The role of microRNA in nutritional control.

MicroRNAs (miRNAs) are one of a growing class of noncoding RNAs that are involved in the regulation of a wide range of metabolic processes including cellular differentiation, cell proliferation and apoptosis. The generation of miRNA is regulated in complex ways, for example by small interfering RNAs (small nucleolar and nuclear RNAs) and various other metabolites. This complexity of control is likely to explain how a relatively small part of the DNA that codes for proteins has enabled the evolution of such complex organisms as mammals. Non-protein-coding DNA is therefore thought to carry the memory of early evolutionary steps that led to progressively complex metabolic controls. Clinically, miRNAs are becoming increasingly important following the recognition that some congenital abnormalities can be traced to defects in miRNA processing. The potential for manipulating metabolism and affecting disease processes by the pharmaceutical or biological targeting of specific miRNA pathways is now being tested. miRNAs are also released into the extracellular milieu after packaging by cells into nano-sized extracellular vesicles. Such vesicles can be taken up by adjacent and possibly more distant cells, thereby allowing coordinated intercellular communication in specific tissues. Extracellular miRNAs found in the blood stream may also serve as novel biomarkers for both diagnosing specific forms of cancer and assessing the likelihood of metastasis, and as powerful prognostic indices for various cancers. Here, we discuss the role of intracellular and extracellular miRNAs in nutritional control of various (patho)physiological processes. In this review, we provide an update of the presentations from the 25th Marabou Symposium (Stockholm, 14-16 June 2013) entitled 'Role of miRNA in health and nutrition', attended by 50 international experts

ATM and RPA in meiotic chromosome synapsis and recombination.

ATM is a member of the phosphatidylinositol 3-kinase (PIK)-like kinases, some of which are active in regulating DNA damage-induced mitotic cell-cycle checkpoints. ATM also plays a role in meiosis. Spermatogenesis in Atm-/- male mice is disrupted, with chromosome fragmentation leading to meiotic arrest; in human patients with ataxia-telangiectasia (A-T), gonadal atrophy is common. Immuno-localization studies indicate that ATM is associated with sites along the synaptonemal complex (SC), the specialized structure along which meiotic recombination occurs. Recombination, preceded by pairing of homologous chromosomes, is thought to require heteroduplex formation between homologous DNA, followed by strand exchange. These early meiotic steps (entailing the formation and processing of meiotic recombination intermediates with DNA-strand interruptions) require ssDNA-binding proteins such as replication protein A (RPA; refs 5-7). In somatic cells, DNA damage induces ATM-dependent phosphorylation of RPA. We demonstrate here that ATM and RPA co-localize along synapsed meiotic chromosomes and at sites where interactions between ectopic homologous chromosome regions appear to initiate. In Atm-/- meiotic prophase spermatocytes, immuno-localization shows that RPA is present along synapsing chromosomes and at sites of fragmentation of the SC. These results suggest that RPA and ATM co-localize at sites where interhomologous-DNA interactions occur during meiotic prophase and where breaks associated with meiotic recombination take place after synapsis, implying a possible functional interaction between these two proteins.

Regulation of the NF-kappa B/rel transcription factor and I kappa B inhibitor system.

The interplay between proteins of the NF-kappa B/rel and I kappa B families is a tightly regulated process that ensures appropriate responses to specific environmental and developmental signals. Various mechanisms are utilized in regulating NF-kappa B/rel and I kappa B activities, some unique to this transcription factor system. All of these regulatory strategies converge towards one purpose, namely the controlled nuclear translocation of activated NF-kappa B/rel protein complexes. The variety of rel-related and ankyrin repeat containing subunits makes regulation of this system both rich and complicated.

A quantitative assay for transformation of bone marrow cells by Abelson murine leukemia virus.

A quantitative Abelson murine leukemia virus (A-MuLV) lymphoid cell transformation assay has been developed using a semisolid agarose culture system. Under these conditions lymphoid cell transformation was shown to vary linearly with the dose of A-MuLV used. The susceptibility of bone marrow cells from different strains of mice to A-MuLV-induced transformation can be estimated using the agarose assay. Strains with bone marrow cells of high, medium, and low susceptibility to A-MuLV can be identified. The assay has been used to study the susceptibility of cells from lymphoid organs of fetal and adult mice to A-MuLV. Cell suspensions from fetal liver, adult bone marrow, and adult spleen are susceptible to A-MuLV, while thymocytes are resistant to A-MuLV-induced transformation. Bovine serum albumin gradient fractionation of bone marrow cells before infection with A-MuLV demonstrates that the majority of A-MuLV-sensitive cells are recovered in a broad band partially overlapping the majority of the nucleated cells. The agarose assay system allows study of A-MuLV-lymphoid cell interaction at the level of single cell-single virus particle interaction.

