Is it time to test biguanide metformin in the treatment of melanoma?

Metformin is the most widely used antidiabetic drug that belongs to the biguanide class. It is very well tolerated and has the major clinical advantage of not inducing hypoglycemia. Metformin decreases hepatic glucose production via a mechanism requiring liver kinase B1, which controls the metabolic checkpoint, AMP‐activated protein kinase‐mammalian target of rapamycin and neoglucogenic genes. The effects of metformin on this pathway results in reduced protein synthesis and cell proliferation. These observations have given the impetus for many investigations on the role of metformin in the regulation of tumor cell proliferation, cell‐cycle regulation, apoptosis, and autophagy. Encouraging results from these studies have shown that metformin could potentially be used as an efficient anticancer drug in various neoplasms such as prostate, breast, lung, pancreas cancers, and melanoma. These findings are strengthened by retrospective epidemiological studies that have found a decrease in cancer risk in diabetic patients treated with metformin. In this review, we have focused our discussion on recent molecular mechanisms of metformin that have been described in various solid tumors in general and in melanoma in particular.     

Microarray and Proteomic Analyses of Myeloproliferative Neoplasms with a Highlight on the mTOR Signaling Pathway

The gene and protein expression profiles in myeloproliferative neoplasms (MPNs) may reveal gene and protein markers of a potential clinical relevance in diagnosis, treatment and prediction of response to therapy. Using cDNA microarray analysis of 25,100 unique genes, we studied the gene expression profile of CD34⁺ cells and granulocytes obtained from peripheral blood of subjects with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF). The microarray analyses of the CD34⁺ cells and granulocytes were performed from 20 de novo MPN subjects: JAK2 positive ET, PV, PMF subjects, and JAK2 negative ET/PMF subjects. The granulocytes for proteomic studies were pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. The number of differentially regulated genes was about two fold larger in CD34⁺ cells compared to granulocytes. Thirty-six genes (including RUNX1, TNFRSF19) were persistently highly expressed, while 42 genes (including FOXD4, PDE4A) were underexpressed both in CD34⁺ cells and granulocytes. Using proteomic studies, significant up-regulation was observed for MAPK and PI3K/AKT signaling regulators that control myeloid cell apoptosis and proliferation: RAC2, MNDA, S100A8/9, CORO1A, and GNAI2. When the status of the mTOR signaling pathway related genes was analyzed, PI3K/AKT regulators were preferentially up-regulated in CD34⁺ cells of MPNs, with down-regulated major components of the protein complex EIF4F. Molecular profiling of CD34⁺ cells and granulocytes of MPN determined gene expression patterns beyond their recognized function in disease pathogenesis that included dominant up-regulation of PI3K/AKT signaling.     

The bicentenary of the research on ‘beautiful’ vavilovia (Vavilovia formosa), a legume crop wild relative with taxonomic and agronomic potential

Vavilovia formosa is a relict, endangered species from the highlands of the Caucasus and the Near East. Described in 1812, it has had an uncertain status and was finally recognized as a separate genus of tribe Fabeae (Fabaceae). Our informal international group was established in 2007 to revive the interest in this species as it had been seriously neglected for decades. Here, we provide an overview of the accumulated knowledge on V. formosa and present the results of the most recent multidisciplinary research. Three expeditions were made to two locations in Armenia in 2009, providing the material for anatomical, morphological, chemical and molecular analysis. Unlike previous attempts, ex situ conservation in Yerevan and in vitro propagation, important for potential interspecific hybridization, were successful. Molecular tools were used to clarify the taxonomic position of V. formosa, often considered the closest to the extinct ancestor of the whole tribe. The analysis of four informative regions of plastid and nuclear DNA showed that V. formosa belongs to the same clade as Lathyrus and Pisum, with a distinct status. Preservation and maintenance of V. formosa remains the only basis for further development of all other scientific aspects, especially breeding and uses in agronomy. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 172 , 524–531.     

Impact of estradiol on insulin signaling in the rat heart

It is well known that variation in the concentration of estrogens affects insulin action. In this study we examine the impact of estradiol (E2) on insulin signaling in the rat heart. Ovariectomized female rats were treated with E2 6 h prior to analysis of basal protein and mRNA content of insulin signaling molecules, and additionally with insulin 30 min before the experiment to delineate E2 effects on phosphorylations and molecular associations relevant for insulin signaling. The results show that E2 decreased insulin receptor (IR) tyrosine phosphorylation, while it did not alter IR protein and mRNA content. E2 administration did not change IR substrate 1 (IRS‐1) protein content and tyrosine phosphorylation, while decreased mRNA content and increased its association with the p85 subunit of phosphatidylinositol 3‐kinase (PI3K). E2 decreased protein and mRNA content of IR substrate 2 (IRS‐2), while did not change IRS‐2 tyrosine phosphorylation and IRS‐2 association with p85. The increase of IRS‐1/p85 is accompanied by increase of p85 protein and mRNA levels, and by stimulation of protein kinase B (Akt) Ser⁴⁷³ phosphorylation. In contrast, Akt protein and mRNA content were not changed. In summary, although in some aspects cardiac insulin signaling is obviously improved by E2 treatment (increase of p85 mRNA and protein levels, enhancement of IRS‐1/p85 association and Ser⁴⁷³Akt phosphorylation), the observed decrease of IR tyrosine phosphorylation, IRS‐2 protein content, and IRSs mRNA contents, suggest very complex interplay of beneficial and suppressive effects of E2, both genomic and non‐genomic, in regulation of heart insulin signaling. Copyright © 2009 John Wiley & Sons, Ltd.     

