Adsorption of endotoxins on glass in the presence of cationic proteins.

Endotoxin activity was detected in empty glass tubes where endotoxins were incubated with lysozyme, histone or RNaseA, indicating adsorption of endotoxins on glass in the presence of cationic proteins. In the case of lysozyme, the recovery of spiked endotoxins (90.0%) using polystyrene tubes for incubation was much greater than the recovery (28.5%) using glass tubes, suggesting that lysozyme-mediated adsorption of endotoxins on glass is a major cause of poor recovery of spiked endotoxins in the LAL assay using glass tubes. In contrast, the recovery of spiked endotoxins (64.7%) using polystyrene tubes in the presence of the non-cationic protein BSA was less than the recovery (103.9%) using glass tubes. The difference in endotoxin recovery using glass or polystyrene tubes in the presence of cationic proteins or BSA can be explained by differences in protein adsorption on the tubes. Consequently, care must be exercised in selecting containers used for the LAL assay of proteins which bind to endotoxins.

Reversed phase HPLC of Met58 oxidized rhIL-11: oxidation enhanced by plastic tubes.

The hydrophobicity of human recombinant interleukin 11 (rhIL-11) with an oxidized Met58 residue is nearly identical to the hydrophobicity of native rhIL-11. Consequently, separation of these species using standard gradient elution or isocratic elution is very difficult. Using an optimized, shallow gradient RP-HPLC method. Met58 oxidized rhIL-11 could be separated sufficiently from native rhIL-11. The identity of the oxidized form detected with this method was confirmed by peptide mapping with trypsin and endoproteinase Asp-N, N-terminal sequencing and mass spectrometric analysis. This method was employed to determine the effect of disposable laboratory plastic tubes for the oxidation. The amounts of Met58 oxidized rhIL-11 were increased when rhIL-11 samples were stored in plastic tubes at 37 degrees C in the dark. Samples stored in polypropylene tubes were oxidized much more than samples stored in polystyrene tubes. Additionally, the oxidation was greatly enhanced when samples were stored in polypropylene tubes exposed to light before rhIL-11 sample storage. The extent of the oxidation was also affected by the sources of polypropylene tubes. A maximum increase in Met58 oxidized rhIL-11 was more than 30% when samples were stored at 37 degrees C for 14 days in polypropylene tubes exposed to a daylight fluorescent lamp for 25 days. Consequently, these results indicate that attention should be paid for selection of suitable plastic tubes used for storage of protein samples, and for protection of the plastic tubes themselves from extended exposure to light while in storage.

Quantitative in vitro bioassay for recombinant human interleukin-11.

A cell culture-based in vitro bioassay was developed to measure the biological activity of recombinant human interleukin-11 (rhIL-11). The bioassay measures induced proliferation of T10 cells, derived from the T1165 murine plasmacytoma line. A colorimetrically detectable formazan product, obtained by cellular reduction of the tetrazolium compound, 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1), was used as an endpoint for response of clone T10 to added rhIL-11. Positions of the samples and the standards in 96-well microplates affected the precision of this bioassay, which was improved by using 2 microplates where serially diluted sample and standard lines were interleaved and their positions were alternated. The coefficient of variation for this bioassay was less than 8%. This method is suitable for quality control of rhIL-11 because of its simplicity, reproducibility, and accuracy.

Adsorption of endotoxin on glass in the presence of rhIL-11.

Poor recovery of spiked endotoxin in the Limulus amebocyte lysate assay (LAL assay) was observed in the presence of recombinant human interleukin-11 (rhIL-11), a cationic, hydrophobic protein. Detection of endotoxin activity remaining in the empty glass tubes in which endotoxin and rhIL-11 mixtures were incubated indicated adsorption of endotoxin on glass. At low concentrations of rhIL-11, a correlation between endotoxin adsorbed on glass and a decrease of endotoxin in solution was observed. Adsorption of rhIL-11 on glass correlated with adsorption of endotoxin, which indicates that rhIL-11 mediates adsorption of endotoxin on glass. Consequently, adsorption of endotoxin on glass may occur in the presence of other substances which bind to both of endotoxin and glass.

Monosaccharide composition analysis of pamiteplase by anion exchange chromatography with pulsed amperometric detection.

