The Gliopeptide ODN, a Ligand for the Benzodiazepine Site of GABAA Receptors, Boosts Functional Recovery after Stroke.

Following stroke, the survival of neurons and their ability to reestablish connections is critical to functional recovery. This is strongly influenced by the balance between neuronal excitation and inhibition. In the acute phase of experimental stroke, lethal hyperexcitability can be attenuated by positive allosteric modulation of GABAA receptors (GABAARs). Conversely, in the late phase, negative allosteric modulation of GABAAR can correct the suboptimal excitability and improves both sensory and motor recovery. Here, we hypothesized that octadecaneuropeptide (ODN), an endogenous allosteric modulator of the GABAAR synthesized by astrocytes, influences the outcome of ischemic brain tissue and subsequent functional recovery. We show that ODN boosts the excitability of cortical neurons, which makes it deleterious in the acute phase of stroke. However, if delivered after day 3, ODN is safe and improves motor recovery over the following month in two different paradigms of experimental stroke in mice. Furthermore, we bring evidence that, during the subacute period after stroke, the repairing cortex can be treated with ODN by means of a single hydrogel deposit into the stroke cavity.SIGNIFICANCE STATEMENT Stroke remains a devastating clinical challenge because there is no efficient therapy to either minimize neuronal death with neuroprotective drugs or to enhance spontaneous recovery with neurorepair drugs. Around the brain damage, the peri-infarct cortex can be viewed as a reservoir of plasticity. However, the potential of wiring new circuits in these areas is restrained by a chronic excess of GABAergic inhibition. Here we show that an astrocyte-derived peptide, can be used as a delayed treatment, to safely correct cortical excitability and facilitate sensorimotor recovery after stroke.

Glial endozepines and energy balance: Old peptides with new tricks.

The contribution of neuroglial interactions to the regulation of energy balance has gained increasing acceptance in recent years. In this context, endozepines, endogenous analogs of benzodiazepine derived from diazepam-binding inhibitor, are now emerging as major players. Produced by glial cells (astrocytes and tanycytes), endozepines have been known for two decades to exert potent anorexigenic effects by acting at the hypothalamic level. However, it is only recently that their modes of action, including the mechanisms by which they modulate energy metabolism, have begun to be elucidated. The data available today are abundant, significant, and sometimes contradictory, revealing a much more complex regulation than initially expected. Several mechanisms of action of endozepines seem to coexist at the central level, particularly in the hypothalamus. The brainstem has also recently emerged as a potential site of action for endozepines. In addition to their central anorexigenic effects, endozepines may also display peripheral effects promoting orexigenic actions, adding to their complexity and raising yet more questions. In this review, we attempt to provide an overview of our current knowledge in this rapidly evolving field and to pinpoint questions that remain unanswered.

Glial Endozepines Reverse High-Fat Diet-Induced Obesity by Enhancing Hypothalamic Response to Peripheral Leptin.

Research on energy homeostasis has focused on neuronal signaling; however, the role of glial cells has remained little explored. Glial endozepines exert anorexigenic actions by mechanisms which remain poorly understood. In this context, the present study was designed to decipher the mechanisms underlying the anorexigenic action of endozepines and to investigate their potential curative effect on high-fat diet-induced obesity. We carried out a combination of physiological, pharmacological, and molecular analyses together to dissect the underlying mechanisms of endozepine-induced hypophagia. To evaluate the potential anti-obesity effect of endozepines, different model of obesity were used, i.e., ob/ob and diet-induced obese mice. We show that the intracerebral administration of endozepines enhances satiety by targeting anorexigenic brain circuitry and induces STAT3 phosphorylation, a hallmark of leptin signaling. Strikingly, endozepines are entirely ineffective at reducing food intake in the presence of a circulating leptin antagonist and in leptin-deficient mice (ob/ob) but potentiate the reduced food intake and weight loss induced by exogenous leptin administration in these animals. Endozepines reversed high fat diet-induced obesity by reducing food intake and restored leptin-induced STAT3 phosphorylation in the hypothalamus. Interestingly, we observed that glucose and insulin synergistically enhance tanycytic endozepine expression and release. Finally, endozepines, which induce ERK activation necessary for leptin transport into the brain in cultured tanycytes, require tanycytic leptin receptor expression to promote STAT3 phosphorylation in the hypothalamus. Our data identify endozepines as potential anti-obesity compounds in part through the modulation of the LepR-ERK-dependent tanycytic leptin shuttle.

