Novel rearrangement of purines

Summary 6-Trichloromethyl-9-methylpurine (1) rearranges to 6-dichloromethyl-9-methyl-8-oxopurine (2) in aqueous mild acidic solution. The rearrangement is rationalized in terms of a reaction involving protonation, covalent hydration, prototropic equilibrium and/or a hydride transfer. An alternative mechanism involving a positive halogen compound and hypochlorous acid as an intermediary is also proposed. Compound1 condenses with 4,5-diaminopyrimidine to give the purine-pyrimidine Schiff base pair4.Acknowledgments. We are deeply indebted to Professor S. Cohen of the Sackler School of Medicine, Tel Aviv University (Ramat Aviv, Isreal) for his advice and encouragement. Support of this research by the Israel Cancer Association, the Ber-Lamsdorf Foundation Switzerland Israel and by the Advancement of Mankind Foundation, is gratefully acknowledged. We thank Proff. D. Arigoni and A. Eschenmoser, ETH Zürich, for their valuable proposals and comments on the mechanism of the rearrangement.     

ALCAM/CD166 adhesive function is regulated by the tetraspanin CD9

ALCAM/CD166 is a member of the immunoglobulin superfamily of cell adhesion molecules (Ig-CAMs) which mediates intercellular adhesion through either homophilic (ALCAM–ALCAM) or heterophilic (ALCAM–CD6) interactions. ALCAM-mediated adhesion is crucial in different physiological and pathological phenomena, with particular relevance in leukocyte extravasation, stabilization of the immunological synapse, T cell activation and proliferation and tumor growth and metastasis. Although the functional implications of ALCAM in these processes is well established, the mechanisms regulating its adhesive capacity remain obscure. Using confocal microscopy colocalization, and biochemical and functional analyses, we found that ALCAM directly associates with the tetraspanin CD9 on the leukocyte surface in protein complexes that also include the metalloproteinase ADAM17/TACE. The functional relevance of these interactions is evidenced by the CD9-induced upregulation of both homophilic and heterophilic ALCAM interactions, as reflected by increased ALCAM-mediated cell adhesion and T cell migration, activation and proliferation. The enhancement of ALCAM function induced by CD9 is mediated by a dual mechanism involving (1) augmented clustering of ALCAM molecules, and (2) upregulation of ALCAM surface expression due to inhibition of ADAM17 sheddase activity.     

Antitumoral effects of 9-cis retinoic acid in adrenocortical cancer

The currently available medical treatment options of adrenocortical cancer (ACC) are limited. In our previous meta-analysis of adrenocortical tumor genomics data, ACC was associated with reduced retinoic acid production and retinoid X receptor-mediated signaling. Our objective has been to study the potential antitumoral effects of 9-cis retinoic acid (9-cisRA) on the ACC cell line NCI-H295R and in a xenograft model. Cell proliferation, hormone secretion, and gene expression have been studied in the NCI-H295R cell line. A complex bioinformatics approach involving pathway and network analysis has been performed. Selected genes have been validated by real-time qRT-PCR. Athymic nude mice xenografted with NCI-H295R have been used in a pilot in vivo xenograft model. 9-cisRA significantly decreased cell viability and steroid hormone secretion in a concentration- and time-dependent manner in the NCI-H295R cell line. Four major molecular pathways have been identified by the analysis of gene expression data. Ten genes have been successfully validated involved in: (1) steroid hormone secretion (HSD3B1, HSD3B2), (2) retinoic acid signaling (ABCA1, ABCG1, HMGCR), (3) cell-cycle damage (GADD45A, CCNE2, UHRF1), and the (4) immune response (MAP2K6, IL1R2). 9-cisRA appears to directly regulate the cell cycle by network analysis. 9-cisRA also reduced tumor growth in the in vivo xenograft model. In conclusion, 9-cisRA might represent a promising new candidate in the treatment of hormone-secreting adrenal tumors and adrenocortical cancer.     

A discoidin domain receptor 1 knock-out mouse as a novel model for osteoarthritis of the temporomandibular joint

Discoidin domain receptor 1 (DDR-1)-deficient mice exhibited a high incidence of osteoarthritis (OA) in the temporomandibular joint (TMJ) as early as 9 weeks of age. They showed typical histological signs of OA, including surface fissures, loss of proteoglycans, chondrocyte cluster formation, collagen type I upregulation, and atypical collagen fibril arrangements. Chondrocytes isolated from the TMJs of DDR-1-deficient mice maintained their osteoarthritic characteristics when placed in culture. They expressed high levels of runx-2 and collagen type I, as well as low levels of sox-9 and aggrecan. The expression of DDR-2, a key factor in OA, was increased. DDR-1-deficient chondrocytes from the TMJ were positively influenced towards chondrogenesis by a three-dimensional matrix combined with a runx-2 knockdown or stimulation with extracellular matrix components, such as nidogen-2. Therefore, the DDR-1 knock-out mouse can serve as a novel model for temporomandibular disorders, such as OA of the TMJ, and will help to develop new treatment options, particularly those involving tissue regeneration.     

