SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

EFFECTS OF CYTOCHALASIN B ON THE UPTAKES OF MONOSACCHARIDES BY RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal uptakes of a number of simple carbohydrates were strongly inhibited by cytochalasin B (K₁= 7-9 × 10⁻⁸M). Phloretin (K₁= 2-4 × 10⁻⁶M) and phloridzin (K₁= 3-4 × 10⁻⁴M) were less inhibitory. Cytochalasin B competitively inhibited the uptakes of carbohydrates with saturable transport kinetics. Inhibition of sugar uptake was immediate on addition of cytochalasin B but was promptly reversed upon removal of the drug. Cytochalasin B had no effect on the efflux of D-glucos-amine or of the phosphorylated sugar, and did not affect intrasynaptosomal hexokinase(s). The synapto-somal uptakes of L-glucose, D-mannitol, L-fucose and the N -acetylated amino sugars were non-saturable and uninhibited by cytochalasin B. In the case of sugars which enter synaptosomes by both saturable and non-saturable processes, cytochalasin B could be used to selectively inhibit the saturable uptake components. The resultant non-saturable cytochalasin-insensitive uptake rates obtained were found to be widely different among the sugars studied, and must be corrected for in order to estimate accurate kinetic constants of the saturable processes.     

Vegetalization of Sea Urchin Larvae Induced with Cycloheximide

The embryos, kept at 20°C for 3 hr–6 hr from the time of fertilization (at the morula stage), were cultured in sea water containing cycloheximide (10–16 mM) for successive 3 hr and then transferred to normal sea water. The embryos, thus treated, became vegetalized larvae. With the same treatment performed at a developmental stage prior to 3 hr of fertilization, most of embryos developed to small blastulae filled with mesenchyme-like cells. The treatment at a stage after 6 hr of fertilization yielded normal plutei. From the embryos exposed to both ¹⁴C-leucine and ³H-thymidine during the treatment, labelled chromatin was isolated. Only in the presumptive vegetalized embryos obtained by the cycloheximide treatment of morulae, ratio of ¹⁴C-radioactivity found in proteins of chromatin to ³H-radioactivity in DNA was markedly lower than that observed in chromatin from control embryos. The rate of ³H-radioactivity-decrease by DNase I treatment was higher in chromatin isolated from the presumptive regetalized embryos than that observed in chromatin isolated from control ones. Probable failure of chromatin structure formation, due to cycloheximide-inhibition of chromatin protein synthesis, seems to disturb the determination in the embryos at the morula stage, resulting in an induction of vegetalized embryos.     

Cyclic AMP-Elevating Agents Inhibit Proliferation of Keratinizing Guinea Pig Epidermal Cells

Cultured guinea pig epidermal cells and dermal fibroblasts were chosen as model systems to study possible growth inhibition by cyclic AMP (cAMP)-elevating drugs. The rate of DNA synthesis was used to assay growth rate in control cultures and those treated with agents which increase intracellular cAMP, including dibutyryl cAMP, the phosphodiesterase inhibitors papaverine and theophylline and agents which stimulate adenylate cyclase, iso-proterenol and prostaglandin E₂ methyl ester. Treatment for 24 h with dibutyryl cAMP (10⁻⁴ to 10⁻² M) inhibited cell growth by 50 to 95%, whereas butyrate(10⁻⁴M) showed essentially no effect. This inhibition could not be attributed to decreased precursor transport or to drug toxicity. Papaverine (10⁻⁶ to 10⁻⁴ M) and theophylline (10⁻⁴ to 10⁻³ M) also gave dose-dependent growth inhibition as did isoproterenol and prostaglandinE₂methyl ester. Radioautographic analysis of grain density after dibutyryl cAMP treatment and ³H-thymidine incorporation indicated no S-phase inhibition. Cyclic AMP-elevating drugs appear to inhibit growth of guinea-pig epidermal cells and dermal flbroblasts by blocking the cell cycle in G₋₂, M₁, or G. ₋₁     

The effect of benzo-18-crown-6, a synthetic ionophore, on stomatal opening and its interaction with abscisic acid

Abstract. Treatment of tomato seedlings with 5 mM benzo-18-crown-6, a potassium ionophore, produced a reduction in transpiration of 40%. Treatments of epidermal strips of Commelina communis with ben-zo-18-crown-6 (1-l0mM) inhibited stomatal opening, and this effect was shown to be reversible. An antagonistic interaction between abscisic acid (10⁻⁷M) and benzo-18-crown-6 (4 × 10⁻³ M) was also observed.     