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl-CoA binding protein.

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl-CoA-dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over-expressed in some algal species, the detailed structure-function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure-function features of the hydrophilic N-terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N-terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N-terminal domains of animal and plant DGAT1s were previously found to bind acyl-CoAs, replacement of CzDGAT1 N-terminus by an acyl-CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N-terminus of the full-length CzDGAT1 could enhance the enzyme affinity for acyl-CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full-length CzDGAT1. Overall, our findings unravel the distinct features of the N-terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane-bound acyl-CoA-dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.

Characterization of Oil Palm Acyl-CoA-Binding Proteins and Correlation of Their Gene Expression with Oil Synthesis.

Acyl-CoA-binding proteins (ACBPs) are involved in binding and trafficking acyl-CoA esters in eukaryotic cells. ACBPs contain a well-conserved acyl-CoA-binding domain. Their various functions have been characterized in the model plant Arabidopsis and, to a lesser extent, in rice. In this study, genome-wide detection and expression analysis of ACBPs were performed on Elaeis guineensis (oil palm), the most important oil crop in the world. Seven E. guineensis ACBPs were identified and classified into four groups according to their deduced amino acid domain organization. Phylogenetic analysis showed conservation of this family with other higher plants. All seven EgACBPs were expressed in most tissues while their differential expression suggests various functions in specific tissues. For example, EgACBP3 had high expression in inflorescences and stalks while EgACBP1 showed strong expression in leaves. Because of the importance of E. guineensis as an oil crop, expression of EgACBPs was specifically examined during fruit development. EgACBP3 showed high expression throughout mesocarp development, while EgACBP1 had enhanced expression during rapid oil synthesis. In endosperm, both EgACBP1 and EgACBP3 exhibited increased expression during seed development. These results provide important information for further investigations on the biological functions of EgACBPs in various tissues and, in particular, their roles in oil synthesis.

Acyl-CoA-Binding Proteins (ACBPs) in Plant Development.

Acyl-CoA-binding proteins (ACBPs) play a pivotal role in fatty acid metabolism because they can transport medium- and long-chain acyl-CoA esters. In eukaryotic cells, ACBPs are involved in intracellular trafficking of acyl-CoA esters and formation of a cytosolic acyl-CoA pool. In addition to these ubiquitous functions, more specific non-redundant roles of plant ACBP subclasses are implicated by the existence of multigene families with variable molecular masses, ligand specificities, functional domains (e.g. protein-protein interaction domains), subcellular locations and gene expression patterns. In this chapter, recent progress in the characterization of ACBPs from the model dicot plant, Arabidopsis thaliana, and the model monocot, Oryza sativa, and their emerging roles in plant growth and development are discussed. The functional significance of respective members of the plant ACBP families in various developmental and physiological processes such as seed development and germination, stem cuticle formation, pollen development, leaf senescence, peroxisomal fatty acid ß-oxidation and phloem-mediated lipid transport is highlighted.

Increased Hypothalamic Levels of Endozepines, Endogenous Ligands of Benzodiazepine Receptors, in a Rat Model of Sepsis.

