Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

Carrier-mediated processes at several rat brain interfaces determine the neuropharmacokinetics of morphine and morphine-6-beta-D-glucuronide

We investigated whether capacity-limited transport processes were involved in morphine and morphine-6-beta-D-glucuronide (M6G) neuropharmacokinetics, at the level of the blood-brain barrier (BBB), the brain extra- and intra-cellular fluids (bECF/bICF), and the bECF/cerebrospinal fluid (CSF) interfaces. We performed transcortical retrodialysis in the rat, by perfusing morphine or M6G through the microdialysis probe in the presence or absence of probenecid. We measured for each compound the in vitro and in vivo (R(D)) probe recoveries. The in vivo R(D), which takes into account the permeability of the tissue surrounding the probe, informs about the morphine and M6G distribution capabilities from bECF to adjacent fluids (bICF, CSF, plasma). We also measured plasma and CSF concentrations at three time points after having added probenecid or not. Finally, we tested several pharmacokinetic models, assuming first-order or capacity-limited processes at each brain interface, to describe experimental morphine and M6G concentrations previously obtained in rat plasma and brain fluids. We found that morphine distributes more easily outside bECF than M6G. Adding probenecid caused a 2-fold decrease and a 1.3-fold increase in morphine and M6G R(D), respectively, and 30 min after adding probenecid, plasma and CSF concentrations increased for M6G but not for morphine. The pharmacokinetic model that gave the best fit included capacity-limited processes at the BBB and bECF/bICF interface for morphine and at the BBB and bECF/CSF interface for M6G. In conclusion, morphine accumulates into brain cells thanks to a probenecid-sensitive transporter located at the bECF/bICF interface, whereas M6G is trapped in bECF thanks to transporters located at the BBB and the bECF/CSF interface.     

Entry of Trypanosoma brucei gambiense into microvascular endothelial cells of the human blood-brain barrier

Using an in vitro model of the human blood-brain barrier consisting of human brain microvascular endothelial cells we recently demonstrated that Trypanosoma brucei gambiense bloodstream-forms efficiently cross these cells via a paracellular route while Trypanosoma brucei brucei crosses these cells poorly. Using a combination of techniques that include fluorescence activated cell sorting, confocal and electron microscopy, we now show that some T.b. gambiense blood stream form parasites have the capacity to enter human brain microvascular endothelial cells. The intracellular location of the trypanosomes was demonstrated in relation to the endothelial cell plasma membrane and to the actin cytoskeleton. These parasites may be a terminal stage within a lysosomal compartment or they may be viable trypanosomes that will be able to exit the brain microvascular endothelial cells. This process may provide an additional transcellular route by which the parasites cross the blood-brain barrier.     

Ouabain-Sensitive Choline Transport System in Capillaries Isolated from Bovine Brain

In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood-brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km = 17.8 +/- 4.8 microM; Vmax = 11.3 +/- 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 microM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10(-4) M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood-brain barrier endothelium is specifically provided with an energy-dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.     

Altered blood-brain barrier permeability and its effect on the distribution of Evans blue and sodium fluorescein in the rat brain applied by intracarotid injection

The aim was to study the blood-brain permeability according to the distribution in the rat brain of Evans blue (EB) and sodium fluorescein (NaFl) administered by an intracarotid injection. Eighteen animals were divided into six groups according to the state of the blood-brain barrier (BBB) at the moment when the dyes were being applied. In the first two groups, the BBB was intact, in groups 3 and 4 the barrier had been opened osmotically prior to the application of the dyes, and in groups 5 and 6 a cellular edema was induced by hyperhydration before administration of the dyes. The intracellular and extracellular distribution of the dyes was studied by fluorescence microscopy. The histological picture thus represented the morphological correlate of the way BBB permeability had been changed before the application of the dyes.     

