bluuebunny/crossref_metadata_2025 huggingface dataset:
Fluorescence and chemiluminescence detection of oxazole‐labelled amines and thiols
Authors: Toyo'oka Toshimasa , Chokshi Hitesh P. , Givens Richard S. , Carlson Robert G. , Lunte Susan M. , Kuwana Theodore
Published: May 2005

Fluorescence and chemiluminescence analyses of amino acids and thiols derivatized with 2‐fluoro‐4,5‐diphenyloxazole (DIFOX) and 2‐chloro‐4,5‐bis(p‐N,N‐dimethylaminosulphonylphenyl)oxazole (SAOX‐CI) were investigated. Thirteen diphenyloxazole (DIOX)‐derivatized amino acids were separated within 38 min by a linear gradient elution from 100% A [0.05 M phosphate (pH 7.0):CH3CN (75:25)] to 100% B [0.05 M phosphate (pH 7.0):CH3CN (1:1)] over 30 min and an isocratic elution of 100% B for 30 min. The detection limits (S/N = 2) with fluorescence detection were in the range of 19–64 fmol. Thiols derivatized with SAOX‐CI were separated by an isocratic elution using 0.1 M H3PO4:CH3CN (65:35) and detected fluorimetrically. The detection limits (S/N = 2) of reduced glutathione, N‐acetylcysteine, 2‐mercaptopropionylglycine, cysteine, homocysteine and captopril were 1.2, 1.5, 1.9, 5.7, 6.4 and 7.9 fmol, respectively. Peroxyoxalate chemiluminescence (CL) intensities of sulphonyl‐5‐N,N‐dimethylaminonaphthalene (DNS), SAOX and DIOX derivatives were compared using three different oxalate esters (DFPO, TCPO and TDPO) by flow injection analysis. The relative chemiluminescence intensity (RCL) of SAOX‐proline and DIOX‐proline were 76–80% and 19–25% of DNS‐proline (100%), respectively. Other SAOX and DIOX derivatives showed lower CL intensities ( < 12%). Extremely low CL intensities were obtained for the fluorescent tagging reagents (<0.11%) and their hydrolysis products (<0.80%). Secondary amino acids and peptides, derivatized with DIFOX in aqueous media at room temperature for 1 h, were detected using DFPO/H2O2. TCPO/H2O2 and TDPO/H2O2 after separation by high performance liquid chromatography. The detection limits (S/N = 2) of hydroxy‐L‐proline, L‐proline, L‐prolyl‐glycyl‐glycine and L‐prolyl‐L‐leucine with the three oxalate esters were 10–17 fmol, 22–34 fmol, 50–61 fmol and 80–123 fmol, respectively.
A sensitive and rapid HPLC‐ECD method for the simultaneous analysis of norepinephrine, dopamine, serotonin and their primary metabolites in brain tissue
Authors: Alburges Mario E. , Narang Neelam , Wamsley James K.
Published: May 2005

This report details a very sensitive, rapid and accurate ion‐pair HPLC‐ECD method for the analysis of biogenic amines and their metabolites in brain tissue. The method described enables detection of picogram amounts of 3‐methoxy‐4‐hydroxy phenethyleneglycol (MHPG) (37 pg), 3,4‐dihydroxy phenylacetic acid (DOPAC) (18 pg), norepinephrine (NE) (12 pg), epinephrine (E) (6 pg), 5‐hydroxyindoleacetic acid (5‐HIAA) (18 pg), 3,4‐dihydroxyphenylethylamine (DA) (6 pg), 3‐methoxy‐4‐hydroxyphenylacetic acid (HVA) (12 pg) and 5‐hydroxytryptamine (5‐HT) (12 pg). The linearity of the method is from 18.75 ng/mL to 300 ng/mL for MHPG; 9.37 to 150 ng/mL for DOPAC and 5‐HIAA; 6.25 to 100 ng/mL for NE, HVA and 5‐HT; and from 3.12 to 100 ng/mL for E and DA. The reproducibility, expressed as coefficient of variance (CV%) within‐run and between‐run groups, was 2.25% and 19.49% for MHPG; 3.84% and 29.84% for DOPAC; 0.89% and 8.97% for NE; 1.26% and 5.61% for E; 1.07% and 28.77% for 5‐HIAA; 2.65% and 10.65% for DA; 5.97% and 24.38% for HVA; and 4.44% and 9.45% for 5‐HT.
Rapid assay of iopanoic acid in dog plasma using a Hisep column
Authors: Andeejani Ahmed M. I. , Hughes Herbert , Feuchuk Danny M. , Aboul‐Enein Hassan Y.
Published: May 2005

A rapid method for the determination of iopanoic acid (IOP) in dog plasma utilizing a Hisep column was developed. A mobile phase of 12% methanol, 88% 0.05 M phosphate buffer pH 3.4 yielded a k′ of 8.5 with no interference from proteins present in plasma. Recoveries of IOP from spiked plasma ranged from 97% to 103% at 270 μmol/L and 1.75 mmol/L respectively. Replication was ±2.8% at 1.75 mmol/L and ±6% at 21 μmol/L. A method utilizing 2,4,6‐triiodobenzoic acid as internal standard was also developed for comparison.
A liquid chromatographic method for the determination of fenoprofen in equine plasma and urine
Authors: Delbeke F. T. , Debackere M.
Published: May 2005

