Autoradiographic detection of uptake of bacterial3H-DNA label by barley seeds and isolated barley embryos

Barley seeds and extirpated embryos were cultivated in³H-DNA fromBacillus subtilis. Control group of embryos was cultivated in³H-thymidine. Uptake of the label from³H-DNA was observed after both methods of application, to halves of seeds and to embryos, and the label was localized almost exclusively in some of the nuclei of meristematic cells.     

The growth of isolated barley embryos cultivated under different conditions

Isolated barley embryos were cultivated aseptically in three different complete media. The growth of the primary root of each embryo was measured during four days of cultivation. Embryos were cultivated on three different consistencies of media: on agar plates, on cellulose tissue moistened with the medium and in liquid shaken cultures. The last way of cultivation yielded the highest degree of the growth of roots. Optimum combination of conditions in shaken liquid medium was selected on the basis of systematic study. The growth of roots of isolated embryos under appropriate conditions approaches that of roots of seedlings cultived on moistened blotting paper.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Studies on the in vitro metabolism of [3H]cortisol and [3H]oestradiol by sheep skin and wool roots.

The in vitro metabolism of [3H]cortisol, [3H]cortisone and [3H]estradiol-17 beta by adult sheep skin and wool follicle tissue (wool roots) was examined. The main metabolic product of the incubation of [3H]cortisol with sheep skin was [3H]cortisone, and the conversion was reversible. Wool roots were unable to carry out detectable interconversion, nor did this tissue give rise to other significant metabolites. Sheep skin and wool roots both rapidly converted [3H]oestradiol-17 beta to [3H]oestrone and the conversion could be carried out by follicle and non-follicle skin structures. It is suggested that sheep skin contains both 11 beta- and 17 beta-hydroxysteroid dehydrogenases, but that wool follicles contain only the latter enzyme.     

Distribution of spring barley roots in Danish soils,of different texture and under different climatic conditions

Summary Spring barley root profiles have been investigated in three years with different climatological conditions during the growing season. In total, 50 root profiles were determined by measuring cm root/ml soil in different 10 cm sections of the profile. The investigations, show that the root density was nearly identical for all soil types within the upper part of the plough layer. The decrease in root density with depth is most pronounced for the sandy soils and less for the loamy soils. The mean max. root depth in the sandy soils was roughly 70 cm, while it was roughly 140 cm for the loamy soils. A comparison between the clay and silt content in the subsoil and the thickness of soil layers with more than given root densities shows that there is no correlation between texture and thickness of soil layers with more than 1.0 cm root/ml soil, while there was a clear, positive correlation between thickness of soil layers with lower root densities and the clay and silt content in the subsoil. The different climatological conditions during the growing season give rise to differences in the root development. Very wet springs seem to impede root development in loamy soils with slowly permeable subsoils, while this is not the case in the sandy soils.     

Radiotoxicities of [3H]thymidine and of [3H]arginine compared in mouse embryos in vitro

Tritium that is bound to organic molecules is of special risk for living systems, in particular when such molecules are components of the cell nucleus. Therefore, [3H]thymidine and [3H]arginine were studied for radiotoxicity in early mammalian embryo development. Starting with the two-cell stage, mouse embryos were incubated in vitro with [3H]thymidine or [3H]arginine at either 370 Bq/ml (10 nCi/ml) or 925 Bq/ml (25 nCi/ml). Development in vitro was followed up to the formation of the inner cell mass at 192 h postconception (p.c.). There was no difference in radiotoxicity of the two substances with respect to cell proliferation; however, formation of blastocysts, hatching of blastocysts, trophoblast outgrowth, and formation of inner cell mass were impaired more strongly by [3H]arginine than by [3H]thymidine when the external exposure concentrations were the same. Similarly, micronuclei were seen in blastocysts at 96 h p.c. at higher frequency after incubation with [3H]arginine. However, uptake of [3H]arginine by the embryos was considerably faster than that of [3H]thymidine, and this most probably accounts for the apparent difference in radiotoxicity.     

