bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytochrome c maturation: a complex pathway for a simple task?
Authors: Thöny-Meyer L.
Published: June 2004

Post-translational maturation of c-type cytochromes involves covalent attachment of haem to the apocytochrome polypeptide by two thioether bonds. In bacteria, haem attachment occurs in the periplasm, after the separate translocation of haem and the polypeptide across the cytoplasmic membrane. In Escherichia coli, delivery and attachment of the cofactor requires eight or nine specific proteins, which are believed to be organized in a membrane protein complex. After transport across the membrane, haem is attached covalently to the haem chaperone CcmE in an unusual way at a single histidine residue. However, haem binding to CcmE is transient and is succeeded by a further transfer to apocytochrome c. Both haem binding to and release from CcmE involve integral membrane proteins, CcmC and CcmF respectively, which carry a conserved tryptophan-rich motif in a periplasmic domain. Apocytochrome c polypeptides are synthesized as precursors and reach the periplasm by sec-dependent translocation. There they are prepared for haem binding by reduction of the cysteine residues in the motif Cys-Xaa-Xaa-Cys-His, which is characteristic of such proteins. This reduction is achieved in a thioreduction pathway, whereby electrons are passed from cytoplasmic thioredoxin to the transmembrane protein DsbD, across the membrane, and on to the specific reductases CcmG/CcmH. The merging of the haem delivery and the thioreduction pathways leads to the stereospecific insertion of haem into various type c cytochromes.
Cobalamin (vitamin B12) biosynthesis in Rhodobacter capsulatus
Authors: McGoldrick H. , Deery E. , Warren M. , Heathcote P.
Published: June 2004

In Rhodobacter capsulatus, cobalamin biosynthesis has been shown to occur when the bacteria are grown either aerobically or anaerobically. However, a comparison of the main cobalamin biosynthetic operon found within R. capsulatus would suggest that the encoded proteins belong to the oxygen-dependent pathway for cobalamin biosynthesis, although, significantly, no homologue of the essential mono-oxygenase CobG has yet been detected. Nonetheless, within this main cob operon is found a large open reading frame termed orf663 that is not found in any other cobalamin biosynthetic operon. When overproduced in Escherichia coli, orf663 was found to encode a 90 kDa integral membrane protein. Some of this protein is cleaved within E. coli to give a soluble N-terminal region that can easily be purified and yields a 50 kDa flavoprotein. When expressed in harness with the genes for precorrin-3a synthesis, ORF663 appears to mediate the transformation of precorrin-3a into a new chromophoric compound. Another open reading frame in close proximity to orf663 is termed orf647, and was found to encode a 2Fe-2S ferredoxin-like protein. We suggest that these two proteins may provide an alternative oxygen-independent mechanism for ring contraction within R. capsulatus.
Pentahaem cytochrome c nitrite reductase: reaction with hydroxylamine, a potential reaction intermediate and substrate
Authors: Rudolf M. , Einsle O. , Neese F. , Kroneck P. M. H.
Published: June 2004

The pentahaem enzyme cytochrome c nitrite reductase catalyses the reduction of nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoreductase, a trimeric protein with eight haem molecules per subunit.
Cytochrome cbb3 oxidase and bacterial microaerobic metabolism
Authors: Pitcher R. S. , Brittain T. , Watmugh N. J.
Published: June 2004

Cytochrome cbb3 oxidase is a member of the haem-copper oxidase superfamily. It is characterized by its high oxygen affinity, while retaining the ability to pump protons. These attributes are central to its proposed role in bacterial microaerobic metabolism. Recent spectroscopic characterization of both the cytochrome cbb3 oxidase complex from Pseudomonas stutzeri and the dihaem ccoP subunit expressed separately in Escherichia coli has revealed the presence of a low-spin His/His co-ordinated c-type cytochrome. The low midpoint reduction potential of this haem (Em < + 100 mV), together with its unexpected ability to bind CO in the reduced state at the expense of the distal histidine ligand, raises questions about the role of the ccoP subunit in the delivery of electrons to the active site.
Molecular and atomic analysis of uranium complexes formed by three eco-types of Acidithiobacillus ferrooxidans
Authors: Merroun M. , Hennig C. , Rossberg A. , Geipel G. , Reich T. , Selenska-Pobell S.
Published: June 2004

A combination of EXAFS, transmission electron microscopy and energy-dispersive X-ray was used to conduct a molecular and atomic analysis of the uranium complexes formed by Acidithiobacillus ferrooxidans. The results demonstrate that this bacterium accumulates uranium as phosphate compounds. We suggest that at toxic levels when the uranium enters the bacterial cells, A. ferrooxidans can detoxify and efflux this metal by a process in which its polyphosphate bodies are involved.
Biosynthesis of iron-sulphur clusters is a complex and highly conserved process
Authors: Frazzon J. , Fick J. R. , Dean D. R.
Published: June 2004

