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A successful pregnancy from the oocyte donation programme in Singapore General Hospital.

A 41-year-old Chinese woman was seen in our centre in June 1989 with 5 years of premature menopause, requesting ovum donation. She was placed on cyclical hormonal therapy and put on the waiting list for ovum donation. Excess ova became available through one of our IVF patients and were fertilised with her husband's semen. Four pre-embryos resulted and these were cryopreserved using 1,2 propanediol. Embryo transfer in her second cycle of trying on 16 March 1990 of 2 pre-embryos was successful and a singleton pregnancy resulted. The pregnancy was supported with estradiol valerate and intramuscular progesterone till 17 weeks of gestation. The pregnancy went on uneventfully and she delivered a healthy baby boy by Caesarean section on 21 December 1990.

Adult↗

[Change in the cleavage site of synthetic substrates of SsoII restriction endonucleases upon introduction of non-nucleotide inserts in the recognition segment].

The cleavage of synthetic DNA duplexes containing 1,3-propanediol, 1,2-dideoxy-D-ribofuranose or 9-[1'-hydroxy-2'-(hydroxymethyl)ethoxy]methylguanine (glG) residues instead of one of dG residues or one of the nucleosides of the central base pair of the recognition site by SsoII restriction endonuclease (decreases CCNGG) has been studied. It is found that the non-nucleotide insertions (except for glG) result in a change of the SsoII cleavage site and an increase of the efficiency of the cleavage. The novel noncanonical cleavage occurs at the phosphodiester bond adjoining the non-nucleotide insert from the 5'-end.

Autoradiography↗

[The first pregnancy and birth after cryopreservation of oocytes in the Czech Republic--importance of the method for reproductive medicine].

OBJECTIVE: The purpose of this work was to develop methods for successful cryopreservation of human oocytes. DESIGN: Case-controlled study and case report. SETTING: Sanatorium Pronatal, Prague. METHODS: Propanediol (PrOH)-sucrose was used as cryoprotectant medium for cumulus-free oocytes and stepwise dilution of cryoprotectant post-thaw. RESULTS: The method was used in three patients (38 denuded oocytes) and yielded excellent survival and fertilization rates (89.5% and 73.5% respectively). In all patients embryo-transfer was possible, one patient got pregnant and delivered a healthy baby. CONCLUSION: Our data show that cryopreservation may ensure that the integrity of the human oocyte is adequate for normal fertilization and embryo development.

Adult↗

Is there any future for felbamate treatment?

Felbamate (2-phenyl-1,3-propanediol dicarbamate), a representative of novel antiepileptic drugs (AESs), proved to have broad-spectrum anticonvulsive activity. Particularly beneficial efficacy was found against partial seizures and Lennox-Gastaut syndrome. Therefore, felbamate started to be indicated not only as an adjunctive antiepileptic drug but also in monotherapy. Unfortunately, it was also evidenced that the drug may induce aplastic anemia or hepatic failure. The former complication was frequently described in patients with previously diagnosed hematopoetic disturbances. Thirty-four cases of well-documented bone marrow suppression, occurred fatal in thirteen cases. Subsequently, felbamate's usage was essentially restricted and at present felbamate is not a first-line AED. However, excluding anemia-prone individuals, new possibilities may open for felbamate position in add-on therapy of drug-resistant epilepsy. Experimental studies provide a good theoretical basis for this kind of treatment.

Anemia, Aplastic↗

Some recent advances in the symptomatic treatment of osteoarthritis.

In over 400 treatments with procaine intravenously, moderate to good improvement was noted in about 60 per cent of patients with osteoarthritis and radiculitis. Definite improvement after a single treatment was noted occasionally, but more often relief was not obtained until six to eight treatments had been given. Symptoms due to osteoarthritic changes in peripheral joints did not respond as well as did those due to spinal arthritis. Curare relieves some of the pain of arthritis due to muscle spasm, but does not bring improvement in motion of the small joints, such as those of the fingers. Preliminary experience with a new synthetic drug, 3-ortho-toloxy-1, 2-propanediol (Tolserol(R)), which produces muscular relaxation differing from that induced by curare, suggests further clinical trial.Intra-articular acidification by injection relieved pain in about 50 per cent of patients with osteoarthritis of the knee.

