Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

A convenient synthesis of adenylyl-(2'----5')-adenylyl-(2'----5')-adenosine (2-5A core)

A simple synthesis of adenylyl-(2'----5')-adenylyl (2'----5')-adenosine (2-5A core) has been achieved on the basis of selective 3'-O-silylation of 5'-O-p-monomethoxytrityladenosine and chemo-selective formation of the 2'-5' internucleotide linkage using N-unprotected nucleosides.     

Analogs of (2'-5')oligo(A). Endonuclease activation and inhibition of protein synthesis in intact cells

Synthetic analogs of (2'-5')oligo(A) were assayed for endonuclease activation in cell extracts and for inhibition of protein synthesis in intact cells. The analogs are triadenylates: (i) methylated in the terminal 3'-OH; (ii) methylated at all three 3'-OH groups; (iii) with different numbers of phosphate groups at the 5' terminus or with a methylene group between the beta- and gamma-phosphate. Only 5'-phosphorylated monomethylated analogs activate an endonuclease in cell extracts and are powerful inhibitors of protein synthesis in intact cells. The analogs with only one 5'-terminal phosphate may require addition of another phosphate for activity since the kinase inhibitor 2-aminopurine prevents endonuclease activation by this compound but not by the di- and triphosphate-terminated triadenylates. These results suggest that two terminal phosphates and one or two free 3'-OH are required for endonuclease activation and inhibition of protein synthesis. The monomethylated analogs are more active than (2'-5')pppA3 because of their resistance to degradation by cellular enzymes. Accordingly, the monomethylated analogs cause a prolonged inhibition of protein synthesis in human fibroblasts treated with nanomolar concentrations of these compounds.     

Lineage(-)Sca1+c-Kit(-)CD25+ cells are IL-33-responsive type 2 innate cells in the mouse bone marrow

IL-33 promotes type 2 immune responses, both protective and pathogenic. Recently, targets of IL-33, including several newly discovered type 2 innate cells, have been characterized in the periphery. In this study, we report that bone marrow cells from wild-type C57BL/6 mice responded with IL-5 and IL-13 production when cultured with IL-33. IL-33 cultures of bone marrow cells from Rag1 KO and Kit(W-sh/W-sh) mice also responded similarly; hence, eliminating the possible contributions of T, B, and mast cells. Rather, intracellular staining revealed that the IL-5- and IL-13-positive cells display a marker profile consistent with the Lineage(-)Sca-1(+)c-Kit(-)CD25(+) (LSK(-)CD25(+)) cells, a bone marrow cell population of previously unknown function. Freshly isolated LSK(-)CD25(+) cells uniformly express ST2, the IL-33 receptor. In addition, culture of sorted LSK(-)CD25(+) cells showed that they indeed produce IL-5 and IL-13 when cultured with IL-33 plus IL-2 and IL-33 plus IL-7. Furthermore, i.p. injections of IL-33 or IL-25 into mice induced LSK(-)CD25(+) cells to expand, in both size and frequency, and to upregulate ST2 and α(4)β(7) integrin, a mucosal homing marker. Thus, we identify the enigmatic bone marrow LSK(-)CD25(+) cells as IL-33 responsive, both in vitro and in vivo, with attributes similar to other type 2 innate cells described in peripheral tissues.     

Hepatic microsomal enzyme induction by 2, 2', 3, 3', 4, 4'- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl

In an attempt to resolve conflicting reports in the literature, the effects of 2, 2', 3, 3', 4, 4'-hexa- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl on the hepatic microsomal drug-metabolizing enzymes were evaluated in the immature male rat. By comparison with the effects of the classical enzyme inducers, phenobarbitone (PB) and 3-methylcholanthrene (MC), 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl, irrespective of its synthetic route, exhibited PB- and MC-type characteristics; the most prominent feature of the latter being a seven-fold increase in 4-chlorobiphenyl hydroxylase activity. 2, 2', 3', 4, 4', 5-Hexachlorobiphenyl also resembled a mixed (PB + MC)-type inducer although its MC-type characteristics were more pronounced than those of 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl.     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.     

Interleukin 10 inhibits transforming growth factor-beta (TGF-beta) synthesis required for osteogenic commitment of mouse bone marrow cells

Interleukin 10 (IL-10) suppressed TGF-beta synthesis in mouse bone marrow cultures. Coincidingly, IL-10 down-regulated the production of bone proteins including alkaline phosphatase (ALP), collagen and osteocalcin, and the formation of mineralized extracellular matrix. The mAb 1D11.16 which neutralizes TGF-beta 1 and TGF-beta 2, induced suppressive effects comparable to IL-10 when administered before the increase of cell proliferation in the culture. It appears that mainly TGF-beta 1 plays a role in this system since (a) TGF-beta 2 levels were undetectable in supernatants from osteogenic cultures, (b) no effect was observed when the anti-TGF-beta 2 neutralizing mAb 4C7.11 was added and (c) the suppressive effect of IL-10 could be reversed by adding exogenous TGF-beta 1. It is unlikely that TGF-beta 1 modulates osteogenic differentiation by changing the proliferative potential of marrow cells since 1D11.16 did not affect [3H]thymidine ([3H]TdR) incorporation or the number of fibroblast colony forming cells (CFU-F) which harbor the osteoprogenitor cell population. Furthermore, 1D11.16 did not alter [3H]TdR uptake by the cloned osteoprogenitor cell lines MN7 and MC3T3. Light and scanning electron microscopy showed that IL-10 and 1D11.16 induced comparable morphological changes in the marrow cultures. Control cultures contained flat adherent cells embedded in a mineralized matrix. In contrast, IL-10 and 1D11.16 treated cultures were characterized by round non-adherent cells and the absence of a mineralized matrix. In this study, the mechanism by which IL-10 suppresses the osteogenic differentiation of mouse bone marrow was identified as inhibition of TGF-beta 1 production which is essential for osteogenic commitment of bone marrow cells.     

