Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans

A systematic method for ¹³C labeling of the glycan of immunoglobulin G for NMR study has been developed. A mouse immunoglobulin of subclass IgG2b has been used for the experiment. On the basis of chemical shift and linewidth data, it has been concluded that (1) the mobility of the carbohydrate chain in IgG2b is comparable to that of the backbone polypeptide chain with the exception of the galactose residue at the nonreducing end of the Man1–3 branch, which is extremely mobile and (2) agalactosylation does not induce any significant change in the mobility. The results obtained indicate that even in the agalactosyl form the glycans are buried in the protein. Biological significance of the NMR results obtained is also briefly discussed.     

Histaminergic Modulation of Hippocampal Acetylcholine Release In Vivo

Abstract: In order to elucidate the modulatory role of the histaminergic neural system in the cholinergic neural system, the acetylcholine release from the CA1-CA3 region in the hippocampus of anesthetized rats was studied by an in vivo microdialysis method coupled with HPLC-electrochemical detection. The mean value for the basal acetylcholine release was 0.98 β 0.04 pmol/20 min. The acetylcholine release was increased to 172% of the basal level when an electrical stimulation at 200 μA was applied to the tuberomammillary nucleus. An administration of α-fluoromethylhistidine (100 mg/kg i.p.) blocked the electrically evoked release of histamine both from the septal-diagonal band complex and the hippocampus, and abolished the electrically evoked release of acetylcholine from the hippocampus. Zolantidine (5 mg/kg i.p.) attenuated the increase in the electrically stimulated acetylcholine release, but pyrilamine (5 mg/kg i.p.) did not attenuate the increase in the acetylcholine release. These drugs showed no significant effect on the basal acetylcholine release. An administration of ( R )-α-methylhistamine (5 mg/kg i.p.) caused a decrease in the acetylcholine release to 48.7% of the basal level, whereas thioperamide (5 mg/kg i.p.) caused an increase in the acetylcholine release 60 min after the injection. These results suggest that the histaminergic system may contribute to the modulation of the activity of the septohippocampal cholinergic system, mainly through H₂ receptprs.     

Energetic coupling in the primary processes of photosynthesis in Chromatium. pH dependence of delayed fluorescence, electron transfer and degree of coupling.

The effects of pH on the thermodynamic properties of the proton-translocating cyclic electron transfer system in a purple photosynthetic bacterium Chromatium vinosum were studied. Two thermodynamic parameters, the flux (Je) and force (deltamue) of the electron transfer process, were analyzed. The rate of electron transfer in the re-reduction of photooxidized reaction-center bacteriochlorophyll was used as Je. deltamue was determined from the intensity of the delayed fluorescence from bacteriochlorophyll. deltamue is composed of the redox potential difference and the electrical potential difference between two electron transfer components. In the steady state under illumination, the flux-to-force ratio is determined by the following relationship: Je = (1--q2)Lee deltamue where q is the "degree of coupling" of electron transfer to proton translocation and Lee is the value of Je/delta-approximately similar e when there is no back pressure by formation of delta approximately muH+ (electrochemical potential difference of H+). The value of (1--q2) Lee increased with increasing pH in the neutral pH range. Uncouplers and ionophores that dissipate delta-approximately muH+ increased Je and decreased deltamue. The effects were more prominent in the lower pH range. Therefore, q must be smaller at higher pH. The coupling is probably tight when redox components are saturated with protons. The experimental results agreed with the theoretical predictions for a system where a hydrogen-translocating component functions as an electron-proton symport carrier.     

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores.

Delayed fluorescence from bacteriochlorophyll in Chromatium vinosum chromatophores was studied at room temperature and under intermittent illuminations. The decay of delayed fluorescence was constituted of two components; a fast component decayed with a half time of about 8 ms, a slow one decayed in parallel with the reduction of photooxidized bacteriochlorophyll (P+) with a half time of 100-200 ms. The biphasic decay of delayed fluorescence indicated that a rapid equilibrium was established between the primary electron acceptor and the secondary acceptor. In the presence of o-phenanthroline, the time course of the decay of delayed fluorescence was identical with that of the reduction of P+ in reaction center-rich subchromatophore particles, although they did not necessarily coincide with each other in "intact" chromatophores. The intensity of the slow component was increased and the decay was accelerated at basic pH values. Reagents that dissipate the proton gradient across the chromatophore membranes such as carbonylcyanide m-chlorophenylhydrazone (CCCP) and nigericin accelerated the decay of the slow component. These effects are probably resulting from changes in internal pH of chromatophore vesicles. Reagents that dissipate the membrane potential such as CCCP and valinomycin decreased the intensity.     

