Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

The synergistic effects of interleukin 2 and interleukin 7 on the proliferation and autologous tumor cell lysis of tumor-associated lymphocytes

Interleukin-7 (IL-7) has an ability to stimulate the proliferation of pre-B cells. It has been shown that IL-7 can also activate T lymphocytes. We here demonstrate that IL-7 in combination with interleukin-2 (IL-2) can drive cell proliferation and enhance the autologous tumor cell lysis by peripheral blood mononuclear cells (PBMC) and autologous mixed lymphocyte tumor cell culture (MLTC)-derived effector cells (MLTC cells). These synergistic effects of IL-2 and IL-7 on the proliferation and the augmentation of autologous tumor cell lysis were found for both effector cells. These effects were inhibited by neutralizing antibodies to IL-2 or IL-7, and by a combination of both antibodies, significantly. In terms of phenotypical expression, CD3 positive cells comprised the vast majority of MLTC cells after culture in medium containing IL-2 and IL-7 with an increase of IL-2 receptor positive cells.Abbreviations CD cluster differentiation - IFN interferon - IL interleukin - JRU Japanese Reference Unit - LAK lymphokine activated killer - mAb monoclonal antibody - MLTC mixed lymphocyte tumor cell culture - PBMC peripheral blood mononuclear cells - TILs tumor infiltrating lymphocytes     

Determination of the NMR solution structure of a specific DNA complex of the Myb DNA-binding domain

Summary The solution structure of a specific DNA complex of the minimum DNA-binding domain of the mouse c-Myb protein was determined by distance geometry calculations using a set of 1732 nuclear Overhauser enhancement (NOE) distance restraints. In order to determine the complex structure independent of the initial guess, we have developed two different procedures for the docking calculation using simulated annealing in four-dimensional space (4D-SA). One is a multiple-step procedure, where the protein and the DNA were first constructed independently by 4D-SA using only the individual intramolecular NOE distance restraints. Here, the initial structure of the protein was a random coil and that of the DNA was a typical B-form duplex. Then, as the starting structure for the next docking procedure, the converged protein and DNA structures were placed in random molecular orientations, separated by 50 Å. The two molecules were docked by 4D-SA utilizing all the restraints, including the additional 66 intermolecular distance restraints. The second procedure comprised a single step, in which a random-coil protein and a typical B-form DNA duplex were first placed 70 Å from each other. Then, using all the intramolecular and intermolecular NOE distance restraints, the complex structure was constructed by 4D-SA. Both procedures yielded the converged complex structures with similar quality and structural divergence, but the multiple-step procedure has much better convergence power than the single-step procedure. A model study of the two procedures was performed to confirm the structural quality, depending upon the number of intermolecular distance restraints, using the X-ray structure of the engrailed homeodomain-DNA complex.Abbreviations rmsd root-mean-square deviation - NOE nuclear Overhauser enhancement - 4D-SA simulated annealing in four-dimensional space - Myb-R2R3 repeats 2 and 3 of the DNA-binding domain of the c-Myb protein - DNA 16 Myb-specific binding DNA duplex with 16 base pairs - IHDD-C residues 3 to 59 of the C-chain of the engrailed homeodomain-DNA complex - DNA11 DNA duplex with base pairs 9 to 19 of the engrailed homeodomain-DNA complex     

Effects of ligation on ethanol-induced Balbiani ring puffing in salivary glands of Chironomus

Treatment of Chironomus larvae with dilute (0.5%–1.0%) ethanol results in puffing changes similar to those obtained with galactose in the Balbiani rings (BRs) of the salivary gland chromosomes. A shift in the relative size of BR1 and BR2 in chromosome 4 of C. pallidivittatus or C. tentans was observed within 1–2 days after ethanol treatment. The exceptional Balbiani ring, BR6 in chromosome 3, began to appear within 1 day after ethanol treatment of C. pallidivittatus and was fully developed after 3–4 days. Prepupae appeared to be refractory to the treatment. To localize possible controls of BR puffing in Chironomus, ligatures were made at various positions along the thorax and the anterior abdominal segments of the ethanoltreated larvae. In surviving larvae, ligated anterior to the brain or posterior to the salivary glands, induction of BR6 could be detected. In contrast, little or no BR6 puff induction was found in animals ligated in the middle of the second segment approximately between the brain and the salivary glands. No shift in the BR1/BR2 relation occurred with any of the ligations combined with ethanol treatment.     

