Exposure to naphthalene induces naphthyl-keratin adducts in human epidermis in vitro and in vivo

We observed naphthyl-keratin adducts and dose-related metabolic enzyme induction at the mRNA level in reconstructed human epidermis in vitro after exposure to naphthalene. Immunofluorescence detection of 2-naphthyl-keratin-1 adducts confirmed the metabolism of naphthalene and adduction of keratin. We also observed naphthyl-keratin adducts in dermal tape-strip samples collected from naphthalene-exposed workers at levels ranging from 0.004 to 6.104 pmol adduct µg^?1 keratin. We have demonstrated the ability of the human skin to metabolize naphthalene and to form naphthyl-keratin adducts both in vitro and in vivo. The results indicate the potential use of keratin adducts as biomarkers of dermal exposure.     

Stereospecific determination,chiral inversion in vitro and pharmacokinetics in humans of the enantiomers of thalidomide

The purposes of this work were (1) to develop a high performance liquid chromatographic (HPLC) assay for the enantiomers of thalidomide in blood, (2) to study their inversion and degradation in human blood, and (3) to study the pharmacokinetics of (+)-(R)- and (?)-(S)-thalidomide after oral administration of the separate enantiomers or of the racemate to healthy male volunteers. The enantiomers of thalidomide were determined by direct resolution on a tribenzoyl cellulose column. Mean rate constants of chiral inversion of (+)-(R)-thalidomide and (?)-(S)-thalidomide in blood at 37°C were 0.30 and 0.31 h^?1, respectively. Rate constants of degradation were 0.17 and 0.18 h^?1. There was rapid interconversion in vivo in humans, the (+)-(R)-enantiomer predominating at equilibrium. The pharmacokinetics of (+)-(R)- and (?)-(S)-thalidomide could be characterized by means of two one-compartment models connected by rate constants for chiral inversion. Mean rate constants for in vivo inversion were 0.17 h^?1 (R to S) and 0.12 h^?1 (S to R) and for elimination 0.079 h^?1 (R) and 0.24 h^?1 (S), i.e., a considerably faster rate of elimination of the (?)-(S)-enantiomer. Putative differences in therapeutic or adverse effects between (+)-(R)- and (?)-(S)-thalidomide would to a large extent be abolished by rapid interconversion in vivo. © 1995 Wiley-Liss, Inc.     

Factors Influencing the Formation of the Carbon Dioxide Radical Anion (CO2−) Spin Adduct of Pbn in the Rat Liver Metabolism of Halocarbons

Spin trapping techniques have been used to detect free radicals generated from the in vitro metabolism by rat liver microsomes of carbon tetrachloride (CCl₄) and bromotrichloromethane (BrCCI) under conditions of varying oxygen tension and pH. Dispersions of rat liver microsomes incubated with ¹²CCl₄, ¹³CCl₄ or Br¹²CCl₃, α-phenyl-tert-butyl nitrone (PBN) and NADPH/NADH in a phosphate buffer varying in pH from 6.6 to 8.0 under varying oxygen tensions produced various amounts of four different PBN adducts: PBN-CCl₃, PBN-L, PBN-OL and PBN-CO^?₂ where L is a carbon-centered lipid type radical and LO is an oxygen-centered lipid type radical. The relative amount of PEN-CO; increases with the absence of oxygen. With the use of ³¹P-NMR in vivo spectroscopy it was possible to detect a pH change from 7.4 to 6.8 in the livers of rats treated with CCl₄, or BrCCl₃. These results suggest that halocarbon metabolism in biological systems may depend on both oxygen tension as well as pH.     

Cloning of structural genes for ß-glucosidase fromCellulomonas biazotea intoE. coli andSaccharomyces cerevisiae using shuttle vector pBLU-D

A Smal genomic library fromCellulomonas biazotea NIAB 442 was constructed inEscherichia coli HB101 using shuttle vector pBLU-D. Three clones with ability to hydrolyse esculin were isolated. These clones were comparedin vivo andin vitro tests to select for hyper-secretion of ß-glucosidase. The recombinant plasmids were transformed to competent cells of a Cir^o yeast.In vivo studies indicated that the genes were fully expressed in yeast as well.     