In vitro effects of zinc on lymphoid cells of the carp, Cyprinus carpio L.

Cells from the spleen, anterior kidney, middle kidney, thymus and peripheral blood of common carp were tested in culture at 22°C. Unlike mammals, zinc was not found to be a mitogen but to have a suppressive effect. This was usually detectable at very low concentrations (10⁻⁷M), although the degree of suppression varied depending on the organ investigated. Direct toxic effects of the metal were only observed at high concentrations (0·5 × 10⁻³ m or greater). The use of carp serum supplement rather than foetal calf serum was important in demonstrating these effects.     

STIMULATORY EFFECTS OF SUBSTANCE P ON NEURITE EXTENSION AND CYCLIC AMP LEVELS IN CULTURED NEUROBLASTOMA CELLS

Abstract— Synthetic substance P initially increased cyclic AMP levels and subsequently induced neurite extension in cultured neuroblastoma N 18 cells. The magnitude of these effects depended on the concentration of fetal calf serum (FCS) in the culture medium, being more evident in the presence of a lower (0.1%) concentration of FCS.
In Eagle's medium containing 0.1% FCS, low concentrations of substance P (10⁻⁷-10⁻⁵ M) increased cyclic AMP levels and stimulated neurite extension.
In Eagle's medium containing 5%FCS, both substance P at concentrations of 10⁻⁵-10⁻³M and dibutyryl cyclic AMP at concentrations of 10⁻⁴-10⁻²M increased cyclic AMP levels and stimulated neurite extension. The activities of acetylcholinesterase, (Na⁺+ K⁺)-, HCO₃⁻ and Mg²⁺ -stimulated-ATPase were also increased. Cell growth was inhibited.
Substance P at concentrations of 10-⁷-10⁻⁵M also stimulated the adenylate cyclase activity of a particulate fraction of N 18 in a concentration-dependent manner.     

The Effects of Insect Juvenile Hormone on the Growth of Some Protozoans in vitro. I. Crithidia sp.

SYNOPSIS. Experiments were designed to investigate the effects of insect juvenile hormone (JH) on the over-all growth and macromolecular synthesis of Crithidia sp. in vitro. Cells grown in the presence of 10⁻⁵M-10⁻³M JH showed a concentration-dependent inhibition of growth, which appeared to result from both a prolongation of generation time and a delay in the onset of logarithmic growth. Juvenile hormone (10⁻³M) inhibited the incorporation of [³H]thymidine, [³H]uridine and [³H] leucine into logarithmically growing cells by 50, 70 and 40% respectively. The incorporation of [³H]uridine into acid insoluble material could be stopped within 1 hr of application of the hormone (10⁻³M). The inhibitory effect was reversible in terms of cell numbers in subcultures of washed cells but an examination of the reversibility of RNA synthesis inhibition suggested that the resumption of RNA synthesis at an optimal level would require a lag period of at least 1–3 hr. It is suggested that JH may act by interfering with RNA synthesis either directly or indirectly by primarily acting at the level of the plasma membrane.     

Nitrilase activity in clubroot diseased plants

Nitrilase activity was detected in desalted extracts of leaves, hypocotyls and roots of swede ( Brassica napus ) but was considerably higher in leaves than in roots. After inoculation with Plasmodiophora brassicae infected roots and hypocotyls showed an increase in nitrilase activity beginning at the early stages of club development before total protein increased significantly. Enzyme activity of infected tissue was partially purified by DEAE ion exchange chromatography and compared to the enzyme extracted from non infected seedlings. It appears that the increase in nitrilase activity is due to an increase of the plant enzyme which is initially present with lower activity. K_m values for the artificial substrate 3-cyanopyridine and for indole-3-acetonitrile were 2.1 × 10⁻³ M and 6.2 × 10⁻⁴M, respectively. The role of nitrilase activity for IAA biosynthesis is discussed.     

