Activin A is an essential cofactor for osteoclast induction

Recently, receptor activator of NF-kappaB ligand (RANKL) was shown to be necessary for osteoclast formation. We now report that activin A, a cytokine enriched in bone matrix and secreted by osteoblasts and osteoclasts, powerfully synergized with RANKL for induction of osteoclast-like cells (OCL) from bone marrow precursors depleted of stromal cells. Moreover, OCL formation in RANKL was virtually abolished by soluble type II A activin receptors (ActR-II(A)), suggesting that activin A is essential for OCL formation. Activin A was most effective when precursors were exposed to RANKL and activin A simultaneously: resistance to OCL-induction that occurs when precursors are pre-incubated in M-CSF was reduced. Incubation on bone matrix also enhanced the sensitivity of precursors to OCL-induction by RANKL; and this was prevented by soluble ActR-II(A). Thus, activin A in bone matrix, or released from osteoblastic or other cells, enhances the osteoclast-forming potential of precursors and synergizes with RANKL in inducing osteoclastic differentiation.     

Drosophila nemo is an essential gene involved in the regulation of programmed cell death

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development.     

The menin tumor suppressor protein is an essential oncogenic cofactor for MLL-associated leukemogenesis

The Mixed-Lineage Leukemia (MLL) protein is a histone methyltransferase that is mutated in clinically and biologically distinctive subsets of acute leukemia. MLL normally associates with a cohort of highly conserved cofactors to form a macromolecular complex that includes menin, a product of the MEN1 tumor suppressor gene, which is mutated in heritable and sporadic endocrine tumors. We demonstrate here that oncogenic MLL fusion proteins retain an ability to stably associate with menin through a high-affinity, amino-terminal, conserved binding motif and that this interaction is required for the initiation of MLL-mediated leukemogenesis. Furthermore, menin is essential for maintenance of MLL-associated but not other oncogene induced myeloid transformation. Acute genetic ablation of menin reverses aberrant Hox gene expression mediated by MLL-menin promoter-associated complexes, and specifically abrogates the differentiation arrest and oncogenic properties of MLL-transformed leukemic blasts. These results demonstrate that a human oncoprotein is critically dependent on direct physical interaction with a tumor suppressor protein for its oncogenic activity, validate a potential target for molecular therapy, and suggest central roles for menin in altered epigenetic functions underlying the pathogenesis of hematopoietic cancers.     

Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent

Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells from both wild-type (WT) and caveolin-1-deficient mice. Activation of PKC by phorbol myristate acetate (PMA) (100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in caveolin-1-deficient monolayers. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA significantly reduced bead binding to 78±6% of controls in WT endothelial cells without any effect in caveolin-1-deficient cells. In WT cells, PMA caused an 86±18% increase in FITC-dextran permeability whereas no increase in permeability was observed in caveolin-1-deficient monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and caveolin-1-deficient MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by the reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1.     

glnE is an essential gene in Mycobacterium tuberculosis

Mycobacterium tuberculosis possesses a homologue of glnE, potentially encoding a regulator of glutamine synthetase activity. We attempted to construct glnE-disrupted mutants using a two-step strategy, whereby a single-crossover strain was first isolated, followed by sacB counterselection to isolate the double-crossover strain. Of 192 sucrose-resistant colonies tested, none were mutants, although the wild-type double crossover could be easily isolated. When a second copy of the wild-type glnE was integrated into the chromosome, we could isolate both wild-type and mutant double-crossover strains. Thus, the chromosomal gene could only be replaced with a disrupted copy when another functional copy of the gene was provided, demonstrating that this gene is essential under the conditions tested.     

There is an alpha-actin skeletal muscle-specific gene in a salamander (Pleurodeles waltlii)

We cloned, from a cDNA library, an alpha-actin sequence from a salamander (Pleurodeles waltlii), which codes for the 125 COOH-terminal amino acid residues of a skeletal muscle actin (without any difference from the corresponding protein of warm blood vertebrates). An important conservation in the 3' untranslated region between this sequence and skeletal alpha-actin genes of chicken and man was noted. These results demonstrate, contrary to what was thought previously, that there exists in salamander a true skeletal alpha-actin gene. The results suggest that striated muscle actin genes in lower vertebrates could be a mosaic of cardiac and skeletal-specific amino acid residues, and that the divergence between these two types of genes is older than the NH2-terminal analysis of actins suggested previously.     

