bluuebunny/crossref_metadata_2025 huggingface dataset:
Exoenzyme S binds its cofactor 14-3-3 through a non-phosphorylated motif
Authors: Hallberg B.
Published: June 2004

14-3-3 proteins belong to a family of conserved molecules, which play a regulatory role and participate in signal transduction and checkpoint control pathways. 14-3-3 proteins bind phosphoserine-phosphorylated ligands, such as the Raf-1 kinase and Bad, through recognition of the phosphorylated consensus motif, RSXpSXP (where pS is phosphoserine). Recently, a phosphorylation-independent interaction has been reported to occur between 14-3-3 and a small number of proteins, for example the 43 kDa inositol polyphosphate 5-phosphatase, glycoprotein Ib, p75NTR-associated cell-death executor (NADE) and the bacterial ADP-ribosyltransferase toxin exoenzyme S (ExoS). It has been suggested that specific residues of 14-3-3 proteins are required for activation of the bacterial toxin ExoS. An unphosphorylated peptide derived from a phage display library, known as the R18 peptide, and a synthetic peptide derived from ExoS inhibit the interaction between ExoS and 14-3-3. In this report we identify the amino acid sequence on ExoS which is responsible for its specific interaction with 14-3-3, both in vitro and in vivo. In addition, we believe that this interaction is critical for the ADP-ribosylation of an endogenous target, Ras, by ExoS both in vitro and in vivo. Loss of the 14-3-3-binding site on ExoS results in an ExoS molecule that is unable to efficiently inactivate Ras and shows a reduced capacity to change the morphology of infected cells, together with reduced killing activity.
Phosphorylation-independent interaction between 14-3-3 protein and the plant plasma membrane H+-ATPase
Authors: Borch J. , Bych K. , Roepstorff P. , Palmgren M. G. , Fuglsang A. T.
Published: June 2004

14-3-3 proteins interact with a novel phosphothreonine motif (Y946pTV) at the extreme C-terminal end of the plant plasma membrane H+-ATPase molecule. Phosphorylation-independent binding of 14-3-3 protein to the YTV motif can be induced by the fungal phytotoxin fusicoccin. The molecular basis for the phosphorylation-independent interaction between 14-3-3 and H+-ATPase in the presence of fusicoccin has been investigated in more detail. Fusicoccin binds to a heteromeric receptor that involves both 14-3-3 protein and H+-ATPase. Binding of fusicoccin is dependent upon the YTV motif in the H+-ATPase and, in addition, requires residues further upstream of this motif. Apparently, 14-3-3 proteins interact with the unusual epitope in H+-ATPase via its conserved amphipathic groove. This implies that very diverse epitopes bind to a common structure in the 14-3-3 protein.
Adrenomedullin: receptor and signal transduction
Authors: Smith D. M. , Coppock H. A. , Withers D. J. , Owji A. A. , Hay D. L. , Choksi T. P. , Chakravarty P. , Legon S. , Poyne D. R.
Published: June 2004

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP1) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP1 receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via Gs and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.
Interaction of calcitonin-gene-related peptide with its receptors
Authors: Conner A. C. , Hay D. L. , Howitt S. G. , Kilk K. , Langel Ü. , Wheatley M. , Smith D. M. , Poyner D. R.
Published: June 2004

The receptor for calcitonin-gene-related peptide (CGRP) is a heterodimer formed by calcitonin-receptor-like receptor (CRLR), a type II (family B) G-protein-coupled receptor, and receptor-activity-modifying protein 1 (RAMP1), a single-membrane-pass protein. It is likely that the first seven or so amino acids of CGRP (which form a disulphide-bonded loop) interact with the transmembrane domain of CRLR to cause receptor activation. The rest of the CGRP molecule falls into three domains. Residues 28–37 and 8–18 are normally required for high-affinity binding, while residues 19–27 form a hinge region. The 28–37 region is almost certainly in direct contact with the receptor; 8–18 may make additional receptor contacts or may stabilize an appropriate conformation of 28–37. It is likely that these regions of CGRP interact both with CRLR and with the extracellular domain of RAMP1.
Biosynthesis of cobalamin (vitamin B12)
Authors: Scott A. I. , Roessner C. A.
Published: June 2004

The biosynthesis of vitamin B12 is summarized, emphasizing the differences observed between the aerobic and anaerobic pathways. The biosynthetic route to adenosylcobalamin from its five-carbon precursor, 5-aminolaevulinic acid, can be divided into three sections: (1) the biosynthesis of uroporphyrinogen III from 5-aminolaevulinic acid, which is common to both pathways; (2) the conversion of uroporphyrinogen III into the ring-contracted, deacylated intermediate precorrin 6 or cobalt-precorrin 6, which includes the primary differences between the two pathways; and (3) the transformation of this intermediate to form adenosylcobalamin.
Role of proteolysis in copper homoeostasis
Authors: Solioz M.
Published: June 2004

The cop operon of Enterococcus hirae controls cytoplasmic copper levels. It encodes two copper ATPases, a repressor, and the CopZ metallo-chaperone. Transcription of these genes is induced by copper. However, at higher copper concentrations, CopZ is degraded by a copper-activated proteolytic activity. This specific proteolysis of CopZ can also be demonstrated in vitro with E. hirae extracts. Growth of the cells in copper increases the copper-inducible proteolytic activity in extracts. Zymography reveals the presence of a copper-dependent protease in crude cell lysates. Copper-stimulated proteolysis of CopZ appears to play an important role in copper homoeostasis by E. hirae.
The response to iron deprivation in Saccharomyces cerevisiae: expression of siderophore-based systems of iron uptake
Authors: Philpott C. C. , Protchenko O. , Kim Y. W. , Boretsky Y. , Shakoury-Elizeh M.
Published: June 2004

