Regulatory Mechanism of Delayed-Type Hypersensitivity in Mice: III. In Vitro Analysis of Memory T Cells Involved in Augmentation of DTH Responses

The memory of delayed-type hypersensitivity (DTH), manifested by the augmented responsiveness upon challenge with alum-absorbed ovalbumin (OA), was induced in mice primed 7 days, 21 days, or 90 days previously with 1 μg of reduced and alkylated OA. The memory cells involved in the augmentation of DTH responses were analyzed in the in vitro induction system of T cells which mediate DTH against OA. Spleen cells from the primed mice generated DTH-effector T cells (DTH-Te) in a significantly accelerated fashion, compared with unprimed spleen cells, when cultured with OA. The accelerated generation of DTH-Te in vitro was induced antigen specifically and was dependent on a certain T cell population in the primed spleen. The T cell population was found in the spleen of primed mice for at least 3 months after priming, corresponding to the persistence of DTH-memory in vivo. Moreover, it was fractionated in the high-density layer by discontinuous bovine serum albumin gradient centrifugation. The high-density cell population decreased in density with increase in the time of culture and developed into DTH-Te, which were separated in the low-density layer on day 4 of culture. These results indicate that the T cells involved in the accelerated generation of DTH-Te in vitro are long-lived DTH-memory T cells, which are probably precursor cells, capable of differentiating into DTH-Te upon challenge with the antigen.     

Newt RAD51: Cloning of cDNA and analysis of gene expression during spermatogenesis

A cDNA encoding a newt homolog of Escherichia coli RecA and yeast RAD51 from a testis cDNA library was isolated. The newt RAD51 (nRAD51) cDNA predicted a 337 amino acid protein with a 95-96% amino acid identity to Xenopus and mammalian RAD51. Northern blot analysis showed that nRAD51 mRNA, 1.7 kb in length, was expressed strongly in the testis and ovary, but weakly in the liver, kidney and brain. In situ hybridization revealed that expression of nRAD51 mRNA was barely observed in primary spermatogonia (one cell in a cyst) and early secondary spermatogonia (two to four cells in a cyst), but increased in late secondary spermatogonia (> or =eight cells in a cyst), reaching a maximum level in leptotene-zygotene spermatocytes, and thereafter declined. These results suggest that nRAD51 is involved in mitotic recombination in spermatogonia as well as in meiotic recombination in spermatocytes.     

Correlation between Production of Inflammatory Substances and Generation of Effector T Cells Mediating Delayed-Type Hypersensitivity in Mice

In vitro exposure to human serum albumin (HSA) of splenic lymphocytes from mice sensitized for delayed-type hypersensitivity (DTH) against HSA resulted in the release of substances that could induce a footpad inflammatory reaction with a maximum 6 hr after injection into normal mice. The substances were fractionated mainly in a molecular weight range of 30,000 to 70,000 daltons on Sephadex G-200. The ability of sensitized lymphocytes to produce the substances was dependent on T cells, was antigen specific, and correlated well with the ability of the lymphocytes to mediate DTH reactions. Moreover, the substances were produced efficiently by the DTH effector cell population generated in the in vitro culture system and also by the effector cell-enriched fractions on discontinuous bovine serum albumin gradients. These results suggest that the substances are produced by DTH-effector cells.     

A new method for determination of parity in optical diffraction patterns from the structures with helical symmetry

In optical diffraction patterns from the structures with helical symmetry, the reflections appear as layer-lines. The Bessel function orders (n) of the layer-lines are not easily determined, because the radial co-ordinates of the principal maxima on the layer-lines cannot be measured precisely. We developed a method to find whether n is even or odd (parity of n) by superposing two identical images of a particle back to back with the two axes aligned. The method was successfully applied to analysis of the structure of straight polyhooks isolated from the mutant SJW880 of Salmonella typhimurium.     

Inhibition of second meiotic division and a switching over to flagellar formation in secondary spermatocytes of newt by cycloheximide

10.0 micro M cycloheximide (CH) was found to completely inhibit the second meiotic division of newt spermatocytes. Under continuous incubation with CH from the beginning of interphase II, secondary spermatocytes fail to initiate chromosomal condensation and thus remain in interphase II. After 12-15 h of incubation, a single motile flagellum, about 5 micrometers in length, was observed on each of the secondary spermatocytes. These flagella grew to a length of 60-80 micrometers, but thereafter ceased to grow, whereas ordinarily spermatids grew flagella up to 500 micrometers in length in the absence of CH [1]. When CH was applied within 2 h following telophase I, the percentage of meiosis II inhibition was almost 100% and when applied even later, it became less, which showed that the early half period during interphase II was sensitive to CH. Regardless of the length of incubation time with CH, flagella were found to grow within a period of 12-15 h following telophase I. Upon removal of CH, even after 60 h of incubation, the flagella of the secondary spermatocytes shortened and disappeared completely. These spermatocytes underwent the second meiotic divisions. Also, flagella grew on the resulting spermatids. The possibility that a particular centriole which participated in the first meiotic division changes into a basal body for flagellar formation under the influence of CH and vice versa upon removal of it, is discussed in the following.     

Absolute configuration of Nδ-benzoyl-γ-hydroxyornithine from Vicia pseudo-orobus

A pair of diastereoisomers of Nδ-benzoyl-γ-hydroxy-l-ornithine was synthesized. By comparison with the two synthetic compounds, the natural     

Molecular Determination of Infection Source of a Sporadic Legionella Pneumonia Case Associated with a Hot Spring Bath

To determine the infection source of a sporadic Legionella pneumonia case associated with a hot spring bath, we used five molecular methods, including repetitive element polymerase chain reaction (rep-PCR), arbitrarily primed PCR (AP-PCR), ribotyping, restriction endonuclease analysis (REA), and macrorestriction endonuclease analysis (MREA) by pulsed-field gel electrophoresis. L. pneumophila serogroup (SG) 3 strain EY 3702, isolated from an intratracheal specimen of a 71-year-old Japanese female who developed pneumonia after nearly drowning in a hot spring spa bath, produced rep-PCR and AP-PCR fingerprints identical to those of L. pneumophila SG 3 strains EY 3768 and EY 3769 isolated from the bath water. Four epidemiologically unrelated L. pneumophila SG 3 strains showed different rep-PCR or AP-PCR fingerprints from those of the three EY strains (EY 3702, 3768, and 3769). The three EY strains were also genotypically indistinguishable by ribotyping with EcoRI and PstI, by REA with EcoBI or HindIII, and by MREA with NotI. Based on these results, we identified the bath water of the hot spring spa as the source of infection of this patient, even though the viable number of the organisms in the bath water was low (3 CFU/100 ml) when determined 27 days after her nearly drowning.