Changes in calmodulin level after fertilization and during first cleavage in the egg of the urodelan amphibian Pleurodeles waltlii

We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.     

Binding of phosphatidylinositol-3,4,5-triphosphate to 45-kDa Sec14-like protein from the rat olfactory epithelium in liposomes

The binding of phosphatidylinositol-3,4,5-triphosphate to a protein with molecular mass of 45 kDa from rat olfactory epithelium (p45) was investigated using a model membrane system. Liposomes containing a mixture of phospholipids (phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol-3,4,5-triphosphate) were used in the study. The binding of the protein to liposomes caused by its interaction with phosphatidylinositol-3,4,5-triphosphate was confirmed by cosedimentation and immunoblotting with chemiluminescent detection using monoclonal antibodies to the native protein p45.     

Uptake and distribution of calcium in rose plantlets as affected by calcium and boron concentration in culture solution

Grafted rose plantlets were grown in a controlled growth room in order to study the effect of Ca and B concentration on Ca uptake, distribution and transport to the apex. There were three Ca levels and three B levels. Root development was affected by Ca and B levels available to the roots. ⁴⁵Ca taken up was readily translocated to the scion, but accumulated in the rootstock in low-Ca plants. Translocation of Ca was increased by increasing B concentration. The relative Ca sink strength of low- and high-transpiring organs was affected by Ca and B concentrations. Increasing Ca level will increase rose growth only if B concentration is adjusted accordingly.     

An experimental study of 226Ra and 45Ca accumulation from the aquatic medium by freshwater turtles (fam. Chelidae) under varying Ca and Mg water concentrations

Snapping turtles Elseya dentata (Gray) from Magela Creek, Northern Territory, were exposed under laboratory conditions for up to 30 days to waters resembling the inorganic composition of Magela Creek water during the Wet season, with background and elevated Ca and Mg concentrations, that were labelled with ²²⁶Ra and ⁴⁵Ca. The resulting concentrations of ⁴⁵Ca in muscle, skin, gut, liver, shell bone and leg bone of E. dentata equilibrated or approached equilibrium by 12–18 days. Among the experiments, the concentrations of ⁴⁵Ca in all six tissues were inversely related to turtle mass. An increase in the Ca water concentration by a factor of 15 increased the ⁴⁵Ca concentration in all six tissues. The arithmetic factors of increase in the concentration in each tissue were proportional or more than proportional to the factor of increase in Ca water concentration; this factor was highest for muscle tissue (26.6). An increase in the Mg water concentration by a factor of 48 reduced the ⁴⁵Ca concentration in all tissues except skin where it increased. The concentration of ²²⁶Ra in each tissue (except the gut) was positively related to its ⁴⁵Ca concentration and inversely related to turtle mass in muscle, skin and liver. With the exception of the skin, the increased Ca water concentration did not reduce the ²²⁶Ra in the tissues but increased the ²²⁶Ra concentration in bone and muscle. The increased Mg water concentration had an inverse effect on the ²²⁶Ra concentrations in all tissues except shell. With the exception of the skin the effects of increased Ca and Mg water concentrations and turtle size on ²²⁶Ra concentrations in the tissues of E. dentata were similar to their effects on ⁴⁵Ca tissue concentrations, indicating the similar metabolic behaviour of ²²⁶Ra and ⁴⁵Ca in E. dentata.Exposures of the species Elseya latisternum (Gray), Emydura signata (Ahl) and Chelodina longicollis (Shaw), which are the same or closely related to species reported to occur in Magela Creek, to ⁴⁵Ca-labelled Sydney tap water for 7 days demonstrated their ability to also accumulate ⁴⁵Ca from their aquatic medium. The patterns of ⁴⁵Ca concentrations in the tissues of these species indicated that they were inversely related to turtle mass, as demonstrated in E. dentata. The concentrations of ⁴⁵Ca accumulated in the tissues were also comparable to those found in single specimens of E. dentata and E. victoriae (Gray) that were exposed for 7 days to simulated Magela Creek water. The data also indicated the larger long-necked C. longicollis accumulated less ⁴⁵Ca per gram of tissue than similar-sized, short-necked species E. signata and E. latisternum, suggesting that long-necked turtles from Magela Creek would accumulate less ²²⁶Ra from their aquatic medium than similar-sized short-necked species.The capacity of E. dentata to accumulate ²²⁶Ra from the aquatic medium is about two orders of magnitude less than that of the tissue of the freshwater mussel Velesunio angasi (Sowerby) exposed under similar experimental conditions.     

