Murine acute leukemia cell line with megakaryocytic differentiation (MK-8057) induced by whole-body irradiation in C3H/He mice: cytological properties and kinetics of its leukemic stem cells

Five cases of murine leukemia with megakaryocytic differentiation were observed among the 417 cases of radiation-induced leukemias which developed in 30% of C3H/HeMs mice exposed at 8 to 10 weeks to 0.5 to 5 gy total body irradiation. Cells from individual leukemic colonies in the spleen of the irradiated mice, and cells from colonies in methylcellulose (MC) culture in vitro, derived from one of these leukemias, MK-8057, were injected into mice; both types of cells caused the deaths of the recipient mice by inducing the same type of leukemia. MK-8057 can be maintained in Dexter-type liquid culture with a feeder layer of irradiated bone marrow cells. There was a linear reciprocal relationship between the increasing number of MK-8057 cells injected versus the survival of the recipient mice. A reciprocal relationship also was seen between an increasing number of leukemic stem cells, corresponding to the number of MK-8057 cells, and the survival of mice injected with MK-8057. Giant nuclear megakaryocytes developed during the course of colony growth in the spleen as they did in the MC culture. Such megakaryocytes were acetylcholinesterase positive, whereas leukemic cells in the peripheral blood showed no sign of platelet production nor of a positive reaction to acetylcholinesterase. Cells maintained in culture were entirely positive in platelet glycoprotein IIb/IIIa when anti-human antibody was used. The larger cells in a splenic cell suspension derived from a moribund mouse were separated and enriched by velocity sedimentation using centrifugal elutriation (CE), and then subjected to flow cytometry using propidium iodide staining. Cells with up to 32N-DNA content were detected. After separating MK-8057 by counter-flow CE, the larger cell fraction (mode at 540 microns3) produced more leukemic colonies when injected into irradiated mice than did the small cell fraction (mode at 240 microns3). A higher percent of the larger cell fraction (61.9%) was killed by the addition of tritiated thymidine cytocide than in the smaller cell fraction (14.9%). Thus, the smaller cell fraction is considered to have more leukemic spleen colony-forming units (L-CFU-s) in the resting state.     

Associative learning and discrimination of motion cues in the harnessed honeybee Apis mellifera L.

We previously studied a conditioning paradigm to associate the proboscis extension reflex (PER) with monochromatic light (conditioned stimulus; CS) in harnessed honeybees. Here, we established a novel conditioning paradigm to associate the PER with a motion cue generated using graphics interchange format (GIF) animations with a speed of 12 mm/s speed and a frame rate of 25 Hz as the CS, which were projected onto a screen consisting of a translucent circular cone that largely covered the visual field of the harnessed bee using two liquid crystal projectors. The acquisition rate reached a plateau at approximately 40% after seven trials, indicating that the bees were successfully conditioned with the motion cue. We demonstrated four properties of the conditioning paradigm. First, the acquisition rate was enhanced by antennae deprivation, suggesting that sensory input from the antennae interferes with the visual associative learning. Second, bees conditioned with a backward-direction motion cue did not respond to the forward-direction, suggesting that bees can discriminate the two directions in this paradigm. Third, the bees can retain memory for motion cue direction for 48 h. Finally, the acquisition rate did not differ significantly between foragers and nurse bees. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Progeny production in the periplasm of Thermosipho globiformans

Thermotogales are rod-shaped, Gram-negative, anaerobic, (hyper) thermophiles distinguished by an outer sheath-like toga, which comprises an outer membrane (OM) and an amorphous layer (AL). Thermosipho globiformans bacteria can transform into spheroids with multiple cells concurrently with AL disintegration during early growth; the cell is defined as the cytoplasmic membrane (CM) plus the entity surrounded by the CM. Spheroids eventually produce rapidly moving periplasmic ‘progenies’ through an unknown mechanism. Here, we used high-temperature microscopy (HTM) to directly observe spheroid generation and growth. Rod OMs abruptly inflated to form ~2 μm-diameter balloons. Concurrently, multiple globular cells emerged in the balloons, suggesting their translocation and transformation from the rod state. During spheroid growth, the cells elongated and acquired a large dish shape by possible fusion. Spheroids with dish-shaped cells further enlarged to ~12 μm in diameter. HTM and epifluorescence-microscopy results collectively indicated that the nucleoids of dish-shaped cells transformed to form a ring shape, which then distorted to form a lip shape as the spheroid enlarged. HTM showed that ‘progenies’ were produced in the spheroid periplasm. Transmission electron microscopy results suggested that the ‘progenies’ represented immature progenies lacking togas, which were acquired subsequently.

