New records of three marine red algae from Japan

Three species in the red algal order Ceramiales, Dasya longifila Masuda et Uwai (Dasyaceae), Endosiphonia horrida (C. Agardh) P. Silva (Rhodomelaceae) and Laurencia flexilis Setchell (Rhodomelaceae), are reported from Japan for the first time, and their morphological features are described along with taxonomic comments. Our findings point to the northernmost limit of geographic distribution of these species in the north‐western Pacific. Dasya longifila is characterized by small, sparsely corticated axes, long pseudolaterals in which intercalary cell divisions take place, and a small number of tetrasporangial stichidia and spermatangial branches per fertile pseudolateral. Endosiphonia horrida is characterized by frequently anastomosing branches that form a bush‐like tuft without a percurrent axis, inner cortical cells becoming the same length as the axial and periaxial cells, and luxuriously developed, unbranched trichoblasts. Laurencia flexilis is characterized by numerous cartilaginous rigid axes developing from a basal disc without creeping branches, the production of 4 periaxial cells per vegetative segment and the absence of longitudinally oriented secondary pit‐connections between contiguous superficial cortical cells.     

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.     

Transmembrane-Domain Trapping: A Novel Method for Isolation of cDNAs Encoding Putative Membrane Proteins

We have developed a method that enables us to isolate cDNAsof putative membrane proteins. The system is designed to isolatea cDNA which can provide the transmembrane domain to the extracellularpart of the IL-2 receptor chain. We constructed a p18Mac vectorby putting part of the IL-2 receptor chain cDNA that encodedits signal sequence and extracellular domain, a cDNA cloningsite and a poly(A) additional signal after a strong promoterSR. If a cloned cDNA provides a transmembrane domain in-frame,the extracellular domain of the IL-2 receptor chain will beexpressed on the surface of the transfected cells. Otherwise,the chimeric protein will be either secreted or retained insidethe transfected cells. We made a cDNA library using p18Mac andscreened for cDNA clones which allowed the expression of theextracellular domain of the IL-2 receptor chain on the cellsurface. Of the 2000 clones screened, 5 clones were scored aspositive. Partial sequence analysis revealed that one cloneencoded the amyloid precursor protein, two others encoded mitochondrialproteins and the rest were new. These results suggest the systemis effective in isolating cDNAs encoding putative membrane proteins.     

Reduced glutathione promotes callus growth and shoot development in a shoot tip culture of apple root stock M26

Reduced glutathione (GSH) was applied to prevent browning in shoot tip explants of apple (Malus pumila Mill.). Development was compared between shoot tips treated either by (a) dipping into 0.1 mm GSH solution prior to culture (dip treatment), (b) dipping into the GSH solution and transferring to a medium containing 0.1 mm GSH (dip-and-add treatment), or (c) without dipping or culturing with GSH (control). In the dip treatment, 100% of shoot tips developed into normal shoots after 120 days, while the results with the dip-and-add treatment and control were 50 and 40%, respectively. The results show that application of antioxidant GSH in the initial phase of culture promoted the normal development of shoot tips. Received: 10 October 1997 / Revision received: 12 January 1998 / Accepted: 24 January 1998     

Identification of conservation measures to protect the Japanese endangered plant species Aster kantoensis

To identify the factors responsible for degrading the habitat of the endangered plant species Aster kantoensis, as well as the vulnerable life stage where this occurs, we carried out sowing experiments. Two natural habitats were simulated, being situated along the floodplains of the Tama River in central Japan. Seeds collected from a natural habitat were sown in two apparently suitable locations (Tomoda and Ishida sites). Germination, survival, growth, and seed production were subsequently monitored from 1993 through to 1997. The Tomoda site was a gravel bar in floodplains formed by flooding in 1991, while the Ishida site (two plots) was one gravel bar where several plants were growing sparsely and another where a population had become extinct in 1992. Seed cohorts completed their life cycle within 3 years at the Ishida site and within 5 years at the Tomoda site. Monitored parameters at Ishida were substantially lower than those at Tomoda. In addition, estimates of population growth indicated an increase at Tomoda and a rapid decrease at Ishida. However, degradation of habitats seemed to occur at Tomoda over the monitored periods. In view of our results, we conclude that natural germination of about 0.13% is needed for increasing population size. The major factors for decreasing population size are believed to be the lack of safe sites for germination and seedling establishment in old habitats (>10years). Conservation measures are suggested based on these findings.     