Helicase Subunit Cdc45 Targets the Checkpoint Kinase Rad53 to Both Replication Initiation and Elongation Complexes after Fork Stalling

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The autophagy gene Wdr45/Wipi4 regulates learning and memory function and axonal homeostasis

WDR45/WIPI4, encoding a WD40 repeat-containing PtdIns(3)P binding protein, is essential for the basal autophagy pathway. Mutations in WDR45 cause the neurodegenerative disease β-propeller protein-associated neurodegeneration (BPAN), a subtype of NBIA. We generated CNS-specific Wdr45 knockout mice, which exhibit poor motor coordination, greatly impaired learning and memory, and extensive axon swelling with numerous axon spheroids. Autophagic flux is defective and SQSTM1 (sequestosome-1)/p62 and ubiquitin-positive protein aggregates accumulate in neurons and swollen axons. Nes-Wdr45^fl/Y mice recapitulate some hallmarks of BPAN, including cognitive impairment and defective axonal homeostasis, providing a model for revealing the disease pathogenesis of BPAN and also for investigating the possible role of autophagy in axon maintenance.     

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.     

Impact of extreme temperatures on daily mortality in Madrid (Spain) among the 45–64 age-group

This paper analyses the relationship between extreme temperatures and mortality among persons aged 45–64 years. Daily mortality in Madrid was analysed by sex and cause, from January 1986 to December 1997. Quantitative analyses were performed using generalised additive models, with other covariables, such as influenza, air pollution and seasonality, included as controls. Our results showed that impact on mortality was limited for temperatures ranging from the 5th to the 95th percentiles, and increased sharply thereafter. During the summer period, the effect of heat was detected solely among males in the target age group, with an attributable risk (AR) of 13.3% for circulatory causes. Similarly, NO₂ concentrations registered the main statistically significant associations in females, with an AR of 15% when circulatory causes were considered. During winter, the impact of cold was exclusively observed among females having an AR of 7.7%. The magnitude of the AR indicates that the impact of extreme temperature is by no means negligible.Julio Díaz is adviser to the Madrid Autonomous University General Foundation, on behalf of the Department of Education for Sustainable Development, Madrid City Council, Madrid, Spain     

Nitric Oxide Regulates Shikonin Formation in Suspension-Cultured Onosma paniculatum Cells

Endogenously occurring nitric oxide (NO) is involved in theregulation of shikonin formation in Onosma paniculatum cells.NO generated after cells were inoculated into shikonin productionmedium reached the highest level after 2 d of culture, whichwas 16 times that at the beginning of the experiment, and maintaineda high level for 6 d. A nitric oxide synthase (NOS) inhibitor,N-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor,sodium azide (SoA), consistent with their inhibition of NO biosynthesis,decreased shikonin formation significantly. This reduction couldbe alleviated or even abolished by exogenous NO supplied bysodium nitroprusside (SNP), suggesting that the inhibition ofNO biosynthesis resulted in decreased shikonin formation. However,when endogenous NO biosynthesis was up-regulated by the elicitorfrom Rhizoctonia cerealis, shikonin production was enhancedfurther, showing a dependence on the elicitor-induced NO burst.Real-time PCR analysis showed that NO could significantly up-regulatethe expression of PAL, PGT and HMGR, which encode key enzymesinvolved in shikonin biosynthesis. These results demonstratedthat NO plays a critical role in shikonin formation in O. paniculatumcells.     

Differential Positioning of C4 Mesophyll and Bundle Sheath Chloroplasts: Recovery of Chloroplast Positioning Requires the Actomyosin System

In C₄ plants, bundle sheath (BS) chloroplasts are arranged inthe centripetal position or in the centrifugal position, althoughmesophyll (M) chloroplasts are evenly distributed along cellmembranes. To examine the molecular mechanism for the intracellulardisposition of these chloroplasts, we observed the distributionof actin filaments in BS and M cells of the C₄ plants fingermillet (Eleusine coracana) and maize (Zea mays) using immunofluorescence.Fine actin filaments encircled chloroplasts in both cell types,and an actin network was observed adjacent to plasma membranes.The intracellular disposition of both chloroplasts in fingermillet was disrupted by centrifugal force but recovered within2 h in the dark. Actin filaments remained associated with chloroplastsduring recovery. We also examined the effects of inhibitorson the rearrangement of chloroplasts. Inhibitors of actin polymerization,myosin-based activities and cytosolic protein synthesis blockedmigration of chloroplasts. In contrast, a microtubule-depolymerizingdrug had no effect. These results show that C₄ plants possessa mechanism for keeping chloroplasts in the home position whichis dependent on the actomyosin system and cytosolic proteinsynthesis but not tubulin or light.