bluuebunny/crossref_metadata_2025 huggingface dataset:
Disopyramide analysis using REMEDi: Comparison with EMIT and conventional high performance liquid chromatographic methods
Authors: Patel V. , McCarthy P. T. , Flanagan R. J.
Published: May 2005

REMEDi (Rapid EMErgency Drug identification; Bio‐Rad) is an automated high performance liquid chromatographic (HPLC) system designed to detect, identify and measure a range of basic and neutral drugs in 0.5‐1.0 mL of urine or plasm/serum. We have evaluated REMEDi in the analysis of the antiarrhythmic drug disopyramide in patient samples. The specimens were also analysed by a conventional HPLC method, based on solvent extraction and UV detection (254 nm), and by EMIT. There were good correlations between the results obtained with each method (r = 0.91 or greater). REMEDi gave a lower mean result than EMIT [means±SD (m/L): REMEDi 2.64±1.10, EMIT 3.14±1.51; t = 4.0, p<0.01; n = 25], but there were no other significant differences in mean results. The principal disopyramide metabolite, mono‐N‐desalkyldisopyramide, did not interfere in any method. Clearly REMEDi can be used for therapeutic drug monitoring of disopyramide provided enough sample is available.
High performance liquid chromatography of spermidine and spermine using a postcolumn reactor of immobilized polyamine oxidase (Aspergillus terreus) Followed by Electrochemical Detection
Authors: Watanabe Noriyuki
Published: May 2005

A highly sensitive and selective method for the determination of spermidine and spermine has been developed. A polyamine oxidase (Aspergillus terreus) immobilized column was used as a postcolumn reactor. The detection limit was 0.2 pmol/injection for both spermidine and spermine with a linear range of three orders of magnitude.
Determination of panaxadiol and panaxatriol in ginseng and its preparations by capillary supercritical fluid chromatography (SFC)
Authors: Yun‐hua Li , Xiu‐lu Li , Lan Hong , Jin‐yao Liu , Mei‐Yi Zhang
Published: May 2005

Capillary supercritical fluid chromatographic (SFC) method has been developed for the determination of panaxadiol and panaxatriol in ginseng and its preparations. 0.1 g ginseng or an appropriate amount of its preparations was hydrolysed by 15% H2SO4 in an ethanol: water (1:1 v/v) solution for 4 h followed by 15% NaOH for 0.5 h. The mixture was extracted by cyclohexane. The cyclohexane extracts were purified by a partition column and concentrated by an adsorption column and then analysed by SFC. Methyltestosterone was used as the internal standard.
Simultaneous determination of paraquat and diquat in serum using capillary electrophoresis
Authors: Tomita Masafumi , Okuyama Toshiko , Nigo Yayoi
Published: May 2005

The use of capillary electrophoresis for simultaneous separation and detection of the two bipyridylium herbicides, paraquat and diquat, was investigated. Both herbicides were extracted from fortified sera with disposable ODS‐silica cartridges. Separation was carried out using a capillary tube (50 μm i.d., 750 mm) of fused silica containing 10 mM glycine–HCl buffer (pH 3.0), 40 mM NaCl and 20% methanol as the carrier. Paraquat and diquat were completely separated in 10 min at an applied potential of 20 kV. On‐column UV monitoring allowed detection of both herbicides simultaneously. The assay sensitivity was 0.05 μg/mL (signal‐to‐noise ratio, 2:1), which probably increases with increase in the sample volume of serum. Analytical recovery of both herbicides added to serum was about 97% at concentrations of 0.5–2.0 μg/mL.
High performance liquid chromatographic strategy suitable for the analysis of exopolysaccharides extracted from pathogenic bacteria
Authors: Radin Livio , Sivori Simona , Eftimiadi Costantino , Lucarelli Claudio
Published: May 2005

A high performance liquid chromatographic (HPLC) method has been developed to permit the rapid comparison of acidic polysaccharides of diverse compositions and the sensitive determination of their constituents. It is based on two combined analyses of the polysaccharide hydrolysates—a separation of the released compounds by ion‐moderated partition chromatography with UV detection at two wavelengths and a separation of the sugar dansylhydrazine derivatives by reversed phase chromatography. The former permits identification and quantitation of uronic and carboxylic acids, the latter permits more sensitive and specific determination of the neutral aldoses. Some bacterial exopolysaccharides have been used to demonstrate the validity of this HPLC procedure for the chemical characterization of uronic acid‐containing polysaccharides. This method appears to be useful for studying capsular polysaccharides, which are involved in the evasion of phagocytosis by pathogenic bacteria.
Quantitative determination of CGS 18102A, a new anxiolytic, in human plasma using capillary gas chromatography/mass spectrometry
Authors: Leal M. , Hayes M. J. , Powell M. L.
Published: May 2005

CGS 18102A is a novel hexahydrobenzopyranopyridine that has a mixed pharmacological profile of 5‐HT‐1A agonist and 5‐HT‐2 antagonist properties. Based upon these mechanisms, the compound is predicted to have anxiolytic efficacy with possible efficacy in depression. Preclinical studies in the rat have shown the drug to be well absorbed and extensively metabolized. Because of the anticipated low plasma levels in humans a gas chromatography/mass spectrometry (GC/MS) analytical method has been developed and validated to determine plasma concentrations of CGS 18102A in early clinical studies. The method utilizes CGS 18416A as the internal standard. Samples (1 mL) were extracted with pentane:ethyl acetate (75:25, v:v). Extracts were then concentrated and analysed directly by GC/MS. Separation was accomplished on a methylsilicone capillary column (30 m × 0.32 mm i.d.). GC/MS was carried out under positive ion ammonia CI conditions, with selected ion monitoring of the [M + H]+ ions (m/z = 262 and 248) for CGS 18102A and CGS 18416A, respectively. The method was successively applied to the analysis of clinical samples from an ascending multidose safety and tolerability study conducted in six normal healthy male volunteers.
A Selective HPLC/RIA for dexamethasone and its prodrug dexamethasone‐21‐sulphobenzoate sodium in biological fluids
Authors: Hochhaus Günther , Hochhaus Renate , Werber Gisela , Derendorf Hartmut , Möllmann Helmut
Published: May 2005

