Clonal nature of murine hemopoietic blast cell colonies

Murine hemopoietic blast cell colonies obtained from spleen cells of 5-fluorouracil (5-FU)-treated mice give rise to many multilineage colonies including granulocyte - erythrocyte - macrophage - megakaryocyte (GEMM) colonies in secondary cultures. Progenitor cells for blast cell colonies are considered to be more primitive than colony forming units (CFU)-GEMM. To determine whether they are clonal, we examined the phosphoglycerate kinase-1 (PGK-1) isozyme type of colonies originally grown from spleen cells of 5-FU-treated mice which had PGK-1 isozyme mosaicism. PGK assays of whole secondary colonies derived from one blast cell colony showed that they were either of type A or type B but not both. These results suggest that murine hemopoietic blast cell colonies are clonal.     

Guanosine 5'-triphosphate, 3'-diphosphate 5'-phosphohydrolase. Purification and substrate specificity

The regulatory nucleotide guanosine 5'-diphosphate, 3'-diphosphate (ppGpp) and its precursor guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) are accumulated during stringent response in bacterial cells. The enzyme pppGpp-5'-phosphohydrolase, which catalyzes the conversion of pppGpp to ppGpp, was partially purified from Escherichia coli. It has Mr = 140,000 and an apparent Km of 0.11 mM for pppGpp. It requires Mg2+ and a monovalent cation. NH4+ is preferred over K+, while Na+ is inactive. The enzyme does not hydrolyze GTP, ATP, pppApp, or ppGpp. It is also not effectively inhibited by these nucleotides. pppGpp-5'-phosphohydrolase hydrolyzes the 3'-monophosphate analog pppGp equally well (apparent Km of 0.13 mM), yielding the recently identified MS III nucleotide (ppGp). pppGpp-5'-phosphohydrolase does not have RNA 5'-terminal gamma-phosphatase activity; however, 5'-terminal phosphates are released by pppGpp-5'-phosphohydrolase when the GTP-terminated RNA chains are first converted into oligonucleotides by RNase A treatment. pppGpp-5'-phosphohydrolase was found to actively hydrolyze the dinucleotide fragment pppGpNp but exhibited very low activity toward longer chain fragments. The 3'-unphosphorylated dinucleotide pppGpN was, however, not hydrolyzed. The ability of pppGpp-5'-phosphohydrolase to hydrolyze pppGpp, pppGp, and pppGpNp, but not pppG and pppGpN, indicates that pppGpp-5'-phosphohydrolase is rather nonspecific toward the 3'-OH substitutions of the substrates although a free, unsubstituted phosphate group at the 3'-OH position is essential.     

Immunological detection of the dystrophin molecule with antibody directed against the synthetic peptide.

We synthesized a peptide designated R8 (amino acid residues 1157-1201) based on the primary structure presumed from the nucleotide sequence of the cDNA clone from the gene for Duchenne muscular dystrophy. Antibody to the synthetic R8 generated by immunization of rabbits was tested on human and mouse skeletal muscle by Western blotting analysis. The antibody reacted with a component of the 400K dystrophin of normal human and mouse skeletal muscles, but not with components of the muscles of Duchenne muscular dystrophy patients and mdx mice. Thus we established that this peptide sequence is in fact missing in the protein product 'dystrophin' encoded by the DMD gene. The antibody may prove useful for the diagnosis of the Duchenne types of muscular dystrophy.     

Physiological polyspermy: Selection of a sperm nucleus for the development of diploid genomes in amphibians

The union between a sperm and an egg nucleus in egg fertilization is necessary to mix genetic materials to create a new diploid genome for the next generation. In most animals, only one sperm is incorporated into the egg (monospermy), but several animals exhibit physiological polyspermy in which several sperms enter the egg during normal fertilization. However, only one sperm nucleus forms the zygote nucleus with the egg nucleus, even in a polyspermic egg. The cellular and molecular mechanisms involved in the selection of sperm nuclei in the egg cytoplasm have been well investigated in urodele amphibians. The principal sperm nucleus develops a larger sperm aster and contacts the egg nucleus to form a zygote nucleus, whereas other accessory sperm nuclei are unable to approach the egg nucleus. The diploid zygote nucleus induces cleavage and participates in embryonic development, whereas the accessory sperm nuclei undergo pyknosis and degenerate. We propose several models to account for the mechanisms of the selection of one sperm nucleus and the degeneration of accessory sperm nuclei. The roles of physiological polyspermy in animal reproduction are discussed by comparison with other polyspermic species.     

