In Vitro Studies on the Immunological Memory for Antibody Response to Bovine Serum Albumin

Immunological memory for T and B cells was studied in an in vitro culture system with spleen cells from mice primed with bovine serum albumin (BSA). Spleen cells taken from mice immunized at various times previously with a single intravenous injection of alum-precipitated (AP) BSA and bacterial endotoxin (ET) were cultured in Marbrook's system with dinitrophenylated (DNP) BSA as the in vitro antigen. In the cultures of spleen cells obtained from mice primed more than 14 days previously, an IgG-predominant anti-BSA response was generated. However, no anti-BSA response was observed in the culture of spleen cells taken from mice primed 7 days previously (day 7 spleen cells). The failure of day 7 spleen cells to generate an antibody response in vitro was shown to be attributable to both the lack of B memory cells and the effect of “suppressive” macrophages induced by ET. On the other hand, anti-BSA memory in the spleen of mice primed with AP-BSA plus ET and 2 months later challenged with AP-BSA matured within 7 days and declined rather quickly by 30 days after the challenge. The difference in the time course of the generation of memory between the spleen cells from primary and from secondary immunized mice might be attributable to the difference in the maturation of memory B cells, since the time course of the development of memory T cells after the secondary immunization was similar to that observed after primary immunization.     

Action Spectra for Inhibition by Light of Accumulation of Bacteriochlorophyll and Carotenoid during Aerobic Growth of Photosynthetic Bacteria

Action spectra for the inhibition by light of the accumulationof photosynthetic pigments during the aerobic growth of a photosyntheticbacterium, Rhodobacter sphaeroides, and an aerobic photosyntheticbacterium, Erythrobacter sp. strain OCh 114, were determinedover the range of wavelengths from 380 to 870 nm. The actionspectra for the inhibition of accumulation of bacteriochlorophyllin both R. sphaeroides and Erythrobacter sp. strain OCh 114indicated that the maximum inhibition occurred at approximately400 nm and a low level of inhibition occurred at 575 and 770nm. In R. sphaeroides, the action spectrum for the inhibitionof accumulation of carotenoid paralleled that for the inhibitionof accumulation of bacteriochlorophyll over the same range ofwavelengths. These results indicate that in both species, grownunder aerobic conditions, the same photoreceptor is involvedin the inhibition. One possible candidate for the relevant photoreceptormay be the precursor(s) to bacteriochlorophyll. It is possiblethat the photoreceptor is decomposed by light absorbed by itselfor by an unidentified photoreceptor that absorbs blue light(a photo-sensitizer). It is suggested that the accumulationof carotenoid is dependent on the stability of the bacteriochlorophyll. (Received September 16, 1988; Accepted March 2, 1989)     

Adenosine 5'-O-(3-thiotriphosphate) hydrolysis by dynein

The interaction of dynein with ATP gamma S, a phosphorothioate analogue of ATP, has been investigated in depth. The hydrolyses of ATP gamma S and of ATP were shown to be mutually competitive. ATP gamma S induced complete dissociation of the microtubule-dynein complex such that the time course of dissociation monitored by stopped-flow light-scattering methods followed a single exponential. The ATP gamma S concentration dependence of the rate of dissociation was hyperbolic, indicating that the dissociation is at least a two-step process: M.D + ATP gamma S in equilibrium M.D.ATP gamma S----M + D.ATP gamma S. The fit to the hyperbola gives an apparent Kd = 0.5 mM for the binding of ATP gamma S to the microtubule-dynein complex, and the maximal rate of 45 s-1 defines the rate of dissociation of the ternary M.D.ATP gamma S complex. Rapid quench-flow experiments demonstrated that the hydrolysis of ATP gamma S by dynein exhibited an initial burst of product formation. The size of the burst was 1.2 mol/10(6) g of dynein, comparable to that in the case of ATP hydrolysis. The steady-state rate of ATP gamma S turnover by dynein was activated by MAP-free microtubules. Because the rate of ATP gamma S turnover is severalfold (4-8) slower than ATP turnover, the rate-limiting step must be release of thiophosphate, not ADP. Thus, microtubules can activate the rate of thiophosphate release. The stereochemical course of phosphoric residue transfer was determined by using ATP gamma S stereospecifically labeled in the gamma position with 18O.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased frequency of 6-thioguanine-resistant peripheral blood lymphocytes in Werner syndrome patients

Summary The frequency of spontaneous 6-thioguanine (TG)-resistant peripheral blood lymphocytes in five unrelated Werner syndrome (WS) patients was determined using an autoradiographic labeling assay. The average frequency of TG-resistant lymphocytes was eightfold higher in WS patients than in sex- and age-matched normal control donors. This finding and previous identification of increased spontaneous chromosomal rearrangements and deletions in WS cells or cell lines suggest that WS is a human genomic instability or mutator syndrome.     

