Generation of multivirus-specific T cells by a single stimulation of peripheral blood mononuclear cells with a peptide mixture using serum-free medium

Background: Restoration of virus-specific immunity by virus specific T cells (VSTs) offers an attractive alternative to conventional drugs, and can be highly effective in immunocompromised patients, including hematopoietic stem cell transplant (HSCT) recipients. However, conventional VSTs manufacture requires preparation of specialized antigen-presenting cells (APCs), prolonged ex vivo culture in serum-containing medium and antigen re-stimulation with viruses or viral vectors to provide viral antigens for presentation on APCs. Methods: To simplify this complex process, we developed a method to generate multiple VSTs by direct stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries in serum-free medium. Results: We generated VSTs that targeted seven viruses (cytomegalovirus [CMV], Epstein-Barr virus [EBV], adenovirus [AdV], human herpesvirus 6 [HHV-6], BK virus [BKV], JC virus [JCV] and Varicella Zoster virus [VZV]) in a single line. The phenotype, growth and specificity of multiple VSTs produced in serum-free medium were equivalent to those generated in conventional serum-containing medium. Discussion: The use of serum-free medium allows this approach to be readily introduced to clinical practice with lower cost, greater reproducibility due to the absence of batch-to-batch variability in serum and without concerns for infectious agents in the serum used. This simplified approach will now be tested in recipients of Human Leukocyte Antigen (HLA)–matched sibling HSCT.     

Alterations in the biomass-specific productivity of periphyton assemblages mediated by fish grazing

1. In an experimental flume, we examined the effects of a biomass reduction and alteration of taxonomic composition, because of grazing by the fish Plecoglossus altivelis, on the net biomass accumulation of periphyton. 2. Grazed and ungrazed assemblages with different biomass and taxonomic composition were first prepared in fish enclosures and exclosures, respectively. These assemblages were then set out in the flume and incubated for 2 days under grazing‐free conditions to examine (i) the relationship between biomass and biomass accumulation rate and (ii) the effect of taxonomic composition on the relationship between these two. 3. The grazed and ungrazed assemblages were dominated by upright filamentous cyanobacteria and diatoms, respectively. The rate of biomass accumulation decreased with increasing periphyton biomass in both the grazed and ungrazed assemblages, and was lower in the grazed than the ungrazed assemblages at any biomass level. 4. The results showed that the reduction in biomass and the alteration of taxonomic composition due to fish grazing have opposite effects on biomass‐specific productivity. Biomass accumulation rate increased in response to biomass reduction, although a shift in dominance from diatoms to upright filamentous cyanobacteria decreased the overall productivity of the periphyton.     

Nitrogen delivery to maize via mycorrhizal hyphae depends on the form of N supplied

Arbuscular mycorrhizal fungi can enhance nutrient acquisition by a plant via their extraradical hyphae. This is particularly true for phosphorus, but the case for nitrogen (N) has been less clear. In our growth systems there was a small air-gap between root and hyphal compartments, which eliminated diffusion of nutrients between compartments. Moreover, our methods allowed us to distinguish between nitrate and ammonium. We found that N transfer to Zea maize L. depends on the sources fed to the hyphae of Glomus aggregatum Schenck & Smith. In experiment 1, despite the fact that plant demand for N was already met, plants received 10 times as much ¹⁵N from ammonium than from nitrate. In experiment 2, 74% of shoot-N was derived from the slow-release urea added to the hyphal compartment while only 2.9% was derived from the nitrate-N. Intraradical hyphae isolated from roots contained a considerable amount of ¹⁵N in the cell wall even when ¹⁵N-nitrate was the source. We conclude that the mycorrhizal fungus can rapidly deliver ammonium-N to the plants, and that while the fungus can absorb nitrate, it apparently lacks the capacity to transfer it to the plant.     

Transition from primary dormancy to secondary dormancy in cocklebur seeds

Abstract The transition from primary dormancy to secondary dormancy was examined using upper cocklebur (Xanthium pennsylvanicum Wallr.) seeds. The non-after-ripened seeds with primary dormancy responded to chilling, anoxia, KCN, and NaN₃ with an increase in germination. However, their maximal responses to these treatments only occurred after a period of water imbibition, probably a reflection of the increasing growth potential of the axial tissue which was accompanied by the increase in the capacities of respiration and ethylene production. On the other hand, the establishment of secondary dormancy was accompanied by a decrease in respiration and ethylene production of seeds, and in the growth potential of both axial and cotyledonary tissues. The decrease in growth potential of these tissues occurred regardless of whether they were excised from after-ripened seeds or non-after-ripened seeds. It is inferred that the primary dormancy of cocklebur seeds is a state maintained in un-germinated seeds for a long time through a spontaneous transition to secondary dormancy.     

Pluripotency of Homozygous-Diploid Mouse Embryos in Chimeras

Pluripotency of mouse uniparental cells (complete homozygous-diploid gynogenetic) produced by embryo manipulation was examined in aggregation chimeras with normally fertilized embryos. A male pronucleus was removed from fertilized eggs by micromanipulation and eggs were diploidized with cytochalasin B. Uniparental cells that developed to 4-cell or more advanced stages were aggregated with normally fertilized 8-cell embryos and transferred to the pseudopregnant female uteri to develop to term. Among the pups, 1 female and 3 males were identified as overt chimeras by their coat color and pigmentation of the retina. Using electophoretic analysis of the isozymes, the contribution of uniparental cells in these chimeras was confirmed by findings in the major organs such as liver, brain, small intestine, kidney, spleen, heart and testis. The female chimera produced offspring derived from oocytes of uniparental origin. Our experiments verified the pluripotency of microsurgically produced mouse uniparental cells.     

