COMPARISON OF NEURONAL AND LENS PHENOTYPE EXPRESSION IN THE TRANSDIFFERENTIATING CULTURES OF NUERAL RETINA WITH DIFFERENT CULTURE MEDIA*

The effects of three different culture media (Eagle's MEM, F-12 and L-15) on the transdifferentiation of 8-day chick embryonic neural retina into lens cells, were examined with respect to the expression of two phenotypes. One type referred to neuronal specificity (as represented by the level of cholineacetyl-transferase, CAT, activity) and the other to lens specificity (as represented by content of α-and δ-crystallin). In 7-day cell cultures before the visible differentiation of lentoid bodies, CAT activity was detected in all media. But, its level was about 9 times higher in cultures with L-15 than in those with MEM and 3 times higher than in F-12. In 26-day cultures, CAT activity was practically undetectable. The production of α-and δ-crystallin was detected in cultures at 26 days. There were quantitative differences in the crystallin content with different media, and it was highest in cultures with L-15. The results indicate that conditions most favourable to the maintenance of the neuronal specificity in cell cultures of neural retina, can also support the most extensive transdifferentiation. The possibility of direct transdifferentiation of once neuronally specified cells into lens cells in cultures with L-15 has been suggested to explain the present results.     

Relation Between Cell Union and Nuclear Reorganization in Triplet Conjugants of Paramecium caudatum and Paramecium multimicronucleatum

SYNOPSIS Triplet conjugants of Paramecium caudatum which appeared naturally in mating mixtures and those of Paramecium multimicronucleatum which were produced by conjugation-inducing chemicals were isolated. Triplet conjugants lasting for more than 3 h were stained to examine macronuclear events. In P. caudatum , only 2 triplets among 182 (1%) contained macronuclear fragmentation in all 3 members. The most frequently occurring triplets (79%) were those producing 1 cell without and 2 cells with macronuclear fragments. There were also triplets (17%) producing 1 cell with, and 2 without macronuclear fragments, and some (3%) with 3 cells that contained no fragments. The length of persistence of the triplet was not responsible for the occurrence of macronuclear fragmentation in the 3rd cell of the triplet. In P. multimicronucleatum , the same 4 classes of triplets occurred, but the most frequently occurring class was that consisting of 3 cells (91%) with macronuclear fragments. Induction of nearly 100% of triplets with 3 such cells was possible by isolating the triplets' from a culture which was treated chemically at about 24 h after the last feeding. Treatment with chemicals in starved cultures resulted in triplets with incompletely fragmented or nonfragmented macronuclei. Further, in P. multimicronucleatum , chemicallyinduced triplets involving only holdfast pairs to which the 3rd cells were uniting often produced 3 cells with fragmented macronuclei.     

Structure of the “Peroxy-Y Base” from Liver tRNA<Superscript>Phe</Superscript>

WE wish to report that reconstituted sperm whale myoglobin prepared by the method of Breslow¹ (except that pH 2 was found sufficient to remove all the haem) (I) crystallizes² in a different habit from those prepared by the method of Rossi-Fanelli et al.³ (II) using haemin of Sigma lot 77B-0220 and our own ⁵⁷Fe photoporphyrin preparation and the native myoglobin (III). Although all three form type A³ monoclinic prisms, the best developed plane is [001] for II and III, it is [100] for I. There seems to be great interest in reconstituted haemoproteins^4,5, so it is important that crystallization habit may be a sensitive test for subtle changes in protein structures.     

Correlation Between the Molecular Structure and the Biological Activity of Co-ARIS, a Cofactor for Acrosome Reaction-Inducing Substance

Two components in the egg jelly are required for inducing the acrosome reaction in starfish; a sulfated glycoprotein called acrosome reaction-inducing substance (ARIS) and its cofactor called Co-ARIS. Three distinct molecules were isolated as the major Co-ARIS' and designated as Co-ARIS' I, II and III. Structural analysis of Co-ARIS' revealed that they are steroidal saponins comprising a sulfated steroid and a pentasaccharide chain. Co-ARIS' I and II differ only in the steroidal side chain. In the presence of ARIS, each Co-ARIS induced the acrosome reaction with a maximal effect at 100–200 μM (Co-ARIS I) or 25–50 μM (Co-ARIS' II and III). Mixtures of Co-ARIS' I, II and III were more effective than the individuals. The activity of Co-ARIS was considerably reduced by solvolytic desulfation but was not affected at all by periodate oxidation. Reduction by NaBH₄ decreased the activity of Co-ARIS I and enhanced that of Co-ARIS II. Treatment of Co-ARIS III with NaBH₄ did not affect the activity as anticipated from its structure. These results suggest that the sulfate moiety and the side chain of steroid are important for the activity of Co-ARIS. The saccharide chain, however, seems not necessarily to be strictly specified for the activity.     

Change in CO2 balance under a series of forestry activities in a cool-temperate mixed forest with dense undergrowth

To evaluate the effects on CO₂ exchange of clearcutting a mixed forest and replacing it with a plantation, 4.5 years of continuous eddy covariance measurements of CO₂ fluxes and soil respiration measurements were conducted in a conifer-broadleaf mixed forest in Hokkaido, Japan. The mixed forest was a weak carbon sink (net ecosystem exchange, −44 g C m⁻² yr⁻¹), and it became a large carbon source (569 g C m⁻² yr⁻¹) after clearcutting. However, the large emission in the harvest year rapidly decreased in the following 2 years (495 and 153 g C m⁻² yr⁻¹, respectively) as the gross primary production (GPP) increased, while the total ecosystem respiration (RE) remained relatively stable. The rapid increase in GPP was attributed to an increase in biomass and photosynthetic activity of Sasa dwarf bamboo, an understory species. Soil respiration increased in the 3 years following clearcutting, in the first year mainly owing to the change in the gap ratio of the forest, and in the following years because of increased root respiration by the bamboo. The ratio of soil respiration to RE increased from 44% in the forest to nearly 100% after clearcutting, and aboveground parts of the vegetation contributed little to the RE although the respiration chamber measurements showed heterogeneous soil condition after clearcutting.     

The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining

The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method. © 1998 Chapman & Hall     

SOME PROPERTIES OF SULFITE REDUCTASE FROM YEAST

The MVH- and NADPH-linked sulfite reducing activities were assayedin extracts obtained from a wild strain of Saccharomyces cerevisiaeand various auxotrophic mutants derived from it. All the extractspossessing the NADPH-linked activity also showed the MVH linkedactivity, and all those lacking the latter also lacked the former.However, extracts from several mutants had the MVH-linked activitywithout having the other. NADPH-sulfite reductase was purified nearly 200-fold from extractsof the wild strain. Throughout the purification steps, the MVH-linkedactivity was also purified in association with the NADPH-linkedactivity, and the ratio between the two remained essentiallyconstant. However, on exposure to heat, low ionic strengthsand PCMB, only the NADPH-linked activity was lost, leaving theMVH-linked activity almost unaffected. Both activities weresensitive to cyanide. The sulfite reductase could also reduce nitrite and hydroxylamine, and the nitrite- and hydroxylamine-reducing activitywas sensitive to the treatments which inhibited the NADPH-sulfitereducing activity. Addition of nitrite or hydroxylamine competitivelyinhibits the NADPH linked sulfite reduction. The enzyme alsoshowed diaphorase activity, reducing DPIP or MV. This activitywas inhibited by the treatments to which the NADPH-linked sulfitereduction was sensitive, but it was not sensitive to cyanide. A tentative schematic model for yeast sulfite reductase is presented. ( Accepted October 8, 1964)