Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

Metabolism of α-aminoisobutyric acid in mungbean hypocotyls in relation to metabolism of 1-aminocyclopropane-1-carboxylic acid

1-Aminocyclopropane-1-carboxylic acid (ACC) is known to be converted to ethylene and conjugated into N-malonyl-ACC in plant tissues. When -amino[1-¹⁴C]isobutyric acid (AIB), a structural analog of ACC, was administered to mungbean (Vigna radiata L.) hypocotyl segments, it was metabolized to ¹⁴CO₂ and conjugated to N-malonyl-AIB (MAIB). -Aminoisobutyric acid inhibited the conversion of ACC to ethylene and also inhibited, to a lesser extent, N-malonylation of ACC and d-amino acids. Although the malonylation of AIB was strongly inhibited by ACC as well as by d-amino acids, the metabolism of AIB to CO₂ was inhibited only by ACC but not by d-amino acids. Inhibitors of ACC conversion to ethylene such as anaerobiosis, 2,4-dinitrophenol and Co²⁺, similarly inhibited the conversion of AIB to CO₂. These results indicate that the malonyalation of AIB to MAIB is intimately related to the malonylation of ACC and d-amino acids, whereas oxidative decarboxylation of AIB is related to the oxidative degradation of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AIB -aminoisobutyric acid - MACC 1-(malonylamino)-cyclopropane-1-carboxylic acid - MAIB -(malonylamino)-isobutyric acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Conversion of 4-aminobutyraldehyde to γ-aminobutyric acid in striatum treated with semicarbazide and kainic acid

4-Aminobutyraldehyde (ABAL) has been shown to cross the blood-brain barrier and to be converted rapidly to -aminobutyric acid (GABA) in various regions of the brain. In this paper, the formation of GABA from ABAL was studied with striatum that had suffered a lesion to GABA synthesis via glutamic acid decarboxylase (GAD). The GABA formation from ABAL was invariably observed in striatum in which GAD was severely inhibited by semicarbazide or kainic acid. Thus, this is another pathway for GABA formation.     

Overexpression of γ－aminobutyric acid transporter subtype I leads to susceptibility to kainic acid－induced seizure in transgenic mice

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter, and the GABAergic synaptic transmission is normally terminated by the rapid uptake through GABA transporters. With transgenic mice ubiquitously overexpressing GABA transporter subtype I (GAT1), the present study explored the pathophysiological role of GAT1 in epileptogenesis. Though displaying no spontaneous seizure activity, these mice exhibit altered electroencephalographic patterns and increased susceptibility to seizure induced by kainic acid. In addition, the GABA(A) receptor and glutamate transporters are up-regulated in transgenic mice, which perhaps reflects a compensatory or corrective change to the elevated level of GAT1. These preliminary findings support the hypothesis that excitatory and inhibitory neurotransmission, and seizure susceptibility can be altered by neurotransmitter transporters.     

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.     

Plant improvement for tolerance to aluminum in acid soils – a review

Development of acid soils that limit crop production is an increasing problem worldwide. Many factors contribute to phytotoxicity of these soils, however, in acid soils with a high mineral content, aluminum (Al) is the major cause of toxicity. The target of Al toxicity is the root tip, in which Al exposure causes inhibition of cell elongation and cell division, leading to root stunting accompanied by reduced water and nutrient uptake. Natural variation for Al tolerance has been identified in many crop species and in some crops tolerance to Al has been introduced into productive, well-adapted varieties. Aluminum tolerance appears to be a complex multigenic trait. Selection methodology remains a limiting factor in variety development as all methods have particular drawbacks. Molecular markers have been associated with Al tolerance genes or quantitative trait loci in Arabidopsis and in several crops, which should facilitate development of additional tolerant varieties. A variety of genes have been identified that are induced or repressed upon Al exposure. Most induced genes characterized so far are not specific to Al exposure but are also induced by other stress conditions. Ectopic over-expression of some of these genes has resulted in enhanced Al tolerance. Additionally, expression of genes involved in organic acid synthesis has resulted in enhanced production of organic acids and an associated increase in Al tolerance. This review summarizes the three main approaches that have been taken to develop crops with Al tolerance: recurrent selection and breeding, development of Al tolerant somaclonal variants and ectopic expression of transgenes to reduce Al uptake or limit damage to cells by Al.     

Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.     

β-nitropropionic acid and nitrite in relation to nitrate formation by Aspergillus flavus

Summary -nitropropionic acid (BNP) was converted to nitrate in media inoculated with A. flavus spores or with replacement cultures of mycelium pregrown in glucose-peptone medium. Conversion by replacement cultures was rapid: 8–30% in 2 days; influenced by pH: most rapid at pH 3.5; and extensive: as much as 80% BNP nitrogen appeared as nitrate after 14 days. Nitrite was detectable in BNP replacement cultures at low levels or not at all, and nitrate was formed in BNP replacement media with or without glucose. Nitrite was not oxidized in growing cultures inoculated with spores, but replacement cultures oxidized over 50% of added nitrite to nitrate in 8 days. No nitrite or nitrate appeared in replacement systems with pyruvic oxime, oxalacetic acid oxime, acetoxime, ketoglutaric acid oxime, or hydroxylamine.Of the three non-nitrifying mutants of A. flavus obtained, all formed nitrate from BNP in replacement but only one oxidized nitrite to nitrate. No accumulation of free or bound hydroxylamine or of nitrite could be detected in the mutants. BNP was detected by qualitative test in cultures of the wild type but not the mutants. Evidence indicates that the pathway in A. flavus is BNPNO₃ ^- rather than BNPNO₂ ^-NO₃ ^-.     

Glutathione-mediated response to acid stress in the probiotic bacterium, Lactobacillus salivarius

Lactobacillus salivarius, a probiotic bacterium, encounters acidic conditions in its passage through the gastrointestinal tract of human and animal hosts. We studied the effect of a rapid downshift in extracellular pH from 6.5 to 4 on cell growth. The maximum growth rate was higher in low pH medium with glutathione supplementation than without. Cells developed a GSH-mediated acid-tolerance response and, when grown with 0.5 mM GSH, reached a higher final density than with other conditions. These findings suggest that the increased growth rate is caused by uptake of GSH which acts as a nutrient source as well as having protective functions, allowing for continued growth.     

Circadian dysfunction in response to in?vivo treatment with the mitochondrial toxin 3-nitropropionic acid

Sleep disorders are common in neurodegenerative diseases including Huntington''s disease (HD) and develop early in the disease process. Mitochondrial alterations are believed to play a critical role in the pathophysiology of neurodegenerative diseases. In the present study, we evaluated the circadian system of mice after inhibiting mitochondrial complex II of the respiratory chain with the toxin 3-nitropropionic acid (3-NP). We found that a subset of mice treated with low doses of 3-NP exhibited severe circadian deficit in behavior. The temporal patterning of sleep behavior is also disrupted in some mice with evidence of difficulty in the initiation of sleep behavior. Using the open field test during the normal sleep phase, we found that the 3-NP-treated mice were hyperactive. The molecular clockwork responsible for the generation of circadian rhythms as measured by PER2::LUCIFERASE was disrupted in a subset of mice. Within the SCN, the 3-NP treatment resulted in a reduction in daytime firing rate in the subset of mice which had a behavioral deficit. Anatomically, we confirmed that all of the treated mice showed evidence for cell loss within the striatum but we did not see evidence for gross SCN pathology. Together, the data demonstrates that chronic treatment with low doses of the mitochondrial toxin 3-NP produced circadian deficits in a subset of treated mice. This work does raise the possibility that the neural damage produced by mitochondrial dysfunction can contribute to the sleep/circadian dysfunction seen so commonly in neurodegenerative diseases.     

β-Hydroxymyristic acid as a chemical marker to detect endotoxins in dialysis water

An analytical chemical method has been developed for determination of β-hydroxymyristic acid (β-HMA), a component of lipopolysaccharides (LPSs/endotoxins) in dialysis water. In our investigation, the β-HMA component was used as a chemical marker for endotoxin presence in dialysis water because it is available in the molecular subunit (lipid A) and responsible for toxicity. It is the most abundant saturated fatty acid in that subunit. The developed method is based on fluorescence derivatization with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD–PZ). A high-performance liquid chromatographic separation of the β-HMA derivative was achieved using an octadecyl silica column in gradient elution. A wide dynamic range of β-HMA was tested and a calibration curve was constructed with accuracy of 90% and variability of less than 10%. The limits of detection and quantification obtained were 2 and 5 μM, respectively. The developed method was applied to detect endotoxins in dialysis water by alkaline hydrolysis of LPS using NaOH (0.25 M) at 60 °C for 2 h. After hydrolysis, free acid was detected as its NBD–PZ derivative using high-performance liquid chromatography/mass spectrometry (HPLC/MS). Good recovery rates ranging from 98 to 105% were obtained for β-HMA in dialysis water.     