Female↗

Integrity of endothelium in cryopreserved human cornea.

The aim of the present study was to elaborate an optimal method for cryopreservation of human donor cornea for transplantation and to follow the morphological changes in the structure of the endothelial cell layer using scanning electron microscopy (SEM). Sixteen groups, with four donor cornea each, were cryopreserved at cooling rates of 1 degree C per min and 5 degree C per min. Four cryoprotectants (glycerol, dimethyl sulfoxide, 1,2-propanediol, polyethylene glycol-400) in two concentrations (5% and 10% v/v) were prepared on the bases of medium Optisol GS supplied with 20% v/v human serum albumin. Four additional human cornea were used as controls. Endothelial cell recovery of the cornea after thawing and 24 hours culture, was calculated as a percent of the preserved recovered cells. Sufficient recovery of the endothelial cell layer, making the cornea suitable for transplantation was obtained using the cryoprotectants dimethyl sulfoxide and especially polyethylene glycol-400.

Adult↗

[Co-expression of gpd1 and hor2 from Saccharomyces cerevisiae in Escherichia coli].

Based on the principle of the pathway engineering, a novel pathway of producing glycerol was built in E. coli. The gpd1 gene encoding glycerol 3-phosphate dehydrogenase and the hor2 gene encoding glycerol 3-phosphatase were cloned from Saccharomyces cerevisiae, respectively. The two genes were inserted into expression vector pSE380 together. A recombinant plasmid pSE-gpd1-hor2 containing polycistron was constructed under the control of the strong trc promoter. Then it was transformed into E. coli BL21. The result showed the recombinant microorganism GxB-gh could convert glucose to glycerol directly. And the recombinant microorganism GxB-gh was incubated to produce glycerol from D-glucose in the fermentor. The maximal concentration of glycerol was 46.67g/L at 26h. Conversion rate of glucose was 42.87%. The study is about "green" producing glycerol by recombinant microorganism and is also useful for further working in recombining microorganism of producing 1,3-propanediol.

Cloning, Molecular↗

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.

Animals↗

[A silicon phthalocyanine axially substituted by nipagin: synthesis, molecular spectroscopic properties, and in vitro photodynamic activities].

A new silicon phthalocyanine axially substituted by Nipagin, i.e. bis(4-methoxycarboxyl phenoxy)phthalocyaninatosilicon, has been synthesized and characterized by IR, NMR, HPLC and elemental analysis. Its electronic absorption spectra and fluorescence spectra in different solvents were investigated. It was found that the compound existed in the form of monomer in a saline solution containing 2% (phi) Cremophor EL and 20% (phi) propanediol. The electronic absorption spectra of the compound in the above saline solution were typical for non-aggregated phthalocyanines, showing an intense and sharp Q band at 683 nm with a molar absorption coefficient of 7.47 x 10(4) mol(-1) x L x cm(-1). The compound exhibited a relatively strong fluorescence emission at 690 nm with a quantum yield of 0.34 and with a fluorescence lifetime of 4.7 ns. The primary in vitro assay showed the compound had photodynamic killing activities against B16 melanoma cells with a LD50(half lethal dose) of 1.2 x 10(-4) mol x L(-1).

Chromatography, High Pressure Liquid↗

Disposition, metabolism, and excretion of the anticancer agent crisnatol in the rat.

Disposition and metabolism of crisnatol (14C-labeled), a novel antitumor agent, was examined after po and iv administration to rats. After both routes of drug administration, there was rapid elimination of the administered radioactivity in the urine (6-12% of the dose) and feces (81-92% of the dose). The drug appeared to be rapidly absorbed after oral dose and there was substantial "first-pass" metabolism. Analysis of the excreta indicated extensive metabolism of crisnatol by the rat, with the intact compound being the major radiolabeled component in the feces (17-20% of dose). Intact drug was not present in urine. Biotransformation of crisnatol by the rat mainly involves oxidation and conjugation pathways. Hydroxylation and dihydrodiol formation in the chrysene ring and oxidation of the propanediol side chain resulted in the formation of the three major fecal metabolites. The principal metabolite in the urine was also a dihydrodiol. Concentrations of intact drug in each tissue assayed exceeded those in plasma, and in the lungs the tissue/plasma ratio approached 300 and 82 at 2 hr after iv and po doses, respectively.

