Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

A field study on the influence of food and immune priming on a bumblebee–gut parasite system

Laboratory experiments are often preferred over field experiments because they allow the control of confounding factors that would otherwise influence the causal effect of a particular focal experimental factor. These confounding factors can, however, significantly alter the response of an organism confronted with a particular situation, which can have great implications. In a field experiment with a bumblebee host–parasite system, we looked at the influence of additional food supply and immune challenge on various colony fitness values and parasite traits. We could confirm the importance of food on the colony fitness, but not on parasite infection probability or parasite genetic diversity. In contrast to the findings of laboratory experiments of this system, challenge of the immune system had no significant effect on colony fitness or parasite infections. These results likely reflect an overriding effect of environmental variation without disproving the concept of a cost of defence per se. But the results also demonstrate that confounding factors purposely controlled for in the laboratory have to be weighed against their ecological relevance, and stress the need for careful analysis before any direct transfer is made of laboratory results to field situations.     

The effects of γ-irradiation on the priming by deoxyribonucleohistone of ribonucleic acid polymerase

1. The effect of gamma-irradiation of solutions of DNA and deoxyribonucleohistone (DNH) on their ability to prime the synthesis of RNA by DNA-dependent RNA polymerase has been studied. 2. The priming ability of both DNA and DNH decreased continuously with increasing radiation dose, but more rapidly with DNH. 3. These decreases have been compared with decreases in molecular weight and with the breakdown of the specific hydrogen-bonded structure of DNA. 4. It is concluded that a process was occurring during gamma-irradiation of DNH that, although involving a decrease in molecular weight, did not diminish and even enhanced its priming ability. This is consistent with previous physicochemical evidence that gamma-irradiation causes dissociation of histone from DNH.     

Effect of culture conditions on mycelial growth and production of γ-linolenic acid by the fungus Mortierella ramanniana

Summary Effect of culture conditions on cell growth, lipid accumulation and -linolenic acid production is reported for four Mortierella species. The highest concentration as well as the highest productivity of -linolenic acid in lipid was determined in strains of M. ramanniana. M. ramanniana CBS 112.08 was used in the studies of the influence of medium composition, concentration of carbon- and nitrogen sources and growth temperature. Several carbon sources provided good growth and a high lipid content in biomass. The highest dry weights (11–12g/l) and lipid contents (24%, w/w), were observed if glucose or fructose was used as carbon source, whereas the highest amount of -linolenic acid (26%) was determined in starch-grown cells. The fatty acid composition in the lipid was influenced by the cultivation time, growth temperature and, to a minor extent, by the carbon source used. In fermentor cultures, both strains of Mortierella ramanniana showed relatively poor growth and incomplete consumption of glucose. M. vinacea, on the other hand, grew well in tower reactors. M. vinacea, which has a different morphology than M. ramanniana strains, also showed higher yields of biomass and lipid and higher yield coefficients than the latter.     

Selective oxidation of imidazole ring in histidine residues by the ascorbic acid — copper ion system

In connection with the physiological actions of active oxygen species on proteins, oxidative modification of histidine residues by the autoxidation of ascorbic acid was determined and the main oxidized compound was identified. Oxidation of imidazole ring by the ascorbic acid — copper ion system was considerably site-specific and assumed to be initiated by the addition of the hydroxyl radical (·OH) at C-2 position in the imidazole ring.     

Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel

A newly isolated strain of Cunninghamella echinulata grown on glucose produced significant quantities of biomass and cellular lipids in media with high C/N ratio. The oil yield from glucose consumed increased after nitrogen exhaustion in the growth medium, but gamma-linolenic acid (GLA) content in cellular oil systematically decreased during the lipid accumulation process. When lipid accumulation was completed, GLA concentration in the cellular lipids progressively increased. The highest GLA production (720 mg/l) was achieved in medium with a C/N ratio equal to 163. C. echinulata was also able to grow on orange peel. The C/N ratio in the orange peel decreased from 50 to 26 during solid-state fermentation. Maximum oxygen uptake was observed during assimilation of reducing sugars, whereas a polygalacturonase activity was detected after reducing sugars had been exhausted. The maximum GLA production was 1.2-1.5 mg/g of fermented peel, calculated on a dry weight basis. After enrichment of the pulp with inorganic nitrogen and glucose, an increase in the production of oil and GLA was observed.     

