The Ultrastructure of the Gastrodermal Gland Cells in the Freshwater Planarian Dugesia gonocephala s.l.

Light and transmission electron microscopy have been used to study the gastrodermal gland cells of the triclad Dugesia gonocephala s.l. The events involved in the ultrastructural transformation and the secretion process in these cells were followed at four different stages in both fasted and fed animals. During the feeding stage their secretory granules are directly discharged into the intestinal lumen by means of a secretion process of the holocrine type that is described in this paper. It is suggested that such secretions contribute to extracellular digestion and that disintegration of the gland cells is accompanied by a differentiation of neoblasts into new gland cells, reflecting a turnover of gland cells during the triclad digestive stages.     

Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells

Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42 degrees C in CHO and 34 degrees C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45 degrees C in CHO and 37 degrees C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5 mM canavanine or 5 mM S-aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 microM cadmium sulfate or 100 microM sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.     

Purification of Potato Leaf Plasma Membrane Protein pp34, a Protein Phosphorylated in Response to Oligogalacturonide Signals for Defense and Development

A potato (Solanum tuberosum L.) plasma membrane protein called pp34, the only known example of a plasma membrane protein that is phosphorylated specifically in response to defined Oligogalacturonide signals in plants, has been purified to apparent homogeneity. Polyclonal antibodies raised in rabbits against the purified pp34 protein immunoprecipitated a single thiophosphorylated protein species from potato plasma membranes, as analyzed by two-dimensional denaturing electrophoresis and fluorography. The pp34 antibodies also recognized a single protein in tomato (Lycopersicon esculentum L.) membranes that is thiophosphorylated in response to Oligogalacturonide elicitors, as demonstrated by western blotting and specific immunoprecipitation. These experiments confirm the identity of the tomato membrane protein as a pp34 homolog and establish the high monospecificity of the pp34 antibodies. This will permit further investigation of the role of protein phosphorylation in oligouronide signaling for defensive genes in potato and tomato plants.     

Myotubes from transgenic mdx mice expressing full-length dystrophin show normal calcium regulation.

A lack of dystrophin results in muscle degeneration in Duchenne muscular dystrophy. Dystrophin-deficient human and mouse muscle cells have higher resting levels of intracellular free calcium ([Ca2+]i) and show a related increase in single-channel open probabilities of calcium leak channels. Elevated [Ca2+]i results in high levels of calcium-dependent proteolysis, which in turn increases calcium leak channel activity. This process could initiate muscle degeneration by further increasing [Ca2+]i and proteolysis in a positive feedback loop. Here, we tested the direct effect of restoration of dystrophin on [Ca2+]i and channel activity in primary myotubes from mdx mice made transgenic for full-length dystrophin. Transgenic mdx mice have been previously shown to have normal dystrophin localization and no muscle degeneration. Fura-2 calcium measurements and single-channel patch recordings showed that resting [Ca2+]i levels and open probabilities of calcium leak channels of transgenic mdx myotubes were similar to normal levels and significantly lower than mdx littermate controls (mdx) that lack dystrophin. Thus, restoration of normal calcium regulation in transgenic mdx mice may underlie the resulting absence of degeneration.     

Heterokaryon myotubes with normal mouse and Duchenne nuclei exhibit sarcolemmal dystrophin staining and efficient intracellular free calcium control.

Duchenne and mdx muscle tissues lack dystrophin where it normally interacts with glycoproteins in the sarcolemma. Intracellular free calcium ([Ca2+]i) is elevated in Duchenne and mdx myotubes and is correlated with abnormally active calcium-specific leak channels in dystrophic myotubes. We fused Duchenne human and normal mouse myoblasts and identified heterokaryon myotubes by Hoechst 33342 staining to measure the degree to which dystrophin introduced by normal nuclei could incorporate throughout the myotube at the sarcolemma and restore normal calcium homeostasis. Dystrophin expression in myotubes was determined by immunofluorescence and confocal laser scanning microscopy. Dystrophin was expressed at the sarcolemma in normal mouse and heterokaryon myotubes, but not in Duchenne myotubes. In heterokaryons, extensive dystrophin localization occurred at the sarcolemma even where only Duchenne nuclei were present, indicating that dystrophin does not exhibit nuclear domains. Heterokaryon, normal mouse and Duchenne myotube [Ca2+]i was measured using fura-2 and fluorescence ratio imaging. Heterokaryon and normal mouse myotubes were found to maintain similar levels of [Ca2+]i. In contrast, Duchenne myotubes had significantly higher [Ca2+]i (p < 0.001). Furthermore, the ability of heterokaryons to maintain normal [Ca2+]i did not depend on greater numbers of normal nuclei than Duchenne being present in the myotube. These results support the view that dystrophin expression in heterokaryons allows for efficient control of [Ca2+]i.     