A high performance liquid chromatographic method to measure plasma and urine fenoprofen levels in equine biofluids is described. Liquid–liquid extraction with diethylether was used to isolate the drug from plasma and urine. The accuracy and reproducibility of the method were within acceptable limits over the concentration range 0–10 μg/mL and 0–20 μg/mL respectively from plasma and urine. Detection limits were 0.05 μg/mL (2 mL plasma) and 0.2 μg/mL (0.5 mL urine). This procedure was applied to ascertain the pharmacokinetics of a 3 g dose of fenoprofen calcium in a horse.
Simultaneous determination of a mixture of organophosphorus and carbamate pesticides by high performance liquid chromatography
Authors: Thapar Sangita , Bhushan Ravi , Mathur R. P.
Published: May 2005

A simple HPLC procedure has been developed for the simultaneous determination of a mixture of standard carbaryl, carbofuran, carbendazim, dimethoate, malathion and methyl parathion from a spiked agricultural soil sample. The pesticide mixture has been extracted using methanol–water. A concave gradient elution with acetonitrile–phosphate buffer for 15 min followed by linear elution for 5 min, using a wavelength of 224 nm for detection, has been found to resolve the mixture efficiently.
High performance liquid chromatographic analysis of polypeptide hormones in transplanted rat islets
Authors: Ohkubo Tatsuo
Published: May 2005

This report describes high performance liquid chromatographic analysis of transplanted pancreatic islets. A reversed phase ODS column made it possible to measure rat insulin I, II, rat C‐peptide I, II and glucagon simultaneously in isolated rat islets without using radioisotopes. Freshly isolated islets contained 118.0 ± 9.7 ng (mean ± SE, n = 6) insulin and 3.01 ± 0.60 ng glucagon per islet. The insulin I/II ratio was 1.22 ± 0.03. Isolated islets were then cultured in vitro or transplanted into mice under the renal capsule. Transplantation induced mild hypoglycemia in the recipients. The graft mean survival time was 7.2 ± 0.4 days (n = 5). Both cultured (n = 7) and transplanted (n = 6) islets showed similar alterations of polypepide hormones on day 4. Insulin decreased to one third and glucagon remained unchanged. The insulin I/II ratio increased twofold. In conclusion, it was suggested that the general fate of isolated islets was caused by ischemia and denervation. Relatively, ischemia may not damage α cells but may damage β cells because α cells are peripherally located. Denervation may release β cells from a resting state under neural tonic inhibition. Mild hypoglycemia and an increased insulin I/II ratio were related to the accelerated insulin synthesis in the isolated islets.
A highly sensitive method to quantify triazolam and its metabolites with liquid chromatography—mass spectrometry
Authors: Senda Naoto , Kohta Kimiyoshi , Takahashi Toshiaki , Shizukuishi Kenichi , Mimura Tadao , Fujita Tomoyuki , Nakayama Mitsuru
Published: May 2005

We established a highly sensitive method to determine triazolam and its major metabolites, α‐hydroxytriazolam and 4‐hydroxytriazolam, in human plasma and urine with a liquid chromatography‐mass spectrometry system which incorporates an atmospheric chemical ionization interface. A plasma sample and a urine sample after solvent extraction were injected into an ODS column of reversed phase with a mobile phase in a linear solvent gradient of initially 50 mM ammonium acetate (pH 4.0) 50%: methanol 50% and 15 min later methanol 100%; quantification limits of as low as 20 pg/mL at an SNR of 3 were obtained for each compound. With diazepam as an internal standard, recovery yields of 84, 81, and 77% were obtained for triazolam, α‐hydroxytriazolam and 4‐hydroxytriazolam, respectively.
Determination of chondroitin sulphates in human whole blood, plasma and blood cells by high‐performance liquid chromatography
Authors: Huang Yong , Toyoda Hidenao , Toida Toshihiko , Imanari Toshio
Published: May 2005

A simple HPLC method for determination of chondroitin sulphates as thier unsaturated disaccharides by chondroitinases has been applied to measure the glycosaminoglycans (GAGs) in human whole blood, plasma and blood cells. This method may be useful for the investigations on the role of blood GAGs and the metabolic fates of the exogenous GAG.
Quantitative determination of 25‐hydroxyvitamin D3 3‐sulphate in human plasma using high performance liquid chromatography
Authors: Shimada Kazutake , Mitamura Kuniko , Kitama Nami
Published: May 2005

The quantitative determination of 25‐hydroxyvitamin D3 3‐sulphate in human plasma was completed using reversed phase high performace liquid chromatography with UV detection (265 nm) and an internal standard method. The vitamin D sulphate fraction was obtained from a plasma specimen with the combined use of a Bond Elut C18 cartridge for solid‐phase extraction and a piperidinohydroxypropyl Sephadex LH‐20 column for lipophilic ion‐exchange chromatography. Separation of the compounds was performed on a YMC‐Pack ODS‐AM column. The limit of quantitation was 5 ng/mL and the assay was linear from 5 to 50 ng/mL. The proposed method is satisfactory in its accuracy and precision.
Chiral separation of naphthalene‐2, 3‐dialdehyde labelled peptides by cyclodextrin‐modified electrokinetic chromatography
Authors: DeSilva Kapila , Jiang Qun , Kuwana Theodore
Published: May 2005

The chiral separation of several dipeptide enantiomers, derivatized by naphthalene‐2,3‐dialdehyde (NDA), was achieved by use of cyclodextrin‐modified micellar electrokinetic chromatography (CD‐MEKC). The dipeptides contained one or two chiral centers. In the case of several dipeptides containing two chiral centers, all four of the optical isomers could be separated and baseline resolved in less than 15 min with 20 mM γ‐CD and 50 mM sodium dodecyl sulphate (SDS) in the background electrolyte. The order of elution of these derivatized dipeptides was explained by considering important chemical equlibria in the SDS, CD and peptide system. Additionally, the influence of geometric location of the chiral center, the structure of the side‐chain and the effect of CD concentration on the resolution are discussed.
Behaviour of tyrosine amino transferase and convertase during the first hours after hepatectomy in rats
Authors: Biondi Roberto , Viola‐Magni Maria Pia
Published: May 2005