The budding yeast Saccharomyces cerevisiae responds to growth in limiting amounts of iron by activating the transcription factor Aft1p and expressing a set of genes that ameliorate the effects of iron deprivation. Analysis of iron-regulated gene expression using cDNA microarrays has revealed the set of genes controlled by iron and Aft1p. Many of these genes are involved in the uptake of siderophore-bound iron from the environment. One family of genes, FIT1, FIT2 and FIT3, codes for mannoproteins that are incorporated into the cell wall via glycosylphosphatidylinositol anchors. These genes are involved in the retention of siderophore-iron in the cell wall. Siderophore-bound iron can be taken up into the cell via two genetically separable systems. One system requires the reduction and release of the iron from the siderophore prior to uptake by members of the Fre family of plasma-membrane metalloreductases. Following reduction and release from the siderophore, the iron is then taken up via the high-affinity ferrous transport system. A set of transporters that specifically recognizes siderophore-iron chelates is also expressed under conditions of iron deprivation. These transporters, encoded by ARN1, ARN2/TAF1, ARN3/SIT1 and ARN4/ENB1, facilitate the uptake of both hydroxamate- and catecholate-type siderophores. The Arn transporters are expressed in intracellular vesicles that correspond to the endosomal compartment, which suggests that intracellular trafficking of the siderophore and/or its transporter may be important for uptake.
Mercury transport and resistance
Authors: Brown N. L. , Shih Y.-C. , Leang C. , Glendinning K. J. , Hobman J. L. , Wilson J. R.
Published: June 2004

Resistance to mercuric ions in bacteria is conferred by mercuric reductase, which reduces Hg(II) to Hg(0) in the cytoplasmic compartment. Specific mercuric ion transport systems exist to take up Hg(II) salts and deliver them to the active site of the reductase. This short review discusses the role of transport proteins in resistance and the mechanism of transfer of Hg(II) between the mercury-resistance proteins.
Novel cytotoxic chelators that bind iron(II) selectively over zinc(II) under aqueous aerobic conditions
Authors: Planalp R. P. , Przyborowska A. M. , Park G. , Ye N. , Lu F. H. , Rogers R. D. , Broker G. A. , Torti S. V. , Brechbiel M. W.
Published: June 2004

To achieve cellular iron deprivation by chelation, it is important to develop chelators with selective metal-binding properties. Selectivity for iron has long been the province of certain oxygen-donor chelators such as desferrioxamine, which target Fe(III) and exploit the strength of a relatively ionic Fe(III)-O interaction. We have been studying novel chelators that possess mechanisms to selectively chelate + 2 biometals, particularly tachpyr [N,N′,N″-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane] and derivatives from N, N′, N″-trialkylation and pyridine ring alkylation. Metal-exchange and metal-binding competition reactions have been conducted at pH 7.4, 37°C and time periods until no further change was observed (generally 24–48 h). Under anaerobic conditions, tachpyr is strongly selective for iron, binding 95±5% Fe(II) versus 5±5% Zn(II) in the forms [Fe(tachpyr)]2+ and [Zn(tachpyr)]2+ respectively. Under aerobic conditions, tachpyr complexes Fe(II) more effectively than Fe(III), forming iminopyridyl complexes [Fe(tachpyrox-n)]2+ (n = 2,4) by O2-induced and ironmediated oxidative dehydrogenation. Complexes [Fe(tachpyr-ox-n)]2+ are also strongly bound forms of iron that are unaffected by an excess of Zn(II) (75 mol zinc:1 mol iron complex). The preference of tachpyr for iron over zinc under aerobic conditions appears to be hindered by oxidation of Fe(II) to Fe(III), such that the proportions bound are 44±10% Fe(II) versus 56±10% Zn(II), in the respective forms [Fe(tachpyr-ox-n)]2+ and [Zn(tachpyr)]2+. However, upon addition of the reducing agent Na2S2O4 that converts Fe(III) to Fe(II), the binding proportions shift to 76±10% Fe(II) versus 24±10% Zn(II), demonstrating a clear preference of tachpyr for Fe(II) over Zn(II). Iron(II) is in the low-spin state in [Fe(tachpyr)]2+ and [Fe(tachpyr-oxn)]2+ (n = 2, 4), which is a likely cause of the observed selectivity. N-methylation of tachpyr [giving (N-methyl)3tachpyr] results in the loss of selectivity for Fe(II), which is attributed to the steric effect of the methyl groups and a resulting high-spin state of Fe(II) in [Fe(N-methyl)3tachpyr)]2+. The relationship of chelator selectivity to cytotoxicity in the tach family will be discussed.
Hyper-IgM syndromes: a model for studying the regulation of class switch recombination and somatic hypermutation generation
Authors: Durandy A.
Published: June 2004