Chemical composition and larvicidal activity of essential oils of three Artemisia species

Aedes aegypti L. (Diptera: Culicidae) is a vector for serious diseases in tropical regions. This pest is mainly controlled by commercial larvicides but the application of such products has led to environmental problems. Essential oils (EO) have been consistently reported as molecules with insecticidal activity and can be used to produce more environmentally friendly larvicides in the control of A. aegypti. In this study, the larvicidal effect of essential oils (EO) from the leaves of three Artemisia species was evaluated against A. aegypti. The oils were obtained from steam distillation and their chemical composition was determined by gas chromatography–mass spectrometry. The EO of Artemisia camphorata was the most active in the screening bioassay and presented LC₅₀ and LC₉₅ of 64.95 and 74.18 μg ml⁻¹, respectively. In addition, we found that germacrene D-4-ol was the constituent responsible for the toxicity of this EO. Artemisia camphorata EO and its major constituent, germacrene D-4-ol, are promising for the development of natural larvicides against A. aegypti.     

Histochemical study of glycoconjugates in the epididymis of the hamster (Mesocricetus auratus)

Summary The glycoconjugates of hamster epididymis were investigated with conventional and lectin histochemistry. A zone of the caput epididymis, with particular histochemical characteristics, has been differentiated. β-Elimination in combination with lectins was used to establish the presence and distribution of N- and O-linked glycoconjugates. The epithelium, spermatozoa and the intertubular matrix were rich in glycoconjugates. The Golgi apparatus and stereocilia of the principal cells were intensely positive with HPA, PNA and SBA lectins. β-limination indicated that these cells contained abundant O-linked glycoconjugates. Apical and clear cells presented a common lectin affinity; their reactivities towards WGA and UEA-I were very positive. These cells probably contain abundant N-glycoconjugates. The spermatozoa were stained by periodic acid-Schiff (PAS) and by all the lectins (especially in the acrosome), except by those with an affinity for α-l-fucosyl residues; the most intense reaction was found with HPA, WGA, PNA and SBA. Changes in the sperm lectin binding along the ductus were observed: sperm flagellum abruptly acquired WGA and PNA labelling from the posterior caput, and HPA reactivity was negative only in the zone between the caput and the corpus.     

Intracellular pH controls the development of new potassium conductance after fertilization of the sea urchin egg

Fertilization of the sea urchin egg leads to a sequence of changes at the egg surface and the interior cytoplasm. Among these changes are the transient elevation of internal calcium levels, alkalization of the cytoplasm and development of new K⁺-conductance. In the series of experiments reported here, we separate the effects on potassium activation of the calcium release and the rise in the intracellular pH. The development of new K⁺-conductance was dependent on alkalization of the egg cytoplasm, and not on a rise of internal calcium levels. The effects of 2,4-dinitrophenol, N-ethylmalemide, antimycin A and oligomycin suggest that the maintenance of the alkaline internal pH of fertilized eggs appears to be dependent on membrane ATPase activity.     

Studies on plating efficiency and estimation of viability of suspensions of Paracoccidioides brasiliensis yeast cells

Mild sonication was used to obtain single cell suspensions of Paracoccidioides brasiliensis. These cells were intact by microscopic criteria. Direct cell counts in a given inoculum and colony formation on various media were used to determine plating efficiency. Sonicated and nonsonicated cell suspensions were used to study plating efficiency and to estimate viability by means of vital dyes. Methylene blue, Erythrosin B, and Janus green were unreliable when used with P. brasiliensis, but vital dyes were accurate when tested with Candida albicans.Acridine orange gave more meaningful results of viability. Estimates of viability, however, changed significantly as a result of relatively minor alterations in the composition of the suspending medium.In initial experiments, the plating efficiency of P. brasiliensis was dismally low. It descended abruptly with increasing dilution of inoculum. Efficiency was much improved if horse serum was added to brain heart infusion plates or if glucose glycine yeast extract (GGY) plates were incubated at room temperature and mycelial colonies were counted. With the technique we report, current plating efficiency of sonicated suspensions is of the order of 25 %. Our results and procedures have an important bearing upon those studies concerned with in vitro killing of P. brasiliensis in suspensions or with isolating this fungus from clinical or environmental specimens.