The activity of rat liver tyrosine amino transferase (TAT) increases after hepatectomy with a first prominent peak at 8 h and a second peak at 18 h. This change in activity is probably due to de novo enzyme synthesis since it is prevented by actinomycin‐D (AMD). In the same period an increase of the lysosomal converting enzyme (convertase) which catalyses the in vitro transition of TAT from form I to form III, has been observed; this is not accompanied by changes of other lysosomal enzymes, such as acid phosphatase and cathepsin L. The activity of convertase is equal to that of the controls (sham operated animals) 2 h after hepatectomy, increases three times at 5 h, maintains the same value at 8 h and then decreases slowly to control level after 24 h. The correlation between the activity changes of the two enzymes strongly suggests a physiological role of convertase in TAT turnover.
Fluorimetry of mitochondria in cells vitally stained with DASPMI or rhodamine 6 GO
Authors: Bereiter‐Hahn Jürgen , Seipel Karl‐Heinz , Vöth Monika , Ploem Johan S.
Published: May 2005

The fluorescent dyes DASPMI and rhodamine 6 GO specifically stain mitochondria in living cells. Dye concentrations from 10−8 to 5 × 10−6 mole l−1 can be used. Excitation and emission spectra, and quantum efficiency of DASPMI depend on solvent characteristics. Spectra of mitochondria in living cells correspond to those in phospholipids (excitation around 470 nm, emission 560–570 nm). Fluorescence intensity of DASPMI is a measure for the energization of mitochondria, as revealed by in vitro studies. In living cells uptake of the dye is strongly influenced by inhibitors of oxidative phosphorylation (i.e. by oligomycin, FCCP). Distribution of fluorescence intensity indicates an intracellular gradient in energy load of endothelial cells. Single mitochondria exhibit oscillations in fluorescence. Mitochondria loaded with DASPMI release the dye suddenly into the cytoplasm on treatment with FCCP. Cyanide and antimycin however, do not diminish fluorescence in vivo under optimal nutritional conditions, while they do so in mitochondrial suspension, indicating different mitochondrial behaviour in vivo and in suspension.
Glycosaminoglycan: Cell interactions; their role in lipoprotein lipase secretion from isolated cardiac muscle cells
Authors: Cryer Anthony , Wusteman Frederick S. , Casey Jill J.
Published: May 2005

When cardiac muscle cells from mature rats were incubated in vitro in the presence of heparin (8.7 nmole ml−1) lipoprotein lipase activity appeared in the incubation medium. The intracellular activity of the enzyme remained unchanged. Other glycosaminoglycans (heparan sulphate, dermatan sulphate, keratan sulphate and chrondroitin 6‐sulphate) at the same or higher concentrations were totally ineffective in producing any enzyme redistribution between cells and medium. The release seen in the presence of heparin was blocked by the presence of cycloheximide. Cycloheximide by contrast had no effect on the release observed in the presence of dexamethasone, The action of endogenous glycosaminoglycans are unlikely therefore to have a significant role to play in the movement of lipoprotein lipase in heart tissue in vivo.
Drug—membrane interactions: Silymarin, silibyn and microsomal membranes
Authors: Parasassi T. , Martellucci A. , Conti F. , Messina B.
Published: May 2005

Silymarin and silibyn are extracted from the seeds of Silybum marianum and used as a liver protectant because of their free radical scavenging. When incorporated into rabbit liver microsomes they cause a small decrease in the flourescence anisotropy of 1,6‐diphenyl‐1,3,5‐hexatrine (DPH) but not of 1‐anilinononaphthalene‐8‐sulphonic acid (ANS), incorporated into the membranes. They do, however, reduce the fluorescence intensity of incorporated ANS without changing the wavelength of maximum intensity. These observations suggest that the drugs are incorporated into the hydrophobic–hydrophilic interface of the microsomal bilayer and perturb the structure by influencing the packing of the acyl chains.
Stimulation of rat renal Na+, K+‐ATPase activity by thyroid hormones
Authors: Pippard Corinna , Baylis P. H.
Published: May 2005

The effect of thyroid hormones (T4, T3 and reverse T3) on rat renal Na+, K+‐ATPase activity was investigated by a cytochemical technique. T3 caused stimulation of Na+, K+‐ATPase activity in the renal medulla but not in the renal cortex. There was a peak in enzyme activity after cultured renal segments had been exposed to T3 for 11 min and this time of maximal stimulation did not vary with the concentration of T3. A rectilinear response in Na+, K+‐ATPase activity was observed over T3 concentration range 10 pmol l−1 to 100 nmol l−1; at higher T3 concentrations, Na+,K+‐ATPase activity was inhibited. The enzyme response was totally blocked by specific T3 antiserum. Addition of T4 and reverse T3 (100 fmol l−1 − 1 mmol l−1) failed to stimulate Na+,K+‐ATPase activity in any part of the kidney. Plasma (neat and diluted 1:10) stimulated the enzyme in parallel with the dose response curve and the stimulatory effect was abolished by prior addition of specific T3 antiserum.
Polyamines in Entamoeba invadens
Authors: Chayen A. , Mirelman D. , Chayen R.
Published: May 2005