Several genetic defects in class switch recombination, which lead to a hyper-IgM syndrome, have been described recently in humans. In addition to the well known role of CD40-ligand-CD40 interaction, these pathologies demonstrate definitively the requirement of CD40-mediated nuclear factor kB activation and the essential role of a recently described molecule, the activationinduced cytidine deaminase in an efficient humoral response, which includes class switch recombination and the production of high-affinity antibodies.
Mechanobiology of the intervertebral disc
Authors: Lotz J. C. , Hsieh A. H. , Walsh A. L. , Palmer E. I. , Chin J. R.
Published: June 2004

Intervertebral disc degeneration has been linked in humans to extreme spinal loading regimens. However, mechanisms by which spinal force influences disc cellularity, morphology and consequently biomechanical function are unclear. To gain insight into mechanobiological interactions within the disc, we developed an in vivo murine tail-compression model. Results from this model demonstrate how deviations in spinal stress induce a cycle of altered cell function and morphology as the disc remodels to a new homoeostatic configuration.
The effect of thymosin β4 on articular cartilage chondrocyte matrix metalloproteinase expression
Authors: Blain E. J. , Mason D. J. , Duance V. C.
Published: June 2004

Mechanical loading is paramount in regulating both the anabolic and catabolic activities of articular cartilage chondrocytes, essential for the matrix to retain its functional integrity. We have identified thymosin β4 as a putative mechanically regulated gene that may mediate load-enhanced synthesis and activation of matrix metalloproteinases (MMPs) 2 and 9 in articular cartilage. The objective of this study was to confirm the mechanical regulation of thymosin β4 and determine its effect on cartilage chondrocyte MMP production. Thymosin β4 mRNA expression, analysed by quantitative PCR, revealed a significant 20-fold increase in cartilage loaded for 10 min which was still evident after 30 min of loading. Treatment of primary chondrocytes with 2 and 4 μg · ml-1 thymosin β4 peptide for 4 h significantly increased pro-MMP 9 expression and activation. We postulate a functional role for load-induced thymosin β4 in modulating the cytoskeletal organization of articular cartilage chondrocytes to affect MMP expression.
Clinical importance of non-specific lipid transfer proteins as food allergens
Authors: van Ree R.
Published: June 2004

Non-specific lipid transfer proteins (nsLTPs) have recently been identified as plant food allergens. They are good examples of true food allergens, in the sense that they are capable of sensitizing, i.e. inducing specific IgE, as well as of eliciting severe symptoms. This is in contrast with most plant food allergens, which are recognized because of primary sensitization to related inhalant allergens (cross-reactivity), i.e. pollen allergens. The basis of the difference between the latter category and strong food allergens such as nsLTPs appears to lie in the sensitivity of the allergens to proteolytic attack and food processing. Stability allows the allergen to reach the gastrointestinal immune system in an immunogenic and allergenic conformation, allowing sensitization and induction of systemic symptoms. Stability also explains the presence of such allergens in processed foods. Together, these characteristics make nsLTPs clinically highly relevant plant food allergens and ideal tools with which to study the mechanisms involved in food allergy.
Structural and functional studies of complement inhibitor C4b-binding protein
Authors: Blom A. M.
Published: June 2004

C4b-binding protein (C4BP) is a potent inhibitor of the classical pathway of the complement system. This large plasma glycoprotein consists of seven identical α-chains and a unique β-chain held together by disulphide bridges. Both types of subunits are composed almost exclusively of complement control protein domains (CCPs). Using homology-based computer modelling and mutagenesis of recombinant proteins we have localized binding sites for several ligands of C4BP: complement factor C4b, heparin and vitamin K-dependent anticoagulant protein S (PS). We found that C4b requires CCP1–3 of the α-chain for binding. The interaction is ionic in nature and mediated by a cluster of positively charged amino acids present on the interface between CCP1 and CCP2 of the α-chain. Loss of C4b-binding resulted in a loss of all inhibitory functions of C4BP within the classical pathway of complement. Binding of heparin required CCPs 1–3 of the α-chain, with CCP2 being the most important, as well as the cluster of positively charged amino acids involved in binding of C4b. The interaction between C4BP and PS is of very high affinity and conveyed by a cluster of surface exposed hydrophobic amino acids localized on CCP1 of the β-chain. Furthermore, C4BP is captured on the surface of several pathogens, which may contribute to their serum resistance and pathogenicity. We have localized interaction of C4BP with Neisseria gonorrhoeae, Bordetella pertussis, Streptococcus pyogenes and Escherichia coli to various regions of the α-chain.
Use of control analysis to study the regulation of plant lipid biosynthesis
Authors: Ramli U. S. , Baker D. S. , Quant P. A. , Harwood J. L.
Published: June 2004

Control analysis is a powerful method to quantify the regulation of metabolic pathways. We have applied it to lipid biosynthesis for the first time by using model tissue culture systems from the important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). By the use of top-down control analysis, fatty acid biosynthesis has been shown to exert more control than lipid assembly under different experimental conditions. However, both parts of the lipid biosynthetic pathway are important, so that attempts to alter oil yield by manipulating the activity of a single enzyme step are very unlikely to produce significant increases.
Investigations into the regulation of lipid biosynthesis in Brassica napus using antisense down-regulation
Authors: Slabas A. R. , White A. , O'Hara P. , Fawcett T.
Published: June 2004

