An Internal View of the Spherical Body of Treponema macrodentium as Revealed by Scanning Electron Microscopy

The osmium-dimethyl sulfoxide-osmium method for clear visualization of intracellular structure was used to observe the detailed inner structure of the spherical bodies produced in vitro by a human oral treponeme. Scanning electron microscopy of the cracked spherical body revealed no morphological differences between the outer and inner surfaces of the spherical body membrane, and that multiple folded or somewhat linear main bodies adhere closely to the inner surface. In addition, axial flagella partially free from the main bodies spread widely within the body to make a network, and a number of blebs ranging from approximately 1 μm to 0.2 μm in diameter were located near the terminal or subterminal areas of the main bodies. The origin of the blebs and the mechanism of spherical body formation are discussed.     

Partial purification and characterization of taurodeoxycholate 7α-monooxygenase

Taurodeoxycholate 7α-monooxygenase was partially purified from rat liver microsomes. The enzyme was solubilized with cholate, fractionated with polyethylene glycol and chromatographed on a Sepharose 4B column with cholate as ligand. The enzyme activity was eluted from the column into the fraction eluted with 50 mM phosphate buffer containing cholate and KCl, whereas the benzphetamine demethylase activity was eluted in the non-bound fraction. Thus it was established that both enzymes are different entities. The taurodeoxycholate 7α-monooxygenase activity was reconstituted from the partially purified cytochrome P-450, highly purified NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine and NADPH.     

Chemical modification of a functional arginine residue of rat liver glycine methyltransferase

Rat liver glycine methyltransferase is inactivated irreversibly by phenylglyoxal in potassium phosphate buffer. The inactivation obeys pseudo-first-order kinetics, and the apparent first-order rate constant for inactivation is linearly related to the reagent concentration. A second-order rate constant of 10.54 +/- 0.44 M-1 min-1 is obtained at pH 8.2 and 25 degrees C. Amino acid analysis shows that only arginine is modified upon treatment with phenylglyoxal. Sodium acetate, a competitive inhibitor with respect to glycine, affords complete protection in the presence of S-adenosylmethionine. Acetate alone has no effect on the rate of inactivation. The value of the dissociation constant for acetate determined from the protection experiment is in good agreement with that obtained by kinetic analysis. Comparison of the amount of [14C]phenylglyoxal incorporated into the protein and the number of arginine residues modified in the presence and absence of protecting ligands indicates that modification of one arginine residue per enzyme subunit eliminates the enzyme activity, and this residue is identified as Arg-175 by peptide analysis. The arginine-modified glycine methyltransferase appears to bind S-adenosylmethionine as the native enzyme does, as seen from quenching of the protein fluorescence by S-adenosylmethionine. These results suggest the requirement of Arg-175 in binding the carboxyl group of the substrate glycine.     

Mechanism of function of dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter in Halobacterium halobium: pH effect

The regulatory roles of medium pH, a transmembrane pH gradient (delta pH), and an electrical potential (delta phi) on the activation of the N,N'-dicyclohexylcarbodiimide-sensitive Na+/H+-antiporter were studied in the membrane vesicle of Halobacterium halobium in the dark. Neither delta pH nor delta phi independently activated the antiporter but a combination could. The initial rate of Na+ extrusion did not proportionally relate to the size of delta microH+ imposed. The delta microH+-coupled Na+ efflux in the presence of delta phi (-140 mV) increased as external pH decreased, regardless of the size of delta pH, suggesting the existence of one external H+-binding site (apparent pKa 4.6) whose protonation determines primarily the Na+/H+-exchange activity. On the other hand, the dependence of the Na+ efflux on cytoplasmic pH varied with the size of delta pH imposed and the apparent pKa for the cytoplasmic H+ increased with elevating delta pH. The resulting pKa difference across the membrane seems to be the key mechanism for the facilitation of Na+-coupled H+ influx. In other words, delta pH modulates Na+/H+-exchange activity through manipulating the H+ affinity on the cytoplasmic regulatory site. The Na+ extrusion was gated by the threshold delta phi of -100 mV regardless of the size of existing delta pH. delta phi acts on the protonated antiporter and converts it into an active state which becomes delta pH reactive.     

Membrane potential in liposomes measured by the transmembrane distribution of 86Rb+, tetraphenylphosphonium or triphenylmethylphosphonium: effect of cholesterol in the lipid bilayer

Valinomycin-induced potassium diffusion potential (delta psi, inside negative) in the liposomes made of phosphatidylcholine and various amounts of cholesterol was measured by uptake of 86Rb+, tetraphenylphosphonium (TPP+) or triphenylmethylphosphonium (TPMP+). In any liposome, the values of membrane potential obtained by 86Rb+ uptake (delta psi Rb) agreed well with those calculated from the imposed potassium concentration gradient using the Nernst equation, and were not affected by the presence of cholesterol. However, both delta psi TPP and delta psi TPMP showed smaller values than delta psi Rb when the cholesterol content in liposomes increased. delta psi TPMP at a stationary state was much smaller than delta psi TPP. The orientational order parameter of the lipids' bilayer with various cholesterol content was estimated from fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The results indicated that the permeation of TPP+ or TPMP+ into liposomes containing a large amount of cholesterol is strongly restricted by the high ordering of phosphatidylcholine acyl chains.     