The polyamine content of Entamoeba was measured by a procedure that involved benzoylation followed by high performance liquid chromatography (h.p.l.c.). A high concentration of putrescine and significant amounts of spermidine and spermine were found in actively growing trophozoites and in the cyst forms of the organism. In contrast, trophozoites in stationary phase had greatly reduced amounts of putrescine and exhibited peaks in h.p.l.c., possibly indicative of acetylated polyamines. α‐D,L‐difluoromethylornithine (DFMO) lowered the concentration of polyamines in growing trophozoites, but did not inhibit the degree of proliferation. There is evidence for pathways of polyamine biosynthesis in Entamoeba other than through ornithine decarboxylase (ODC).
Immunofluorescent staining method for use with monoclonal antibodies
Authors: Costanzo Catherine , Raza Azra
Published: May 2005

This note describes an immunofluorescent staining method for cells in the S‐phase which have been allowed to take up bromodeoxyuridine into their DNA in place of thymine. The technique involves the use of fluorescinated monoclonal antibodies against bromodeoxyuridine and allows rapid and accurate estimation of cells in the S‐phase, the technique does not require interpretation by skilled technicians.
Comparative subcellular fractionation of control and cold‐adapted rat brown and white adipose tissue with special reference to peroxisomal and mitochondrial distributions
Authors: Karmali Anis , Montague David J. , Peters Timothy J. , Holloway Brian R.
Published: May 2005

A new technique for single‐step subcellular fractionation of adipose tissue homogenates by analytical sucrose density gradient centrifugation in a vertical pocket reorientating rotor is described. The density gradient distributions of mitochondrial and peroxisomal marker enzymes in brown and white adipose tissue of control and cold exposed rats are compared. The equilibrium density of brown fat mitochondria was found to be significantly increased compared with white fat mitochondria. GDP binding activity was localized solely to the mitochondria in both control and cold‐adapted brown adipose tissue. Brown and white fat mitochondria fractions were isolated by differential centrifugation and the specific activities of various enzymes in the homogenate and mitochondrial preparations determined. The specific activity of creatine kinase in brown adipose tissue was found to be ten‐fold higher than in white fat and subcellular fractionation studies showed the activity to have an exclusively cytosolic distribution in both tissues. GDP binding activity and some of the mitochondrial enzymes showed, in brown adipose, a striking increase in total activity in cold adapted rats compared to control animals. For some enzyme activities there was a small increase when expressed per mg tissue or per mg mitochondrial protein. When expressed per mg DNA i.e. per cell, there was a reduced specific activity of the mitochondrial and peroxisomal enzymes in both brown and white adipose tissue on cold adaptation.
Adenine nucleotides in thrombocytes of birds
Authors: Wachowicz Barbara
Published: May 2005

Analysis of free nucleotide composition of both avian thrombocytes and pig platelets showed quantitative differences in the level of adenine nucleotides. 3H‐adenine taken up by turkey thrombocytes was metabolized mainly to adenine nucleotides was not released after thrombin action. Thrombin liberated non‐radioactive adenine nucleotides (18.2 ± 1.5 %, 20.6 ± 1.9%) of the total, probably localized in a storage pool. Malonyldialdehyhyde (MDA) production due to thrombin was observed in both platelets and thrombocytes.
Shape change of human erythrocytes induced by phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths
Authors: Fujii Tatsuzo , Tamura Akira
Published: May 2005

Intact human erythrocytes were treated, under non‐haemolytic conditions at 37°C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8 ∼ 18 even‐numbered carbon atoms (C8 ∼ C18‐PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8 ∼ 18 carbon atoms (C8 ∼ C18‐lysoPC). The C8 ∼ C14‐PC and C12 ∼ C18‐lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16‐PC, C18‐PC, C8‐lysoPC and C10‐lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.
Iodide accumulation into thyroid golgi vesicles
Authors: Bastiani P. , Rognoni J. B.
Published: May 2005

Pig thyroid Golgi vesicles incubated in a suitable medium were able to concentrate iodide from the medium. This trapping required the integrity of the vesicles, was time‐ and temperature‐dependent, and was inhibited by a competitive inhibitor of iodide active transport (perchlorate), suggesting a facilitated transport mechanism.
Phospholipids in plant and animal chromatin
Authors: Viola‐Magni M. P. , Gahan P. B. , Pacy J.
Published: May 2005

Isolated hepatic nuclei and hepatic chromatin have been analysed for their DNA, RNA, protein and phospholipid content. The protein/DNA ratio is 3 for nuclei and 1·95 for chromation extracted from Triton X‐100 treated nuclei. The phospholipids, (2·36 ± 0·91 (S.D.) per cent of the total nuclear material), are lost during the chromatin preparation mainly during the Triton X‐100 washings of the nuclei. Nevertheless, 10 per cent of the total nuclear phospholipids and fatty acid composition. Thus, the chromatin‐associated phospholipid cannot be attributed simply to contaminating nuclear membrane.

This is supported by the autoradiographic study of semi‐thin sections of interphase nuclei from root apices of Vicia faba in which [3H] ethanolamine is clearly localized in the chromatin and nucleolar regions of the nuclei.
Partial immunochemical identity between (Na+ + K+)‐atpase and a membrane component extractable with chloroform: Methanol
Authors: Addis John S. , Tenbarge Karen M. , Barrnett Russell J.
Published: May 2005

An IgG fraction prepared from an antiserum against a holoenzyme preparation of (Na+ + K+)‐ATPase precipitated a single antigen when samples of holoenzyme were subjected to crossed immunoelectrophoresis but precipitated an additional, immunochemically‐related antigen when a plasma membrane‐enriched fraction was subjected to crossed immunoelectrophoresis under the same conditions. The immunochemically‐related antigen could be extracted from the plasma membrane fraction with CHCl3:CH3OH.
Role of transferrin in metal uptake by human lymphoblasts in vitro
Authors: Planas‐Bohne Felicitas , Taylor David M. , Duffield John R.
Published: May 2005