Inhibition of alcohol dehydrogenase by disulfiram; possible relation to the disulfiram-ethanol reaction

Disulfiram inhibited mouse and rat liver alcohol dehydrogenase (LADH) $\text{in}$$\text{vitro}$. Inhibition of LADH by disulfiram appears to be non-competitive. The inhibition constants (Ki) were about 1.5 × 10^?4 M and 4.3 × 10^?5 M for mouse and rat LADH respectively. Ethanol elimination was significantly reduced in mice pretreated with disulfiram. At identical time intervals after ethanol administration, the concentration of ethanol in blood from disulfiram-, cyanamide-, or dimethyl formamide-treated mice were significantly higher than the ethanol concentration in blood from control mice. Both cyanamide and dimethyl formamide (DMF) can precipitate a disulfiram-like reaction in man when ethanol is ingested. These and previous experiments suggest that elevated concentrations of ethanol should be considered in the etiology of some of the symptoms seen in the disulfiram-ethanol reaction.     

Carboxymethylated beta-1,3-glucan inhibits the binding and degradation of acetylated low density lipoproteins in macrophages In Vitro and modulates their plasma clearance In Vivo

In atherosclerotic lesions, macrophages are transformed into foam cells accumulating modified low density lipoproteins (LDL) via the scavenger receptor pathway. We have investigated the effects of carboxymethylated beta-1,3-glucan (CMG) on acetylated LDL (AcLDL) metabolism in murine peritoneal macrophages in vitro and upon the clearance of AcLDL by rat liver in vivo. In cultured murine peritoneal macrophages, CMG reduced substantially the AcLDL-induced synthesis of cholesteryl esters, decreased the binding and degradation of [¹²⁵I]-AcLDL in a dose-dependent manner with complete inhibition at 20–30 nM , but had no effect on the binding and degradation of native [¹²⁵I]–LDL. In contrast, other polysaccharides studied, namely zymosan, lipopolysaccharide, non-modified glucan and mannan Rhodexman, had a slight effect at concentrations significantly exceeding the concentrations of CMG. [¹²⁵I]-AcLDL injected intravenously into rats was cleared from the blood with a half-life of 3.7 min. About 56 per cent of the label of injected [¹²⁵I]-AcLDL was recovered in the liver 15 min after administration. Co-injection of the labelled AcLDL with CMG (25 mg kg^?1 b.w.) decreased the rate of AcLDL clearance so that the half-life increased to 6.0 min. Injections of CMG (25 mg kg^?1 b.w.) 48 and 24 h before the determination increased the rate of [¹²⁵I]-AcLDL clearance (with a half-life of about 2.3 min) and increased the uptake of AcLDL by the liver. We suggest that CMG competed with AcLDL for scavenger receptors in vitro and in vivo and repeated CMG injections before the measurements of AcLDL resulted in the induction of scavenger receptor function.     

miR-146a Ameliorates Liver Ischemia/Reperfusion Injury by Suppressing IRAK1 and TRAF6

A critical role of the Toll-like receptor(TLR) and its downstream molecules, including IL-1 receptor-associated kinase 1(IRAK1) and tumor necrosis factor receptor– associated factor 6(TRAF6), in the pathogenesis of liver ischemia/reperfusion (I/R) injury has been documented. Recently a microRNA, miR-146a, was identified as a potent negative regulator of the TLR signaling pathway. In this study, we investigated the role of miR-146a to attenuate TLR signaling and liver I/R injury in vivo and in vitro. miR-146a was decreased in mice Kupffer cells following hepatic I/R, whereas IRAK1 and TRAF6 increased. Overexpression of miR-146a directly decreased IRAK1 and TRAF6 expression and attenuated the release of proinflammatory cytokines through the inactivation of NF-κB P65 in hypoxia/reoxygenation (H/R)-induced macrophages, RAW264.7 cells. Knockdown experiments demonstrated that IRAK1 and TRAF6 are two potential targets for reducing the release of proinflammatory cytokines. Moreover, co-culture assays indicated that miR-146a decreases the apoptosis of hepatocytes after H/R. In vivo administration of Ago-miR-146a, a stable version of miR-146a in vivo, protected against liver injury in mice after I/R via inactivation of the TLR signaling pathway. We conclude that miR-146a ameliorates liver ischemia/reperfusion injury in vivo and hypoxia/reoxygenation injury in vitro by directly suppressing IRAK1 and TRAF6.     

