Two degradation pathways of endoplasmic reticulum in fish hepatocytes

Summary— Zebrafish hepatocytes respond to stimulation with estradiol 17β (E2) with an extreme enlargement of the endomembrane system, especially the endoplasmic reticulum (ER), and when stimulation is stopped with a rapid degradation of enlarged endomembranes. Two pathways for degradation of ER were studied: a) the autophagy which was evaluated by stereological measurement; and b) the activity of cytosolic phospholipase B (PL-B) measured by a titration method. After a 30-day treatment with E2 a six-fold increase of the surface density of ER was accompanied by an increase of both autophagy and PL-B activity. 2 days after stopping the stimulation with E2 the ER vesiculated and its surface density decreased to the half value. Interestingly, at the same time autophagic vacuoles (AV) almost disappeared from hepatocytes, while the activity of PL-B reached its maximum at which it persisted for a further 4 days. After 4–6 days without E2 the cistern of ER became flattened again and new AVs reappeared. The data suggest that the regulation mechanisms of endomembrane degradation by PL-B and autophagy do not depend on each other and also that the appearance of AV is strongly related to the shape of ER.     

Correlation between suckling-induced changes in the ultrastructure of mammotrophs and prolactin release

Effects of suckling on the structure of mammotrophs and the release of prolactin, were studied in rats on the 10th day of lactation with the use of electron microscopy and radioimmunoassay techniques. Nursing animals were separated from their young for 8 hr and subsequently united and permitted to nurse for 1, 5, 15, 30 min; or 1, 2 and 4 hr. Blood samples were obtained prior to and throughout the suckling interval and pituitary glands were processed for electron microscopy. Control animals consisted of normal lactating females and animals separated from their young for 8 hr. Normally lactating controls had high prolactin serum levels (501 +/- 95 ng/ml) and synthetically active appearing mammotrophs. An 8 hr separation from the pups induced a dramatic lowering of serum prolactin (32 +/- 5 ng/ml), an increase in secretory granule storage, and a great dilation of rough endoplasmic reticulum (RER) cisternae. Five min of renewed suckling resulted in a rise of plasma prolactin levels (605 +/- 183 ng/ml) which remained high thereafter. The major ultrastructural changes observed during the first 30 min of suckling were as follows: 1) at 1 min, the RER became cmone?); 2) AT 5 MIN, AND MUCH MORE OBVIOUSLY AT 15 AND 30 MIn, a massive discharge of secretory granules was observed; and 3) at 15 min, the collapsed RER underwent transformation for 1,2 and 4 hr) induced new hormone synthesis as suggested by the presence of hypertrophied Golgi elements and numerous immature granules. This was accompanied by a new transformation of the RER from the vesicular into a lamellar form now consisting of very slender cisternae lined with numerous ribosomes, presumably involved in the renewal of the synthetic process. The morphologic findings described correlate well with the time table of prolactin release. In addition, the dramatic early changes in the structure of the RER suggest a possible involvement of this organelle in the storage and release of a proposed rapidly releasable pool of prolactin.     

Porphyromonas gingivalis and Treponema denticola Exhibit Metabolic Symbioses

Porphyromonas gingivalis and Treponema denticola are strongly associated with chronic periodontitis. These bacteria have been co-localized in subgingival plaque and demonstrated to exhibit symbiosis in growth in vitro and synergistic virulence upon co-infection in animal models of disease. Here we show that during continuous co-culture a P. gingivalis:T. denticola cell ratio of 6∶1 was maintained with a respective increase of 54% and 30% in cell numbers when compared with mono-culture. Co-culture caused significant changes in global gene expression in both species with altered expression of 184 T. denticola and 134 P. gingivalis genes. P. gingivalis genes encoding a predicted thiamine biosynthesis pathway were up-regulated whilst genes involved in fatty acid biosynthesis were down-regulated. T. denticola genes encoding virulence factors including dentilisin and glycine catabolic pathways were significantly up-regulated during co-culture. Metabolic labeling using ¹³C-glycine showed that T. denticola rapidly metabolized this amino acid resulting in the production of acetate and lactate. P. gingivalis may be an important source of free glycine for T. denticola as mono-cultures of P. gingivalis and T. denticola were found to produce and consume free glycine, respectively; free glycine production by P. gingivalis was stimulated by T. denticola conditioned medium and glycine supplementation of T. denticola medium increased final cell density 1.7-fold. Collectively these data show P. gingivalis and T. denticola respond metabolically to the presence of each other with T. denticola displaying responses that help explain enhanced virulence of co-infections.     

Phenethyl isothiocyanate potentiates anti‐tumour effect of doxorubicin through Akt‐dependent pathway

The present study aims to investigate the in vivo and in vitro anti‐tumour properties of phenethyl isothiocyanate (PEITC) alone and in combination with doxorubicin (Dox). The anti‐tumour activity was evaluated in vitro by MTT assay using cultured human breast cancer cell line (MCF‐7) and human hepatoma cell line (HepG‐2) cell lines. In vivo, Ehrlich solid tumour model was used. Tumour volume, weight and antioxidant parameters were determined. Immunohistochemistry analysis for active (cleaved) caspase‐3 was also performed. We tested the effect of PEITC treatment on pAkt/Akt ratio, NF‐κB p65 DNA binding activity and caspase‐9 enzyme activity in both MCF‐7 and HepG‐2 cell lines. Effect of PEITC treatment on cell migration was assessed by wound healing assay. PEITC and/or Dox treatment significantly inhibited solid tumour volume and tumour weight when compared with control mice. PEITC treatment significantly reduced oxidative stress caused by Dox treatment as indicated by significant increase in total antioxidant capacity and decrease in malondialdehyde level. Microscopic examination of tumour tissues showed a significant increase in active (cleaved) caspase‐3 expression in PEITC and/or Dox treated groups. PEITC showed a dose‐dependent inhibition of MCF‐7 and HepG‐2 cellular viability. PEITC inhibited Akt and NF‐κB activation and increased caspase‐9 activity in a dose‐dependent manner. PEITC treatment effectively inhibited both MCF‐7 and HepG‐2 cell migration. We can conclude that PEITC acts via multiple molecular targets to elicit anti‐carcinogenic activity. PEITC/Dox combination therapy might be a potential novel strategy, which may benefit patients with breast and liver cancers. Copyright © 2015 John Wiley & Sons, Ltd.     

