Administration of isothiocyanates enhances heat tolerance in Arabidopsis thaliana

Although it has been documented that plants generate isothiocyanates (ITCs) through the glucosinolate-myrosinase system to defend against biotic stresses, the roles of ITCs in defending against abiotic stresses have scarcely been studied. Here, we report that exogenously applied ITCs enhance the heat tolerance of Arabidopsis thaliana. Pre-administration of phenethyl ITC to Arabidopsis plants mitigated growth inhibition after heat stress at 55?°C for 1?h. Although methyl ITC and allyl ITC also tended to reduce the growth inhibition that the same heat treatment caused, the reduction effects were weaker. The expression levels of heat shock protein 70 genes in Arabidopsis were elevated after phenethyl ITC treatment. These results suggest that ITCs may act as heat-tolerance enhancers in plants.     

Regional distribution study of brain perfusion and metabolic agents in normal and tumour bearing rat brains using autoradiographic technique

Regional distribution of brain perfusion imaging agents, [¹³¹I]N,N,N′-trimethyl-N′-[2-hydroxy-3-methyl-5-iodobenzyl]1,3 propanediamine (HIPDM) and [¹³¹I]N-isopropyl-p-iodoamphetamine (IMP), was compared with the distribution of patterns of [¹⁴C]l-methionine and [¹⁴C]d-glucose in normal and tumour bearing rat brains using autoradiographic technique. There was higher concentration of the radiopharmaceutical in grey than white matter in normal rat brain. Autoradiographs of brain tumour sections showed very low uptake of [¹³¹I]HIPDM and [¹³¹I]IMP as compared to normal brain tissue. There was moderate concentration of [¹⁴C]d-glucose and avid uptake of [¹⁴C]l-methionine in tumours. Autoradiographic study is useful for evaluating distribution patterns of radiopharmaceuticals.     

Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells

To characterize the function of the sodium/inositol symporter SMIT2 in skeletal muscle, human SMIT2 cDNA was transfected into L6 myoblasts using pcDNA3.1 expression vector. Compared with the pcDNA3.1 vector only transfection, this overexpression increased the uptake of [³H]d-chiro-inositol (DCI) by 159-fold. [³H]myo-Inositol uptake increased by 37-fold. In contrast, [¹⁴C]d-glucose, [¹⁴C]2-deoxy-d-glucose, or [¹⁴C]3-O-methyl-d-glucose uptake remained unchanged in the presence of either 0, 5.5, or 25 mM unlabeled glucose. The K_m of DCI and myo-inositol for DCI uptake was 111.0 and 158.0 μM, respectively, whereas glucose competed for DCI uptake with a K_i of 6.1 mM. Insulin treatment of non-transfected L6 cells (2 μM for 24 h) increased [³H]DCI specific uptake 18-fold. DCI transport is up regulated by insulin and competitively inhibited by millimolar levels of glucose. Therefore, expression and/or function of SMIT2, a high affinity transporter specific for DCI and myo-inositol, may be reduced in diabetes mellitus, insulin resistance and polycystic ovary syndrome causing the abnormal DCI metabolism observed in these conditions.     

Reactive oxygen species modulate itraconazole-induced apoptosis via mitochondrial disruption in Candida albicans

Itraconazole (ITC), a well-known fungistatic agent, has potent fungicidal activity against Candida albicans. However, its mechanism of fungicidal activity has not been elucidated yet, and we aimed to identify the mechanism of ITC against C. albicans. ITC caused cell shrinkage via potassium leakage through the ion channel. Since shrunken cells could indicate apoptosis, we investigated apoptotic features. Annexin V-FITC and TUNEL assays indicated that fungicidal activity of ITC was involved in apoptosis. Subsequently, we confirmed an intracellular factor that could cause apoptosis. ITC treatment caused reactive oxygen species (ROS) accumulation. To confirm whether ROS is related with ITC-triggered cell death, cell viability was examined using the ROS scavenger N-acetylcysteine (NAC). NAC pretreatment recovered ITC-induced cell death, indicating that antifungal activity of ITC is associated with ROS, which is also confirmed by impaired glutathione-related antioxidant system and oxidized intracellular lipids. Moreover, ITC-induced mitochondrial dysfunction, in turn, triggered cytochrome c release and metacaspase activation, leading to apoptosis. Unlike the only ITC-treatment group, cells with NAC pretreatment did not show significant damage to mitochondria, and attenuated apoptotic features. Therefore, our results suggest that ITC induces apoptosis as fungicidal mechanism, and intracellular ROS is major factor to trigger the apoptosis by ITC in C. albicans.     