Relationships between nitrate level,nitrate reductase activity and anaerobic nitrite production inPisum sativum leaf tissue

Anaerobic nitrite production (thein vivo NO₃-R activity) in an incubation medium lacking exogenous nitrate but containing 0.5%n-propanol and 0.1% Triton X-100 showed higher correlation (y - ax ^b) with the level of endogenous nitrate inPisum sativum L. leaves than thein vitro nitrate reductase activity. Thein vivo NO₃-R activity correlated well with thein vitro activity up to the 50 ppm NO₃-N level of endogenous nitrate. The ratioin vivo: in vitro activity slightly decreased with increasing level of endogenous nitrate in leaf tissue.     

Alterations at the Cross-Bridge Level Are Associated with a Paradoxical Gain of Muscle Function In Vivo in a Mouse Model of Nemaline Myopathy

Nemaline myopathy is the most common disease entity among non-dystrophic skeletal muscle congenital diseases. The first disease causing mutation (Met9Arg) was identified in the gene encoding α-tropomyosin_slow gene (TPM3). Considering the conflicting findings of the previous studies on the transgenic (Tg) mice carrying the TPM3 ^Met9Arg mutation, we investigated carefully the effect of the Met9Arg mutation in 8–9 month-old Tg(TPM3)^Met9Arg mice on muscle function using a multiscale methodological approach including skinned muscle fibers analysis and in vivo investigations by magnetic resonance imaging and 31-phosphorus magnetic resonance spectroscopy. While in vitro maximal force production was reduced in Tg(TPM3)^Met9Arg mice as compared to controls, in vivo measurements revealed an improved mechanical performance in the transgenic mice as compared to the former. The reduced in vitro muscle force might be related to alterations occuring at the cross-bridges level with muscle-specific underlying mechanisms. In vivo muscle improvement was not associated with any changes in either muscle volume or energy metabolism. Our findings indicate that TPM3(Met9Arg) mutation leads to a mild muscle weakness in vitro related to an alteration at the cross-bridges level and a paradoxical gain of muscle function in vivo. These results clearly point out that in vitro alterations are muscle-dependent and do not necessarily translate into similar changes in vivo.     

Age-Related Features of Ceruloplasmin Biosynthesis and Distribution in Rats

In vivo biosynthesis of ceruloplasmin (Cp), a copper-containing glycoprotein, which plays an important role in copper transfer between organs and bidirectional iron transport in vertebrates, was studied in 7-day old rats, which are characterized by the embryonic type of copper metabolism. In addition to the liver, Cp is synthesized in the lungs, brain, and kidneys. In pulse-chase experiments it was demonstrated that [¹⁴C]-Cp appearance in the blood coincides with the secretion ofde novo synthesized Cp from the liver. [¹⁴C]-Cp is taken up from the blood stream by cells of the heart, lung, and kidneys and binds to red blood cells, while Cp polypeptide chain is not taken up by the brain cells. Immunoreactive polypeptides of the Cp receptor were found using immunoblotting in plasma membranes of the heart, liver, kidneys, and red blood cells, rather than in the brain. Using the RT-PCR method with selective primers, it was shown that these cells contain molecular forms of Cp-mRNAs programming the synthesis of both secretory Cp and Cp bound to the plasma membrane via a glycosyl phosphatidylinositol anchor. After switching to the adult type of copper metabolism, the blood serum contents of copper and Cp sharply increase, while the Cp content in the cerebrospinal fluid, as measured according to the oxidase and antigen activities, and copper concentration, as determined by atomic absorption spectroscopy, remain low. Ontogenetic features of the system ensuring the copper homeostasis in mammals are discussed.     

Biodegradation of pentachlorophenol by white rot fungi under ligninolytic and nonligninolytic conditions

The roles of lignin peroxidase, manganese peroxidase, and laccase were investigated in the biodegradation of pentachlorophenol (PCP) by several white rot fungi. The disappearance of pentachlorophenol from cultures of wild type strains,P. chrysosporium, Trametes sp. andPleurotus sp., was observed. The activities of manganese peroxidase and laccase were detected inTiametes sp. andPleurotus sp. cultures. However, the activities of ligninolytic enzymes were not detected inP. chrysosporium cultures. Therefore, our results showed that PCP was degraded under ligninolytic as well as nonligninolytic conditions. Indicating that lignin peroxidase, manganese peroxidase, and laccase are not essential in the biodegradation of PCP by white rot fungi.     

