Hydromorphone-3-glucuronide: Biochemical synthesis and preliminary pharmacological evaluation

Hydromorphone-3-glucuronide (H3G) was synthesized biochemically using rat liver microsomes, uridine-5'-diphosphoglucuronic acid (UDPGA) and the substrate, hydromorphone. Initially, the crude putative H3G product was purified by ethyl acetate precipitation and washing with acetonitrile, Final purification was achieved using semi-preparative high-performance-liquid-chromatography (HPLC) with ultraviolet (UV) detection. The purity of the final H3G product was shown by HPLC with electrochemical and ultraviolet detection to be > 99.9% and it was produced in a yield of approximate to 60% (on a molar basis). The chemical structure of the putative H3G was confirmed by enzymatic hydrolysis of the glucuronide moiety using P-glucuronidase, producing a hydrolysis product with the same HPLC retention time as the hydromorphone reference standard. Using HPLC with tandem mass spectrometry (HPLC-MS-MS) in the positive ionization mode, the molecular mass (M+1) was found to be 462 g/mol, in agreement with H3G's expected molecular weight of 461 g/mol. Importantly, proton-NMR indicated that the glucuronide moiety was attached at the 3-phenolic position of hydromorphone. A preliminary evaluation of H3G's intrinsic pharmacological effects revealed that following icy administration to adult male Sprague-Dawley rats in a dose of 5 mu g, H3G evoked a range of excitatory behavioural effects.including chewing, rearing, myoclonus, ataxia and tonic-clonic convulsions, in a manner similar to that reported previously for the glucuronide metabolites of morphine, morphine-3-glucuronide and normorphine-3-glucuronide.

Hepatic disposition and metabolite kinetics of a homologous series of diflunisal esters

The hepatic disposition and metabolite kinetics of a homologous series of diflunisal O-acyl esters (acetyl, butanoyl, pentanoyl, anti hexanoyl) were determined using a single-pass perfused in situ rat liver preparation. The experiments were conducted using 2% BSA Krebs-Henseleit buffer (pH 7.4), and perfusions were performed at 30 mL/min in each liver. O-Acyl esters of diflunisal and pregenerated diflunisal were injected separately into the portal vein. The venous outflow samples containing the esters and metabolite diflunisal were analyzed by high performance liquid chromatography (HPLC). The normalized outflow concentration-time profiles for each parent ester and the formed metabolite, diflunisal, were analyzed using statistical moments analysis and the two-compartment dispersion model. Data (presented as mean +/- standard error for triplicate experiments) was compared using ANOVA repeated measures, significance level P < 0.05. The hepatic availability (AUC'), the fraction of the injected dose recovered in the outflowing perfusate, for O-acetyldiflunisal (C2D = 0.21 +/- 0.03) was significantly lower than the other esters (0.34-0.38). However, R-N/f(u), the removal efficiency number R-N divided by the unbound fraction in perfusate f(u), which represents the removal efficiency of unbound ester by the liver, was significantly higher for the most lipophilic ester (O-hexanoyldiflunisal, C6D = 16.50 +/- 0.22) compared to the other members of the series (9.57 to 11.17). The most lipophilic ester, C6D, had the largest permeability surface area (PS) product (94.52 +/- 38.20 mt min-l g-l liver) and tissue distribution value VT (35.62 +/- 11.33 mL g(-1) liver) in this series. The MTT of these O-acyl esters of diflunisal were not significantly different from one another. However, the metabolite diflunisal MTTs tended to increase with the increase in the parent ester lipophilicity (11.41 +/- 2.19 s for C2D to 38.63 +/- 9.81 s for C6D). The two-compartment dispersion model equations adequately described the outflow profiles for the parent esters and the metabolite diflunisal formed from the O-acyl esters of diflunisal in the liver.

Arterial heparan sulfate proteoglycans inhibit vascular smooth muscle cell proliferation and phenotype change in vitro and neointimal formation in vivo