The uptake of organic molecules by the ventral tube of Tomocerus flavescens (Tullberg, 1871) (Insecta: Collembola)

The net influx of water-soluble organic molecules by the ventral tube of Tomocerus flavescens, a soil litter-inhabiting Collembolan, was investigated. The following substances were tested: [¹⁴C]urea, [¹⁴C]glycerol, [¹⁴C]erythritol, [¹⁴C]l-leucine, [¹⁴C]d-glucose, [³H]inulin. The animals were exposed to moist filter paper containing a specific test solution. When they evert their ventral-tube vesicles, they absorb water and solutes through the cuticle and the transport epithelium into the body haemolymph. Contamination by radioactive substances and oral solute uptake was avoided by an experimental device. It is evident that the uptake rates decrease with increasing molecular mass especially in a range of 100–200. Further, the rates correlate with the radius of hydrated molecules and their lipid solubility. Significant differences in urea uptake have been shown for animals less than 10 days in culture (“field” animals) and more than 10 days in culture (“laboratory” animals). Whether changes in cuticle permeability could be affected by abrasion is discussed. There is a high deviation amongst uptake values in all experimental series. It seems probably that, besides individual differences caused by abrasion, the animals differ physiologically, e.g. during the moulting cycle and seasonally. A nutritive function of the ventral tube seems to be unlikely. Calculation reveals that the absorbed glucose provides only 0.013% of the amount the animals need for respiration.     

Metabolism of phenylalanine and tyrosine in tobacco cell lines resistant and sensitive to p-fluorophenylalanine

Chromatography of soluble polyphenols of p-fluorophenylalanine-sensitive and -resistant tobacco cells revealed that the 10-fold increased level found in the resistant line was largely due to the accumulation of two unidentified polyphenols. The uptake of Phe-[U-¹⁴C] and Tyr-[U-¹⁴C] by the resistant line was ca 10 % that by the sensitive line. About 90 % of the phenylalanine-[¹⁴C] which was taken up by both cell lines could be accounted for as free phenylalanine in protein, soluble polyphenols or CO₂. The fate of Tyr-[¹⁴C] was similar to that of phenylalanine except that the incorporation was into insoluble polymeric forms of polyphenols rather than into soluble polyphenols. The resistant line incorporated 9-times more phenylalanine-[¹⁴C] into soluble polyphenols than did the sensitive line. The different ¹⁴C-aromatic amino acid accumulation and incorporation patterns noted with the two cell lines indicates that there are different active pools. Differential uptake rates by the two cell lines might affect the distribution of the absorbed amino acid among the different pools.     

Pathway of Phloem unloading of sucrose in corn roots

The pathway of phloem unloading and the metabolism of translocated sucrose were determined in corn (Zea mays) seedling roots. Several lines of evidence show that exogenous sucrose, unlike translocated sucrose, is hydrolyzed in the apoplast prior to uptake into the root cortical cells. These include (a) presence of cell wall invertase activity which represents 20% of the total tissue activity; (b) similarity in uptake and metabolism of [¹⁴C]sucrose and [¹⁴C]hexoses; and (c) randomization of ¹⁴C within the hexose moieties of intracellular sucrose following accumulation of [¹⁴C] (fructosyl)sucrose. Conversely, translocated sucrose does not undergo apoplastic hydrolysis during unloading. Asymmetrically labeled sucrose ([¹⁴C](fructose)sucrose), translocated from the germinating kernels to the root, remained intact indicating a symplastic pathway for unloading. In addition, isolated root protoplasts and vacuoles were used to demonstrate that soluble invertase activity (V_max = 29 micromoles per milligram protein per hour, K_m = 4 millimolar) was located mainly in the vacuole, suggesting that translocated sucrose entered via the symplasm and was hydrolyzed at the vacuole prior to metabolism.     

