Pluripotency of mouse embryonic cells on germline at 3.5–8.5 and 11.5 days post-coitum after aggregation with precompacted embryos

Albino mouse embryonic cells (Gpi-la/a) at 3.5–8.5 and 11.5 days were aggregated with zona cut 8–16 cell stage embryos from F₁ females (Gpi-1 b/b), respectively. The aggregated embryos were transferred to pseudopregnant female mice. The recipients were allowed to go to term or were dissected at mid-gestation to assess the donor contribution in the conceptuses using glucose phosphate isomerase (GPI) analysis. The donor cells, which were previously labeled with fluorescent latex microparticles, were aggregated with embryos, and the allocation of the donor cells at the compacted morula and blastocyst stages were observed under a fluorescence microscope. When 3.5 and 45 day old inner-cell-mass (ICM) cells were used, fertile chimeric mice were obtained (50 and 19%, respectively), and when 5.5 days old primitive ectoderm cells were aggregated, they did not form chimeras but contributed to the fetuses, placenta and membrane after 13.5 days of pregnancy. However, cells from further stages never contributed to the conceptuses even though they were analyzed after 10.5 days of pregnancy. The labeled donor cells at these stages were not positively incorporated in the interior part of the compacted morula and the ICM of the blastocyst stage unlike the ICM at 3.5 days post-coitum after overnight culture.     

Molecular cloning and functional characterization of a cDNA encoding nucleosome assembly protein 1 (NAP-1) from soybean

NAP-1, a protein first isolated from mammalian cells, can introduce supercoils into relaxed circular DNA in the presence of purified core histones. Based on its in vitro activity, it has been suggested that NAP-1 may be involved in nucleosome assembly in vivo. We isolated a cDNA clone encoding a soybean NAP-1 homolog, SNAP-1. The SNAP-1 cDNA contains an open reading frame of 358 amino acid residues with a calculated molecular weight of 41 kDa. The deduced amino acid sequence of SNAP-1 shares sequence similarity with yeast NAP-1 (38%) and human hNRP (32%). Notable features of the deduced sequence are two extended acidic regions thought to be involved in histone binding. SNAP-1 expressed in Escherichia coli induces supercoiling in relaxed circular DNA, suggesting that SNAP-1 may have nucleosome assembly activity. The specific activity of SNAP-1 is comparable to that of HeLa NAP-1 in an in vitro assay. Western analysis reveals that SNAP-1 is expressed in the immature and young tissues that were examined, while mature tissues such as old leaves and roots, show very little or no expression. NAP-1 homologs also appear to be present in other plant species.     

Oviposition deterrents from the egg masses of the adzuki bean borer, Ostrinia scapulalis and Asian corn borer, O. furnacalis

Bioassays using gravid females of the adzuki bean borer, Ostrinia scapulalis (Walker), and the Asian corn borer, O. furnacalis (Guenée) (Lepidoptera: Crambidae), showed that the presence of an egg mass of a conspecific deters oviposition. Volatile chemicals emanating from the egg mass were responsible for the deterrence, and these deterrents could be extracted from the egg mass with hexane. When fractionated using a Sep‐Pak® Plus NH₂ cartridge, the deterrents were eluted with a 98 : 2 mixture of diethyl ether and acetic acid (polar lipid fraction). The polar lipid fraction contained free fatty acids with 14–20 carbons, and palmitic acid, palmitoleic acid, and oleic acid were predominant. A blend of all identified fatty acids, a blend of six major fatty acids (palmitic, palmitoleic, stearic, oleic, linoleic, and linolenic acids), a blend of the two Z‐9‐alkenoic acids (palmitoleic and oleic acids), palmitoleic acid alone, and oleic acid alone showed deterrence against O. scapulalis which was comparable to that provoked by the full egg extract. The dose‐dependency of the deterrent effects of palmitoleic acid and oleic acid was verified in O. scapulalis. The binary blend of palmitoleic acid and oleic acid was also confirmed to deter oviposition in O. furnacalis.     

Distribution and chemical speciation of aluminum in the Al accumulator plant,Melastoma malabathricum L.