Steady-state kinetic study on the inhibition of the adenosinetriphosphatase activity of dynein from Tetrahymena cilia by glycerol

The characteristics of glycerol-induced inhibition of the dynein ATPase extracted from Tetrahymena cilia were investigated. Fifty percent inhibition was observed at about 15% (v/v) glycerol with the 22S dynein Mg-ATPase. Ethylene glycol was equally inhibitory, while sucrose, a kind of polyol, was less effective. The glycerol-induced inhibition of the 22S dynein Mg-ATPase was not influenced by pH or by raising the ionic strength of the assay solution. An aqueous glycerol solution treated with anion or cation exchanger or charcoal was equally inhibitory to a non-treated solution. The inhibition was most likely to be due to glycerol or ethylene glycol itself, not to a contaminant. The inhibition of the 22S dynein Mg-ATPase was apparently noncompetitive: only the Vmax was reduced without a significant change in the apparent Km. The dynein ATPase is known to be inhibited potently by vanadate. Glycerol reduced the sensitivity of the dynein ATPase to the vanadate-induced inhibition. Glycerol exhibited a decelerating effect on the rate of the oxygen exchange between phosphate and water catalyzed by 22S dynein in the presence of ADP and Mg2+. If it is assumed that the rate constants of the ATP hydrolysis step are not affected by glycerol, it may be implied that the phosphate release from the E.ADP.P1 intermediate was decelerated by glycerol and that the deceleration of the phosphate release paralleled the reduction of the overall ATPase activity over a wide range of glycerol concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Some Properties of Soluble Cytochromes, Cytochrome c-551 and Cytochrome c-550, from a Facultative Methylotroph, Protaminobacter ruber strain NR-1

Two soluble cytochromes of the C-type, cytochrome c-551 andcytochrome c-550, were purified from the bacteriochlorophyll-containingcells of a facultative methylotroph, Protaminobacter ruber StrainNR-1, by ion-exchange chromatography and gel-filtration. Cytochrome c-551 had absorption maxima at 551, 522 and 416 nmin the reduced form, and at 525, 410 and 273 nm in the oxidizedform. This cytochrome was a slightly basic protein with an isoelectricpoint of 8.4. It had a mid-point redox potential of 272 mV atpH 7.0. The molecular weight of this protein was 13,500 and13,700 by sodium dodecylsulfate polyacrylamide gel electrophoresis(SDS-PAGE) and gel-filtration, respectively. Cytochrome c-550 had absorption maxima at 550, 522 and 415 nmin the reduced form, and at 527, 409 and 278 nm in the oxidizedform. This cytochrome was acidic, having an isoelectric pointof 4.3. It had a mid-point redox potential of 227 mV at pH 7.0.Its molecular weight was 19,500 and 22,000 by SDS-PAGE and gel-filtration,respectively. (Received August 4, 1984; Accepted October 22, 1984)     

Structure and inherent properties of the bacteriophage lambda head shell. III. Spectroscopic studies on the expansion of the prohead

The head shell of bacteriophage lambda expands by about 20% in diameter when it packages the DNA molecule in vivo. The expansion reaction is essentially a conformational change of the major head protein molecules to a state of lower free energy and can also be triggered in vitro by treatment with 4 M-urea. In order to investigate the conformational change, we have measured the circular dichroism, fluorescence and difference absorption spectra of the lambda head shell before and after the expansion by the treatment with urea. The far-ultraviolet circular dichroism spectra and the fluorescence spectra show that the expansion is not accompanied by a great change in the secondary structure (29% alpha-helix, 23% beta-structure) and the environment (non-polar) of the tryptophan residues of the major head protein molecule. On the other hand, by measurements of the circular dichroism and difference absorption spectra in the near-ultraviolet region as well as by chemical modification experiments with tetranitromethane, we have found that one or two tyrosine residues of the major head protein are transferred from a polar, solvent-exposed to a non-polar, solvent-unexposed environment during the expansion. Judging from these results, the conformational change seems to be mainly intermolecular or interdomainal rather than intradomainal.     

Isolation and Purification of Cytochrome b561 from a Photosynthetic Bacterium, Rhodopseudomonas sphaeroides

Cytochrome b₅₆₁ was removed from chromatophores of a photoanaerobicallygrown Rhodopseudomonas sphaeroides by deoxycholate-cholate andTriton X-100 treatments of the chromatophores. The cytochromewas purified by ammonium sulfate fractionation and gel filtration.Its molecular weight was 45,000 (45 kD) and it was composedof three subunits with molecular weights of 23 kD, 19 kD andless than 6 kD. The cytochrome preparation had absorption maximaat 414 nm in the oxidized form, and at 428, 530 and 561 nm inthe reduced form. Its pi was 4.8. The midpoint potential ofthis cytochrome was 153 mV at pH 7.0. The compound was autooxidizable,and it had cytochrome c oxidase activity. (Received May 16, 1983; Accepted September 8, 1983)     

Structure and inherent properties of the bacteriophage lambda head shell. IV. Small-head mutants

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.     

Peroxidatic activity of catalase in the cortical granules of sea urchin eggs

The presence of peroxidatic activity of catalase in eggs of the sea urchins Hemicentrotus pulcherrimus and Temnopleurus toreumaticus was investigated by the ultrastructural cytochemical techniue and by biochemical assay on homogenates of eggs from before fertilization to the 2-cell stage. Biochemical assays showed that the unfertilized eggs had strong catalase activity whereas fertilized eggs had weak activity owing to the rapid decrease of activity after fertilization. The activity did not change from immediately after fertilization to the 2-cell stage. Cytochemical examination showed that the peroxidatic activity of catalase was mainly localized in the lamellae in the cortical granules. Disintegrated cortical granules with no lamellae and substances in the perivitelline space derived from breakdown of the cortical granules had no peroxidatic activity of catalase.