Sink Metabolism in Tomato Fruit : II. Phloem Unloading and Sugar Uptake

Analysis of [³H]-(fructosyl)-sucrose translocation in tomato (Lycopersicon esculentum Mill.) indicates that phloem unloading in the fruit occurs, at least in part, to the apoplast followed by extracellular hydrolysis. Apoplastic sucrose, glucose, and fructose concentrations were estimated as 1 to 7, 12 to 49, and 8 to 63 millimolar, respectively in the tomato fruit pericarp tissue. Hexose concentrations were at least four-fold greater than sucrose at all developmental stages. Short-term uptake of [¹⁴C]sucrose, -glucose, and -fructose in tomato pericarp disks showed first order kinetics over the physiologically relevant concentration range. The uptake rate of [¹⁴C]-(glucosyl)-1′-fluorosucrose was identical to the rate of [¹⁴C]sucrose uptake, suggesting sucrose may be taken up directly without prior extracellular hydrolysis. Short-term uptake of all three sugars was insensitive to 10 micromolar carbonyl cyanide m-chlorophenylhydrazone and to 10 micromolar p-chloromercuribenzene sulfonic acid. However, long-term accumulation of glucose was sensitive to carbonyl cyanide m-chlorophenylhydrazone. Together these results suggest that although sucrose is at least partially hydrolyzed in the apoplast, sucrose may enter the metabolic carbohydrate pool directly. In addition, sugar uptake across the plasma membrane does not appear to be energy dependent, suggesting that sugar accumulation in the tomato fruit is driven by subsequent intracellular metabolism and/or active uptake at the tonoplast.     

Metabolic pools associated with monoterpene biosynthesis in higher plants

Partial degradations of (+)-isothujone biosynthesised in Tanacetum vulgare after feeding IPP-[4-¹⁴C], DMAPP-[4-¹⁴C] or 3,3-dimethylacrylate-[Me-¹⁴C], and of geraniol and (+)-pulegone formed in Pelargonium graveolens and Mentha pulegium respectively after uptake of 3,3-dimethylacrylate-[Me-¹⁴C], indicated that none of these metabolites was a direct source of the part of the monoterpene skeleton derived hypothetically from DMAPP. Uptake of glucose-[U¹⁴C] into P. graveolens led, in contrast, to both IPP and DMAPP-derived moieties of geraniol being extensively labelled. Feeding of l-valine-[U-¹⁴C] and l-leucine-[U-¹⁴C] to all three plants resulted in negligible incorporation of tracer into monoterpenes. A soluble enzyme system prepared from foliage of T. vulgare that had been exposed to CO₂-[¹⁴C] for 20 days converted isotopically-normal IPP into GPP with the DMAPP-derived portion containing essentially all (>98%) of the radioactivity present. These observations and those previously obtained from feeding experiments with other [¹⁴C]-labelled precursors on the same plant species are consistent with the occurrence of two metabolic pools of intermediates for monoterpene biosynthesis, one of which is probably protein-bonded.     

Release of soluble factors from lymph nodes containing mycobacterial granulomas and their effect on fibroblast function in vitro

A study has been made of the action of culture supernatants from guinea pig lymph nodes containing mycobacterial granulomas on protein and DNA synthesis of homologous fibroblast cultures. Supernatants from both the Bacillus Calmette-Guerin (BCG) and Mycobacterium leprae granulomas release soluble nondialysable factors in vitro which stimulate [¹⁴C]proline and [¹⁴C]leucine incorporation by fibroblasts and depress their [³H]thymidine uptake. These supernatants did not show any detectable migratory inhibitory activity in vitro. On the other hand, supernatants from sensitized lymphocytes incubated with purified protein derivative (positive migratory inhibitory activity) had no effect on fibroblast function. Thus, the effect of granuloma supernatants is unlikely to be due to lymphokines. However, supernatants from dinitrofluorobenzene-sensitized lymph nodes also showed a stimulation of [¹⁴C]proline incorporation into total protein synthesised by fibroblasts and depressed the [³H]thymidine uptake. Furthermore, supernatants from live BCG organisms in culture on addition to fibroblasts enhanced their [³H]thymidine uptake in vitro. It would appear therefore that fibroblast activation in lymph nodes containing mycobacterial granulomas could result from the release of soluble factors of lymphocyte origin rather than from cells of the mononuclear phagocyte system. These factors appear to be independent of classical lymphokines that act on macrophages in vitro. An additional factor may be derived from the mycobacteria themselves.     

