bluuebunny/crossref_metadata_2025 huggingface dataset:
Homologous recombination in Archaea: new Holliday junction helicases
Authors: Bolt E.L. , Guy C.P.
Published: June 2004

Homologous recombination (HR) maintains genome stability by promoting high fidelity DNA repair. Several recent reports have established that the primary function of HR enzymes is to underpin DNA replication, resetting forks that are blocked or collapsed at sites of DNA damage remote from replication origins. These functions are crucial to ensuring that genomes are transmitted successfully into subsequent generations of cells. Enzymes of HR have been unearthed in all three domains of life: bacteria, Archaea and eukarya. Helicases that specifically unwind branched DNA molecules are pivotal in linking HR and DNA replication in bacteria. However, knowledge of helicases with these functions in eukaryotes is vague and is wholly absent in Archaea. We are using the archaeal species Methanothermobacter thermautotrophicus to identify new DNA helicases of homologous recombination.
Exploring the structure and function of zinc metallopeptidases: old enzymes and new discoveries
Authors: Turner A.J.
Published: June 2004

Neprilysin [or neutral endopeptidase (NEP)] and angiotensin-converting enzyme (ACE) are zinc metallopeptidases involved in the extracellular metabolism of biologically active peptides. Recent genomic advances have led to the identification of novel homologues of each of these ectoenzymes and new physiological and pathological roles are emerging for them. The structures of each of these peptidases have recently been solved providing insight into their distinct catalytic sites. In addition to its originally identified role in neuropeptide metabolism in the nervous system, NEP is implicated in regulation of the cardiovascular system and is protective in prostate and certain other cancers. Hence the cellular concentration of NEP is critical to tissue homoeostasis. Most recently, NEP has been shown to exert neuroprotective actions, principally through its ability to catabolize the neurotoxic Alzheimer's amyloid peptide. The only known homologue of ACE, termed ACE2, is critical to cardiovascular function, but its physiological substrates and precise metabolic roles remain to be elucidated. Other members of these growing metallopeptidase families await further characterization and possible exploitation as therapeutic targets.
How to tell the time: the regulation of phosphoenolpyruvate carboxylase in Crassulacean acid metabolism (CAM) plants
Authors: Nimmo H.G.
Published: June 2004

Crassulacean acid metabolism (CAM) plants exhibit persistent circadian rhythms of CO2 metabolism. These rhythms are driven by changes in the flux through phosphoenolpyruvate carboxylase, which is regulated by reversible phosphorylation in response to a circadian oscillator. This article reviews progress in our understanding of the circadian expression of phosphoenolpyruvate carboxylase kinase.
Clathrin-coated vesicle formation: a paradigm for coated-vesicle formation
Authors: Smythe E.
Published: June 2004

Clathrin-coated pits are the major ports of entry into the cell and are responsible for the internalization of a variety of biologically important macromolecules. These transport intermediates form as a result of the co-ordinated assembly of a number of cytosolic proteins on to the membrane which results in specific cargo recruitment. We have used a variety of approaches including permeabilized cell assays and light and electron microscopy to identify and characterize the proteins and enzymes involved in coated vesicle formation.
Chromatin-modifying enzymes in transcription and cancer
Authors: Kouzarides T.
Published: June 2004

In recent years, our view of how gene expression is controlled has changed dramatically. The discovery of enzymes that modify histones has revealed that transcription is an enzymically driven process. Such modifications can recruit specific proteins and mediate chromatin changes that affect transcription either positively or negatively. Important biological pathways leading to cell proliferation are under the control of these enzymes, and several of them are found deregulated in cancer. The hope is that chromatin-modifying enzymes will be a rich source of targets for drug discovery.
Biochemistry and genetics of mannan-binding lectin (MBL)
Authors: Presanis J.S. , Kojima M. , Sim R.B.
Published: June 2004

Mannose- or mannan-binding lectin (MBL) is a member of the collectin protein family, which includes lung surfactant proteins SP-A and SP-D. Each member consists of similar or identical polypeptide chains with a region of collagen-like sequence followed by a C-type lectin domain. The polypeptides associate in threes to form a subunit containing a collagen-like helix, with three clustered lectin domains. These subunits associate into larger structures, usually with 12–18 polypeptides. The collectins bind to patterns of neutral sugars on surfaces (e.g. of micro-organisms) and mediate effector functions associated with killing/phagocytosis. MBL is the only collectin which activates complement. It resembles in quaternary structure the complement protein C1q, which recognizes targets via charge clusters. Binding of MBL to a surface activates MBL-associated serine proteases (MASPs) attached to MBL, and MASP-2 activates complement proteins C4 and C2. The MASPs are homologous to the C1q-associated proteases, C1r and C1s. MBL therefore activates complement by a mechanism very similar to C1q, and engages the opsonic activity of complement to clear micro-organisms. The serum concentration of MBL is very variable in humans. The variability is largely associated with mutations leading to amino acid substitutions in the collagen-like region which decrease MBL assembly and stability. Many studies demonstrate that MBL deficiency is associated with susceptibility to a range of infectious and inflammatory diseases.
Psp and Smgb: a model for developmental and functional regulation in the rat major salivary glands
Authors: Ball W.D. , Mirels L. , Hand A.R.
Published: June 2004