The Al accumulation mechanisms in an Al accumulator plant, Melastoma malabathricum L. (Melastoma), was investigated. Al was located in the upper epidermal cells and also distributed in mesophyll cells in leaf sections. In root sections, Al was found in all the root tissues, particularly in the epidermis and endodermis. Al concentrations in young leaves, mature leaves, old leaves, and roots were 8.0, 9.2, 14.4, and 10.1 mg g¹, respectively. Approximately 45% of total Al in oldest leaves, and approximately 60% of total Al in leaves of other positions and roots were extracted in Tris-HCl buffer (pH 7.0). Since Al in the residual parts was mostly dissolved in hot 0.5 M H₂SO₄ containing 2% cetyl trimethylammonium bromide, residual Al seemed to consist mainly of monomeric Al and Al bound to pectic substances and hemicellulose. Al in the Tris-HCl extract consisted of non-monomeric Al (complexed form). Oxalate concentration in the Tris-HCl extract in leaves was significantly higher in the +Al treatment than in the –Al treatment and there was a positive correlation between the Al concentration and oxalate concentration. ²⁷Al NMR spectrum of fresh leaves indicated the presence in the order of monomeric Al, Al-oxalate, Al-(oxalate)₂, and Al-(oxalate)₃ in intact leaves.     

Correlative Regulation of Nerve Growth Factor Level and Choline Acetyltransferase Activity by Thyroxine in Particular Regions of Infant Rat Brain

Abstract: Effects of thyroxine (T₄) on nerve growth factor (NGF) level and choline acetyltransferase (ChAT) activity of rat brains were investigated. Repetitive intraperitoneal administration of T₄ caused increases in both NGF level and ChAT activity in the frontal cortex, septum, hippocampus, and striatum and decreases in the cerebellum in 2-day-old rats. Only ChAT activity was elevated in the olfactory bulb, and the NGF level remained unchanged there. No changes were observed in the midbrain and pons/medulla. Furthermore, T₄ was effective on the post-natal rats only up to day 11. These results suggest that T₄ plays a role in the developmental regulation of NGF level and ChAT activity in rat brain in a region- and/or stage-specific manner. That (1) changes in NGF level and ChAT activity occurred in regions nearly identical to those that contained NGF-responding neurons, and (2) the change in NGF level in the hippocampus and frontal cortex was followed by the change of ChAT activity after a single injection of T₄ suggest that the effects of T₄ on cholinergic differentiation are, at least in part, mediated via NGF, which itself is quantitatively regulated by T₄.     

Detection of a novel HLA-DQ specificity: serological and immunochemical analyses by a monoclonal antibody

A monoclonal antibody (mAb) with a novel human B-cell allospecificity was produced by immunizing a C3H/He mouse with the human B lymphoblastoid cell line EBV-Wa (HLA-DR4/Dw15/DQblank homozygous). The mAb, termed HU-46, reacted with B cells from not only DR4/Dw15-positive individuals but also certain DRw8/Dw8-positive ones whose DQ phenotypes had not yet been defined. Two-dimensional gel analyses indicated that the mAb recognized class II antigens which were encoded by the HLA-DQ locus. Furthermore, in genetic analysis, the gene encoding the class II antigen detected by HU-46 met the Hardy-Weinberg condition as a fourth allele of the DQ locus. We provisionally labeled this novel DQ specificity DQWa.     

Sperm competition ofMacrocheles muscaedomesticae (Scopoli) (Acarina: Mesostigmata: Macrochelidae), with special reference to precopulatory mate guarding behavior

The adult males of the manure-inhabiting predatory mite,Macrocheles muscaedomesticae, mounted on the backs of pharate female deutonymphs and guarded them from other males for several hours until female emergence and ensuing copulation. I assumed that an adaptive significance of such characteristic precopulatory mate guarding behavior was closely related to inter-male sperm competition. An existence of 2 sexually compatible strains having different esterase isozyme patterns were found through polyacrylamidegel electrophoresis. A double copulation experiment utilizing these patterns as genetic markers was conducted. In 410 F₁ progeny from doubly copulated mothers examined, 409 daughters were fathered by the first males, and only 1 was fathered by the second male (F₁ males were eliminated because of the arrhenotokous sex determination system). These results seem to indicate that males ofM. muscaedomesticae guard the immature females in order to secure virgin individuals to mate with. Contribution No. 221 from Lab. Entomol., Kyoto Pref. Univ.     