Autoradiographic detection of the uptake of the label from bacterial3H-DNA in relation to the kinetics of the mitotic cycle in barley embryos

Extirped barley embryos were pre-cultivated in aerated liquid nutrient solution for 24 h and then cultivated for 6 h in nutrient solution containing either³H-DNA fromBacillus subtilis or³H-thymidine. After this treatment the embryos were thoroughly washed and transferred to the fresh nutrient medium. Samples were fixed at different intervals up to 24 h. Feulgen squashes were made and covered with autoradiographic emulsion. Microautodiagrams of different parts of the embryos (root meristem, shoot apex plus meristem of the third leaf, second leaf meristem, coleoptile, scutelum) were observed. Labelling of the nuclei after the application of both³H-DNA and³H-thymidine was found in the proliferating parts of the embryos but no label was found in the scutelum. The labelling index values were almost similar in different embryo organs after the treatment with³H-DNA and³H-thymidine. Labelling index and the fraction of labelled mitoses at different intervals after the application of the labelled substances were almost similar after treatment with³H-DNA and³H-thymidine, except some variations due to irrelevant differences in the kinetics of the mitotic cycle. No disappearance of the activity of³H-DNA was observed at different intervals after removal from the labelled solutions during cultivation for other 24 h in non-labelled nutrient medium either containing DNA fromBacillus subtilis or without it. The embryos which were immersed into 0.2% NaCl solution with either one of the labelled compounds did not show any initiation of the S phase nor uptake of³H-DNA. All these results demonstrate that the label from³H-DNA is localized in those cell nuclei which were in the S phase during treatment but they do not yet distinguish unambiguously between the adsorbtion of polymerous DNA or its degradation and reutilization of low-molecular weight products.     

Uptake and binding of tritium from [chloroethyl 3H] cyclophosphamide by rat embryos in vitro

Day 10 rat embryos were exposed in vitro to [chloroethyl 3H] cyclophosphamide (3H-CP) at 4 micrograms/ml over a 24-hour period and the uptake and binding of labeled drug were monitored autoradiographically and biochemically. Autoradiographic analysis revealed that embryos exposed to 3H-CP and a complete activating system exhibited radioactivity distributed throughout the embryo. Subsequent analysis indicated that the distribution of autoradiographic grains on a per cell basis ranged from 7.7 in surface ectoderm to 13.4 in the neuroepithelium. No correlation was found between the sensitivity of various embryonic tissues to the cytotoxic effects of CP and the number of grains per cell. Direct radiochemical analysis of the amount of tritium taken up and bound by embryos under bioactivating conditions (3H-CP + S-9 + cofactors) confirmed the autoradiographic analysis. Autoradiographic and radiochemical analyses demonstrate that embryos exposed under bioactivating conditions take up and bind approximately three times more tritium than embryos exposed under nonactivating conditions (3H-CP + S-9 without cofactors). Additional studies have demonstrated that uptake and binding of tritium from bioactivated 3H-CP only are linear over the first 10 hours of incubation with no detectable increases thereafter.     

Different specific binding sites of [3H]glycine and [3H]strychnine in synaptosomal membranes isolated from frog retina

Synaptosomal fractions were isolated from frog retina: a fraction enriched in photoreceptor terminals (P1) and a second one (P2) containing interneurons terminals. We compared the binding of [³H]glycine and [³H]strychine to membranes of these synaptosomes. The binding of both radioactive ligands was saturable and Na⁺-independent. [³H]Glycine bound to a single site in P1 and P2 synaptosomal fractions, with K_D=12 and 82 nM and B_Max=3.1 and 3.06 pmol/mg protein respectively. [³H]Strychnine bound to two sites in each one of the synaptosomal fractions. For P1 K_D values were 3.9 and 18.7 nM, and B_Max values were 1.1 and 7.1 pmol/mg protein, respecitively. Membranes from the P2 synaptosomal fraction showed K_D's of 0.6 and 48 nM and B_Max's of 0.4 and 4.5 pmol/mg. Specific [³H]glycine binding was displaced by -alanine, l-serine, d-serine and HA966, but not by strychnine 7-chlorokynurenic or 5,7-dichloro-kynurenic acids. Specific [³H]strychnine, binding was partially displaced by glycine and related aminoacids and totally displaced only by 2-NH₂-strychnine. Our results indicate the presence of high affinity binding sites for glycine and strychnine in frog retinal synaptosomal membranes. The pharmacological binding pattern indicates the presence of the strychnine sensitive glycine receptor as well as other sites. These might not include the NMDA receptor-associated glycine site.     