The uptake and binding of 59Fe, 67Ga and 239Pu complexed with citrate of transferrin (Tf) and of 125I‐labelled Fe–Tf by human lymphoblasts (WI‐L2 cells) have been studied. Uptake kinetics of 59Fe–Tf and [125I]‐Tf point to internalization by receptor mediated endocytosis. 67Ga binding and uptake is always less. This may be explained by a lower affinity of Ga‐complexes for the cell surface. Factors which influence Fe uptake have a similar effect on Ga. 239Pu uptake and binding, however, are different, especially in that Tf does not stimulate 239Pu uptake and may actually decrease it.
Characterization of insulin receptors in isolated epithelial cells of rat ventral prostate: Effect of fasting
Authors: Carmena M. J. , Fernandez‐Moreno M. D. , Prieto J. C.
Published: May 2005

Insulin receptors have been characterized in rat prostatic epithelial cells by using [125I]insulin and a variety of physicochemical conditions. The binding data at equilibrium (2h at 15°C) could be interpreted in terms of two populations of insulin receptors: a class of receptors with high affinity (Kd = 2·16 nM) and low binding capacity (28·0 fmol mg−1 protein), and another class of receptors with low affinity (Kd = 0·29 μM) and high binding capacity (1·43 pmol mg−1 protein). Proinsulin exhibited a 63‐fold lower affinity than insulin for binding sites whereas unrelated peptides were ineffective. The specific binding of insulin increased by about 50 per cent after 96 h of fasting; this increase could be explained by an increase of both the number of the high affinity–low capacity sites and the affinity of the low affinity–high capacity sites. These results together with previous studies on insulin action at the prostatic level strongly suggest that insulin may exert a physiological role on the prostatic epithelium.
Effects of antioxidants and haemoglobin status on the t‐butyl hydroperoxide‐induced oxygen uptake by red blood cells
Authors: Lissi E. , Franz R. , Cabezas J. , Fernández V. , Videla L. A.
Published: May 2005

Oxygen uptake by erythrocytes exposed to t‐butyl hydroperoxide (t‐BHP) exhibited an induction period. The rate of oxygen consumption can be reduced by antioxidants and blood plasma. The induction time was not appreciably modified by the antioxidants tested, however, plasma increased it by a factor of two. The in vivo pretreatment with diethyl maleate (0·6 g kg−1) produced increased rates of oxygen uptake without changes in the induction period, while vitamin E (12·5 mg kg−1) elicited lower oxygen consumption rates and longer induction times, compared to those observed in cells from control rats upon addition of the hydroperoxide. These results suggest that the antioxidants tested on the t‐BHP lipid peroxidation in erythrocyte suspensions act as inhibitors and/or retarders of the process. Furthermore, lipid peroxidation induced in these conditions seems to depend upon the haemoglobin status of the cells as oxygen uptake, malondialdehyde production and chemiluminescence were significantly higher in methaemoglobin‐containing cells than in those containing oxyhaemoglobin.
Flow cytometric determination of cell cycle phase‐specific changes in cellular phosphatase and glycosidase activities
Authors: Britten R. A. , Dyson J. E. D.
Published: May 2005

The activities of two phosphatases (E. C. 3.1.3.1 and 3.1.4.1) and four glycosidases (E.C. 3.2.1.21, 3.2.1.30, 3.2.1.31 and 3.2.1.51) were measured by fluorescence spectrophotometry, and flow cytometry, in mitogenstimulated lymphocytes, and in cultures of Molt‐4‐F and F‐89 cell lines, syncronized by hydroxyurea or thymidine.

All enzymes were active throughout the cycle but the activities of three enzymes were elevated at specific points in the cycle, alkaline phosphatase activity increased at G2 + M/G1 boundary and in early S‐phase, the activity of β‐L fucosidase was elevated in G1 and late S‐phase. Orthophosphate diesterase activity was elevated at the G1/S boundary, and during G2 + M. The increase in β‐L fucosidase activity was due to an increased number of cells showing activity, whilst the increase in orthophosphate diesterase activity was attributable to an increase in cellular enzyme activity.

Only the activities of orthophosphate diesterase and β‐L fucosidase were measurable by flow cytometry, alkaline phosphatase activity was mainly extracellular, and therefore not detectable by flow cytometric methods employed.
Calcium transport from blood into the bile in normal and regenerating rat liver
Authors: Bachs Oriol , Soriano Montserrat , Piñol M. Rosa , Serratosa Joan , Enrich Carlos
Published: May 2005

We have studied calcium movement from blood into the bile by injecting 45Ca2+ intravenously and measuring the radioactivity appearing in the bile. 45Ca2+ started to appear in the bile at 3 min and maximum values were observed at 5 min after its administration. The amount of calcium secreted into the bile was proportional to the blood calcium concentration indicating that the main pathway involved in calcium movement behaved as a non‐saturable system.

We have also studied the 45Ca2+ circulation from blood into the bile in rats subjected to a partial hepatectomy. Thereafter, the calcium transported into the bile per gram of liver increased by about 50 per cent. Since bile flow behaved in a similar way, the biliar calcium concentration remained unmodified after hepatectomy.

