Ascorbate Synthesis Pathway: DUAL ROLE OF ASCORBATE IN BONE HOMEOSTASIS*

Using mouse gene knock-out models, we identify aldehyde reductase (EC 1.1.1.2, Akr1a4 (GR)) and aldose reductase (EC 1.1.1.21, Akr1b3 (AR)) as the enzymes responsible for conversion of d-glucuronate to l-gulonate, a key step in the ascorbate (ASC) synthesis pathway in mice. The gene knock-out (KO) mice show that the two enzymes, GR and AR, provide ∼85 and ∼15% of l-gulonate, respectively. GRKO/ARKO double knock-out mice are unable to synthesize ASC (>95% ASC deficit) and develop scurvy. The GRKO mice (∼85% ASC deficit) develop and grow normally when fed regular mouse chow (ASC content = 0) but suffer severe osteopenia and spontaneous fractures with stresses that increase ASC requirements, such as pregnancy or castration. Castration greatly increases osteoclast numbers and activity in GRKO mice and promotes increased bone loss as compared with wild-type controls and additionally induces proliferation of immature dysplastic osteoblasts likely because of an ASC-sensitive block(s) in early differentiation. ASC and the antioxidants pycnogenol and resveratrol block osteoclast proliferation and bone loss, but only ASC feeding restores osteoblast differentiation and prevents their dysplastic proliferation. This is the first in vivo demonstration of two independent roles for ASC as an antioxidant suppressing osteoclast activity and number as well as a cofactor promoting osteoblast differentiation. Although humans have lost the ability to synthesize ASC, our mouse models suggest the mechanisms by which suboptimal ASC availability facilitates the development of osteoporosis, which has important implications for human osteoporosis.     

Identification of genes and proteins involved in the pleiotropic response to arsenic stress in Caenibacter arsenoxydans, a metalloresistant beta-proteobacterium with an unsequenced genome

The effect of high concentrations of arsenic has been investigated in Caenibacter arsenoxydans, a beta-proteobacterium isolated from an arsenic contaminated environment and able to oxidize arsenite to arsenate. As the genome of this bacterium has not yet been sequenced, the use of a specific proteomic approach based on nano-high performance liquid chromatography tandem mass spectrometry (nanoLC-MS/MS) studies and de novo sequencing to perform cross-species protein identifications was necessary. In addition, a random mutational analysis was performed. Twenty-two proteins and 16 genes were shown to be differentially accumulated and expressed, respectively, in cells grown in the presence of arsenite. Two genes involved in arsenite oxidation and one in arsenite efflux as well as two proteins responsible for arsenate reduction were identified. Moreover, numerous genes and proteins belonging to various functional classes including information and regulation pathways, intermediary metabolism, cell envelope and cellular processes were also up- or down-regulated, which demonstrates that bacterial response to arsenic is pleiotropic.     

Characterization of Pharmacologically Active Anti-Peptide Antibodies Directed Against the First and Second Extracellular Loops of the Serotonin 5-HT1A Receptor

