Repairable and unrepairable DNA strand breaks induced by decay of3H and125I incorporated into DNA of mammalian cells

Summary Chinese hamster cells (Cl : 1) were labelled with³H-thymidine or¹²⁵Iododeoxyuridine for 18 h and after 3 h in non-radioactive medium they were stored at 0° C up to 6 h. The number of DNA strand breaks observed after the labelling period (37° C) or after treatment at 0° C was determined using the DNA-unwinding technique.¹²⁵I-decays in DNA were significantly more efficient than³H-decays in introducing unrepairable DNA strand breaks during the labelling period. 32% of¹²⁵I-induced and 3% of³H-induced DNA strand breaks were unrepaired after 21 h at 37° C. Comparison between the effects of¹²⁵I- or 3H-disintegrations in DNA in three different ways shows 7–12 times more pronounced effects for¹²⁵I-decays. For¹²⁵I-labelled cells 3–4 DNA strand breaks were found per decay and the corresponding value for³H- labelled cells was 2.     

Sites of uptake of3H-5-hydroxytryptamine in rat isolated lung

Summary The localisation of radioactivity in rat lungs after perfusion of³H-5-hydroxytryptamine (5-HT) was studied by autoradiography. Rat isolated lungs, perfused with Krebs bicarbonate solution, via the pulmonary circulation, were rapidly frozen after the infusion of³H-5-HT. All subsequent handling was carried out at −30 to −40°C. Developed sections were examined by the light microscope and showed that radioactivity was concentrated around the alveoli with little or no label in other parts of the lung. Lungs perfused with³H-5-HT in the presence of mebanazine, a monoamine oxidase inhibitor, showed label in the endothelial cells of arteries and arterioles as well as the alveolar label. Lungs treated with amitriptyline were essentially devoid of label. These results indicate that the site of the avid uptake and metabolism of 5-HT in the lung is the endothelial cells of the vasculature.     

Effect of 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) on monoamine neurotransmitters in mouse brain & heart

The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was studied on dopamine (DA), norepinephrine (NE), serotonin (5HT) and γ-aminobutyric acid (GABA) neurons in mouse brain and on NE neurons of mouse heart. MPTP (45 mg/kg) was administered s.c. to mice twice daily for 2 consecutive days. This dosage regimen produced a decrease in the forebrain concentrations of DA and NE at 7 and 20 days after injection. In contrast, the forebrain concentrations of 5HT and GABA were not significantly decreased at either time. MPTP administration also produced a marked decrease in the uptake of ³H-DA into striatal slices and ³H-NE into cerebral cortical slices. In contrast, the uptake of ³H-NE into hypothalamic slices and the uptake of ³H-5HT into slices from several brain regions were not altered. MPTP initially reduced the concentration of NE in the heart, but unlike the persistent decreases in the forebrain concentrations of NE and DA, the NE concentration in the heart returned to control levels at approximately 20 days after MPTP administration. These results, showing that MPTP can produce a long lasting and selective decrease in the forebrain concentrations of NE and DA and in the uptake of radioactive DA and NE into brain slices, suggest that MPTP can cause the destruction of catecholamine neurons in mouse brain. In contrast, MPTP administration does not appear to produce long term changes in either 5HT or GABA neurons.     

Metabolism ofl-proline toN-acetyl-d-glucosamine during germ tube formation ofCandida albicans

The metabolism ofl-proline toN-acetyl-d-glucosamine (GlcNAc) during germ tube formation ofCandida albicans (C. albicans) ATCC 1002 was studied. In uptake experiments, 6.9 nmol ofl-[¹⁴C]proline were taken up by 1×10⁶ cells during 3 h of incubation at 37°C. The percentage of germ tube formation was 94 under the same condition. The presence of GlcNAc reduced the uptake ofl-proline to 3.0 nmol. The percentage of germ tube formation was 95 in the presence and absence of GlcNAc. The [³H]GlcNAc uptake was 3.0 nmol and was constant whetherl-proline was present or not. After the preparation of a chitin fraction from germ tubes that were labeled withl-[¹⁴C]proline, the radioactivity froml-proline was detected in the glucosamine (GlcN) fraction by thin-layer chromatography (TLC). The metabolism ofl-proline to GlcNAc in chitin during germ tube formation was confirmed in this experiment.     