We have generated antisense plants of Brassica napus, targeting either the cytoplasmic Type I acetyl-CoA carboxylase or the β-ketoacyl-acyl carrier protein reductase of the Type II dissociable fatty acid synthase, in order to investigate the importance of these components in regulating lipid metabolism in plants. Using a range of down-regulated plants, it has become clear that down-regulation of these genes also causes down-regulation of other components of lipid metabolism, at the activity, translational and transcriptional levels. These plants exhibit effects, not immediately predicted, on oil yield and carbon resource allocation in seeds.
Regulation of mammalian acetyl-CoA carboxylase
Authors: Munday M. R.
Published: June 2004

Acetyl-CoA carboxylase (ACC) plays a critical role in the regulation of fatty acid metabolism and its two isoforms, ACCα and ACCβ, appear to have distinct functions in the control of fatty acid synthesis and fatty acid oxidation, respectively. They are regulated by similar short-term mechanisms of allosteric activation by citrate, and reversible phosphorylation and inactivation, and there is clearly interaction between these mechanisms. AMP-activated protein kinase is the important physiological ACC kinase for both isoforms and yet there is a potential physiological role for cAMP-dependent protein kinase in the hormonally mediated inactivation of ACCα, and phosphorylation of ACCβ in its unique N-terminus.
Acyl-CoA measurements in plants suggest a role in regulating various cellular processes
Authors: Graham I. A. , Li Y. , Larson T. R.
Published: June 2004

Acyl-CoA esters have been shown to be involved in regulating metabolism and cell signalling in bacteria, yeast and mammalian cells, but little is known about their role in plants. Using a new method for the sensitive detection and quantification of acyl-CoA esters, we have recently shown that acyl-CoA pools can be dramatically altered in transgenic oilseed rape embryos, engineered to produce medium-chain fatty acids, and in mutant Arabidopsis seedlings that are unable to mobilize storage lipid. The consequences of these alterations are discussed in the context of oil yield and organelle biogenesis and the possible role of acyl-CoAs in regulating these processes.
The hairpin ribozyme: from crystal structure to function
Authors: Ferré-D'Amaré A. R. , Rupert P. B.
Published: June 2004

The hairpin ribozyme is one of four known natural catalytic RNAs that carry out sequence-specific cleavage of RNA. It is of particular biochemical interest because, unlike ‘classic’ ribozymes, such as the group I intron, it appears not to employ metal ions as catalytic cofactors. We have determined the crystal structure of a hairpin-ribozyme-inhibitor complex at a resolution of 2.4 Å. The active site of the ribozyme results from docking of two irregular double helices. Docking results in major structural rearrangements of the helices, including a distortion of the substrate strand that brings it into a reactive conformation. It remains to be established whether this RNA enzyme relies exclusively on binding energy to carry out catalysis, or whether some other mechanism, such as general acid-base catalysis, is being employed.
RNase P RNA-mediated catalysis
Authors: Kirsebom L. A.
Published: June 2004

The endoribonuclease RNase P is involved in the processing of tRNA precursors to generate mature 5′ termini. The catalytic activity of RNase P is associated with an RNA, RNase P RNA. A specific interaction between the 3′ end of the substrate and RNase P RNA, to form an RNase P RNA-substrate complex, is referred to as the ‘73–294-interaction’. This interaction has an important role for efficient and correct cleavage to occur. Here our understanding of the contribution of the 73–294-interaction and metal ions, with respect to efficient and correct cleavage in RNase P RNA-mediated catalysis, will be discussed.
Glycoprotein hormone GalNAc-4-sulphotransferase
Authors: Baenziger J.U.
Published: June 2004

The glycoprotein hormones lutropin (LH) and thyrotropin and a limited number of additional glycoproteins bear carbohydrate structures terminating with the unique sequence SO4-4-GalNAcβ1,4GlcNAcβ that has been conserved in the glycoprotein hormones of all vertebrate species. Synthesis of these structures is mediated by a protein-specificβ1,4GalNAc-transferase and a GalNAc-4-sulphotransferase (GalNAc-4-ST1). GalNAc-4-ST1 is a member of a family of sulphotransferases that includes HNK-1 sulphotransferase, chondroitin-4-sulphotransferases-1–3 and dermatan-4-sulphotransferase-1. With the exception of HNK-1-ST, these sulphotransferases add sulphate to the C-4 hydroxy group of either terminal or non-terminalβ1,4-linked GalNAc. GalNAc-4-ST1 is most highly expressed in pituitary, cerebellum and other regions of the brain. The terminal GalNAcSO4 on LH is recognized by the cysteine-rich domain of the mannose/GalNAc-4-SO4 receptor located in hepatic endothelial cells. Each cysteine-rich domain binds a single terminal GalNAc-4-SO4, and the receptor must form non-covalently associated homodimers in order to simultaneously engage two GalNAc-4-SO4 moieties on separate oligosaccharides with sufficient affinity to form stable complexes. The receptor mediates the clearance of LH from the blood. This clearance, in conjunction with the stimulated release of hormone from dense core granules in pituitary gonadotroph cells, is required to produce the episodic rise and fall in LH levels needed for optimal oestrogen production during the implantation of embryos in the uterus.
Heparan sulphate sulphotransferase expression in mice and Caenorhabditis elegans
Authors: Turnbull J. , Drummond K. , Huang Z. , Kinnunen T. , Ford-Perriss M. , Murphy M. , Guimond S.
Published: June 2004