Iron-sulphur ([Fe-S]) clusters are simple inorganic prosthetic groups that are contained in a variety of proteins having functions related to electron transfer, gene regulation, environmental sensing and substrate activation. In spite of their simple structures, biological [Fe-S] clusters are not formed spontaneously. Rather, a consortium of highly conserved proteins is required for both the formation of [Fe-S] clusters and their insertion into various protein partners. Among the [Fe-S] cluster biosynthetic proteins are included a pyridoxal phosphate-dependent enzyme (NifS) that is involved in the activation of sulphur from L-cysteine, and a molecular scaffold protein (NifU) upon which [Fe-S] cluster precursors are formed. The formation or transfer of [Fe-S] clusters appears to require an electron-transfer step. Another complexity is that molecular chaperones homologous to DnaJ and DnaK are involved in some aspect of the maturation of [Fe-S]-cluster-containing proteins. It appears that the basic biochemical features of [Fe-S] cluster formation are strongly conserved in Nature, since organisms from all three life Kingdoms contain the same consortium of homologous proteins required for [Fe-S] cluster formation that were discovered in the eubacteria.
Microbial siderophore-mediated transport
Authors: Winkelmann G.
Published: June 2004

Microbial iron chelates, called siderophores, are synthesized by bacteria and fungi in response to low iron availability in the environment. The present review summarizes structural details of siderophore ligands with respect to their transport properties. This presentation is largely centred on the occurrence and function of siderophores in the various bacterial and fungal genera.
Siderophore production by Fusarium venenatum A3/5
Authors: Wiebe M. G.
Published: June 2004

Fusarium venenatum A3/5 was grown in iron-restricted batch cultures and iron-limited chemostat cultures to determine how environmental conditions affected siderophore production. The specific growth rate in iron-restricted batch cultures was 0.22 h−1, which was reduced to 0.12 h−1 when no iron was added to the culture. Derit in iron-limited chemostat culture was 0.1 h−1. Siderophore production was correlated with specific growth rate, with the highest siderophore production occurring at D = 0.08 h−1 and the lowest at D = 0.03 h−1. Siderophore production was greatest at pH 4.7 and was significantly reduced at pHs above 6.0. Siderophore production could be enhanced by providing insoluble iron instead of soluble iron in continuous flow cultures.
Transferrin-mediated iron acquisition by pathogenic Neisseria
Authors: Evans R. W. , Oakhill J. S.
Published: June 2004

The pathogenic Neisseria have a siderophore-independent iron-uptake system reliant on a direct interaction between the bacterial cell and transferrin. In the meningococcus this uptake system is dependent on two surface-exposed transferrin-binding proteins. This short account will review our current knowledge of the transferrin-mediated iron-acquisition system of pathogenic Neisseria.
Bipartite gating in the outer membrane protein FecA
Authors: van der Helm D. , Chakraborty R. , Ferguson A. D. , Smith B. S. , Esser L. , Deisenhofer J.
Published: June 2004

The recent structure determination of FecA, with and without ligand, shows the existence of two gates. These are the extracellular loops closing over the binding site and the plug located inside the barrel. It indicates a process which is described as bipartite gating and allows for a rational distinction between the binding event and the transport process.
Investigations of iron uptake in Halobacterium salinarum
Authors: Hubmacher D. , Matzanke B. F. , Anemüller S.
Published: June 2004

The iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum JW5 was investigated. Experiments to detect endogenous siderophores from H. salinarum failed, but it was able to utilize exogenous siderophores. Measurement of the uptake of 55Fe and [14C]citrate gave evidence only for the accumulation of iron. Two additional membrane proteins could be detected in iron-starved cells, one in iron-repleted membranes and one that is up-regulated there. Respiratory rates of iron-starved membranes after the addition of succinate and NADH differed considerably from iron-repleted ones. Furthermore, both types of membrane exhibited different degrees of inhibition by cyanide.
Characterization of a non-haem ferritin of the Archaeon Halobacterium salinarum, homologous to Dps (starvation-induced DNA-binding protein)
Authors: Reindel S. , Schmidt C. L. , Anemüller S. , Matzanke B. F.
Published: June 2004

An iron-rich protein was isolated from the Archaeon Halobacterium salinarum sharing a sequence identity of 35% with the starvation-induced DNA-binding protein, DpsA, of Synechecoccus sp. PCC 7942. It consists of 20 kDa subunits, forming a dodecameric structure. The protein exhibits a ferric iron loading of up to 103 Fe ions/mol of holoprotein. CD spectra are consistent with an α-helical contribution of 58%. The UV/visible spectrum provides no evidence for the presence of haem groups. This protein exhibits features of a non-haem-type bacterial ferritin although it shares only little sequence homology with non-haem bacterial ferritin.
Mercury resistance (mer) operons in enterobacteria
Authors: Hobman J. L. , Essa A. M. M. , Brown N. L.
Published: June 2004

Mercury resistance is found in many genera of bacteria. Common amongst enterobacteria are transposons related to Tn21, which is both mercuric ion- and streptomycin-/spectinomycin-and sulphonamide-resistant. Other Tn21-related transposons often have different antibiotic resistances compared with Tn21, but share many non-antibiotic-resistance genes with it. In this article we discuss possible mechanisms for the evolution of Tn21 and related genetic elements.
Molecular evidence for the role of a ferric reductase in iron transport
Authors: McKie A. T. , Latunde-Dada G. O. , Miret S. , McGregor J. A. , Anderson G. J. , Vulpe C. D. , Wrigglesworth J. M. , Simpson R. J.
Published: June 2004