Carrier-mediated lactate entry into isolated hepatocytes from fed and starved rats: Zonal distribution and temperature dependence

We examined the possibility of quantitative differences in lactate entry into periportal and perivenous hepatocytes under different nutritional states. The rate of¹⁴C-L(+)-lactate uptake was determined after 15-second incubations with freshly isolated zonally separated hepatocytes using a centrifuge stop technique at 37 °C and 4 °C, in the presence or absence of either differing amounts of unlabelled lactate or of a hepatocyte lactate transport inhibitor,-cyano-3-hydroxycinnamate. Total entry as well as carrier mediated entry of¹⁴C-L(+)-lactate into the isolated cell populations was found to be similar in periportal and perivenous hepatocytes, irrespective of the nutritional state of the animal. Periportal and perivenous hepatocytes showed a greater tendency to transport lactate when isolated from starved animals, in agreement with previously reported data from non-zonally separated isolated hepatocytes. The activity of the hepatocyte plasma-membrane lactate transporter was diminished between fourfold and eightfold in transport studies conducted at 4 °C; similar results were obtained in unseparated and zonally separated suspensions. Temperature dependence of the hepatocyte transporter is markedly less than that reported for the erythrocyte transporter.     

Melatonin synthesis by rabbit platelets

Platelets of reserpinized rabbits, incubated in buffer containing tritiated 5-hydroxytryptamine (³H-5HT), have the ability to convert ³H-5HT into labelled compound (s) extractable with alkaline-chloroform. The bulk of the chloroform-extractable radioactivity showed a Rf value similar to authentic melatonin on silicagel thin-layer chromatography.The labelled product eluted with ethanol from the silicagel area where melatonin was suspected to reside was indentified as melatonin by mass spectrometry and radioimmunoassay.Our in vitro experiments demonstrate that rabbit platelets are capable of converting 5-HT into melatonin apparently because they possess the enzymatic equipment necessary for this biosynthesis i.e. serotonin N-acetyltransferase and hydroxyindole-O-methyltransferase.     

STUDIES OF AMINES IN THE STRIATUM IN MONKEYS WITH NIGRAL LESIONS

The effects of ventromedial tegmental lesions on the biosynthesis and disposition of biogenic amines in the striatum of monkeys were investigated. The concentrations of endogenous dopamine and of the intraventricularly injected [³H]dopamine were distinctly lower in the striatum on the lesion side than on the intact side. The storage of [³H]dopamine in the caudate nucleus was impaired to a much greater extent than the storage of the newly synthesized [³H]norepinephrine. The concentrations of endogenous serotonin and of the intraventricularly injected [¹⁴C]serotonin were lower in the striatum on the lesion side than on the intact side. However following MAO inhibition, the concentration of [¹⁴C]serotonin did not differ significantly on the two sides of the caudate nucleus. The in vivo biosynthesis of dopamine from tyrosine was significantly reduced in the striatum on the lesion side. Tyrosine hydroxylase and DOPA decarboxylase activities were decreased on the lesion side of the striatum as compared with the intact side. Thus, the ventromedial tegmental lesions affect the storage and the synthesis of dopamine and serotonin in the ipsilateral striatum.     