Overexpression of γ-aminobutyric acid transporter subtype I leads to susceptibility to Kainic acid-induced seizure in transgenic mice

INTroDUCTION.7-aminobutyric acid (GABA), a major in-hibitory neurotransmitter in vertebrate brain, in--duces neuronal inhibition via GABA receptors. Adeficiency of GABAergic inhibition has been hy-pothesized to be a principal factor in the patho-genesis of epilepsy[1]. GABA transporters are im-portant components of the GABAergic system, andfunction in part to terminate the GABA transmis-sion through rapid re-uptake of GABA into thepresynaptic neurons and surrounding gliaJ cells[2…     

Oleic acid activates peroxisome proliferator-activated receptor δ to compensate insulin resistance in steatotic cells

Nonalcoholic fatty liver disease is frequently associated with type 2 diabetes; however, this idea is challenged by recent studies because hepatic steatosis is not always associated with insulin resistance (IR). Oleic acid (OA) is known to induce hepatic steatosis with normal insulin sensitivity; however, the mechanism is still unknown. Previous studies depict that activation of peroxisome proliferator-activated receptor δ (PPARδ?) improves hepatic steatosis and IR, whereas the role of PPARδ in the improvement of insulin sensitivity by OA is unknown. Here we induced steatosis in HepG2 cells by incubation with OA and OA significantly increased the expression of PPARδ through a calcium-dependent pathway. OA also induced the expression of G protein-coupled receptor 40 (GPR40), and deletion of GPR40 by small interfering ribonucleic acid transfection partially reversed the effect of OA on PPARδ?. Inhibition of phospholipase C (PLC) by U73122 also reversed OA-induced PPARδ expression. Otherwise, deletion of PPARδ augmented the OA-induced steatosis in HepG2 cells. Furthermore, IR was developed in OA-treated HepG2 cells with PPARδ deletion, while insulin-related signals and insulin-stimulated glycogen synthesis were reduced through increase of phosphatase and tensin homolog (PTEN) expression. In conclusion, OA activates GPR40-PLC-calcium pathway to increase the expression of PPARδ and PPARδ further decreased the expression of PTEN to regulate insulin sensitivity in hepatic steatosis.     

Is adamantane a suitable substituent to pre-organize the acid orientation in E-selectin antagonists?

The selectins play a key role in the inflammatory process, that is, the recruitment of leukocytes from blood vessels into inflamed tissue. Because excessive infiltration of leukocytes can induce acute or chronic reactions, the control of leukocyte extravasation is of great pharmaceutical interest. All physiological ligands of the selectins contain the tetrasaccharide epitope sialyl Lewis(x), which therefore became the lead structure in selectin antagonist research. Previous studies indicated that an important factor for the affinity of sLe(x) is the fact that in solution its pharmacophores are already conformationally pre-organized in the bioactive orientation. In mimics where the GlcNAc- and the NeuNAc-moieties of sLe(x) were replaced by (R,R)-cyclohexane-1,2-diol and (S)-cyclohexyllactic acid, respectively, an optimized pre-organization of the pharmacophores could be realized, leading to antagonists with improved affinities. To further optimize the pre-organization of the carboxylic acid, a pharmacophore essential for binding, the replacement of NeuNAc by bulky (R)- and (S)-adamantyl-lactic acid was studied. Although antagonist (S)-7 showed a slightly reduced affinity, the expected beneficial effect of the (S)-configuration at C-2 of the lactate could be confirmed.     