Animals↗

[Synthesis of ethriolophospholipids of acetal type].

New analogues of acetal-type phospholipids were obtained on the basis of ethriol (2-hydroxymethyl-2-ethyl-1,3-propanediol). The starting triol originally was condensed with decanal or dodecanal to form acetals, which were then phosphorylated with tetraethyldiamidophosphorous acid chloride. The amidophosphites were further oxidized with iodosobenzene or sulfurized to the corresponding acetal-type phospholipids and their thio analogues.

Acetals↗

Optimal conditions for assaying human lactate dehydrogenase by the lactate-to-pyruvate reaction: Arrhenium relationships for lactate dehydrogenase isoenzymes 1 and 5.

Optimal reaction conditions to sassay human lactate dehydrogenase (lactate-to-pyruvate) were established for isoenzymes 1 and 5 at 25, 30, and 37 degrees C in diethanolamine and 2-amino-2-methyl-1,3-propanediol. Different substrate concentrations are required at each temperature. The conditions permit measurement of lactate dehydrogenase 1 and 5 with the lowest substrate concentrations that allow for the highest equal sustainable efficiency in measuring both isoenzymes. About 95% of each isoenzyme activity is measured if the assay is performed within the first minute after the reaction is initiated even for activities as high as triple the upper limit of normal. The Arrhenius relationship is different for each isoenzyme, but results obtained for each at one temperature can be compared with results at another temperature by use of simple conversion equations. Assays at 25 and 30 degrees C are more economical and less variable than assays at 37 degrees C.

Buffers↗

A procedure for cryopreservation of hamster oocytes yielding highly conserved oocytes suitable for sperm penetration tests.

Hamsters were superovulated and their oocytes frozen in Dulbecco's phosphate-buffered saline containing 10% fetal calf serum and 1.5 molar 1,2-propanediol. In several experiments, 94% of the hamster oocytes survived freezing and were equivalent to fresh oocytes in sperm penetration. In routine use of the procedure described, also 94% of 1,340 frozen-thawed oocytes were satisfactory for sperm penetration of several species. Frozen oocytes eliminate problems and costs of maintaining hamster colonies with variable responses on individual days.

Analysis of Variance↗

Fatal propylene glycol toxicosis in a horse.

Toxicosis attributable to propylene glycol (1,2-propanediol) was suspected in an 8-year-old 450- to 500-kg male Quarter Horse. Clinical signs of toxicosis developed within 15 minutes of the accidental iatrogenic oral administration of 3.8 L of propylene glycol. Clinical signs of toxicosis included salivation, sweating, ataxia, and signs of pain. Additionally, at 24 hours after propylene glycol ingestion, the horse became increasingly atactic, had an abnormal breath odor, developed rapid shallow breathing, and was cyanotic. The horse died of apparent respiratory arrest 28 hours after the propylene glycol ingestion. Analysis of serum and combined urine and blood from the kidneys confirmed the presence of propylene glycol. Propylene glycol is used for the treatment and prevention of bovine ketosis, and is similar in appearance to mineral oil. The accidental administration of propylene glycol to horses may result in fatal poisoning.

Animals↗

Propylene glycol ingestion causes D-lactic acidosis.

