The adipose tissue is the site of expression and secretion of a range of biologically active proteins, called adipokines, for example, leptin, adiponectin, and resistin. Leptin has previously been shown to be expressed in osteoblasts and to promote bone mineralization, whereas adiponectin expression is enhanced during osteoblast differentiation. In the present study we explored the possible role of resistin in bone metabolism. We found that resistin is expressed in murine preosteoclasts and preosteoblasts (RAW 264.7, MC3T3-E1), in primary human bone marrow stem cells and in mature human osteoblasts. The expression of resistin mRNA in RAW 264.7 was increased during differentiation and seemed to be regulated through PKC- and PKA-dependent mechanisms. Recombinant resistin increased the number of differentiated osteoclasts and stimulated NFkappaB promoter activity, indicating a role in osteoclastogenesis. Resistin also enhanced the proliferation of MC3T3-E1 cells in a PKA and PKC-dependent manner, but only weakly interfered with genes known to be upregulated during differentiation of MC3T3-E1 into osteoblasts. All together, our results indicate that resistin may play a role in bone remodeling.
C are repaired by the AfAlkA base excision repair glycosylase of Archaeoglobus fulgidus, suggesting a different repair mechanism for these lesions in the third domain of life. In addition, AfAlkA was found to effect a robust excision of 1,N 6 -ethenoadenine. We present a high-resolution crystal structure of AfAlkA, which, together with the characterization of several sitedirected mutants, forms a molecular rationalization for the newly discovered base excision activity.
Uracil arises in DNA by hydrolytic deamination of cytosine (C) and by erroneous incorporation of deoxyuridine monophosphate opposite adenine, where the former event is devastating by generation of C → thymine transitions. The base excision repair (BER) pathway replaces uracil by the correct base. In human cells two uracil-DNA glycosylases (UDGs) initiate BER by excising uracil from DNA; one is hSMUG1 (human single-strand-selective mono-functional UDG). We report that repair initiation by hSMUG1 involves strand incision at the uracil site resulting in a 3′-α,β-unsaturated aldehyde designated uracil-DNA incision product (UIP), and a 5′-phosphate. UIP is removed from the 3′-end by human apurinic/apyrimidinic (AP) endonuclease 1 preparing for single-nucleotide insertion. hSMUG1 also incises DNA or processes UIP to a 3′-phosphate designated uracil-DNA processing product (UPP). UIP and UPP were indirectly identified and quantified by polyacrylamide gel electrophoresis and chemically characterised by matrix-assisted laser desorption/ionisation time-of-flight mass-spectrometric analysis of DNA from enzyme reactions using 18O- or 16O-water. The formation of UIP accords with an elimination (E2) reaction where deprotonation of C2′ occurs via the formation of a C1′ enolate intermediate. A three-phase kinetic model explains rapid uracil excision in phase 1, slow unspecific enzyme adsorption/desorption to DNA in phase 2 and enzyme-dependent AP site incision in phase 3.
Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea. Here we report a different type of BER performed by extract prepared from cells of the euryarchaeon Archaeoglobus fulgidus. Although immunodepletion showed that the monofunctional family 4 type of uracil-DNA glycosylase (UDG) is the principal and probably only UDG in this organism, a -elimination mechanism rather than a hydrolytic mechanism is employed for incision of the abasic site following uracil removal. The resulting 3 remnant is removed by efficient 3-phosphodiesterase activity followed by single-nucleotide insertion and ligation. The finding that repair product formation is stimulated similarly by ATP and ADP in vitro raises the question of whether ADP is more important in vivo because of its higher heat stability.After depurination, hydrolytic deamination of cytosine to uracil is the most frequent event that damages DNA (36), and it results in G ⅐ C-to-A ⅐ T transition mutations if the damage is not repaired. In addition, some dUTP molecules escape hydrolysis by dUTPase, which results in a certain amount of dUMP introduced into DNA opposite adenine during replication (32). Irrespective of the mode of appearance, all cells contain uracil-DNA glycosylase (UDG) (EC 3.2.2.3) enzymes to remove uracil from DNA (17). The resulting abasic or apurinic/apyrimidinic (AP) site can subsequently be removed, and the integrity of the DNA can be restored by the so-called base excision repair (BER) pathway, which consists in its simplest form of the sequential actions of 5Ј-acting AP endonuclease, 5Ј-deoxyribose phosphate (dRP) lyase, DNA polymerase, and DNA ligase. The BER pathway can be initiated by one of several DNA glycosylases with different substrate specificities (17,36,57), and quantitatively it is the most important repair mechanism for the removal of spontaneously generated base modifications. Genes encoding bacterial and eukaryotic UDGs exhibiting significant selectivity for uracil have been cloned and sequenced in the last 2 decades, and the results have demonstrated that there is a high degree of conservation between distantly related species. Family 1 UDGs (for a review of UDG families 1 to 3, see reference 44), typified by the Escherichia coli Ung enzyme (37), recognize uracil in an extrahelical or flipped-out conformation in double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Several family 1 enzymes have been extensively characterized, both structurally and at the cell and organism levels. Family 2 UDGs, which includes E. coli Mug and mammalian thymine-DNA glycosylase, are mismatch specific and recognize guanine on the complementary strand...