The effect of helper virus on Abelson virus-induced transformation of lymphoid cells.

Abelson murine leukemia virus (A-MuLV)-transformed fibroblast nonproducer cells were used to prepare A-MuLV stocks containing a number of different helper viruses. The oncogenicity of the A-MuLV stocks was tested by animal inoculation and their ability to transform normal mouse bone marrow cells was measured in vitro. All of the A-MuLV stocks transformed fibroblast cells efficiently. However, only A-MuLV stocks prepared with helper viruses that are highly oncogenic were efficient in vivo and in vitro in hematopoietic cell transformation. In addition, inefficient helpers did not establish a stable infection in lymphoid nonproducer cells. Thus, helper virus has a more central role in lymphoid cell transformation than in fibroblast cell transformation.

Virus-transformed pre-B cells show ordered activation but not inactivation of immunoglobulin gene rearrangement and transcription.

Virus-transformed pre-B cells undergo ordered immunoglobulin (Ig) gene rearrangements during culture. We devised a series of highly sensitive polymerase chain reaction assays for Ig gene rearrangement and unrearranged Ig gene segment transcription to study both the possible relationship between these processes in cultured pre-B cells and the role played by heavy (H) chain (mu) protein in regulating gene rearrangement. Our analysis of pre-B cell cultures representing various stages of maturity revealed that transcription of each germline Ig locus precedes or is coincident with its rearrangement. Cell lines containing one functional rearranged H chain allele, however, continue to transcribe and to rearrange the allelic, unrearranged H chain locus. These cell lines appear to initiate but not terminate rearrangement events and therefore provide information about the requirements for activating rearrangement but not about allelic exclusion mechanisms.

Elevated myc expression and c-myc amplification in spontaneously occurring B lymphoid cell lines.

Recently, a minor subpopulation of murine B lymphocytes, Ly-1+ B cells, has been distinguished by its unique ontogeny, tissue distribution, and prominence in certain autoimmune and neoplastic B cell diseases. We have previously described a simple murine spleen culture system that results in the spontaneous and exclusive outgrowth of long-term Ly-1+ B cell lines (B Ly-1 cells). Here, we report that the immortal growth property of B Ly-1 cells correlates with a 10-45-fold elevation of steady-state myc RNA and 2-10-fold amplification of the c-myc locus. While c-myc amplification has been observed in malignant cell lines derived from several tissues of origin, its occurrence in lymphoid cells has not been previously reported. The consistent c-myc amplification in B Ly-1 cells may reflect a unique state of this locus in the Ly-1+ B lymphocyte lineage, and contribute to the spontaneous immortalization of this B cell population in vitro, and its apparent predilection for malignant transformation in vivo.

Terminal deoxynucleotidyl transferase is found in prothymocytes.

Terminal deoxynucleotidyl transferase is an enzyme which has the unique property of polymerizing polydeoxynucleotides onto a primer in the absence of a template (1,2). This enzyme is found both in the thymus and the bone marrow of birds, rodents, and humans (3-7). Whether the marrow cells that contain terminal transferase are related to thymocytes, or are on a separate pathway of differentiation, is not yet known (7,8). To determine the lineage of the murine bone marrow cells that have terminal transferase, we have investigated whether these cells have the antigen Thy-1 induced on the cells by treatment with thymopoietin (9). Thymopoietin is known to induce a set of characteristic T-cell markers including the Thy-1 alloantigen on the surface of a subpopulation of bone marrow cells committed to T-cell differentiation (prothymocytes) (10). Destruction of Thy- 1-positive cells after exposure to thymopoietin allows elimination of a substantial fraction of those bone marrow cells that can repopulate an irradiated thymus (11). We find that such an elimination after induction with the thymic polypeptide removes a substantial amount of terminal transferase from the bone marrow cell population, suggesting that at least one-half of the marrow cells bearing this enzyme are related to those found in the thymus.

