Tubular extensions of the plasmalemma in leaf cells of Zea mays L.

Leaf tissues of Zea mays were examined with a transmission electron microscope and a high-voltage electron microscope. Tubular extensions (invaginations) of the plasmalemma were found in vascular parenchyma cells and thick-walled, lateformed sieve elements of intermediate and small veins, and in epidermal, mesophyll, and sheath cells of all leaves examined. No continuity seems to exist between the tubules and other cellular membranes.     

Isolation of somatic hybrids by cloning Nicotiana heterokaryons in nurse cultures

Fusion of Nicotiana knightiana Goodsp. and kanamycin resistant Nicotiana sylvestris Speg. et Com. protoplasts was induced by polyethylene glycol treatment. Heterokaryons were isolated by micropipette and transferred to nurse cultures of albino cells. Colonies originating from the heterokaryons could subsequently be distinguished by their green colour. The somatic hybrid nature of four such colonies was confirmed by isoenzyme pattern, kanamycin resistance and restored morphogenic potential. An additional kanamycin resistant line with characteristic Nicotiana knightiana isoenzymes was also found indicating that the drug resistance in the kanamycin resistant parent is under cytoplasmic control.Abbreviations ADH alcohol dehydrogenase - Nk Nicotiana knightiana     

Inositol 1,4,5-Trisphosphate 3-Kinase mRNA: High Levels in the Rat Hippocampal CA1 Pyramidal and Dentate Gyrus Granule Cells and in Cerebellar Purkinje Cells

The distribution of inositol 1,4,5-trisphosphate (InsP3) 3-kinase mRNA in the rat brain is reported using oligonucleotides based on a cDNA clone sequence that encodes rat brain InsP3 3-kinase and the in situ hybridization technique. Moderate levels were found in CA2-4 pyramidal neurons, in the cortex, and in the striatum. The cerebellar granule cells, thalamus, hypothalamus, brainstem, spinal cord, and white matter tracts were almost negative. The levels of InsP3 3-kinase mRNA were highest in the hippocampal CA1 pyramidal neurons, granule cells of the dentate gyrus, and cerebellar Purkinje cells. These results contrast with the lower concentration of the InsP3 receptor already reported in the hippocampus versus the Purkinje cells and suggest a special role for inositol 1,3,4,5-tetrakisphosphate in Ammon's horn.     

Enhancement by Cytidine of Membrane Phospholipid Synthesis

Cytidine, as cytidine 5'-diphosphate choline, is a major precursor in the synthesis of phosphatidylcholine in cell membranes. In the present study, we examined the relationships between extracellular levels of cytidine, the conversion of [14C]choline to [14C]phosphatidylcholine, and the net syntheses of phosphatidylcholine and phosphatidylethanolamine by PC12 cells. The rate at which cytidine (as [3H]cytidine) was incorporated into the PC12 cells followed normal Michaelis-Menten kinetics (Km = 5 microM; Vmax = 12 x 10(-3) mmol/mg of protein/min) when the cytidine concentrations in the medium were below 50 microM; at higher concentrations, intracellular [3H]cytidine nucleotide levels increased linearly. Once inside the cell, cytidine was converted mainly into cytidine triphosphate. In pulse-chase experiments, addition of cytidine to the medium caused a time- and dose-dependent increase (by up to 30%) in the incorporation of [14C]choline into membrane [14C]-phosphatidylcholine. When the PC12 cells were supplemented with both cytidine and choline for 14 h, small but significant elevations (p less than 0.05) were observed in their absolute contents of membrane phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine, all increasing by 10-15% relative to their levels in cells incubated with choline alone. Exogenous cytidine, acting via cytidine triphosphate, can thus affect the synthesis and levels of cell membrane phospholipids.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Opioid Signal Transduction in Intact and Fragmented SH-SY5Y Neural Cells