Domain-based assays of individual antibody concentrations in an oligoclonal combination targeting a single protein

Quantitation of individual monoclonal antibodies (mAbs) within a combined antibody drug product is required for preclinical and clinical drug development, including pharmacokinetic (PK), toxicology, stability, and biochemical characterization studies of such drugs. We have developed an antitoxin, XOMA 3AB, consisting of three recombinant mAbs that potently neutralize the known subtypes of type A botulinum neurotoxin (BoNT/A). The three mAbs bind nonoverlapping BoNT/A epitopes with high affinity. XOMA 3AB is being developed as a treatment for botulism resulting from BoNT/A. To develop antibody-specific assays, we cloned, expressed, and purified BoNT/A domains from Escherichia coli. Each mAb bound only to its specific domain with affinity comparable to the binding to holotoxin. mAb-specific domains were used to develop an enzyme-linked immunosorbent assay (ELISA) for characterization of the integrity and binding activity of the three mAbs in the drug product. An electrochemiluminescence bridging assay that is robust to interference from components in serum was also developed, and we demonstrate that it can be used for PK assays. This type of antigen engineering to generate mAb-specific domains is a general method allowing quantitation and characterization of individual mAbs in a mAb cocktail that binds the same protein and is superior to anti-idiotype approaches.     

Mast cell degranulation mediates compound 48/80-induced hyperalgesia in mice

Mast cells mediate allergies, hypersensitivities, host defense, and venom neutralization. An area of recent interest is the contribution of mast cells to inflammatory pain. Here we found that specific, local activation of mast cells produced plantar hyperalgesia in mice. Basic secretagogue compound 48/80 induced plantar mast cell degranulation accompanied by thermal hyperalgesia, tissue edema, and neutrophil influx in the hindpaws of ND4 Swiss mice. Blocking mast cell degranulation, neutrophil extravasation, and histamine signaling abrogated these responses. Compound 48/80 also produced edema, pain, and neutrophil influx in WT C57BL/6 but not in genetically mast cell-deficient C57BL/6-Kit(W-sh)(/)(W-sh) mice. These responses were restored following plantar reconstitution with bone marrow-derived cultured mast cells.     

A modular approach to the design of protein-based smart gels

The modular nature of repeat proteins makes them a versatile platform for the design of smart materials with predetermined properties. Here, we present a general strategy for combining protein modules with specified stability and function into arrays for the assembly of stimuli-responsive gels. We have designed tetratricopeptide repeat (TPR) arrays which contain peptide-binding modules that specify the strength and reversibility of network crosslinking in combination with spacer modules that specify crosslinking geometry and overall stability of the array. By combining such arrays with multivalent peptide ligands, self-supporting stimuli-responsive gels are formed. Using microrheology, we characterized the kinetics of gelation as a function of concentration and stoichiometry of the components. We also show that such gels are effective in encapsulating and releasing small molecules. Moreover, TPR gels alone are fully compatible with cell growth, whereas gels loaded with an anticancer compound release the compound, resulting in cell death. Thus, we have demonstrated that this new class of tunable biomaterials is ripe for further development as tissue engineering and drug delivery platform.     

Determination of the target nucleosides for members of two families of 16S rRNA methyltransferases that confer resistance to partially overlapping groups of aminoglycoside antibiotics

The 16S ribosomal RNA methyltransferase enzymes that modify nucleosides in the drug binding site to provide self-resistance in aminoglycoside-producing micro-organisms have been proposed to comprise two distinct groups of S-adenosyl-l-methionine (SAM)-dependent RNA enzymes, namely the Kgm and Kam families. Here, the nucleoside methylation sites for three Kgm family methyltransferases, Sgm from Micromonospora zionensis, GrmA from Micromonospora echinospora and Krm from Frankia sp. Ccl3, were experimentally determined as G1405 by MALDI-ToF mass spectrometry. These results significantly extend the list of securely characterized G1405 modifying enzymes and experimentally validate their grouping into a single enzyme family. Heterologous expression of the KamB methyltransferase from Streptoalloteichus tenebrarius experimentally confirmed the requirement for an additional 60 amino acids on the deduced KamB N-terminus to produce an active methyltransferase acting at A1408, as previously suggested by an in silico analysis. Finally, the modifications at G1405 and A1408, were shown to confer partially overlapping but distinct resistance profiles in Escherichia coli. Collectively, these data provide a more secure and systematic basis for classification of new aminoglycoside resistance methyltransferases from producers and pathogenic bacteria on the basis of their sequences and resistance profiles.     

Complete genome sequence of Slackia heliotrinireducens type strain (RHS 1)

Stackebrandtia nassauensis Labeda and Kroppenstedt (2005) is the type species of the genus Stackebrandtia, and a member of the actinobacterial family Glycomycetaceae. Stackebrandtia currently contains two species, which are differentiated from Glycomyces spp. by cellular fatty acid and menaquinone composition. Strain LLR-40K-21(T) is Gram-positive, aerobic, and nonmotile, with a branched substrate mycelium and on some media an aerial mycelium. The strain was originally isolated from a soil sample collected from a road side in Nassau, Bahamas. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of the actinobacterial suborder Glycomycineae. The 6,841,557 bp long single replicon genome with its 6487 protein-coding and 53 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Complete genome sequence of Halorhabdus utahensis type strain (AX-2)

Halorhabdus utahensis Wainø et al. 2000 is the type species of the genus, which is of phylogenetic interest because of its location on one of the deepest branches within the very extensive euryarchaeal family Halobacteriaceae. H. utahensis is a free-living, motile, rod shaped to pleomorphic, Gram-negative archaeon, which was originally isolated from a sediment sample collected from the southern arm of Great Salt Lake, Utah, USA. When grown on appropriate media, H. utahensis can form polyhydroxybutyrate (PHB). Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the a member of halobacterial genus Halorhabdus, and the 3,116,795 bp long single replicon genome with its 3027 protein-coding and 48 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.