Duodenal cytochrome b (Dcytb) is a haem protein similar to the cytochrome b561 protein family. Dcytb is highly expressed in duodenal brush-border membrane and is implicated in dietary iron absorption by reducing dietary ferric iron to the ferrous form for transport via Nramp2/DCT1 (divalent-cation transporter 1)/DMT1 (divalent metal-transporter 1). The protein is expressed in other tissues and may account for ferric reductase activity at other sites in the body.
Relationship between intestinal iron-transporter expression, hepatic hepcidin levels and the control of iron absorption
Authors: Anderson G.J. , Frazer D. M. , Wilkins S.J. , Becker E. M. , Millard K. N. , Murphy T. L. , McKie A. T. , Vulpe C. D.
Published: June 2004

Hepcidin is an anti-microbial peptide predicted to be involved in the regulation of intestinal iron absorption. We have examined the relationship between the expression of hepcidin in the liver and the expression of the iron-transport molecules divalent-metal transporter 1, duodenal cytochrome b, hephaestin and Ireg1 in the duodenum of rats switched from an iron-replete to an iron-deficient diet or treated to induce an acute phase response. In each case, elevated hepcidin expression correlated with reduced iron absorption and depressed levels of iron-transport molecules. These data are consistent with hepcidin playing a role as a negative regulator of intestinal iron absorption.
Down-regulation of liver iron-regulatory protein I in haemochromatosis
Authors: Neonaki M. , Graham D. Cunninghame , White K. N. , Bomford A.
Published: June 2004

Cellular iron homoeostasis is maintained by iron sensor proteins known as iron-regulatory proteins (IRPs), which act post-transcriptionally by binding RNA stem-loop structures, termed iron-responsive elements (IREs), present on the mRNAs of proteins involved in iron storage, utilization and transport. IRP1 is a bifunctional protein that can act either as a cytoplasmic aconitase or as an IRE-binding protein. The RNA-binding activity of IRP1 is regulated post-translationally by the insertion or extrusion of a 4Fe-4S cluster, without changes in the levels of protein. In hereditary haemochromatosis (HH) accumulation of iron in parenchymal tissues, including the liver, occurs, possibly through dysfunctional IRP1. Investigation of IRP1 expression in liver biopsies from HH patients showed that the protein is completely absent or markedly reduced in heavily iron-loaded HH patients. Real-time PCR was then conducted in an attempt to investigate the mRNA levels and establish the underlying mechanism behind the disappearing act of IRP1. The two possibilities are: transcriptional regulation (through the inhibition of transcription) or post-transcriptional regulation (either through increased turnover of protein or inhibition of translation) of IRP1. Preliminary data suggest that transcription of IRP1 is not affected by chronic iron overload, and down-regulation may be attributable instead to degradation of the protein.
Iron targeting to mitochondria in erythroid cells
Authors: Ponka P. , Sheftel A. D. , Zhang A.-S.
Published: June 2004

Immature erythroid cells have an exceptionally high capacity to synthesize haem that is, at least in part, the result of the unique control of iron metabolism in these cells. In erythroid cells the vast majority of Fe released from endosomes must cross both the outer and the inner mitochondrial membranes to reach ferrochelatase, which inserts Fe into protoporphyrin IX. Based on the fact that Fe is specifically targeted into erythroid mitochondria, we have proposed that a transient mitochondria-endosome interaction is involved in Fe transfer to ferrochelatase [Ponka (1997) Blood 89, 1–25]. In this study, we examined whether the inhibition of endosome mobility within erythroid cells would decrease the rate of 59Fe incorporation into haem. We found that, in reticulocytes, the myosin light-chain kinase inhibitor, wortmannin, and the calmodulin antagonist, W-7, caused significant inhibition of 59Fe incorporation from 59Fe-transferrin-labelled endosomes into haem. These results, together with confocal microscopy studies using transferrin and mitochondria labelled by distinct fluorescent markers, suggest that, in erythroid cells, endosome mobility, and perhaps their contact with mitochondria, plays an important role in a highly efficient utilization of iron for haem synthesis.
The Wilson's disease protein expressed in Sf9 cells is phosphorylated
Authors: Vanderwerf S. M. , Lutsenko S.
Published: June 2004

The Wilson's disease protein (WNDP), a copper transporter, is a crucial mediator of copper homoeostasis in mammalian cells. We recently found that changes in copper concentration regulate the phosphorylation level of WNDP. WNDP phosphorylation was observed in several mammalian cell lines, suggesting that a common phosphorylation pathway exists in these cells. Here we demonstrate that WNDP expressed in Sf9 insect cells is also phosphorylated, as evidenced by metabolic labelling of these cells with [32P]Pi. Because the baculovirus system allows us to generate large amounts of protein, we are using this expression method to isolate WNDP and map the sites of WNDP phosphorylation. The identification of phosphorylation sites is the first step towards understanding the physiological role of WNDP phosphorylation.
Copper and prion diseases
Authors: Brown D. R.
Published: June 2004

Transmissible spongiform encephalopathies are diseases of animals and humans that are also termed prion diseases. These diseases are linked together because a normal brain glycoprotein termed the prion protein is converted to a readily detectable protease-resistant isoform. There is now strong evidence to suggest that apart from this difference in resistance a major difference between the isoforms is that the normal prion protein binds copper and has an anti-oxidant function. Brains from Creutzfeldt-Jakob disease patients and brains from mice with experimental mouse scrapie have been shown to have changes in the levels of both copper and manganese. There is growing evidence that links prion diseases to disturbances of metal metabolism.
Toxicity of myoglobin and haemoglobin: oxidative stress in patients with rhabdomyolysis and subarachnoid haemorrhage
Authors: Reeder B. J. , Sharpe M. A. , Kay A. D. , Kerr M. , Moore K. , Wilson M. T.
Published: June 2004

