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Cryopreservation of equine embryos with glycerol plus sucrose and glycerol plus 1,2-propanediol.

Six or 7-day-old equine embryos were divided into 4 groups; Group 1, n = 15, Day 7 embryos destined for immediate transfer; Group 2, n = 15, Day 6 embryos destined for deep-freezing with glycerol plus sucrose as cryoprotectant; Group 3, n = 10, Day 6 embryos destined for deep-freezing with glycerol plus 1,2-propanediol as cryoprotectant and Group 4, n = 3, fresh embryos destined for ultrastructural analysis. All the frozen/thawed embryos were transferred to recipient mares, except 3 embryos in Group 3 that were subjected to ultrastructural analysis. After thawing the cryoprotectants were removed by successive dilutions in PBS + 15% v:v fetal calf serum (FCS) containing decreasing concentrations of the cryoprotectants. Pregnancy was diagnosed ultrasonographically in 53.3%, 13.3% and 0% of the mares in Groups 1, 2 and 3 respectively. Ultrastructural analysis showed differences between frozen/thawed and fresh embryos. In the former, embryonic cells were deformed and showed dilation of the intercellular and perivitelline spaces, a decrease of desmosome number in the junctional complexes, few microvilli on the apical surface of the trophectoderm and an almost total absence of pinocytotic vesicles. Most of the mitochondria showed regions containing dilation and irregularities on the cristae, which appeared electron-dense. The results obtained with Groups 2 and 3 embryos showed that the cryoprotectants employed were not effective in protecting the embryos against damage during freezing and thawing. Indeed, the ultrastructural changes observed in the Group 3 embryos explained the absence of any established pregnancies in this group of mares.

Animals↗

PGE2 glycerol ester, a COX-2 oxidative metabolite of 2-arachidonoyl glycerol, modulates inhibitory synaptic transmission in mouse hippocampal neurons.

The oxygenation of endogenous cannabinoids (eCBs) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide by cyclooxygenase-2 (COX-2) produces novel types of prostanoids: prostaglandin glycerol esters (PG-Gs) and prostaglandin ethanolamides (PG-EAs). However, the physiological function of COX-2 oxidative metabolites of eCBs is still unclear. Here we demonstrate that PGE2-G, a COX-2 oxidative metabolite of 2-AG, induced a concentration-dependent increase in the frequency ofminiature inhibitory postsynaptic currents (mIPSCs) in primary cultured hippocampal neurons, an effect opposite to that of 2-AG. This increase was not inhibited by SR141716, a CB1 receptor antagonist, but was attenuated by an IP3 or MAPK inhibitor. In addition, we also examined the effects of other prostanoids derived from COX-2 oxygenation of eCBs on mIPSCs. PGD2-G, PGF2alpha-G and PGD2-EA, but not PGE2-EA or PGF2alpha-EA, also increased the frequency of mIPSCs. The eCB-derived prostanoid-induced responses appeared to be different from those of corresponding arachidonic acid-derived prostanoids, implying that these effects are not mediated via known prostanoid receptors. We further discovered that the inhibition of COX-2 activity reduced inhibitory synaptic activity and augmented depolarization-induced suppression of inhibition (DSI), whereas the enhancement of COX-2 augmented the synaptic transmission and abolished DSI. Our results, which show that COX-2 oxidative metabolites of eCBs exert opposite effects to their parent molecules on inhibitory synaptic transmission, suggest that alterations in COX-2 activity will have significant impact on endocannabinoid signalling in hippocampal synaptic activity.

Animals↗

Glycerol Jelly (GJ) mount: a new and simple method for routine stool examination using a modified glycerol jelly reagent.

Wet mount is the basic primary technique for stool examination in laboratories, allowing only the use of X10 and X40 objectives which do not sometimes reveal relevant details to make an exact identification of certain protozoa. In a modification of the Glycerol Jelly (GJ) reagent, which is used in permanent preparation of helminths, five concentrations were prepared and tested for fixing the cover glass instantly while maintaining the high translucency of the fecal films. GJ reagent (7 gm gelatine dissolved in 50 ml boiling water added to 10 ml glycerol) gave satisfactory results especially with iodine and alkaline Methylene blue mounts which stained the cytological structures of protozoa while the GJ reagent enabled the examiner to use X100 oil immersion objective immediately and consequently identify protozoa with certainty and make an accurate identification. Identification of Cryptosporidium parvum oocysts by GJ wet mount, inspite of its small size, was the most impressive. GJ fecal films were examined up to 8 weeks of preparation and they were valid and reliable. GJ mount is an easy, fast and cheap technique for examining the fecal direct smear with the oil-immersion lense.