Heterotrophic Uptake Experiments with 14C-Labeled Histidine in a Histidine-Limited Chemostat

The histidine uptake by bacterial strain HIS 42 was determined with [U-¹⁴C]histidine and through oxygen uptake experiments on samples taken from a histidine-limited chemostat. The uptake of [U-¹⁴C]histidine was characterized by a saturation constant of 12.8 to 78.6 nM histidine. At higher growth rates, the measured maximum uptake rate of histidine was lower than the actual uptake rate in the culture. The percentage of respired substrate (76 to 93%) was about 30 to 40% higher than the comparable value for the culture. The uptake of histidine as analyzed through the measurement of oxygen uptake rates was characterized by a saturation constant of 1.7 to 10.5 μM histidine; the maximum uptake rate was always greater than the actual histidine uptake rate in the culture. By the application of the two cited methods, set up to determine the histidine uptake kinetics, two different uptake processes were analyzed. It appeared that the determination of the histidine uptake through measurement of the oxygen uptake rate showed a better reflection of the actual uptake process of histidine in the culture. With the available data it was impossible to assess a correlation between the uptake of histidine, as determined with [U-¹⁴C]histidine, and the actual metabolism of the bacterial population.     

Mechanisms of citrate transport and exchange in corn mitochondria

Previous work (Birnberg, Jayroe, Hanson 1982 Plant Physiol 70: 511-516) demonstrated that corn mitochondria (Zea mays L.) can accumulate citrate by a malate- and phosphate-independent proton symporter. This uptake and symport of other ions were investigated. Passive swelling experiments indicated that corn mitochondria can accumulate several other anions by proton symport, but only isocitrate is taken up nearly as effectively as citrate. At the optimal pH (4.5), active uptake of carrier-free [¹⁴C]citrate in 50 micromolar mersalyl is inhibited by fourteen anions, but only the I₅₀ (the concentration of inhibitor required to reduce uptake of carrier-free [¹⁴C]citrate by 50%) values of citrate (0.08 millimolar) and d-and l-isocitrate (0.5 millimolar) are less than 4 millimolar. Isocitrate is a competitive inhibitor of citrate uptake and [¹⁴C]isocitrate is accumulated with a K_m similar similar to its I₅₀. Valinomycin reduces net active citrate accumulation at pH 7.5, consistent with the relatively low V_max for citrate uptake. At pH 4.5, mersalyl reduces the rate of citrate uptake without changing the affinity of the carrier for citrate. Thus, the corn mitochondria have a high-affinity, mersalyl-insensitive carrier selective for citrate that also transports isocitrate.     

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-d-glucosamine

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[¹⁸F]fluoroacetyl-d-glucosamine (¹⁸F-GlcNFAc). The uptake of ¹⁸F-GlcNFAc was slightly higher (P < 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. ¹⁸F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-d-[l-¹⁴C]glucosamine, 2-deoxy-d-[l-¹⁴C]glucose and [6-³H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.     

Induction of cell DNA replication in G1-specific ts mutants by microinjection of SV40 DNA

Treatment of Xenopus laevis follicles with 50–100 units/ml of human chorionic gonadotropin causes rapid stimulation of [¹⁴C]glucose uptake. Studies with these follicles showed that the stimulation of uptake occurred with a wide range of concentrations of [¹⁴C]glucose or its nonmetabolizable analog [¹⁴C]3-O-methylglucose. Approx. 70% of the glucose taken up in both hormone-treated and control cells becomes incorporated into glycogen within 1 h. The uptake of sugar by these follicles was also stimulated by bovine-luteinizing hormone—but not by folliclestimulating hormone, progesterone or insulin. Human chorionic gonadotropin stimulated sugar uptake by follicles containing medium-sized oocytes (stages 3,4 and 5 according to Dumont) which cannot be induced to undergo meiotic maturation by this hormone. After 4–6 h treatment of fully grown X. laevis follicles with either progesterone or human chorionic gonadotropin, glucose uptake suffers a drastic decrease to below basal levels. This inhibition of uptake is coincident with the breakdown of the germinal vesicle of the oocyte and is clearly related to meiotic maturation, since it is not observed with medium-sized follicles which cannot mature.     

