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PubMed2003

[Application of gas chromatography-mass spectrometry analysis on urine filter paper in the high-risk screening and diagnosis of inherited metabolic diseases].

OBJECTIVE: To establish a specific procedure for the high-risk screening and diagnosis of organic acidurias and other inherited metabolic diseases in China. METHODS: A nation-wide network for the high-risk screening and diagnosis of genetic metabolic diseases was formed to facilitate the collaboration. Urine samples were collected using filter paper from patients with clinical symptoms suspicious of inherited metabolic diseases. The samples were eluted with distilled water and internal standards were added. Samples were treated with hydroxylamine hydrochloride to form oximes to improve the recoveries of 2-ketoacids. Urinary organic acids were extracted with ethyl acetate and diethyl ether under acidic condition. After dehydration, the combined organic phase was evaporated to dryness with nitrogen. The residues were added with BSTFA + 1%TMCS and heat incubated to form the trimethylsilyl derivatives, and then were analyzed on an Agilent 5890/5973N gas chromatography-mass spectrometer (GC-MS), with a 7683 series auto-sampler. The peaks were identified by reference to a mass spectral library. RESULTS: Totally 352 samples were collected from the network collaborating hospitals since 2001. Thirty-four (9.66%) cases of various inherited metabolic diseases were diagnosed with an age range of 2 days to 14 years. The disease profile was consisted of methylmalonic acidemias (6), alpha-keto-glutaric aciduria (5), tyrosinemia type I (4), dicarboxylic aciduria (4), multiple carboxylase deficiency (3), phenylketonuria (3), lactic acidemia (3), propionic acidemia (2), ornithine transcarbamoylase deficiency (1), ethylmalonic-adipic aciduria (1), glutaric aciduria type II (1) and 3-methylcrotyl CoA carboxylase deficiency (1). The most common clinical symptoms and signs included mental and developmental retardation, convulsion, musculotonic abnormality and jaundice. Routine laboratory tests often revealed metabolic acidosis, hypoglycemia and hyperammonemia, etc. CONCLUSION: Urine organic acids analysis by GC-MS remains to be the most important technique for the high-risk screening and diagnosis of inherited metabolic diseases. Use of urine filter paper for sample collection and analysis in advanced genetic metabolic centers is a practical approach to extend the diagnostic capacity and improve the management of such diseases in China. Collaborative network played a critical role in the success of the program.

Adolescent↗
PubMedDate not supplied

[Giant retroperitoneal lipoma].

70 retroperitoneal tumors (10 with an adipous origin out of which 8 were lipomas) have been operated upon in our clinic since 1985. Four cases of giant lipomas are presented in this paper. They weighed 4.5-7.5 kg. and had a diameter over 15 centimeters. Their close proximity with the kidney, duodenum, colon, inferior vena cava etc made there extirpate difficult. The post-operative condition of the patients was good. Although the histopathological tests proved that all these giant lipomas were benign after two to four years, their relapse required a new surgical intervention. The histopathology of the relapsed lipomas revealed a malign degeneration in 3 out of the four cases. The origin of this malign degeneration was in the fibrous part of the tumors.

Female↗
PubMed1992

Yeast lacking superoxide dismutase. Isolation of genetic suppressors.

Null mutants of superoxide dismutase (SOD) in Saccharomyces cerevisiae are associated with a number of biochemical defects. In addition to being hypersensitive to oxygen toxicity, strains containing deletions in both the SOD1 (encoding Cu/Zn-SOD) and SOD2 (encoding Mn-SOD) genes are defective in sporulation, are associated with a high mutation rate, and are unable to biosynthesize lysine and methionine. The sod-linked defect in lysine metabolism was explored in detail and was found to occur at an early step in lysine biosynthesis, evidently at the level of the alpha-amino adipate transaminase. To better understand the role of SOD in cell metabolism, our laboratory has isolated yeast suppressors that have bypassed the SOD defect ("bsd" strains), that is, S. cerevisiae cells lacking SOD, yet resistant to oxygen toxicity. Two nuclear bsd complementation groups have been identified, and both suppress a variety of biological defects associated with sod1 and sod2 null mutants. These results demonstrate that a single gene mutation can alleviate the requirement for SOD in cell growth. Both bsd complementation groups are unable to utilize many non-fermentable carbon sources, suggesting a possible suppressor-linked defect in electron transport.