Characterization of the interaction between fibronectin andTreponema pallidum

The interaction betweenTreponema pallidum and rabbit plasma fibronectin was characterized. Fibronectin was isolated from rabbit plasma and radioiodinated by the lactoperoxidase method. Fibronectin bound to the surface ofT. pallidum, reaching saturation at approximately 54 g/ml. The association affinity constant was 2.85×10⁷ M ^–1, much lower than that ofStaphylococcus aureus (5.6×10⁹ M ^–1) Fibronectin binding plateaued within 15 min at 20° and 37°C, with some reelution at 37°C by 30 min. Little fibronectin, bound toT. pallidum at 4°C. The greatest amount of fibronectin was bound at the lowest pH tested (pH 6.0); the poorest binding was at pH 7.5. Approximately 90% of the binding was reversible in the presence of excess unlabeled fibronectin. The data indicate a more dynamic and weaker interaction betweenT. pallidum and fibronectin than that seen withS. aureus.     

Effect of exogenous hormones on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L.

Inclusion of IAA in the vase water had little effect on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L. while kinetin delayed leaf yellowing at 10^–4M (continuous treatment). Chlorophyll was effectively retained by 10^–7M gibberellic acid (GA) in the vase water or by a 20h pulse at 5°C with 10^–5/10^–4M GA. After 16h of ¹⁴C-GA₃ uptake at 20°C relatively high levels of ¹⁴C were found in leaves and low levels in stems and flowers. After this treatment about half of the ¹⁴C-GA₃ in leaves was metabolized into unknown compounds.     

Adult development of the hippocampal-serotonin system of C57BL/6N mice; analysis of high-affinity uptake of 3H-5HT in slices and synaptosomes

The saturable and specific high-affinity uptake of [³H]serotonin ([³H]5HT) (5 × 10^?8 M) was studied in slices from the hippocampus, parietal cortex, septum-preoptic area, and hypothalamus of male 2, 6, 12 and 24–32 month old C57BL/6N mice. Hippocampal [³H]5-HT uptake showed a significant biphasic relationship to age, with lower values in the 2 and 24–32 month old mice compared to 6 month old mice. No significant age effects were seen in the other regions, or in [³H]norepinephrine high-affinity uptake in the hippocampus.Studies of the high-affinity uptake mechanism in synaptosomal preparations were made in a subgroup of 12 and 24 month old mice. A micro-assay using a tissue-harvester measured high-affinity uptake on 8–30 μl of the P₂ suspension (crude-synaptosomal preparation). The high-affinity uptake was linear for 4 min at 37°C and inhibited in both the adult and aged tissue by 10^?5 M cold 5-HT (83 and 78% respectively), 10^?5 M fluoxetine (85 and 82% respectively) and 10^?3 M NaCN (57 and 57% respectively). Kinetic analysis of the [³H]5HT high-affinity uptake in the hippocampus (3 min, 37°C) revealed the same apparent K_m for serotonin at both ages (6.7 x 10^?8 M), but a 44% decrease in V_max in the aged hippocampal synaptosomal high-affinity uptake compared to adults (120 vs 215 pmol of 5-HT/g-tissue/3 min).These results are discussed in relationship to the reported age effects on the intrinsic neurons of the hippocampus.     

Effects of ascorbic acid on the uptake of serotonin in differentiated neuroblastoma cells

Ascorbic acid causes concentration-dependent and time-dependent effects on [³H]-serotonin (³H-5HT) uptake into differentiated neuroblastoma N-2a cells. Preincubation of cells with ascorbic acid inhibits both passive diffusion and active transport of ³H-5HT (0.1 μM). The kinetic characteristics of the active uptake process change with ascorbic acid treatment, resulting in an increase in the Km from 0.27 μM to 3.0 μM and in the Vmax from 453 to 2369 fmol/min/10⁶ cells. This inhibitory effect of ascorbic acid appears to be due to its reducing properties.     