Murine terminal deoxynucleotidyl transferase: cellular distribution and response to cortisone.

The mouse thymus contains two forms of terminal deoxynucleotidyl transferase (TdT) which are distinguishable by the salt concentration necessary to elute them from a phosphocellulose column, by their distrubtion among the thymocyte subpopulations, and by their sensitivity to cortisone treatment. In the whole thymus the later eluting peak (peak II) is the predominant one with about 3-10% of the total activity appearing in peak I. Both peak I and peak II activities are most sensitively assayed by the polymerization of dGMP onto an oligo(dA) primer. The minor population of thymocytes which is less dense and cortisone-resistant contains a higher specific activity of peak I TdT. The majority of TdT activity is, however, found in the major population of thymocytes which occurs in the center region of a bovine serum albumin gradient and is cortisone-sensitive. A very low level of an activity indistinguishable from peak II TdT activity is also detected in the mouse bone marrow. Other tissues, such as spleen, liver, heart, and brain lack detectable amounts of TdT activity.

Lipopolysaccharide is a potent monocyte/macrophage-specific stimulator of human immunodeficiency virus type 1 expression.

Lipopolysaccharide (LPS) potently stimulates human immunodeficiency virus type 1-long terminal repeat (HIV-1-LTR) CAT constructs transfected into monocyte/macrophage-like cell lines but not a T cell line. This effect appears to be mediated through the induction of nuclear factor kappa B (NF-kappa B). Electrophoretic mobility shift assays demonstrate that LPS induces a DNA binding activity indistinguishable from NF-kappa B in U937 and THP-1 cells. LPS is also shown to dramatically increase HIV-1 production from a chronically infected monocyte/macrophage-like cloned cell line, U1, which produces very low levels of HIV-1 at baseline. The stimulation of viral production from this cell line occurs only if these cells are treated with granulocyte/macrophage colony-stimulating factor (GM-CSF) before treatment with LPS. This stimulation of HIV-1 production is correlated with an increase in the level of HIV-1 RNA and and activation of NF-kappa B. LPS is not able to induce HIV-1 production in a cloned T cell line. The effect of LPS on HIV-1 replication occurs at picogram per milliliter concentrations and may be clinically significant in understanding the variability of the natural history of HIV-1 infection.

p50-NF-kappaB complexes partially compensate for the absence of RelB: severely increased pathology in p50(-/-)relB(-/-) double-knockout mice.

RelB-deficient mice (relB(-/-)) have a complex phenotype including multiorgan inflammation and hematopoietic abnormalities. To examine whether other NF-kappaB/Rel family members are required for the development of this phenotype or have a compensatory role, we have initiated a program to generate double-mutant mice that are deficient in more than one family member. Here we report the phenotypic changes in relB(-/-) mice that also lack the p50 subunit of NF-kappaB (p50(-/-)). The inflammatory phenotype of p50(-/-)relB(-/-) double-mutant mice was markedly increased in both severity and extent of organ involvement, leading to premature death within three to four weeks after birth. Double-knockout mice also had strongly increased myeloid hyperplasia and thymic atrophy. Moreover, B cell development was impaired and, in contrast to relB(-/-) single knockouts, B cells were absent from inflammatory infiltrates. Both p50(-/-) and heterozygous relB(-/+) animals are disease-free. In the absence of the p50, however, relB(-/+) mice (p50(-/-)relB(-/+)) had a mild inflammatory phenotype and moderate myeloid hyperplasia. Neither elevated mRNA levels of other family members, nor increased kappaB-binding activities of NF-kappaB/Rel complexes could be detected in single- or double-mutant mice compared to control animals. These results indicate that the lack of RelB is, in part, compensated by other p50-containing complexes and that the "classical" p50-RelA-NF-kappaB activity is not required for the development of the inflammatory phenotype.

Synthetic phospholipid vesicles containing a purified viral antigen and cell membrane proteins stimulate the development of cytotoxic T lymphocytes.