A combined high performance liquid chromatography/radioimmunoassay procedure is described for the simultaneous determination of dexamethasone (DEX) and its prodrug dexamethasone‐21‐sulphobenzoate sodium (DSS) in plasma. After precipitation of the plasma proteins by acetonitrile, the protein‐free supernatant was injected onto a C18 reversed phase liquid chromatographic system and DSS‐ and DEX‐containing fractions were collected. Hydrolysis of DSS by 0.01 N NaOH, followed by fractions extraction of both hydrolysed DSS and DEX fractions with ethyl acetate allowed the use of a dexamethasone‐specific radioimmunoassay for the selective determination of both compounds. The method is accurate and reproducible (intraday variability for DSS and DEX<6%, interday variability for DEX 14%), allowing quantification of DEX and DSS as low as 0.3 ng/mL and 0.7 ng/mL, respectively.
Measurement of ascorbic acid and dehydroascorbic acid in mammalian tissue utilizing HPLC and electrochemical detection
Authors: Schell Debra A. , Bode Ann M.
Published: May 2005

Reliable measurement of the reduced and oxidized forms of ascorbic acid (AA) is challenging because they are highly reactive and unstable compounds. Detection of small amounts of AA and dehydroascorbic acid (DHAA) is essential for determining the biochemical function of the vitamin. While a variety of techniques exist for measurement of AA with detection limits in the millimolar range, a need exists for highly reliable assessment of picomolar levels of AA and DHAA in tissues. The present study presents a method for measuring AA and DHAA that combines high performance liquid chromatography with the advantages and increased detection limits and selectivity available with coulometric electrochemical detection. The difference between AA and „total AA”︁ in tissue samples gives an assessment of DHAA concentration. Verification of the reliability of assay is by the successful linear recovery of exogenously added AA and DHAA in tissue homogenate. Optimal conditions for reducing DHAA in tissue samples include a pH of 7.2, reaction time of 10 min, reaction temperature equal to room temperature, and a 10 mM concentration of the reducing agent, β‐mercaptoethanol. AA and DHAA are measured in several mammalian tissues using the method presented.
Chiral separation of amines by high‐performance liquid chromatography after tagging with 4‐(N,N‐dimethylaminosulphonyl)‐7‐(2‐chloroformylpyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole
Authors: Toyo'oka Toshimasa , Liu Yi‐Ming , Jinno Hideto , Hanioka Nobumitsu , Ando Masanori , Imai Kazuhiro
Published: May 2005

Chiral tagging reagents, 4‐(N,N‐dimethylaminosulphonyl)‐7‐(2‐chloroformylpyrrolidin‐1‐yl)‐2,1,3‐benzoxadiazole (R(+)‐DBD‐Pro‐COCl and S(−)‐DBD‐Pro‐COCI), react with mirror image enantiomers of amines to produce corresponding diastereomers in the presence of pyridine as a catalyst. The maximal excitation and emission wavelengths of the resulting diastereomers were ca. 450 nm and 560 nm, respectively. The diastereomers derived from some aliphatic amines were resolved by a reversed‐phase chromatography with water–acetonitrile or normal‐phase chromatography with n‐hexane–ethyl acetate as the eluent. The reactivities of both enantiomers of DBD‐Pro‐COCI to chiral amines were almost comparable, whereas a slight difference of fluorescence intensity was observed with S(−)‐DBD‐Pro‐COCI. When S(−)‐DBD‐Pro‐COCI was used as the derivatization reagent, amines corresponding to S‐configuration were eluted faster than R‐configuration. The opposite elution order was obtained with the use of R(+)‐DBD‐Pro‐COCI, instead of S(−)‐DBD‐Pro‐COCI. The Rs values obtained from 1‐cyclohexylethylamine (CEA) having aliphatic ring structure was larger than those of amines (1‐(1‐naphtyl)ethylamine (NEA) and 1‐phenylethylamine (PEA)) having aromatic ring structures.
Reversed‐phase HPLC separation and chromatographic–spectral characterization of 17β‐(2′‐thiazolyl)androst‐5‐en‐3β‐ols and their acetates
Authors: Drašar P. , Urbanský M.
Published: May 2005

Simple isocratic HPLC separation of a series of thiazolyl steroids with the 17β‐2′ linkage is described. The chromatographic and spectral characterization utilizes both on‐the‐fly UV spectral maxima and absorbances changes.
A Novel electrophilic reagent, 4‐(N‐chloroformylmethyl‐N‐methyl)amino‐7‐N,N‐dimethylaminosulphonyl‐2, 1,3‐benzoxadiazole (DBD‐COCI) for fluorometric detection of alcohols, phenols, amines and thiols
Authors: Imai Kazuhiro , Fukushima Takeshi , Yokosu Hirochika
Published: May 2005

A novel electrophilic reagent for alcohols, phenols, amines and thiols, 4‐(N‐chloroformylmethyl‐N‐methyl)amino‐7‐N,N‐dimethylaminosulphonyl‐2, 1, 3‐benzoxadiazole (DBD‐COCI) was synthesized. The reactivity of the reagent to these nucleophiles was studied using high‐performance liquid chromatography (HPLC) with precolumn derivatization techniques.