Kinetic analysis of 3H-serotonin accumulation in four regions of rat brain after lesions in the medial forebrain bundle

Kinetic analysis of ³H-serotonin accumulation by crude synaptosomal suspensions of neocortex, hippocampus and caudate or by whole homogenates of cerebellum revealed the presence of a high affinity uptake component having an apparent K_m for serotonin which ranged from 2.8 to 6.0 × 10^?8 M. A second, low affinity, uptake component with an apparent K_m of 7 × 10^?6 M was present in caudate. A comparable low affinity uptake component for serotonin was not observed in neocortex, hippocampus or cerebellum. Lesions in the medial forebrain bundle produced significant decreases in serotonin comtent of neocortes, hippocampus and caudate (66 to 75%) and a significant increase in serotonin content of cerebellum (25%). The lesions did not affect the apparent K_m of the high affinity uptake system but did produce change in V_max which paralleled the changes in content of serotonin. The lesions also produced decreases in dopamine and norepinephrine content of caudate and a comparable decrease in the V_max of the low affinity uptake system with no change in the apparent K_m. There was a correlation of 0.97 between the endogenous content of serotonin and the V_max of the high affinity uptake system. These results support the view that the high and low affinity components of serotonin uptake represent accumulation into serotonergic and catecholaminergic neurons, respectively.     

[3H]Serotonin binding sites in goldfish retinal membranes

Serotonin (5HT) binding sites were studied in goldfish retinal membranes by radioligand experiments. The binding site of [³H]5HT was sensitive to pre-treatment of the membranes at 40° or 60° C. 5HT and 5-methoxy-N,N-dimethyltryptamine were the best inhibitors of [³H]5HT binding to retinal membranes. The 5HT₂ agonist, 1-(-naphtyl)piperazine, was also a potent inhibitor, however, (+)-1-2,5-dimethoxy-4-iodopheny1-2-aminopropane was less efficient. The catecholaminergic agents haloperidol and clonidine did not display an important inhibition. Propranolol, also reported as 5HT_1B antagonist, was a relatively potent blocker. Monoamine uptake blockers did not show potent inhibition. The GTP analog, GppNHp, inhibited the binding. The iterative analysis of saturation curves revealed two classes of binding sites, a high affinity component (B_max 2.45 pmol/mg of protein, k_d 6.86 nM), and a low affinity component (B_max 53.46 pmol/mg of protein, K_d 232.07 nM). Analysis of the association and dissociation kinetics suggested a binding site (K_d 2 nM). The semilogarithmic plot of the dissociation kinetics gave curves concave to the upper side. The selectivity of the binding and the inhibition by GppNHp suggest the existance of 5HT₁ receptors in goldfish retina. The low affinity interaction probably represents the transporter of 5HT or a suptype of receptor expressed in glial cells.Abbreviations used B _max maximum binding capacity - CPP, 1 (3 chlorophenyl)piperazine - CLN clonidine - DMI desimipramine - DMT 5-methoxy-N,N-dimethyltryptamine - DOI (+)-1-(2,5-dimethoxy-4-iodophenyl-2-aminopropane - DPAT (+)-8-hydroxy-2-(D1-N-propylamino)tetralin - GppNHp 5-guanylylimidodiphosphate - HAL haloperidol - 5HT serotonin - IC₅₀ concentration of drug producing 50% inhibition of binding - IMI imioramine - K_d equilibrium dissociation constant - MIAN mianserin - NOM nomifensin - NP 1-(1-napthyl)piperazine - PRP propranolol In memory of Dr. Boris Druian who died on Dec. 24, 1991.     

The effect of temperature on glycollate decarboxylation in leaf peroxisomes

[1-¹⁴C]glycollate was oxidised to¹⁴CO₂ by peroxisomes isolated from leaves of spinach beet about 3 times as rapidly at 35°C as at 25°C; the rate was further increased with rise in temperature to a maximum at 55°C. These increases are shown to be mainly due to the increased H₂O₂ available to oxidise glyoxylate non-enzymically as a result of the higher temperature coefficient of glycollate oxidase activity relative to that of catalase. These results are compared with similar increases in the rate of¹⁴CO₂ release between 25°C and 35°C when [1-¹⁴C]glycollate was supplied to leaf discs in light or darkness. The role of these reactions in accounting for the temperature effect on the release of photorespiratory CO₂ is discussed.Abbreviations PHMS Pyrid-2-yl--hydroxymethane sulphonate - FMN flavin mononucleotide     