Purpose: The aim of this study was to determine whether heparan sulfate proteoglycans (HSPGs) from the normal arterial wall inhibit neointimal formation after injury in vivo and smooth muscle cell (SMC) phenotype change and proliferation in vitro. Methods: Arterial HSPGs were extracted from rabbit aortae and separated by anion-exchange chromatography. The effect of HSPGs, applied in a periadventitial gel, on neointimal formation was assessed 14 days after balloon catheter injury of rabbit carotid arteries. Their effect on SMC phenotype and proliferation was measured by point-counting morphometry of the cytoplasmic volume fraction of myofilaments (Vvmyo) and H-3-thymidine incorporation in SMCs in culture. Results: Arterial HSPGs (680 mu g) reduced neointimal formation by 35% at 14 days after injury (P =.029), whereas 2000 mu g of the low-molecular-weight heparin Enoxaparin was ineffective. HSPGs at 34 mu g/mL maintained subconfluent primary cultured SMCs with the same high Vvmyo (52.1% +/- 13.8%) after 5 days in culture as did cells freshly isolated from the arterial wall (52.1% +/- 15.1%). In contrast, 100 mu g/mL Enoxaparin was ineffective in preventing phenotypic change over this time period (Vvmyo 38.9% +/- 14.6%, controls 35.9% +/- 12.8%). HSPGs also inhibited 3H-thymidine incorporation into primary cultured SMCs with an ID50 value of 0.4 mu g/mL compared with a value of 14 mu g/ml; for Enoxaparin (P

Biomolecular interaction analysis of IFN gamma-induced signaling events in whole-cell lysates: Prevalence of latent STAT1 in high-molecular weight complexes

The basic framework for the JAK/STAT pathway is well documented. Recruitment of latent cytoplasmic STAT transcription factors to tyrosine phosphorylated docking sites on cytokine receptors and their JAK-mediated phosphorylation instigates their translocation to the nucleus and their ability to bind DNA, The biochemical processes underlying recruitment and activation of this pathway have commonly been studied in reconstituted in vitro systems using previously defined recombinant signaling components. We have dissected the Interferon gamma (IFN gamma) signal transduction pathway in crude extracts from wild-type and STAT1-negative mutant cell Lines by real-time BIAcore analysis, size-exclusion (SE) chromatography and immune-detection. The data indicate that in detergent-free cell extracts: (1) the phospho-tyrosine (Y440P)-containing peptide motif of the IFN gamma-receptor ct-chain interacts directly with STAT1, or STAT1 complexes, and no other protein; (2) nonactivated STAT 1 is present in a higher molecular weight complex(es) and, at least for IFN gamma-primed cells, is available for recruitment to the activated IFN gamma-receptor from only a subset of such complexes; (3) activated STAT1 is released from the receptor as a monomer.

Analysis of immunosuppressive proteins in serum of liver-transplanted rats by using an anti-LSF-1 affinity column

Liver suppressor factor one (LSF-1) is a 40-kDa immunosuppressive protein in the serum of rats 60 days after orthotopic liver transplantation (OLT) between the nonrejector combination of DA donors into PVG; recipients. In the present study, the purification of proteins from rat OLT serum taken 60 days after transplantation Mras performed by affinity chromatography using the anti-LSF-1 polyclonal antibody (pAb). The assessment of column eluates using anti-LSF-1 and OLT serum was studied using rat heart and liver transplantation models. Rejection was not suppressed by the administration of OLT serum in heart or liver allografts. However, heart allografts treated with peak eluates (450 mu g single shot im, dissolved in Intralipos) taken from the affinity OLT serum survived significantly longer than untreated rats (median = 36.5 days; n = 7 vs 6.5 days; n = 5, respectively, P = 0.011). The same treatment with anti-LSF-1 column eluates also prolonged liver allografts significantly (>200 days) than those in either the untreated group (median = 11 days; n = 7) or those which received only Intralipos (median = 10.5 days; n = 5, P = 0.019). Subsequent analysis of the N-terminal sequences of some of the proteins which were eluted from the affinity column revealed that the homology of a 30-kDa protein was identical to hemoglobin alpha-chain, a 59-kDa protein to granulocyte inhibitory factor, a 70-kDa and a 90-kDa to albumin and its precursor, respectively. Although the specific immunosuppressive component has not been isolated, our results suggested that the anti-LSF-1 column can extract immunosuppressive moiety of LSF-1 from OLT serum. (C) 1998 Academic Press.

High level of aspartic acid-bond isomerization during the synthesis of an N-linked tau glycopeptide

An increased degree of utilization of the potential N-glycosylation site In the fourth repeat unit of the human tau protein may be involved in the inability of tau to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with, the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a beta-peptide. The anomalous chromatographic behavior of the acetylated beta-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated tau into the paired helical filaments in the affected brains. Copyright (C) 1999 European Peptide Society and John Wiley & Sons, Ltd.