Determination of the activities of the Ca2+ transporting systems in isolated plasma membrane fractions from regenerating livers showed no modification in these activities suggesting that the elevation in calcium movement observed after hepatectomy is not due to an increase in the circulation of Ca2+ through the transhepatocyte pathway, an observation compatible with the absence of saturation in the transport.
Lipases operative at the fat cell surface: Attempt at an integrated approach
Authors: Verine Alain , Boyer Jean
Published: May 2005

Extracellular acylglycerols are hydrolysed by lipases active at the surface of intact fat cells isolated from rat or human adipose tissue. During short‐term incubation, rat fat cells hydrolyse di‐[3H]oleyl‐[14C]glycerol at a rate of 70 ± 7·7 mU/106 cells (means ± S.E.) versus 440 ± 62 mU/106 cells for the hydrolysis of mono‐[3H]oleylglycerol; these relatively high lipolytic potencies may serve, among other functions, to counteract the cytolytic effect of both esters. Reaction rates with both substrates are unchanged by addition of various apolipoproteins C and by the nutritional state of the animals. Fat cells incorporate 15–20 per cent of the total [3H]‐oleic chains liberated by hydrolysis, with no correlation between uptake and hydrolysis rates. [3H]‐oleic chains in cell lipids are found mainly as diacylglycerol (15 per cent) and triacylglycerol (80 per cent). Both lipolytic processes differ from the hydrolysis of trioleyglycerol by cell‐bound lipoprotein lipase, which occurs at lower rates (6·5 ± 0·6 mU/106 cells) and depends on apolipoprotein C‐II and nutritional state of the animals. The results support the accepted view that lipoprotein lipase and monoacylglycerol lipase are distinct enzymes. Differences between lipoprotein lipase and diacylglycerol lipase activities raise the possibility of different catalytic entities. In conclusion, isolated fat cells in suspension hydrolyse and incorporate lipids. This model should approximate physiological conditions more closely than the use of lipases in the free state.
Prolyl hydroxylase activity as an index of liver damage induced by ethanol
Authors: Bańkowski Edward , Pawlicka Elżbieta , Jodczyk Kazimierz J.
Published: May 2005

It was found that chronic intoxication of rats with ethanol results in an increase of prolyl hydroxylase activity in liver and serum of the experimental animals. The increase of enzyme activity precedes the morphological symptoms of liver damage. The possibility arises that the assay of prolyl hydroxylase in serum or in liver biopsy samples could be useful for the diagnosis of the tendency of some individuals to develop liver cirrhosis induced by ethanol.
Introduction and metabolism of pentose and hexose phosphates in permeabilized morris hepatoma 5123TC cells
Authors: Arora Krishan K. , Williams John F.
Published: May 2005

Metabolism of arabinose 5‐P, ribose 5‐P and glucose 6‐P in permeabilized and resealed Morris hepatoma 5123TC cells was investigated by measuring the contribution of these compounds to nucleic acid biosynthesis. The level of [14C]‐arabinose (non‐phosphorylated) incorporation into nucleic acids was slight, presumably due to the low activity of the transport system or the absence or low activity of a specific ‘kinase’ enzyme. The permeabilizing procedure involved the brief treatment of Morris hepatoma 5123TC cells with lysolecithin and resulted in a cell population which was permeable to charged compounds i.e. Sugar phosphates and nucleotides, that otherwise could not cross the plasma membrane. The permeabilized (and resealed cells) retained normal cellular morphology and intactness of specific organelles as judged by the maintenance of functional properties. Following permeabilization, these cells resealed when transferred back to normal growth medium, and continued to divide and increase at the same rates as control non‐permeabilized cell cultures. The permeabilized cells incorporated deoxyribonucleotides ([methyl ‐3H]‐TTP) into DNA at a linear rate of 0·047 nmol per 107 cells min−1, representing 90–100 per cent of the DNA synthesis rate in vivo. The permeabilization technique, when coupled with procedures to establish cell synchrony, permitted the comparative estimate of the contributions of [14C]‐labelled arabinose 5‐P, ribose 5‐P and glucose 6‐P to RNA, DNA, amino acids, CO2, lactate and sugar mono‐ and bisphosphates. The percentage of [14C]‐isotope incorporated into total nucleic acids by these three labelled sugar phosphates were 2·3, 4·9 and 6·3 respectively. Possible reasons for the lower incorporation of 14C from arabinose 5‐P are given. The results are consistent with the proposal that arabinose 5‐P, an intermediate of the L‐type pentose pathway activity of 5123TC cells, was incorporated into nucleic acids by its interconversion with ribulose 5‐P and ribose 5‐P and thus into PRPP. This study represents the first report of sugar phosphate as opposed to free sugar metabolism by tumour cells in culture.
Pyruvate kinase activity and response to allosteric effectors in rat erythrocytes and reticulocytes fractionated by multiple partitioning in aqueous two‐phase systems
Authors: Pinilla Montserrat , Rodriguez‐Horche Paz , Luque Jose
Published: May 2005

Specific activity of pyruvate kinase decreases as the age of rat erythrocytes increases in fractions obtained by counter‐current distribution in dextran‐polyethylene glycol biphasic systems; the enzyme is inhibited by ATP and activated by fructose‐1,6‐bisphosphate at low phosphoenol pyruvate concentrations. Specific activity does not change in fractions from > 95 per cent‐rich reticulocytes (anaemic rats); the enzyme is inhibited by ATP but not activated by fructose‐1,6‐bisphosphate. These results can be explained on the basis of different pyruvate kinase isozymes and suggest that decrease in activity is not affecting regulatory properties during erythrocytes aging.
Ehrlich ascites tumour cells become refractory to α‐difluoromethylornithine at a certain stage of growth
Authors: Anehus Siw , Oredsson Stina M. , Heby Olle
Published: May 2005

