The role of embolisation in ENT: an update.

OBJECTIVE: To provide an update on the ever-increasing role that embolisation plays in the practice of otolaryngology. METHOD: A literature search was performed during November 2008. The Medline, Embase, PubMed and Cochrane databases were searched. This resulted in 285 papers relevant for review. CONCLUSION: The role of embolisation has expanded greatly to include the management of refractory epistaxis, pre-operative preparation of vascular tumours, vascular injuries and as an adjunct in skull base surgery.

Glycosylasparaginase inhibition studies: competitive inhibitors, transition state mimics, noncompetitive inhibitors.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond between asparagine and N-acetylglucosamine in the catabolism of N-linked glycoproteins. Previously only three competitive inhibitors, one noncompetitive inhibitor, and one irreversible inhibitor of glycosylasparaginase activity had been reported. Using human glycosylasparaginase from human amniotic fluid, L-aspartic acid and four of its analogues, where the alpha-amino group was substituted with a chloro, bromo, methyl or hydrogen, were competitive inhibitors having Ki values between 0.6-7.7 mM. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction. A proposed phosphono transition state mimic and a sulfo transition state mimic were competitive inhibitors with Ki values 0.9 mM and 1.4 mM, respectively. These results support a mechanism for the enzyme-catalyzed reaction involving formation of a tetrahedral high-energy intermediate. Three analogues of the natural substrate were noncompetitive inhibitors with Ki values between 0.56-0.75 mM, indicating the presence of a second binding site that may recognize (substituted)acetamido groups.

Glycosylasparaginase activity requires the alpha-carboxyl group, but not the alpha-amino group, on N(4)-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine.

Glycosylasparaginase catalyzes the hydrolysis of the N-glycosylic bond in N(4)-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine in the catabolism of N-linked oligosaccharides. A deficiency, or absence, of enzyme activity gives rise to aspartylglycosaminuria, the most common disorder of glycoprotein metabolism. The enzyme catalyzes the hydrolysis of a variety of asparagine and aspartyl compounds containing a free alpha-carboxyl group and a free alpha-amino group; computational studies suggest that the alpha-amino group actively participates in the catalytic mechanism. In order to study the importance of the alpha-carboxyl group and the alpha-amino group on the natural substrate to the reaction catalyzed by the enzyme, 14 analogues of the natural substrate were studied where the structure of the aspartyl group of the substrate was changed. The incremental binding energy (DeltaDeltaGb) for those analogues that were substrates was calculated. The results show that the alpha-amino group may be substituted with a group of comparable size, for the alpha-amino group contributes little, if any, to the transition state binding energy of the natural substrate. The alpha-amino group position acts as an "anchor" in the binding site for the substrate. On the other hand, the alpha-carboxyl group is necessary for enzyme activity; removal of the alpha-carboxyl group or changing it to an alpha-carboxamide group results in no hydrolysis reaction. Also, N-acetyl-D-glucosamine is not sufficient for binding to the active site for efficient hydrolysis by the enzyme. These results provide supporting evidence for a proposed intramolecular autoproteolytic activation reaction for the enzyme. However, the results raise a question as to an important role for the alpha-amino group in the catalytic mechanism as indicated in computational studies.

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues. n-Butyramide, 3-chloropropionamide, 3-aminopropionamide, and isovaleramide analogues.

The syntheses of four analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. Activated carboxylic acids were reacted with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. n-Butyric anhydride gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-n-butyramide. 3-Chloropropionic anhydride was synthesized from 3-chloropropionic acid and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-chloropropionamide. Equilibration of the latter with ammonium bicarbonate gave N1-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-aminopropionamide. Succinimidyl isovalerate was synthesized and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-isovaleramide.

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues: succinamide, L-2-hydroxysuccinamide, and L-2-hydroxysuccinamic acid hydrazide analogues.