Pattern and Time Course of Cleavages in Early Development of the Ovoviviparous Pond Snail, Sinotaia quadratus historica

The pattern and time course of cleavage during early development of the ovoviviparous pond snail, Sinotaia quadratus historica , in in vitro culture were investigated. The fertilized egg, 25–30 μm in diameter, underwent cleavage by repeated constriction and compaction as in blastocyst formation in mammalian embryos. The cleavage was slightly unequal and dextrally spiral, although a slight time lag in cleavages of blastomeres was observed after the 2nd cleavage. A small polar lobe was formed at the 1st cleavage, but not at the 2nd cleavage. Each cleavage proceeded very slowly under the experimental conditions, the 1st, 2nd, 3rd and 4th cleavages taking 22 hr or more, 9 hr and 10 hr, respectively. The cleavage pattern in in vitro cultures observed by light microscopy was confirmed by scanning electron microscopy.     

Phylogeny of Regulatory Regions of Vertebrate Tyrosinase Genes

Highly homologous DNA elements were found to be shared by the upstream regions of the mouse tyrosinase and tyrosinase related protein (TRP-1) genes. Several nuclear proteins were shown to bind to both of these upstream regions. Shared homologous DNA elements were also found in the 5’ flanking sequences of Japanese quail and snapping turtle tyrosinase genes. Shared homologous nucleotide sequences were found to be scattered like an archipelago in the 5’ upstream regions of mouse and human tyrosinase genes. Comparisons between Japanese quail and snapping turtle tyrosinase genes gave similar results. On the contrary, mammalian (mouse and human) and nonmammalian (quail and snapping turtle) tyrosinase genes did not show significant homology in their 5’ upstream regions. In contrast, coding sequences in the first exons of vertebrate tyrosinase genes and their deduced amino acid sequences were found to be highly conserved except for their putative leader sequence-coding regions.     

INDUCTIVE CAPACITIES OF GLOMERULAR BASEMENT MEMBRANES AND DENTINE MATRIX

The inductive capacities of the basement membranes of calf kidney glomeruli and the dentine matrix of the incisors of 23-day rabbit fetuses were examined on the presumptive ectoderm of Triturus gastrulae. The basement membranes caused almost entirely neural induction and the dentine matrix caused mesodermal induction. These findings suggest that intercellular substances play an important role in the inductive effects of heterologous tissues.     

CELL CYCLE STUDY UP TO THE TIME OF HATCHING IN THE EMBRYOS OF THE SEA URCHIN,HEMICENTROTUS PULCHERRIMUS*

Cell cycles have been analyzed in 10 divisions up to the time of hatching in the embryos of the sea urchin, Hemicentrotus pulcherrimus. In the first 5 cleavages, division synchrony is very high. On the average, T_GC= 55.4 min, T_G1= 0 min, T_s= 12 min, T_G2=±0 min, T_M= 42 min. In the remaining 5 cleavages, T_GC becomes longer: 70 min for the 7th to 246 min for the 10th cleavage. G₁ and G₂ become definitely recognizable and become longer along with T_s. T_M stays more or less constant. Plots of the changing lengths of the four compartments (G₁, S, G₂, M) on the Y-axis against T_GC (X-axis) can be fitted to the following 4 regression equations; T_G1= 0.28T_GC - 19.7, T_s= 0.609T_GC - 15.2, T_G2= 0.104T_GC - 4.72 and T_M= 0.007T_GC+ 39.6.     

EFFECTS OF THE SURFACTANTS ON THE CLEAVAGE AND FURTHER DEVELOPMENT OF THE SEA URCHIN EMBRYOS II. DISTURBANCE IN THE ARRANGEMENT OF CORTICAL VESICLES AND CHANGE IN CORTICAL APPEARANCE

After fertilization, two types of cortical vesicles ware examined under the electron microscope (the cortical vesicle I and II) and the light microscope (pigment granules and another kind of vesicles). The cortical vesicle I corresponds to the pigment granule and the cortical vesicle II does to the other vesicle.
The unequal division of the sea urchin embryo which occurs at the fourth cleavage was modified to an equal cleavage pattern by the treatment with sodium lauryl sulfate (SLS) or cetyl trimethyl ammonium bromide (CTAB). But other surfactants such as sodium deoxycholate, Tween 80, Lubrol PX did not have such an effect. The cell surface of the embryo which had been treated either SLS or CTAB became rough or smooth. Cortical vesicles and pigment granules disappeared and/or were dislocated from the cortex. However, cell organelles were as normal as the control. On the other hand, the cortical appearance of other surfactant-treated embryos showed no disturbance and cell organelles were also more or less normal. Therefore, the equalization of unequal cleavage is caused by the disturbance in the cortex and thus the cortex plays a major role on the micromere formation at the 16-cell stage and on the further sea urchin development.