granules.

The presence of secretory protein-I (SP-I) or chromogranin A (CGA) in granules isolated from the granular cells of the amphibian urinary bladder epithelium was investigated using ultraimmunohistochemistry. Granules were isolated by cell fractionation using Percoll density gradients. SP-I was isolated and purified from bovine parathyroid glands. Antibodies were raised in rabbits and purified by affinity chromatography. Ultraimmunocytochemistry, employing the avidin-biotin-peroxidase (ABC-complex) procedure, was used to localize SP-I on thin sections of isolated granules. About 27% of the granules from control (-ADH) cells were SP-I+, while 51% of the granules fractionated from hormone treated (+ADH) cells were positive for this protein (p less than 0.0001). Accordingly, granules from ADH-treated cells also showed a significant (p less than 0.0001) increase in total protein.