The syntheses of three analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. N-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosylamine was synthesized, and the complete assignment of the 1H NMR spectrum is given. Reaction of the protected beta-D-glycosylamine with L-malic acid chloralid in the presence of a coupling agent (EEDQ) gave N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-L-malamic acid chloralid that was deprotected two ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-2-hydroxysuccinamide, and (2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-1-D-glucopyranosyl)-L-2-hydroxysuccinamic acid hydrazide.

Double cardiomyopathy: coexistent cardiac amyloidosis and hypertrophic obstructive cardiomyopathy.

A combination of hypertrophic obstructive cardiomyopathy (HOCM) and cardiac amyloidosis in the same patient is very rare. Clinical diagnosis could be extremely difficult and may require myocardial biopsy. We are reporting a patient with this combination who was referred to our institution because of features of HOCM based on clinical, echocardiographic and Doppler criteria. Cardiac amyloidosis was only recognized after myocardial biopsy that failed to reveal evidence of HOCM. Only after the patient expired from severe, intractable heart failure did the autopsy findings confirm the association of HOCM. We believe that the combination of the two cardiomyopathic processes is very rare and makes treatment extremely difficult.

Influence of chronic energy intake restriction on intestinal alkaline phosphatase in C3H/Bi mice and autoimmune-prone MRL/lpr,lpr mice.

The influence of chronic energy intake restriction (CEIR) on the level and activity of intestinal alkaline phosphatase was investigated in mice of the autoimmune-prone MRL/lpr,lpr strain and in mice of the autoimmune-resistant C3H/Bi strain. In both strains of mice, CEIR of 40% resulted in a significant increase in intestinal alkaline phosphatase (IAP) specific activity in MRL/lpr,lpr mice after 10 wk of feeding, and in C3H/Bi mice after 6 wk of feeding. An increase in the amount of immunoreactive alkaline phosphatase antigen was also found to be associated with the increased enzyme activity in CEIR mice. These results suggest that a specific induction of an intestinal enzyme occurs or, alternatively, that there is a specific relative decrease in synthesis of intestinal proteins other than IAP as a function of CEIR. Thus, CEIR appears to regulate the expression of proteins in the small intestine in a specific manner.

Carbohydrate removal fails to eliminate the heterogeneity of human prostatic acid phosphatase.

Human prostatic acid phosphatase is known to display considerable charge heterogeneity upon isoelectric focusing. The structural basis of this heterogeneity is not known, although it has been widely attributed to variations in the nature of the carbohydrate chains or to substituents on the carbohydrate chains of the glycoprotein. In this study, the role of the carbohydrate chains in the charge heterogeneity of the protein was examined. First, sialic acid residues were removed by treatment of the acid phosphatase with neuraminidase. The desialo enzyme was fractionated and purified by L-tartramic acid affinity chromatography. Then, after the protein oligosaccharide linkages were made accessible by the presence of NP-40 or by denaturing the protein, the protein was completely deglycosylated by endo-beta-N-acetylglucosaminidase F at pH 4.5 and 9.3. Two discrete intermediates were clearly resolved by SDS gel electrophoresis during the deglycosylation of the denatured protein at pH 9.3, indicating the existence of three sites of glycosylation on the protein. Peptide mixtures were obtained by digestion of carboxymethylated and citraconylated derivatives of the enzyme with trypsin and the glycopeptides were isolated. The amino acid compositions of the glycopeptides were consistent with the interpretation that there are a minimum of two sites of glycosylation on each peptide subunit of the enzyme. Isoelectric focusing experiments on the native, desialo, and denatured, deglycoso acid phosphatase showed that the heterogeneity of the protein is not eliminated either by desialylation or by deglycosylation. Thus, the electrophoretic heterogeneity of human prostatic acid phosphatase does not lie primarily in the oligosaccharide part of the glycoprotein or in altered conformational states of the protein, but in structural variations of the polypeptide itself. The heterogeneity may be due to variations at the C-terminus, partial deamidation, phosphorylation, sulfation or other posttranslational modifications of the protein chain.