DBD‐COCI reacted in benzene solution at the room temperature or 60°C with androsterone (a representative of hydroxyls), (−)‐mandelic and D,L‐lactic acid (hydroxy acid), estrone (phenol), benzylamine (primary amine), phenetidine (aromatic amine) and α‐mercapto‐N,2‐naphthylacetamide (thiol) to give fluorescent products bearing fluorescence wavelengths at between 543 nm and 555 nm (excitation at between 437 nm and 445 nm). The base catalyst, quinuclidine was required to complete the reaction with the nucleophiles except aromatic amines. The reaction solution was subjected to a reversed phase HPLC and the detection limits of the derivatives were at the femto mol range on column.
Polymeric reagents for derivatizations in micellar electrokinetic chromatography
Authors: Szulc Michael , Krull Ira S.
Published: May 2005

A polymer immobilized o‐nitrobenzophenone reagent was prepared for analysis of amine drugs in micellar electrokinetic chromatography (MEKC). A model compound, propylamine, was used to characterize the reagent's performance in MEKC. Derivatizations were performed on the CE instrument with reagent in the sample vial. The yielded derivative was directly sampled from the reaction mixture, and directly injected onto the MEKC system. The derivatization reagent was also applied to the derivatization of n‐alkyl amine mixtures and amino acids. The method was validated for adamantanamine in urine and amino acids. The method was validated for adamantanamine in urine and in plasma by single‐blind spike analysis. Precisions and accuracies for all samples were less than 6.0% for urine samples and 10% for plasma samples. The procedure was a direct injection technique requiring minimal sample preparation for the analysis of drugs in biofluids.
Determination of chloral hydrate and its metabolites (trichloroethanol and trichloracetic acid) in human plasma and urine using electron capture gas chromatography
Authors: Humbert L. , Jacquemont M. C. , Leroy E. , Leclerc F. , Houdret N. , Lhermitte M.
Published: May 2005

Capillary gas chromatography with electron capture detection is described for the quantification of chloral hydrate (CH) and is metabolites trichloroethanol (TCE) and trichloroacetic acid (TCA) in 0.1–1 mL of plasma samples. The method, with 2,2′‐dichloroethanol (DCE) as internal standard, involved extraction of chloral hydrate and trichloroethanol with diethyl ether and methylation of trichloroacetic acid with 3‐methyl‐1‐tolyltriazene (MTT), followed by diethyl ether extraction. The method has a detection limit of 5 ng/mL for CH and TCE and 10 ng/mL for TCA and also allows the determination of TCE‐glucuronide in 0.1–1 mL of plasma samples. It exhibits good linearity and precision. The method was applied to samples of plasma from a neonate after a single dose of 40 mg/kg of chloral hydrate and from an adult after a single dose of 6.25 mg/kg.
Gas chromatographic determination of enantiomers as diastereomers following pre‐column derivatization and applications to pharmacokinetic studies: A review
Authors: Srinivas Nuggehally R. , Shyu Wen Chyi , Barbhaiya Rashmi H.
Published: May 2005

The introduction of chiral chromatographic methods has revolutionized the art of separationand quantitation of chiral drugs in biological fluids. A large number of chiral derivatization reagents for various funtional grups are available commercially. Therefore, Pre‐column derivation methods have become attractive and simple for the gas chromatographic assays in biologcal fluids. The intent of this article is to review the pre‐column chiral derivatization reagents employed in gas chromatographic separations and analyses of enantiomers. A discussion of numerous procedures necessary to develop a quantitative gas chromatographic assay method for drug enantiomers is presented. In this regard, quantitative gas chromatographic assays based on this approach have been applied to investigate the stereoselective pharmacokinetics of several chiral drug such as fenfluramine, norfenfluramine, methylphenidate, etodolac, propranol, suprofen and methoxyphenamine.
Enantiomeric separation and sensitive determination of D, L‐amino acids derivatized with fluorogenic benzofurazan reagents on pirkle type stationary phases
Authors: Fukushima Takeshi , Kato Masaru , Santa Tomofumi , Imai Kazuhiro
Published: May 2005

The enantiomeric separations of D, L‐amino acids derivatized with fluorogenic reagents, 4‐fluoro‐7‐nitro‐2,1,3‐benzoxadiazole (NBD‐F), 4‐(N,N‐dimethylaminosulphonyl)‐7‐fluro‐2,1‐3,‐benzoxadiazole (DBD‐F) and 4‐aminosulphonbly‐7‐fluoro‐2,1,3‐ benzoxadiazole (ABD‐F) by high‐performance liquid chromatography (HPLC) ON various Pirkle type chiral stationary phases (CSPs, Sumichiral OA series) with citric acid in methanol as a mobile phase were studied. Since the least retention and no separation was observed for the derivatives of racemic phenylalanine methyl‐ester,‐amide and a drug without an α‐carboxyl group, the carboxylic acid group of the amino acid derivatives seemed to contribute to the enantioselective fixation of the derivatives through hydrogen bonding on the N‐acyl‐amino acid amide moiety of the CSP. The enantioselective retention of the derivatives was attained through the (S) or (R) configuration of valine, phenylglycine, naphthylglycine, naphthylethylamine or the tert‐leucine moiety in the CSP. The 2, 1, 3‐benzoxadazole (benzofurazan) moiety in the derivatives helps the effective fixation of the derivatives through a π‐πinteraction with an aromatic moiety such as a 3, 5‐dinitrophenyl group in the Pirkle type chiral stationary phases.