When Ehrlich ascites tumour cells are induced to proliferate by serum stimulation, the ornithine decarboxylase (ODC) activity increases rapidly and reaches two to three peaks during the first 24 h. Inhibition of the first peak in ODC activity (occurring at 4 h) by adding α‐difluoromethylornithine (DFMO) within 2 h of serum stimulation, results in maximal growth inhibition. Under these conditions, similar degrees of polyamine depletion are achieved. When DFMO is added 3 h after seeding, however, enough polyamines have already accumulated during the initial burst in ODC activity to reduce the antiproliferative effect of the drug. The antiproliferative effect is further reduced when DFMO is added 6 h after seeding. When DFMO is added 23 h after seeding, i.e. after maximal accumulation of polyamines, there is no inhibition of cell proliferation. These findings are important to consider both when designing experimental as well as clinical regimens for this drug.
Immunofluorescent localization of transglutaminase in rat small intestine
Authors: Sharp Gaynor , Grindley Helen , Lorand L. , Peters Timothy J.
Published: May 2005

The distribution of intestinal transglutaminase was investigated by immunofluorescence microscopy using rabbit anti‐guinea pig transglutaminase immunoglobulin. Transglutaminase‐related antigen was demonstrated principally in the cytoplasm of villous core interstitial cells with some activity in the brush border region of the villous epithelial cells. Implications for the pathogenesis of coeliac disease are discussed.
Purine and pyridine nucleotides in rabbit red blood cells of different maturity
Authors: Werner Andreas , Siems Werner , Gerber Gerhard
Published: May 2005

Using reversed‐phase high‐performance liquid chromatography purine nucleotides, nucleosides and nucleobases as well as pyridine nucleotides were determined in extracts of reticulocytes and mature red blood cells of rabbits. The concentrations of almost all compounds measured decrease during the last phase of red blood cell maturation.

These changes were interpreted with respect to the loss of mitochondria, accompanied by shifting the energy production from the preferentially oxidative mode to the exclusively glycolytic one and variations in the concentrations of purine compounds in blood plasma during reticulocytosis.
Phosphate transport across the basolateral membrane of chick kidney proximal tubule cells
Authors: Myint Sabai , Butterworth Peter J.
Published: May 2005

Characterization of the phosphate transport system across the basolateral membrane of renal proximal tubule has been attempted using isolated proximal tubule cells prepared from chicks. The Pi efflux system is independent of Na+ ions and is not influenced by the nature of the chief anion present in the bathing medium. Pi efflux is not sensitive to DIDS and it is concluded that a generalized anion transporter of band III type is not the chief agent for facilitating Pi exit from the cell across the basolateral membrane. Inhibition of efflux by vanadate is evidence for a specific carrier protein in the membrane. The carrier probably possesses thiol group(s) that are essential for activity. The carrier may effect electroneutral transport of Pi possibly in exchange for OH− ions. The activity of the transport process is not stimulated by depleting the cells of phosphate or inhibited by rearing the chicks on a vitamin D‐deficient diet. The system is unlikely to be of great importance for the expression of various regulatory mechanisms that act on the kidney to control the excretion of Pi. The activity declines as the chicks mature however.
A prostaglandin oligomeric derivative inhibits activities of phospholipase and protease: A possible mechanism of membrane protection during ischemia
Authors: Ohnishi S. Tsuyoshi , Katsuoka Masayuki , Hidaka Saburo
Published: May 2005

A prostaglandin oligomeric derivative was synthesized by alkaline treatment of prostaglandin E1. This compound protected the perfused rat heart from global ischemia. This compound was found to inhibit several lipolytic and proteolytic enzymes in vitro. When phospholipase A2 from Naja naja venom was used as an enzyme and phosphatidylcholine was used as a substrate, 50 per cent inhibition was achieved at 50 μM of the prostaglandin derivative. When trypsin and casein were used as enzyme and substrate, 50 per cent inhibition was obtained at 80 μM. A possible mechanism of beneficial effect of this compound in protecting membranes during ischemia is discussed.
Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex
Authors: Żelewski Mateusz , Świerczyṅski Julian
Published: May 2005

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ.

The increase in activity of malic enzyme in the 13 000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate‐treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria.

Calculations showed that 0·57 and 0·53 μmoles min−1 g−1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate‐treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.
Methotrexate: Studies on cellular metabolism. III.‐Effect on the transplasma‐membrane redox activity and on ferricyanide‐induced proton extrusion by HeLa cells
Authors: Oliveira Ma. Benigna M. , Campello Annibal P. , Klüppel Ma. Lúcia W.
Published: May 2005

The effect of methotrexate (MTX) on transplasma‐membrane electron transport and ferricyanide‐induced proton extrusion by HeLa cells was studied. Both systems were inhibited by MTX. It is suggested that inhibition of electron transport and proton extrusion caused by MTX could be associated with other metabolic alterations such as response to the increase in NADH levels and decrease in intracellular pH.
Effect of orally‐administered epidermal growth factor on intestinal iron absorption and mucosal permeability
Authors: Raja K. B. , Simpson R. J. , Gregory H. , Peters T. J.
Published: May 2005

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3 d and appeared to be dose‐dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO‐feeding increased mucosal permeability (evaluated with [51Cr]‐EDTA): the latter could account for the increase in iron absorption. Sialodenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.
Effect of silybin dihemisuccinate on the ethanol metabolizing systems of the rat liver
Authors: Valenzuela Alfonso , Bustamante Juan C. , Videla Carlos , Guerra Ricardo
Published: May 2005

Silybin dihemisuccinate produces a decrease in the ethanol metabolic rate of rats. This effect is ascribed to an inhibition of the microsomal ethanol oxidizing system (MEOS). Alcohol dehydrogenase activity, catalase activity and NADPH cytochrome c reductase activity are not affected by the flavonoid. It is proposed that the inhibition of MEOS by silybin dihemisuccinate is related to its antioxidant properties, acting as a scavenger of the free radicals involved in ethanol metabolism by this enzymatic system. This observation may have therapeutical implications because microsomal lipid peroxidation induced by hydroxyl free radicals has been related to the etiology of hepatic alcoholic disease.
Microsomal membrane fluidity and phosphatidylcholine synthesis in rabbit lung under high oxygen tension
Authors: Casals Cristina , Herrera Luz , Gasset Maria , Garcia‐Barreno Pedro , Municio Angel M.
Published: May 2005

Phosphatidylcholine metabolism and membrane fluidity were studied in microsomes isolated from rabbit lung, which had been exposed to high oxygen tension for 30 min.