Electron-atom source as a primary radiometric standard for the EUV spectral region.

A compact, portable radiometric standard for the EUV wavelength region utilizing single photon counting is described. An energetic beam of electrons is passed through a thin atomic or molecular gas target inducing radiative transitions. The absolute flux of radiation per unit length of beam emitted in a spectral line can be calculated from the current of electrons and the number density of the target gas if the relevant electronimpact photoemission cross section is known at the electron-impact energy used. In typical operating conditions and for a photoemission cross section of 10(-22) m(2), the emitted flux of photons can be 10(10) photons/s/cm of beam length resulting in a count rate of 500 counts/s into an f/6 spectrometer-detector system. Assessment of previously measured electron-impact photoemission cross sections indicates that, once precision benchmark cross-sectional measurements are available, ultimate uncertainties as low as 3% could be obtained for this standard.

Structures of the carbohydrate moieties of human prostatic acid phosphatase elucidated by H1 nuclear magnetic resonance spectroscopy.

The structures of the oligosaccharides comprising the carbohydrate moieties of human prostatic acid phosphatase were elucidated by 1H NMR spectroscopy. Homogeneous enzyme was digested with Pronase P, and three asparagine-linked carbohydrate moieties were obtained upon fractionation of the digest using a concanavalin A-Sepharose affinity column. One fraction did not bind to the column, while the portion that did bind was separated into two fractions by elution with two concentrations of mannose. The high-resolution 1H NMR spectra for the three fractions were recorded at 470 MHz. From these data, the structures were deduced to be high mannose, partially sialylated and fucosylated biantennary complex, and fucosylated, partially sialylated triantennary complex oligosaccharides. No O-linked carbohydrate moiety was detected, although the possible presence of small O-linked oligosaccharides cannot be completely discounted from these data.

Comparison of methods for determining the second-order detection efficiency of a VUV spectrometer.

Four independent procedures were developed and tested to measure the apparatus response function of a VUV spectrometer-detector system for unpolarized 46-nm radiation dispersed in second order. These measurements were made to allow the use of continuum synchrotron radiation for the calibration of the response of the spectrometer-detector system for dispersion of 92-nm radiation in first order with full correction for the effects of synchrotron radiation dispersed in second order. In the first method, synchrotron radiation was used in combination with a thin Al foil to block out synchrotron radiation at 92 nm while allowing 46-nm radiation to enter the spectrometer. In the second as well as the third method NeII 46-nm line radiation was used to measure the response function in first and second order. The line radiation was produced by (1) an electron beam exciting a Ne gas target for which the resulting VUV light illuminated the entire grating and (2) a duoplasmatron VUV light source operating with Ne gas producing a small spot of radiation that was scanned across the surface of the spectrometer grating. In the fourth method the difference in the spectral distributions of synchrotron radiation produced by electrons with different kinetic energies was employed to deduce the second-order detection efficiency. The ratio of the second- to firstorder response function for 46-nm radiation could be determined to a precision of 6% using the bandpass filter and electron-beam methods, 10% using the duoplasmatron method, and 250% using the multiple electron energy method.

Hydrolysis of S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721).

The hydrolysis reaction of S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721), a radioprotective agent currently undergoing clinical trials, was studied under a variety of experimental conditions in order to provide more complete data and to reconcile significant differences found between two previous studies. 31P NMR spectroscopy was primarily used to follow the reaction, but comparable results were also obtained in parallel studies using a spectrophotometric technique and a technique involving liquid chromatography with electrochemical detection, in which the free sulfhydryl product, 2-(3-aminopropylamino)ethanethiol (WR-1065), was measured. Upon hydrolysis, inorganic phosphate and the free sulfhydryl group were formed by cleavage of the P-S bond. The reaction rate versus pH profile at 30 degrees in 42.5 mM buffer, mu = 127.5 mM, showed primarily hydrolysis of the monoanion, with an acid-catalyzed reaction below pH 1.5 to 2.0 involving the neutral species of the ester. The energy of activation at pH 4.0 in 42.5 mM acetate buffer was 25.7 kcal/mole (23.1 kcal/mole by liquid chromatography with electrochemical detection). The entropy of activation at pH 4.0, 36 degrees was positive, and there was a deuterium isotope effect on the reaction. A small buffer effect on the rate of the reaction at pH 4.0 and pH 5.0 was found to include contributions from both general acid and general base catalysis. These data are consistent with a mechanism for hydrolysis of the monoanion involving a partially rate-determining proton transfer to the sulfur atom and the formation of metaphosphate ion, which is rapidly hydrolyzed to inorganic phosphate.