Neurochemical Changes in Cerebellum of Goldfish Exposed to Various Temperatures

Acclimation of goldfish at 35°C increased the cerebellar content of aspartate, glutamate, and taurine and [³H]glutamate uptake. Acclimation at 4°C increased the levels of glutamine, serine, and alanine and glutamine synthetase (GS) activity. Adenosine content increased in cerebellum of fish acclimated to warm temperature. K⁺-evoked release of endogenous and exogenous glutamate from cerebellar slices increased in fish acclimated at 35°C compared to 4°C. The basal level of cyclic adenosine 3:5-monophosphate (cAMP) in perfused cerebellar slices in fish acclimated at 35°C was much higher than in fish acclimated at 5° and 22°C. It is concluded that variations of environmental temperature produces large neurochemical changes in goldfish cerebellum.     

Ultrastructural relationship between monoamine- and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section

Summary The correlation of dopamine (DA)-, noradrenaline (NA)- or serotonin (5HT)-containing neurons and thyrotropin releasing hormone (TRH)-containing neurons in the median eminence of the rat, as well as the coexistence of monoamines (MA) and TRH in the neurons, were examined by subjecting ultrathin sections to a technique that combines MA autoradiography and TRH immunocytochemistry. The distribution and localization of silver grains after ³H-MA injection were examined by application of circle analysis on the autoradiographs.TRH-like immunoreactive nerve terminals containing the immunoreactive dense granular vesicles were found to have an intimate contact with monoaminergic terminals labeled after ³H-DA, ³H-NA or ³H-5HT infusion in the vicinity of the primary portal capillaries in the median eminence. Synapses between TRH-like immunoreactive axons and MA axons labeled with silver grains, however, have not been observed to date. Findings suggesting the coexistence of TRH and MA in the same nerve terminals or the uptake of ³H-MA into TRH-like immunoreactive nerve terminals, where silver grains after ³H-MA injection were concurrently localized in TRH-like immunoreactive nerve terminals, were rarely observed in the median eminence. Percentages of the nerve terminals containing both immunoreactive granular vesicles and silver grains after ³H-MA injection to total nerve terminals labeled after ³H-MA infusion silver grains were equally very low in ³H-DA, ³H-NA or ³H-5HT, amounting to less than 6.1%.This work was supported in part by grant-in-aid for scientific research from the Japan Ministry of Education (No. 557018).     

Einfluß kurzer Dunkelperioden auf CO2-Aufnahme und Phosphoenolpyruvat-Carboxylierung während der Photosynthese-Induktion bei Chlorella vulgaris

Zusammenfassung Nach einer Dunkelperiode von 40 min und 40 sec wurden die CO₂-Aufnahme und die ¹⁴C-markierten Produkte während der Photosynthese-Induktion bei Chlorella vulgaris (211-11f) bestimmt. Die mit Preßluft (0,03 Vol.-% CO₂) begasten Algen sind bei +27°C kultiviert und bei +10° oder +25°C gemessen worden. Ein Induktionseffekt der photosynthetischen CO₂-Aufnahme konnte nur nach einer längeren Dunkelperiode (>3 min) beobachtet werden. Unter diesen Bedingungen wurde ¹⁴CO₂ am Anfang der Belichtung in Malat, Aspartat und 3-Phosphoglycerat eingebaut. Nach einer kurzen Dunkelperiode (40 sec) waren zu Beginn der Belichtung vor allem die Produkte des Calvin-Cyclus markiert. Die Wirkung von Intermediaten auf die Ausbildung der Induktionseffekte wird diskutiert.