New advances in coenzyme Q biosynthesis

Summary Coenzyme Q (or ubiquinone) is the product of two distinct biosynthetic pathways: the lipid tail of coenzyme Q is formed via the isoprene biosynthetic pathway, and the quinone ring derives from the metabolism of either shikimic acid or tyrosine. In general, eukaryotic organisms use the classical mevalonate pathway to form isopentenyl- and dimethylallyl-diphosphate, the five carbon building blocks of the polyisoprenoid tail, and prokaryotes use 1-deoxy-D-xylulose-5-phosphate, formed via the Rohmer pathway. The quinone ring precursor is 4-hydroxybenzoic acid, which is formed directly from chorismate inSaccharomyces cerevisiae andEscherichia coli, or from tyrosine in animal cells. Ring modification steps including prenylation, decarboxylation, and successive hydroxylation and methylation steps form the fully substituted benzoquinone ring of coenzyme Q. Many of the genes and polypeptides involved in coenzyme Q biosynthesis have been isolated and characterized by utilizing strains ofE. coli andS. cerevisiae with mutations in theubi andCOQ genes, respectively. This article reviews recent progress in characterizing the biosynthesis of coenzyme Q inE. coli, S. cerevisiae, and other eukaryotic organisms.     

Purification, Characterization, andin VitroPhosphorylation of the Neuron-Specific Membrane-Associated Protein SCG10

SCG10 is a neuron-specific, developmentally regulated protein which is highly enriched in growth cones. Sequence homology indicates that it is related to the phosphoprotein stathmin or Op18, anin vitroandin vivosubstrate for several serine/threonine kinases which are involved in a variety of signaling pathways. As a first step to examine the biochemical properties of SCG10, the protein was expressed inEscherichia coliand purified to apparent homogeneity. The purified protein was used inin vitrophosphorylation assays. SCG10 was phosphorylated by MAP kinase, cAMP-dependent protein kinase, cGMP-dependent protein kinase, p34^cdc2kinase, DNA-dependent protein kinase, Ca²⁺/calmodulin kinase II, and casein kinase II. The protein was not a substrate for casein kinase I and protein kinase C. SCG10 was phosphorylated by src tyrosine kinase, which demonstrates that the protein can be phosphorylatedin vitroon a tyrosine residue. Our data suggest that SCG10 is a phosphoprotein which might be involved in signal transduction in neurons.     

Chemical- and cuticular phenoloxidase- mediated synthesis of cysteinyl–catechol adducts

N-Acetylcysteine adducts of o-benzoquinones derived from catechol, 4-methylcatechol, and N-acetyldopamine were chemically synthesized and characterized by a combination of UV, IR, and NMR spectral studies. Oxidation of catechol, 4-methylcatechol, and N-acetyldopamine by cuticle-bound phenoloxidase from Sarcophaga bullata in the presence of N-acetylcysteine resulted in the formation of covalent adducts between catecholic compounds and N-acetylcysteine. Structural identities of these adducts were established by comparison of their HPLC retention time and UV spectra with those of synthetic adducts and by cochromatography with authentic samples. Although insect cuticle is known to contain only trace amounts of cysteine, the in vitro synthesis of quinone cysteine adducts mediated by cuticular phenoloxidase strongly indicates the occurrence of similar reactions in vivo as well and is in support of Pryor's quinone tanning hypothesis.     

Altered metabolism of ppGpp in quinone-treated E. coli: Possible role of aminoacyl-tRNA synthetases

The bacteriostatic quinone 6-amino-7-chloro-5,8-dioxoquinoline inhibits leucyl-tRNA synthetase in vivo and in vitro (Ogilvie et al. Biochim. Biophys. Acta $\text{407}$, 357–364; 1975). In this report it is shown that the quinone also interferes with the metabolism of ppGpp. Quinone treatment of E. coli MRE 600 causes the same phenotypic pattern as found in spoT^? mutants: overproduction of ppGpp and a drastic increase of its half-life; the formation of pppGpp, the possible degradation product of ppGpp, is blocked. A model is discussed to explain how the inhibition of leucyl-tRNA synthetase could account for the altered metabolism of ppGpp.     