D‐Amino acids in biological samples were easily determined utilizing the present derivatization with NBD‐F, enantiomeric separation and fluorometric detection (530 nm em/470nm ex) following deproteinization of biological samples (serum or brain homogenate) with methanol and centrifugation. The applications of the method were clearly demonstrated by the following results; D‐Ala was detected in sera of healthy volunteers at a level of 0.48‐3.10μM. D‐Lys was found in the serum of a patient with myeloma and requiring renal dialysis, and D‐Ser was found in rat and bovine cerebrum. Peak identification was performed by use of different types of stationary phases especially those bearing the opposite configuration to that of the chiral centre.
Determination of 5‐hydroxytryptamine, 5‐hydroxyindoleacetic acid and tryptophan in plasma and urine by HPLC with fluorimetric detection
Authors: Bearcroft C. P. , Farthing M. J. G. , Perrett D.
Published: May 2005

Using native fluorescence detection, 5‐hydroxytryptamine (5‐HT), 5‐hydroxyindoleacetic acid (5‐HIAA) and tryptophan were resoved from themselves and other naturally occurring compounds using reversed‐Phase HPLC within 5 min. Deproteinated platelet‐poor plasma (ppp) and crude diluted urine were injected directly into the chromatograph. Careful selection of the HPLC column is important and various octadecyl silica (ODS) and base deactivated silic (BDS) columns were evaluated. Pre‐treatment of an ODS column with tetrabutylammonium ions gave good selectivity. Between pH5 and 6 the compounds were well resolved from each other. The limit of quantitatiave detection of 5‐HT and 5‐HIAA was 3.5 nmol/L. The overal chromatogram obtained using native fluorescence is cleaner than that obtained with the more commonly employed electrochemical (EC) systems although the chromatography is effectively the same. For analysis of 5‐HT in plasma, collection in EDTA was more efficient than lithium heparin. Plasma 5‐HT in healthy volunteers was mean 61 (SD=±73) nmol/L, n=20; urine 5‐HIAA gave mean 28.95 (SD=±0.98)μmol/L, (n=12). Whole blood 5‐HT analysis is unreliable in comparison with platelet‐poor plasma.
N‐glycosylation site mapping of recombinant tissue plasminogen activator by micellar electrokinetic capillary chromatography
Authors: Taverna M. , Baillet A. , Schlüter M. , Baylocq‐Ferrier D.
Published: May 2005

This report describes the N‐glycosylation mapping of recombinant tissue plasminogen activator (rt‐PA) using micellar electrokinetic capillary chromatography. The carbohydrate structures were tentatively assigned by comparison with the anion‐exchange fractionated oligosaccharides and by a comparison with previously reported data. The separation was shown to rely mainly on the degree of sialylation of the oligosaccharides, allowing a quantitative determination of the proportion of neutral and mono‐ to tetrasialylated structures. Significant differences in the oligosaccharide distribution of the two variants of rt‐PA, which differ by the presence (type I) or the absence (type II) of oligosaccharides at the Asn‐184 site, were observed. The distribution of the oligosaccharides at each of the rt‐PA glycosylation sites was then determined. Glycopeptides were prepared by tryptic digestion of rt‐PA and isolated using two consecutive chromatographic procedures. The glycopeptides were finally treated with N‐glycanase, and the resulting oligosaccharides were analysed by capillary electrophoresis. Oligosaccharide mapping revealed that the Asn‐448 and Asn‐184 sites carry the same population of complex‐type oligosaccharides but that the relative amounts of each oligosaccharide vary markedly. High‐pH anion‐exchange chromatography performed on the desialylated oligosaccharides at each glycosylation site showed that the degree of microheterogeneity was related not only to the degree of sialylation but also to structural differences in the oligosaccharide sequences. From the results as a whole, we concluded that the Asn‐448 site contains a greater proportion of heavily sialylated structures and has a higher degree of microheterogeneity. The Asn‐117 site was demonstrated to contain a significant number of monosialylated structures (18.4%) in addition to the high‐mannose‐type oligosaccharides.
Occurrence of D‐aspartic acid in rat brain pineal gland
Authors: Imai Kazuhiro , Fukushima Takeshi , Hagiwara Kenichi , Santa Tomofumi
Published: May 2005

We found a high concentration (1030 pmol per pineal gland) of D‐aspartic acid (D‐Asp) in the pineal gland of 6‐week‐old Sprague‐Dawley (SD) rats. The content of D‐Asp decreased with age, being 210 and 33 pmol per pineal gland in 28‐ and 45‐week‐old rats respectively. The proportion of D‐Asp [(D‐Asp/total Asp) × 100] also decreased with age, declining from 66% to 10% between 6 and 45 weeks after birth. The proportion of D‐Asp did not differ between the sexes. The concentration of D‐Asp was higher at night (at 2.00 a.m. 2830±485 pmol per pineal gland) than during the day (at 10.00 a.m. 1030±200 and at 3:00 p.m. 682±194 pmol per pineal gland), suggesting that biosynthesis of D‐Asp in the pineal gland occurs at night. D‐Asp was found to be distributed in the cytosol of pinealocytes, but its biological role remains unclear.
Determination of medetomidine, atipamezole and midazolam in pig plasma by liquid chromatography‐mass spectrometry
Authors: Kanazawa Hideko , Nishimura Ryohei , Sasaki Nobuo , Takeuchi Akira , Takai Nobuharu , Nagata Yoshiko , Matsushima Yoshikazu
Published: May 2005

Medetomidine, atipamezole and midazolam in pig plasma were determined by liquid chromatography‐mass spectrometry (LCMS) with an atmospheric pressure chemical ionization interface system by the use of detomidine as an internal standard. The method was applied to studies of pharmacokinetic behaviour of these drugs.
Chromatographic modelling of interactions between melanin and phenothiazine and dibenzazepine drugs
Authors: Radwa Aleksandra , Fra̧ckowiak Teresa , Ibrahim Houda , Aubry Anne‐Françoise , Kaliszan Roman
Published: May 2005