Retinoic acid inhibits the growth of bone marrow mesenchymal stem cells and induces p27Kip1 and p16INK4A up-regulation

The importance of bone marrow mesenchymal stem cells in hemopoiesis has been definitely demonstrated. Thus, their impairment might cause profound alteration on production and maturation of blood cells. In the present paper, we investigated, for the first time, the effect of retinoic acid, an important antileukemic molecule, on the proliferation of primary cultures of human bone marrow mesenchymal stem cells. We demonstrated that retinoic acid, at a pharmacological concentration, hampers strongly the growth of the cells, without inducing osteoblastic differentiation. The analysis of cell division cycle machinery showed that the antiproliferative effect is associated with (i) the up-regulation of two cyclin-dependent kinase inhibitors, namely p27^Kip1 and p16^INK4A, and (ii) the down-regulation of cyclin-dependent kinase 2 activity and pRB phosphorylation. The reported findings represent novel insights into the antileukemic effects of the drug and contribute in clarifying the molecular mechanism of its pharmacological activity.     

New synthesis of 2β-hydroxy-19-oxoandrost-4-ene-3,17-dione and its 2β-O analog

Treatment of 19-[oxygenated]-androst-4-ene-3,17-dione with Mn(AcO)₃ and ClCH₂COOH in benzene gave epimeric mixtures of the corresponding 2ξ-chloroacetates and 2ξ-acetates. The products were processed to give the title compound. For the synthesis of the 2-¹⁸O analog, ClCH₂C¹⁸OOH was used, which was prepared from ClCH₂COCl.     

A subpopulation of t(2;14)(p11;q32) cells in ataxia telangiectasia B lymphocytes

Summary Partially purified B cells from ataxia telangiectasia (A-T) patients and normal individuals were stimulated with Staphylococcus aureus Cowan I organisms (SAC). High levels of apparently random rearrangements were seen in the A-T B cells only. In addition a t(2;14)(p11;q32) rearrangement was identified in B cells from more than one patient.     

Activity of a nitroalkene derivative, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene, in the Salmonella/microsome assay and the mouse bone marrow micronucleus test

Mutagenicity of a substituted nitroalkene, 1-(5-bromofur-2-il)-2-bromo-2-nitroethene (BNF) was tested in the Salmonella/microsome assay using the strains TA 98, TA 100 and TA 100NR (nitroreductase deficient). BNF was a direct mutagen in TA 98 and TA 100; the response was lowered when exogenous metabolic activation (S9) was used. A further decrease in mutagenicity was observed in strain TA 100NR, as compared to the parental TA 100, which showed the involvement of nitroreduction in the overall response elicited by BNF. The micronucleus assay was carried out in Swiss male mice which were given a single i.p. dose of 10–20 mg/kg of BNF dissolved in peanut oil, bone marrow being sampled 24 and 48 h later. The micronucleated polychromatic erythrocyte counts (MNPCE) showed a weak response in the dose range of 10–17.5 mg/kg at the second sampling (48 h) and a significant rise for 20 mg/kg at 24 and 48 h.     

Characterization of new bone morphogenetic protein (Bmp)‐2 regulatory alleles

Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell proliferation, differentiation, and apoptosis. Key events requiring precise Bmp2 regulation include heart specification and morphogenesis and neural development. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence pattern formation and organogenesis, the mechanisms that regulate Bmp2 are crucial. A sequence within the 3'UTR of the Bmp2 mRNA termed the “ultra‐conserved sequence” (UCS) has been largely unchanged since fishes and mammals diverged. Cre‐lox mediated deletion of the UCS in a reporter transgene revealed that the UCS may repress Bmp2 in proepicardium, epicardium, and epicardium‐derived cells (EPDC) and in tissues with known epicardial contributions (coronary vessels and valves). The UCS also repressed the transgene in the aorta, outlet septum, posterior cardiac plexus, cardiac and extra‐cardiac nerves, and neural ganglia. We used homologous recombination and conditional deletion to generate three new alleles in which the Bmp2 3'UTR was altered as follows: a UCS flanked by loxP sites with or without a neomycin resistance targeting vector, or a deleted UCS. Deletion of the UCS was associated with elevated Bmp2 mRNA and BMP signaling levels, reduced fitness, and embryonic malformations.