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.     

Binding of [3H]ctyochalasin B and [3H]colchicine to isolated liver plasma membranes.

The binding to isolated hepatocyte plasma membranes of radioactively labelled inhibitors of microfilamentous and microtubular protein function ([3H]cytochalasin B and [3H]colchicine, respectively) was studied as one means of assessing the degree of association of these proteins with cell surface membranes. [3H]Cytochalasin B which behaved identically to the unlabelled compound with respect to binding to these membranes was prepared by reduction of cytochalasin A with NaB3H4. The binding was rapid, readily reversible, proportional to the amount of membrane and relatively insensitive to changes of pH or ionic strength. At 10(-6) M [3H]cytochalasin B, glucose of p-chloromercuribenzoate, an inhibitor of glucose transport inhibited binding by about 20%; treatment of membranes with 0.6 M KI which depolymerizes F actin to G actin caused about 60% inhibition of binding. These two types of inhibition were additive indicating two separate classes of binding sites, one associated with sugar transport and one with microfilaments. Filamentous structures with the diameter of microfilaments (50 A) were seen in electron micrographs of thin sections of the membranes. At concentrations greater than 10(-5) M [3H]cytochalasin B, binding was proportional to drug concentration, characteristic of non-specific adsorption or partitioning. Intracellular membranes of the hepatocyte also bound [3H]cytochalasin B, those of the smooth endoplasmic reticulum to a greater extent than plasma membranes. [3H]Colchicine bound to plasma membranes in proportion to the amount of membrane and at a rate compatible with binding to tubulin. However, other properties of the binding including effects of temperature, drug concentration and antisera against tubulin were different from those of binding to tubulin. Hence, no evidence was obtained for association of microtubular elements with these membranes. Despite this there appeared to be an interdependence between microtubule and microfilament inhibitors: vinblastine sulfate stimulated [3H]cytochalasin B binding and cytochalasin B stimulated 3H colchicine binding. [3H]Colchicine also bound to intracellular membranes, especially smooth microsomes.     

Utilization by the isolated perfused rat liver of N-acetyl-D-[1-14C]galactosamine and N-[3H]acetyl-D-galactosamine for the biosynthesis of glycoproteins.

The isolated perfused rat liver system has been used to monitor the utilization of N-[3H]acetyl-D-galactosamine and N-acetyl-D-[1-14C]galactosamine for the biosynthesis of radiolabelled glycoproteins, which are subsequently secreted into the plasma. Both radiolabels appear in a number of different glycoproteins, predominantly as sialic acid and N-acetylglucosamine. The ratio of labelled sialic acid to labelled N-acetylglucosamine varies for different glycoproteins, but the bulk of N-acetyl-D-galactosamine is incorporated without deacetylation.     

Effect of pressure on [3H]GABA release by synaptosomes isolated from cerebral cortex

High hydrostatic pressure has been shown to produce neurological changes in humans which manifest, in part, as tremor, myoclonic jerks, electroencephalographic changes, and convulsions. This clinical pattern has been termed high-pressure nervous syndrome (HPNS). These symptoms may represent an alteration in synaptic transmission in the central nervous system with the inhibitory neural pathways being affected in particular. Since gamma-aminobutyric acid (GABA) transmission has been implicated in other seizure disorders, it was of interest to study GABAergic function at high pressure. Isolated synaptosomes were used to follow GABA release at 67.7 ATA of pressure. The major observation was a 33% depression in total [3H]GABA efflux from depolarized cerebrocortical synaptosomes at 67.7 ATA. The Ca2+-dependent component of release was found to be completely blocked during the 1st min of [3H]GABA efflux with a slow rise over the subsequent 3 min. These findings lead us to conclude that high pressure interferes with the intraterminal cascade for Ca2+-dependent release of GABA.     