The impact of interventions in the global land and agri‐food sectors on Nature’s Contributions to People and the UN Sustainable Development Goals

Interlocked challenges of climate change, biodiversity loss, and land degradation require transformative interventions in the land management and food production sectors to reduce carbon emissions, strengthen adaptive capacity, and increase food security. However, deciding which interventions to pursue and understanding their relative co‐benefits with and trade‐offs against different social and environmental goals have been difficult without comparisons across a range of possible actions. This study examined 40 different options, implemented through land management, value chains, or risk management, for their relative impacts across 18 Nature's Contributions to People (NCPs) and the 17 Sustainable Development Goals (SDGs). We find that a relatively small number of interventions show positive synergies with both SDGs and NCPs with no significant adverse trade‐offs; these include improved cropland management, improved grazing land management, improved livestock management, agroforestry, integrated water management, increased soil organic carbon content, reduced soil erosion, salinization, and compaction, fire management, reduced landslides and hazards, reduced pollution, reduced post‐harvest losses, improved energy use in food systems, and disaster risk management. Several interventions show potentially significant negative impacts on both SDGs and NCPs; these include bioenergy and bioenergy with carbon capture and storage, afforestation, and some risk sharing measures, like commercial crop insurance. Our results demonstrate that a better understanding of co‐benefits and trade‐offs of different policy approaches can help decision‐makers choose the more effective, or at the very minimum, more benign interventions for implementation.     

Ontogeny of pituitary cell-types and the hypothalamo-hypophysial relationship during early development of chum salmon,Oncorhynchus keta

Tissue non-specific alkaline phosphatase is a membrane-bound glycoprotein enzyme which is characterized by its phosphohydrolytic, protein phosphatase, and phosphotransferase activities. This enzyme is distributed virtually in all mammalian tissues, particularly during embryonic development. Its expression is stagespecific and can be demonstrated in the developing embryo as early as the 2-cell stage. It has been suggested that tissue non-specific alkaline phosphatase might play a role in tissue formation. In the study reported here, a genetransfer approach was employed to investigate possible roles for this enzyme by inserting the cDNA for rat tissue non-specific alkaline phosphatase into CHO and LLC-PK1 cells. Permanently transfected cell-lines expressing varying levels of alkaline phosphatase were estblished. The data showed that functional enzyme was expressed in the transfected cells. Cell spreading and attachment were enhanced in transfected CHO cells expressing high levels of tissue non-specific alkaline phosphatase but not in the LLC-PK1 cells. Further, in CHO cells, proliferation was shown to be inversely proportional to the level of the tissue non-specific alkaline phosphatase expression. Homotypic cell association was demonstrated in both alkaline phosphatase-positive and alkaline phosphatase-negative cells in both CHO and LLC-PK1 celllines. Taken together, these findings suggest that in addition to a role in mineralization of bone, tissue nonspecific alkaline phosphatase might also play a role in other cell activities, including those related to differentiation, such as cell-cell or cell-substrate interaction and proliferation.     

Sequence rearrangement in JC virus DNAs molecularly cloned from immunosuppressed renal transplant patients.