Haemolytic events, such as those following rhabdomyolysis and subarachnoid haemorrhage, often result in pathological complications such as vasoconstriction. Haem-protein cross-linked myoglobin and haemoglobin are generated by ferric-ferryl redox cycling, and thus can be used as markers of oxidative stress. We have found haem-protein cross-linked myoglobin in the urine of patients suffering from rhabdomyolysis and haem-protein cross-linked haemoglobin in the cerebrospinal fluid of patients following subarachnoid haemorrhage. These findings provide strong evidence that these respiratory haem proteins can be involved in powerful oxidation processes in vivo. We have previously proposed that these oxidation processes in rhabdomyolysis include the formation of potent vasoconstrictor molecules, generated by the myoglobin-catalysed oxidation of membranes, inducing nephrotoxicity and renal failure. Haem-protein cross-linked haemoglobin in cerebrospinal fluid suggests that a similar mechanism of lipid oxidation is present and that this may provide a mechanistic basis for the delayed vasospasm that follows subarachnoid haemorrhage.
Design of therapeutic chelating agents
Authors: Hider R. C.
Published: June 2004

The successful design of orally active non-toxic selective metal chelators is a much sought-after goal. In order to identify an ideal chelator for clinical use, a range of specifications must be considered, such as metal selectivity and affinity, kinetic stability of the complex, bioavailability and toxicity. In this overview the comparative properties of ligands capable of endowing complexes with such properties will be discussed.
Manganese transport and its regulation in bacteria
Authors: Bhattacharyya-Pakrasi M. , Pakrasi H. B. , Ogawa T. , Aurora R.
Published: June 2004

Regulation of manganese acquisition by bacteria occurs by both biochemical regulation of the activity of the transporters and transcriptional regulation of gene expression. Structural analysis suggests that calcium ions may regulate the function of an Mn ATP-binding cassette (ABC)-permease in Synechocystis 6803, a cyanobacterium, as well as in a number of other bacteria. The expression of genes encoding the manganese transporter in Synechocystis 6803 is regulated by a twocomponent signal-transduction mechanism that has not been previously observed for manganese and zinc transport in bacteria.
Structural studies of the Fur protein from Rhizobium leguminosarum
Authors: Kolade O. O. , Bellini P. , Wexler M. , Johnston A. W. B. , Grossmann J. G. , Hemmings A. M.
Published: June 2004

The X-ray crystal structure of the apo-form of the Fur protein from Rhizobium leguminosarum has been solved at 2.7 å resolution. Small-angle X-ray scattering was used to give information on the solution conformation of the protein. The Fur homodimer folds into two domains. The N-terminal domain is formed from the packing of two helix-turn-helix motifs while the C-terminal domain appears primarily to stabilize the dimeric state of the protein.
c-Myc represses the murine Nrampl promoter
Authors: Bowen H. , Biggs T. E. , Baker S. T. , Phillips E. , Perry V. H. , Mann D. A. , Barton C. H.
Published: June 2004

The Nrampl (natural resistance-associated macrophage protein 1) gene modulates the growth of intracellular pathogens and encodes a divalent cation transporter within lysosomes/late endosomes of macrophages. Nrampl modulates the cytoplasmic iron pool. Wu, Polack and Dalla-Favera [(1999) Science 283, 676–679] showed reciprocal control of H-ferritin and IRP2 by c-Myc, and suggest that c-Myc regulates genes to increase cytoplasmic iron. A role for c-Myc in Nrampl regulation was evaluated. Co-transfection studies show that c-Myc represses Nrampl promoter function. Five non-canonical Myc-max binding sites (E-box) identified within the Nrampl 5′-flanking sequence are not responsible for the inhibitory effects of c-Myc on Nrampl expression. An initiator(s) adjacent to the transcriptioninitiation site is a candidate for the inhibition observed. Results are consistent with a role for Nrampl removing iron from the cytosol and antagonizing c-Myc function.
A flexible technology platform to explore valuable drug targets
Authors: Connick J. H.
Published: June 2004

The high-throughput screening platform implemented for drug discovery is driven by the therapeutic areas of interest. Therefore the speed and information derived is governed by these areas. Multiple technologies are needed to exploit this and it is also important to show reactivity to new advances in technology. In contrast with a drive over the last few years towards higher throughput and speed, higher information content will be instrumental in driving lead discovery in the future.
Strategies to identify ligands for orphan G-protein-coupled receptors
Authors: Milligan G.
Published: June 2004

G-protein-coupled receptors are the most tractable class of protein targets for small molecule drug design. Sequencing of the human genome allied to bio-informatic analysis has identified a large number of putative receptors for which the natural ligands remain undefined. A range of currently employed and developing strategies to identify ligands that interact with these orphan receptors and to validate them as drug targets are described and discussed.
High-throughput screening in the diagnostics industry
Authors: Wilson S. , Howell S.
Published: June 2004