A Study of Babylonian Observations of Planets Near Normal Stars

The present paper is an attempt to describe the observational practices behind a large and homogeneous body of Babylonian observation reports involving planets and certain bright stars near the ecliptic (Normal Stars). The reports in question are the only precise positional observations of planets in the Babylonian texts, and while we do not know their original purpose, they may have had a part in the development of predictive models for planetary phenomena in the second half of the first millennium B.C. The paper is organized according to the following topics: (I) Sections 1–3 review the format of the observations and the texts in which they are found; (II) Sections 4–6 discuss the composition of the Normal Star list; (III) Sections 7–8 concern the orientation of the reported celestial directions from star to planet; (IV) Sect. 9 concerns the relationship between the reported distances and the actual angular distances between planet and star; and (V) Sect. 10 discusses the reports of planetary stations, which are the most common reports giving precise locations of planets when they are not near their closest approach to stars, and draws some brief general conclusions about the utility of the Babylonian observations for estimating planetary longitudes and calibrating models in antiquity.I wish to thank Lis Brack-Bernsen, John Britton, Peter Huber, Hermann Hunger, Teije de Jong, Norbert Roughton, John Steele, and Noel Swerdlow for comments on drafts of the paper, for access to work before publication, and for help in various forms.     

Post-transcriptional gene silencing of ribosomal protein S6 kinase 1 restores insulin action in leucine-treated skeletal muscle

Excessive nutrients, especially amino acids, impair insulin action on glucose metabolism in skeletal muscle. We tested the hypothesis that the branched-chain amino acid leucine reduces acute insulin action in primary myotubes via a negative feedback mechanism involving ribosomal protein S6 kinase 1 (S6K1). The effect of S6K1 on glucose metabolism was determined by applying RNA interference (siRNA). Leucine (5 mM) reduced glucose uptake and incorporation to glycogen by 13% and 22%, respectively, compared to the scramble siRNA-transfected control at the basal level. Leucine also reduced insulin-stimulated Akt phosphorylation, glucose uptake and glucose incorporation to glycogen (39%, 39% and 37%, respectively), and this reduction was restored after S6K1 silencing. Depletion of S6K1 enhanced basal glucose utilization and protected against the development of impaired insulin action, in response to excessive leucine. In conclusion, S6K1 plays an important role in the regulation of insulin action on glucose metabolism in skeletal muscle. Received 22 December 2008; received after revision 19 February 2009; accepted 23 February 2009     

CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure

CSTX-9 (68 residues, 7530.9 Da) is one of the most abundant toxic polypeptides in the venom of the wandering spider Cupiennius salei. The amino acid sequence was determined by Edman degradation using reduced and alkylated CSTX-9 and peptides generated by cleavages with endoproteinase Asp-N and trypsin, respectively. Sequence comparison with CSTX-1, the most abundant and the most toxic polypeptide in the crude spider venom, revealed a high degree of similarity (53% identity). By means of limited proteolysis with immobilised trypsin and RP-HPLC, the cystine-containing peptides of CSTX-9 were isolated and the disulphide bridges were assigned by amino acid analysis, Edman degradation and nanospray tandem mass spectrometry. The four disulphide bonds present in CSTX-9 are arranged in the following pattern: 1-4, 2-5, 3-8 and 6-7 (Cys₆-Cys₂₁, Cys₁₃-Cys₃₀, Cys₂₀-Cys₄₈, Cys₃₂-Cys₄₆). Sequence comparison of CSTX-1 with CSTX-9 clearly indicates the same disulphide bridge pattern, which is also found in other spider polypeptide toxins, e.g. agatoxins (ω-AGA-IVA, ω-AGA-IVB, μ-AGA-I and μ-AGA-VI) from Agelenopsis aperta, SNX-325 from Segestria florentina and curtatoxins (CT-I, CT-II and CT-III) from Hololena curta. CSTX-1/CSTX-9 belong to the family of ion channel toxins containing the inhibitor cystine knot structural motif. CSTX-9, lacking the lysine-rich C-terminal tail of CSTX-1, exhibits a ninefold lower toxicity to Drosophila melanogaster than CSTX-1. This is in accordance with previous observations of CSTX-2a and CSTX-2b, two truncated forms of CSTX-1 which, like CSTX-9, also lack the C-terminal lysine-rich tail. Received 23 July 2001; accepted 31 July 2001     

Genetic analysis of interspecific crosses Mus musculus L. x Mus spretus Lataste: linkage of Adh-1 with Amy-1 on chromosome 3 and Es-14 with Mod-1 on chromosome 9]

192 offsprings from interspecific back-crosses (male M. spretus x female BALB/c) F1 x male BALB/c or (male M. spretus x female C57BL6) F1 x male C57BL6 were analysed at thirteen structural loci. Linkage of ES-14 with Mod-1 on chromosome 9 and that of Adh-1 with Amy-1 on chromosome 3 are shown. The following order centromere/Car-2/Amy-1 is tentatively proposed for these loci on chromosome 3.     

