Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos: VI. Occurrence in mature oocytes and unfertilized eggs

Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [³H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.     

Characterization of a Xenopus laevis skin peptidylglycine alpha-hydroxylating monooxygenase expressed in insect-cell culture.

The C-terminal amide structure of peptide hormones and neurotransmitters is synthesized via a two-step reaction catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase. A Xenopus laevis PHM expressed in insect-cell culture by the baculovirus-expression-vector system was purified to homogeneity and characterized. Using a newly established assay system for PHM, the kinetic features of this enzyme were investigated. As expected, the enzyme required copper ions, L-ascorbate and molecular oxygen for turnover. Salts like KI and KCl, and catalase stabilized the enzyme in the presence of L-ascorbate. The optimum pH value for the enzyme reaction was around six when Mes buffer was used and around seven when phosphate buffer was used under the same assay condition. Below pH 6, acetate, iodide and chloride ions activated the reaction. The kinetic analysis is consistent with a ping-pong mechanism with respect to peptide and L-ascorbate, and the peptide showed substrate inhibition. The substrate specificity of the enzyme at the penultimate position was examined by competitive assay using tripeptides with glycine at the C-termini and the inhibitory potency of these peptides in descending order was methionine > aromatic > non-polar amino acids.     

Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology

Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding the basic mechanisms of somatic embryogenesis. Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

A role of membranes in the activation of a new multifunctional protein kinase system.

A new multifunctional protein kinase, which normally exists as an inactive form in the soluble fraction in mammalian tissues, attaches to membranes to exhibit full enzymatic activity. A low concentration of Ca2+ is absolutely necessary for this activation. This process is reversible. cAMP shows no effect. The active factors in membranes are phosphatidylinositol, phosphatidylserine, phosphatidic acid, diphosphatidylglycerol, and phosphatidylethanolamine in that order. Phosphatidylcholine and sphingomyelin are far less effective. Cytoplasmic as well as other membrane fractions from various tissues are active in supporting the enzymatic activity. A possible role of this Ca2+ and phospholipid-activated protein kinase system in transmembrane control is proposed.     

Proline in the Osmoregulation of Brevibacterium lactofermentum

Osmoregulation in Brevibacterium lactofermentum was studied. Proline was accumulated up to approximately 35mg/g dry cell weight in the cells of a wild strain of the bacterium grown under osmotic stress. The osmotic tolerance of a proline auxotroph mutant obtained from the bacterium was lower than that in the wild strain. The activity of pyrroline-5-carboxylate reductase, one of the enzymes in the proline biosynthetic pathway, increased about 3-fold when the cells of B. lactofermentum were grown under osmotic stress. These data indicated that proline is important in osmoregulation in the bacterium.     

Quantitative Proteomic Profiling Identifies DPYSL3 as Pancreatic Ductal Adenocarcinoma-Associated Molecule That Regulates Cell Adhesion and Migration by Stabilization of Focal Adhesion Complex

Elucidation of how pancreatic cancer cells give rise to distant metastasis is urgently needed in order to provide not only a better understanding of the underlying molecular mechanisms, but also to identify novel targets for greatly improved molecular diagnosis and therapeutic intervention. We employed combined proteomic technologies including mass spectrometry and isobaric tags for relative and absolute quantification peptide tagging to analyze protein profiles of surgically resected human pancreatic ductal adenocarcinoma tissues. We identified a protein, dihydropyrimidinase-like 3, as highly expressed in human pancreatic ductal adenocarcinoma tissues as well as pancreatic cancer cell lines. Characterization of the roles of dihydropyrimidinase-like 3 in relation to cancer cell adhesion and migration in vitro, and metastasis in vivo was performed using a series of functional analyses, including those employing multiple reaction monitoring proteomic analysis. Furthermore, dihydropyrimidinase-like 3 was found to interact with Ezrin, which has important roles in cell adhesion, motility, and invasion, while that interaction promoted stabilization of an adhesion complex consisting of Ezrin, c-Src, focal adhesion kinase, and Talin1. We also found that exogenous expression of dihydropyrimidinase-like 3 induced activating phosphorylation of Ezrin and c-Src, leading to up-regulation of the signaling pathway. Taken together, the present results indicate successful application of combined proteomic approaches to identify a novel key player, dihydropyrimidinase-like 3, in pancreatic ductal adenocarcinoma tumorigenesis, which may serve as an important biomarker and/or drug target to improve therapeutic strategies.