Ribonucleic acid–deoxyribonucleic acid hybridization in aqueous solutions and in solutions containing formamide

Hybridization in 6xSSC (SSC, 0.15m-sodium chloride-0.015m-sodium citrate) at 66 degrees C was compared with hybridization in formamide-6xSSC (1:1, v/v) at 35 degrees C. As expected, the RNA hybridization potential was labile in the former system and stable in the latter. DNA retention by filters was poor in the formamide system, but could be improved. Several other properties of the hybridization reaction were explored and it was concluded that the formamide system is generally superior.     

The synthesis of ribonucleic acid in immature rat uterus responding to oestradiol-17β

Stimulation of incorporation of labelled precursors into the RNA of immature rat uterus is an early result of oestradiol-17beta action. However, the extent of the increased incorporation varies with the mode of administration of the labelled precursors and with the weight of the rat. At the age and weight range normally used response is maximal at ten times control incorporation, 4h after the administration of 0.3mug or more of oestradiol-17beta. Under these conditions the stimulation of incorporation into the acid-soluble fraction is only 2-2.5-fold. When the purified RNA is separated on polyacrylamide gels the major increase in incorporation of radioactive precursor is found in rRNA and 4S RNA; the formation of the former has been followed from the 45S precursor. Preceding these events by at least 30min, however, is an increase in the incorporation of precursor into RNA species of very high molecular weight, which remained in the first few slices of the gel. The possible significance of these findings is discussed. The increased synthesis of rRNA in response to oestradiol-17beta is more strongly inhibited by actinomycin D than the synthesis of other RNA species. Cycloheximide, depending on time of administration and dosage, inhibits either RNA synthesis or the maturation of rRNA.     

Does engineering abscisic acid biosynthesis in Nicotiana plumbaginifolia modify stomatal response to drought?

The consequences of manipulating abscisic acid (ABA) biosynthesis rates on stomatal response to drought were analysed in wild‐type, a full‐deficient mutant and four under‐producing transgenic lines of N. plumbaginifolia. The roles of ABA, xylem sap pH and leaf water potential were investigated under four experimental conditions: feeding detached leaves with varying ABA concentration; injecting exogenous ABA into well‐watered plants; and withholding irrigation on pot‐grown plants, either intact or grafted onto tobacco. Changes in ABA synthesis abilities among lines did not affect stomatal sensitivity to ABA concentration in the leaf xylem sap ([ABA]_xyl), as evidenced with exogenous ABA supplies and natural increases of [ABA]_xyl in grafted plants subjected to drought. The ABA‐deficient mutant, which is uncultivable under normal evaporative demand, was grafted onto tobacco stock and then presented the same stomatal response to [ABA]_xyl as wild‐type and other lines. This reinforces the dominant role of ABA in controlling stomatal response to drought in N. plumbaginifolia whereas roles of leaf water potential and xylem sap pH were excluded under all studied conditions. However, when plants were submitted to soil drying onto their own roots, stomatal response to [ABA]_xyl slightly differed among lines. It is suggested, consistently with all the results, that an additional root signal of soil drying modulates stomatal response to [ABA]_xyl.     

Abcb11 deficiency induces cholestasis coupled to impaired β-fatty acid oxidation in mice

The bile salt export pump (BSEP) is an ATP-binding cassette transporter that serves as the primary system for removing bile salts from the liver. In humans, deficiency of BSEP, which is encoded by the ABCB11 gene, causes severe progressive cholestatic liver disease from early infancy. In previous studies of Abcb11 deficiency in mice generated on a mixed genetic background, the animals did not recapitulate the human disease. We reasoned that ABCB11 deficiency may cause unique changes in hepatic metabolism that are predictive of liver injury. To test this possibility, we first determined that Abcb11 knock-out (KO) C57BL/6J mice recapitulate human deficiency. Before the onset of cholestasis, Abcb11 KO mice have altered hepatic lipid metabolism coupled with reduced expression of genes important in mitochondrial fatty acid oxidation. This was associated with increased serum free-fatty acids, reduced total white adipose, and marked impairment of long-chain fatty acid β-oxidation. Importantly, metabolomic analysis confirmed that Abcb11 KO mice have impaired mitochondrial fatty acid β-oxidation with the elevated fatty acid metabolites phenylpropionylglycine and phenylacetylglycine. These metabolic changes precede cholestasis but may be of relevance to cholestatic disease progression because altered fatty acid metabolism can enhance reactive oxygen species that might exacerbate cholestatic liver damage.