Fine Structural Changes in the Acrosome Reaction of the Japanese Abalone, Haliotis disus

The spermatozoon of the Japanese abalone, Haliotis discus , and its structural changes during the acrosome reaction were observed by electron microscopy. The spermatozoon has a huge acrosome in the shape of a hanging bell or a forefinger with a deep fossa at the posterior end being filled with a bundle of microfilaments. The membranes of the acrosomal apex, the so-called trigger region, are structurally discernible from those of other acrosomal regions. Following the trigger region, a unique structure under the acrosomal membrane covers the surface of the acrosomal content in the form of a truncated cone.
The acrosome reaction occurs in the jelly layer very close to the egg envelope. First, the membranes at the apex of the acrosome are vesiculated, followed by the formation of a narrow gap between the outer acrosomal membrane and the acrosomal content. Next, the bundle of micro-filaments elongates, running through the center of the acrosome, reaching the trigger region and protruding out of the acrosomal top. Then release of the acrosomal content occurs in two steps, disclosing the "membrane undercoating structure" that comprises globular particles with a fuzzy material connecting them. This resembles the undercoat network found in erythrocytes.     

Modelling various sculptures in the Cretaceous bivalve Inoceramus hobetsensis

The geometry of the external shell sculpture in the Late Cretaceous inoceramid bivalve Inoceramus hobetsensis Nagao & Matsumoto, 1939 was studied both empirically and theoretically. A large sample, collected from the Upper Cretaceous of Hokkaido, Japan, shows remarkably high intraspecific variation in the shell sculptural pattern. Quasi-commarginal ribs, slightly oblique to the external growth increments, occur in some specimens. These sculptures are commonly irregular in strength and spacing, and their features are successfully modelled by computer simulations when the commarginal ribs are superposed with nearly concentric divaricate rib. Computer models indicate that the divergent sculpture element, often found in other inoceramids, was present throughout the evolution of I. hobetsensis and was developing in the evolutionary lineage from I. hobetsensis nonsulcatus to I. hobetsensis hobetsensis, although it was only weakly expressed. The results also suggest that some apparently distinct sculptural patterns of I. hobetsensis are the result of minor changes in the morphogenetic program.     

Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots

Putrescine N-methyltransferase, a new enzyme catalyzing theformation of N-methylputrescine from putrescine and S-adenosyl-L-methioninewas found in roots of tobacco plants. The enzyme was purified30-fold from crude extracts of tobacco roots. NMethylputrescinewas identified as the reaction product by comparison with theauthentic compound. The enzyme had a pH optimum between pH 8and 9, and a molecular weight of about 60,000, as determinedby gel filtration. Km values for putrescine and 5-adenosyl-L-methioninewere 4.0 x 10^–4 M and 1.1 x 10^–4 M, respectively.Enzyme activity was inhibited by N-chloromercuribenzoate andAg⁺. No cofactors were required. Of the various substrates tested,only putrescine served as a methyl acceptor. The enzyme waslocalized exclusively in the roots and its activity was greadyenhanced by decapitation. The presence of putrescine N-methyltransferase in tobacco rootsstrongly suggests that N-methylputrescine participates as anintermediate in nicotine biosynthesis. (Received March 2, 1971; )     

Differential anatomical responses to elevated CO2 in saplings of four hardwood species

To determine whether an elevated carbon dioxide concentration ([CO₂]) can induce changes in the wood structure and stem radial growth in forest trees, we investigated the anatomical features of conduit cells and cambial activity in 4‐year‐old saplings of four deciduous broadleaved tree species – two ring‐porous (Quercus mongolica and Kalopanax septemlobus) and two diffuse‐porous species (Betula maximowicziana and Acer mono) – grown for three growing seasons in a free‐air CO₂ enrichment system. Elevated [CO₂] had no effects on vessels, growth and physiological traits of Q. mongolica, whereas tree height, photosynthesis and vessel area tended to increase in K. septemlobus. No effects of [CO₂] on growth, physiological traits and vessels were seen in the two diffuse‐porous woods. Elevated [CO₂] increased larger vessels in all species, except B. maximowicziana and number of cambial cells in two ring‐porous species. Our results showed that the vessel anatomy and radial stem growth of Q. mongolica, B. maximowicziana and A. mono were not affected by elevated [CO₂], although vessel size frequency and cambial activity in Q. mongolica were altered. In contrast, changes in vessel anatomy and cambial activity were induced by elevated [CO₂] in K. septemlobus. The different responses to elevated [CO₂] suggest that the sensitivity of forest trees to CO₂ is species dependent.     

