Promotion of plantlet formation from somatic embryos of carrot treated with a high molecular weight extract from a marine cyanobacterium

Summary Somatic embryos of Daucus carota L. developed into plantlets at high frequency after addition of an extract from a marine cyanobacterium, Synechococcus sp. NKBG 042902. High molecular weight, nondialyzing fraction, separated from the extract, possessed enhanced plantlet formation promoting activity. Plantlet formation frequency was 60 % after addition of nondialysate (100 mg/l) compared to 28 % without addition. Embryos treated with the nondialysate contained five times more chlorophyll than nontreated embryos after 6 days of culture. The chlorophyll a/b ratio of 4-day old treated somatic embryos was found to be similar to that of zygotic embryos. However, the chlorophyll a/b ratio of plantlets induced from nontreated somatic embryos was variable. Nondialysate was fractionated by ultracentrifugation and an active component obtained, which gave a maximum plantlet formation frequency of 71 %, and induced rapid greening of shoots.Abbreviations MS Murashige and Skoog medium - 2,4D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - E Einstein - Chl Chlorophyll - vvm volume of air, volume of medium per minute     

Differentiation of restriction sites in ribosomal DNA in the genusApodemus

Southern blot analysis of ribosomal DNA (rDNA) from seven species ofApodemus was carried out in order to examine the genetic relationships between the species. Analysis of heterogeneity in rDNA spacers inA. sylvaticus, A. flavicollis, A. semotus, A. agrarius, A. argenteus, A. speciosus, andA. peninsulae, using 13 different restriction enzymes and cloned mouse rDNA probes, revealed that the families of rDNA in these species can be characterized by restriction maps which show the major constituents of rDNA repeating units (repetypes). Based on differences in the arrangement of restriction sites, sequence divergence among the different major repetypes was estimated. Among the seven species ofApodemus examined, the major repetypes ofA. flavicollis andA. sylvaticus were the most closely related, having only 1.0% sequence divergence. These repetypes and those of the remaining five species differ substantially from one another, with 4.3–8.5% divergence.This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.     

Growth Competition between W Mutant and Wild-type Cells in Mouse Aggregation Chimeras

The dominant spotting (W) locus of the mouse has been demonstrated to be identical with the c-kit proto-oncogene. The c-kit is strongly expressed in hematopoietic organs and the brain of mice. In homozygotes and double heterozygotes of the W mutant alleles (hereafter W mutant), development of erythrocytes, mast cells, melanocytes and germ cells is deficient. The deficiency of erythrocytes, mast cells and melanocytes is attributed to a defect of precursor cells, but the cause of the germ cell deficiency is not clear. We investigated the effect of the W mutation on proliferative potential of cells composing various organs by examining aggregation chimeras between W mutant and wild-type (+/+) embryos. Proportions of +/+ components were significantly greater in the male germ cells and hematopoietic cells. In contrast, the average proportions of +/+ components were comparable to those of W mutant components in other organs including the brain. The present result suggests that the W (c-kit ) gene plays an important role in development of the male germ cells and hematopoietic cells and that it does not promote the proliferation of major cell population in the brain, in spite of the strong expression of the W (c-kit ) gene in the brain.     

Fatty acid effects on calcium influx and efflux in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

Low concentrations of fatty acids inhibited initial Ca uptake by sarcoplasmic reticulum vesicles, the extent of inhibition varying with chain length and unsaturation in a series of C₁₄–C₂₀ fatty acids. Oleic acid was a more potent inhibitor of initial Ca uptake than stearic acid at 25°C, whereas at 5°C there was less difference between the inhibitory effects of low concentrations of these fatty acids. When the fatty acids were added later, during the phase of spontaneous Ca release that follows Ca uptake in reactions carried out at 25°C, 1–4 μM oleic and stearic acids caused Ca content to increase. This effect was due to marked inhibition of Ca efflux and slight stimulation of Ca influx. At concentrations of >4 μM, both fatty acids inhibited the Ca influx that occurs during spontaneous Ca release; in the case of oleic acid, this inhibition resembled that of initial Ca uptake at 5°C. The different effects of fatty acids at various times during Ca uptake reactions may be explained in part if alterations in the physical state of the membranes occur during the transition from the phase of initial Ca uptake to that of spontaneous Ca release.     