Expression of cell cycle regulating factor mRNA in small cell lung cancer xenografts

We have investigated the expression of cyclins, cyclin dependent kinases (CDK), and CDK inhibitors (CKI) at the mRNA level in a panel of small-cell lung cancer (SCLC) cell linesin vitro andin vivo as xenografts in nude mice. The results showed that the cell lines expressed varying amounts of most cyclin and CDK’s but only a few of the cell lines expressed cyclin D₁ and/or D₂ and some lacked expression of CDK6. Most cell lines expressed mRNA for the CKI’s but two cell lines lacked expression of p15^INK4B and p16^INK4A. The mRNA expression differed for a few of the cell lines regarding cyclin D₂ and CDK6 whenin vitro andin vivo data were compared. Two of the cell lines that express the retinoblastoma (Rb) protein had no sign of a deregulated Rb pathway but further studies at the protein level are necessary to demonstrate whether these two cell lines should have a normal Rb pathway or whether they will join the majority of cell lines with deregulated Rb pathway.     

N-Acetylneuraminic acid biosynthesis in rat liver and kidney

Rat liver and kidney tissue slices incubated withN-acetyl [³H]mannosamine incorporated radioactivity into free and boundN-acetylneuraminic acid and CMP-N-acetylneuraminic acid (CMP-NeuAc). Liver and kidney also incorporated radioactivity from intravenously injected [³H]ManNAc intoN-acetylneuraminic acid and CMP-NeuAc. From the decrease in the specific radioactivity of CMP-NeuAc after a single injection ofN-acetyl[³H]mannosamine the half-life of CMP-NeuAc was determined. From this half-life and the pool size of CMP-NeuAc a synthesis rate of CMP-NeuAc was calculated, being 1.2 nmol/min/g wet weight of kidney. In previous experiments a value of 1.0 nmol/min/g wet weight was determined for liver [Ferwerdaet al. (1983) Biochem J 216: 87–92]. The synthesis rate of CMP-NeuAcin vivo was in the same range as the synthesis rate calculated from the turnover of boundN-acetylneuraminic acid, which was 2.7 and 0.4 nmol/min/g wet weight for liver and kidney respectively.The assay conditions for UDP-N-acetylglucosamine 2-epimerase andN-acetylmannosamine kinase were adapted to measure low activitiesin vitro. It appeared that the kinase activity detected in kidney can synthesizeN-acetylmannosamine6-phosphate at a rate sufficient for the observed production ofN-acetylneuraminic acidin vivo. Also a low, but measurable activity of UDP-N-acetylglucosamine 2-epimerase was detected in kidneyin vitro, suggesting that the biosynthetic pathway ofN-acetylneuraminic acid in kidney is the same as in liver. The synthesis rate ofN-acetylneuraminic acid in liver determinedin vivo is approximately 12 times slower than the maximal potential rate calculated from the activities of theN-acetylneuraminic acid (precursor-) forming enzymes as detectedin vitro. This indicates that in liverin vivo the enzymes are working far below their maximal capacity.     

The effects of extremely alkaline water (pH 9·5) on rainbow trout gill function and morphology

Rainbow trout that were held under control conditions, at pH8·0, in moderately hard Hamilton tap water, had Cl^? and Na⁺ influx rates (J^CLin and J^Na, respectively) of 270 and 300 μmol kg^?1 h^?1, respectively. Exposure to pH 9·5 water led to an immediate 67% decline in J^CL_in and a 45% reduction in J^Na_in at 0–1 h. Influx rates declined further and by 4–5 h the net decreases in both J^CL_in and J^Na_in approximated 80%. By 24 h J^CL_in had recovered to rates not significantly different from those at pH 8·0; while J^Na_in only partially recovered and remained about 50% lower than control measurements through 72 h. The complete recovery of J^CL_in and partial recovery of J^Na_in may have been related to a fourfold greater branchial chloride cell (CC) fractional surface area observed in rainbow trout exposed to pH 9·5 for 72 h. Ammonia excretion (J_Amm) was about 170 μmol N kg^?1 h^?1 at pH 8·0 but was initially reduced by 90% over the first hour of high pH exposure. J_Amm rapidly recovered and by 24 h it had returned to pre-exposure levels. This recovery tended to parallel the partial recovery of J^Na_in. However, subsequent addition of amiloride (10^?4M) to the water at 75 h led to no change in J_Amm, despite a 50% reduction in J^Na_in. Thus, it does not appear that there is a linkage between Na⁺ influx and the recovery of ammonia excretion under highly alkaline conditions.     