A comparative study of the effect of 17β-estradiol and estriol on peripheral pain behavior in rats

Although estradiol has been reported to influence pain sensitivity, the role of estriol (an estradiol metabolite and another widely used female sex hormone) remains unclear. In this study, pain behavior tests, whole-cell patch clamp recording and Western blotting were used to determine whether estriol plays a role in pain signal transduction and transmission. Either systemic or local administration of 17β-estradiol produced a significant rise of mechanical pain threshold, while estriol lacked this effect in normal and ovariectomized (OVX) rats following estriol replacement. Local administration of 17β-estradiol or estriol significantly decreased ATP-induced spontaneous hind-paw withdrawal duration (PWD), which was blocked by an estrogen receptor antagonist, ICI 182, 780. However, systemic application of estriol in normal or OVX rats lacked this similar effect. In cultured dorsal root ganglion neurons, estriol attenuated α,β-methylene ATP-induced transient currents which were blocked by ICI 182, 780. In complete Freund's adjuvant treated (CFA) rats, systemic application of 17β-estradiol or estriol decreased the mechanical pain threshold significantly, but did not change the inflammatory process. Similar effects were observed after estriol replacement in OVX rats. The expression of c-fos in lumbosacral spinal cord dorsal horn (SCDH) was increased significantly by administration of 17β-estradiol but not estriol, and not by estriol replacement in OVX rats. These results suggest that 17β-estradiol but not estriol plays an anti-hyperalgesic role in physiological pain. However, both peripheral 17β-estradiol and estriol play anti-hyperalgesic roles in ATP-induced inflammatory pain. Systemic application of estriol as well as 17β-estradiol plays hyperalgesic roles in CFA-induced chronic pain.     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Transferrin-receptor interaction and iron uptake by reticulocytes of vertebrate animals — a comparative study

Summary Transferrin-receptor interactions and iron uptake were studied in eleven different species of vertebrate animals (3 eutherian mammals, 3 marsupials, 2 reptiles and 1 bird, amphibian and bony fish). In the initial experiments it was shown that the uptake of transferrin-bound iron by immature erythroid cells from marsupial and reptilian species occurs by receptor-mediated endocytosis as in other vertebrate animals.Reticulocytes were incubated with¹²⁵I-⁵⁹Fe-labelled transferrins from heterologous species and the results for iron and transferrin uptake compared with those obtained with the homologous protein. Cells from eutherian mammals were able to take up transferrin and iron from other eutherians and from the bob-tailed lizard but not from marsupials and other submammalian species. With marsupials and reptiles a similar specificity was observed, and the marsupial cells could also utilize chicken transferrin but not vice versa.The results were extended by performing competition experiments in which the cells were incubated with radiolabelled homologous transferrin in the presence of increasing concentrations of non-radioactive heterologous transferrins. From the ability of the heterologous proteins to inhibit uptake of the homologous protein relative association constants (K _a ¹) for the transferrin-receptor interactions could be calculated. TheseK _a ¹ values reflected the patterns observed in the first series of experiments.These studies demonstrate that, although specificity exists in transferrin-receptor interactions throughout the range of vertebrate animals, in several instances reactivity between widely divergent species is also observed. Hence, structural similarities have been maintained throughout evolution. Nevertheless, no evidence of interaction between transferrin and its receptor from the two divisions of the Mammalia, the eutherians and the marsupials, was observed.Abbreviations BSS Hanks balanced salt solution - PBS phosphate-buffered saline - RRS Rana Ringer solution     

Effect of cadmium on physiological parameters of cereal and millet plants—A comparative study

Metal load is an abiotic stress that becomes stronger by continual industrial production, wastage, and long-range transport of contaminants. It deteriorates the conditions of agricultural soil that leads to lower growth of cereals as well as decreasing nutritional value of harvested grains. Cadmium (Cd) entry by food chain also affects the health of population. The present study is focused on finding out the superior cereal variety under increasing Cd regime. The plants were grown in increasing Cd levels (0–1000 µM) in the medium and were investigated on 15th day of the exposure. Various parameters like antioxidative enzymes and osmoprotectant levels were studied in both roots and shoots. Cd accumulation in plant organs was determined by atomic absorption spectrophotometry (AAS). Analysis of stress tolerance mechanisms through reactive oxygen species (ROS) scavenging and better partitioning of Cd in roots indicated kodo millet to be more stress tolerant than wheat.     

Improvement of γ-decalactone production by stimulating the import of ricinoleic acid and suppressing the degradation of γ-decalactone in Saccharomyces cerevisiae

A number of yeast species can transform ricinoleic acid into γ-decalactone, a high-value compound with fruity aroma, through β-oxidation. This study investigated the effect of l-carnitine on γ-decalactone production by Saccharomyces cerevisiae MF013 to increase the β-oxidation rate. Results showed that l-carnitine shortened the biotransformation period by approximately 10?h and increased γ-decalactone production by 19.5%. γ-Caprolactone, γ-octalactone, and γ-dodecalactone were separately added to the medium to prevent γ-decalactone degradation by yeast cells at the end of biotransformation. γ-Octalactone competitively inhibited γ-decalactone from binding to lactonase, resulting in an 11% increase in γ-decalactone production. This research proposed an effective approach to improve the γ-decalactone production rate, shorten the biotransformation period, and suppress the γ-decalactone degradation in S. cerevisiae.     