The complementary membranes forming the blood-brain barrier

Brain capillary endothelial cells form the blood-brain barrier. They are connected by extensive tight junctions, and are polarized into luminal (blood-facing) and abluminal (brain-facing) plasma membrane domains. The polar distribution of transport proteins allows for active regulation of brain extracellular fluid. Experiments on isolated membrane vesicles from capillary endothelial cells of bovine brain demonstrated the polar arrangement of amino acid and glucose transporters, and the utility of such arrangements have been proposed. For instance, passive carriers for glutamine and glutamate have been found only in the luminal membrane of blood-brain barrier cells, while Na-dependent secondary active transporters are at the abluminal membrane. This organization could promote the net removal of nitrogen-rich amino acids from brain, and account for the low level of glutamate penetration into the central nervous system. Furthermore, the presence of a gamma-glutamyl cycle at the luminal membrane and Na-dependent amino acid transporters at the abluminal membrane may serve to modulate movement of amino acids from blood-to-brain. Passive carriers facilitate amino acid transport into brain. However, activation of the gamma-glutamyl cycle by increased plasma amino acids is expected to generate oxoproline within the blood-brain barrier. Oxoproline stimulates secondary active amino acid transporters (Systems A and B(o)+) at the abluminal membrane, thereby reducing net influx of amino acids to brain. Finally, passive glucose transporters are present in both the luminal and abluminal membranes of the blood-brain barrier. Interestingly, a high affinity Na-dependent glucose carrier has been described only in the abluminal membrane. This raises the question whether glucose entry may be regulated to some extent. Immunoblotting studies suggest more than one type of passive glucose transporter exist in the blood-brain barrier, each with an asymmetrical distribution. In conclusion, it is now clear that the blood-brain barrier participates in the active regulation of brain extracellular fluid, and that the diverse functions of each plasma membrane domain contributes to these regulatory functions.     

Proteins in cerebrospinal fluid and plasma of postnatal Monodelphis domestica (grey short-tailed opossum)

1. The protein composition and concentrations of total protein in cerebrospinal fluid (CSF) and plasma of opossum (Monodelphis domestica) from birth until adulthood have been estimated. 2. Total protein in CSF increased from birth to a peak concentration between 5 and 10 days (500 mg/100 ml) after which it declined rapidly. 3. Total protein in plasma started at a low level at birth (below 400 mg/100 ml) and increased progressively to the adult value (8500 mg/100 ml). 4. Proteins identified in CSF and plasma were: albumin, alpha 1 antitrypsin, transferrin, alpha 2 macroglobulin, lipoproteins and immunoglobulin G. A fetal protein, probably alpha-fetoprotein, has been identified. 5. The only plasma proteins found within the brain, even in the very immature newborn of this species, appeared to have an intracellular rather than an extracellular distribution. This indicates that the blood-brain barrier is present very early in development and that there is also a CSF-brain barrier that appears to exclude CSF protein from brain extracellular space.     

Electrochemical monitoring of brain ascorbic acid changes associated with hypoxia,spreading depression,and seizure activity

In vivo electrochemistry has been a valuable tool in detecting real time neurochemical changes in extracellular fluid. Absolute selectivity has been difficult to achieve previously, but we report here a carbon fiber electrode and measurement technique which is specific for one oxidizable species: ascorbic acid. Ascorbic acid is highly concentrated in extra- as well as intracellular brain spaces, and appears to undergo dynamic changes in response to a variety of physiological and pathophysiological circumstances. Recent studies have implicated glutamatergic mechanisms which give rise to extracellular changes in brain ascorbate, and we confirm and extend these observations. Preliminary studies, directed towards examining ascorbic acid as an index and/or result of hypoxia, spreading depression, and seizure activity, have been undertaken and the results are reported herein.Special issue dedicated to Dr. Frederick E. Samson.     