Proton correlation nuclear magnetic resonance study of anaerobic metabolism of Escherichia coli.

Proton correlation nuclear magnetic resonance has been used to investigate anaerobic metabolism of glucose in Escherichia coli cells. The time course of the concentrations of six metabolites (ethanol, lactate, acetate, pyruvate, succinate, and formate) has been followed at the very early state of fermentation, and used to discuss dynamical aspects of the mixed-acid fermentation of glucose by E. coli.     

Acceleration of the ATPase activity of glycerol-treated muscle fibers by repeated stretch-release cycles.

Glycerol-treated muscle fiber bundles were fixed at their rest length in 50 mM KC1, 2 mM MgC1(2), and 10 micron CaC1(2) at pH 7.8 and 0 degrees C in the presence of sufficient amounts of ATP, creatine kinase, and creatine phosphate. The fiber bundles were stretched linearly with time for 0.3 s at a constant amplitude, suddenly released, then fixed at the rest length for a constant time interval (alpha seconds). The stretch-release cycle was repeated, and the ATPase activity (the rate of ADP liberation) [EC 3.6.1.3] was measured. It was found that: 1. ATPase was activated by repeated stretch-release. As repetitive stretch-release of 1--2% of the rest length caused maximum activation, we usually selected a value of 2.5% of the rest length. The activation of ATPase was found to be a function of the duration, alpha, of the isometric phase after sudden release from stretching. The ATPase activity of fiber bundles was almost unaffected when they were oscillated by a simple stretch-release without an isometric phase after the sudden release (alpha=0). 2. The ATPase activity of oscillated muscle fibers increased with increase in the value of alpha, reached a maximal level, then decreased gradually with further increase of alpha to a value slightly larger than that of static fibers. At 0 degrees C, the value of alpha for the maximum activation was observed at about 2 s, and the maximum activity was about 2.5 times that of static fibers. At 20 degrees C, the alpha value for maximum activation was about 0.5 s, and the maximum activity was about 1.8 times that of static fibers. 3. The time course of ADP liberation after one stretch-release cycle could be easily calculated from the ATPase activity of the summed durations of the isometric phase, alpha, assuming that the ATPase activation was turned off and on by the stretching and release, respectively, and that the state of cross-bridges immediately after the stretch-release was independent of alpha of the cycle. The rate of ADP liberation after stretch-release thus obtained showed a short lag phase, a sigmoidal increase, a decrease to almost zero, then a return to nearly the original level (the rate of static fibers). About 1.3 mol of ATP per mol of myosin was hydrolyzed at both 0 degrees C and 20 degrees C during one cycle of the changes in the rate of ADP liberation.     

Thermodynamics of Malate Transport across the Tonoplast of Leaf Cells of CAM Plants

The electrochemical potential difference for each dissociationstate of malic acid across the tonoplast of leaf cells was examinedin two CAM plants, Graptopetalum paraguayense and Kalanchoëdaigremontiana. The concentration of malic acid in each dissociationstate was estimated from an analysis of pH and concentrationsof ionic species that included calcium, malate and isocitrate.The vacuoles contained 30–40 mM isocitrate and 50–70mM calcium in G. paraguayense, and 20–30 mM isocitrateand 70–100 mM calcium in K. daigremontiana. For the calculationof the pattern of dissociation of malic acid, the formationof chelates of calcium with malate and isocitrate, which havedifferent stability constants depending on the dissociationof the acids, were also taken into consideration. The vacuolarconcentrations of the divalently dissociated form of malic acid(mal^2– were 4–7 mM and 1-3 mM in G. paraguayenseand in K. daigremontiana, respectively. To obtain informationabout the cytoplasmic concentration of malate, the apparentinhibition constant for malate of phosphoenolpyruvate carboxylasewas measured. It was about 330 µM in the dark period and60 µM in the light period. Considering an inside-positivemembrane potential, we conclude that mal^2– can be takenup passively into the vacuole during the dark period and canbe released passively from the vacuole during the light period.Two types of channel (the "SV-type" channel and a novel "MU-type"channel) which we found recently in G. paraguayense [Iwasakiet al. (1992) Plant Physiol. 98: 1494] are probably involvedin the uptake and the release of malate in the diurnal CAM rhythm.The existence of a large pH-buffering capacity due to isocitricacid in the vacuole allows the accumulation of a large amountof malic acid during the diurnal CAM rhythm. (Received February 12, 1992; Accepted July 10, 1992)     