Implication of protein kinase R Gene quantification in hepatitis C Virus Genotype 4 induced Hepatocarcinogenesis

ABSTRACT: BACKGROUND: Protein kinase RNA (PKR-regulated) is a double-stranded RNA activated protein kinase whose expression is induced by interferon. The role of PKR in cell growth regulation is controversial, with some studies supporting a tumour suppressor function and others suggesting a growth-promoting role. However, it is possible that the function of PKR varies with the type of cancer in question. METHODS: We report here a detailed study to evaluate the function of PKR in hepatitis C virus genotype 4 (HCV-4) infected patients. PKR gene was quantitated in HCV related malignant and non-malignant liver tissue by RT-PCR technique and the association of HCV core and PKR was assessed. RESULTS: If PKR functions as a tumour suppressor in this system, its expression would be higher in chronic hepatitis tissues. On the contrary our study demonstrated the specific association of HCV-4 with PKR expressed in hepatocellular carcinoma (HCC) tissues, leading to an increased gene expression of the kinase in comparison to chronic hepatitis tissues. This calls into question its role as a tumour suppressor and suggests a positive regulatory role of PKR in growth control of liver cancer cells. One limitation of most of other studies is that they measure the levels rather than the quantitation of PKR gene. CONCLUSION: The findings suggest that PKR exerts a positive role in cell growth control of HCV-4 related HCC, obtaining a cut-off value for PKR expression in liver tissue provides the first evidence for existence of a viral activator of PKR. Virtual Slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1267826959682402.     

Einfluß der natürlichen Cytokinine vonPinus sylvestris L. und anderer Wuchsstoffe auf das Myzelwachstum vonBoletus edulis var.pinicolus Vitt.

Ohne Zusammenfassung     

Supplementary CaCl2 ameliorates wheat tolerance to NaCl

Nine-day-old seedlings of two wheat cultivars (Misr1 and Sakha93) were treated with NaCl at 75, 150 and 225 mM for 15 days with or without the presence of 10 mM CaCl₂. All concentrations of NaCl led to significant decreases in fresh and dry weights of only Sakha93; however, Misr1 seemed to be affected only at the highest concentration. Nonetheless, growth parameters of both cultivars under normal conditions were most likely similar. On the other hand, lipid peroxides (as MDA) and H₂O₂ were greatly accumulated particularly in Sakha93; significant increases were detected in Misr1 treated only at 225 mM. Also, all concentrations of NaCl decreased GSH content in Sakha93; nevertheless, there were no great differences among both cultivars under normal conditions. On the other hand, the activities of the enzymatic antioxidants, GR, GST, CAT and POD were unaffected in Misr1 by all concentrations but inhibited in Sakha93. AOX responded differently to NaCl, there were decreases in Misr1 by 75 and 225 mM and in Sakha93 by 75 and 150 mM. However, the application of CaCl₂ alleviated the impacts of NaCl; there was a retraction in growth reduction in Misr1 to reach most likely those of the control. In addition, the accumulated MDA and H₂O₂ were greatly counterbalanced. On the contrary, the decreased GSH contents seemed unrecovered in Sakha93 in spite of the alleviations in magnitudes. Moreover, there were recoveries in the activities of GR and POD in Sakha93; nevertheless, GST and CAT activities remained significantly inhibited. These findings suggest that Misr1 is a more tolerant cultivar to NaCl than Sakha93. Moreover, the results reveal that ROS scavenging is efficient and became more inducible in the less susceptible than in the more susceptible cultivar. The response of AOX appeared to coincide with antioxidants so that the damage which was inflicted by NaCl can be ameliorated by over-expression of antioxidants especially with the presence of CaCl₂.     

Specific effect of 5,6-dihydroxytryptamine on the monoamine fluorophore of the frog's gustatory cells

Summary A specific formaldehyde-induced yellow fluorescence, suggesting the presence of serotonin-like monoamine has been demonstrated in the gustatory cells of the frog.The fungiform papillae of frogs were examined fluorescence-histochemically after intraperitoneal injection of 5,6-dihydroxytryptamine. The results indicated that the fluorophore of gustatory cells was affected selectively by the drug injection: the yellow fluorescence was transiently enhanced 3 hours after the drug injection, thereafter being reduced rapidly. The effect of 5,6-dihydroxytryptamine was long-lasting with the reduction of the yellow fluorophore persisting at least for the experimental duration of 14 days. A single injection of 6-hydroxydopamine induced a complete depletion of noradrenaline fluorescence from adrenergic nerve terminals, while the fluorescence of gustatory cells was not affected by a high dose of the drug.The present results with pharmacologic treatments further support the view that the gustatory cell of the frog contains a serotonin-like monoamine.