Tissue-type plasminogen activator is a regulator of monocyte diapedesis through the brain endothelial barrier

Inflammatory cell trafficking into the brain complicates several neurological disorders including multiple sclerosis. Normally, reliable brain functioning is maintained and controlled by the blood-brain barrier (BBB), which is essential to restrict the entry of potentially harmful molecules and cells from the blood into the brain. The BBB is a selective barrier formed by dedicated brain endothelial cells and dependent on the presence of intracellular tight junctions. In multiple sclerosis, a severe dysfunction of the BBB is observed, which is key to monocyte infiltration and inflammation in the brain. Proteolytic activity has been associated with these inflammatory processes in the brain. Our studies in plasma of rats indicated that the extracellular protease tissue-type plasminogen activator (tPA) correlates with the clinical signs of experimental allergic encephalomyelitis, a rat model of multiple sclerosis. In this study, we studied the function of the tPA during diapedesis of monocytes through a rat and human brain endothelial barrier. Monocyte-brain endothelial cell coculture experiments showed that monocytes induce the release of tPA by brain endothelial cells, which subsequently activates the signal transduction protein extracellular signal related kinase (ERK1/2), both involved in monocyte diapedesis. Importantly, live imaging and immunoblot analyses of rat brain endothelial cells revealed that tPA and ERK1/2 control the breakdown of the tight junction protein occludin. These studies identify tPA as a novel and relevant pathological mediator of neuroinflammation and provide a potential mechanism for this.     

Physico-chemical parameters influencing DNase activity of the cyanobacterium Spirulina platensis

The effects of temperature, Mg2+, EDTA concentration and rinsing on extra- and intra-cellular DNase activity of Spirulina platensis strain SSP-14, were investigated. The results indicate that the tested strain contains very high extra- and intracellular DNase activity, which actually hinders the transfer of foreign gene(s) to S. platensis, a cyanobacterium with multiple economic potentials. The extracellular DNase activity could easily be removed by rinsing the cells with Zarrouk medium more than once. The intracellular DNase activity could also be inhibited by (1) removal of Mg2+, (2) maintaining EDTA concentration above 1 mmol l(-1), and (3) manipulating below 0-4 degrees C, during all the incubation procedures. We suggest that, by using one or more of, or combining, all those experimental conditions, the chances of foreign DNA attempted to be introduced into S. platensis without being digested would be increased.     

Simultaneous NMR microdialysis study of brain glucose metabolism in relation to fasting or exercise in the rat.

To study the impact of exercise or fasting and of subsequent glucose supplementation on glucose metabolism in rats, a spectrophotometric method was used to determine peripheral blood glucose; a technique associating (1)H-NMR spectroscopy and cortical microdialysis was also used to observe intra- plus extracellular and extracellular brain glucose variations, respectively. Compared with control animals (204 +/- 19 microM in dialysate, n = 10), exercise increased brain extracellular glucose levels to 274 +/- 22 microM (n = 8; P < 0.05), whereas fasting induced a drop in glucose levels down to 140 +/- 9 microM (n = 8; P < 0.05). After fasting, glucose supplemented by infusion increased glycemia from 7.4 +/- 0.4 to 19.9 +/- 0.8 mM (n = 10; P < 0.001), as well as extracellular and extra- plus intracellular brain glucose to 263 +/- 20% (n = 8; P < 0.001) and 342 +/- 28% (n = 8; P < 0.001), respectively, over basal for that group. After exercise, a similar infusion increased glycemia from 7. 3 +/- 0.3 to 16.8 +/- 1.1 mM (n = 10; P < 0.001), as well as extracellular and extra- plus intracellular brain glucose to 178 +/- 19% (n = 8; P < 0.001) and 244 +/- 20% (n = 8; P < 0.001), respectively, over basal for that group. These results confirmed the existence of a link between glucose level variations in peripheral and cerebral areas but also showed that exercise increased extracellular brain glucose levels despite peripheral hypoglycemia, suggesting a specific regulation mechanism of cerebral glucose metabolism during exercise.     