BACKGROUND: The mechanisms involved in septic anorexia are mainly related to the secretion of inflammatory cytokines. The term endozepines designates a family of neuropeptides, including the octadecaneuropeptide (ODN), originally isolated as endogenous ligands of benzodiazepine receptors. Previous data showed that ODN, produced and released by astrocytes, is a potent anorexigenic peptide. We have studied the effect of sepsis by means of a model of cecal ligation and puncture (CLP) on the hypothalamic expression of endozepines (DBI mRNA and protein levels), as well as on the level of neuropeptides controlling energy homeostasis mRNAs: pro-opiomelanocortin, neuropeptide Y, and corticotropin-releasing hormone. In addition, we have investigated the effects of two inflammatory cytokines, TNF-α and IL-1ß, on DBI mRNA levels in cultured rat astrocytes. METHODS: Studies were performed on Sprague-Dawley male rats and on cultures of rat cortical astrocytes. Sepsis was induced using the CLP method. Sham-operated control animals underwent the same procedure, but the cecum was neither ligated nor incised. RESULTS: Sepsis caused by CLP evoked an increase of DBI mRNA levels in ependymal cells bordering the third ventricle and in tanycytes of the median eminence. CLP-induced sepsis was also associated with stimulated ODN-like immunoreactivity (ODN-LI) in the hypothalamus. In addition, TNF-α, but not IL-1ß, induced a dose-dependent increase in DBI mRNA in cultured rat astrocytes. An increase in the mRNA encoding the precursor of the anorexigenic peptide α-melanocyte stimulating hormone, the pro-opiomelanocortin, and the corticotropin-releasing hormone was observed in the hypothalamus. CONCLUSION: These results suggest that during sepsis, hypothalamic mRNA encoding endozepines, anorexigenic peptide as well as stress hormone could play a role in the anorexia/cachexia associated with inflammation due to sepsis and we suggest that this hypothalamic mRNA expression could involve TNF-α.

A novel role for central ACBP/DBI as a regulator of long-chain fatty acid metabolism in astrocytes.

Acyl-CoA-binding protein (ACBP) is a ubiquitously expressed protein that binds intracellular acyl-CoA esters. Several studies have suggested that ACBP acts as an acyl-CoA pool former and regulates long-chain fatty acids (LCFA) metabolism in peripheral tissues. In the brain, ACBP is known as Diazepam-Binding Inhibitor, a secreted peptide acting as an allosteric modulator of the GABAA receptor. However, its role in central LCFA metabolism remains unknown. In the present study, we investigated ACBP cellular expression, ACBP regulation of LCFA intracellular metabolism, FA profile, and FA metabolism-related gene expression using ACBP-deficient and control mice. ACBP was mainly found in astrocytes with high expression levels in the mediobasal hypothalamus. We demonstrate that ACBP deficiency alters the central LCFA-CoA profile and impairs unsaturated (oleate, linolenate) but not saturated (palmitate, stearate) LCFA metabolic fluxes in hypothalamic slices and astrocyte cultures. In addition, lack of ACBP differently affects the expression of genes involved in FA metabolism in cortical versus hypothalamic astrocytes. Finally, ACBP deficiency increases FA content and impairs their release in response to palmitate in hypothalamic astrocytes. Collectively, these findings reveal for the first time that central ACBP acts as a regulator of LCFA intracellular metabolism in astrocytes. Acyl-CoA-binding protein (ACBP) or diazepam-binding inhibitor is a secreted peptide acting centrally as a GABAA allosteric modulator. Using brain slices, cortical, and hypothalamic astrocyte cultures from ACBP KO mice, we demonstrate that ACBP mainly localizes in astrocytes and regulates unsaturated but not saturated long-chain fatty acids (LCFA) metabolism. In addition, ACBP deficiency alters FA metabolism-related genes and results in intracellular FA accumulation while affecting their release. Our results support a novel role for ACBP in brain lipid metabolism. FA, fatty acids; KO, knockout; PL, phospholipids; TAG, triacylglycerol.

The Arabidopsis cytosolic Acyl-CoA-binding proteins play combinatory roles in pollen development.

In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been demonstrated to function in plant stress responses and development. Three of these AtACBPs (AtACBP4-AtACBP6) are cytosolic proteins and all are expressed in floral organs as well as in other tissues. The roles of cytosolic AtACBPs in floral development were addressed in this study. To this end, a T-DNA insertional knockout mutant of acbp5 was characterized before use in crosses with the already available acbp4 and acbp6 T-DNA knockout mutants to examine their independent and combinatory functions in floral development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuole accumulation in the acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen was the most severe abnormality occurring in the double and triple mutants. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant, which also displayed reduced ability in in vitro pollen germination. Transgenic Arabidopsis expressing ß-glucuronidase (GUS) driven from the various AtACBP promoters indicated that AtACBP6pro::GUS expression overlapped with AtACBP4pro::GUS expression in pollen grains and with AtACBP5pro::GUS expression in the microspores and tapetal cells. Taken together, these results suggest that the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development.