TaqI polymorphism in the LDL receptor gene and a TaqI 1.5-kb band associated with familial hypercholesterolemia

Summary The low density lipoprotein (LDL) receptor gene was analyzed in 67 unrelated healthy Japanese and 38 members of six consecutive families with familial hypercholesterolemia (FH) by Southern blot hybridization with TaqI, an LDL receptor cDNA fragment containing exons 1 to 8 being used as a probe. A new TaqI RFLP at the LDL receptor locus was detected with allele frequencies of 0.67 and 0.33. The data obtained with smaller cDNA subfragment probes revealed that the TaqI RFLP site is located within 1.1 kb of the 5 side of the EcoRI site of exon 5. The TaqI RFLP was in linkage disequilibrium with the PstI RFLP but showed no significant linkage disequilibrium with the RFLPs for AvaII, ApaLI/I15, PvuII, NcoI, and ApaLI/3. Among the seven RFLPs at the LDL receptor locus, the TaqI RFLP was the only useful genetic marker in one of the six families with FH. Furthermore, the association of an additional TaqI 1.5-kb band with a mutant LDL receptor gene was observed in another family with FH in which the proband was homozygous for all of the seven RFLPs. The data obtained with various restriction enzymes and smaller cDNA subfragments probes suggested that a minor change in nucleotide sequences in the region including exons 5 to 8 is present in the mutant gene. These data suggest that the TaqI RFLP is a useful genetic marker at the LDL receptor locus and that TaqI serves for the analysis of some mutant LDL receptor genes, when used with small LDL receptor cDNA probes.     

Light Actions in the Germination of Cocklebur Seeds II. Possible Origin of the Diversity of Light Responses in Seed Germination

In negatively photoblastic, lower seeds of cocklebur (Xanthiumpennsylvanicum Wallr.), the respective germination-inhibitingeffects of red (R) and far-red (FR) lights were found in theproximal and near-tip zones of the axial tissues. In contrast,the germination-stimulating effect of R in positively photoblastic,upper cocklebur seeds was manifested in the near-tip zone ofthe axes, the R effect being reversed when FR was given to thezone. The R-sensitive zone in the upper seeds, however, shiftedtowards the more proximal zone as the period of pre-soakingat low temperatures increased. This shift was accompanied bythe ability to germinate in the dark in the upper seeds. In the lower seeds, R inhibited axial growth in the near-tipzone, whereas FR inhibited it in the proximal zone. In contrast,axial growth in the near-tip zone of the upper seeds was promotedby R. In both seeds, light had little effect on the growth ofthe radicle tip. Pre-soaking at low temperatures induced dark-germinationby hastening the axial growth of the upper seeds, thus allowingthe upper seed to resemble the lower one. We therefore proposea hypothesis that explains the diversification of photoresponsesin seed germination. (Received August 7, 1984; Accepted December 24, 1984)     

Effect of High Temperature on the Development of Cyanide-Sensitive Respiration in the Germination Process of Spinach Seeds

Germination of spinach (Spinacia oleracea L. var. grabra cv.Nobel) seeds was inhibited at a high temperature (35?C). Effectsof KCN on the respiration of seeds incubated at 20 and 35?Cwere compared in order to investigate the mechanism of inhibitionof seed germination by high temperature. Respiration of germinatingseeds incubated at 20?C was inhibited about 50% by 5 mM. KCNsolution, whereas it hardly inhibited the weak respiration ofthe seeds at 35?C. Germination of seeds was delayed by exogenousKCN. When the KCN solution was renewed daily, germination wascompletely inhibited. Pericarp removal promoted germinationat 35?C, but atypical germination (cotyledons emerging earlierthan a radicle) took up more than half of the total germination.The inhibitory action of KCN on the respiration of seeds wasnot altered by pericarp removal. A KCN addition, even at 20?C,elicited atypical germination in the pericarp-less seeds. Theseresults show that cyanide-sensitive respiration is needed toinduce typical spinach seed germination (root emergence), butis rendered inoperative by high temperatures thus bringing aboutpoor germination and atypical germination. (Received December 1, 1984; Accepted February 8, 1985)