Defined nutrient medium for the in vitro maintenance ofXenopus laevis oocytes

Summary A procedure is described for the isolation and culture of large numbers of follicle cell-freeXenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenance of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morphological and functional characteristics of oocytes for periods up to 3 weeks. Research supported by grant BMS 74-18790 from the National Science Foundation to JJE. Operated by Union Carbide Corporation for the U.S.E.R.D.A.     

Screening procedure for complementation-dependent mutants of vesicular stomatitis virus.

To isolate new types of vesicular stomatitis virus (VSV) mutants, a four-stage screen was developed which identifies and characterizes mutants capable of complementing the defect in the VSV temperature-sensitive mutant tsG11. Two types of mutants of VSV, Indiana serotype, have been found by using the screen; they are new temperature-sensitive mutants which are, of necessity, not in complementation group I and mutants which do not produce plaques under conditions of single infection at 31 C (the normal permissive temperature) and are, therefore, called complementation-dependent mutants. The newly isolated, temperature-sensitive mutants fall into three complementation groups, two of which are congruent with known complementation groups; the newly identified group extends to six the number of complementation groups of VSV Indiana. The nature of the complementation-dependent mutants has not been established, but one was shown to not contain a significant deletion in its nucleic acid.     

Transfer RNA and protein synthesis

The three-dimensional structure of yeast phenylalanine transfert RNA has been determined in orthorhombic crystals. The current status of this work is reviewed together with the relationship of the transfer RNA structure in the crystal to its biologically active form. In addition some speculations are put forward regarding the mode of interaction of tRNA molecules in the ribosome and the manner in which tRNA interacts with aminoacyl synthetase.     

JmjC-KDMs KDM3A and KDM6B modulate radioresistance under hypoxic conditions in esophageal squamous cell carcinoma

Esophageal squamous cell carcinoma (ESCC), the most frequent esophageal cancer (EC) subtype, entails dismal prognosis. Hypoxia, a common feature of advanced ESCC, is involved in resistance to radiotherapy (RT). RT response in hypoxia might be modulated through epigenetic mechanisms, constituting novel targets to improve patient outcome. Post-translational methylation in histone can be partially modulated by histone lysine demethylases (KDMs), which specifically removes methyl groups in certain lysine residues. KDMs deregulation was associated with tumor aggressiveness and therapy failure. Thus, we sought to unveil the role of Jumonji C domain histone lysine demethylases (JmjC-KDMs) in ESCC radioresistance acquisition. The effectiveness of RT upon ESCC cells under hypoxic conditions was assessed by colony formation assay. KDM3A/KDM6B expression, and respective H3K9me2 and H3K27me3 target marks, were evaluated by RT-qPCR, Western blot, and immunofluorescence. Effect of JmjC-KDM inhibitor IOX1, as well as KDM3A knockdown, in in vitro functional cell behavior and RT response was assessed in ESCC under hypoxic conditions. In vivo effect of combined IOX1 and ionizing radiation treatment was evaluated in ESCC cells using CAM assay. KDM3A, KDM6B, HIF-1α, and CAIX immunoexpression was assessed in primary ESCC and normal esophagus. Herein, we found that hypoxia promoted ESCC radioresistance through increased KDM3A/KDM6B expression, enhancing cell survival and migration and decreasing DNA damage and apoptosis, in vitro. Exposure to IOX1 reverted these features, increasing ESCC radiosensitivity and decreasing ESCC microtumors size, in vivo. KDM3A was upregulated in ESCC tissues compared to the normal esophagus, associating and colocalizing with hypoxic markers (HIF-1α and CAIX). Therefore, KDM3A upregulation in ESCC cell lines and primary tumors associated with hypoxia, playing a critical role in EC aggressiveness and radioresistance. KDM3A targeting, concomitant with conventional RT, constitutes a promising strategy to improve ESCC patients’ survival.Subject terms: Predictive markers, Cancer     

Prolactin-induced cytologic changes in the mucosa of the pigeon crop during crop-“milk” formation

Summary Light and electron microscope observations reveal two basic histo- and cytologic changes which take place in the pigeon crop during the course of prolaction administration to both sexes; (1) an increase in mitotic activity resulting in hyperplasia of the lateral lobes of the crop and (2) the accumulation of lipid in cells of these lobes. The latter process begins about twelve hours following prolactin injection. Histologic and cytochemical tests, as well as thin layer chromatographic analysis, reveal that these lipids may be classified as neutral unsaturated triglycerides. Tissues of control and prolactin-treated groups examined during the course of this study do not reveal any striking changes in organelle systems — aside from the accumulation of large lipid droplets. It does appear, however, that while ribosomes exist primarily as single units in non-stimulated cells, they appear primarily as polysomes in stimulated cells actively engaged in lipid synthesis. The origin of the cytoplasmic lipid inclusions is of interest. Morphologic evidence suggests that (1) they are not the result of cyto-pathologic changes, (2) they do not result from mitochondrial transformation and (3) they are not incorporated as discrete triglyceride micelles into these cells. The availability of precursors to the cell is dicussed in light of the observation that intercellular channels increase in width, and microvilli and desmosomes decrease in number throughout the experimental series. The incorporation of possible precursors into the cytoplasm may occur via two distinct classes of vesicles. One class is thought to arise through micropinocytotic activity. These vesicles have filamentous boundaries which are thought to render them specific for certain classes of compounds. The second class is somewhat larger in size and differs morphologically from micropinocytotic vesicles. These are thought to arise by the fusion of tips of microvilli and the plasma membrane, thus inpounding and internalizing materials from the intercellular canals into the cytoplasm.The author is indebted to Dr. Everett Anderson for his valuable criticism and encouragement offered during the course of this work. The investigation was supported in part by a Pre-Doctoral Fellowship (1-F1-GM-20, 296-01) from the National Institutes of Health and in part by grant GM-08776 from the National Institutes of Health awarded to Dr. Everett Anderson.