Endogenous benzodiazepine site peptide ligands operating bidirectionally in vivo in neurogenesis and thalamic oscillations.

By binding to the benzodiazepine site, diazepam binding inhibitor (DBI) is associated with negative allosteric modulation (NAM) of GABAA receptors (Costa and Guidotti in Life Sci 49:325-344, 1991). However, the demonstration of a true physiological role of DBI and its fragments has only recently been reported. Based on DBI gain- and loss-of-function experiments in vivo, DBI and its fragment ODN were found to promote neurogenesis in the subventricular zone in vivo. Acting as NAM on GABAA receptors of precursor cells, DBI counteracted the inhibitory effect of GABA and thereby enhanced the proliferation of these cells (Alfonso et al. in Cell Stem Cell 10:76-87, 2012). Conversely and most remarkably, in similar gain- and loss-of-function experiments in the thalamus, the DBI gene products acted as positive allosteric modulators (PAM) of GABAA receptors in prolonging the duration of IPSCs, an effect which was specific for GABA transmission within the reticular nucleus (nRT) (Christian et al. in Neuron 78:1063-1074, 2013). Since intra-nRT potentiation of GABA transmission by benzodiazepine drugs exerts powerful anti-oscillatory effects, DBI might be endogenously effective by modulating seizure susceptibility. It remains to be seen by which mechanism both NAM and PAM activity can arise from the Dbi gene. Nevertheless, the results open new perspectives on the regionally distinct endogenous modulation of GABA transmission via the benzodiazepine site.

Endogenous positive allosteric modulation of GABA(A) receptors by diazepam binding inhibitor.

Benzodiazepines (BZs) allosterically modulate γ-aminobutyric acid type-A receptors (GABAARs) to increase inhibitory synaptic strength. Diazepam binding inhibitor (DBI) protein is a BZ site ligand expressed endogenously in the brain, but functional evidence for BZ-mimicking positive modulatory actions has been elusive. We demonstrate an endogenous potentiation of GABAergic synaptic transmission and responses to GABA uncaging in the thalamic reticular nucleus (nRT) that is absent in both nm1054 mice, in which the Dbi gene is deleted, and mice in which BZ binding to α3 subunit-containing GABAARs is disrupted. Viral transduction of DBI into nRT is sufficient to rescue the endogenous potentiation of GABAergic transmission in nm1054 mice. Both mutations enhance thalamocortical spike-and-wave discharges characteristic of absence epilepsy. Together, these results indicate that DBI mediates endogenous nucleus-specific BZ-mimicking ("endozepine") roles to modulate nRT function and suppress thalamocortical oscillations. Enhanced DBI signaling might serve as a therapy for epilepsy and other neurological disorders.

In search of the ever-elusive positive endozepine.

In this issue of Neuron, Christian et al. (2013) provide functional evidence for positive endozepines (positive allosteric modulators of GABAARs) within the thalamic reticular nucleus. These molecules are encoded by the Dbi gene and modulate thalamocortical oscillations.

Gliotransmission and brain glucose sensing: critical role of endozepines.

Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.

Novel anti-Cryptosporidium activity of known drugs identified by high-throughput screening against parasite fatty acyl-CoA binding protein (ACBP).