Enhancement in dopamine uptake and release induced by monosodium l-glutamate from caudate nucleus under in vitro conditions

l-Glutamate has an excitatory and cytotoxic effect on the central nervous system. It was shown previously that norepinephrine and dopamine uptake and release were affected by in vivo administration of glutamate to adult rats. The kinetic parameters, K_m and V_max of [¹⁴C]DA uptake and release were measured on synaptosomal and slices from caudate nucleus under in vitro conditions at different glutamate concentrations. Results showed an important increase in [¹⁴C]DA uptake on synaptosomal (> 100%) and slices by lower glutamate concentrations, the affinity for transport system was increased (100%) and its release of high potassium evoked was also increased at 0.5 μM of glutamate. The results suggest the possibility that glutamate may modify DA uptake and release interacting with the DA transporter complex at the synaptic level.     

Characterization of the system L amino acid transporter in T24 human bladder carcinoma cells

System L is a major nutrient transport system responsible for the Na⁺-independent transport of large neutral amino acids including several essential amino acids. In malignant tumors, a system L transporter L-type amino acid transporter 1 (LAT1) is up-regulated to support tumor cell growth. LAT1 is also essential for the permeation of amino acids and amino acid-related drugs through the blood-brain barrier. To search for in vitro assay systems to examine the interaction of chemical compounds with LAT1, we have investigated the expression of system L transporters and the properties of [¹⁴C]l-leucine transport in T24 human bladder carcinoma cells. Northern blot, real-time quantitative PCR and immunofluorescence analyses have reveled that T24 cells express LAT1 in the plasma membrane together with its associating protein 4F2hc, whereas T24 cells do not express the other system L isoform LAT2. The uptake of [¹⁴C]l-leucine by T24 cells is Na⁺-independent and almost completely inhibited by system L selective inhibitor BCH. The profiles of the inhibition of [¹⁴C]l-leucine uptake by amino acids and amino acid-related compounds in T24 cells are comparable with those for the LAT1 expressed in Xenopus oocytes. The majority of [¹⁴C]l-leucine uptake is, therefore, mediated by LAT1 in T24 cells. Consistent with LAT1 in Xenopus oocytes, the efflux of preloaded [¹⁴C]l-leucine is induced by extracellularly applied substrates of LAT1 in T24 cells. This efflux measurement has been proven to be more sensitive than that in Xenopus oocytes, because triiodothyronine, thyroxine and melphalan were able to induce the efflux of preloaded [¹⁴C]l-leucine in T24 cells, which was not detected for Xenopus oocyte expression system. T24 cell is, therefore, proposed to be an excellent tool to examine the interaction of chemical compounds with LAT1.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Distinct patterns of entry of two non-metabolizable amino acids into brain and other organs of infant guinea pigs

Abstract— Entry of [3-¹⁴C] α-aminoisobutyric acid (AIB) and [1-¹⁴C] 1-aminocyclopentanecarboxylic acid (cycloleucine) into the brain and other organs of the infant guinea pig has been investigated in vivo. The entry of [¹⁴C]AIB into brain was markedly restricted in comparison to its entry into other organs. The mean distribution ratio (¹⁴C in tissue water/¹⁴C in plasma water) achieved in brain at 45 min after administration of a pulse of [¹⁴C]AIB was 0.3. All other organs studied concentrated [¹⁴C]AIB from the blood stream, with the greatest uptake occurring in liver and kidney, in which distribution ratios reached values of 5–10. In contrast to AIB, [¹⁴C]cycloleucine entered the brain at a rate approximately the same as that into other organs. Distribution ratios for [¹⁴C]cycloleucine ranged between 0.5 and 2.0 for all organs. During the first few days of postnatal life, there was a sharp increase of concentrative uptake of [¹⁴C]AIB into liver and kidney. The entry of [¹⁴C]AIB into brain remained unchanged during this period. There was a small (35 percent) decrease in the rate of entry of [¹⁴C]cycloleucine into brain during the first 3 days of postnatal life. Since [¹⁴C]AIB is known to be concentrated from the surrounding medium by brain slices in vitro, we concluded that the locus of restriction of the entry of [¹⁴C]AIB into the brain in vivo is at the blood-brain barrier. We hypothesize that this property of the barrier is important in preventing concentrative uptake of pharmacologically active and potentially harmful amino acids by brain tissue.