Differences in drug‐melanin interactions were determined for 13 phenothiazine neuroleptics and 2 dibenzazepine thymoleptics by means of high‐performace liquid chromatography. The chromatographic column was packed with a stationary phase obtained by chemical immobilization of synthetic L‐dopa melanin on silica particles. For six phenothiazines the melanin‐binding parameters were also determined by an ultrafiltration method. Correlation between measures of drug‐melanin interaction determined chromatographically and by the standard slow‐equilibrium method was significant, however moderate. The chromatographic method of assessing interactions between drugs‐melanin interaction determined chromatographically and by the standard slow‐equilibrium method was significant, however moderate. The chromatographic method of assessing interactions between drugs and melanin permitted reliable and quantitatively comparable data for representative series of solutes to be readily obtained. Such data were subjected to the analysis of quantitative structure‐retention relationships (QSRR). It was found that retention of the agents on the immobilized melanin column could be described by two‐parameter regression equations comprising the energy of the lowest unoccupied molecular orbital and either the water‐accessible surface area of a drug molecule or its hydrophobicity parameter, determined chromatographically on an immobilized artificial membrane column. The QSRR equation derived allows for the estimation of melanin binding based on the structure of a compound candidate, and thus rationalizes predictions of potential toxicity of drugs or drug candidates.
Resonance raman study on axial ligands of heme irons in cytochrome bd‐type ubiquinol oxidase from Escherichia coli
Authors: Hirota Shun , Mogi Tatsushi , Anraku Yasuhiro , Gennis Robert B. , Kitagawa Teizo
Published: May 2005

Resonance Raman (RR) spectra of cytochrome bd‐type ubiquinol oxidase enriched with Fe‐isotopes were investigated with excitation at 406.7, 424.0, 427.0, 441.6, and 647.1 nm. The bands of reduced form at 252 and 400 cm−1 were tentatively assigned to vFeHis of high‐spin b595 and vFeMet of low‐spin b558, respectively. The v and δFeOO bands of heme d‐O2 were observed at 566 and ∼445 cm−1, respectively. The vFeCO frequency of heme d‐CO was unusually low (477 cm−1) but the v4 frequency was not of P‐450 type. Therefore, the axial ligand of heme d would not be an ordinary histidine or cysteine. © 1995 John Wiley & Sons, Inc.
Competition of congo red and thioflavin S binding to amyloid sites in alzheimer's diseased tissue
Authors: Elhaddaoui A. , Pigorsch E. , Delacourte A. , Turrell S.
Published: May 2005

Though the strong affinity of Congo red and thioflavin S for amyloid deposits has been known for a long time, the binding mechanism of these biological dyes to amyloid sites is still not understood. In the present work, the competition of these two dyes for binding in Alzheimer amyloid is investigated. Analyses by optical microscopy and microspectrofluorimetry were performed on several series of stained diseased brain sections. Microspectrofluorimetric analyses showed that bound Congo red and bound thioflavin S have characteristic spectra in which the fluorescence maxima are distinct of those for the free dyes. Both, optical microscopic analyses and fluorescence measurements of stained brain sections indicated that Congo red and thioflavin S compete with each other at the same interacting site on diseased cerebral tissue. The staining efficiency of the replacing dye seems to be a maximum for a concentration of about 1%. © 1995 John Wiley & Sons, Inc.
Subcellular localization of acetaldehyde dehydrogenase in human liver
Authors: Jenkins William J. , Peters Timothy J.
Published: May 2005

The subcellular distribution of aldehyde dehydrogenase activity was determined in human liver biopsies by analytical sucrose density‐gradient centrifugation. There was bimodal distribution of activity corresponding to mitochondral and cytosolic localizations. At pH 9.6 cytosolic aldehyde dehydrogenase had a lower apparent K for NAD (0.03 mmol l−1), than the mitochrondrial enzyme (K NAD = 1.1 mmol l−1). Also, the pH optimum for cytosolic aldehyde dehydrogenase activity (pH 7.5) was lower than that for the mitochondrial enzyme activity (pH 9.0), and the cytosolic enzyme activity was more sensitive to inhibition by disulfiram in vitro. Disulfiram (40 μmol l−1) caused a 70% reduction in cytosolic aldehyde dehydrogenase activity, but only a 30% reduction in mitochondrial enzyme activity after 10 min incubation. The liver cytosol may therefore be the major site of acetaldehyde oxidation in vivo in man.
Lipid peroxidation in hepatomas of different degrees of deviation
Authors: Rossi Maria Armida , Cecchini Giovanni
Published: May 2005

Lipid peroxidation rate in four different hepatomas is quite different and seems to be related to their degree of deviation, low deviation tumours displaying higher peroxidative ability. Moreover, the supernatant of the highly anaplastic Yoshida hepatoma is able to decrease the peroxidation rate in normal liver microsomes. This antioxidant ability is not dependent upon an increased level of glutathione. The concentration of reduced glutathione (GSH) declines strongly during incubation in conditions favouring lipid peroxidation. Unlike normal liver homogenates, this decline of GSH in hepatomas is not due to the transformation of GSH into oxidized glutathione (GSSG) but mostly to the increased activity of the γ‐glutamyl‐transpeptidase pathway.
Dispersed sensory ganglion cells cleave an active entity from nerve growth factor coupled to Sepharose
Authors: Khan A. A. , Pearce F. L. , Vernon C. A.
Published: May 2005

Dispersed sensory ganglion cells from the embryonic chick, but not cells from other tissues, liberate biologically active material from nerve growth factor coupled to Sepharose beads. Experiments with such insolubilized material cannot then be taken as evidence for the existence of an external membrane receptor for the factor and may indicate that responsive cells have the ability to cleave an active fragment from the polypeptide.
Biochemistry of the polyamines
Authors: Allen John C.
Published: May 2005