Diploidy↗
PubMed2005

Immunoprotective potential of polysaccharide-tetanus toxoid conjugate in Klebsiella pneumoniae induced lobar pneumonia in rats.

The polysaccharide (PS) derived from K. pneumoniae NCTC 5055 lipopolysaccharide (LPS) was covalently linked to tetanus toxoid by using carbodimide with adipic acid dihydrazide as a spacer molecule. The conjugate was found to be non-toxic and non-pyrogenic at 100 microg dose level. At a similar dose, the conjugate did not elicit any local skin reaction on intradermal preparatory injection in rabbits. The conjugate was immunoprotective as was evident from the decrease in relative colonization of bacteria in lungs of immunized rats as compared to the control animals. Immunization with the conjugate resulted in alveolar macrophage activation in terms of their ability to phagocytose bacteria in vitro.

Animals↗
PubMed2004

[Infrared absorption spectroscopy of optical waveguide in proton-exchanged LiNbO3].

Z-cut proton-exchanged LiNbO3 optical waveguides exchanged with different proton sources mixed with adipic acid and benzoic acid in different mol ratios were analyzed by infrared absorption spectroscopy. The result shows that there is an absorption peak of OH- free group at 3500 cm(-1) with the characteristic of substitute protons, and the absorption peak of H-bond is nearby 700 cm(-1) with the characteristic of packing protons. The different mixing ratios and different concentrations of H+ in the proton source, result in different proton-exchanged velocities and bring on the change in these absorption peaks. It is very important to study the change in these absorption peaks for making high quality LiNbO3 optical waveguide.

English Abstract↗
PubMed2005

Inhibitory effects of some possible endocrine-disrupting chemicals on the isozymes of human 11beta-hydroxysteroid dehydrogenase and expression of their mRNA in gonads and adrenal glands.

The in vitro inhibitory effects of 23 compounds designated as possible endocrine-disrupting chemicals, such as alkylphenols, phthalate esters, phytoestrogens, organotins, and pesticides, on the enzyme activity of the isozymes of human 11beta-HSD, type 1 and 2, were examined. Human liver microsomal 11beta-HSD type 1 was inhibited by 4-nonylphenol (4-NP) and diethylhexyl adipate (IC50 = 49.3 and 49.4 mM, respectively). Further, human kidney microsomal 11beta-HSD type 2 was inhibited by triphenyltin, tributyltin, 4-t-octylphenol, 4-NP, 4-n-octylphenol, 4-n-nonylphenol, and dicyclohexyl phthalate (IC50 = 3.3, 16.5, 8.9, 20.3, 23.5, 26.2 and 46.5 microM, respectively). The other chemicals studied did not significantly inhibit the enzyme at concentrations lower than 50 microM. The expression of mRNA of the isozymes in gonads, accessory genital glands, and adrenal glands were investigated by RT-PCR. 11beta-HSD type 1 was detected in human testis, ovary, and prostate. The level in the adrenal gland(s) was similar to that in the liver, which is known to be expressed in large quantities. In contrast, except for the prostate, only small amounts of the 11beta-HSD type 2 isozyme were detected in these human tissues compared to kidney.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMedDate not supplied

Hepatomegaly is an early biomarker for hepatocarcinogenesis induced by peroxisome proliferators.

