Characteristics of the Deoxyribonucleic Acid of Tφ3, a Bacteriophage for Bacillus stearothermophilus

It is shown that the individual strands of bacteriophage Tphi3 DNA are intact and that heat-denatured Tphi3 DNA forms a bimodal distribution in a neutral CsCl density gradient.     

Use of a Base-Labile Protected Derivative of 6-Mercaptohexanol for the Preparation of Oligonucleotides Containing a Thiol Group at the 5′-End

Abstract

The preparation of a base-labile (Dnpe) protected derivative of 6-mercaptohexanol is described. The use of the phosphoramidite derivative of this compound improves both yields and the time needed for the preparation of oligonucleotides containing a thiol group at the 5′-end.     

Membrane-associated Activation of Cholesterol ��-Glucosyltransferase, an Enzyme Responsible for Biosynthesis of Cholesteryl-��-d-Glucopyranoside in Helicobacter pylori Critical for Its Survival

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-d-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme’s localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.     

Deorphanization of GPR109B as a Receptor for the ��-Oxidation Intermediate 3-OH-octanoic Acid and Its Role in the Regulation of Lipolysis

The orphan G-protein-coupled receptor GPR109B is the result of a recent gene duplication of the nicotinic acid and ketone body receptor GPR109A being found in humans but not in rodents. Like GPR109A, GPR109B is predominantly expressed in adipocytes and is supposed to mediate antilipolytic effects. Here we show that GPR109B serves as a receptor for the β-oxidation intermediate 3-OH-octanoic acid, which has antilipolytic activity on human but not on murine adipocytes. GPR109B is coupled to G_i-type G-proteins and is activated by 2- and 3-OH-octanoic acid with EC₅₀ values of about 4 and 8 μm, respectively. Interestingly, 3-OH-octanoic acid plasma concentrations reach micromolar concentrations under conditions of increased β-oxidation rates, like in diabetic ketoacidosis or under a ketogenic diet. These data suggest that the ligand receptor pair 3-OH-octanoic acid/GPR109B mediates in humans a negative feedback regulation of adipocyte lipolysis to counteract prolipolytic influences under conditions of physiological or pathological increases in β-oxidation rates.Triacylglycerols stored in the white adipose tissue serve as the major energy reserve in higher eukaryotes (1). Although they are constantly turned over by lipolysis and re-esterification, their mobilization and storage are precisely balanced by various hormones and other factors depending on the nutritional state (2). The net rate of lipolysis is increased during fasting or periods of increased energy demand. Fatty acids generated via lipolysis undergo β-oxidation in the muscle and liver to serve directly as a source of energy or as a precursor for ketone bodies (3). The major intracellular regulator of lipolysis is cyclic AMP, which stimulates cAMP-dependent kinase to activate lipolytic enzymes (2, 4–6). This lipolytic pathway is induced, for example, via β-adrenergic receptors that couple to the G-protein G_s and thereby stimulate adenylyl cyclase (7, 8). To adjust lipolysis at the appropriate rate, the effects of prolipolytic stimuli are balanced by various antilipolytic influences. Besides insulin, which promotes the degradation of cAMP via activation of phosphodiesterase 3B (2, 5, 7), several antilipolytic stimuli decrease cAMP levels by activation of G_i-coupled receptors, which mediate an inhibition of adenylyl cyclase (5, 8). One of these receptors, GPR109A, has recently been shown to mediate the anti-lipolytic effects of high concentrations of the ketone body 3-OH-butyrate thereby providing a negative feedback mechanism during fasting (9, 10). GPR109A also binds nicotinic acid (11–13) and mediates the anti-lipolytic effects of this anti-dyslipidemic drug (12).GPR109B, a close relative of GPR109A, is the result of a recent gene duplication being present in humans but not in rodents and most other mammals (14). GPR109B differs from GPR109A in an extended C-terminal tail as well as in 16 amino acids (11, 13). Despite its high homology to GPR109A, GPR109B does not bind nicotinic acid or 3-OH-butyrate with reasonable affinity (10, 11, 13). Because GPR109A and GPR109B have very similar expression patterns (11, 13, 15) and are likely to have the same basic signaling properties, agonists of GPR109B are expected to have physiological and pharmacological effects comparable with those of the GPR109A agonist 3-OH-butyrate and nicotinic acid, respectively. Recently, several synthetic compounds as well as various aromatic d-amino acids have been shown to be selective agonists at GPR109B (16–18). However, endogenous physiological anti-lipolytic ligands of GPR109B are unknown.In this study we tested endogenous carboxylic acids for their ability to activate GPR109B. We found that the fatty acid β-oxidation intermediate 3-OH-octanoic acid is a highly specific agonist of GPR109B. 3-OH-octanoic acid has anti-lipolytic activity, and its plasma concentration in humans reflects the β-oxidation flux. Our data suggest that 3-OH-octanoic acid and GPR109B mediate a negative feedback regulation of adipocyte lipolysis.     