Development of Melanocyte Progenitors in Murine Steel Mutant Neural Crest Explants Cultured With Stem Cell Factor,Endothelin-3, or TPA

Stem cell factor (SCF) has been suggested to be indispensable for the development of neural crest cells into melanocytes because Steel mutant mice (i.e., Sl/Sf¹) have no pig-mented hairs. On the other hand, it has been demonstrated that the addition of endothelin 3 (ET-3) or TPA to neural crest cell cultures can induce melanocyte differentiation without addition of extrinsic SCF. In this study, we excluded the influence of intrinsic SCF by using SI/SI mouse embryos to study more precisely the effects of natural cytokines, such as extrinsic soluble SCF or ET-3, or chemical reagents, such as TPA or cholera toxin. We found that SCF is supplied within the wild-type neural crest explants and that ET-3 cannot induce melanocyte differentiation or proliferation without SCF. These results indicate that SCF plays a critical role in survival or G1/S entry of melanocyte progenitors and that SCF initially stimulates their proliferation and then ET-3 accelerates their proliferation and differentiation. TPA has the ability to elicit neural crest cell differentiation into melanocytes without exogenously added SCF but it is not as effective as SCF because many more melanocytes developed in the wild-type neural crest explants cultured with TPA.     

Effect of Arbutin on Melanogenic Proteins in Human Melanocytes

The inhibitory effect of arbutin, a naturally occurring β-D-glucopyranoside derivative of hydroquinone, on melanogenesis was studied biochemically by using human melano-cytes in culture. Cells were cultured in the presence of different concentrations of arbutin. The maximum concentration of arbutin that was not inhibitory to growth of the cells was 100 ug/ml. At that concentration, melanin synthesis was inhibited significantly by ～20% after 5 days, compared with untreated cells. This phenotypic change was associated with the inhibition of tyrosinase and DHICA polymerase activities, and the degree of inhibition was dose dependent. No significant difference in DOPAchrome tautomerase (DT) activity was observed before or after arbutin treatment. Western blotting experiments revealed there were no changes in protein content or in molecular size of tyrosinase, TRP-1 or TRP-2, indicating that inhibition of tyrosinase activity by arbutin might be due to effects at the post-translational level.     

Co-sedimentation of Actin, Tubulin andMembranes in the Cytoskeleton Fractions from Peas and Mouse 3T3 Cells

Pea stem tissue (Pisum sativum L. var. Alaska) was homogenizedin a recently-developed cytoskeleton-stabilizing buffer, CSB,(Abeand Da vies, 1991) and homogenates electrophoresed and blottedon to membranes. Blots probed individually withantibodies toactin, alpha-tubulin, and beta-tubulin, revealed bands withapparent molecular weights of 42, 46, and 48–50 kDa,respectively.Blots probed with all three antibodies simultaneously revealedall three bands which could be distinguished in thesame lane.Homogenates of mouse 3T3 cells yielded an actin band at about42 kDa, but both alpha- and beta-tubulin appeared atabout 50kDa and thus could not be distinguished on blots probed simultaneously.This ‘triple-blotting technique’ was, therefore,suitablefor pea tissue, but not for mouse tissue. In pea tissue, sedimentabletubulin and actin were found maximally in the 4000 xg pelletand less in successive 15000 and l00000xg pellets. Both EGTAand Mg²⁺ which had been found earlier to beessential for stabilityof the actin cytoskeleton as revealed by fluorescence microscopy,were essential for co-sedimentation of actinand tubulin. Incontrast to the results with pea stems, only the actin componentof the cytoskeleton could be isolated from mouse 3T3 cells usingCSB. Pea tissue was homogenized in CSB without PTE and the resultingcytoskeletal pellets resuspended in actin- or tubulin-solubilizingbuffers with and without PTE. In the absence of PTE, the bufferssolubilized their appropriate cytoskeletal protein, but littleof the other protein, while in the presence of PTE both proteinswere quite effectively solubilized by both buffers. Incontrast,in CSB with or without PTE, both proteins remained in the sedimentablefraction. These results, taken together withother evidence,indicate that microtubules, as well as microfilaments are importantcomponents of the sedimentable cytoskeletonfraction of peasand that the membrane system is intimately involved in organizationof the cytoskeleton in peas. Key words: Actin, tubulin, membranes, detergent, Ca²⁺, Mg²⁺, cytoskeleton     

Emergence of the Extrachromosomal Circular DNA Complexes as One of the Earliest Signals of Cellular Differentiation in the Early Development of Mouse Embryo

A small number of mouse embryos and embryonal carcinoma cells were pressed by mica sheet; then the extruded DNA complexes were adsorbed to mica and processed for electron microscopy. Extrachromosomal circular DNA complexes longer than 1 μm emerged during the compaction process of mouse embryos and during the differentiation of embryonal carcinoma cells induced with retinoic acid. These DNA molecules are discussed as possible products of developmental gene rearrangements occurring in the chromosomal DNA.