AN IN VITRO NITRATE REDUCTASE ASSAY FOR MARINE MACROALGAE: OPTIMIZATION AND CHARACTERIZATION OF THE ENZYME FOR FUCUS GARDNERI (PHAEOPHYTA)1

Measurement of the activity of the enzyme nitrate reductase (NR) may provide a useful index of nitrogen metabolism in marine macroalgae. In several species, including Fucus gardneri P. C. Silva, in vitro assays previously failed to detect NR activity, necessitating the use of in situ (or so-called“in vivo”) assays, which are more loosely controlled and lead to dafficulties in assessing enzyme characteristics such as the half-saturation constant (K_m). In this paper, we describe an in vitro NR assay developed for F. gardneri, in which tissue was homogenized using liquid nitrogen prior to the assay. In contrast to previous studies, enzyme activity was always detectable in F. gardneri collected directly from the field at levels up to 30 nmol nitrate converted to nitrite·min^?1·g^?1 wet weight. The effect of a variety of compounds, commonly added to NR extraction buffers, were tested. Additions of protease inhibitors, bovine serum albumin, and ethylenediamine tetraacetic acid had no consistent effects on NR activity, while polyvinyl pyrrolidone, potassium ferricyanide, and flavin adenine dinucleotide significantly decreased activity. The half-saturation constant (K_m) for NADH was 0.18 (± 0.05) mM and for nitrate, K_m=0.99 (±0.41) mM. Significant NR activity was detected without the addition of nitrate, suggesting that internal pools of nitrate averaging approximately 20 μmol NO₃^?·g^?1 wet weight were present in F. gardneri in February. The distribution of NR activity within the plant was highly variable between individuals, but activities were approximately 5-fold lower in the stipe than in midregions. In plants freshly sampled from the field, NR activity increased 7-fold from February to March, then fell to near-February levels by April. These changes in activity may correspond to seasonal changes in growth rate. The assay, optimized for F. gardneri, was used in several different macroalgal species from different taxa: Porphyra sp., Coralina vancouveriensis Yendo, Ulva sp., Enteromorpha intestinalis (Linnaeus) Nees, Macrocystis integrifolia Bory; and Costaria costatum (C. Agardh) Saunders. For all species tested, NR activity was detectable and, except for one species (Porphya sp.), was equal to or greater than activities measured by other workers using in vivo or in vitro assays for plants under similar conditions.     

Labelling of high molecular weight hyaluronan with125I-tyrosine: studiesin vitro andin vivo in the rat

Studies on the metabolism of the polysaccharide hyaluronan has previously been hampered by the lack of radioactive hyaluronan of high molecular weight (MW) and high specific activity. In the present study¹²⁵I-tyrosine (T)-labelled hyaluronan was produced after CNBr-activation of the polysaccharide. A specific activity of approximately 0.1 MBq µg^–1 was achieved using 100 µg of 0.5×10⁶ Da hyaluronan labelled for 2 h with 18 MBq¹²⁵I. The¹²⁵I-T-hyaluronan kept a high MW-profile upon gel filtration chromatography and was found to be cleared from the circulation with the kinetics and organ distribution reported for biosynthetically labelled hyaluronan of high MW. The¹²⁵I-labelled polysaccharide is also taken up by liver endothelial cells bothin vivo andin vitro, indicating that the labelling does not interfere with the binding to specific cell-surface receptors found on these cells. The intracellular degradation is slower than that earlier reported for biosynthetically labelled hyaluronan and seems to be halted at the level of low MW oligo- or mono-saccharides that eventually leave the organism via the urine. Scintigraphic images of rats after intravenous injection of¹²⁵I-T-hyaluronan showed rapid uptake in the liver and a redistribution of radioactivity from liver to urine with time. Our results indicate that the¹²⁵I-T-hyaluronan is suitable for studies of hyaluronan-metabolism in a number of ways. The gamma emitters¹²⁵I and¹³¹I are easy to monitor and can be used also forin vivo 3D-imaging using single photon emission computer tomography.Abbreviations CNBr cyanogen bromide - T-HA tyrosine-labelled hyaluronan