Activation of AMP-activated protein kinase alleviates High-glucose-induced dysfunction of brain microvascular endothelial cell tight-junction dynamics

The blood-brain barrier, formed by specialized brain endothelial cells that are interconnected by tight junctions, strictly regulates paracellular permeability to maintain an optimal extracellular environment for brain homeostasis. Diabetes is known to compromise the blood-brain barrier, although the underlying mechanism remains unknown. The aim of this study was to elucidate the molecular mechanisms underlying disruption of the blood-brain barrier in diabetes and to determine whether activation of AMP-activated protein kinase prevents diabetes-induced blood-brain barrier dysfunction. Exposure of human brain microvascular endothelial cells to high glucose (25mmol/L d-glucose), but not to high osmotic conditions (20mmol/L l-glucose plus 5mmol/L d-glucose), for 2h to 1 week significantly increased the permeability of the blood-brain barrier in parallel with lowered expression levels of zonula occludens-1, occludin, and claudin-5, three proteins that are essential to maintaining endothelial cell tight junctions. In addition, high glucose significantly increased the generation of superoxide anions. Adenoviral overexpression of superoxide dismutase or catalase significantly attenuated the high-glucose-induced reduction of endothelial cell tight-junction proteins. Furthermore, administration of apocynin reversed the effects of high glucose on endothelial cell tight-junction proteins. Finally, activation of AMP-activated protein kinase with 5-amino-4-imidazole carboxamide riboside or adenoviral overexpression of constitutively active AMP-activated protein kinase mutants abolished both the induction of NAD(P)H oxidase-derived superoxide anions and the tight-junction protein degradation induced by high glucose. We conclude that high glucose increases blood-brain barrier dysfunction in diabetes through induction of superoxide anions and that the activation of AMP-activated protein kinase protects the integrity of the blood-brain barrier by suppressing the induction of NAD(P)H oxidase-derived superoxide anions.     

Intra- and extracellular pH of the brain in vivo studied by 31P-NMR during hyper- and hypocapnia.

Studies were performed to determine the pH relationships among the extracellular, intracellular, and arterial blood compartments in the brain in vivo. Resolution of the extracellular monophosphate resonance peak from the intracellular peak in 31P nuclear magnetic resonance (NMR) spectra of sheep brain with the calvarium intact enabled pH measurement in these respective compartments. Sheep were then subjected to both hyper- and hypoventilation, which resulted in a wide range of arterial PCO2 and pH values. Linear regression analysis of pH in these compartments yielded slopes of 0.56 +/- 0.05 for extracellular pH (pHe) vs. arterial pH, 0.43 +/- 0.078 for intracellular pH (pHi) vs. pHe, and 0.23 +/- 0.056 for pHi vs. arterial pH. These data indicate that CO2 buffering capacity is different and decreases from the intracellular to extracellular to arterial blood compartments. Separation of the extracellular space from the vascular space may be a function of the blood-brain barrier, which contributes to the buffering capability of the extracellular compartment. A marked decrease in the pH gradient between the extracellular and intracellular space occurs during hypercarbia and may influence mechanisms of central respiratory control.     

Sigma-1 receptor as regulator of neuronal intracellular Ca2+: clinical and therapeutic relevance

Preserving brain function and cognitive faculties during aging and psychiatric diseases (e.g. psychotic, anxiety and affective disorders, dementia) is essential for the self-reliance and quality of life of patients. Cognitive loss involves not only memory, but also motor function. The decrease of catecholaminergic and excitatory neurotransmissions, as well as of protein phosphorylation, have currently been identified as prominent biological markers of the above-mentioned diseases. Such deleterious biological events are well known to occur downstream of a progressive decline of intracellular Ca2+ signalling. This latter constitutes a key target for the neuronal plasticity that has also been reported during aging and psychiatric disorders. Most of the medicines used in psychiatry are active on the sigma-1 receptor. This membrane bound receptor is widely distributed in memory-associated cortical and motor-related brainstem areas, prompting the hypothesis that it might contribute to the pathophysiology of these behavioural brain diseases. The sigma-1 receptor is characterized by a unique mode of action by regulating both Ca2+ entry at the plasma membrane level (i.e. via potassium channels, voltage-sensitive Ca2+ channels) and Ca2+ mobilization from endoplasmic stores [i.e. via Ins(1,4,5)P3 receptors]. This review presents recent data supporting the notion that drugs acting via the endoplasmic reticulum-coupled sigma-1 receptor might reverse these deleterious events by restoring both extra- and intra-cellular Ca(2+)-dependent neuronal responses.     

