Estimation of rRNA synthesis and degradation rates in senescing wheat leaves

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.     

Characterization of glucocorticoid inhibition of antigen-induced inositolphosphate formation by rat basophilic leukemia cells: possible involvement of phosphatases.

The suppressive effect of glucocorticoids (GC) upon antigen-induced phosphatidylinositol phospholipase C (PI-PLC) activity and inositol phosphate formation by rat basophilic leukemia cells (RBL-2H3) has been characterized. Addition of antigen for a period of 1-30 min enhanced production of [3H]inositol monophosphate (IP1), inositol 1,4-bisphosphate (IP2) and inositol 1,4,5-trisphosphate (IP3) by about 5-10-fold. Pretreatment with hydrocortisone (HC) reduced formation of the various inositol phosphates (IPs) and degradation of phosphatidylinositol 4,5-bisphosphate (PIP2) by an average of 50%. Maximal inhibition of hydrolysis of PIP2 and reduction in stimulation of IP3 formation was reached after 4 h of preincubation with 2.10(-6) M of HC. Cycloheximide and RU486, a GC receptor antagonist, completely prevented the inhibitory effect of HC on IP formation. Other GC, dexamethasone (DEX) and triamcinolone (each at 2.10(-7) M) markedly suppressed antigen induced IP3 production, while aldosterone and sex steroids such as estradiol and progesterone (each at 2.10(-6) M) were virtually inactive. Antigen-stimulated phosphorylation of a 18 kDa and other proteins was inhibited by about 60% following pretreatment with the GC. This inhibition was in turn prevented by cycloheximide. DEX also doubled the activity of cellular acid phosphatase activity. The results suggest that the inhibitory effect of GC is specific, receptor-mediated, dependent on protein synthesis and possibly mediated by protein phosphatase activity.     

DNA repair and cell cycle checkpoint defects in a mouse model of ‘BRCAness’ are partially rescued by 53BP1 deletion

‘BRCAness’ is a term used to describe cancer cells that behave similarly to tumors with BRCA1 or BRCA2 mutations. The BRCAness phenotype is associated with hypersensitivity to chemotherapy agents including PARP inhibitors, which are a promising class of recently-licensed anti-cancer treatments. This hypersensitivity arises because of a deficiency in the homologous recombination (HR) pathway for DNA double-strand break repair. To gain further insight into how genetic modifiers of HR contribute to the BRCAness phenotype, we created a new mouse model of BRCAness by generating mice that are deficient in BLM helicase and the Exo1 exonuclease, which are involved in the early stages of HR. We find that cells lacking BLM and Exo1 exhibit a BRCAness phenotype, with diminished HR, and hypersensitivity to PARP inhibitors. We further tested how 53BP1, an important regulator of HR, affects repair efficiency in our BRCAness model. We find that deletion of 53BP1 can relieve several of the repair deficiencies observed in cells lacking BLM and Exo1, just as it does in cells lacking BRCA1. These results substantiate the importance of BRCAness as a concept for classification of cancer cases, and further clarify the role of 53BP1 in regulation of DNA repair pathway choice in mammalian cells.     

Co-action between phytochrome B and HY4 in Arabidopsis thaliana

A combination of physiological and genetic approaches was used to investigate whether phytochromes and blue light (BL) photoreceptors act in a fully independent manner during photomorphogenesis of Arabidopsis thaliana (L.) Heynh. Wild-type seedlings and phyA, phyBand hy4 mutants were daily exposed to 3 h BL terminated with either a red light (R) or a far-red light (FR) pulse. In wild-type and phyA-mutant seedlings, BL followed by an R pulse inhibited hypocotyl growth and promoted cotyledon unfolding. The effects of BL were reduced if exposure to BL was followed by an FR pulse driving phytochrome to the R-absorbing form (Pr). In the wild type, the effects of R versus FR pulses were small in seedlings not exposed to BL. Thus, maximal responses depended on the presence of both BL and the FR-absorbing form of phytochrome (Pfr) in the subsequent dark period. Impaired responses to BL and to R versus FR pulses were observed in phyB and hy4 mutants. Simultaneous irradiation with orange light indicated that BL, perceived by specific BL photoreceptors (i.e. not by phytochromes), required phytochrome B to display a full effect. These results indicate interdependent co-action between phytochrome B and BL photoreceptors, particularly the HY4 gene product. No synergism between phytochrome A (activated by continuous or pulsed FR) and BL photoreceptors was observed.Abbreviations BL blue light - D darkness - FR far-redlight - FRc continuous FR - Pfr FR-absorbing form of phytochrome - Pfr/P proportion of phytochrome as Pfr - phyA phytochrome A - phyB phytochrome B - R red light - WT wild type We thank Professors R.E. Kendrick and M. Koornneef (Wageningen Agricultural University, The Netherlands), Professor J. Chory (Salk Institute, Calif., USA) and the Arabidopsis Biological Resource Center (Ohio State University, Ohio, USA) for their kind provision of the original seed batches. This work was financially supported by CONICET, Universidad de Buenos Aires (AG 040) and Fundación Antorchas (A-12830/1 0000/9)     