Spiral conformation of Vibrio cholerae as determined by scanning electron microscopy of elongated cells induced by cephalexin treatment.

The elongated cells of Vibrio spp. induced by cephalexin treatment were examined by scanning electron microscopy. The results showed that Vibrio cholerae has a twisted cell body and a right-handed spiral conformation and that the cell bodies of V. parahaemolyticus and V. alginolyticus are straight rather than curved.     

Preliminary crystallographic data for cross-linked (lysine7-lysine41)-ribonuclease A

A cross-linked derivative of ribonuclease A, N^ε,N^ε′-(2,4-dinitrophenylene-1,5)-(lysine⁷-lysine⁴¹)-RNase A, has been crystallized by dialysis against 30% ($\text{v}\text{v}$) ethanol/water mixtures buffered at high pH. Single crystals belong to the orthorhombic space group P2₁2₁2₁, $\text{a = 37.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}\text{, b = 41.2}\text{A}\text{?}$, with one molecule in the Crystallographic asymmetric unit.     

Genotoxicity of carcinogenic N-nitrosopropylamine derivatives in the hepatocyte primary culture/DNA-repair test

The genotoxicity of N-nitrosodipropylamine, 8 of its oxidized derivatives and N-nitroso-2,6-dimethylmorpholine was examined in the hepatocyte primary culture (HPC)/DNA repair test. Nine N-nitrosamines which are known to be carcinogenic and mutagenic were clearly positive in the HPC/DNA-repair test. N-Nitroso(2,3-dihydroxypropyl) (2-hydroxypropyl)amine did not elicit DNA repair, but showed a borderline mutagenic response in the Salmonella/microsome test. Thus, the HPC/DNA-repair test displays a comparable capacity to the bacterial mutagenesis test for detecting the genotoxic effects of this class of carcinogens.     

Alleviation of Aluminum Stress and Stimulation of Tea Pollen Tube Growth by Fluorine

Aluminum (Al) and fluorine (F) were found to affect tea pollentube growth on an agar medium. Not only was the growth stronglyrepressed by increasing Al content of the medium but it wasalso distinctly affected by declining pH from 5.2 to 4.4. Additionof 0.2 mM Al as Al₂(SO₄)₃ to a pH 4.6 medium containing 1.2%agar, 8% sucrose and 17 ppm boron remarkably repressed tea pollentube growth. However, NaF added to medium containing Al clearlyalleviated the growth inhibition. This effect was observed with0.2 mM and 0.4 HIM NaF, and the presence of 0.6 mM and 1.2 DIMNaF with 0.2 mM Al even produced a stimulatory effect. Treatmentwith NaF alone significantly stimulated growth at pH 4.6 and5.2. These results indicate that Al-F complexes have a favorablerather than adverse effect on tea pollen tube growth. (Received November 22, 1982; Accepted April 20, 1983)     

Phenethyl Alcohol Resistance in ESCHERICHIA COLI. III. a Temperature-Sensitive Mutation (dnaP) Affecting DNA Replication

A temperature-sensitive DNA replication mutant of E. coli K-12 was isolated among the mutants selected for phenethyl alcohol resistance at low temperatures. This mutation, designated as dnaP18, affects sensitivity of the cell to phenethyl alcohol, sodium deoxycholate and rifampicin, presumably due to an alteration in the membrane structure. At high temperatures (e.g., 42 degrees ), synthesis of DNA, but not RNA or protein, is arrested, leading to the formation of "filaments" in which no septum formation is apparent. Nucleoids observed under electron microscope seem to become dispersed and DNA fibrils less condensed, which may explain the loss of viability under these conditions. Genetic analyses, including reversion studies, indicate that a recessive dnaP mutation located between cya and metE on the chromosome is responsible for both alterations of the membrane properties and temperature sensitivity. The dnaP18 mutation does not affect growth of phage T4 or lambda under conditions where host DNA replication is completely inhibited. Kinetic studies of DNA replication and cell division in this mutant after the temperature shift from 30 to 42 degrees , and during the subsequent recovery at 30 degrees , accumulated evidence suggesting that DNA replication comes to a halt at 42 degrees upon completion of a cycle already initiated before the temperature shift. Since the recovery of DNA synthesis after exposure to 42 degrees does not depend on protein or RNA synthesis or other energy-requiring processes, the product of the mutant dnaP gene appears to be reversibly inactivated at 42 degrees . Taken together with the recessive nature of the present mutation, it was suggested that one of the membrane proteins involved in initiation of DNA replication is affected in this mutant.