Description of Gluconacetobacter sacchari sp. nov., a new species of acetic acid bacterium isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug

A new species of the genus Gluconacetobacter, for which the name Gluconacetobacter sacchari sp. nov. is proposed, was isolated from the leaf sheath of sugar cane and from the pink sugar-cane mealy bug, Saccharicoccus sacchari, found on sugar cane growing in Queensland and northern New South Wales, Australia, The nearest phylogenetic relatives in the alpha-subclass of the Proteobacteria are Gluconacetobacter liquefaciens and Gluconacetobacter diazotrophicus, which have 98.8-99.3% and 97.9-98.5% 16S rDNA sequence similarity, respectively, to members of Gluconacetobacter sacchari. On the basis of the phylogenetic positioning of the strains, DNA reassociation studies, phenotypic tests and the presence of the Q10 ubiquinone, this new species was assigned to the genus Gluconacetobacter. No single phenotypic characteristic is unique to the species, but the species can be differentiated phenotypically from closely related members of the acetic acid bacteria by growth in the presence of 0.01% malachite green, growth on 30% glucose, an inability to fix nitrogen and an inability to grow with the L-amino acids asparagine, glycine, glutamine, threonine and tryptophan when D-mannitol was supplied as the sole carbon and energy source. The type strain of this species is strain SRI 1794(T) (= DSM 12717(T)).

Chemical synthesis, antibacterial activity and conformation of diptericin, an 82-mer peptide originally isolated from insects

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side-chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid-phase step-by-step synthesis of diptericin, an 82-mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed-phase high performance liquid chromatography, and its activity spectrum was compared to that Of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N-terminal domain. Diptericin appeared to have antibacterial activity:for only a limited number of Gram-negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not,necessary to kill bacteria. Neither the N-terminal, drosocin-analog fragment, nor the C-terminal, glycine-rich attacin-analog region was active against any of the bacterial strains studied, regardless of whether the Gal-GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two-dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within I h, considerably faster than drosocin and the attacins; unlike some other, fast-acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.

Can tissue drug concentrations be monitored by microdialysis during or after isolated limb perfusion for melanoma treatment?

Isolated limb perfusion (ILP) with melphalan is used to treat recurrent melanoma. This study aimed to develop a microdialysis technique for melphalan tissue concentration measurement during ILP. The effects of melphalan concentration (50-600 mu g/ml), microdialysis flow rate (0.55-17.5 mu l/min), probe length (5-50 mm) and temperature (25-41.5 degrees C) on in vitro recovery were studied. In addition, in vivo recovery was measured in rat hindlimbs perfused with melphalan using 50 mm microdialysis probes implanted subcutaneously and into muscle. Both dialysate and tissue sample melphalan concentrations were determined by high performance liquid chromatography. The in vitro recovery of melphalan was not affected by melphalan concentration or temperature, but increased with probe length and decreased with flow rate. The melphalan concentrations in subcutaneous and muscle dialysates were not significantly different. A linear relationship was found between tissue dialysate concentrations and actual tissue concentrations of melphalan (r(2) = 0.97). Microdialysis is a potential method for tissue drug monitoring which may assist in the efficacious use of cytotoxics in human ILP. (C) 2000 Lippincott Williams & Wilkins.

Shared and unique environmental factors determine the ecology of methanogens in humans and rats

OBJECTIVE: This study ascertains the relative contributions of genetics and environment in determining methane emission in humans and rats. There is considerable interest in the factors determining the microbial species that inhabit the colon. Methanogens, which are archaebacteria, are an easily detected colonic luminal bacteria because they respire methane. They are present in some but not all human colons and lower animal hindguts. Opinion varies on the nature of the factors influencing this ecology with some studies proposing the existence of host genetic influences. METHODS: Methane emission was measured in human twin pairs by gas chromatography, and structural equation modeling was used to determine the proportion of genetic and environmental determinants. The importance of the timing of environmental effects and rat strain on the trait of methane emission were ascertained by experiments with cohabiting methanogenic and nonmethanogenic rats. RESULTS: Analysis of breath samples from 274 adolescent twin pairs and their families indicated that the major influences on the trait of methane emission are the result of shared (53%, 95% confidence interval 39-61) and unique environmental (47%, 95% confidence interval 38-56) effects. No significant autosomal genetic effects were detected, but as observed in other studies, men (37%) were less likely to excrete methane in their breath than women (63%). Investigation of methane emission in rats indicated that environmental effects in this animal are most potent during the weaning period, with stable gut microbial ecology thereafter for some but not all rat strains. CONCLUSIONS: These results are consistent with shared and unique environmental factors being the main determinants of the ecology of this colonic microbe. (Am J Gastroenterol 2000;95:2872-2879. (C) 2000 by Am. Coll. of Gastroenterology).