Sublethal effects of imidacloprid and pymetrozine on population growth parameters of cabbage aphid, Brevicoryne brassicae on rapeseed, Brassica napus L.

Efficiency of imidacloprid and pymetrozine on population growth parameters of cabbage aphid, Brevicoryne brassicae L. （Homoptera： Aphididae） was determined using demographic toxicology by leaf dip method. At first, bioassay tests were performed. The LC50 value and confidence limit for imidacloprid and pymetrozine were 1.61 × 10^-5 mol/L （0.74 × 10^-5-2.66 × 10^-5） and 2.14 × 10^-4 mol/L （1.24 × 10^-4-3.40 × 10^-4）, respectively. To evaluate the sublethal effect of two insecticides on population growth parameters of cabbage aphid, LC30 concentrations of imidacloprid and pymetrozine were used at 5 mol/L and 30 mol/L. The experiments were carried out in a incubator at 20±1℃, 60% ± 5% RH and 16：8 （L：D） photoperiod on canola seedlings, Brassica napus L. var. ＇PF＇. Net fecundity rate decreased in both insecticide-treated populations. Intrinsic rates of increase （rm） were lower in imidacloprid and pymetrozine treatments than in controls. Intrinsic birth rates also decreased in treated populations. There was a relative increase in intrinsic death rates of treated populations. The mean generation times and doubling time were also lower in populations treated with insecticides than in controls. There was a considerable reduction in the average numbers of nymphs reproduced per female as compared with the control. The average longevity of female adults in the control was significantly different from those treated with imidacloprid and pymetrozine. However, there was no significant differences in aphid life-table parameters between the two insecticide-treated populations （P 〉 0.01）.     

PREVENTION BY HYDROXYUREA OF VEGETALIZATION OF SEA URCHIN LARVAE INDUCED BY cAMP PHOSPHODIESTERASE INHIBITORS

During 3-hr treatment of the morulae of sea urchin with cAMP phosphodiesterase (PDE) inhibitors, which produce vegetalized larvae, incorporation of ³H-thymidine into DNA occurs at almost the same rate as in control embryos. DNase I digests the newly synthesized DNA in chromatin isolated from morulae treated with PDE-inhibitor (caffeine) faster than that isoloated from normal morulae whereas it dogests DNA isolated from chromatin in caffeine treated embryos at almost the same rate as that in normal embryos. Hydroxyurea, an inhibitor of nucleside diphosphate reductase, prevents the vegetalizing effect of PDE-inhibitor on the development of sea urchin embrys.     

Chlorsulfuron influence on garden pea reproduction

The influence of chlorsulfuron on the reproduction of green pea ( Pisum sativum ) was examined by exposing plants at 3 different stages of development to 3 different exposure levels (46, 92, and 180 mg ha⁻¹; corresponding to 2 × 10⁻³, 4 × 10⁻³, and 8 × 10⁻³ of the recommended field application rates for small grain crops). Reproduction was only influenced by the two higher rates. The most susceptible stage of development was when plants possessed 6 expanded leaves and one visible flower bud. At that stage, an application rate of 180 mg ha⁻¹ (0.8% of the recommended field rate) reduced the seed yield of treated plants to 1% of that of the control plants without severely altering the height or appearance of mature plants. When correspondingly low application rates of atrazine. glyphosate, and 2.4-D were administered at this same developmental stage there were no effects on either growth or reproduction. Thus, chlorsulfuron had an influence on plant reproduction that was not produced by other herbicides administered at low levels.     