The diagnostics industry is constantly under pressure to bring innovation quicker to market and so the impetus to speed up product-development cycle times becomes greater. There are a number of steps in the product-development cycle where the application of high-throughput screening can help. In the case of lateral-flow immunodiagnostics the selection of antibody reagents is paramount. In particular, rapid identification of antibody pairs that are able to ‘sandwich’ around the target antigen is required. One screen that has been applied successfully is the use of surface plasmon resonance biosensors like Biacore®. Using such a system one can evaluate over 400 antibody pairings in under 5 days. Conventional approaches to screen this number of antibody pairs would take many months. Other automated screening systems like DELFIA® can be used in processing the vast amount of tests required for clinical trials. In addition, the use of robotics to automate routine product testing can be used to shorten the product-development cycle.
Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery
Authors: Daniele N. , Halse R. , Grinyo E. , Yeaman S.J. , Shepherd P. R.
Published: June 2004

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.
Miniaturized screening technologies for drug discovery
Authors: Cooper J. M. , Cass A. E. G.
Published: June 2004

Adaptive Profiling (APL) and other biochip companies aim to harness the power of microsystems technology together with advances in chemistry and molecular biology, to become service and technology providers to organizations involved in pharmaceutical research and development. By supplying a unique range of decisionmaking tools that aid an earlier identification of qualified drug candidates for clinical development, the company should gain a significant share of the US$10 billion biological screening, bioavailability and toxicity assessment market.
B-cell development and antigen receptor signalling
Authors: Turner M.
Published: June 2004

B-cell development and function requires the products of the Vav family of genes. Vav proteins act as key components of the antigen receptor signal transduction machinery by integration of signals that control the cell cycle, differentiation and apoptosis. At the molecular level, Vav proteins regulate small-molecular-mass GTPases, phosphoinositide 3-kinase, TEC family tyrosine kinases and intracellular calcium flux.
Consequences of Fcγ receptor type III reactivity in non-organ-specific autoimmune diseases
Authors: Youinou P. , Renaudineau Y.
Published: June 2004

Polymorphonuclear neutrophils (PMNs) express constitutively the Fcγ receptors (FcγRs) FcγRIIIb and FcγRIIa for the Fc part of IgG, and soluble FcγRIIIb (sFcγRIIIb) has been identified. Elevated levels of sFcγRIIIb were detected in non-organ-specific autoimmune conditions, and there was a considerably decreased number of PMNs undergoing apoptosis in the presence of sFcγRIIIb. Anti-FcγRIIIb autoantibodies (autoAbs) have also been described in such patients. They are not cytotoxic to these cells, but they extend their survival. The anti-apoptotic signal can be transduced through FcγRIIa and/or CD11b, the β-chain of the complement receptor 3. However, FcγRIIIb appears to be also competent. Anti-FcγRIIIb-conditioned supernatant from cultured PMNs induces the transcription of messenger RNA for granulocyte colonystimulating factor and granulocyte/macrophage colony-stimulating factor, followed by protein synthesis. The delay in apoptosis may be generated by a downregulation of the death promoter Bax. Inflammation might thus be modulated by sFcγRIIIb and anti-FcγRIIIb autoAbs in systemic diseases.
B-lymphocyte depletion therapy in rheumatoid arthritis and other autoimmune disorders
Authors: Edwards J. C. W. , Leandro M.J. , Cambridge G.
Published: June 2004

B-lymphocyte depletion therapy is being explored in a wide range of autoimmune disorders. In many, there is early evidence for efficacy, and immunosuppression has not been a major problem. The mechanism of action is unclear, but appears to be consistent with the lowering of autoantibody levels, where relevant antibodies are quantifiable. An interesting finding is the persistence of clinical improvement for periods of 1 year or more after B-lymphocyte return, which supports the concept that stochastic generation of rare pathogenic B-lymphocyte subsets may be a rate-limiting step in pathogenesis.
Expression of type II procollagens during development of the human intervertebral disc
Authors: McAlinden A. , Zhu Y. , Sandell L. J.
Published: June 2004

Mice lacking type II collagen fail to develop intervertebral discs. The present study describes the distribution of the developmentally expressed type IIA procollagen molecule, as well as types I and III collagens, in human IV disc specimens ranging from 42 to 101 days gestation. Type IIA procollagen contains the alternatively spliced exon 2 which encodes a 69-amino-acid cysteinerich domain. By radioactive in situ hybridization and fluorescence immunohistochemistry, we identified changes in the localization patterns of type IIA procollagen, particularly between days 54 and 101. At day 54, the developing disc was divided into the outer annulus containing types I and III collagens, the inner annulus containing type IIA procollagen and the notochord consisting of all three fibrillar collagens. Specifically, the IIA N-terminal propeptide was localized in the extracellular matrix at day 54 but, by day 101, was only observed in the cytoplasm of the inner annulus cells. A functional role for the IIA N-terminal propeptide during this specific stage of disc development seems apparent. This function may involve regulation of growth factors since the exon 2-encoded domain of type IIA procollagen has previously been shown to bind to bone morphogenetic protein-2 and transforming growth factor-β. We aim to explore this mechanism further.
The role of disc cell heterogeneity in determining disc biochemistry: a speculation
Authors: Oegema T. R.
Published: June 2004