Structures of the carbohydrate moiety attached to one site in the first domain of turkey ovomucoid: elucidation by 1H-n.m.r. spectroscopy.

1H-N.m.r. spectroscopy was used to elucidate the primary structures of the carbohydrate moiety attached to asparagine at residue 53 in the first domain of turkey ovomucoid, a serine proteinase inhibitor. The carbohydrate moiety is a heterogeneous mixture of three structurally closely related complex-type oligosaccharides. Of the total carbohydrate moiety, 61% is tetra-antennary with terminal galactose and with an intersecting N-acetylglucosamine, and containing an additional N-acetylglucosamine (10') attached to mannose (4'). Another 23% is tri-antennary with terminal galactose and with an intersecting N-acetylglucosamine. The remaining 16% is tri-antennary with terminal galactose (6 and 8 only), with an intersecting N-acetylglucosamine.

1H NMR evidence that almond "peptide: N-glycosidase" is an amidase. Kinetic data and trapping of the intermediate.

The enzyme from almond that catalyzes the hydrolysis of the N-glycosidic linkage between asparagine and the oligosaccharide chain of glycopeptides and glycoproteins has been variously termed an N-glycosidase and an amidase enzyme. Using turkey ovomucoid glycopeptide as a substrate for the enzyme, we followed the hydrolysis reaction by 1H NMR spectroscopy. These kinetic data revealed a rapid hydrolysis of the substrate but a delayed appearance of the final product. This implied that an intermediate, most likely a 1-aminooligosaccharide, was formed during the reaction. Identification of the intermediate as a 1-beta-amino-N-acetylglucosamine-oligosaccharide was achieved by trapping it as the 1-acetamido derivative using acetic anhydride and subsequent analysis by 1H NMR. The data conclusively demonstrate that the enzyme catalyzes the hydrolysis of the glycopeptide to form an aspartic acid-containing polypeptide and an intermediate oligosaccharide amine. The latter derivative is hydrolyzed nonenzymatically to yield the final carbohydrate product. Thus, the enzyme is in fact an amidohydrolase (amidase) and not an N-glycosidase. The trivial name glycopeptidylamidase is suggested.

Structural and immunological relationships among mammalian arylsulfatase A enzymes.

Structural and immunological properties of numerous arylsulfatase A enzymes (EC 3.1.6) were examined in order to assess the relationships among these enzymes in animals. Arylsulfatase A enzymes from all animals bind to a Concanavalin A-Sepharose column, consistent with the conclusion that they are all glycoproteins. At pH 7.5 the apparent mol. wts of the enzymes are 80-182 kDa, while at pH 4.5 the mammalian arylsulfatase A enzymes dimerize and exhibit apparent mol. wts in the range of 297-348 kDa, but the enzymes from opossum and other lower classes of animals do not aggregate at pH 4.5. The mammalian arylsulfatase A enzymes, which aggregate at pH 4.5, also bind to rabbit liver arylsulfatase A monomers immobilized on an Affi-Gel 10 matrix. The arylsulfatase A enzymes that were studied all exhibit the anomalous kinetic behavior regarded as characteristic of these enzymes. However, not all of the inactivated enzymes are reactivated by sulfate ions. Goat antiserum raised against homogeneous rabbit liver arylsulfatase A cross-reacts with all of the mammalian enzymes in Ouchterlony gel diffusion experiments, whereas the enzymes from lower classes of animals do not cross-react. Quantitative immunoprecipitation experiments demonstrate that the mammalian enzymes are very similar to each other, with greater than 60% primary sequence homology indicated, while arylsulfatase A from opossum and other lower classes of animals show only a partial immunological similarity with the mammalian enzymes. Taken together, the data suggest that the active site of the enzyme and the structural features of the protein are highly conserved during the evolution of the enzyme molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of oxygen exchange at the anomeric carbon atom of D-glucose and D-erythrose using the oxygen-18 isotope effect in carbon-13 nuclear magnetic resonance spectroscopy.