Animals↗

Comparative study of the clinical effects of vincamine + glycerol versus glycerol + placebo in the acute phase of stroke.

The treatment of the acute phase of stroke creates a difficult problem to the clinician. The presently used drugs lead to controversial results. The progress in knowledge of the pathogenesis of cerebral damage underlines the determinant role of the metabolic deficits in the ischemic areas. The effect on the clinical symptomatology of patients in the acute phase of stroke was studied during a double-blind comparative clinical trial with an alcaloid of Vinca minor (Pervincamine) which acts favorably against disturbances of oxydative glucose metabolism and of cerebral microcirculation. 20 patients divided into two homogeneous groups received during 5 days either Pervincamine (4 ampoules of 3 ml = 60 mg vincamine p.d.) + glycerol, or glycerol + placebo administered by i.v. infusion. Clinically the results indicate a greater improvement of the neurological status (objectivated by a neurological scale) with vincamine treatment than with placebo. Statistically the analysis confirms the highly significant effect of vincamine on motility of lower and upper limbs (p less than 0.02) and the significant effect on cranial nerves (homonymous hemianopsia and conjugated deviation of eyes) (p less than 0.05) and on the sphincter control level (p less than 0.05).

Adult↗

Cloning of glycerol-3-phosphate dehydrogenase genes (ZrGPD1 and ZrGPD2) and glycerol dehydrogenase genes (ZrGCY1 and ZrGCY2) from the salt-tolerant yeast Zygosaccharomyces rouxii.

The ZrGPD1 and ZrGPD2 genes encoding putative glycerol-3-phosphate dehydrogenases were isolated from the salt-tolerant yeast, Zygosaccharomyces rouxii. Both genes are homologous to GPD1 of Saccharomyces cerevisiae and are constitutively expressed in Z. rouxii cells. Putative glycerol dehydrogenase genes, ZrGCY1 and ZrGCY2, which are highly homologous to GCY1 of S. cerevisiae, were also isolated. Since the level of transcripts of ZrGCY1 and ZrGCY2 increased in Z. rouxii cells subjected to salt stress, it is suggested that the pathway of the signal transduction of salt stress controls the expression of these genes. The Accession Nos of these sequences in GenBank are as follows: ZrGPD1, AB047394; ZrGPD2, AB047395; ZrGCY1, AB047396; ZrGCY2, AB047397.

Amino Acid Sequence↗

Occurrence of monoacyl-diglucosyl-diacyl-glycerol and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol in membranes of Acholeplasma laidlawii strain B-PG9.

It is shown by thin-layer and high-performance liquid chromatography that the two membrane lipids monoacyl-diglucosyl-diacyl-glycerol (MADGlcDAG) and monoacyl-bis-glycerophosphoryl-diglucosyl-diacyl-glycerol are synthesized by Acholeplasma laidlawii strain B-PG9 when the cells are grown in two different growth media. The two lipids are also synthesized by A. laidlawii strain A-EF22 and their chemical structures have been determined previously by NMR spectroscopy. Since a reversed hexagonal phase is the only liquid-crystalline phase formed by MADGlcDAG, it is concluded that A. laidlawii strain B-PG9, in resemblance to strain A-EF22, synthesizes three membrane lipids that are able to form reversed nonlamellar phases. A comparison of the membrane lipids from the two strains shows that there is essentially one lipid from each strain that differs. However, both these lipids have common physico-chemical properties, namely the ability to form reversed nonlamellar phases. Finally, it is also shown that novel lipids may be synthesized by A. laidlawii through long-time adaptation to altered growth conditions.

Acholeplasma laidlawii↗

Identification of a mitochondrial glycerol-3-phosphate dehydrogenase from Arabidopsis thaliana: evidence for a mitochondrial glycerol-3-phosphate shuttle in plants.