Heparan sulphate (HS) acts as a multifunctional cell regulator, with specific sulphated saccharide sequences designed for selective interactions with many proteins. Functionally, these interactions result in regulation of the protein activities, and there is growing evidence that cells can dynamically alter the structure of HS sequences that they display. HS biosynthesis involves the action of a complex set of enzymes with polymerase, epimerase and sulphotransferase (ST) activities. In higher organisms, multiple isoforms of STs decorate the nascent HS chains with specific patterns of sulphation that confer selective biological functions. The study of HSSTs in model organisms provides a valuable opportunity to examine the expression of these enzymes in relation to the structure and activities of the HS produced. Here we describe that, in mice, there are stage-specific combinations of HSST isoenzymes that underlie the synthesis of different HS species at different times in the developing brain. This differential expression of HSSTs results in the synthesis of structurally variant HS species that form functional signalling complexes with specific fibroblast growth factors and their receptors. Regulated synthesis of specific HS species could be a mechanism for the regulation of proliferation and differentiation in the developing brain. We also describe evidence that a Caenorhabditis elegans orthologue of the mammalian 2OST enzyme, called HST-2, is essential for the normal development of this nematode. Together, these studies emphasize the importance of HSSTs in the biosynthesis of functionally variant HS proteoglycans, and demonstrate the importance of these complex regulatory molecules in developmental processes.
Acrolein produced from polyamines as one of the uraemic toxins
Authors: Sakata K. , Kashiwagi K. , Sharmin S. , Ueda S. , Igarashi K.
Published: June 2004

It is well known that the addition of spermine or spermidine to culture medium containing ruminant serum inhibits cellular proliferation. This effect is caused by the products of oxidation of polyamines that are generated by serum amine oxidase. Among the products, we found that acrolein is a major toxic compound produced from spermine and spermidine by amine oxidase. We then analysed the level of polyamines (putrescine, spermidine and spermine) and amine oxidase activity in plasma of patients with chronic renal failure. It was found that the levels of putrescine and the amine oxidase activity were increased, whereas spermidine and spermine were decreased in plasma of patients with chronic renal failure. The levels of free and protein-conjugated acrolein were also increased in plasma of patients with chronic renal failure. An increase in putrescine, amine oxidase and acrolein in plasma was observed in all cases such as diabetic nephropathy, chronic glomerulonephritis and nephrosclerosis. These results suggest that acrolein is produced during the early stage of nephritis through kidney damage and also during uraemia through accumulation of polyamines in blood due to the decrease in their excretion into urine.
Polyamines and colon cancer
Authors: Milovic V. , Turchanowa L.
Published: June 2004

In colon cancer, the activities of polyamine-synthesizing enzymes and polyamine content are increased 3–4-fold over that found in the equivalent normal colonic mucosa, and polyamines have even been attributed as markers of neoplastic proliferation in the colon. Furthermore, and in contrast with all other cell systems in the body, normal and neoplastic cells in the colon are exposed to high concentrations of putrescine from the lumen, synthesized by colonic microflora. While such a high polyamine supply may be of benefit in non-neoplastic colonic mucosal growth, the role of luminal polyamines in colon cancer is a clear concern. Luminal polyamines are readily taken up by neoplastic colonocytes, they are utilized in full to support neoplastic growth, and their uptake is strongly up-regulated by the mitogens known to play an important role in colonic carcinogenesis. Inhibition of polyamine synthesis and their uptake, impaired utilization of exogenous polyamines, and enhanced catabolism of polyamines in neoplastic colonocytes are therefore logical approaches in the chemoprevention of colorectal cancer.
The biological activities of new polyamine derivatives as potential therapeutic agents
Authors: Kong Thoo Lin P. , Dance A.M. , Bestwick C. , Milne L.
Published: June 2004

A number of polyamine derivatives have demonstrated potential as therapeutic agents. For example, 1,12-bisethylspermine and bisnaphthalimide (elinafide) are currently in phase I clinical trials for the treatment of certain cancers. Here, the biological activities of two new groups of polyamine derivative, namely the oxa-polyamines and the bisnaphthalimides, are presented. The most active compounds in the oxa-polyamine and bisnaphthalimido series possessed IC50 values of 2.93 and 1.38 μM, respectively, against MCF7 cells after 48 h of exposure. The structure–relationship activities of each group of compounds are discussed. Bisnaphthalimido compounds are DNA-binding agents. Addition of the bisnaphthalimides PK3, PK4, PK5, PK6 and PK7, at a concentration of 10 μM, to the calf thymus DNA duplex increased the Tm of DNA by 11.55±0.56, 14.545±1.59, 6.23±2.45, 12.56±1.84 and 16.45±0.39°C respectively. With the exception of PK5, all compounds bind to DNA by intercalation as judged by effect of compounds on DNA mobility. Ethidium bromide displacement assay showed that all the compounds have significant affinity for calf thymus DNA (the drug concentration required to reduce the fluorescence of initially DNA-bound ethidium bromide by 50%, C50, was 1.21–17.33 μM). The order of DNA-binding strength was PK4 > PK3 > PK7 > PK6 > PK5. In HL-60 promyelocytic leukaemia cells, oxa-polyamine and bisnaphthalimido treatment resulted in a decline in cell proliferation and viability. The assays performed suggested that apoptosis was not the principal cell death mechanism involved in oxa-polyamine cytotoxicity. In contrast, HL-60 cell death induced by the bisnaphthalimido series was characterized by early exposure of phosphatidylserine exclusive from membrane damage, elevated caspase-3 activity, increased DNA instability and, ultimately, DNA fragmentation. Thus the principal cytotoxic members of the bisnaphthalimido series appear to induce apoptosis.
Centenarians as a model for healthy aging
Authors: Franceschi C. , Bonafè M.
Published: June 2004