Hydrocortisone inhibits antigen-induced rise in intracellular free calcium concentration and abolishes leukotriene C4 production in leukemic basophils

Antigenic stimulation of rat basophilic leukemia cells (RBL-3H3) elevates intracellular free Ca2+ concentration ([Ca2+]i) and induces production of leukotriene C4 (LTC4). This model was used to examine the role of Ca2+ in LTC4 formation, and inhibition by hydrocortisone (HC). HC, at a physiological concentration (2 x 10(-7) M), selectively prevented the stimulatory effect of the antigen on LTC4 production whereas the response to calcium ionophore (A23187) remained unimpaired. The inhibition by HC was time-dependent: half maximal response was reached at 2 hour and maximal response at 3 hours. Addition of arachidonic acid (3 micrograms/ml) did not overcome the inhibitory action of HC. An elevated [Ca2+]i is known to be essential for the activation of both 5-lipoxygenase and phospholipase A2. The stimulatory effect of the antigen on LTC4 production was abolished when the cells were incubated in Ca2+-deficient medium. Likewise, calcium ionophore stimulation shows dependence on extracellular Ca2+. Half maximal stimulation by the antigen and calcium ionophore was observed at external Ca2+ concentration of 150 microM and 40 microM respectively. Treatment with HC largely prevented the antigen-induced rise in [Ca2+]i, measured by Quin 2. In addition, HC reduced by 70% the accumulation of 45Ca2+ induced by the antigen. Collectively, these results demonstrate for the first time that HC reduces antigen-induced elevation of [Ca2+]i, and this may be associated with the inhibitory action of HC on LTC4 formation. This property could be partly responsible for the antiallergic and antiinflammatory activities of HC.     

Thioacetamide induced liver damage in zebrafish embryo as a disease model for steatohepatitis

Summary Steatohepatitis has recently been increasing as a cofactor influencing the progression of fibrosis, cirrhosis, adenoma and carcinoma in liver; however, the mechanisms by which it contributes to liver injury remain uncertain. We induced steatohepatitis in zebrafish embryos using thioacetamide (TAA). TUNEL assay revealed significant increasing of apoptosis in liver after 5 days post fertilization and the increasing of apoptosis was observed to be associated with the up-regulation of apoptotic genes such as, bad, bax, P-38a, caspase-3 and 8, and JNK-1. Histological sections by oil red O stain showed the accumulation of fatty droplets which causes the pushing of the nucleus towards one side. Up-regulation of steatosis markers such as, ACC, adiponectin, PTL, CEBP- and , SREBP-1 was also observed. Furthermore, the elevation of glutathione peroxidase in TAA treated embryos indicated that TAA induces lipid peroxidation which leads to causes liver damage. Zebrafish has already been considered as a good human disease model and in this context; TAA-treated zebrafish may serve as a good animal model to study the molecular pathogenesis of steatohepatitis. Moreover, non-availability of specific drugs to prevent steatohepatitis, this animal model may serve as a powerful preclinical platform to study the therapeutic strategies and for evaluating chemoprevention strategies for this disease.     

Ecology and transmission of Listeria monocytogenes infecting ruminants and in the farm environment

A case-control study involving 24 case farms with at least one recent case of listeriosis and 28 matched control farms with no listeriosis cases was conducted to probe the transmission and ecology of Listeria monocytogenes on farms. A total of 528 fecal, 516 feed, and 1,012 environmental soil and water samples were cultured for L. monocytogenes. While the overall prevalence of L. monocytogenes in cattle case farms (24.4%) was similar to that in control farms (20.2%), small-ruminant (goat and sheep) farms showed a significantly (P < 0.0001) higher prevalence in case farms (32.9%) than in control farms (5.9%). EcoRI ribotyping of clinical (n = 17) and farm (n = 414) isolates differentiated 51 ribotypes. L. monocytogenes ribotypes isolated from clinical cases and fecal samples were more frequent in environmental than in feed samples, indicating that infected animals may contribute to L. monocytogenes dispersal into the farm environment. Ribotype DUP-1038B was significantly (P < 0.05) associated with fecal samples compared with farm environment and animal feedstuff samples. Ribotype DUP-1045A was significantly (P < 0.05) associated with soil compared to feces and with control farms compared to case farms. Our data indicate that (i) the epidemiology and transmission of L. monocytogenes differ between small-ruminant and cattle farms; (ii) cattle contribute to amplification and dispersal of L. monocytogenes into the farm environment, (iii) the bovine farm ecosystem maintains a high prevalence of L. monocytogenes, including subtypes linked to human listeriosis cases and outbreaks, and (iv) L. monocytogenes subtypes may differ in their abilities to infect animals and to survive in farm environments.