Abstract: The immunological properties and the functional role of the first (loop I) and second (loop II) extracellular loops of the human serotonin 5-HT_1A receptor were studied with three populations of anti-peptide antibodies: Ab-1 (loop I; sequence Y-Q-V-L-N-K-W-T-L-G-Q-V-T-C-D-L; residues 96–111), Ab-2 (loop II; sequence G-W-R-T-P-E-D-R-S-D-P-D-A-C-T-I-S-K-D-H-G; residues 173–193), and Ab-12 (produced against loop I but cross-reacting with loop II). Chemical modification of peptide amino acid residues revealed the importance of the polyanionic stretch near the N-terminal domain of loop II for Ab-2 antibody binding and the role of the cysteine residues in both loops for the binding of Ab-1 and Ab-12 antibodies. Antibodies Ab-2 and Ab-12 recognized only the nonglycosylated form of the receptor (42 kDa) on immunoblots with transfected HeLa cells expressing the human 5-HT_1A receptor but recognized the glycosylated forms (55 and 65 kDa) of rat 5-HT_1A receptor from hippocampus membranes. The Ab-1 antibodies recognized no protein band from any cell type studied. Preincubation of transfected HeLa cell membranes with Ab-2 antibodies revealed two affinity binding sites of the 5-HT_1A receptor (K_DH = 0.54 ± 0.09 nM and K_DL = 13.74 ± 4.9 nM) for the agonist 8-hydroxy-2-(di-n-[³H]propylamino)tetralin ([³H]8-OH-DPAT) binding, but Ab-1 and Ab-12 revealed only one site (K_D of ≈2.5 nM). In contrast to the Ab-2 antibodies, Ab-1 and Ab-12 antibodies decreased the B_max of the [³H]8-OH-DPAT binding to 42 and 31%, respectively. These findings suggest that there are at least two epitopes on the extracellular loops: one inducing a high-affinity state for agonist binding and the other interfering with the accessibility of the ligand binding pocket.     

Apoptotic suppression by baculovirus P35 involves cleavage by and inhibition of a virus-induced CED-3/ICE-like protease.

Baculovirus p35 prevents programmed cell death in diverse organisms and encodes a protein inhibitor (P35) of the CED-3/interleukin-1 beta-converting enzyme (ICE)-related proteases. By using site-directed mutagenesis, we have identified P35 domains necessary for suppression of virus-induced apoptosis in insect cells, the context in which P35 evolved. During infection, P35 was cleaved within an essential domain at or near the site DQMD-87G required for cleavage by CED-3/ICE family proteases. Cleavage site substitution of alanine for aspartic acid at position 87 (D87A) of the P1 residue abolished P35 cleavage and antiapoptotic activity. Although the P4 residue substitution D84A also caused loss of apoptotic suppression, it did not eliminate cleavage and suggested that P35 cleavage is not sufficient for antiapoptotic activity. Apoptotic insect cells contained a CED-3/ICE-like activity that cleaved in vitro-translated P35 and was inhibited by recombinant wild-type P35 but not P1- or P4-mutated P35. Thus, baculovirus infection directly or indirectly activates a novel CED-3/ICE-like protease that is inhibited by P35, thereby preventing virus-induced apoptosis. Our findings confirmed the inhibitory activity of P35 towards the CED-3/ICE protease, including recombinant mammalian enzymes, and were consistent with a mechanism involving P35 stoichiometric interaction and cleavage. P35's inhibition of phylogenetically diverse proteases accounts for its general effectiveness as an apoptotic suppressor.     

Serotonin content of the interscapular brown adipose tissue of growing rats.

In brown fat of newborn rats the serotonin (5HT) content is high during the first five days of life. This may play a part in the lipid repletion of the tissue, lipids being the main fuel for nonshivering thermogenesis. Subsequently 5HT increases more in young rats reared at 16 degrees C than in those reared at 28 degrees C but to a lesser extent than norepinephrine (NE) content. A possible role of 5HT in thermoregulation of the rat during the early postnatal period is discussed.     

Death-effector Filaments: Novel Cytoplasmic Structures that Recruit Caspases and Trigger Apoptosis

The death-effector domain (DED) is a critical protein interaction domain that recruits caspases into complexes with members of the TNF-receptor superfamily. Apoptosis can also be induced by expressing certain DED-containing proteins without surface receptor cross-linking. Using Green Fluorescent Protein to examine DED-containing proteins in living cells, we show that these proteins cause apoptosis by forming novel cytoplasmic filaments that recruit and activate pro-caspase zymogens. Formation of these filaments, which we term death-effector filaments, was blocked by coexpression of viral antiapoptotic DED-containing proteins, but not by bcl-2 family proteins. Thus, formation of death-effector filaments allows a regulated intracellular assembly of apoptosis-signaling complexes that can initiate or amplify apoptotic stimuli independently of receptors at the plasma membrane.