The glucose transporters GLUT-1 and GLUT-3 are targets of the hypoxia-inducible transcription factor HIF-1alpha and it has been shown that nucleus pulposus (NP) cells in rat intervertebral discs (IVD) express both HIF-1alpha and GLUT-1. However, there is limited data on the expression of HIF-1alpha and GLUTs in human IVD. The aim here was to (1) determine whether, like articular chondrocytes, human IVD cells express GLUT-1, 3 and 9 and whether there was any co-expression with HIF-1alpha; and (2) to localise expression of the GLUT isoforms in the disc and identify any changes during degeneration. Real-time PCR was used to identify expression of GLUT1, 3, 9 and HIF-1alpha mRNAs and immunohistochemistry was used to analyse protein expression and localisation of GLUTs in normal and degenerate IVD biopsies. Results confirmed HIF-1alpha, GLUT1, 3 and 9 mRNA expression in NP and AF and co-expression of each GLUT isoform with HIF-1alpha in the NP, but not the AF. Immunohistochemistry demonstrated regional differences in GLUT expression, with the highest expression being in the NP. GLUT expression also changed as degeneration progressed. This study demonstrates that NP and AF cells have different GLUT expression profiles that suggest regional differences in the metabolic nature of the human IVD and that this environment changes during degeneration.

Twonew /3-lactams have been isolated from strains of Serratia and Erwinia sp. and identified as (3R95R)-and (SS^iQ-carbapenam-S-carboxylic acid. These novel carbapenams lack antibacterial activity, are resistant to both /5-lactamases I and II from Bacillus cereus and are not detected by the lactamase induction assay. Radiolabelled and stable isotope experiments have established that both metabolites together with the antibiotic 52?-carbapenem-3-carboxylic acid are glutamate and acetate derived. A number of possible pathways for the biosynthesis of these compounds as well as their relationship to the morecomplexmembers of the carbapenem family of /5-lactam antibiotics are discussed.The /3-lactam antibiotics thienamycins (1) and the olivanic acids (2) which possess the carbapenem ring system are nowrepresentative of a substantial family of naturally occurring compounds1~4). These have mainly been isolated from Streptomyces species and to date biosynthetic studies on this important group of antibiotics have been restricted by the complexity, low yield and relative instability of the carbapenems produced. Radioactive and stable isotope studies with Streptomyces cattleya have demonstrated that, in thienamycin (1), the C6 and C7 of the /3-lactam ring are derived from acetate, the cysteaminyl side-chain from cysteine and both carbon atoms of the hydroxyethyl substituent at C6 from the methyl of methionine5>6). Anearlier preliminary account7) suggested that glutamate was the source of the carbon atoms of the pyrroline moiety. Onthe basis of these observations it has been proposed that the parent ring system (3) is formed from the interaction of an acetate unit with a yactivated form of glutamate and that this intermediate acts as the precursor of all the carbapenems5). The sequence of events leading to the introduction of the substituents at C2 and C6 remains somewhat obscure, although the Michael-type addition of thiol derivatives at C2 and subsequent oxidation have been implicated4>5>8~10\ The compound, sodium carbapen-2-em-3-carboxylate (3a) has been isolated from species of the bacteria Erwinia and Serratia, as its /?-nitrobenzyl ester (3b)1D, and recently shown to possess the same absolute configuration at C-5 as the products from Streptomyces1^.In a recent communication1^we described the isolation and characterisation of the novel carbapenamcompounds4 and 5 as their /?-nitrobenzyl esters, which were detected as a consequence of a study on the role of L-glutamate in the biosynthesis of the parent carbapenem 3a. This paper describes the details of the incorporation of radiolabelled glutamate into the $-lactams 3, 4 and 5, as well as the incorporation of [l,2-13C2]acetate into 3 and 4.

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Labelling experiments are described that identify three new compounds, N*-(2-carboxyethyl)arginine, 5-guanidino-(2-oxoazetidin-l-yl)pentanoic acid, and 3-hydroxy-5-guanidino-2-( 2-oxoazetidin-I -yl)pentanoic acid as biosynthetic precursors of proclavaminic acid and hence clavulanic acid in Streptom yces clavuligerus ATCC 27064 and a new amidino hydrolase, which hydrolyses 3-hydroxy-5-guanidino-2-(2-oxoazetidin-l -yl)pentanoic acid to proclavaminic acid has been characterised. As part of our goal to elucidate the biosynthesis of clavulanicAlso we reported4 on the production of two new arginine acid 11** we demonstrated3 that arginine 2 and not ornithine is derivatives 3 and 4 by the mutant S . clavuligerus dclH 65 which the amino acid that is processed into the biosynthetic pathway.is blocked in clavulanic acid biosynthesis. In addition, sequencing of the DNA of the clavulanic acid gene cluster identified an open reading frame, which showed homology to arginaseaS From these data we can postulate a number of possible biosynthetic sequences prior to prOClaVaminiC acid 5 which are shown in Scheme 1.

H and 13C n.m.r. spectroscopy combined with molecular modelling techniques were used to show that the major conformation of erythromycin A (1) in CDCI, solution is very similar t o the crystalline state conformation (A) of erythromycin A hydroiodide dihydrate. However, ' H nuclear Overhauser enhancement (n.O.e.), variable-temperature and variable-solvent n.m.r. experiments showed that, in contrast to previous reports, the major conformation of (1) is in fast equilibrium with a second, minor conformation. This minor conformation is related to the major conformation via a reorganisation of the macrocyclic lactone ring in the C-2 to C-9 region.13C N.m.r. relaxation measurements showed that the desosamine sugar possesses more conformational freedom than the cladinose sugar, in agreement both with previous results and with energy calculations based on the crystal structure. In addition, the relaxation experiments indicated that some methyl groups in ( I ) are sterically hindered whereas others possess motional freedom. A good agreement was found between the n.m.r. relaxation results and calculations of methyl group rotational energy barriers in the crystal structure. This good agreement provided further evidence of the similarity between the major solution-state conformation of (1 ) and the crystalline-state conformation A.