Role of methionine in the regulation of serine hydroxymethyltransferase in Eschericia coli.

Significant derepression of serine hydroxymethyltransferase is observed when metE or metF mutants of Escherichia coli K-12 are grown on D-methionine sulfoxide instead of L-methionine. The derepression is not prevented by addition of glycine, adenosine, guanosine, guanosine, and thymidine to the growth medium of methionine-limited metF cells showing that the effect is not due to a secondary deficiency of these nutrients. On the other hand, methionine-limited growth of a metA mutant leads to derepression of met regulon enzymes, but only a marginal increase in serine hydroxymethyltransferase activity. A prototrophic metJ strain grown on minimal medium has about the same serine hydroxymethyltransferase as the wild type. The enzyme activity of the metJ strain is not influenced by methionine, but it is partially repressed by glycine, adenosine, and thymidine. metK strains have about twice as much serine hydroxymethyltransferase activity as wild-type cells when grown on minimal medium; but when both types of cells are grown on medium supplemented with glycine, adenosine, guanosine, and thymidine, their enzyme activities are about the same. The results show that methionine limitation can lead to depression of serine hydroxymethyltransferase, but that the regulatory system is different from the one which controls the methionine regulon.     

Formation of internucleotide 3''-5'' phosphoramidate links by direct coupling of phosphoryl and amino groups.

The stepwise synthesis of oligomers derived from 5'-amino-5'-deoxythymidine 3'-phosphate and 5'-amino-5'-deoxy-3'-O-mono-p-methoxytrityl-thymidine is described. The internucleotide phosphoramidate links were formed by condensation of nucleoside 3'-phosphates with aminonucleosides by means of triphenylphosphine and dipridyl disulfide.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

The NF-kappa B-inducing kinase induces PC12 cell differentiation and prevents apoptosis

NF-kappa B has been implicated in the survival and differentiation of PC12 cells. In this study, we examined the effect of the NF-kappa B-inducing kinase (NIK) on these processes. When inducibly expressed in PC12 cells, a kinase-proficient but not -deficient form of NIK promoted neurite process formation and mediated anti-apoptotic signaling. As expected, NIK expression led to I kappa B kinase activation and induced nuclear translocation of NF-kappa B. However, NIK-induced neurite outgrowth was only partially blocked by concomitant expression of a nondegradable form of I kappa B alpha that completely blocks NF-kappa B induction. In search of additional signaling pathways activated by NIK, we now demonstrate that NIK activates MEK1 phosphorylation and induces the Erk1/Erk2 MAPK pathway. Treatment of PC12 cells with PD98059, a MEK1 inhibitor, potently blocked neurite process formation; however, a dominantly interfering mutant of the upstream Shc adapter failed to alter this response. These findings reveal a new function for NIK as a MEK1-dependent activator of the MAPK pathway and implicate both the I kappa B kinase and MAPK signaling cascades in NIK-induced differentiation of PC12 cells.     

Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.     

Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

Syndecan-4 associates with alpha-actinin

Cell adhesion to the extracellular matrix influences many cellular functions. The integrin family of matrix receptors plays major roles in the formation of adhesions, but other proteins modulate integrin signaling. Syndecan-4, a transmembrane proteoglycan, cooperatively signals with integrins during the formation of focal adhesions. To date, a direct link between syndecan-4 and the cytoskeleton has remained elusive. We now demonstrate by Triton X-100 extraction immunoprecipitation and in vitro binding assays that the focal adhesion component alpha-actinin interacts with syndecan-4 in a beta-integrin-independent manner.