Identification of (3R,4R)-gD 1(6) as an isolation artefact of cannabinoid acids formed by callus cultures ofCannabis sativa L

Summary For the first time we have isolated a major psychoactive cannabinoid, (3R, 4R)- ¹⁽⁶⁾-tetrahydrocannabinol3 from callus cultures ofCannabis sativa L.3 was obtained as an artefact of the actually formed (3R, 4R)- ¹-tetrahydrocannabinol-3- and/or 5-carboxylic acids1 and2 by subjecting the culture material to a decarboxylation step prior to extraction. No attempt was made to isolated acids1 and2. The identity of3 was confirmed by comparison with an authentic sample of (3R, 4R)- ¹-tetrahydrocannabinol. Culture conditions, isolation procedure and identification of the cannabinoid are described.Acknowledgment. The authors are greatly indebted to Dr W. Heller and Dr G. Schroeder-Frey for deriving the callus cultures from seedlings ofC. sativa and to Prof. N. Amrhein for valuable gifts of culture media and growth regulators.     

Protective effects of the PARP-1 inhibitor PJ34 in hypoxic-reoxygenated cardiomyoblasts

To clarify the role of poly(ADP-ribose)polymerase-1 (PARP-1) in myocardial ischemia-reperfusion injury, we explored some effects of PJ34, a highly specific inhibitor of this enzyme, in hypoxic-reoxygenated (HR) H9c2 cardiomyoblasts. Compared to the control, HR cells showed signs of oxidative stress, marked PARP-1 activation, NAD⁺ and ATP depletion and impaired mitochondrial activity. HR cardiomyoblasts were affected by both necrosis and apoptosis, the latter involving the nuclear translocation of apoptosis-inducing factor. In HR cardiomyoblasts treated with PJ34, oxidative stress and PARP-1 activity were decreased, and NAD⁺ and ATP depletion, as well as mitochondrial impairment, were attenuated. Above all, PJ34 treatment improved the survival of HR cells; not only was necrosis significantly diminished, but apoptosis was also reduced and shifted from a caspase-independent to a caspase-dependent pathway. These results suggest that PARP-1 modulation by a selective inhibitor such as PJ34 may represent a promising approach to limit myocardial damage due to post-ischemic reperfusion. Received 27 July 2006; accepted 26 October 2006     

Chromogranin A binds to αvβ6-integrin and promotes wound healing in mice

Chromogranin A (CgA), a secretory protein expressed by many neuroendocrine cells, neurons, cardiomyocytes, and keratinocytes, is the precursor of various peptides that regulate the carbohydrate/lipid metabolism and the cardiovascular system. We have found that CgA, locally administered to injured mice, can accelerate keratinocyte proliferation and wound healing. This biological activity was abolished by the Asp(45)Glu mutation. CgA and its N-terminal fragments, but not the corresponding Asp(45)Glu mutants, could selectively recognize the αvβ6-integrin on keratinocytes (a cell-adhesion receptor that is up-regulated during wound healing) and regulate keratinocyte adhesion, proliferation, and migration. No binding was observed to other integrins such as αvβ3, αvβ5, αvβ8, α5β1, α1β1, α3β1, α6β4, α6β7 and α9β1. Structure-activity studies showed that the entire CgA(39-63) region is crucial for αvβ6 recognition (K(i) = 7 nM). This region contains an RGD site (residues CgA(43-45)) followed by an amphipathic α-helix (residues CgA(47-63)), both crucial for binding affinity and selectivity. These results suggest that the interaction of the RGD/α-helix motif of CgA with αvβ6 regulates keratinocyte physiology in wound healing.     

Cannabiripsol: a novel Cannabis constituent.

Cannabiripsol [(-) (6aR, 9S, 10S, 10aR)9,10-dihydroxy-hexahydrocannabinol] (1), a new cannabinoid was isolated from a South African Cannabis variant. The structure was determined by spectral means and by synthesis.     