Gas chromatography with surface ionization detection: a highly sensitive method for determining underivatized codeine and dihydrocodeine in body fluids

Underivatized codeine and dihydrocodeine in human plasma and urine have been determined with a high degree of accuracy by capillary gas chromatography (GC) with surface ionization detection (SID). The drugs were extracted with the aid of Sep-Pak C₁₈ cartridges. Recovery of both drugs was 90%. The calibration curves obtained with dimemorfan as an internal standard showed linearity in the range 4.5–72.3 and 3.0–75.5 ng/ml of plasma for codeine and dihydrocodeine, respectively. The detection limit was about 100 pg on column (2.5 ng/ml sample). Codeine was determined quantitatively in plasma and urine obtained from a volunteer who had received 10 mg codeine phosphate orally 3 h before the sampling: the levels were found to be 14.1 and 142 ng/ml, respectively. The present GC-SID method has been compared carefully with GC-NPD (nitrogen-phosphorus detection) using the same extracts; the sensitivity of GC-SID was more than ten times greater than that of GC-NPD, with background noise correspondingly lower.     

Calcium Channels in the GABAergic Presynaptic Nerve Terminals Projecting to Meynert Neurons of the Rat

Abstract : Effects of selective Ca²⁺ channel blockers on GABAergic inhibitory postsynaptic currents (IPSCs) were studied in the acutely dissociated rat nucleus basalis of Meynert (nBM) neurons attached with nerve endings, namely, the “synaptic bouton” preparation, and in the thin slices of nBM, using nystatin perforated and conventional whole-cell patch recording modes, respectively. In the synaptic bouton preparation, nicardipine (3 × 10^-6M) and ω-conotoxin-MVIIC (3 × 10^-6M) reduced the frequency of spontaneous postsynaptic currents by 37 and 22%, respectively, whereas ω-conotoxin-GVIA had no effect. After blockade of L- and P/Q-type Ca²⁺ channels, successive removal of Ca²⁺ from external solution had no significant effect on the residual spontaneous activities, indicating that N-, R-, and T-type Ca²⁺ channels are not involved in the spontaneous GABA release. Thapsigargin, but not ryanodine, increased the frequency of spontaneous IPSCs in both the synaptic bouton and slice preparations, suggesting the partial contribution of the intracellular Ca²⁺ storage site to the spontaneous GABA release. In contrast, ω-conotoxin-GVIA (3 × 10^-6M) and ω-conotoxin-MVIIC (3 × 10^-6M) suppressed the evoked IPSCs by 31 and 37%, respectively, but nicardipine produced no significant effect. The residual evoked currents were abolished in Ca²⁺-free external solution but not in the external solution containing 10^-5M Ni²⁺, suggesting the involvement of N-, P/Q-, and R-type Ca²⁺ channels but not L- and T-type ones in the evoked IPSCs. Neither thapsigargin nor ryanodine had any significant effects on the evoked IPSCs. It was concluded that Ca²⁺ channel subtypes responsible for spontaneous transmitter release are different from those mediating the transmitter release evoked by nerve stimulation.     

Interaction of Poliovirus with Its Receptor Affords a High Level of Infectivity to the Virion in Poliovirus Infections Mediated by the Fc Receptor

Poliovirus infects susceptible cells through the poliovirus receptor (PVR), which functions to bind virus and to change its conformation. These two activities are thought to be necessary for efficient poliovirus infection. How binding and conformation conversion activities contribute to the establishment of poliovirus infection was investigated. Mouse L cells expressing mouse high-affinity Fcγ receptor molecules were established and used to study poliovirus infection mediated by mouse antipoliovirus monoclonal antibodies (MAbs) (immunoglobulin G2a [IgG2a] subtypes) or PVR-IgG2a, a chimeric molecule consisting of the extracellular moiety of PVR and the hinge and Fc portion of mouse IgG2a. The antibodies and PVR-IgG2a showed the same degree of affinity for poliovirus, but the infectivities mediated by these molecules were different. Among the molecules tested, PVR-IgG2a mediated the infection most efficiently, showing 50- to 100-fold-higher efficiency than that attained with the different MAbs. A conformational change of poliovirus was induced only by PVR-IgG2a. These results strongly suggested that some specific interaction(s) between poliovirus and the PVR is required for high-level infectivity of poliovirus in this system.