The nucleus pulposus is a key player in very early disc degeneration. In the young disc, by acting as a water-like fluid, as opposed to a solid, it resists compression and instantaneously distributes forces evenly in all directions to the inner annulus. The disc anlage notochordal cells contribute not only to how the disc develops, but also to the matrix of the young disc at a time when the nucleus is at its most fluid-like. In humans, the notochordal cells disappear early, when there is a transformation of the nucleus into a more solid cartilaginous tissue. In cell culture, the co-cultures of the notochordal cells and chondrocytic cells enhance proteoglycan synthesis by the opposite cell type due, at least partly, to soluble factors. The continued presence of notochordal cells in vivo may provide protection. In work by others, in vivo reinsertion of notochordal-rich nucleus pulposus in a damaged disc will delay annular degeneration. The notochordal cells in the nucleus may have a different phenotype from when they are in the notochord and they may go through a changing programme of expression critical to disc development and maintaining a fluid-like nucleus. Little is known about why, in many species, the notochordal cells die early during growth and only the chondrocytic cells persist. This area offers an interesting avenue of research that may lead to very early intervention in disc degeneration.
Elastic tissues of the intervertebral disc
Authors: Yu J.
Published: June 2004

Elastic fibres have been generally considered to play no significant role in the mechanical functioning of the intervertebral disc since earlier studies reported that the elastic fibre network was sparse and irregular. However, a recent study has reported that the network is highly organized and that the distribution and orientation of elastic fibres varies from region to region. In the annulus, elastic fibres appear densely distributed in the region between the lamellae and also in ‘bridges’ across the lamellae. They are also organized in the nucleus where long straight fibres are radially oriented and anchor perpendicularly or obliquely into the cartilaginous endplate. Immunohisto-chemistry using specific antibodies indicates that elastin is present in the network, as is fibrillin. Biochemical studies show, however, that the amino acid composition of the residue remaining after alkaline (NaOH) extraction or CNBr digestion contains a higher concentration of polar amino acids than ligamentum nuchal elastin. The composition of the residue suggests that disc elastin may cross-link strongly with some other matrix components. With such coupling, it is thought that elastic fibres could play a significant mechanical role even though overall elastin is less than 5% of the total dry weight of the disc.
The role of the physicochemical environment in determining disc cell behaviour
Authors: Urban J. P. G.
Published: June 2004

The cells of the intervertebral disc exist in an unusual environment. They are embedded in a dense matrix containing a high concentration of aggrecan whose fixed negative charges regulate the extracellular ionic composition and osmolarity; both extracellular cation concentrations and osmolarity are considerably higher than those experienced by most cell types. The disc also is avascular. Oxygen levels in the centre of the nucleus, where cells may be 6–8 mm from the blood supply, are very low. Since metabolism is mainly by glycolysis, lactic acid is produced at high rates and hence the pH is acidic. Finally, the disc is subjected to mechanical forces at all times; these vary with posture and activity. In particular, because the disc is under low loads during rest and high loads during the day's activities, it loses and regains around 25% of its fluid over a diurnal cycle with consequent changes to the concentrations of extracellular matrix macromolecules and ions and hence extracellular osmolality. Here we will briefly review these factors and discuss the influence of changes in the physicochemical environment on cellular activity, in particular on the rate at which disc cells synthesize and degrade matrix macro-molecules.
Disc morphology in health and disease
Authors: Roberts S.
Published: June 2004

The morphology of the intervertebral disc is dependent on the type of components present and the manner in which they are assembled. This, in turn, will determine how the tissue carries out its primary physiological functions of load bearing and allowing movement in all directions of the otherwise rigid spine. The disc starts its life in an ordered fashion, with the outer annulus fibrosus consisting of a series of regular concentric bundles of collagen fibres around the central gelatinous nucleus pulposus. With advancing age there is increased complexity of lamellae, with more bifurcations, interdigitations and irregularity in number and size of lamellar bands, probably as a result of the disc cells' synthetic response to variations in incident loads. This change in organization itself will lead to altered load bearing, hence possibly establishing a self-perpetuating cycle of disruption to disc morphology, which, once started, may be irreversible. There are also alterations to cell organization with disease and degeneration. There may be increased cell number, with clusters of cells forming by cell proliferation, or increased cell death, whether by necrosis or apoptosis, in degenerate or deformed discs such as are found in spinal deformities; for example, scoliosis. Any change in cell number is likely to alter the nutritive requirements and concentration gradients of both nutrients and metabolites. The normally avascular disc in the healthy adult can become increasingly vascularized and innervated with degeneration and disease. This may lead to an increased supply of oxygen and nutrients to the disc, but can also introduce other cells types and molecules such as cytokines and growth factors.
The role of proteoglycans in aging, degeneration and repair of the intervertebral disc
Authors: Roughley P. J. , Alini M. , Antoniou J.
Published: June 2004

The ability of the nucleus pulposus of the intervertebral disc to resist compressive loads is due to its high content of the proteoglycan aggrecan. Degeneration of the intervertebral disc is preceded and accompanied by a loss of aggrecan due to proteolysis. Biological repair of intervertebral disc degeneration should strive to restore aggrecan content to its optimal functional level. One approach to such repair is to supplement the degenerate nucleus with cells that are capable of aggrecan synthesis. Such cells can be supported in a biomolecular scaffold, but it is essential that the scaffold is compatible with high aggrecan retention if a functional tissue is to be attained.
Erythropoietin and interleukin-1β modulate nitrite production in a Swiss 3T3 cell model of rheumatoid synovial fibroblasts
Authors: Baig S. , Patel Y. , Coussons P. , Grant R.
Published: June 2004

