Unique Pattern of Gene Expression in the Erythroid Precursor Cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU-E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt-genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt-genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU-E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt-genes may be induced after the stimulation of erythropoietin.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

Reactivation of the Hill reaction of Tris-washed chloroplasts

The Hill reaction of chloroplasts was inhibited by washing themwith 0.8 M Tris buffer. This inhibition was further promotedby adding ferricyanide in the washing medium. When a reducingreagent, such as the 2,6-dichlorophenol indophenol (DCPIP)-ascorbatesystem or the hydroquinone (HQJ-ascorbate system, had been addedto the Tris buffer, Hill reaction activity was unaffected. Hill reaction activity of Tris-washed chloroplasts recoveredup to 70% of the initial level by re-washing the chloroplastswith a preparation medium containing theabove reducing reagents. Photobleaching of carotenoid and chlorophyll is characteristicof Tris-washed chloroplasts. However, reactivated chloroplastsshowed no photobleaching as in the case with intact chloroplasts. (Received April 20, 1970; )     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

Non-cyclic photophosphorylation by spinach grana treated with 0.8 M Tris buffer

1. Photophosphorylation was measured with spinach grana sampleswashed by 0.8 M Tris buffer at pH 8.0, which no longer catalyzedthe ferricyanide and NADP HILL reactions with water as the electrondonor. The photophosphorylation with the reaction mixture containing2 10^–4 M 2,6-dichlorophenol indophenol (DCPI) plus above2 10^–3 M ascorbate as the electron donor system insteadof water under anaerobic conditions was, in the most part, dependenton the addition of both PPNR (a nonheme iron protein requisitefor photosynthetic pyridine nucleotide reduction ; spinach ferredoxin)and NADP as the electron acceptor system. However, when ascorbateconcentration only was lowered to 2 10^–4 M, the entirephotophosphorylation proceeded, even in the absence of the electronacceptor system. 2. When the NADP added in the reaction mixture had been reducedby glucose-6-phosphate and glucose-6-phosphate dehydrogenasebefore illumination, the photophosphorylation with 2 10^–4M DCPI plus 6.7 10^–3 M ascorbate decreased to aboutthe same rate as that obtained without NADP. 3. The time course for photophosphorylation in the presenceof NADP was consistent with the time course for the photoreductionof NADP: On the complete reduction of NADP, the photophosphorylationstopped. 4. In the presence of 6.7 10^–3 M dichloropheny 1.1,1-dimethylureaor 3 10^–4 M o-phenanthroline, non-cyclic photophosphorylationwith 2 10^–4 M DCPI plus 6.7 10^–3 M ascorbateas the electron donor system decreased to about half that ofthe control, and the remaining activities were hardly affectedeven at higher concentrations of both inhibitors. The P/2e^–ratios of non-cyclic photophosphorylation in the absence andpresence of ophenanthroline were 0.74 and 0.48, respectively. ¹Present address: Department of Biology, University of California,San Diego, La Jolla, California 92037, U. S. A.     

POLYOL ACCUMULATION IN IN VITRO SYSTEMS OF BOMBYX EGGS

The mechanism of polyol accumulation in diapausing Bombyx eggs, conversion of [6-¹⁴C] glucose-6-phosphate into polyols and other neutral sugars was investigated in in vitro reaction systems. When a crude homogenate or a press juice of the eggs was incubated with [6-¹⁴C]glucose-6-P, the labelled trehalose, sorbitol and glycerol accumulated in the reaction mixture. In the press juice incubation system of developing eggs at day 1, ¹⁴C-sorbitol was detected in appreciable amounts, but it decreased rapidly with the development of the embryos. When the press juice was prepared from eggs in diapause, the formation of ¹⁴C-sorbitol was 3–5 times greater in eggs at early stages (day 2 to day 4) than in developing eggs.     

SPICULE FORMATION IN VITRO BY THE DESCENDANTS OF PRECOCIOUS MICROMERE FORMED AT THE 8-CELL STAGE OF SEA URCHIN EMBRYO*

The micromeres at the 16-cell stage of sea urchin embryo have already been endowed with a faculty to self-differentiate into spicule-forming cells (11). The present experiment was designed to test whether the factor(s) necessary for such self-differentiation had already been localized at the 8-cell stage in an area corresponding to the presumptive micromere region in Hemicentrotus pulcherrimus. Since the blastomeres at the 8-cell stage are all equal in size in normal embryo, unequal 3rd cleavage, by which small blastomeres are pinched off toward the vegetal pole (precocious micromeres), was experimentally induced either by treatment with 4NQO (4-nitroquinoline-1-oxide) at the 2-cell stage or by continuous culture in Ca-free sea water. The precocious micromeres were cultured in vitro in natural sea water containing horse serum. Descendants of the precocious micromeres formed spicules. In comparison their spicule formation with that by the descendants of the micromere of normal embryo, no differences were found regarding 1) time of initiation of spicule formation, 2) rate of growth of spicule, 3) size and shape of resultant spicule and 4) percentage of clones which formed spicule. The fact indicates that factor(s) indispensable for self-differentiation into spicule-forming cells have already been localized near the vegetal pole as early as the 8-cell stage.     

Cyclic Changes in Shape of A Non-Nucleate Egg Fragment of Tubifex (Annelida, Oligochaeta)

A non-nucleate fragment separated from the fertilized Tubifex egg at metaphase of the second meiosis showed temporary surface deformation at 3–3.5 hr intervals, i.e. , synchronously with the onset of formation of the second polar body and early cleavages in control eggs. From the two-cell stage on, the periodicity of the surface activity in the non-nucleate fragment was found to be synchronous with the cleavage cycles of the CD-cell and its descendants, but not with those of the AB-cell. This surface deformation was completely inhibited by cytochalasin B (50 μg/ml). Electron microscopy shows that microfilaments are present exclusively in the cortical layer of the deforming fragments. Cycloheximide-treated egg fragments commenced surface deformation after a delay of 1–2 hrs; pulse-treatment indicated that the surface deformation requires proteins synthesized specifically during the period of the previous surface deformation. These results are discussed in relation to the nucleus-independent cytoplasmic rhythm and asynchronous cleavage of Tubifex eggs.     

CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES FROM SILKWORM EGGS: OCCURRENCE OF TWO CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES, CHANGES IN THEIR ACTIVITIES AND LEVELS OF CYCLIC NUCLEOTIDES DURING DEVELOPMENT OF THE EGGS

Two cyclic nucleotide-dependent protein kinases, designated as protein kinase-I and -II, were obtained from the eggs of the silkworm, Bombyx mori . Protein kinase-I is highly dependent on cGMP, whereas protein kinase-II is dependent on cAMP. In developing non-diapause eggs, the level of cyclic nucleotide-dependent protein kinase activity is quite high but that in the diapause eggs is not. The developmental changes in the two protein kinases and the level of cyclic nucleotides were also studied during the development of the eggs.     

A role for oxalic acid generation in ozone‐induced signallization in Arabidopis cells

Ozone (O₃) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O₃ results in ascorbate‐derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca²⁺ concentration, generation of reactive oxygen species and cell death. We confirmed that O₃ reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O₃‐induced signalling pathways that trigger programmed cell death.