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.     

Construction and characterization of Escherichia coli disruptants defective in the yaeM gene.

Escherichia coli disruptants defective in the yaeM gene, which is located at 4.2 min on the chromosome map, were constructed and characterized. The disruptants showed auxotrophy for 2-C-methylerythritol, a free alcohol of 2-C-methyl-D-erythritol 4-phosphate that is a biosynthetic precursor in the nonmevalonate pathway. This result clearly shows that the yaeM gene is indeed involved in this pathway in E. coli.     

LiCl treatment releases a nickase implicated in genetic transformation of Streptococcus pneumoniae.

When cells competent for genetic transformation of Streptococcus pneumoniae which could bind and enable entry of extracellular DNA molecules were treated with LiCl, they released a nickase that introduced nicks into a double-stranded DNA in the presence of EDTA. The nickase was specific for competent cells and coupled with DNA-binding activity. Furthermore, when noncompetent cells were treated with LiCl, they released the putative receptors for the competence activator.     

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)     

Molecular cloning of the gene coding for the human T cell differentiation antigen CD7

The CD7 molecule is a differentiation antigen found on the surface of T lymphocytes and also on a very minor fraction of acute nonlymphocytic leukemia (ANLL). To study the genomic structure of the CD7 gene, two clones (SY4 and SY22) were isolated by screening a genomic library with a CD7 cDNA probe. Restriction mapping of these two phage clones showed that both overlapped each other, covering a total length of 23 kilobases (kb). Transfection of mouse L cells demonstrated that SY22 contains the gene expressing the CD7 antigen reactive with monoclonal CD7 antibody (Tp40), while SY4 does not. Subcloning of a 10.5 kb fragment from a 14.4 kb insert of SY22 contained the structural gene for the CD7 antigen. Detailed restriction mapping and partial sequence analysis revealed the CD7 gene to consist of four exons. By RNase protection assay, multiple initiation sites — 122 base pairs (bp) to — 38 bp from ATG translation initiation site were demonstrated. The promoter region had high G+C content and contained two SP1 binding sites (CCGCCC) and an AP2 binding site (CCCCAGGC), but lacked CAAT and TATA motifs.     

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)     

Glycolipid composition of ascitic fluids from patients with cancer

The glycolipid composition of ascitic fluids from nine patients with cancer and one pleural effusion from a hepatoma patient was studied. Glucosylceramide, lactosylceramide, globotriaosylceramide, and globotetraosylceramide were found in all samples and also in normal human serum. These glycolipids accounted for more than 90% of the neutral glycolipid fraction and the composition in ascitic fluids was similar to that in normal human serum. From ascitic fluids, several minor glycolipids, which could not be detected in normal human serum, were isolated and characterized by exoglycosidase treatment. Lactoneotetraosylceramide was found in eight samples of ascitic fluids, and globopentaosylceramide was detected in two samples from hepatoma and one from pancreatic cancer. A fucolipid which was converted to lactoneotetraosylceramide by alpha-L-fucosidase treatment was recognized in two samples from hepatoma patients. In the ganglioside fraction, GM3 was the predominant component both in normal human serum and in ascitic fluid. The GM2 content in ascitic fluids was much higher than that in normal human serum. From these results, lactoneotetraosylceramide and GM2 are possible candidates as cancer markers, because they seemed to be derived from cancer tissues by shedding.     

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.