Studies on the uptake of [3H]dopamine by synaptic vesicle fraction isolated from bovine brain

1. A synaptic vesicle fraction isolated from bovine caudatolenticular nuclei showed enzymic activities of tyrosine hydroxylase [EC 1.14.16.2] and dopamine beta-hydroxylase [EC 1.14.17.1]. Tyrosine hydroxylase, whose subcellular localization is uncertain, appeared to be associated with the synaptic vesicles. 2. The vesicle fraction took up [3H]dopamine increasingly with time without the aid of ATP (6.3 pmol of [3H]dopamine per mg of vesicle proteins, or 3-4% of the added dopamine, at 30 min). In the presence of ATP, a transient accumulation of the amine was observed, reaching the highest level at 7-10 min (5.6 pmol dopamine/mg protein), and then a rapid release of the amine took place, obeying first-order kinetics with respect to the amine concentration. The amine uptake was strongly inhibited with NEM, regardless of the presence or absence of ATP. 3. The vesicle fraction also exhibited a weak ability to translocate protons inward and ATP-dependently, as monitored by an increase in the fluorescence intensity of ANS. The fluorescence enhancement persisted for at least 30 min and this time-dependent change was not consistent with that of the transient accumulation of [3H]dopamine mentioned above. Since the present vesicle preparation contained a small amount of mitochondrial ATPase (18-20% of the total activity), the proton translocating ability could be attributable to contaminating submitochondrial particles. 4. Therefore these results made it impossible to conclude that the transient uptake of [3H]dopamine by the synaptic vesicles was coupled to ATP hydrolysis.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Delta pH-dependent and -independent uptake of [3H]dopamine by synaptic vesicles isolated from rat brain

The dopamine (DA)-translocating mechanism of synaptic vesicles isolated from rat brain has been studied in the presence of an artificially imposed delta pH on the vesicle membranes (acidic inside with respect to the external medium) without the aid of ATP-Mg2+. Under the experimental conditions, [3H]DA uptake by the synaptic vesicles was driven by two different, i.e. delta pH-dependent and -independent processes. Both processes appeared to be carrier-mediated based on the inhibition by NEM (N-ethylmaleimide), an -SH reagent, and by nomifensine, a DA uptake blocker at nerve terminals. The DA carrier of the vesicles was similar to that of the nerve terminal plasma membrane with respect to their susceptibility to nomifensine. The delta pH-dependent uptake was transient and most of the incorporated DA was easily lost from the vesicles. On the other hand, the delta pH-independent uptake increased with time and the amine was retained in the vesicles. The initial rate of the delta pH-independent uptake was lower than that of the delta pH-dependent one but their extents were comparable with each other. These results indicate that rat brain synaptic vesicles have a DA uptake system requiring no ATP hydrolysis. A preparation of synaptic vesicles used here exhibited an inwardly directed proton translocation in the presence of ATP-Mg2+ when monitored by following changes in the fluorescence of ANS (8-anilino-1-naphthalene sulfonate). However, the time course of the delta pH-generation was not influenced by the addition of 1 mM DA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition by Anandamide and Synthetic Cannabimimetics of the Release of [3H]d-Aspartate and [3H]GABA from Synaptosomes Isolated from the Rat Hippocampus

Cannabinoids (CB) can act as retrograde synaptic mediators of depolarization-induced suppression of inhibition or excitation in hippocampus. This mechanism may underlie the impairment of some cognitive processes produced by these compounds, including short-term memory formation in the hippocampus. In this study, we investigated several compounds known to interact with CB receptors, evaluating their effects on K(+)-evoked release of [3H]D-aspartate ([3H]D-ASP) and [3H]GABA from superfused synaptosomes isolated from the rat hippocampus. [3H]D-ASP and [3H]GABA release were inhibited to different degrees by the synthetic cannabinoids WIN 55,212-2; CP 55,940, and arachidonyl-2'-chloroethylamide/N-(2-chloroethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (ACEA), as well as by the endocannabinoids, anandamide (AEA), and 2-arachidonoylglycerol (2-AG). Both types of release were also inhibited by capsaicin. The inhibition produced by each of the cannabinoid compounds and capsaicin was unaffected by capsazepine or by the CB1-receptor antagonists AM-251 and SR141716A. The mechanism underlying AEA- and synthetic CB-induced inhibition of the release of [3H]GABA and [3H]D-ASP from rat hippocampal synaptosomes might not involve activation of presynaptic CB1 receptors.