For over 10 years we have studied centenarians as a model to address the biological basis of aging and longevity, with particular attention to immunology and genetics. The most important findings can be summarized as follows. (i) Human immunosenescence represents a complex remodelling, whereby clonotypical immunity deteriorates, while ancestral, innate immunity is largely preserved. (ii) Continuous exposure to antigens causes a lifelong, chronic antigenic stress, which is responsible, together with the involution of the thymus, for the accumulation of memory/effector T cells and the exhaustion of naïve T cells. (iii) Aging is characterized by a peculiar chronic inflammatory status that we propose to call ‘inflammaging’, which appears to be under genetic control, is detrimental for longevity and is more evident in men than in women. Inflammaging, i.e. the up-regulation of a variety of anti-stress responses at the cellular and molecular level, is the consequence of the ability of the body to adapt to and counteract the effects of a variety of stressors, which causes the accumulation of molecular and cellular scars. Inflammaging is considered the common and most important driving force of age-related pathologies, such as neurodegeneration, atherosclerosis, diabetes and sarcopenia, among others, all of which share an inflammatory pathogenesis. (iv) Possible strategies to counteract the major effects of immunosenescence and inflammaging, such as the systematic reduction of the lifelong antigenic load, the elimination of chronic infections, thymic rejuvenation and preventative treatment with anti-inflammatory drugs in people with a pro-inflammatory genotype, are envisaged.
Calmodulin-dependent regulation of mammalian nitric oxide synthase
Authors: Daff S.
Published: June 2004

The nitric oxide synthases are large, modular, dimeric enzymes composed of a reductase domain, which is related to cytochrome P450 reductase, and a structurally unique oxygenase domain containing a Cys-ligated haem. Both the neuronal and endothelial isoforms are activated by the reversible binding of calmodulin (CaM) at elevated intracellular Ca2+ levels to produce NO as part of a number of cell signalling pathways. CaM binds to the linker region between the two domains and activates the enzyme by inducing intramolecular electron transfer. Protein-engineering experiments have shown that a series of unusual autoinhibitory inserts found only in the CaM-dependent NOS isoforms control both CaM binding and the structural rearrangement it induces. These lie in the reductase domain of the enzyme and include a 40-amino-acid autoinhibitory loop in the FMN-binding module, a 30-amino-acid extension to the C-terminus and the CaM-binding site itself. The substrate (NADPH) also plays an important role in defining the CaM-dependence of the reductase domain by inducing a tight conformational lock in the absence of CaM. Both the substrate and the conformational lock appear to be released on CaM binding; the resultant domain mobility leads to activation.
The tuberous sclerosis complex (TSC) pathway and mechanism of size control
Authors: Potter C.J. , Pedraza L.G. , Huang H. , Xu T.
Published: June 2004

We have identified three groups of growth-constraint genes using mosaic genetic screens in Drosophila melanogaster, including PTEN (phosphatase and tensin homologue deleted on chromosome 10), and the tuberous sclerosis complex (TSC) genes, Tsc1 and Tsc2. Our studies show that all three groups of genes participate in mechanisms that regulate organ and organism size in animals. We propose that mechanisms of organ size control are critical targets for diseases, such as tumorigenesis, which require an increase in tissue size and total mass, and for evolutionary events that alter the size of organisms. Using genetic and biochemical methods, we have shown that Tsc1 and Tsc2 function in the insulin/phosphoinositide 3-kinase (PI3K)/Akt pathway. We have shown that Akt regulates the Tsc1–Tsc2 complex by directly phosphorylating Tsc2. We have shown further that S6 kinase (S6K) is a downstream component of the PI3K/Akt/TSC pathway and reduction of S6K activity can block TSC defects. Recent studies from many laboratories have now confirmed our findings in mice, rats and human patients, and have shown that drugs that antagonize S6K activities, such as rapamycin, diminish tumours in TSC-deficient mice and rats. Clinical trials based on these findings have begun. Given that other components of the pathway, such as PTEN, are also mutated in a large number of cancer patients and that these components regulate intracellular insulin signalling, therapeutics based on the knowledge of the pathway could have effects beyond the TSC patient population.
DNA replication in the hyperthermophilic archaeon Sulfolobus solfataricus
Authors: Dionne I. , Robinson N.P. , McGeoch A.T. , Marsh V.L. , Reddish A. , Bell S.D.
Published: June 2004

Studies of the DNA-replication machinery of Archaea have revealed striking similarities to that of eukaryotes. Indeed, it appears that in most cases Archaea possess a simplified version of the eukaryotic replication apparatus. Studies of Archaea are therefore shedding light on the fundamental processes of DNA replication in both domains of life.
It can be a complicated life being an enzyme
Authors: Tipton K.F. , O'Sullivan M.I. , Davey G.P. , O'Sullivan J.
Published: June 2004

It is becoming increasingly apparent that many well-known enzymes have alternative non-enzymic functions. Similarly, several proteins that were identified as having non-catalytic functions were subsequently found to have enzyme activities. Some examples are considered to illustrate the diversity of alternative functions. The semicarbazide-sensitive amine oxidase (EC 1.4.3.6) is considered in more depth as an example. It was originally believed to be a detoxifying enzyme, but the reaction products may have important signalling functions. Furthermore, this enzyme, from some sources, also behaves as a vascular-adhesion protein. Finally, the challenges posed by such multiplicity of functions for the interpretation of genetic deletion, in vivo inhibition and the development of functional protein databases are briefly considered.
Mannan-binding lectin (MBL) production from human plasma
Authors: Laursen I.
Published: June 2004