The naturally‐occurring polyamines exist in the free form, as N‐acetyl derivatives and bound to protein. Their biosynthesis is subject to sensitive control, particularly of ornithine decarboxylase. This enzyme may be multifunctional and a key regulatory protein. Studies, principally with selective inhibitors, have elucidated the roles of polyamines in cell proliferation. Oxidized polyamines, in contrast, can be potent mitotic inhibitors. These effects are reviewed in terms of their chemistry and biochemistry. Their principal distinctions are that they can be made or degraded intracellularly, they can associate electrostatically with macromolecules by means of their spaced cationic groups, and these can be readily converted to covalent bonds.
Surface of articular cartilage: Immunohistological studies
Authors: Duance V. C.
Published: May 2005

Using several physical techniques the surface of articular cartilage has been reported to be structurally different from the deeper layers. In this paper using immunohistochemical methods, the surface has been shown to contain a characteristically different collagen, Type I in contrast to Type II which is the major collagen of cartilage. These results support previous proposals for a surface layer, or lamina splendens, the presence of which would be of considerable importance in understanding the degradation of cartilage in arthritides.
Quantitative cytochemical analyses of ovarian luteal and interstitial cell RNA in relation to plasma progestins during pseudopregnancy in the rat
Authors: Doebler Jeffrey A. , Wickersham Edward W. , Anthony Adam
Published: May 2005

Luteal and interstial cell RNA contents and circulating progesterone (P) and 20α‐hydroxypregn‐4‐ene‐3‐one (20α‐OH P) levels were measured during pseudopregnancy in order to characterize relationships between ovarian 20α‐OH P secretion and luteal regression. Functional luteolysis, as manifested in depressed P levels, was not associated with concurrent elevations in 20α‐OH P. Rather, augmented 20α‐OH P levels were evidenced in periovulatory periods at the onset and termination of pseudopregnancy, subsequent to RNA accumulation in both luteal and interstitial compartments. It is postulated that 20α‐OH P, the putative inactive metabolite of P, is produced by both ovarian compartments in a cyclic manner and in response to gonadotrophin released in the preovulatory period.
Effects of diterpene forskolin on the release reaction and protein phosphorylation of human platelets
Authors: González A. , Aranda E. , Mezzano D. , Garrido J.
Published: May 2005

The effects of diterpene forskolin on the human platelet release reaction and on platelet protein phosphorylation were studied. This drug is shown to have the same effects as other agents which increase cAMP levels, namely, it inhibits the secretory response to diverse agonists and causes changes in the phosphorylation of several specific proteins. An increase of the 32P content is seen in the MW 47 000, 24 000 and 21 000 polypeptides while a decrease is observed in the MW 41 000 and 27 000 and 20 000 species. Forskolin also inhibits the changes in protein phosphorylation pattern induced by the powerful platelet secretagogue, thrombin. Our results relate the effects of antagonists of platelet secretion such as prostaglandins more closely to changes in cAMP levels and in protein phosphorylation than to other possible effects of the receptor–ligand interaction, which is by‐passed by the use of forskolin. Our results also provide additional evidence involving these changes in the mechanisms which regulate the secretory process in platelets.
Altered distribution profiles of endoplasmic reticulum subfractions after incubation of Krebs II ascites cells with different concentrations of cytochalasin B
Authors: Fjose Anders , Pryme Ian F.
Published: May 2005

Information on the interaction between endoplasmic reticulum (ER) membranes and components of the skeletal network of the cell was gained by treating cells with the antimicrofilament agent cytochalasin B prior to cell disruption by nitrogen cavitation. Treatment of Krebs II ascites cells with cytochalasin B (5–10 μg ml−1) resulted in an increased yield of three ER membrane subfractions — heavy rough (HR), light rough (LR) and smooth (S) membranes, as judged by 3H‐choline incorporation in gradient fractions following discontinuous sucrose gradient centrifugation. The major increase was observed in the HR fraction. These results indicate that the actual yield of the respective ER membrane subfractions after cell disruption is dependent on the degree of direct and/or indirect interaction between individual ER membranes and actin containing filaments of the cytoskeleton in the intact cell.
Studies on rat liver mitochondria:4. Enzyme activities in mitochondria preserved at 0—4°C
Authors: da Silva Patricia Teixeira Padilha , Higuti Ilma Hiroko , Stencel Mario , Campello Annibal de Paiva , do Nascimento Aguinaldo José
Published: May 2005

Rat liver mitochondria, stored with the energy‐linked functions preserved or in aging conditions, were used to assay the activity of various enzymes during five days. The preservation of energy‐linked functions was monitored by the respiratory control coefficient. ATPase, cytochrome oxidase and NADH dehydrogenase showed increased activity when the energy‐linked functions were preserved. In aging conditions, cytochrome oxidase, NADH dehydrogenase and ATPase showed decreased activity. The ATPase activity increased only when mitochondria were stored in the presence of inhibitors of the electron transport chain. The activity of NADH oxidase did not change, and succinate oxidase and succinate dehydrogenase showed a small decrease in their activity. The enzymes of the matrix, α‐ketoglutarate dehydrogenase, malate dehydrogenase and aspartate aminotransferase showed little decrease in activity under either of the conditions of storage. The total protein content decreased slightly under both conditions of storage. These results show that the activity of the enzymes analysed was maintained at reasonable levels, when the energy‐linked functions of isolated mitochondria were preserved.
Hydrolysis of thyroglobulin inside isolated rat thyroid and liver lysosomes
Authors: Fouchier Francis , Dang Josseline , Bastiani Paul , Peyron Claude , Mego John L.
Published: May 2005

