Peripheral plasma levels of oestradiol-17 beta and progesterone in the bitch during the oestrous cycle, in normal pregnancy and after dexamethasone treatment.

Plasma oestradiol-17 beta concentrations in Labradors increased during pro-oestrus to an average maximal concentration of of 79-7 +/- 10-9 (S.D.) pg/ml, and then fell rapidly. In 6/7 bitches the peak occurred within 1 day of oestrus. No consistent changes in plasma oestradiol levels were observed during pregnancy and at parturition and the values were similar to those in late anoestrus. Plasma progesterone levels did not increase markedly during pro-oestrus. At oestrus, progesterone values rose and maximal concentrations, which varied from about 20 to about 55 ng/ml, were reached within a few days of the oestradiol peak. Plasma progesterone decreased in late pregnancy and in one of the three bitches studied in detail low or undetectable levels were reached 10 days before parturition. In the other two bitches an abrupt decrease in progesterone occurred just before parturition. Dexamethasone treatment (2 X 5 mg daily for 10 days) from Day 30 of pregnancy resulted in intrauterine death and resorption of the fetuses in the two bitches studied. Treatment from about Day 45 resulted in the birth of dead fetuses at Days 55 and 59 of pregnancy. The changes in plasma oestradiol levels were very small. No changes in plasma progesterone levels were seen when dexamethasone was given in late pregnancy, but an accelerated decline occurred after treatment in mid-pregnancy.     

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.     

Are mice calorically restricted in nature?

An important question about traditional caloric restriction (CR) experiments on laboratory mice is how food intake in the laboratory compares with that of wild mice in nature. Such knowledge would allow us to distinguish between two opposing views of the anti-aging effect of CR--whether CR represents, in laboratory animals, a return to a more normal level of food intake, compared with excess food consumption typical of laboratory conditions or whether CR represents restriction below that of animals living in nature, i.e. the conditions under which house mice evolved. To address this issue, we compared energy use of three mouse genotypes: (1) laboratory-selected mouse strains (= laboratory mice), (2) house mice that were four generations or fewer removed from the wild (= wild-derived mice) and (3) mice living in nature (= wild mice). We found, after correcting for body mass, that ad libitum fed laboratory mice eat no more than wild mice. In fact, under demanding natural conditions, wild mice eat even more than ad libitum fed laboratory mice. Laboratory mice do, however, eat more than wild-derived mice housed in similar captive conditions. Therefore, laboratory mice have been selected during the course of domestication for increased food intake compared with captive wild mice, but they are not particularly gluttonous compared with wild mice in nature. We conclude that CR experiments do in fact restrict energy consumption beyond that typically experienced by mice in nature. Therefore, the retarded aging observed with CR is not due to eliminating the detrimental effects of overeating.     

The mRNA-binding site of annexin A2 resides in helices C-D of its domain IV

Annexin A2 (AnxA2) is a Ca(2+)-binding and phospholipid-binding protein involved in different intracellular processes including exocytosis, endocytosis and membrane-cytoskeleton movements. We have previously identified AnxA2 as an mRNA-binding protein present in cytoskeleton-bound polysomes, that binds to a specific approximately 100 nucleotide region in the 3'-untranslated region of c-myc and its cognate mRNA. In the present study, we show by UV cross-linking assays and surface plasmon resonance analyses that the mRNA-binding site of AnxA2 resides in its domain IV. Furthermore, the interaction of full-length AnxA2 with the 3'-untranslated region of anxA2 mRNA is Ca(2+)-dependent. By contrast, the interaction is Ca(2+)-independent for the isolated domain IV of AnxA2, suggesting that the mRNA-binding site is masked in Apo-AnxA2 and gains exposure through a Ca(2+)-induced conformational change of AnxA2 generating a favourable mRNA-binding site. The AnxA2-mRNA interaction is specific and involves helices C and D in domain IV of AnxA2, since point mutagenesis of several charged and polar exposed residues of these helices in the full-length protein strongly reduce RNA binding. The interaction appears to be sequential involving an initial phase of recognition dominated by electrostatic interactions, most likely between lysine residues and the phosphate backbone of RNA, followed by a second phase contributing to the specificity of the interaction.     

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication. Overproduction of DnaA protein, which in wild-type cells leads to over-replication, had no effect in the dnaN(G157C) mutant. Origins already opened by DnaA seemed to remain open for a prolonged period, with a stage of initiation involving β clamp loading, presumably limiting the initiation process. The existence of opened origins led to a moderate SOS response. Lagging strand synthesis, which also requires loading of the β clamp, was apparently unaffected. The result indicates that some aspects of β clamp activity are specific to the origin. It is possible that the origin specific activities of β contribute to regulation of initiation frequency.