Production of normal young following transfer of mouse embryos obtained by in vitro fertilization using cryopreserved spermatozoa

Spermatozoa from cauda epididymis of mature mice were suspended in preservation solution (Dulbecco's PBS containing raffinose in combination with glycerol, DMSO or skim milk as freezing protective agents). The suspension was frozen by the dry ice-alcohol method and preserved for 1-120 days in liquid nitrogen (-196 degrees C). Highest sperm viability after thawing was obtained with a combination of 10% raffinose and 5% glycerol or with a combination of 10% raffinose and 10% DMSO. These frozen thawed sperm were found to have fertilizing capacity when used for in vitro fertilization. The 2-cell embryos obtained through the above procedures developed into normal pups at a high rate when transferred into the oviducts of pseudopregnant female mice.     

The actin-depolymerizing factor destrin has an actin-stabilizing domain.

Destrin is a 19 kDa actin-depolymerizing protein of the ADF-cofilin family. Destrin was digested with trypsin to a structurally stable 9.2 kDa fragment that contains the actin-binding sequence. The purified 9.2 kDa fragment has an actin filament stabilizing activity, rather than an actin filament depolymerizing activity. The deleted region is probably essential for the actin filament depolymerizing activity of intact destrin. Surprisingly, the 9.2 kDa fragment also has an assembly-promoting activity in the absence of ATP.     

Three phases in transformation of human lymphocytes by Epstein-Barr virus, as revealed by colony formation in soft agar

Human cord lymphocytes were examined for colony formation in soft agar medium at various times after infection with Epstein-Barr virus (EBV). The transformation process could be divided into the following three sequential phases: (1) A pre-early phase, lasting about 10 hr post infection (pi), during which the number of transformed colonies was virus dose dependent. (2) An early phase, between about 10 and 60 hr pi, during which cloning efficiency was markedly lower than that of the pre-early phase. (3) A later phase, starting at about 60 hr pi, during which cloning efficiency was restored and the number of colony formers increased in an exponential fashion. EBV-associated nuclear antigen-positive cells first appeared at the beginning (12 hr pi) of the early phase and increased continuously thereafter. Increased DNA synthesis began at about 48 hr pi. These results suggest that the cloning efficiency of the infected cells changes during the transformation process, possibly reflecting altered cell sensitivity to agar.     

Retinoic acid receptor stimulation protects midbrain dopaminergic neurons from inflammatory degeneration via BDNF-mediated signaling

Functions of retinoic acid receptors (RARs) in adult CNS have been poorly characterized. Here we investigated potential neuroprotective action of tamibarotene (Am80), an RARα/β agonist available for the treatment of acute promyelocytic leukemia, on midbrain dopaminergic neurons. Am80 protected dopaminergic neurons in rat midbrain slice culture from injury mediated by lipopolysaccharide-activated microglia, without affecting production of nitric oxide, a key mediator of cell injury. The effect of Am80 was mimicked by another RAR agonist, TAC-101, but not by a retinoid X receptor agonist, HX630, and HX630 did not synergize with Am80. We observed neuronal expression of RARα and RARβ in midbrain slice culture and also found that Am80 increased tissue level of brain-derived neurotrophic factor (BDNF) mRNA. Exogenous BDNF prevented dopaminergic neurodegeneration, and the neuroprotective effect of Am80 was suppressed by a TrkB inhibitor, K252a, or by anti-BDNF neutralizing antibody. These results reveal a novel action of RARs mediated by enhancement of BDNF expression. Finally, oral administration of Am80 prevented dopaminergic cell loss in the substantia nigra induced by local injection of lipopolysaccharide in mice, indicating that RARs are a promising target of therapeutics for neurodegenerative disorders.     

Cell volume change of Escherichia coli under stringent control apparent increase of K in rel cells

With several pairs of rel⁺ and rel⁻ strains of Escherichia coli, the effects of amino acid starvation on the intracellular concentration of K⁺ and the rate of uptake of ⁴²K⁺ were investigated. In the early phase of the experiments, the intracellular concentration of K⁺ was estimated by the conventional method in which the cell volume per A₆₆₀ value of the culture was assumed to be constant, being not influenced by the variation of growth condition and strain. Apparently, the K⁺ concentration of rel⁺ cells was kept almost constant, while that of rel⁻ cells increased about 1.5-fold 2 h after the exposure to amino acid starvation. Unexpectedly, however, the above assumption was found not to be valid in the present study. The cell volume per A₆₆₀ changed only slightly in CP78 (rel⁺) cells, while it increased markedly in CP79 (rel⁻) cells after the exposure to amino acid starvation. Reestimation of the K⁺ concentrations based on the estimated respective values of cell volumes per A₆₆₀ revealed no significant difference between both strains. After all, the above apparent phenomenon was found to be due to the fact that the increase in cell volume of the rel⁺ cells was arrested upon amino acid starvation whereas that in the rel⁻ cells was not. The ⁴²K⁺ uptake by the rel⁺ cells was depressed upon amino acid starvation, whereas that by the rel⁻ cells increased. Some regulatory mechanism was suggested to operate in both strains to keep their K⁺ concentrations constant. When intracellular concentration of a metabolite is to be determined, importance of measurement of cell volume under the respective conditions, without assuming the constancy of the cell volume per A₆₆₀ of the culture, was pointed out.