Eosinophils and Megakaryocytes Support the Early Growth of Murine MOPC315 Myeloma Cells in Their Bone Marrow Niches

Multiple myeloma is a bone marrow plasma cell tumor which is supported by the external growth factors APRIL and IL-6, among others. Recently, we identified eosinophils and megakaryocytes to be functional components of the micro-environmental niches of benign bone marrow plasma cells and to be important local sources of these cytokines. Here, we investigated whether eosinophils and megakaryocytes also support the growth of tumor plasma cells in the MOPC315.BM model for multiple myeloma. As it was shown for benign plasma cells and multiple myeloma cells, IL-6 and APRIL also supported MOPC315.BM cell growth in vitro, IL-5 had no effect. Depletion of eosinophils in vivo by IL-5 blockade led to a reduction of the early myeloma load. Consistent with this, myeloma growth in early stages was retarded in eosinophil-deficient ΔdblGATA-1 mice. Late myeloma stages were unaffected, possibly due to megakaryocytes compensating for the loss of eosinophils, since megakaryocytes were found to be in contact with myeloma cells in vivo and supported myeloma growth in vitro. We conclude that eosinophils and megakaryocytes in the niches for benign bone marrow plasma cells support the growth of malignant plasma cells. Further investigations are required to test whether perturbation of these niches represents a potential strategy for the treatment of multiple myeloma.     

Thiostrepton-resistant mutants of Bacillus subtilis: Localization of resistance to the 50S subunit

Summary Sexual crosses were studied between mutants ofPhycomyces blakesleeanus with abnormal phototropism (phenotypemad). Recombination frequencies were determined among five genesmadA tomadE. No clear evidence was found for linkage between any of the genes. Inconsistent results in crosses involvingmadC are attributed to nonisogenicity between the particular strains used. Onemad strain was discovered to be a double mutant. A new gene, tentatively designatedmadG, was segregated from a cross involving that strain.     

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of (S)-aspartate semialdehyde and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). l-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper-l-lysine producing bacterial strains.     

The Mycobacterium tuberculosis High-Affinity Iron Importer,IrtA, Contains an FAD-Binding Domain

Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.′Mycobacterium tuberculosis, the causative agent of human tuberculosis, like most organisms, requires iron to sustain essential cellular functions. Due to the poor aqueous solubility of the ferric ion (Fe³⁺) in aerobic and neutral pH conditions, free ferric iron is not found in the mammalian host but is bound to iron-binding proteins such as transferrin, lactoferrin, and ferritin (30). A common mechanism by which bacteria acquire iron is the synthesis and secretion of siderophores (high-affinity iron chelators) that can solubilize iron in the environment or remove it from iron-binding proteins of the mammalian host. Fe³⁺-siderophore complexes are recognized by specific surface receptors and translocated through the plasma membrane by ABC-type transporters, using the energy generated by ATP hydrolysis (13). Dissociation of iron from the incorporated siderophore complex can occur via cleavage of the siderophore or by the action of a ferric reductase (13). Reduction of Fe³⁺ results in a weaker binding of Fe²⁺ to the siderophore, allowing release of iron that can then be utilized (21).To overcome iron limitation, M. tuberculosis synthesizes siderophores named mycobactin and exomycobactin. Mycobactin is very hydrophobic and remains cell associated, whereas exomycobactin (ExMB, also known as carboxymycobactin) is more hydrophilic and is secreted to the medium (8, 16). Fe³⁺-ExMB complexes can deliver iron to the cell by transfer of iron to mycobactin (7) or by a pathway that is mycobactin independent (17). Previously, we showed that inactivation of M. tuberculosis irtA (Rv1348) or irtB (Rv1349) genes, which encode membrane proteins of the ABC transporter family (2), results in decreased ability of M. tuberculosis to replicate in low-iron medium and to utilize Fe³⁺-ExMb as the sole iron source. Because IrtA and IrtB each encode a membrane protein with one permease domain fused to an ATPase domain, and irtA and irtB are organized in an operon, we postulated that these two proteins associate to form one ABC transporter necessary for iron acquisition in vitro and also for normal replication of M. tuberculosis in human macrophages and in infected mice lungs (18). We provide here evidence that supports a role for IrtAB as an iron importer and unveils essential properties of the amino-terminal domain (NTD) of IrtA. We propose a model by which IrtA-NTD couples iron transport to assimilation.     