We report molecular characterization of an Arabidopsis gene encoding a mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (FAD-GPDH) that oxidizes glycerol-3-phosphate (G-3-P) to dihydroxyacetone phosphate. We demonstrate through in vitro targeting assays that the encoded gene product can be imported into mitochondrial membrane systems. Enzyme activity of the protein was confirmed through heterologous expression in Escherichia coli. The Arabidopsis gene is expressed throughout plant development, but at the highest level during seed germination. We also show that expression of the Arabidopsis FAD-GPDH gene is coupled to oxygen consumption and affected by ABA and stress conditions. Together with an NAD(+)-dependent GPDH, this enzyme could form a G-3-P shuttle, as previously established in other eukaryotic organisms, and links cytosolic G-3-P metabolism to carbon source utilization and energy metabolism in plants.

Amino Acid Sequence↗

Triacylglycerol and cholesterol transport during absorption of glycerol trioleate vs. glycerol trielaidate.

We used conscious, chronic lymph-fistula rats to compare intestinal lymphatic transport of glycerol trioleate (TO) vs. glycerol trielaidate (TE) and to determine the effect of TO vs. TE on absorption and transport of cholesterol. Rats were implanted with intestinal lymph fistulas and duodenal cannulas and then given intraduodenal infusions of lipid emulsions containing purified TO or TE (40 mumol/h) and cholesterol (7.8 mumol/h + 2 microCi [14C]cholesterol). Lymph samples were collected at 0, 2, 4, 5, 6, 7, and 8 h after the start of lipid infusion. Lymphatic output and luminal and gut wall recovery of radioactive lipid at 8 h were quantified. Triacylglycerol (TG) fatty acid isomers did not affect lymphatic output of TG; lymph TG fatty acid composition and output reflected infusate composition. Lymphatic output of cholesterol (mass and radioactivity) did not differ between groups; luminal and gut wall recovery of [14C]cholesterol was also similar between groups. Similar lymphatic transport of TG and cholesterol between triolein- and trielaidin-infused rats was maintained for up to 16 h after the cessation of an infused lipid load. These results indicate that TO and TE are transported into lymph similarly, and that cholesterol absorption and transport are similar irrespective of whether TO or TE is the TG source. The data suggest that trans fatty acid-induced hypercholesterolemia is not due to altered intestinal absorption and transport of cholesterol.

Animals↗

Crenarchaeol: the characteristic core glycerol dibiphytanyl glycerol tetraether membrane lipid of cosmopolitan pelagic crenarchaeota.

The basic structure and stereochemistry of the characteristic glycerol dibiphytanyl glycerol tetraether (GDGT) membrane lipid of cosmopolitan pelagic crenarchaeota has been identified by high field two-dimensional (2D)-NMR techniques. It contains one cyclohexane and four cyclopentane rings formed by internal cyclisation of the biphytanyl chains. Its structure is similar to that of GDGTs biosynthesized by (hyper)thermophilic crenarchaeota apart from the cyclohexane ring. These findings are consistent with the close phylogenetic relationship of (hyper)thermophilic and pelagic crenarchaeota based 16S rRNA. The latter group inherited the biosynthetic capabilities for a membrane composed of cyclopentane ring-containing GDGTs from the (hyper)thermophilic crenarchaeota. However, to cope with the much lower temperature of the ocean, a small but key step in their evolution was the adjustment of the membrane fluidity by making a kink in one of the bicyclic biphytanyl chains by the formation of a cyclohexane ring. This prevents the dense packing characteristic for the cyclopentane ring-containing GDGTs membrane lipids used by hyperthermophilic crenarchaeota to adjust their membrane fluidity to high temperatures.

Chromatography, High Pressure Liquid↗

Synthesis and hypolipidemic activity of some new esters of glycerol and 2-O-methyl-glycerol with nicotinic and 5-fluoro-nicotinic acids.

A series of new esters of glycerol and 2-O-methyl-glycerol with nicotinic and 5-fluoro-nicotinic acids were synthesized and their hypolipidemic activities were comparatively tested. The two most interesting compounds, 5 and 12, show a higher activity than both nicotinic and 5-fluoro-nicotinic acids in the following experimental models: Triton and olive oil hyperdyslipemia and tests on old rats.