Physicochemical properties of an extracellular polysaccharide isolated from culture media of Bacillus amyloliquefaciens]

A new extracellular polysaccharide has been isolated by chromatography on anion exchanger of a fraction obtained from highly viscous culture media of Bacillus amyloliquefaciens. This polysaccharide is characterised by high molecular weight (1,000,000 dalton) and intrinsic viscosity (323 ml/g). It contains 24% neutral sugar (galactose and mannose 5:1), 35% glucuronic acid and 51.5% N-acetylhexosamines (N-actylglucosamine, N-acetylgalactosamine and N-acetylbacillosamine 6:9:1).     

BMP9 is produced by hepatocytes and circulates mainly in an active mature form complexed to its prodomain

Bone Morphogenetic Protein 9 (BMP9) has been recently found to be the physiological ligand for the activin receptor-like kinase 1 (ALK1), and to be a major circulating vascular quiescence factor. Moreover, a soluble chimeric ALK1 protein (ALK1-Fc) has recently been developed and showed powerful anti-tumor growth and anti-angiogenic effects. However, not much is known concerning BMP9. This prompted us to investigate the human endogenous sources of this cytokine and to further characterize its circulating form(s) and its function. Analysis of BMP9 expression reveals that BMP9 is produced by hepatocytes and intrahepatic biliary epithelial cells. Gel filtration analysis combined with ELISA and biological assays demonstrate that BMP9 circulates in plasma (1) as an unprocessed inactive form that can be further activated by furin a serine endoprotease, and (2) as a mature and fully active form (composed of the mature form associated with its prodomain). Analysis of BMP9 circulating levels during mouse development demonstrates that BMP9 peaks during the first 3 weeks after birth and then decreases to 2 ng/mL in adulthood. We also show that circulating BMP9 physiologically induces a constitutive Smad1/5/8 phosphorylation in endothelial cells. Taken together, our results argue for the role of BMP9 as a hepatocyte-derived factor, circulating in inactive (40%) and active (60%) forms, the latter constantly activating endothelial cells to maintain them in a resting state.     

Molecular basis of homocysteine toxicity in humans

Because of its similarity to the protein amino acid methionine, homocysteine (Hcy) can enter the protein biosynthetic apparatus. However, Hcy cannot complete the protein biosynthetic pathway and is edited by the conversion to Hcy-thiolactone, a reaction catalyzed by methionyl-transfer RNA synthetase in all organisms investigated, including human. Nitrosylation converts Hcy into a methionine analogue, S-nitroso-Hcy, which can substitute for methionine in protein synthesis in biological systems, including cultured human endothelial cells. In humans, Hcy-thiolactone modifies proteins posttranslationally by forming adducts in which Hcy is linked by amide bonds to -amino group of protein lysine residues (Hcy-N-Lys-protein). Levels of Hcy bound by amide or peptide linkages (Hcy-N-protein) in human plasma proteins are directly related to plasma total Hcy levels. Hcy-N-hemoglobin and Hcy-N-albumin constitute a major pool of Hcy in human blood, larger than total Hcy pool. Hcy-thiolactone and Hcy-thiolactone-hydrolyzing enzyme, a product of the PON1 gene, are present in human plasma. Modification with Hcy-thiolactone leads to protein damage and induces immune response. Autoantibodies that specifically recognize the Hcy-N-Lys-epitope on Hcy-thiolactone-modified proteins occur in humans. The ability of Hcy to interfere with protein biosynthesis, which causes protein damage, induces cell death and elicits immune response, is likely to contribute to the pathology of human disease.Received 30 May 2003; received after revision 21 July 2003; accepted 15 August 2003     

Non-incorporation de tritium dans la dihydroménaquinone-9 deMycobacterium phlei au cours des oxydations phosphorylantes

Summary During oxidative phosphorylation with cell-free extracts ofMycobacterium phlei, carried out in the presence of tritiated water, only negligible incorporation of tritium has been found either into endogenous MK-9(H2) or into phylloquinone added to the irradiated extract. These results rule out previously postulated mechanisms involving the enzymatic isomerization of menaquinones into quinone-methides 2.     

Behaviour of intrinsically disordered proteins in protein–protein complexes with an emphasis on fuzziness

Intrinsically disordered proteins (IDPs) do not, by themselves, fold into a compact globular structure. They are extremely dynamic and flexible, and are typically involved in signalling and transduction of information through binding to other macromolecules. The reason for their existence may lie in their malleability, which enables them to bind several different partners with high specificity. In addition, their interactions with other macromolecules can be regulated by a variable amount of chemically diverse post-translational modifications. Four kinetically and energetically different types of complexes between an IDP and another macromolecule are reviewed: (1) simple two-state binding involving a single binding site, (2) avidity, (3) allovalency and (4) fuzzy binding; the last three involving more than one site. Finally, a qualitative definition of fuzzy binding is suggested, examples are provided, and its distinction to allovalency and avidity is highlighted and discussed.