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Effect of short dark periods on CO₂ uptake and carboxylation of phosphoenolpyruvate during the photosynthetic induction period in Chlorella vulgaris

Summary CO₂ exchange, ¹⁴CO₂ fixation and ¹⁴C labelled products of Chlorella vulgaris (strain 211-11f) were studied during the photosynthetic induction period at +10° and +25°C after a dark period of 40 min and 40 sec. The algae were grown under normal aerated conditions (0.03 vol.-% CO₂) at +27°C. Transient changes in CO₂ uptake, measured with an infrared gas analyzer, could be observed only after a dark period of >3 min; no such changes occurred after a dark period of 40 sec. The autoradiographic studies of the kinetics of the appearance of labelled products at +10° and +25°C showed that after a long dark period (40 min) at the beginning of illumination ¹⁴CO₂ was incorporated into malate, aspartate and 3-phosphoglycerate. Under these conditions, the intermediates of the Calvin cycle were labelled after 30 sec (+25°C) or 2 min (+10°C) of photosynthesis. After a dark period of 40 sec (at +10° and +25°C), however, ¹⁴C incorporation into malate and aspartate was rather low at the beginning of illumination; moreover, the intermediates of the Calvin cycle appeared earlier and were more strongly labelled after this short dark period. The results are discussed with reference to the influence of intermediates on the formation of the transient changes of CO₂ uptake in Chlorella.

     

The effect of temperature on the uptake and loss of anions by seedling roots ofZea mays L

Intact maize seedlings were examined for the uptake and leakage of labelled sulphate and phosphate anions affected by temperature. Control plants, grown at 25 °C were compared from the aspects of uptake capacity and leakage with plants incubated in nutrient solutions cooled to 15 °C and 5 °C, respectively. Short time intervals as well as 1–7 d exposure to cooling were used. Already after 1 h exposure at 5 °C and 5 h cooling at 15 °C and at 5 °C, considerable changes were manifested in anion uptake and leakage. The uptake of³²P declined more than that of³⁵S. So, after a 30 min uptake interval the uptake of³⁵S decreased at 15 °C to 49.84% and at 5 °C to 6.05% comparing with the uptake at 25 °C, while the uptake of³²P declined to 28.64% at 15 °C and to 4.45% at 5 °C. The leakage of both anions was the highest at 25 °C in absolute rates, but relatively most of the uptaken³⁵S and³²P was released at 5 °C. Longer exposure to a chilling temperature of 5 °C (1–7 days) resulted in two patterns of sulphate and phosphate uptake.     

Einfluß der Temperatur auf die Lichtatmung der Blaualge Anacystis nidulans

Summary CO₂ exchange, ¹⁴CO₂ fixation and ¹⁴C-products of Anacystis nidulans (strain L 1402-1) were studied during the induction period at temperatures of +15°C and+35°C. At+15°C the stationary rates of CO₂ uptake and respiration were reached directly. At+35°C a maximum of CO₂ uptake could be observed at the beginning of the illumination period followed by a lower steady rate of photosynthesis. In the following dark period a CO₂ gush appeared at+35°C. The magnitude of the CO₂ outburst is relatively independent of the photosyntbetic period. The autoradiographic studies showed that the Calvin cycle is the main carboxylation pathway in Anucystis. At a temperature of +35°C serine was labelled after 20 sec of photosynthesis. At+15°C, on the other hand, a low radio-activity appeared in serine after 5 min of photosynthesis. The results show that photorespiration of Anacystis is stimulated by high temperatures.     

Effects of acute and chronic toluene inhalation on behavior and (3H)-serotonin binding in rat

Toluene inhalation (0.7 % in air) induced in rats abnormal neurological states resembling the serotonin syndrome, such as hindlimb abduction, resting tremor and head weaving. The frequency and intensity of these responses were unchanged after two weeks of exposure (0.7 % in air, 15 min/day for 14 days), indicating an absence of tolerance development. An examination of specific serotonin (³H-5HT) binding to crude synaptic membranes prepared from brains of rats subjected to acute and chronic toluene exposure revealed that while no changes in either apparent Kd or apparent Bmax occured in acutely exposed animals, in chronically treated animals specific (³H)-5HT binding decreased in hippocampus and pons + medulla oblongata. These results indicate that serotonergic mechanisms may play a role in some of the effects of toluene inhalation in rats, but cannot explain the absence of tolerance development after chronic exposure to toluene.