Animals↗

Mechanism for adaptive modification during cold acclimation of phospholipid acyl chain composition in Tetrahymena. II. Activities of 2-acyl-sn-glycerol-3-phosphorylcholine and 2-acyl-sn-glycerol-3- phosphorylethanolamine acyltransferases involving the reacylation.

The deacylation-reacylation process is very important for the alteration of phospholipid fatty acyl composition on lowering of growth temperature in Tetrahymena pyriformis (Kameyama, Y., Yoshioka, S. and Nozawa, Y., (1984) Biochim. Biophys. Acta 793, 28-33). Microsomes isolated from Tetrahymena cells have reacylation activities not only for 1-acyl-sn-glycerol-3-phosphorylcholine (1-acyl-GPC) and 1-acyl-sn-glycerol-3-phosphorylethanolamine (1-acyl-GPE) but also for 2-acyl-GPC and 2-acyl-GPE. Unsaturated fatty acyl-CoAs were in general much better substrates than saturated fatty acyl-CoAs for acylations of 1-acyl-GPC and 1-acyl-GPE. The acylation rates for 1-acyl-GPE were almost the same in palmitoleoyl-CoA, oleoyl-CoA, linoleoyl-CoA and gamma-linoleoyl-CoA. However, the acylation activity for 1-acyl-GPC was more than 2-fold higher with palmitoleoyl-CoA than with any other unsaturated fatty acyl-CoAs. In contrast, both 2-acyl-GPC and 2-acyl-GPE acyltransferases did not show a distinct preference for various acyl-CoAs, although palmitoyl-CoA was incorporated into both 2-acylphospholipids at higher rates than into 1-acylphospholipids. These specificities for various acyl-CoAs of 1-acyl- and 2-acyl-GPC and 1-acyl- and 2-acyl-GPE acyltransferases were not changed in the microsomes isolated from cells grown isothermally at 39 degrees C and 15 degrees C and cells shifted from 39 degrees C to 15 degrees C. However, the acylating ratio of linoleoyl-CoA to palmitoyl-CoA, which were chosen as typical unsaturated and saturated fatty acyl-CoAs, in the microsomes from cells grown at 15 degrees C was 1.5-3.0-times higher than in the microsomes from 39 degrees C-grown cells in four acyltransferase activities. These results suggest that the changes of acyl-CoA specificities in reacylation enzyme activities during temperature down-shift would make little contribution to the increase in unsaturated fatty acids in phospholipids, although reacylating enzymes from isothermally grown cells at lower temperature would partially participate in those increases.

Acclimatization↗

Application of glycerol to freezing bovine pancreas. Part 2. Effects of glycerol and grinding on frozen pancreas quality.

Effects of glycerol on the quality of ground and whole pancreas stored frozen at -30 degrees C were studied. The quality was assessed from quantitative changes in soluble proteins, proteolytic enzyme activity, and insulin content. Grinding was found to enhance the effects of the cryoprotector due to its uniform distribution in the tissue. A relationship between proteolytic enzyme activity and protein solubility was demonstrated.

Animals↗

Uncoupled glycerol distribution as the origin of the accumulation of 3-hydroxypropionaldehyde during the fermentation of glycerol by enterobacter agglomerans CNCM 1210

Batch fermentation of glycerol to 1,3-propanediol (1,3PPD) by Enterobacter agglomerans CNCM 1210 showed the lethal accumulation of 3-hydroxypropionaldehyde (3-HPA) when performed under initial substrate content higher than 40 g/L. Assigned to the inhibition by the NAD/NADH ratio of the 3-HPA converting enzyme: 1,3PPD dehydrogenase, intracellular assays were conducted in an attempt to identify the metabolic mechanisms involved in the increase of that ratio. An overflow metabolism through the 1,3PPD formation pathway was established, while a catabolic limitation in the oxidative branch at the level of glyceraldehyde-3-phosphate dehydrogenase occurred. Uncoupled activities of synthesis and consumption of reducing equivalents are thus suspected to provoke the increase of the NAD/NADH ratio and the subsequent accumulation of 3-HPA. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗

Thermal behaviour of cubic phases rich in 1-monooleoyl-rac-glycerol in the ternary system. 1-monooleoyl-rac-glycerol/n-octyl-beta-D-glucoside/water.