The relationship between hepatomegaly and the hepatocarcinogenesis associated with by peroxisome proliferators was examined. (1) Male F-344 rats were maintained on diets containing clofibrate, ciprofibrate, nafenopin, gemfibrozil, Wy-14, 643, di(2-ethylhexyl)phthalate (DEHP), or di(2-ethylhexyl)adipate (DEHA) at carcinogenic doses for 1 week. A close correlation between relative liver weights and hepatocarcinogenicity was observed (r = 0.910). (2) Administration of perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), simfibrate, or DL-040, for which hepatocarcinogenicity is not known, resulted in hepatomegaly in all treated groups, this being especially marked in the PFOA case. Therefore, PFOA may have strong hepatocarcinogenic potential. (3) Administration of the antioxidants butylated hydroxyanisole (BHA) or vitamin E (VE) did not affect the hepatomegaly induced by DEHP. These results suggest that the hepatomegaly may be an early biomarker for prediction of the potential hepatocarcinogenicity of peroxisome proliferators. However, this requires further clarification in terms of its relation to the oxidative stress thought to be involved in peroxisome proliferator-induced hepatocarcinogenesis.

Animals↗
PubMed1990

[Pathophysiology of intraocular neovascularization].

The pathology of intraocular neovascularization was studied in human and animal eyes by means of electron microscopy and histochemistry, and also by tissue culture of bovine retinal small vessels. The newly formed vessels in the vitreous obtained at the time of vitrectomy from the eyes with proliferative diabetic retinopathy and retinal vein occlusion lacked tight junction and formed fenestration in the endothelial cells. When 1 microgram of the basic fibroblast growth factor (b-FGF) enveloped in ethylene vinyl acetate copolymer was implanted into the vitreous of monkey eyes, new vessels were formed in the iris in all eyes and in the vitreous in 12 eyes out of 14 experimental eyes. The origins of new vessels were the iris vessels in the iris, and both stromal vessels of the ciliary body and retinal vessels in the vitreous. These vessels showed fenestration in the endothelial cells. The activity of the lysosomal enzyme detected by acid phosphatase increased in the epithelial layer of the ciliary body. The new vessels in the vitreous of rabbits were seen in 9 out of 10 eyes when b-FGF was implanted in the vitreous with an intravitreal injection of amino adipic acid solution (1 mg in 0.2 ml of physical saline), although none of the 8 eyes formed new vessels when sodium iodate was injected intravenously after implantation of b-FGF with aminoadipic acid. Corneal neovascularization was formed by implantation of 250 ng of b-FGF into the corneal micropockets of the guinea pigs, and regressed after the b-FGF was removed. The endothelial budding and protrusion were frequently seen during the course of neovascularization. Immunohistochemical detections showed positive stainings for fibronectin in most front-end lesions, for laminin and type 4 collagen associated with the endothelial cells, and for factor VIII only on the endothelial cells which formed the vascular lumen. The acid phosphatase activity was detected on the leucocytes infiltrating in the corneal stroma. In the course of regression of corneal neovascularization, initial pathological change was thrombus formation followed by disappearance of endothelial cells, although the basement membrane of the endothelial cells remained. Fibronectin reduced its activity in the early stage of regression, laminin and type 4 collagen remained even after the vascular lumen had subsided, and factor VIII was stained in a geographically irregular manner. Migrating activity of the cultured-bovine retinal small vessels was accelerated by fibronectin and fetal bovine serum.

Animals↗
PubMed1991

Study of the acetylcholinesterase activity of Ascaridia galli: kinetic properties and the effect of anthelmintics.

Acetylcholinesterase (EC 3.1.1.7) activity was demonstrated in whole worm homogenates of adult Ascaridia galli with acetylthiocholine as substrate. The pH optimum was not measurable because of an autohydrolysis of the substrate. The Michaelis constant (Km) was 4 mM with saturation by excess substrate. Optimum enzyme activity was noted at a protein concentration of 200 mg/ml assay medium and at a temperature of 37 degrees C. Arrhenius plot of temperature dependence of the enzyme activity showed an energy of activation (delta Ea) of 28.962 K joule/mole above, and 25.448 K joule/mole below, the transition temperature (37 degrees C). Complete inhibition by eserine (physostigmine), a specific and classical acetylcholinesterase inhibitor, established the identity of the enzyme. A marginally higher enzyme activity was observed in females than in males as well as in homogenates from worms of mixed sexes. The enzyme was markedly activated by divalent metal cations such as Fe2+, Mg2+, Cd2+, Cu2+, Zn2+ and Ca2+, while Co2+ and Mn2+ inhibited the activity. Piperazine adipate at a concentration of 10(-3) M caused 45.5% and albendazole, a benzimidazole anthelmintic, 37.5% inhibition in the enzyme activity, while levamisole and mebendazole proved to be practically ineffective, causing an inhibition of 12 and 9%, respectively.