The 18O isotope induced shift in 13C nuclear magnetic resonance (NMR) spectroscopy affords a new and convenient method for the study of oxygen exchange at the anomeric carbon atom of simple sugars. The efficacy of the technique was confirmed by a study of the oxygen exchange reaction of D-[1-13C] glucose. At pH 7.0 and 61 degrees C, the incorporation of 18O from solvent H2(18)O onto the C-1 carbon atom of the diastereomeric alpha- and beta-pyranose sugars was followed by 13C NMR spectroscopy in a continuous assay mode. The pseudo-first-order rate constant for exchange of both the alpha and the beta anomers was 9.5 X 10(-5) s-1, which is in agreement with a rate constant obtained in a previous study by a chemical conversion-mass spectrometry technique. The new technique was applied to a study of the oxygen exchange at the anomeric carbon atom of D-[1-13C]erythrose, a furanose sugar for which no experimental data were available. In unbuffered, aqueous solutions the incorporation of the 18O label from the medium (H2(18)O) onto the C-1 carbon atom of the alpha- and beta-D-[1-13C]erythrose and the D-[1-13C]erythrose hydrate forms was followed by 13C NMR at 10, 23, and 36 degrees C. From analysis of the data for the alpha and beta diastereomers, the pseudo-first-order rate constants for exchange were 1.4 X 10(-4) s-1 at 10 degrees C, 4.8 X 10(-4) s-1 at 23 degrees C, and 8 X 10(-4) s-1 at 36 degrees C, and the apparent energy of activation for the exchange reaction was 12.1 kcal/mol. Particularly in conjunction with the use of specifically 13C-enriched sugars, the new technique for studying oxygen exchange reactions of carbohydrates has many distinct advantages over earlier approaches, including the ability to follow simultaneously the exchange reactions of all of the sugar species for which a 13C NMR signal can be detected, the continuity of the assay, the avoidance of possible artifacts due to incomplete or selective derivatization reactions, and the simplicity of the data analysis.

Phosphate (oxygen)-water exchange reaction catalyzed by human prostatic acid phosphatase.

Conclusive evidence is presented that an acid phosphatase catalyzes phosphate (oxygen)-water exchange. Studies conducted with human prostatic acid phosphatase by two independent methods have established that, despite earlier reports to the contrary, the enzyme catalyzes an exchange reaction between oxygen atoms of phosphate ion and of water. Kinetic data were obtained both by chemical conversion to trimethyl phosphate followed by mass spectroscopy and by a totally independent method involving 31P isotope shift nuclear magnetic resonance spectroscopy. Analysis showed that the enzyme catalyzes the exchange in a random, noncoupled process. If any coupled exchange occurs, it must represent less than 10% of the total. By mass spectral analysis, catalytic rate constants kcat = 0.14 sec-1 (4 degrees) and 1.8 sec-1 (37.5 degrees) were obtained. By 31P nuclear magnetic resonance kcat = 1.6 sec-1 (31 degrees) was obtained. The energy of activation for the exchange reaction is approximately 13kcal mol-1. The kcat value for exchange is about 10-fold greater than that observed with Escherichia coli alkaline phosphatase.