Expression, purification, and characterization of biol: A carbon-carbon bond cleaving cytochrome P450 involved in biotin biosynthesis in Bacillus subtilis

Pimelic acid formation for biotin biosynthesis in Bacillus subtilis has been proposed to involve a cytochrome P450 encoded by the gene biol. We have subcloned bioI and overexpressed the encoded protein, BioI. A purification protocol was developed utilizing ion exchange, gel filtration, and hydroxyapatite chromatography, Investigation of the purified BioI by UV-visible spectroscopy revealed spectral properties characteristic of a cytochrome P450 enzyme. BioI copurifies with acylated Escherichia coil acyl carrier protein (ACP), suggesting that in vivo a fatty acid substrate may be presented to BioI as an acyl-ACP. A combination of electrospray mass spectrometry of the intact acyl-ACP and GCMS indicated a range of fatty acids were bound to the ACP. A catalytically active system has been established employing E. coli flavodoxin reductase and a novel, heterologous flavodoxin as the redox partners for BioI. In this system, BioI cleaves a carbon-carbon bond of an acyl-ACP to generate a pimeloyl-ACP equivalent, from which pimelic acid is isolated after base-catalyzed saponification. A range of free fatty acids have also been explored as potential alternative substrates for BioI, with C16 binding most tightly to the enzyme. These fatty acids are also metabolized to dicarboxylic acids, but with less regiospecificity than is observed with acyl-ACPs. A possible mechanism for this transformation is discussed. These results strongly support the proposed role for BioI in biotin biosynthesis. In addition, the production of pimeloyl-ACP explains the ability of BioI to function as a pimeloyl CoA source in E. coli, which, unlike B. subtilis, is unable to utilize free pimelic acid for biotin production. (C) 2000 Academic Press.

Semiochemicals and social signaling in the wild European rabbit in Australia: I. Scent profiles of chin gland secretion from the field

The European rabbit (Oryctolagus cuniculus) uses the secretion of the chin gland in the maintenance of social status. Previous work has concentrated on secretion collected directly from the animal. In this study, the analysis was conducted by collecting scent marks made by free-ranging animals. Scent marks were found to be concentrated at the center of the area controlled by a social group, and at the boundaries between two adjacent social groups. Only the mark from dominant animals could be identified. Marks were also collected from the skin of rabbits, where they had been placed by the dominant individual. The mark found on the head of a subordinate animal may, in the future, be used to identify the dominant animal of the social group, who placed the mark.

Mimicry of host cuticular hydrocarbons by salticid spider Cosmophasis bitaeniata that preys on larvae of tree ants Oecophylla smaragdina

The salticid spider Cosmophasis bitaeniata preys on the larvae of the green tree ant Oecophylla smaragdina. Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) reveal that the cuticle of C. bitaeniata mimics the mono- and dimethylalkanes of the cuticle of its prey. Recognition bioassays with extracts of the cuticular hydrocarbons of ants and spiders revealed that foraging major workers did not respond aggressively to the extracts of the spiders or conspecific nestmates, but reacted aggressively to conspecific nonnestmates. Typically, the ants either failed to react (as with control treatments with no extracts) or they reacted nonaggressively as with conspecific nestmates. These data indicate that the qualitative chemical mimicry of ants by C. bitaeniata allows the spiders to avoid detection by major workers of O. smaragdina.

Semiochemicals and social signaling in the wild European rabbit in Australia: II. Variations in chemical composition of chin gland secretion across sampling sites

The volatile components of the chin gland secretion of the wild European rabbit, Oryctolagus cuniculus (L.), were investigated with the use of gas chromatography. Studies of the chemical nature of this secretion by previous workers demonstrated that it was important in the maintenance of social structure in this species. This study identified 34 different volatile components that consist primarily of aromatic and aliphatic hydrocarbons. Especially common are a series of alkyl-substituted benzene derivatives that provide most of the compound diversity in the secretion. Samples of chin gland secretion collected from animals at three different geographical locations, separated by more than 100 km, showed significant differences in composition. This work suggests that variation among populations needs to be considered when undertaking semiochemical research. Alternate nonparametric methods are also used for the analysis of chromatographic data.

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins. (C) 2003 Elsevier Science (USA). All rights reserved.