Molecular dynamics simulation of metal free structure of Lmb,a laminin-binding adhesin of Streptococcus agalactiae: metal removal and its structural implications

Metal-binding receptors are one of the extracellular components of ATP-binding cassette transporters that are essential for regulation of metal homeostasis in bacteria. Laminin-binding adhesin (Lmb) of Streptococcus agalactiae falls under this class of solute binding proteins. It binds to zinc with a high affinity. Crystal structure of Lmb solved previously by our group reveals that the zinc is tetrahedrally coordinated by three histidines and a glutamate at the interdomain cleft. Lmb contains a long disordered loop close to the metal-binding site whose precise function is unknown. Several experimental attempts to produce apo-Lmb failed and this prompted us to carry out in silico studies to analyse the structural importance of the metal in Lmb. Here, we present the results of the molecular dynamics (MD) simulation studies of native, apo-(metal removed) and the long loop truncated Lmb models along with a homologous protein, TroA from Treponema pallidum that was taken up for validating the MD results of Lmb. Absence of a metal results in significant structural changes in Lmb, particularly at the metal-binding pocket and with the long loop, although the overall fold is retained. This study thus revealed that the Lmb can exist in different conformational states with subtle differences in the overall fold based on the presence or absence of the metal. This could be functionally important for a putative metal uptake and release and also for the adhesive function of Lmb in recognizing laminin, which contains a high number of zinc finger motifs.     

A Novel Anticancer Agent, 8-Methoxypyrimido[4′,5′:4,5]thieno(2,3-b) Quinoline-4(3H)-One Induces Neuro 2a Neuroblastoma Cell Death through p53-Dependent,Caspase-Dependent and -Independent Apoptotic Pathways

Neuroblastoma is the most common cancer in infants and fourth most common cancer in children. Despite recent advances in cancer treatments, the prognosis of stage-IV neuroblastoma patients continues to be dismal which warrant new pharmacotherapy. A novel tetracyclic condensed quinoline compound, 8-methoxypyrimido [4′,5′:4,5]thieno(2,3-b) quinoline-4(3H)-one (MPTQ) is a structural analogue of an anticancer drug ellipticine and has been reported to posses anticancer property. Study on MPTQ on neuroblastoma cells is very limited and mechanisms related to its cytotoxicity on neuroblastoma cells are completely unknown. Here, we evaluated the anticancer property of MPTQ on mouse neuro 2a and human SH-SY5Y neuroblastoma cells and investigated the mechanisms underlying MPTQ-mediated neuro 2a cell death. MPTQ-mediated neuro 2a and SH-SY5Y cell deaths were found to be dose and time dependent. Moreover, MPTQ induced cell death reached approximately 99.8% and 90% in neuro 2a and SH-SY5Y cells respectively. Nuclear oligonucleosomal DNA fragmentation and Terminal dUTP Nick End Labelling assays indicated MPTQ-mediated neuro 2a cell death involved apoptosis. MPTQ-mediated apoptosis is associated with increased phosphorylation of p53 at Ser15 and Ser20 which correlates with the hyperphosphorylation of Ataxia-Telangiectasia mutated protein (ATM). Immunocytochemical analysis demonstrated the increased level of Bax protein in MPTQ treated neuro 2a cells. MPTQ-mediated apoptosis is also associated with increased activation of caspase-9, -3 and -7 but not caspase-2 and -8. Furthermore, increased level of caspase-3 and cleaved Poly (ADP Ribose) polymerase were observed in the nucleus of MPTQ treated neuro 2a cells, suggesting the involvement of caspase-dependent intrinsic but not extrinsic apoptotic pathway. Increased nuclear translocation of apoptosis inducing factor suggests additional involvement of caspase-independent apoptosis pathway in MPTQ treated neuro 2a cells. Collectively, MPTQ-induced neuro 2a cell death is mediated by ATM and p53 activation, and Bax-mediated activation of caspase-dependent and caspase-independent mitochondrial apoptosis pathways.     

1-(1-acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.     

Methane production in rice soil is inhibited by cyanobacteria

The present study was aimed at understanding the role of cyanobacteria and Azolla in methane production and oxidation in laboratory simulation experiments using soil samples from rice fields. All the seven cyanobacterial strains tested effected a significant decrease in the headspace concentration of methane in flooded soil, incubated under light. Synechocystis sp. was the most effective in retarding methane concentration by 10-20 fold over that in controls without cyanobacteria. The decrease in the headspace concentration of methane was negligible in nonsterile soil samples, inoculated with Synechocystis sp. and then incubated under dark. Moist soil cores (0-5 cm depth), collected from rice fields that had been treated with urea in combination with a cyanobacterial mixture, Azolla microphylla, or cyanobacterial mixture plus A. microphylla, effected distinctly more rapid decrease in the headspace concentration of methane added at 200 microl(-1) than did the soil cores from plots treated with urea alone (30, 60, 90 and 120 kg N ha(-1)), irrespective of the rate of chemical nitrogen applied to rice fields. Besides, soil cores from plots treated with urea alone at 60, 90 and 120 kg N ha(-1) oxidised methane more rapidly than did the core samples from plots treated with urea alone at 30kg N ha(-1). Cyanobacteria and A. microphylla, applied to flood water, appear to play a major role in mitigation of methane emission from rice fields-through enhanced methane oxidation.