Construction of a rat genetic map by using randomly amplified microsatellite polymorphism (RAMP) markers

Many rat strains have been employed in the genetic study of quantitative traits such as blood pressure. In such genetic studies, it is essential to prepare rat genetic maps fine enough to identify the genes regulating quantitative traits. However, it is not an easy task to isolate a sufficient number of genetic markers polymorphic between a particular pair of rat strains. In this study, we applied the randomly amplified microsatellite polymorphism (RAMP) method, a simple method to identify co-dominant markers (Wu et al. Nucleic Acids Res 22, 3257, 1994), to isolate markers polymorphic between the stroke-prone spontaneously hypertensive rat and the Wistar-Kyoto rat, a genetically hypertensive strain and its normotensive control strain, which share a common genetic background. We successfully identified 111 RAMP markers distributed throughout the rat genome after screening 3046 sets of primers. We also showed that we could isolate ordinary simple-sequence-length-polymorphism markers by cloning RAMP markers. The RAMP method is a simple and efficient way to identify co-dominant genetic markers on mammalian genomes. Received: 10 October 1997 / Accepted: 16 March 1998     

Polyacrylamides containing sugar residues: synthesis,characterization and hepatocyte attachment studies

Homo polymers of acrylamide having glucose (PAAm-glucose) and galactose (PAAm-galactose) as pendent groups were synthesized. Tissue culture polystyrene (TCPS) plates coated with these polymers showed increased surface wettability. Coating of PAAm-glucose and PAArn-galactose on to TCPS plates was also confirmed by X-ray photoelectron spectroscopic (XPS) characterization. Rat hepatocytes in primary culture attached to the surfaces of PAArn-galactose homopolymer, but not to those of PAAm-glucose homopolymer. © Rapid Science Ltd. 1998     

Mechanism of Benzyladenine-Induced Stimulation of the Synthesis of 5-Aminolevulinic Acid in Greening Cucumber Cotyledons: Benzyladenine Increases Levels of Plastid tRNAGlu

The mechanism of the stimulatory effect of a cytokinin, namely,benzyladenine (BA), on the synthesis of 5-aminolevulinic acid(ALA) in cucumber cotyledons was studied. The rate of synthesisof ALA by plastids isolated from BA-treated cotyledons was twicethat by plastids from untreated controls. Western blot analysisof stromal proteins showed that BA did not affect the levelof glutamyl-tRNA synthetase or of glutamate l-semialdehyde (GSA)aminotransferase. Analysis of free amino acids revealed thatBA did not increase the level of glutamate in the stroma. However,the amount of total plastidic RNA was doubled in BA-treatedcotyledons. Northern blot analysis showed that the level ofplastid tRNA^Glu was increased by treatment with BA to the sameextent as that of another plastid tRNA, reflecting an increasein total plastidic RNA. The rate of formation of glutamyl-tRNAwas also doubled in plastids from BA-treated cotyledons. Theresults indicate that stimulation of the synthesis of ALA byBA is due to an increased level of tRNA^Glu in plastids. (Received June 6, 1993; Accepted November 26, 1993)     

Evidence against DNA polymerase β as a candidate gene for Werner syndrome

Werner syndrome (WS) is a rare autosomal recessive disorder of humans characterized by the premature onset and accelerated rate of development of several major age-related disorders. An aberration in DNA replication or repair is suggested by the evidence of genome instability. Since the structural gene for DNA polymerase maps within the region of the WS mutation on the short arm of chromosome 8 and is involved in both DNA repair and DNA replication, we evaluated its candidacy as the WS gene. Several independent lines of evidence did not support that hypothesis: (1) activity gels showed normal enzyme activity and electrophoretic mobility; (2) nucleotide sequence analysis of the entire coding region failed to reveal mutations (although indicated mistakes in the published sequence); (3) single-strand conformation polymorphism (SSCP) and heteroduplex analyses failed to reveal evidence of mutations in the promoter region; (4) a newly discerned polymorphism failed to reveal evidence of homozygosity by descent in a consanguineous patient; and 5) fluorescence in situ hybridization (FISH) analysis placed the DNA polymerase gene centromeric to D8S135 at 8p11.2 and thus beyond the region of peak LOD scores for WS.