In these microsomes the incorporation of [3H]‐palmitate into phosphatidylcholine increased whereas the incorporation of [14C]‐glycerol and [14C]‐choline from CDP‐[methyl‐14C]‐choline remained unchanged in comparison to the control microsomes. The enhanced [3H]‐palmitate incorporation may be explained by an increase of the specific activity of acyl‐CoA:lysophosphatidylcholine acyltransferase which was measured in microsomes from hyperoxic lung.

Although microsomal parameters influencing membrane fluidity, such as the cholesterol/phospholipid molar ratio, unsaturation degree of phospholipid acyl chains and lipid/protein ratio, are altered after oxygen treatment in vivo, no change of fluorescence polarization (PDPH) and lipid structural order parameter (SDPH) could be measured. Probably, the membrane maintains its fluidity by counteracting effects on different factors on which the fluidity depends.
Balancing of energy‐consuming processes of rat hepatocytes
Authors: Schneider W. , Siems W. , Grune T.
Published: May 2005

A method for the quantification of energy consuming processes described by Siems et al.6 for reticulocytes and by Müller et al.10 for ascites tumour cells was applied to balance the ATP‐consumption of isolated rat hepatocytes. On the basis of decreased coupled respiration rates following the specific inhibition of energy‐requiring reactions, the energy demands of protein turnover, nucleic acid synthesis, Na+/K+‐ATPase and Ca2+‐transport of hepatocytes in different incubation media were assessed. These processes together with urea synthesis account for about 60 per cent of the total energy consumption in a glucose and amino acid‐enriched Eagle/Borsook medium. The metabolic flux rates of total ATP‐consumption and ATP‐consumption of single energy‐requiring processes in hepatocytes are compared with those in reticulocytes and different tumour cell types.
Stimulus‐secretion coupling of arginine‐induced insulin release: Significance of changes in extracellular and intracellular pH
Authors: Malaisse Willy J. , Plasman Pierre O. , Blachier François , Herchuelz Andre , Sener Abdullah
Published: May 2005

The possible relevance of changes in extracellular and/or intracellular pH to the insulinotropic action of L‐arginine and L‐homoarginine was investigated in rat pancreatic islets. A rise in extracellular pH from 7·0 to 7·4 and 7·8 augmented the secretory response to these cationic amino acids whilst failing to affect the uptake of L‐arginine by islet cells and whilst decreasing the release of insulin evoked by D‐glucose. Under these conditions, a qualified dissociation was also observed between secretory data and 45Ca net uptake. Moreover, at high extracellular pH, the homoarginine‐induced increase in 86Rb outflow from prelabelled islets rapidly faded out, despite sustained stimulation of insulin release. The cationic amino acids failed to affect the intracellular pH of islet cells, whether in the absence or presence of D‐glucose and whether at normal or abnormal extracellular pH. These findings argue against the view that the secretory response to L‐arginine would be related to either a change in cytosolic pH or the accumulation of this positively charged amino acid in the β‐cell. Nevertheless, they suggest that the yet unidentified target for L‐arginine and its non‐metabolized analogue in islet cells displays pH‐dependency with optimal responsiveness at alkaline pH.
The assay of uridine diphosphoglucose dehydrogenase activity: Discrimination from xanthine dehydrogenase activity
Authors: Mehdizadeh S. , Bitensky Lucille , Chayen J.
Published: May 2005

The biochemical and quantitative cytochemical assays of the activity of uridine diphosphoglucose dehydrogenase (UDPG‐D) have produced perplexing results. It is now shown that the perplexity may be due to the possibility that the coenzyme (NAD) required for UDPG‐D activity, may be acting as a substrate for a second dehydrogenase, namely xanthine dehydrogenase, which may utilize NAD as its substrate. The activity of UDPG‐D can be distinguished selectively by the pH of its optimal activity and by decreasing the concentration of the coenzyme used in the assay.
Variation in the isoenzymes of N‐acetyl‐β‐D‐glucosaminidase and protein excretion in aminoglycoside nephrotoxicity in the rat
Authors: Sanchez‐Bernal C. , Vlitos M. , Cabezas J. A. , Price R. G.
Published: May 2005

Three aminoglycosidic antibiotics: tobramycin, amikacin and sisomicin were administred to rats. There was an increase in the activity of N‐acetyl‐β‐D‐glucosaminidase (NAG) excreted in the urine and this was characterized by a change in the isoenzyme profiles eluted from DEAE–cellulose. The largest increase in NAG activity was observed following sisomicin administration due mainly to an increase in the B‐form of NAG with a concomitant fall in the intermediate (I‐form). Separation of urinary proteins by SDS‐polyacrylamide gel electrophoresis demonstrated a mixed tubular and glomerular proteinuria following administration of sisomicin. It is concluded that the separation of NAG isoenzymes and urinary proteins provides valuable additional information on the nature and severity of antibiotic nephrotoxicity.