MORPHOGENETIC SUBSTANCES FOUND IN THE EMBRYOS OF SEA URCHIN, WITH SPECIAL REFERENCE TO THE ANTI-VEGETALIZING SUBSTANCE

When sea urchin embryos up to the 2-cell stage are treated with 5 × 10⁻³ M chloramphenicol for a short period, small blastulae filled with mesenchyme-like cells (type A) are formed. If a homogenate of the embryos up to the 2-cell stage is introduced just after the chloramphenicol treatment, almost all embryos developed to normal plutei. Chloramphenicol treatment, started at the 8 ∽ 32-cell stages, induces vegetalized larvae (type B), and presumptive vegetalized ones develop normally after treatment with a homogenate of the embryos at the 16 ∽ 64-cell stages. If embryos are treated in the same manner at 7 hr after insemination, abnormal embryos which seem to be bipolar ones (type C) are observed at 40 hr after the treatment. These also develop normally, if a homogenate of embryos at the stages from unfertilized egg to gastrula is added to them at the end of the chloramphenicol treatment. The substances having these activities are named morphogenetic substances α, β and γ, respectively. The morphogenetic substance β (anti-vegetalizing substance) is heat stable and its molecular weight is less than 10,000.     

A light intensity threshold for schooling in the Atlantic mackerel, Scomber scombrus

The schooling behaviour of Atlantic mackerel was studied in a large tank at different light intensities in the range 12.6–1.8 × 10⁻¹⁰μEs⁻¹ m⁻². Variable light intensity was produced by accurately controlling the current to a green light-emitting diode (LED) 3 m above the experimental tank. Under high light levels (1.8 × 10⁻⁶μEs⁻¹ m⁻²) mackerel always formed a single school, whereas at lower levels (1.8 × 10⁻⁸μEs⁻¹ m⁻²) they swam as individuals. At light levels down to 1.0 × 10⁻⁶μEs⁻¹ m⁻² the mean nearest neighbour distance in a school remained relatively constant (0.3–0.9 body lengths), and individual mackerel swam along a path which deviated from the position of their nearest neighbours by less than 14°. As light dropped below 1.8 × 10⁻⁷μEs⁻¹ m⁻², both nearest neighbour distance and heading angle between nearest neighbours increased, with mean values of 1–1.8 body lengths and 23–92°, respectively, at 1.8 × 10⁻⁹μEs⁻¹ m⁻². The results are discussed in terms of ambient light conditions in the sea.     

Purification and Characterization of Trypsin like Enzyme of Sperm of the Sea Urchin, Hemicentrotus pulcherrimus

Trypsin like enzyme has been isolated from sperm of the sea urchin, Hemicentrotus pulcherrimus , using tryptophane methyl ester-Sepharose 4B and soybean trypsin inhibitor-Sepharose 4B affinity chromatographies.
The isolated enzyme preparation is homogenous in polyacrylamide gel electrohoresis at pH 2.3. The molecular weight of the enzyme estimated by gel filtration is about 33,000, and the enzyme separates into two subunits of 10,900 and 20,500 on sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence of β-mercaptoethanol.
This enzyme is active to N-α-benzoyl arginine ethyl ester (BAEE), N-α-toluenesulfonyl-L-arginine ethyl ester (TAME), and N-α-benzoyl-DL-arginine-p-nitroanilide, but not N-acetyl-L-tyrosine ethyl ester, N-benzoyl-L-tyrosine ethyl ester, Hippuryl-L-arginine, and Hippuryl-L-phenylalanine. The optimal pH of this enzyme is about 8.0. The Michaelis constants for BAEE and TAME are 3.3 × 10⁻⁶M, and 8.2 × 10⁻⁵M, respectively.
Soybean trypsin inhibitor and lima bean trypsin inhibitor completely inhibit the activity of this enzyme, while N-α-tosyl-L-lysine chloromethyl ketone, ovomucoid trypsin inhibitor, and α-1-antitrypsin partially inhibit. L-1-tosylamide-2-phenyl chloromethyl ketone, chyrnostatin, and aporotinine are without effect.
This enzyme is stable at pH 2.0–3.0 and labile at pH 8.0. Ca²⁺ and Mg²⁺ activate this enzyme, but do not stabilize at pH 8.0. Seawater, NaCl, and KCl inhibit this enzyme activity.
Release of this enzyme from the acrosomal vesicle is suggested.     