Synthetic phospholipid vesicles (liposomes) containing the purified glycoprotein (G) of vesicular stomatitis virus (VSV) and solubilized membrane proteins from cells of the appropriate H-2 haplotype elicited H-2-restricted cytotoxic T lymphocytes (CTL) that lysed VSV-infected target cells. The CTL were elicited by intact liposomes, not by released components. Thus, when spleen cells from VSV-primed H-2d X H-2b hybrid mice were stimulated with liposomes having G protein + membrane proteins from cells with one of the parental H-2 haplotypes, the resulting CTL lysed only VSV-infected target cells with that parent's H-2 type. This result argues against the view that T cells in general recognize only processed antigenic fragments on macrophages. Moreover, liposomes were only effective when G protein and cell membrane proteins were included in the same vesicles. This result suggests that for effective interaction with CTL precursors the antigen (G protein) and products of the H-2 complex must be closer to each other than 600--1,000 angstrom, the diameter of the lipid vesicles used in this study.

Chromosomal location of structural genes encoding murine immunoglobulin lambda light chains. Genetics of murine lambda light chains.

To determine the chromosomal localization of murine lambda light (L) chain structural genes, DNA from a panel of 11 mouse x hamster somatic cell hybrids was scored for the presence of sequences homologous to cloned lambda DNA probe molecules. Six of the hybrids had detectable lambda I and lambda II gene sequences. In all six, the full complement of murine sequences was present, and in its germline configuration. The remaining hybrids lacked any detectable murine lambda L chain gene sequences. The only mouse chromosome present in all of the positive hybrids and absent from the negative ones was number 16, allowing the assignment of lambda L chain structural genes to this chromosome. Together with the previous assignments of the kappa L chain genes to chromosome 6 and heavy chain genes to chromosome 12, this finding completes the mapping of Ig structural genes in the mouse at the chromosomal level.

Severe B cell deficiency in mice lacking the tec kinase family members Tec and Btk.

The cytoplasmic protein tyrosine kinase Tec has been proposed to have important functions in hematopoiesis and lymphocyte signal transduction. Here we show that Tec-deficient mice developed normally and had no major phenotypic alterations of the immune system. To reveal potential compensatory roles of other Tec kinases such as Bruton's tyrosine kinase (Btk), Tec/Btk double-deficient mice were generated. These mice exhibited a block at the B220(+)CD43(+) stage of B cell development and displayed a severe reduction of peripheral B cell numbers, particularly immunoglobulin (Ig)M(lo)IgD(hi) B cells. Although Tec/Btk(null) mice were able to form germinal centers, the response to T cell-dependent antigens was impaired. Thus, Tec and Btk together have an important role both during B cell development and in the generation and/or function of the peripheral B cell pool. The ability of Tec to compensate for Btk may also explain phenotypic differences in X-linked immunodeficiency (xid) mice compared with human X-linked agammaglobulinemia (XLA) patients.

HIV-1 directly kills CD4+ T cells by a Fas-independent mechanism.

The mechanism by which HIV-1 induces CD4(+) T cell death is not known. A fundamental issue is whether HIV-1 primarily induces direct killing of infected cells or indirectly causes death of uninfected bystander cells. This question was studied using a reporter virus system in which infected cells are marked with the cell surface protein placental alkaline phosphatase (PLAP). Infection by HIV-PLAP of peripheral blood mononuclear cells (PBMCs) and T cell lines leads to rapid depletion of CD4(+) T cells and induction of apoptosis. The great majority of HIV-induced T cell death in vitro involves direct loss of infected cells rather than indirect effects on uninfected bystander cells. Because of its proposed role in HIV-induced cell death, we also examined the Fas (CD95/Apo1) pathway in killing of T cells by HIV-1. Infected PBMCs or CEM cells display no increase in surface Fas relative to uninfected cells. In addition, HIV-1 kills CEM and Jurkat T cells in the presence of a caspase inhibitor that completely blocks Fas-mediated apoptosis. HIV-1 also depletes CD4+ T cells in PBMCs from patients who have a genetically defective Fas pathway. These results suggest that HIV-1 induces direct apoptosis of infected cells and kills T cells by a Fas-independent mechanism.

Exclusive development of T cell neoplasms in mice transplanted with bone marrow expressing activated Notch alleles.