BACKGROUND: Cryptosporidium parvum causes an opportunistic infection in AIDS patients, and no effective treatments are yet available. This parasite possesses a single fatty acyl-CoA binding protein (CpACBP1) that is localized to the unique parasitophorous vacuole membrane (PVM). The major goal of this study was to identify inhibitors from known drugs against CpACBP1 as potential new anti-Cryptosporidium agents. METHODS: A fluorescence assay was developed to detect CpACBP1 activity and to identify inhibitors by screening known drugs. Efficacies of top CpACBP1 inhibitors against Cryptosporidium growth in vitro were evaluated using a quantitative RT-PCR assay. RESULTS: Nitrobenzoxadiazole-labelled palmitoyl-CoA significantly increased the fluorescent emission upon binding to CpACBP1 (excitation/emission 460/538 nm), which was quantified to determine the CpACBP1 activity and binding kinetics. The fluorescence assay was used to screen a collection of 1040 compounds containing mostly known drugs, and identified the 28 most active compounds that could inhibit CpACBP1 activity with sub-micromolar IC(50) values. Among them, four compounds displayed efficacies against parasite growth in vitro with low micromolar IC(50) values. The effective compounds were broxyquinoline (IC(50) 64.9 µM), cloxyquin (IC(50) 25.1 µM), cloxacillin sodium (IC(50) 36.2 µM) and sodium dehydrocholate (IC(50) 53.2 µM). CONCLUSIONS: The fluorescence ACBP assay can be effectively used to screen known drugs or other compound libraries. Novel anti-Cryptosporidium activity was observed in four top CpACBP1 inhibitors, which may be further investigated for their potential to be repurposed to treat cryptosporidiosis and to serve as leads for drug development.

Diazepam enhances production of diazepam-binding inhibitor (DBI), a negative saliva secretion regulator, localized in rat salivary gland.

Peripheral-type benzodiazepine receptor (PBR) and central-type benzodiazepine receptor (CBR) in salivary gland play a role in the inhibitory regulation of salivary secretion in rodents. Diazepam-binding inhibitor (DBI), an endogenous ligand for PBR, produces neurosteroids, which modulate CBR activity. In this study, we investigated the effect of repetitive administration of diazepam (DZP) on salivary secretion and expression of DBI mRNA and peptide. Moreover, mRNA expression of PBR and pituitary adenylate cyclase-activating polypeptide (PACAP), a transcriptional regulator for DBI promoter, was evaluated after repetitive administration of DZP. Repetitive administration, but not single administration, of 0.4 mg/kg DZP caused inhibition of salivary secretion and enhanced expression of DBI, PACAP, and PBR mRNA in rat salivary gland, with an increase in production of DBI peptide. These results suggest that repetitive administration of DZP stimulates DBI production, which may result in an increase in the suppressive effect of DZP on salivary secretion.

Involvement of low molecular mass soluble acyl-CoA-binding protein in seed oil biosynthesis.

Acyl-CoA-binding protein (ACBP), a low molecular mass (m) (∼ 10 kDa) soluble protein ubiquitous in eukaryotes, plays an important housekeeping role in lipid metabolism by maintaining the intracellular acyl-CoA pool. ACBP is involved in lipid biosynthesis and transport, gene expression, and membrane biogenesis. In plants, low m ACBP and high m ACBPs participate in response mechanisms to biotic and abiotic factors, acyl-CoA transport in phloem, and biosynthesis of structural and storage lipids. In light of current research on the modification of seed oil, insight into mechanisms of substrate trafficking within lipid biosynthetic pathways is crucial for developing rational strategies for the production of specialty oils with the desired alterations in fatty acid composition. In this review, we summarize our knowledge of plant ACBPs with emphasis on the role of low m ACBP in seed oil biosynthesis, based on in vitro studies and analyses of transgenic plants. Future prospects and possible applications of low m ACBP in seed oil modification are discussed.

Pregnenolone sulfate and cortisol induce secretion of acyl-CoA-binding protein and its conversion into endozepines from astrocytes.

Acyl-CoA-binding protein (ACBP) functions both intracellularly as part of fatty acid metabolism and extracellularly as diazepam binding inhibitor, the precursor of endozepine peptides. Two of these peptides, ODN and TTN, bind to the GABA(A) receptor and modulate its sensitivity to gamma-aminobutyric acid (GABA). We have found that depolarization of mouse primary astrocytes induces the rapid release and processing of ACBP to the active peptides. We previously showed that ODN can trigger the rapid sporulation of the social amoeba Dictyostelium. Using this bioassay, we now show that astrocytes release the endozepine peptides within 10 min of exposure to the steroids cortisol, pregnenolone, pregnenolone sulfate, or progesterone. ACBP lacks a signal sequence for secretion through the endoplasmic reticulum/Golgi pathway and its secretion is not affected by addition of brefeldin A, a well known inhibitor of the classical secretion pathway, suggesting that it follows an unconventional pathway for secretion. Moreover, induction of autophagy by addition of rapamycin also resulted in rapid release of ACBP indicating that this protein uses components of the autophagy pathway for secretion. Following secretion, ACBP is proteolytically cleaved to the active neuropeptides by protease activity on the surface of astrocytes. Neurosteroids, such as pregnenolone sulfate, were previously shown to modulate the excitatory/inhibitory balance in brain through increased release of glutamate and decreased release of GABA. These effects of steroids in neurons will be reinforced by the release of endozepines from astrocytes shown here, and suggest an orchestrated astrocyte-neuron cross-talk that can affect a broad spectrum of behavioral functions.