In vitro, poorly iodinated thyroglobulin (Tg) is hydrolysed at the same rate whether it is enclosed in thyroid or in liver lysosomes, while fully iodinated Tg is degraded faster inside liver lysosomes. After in vivo TSH administration, thyroid lysosomes hydrolyse fully iodinated Tg as fast as do liver lysosomes.
Distribution of non‐histone proteins between micrococcal nuclease sensitive and nuclease resistant chromatin from chicken cells with active and inactive genomes
Authors: Kiliańska Z. , Kłyszejko‐Stefanowicz L.
Published: May 2005

Chicken liver and erythrocyte nuclei were separated by mild treatment with micrococcal nuclease into nuclease sensitive (NS) and nuclease resistant (NR) fractions, differing in chemical composition and transcriptional activity in vitro. Nuclei, NS and NR fractions of both tissues were fractionated by hydroxyapatite chromatography into three groups of non‐histone chromatin proteins (NHCP) and characterized by SDS‐polyacrylamide gel electrophoresis. Some differences in the molecular distribution of non‐histone proteins of chicken liver and erythrocytes between nuclease sensitive and resistant parts of chromatin have been described.
Behaviour of cyclic nucleotides and Ca2+ levels in liver tissue of rats poisoned by white phosphorus and trichlorobromomethane
Authors: Paradisi L. , Panagini C. , Negro F. , Parola M. , Torrielli M. V.
Published: May 2005

The content of hepatic cyclic AMP was increased soon after intoxication by white phosphorus. Its level reached a maximum 4 h after poisoning, but in subsequent phases tended to return to normal. In contrast, the cyclic GMP concentration was altered only 24 and 36 h after treatment with the same hepatotoxin. Similar modifications of cAMP and cGMP content were also detected after poisoning by trichlorobromomethane (CBrCl3). As a consequence, an altered cGMP/cAMP ratio was found in both experimental conditions. Further, the modification of cAMP content after white phosphorus was detected prior to liver damage (steatosis and necrosis), while the highest concentration of the cyclic nucleotide in CBrCl3‐poisoned rats was found when fatty liver was already evident. In addition, in phosphorus‐poisoned rats, the hepatic content of Ca2+ was found to be unmodified during all phases of the intoxication, while after CBrCl3 a phasic increase of the Ca2+ level was observed at 4, 24 and 36 h.
Cyclic nucleotides and haemoglobin concentration in rabbit bone marrow cell suspensions stimulated by purified erythropoietin
Authors: Fanò G. , Menchetti G. , Secca T. , Marsili V. , Torre G. Della
Published: May 2005

This study was conducted to determine the possible correlations between cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanine monophosphate (cGMP), and haemoglobin (Hb) concentration in nucleated cell suspensions of rabbit bone marrow incubated with erythropoietin (Ep). The levels of cAMP and cGMP were measured following the addition of different Ep concentrations to the suspensions. The Hb concentration was also measured in suspensions treated with Ep, dibutyryl cAMP (db‐cAMP) or dibutyryl cGMP (db‐cGMP), respectively. The following results were obtained: (1) upon the addition of 1 IU ml−1 Ep, an increase of cAMP levels was related to an increase in Hb concentration; while a decrease of Hb concentration was related to an increase of cGMP levels obtained when 0.1 IU ml−1 Ep was present in the incubation mixture. (2) A mimetic effect on Hb concentration was obtained upon the addition of db‐cAMP or db‐cGMP to the suspensions. (3) A quantitative correlation was found between the cAMP/cGMP ratio and Hb levels in cellular suspensions. This rapport was reviewed with respect to the controls as a decrease in Hb concentration when the ratio is less than one and an increase in Hb concentration when the ratio is greater than one.
Metabolic and secretory effects of methylamine in pancreatic islets
Authors: Gomis R. , Deleers M. , Malaisse‐Lagae F. , Sener A. , Garcia‐Morales P. , Rovira A. , Valverde I. , Malaisse W. J.
Published: May 2005

The metabolic and secretory effects of methylamine in rat pancreatic islets were investigated. Methylamine accumulated in islet cells, was incorporated into endogenous islet proteins, and inhibited the incorporation of [2,5‐3H] histamine into either N,N‐dimethylcasein or endogenous islet proteins. Methylamine (2 mM) did not affect the oxidation of glucose or endogenous nutrients or the intracellular pH in islet cells. Glucose did not affect the activity of transglutaminase in islet homogenates, the uptake of 14C‐methylamine by intact islets or its incorporation into endogenous islet proteins. Methylamine inhibited insulin release evoked by glucose, other nutrient secretagogues, and non‐nutrient insulinotropic agents such as L‐arginine or gliclazide. The inhibitory effect of methylamine upon insulin release was diminished in the presence of cytochalasin B or at low extracellular pH. Methylamine retarded the conversion of proinsulin to insulin. Trimethylamine (0.7 mM) was more efficiently taken up by islet cells than methylamine (2.0 mM), and yet caused only a modest inhibition of insulin release. These findings suggest that methylamine interferes with a late step in the secretory sequence, possibly by inhibiting the access of secretory granules to their exocytotic site.
Characterization of insulin uptake into subcellular fractions of perfused rat liver using two different iodinated tracers
Authors: Christensen Jillian R. , Smith Geoffrey D. , Peters Timothy J.
Published: May 2005

The binding and uptake of insulin in perfused rat liver has been investigated with specifically labelled 125I‐A14‐tyrosyl insulin as a tracer and compared with a commercially available iodo‐insulin preparation. The commercial preparation did not show saturation uptake kinetics and the clearence from the perfusate remained low and constant throughtout a wide concentration range. A14 labelled insulin showed saturation kinetics and high clearence at low carrier concentration, falling rapidly with increasing carrier concentration and reaching a steady state value of 1 ml/min. These results emphasize the importance of using specifically labelled insulin in physiological and biochemical studies of hepatic insulin metabolism.