Individuals with low levels of mannan-binding lectin (MBL) appear to be susceptible to infectious diseases. This suggests that substitution therapy with MBL might be a beneficial treatment of patients with MBL deficiency. A production process for an MBL product has been developed from a fraction II+III precipitate obtained by ethanol fractionation of plasma. The MBL process includes three chromatographic steps, where the first and key step is affinity chromatography on a cross-linked agarose matrix selecting for oligomeric, carbohydrate-binding MBL. The yield from the production process is about 25% of the plasma MBL content, and the purity is about 65%. The MBL product shows mannan-binding activity and complement-activating ability. A safety study has shown this plasma-derived MBL to be safe and well tolerated in adult MBL-deficient volunteers.
Clinical potential of mannose-binding lectin-replacement therapy
Authors: Summerfield J.A.
Published: June 2004

Mannose-binding lectin (MBL; also known as mannan-binding lectin) is an important component of innate immunity. MBL levels are mainly genetically determined. Low serum MBL levels and their cognate haplotypes have been associated with a wide range of infections. However, most subjects with MBL deficiency remain healthy. MBL deficiency is also associated with non-infectious diseases including systemic lupus erythematosus, rheumatoid arthritis, cystic fibrosis and common variable immunodeficiency. MBL deficiency may affect susceptibility to (e.g. meningococcal disease), or alter the natural history of (e.g. rheumatoid arthritis, cystic fibrosis), a disease. MBL (plasma-derived or recombinant) therapy has yet to be shown to be safe and effective. Potentially it may be useful in MBL-deficient patients to reduce susceptibility to, or enhance recovery from, bacterial infection or to alter the natural history of a disease (disease-modifying drug). In practise the place of MBL therapy may be as a disease-modifying drug to reduce the severity of rheumatoid arthritis and to preserve lung and liver function in cystic fibrosis. MBL therapy may also ameliorate various immunodeficiency syndromes. A potential hazard of MBL therapy is enhanced complement-mediated host damage. The place of MBL therapy will await results of randomized controlled clinical trials.
Visualizing membrane trafficking using total internal reflection fluorescence microscopy
Authors: Beaumont V.
Published: June 2004

There is a dizzying array of fluorescent probes now commercially available to monitor cellular processes, and advances in molecular biology have highlighted the ease with which proteins can now be labelled with fluorophores without loss of functionality. This has led to an explosion in the popularity of fluorescence microscopy techniques. One such specialized technique, total internal reflection fluorescence microscopy (TIR-FM), is ideally suited to gaining insight into events occurring at, or close to, the plasma membrane of live cells with excellent optical resolution. In the last few years, the application of TIR-FM to membrane trafficking events in both non-excitable and excitable cells has been an area of notable expansion and fruition. This review gives a brief overview of that literature, with emphasis on the study of the regulation of exocytosis and endocytosis in excitable cells using TIR-FM. Finally, recent applications of TIR-FM to the study of cellular processes at the molecular level are discussed briefly, providing promise that the future of TIR-FM in cell biology will only get brighter.
Requirement for N-ethylmaleimide-sensitive factor for exocytosis of insulin-containing secretory granules in pancreatic β-cells
Authors: Vikman J. , Ma X. , Tagaya M. , Eliasson L.
Published: June 2004

N-Ethylmaleimide-sensitive factor (NSF) has an important role in fusion processes within intracellular compartments and at the plasma membrane, but the exact role of this protein in the exocytotic machinery has not yet been determined. NSF was found to be present in the cytosol of rat pancreatic β-cells and rat insulinoma INS-1 cells. Capacitance measurements revealed that exocytosis of primed granules was not affected by the presence of a monoclonal antibody against NSF, mAb 2E5, suggesting that NSF is not involved in the fusion process. The antibody markedly decreased rapid refilling of new granules from a reserve pool during a first stimulation. However, slow refilling of primed granules occurred within a 2 min period between the first and second stimulations. We conclude that NSF is required in the exocytotic process in order to obtain a complete exocytotic response. Possible mechanisms by which NSF takes part in this process in insulin-secreting rat β-cells are discussed.
Role of Munc18-1 in synaptic vesicle and large dense-core vesicle secretion
Authors: Toonen R.F.G.
Published: June 2004

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex formation between a vesicle and the target membrane is a central aspect of probably all vesicle fusion reactions. The sec1/munc18 (SM) protein family is also involved in membrane trafficking and fusion events. However, in contrast with the consensus on SNARE protein function, analysis of SM proteins in different systems has produced different ideas about their exact role, their site of action and their relationship to SNARE proteins. Deletion of the SM protein involved in secretory vesicle release in mice, Munc18-1, results in a complete block of exocytosis. Manipulation of Munc18-1 protein levels in neurons and adrenal chromaffin cells argues for a positive role of this protein in vesicle secretion, as overexpression results in an increase in vesicle secretion. A decrease in Munc18-1 protein levels, on the other hand, leads to a decrease in vesicle secretion.
Assembly of N-methyl-d-aspartate (NMDA) receptors
Authors: McIlhinney R.A.J. , Philipps E. , Le Bourdelles B. , Grimwood S. , Wafford K. , Sandhu S. , Whiting P.
Published: June 2004

