Purification of Protein Body-I of Rice Seed and its Polypeptide Composition

Protein body type one (PB-I) was isolated and purified fromdeveloping rice grain by a combination of sucrose density gradientcentrifugation and treatment with pepsin. SDS-PAGE analysisshowed that isolated PB-I contains several polypeptide groups,the largest having an apparent molecular size of 13 kDa andtwo smaller ones of 10 kDa and 16 kDa. The 13-kDa group wasfound to be composed of two polypeptides of slightly differentmolecular sizes, 13a (larger component) and 13b (smaller component).Most of the 13a and 13b polypeptides were shown to be largelyprolamins, although there were also some salt- and alcohol-insolublepolypeptides with an apparent molecular size of 13 kDa. It wasconcluded that PB-I is the accumulation site of rice prolamin.It was further estimated that the protein amount in PB-I accountedfor about 20% of the total protein of rice endosperm. (Received March 20, 1987; Accepted September 8, 1987)     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Allelopathy due to purine alkaloids in tea seeds during germination

Summary During imbibition of whole tea seeds (6 days) two purine alkaloids, caffeine and theobromine, did not decrease in the seed coats and there was no increase in the seeds. In parallel with and after the breaking of seed coats there was a gradual release of caffeine from coats of germinating seeds. By contrast, when the seed was freed from the outer seed coat and soaked, imbibition of the seed required only 2 days and simultaneously caffeine was released from the inner seed coat. In such seeds, but not in whole seeds, growth of embryonic tissues (roots and shoots) was inhibited after the breaking of the inner seed coats. Nevertheless, caffeine increased more in such roots of the seedlings of decoated seeds than in roots of normal seedlings.     

Influence of leaf age on photosynthesis, enzyme activity, and metabolite levels in wheat

The rate of photosynthesis under high light (1000 micromole quanta per square meter per second) and at 25°C was measured during development of the third leaf on wheat plants and compared with the activity of several photosynthetic enzymes and the level of metabolites. The rate of photosynthesis reached a maximum the 7th day after leaf emergence and declined thereafter. There was a high and significant correlation between the rate of photosynthesis per leaf area and the activities of the enzymes ribulose 5-phosphate kinase (r = 0.91), ribulose 1,5-bisphosphate (RuBP) carboxylase (r = 0.94), 3-phosphoglycerate (PGA) kinase (r = 0.82), and fructose 1,6-bisphosphatase (r = 0.80) per leaf area. There was not a significant correlation of photosynthesis rate with chlorophyll content. The rate of photosynthesis was strongly correlated with the level of PGA (r = 0.85) and inversely correlated with the level of triose phosphate (dihydroxyacetone phosphate and glyceraldehyde 3-phosphate) (r = 0.92). RuBP levels did not change much during leaf development; therefore photosynthesis rate was not correlated with the level of RuBP. The rate of photosynthesis was at a maximum when the ratio of PGA/triose phosphate was high, and when the ratio of RuBP/PGA was low. Although several enzymes change in parallel with leaf development, the metabolite changes suggest the greatest degree of control may be through RuBP carboxylase. The sucrose content of the leaf was highest under high rates of photosynthesis. There was no evidence that later in leaf development, photosynthesis (measured under high light and at 25°C) was limited by utilization of photosynthate.     

Induction and subsequent repair of DNA damage by fast neutrons in cultured mammalian cells

The induction and repair of DNA damage were studied by a DNA unwinding method in mouse L5178Y cells exposed to fast neutrons. DNA lesions induced by fast neutrons were classified into three types from their repair profiles: fast-reparable breaks (T1/2 = 3-5 min), slow-reparable breaks (T1/2 = 70 min), and nonreparable breaks. The repair rates of both fast-reparable and slow-reparable breaks were almost the same as those of corresponding damage induced by low-LET radiation. Neutrons induced a smaller amount of fast-reparable damage, an almost equal amount of slow-reparable damage, and a larger amount of nonreparable damage than those induced by equal doses of gamma rays or X rays. RBEs for fast- and slow-reparable damage were 0.3 and 0.9, respectively. The RBE for nonreparable damage was dose dependent and was 1.4 at the level of 100 breaks/10(12) Da DNA. Among the three types of lesions, only the nonreparable damage levels correlated with the linear-quadratic shape of the survival curves and with the enhanced killing effectiveness of neutrons (RBE = 1.7 at D0).     

Positional heterogeneity of interaction between fragments of avian limb bud in organ culture

We have previously succeeded in culturing whole leg bud from stage 21-23 chick embryos and observed a leg structure with typical cartilage pattern in vitro. In the present study, we have attempted the organ culture of the fragmented leg bud and investigated its capacity to form cartilage. Leg buds from stages 17-21 chick embryos were dissected into four pieces in the anteroposterior sequence (named 1, 2, 3, and 4, respectively) and cultured on a membrane filter in a medium consisting of Ham's F-12, chick serum, and chick embryo extract. After 6 days in culture, two central fragments (2 and 3) developed into large cartilaginous masses, while anterior (1) and posterior (4) fragments formed few or small cartilaginous masses. In addition, when these less chondrogenic fragments were combined, pinned together, and cultured, large cartilaginous masses were formed from 1 + 4 combinations but not from 1 + 1 or 4 + 4 combinations. These observations were analyzed quantitatively by measurement of 35SO4 incorporation into the sulfated glycosaminoglycan (S-GAG) and of final DNA content per explant, and by histological reconstruction of the chick-quail chimera explant. The results showed that (a) the 1 + 4 combination resulted in higher S-GAG synthesis and final DNA content than the 1 + 1 or 4 + 4 combinations in stage 18 and 21 leg buds (P less than 5%); (b) removal of ectoderm from the leg bud inhibited the increase observed for the 1 + 4 combination; c) in chick-quail chimera explants the cartilage formed from the 1 + 4 combination was largely of fragment 1 origin. These results demonstrate, first, the presence of a difference in chondrogenic capacity along the anteroposterior axis in the leg bud and, second, the occurrence of an interaction between anterior and posterior fragments which mimics the effects of grafting a zone of polarizing activity (ZPA). The mechanism of ZPA function is still unknown but the ectoderm may play some role. Some roles for ectoderm in ZPA function and differences in mesodermal responsiveness to ZPA factor(s) are suggested.