Perfusion with A14 tyrosine‐labelled insulin at 4°C showed apparent saturation with binding to the plasma membrane fraction. Perfusion at 37°C also showed apparent saturation with uptake predominantly to the ligandosome fraction. These results implicate the plasma membrane‐ligandosome pathway in the hepatic uptake of insulin at both physiological and pharmacological concentrations of the hormone.
Incorporation of antigen 125I IgG into particualr cell compartments: Binding by chromatin
Authors: Rakowicz‐Szulczyńska Ewa M. , Horst Antoni
Published: May 2005

Incorporation of [125I]IgG into spleen cells was studied in vivo and in vivo, the antigen after uptake into the cytoplasm migrated into cell nuclei, where it was bound to chromatin up to the saturation level. One day after immunization the constant level of [125I]IgG was 1·3 × 1012 molecules per spleen (108 cells). The same number of [125I]IgG molecules were bound to chromatin in cell cultures. The uptake of [125I]IgG was competitively inhibited by non‐labelled IgG. Binding of [125I]IgG molecules reextracted from cytoplasm and chromatin with specific anti‐human IgG serum argues against the uptake of degraded [125I]IgG molecules. [125I]IgG was tightly bound to DNA. Approximately 50 per cent of [125I]IgG was present in the residual chromatin fractin (after removal of 0·35 M and 2 M NaCl‐soluble frations) and 40 per cent was complexed with DNA (after removal of histones and non‐histones AP1, AP2, AP3 and AP4).

Binding of [125I]IgG by isolated chromatin was inhibited by the cytoplasmic fraction but not by BSA. Binding of [125I]IgG by fractionated chromatin, (chromatins remaining after removal of 0·35M, and 2M NaCl‐soluble fractions or histones + non‐histones AP1 + AP2 + AP3 + AP4) occurred at a level similar to that observed with native chromatin. The results suggest that interaction of antigen with immunocompetent cells is not restricted to the cell surface but that antigen seems to be taken up into cytoplasm, migrates to the nuclei and is bound to chromatin, probably directly to DNA. The results are discussed in relation to the induction of the immune reaction.
Induction of plasma membrane alkaline phosphatase in rat liver
Authors: Bachs Oriol , Enrich Carles , Soriano Montserrat , Piñol Rosa , Domingo Jordi
Published: May 2005

We have determined alkaline phosphatase activity in total liver plasma membrane fractions from rats subjected to a partial hepatectomy and sham operated with or without manipulation of the liver. In all these cases, an increase of the enzyme activity was observed. Kinetic studies of alkaline phosphatase activity performed on plasma membrane fractions from rats subjected to a partial hepatectomy suggest that alkaline phosphatase increase is produced by de novo biosynthesis of enzyme molecules. Determination of alkaline phosphatase activity in purified plasma membrane subfractions corresponding to each of the three functional regions of the hepatocyte surface (blood sinusoidal, lateral and bile canalicular), indicates that the increase of the enzyme activity observed after partial hepatectomy is selectively induced in the bile canalicular domain of the hepatocyte plasma membrane.
Hormone‐Independent polyamine metabolism of squamous cell carcinoma of the prostate
Authors: Shukla Girja S. , Singhal R. L.
Published: May 2005

The levels of polyamines and their synthesizing enzymes in squamous cell carcinoma of prostate implanted in intact as well as castrated male rats were determined after certain hormonal manipulations. The tumour was found to grow with an identical rate in non‐castrated and castrated rats. Polyamine content and activities of polyamine synthesizing enzymes in the tumour were found to be much lower compared to their values in ventral prostate. Moreover, the levels of these parameters were comparable in tumours whether implanted in non‐castrated or gonadectomized animals. The sequential analyses of putrescine and spermidine and activities of L‐ornithine decarboxylase and S‐adenosyl‐L‐methionine decarboxylase of tumours at different time intervals showed a significant reduction in their levels at 30 days compared to 10 days post implantation in non‐castrated as well as castrated rats. Daily intramuscular administration of tumour‐bearing intact or castrated animals with testosterone (50 μg/g), β‐estradiol (2 μg/g) or cyproterone (12·5 μg/g) for 10 days did not influence polyamine metabolism in tumour tissue. However, either β‐estradiol and cyproterone treatments or castration were found to decrease polyamine synthesis in ventral prostate. At the same time, the testosterone replacement therapy did not allow polyamine levels or activities of polyamine synthesizing enzymes to decline in the ventral prostate of castrated rats. Our results demonstrated that contrary to ventral prostate, the polyamine metabolism in squamous cell carcinoma of prostate is independent of hormonal control. The loss of hormonal sensitivity of polyamine metabolism in the prostatic tumour could be the result of qualitative changes that occurred during transformation.
The interaction between gluconeogenic metabolism and accumulation of phosphate by chick kidney tubule cells
Authors: Grahn Michael F. , Parveen Riffat , Butterworth Peter J.
Published: May 2005

Pyruvate promotes both phosphate uptake and glucose synthesis by isolated chick kidney proximal tubule cells. 3‐Mercaptopicolinate inhibits both glucose synthesis and the promoted phosphate accumulation to the same extent. Glycerol also stimulates glucose synthesis, but does not affect phosphate accumulation. Oxygen utilization by the tissue is slightly stimulated by glycerol and pyruvate, but the enhancement of uptake by pyruvate is unlikely to result from raised cellular oxidative phosphorylation. The action of pyruvate is not a direct effect on the phosphate transporter, or on the transport of phosphate across the basolateral membrane, but entails an obligatory flux to triose phosphate.