Inhibition by Mg2+ of the interaction of Ca2+ with spin-labeled g2 bound to myosin.

According to the measurement of ESR spectrum, Ca2+ induced conformational change of spin-labeled g2 bound to myosin in the presence of 1 mM Mg2+. The half-maximal changes were observed at pCa 6.8 and at pCa 3.7. Spin-labeled phosphorylated g2 bound to myosin showed one transition at pCa 4.5, which shifted to pCa 6.5 after the dephosphorylation with E. coli alkaliphosphatase.     

Calcium dependent action potentials in skeletal muscle fibres of a beetle larva, Xylotrupes dichotomus

The ionic requirement for generating action potentials in ventral longitudinal muscle fibers dissected from beetle larvae was examined by conventional electrophysiological techniques. Muscle fibers that generated only graded responses in physiological saline were able to generate an all-or-none action potential when the potassium permeability of the membrane was inhibited by tetraethylammonium⁺ added to the saline. The peak of the action potential thus elicited was intimately related to the external Ca⁺⁺ concentration. The action potential was blocked by Co⁺⁺ which is known as a competitive inhibitor of Ca-spikes. Neither tetrodotoxin (3 μM) nor a Na-free condition effectively blocked the generation of the action potential. Mg⁺⁺ induced a shift in the peak of the action potential; this was, however, due to the stabilizing action of Mg⁺⁺ but not due to the penetration of Mg⁺⁺ through the muscle membrane. No action potential was elicited in the muscle fiber when immersed in a Ca-free, EGTA saline even when a high concentration of either Mg⁺⁺, Na⁺, or tetraethylammonium⁺ was present. The action potential of the larval muscle fiber was thus concluded to be a Ca-spike, through the channel of which Na⁺ or Mg⁺⁺ did not penetrate.     

Effect of hydrophobic probes on the higher structure of D-amino acid oxidase.

1. The holoenzyme of D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] was found to combine with 1-anilinonaphthalene-8-sulfonate without liberation of its coenzyme, FAD. No energy transfer interaction was found to occur between the bound dye and FAD of the holoenzyme. On the other hand, when the apoenzyme was bound to the dye and then to FAD, energy transfer interaction between the bound dye and bound FAD was observed. In both cases, the dye competes with the substrate, D-alanine. It is concluded that the dye bound to the holoenzyme is oriented in such a special manner that the mutual orientation factor between the dye and FAD becomes very small in magnitude. 2. When the apoenzyme combined with the dye, the monomer-dimer equilibrium of the apoenzyme shifted towards the dimer. On the other hand, 4-monobenzoylamido-4'-aminostilbene-2,2'-disulfonate combined with the apoenzyme to induce monomerization.     

The effects of N-substitution of chitosan and the physical form of the products on the rate of hydrolysis by chitinase from Streptomyces griseus

N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH₃ > CH₃CH₂ > H > CH₃CH₂CH₂ > (CH₃)₂CH > NH₂CH₂ > ClCH₂. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C₅C₁₈) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.     

Structural changes in thin filaments of crab striated muscle.

Low angle X-ray diffraction patterns were recorded from crab leg muscle in living resting state and in rigor (glycerol-extracted). Both resting and rigor patterns showed a series of layer-lines arising from a helical arrangement of actin subunits in the thin filaments. In the resting state, the crossover repeat of the long-pitch actin helices was 36.6 nm, and the symmetry of the genetic actin helix was an intermediate between $\text{26}\text{12}$ and $\text{28}\text{13}$. When the muscle went into rigor, the crossover repeat changed to 38.3 nm and the helical symmetry to $\text{28}\text{13}$.In the living resting pattern, six other reflections were observed on the meridian and in the near-meridional region. These were indexed as orders of 2 × 38.2 nm and could be assigned to troponin molecules; the spacings and the intensity distributions of these reflections could be explained by the model proposed by Ohtsuki (1974) for the arrangement of troponin molecules in the thin filaments.The muscle in rigor gave meridional and near-meridional reflections at orders of 2 × 38.3 nm. These were identified as the same series of reflections as was assigned to troponin in the living resting pattern, but were more intense and could be seen up to higher orders. We consider that the myosin heads attached to the thin filament at regular intervals along its axis also contribute to these reflections in the rigor pattern.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.