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.     

Ultrastructural localization of hCG in the placenta and in testicular germ cell tumors

Human chorionic gonadotrophin(hCG)-producing cells in the human placenta and in testicular germ cell tumors have been investigated by ultrastructural immunohistochemistry using labeled Fab'fragments of anti-hCG-alpha and anti-hCG-beta. Most reaction products were demonstrated in the rough endoplasmic reticulum (rER) and perinuclear spaces of syncytiotrophoblastic giant cells ( STGC ) occurring in embryonal carcinomas and in syncytial cells observed in choriocarcinomas and in normal placenta. The rER of placental and choriocarcinomatous syncytial cells was highly developed and invariably showed a positive reaction. However, the rER of STGCs varied both with regard to its degree of development and its immunoreactivity with hCG. Localization of hCG in the Golgi apparatus and in cytoplasmic granules was indefinite. Mononuclear cells reacting positively with anti-hCG-alpha or -beta were also demonstrated. Cytotrophoblast-like cells which failed to react with anti-hCG-alpha or -beta were noted adjacent to sTGCs and the significance of these cells is discussed.     

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.