Hydrolytic deamination of cytosine to uracil in DNA is increased in organisms adapted to high temperatures. Hitherto, the uracil base excision repair (BER) pathway has only been described in two archaeons, the crenarchaeon Pyrobaculum aerophilum and the euryarchaeon Archaeoglobus fulgidus, which are hyperthermophiles and use single-nucleotide replacement. In the former the apurinic/apyrimidinic (AP) site intermediate is removed by the sequential action of a 5-acting AP endonuclease and a 5-deoxyribose phosphate lyase, whereas in the latter the AP site is primarily removed by a 3-acting AP lyase, followed by a 3-phosphodiesterase. We describe here uracil BER by a cell extract of the thermoacidophilic euryarchaeon Thermoplasma acidophilum, which prefers a similar short-patch repair mode as A. fulgidus. Importantly, T. acidophilum cell extract also efficiently executes ATP/ADP-stimulated long-patch BER in the presence of deoxynucleoside triphosphates, with a repair track of ϳ15 nucleotides. Supplementation of recombinant uracil-DNA glycosylase (rTaUDG; ORF Ta0477) increased the formation of short-patch at the expense of long-patch repair intermediates, and additional supplementation of recombinant DNA ligase (rTalig; Ta1148) greatly enhanced repair product formation. TaUDG seems to recruit AP-incising and -excising functions to prepare for rapid singlenucleotide insertion and ligation, thus excluding slower and energy-costly long-patch BER.In all cells, deamination of cytosine to uracil is only surpassed by depurination as the most common hydrolytic DNAdamaging event (38). While the apurinic/apyrimidinic (AP) site is primarily a cytotoxic lesion inhibiting replication or transcription, deaminated cytosines result in G ⅐ C to A ⅐ T transition mutations if they are not repaired before DNA synthesis. In addition, some dUTP escapes hydrolysis by dUTPase causing a certain amount of uracil introduced into DNA opposite adenine during replication (5, 32).Uracil is excised from DNA by a specific monofunctional uracil-DNA glycosylase (UDG) enzyme in virtually all organisms, including those living at the highest temperatures (22,33,47). After such enzyme-catalyzed, as well as spontaneous, base removal, a single nucleotide is reinserted by the sequential action of a 5Ј-acting AP endonuclease, 5Ј-deoxyribose phosphate (5Ј-dRP) lyase, DNA polymerase, and DNA ligase (22, 32), which constitute the short-patch mode of the base excision repair (BER) pathway. In eukaryotes, like mammals, the 5Ј-dRP lyase activity (35, 41) is a function of DNA polymerase  (41, 62). BER is quantitatively the most important repair mechanism for the removal of spontaneously generated base modifications in DNA and is thus necessary to secure genomic integrity (38). The 5Ј-AP site remnant following strand incision blocks ligation, and when it is chemically modified in such a way that efficient removal by the 5Ј-dRP lyase is obstructed, polymerization will continue past one nucleotide and the downstream DNA strand is displaced. This long-patch mode of BER ...
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