β-Adrenergic antagonists and carp (Cyprinus carpio L.) sperm motility

We studied the effects of two β-adrenergic antagonists, atenolol and propranolol, on carp sperm motility. Atenolol (10⁻³−10⁻⁸ m ) has no appreciable effects while propranolol (6 × 10⁻⁵−3 × 10⁻⁶ m ) affects the percentage of sperm motility in a dose-dependent fashion.     

The detection of dissolved amino acids by the Atlantic cod, Gadus morhua L.

A classical conditioning technique was employed with cod, Gadus morhua L., to determine thresholds for the detection of the L-forms of some α amino acids which are thought to be attractants or feeding stimulants for fish. The amino acids investigated, in order of effectiveness, were tyrosine, cysteine, phenylalanine, glycine and methionine with mean threshold response levels ranging from 2·5 × 10^-8M to 7·4 × 10^-8M. Histidine and lysine resulted in similar thresholds with a mean value of approximately 3 ± 10^-7M while taurine and leucine were least effective with mean threshold levels of 2·1 × 10^-6M and 2·1 × 10^-5M respectively. Comparison is made with electrophysiological and behavioural response data from other species. The effect of raising the background level of glycine on the threshold to glycine for cod is described. The results are discussed with reference to data on levels of dissolved free amino acids in shallow sea waters which may have a bearing on determining chemosensory threshold levels. To detect a specific amino acid against a background level of the same substance the difference in level for detection is proportionally greater for higher background concentrations.     

Protoporphyrinogen oxidation coupled to nitrite reduction with membranes from Desulfovibrio gigas

Abstract The oxidation of protoporphyrinogen by membranes from Desulfovibrio gigas with nitrite as the electron acceptor proceeds at the ratio of 1 mol protoporphyrin produced for each mol of nitrite reduced. Coupled to the protoporphyrinogen-nitrite reaction is the esterification of ortho-phosphate with a P/2e⁻ value of 0.92. Pentachlorophenol, 3 × 10⁻⁵ M, and carbonyl cyanide m -chlorophenyl hydrazone, 3 × 10⁻⁶ M, serve as uncouplers of phosphorylation while rotenone, 9 × 10⁻⁶ M, and hydroxyquinoline- N -oxide, 0.1 × 10⁻⁶ M, inhibit electron flow from protoporphyrinogen oxidase to nitrite reductase.     

Effect of angiotensin II on transepithelial potential in the isolated perfused flounder (Platichthys flesus) gill

Angiotensin II (Asp¹, Val⁵) perfused through isolated flounder gills inhibited the transepithelial potential by up to 25 per cent at a concentration of 10⁻⁹M. There was no effect on gill haemodynamics and the subsequent response to 10⁻⁵ M adrenaline was normal.     

CHARACTERIZATION OF A DOPAMINE-SENSITIVE ADENYLATE CYCLASE IN THE RAT CAUDATE NUCLEUS

Abstract— An adenylate cyclase present in the caudate nucleus of rat brain, which is selectively stimulated by low concentrations of dopamine, and which is believed to mediate dopaminergic synaptic transmission, has been characterized with respect to several properties. The parameters studied included temperature, pH, ATP concentration, Mg/ATP ratio, and metal ion specificity. The effects of other compounds, including EGTA, NaF and several guanosine nucleotides, were also tested on the dopa-mine-sensitive adenylate cyclase. In addition, the subcellular distribution of the enzyme was studied. The highest specific activity was found in subcellular fractions enriched in nerve endings. A half-maximal increase in the activity of the enzyme in a subcellular fraction occurred in the presence of 4 × 10⁻⁶ M dopamine. Fluphenazine, a dopamine antagonist, competitively inhibited the activity of the enzyme in this fraction, with a calculated inhibition constant ( K_i ) of 8 × 10⁻⁹M.