Use of a surface plasmon resonance biosensor for the determination of binding constants of transiently expressed recombinant antibody to its antigen

Summary By using a commercially available surface plasmon resonance (SPR) biosensor, the values of the association rate constant (kass), dissociation rate constant (kdiss), and association constant (KA = kass / kdiss) for binding to the antigens were determined. They were almost the same for the recombinant antibody expressed in COS cells, CHO cells, and mouse hybridoma cells. The system of transient expression of the recombinant antibody (Ab) in COS cells and SPR analysis of the supernatant should be useful for rapid expression and evaluation of the binding ability of large numbers of engineered Abs.     

A sensitive method to screen for hydroxyl radical scavenging activity in natural food extracts using competitive inhibition ELISA for 8-hydroxy deoxyguanosine

Summary Representative endogenous antioxidants and natural food extracts were screened for hydroxyl radical scavenging activity by an ELISA. Whereas conventional assays for hydroxyl radical scavenging activity use either spin traps following the induction of Fenton reaction or measure thiobarbituric acid-reactive substances, this assay measures 8-hydroxy deoxyguanosine liberated from the hydroxylation of deoxyguanosine by Cu²⁺/ascorbate system.     

Acute effect of cigarette smoke on cytoplasmic motility of alveolar macrophages in dogs

To study effects of cigarette smoke on the cytoplasmic motility (CM) of alveolar macrophages (AM), we measured remanent field strength (RFS) in dogs in vivo. Four days after instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the right lower lobe bronchus, RFS was measured at the body surface immediately after magnetization of the Fe3O4 particles by an externally applied magnetic field. RFS decreased with time due to particle rotation (relaxation), which is thought to be inversely related to CM of AM (J. Appl. Physiol. 55: 1196-1202, 1983). The initial relaxation curve was fitted to an exponential function. The relaxation rate (lambda 0) increased during cigarette smoke inhalation and returned to base-line values within 15 min. With the inhalation of the smoke of up to five cigarettes, peak lambda 0 was increased; with a further increase in the number of cigarettes, the effect of cigarette smoke decreased or disappeared. Nicotine injection and acetylcholine inhalation increased respiratory resistance to a degree similar to that observed with cigarette smoke but did not change lambda 0. However, either substance P (SP) or capsaicin injection increased lambda 0 in a fashion similar to that noted with cigarette smoke inhalation. Repeated administration of SP produced a significant tachyphylaxis of the effect, and capsaicin did not increase lambda 0 after the cigarette smoke-induced tachyphylaxis of the effect. Colchicine inhibited the cigarette smoke-induced increase in lambda 0. These results suggest that cigarette smoke increases CM of AM, probably through the release of tachykinins including SP from sensory nerves in the lung.     

Calmodulin-Like Activity Associated with Chromatin from Pea Buds

NAD kinase and cyclic AMP phosphodiesterase were activated bya factor prepared from pea chromatin. About 62% of the originalamount of the factor in the purified chromatin was recoveredin the reassociated chromatin. The NAD kinase- and cyclic AMP phosphodiesterase-activatingfactor was released from the chromatin by heat treatment withethylene glycol-bis(ß-aminoethyl ether)- N,N,N',N'-tetraaceticacid (EGTA) then adsorbed on an affinity gel of phenothiazineagarosederivatives in the presence of excess Ca²⁺ over EGTA, afterwhich it was eluted by a flush of EGTA. Activation of NAD kinaseand cyclic AMP phosphodiesterase by this factor depended onthe presence of Ca²⁺. The NAD kinase-activating factor and chromatin were coelutedwhen soluble chromatin was applied to a Bio-Gel A50 column.When chromatin was chromatographed on the same column afterdigestion by DNase I, the factor was eluted in association withthe digested products of the chromatin. The activation propertiesof this factor indicate that a calmodulin-like activity existsin association with pea chromatin. The activation curves of cyclic AMP phosphodiesterase with thepea bud factor and with bovine brain calmodulin were compared.The amount of the factor in the chromatin fraction that correspondedto authentic calmodulin was calculated as 5.7 µg per mgDNA. (Received August 6, 1982; Accepted February 17, 1983)     

Action of Corn and Rice-inactivating Proteins on a Purified Nitrate Reductase from Chlorella vulgaris

When nitrate reductase (NR) purified from Chlorella was incubated with NR-inactivating proteins purified from corn roots and rice cell suspension cultures or with trypsin there was a loss in NADH-NR and NADH cytochrome c reductase (NADH-CR) activities with time whereas the reduced methylviologen NR (MV-NR) remained active. When NADH-NR and NADH-CR activities were inactivated completely by the incubation with corn protein, the major protein band obtained by polyacrylamide gel electrophoresis shifted from an R_F value of 0.12 to an R_F of 0.25 and reduced MV-NR activity moved to the new position on the gel. When NADH-NR and NADH-CR activities were partially inactivated by the corn protein, NADH-NR activity was detected in an intermediate position (R_F value of 0.18). Incubation with trypsin also caused a change in the NR protein migration pattern (R_F value of 0.20). This protein band also had reduced MV-NR activity. Thus, the corn inactivator degrades NR in a fashion similar to but not identical with trypsin. The incubation of NR with rice inactivating protein resulted in a loss of NADH-NR but had no effect on the migration of NR protein or on the reduced MV-NR activity or mobility suggesting that the rice protein binds to Chlorella NR.     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

PI4P-signaling pathway for the synthesis of a nascent membrane structure in selective autophagy

Phosphoinositides regulate a wide range of cellular activities, including membrane trafficking and biogenesis, via interaction with various effector proteins that contain phosphoinositide binding motifs. We show that in the yeast Pichia pastoris, phosphatidylinositol 4'-monophosphate (PI4P) initiates de novo membrane synthesis that is required for peroxisome degradation by selective autophagy and that this PI4P signaling is modulated by an ergosterol-converting PpAtg26 (autophagy-related) protein harboring a novel PI4P binding GRAM (glucosyltransferase, Rab-like GTPase activators, and myotubularins) domain. A phosphatidylinositol-4-OH kinase, PpPik1, is the primary source of PI4P. PI4P concentrated in a protein-lipid nucleation complex recruits PpAtg26 through an interaction with the GRAM domain. Sterol conversion by PpAtg26 at the nucleation complex is necessary for elongation and maturation of the membrane structure. This study reveals the role of the PI4P-signaling pathway in selective autophagy, a process comprising multistep molecular events that lead to the de novo membrane formation.     

Structural bases of transfer RNA-dependent amino acid recognition and activation by glutamyl-tRNA synthetase

Glutamyl-tRNA synthetase (GluRS) is one of the aminoacyl-tRNA synthetases that require the cognate tRNA for specific amino acid recognition and activation. We analyzed the role of tRNA in amino acid recognition by crystallography. In the GluRS*tRNA(Glu)*Glu structure, GluRS and tRNA(Glu) collaborate to form a highly complementary L-glutamate-binding site. This collaborative site is functional, as it is formed in the same manner in pretransition-state mimic, GluRS*tRNA(Glu)*ATP*Eol (a glutamate analog), and posttransition-state mimic, GluRS*tRNA(Glu)*ESA (a glutamyl-adenylate analog) structures. In contrast, in the GluRS*Glu structure, only GluRS forms the amino acid-binding site, which is defective and accounts for the binding of incorrect amino acids, such as D-glutamate and L-glutamine. Therefore, tRNA(Glu) is essential for formation of the completely functional binding site for L-glutamate. These structures, together with our previously described structures, reveal that tRNA plays a crucial role in accurate positioning of both L-glutamate and ATP, thus driving the amino acid activation.