Using synchrotron X-ray diffraction the thermal behaviour was studied of the cubic phases in the 1-monooleoyl-rac-glycerol (MO)/n-octyl-beta-d-glucopyranoside (OG)/2H2O system with 58 or 45 wt % MO concentration and varying OG/2H2O contents. These MO contents correspond to a Pn3m cubic single-phase or a Pn3m cubic phase in excess water on the binary MO/water axis of the ternary phase diagram. The cubic liquid crystalline phases are stable with small fractions of OG, while higher OG concentrations trigger a cubic-to-lamellar phase transition. Moreover, with increasing OG concentration the initial Pn3m structure is completely converted to an Ia3d structure prior to the Lalpha phase being formed. Upon heating this effect is reversed, resulting in an Ia3d-to-Pn3m phase transition. For some samples additional peaks were observed in the diffractograms upon heating, resulting from the metastability notoriously shown by bicontinuous cubic phases. This judgement is supported by the fact that upon cooling these peaks were absent. Remarkably, both the Ia3d and the Pn3m cubic structures could be in equilibrium with excess water in this ternary system. A comparison is made with previous results on n-dodecyl-beta-d-maltoside (DM), showing that cubic phases with OG have higher thermal and compositional stability than with DM.

Detergents↗

Involvement of a glycerol-3-phosphate dehydrogenase in modulating the NADH/NAD+ ratio provides evidence of a mitochondrial glycerol-3-phosphate shuttle in Arabidopsis.

A mitochondrial glycerol-3-phosphate (G-3-P) shuttle that channels cytosolic reducing equivalent to mitochondria for respiration through oxidoreduction of G-3-P has been extensively studied in yeast and animal systems. Here, we report evidence for the operation of such a shuttle in Arabidopsis thaliana. We studied Arabidopsis mutants defective in a cytosolic G-3-P dehydrogenase, GPDHc1, which, based on models described for other systems, functions as the cytosolic component of a G-3-P shuttle. We found that the gpdhc1 T-DNA insertional mutants exhibited increased NADH/NAD+ ratios compared with wild-type plants under standard growth conditions, as well as impaired adjustment of NADH/NAD+ ratios under stress simulated by abscisic acid treatment. The altered redox state of the NAD(H) pool was correlated with shifts in the profiles of metabolites concerning intracellular redox exchange. The impairment in maintaining cellular redox homeostasis was manifest by a higher steady state level of reactive oxygen species under standard growth conditions and by a significantly augmented hydrogen peroxide production under stress. Loss of GPDHc1 affected mitochondrial respiration, particularly through a diminished capacity of the alternative oxidase respiration pathway. We propose a model that outlines potential involvements of a mitochondrial G-3-P shuttle in plant cells for redox homeostasis.

Abscisic Acid↗

Divergent transcription of the sn-glycerol-3-phosphate active transport (glpT) and anaerobic sn-glycerol-3-phosphate dehydrogenase (glpA glpC glpB) genes of Escherichia coli K-12.

The glpTQ operon and the glpA and glpB genes are located adjacent to one another near min 49 of the linkage map of Escherichia coli K-12. The positions and directions of transcription of the glpA and glpB genes with respect to the glpTQ operon were determined in the present work. Strains harboring Mu d1(Ap lac) fusions in either glpA or glpB were converted to the respective lambda p1(209) lysogens. Induction of these lysogens with mitomycin C resulted in production of Lac+ phage progeny which carried adjacent chromosomal DNA. Genetic crosses with a collection of glpT mutant strains were performed with several such phage lines. A fine-structure deletion map of the glpT gene was thus constructed. All phages used for this mapping carried DNA starting with the promoter-proximal end of glpT. This indicated that the glpTQ operon and the glpA and glpB genes are transcribed divergently. Additional evidence supporting this conclusion was obtained by physical mapping of restriction endonuclease cleavage sites in plasmids carrying these genes and in plasmids carrying glpA-lacZ or glpB-lacZ fusions. A new designation (glpC) for the gene encoding the 41,000-Mr subunit of the anaerobic sn-glycerol-3-phosphate dehydrogenase was proposed to distinguish it from the glpA gene, which encodes the 62,000-Mr subunit of the dehydrogenase, and the glpB gene, which encodes a membrane anchor subunit of the dehydrogenase. These three genes were present in an operon transcribed in the order glpA glpC glpB in the clockwise direction on the linkage map of E. coli.

Alleles↗