Parameters of ligand binding, stimulation of low-Km GTPase, and inhibition of adenylate cyclase were determined in intact human neuroblastoma SH-SY5Y cells and in their isolated membranes, both suspended in identical physiological buffer medium. In cells, the mu-selective opioid agonist [3H]Tyr-D-Ala-Gly(Me)Phe-Gly-ol ([3H]DAMGO) bound to two populations of sites with KD values of 3.9 and 160 nM, with less than 10% of the sites in the high-affinity state. Both sites were also detected at 4 degrees C and were displaced by various opioids, including quaternary naltrexone. The opioid antagonist [3H]naltrexone bound to a single population of sites, and in cells treated with pertussis toxin the biphasic displacement of [3H]naltrexone by DAMGO became monophasic with only low-affinity binding present. The toxin specifically reduced high-affinity agonist binding but had no effect on the binding of [3H]naltrexone. In isolated membranes, both agonist and antagonist bound to a single population of receptor sites with affinities similar to that of the high-affinity binding component in cells. Addition of GTP to membranes reduced the Bmax for [3H]DAMGO by 87% and induced a linear ligand binding component; a low-affinity binding site, however, could not be saturated. Compared with results obtained with membranes suspended in Tris buffer, agonist binding, including both receptor density and affinity, in the physiological medium was attenuated. The results suggest that high-affinity opioid agonist binding represents the ligand-receptor-guanine nucleotide binding protein (G protein) complex present in cells at low density due to modulation by endogenous GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Investigation of reduced serum and serum-free media for the cultivation of insect cells (Bm5) and the production of baculovirus (BmNPV)

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium. (c) 1992 John Wiley & Sons, Inc.     

Advances in cereal protoplast research

Beginning in 1986, plants have been regenerated from protoplasts of all of the important cereal species, including wheat, rice, maize, and barley, and grasses such as sugarcane. In addition, somatic hybrids/cybrids as well as transgenic plants with introduced useful agronomic traits have been obtained in several instances. This rapid and impressive progress in the genetic manipulation of cereals has been made possible by two critical technical advances during the past decade: the establishment of embryogenic suspension cultures as a source of totipotent protoplasts and the direct delivery of DNA into protoplasts for genetic transformation.     

DNA radiation damage and asymmetric somatic hybridization: Is UV a potential substitute or supplement to ionising radiation in fusion experiments?

Recent reports have revealed that the asymmetric nature of the nuclear genome of somatic hybrids, produced following the irradiation of one of the parents with X- or gamma rays, is generally much less than had been anticipated. As a consequence, we have begun to investigate whether UV radiation might be used as an alternative or indeed a supplement to the presently-used ionising radiation techniques in such experiments. Cell culture studies have revealed that UV radiation induces the desired physiological effects in sugar beet ( Beta vulgaris ) protoplasts, namely, a prevention of cell division without immediate cytotoxicity. Preliminary studies using denaturing and pulsed field gel electrophoresis have shown that UV can also induce substantial physical fragmentation of DNA. When using the same techniques, less breakdown was observed following gamma radiation. All results were highly reproducible. Such results augur well for the potential use of UV in asymmetric somatic cell fusion experiments.     

Ploidy reduction and genome segregation in cultured carrot cell lines. I. Prophase chromosome reduction

Cytological analysis of different carrot cell lines in culture has shown various cytogenetic anomalies generating new levels of ploidy and novel chromosome numbers. Polyploidy may be considered a reservoir of variability that can be released in the form of distinct new segregants of different ploidy. Mechanisms alternative to mitosis (reductional grouping, prophase chromosome reduction) operate from a polyploid state (possibly reached by means of endopolyploidy, endomitosis, nuclear fusion, or restitution nuclei) to generate new levels of ploidy and novel chromosome numbers necessary for selection to operate in vitro. The segregational phenomena require chromosome recognition in haploid set complements and abnormal behaviour of mitoses; the resulting chromosome variability suggests that chromosomes are arranged, in the resting nuclei, in an orderly and predictable manner.The knowledge of the molecular events governing these mechanisms, and how to control them, would be of great help for future applications of plant cell culture.