Disruption of the plasma membrane leads to an influx of Na+ and Cl- but not an efflux of K+ in mouse hepatocytes and erythrocytes

To determine what mechanisms might be involved in the maintenance of the known extra-/intracellular concentration gradients of Na+, Cl- and K+, small pieces of mouse liver and heparinized blood were appropriately cryofixed. The tissues were cryosectioned and cryosorbed at -100 degrees C or at -40 degrees C. The former temperature prevented diffusion of all ions as measured by electron probe x-ray microanalysis of the thin (0.1 micron) cryosorbed sections of the cells while the latter temperature allowed significant diffusion of Na+ and Cl- into the hepatocytes and erythrocytes but did not allow diffusion of K+ from the hepatocytes or the erythrocytes. These results indicate that the plasma membrane is involved in maintenance of the extra-/intracellular gradients of Na+ and Cl- but that intracellular association of K+ with macromolecules is the main mechanism responsible for maintenance of the extra-/intracellular K+ concentration gradient.     

Investigation into the Role of N-Acetylaspartate in Cerebral Osmoregulation

Abstract: Marked abnormalities of the magnetic resonance intensity of N -acetylaspartate (NAA) have been reported in patients with various neurological disorders, but the neurochemical consequences of these alterations are difficult to assess because the function of NAA remains speculative. The purpose of this study was to examine whether NAA plays a role in protecting neurons against osmotic stress. Intracerebral microdialysis was used to expose a small region of the rat dorsolateral striatum to an increasingly hyposmotic environment and to measure resulting changes in NAA extracellular concentrations. NAA changes in the extracellular fluid (ECF) were compared with those of the amino acids, in particular, taurine, known to be involved in brain osmoregulation. Stepped increases in cellular hydration produced by hyposmotic perfusion media induced a marked increase in ECF NAA, reflecting a redistribution of NAA from intra-to extracellular space. Parallel experiments showed that, of all the extracellular amino acids measured, only taurine markedly increased with hyposmolar perfusion medium, indicating that the ECF NAA increase associated with hyposmotic stress was a specific response and not passive leakage out of the cells. As NAA is predominantly neuronal, it may contribute to the protection of neurons against swelling (i.e., regulatory volume decrease). In conditions with impaired blood-brain barrier and cytotoxic oedema, efflux of intracellular NAA subsequent to sustained cellular swelling might lead to a reduction in total brain NAA detectable by magnetic resonance spectroscopy. Alternatively, redistribution of NAA from intra-to extracellular space implies changes in its chemical environment that may alter its magnetic resonance visibility.     

Extracellular ribosomes during metamorphosis of the blowfly Calliphora vicina R. D. -- A. reappraisal of their authenticity.

Extracellular ribosomes during adult development of the blowfly $\text{Calliphora}\text{vicina}$ were previously considered to occur naturally $\text{in}\text{vivo}$. A variety of radioisotopic experiments performed at different stages of metamorphosis now demonstrates that the specific activities of extra- and intracellular ribosomes are consistently very similar. Further, in addition to previously described physico-chemical similarities, extra- and intra-cellular ribosomal protein profiles are now shown to be essentially identical. These results vitiate the notion of a natural pool of extracellular ribosomes, the occurrence of which is now ascribed to an experimental artifact, resulting from unusual cell fragility.     