Nucleosides and Nucleotides. 165. Chemical Ligation of Oligodeoxynucleotides Having a Mercapto Group at the 5-Position Of 2′-Deoxyuridine via a Disulfide Bond #

Abstract

We describe the nonenzymatic ligation of oligodeoxynucleotides (ODNs) containing a mercapto group at the 5-position of 2′-deoxyuridine via a disulfide bond. Two ODNs containing different sequences were efficiently ligated in the presence of a template by this method.

     

Structural Requirements of the Photoreceptor Phosphodiesterase ��-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its γ-subunit (Pγ), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of Pγ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (Pγ78–87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the Pγ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of Pγ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63–70 and/or the intact α2 helix of Pγ stabilize binding of C-terminal Pγ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the Pγ molecule and tested for activation by transducin, we found that the C-terminal region (Pγ63–87) by itself could not be displaced but that transducin could relieve inhibition of certain Pγ truncation mutants. Our results are consistent with two distinct mechanisms of Pγ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of Pγ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of Pγ to effectively relieve its inhibitory constraint.     

Development of a Cell-Based Assay Measuring the Activation of FcγRIIa for the Characterization of Therapeutic Monoclonal Antibodies

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.     

DNA stabilization at the Bacillus subtilis PolX core��a binding model to coordinate polymerase, AP-endonuclease and 3��-5�� exonuclease activities

Family X DNA polymerases (PolXs) are involved in DNA repair. Their binding to gapped DNAs relies on two conserved helix-hairpin-helix motifs, one located at the 8-kDa domain and the other at the fingers subdomain. Bacterial/archaeal PolXs have a specifically conserved third helix-hairpin-helix motif (GFGxK) at the fingers subdomain whose putative role in DNA binding had not been established. Here, mutagenesis at the corresponding residues of Bacillus subtilis PolX (PolXBs), Gly130, Gly132 and Lys134 produced enzymes with altered DNA binding properties affecting the three enzymatic activities of the protein: polymerization, located at the PolX core, 3′-5′ exonucleolysis and apurinic/apyrimidinic (AP)-endonucleolysis, placed at the so-called polymerase and histidinol phosphatase domain. Furthermore, we have changed Lys192 of PolXBs, a residue moderately conserved in the palm subdomain of bacterial PolXs and immediately preceding two catalytic aspartates of the polymerization reaction. The results point to a function of residue Lys192 in guaranteeing the right orientation of the DNA substrates at the polymerization and histidinol phosphatase active sites. The results presented here and the recently solved structures of other bacterial PolX ternary complexes lead us to propose a structural model to account for the appropriate coordination of the different catalytic activities of bacterial PolXs.     

Cordycepin Analogs of 2–5 a as Activators of RNase L: Study of the Structural Requirements for RNase L Activation

Abstract

Enzymatically and chemically synthesized cordycepin analogs of 2–5A^? trimer and tetramer were found to be biologically active as protein synthesis inhibitors in intact cultured human fibroblast and murine L929 cells ^1,2. In rabbit reticulocyte lysates, the cordycepin tetramer analog of 2–5A inhibits protein synthesis through binding to and activation of RNase L³. Our present results using L929 cell extracts provide direct evidence that the cordycepin analogs of 2–5A can bind to and activate RNase L.     

Are Roots a Source of Abscisic Acid for the Shoots of Flooded Pea Plants?

Flooding the soil for 2–5 d decreased stomatal conductancesof pea plants (Pisum sativum L., cv. Sprite) with six or sevenleaves. This coincided with slower transpiration, increasedleaf water potentials and increased concentrations of abscisicacid (ABA) in the leaves. No increase in ABA was found in theterminal 20 mm of roots of flooded plants over the same timeperiod. Small stomatal conductances associated with increases in foliarABA were also found in plants grown in nutrient solution whenaeration was halted, causing the equilibrium partial pressuresof dissolved oxygen to fall below 05 It Pa. No increase in ABAconcentration in young secondary roots of the non-aerated plantswas detected after 24, 48 or 72 h, even when the shoot, thepresumed site of deposition for any ABA from the roots, wasremoved 5–6 h before analysis. Similarly, ABA concentrations in roots were not increased whenthe nutrient solution was de-oxygenated by continuous purgingwith nitrogen gas. The abscisic acid concentration in leaf epidermis,the tissue most likely to be the recipient of any ABA movingin the transpiration stream from oxygen-deficient roots, waslower than in the remaining parts of the leaf when examinedin the mutant Argenteum which possesses easily removable epidermallayers. It is concluded that the leaves of plants subjectedto flooding of the soil or oxygen shortage in the root environmentare not enriched substantially with ABA from the roots. A moreprobable source of this growth regulator is the leaf itself. Key words: Pisum sativum, flooding, roots, hormones, aeration stress, abscisic acid, Argenteum mutant