Propylene glycol (1,2-propanediol) is a solvent in numerous pharmaceuticals and a major preservative and source of carbohydrates in processed foods. In mammals, propylene glycol is metabolized similar to ethanol, proceeding via hepatic alcohol and aldehyde dehydrogenases to lactate, which can then enter gluconeogenesis. We observed that cats ingesting 1.6 gm of propylene glycol/kg body weight/day developed increased anion gap. To investigate this further, we measured D- and L-lactate concentrations in these cats; we also measured D-lactate in cats ingesting high doses of propylene glycol (8.0 gm/kg). While L-lactate actually decreased throughout the 35-day course of propylene glycol feeding, D-lactate levels were significantly increased on a dose-dependent basis and correlated positively with anion gap. In cats ingesting the high dose of propylene glycol, D-lactate concentrations were as high as 7 mmol/liter, levels associated with encephalopathy in humans. Indeed, this group of cats developed depression and ataxia, consistent with intoxication by D-lactate. These findings are significant not only for animals ingesting diets which contain propylene glycol, but for humans who receive propylene glycol-containing medications.

Acidosis, Lactic↗

The embryo freezing programme at Groote Schuur Hospital. First results.

'Surplus' embryos from 28 patients on the in vitro fertilisation programme at Groote Schuur Hospital were frozen for transfer in later cycles. Depending on the stage of the embryos, either propanediol or glycerol was used as a cryoprotectant. In 20 patients embryos survived the thawing procedure and were transferred. Five pregnancies resulted, 2 twin and 3 singleton. The pregnancy rate per embryo transfer in this group of patients in thus 25%. The pregnancy rate per freezing procedure is 19.2%.

Cryopreservation↗

[Comparison of various methods of cryopreservation of human pre-embryos].

A human egg and embryo freezing program for non-fertilized eggs and spare embryos has been established in our in vitro fertilization clinic. In the following we report on our first experience with different methods of human pre-embryo cryopreservation. In a simple evaluation we have compared different conventional freezing techniques using cryoprotectant substances such as glycerol, DMSO and 1.2 propanediol and an "ultrarapid" technique. So far we have achieved 6 normal ongoing and 2 biochemical pregnancies out of a total number of 61 embryo transfers. This equals 9.8% pregnancy rate per embryo transfer. Two methods seem preferable, i.e. sucrose and 1.2 propandiol which has the highest success rate and the ultrarapid method which gives a good rate of embryo survival on the one hand and a simple handling on the other.

Embryo Transfer↗

Hydrogen peroxide mediates the oxidative inactivation of enzymes following the switch from anaerobic to aerobic metabolism in Klebsiella pneumoniae.

Klebsiella pneumoniae utilizes distinct pathways for the anaerobic and aerobic metabolism of glycerol. During anaerobic growth, glycerol is first converted to dihydroxyacetone by glycerol dehydrogenase; subsequent phosphorylation yields dihydroxyacetone phosphate. During aerobic growth, glycerol is initially phosphorylated to yield glycerol 3-phosphate; subsequent reduction then gives dihydroxyacetone phosphate. A coordinated response occurs when anaerobically growing cells are switched to aerobic conditions. Synthesis of glycerol dehydrogenase is repressed, glycerol dehydrogenase is inactivated, and the protein is degraded. Ethanol dehydrogenase and propanediol oxidoreductase are also inactivated when cells are exposed to oxygen (Johnson, E. A., Levine, R. L., and Lin, E. C. C. (1985) J. Bacteriol. 164, 479-483). Exposure of anaerobically growing cells to low concentrations of hydrogen peroxide also inactivated these three enzymes and led to rapid degradation of glycerol dehydrogenase. Glycerol dehydrogenase was purified and characterized after in vivo oxidative modification initiated by hydrogen peroxide. No differences in molecular weight, amino acid composition, or Km were detected between the native and oxidatively modified forms, although the modified enzyme had only 10% of the catalytic activity of the native form. The oxidatively modified enzyme was very susceptible to degradation by subtilisin while the native enzyme was resistant. Chloramphenicol prevented the inactivation and degradation of glycerol dehydrogenase caused by exposure to oxygen but did not block that caused by hydrogen peroxide. Thus, protein synthesis appears necessary for in vivo oxidative modification caused by exposure to oxygen but is not necessary when the process is initiated by exposure to hydrogen peroxide. The newly synthesized protein(s) presumably catalyzes the production of hydrogen peroxide which is required for the metal-catalyzed oxidative modification of susceptible enzymes.

Aerobiosis↗