Control of the citric acid cycle by glyoxylate: Mechanism of the inhibition by oxalomalate and γ-hydroxy-α-oxoglutarate

1. Hydroxyoxoglutarate was obtained by three methods: decarboxylation of oxalomalic acid, and synthesis from glyoxylate and pyruvate by using either Mg²⁺ or an enzyme from rat liver as catalysts. 2. The inhibitory effects of oxalomalate and hydroxyoxoglutarate upon aconitate hydratase, isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase were investigated. 3. Oxalomalate at low concentrations (1mm) inhibited almost completely both aconitate hydratase and isocitrate dehydrogenase. Hydroxyoxoglutarate also inhibited these enzymes, but at concentrations approximately tenfold that of oxalomalate. 4. Oxalomalate and hydroxyoxoglutarate, at the higher concentrations, inhibited oxoglutarate dehydrogenase to approximately the same extent. 5. It is suggested that the ability of glyoxylate to control reaction rates in the tricarboxylic acid cycle must in some degree be due to its condensation with oxaloacetate and pyruvate to form enzyme inhibitors.     

Implementation of a dose–response curve for γ-radiation in the Portuguese population by use of the chromosomal aberration assay

An in vitro dose–response curve following exposure to γ-radiation was determined at the IST/ITN, by use of the chromosomal aberration assay. This is the first study of this kind carried out among the Portuguese population. Un-irradiated and γ-irradiated peripheral blood lymphocytes from 16 healthy donors were cultured. A total of 22,395 metaphases were analyzed for frequency and distribution of dicentrics and centric rings, as a function of the radiation dose. The dose–response data for dicentrics and dicentrics plus centric rings were fitted by use of a linear–quadratic model: Y_dic = (0.0011 ± 0.0006) + (0.0105 ± 0.0035)D + (0.0480 ± 0.0019)D² and Y_{dic + rings} = (0.0011 ± 0.0006) + (0.0095 ± 0.0036)D + (0.0536 ± 0.0020)D². Also, calibration curves related to age and gender were determined, but no significant differences were found. Following the establishment of the dose–response curves, a validation experiment was carried out with three individuals. Real and estimated doses, obtained with the dose–response curves, were in agreement. These results give us confidence to apply both dose–response calibration curves in future biological dosimetry requirements.     

A study of the metabolism of l-αγ-diaminobutyric acid in a Xanthomonas species

1. l-αγ-Diaminobutyric acid is metabolized in Xanthomonas sp. to aspartic β-semialdehyde, aspartic acid and oxaloacetic acid. 2. Aspartic β-semialdehyde is formed from diaminobutyric acid by a pyruvate-dependent γ-transamination. 3. The transaminase has a pH optimum of 9 and exhibits a high degree of substrate specificity, as analogues of diaminobutyric acid and pyruvate are inert in the system. The transaminase is inhibited by carbonyl-binding agents such as hydroxylamine. 4. Aspartic acid is formed from aspartic β-semialdehyde by an NAD⁺-dependent dehydrogenation. 5. The dehydrogenase has a pH optimum of 8·5 and is a thiol enzyme. It is specific for aspartic β-semialdehyde but analogues of NAD⁺ such as 3-acetylpyridine–adenine dinucleotide and deamino-NAD are partly active in the system. 6. The significance of these reactions is discussed in relation to diaminobutyric acid metabolism in plants and mammalian systems.     

Electrophysiological characterization of ivermectin triple actions on Musca chloride channels gated by l-glutamic acid and γ-aminobutyric acid

Ivermectin (IVM) is a macrocyclic lactone that exerts antifilarial, antiparasitic, and insecticidal effects on nematodes and insects by acting on l-glutamic acid-gated chloride channels (GluCls). IVM also acts as an allosteric modulator of various other ion channels. Although the IVM binding site in the Caenorhabditis elegans GluCl was identified by X-ray crystallographic analysis, the mechanism of action of IVM in insects is not well defined. We therefore examined the action of IVM on the housefly (Musca domestica) GluCl and γ-aminobutyric acid (GABA)-gated ion channel (GABACl). For both channels, IVM induced currents by itself, potentiated currents induced by low concentrations of agonists, and inhibited currents induced by high concentrations of agonists. Despite exerting common actions on both types of channels, GluCls were more susceptible to IVM actions than GABACls, indicating that GluCls are the primary target of IVM. Substitution of an amino acid residue in the third transmembrane segment (G312M in GluCls, and G333A and G333M in GABACls) resulted in significantly reduced levels or loss of activation, potentiation, and antagonism of the channels, indicating that these three actions result from the interaction of IVM with amino acid residues in the transmembrane intersubunit crevice.