The monosaccharides (neutral and amino sugars) of palmiteplase (recombinant modified human tissue plasminogen activator) were analyzed by high performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Since the palmiteplase formulation contains sucrose, it was removed by reverse-phase high-performance liquid chromatography (RP-HPLC) prior to analysis. Acid hydrolysis with TFA was performed at 100 degrees C for 4 h. Fucose, glucosamine, galactose and mannose were detected by HPAEC-PAD analysis after hydrolysis. The linearity range of HPAEC-PAD analysis was 20-200 pmol/ml(-1) (r > 0.999) and the RSD value for repeatability was less than 7%. The recovery of each monosaccharide spiked into samples was more than 90%. The monosaccharide composition of palmiteplase suggests that it has complex-type oligosaccharides lacking in high-mannose-type oligosaccharides.

Magnesium-mediated conversion of an inactive form of a hammerhead ribozyme to an active complex with its substrate. An investigation by NMR spectroscopy.

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson-Crick 3-base pair duplex and a 4-base pair duplex that included a wobble G:U base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. Elevated concentrations (1.0 M) of monovalent Na+ ions could not induce the formation of the R32-RdC11 complex. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes, but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.

Metabolism of tamsulosin in rat and dog.

1. The metabolism of tamsulosin hydrochloride (TMS), a potent alpha 1-adrenoceptor blocking agent, was studied after a single oral administration to rat and dog. 2. Eleven metabolites (1, 2, 3, 4 and their glucuronides, sulphates of 1 and 3, and A-1) were identified from the urine and bile of rat and dog administered TMS. 3. Unchanged drug and metabolites in urine and bile were quantified in rat and dog dosed with 14C-TMS(1 mg/kg). In rat the main metabolic routes were de-ethylation of the o-ethoxyphenoxy moiety, demethylation of the methoxybenzenesulphonamide moiety, and conjugation of the resultant metabolites by glucuronic acid and sulphuric acid. In dog the main pathways were de-ethylation of the ethoxyphenoxy moiety, conjugation of the de-ethylated product by sulphuric acid, and oxidative deamination of the side chain. 4. The organ responsible for the metabolism of TMS in rat was estimated using 9000g supernatants of liver, kidney, small and large intestine homogenate and plasma. The drug was rapidly metabolized in liver but hardly metabolized in the other organs or plasma.

Kalimantacin A, B, and C, novel antibiotics produced by Alcaligenes sp. YL-02632S. II. Physico-chemical properties and structure elucidation.

Kalimantacin A, B and C are new antibiotics produced by Alcaligenes sp. YL-02632S. Their structures were elucidated to be novel long chain structure compounds containing O-carbamoyl, amide and carboxylic acid moieties based on various 2D NMR experiments and MS analysis.

YM-47141 and YM-47142, new elastase inhibitors produced by Flexibacter sp. Q17897. II. Structure elucidation.

YM-47141 and YM-47142 are new elastase inhibitor produced by Flexibacter sp. Q17897. These structures were elucidated by MS and NMR spectral analysis. YM-47141 and YM-47142 were the cyclic peptides containing tricarbonyl moiety hydrated on the center carbonyl carbon in DMSO-d6.

Magnesium is essential for formation of an active complex of a hammerhead ribozyme with its substrate: an investigation by NMR spectroscopy.

The effects of magnesium ions on a 32-mer ribozyme (R32) were examined by high-resolution NMR spectroscopy. In solution, R32 (without its substrate) consisted of a GAAA loop, stem II, a non-Watson Crick three-base-paired duplex and a four-base-paired duplex that included a wobble base pair. When an uncleavable substrate RNA (RdC11) was added to R32 without Mg2+ ions, a complex did not form between R32 and RdC11 because the substrate-recognition regions of R32 formed intramolecular base pairs (the recognition arms were closed). By contrast, in the presence of Mg2+ ions, the R32-RdC11 complex was formed. Moreover, titration of mixtures of R32 and RdC11 with Mg2+ ions also induced the ribozyme-substrate interaction. These data suggest that Mg2+ ions are not only important as the true catalysts in the function of ribozyme-type metalloenzymes but they also induce the structural change in the R32 hammerhead ribozyme that is necessary for establishment of the active form of the ribozyme-substrate complex.