Iron: an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

The activity of the ethylene-forming enzyme (EFE) in suspension-cultured tomato (Lycopersicon esculentum Mill.) cells was almost completely abolished within 10 min by 0.4 mM of the metal-chelating agent 1,10-phenanthroline. Subsequent addition of 0.4 mM FeSO₄ immediately reversed this inhibition. A partial reversion was also obtained with 0.6 mM CuSO₄ and ZnSO₄, probably as a consequence of the release of iron ions from the 1,10-phenanthroline complex. The inhibition was not reversed by Mn²⁺ or Mg²⁺. Tomato cells starved of iron exhibited a very low EFE activity. Addition of Fe²⁺ to these cells caused a rapid recovery of EFE while Cu²⁺, Zn²⁺ and other bivalent cations were ineffective. The recovery of EFE activity in iron-starved cells was insensitive to cycloheximide and therefore does not appear to require synthesis of new protein. The EFE activity in tomato cells was induced by an elicitor derived from yeast extract. Throughout the course of induction, EFE activity was blocked within 10–20 min by 1,10-phenanthroline, and the induced level was equally rapidly restored after addition of iron. We conclude that iron is an essential cofactor for the conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene in vivo.     

Cdk1 is essential for mammalian cyclosome/APC regulation

The cyclosome/APC (anaphase-promoting complex), the major component of cell-cycle-specific ubiquitin-mediated proteolysis of mitotic cyclins and of other cell cycle proteins, is essential for sister chromatid separation and for exit from mitosis. Cyclosome activity and substrate specificity are modulated by phosphorylation and by transient interactions with Fizzy/cdc20 (Fzy) and Fizzy-related/Hct1/Cdh1 (Fzr). This regulation has been studied so far in Drosophila embryos, in yeast, and in cell-free extracts in vitro. Studying cyclosome regulation in mammalian cells in vivo we found that both Fzr overexpression and Cdk1 inhibition can override the prometaphase checkpoint. We further show that Fzr activation of the cyclosome is negatively regulated by Cdk1. Finally, we show that the mammalian cdc14 phosphatase, like its budding yeast homologue, plays a role in cyclosome pathway regulation. These results suggest that Cdk1 is essential for coupling various activities of the cyclosome and in particular for preventing Fzr from short-circuiting the spindle pole checkpoint. Cdk1-cyclin B is thus an inhibitor, activator, and substrate of the cyclosome.     

IL-1 receptor-mediated signal is an essential component of MyD88-dependent innate response to Mycobacterium tuberculosis infection

MyD88, the common adapter involved in TLR, IL-1, and IL-18 receptor signaling, is essential for the control of acute Mycobacterium tuberculosis (MTB) infection. Although TLR2, TLR4, and TLR9 have been implicated in the response to mycobacteria, gene disruption for these TLRs impairs only the long-term control of MTB infection. Here, we addressed the respective role of IL-1 and IL-18 receptor pathways in the MyD88-dependent control of acute MTB infection. Mice deficient for IL-1R1, IL-18R, or Toll-IL-1R domain-containing adaptor protein (TIRAP) were compared with MyD88-deficient mice in an acute model of aerogenic MTB infection. Although primary MyD88-deficient macrophages and dendritic cells were defective in cytokine production in response to mycobacterial stimulation, IL-1R1-deficient macrophages exhibited only a reduced IL-12p40 secretion with unaffected TNF, IL-6, and NO production and up-regulation of costimulatory molecules CD40 and CD86. Aerogenic MTB infection of IL-1R1-deficient mice was lethal within 4 wk with 2-log higher bacterial load in the lung and necrotic pneumonia but efficient pulmonary CD4 and CD8 T cell responses, as seen in MyD88-deficient mice. Mice deficient for IL-18R or TIRAP controlled acute MTB infection. These data demonstrate that absence of IL-1R signal leads to a dramatic defect of early control of MTB infection similar to that seen in the absence of MyD88, whereas IL-18R and TIRAP are dispensable, and that IL-1, together with IL-1-induced innate response, might account for most of MyD88-dependent host response to control acute MTB infection.     

Pap1-dependent regulation of the GSTII gene from the fission yeast

The genomic DNA encoding a second glutathione S-transferase (GSTII) was previously isolated from the fission yeast Schizosaccharomyces pombe. Its expression was shown to be induced by menadione, mercuric chloride, o-dinitrobenzene, and NO-generating S-nitroso-N-acetylpenicillamine using the GSTII-lacZ fusion harboring the 910 bp upstream region from the translational initiation point. In this study, the additional fusion plasmids pGST50-590 and pGST50-6R-590 were constructed to carry the 590 bp upstream region in the vectors YEp357 and YEp367R, respectively. The synthesis of beta-galactosidase from the fusion plasmid pGST50-590 was about 3-fold higher than that from the fusion plasmid pGST50-F, indicating the presence of negatively activating sequence in the -910 to approximately -590 region. It was also enhanced by the same agents, which induced the synthesis of beta-galactosidase from the fusion plasmid pGST50-F. The synthesis of beta-galactosidase from both fusion plasmids pGST50-F and pGST50-590 was enhanced by the overexpressed Pap1 protein. The synthesis of beta-galactosidase from the two YEp367R derivatives pGST50-6R-F and pGST50-6R-590 was greatly decreased in the Pap1-negative strain TP108-3C. These results propose the Pap1-dependent regulation of the GSTII gene from the fission yeast.