Erythropoietin (EPO), a haemopoietic growth factor and a primary regulator of erythropoiesis, is widely used to treat anaemia in various chronic complications of rheumatoid arthritis (RA). Fibro-blast-like cells, found in the pannus tissue of joints, are thought to contribute to the inflammatory pathology of RA. Thus for the current study we investigated the effects of recombinant human EPO (rHuEPO) on NO metabolism, using an interleukin-1β (IL-1β)-stimulated Swiss 3T3 fibroblast monolayer as a model for fibroblast activity in RA. The results show that, over 3 days, both alone and in combination with the pro-inflammatory cytokine IL-1β (10 ng/ml), rHuEPO (25 μ-units/ml) induced significant production of nitrite in cell culture supernatants. This is an indicator of NO production by nitric oxide synthase (NOS), which is a well-documented mediator of metalloproteinase-mediated tissue remodelling in RA. It therefore appears that, through modulation of NOS-dependent NO production, rHuEPO may influence remodelling of connective tissue in RA, independently of its established erythropoietic role.
Tumour necrosis factor α up-regulates protein kinase R (PKR)-activating protein (PACT) and increases phosphorylation of PKR and eukaryotic initiation factor 2-α in articular chondrocytes
Authors: Gilbert S. J. , Duance V. C. , Mason D. J.
Published: June 2004

Our previous analysis of the genes regulated in cartilage at the onset of spontaneous osteoarthritis in the guinea pig knee revealed up-regulation of the gene for protein kinase R (PKR)-activating protein (PACT), which encodes the cellular activator of the protein kinase, PKR. PACT and PKR are upstream components of a number of signal transduction and gene transcription pathways used by pro-inflammatory cytokines. We have investigated the role of PACT and PKR in articular cartilage degradation using cytokine treatment of bovine primary chondrocytes and cartilage explants. Tumour necrosis factor α increased expression of PACT protein after 3 h of treatment. Furthermore, increased phosphorylation of PKR and eukaryotic initiation factor 2-α was observed. The known role of PKR in cytokine-induced signalling pathways, together with our data showing cytokine regulation of PACT and PKR in chondrocytes, reveals a novel mechanism of cartilage degradation that may be important in the pathogenesis of arthritic diseases.
The glutamate transporter GLAST-I (EAAT-I) is expressed in the plasma membrane of osteocytes and is responsive to extracellular glutamate concentration
Authors: Huggett J. F. , Mustafa A. , O'Neal L. , Mason D. J.
Published: June 2004

The glutamate/aspartate transporter GLAST-1 is expressed in bone in vivo and also exists as a splice variant (GLAST-1a) in which exon 3 is excluded. Since GLAST-1 expression is regulated in bone in response to osteogenic mechanical stimuli in vivo and binding of glutamate to receptors on osteoblasts increases osteoblast number and activity in vitro, control of extracellular glutamate concentrations may be critical for balanced bone remodelling. To determine whether GLAST isoforms may act to regulate extracellular glutamate concentration in bone we investigated whether their pattern or level of expression is responsive to glutamate concentration in bone cells. GLAST-1a mRNA is expressed at lower levels than GLAST-1 mRNA in all cells examined. The GLAST-1a/GLAST-1 mRNA ratio is greater in MLO-Y4 osteocytes than in SaOS-2 osteoblast-like cells, although this does vary in SaOS-2 cells in response to extracellular glutamate concentration. Transfection of MLO-Y4 cells with green fluorescent protein (GFP)-tagged GLAST isoforms revealed a plasma membrane localization of GLAST-1, consistent with its transporter function, whereas GLAST-1a appeared to be expressed within internal vesicles. Interestingly, low extracellular glutamate concentrations redistributed GLAST-1-GFP into a similar internal expression pattern. Regulation of the expression and distribution of GLAST-1 by extracellular glutamate in bone cells indicates that it may regulate glutamate signalling in bone, consistent with its operation in the central nervous system.
Stability of recombinant 2 S albumin allergens in vitro
Authors: Murtagh G. J. , Dumoulin M. , Archer D. B. , Alcocer M. J.
Published: June 2004

Two well known 2 S albumins, Ber e 1 from brazil nut and sunflower 2 S albumin 8 (SFA-8), have been expressed in a eukaryotic system and purified. Analysis of recombinant versions of Ber e 1 and SFA-8 revealed them to be significantly more resistant to digestion by pepsin than BSA, and to be stable for up to 30 min in simulated gastric fluid. Unfolding monitored by CD indicated that both proteins were also very resistant to denaturation induced by heat and low pH. These results suggest that, although the ability of 2 S albumins to reach the circulatory system may be a prerequisite for the allergenicity of this group of proteins, stability is just one of a number of characteristics that provoke a selective immune response.
Interaction between protein allergens and model gastric emulsions
Authors: Burnett G. R. , Wickham M. , Fillery-Travis A. , Robertson J. A. , Belton P. S. , Gilbert S. M. , Tatham A. S. , Shewry P. R. , Mills E. N. C.
Published: June 2004