A new alkaloid antibiotic tetrazomine. Structure determination.

A new alkaloid antibiotic tetrazomine was isolated from the culture broth of Saccharothrix mutabilis subsp. chichijimaensis subsp. nov., and its structure was determined to be I by means of spectroscopic measurements. It has an unusual structure which consists of six rings, including piperidine, piperazine, oxazole, and pyrrolidine rings.

Novel antitumor antibiotic phospholine. 2. Structure determination.

Phospholine, an antitumor antibiotic, has the molecular formula of C25H40NO8P and possesses a delta-lactone and a phosphoric acid ester as functional groups. Its structure was determined based on interpretation of fast atom bombardment MS, 1H NMR, 13C NMR, 1H-1H correlation spectroscopy (COSY), 13C-1H COSY, heteronuclear multiple bond correlation spectroscopy, elemental analysis and chemical modifications.

Okilactomycin, a novel antibiotic produced by a Streptomyces species. II. Structure determination.

Structure of a novel antibiotic, okilactomycin, was determined by a combination of spectroscopic and X-ray crystallographic studies. Okilactomycin has a unique structure containing 13-membered ring cyclized by carbon-carbon bond.

Disposition and metabolism of indeloxazine hydrochloride, a cerebral activator, in rats.

1. The disposition and metabolism of indeloxazine hydrochloride ((+/-)-2-[(inden-7-yloxy)methyl]morpholine hydrochloride) were studied in male Sprague-Dawley rats. 2. After oral administration of 14C-indeloxazine hydrochloride, the plasma concentration of total radioactivity reached a maximum at 15 min and declined with an apparent half-life of 2.2 h in the first 6 h period and declined more slowly thereafter. Unchanged drug in the plasma represented 13.5%, 5.9% and 0.4% of the total radioactivity at 15 min, 1 h and 6 h respectively after administration and levels decayed with a half-life of 0.9 h. 3. After oral and i.v. administration of the labelled compound, the urinary and faecal excretion of radioactivity in 72 h were 61-65% and 31-36% of the dose, respectively. Biliary excretion in bile duct-cannulated animals amounted to 49% of the dose in 72 h. 4. Seven metabolites have been isolated from the plasma or urine and characterized by i.r., n.m.r. and mass spectrometry. They were derived through dihydrodiol formation in the indene ring, hydroxylation of the indene ring and N-acetylation, oxidation and oxidative degradation of the morpholine ring. Some metabolites were excreted as their glucuronic acid or glucose conjugates. The major metabolite appeared to the trans-indandiol analogue of indeloxazine. 5. Possible metabolic pathways of degradation of the morpholine ring are discussed.

Disposition and metabolism of amosulalol hydrochloride, a new combined alpha- and beta-adrenoceptor blocking agent, in rats, dogs and monkeys.

The disposition and metabolism of amosulalol hydrochloride, a combined alpha- and beta-adrenoceptor blocking agent, were studied in rats, dogs and monkeys. After oral administration of [14C]amosulalol hydrochloride, the plasma concentration of radioactivity reached a maximum at 0.5 to 1 h in all species and declined with half-lives of about 2 h in both rats and monkeys, and of about 4 h in dogs. The ratios of unchanged drug to total radioactivity in the rat and dog plasma were 8 and 43% at 0.5 h after administration, respectively. The radioactivity in the rat tissues was high in the liver, kidney, blood and pancreas after oral administration. Following oral dosage, the urinary excretion of radioactivity was 26-34% of the dose in rats, 45% in dogs and 46% in monkeys in 48 h. The biliary excretion after oral dosage amounted to 66% and 41% in rats and dogs, respectively. Six metabolites were isolated and identified from the urine of rats and dogs. They were derived from one or two of the following pathways: I, hydroxylation of the 2-methyl group of the methylbenzenesulphonamide ring; II, demethylation of the o-methoxy group of the methoxyphenoxy ring; III, hydroxylation at the 4 or 5 position of the methoxyphenoxy ring; IV, oxidative cleavage of the C-N bond yielding o-methoxyphenoxy acetic acid. Moreover, some metabolites were metabolized to glucuronide or sulphate.