Notch is a highly conserved transmembrane protein that is involved in cell fate decisions and is found in organisms ranging from Drosophila to humans. A human homologue of Notch, TAN1, was initially identified at the chromosomal breakpoint of a subset of T-cell lymphoblastic leukemias/lymphomas containing a t(7;9) chromosomal translocation; however, its role in oncogenesis has been unclear. Using a bone marrow reconstitution assay with cells containing retrovirally transduced TAN1 alleles, we analyzed the oncogenic potential of both nuclear and extranuclear forms of truncated TAN1 in hematopoietic cells. Although the Moloney leukemia virus long terminal repeat drives expression in most hematopoietic cell types, retroviruses encoding either form of the TAN1 protein induced clonal leukemias of exclusively immature T cell phenotypes in approximately 50% of transplanted animals. All tumors overexpressed truncated TAN1 of the size and subcellular localization predicted from the structure of the gene. These results show that TAN1 is an oncoprotein and suggest that truncation and overexpression are important determinants of transforming activity. Moreover, the murine tumors caused by TAN1 in the bone marrow transplant model are very similar to the TAN1-associated human tumors and suggest that TAN1 may be specifically oncotropic for T cells.

Assignment of the receptor for ecotropic murine leukemia virus to mouse chromosome 5.

The gene for the receptor for ecotropic murine leukemia virus (Rev) has been assigned to mouse chromosome 5. This determination was made possible by an analysis of somatic cell hybrids between mouse and Chinese hamster cells. The parents of these hybrids were A/HeJ or Mus poschiavinus peritoneal exudate cells or BALB/c primary embryo fibroblasts and E36, a Chinese hamster lung fibroblast deficient in hypoxanthine guanine phosphoribosyltransferase. Segregation of mouse chromosomes in these hybrids was analyzed by chromosome banding and isozyme expression. Cells were tested for their ability to absorb and replicate vesicular stomatitis virus (murine leukemia virus [MuLV]) pseudotype particles and ecotropic MuLV as measured by the XC test. The presence of chromosome 5 was essential for receptor expression as determined by three statistical procedures. Segregation of the receptor for ecotropic murine leukemia virus was also followed in two series of subclones. In both, receptor expression was syntenic with phosphoglucomutase-1, an isozyme which has been mapped to mouse chromosome 5.

Signalling through SH2 and SH3 domains.

In 1986, Pawson's group recognized a region of homology between two oncogenic tyrosine kinases that lay outside the catalytic domain. They termed this the Src homology 2, or SH2, domain. In the ensuing years, SH2 domains have been found in an impressive variety of proteins, as has a second region of homology, inevitably termed SH3. These domains appear to mediate controlled protein-protein interactions. Many proteins that contain SH2 and SH3 domains are involved in signal transduction, suggesting a new paradigm for regulation of intracellular signalling pathways.

The COOH terminus of the c-Abl tyrosine kinase contains distinct F- and G-actin binding domains with bundling activity.

The myristoylated form of c-Abl protein, as well as the P210bcr/abl protein, have been shown by indirect immunofluorescence to associate with F-actin stress fibers in fibroblasts. Analysis of deletion mutants of c-Abl stably expressed in fibroblasts maps the domain responsible for this interaction to the extreme COOH-terminus of Abl. This domain mediates the association of a heterologous protein with F-actin filaments after microinjection into NIH 3T3 cells, and directly binds to F-actin in a cosedimentation assay. Microinjection and cosedimentation assays localize the actin-binding domain to a 58 amino acid region, including a charged motif at the extreme COOH-terminus that is important for efficient binding. F-actin binding by Abl is calcium independent, and Abl competes with gelsolin for binding to F-actin. In addition to the F-actin binding domain, the COOH-terminus of Abl contains a proline-rich region that mediates binding and sequestration of G-actin, and the Abl F- and G-actin binding domains cooperate to bundle F-actin filaments in vitro. The COOH terminus of Abl thus confers several novel localizing functions upon the protein, including actin binding, nuclear localization, and DNA binding. Abl may modify and receive signals from the F-actin cytoskeleton in vivo, and is an ideal candidate to mediate signal transduction from the cell surface and cytoskeleton to the nucleus.

I kappa B: a specific inhibitor of the NF-kappa B transcription factor.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein (I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated cells--in a specific, saturable, and reversible manner. The cytoplasmic localization of the complex of NF-kappa B and I kappa B was supported by enucleation experiments. An active phorbol ester must therefore, presumably by activation of protein kinase C, cause dissociation of a cytoplasmic complex of NF-kappa B and I kappa B by modifying I kappa B. this releases active NF-kappa B which can translocate into the nucleus to activate target enhancers. The data show the existence of a phorbol ester-responsive regulatory protein that acts by controlling the DNA binding activity and subcellular localization of a transcription factor.