The N-methyl-d-aspartate receptor (NMDAR) requires both NR1 and NR2 subunits to form a functional ion channel. Despite the recent advances in our understanding of the contributions of these different subunits to both the function and pharmacology of the NMDAR, the precise subunit stoichiometry of the receptor and the regions of the subunits governing subunit interactions remain unclear. Since NR2 subunits are not transported to the cell surface unless they associate with NR1 subunits, cell-surface expression of NR2A can be used to monitor the association of the different subunits in cells transfected with N- and C-terminally truncated NR1 subunits. By combining measurements of cell-surface expression of NR2A with co-immunoprecipitation experiments, and by using Blue Native gel electrophoresis to determine the oligomerization status of the subunits, we have shown that regions of the N-terminus of NR1 are critical for subunit association, whereas the truncation of the C-terminus of NR1 before the last transmembrane region has no effect on the association of the subunits. Evidence from the Blue Native gels, sucrose-gradient centrifugation and size exclusion of soluble NR1 domains suggests that NR1 subunits alone can form stable dimers. Using a cell line, which can be induced to express the NMDAR following exposure to dexamethasone, we have shown that NMDARs can be expressed at the cell surface within 5 h of the recombinant gene induction, and that there appears to be a delay between the first appearance of the subunits and their stable association.
Single-molecule studies of DNA and RNA four-way junctions
Authors: McKinney S.A. , Tan E. , Wilson T.J. , Nahas M.K. , Déclais A.-C. , Clegg R.M. , Lilley D.M.J. , Ha T.
Published: June 2004

Branched helical junctions are common in nucleic acids. In DNA, the four-way junction (Holliday junction) is an essential intermediate in homologous recombination and is a highly dynamic structure, capable of stacking conformer transitions and branch migration. Our single-molecule fluorescence studies provide unique insight into the energy landscape of Holliday junctions by visualizing these processes directly. In the hairpin ribozyme, an RNA four-way junction is an important structural element that enhances active-site formation by several orders of magnitude. Our single-molecule studies suggest a plausible mechanism for how the junction achieves this remarkable feat; the structural dynamics of the four-way junction bring about frequent contacts between the loops that are needed to form the active site. The most definitive evidence for this is the observation of three-state folding in single-hairpin ribozymes, the intermediate state of which is populated due to the intrinsic properties of the junction.
Dietary fatty acids and chylomicron synthesis and secretion
Authors: Williams C.M. , Bateman P.A. , Jackson K.G. , Yaqoob P.
Published: June 2004

There is currently considerable interest in potential atherogenic and thrombogenic consequences of elevated concentrations of triacylglycerols, especially in the post-prandial state. Despite this, there is limited information on the effects of dietary fatty acids on the synthesis, secretion and metabolism of chylomicrons, the large triacylglycerol-rich lipoproteins synthesized in the enterocyte following the digestion and absorption of dietary fat. This brief review considers current approaches to the investigation of chylomicron synthesis and summarizes some of the human, cell and animal studies that have investigated effects of different fatty acids on these pathways. Potential sites for modulatory effects of dietary fatty acids on the molecular events of chylomicron synthesis are proposed in the light of the recent model that has been developed from cell and animal studies and observations based on abnormalities in chylomicron formation in human inherited autosomal recessive diseases.
Regulation of lipoprotein trafficking in the kidney: role of inflammatory mediators and transcription factors
Authors: Ruan X.Z. , Moorhead J.F. , Fernando R. , Wheeler D.C. , Powis S.H. , Varghese Z.
Published: June 2004

Inflammation and dyslipidaemia both play important roles in the development of glomerular atherosclerosis in renal diseases. We have demonstrated that inflammatory mediators induced Scr (scavenger receptor) expression and the formation of foam cells, and that AP-1 (activator protein 1)/ets were necessary transcriptional factors for Scr induction in HMCs (human kidney mesangial cells). Most cells are protected from excessive native LDL (low-density lipoprotein) accumulation by tight feedback regulation of the LDLr (LDL receptor). However, we observed that HMCs formed foam cells via the LDLr pathway when incubated with IL-1β (interleukin-1β; 5 ng/ml) and unmodified LDL (200 μg/ml), suggesting that inflammatory mediators may disrupt the cholesterol-mediated feedback regulation. This feedback involves cholesterol-mediated down-regulation of LDLr controlled by SCAP [SREBP (sterol responsive element-binding protein) cleavage-activating protein]. We have also demonstrated that both tumour necrosis factor α and IL-1β increased nuclear SREBP-1 levels by increasing SCAP mRNA expression, even in the presence of a high concentration of LDL. Since intracellular lipid content is governed by both influx and efflux mechanisms, we set out to examine the impact of inflammatory cytokines on cholesterol efflux, a process mediated by the protein ABCA1 (ATP binding cassette A1). IL-1β inhibited [3H]cholesterol efflux from HMCs by inhibition of the peroxisome-proliferator-activated receptor/LXR (liver X receptor)/ABCA1 pathway. Taken together, our results suggest that inflammatory mediators increase lipid accumulation in HMCs not only by promoting increased lipoprotein uptake by Scr and LDLr, but also by inhibiting ABCA1-mediated cholesterol efflux to high-density lipoprotein.