From nonimmunocompromised individuals, we have recently identified a possible archetypal JC virus DNA sequence from which various regulatory sequences of JC virus isolates derived from patients with progressive multifocal leukoencephalopathy (PML) could have evolved. In this study, we analyzed the regulatory sequences of JCV DNAs cloned from urine samples of a PML risk group (renal transplant patients on immunosuppressive therapy). A number of JC virus DNAs were molecularly cloned from virions excreted in the urine of eight patients. Furthermore, fragments containing the regulatory region were amplified by the polymerase chain reaction and subsequently molecularly cloned from cell-associated JC virus excreted in the urine of two patients. The regulatory regions in all clones were analyzed with restriction enzymes, and those in representative clones were sequenced. We found that clones with the archetypal regulatory sequence were predominant in all urine samples, but a few clones carried regulatory sequences that diverged from the archetypal sequence by deletion or duplication. The finding that sequence rearrangement in the archetypal regulatory region occurs in the course of infection in immunosuppressed hosts is consistent with the adaptation hypothesis which has been put forward to explain the divergence of the regulatory regions in PML-derived JC virus isolates.     

Hypoxia-Inducible Factor 1 Regulation through Cross Talk between mTOR and MT1-MMP

Hypoxia-inducible factor 1 (HIF-1) plays a key role in the cellular adaptation to hypoxia. Although HIF-1 is usually strongly suppressed by posttranslational mechanisms during normoxia, HIF-1 is active and enhances tumorigenicity in malignant tumor cells that express the membrane protease MT1-MMP. The cytoplasmic tail of MT1-MMP, which can bind a HIF-1 suppressor protein called factor inhibiting HIF-1 (FIH-1), promotes inhibition of FIH-1 by Mint3 during normoxia. To explore possible links between HIF-1 activation by MT1-MMP/Mint3 and tumor growth signals, we surveyed a panel of 252 signaling inhibitors. The mTOR inhibitor rapamycin was identified as a possible modulator, and it inhibited the mTOR-dependent phosphorylation of Mint3 that is required for FIH-1 inhibition. A mutant Mint3 protein that cannot be phosphorylated exhibited a reduced ability to inhibit FIH-1 and promoted tumor formation in mice. These data suggest a novel molecular link between the important hub proteins MT1-MMP and mTOR that contributes to tumor malignancy.     

Contribution of endogenous G-protein-coupled receptor kinases to Ser129 phosphorylation of α-synuclein in HEK293 cells

The majority of α-synuclein (αS) deposited in Lewy bodies, the pathological hallmark of Parkinson’s disease (PD), is phosphorylated at serine 129 (Ser129). Ser129 phosphorylation of αS has been demonstrated to enhance the αS toxicity to dopaminergic neurons in a Drosophila model of PD. Phosphorylation of αS at Ser129 seems to play a crucial role in the pathogenesis of PD. Here, we assessed the contribution of ubiquitously expressing members of the G-protein-coupled receptor kinase family (GRK2, GRK3, GRK5, and GRK6) to Ser129 phosphorylation of αS in HEK293 cells. To selectively reduce the endogenous expression of each member of the GRK family in cells, we used small interfering RNAs. Knockdown of GRK3 or GRK6 significantly decreased Ser129 phosphorylation of αS; however, knockdown of GRK2 or GRK5 did not decrease αS phosphorylation. The results indicate that endogenous GRK3 and GRK6, but not GRK2 or GRK5, contribute to Ser129 phosphorylation of αS in HEK293 cells.     

The E3 ligase HACE1 is a critical chromosome 6q21 tumor suppressor involved in multiple cancers

Transformation and cancer growth are regulated by the coordinate actions of oncogenes and tumor suppressors. Here, we show that the novel E3 ubiquitin ligase HACE1 is frequently downregulated in human tumors and maps to a region of chromosome 6q21 implicated in multiple human cancers. Genetic inactivation of HACE1 in mice results in the development of spontaneous, late-onset cancer. A second hit from either environmental triggers or genetic heterozygosity of another tumor suppressor, p53, markedly increased tumor incidence in a Hace1-deficient background. Re-expression of HACE1 in human tumor cells directly abrogates in vitro and in vivo tumor growth, whereas downregulation of HACE1 via siRNA allows non-tumorigenic human cells to form tumors in vivo. Mechanistically, the tumor-suppressor function of HACE1 is dependent on its E3 ligase activity and HACE1 controls adhesion-dependent growth and cell cycle progression during cell stress through degradation of cyclin D1. Thus, HACE1 is a candidate chromosome 6q21 tumor-suppressor gene involved in multiple cancers.