     

Structural Basis for Antibody Recognition in the Receptor-binding Domains of Toxins A and B from Clostridium difficile

Clostridium difficile infection is a serious and highly prevalent nosocomial disease in which the two large, Rho-glucosylating toxins TcdA and TcdB are the main virulence factors. We report for the first time crystal structures revealing how neutralizing and non-neutralizing single-domain antibodies (sdAbs) recognize the receptor-binding domains (RBDs) of TcdA and TcdB. Surprisingly, the complexes formed by two neutralizing antibodies recognizing TcdA do not show direct interference with the previously identified carbohydrate-binding sites, suggesting that neutralization of toxin activity may be mediated by mechanisms distinct from steric blockage of receptor binding. A camelid sdAb complex also reveals the molecular structure of the TcdB RBD for the first time, facilitating the crystallization of a strongly negatively charged protein fragment that has resisted previous attempts at crystallization and structure determination. Electrospray ionization mass spectrometry measurements confirm the stoichiometries of sdAbs observed in the crystal structures. These studies indicate how key epitopes in the RBDs from TcdA and TcdB are recognized by sdAbs, providing molecular insights into toxin structure and function and providing for the first time a basis for the design of highly specific toxin-specific therapeutic and diagnostic agents.     

Effects of ingested phytoecdysteroids on the growth and development of two Lepidopterous larvae

The effects of ingested or injected 20-hydroxyecdysone on silkworm larvae (Bombyx mori) including death without moulting, death following completion of promoted moulting, death during promoted moulting (ecdysis inhibition) and inhibition in growth with and without effects on moulting, are dependent upon the concentration of exogenous hormone, the precise developmental stage of the treated larvae, and the duration of exposure to the exogenous ecdysteroid. Comparisons of 20-hydroxyecdysone with other phytoecdysteroids in the silkworm and pink bollworm, Pectinophora gossypiella, show a similar but more potent effect induced by ponasterone A, while cyasterone causes an ‘antiecdysone’ effect.     

The cytoplasmic male-sterile type and normal type mitochondrial genomes of sugar beet share the same complement of genes of known function but differ in the content of expressed ORFs

The complete nucleotide sequence (501,020 bp) of the mitochondrial genome from cytoplasmic male-sterile (CMS) sugar beet was determined. This enabled us to compare the sequence with that previously published for the mitochondrial genome of normal, male-fertile sugar beet. The comparison revealed that the two genomes have the same complement of genes of known function. The rRNA and tRNA genes encoded in the CMS mitochondrial genome share 100% sequence identity with their respective counterparts in the normal genome. We found a total of 24 single nucleotide substitutions in 11 protein genes encoded by the CMS mitochondrial genome. However, none of these seems to be responsible for male sterility. In addition, several other ORFs were found to be actively transcribed in sugar beet mitochondria. Among these, Norf246 was observed to be present in the normal mitochondrial genome but absent from the CMS genome. However, it seems unlikely that the loss of Norf246 is causally related to the expression of CMS, because previous studies on mitochondrial translation products failed to detect the product of this ORF. Conversely, the CMS genome contains four transcribed ORFs (Satp6presequence, Scox2-2 , Sorf324 and Sorf119) which are missing from the normal genome. These ORFs, which are potential candidates for CMS genes, were shown to be generated by mitochondrial genome rearrangements.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by R. Hagemann     

Ultrastructural studies on early events in fertilization of the bivalveLaternula limicola

Summary Ultrastructural studies on sperm-egg interaction at the time of fertilization inLaternula limicola were performed. The temporary-acrosome did not change morphologically while the sperm passed through the egg investments. At the onset of sperm entrance into the egg, however, the temporary-acrosome and mitochondria were eliminated from the sperm. Afterwards the sperm was engulfed by the egg surface without membrane fusion of the gametes. After entry the sperm nucleus was surrounded by four membranes: the plasma membranes of the egg and of the sperm, and the membranes of the sperm nuclear envelope. As the sperm nucleus differentiated into the male pronucleus, the plasma membranes of both the sperm and egg were initially vesiculated, then dispersed into the egg cytoplasm. Finally, the sperm nuclear envelope changed into the male pronuclear membrane accompanying sperm chromatin dispersion.     

Immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice

Studies on the immediate and long-term effects of radiation on the immune system of specific-pathogen-free mice are summarized in this paper. There was a striking difference in the radiation response of lymphocyte subsets; B cells consist of a fairly radiosensitive homogeneous population, whereas T cells consist of a large percentage (greater than 90 per cent) of radiosensitive and a small percentage (less than 10 per cent) of extremely radioresistant subpopulations. Ly 1+ and Ly 2+ lymphocytes appear equally radiosensitive, although the percentage of radioresistant cells was slightly larger for the former (approximately 5.5 per cent) than the latter (approximately 2.5 per cent). There was a significant strain difference in the radiosensitivity of immune-response potential in mice; immunocompetent cells of C3H mice were more radioresistant than those of BALB/c, C57BL/6, and B10.BR mice. Studies on the long-term effect of radiation on immune system in mice indicated no evidence for accelerated ageing of the immunologic functions when radiation exposure was given to young adults. Preliminary results on the enhancing effect of low dose radiation on cytotoxic T cell response in vitro are also discussed.