The observed resistance to pepsinolysis of known food allergens has been suggested as a predictor of their allergenic risk. Consequently, resistance to pepsinolysis has become incorporated into decision tree assessment for potential allergenic risk posed by novel foods. However, existing methods take little account of the interaction between food structure and physiological conditions existing during digestion in vivo. Here we show that a range of protein allergens can adsorb to model stomach emulsions, providing a further means of resisting digestion. We also show that raising the pH and the addition of bile salts to a model stomach emulsion, thereby mimicking the duodenal environment, has the effect of desorbing the adsorbed protein.
Solution structure of allergenic 2 S albumins
Authors: Pantoja-Uceda D. , Bruix M. , Santoro J. , Rico M. , Monsalve R. , Villalba M.
Published: June 2004

The NMR solution structures at different levels of refinement of three different 2 S albumin seed proteins, the recombinant pronapin precursor from Brassica napus, the recombinant RicC3 from Ricinus communis and the methionine-rich protein from sunflower (Helianthus annuus), are described. The resulting common structure consists of a bundle of five α-helices, folded in a right-handed superhelix. The structure is very similar to that of other plant proteins: the hydrophobic protein from soybean, non-specific lipid transfer proteins and amylase/trypsin inhibitors. Analogies and differences in the structures of these families, as well as their possible relationship to allergenicity, are discussed.
Pathogenesis-related (PR)-proteins identified as allergens
Authors: Hoffmann-Sommergruber K.
Published: June 2004

Type 1 allergies are recognized as an important disease affecting around 25% of the population of industrialized countries of the Northern hemisphere. Allergic patients produce specific IgE antibodies after frequent exposure to either inhaled or nutritive allergens. Of the plant allergens listed in the Official Allergen Database of the International Union of Immunological Societies, approx. 25% belong to the group of pathogenesis-related proteins (PR-proteins). PR-proteins are defined as proteins that are induced upon stress, pathogen attack and abiotic stimuli. This inhomogeneous group of proteins has been classified into 14 PR-protein families. So far, plant-derived allergens have been identified with sequence similarities to PR-protein families 2, 3, 4, 5, 8, 10 and 14. In general, both protein groups, i.e. PR-proteins and allergens, comprise rather small proteins, which are stable at low pH and resistant to proteolysis. These features, and their level of expression, make PR-proteins good candidates for evoking an immune response in predisposed humans, when coming into contact with mucosal surfaces. The identification of PR-proteins with allergenic potential and their homologues is of importance for the allergic patient and the management of this disease. Firstly, plant foods derived from genetically modified plants could represent new allergen sources, and therefore should be evaluated carefully for their potential allergenicity. Secondly, complex plant-derived foods should be analysed for hidden allergens and labelled accordingly.
The latex-fruit syndrome
Authors: Wagner S. , Breiteneder H.
Published: June 2004

Approximately 30–50% of individuals who are allergic to natural rubber latex (NRL) show an associated hypersensitivity to some plant-derived foods, especially freshly consumed fruits. This association of latex allergy and allergy to plant-derived foods is called latex-fruit syndrome. An increasing number of plant sources, such as avocado, banana, chestnut, kiwi, peach, tomato, potato and bell pepper, have been associated with this syndrome. The prevailing hypothesis is that allergen cross-reactivity is due to IgE antibodies that recognize structurally similar epitopes on different proteins that are phylogenetically closely related or represent evolutionarily conserved structures. Several types of proteins have been identified to be involved in the latex-fruit syndrome. Two of these are plant defence proteins. Class I chitinases containing an N-terminal hevein-like domain cross-react with hevein (Hev b 6.02), a major IgE-binding allergen for patients allergic to NRL. A β-1,3-glucanase was identified as an important latex allergen which shows cross-reactivity with proteins of bell pepper. Another important NRL allergen, Hev b 7, is a patatin-like protein that shows cross-reactivity with its analogous protein in potato. Furthermore, patients with allergy to plant-derived foods and associated pollinosis show a high frequency of IgE reactivity to the pan-allergen profilin, which may cause positive serum IgE determinations to NRL. Although there is much information about the plant-derived foods and some data about the allergens involved in the latex-fruit syndrome, it is not always clear whether latex sensitization precedes or follows the onset of food allergy.
Industrial dimensions of food allergy
Authors: Crevel R.
Published: June 2004

Serious attempts to estimate the impact of allergic reactions to foods on public health did not begin until the 1980s. Until about 15 years ago, food allergy was considered a minor aspect of food safety. Two developments probably prompted a radical re-appraisal of that situation. The first was the apparently inexorable rise in the prevalence of atopic diseases, of which food allergy forms a part, with its possible consequences highlighted by some well publicised severe reactions. The second was the growth of genetic modification technology, manifested by the commercialization of transgenic crops. Each of these developments impacted on the food industry in distinct ways. On the one hand, food-allergic consumers had to be enabled to avoid specific allergens in products formulated with existing ingredients. Food manufacturers therefore had to identify those specific allergens down to trace amounts in all the ingredients forming the product, and label or remove them. On the other hand, the introduction of products using ingredients from novel sources required an assessment of the allergenicity of these ingredients as an integral part of safety assurance. The approaches used by the food industry to protect existing allergic consumers and those at potential risk of sensitization by novel proteins will be illustrated, emphasizing how they need to be built into every stage of the life cycle of a product.