Localization of organic anion transporting polypeptide 3 (oatp3) in mouse brain parenchymal and capillary endothelial cells

Organic anion transporting polypeptide 3 (oatp3) transports various CNS-acting endogenous compounds, including thyroid hormones and prostaglandin E2, between extra- and intracellular spaces, suggesting a possible role in CNS function. The purpose of this study was to clarify the expression and localization of oatp3 in the mouse brain. RT-PCR analysis revealed that oatp3 mRNA is expressed in brain capillary-rich fraction, conditionally immortalized brain capillary endothelial cells, choroid plexus, brain and lung, but not in liver or kidney, where oatp1, 2 and 5 mRNAs were detected. Immunohistochemical analysis with anti-oatp3 antibody suggests that oatp3 protein is localized at the brush-border membrane of mouse choroid plexus epithelial cells. Furthermore, intense immunoreactivity was detected in neural cells in the border region between hypothalamus and thalamus, and in the olfactory bulb. Immunoreactivity was also detected in brain capillary endothelial cells in the cerebral cortex. These localizations in the mouse brain suggest that oatp3 plays roles in blood-brain and -cerebrospinal fluid barrier transport of organic anions and signal mediators, and in hormone uptake by neural cells.     

In Vivo Distribution of CGS-19755 Within Brain in a Model of Focal Cerebral Ischemia

The blood-brain barrier permeability of the competitive N-methyl-D-aspartate receptor antagonist CGS-19755 [cis-4-(phosphonomethyl)-2-piperidine carboxylic acid] was assessed in normal and ischemic rat brain. The brain uptake index of CGS-19755 relative to iodoantipyrine was assessed using the Oldendorf technique in normal brain. The average brain uptake index in brain regions supplied by the middle cerebral artery was 0.15 +/- 0.35% (mean +/- SEM). The unidirectional clearance of CGS-19755 from plasma across the blood-brain barrier was determined from measurements of the volume of distribution of CGS-19755 in brain. These studies were performed in normal rats and in rats with focal cerebral ischemia produced by combined occlusion of the proximal middle cerebral artery and ipsilateral common carotid artery. In normal rats the regional plasma clearance across the blood-brain barrier was low, averaging 0.015 ml 100 g-1 min-1. In ischemic rats this clearance value averaged 0.019 ml 100 g-1 min-1 in the ischemic hemisphere and 0.009 ml 100 g-1 min-1 in the nonischemic hemisphere. No significant regional differences in plasma clearance of CGS-19755 were observed in either normal or ischemic rats except in cortex injured by electrocautery where a 14-fold increase in clearance across the blood-brain barrier was measured. We conclude that CGS-19755 crosses the blood-brain barrier very slowly, even in acutely ischemic tissue.     

Minireview. Peptides and the blood-brain barrier

Most neuropeptides are known to occur both in the central nervous system and in blood. This, as well as the occurrence of central nervous peptide effects after peripheral administration, show the importance of studying the relationships between the peptides in the two compartments. For many peptides, such as the enkephalins, TRH, somatostatin and MIF-1, poor penetration of the blood-brain barrier was shown. In other cases, including beta-endorphin and angiotensin, peptides are rapidly degraded during or just after their entry into brain or cerebrospinal fluid. Some peptides, such as insulin, delta-sleep-inducing peptide, and the lipotropin-derived peptides, enter the cerebrospinal fluid to a slight or moderate extent in the intact form. Many peptide hormones, such as insulin, calcitonin and angiotensin, act directly on receptors in the circumventricular organs, where the blood-brain barrier is absent. Oxytocin, vasopressin, MSH, and an MSH-analog alter the properties of the blood-brain barrier, which may result in altered nutritient supply to the brain. In conclusion, the diffusion of most peptides across the brain vascular endothelium seems to be severely restricted. There are, however, several alternative routes for peripheral peptides to act on the central nervous system. The blood-brain barrier is a major obstacle for the development of pharmaceutically useful peptides, as in the case of synthetic enkephalin-analogs.