Lysyl oxidase activates the transcription activity of human collagene III promoter. Possible involvement of Ku antigen

Lysyl oxidase is an extracellular enzyme that controls the maturation of collagen and elastin. Lysyl oxidase and collagen III often show similar expression patterns in fibrotic tissues. Therefore, we investigated the influence of lysyl oxidase overexpression on the promoter activity of human COL3A1 gene. Our results showed that when COS-7 cells overexpressed the mature form of lysyl oxidase, the activity of the human COL3A1 promoter was increased up to an average of 12 times when tested by luciferase reporter assay. The effect was specific, because other promoters were not affected. Moreover, lysyl oxidase effect was abolished by beta-aminopropionitrile, a specific inhibitor of its catalytic activity. Electrophoretic mobility shift assay showed a binding activity in the region from -101 to -77 that was significantly increased by lysyl oxidase overexpression. The binding was specifically competed by the cold probe, and the mutagenesis of this region abolished both the binding activity in gel retardation and lysyl oxidase stimulation of COL3A1 promoter in transfection experiments. We identified the binding activity as Ku antigen in its two components: Ku80 and Ku70. This study suggests a new coordinated mechanism by which lysyl oxidase might control the development of fibrosis.     

Lethal effect of the expression of a killer gene SMK1 in Saccharomyces cerevisiae

Expression of the SMK1 gene which encodes the yeast killer toxin SMKT is lethal in Saccharomyces cerevisiae. Effects of deletion and site-directed mutagenesis of SMK1 on the lethality and the secretion of the gene products were examined. Deletion of the interstitial gamma peptide or the C-terminal loop from Ala208 to the C-terminal Asp222 had no effect on the lethality. Those SMK1 products that lacked either the gamma peptide or the C-terminal loop were expressed in the cells but were not secreted into the culture medium, suggesting that these peptides may have a role in secretion or in protein stability. On the other hand, deletion of the signal sequence resulted in complete loss of the lethal activity. Entering the secretory pathway may be critical for the lethality. Further, deletion of the region from the C-terminus to Leu207 resulted in loss of the lethal activity. Leu207 is located at the C-terminus of the central strand of the beta-sheet structure of SMKT and its side chain is thrust into a hydrophobic environment between the beta-sheet and the alpha-helices. The result obtained upon substitutions of Ala, Ser or Glu for Leu207 suggested that the side chain of Leu207 stabilizes the hydrophobic environment that contributes to the overall structure of the SMK1 product.     

Characterization of plant Aurora kinases during mitosis

The Aurora kinase family is a well-characterized serine/threonine protein kinase family that regulates different processes of mitotic events. Although functions of animal and yeast Aurora kinases have been analyzed, plant aurora kinases were not identified and characterized. We identified three Aurora kinase orthologs in Arabidopsis thaliana and designated these as AtAUR1, AtAUR2, and AtAUR3. These AtAURs could phosphorylate serine 10 in histone H3, in vitro. Dynamic analyses of GFP-fused AtAUR proteins revealed that AtAUR1 and AtAUR2 localized at the nuclear membrane in interphase and located in mitotic spindles during cell division. AtAUR1 also localized in the cell plates. AtAUR3 showed dot-like distribution on condensed chromosomes at prophase and then localized at the metaphase plate. At late anaphase, AtAUR3 is evenly localized on chromosomes. The localization of AtAUR3 during mitosis is very similar to that of phosphorylated histone H3. Interestingly, an overexpression of AtAUR3 induces disassembly of spindle microtubules and alteration of orientation of cell division. Our results indicate that plant Aurora kinases have different characters from that of Aurora kinases of other eukaryotes.†These authors equally contributed to this work