Acute food deprivation reduces expression of diazepam-binding inhibitor, the precursor of the anorexigenic octadecaneuropeptide ODN, in mouse glial cells.

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.

An Arabidopsis family of six acyl-CoA-binding proteins has three cytosolic members.

In Arabidopsis thaliana, a gene family of six members encodes acyl-CoA-binding proteins (ACBPs). These Arabidopsis ACBPs (designated ACBP1 to ACBP6) range in size from 10.4kDa to 73.1kDa and display varying affinities for acyl-CoA esters, suggesting that they have different roles in plant lipid metabolism. In contrast, only the 10-kDa ACBPs have been well-characterized from other eukaryote species. Our previous studies have revealed that ACBP1 and ACBP2 are membrane-associated proteins, while ACBP3 is extracellularly-targeted. More recently, we have reported that the remaining three members in this protein family (namely ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol in Arabidopsis. The subcellular localizations of ACBP4, ACBP5 and ACBP6 in the cytosol were demonstrated using a number of different approaches incorporating biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immunoelectron microscopy using ACBP-specific antibodies. Our results indicate that all three ACBPs in the cytosol are potential candidates for acyl-CoA binding and trafficking in plant cells. In this review, the functional redundancy and differences among the three cytosolic ACBPs are discussed by comparison of their light-regulated expression and substrate affinities to acyl-CoA esters, and from biochemical analyses on their knockout mutants and/or overexpression in transgenic Arabidopsis. The transcriptionally light-induced ACBP4 and ACBP5, which encode the two largest forms of Arabidopsis ACBPs, bind oleoyl-CoA esters and likely transfer oleoyl-CoAs from the plastids (the site of de novo fatty acid biosynthesis) to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids in Arabidopsis.

Natural endogenous ligands for benzodiazepine receptors in hepatic encephalopathy.

Benzodiazepines of natural origin (NBZDs) have been found in human blood and brains as well as in medicinal plants and foods. In plasma and brain tissue there are i.e. diazepam and nordiazepam equal to commercial drugs but there are also other benzodiazepine-like compounds termed "endozepines", which act as agonists at the benzodiazepine receptors of central type (CBR). A synthetic pathway for the production of NBZDs has not yet been found, but it has been suggested that micro-organisms may synthesize molecules with benzodiazepine-like structures. Hence NBZDs could be of both endogenous and exogenous source and be considered as natural anxyolitic and sedative. Interestingly there are also natural compounds, such as the polypeptide Diazepam Binding Inhibitor (DBI) acting as an "inversive agonist" implicated in fair and panic disorders. It has been suggested that NBZDs may play a role in the pathogenesis of hepatic encephalopathy (HE). Multidirectional studies evaluated NBZDs levels (1) in the blood of normal subjects, of cirrhotic with or without HE and in commercial benzodiazepine consumers; (2) in the blood of cirrhotic treated or not with a non-absorbable antibiotic; (3) in several constituents of our diet. In conclusion, NBZDs increase sometime in cirrhotics with or without HE but they reach concentrations not higher than those found in commercial benzodiazepines consumers. Hence NBZDs must be considered as occasional precipitating factor of HE and benzodiazepine antagonists only symptomatic drugs. The finding that NBZDs may be in part synthesized by intestinal bacterial flora and in part constituent of our diet underlines the importance to feed cirrhotic patients with selected food.