Acetylcholinesterase↗
PubMed1990

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes.

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.

Amino Acids↗
PubMed1990

[10-Acyldithranol dimers; new derivatives of the antipsoriatic dithranol; synthesis, characterization and biochemical properties].

With regard to the synthesis of 10-(omega-carboxyacyl)-derivatives dithranol 1 was reacted with the carboxylic acid dichlorides of succinic acid, glutaric acid, adipic acid and pimelic acid in toluene and collidin as a base. Instead of the expected derivatives the 10-acyldithranol dimers 5-7 were isolated as main products except for the reaction of succinyl chloride where only lactone 2 was formed. Moreover the 6-ring lactone 3 as a side product and traces of the 7-ring lactone 4 could also be isolated and characterized. All compounds revealed to be inhibitors of the enzyme glucose-6-phosphate dehydrogenase, indicating antipsoriatic activity.

Anthralin↗
PubMed1989

Leydig cell tumor of testis with adipose metaplasia.

A Leydig cell tumor of testis was discovered during evaluation of infertility in a 36-year-old white man with azoospermia and high plasma estrogen level. Histological examination revealed multiple foci of calcification and massive adipose metaplasia. This is the first time that adipous metaplasia is observed in association with Leydig cell tumor of testis.

Adipose Tissue↗
PubMed1989

[Analysis of the effect of quisqualate, N-methyl-D- aspartate and several blockers of amino acid receptors on synaptic transmission in the ampullae of Lorenzini].

Effects of bath-application of quisqualate (Q), N-methyl-D-aspartate (NMDA) and antagonists of NMDA-receptors: D-amino-adipate (AA), 2-amino-4-phosphonobutyrate (APB), 2-amino-5-phosphonovalerate (APV) and Mg2+ as well as acidic amino acid antagonist: D-glutamylglycine (DGG) on the synapse between the electroreceptor cells and afferent fibres were studied in the ampullae of Lorenzini. Q (threshold concentration 10(-8) M) and NMDA (threshold concentration 10(-5) M) strongly excited afferent fibres. Neither AA nor APB influenced the resting and evoked activities. APV blocked the synaptic transmission. Mg2+ (30-50 mM) blocked the responses to NMDA, while Q-induced responses were not affected, APV preferentially blocked NMDA-induced responses and in lesser degree--L-aspartate (L-ASP)-induced responses. DGG blocked the synaptic transmission. It is supposed that the synaptic receptor could represent a homogeneous receptor with different binding sites to the known agonists.

2-Amino-5-phosphonovalerate↗
PubMed1989

Cyclic hexapeptides bearing carboxyl groups. Interaction with metal ions and lipid membrane.

Three cyclic hexapeptides bearing carboxyl groups, cyclo(L-Asp-L-Phe-L-Pro)2, cyclo(L-Aad-L-Phe-L-Pro)2 (Aad represents alpha-amino adipic acid residue), and cyclo(D-Asp-D-Phe-L-Pro)2 were synthesized and investigated on conformation, complexation with metal ions, and interaction with lipid membrane. These cyclic hexapeptides were found to take several different conformations, though their reference compounds, cyclo(L-Leu-L-Phe-L-Pro)2 and cyclo[D-Asp(OMe)-D-Phe-L-Pro]2, took a single C2 symmetric conformation. Cyclo(D-Asp-D-Phe-L-Pro)2 formed complexes with Ba2+ and Ca2+, whereas cyclo(L-Asp-L-Phe-L-Pro)2 and cyclo(L-Aad-L-Phe-L-Pro)2 did not. The latter two take amphiphilic structures and were distributed to lipid membrane more easily than cyclo(D-Asp-D-Phe-L-Pro)2. Cyclo(D-Asp-L-Phe-L-Pro)2 binds Ca2+ on the lipid membrane.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1987

Scanning electron microscopy of human and murine melanoma cells exposed to medium chain-length (C6-C12) dicarboxylic acids in tissue culture.

Human and murine (Harding-Passey and Cloudman) melanoma cells were exposed to various concentrations (1 x 10(-3) M-1 x 10(-1) M) of adipic (C6), azelaic (C9), and dodecanedioic (C12) acids for 1-6 hours in tissue culture, and the effects on shape and surface topography were examined by scanning electron microscopy. Effects, i.e., rounding up, concentration of microvilli, blebbing, and prominence of retraction fibrils were time and dose dependent, and for the same concentrations and exposure times, C12 had a greater effect than C9, and both a significantly greater effect than C6. These differential reactions to the three diacids parallel previously reported effects on cell kinetics and viability. The changes could be due to a prime effect on the cell membrane, or they might reflect phases of the cell cycle directed by action of the diacids on the nucleus; this latter seems unlikely. An effect on the cytoskeleton is possibly involved.

Animals↗
PubMed1988

Enhanced binding of capsular polysaccharides of Cryptococcus neoformans to polystyrene microtitration plates for enzyme-linked immunosorbent assay.

A sensitive enzyme-linked immunosorbent assay (ELISA) to measure antibodies against capsular polysaccharide was developed, based on the enhanced binding of polysaccharide to polystyrene microtitration plates. The wells of the microtitration plate were primed with an adipic acid dihydrazide derivative of bovine serum albumin (AH-BSA) (100 micrograms/mL, 0.01 M NaPO4-0.14 M NaCl, pH 7.2 (PBS]. Capsular polysaccharide, the glucuronoxylomannan of Cryptococcus neoformans serotype A, was oxidized with NaIO4 for 5 min; the reaction was then quenched with ethylene glycol. The partially oxidized polysaccharide was dialyzed vs. PBS, and its concentration was adjusted to 50 micrograms/mL with PBS. This solution (100 microL/well) was covalently bound to the AH-BSA primed microtitration plates through formation of a Schiff base between the hydrazide group on the AH-BSA and the aldehyde groups on the polysaccharide. Antimouse IgG-alkaline phosphatase conjugate was used in an indirect ELISA to measure captured murine monoclonal antibodies directed against glucuronoxylomannan. Mean absorbances, after 15 min, were 0.13 in negative control wells, and greater than 0.7 in test wells. No intermediate steps were required to block nonspecific binding of antibody.

Antibodies↗
PubMed1986

[A high-capacity affinity gel for the purification of the testicular lutropin receptor].

The lutropin (LH) receptor from porcine testes was solubilized with 1% Triton X-100. The final solution was centrifuged at 100,000 X g and the supernatant was concentrated on a PM30 Amicon membrane. The concentrate was then specifically retained on an affinity gel with human choriogonadotropin (hCG) covalently grafted by its sugar moiety. The agarose affinity gel bore adipic acid hydrazide spacer arms (6 carbon length). Prior to coupling, the subunits of hCG were crosslinked with 1-ethyl-3-(dimethylamino propyl) carbodiimide and the sialic acid residues were subjected to periodate oxidation to yield aldehyde groups which could react with the hydrazides of the gel. 90% of the receptor contained in the solution incubated with this gel was retained and eluted at pH 3.2 with a purification factor greater than 700, versus a 26% yield and a purification factor of 40 with an affinity gel bearing hCG linked through its lysine epsilon-amines.

Animals↗
PubMed1986

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase.

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.

1-Pyrroline-5-Carboxylate Dehydrogenase↗