This paper summarizes past work detailing the developmental expression, cell and organ localization and biochemical features of the proteins parotid secretory protein (PSP) and isoforms of submandibular gland protein B (SMGB), and describes the molecular characterization of the genes that encode them, Psp and Smgb. These genes appear to be related to the BPI (bactericidal/permeability-increasing protein)/LBP (lipopolysaccharide-binding protein)/PLUNC (palate, lung and nasal epithelial clone) gene family found in the oral and respiratory organs of humans, rodents and cattle. We have emphasized the diverse patterns of expression of these genes among the submandibular, sublingual and parotid salivary glands of the rat, and their potential usefulness in defining and identifying genomic regulatory mechanisms of salivary development. While Psp is expressed similarly in the mouse, the putative Smgb gene of the mouse seems not to be expressed, apparently due to the insertion, between exons 1 and 2, of a gene for a retroviral protein.
Bovine parotid secretory protein: structure, expression and relatedness to other BPI (bactericidal/permeability-increasing protein)-like proteins
Authors: Wheeler T.T. , Hood K. , Oden K. , McCracken J. , Morris C.A.
Published: June 2004

Members of the family of BPI (bactericidal/permeability-increasing protein)-like proteins are as yet incompletely characterized, particularly in cattle, where full-length sequence information is available for only three of the 13 family members known from other species. Structural bioinformatic analyses incorporating bovine homologues of several members of the BPI-like protein family, including two forms of bovine parotid secretory protein (PSP), showed that this family is also present in cattle. Expression analyses of several members of the BPI-like protein family in cattle, including PSP (Bsp30), von Ebner's minor salivary gland protein (VEMSGP) and lung-specific X protein (LUNX), showed a restricted pattern of expression, consistent with earlier hypotheses that these proteins function in the innate immune response to bacteria. The possible role of bovine PSP in susceptibility to pasture bloat in cattle is discussed.
Bactericidal/permeability-increasing protein (BPI) and lipopolysaccharide-binding protein (LBP): structure, function and regulation in host defence against Gram-negative bacteria
Authors: Weiss J.
Published: June 2004

Lipopolysaccharide-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI) are closely related endotoxin-binding proteins that function in a co-ordinated manner to facilitate an integrated host response to invading Gram-negative bacteria. Differences in the structure and function of BPI and LBP, as well as differences in their mobilization, permit highly sensitive pro-inflammatory responses to small numbers of bacteria at the onset of bacterial invasion and, later, efficient elimination of viable bacteria and their remnants and of endotoxin-driven inflammation.
Structure of human BPI (bactericidal/permeability-increasing protein) and implications for related proteins
Authors: Beamer L.J.
Published: June 2004

Human bactericidal/permeability-increasing protein (BPI) belongs to a family of mammalian lipopolysaccharide-binding and lipid transport proteins. Recent sequence database searches indicate that several other protein families, including the palate, lung and nasal epithelial clone (PLUNC), parotid secretory protein (PSP) and BPI-like proteins, are likely to share the BPI fold, which was determined through X-ray crystallographic studies. As the single representative of its fold family of known structure, the three-dimensional model of BPI suggests structural features that are likely to be conserved across this large and varied group of proteins.
Four BPI (bactericidal/permeability-increasing protein)-like genes expressed in the mouse nasal, oral, airway and digestive epithelia
Authors: LeClair E.E.
Published: June 2004

A cluster of related genes whose products show structural identity with bactericidal permeability-increasing protein (BPI) has been identified in the genomes of both mice (on chromosome 2) and humans (on chromosome 20). Genes in the cluster include those encoding parotid secretory protein (PSP), von Ebner minor salivary gland protein (VEMSGP) and sequences in the PLUNC (palate, lung and nasal epithelium clone) family, among others. This mini-review addresses the tissue-specific expression of these genes in the mouse.
PLUNC (palate, lung and nasal epithelial clone) proteins in human nasal lavage fluid
Authors: Ghafouri B. , Kihlström E. , Ståhlbom B. , Tagesson C. , Lindahl M.
Published: June 2004

PLUNC (palate, lung and nasal epithelial clone) is a newly discovered gene that is expressed in the upper respiratory tract and is suggested to be of importance in host defence against bacteria. We have identified two forms of the PLUNC protein in human nasal lavage fluid (NLF) using two-dimensional gel electrophoresis (2-DE) and MS. The apparent molecular masses and isoelectric points of these forms are 24.8 kDa/pI 5.4 and 25.1 kDa/pI 5.5. Notably, the 24.8 kDa/pI 5.4 form of PLUNC is an abundant protein in the 2-DE protein patterns of NLF from healthy subjects. Decreased levels of PLUNC were found in NLF from smokers and workers exposed to reactive epoxy chemicals, indicating that long-term exposure to airway irritants impairs the production of PLUNC in the upper respiratory tract. We have also investigated the presence of lipopolysaccharide (LPS)-binding proteins in NLF. Five proteins were found to adsorb to a LPS-coated surface; two of these proteins correspond to the two PLUNC forms, as judged by 2-DE pattern matching. For comparison, human saliva was found to contain a set of LPS-binding proteins with similar 2-DE spot positions (the same pIs but somewhat lower apparent molecular masses of ≈20 kDa). These results indicate that PLUNC may be a new marker of airway inflammation and may play a part in the innate immune response, and that human saliva contains yet other members of the family of LPS-binding proteins.
Expression and anti-bacterial activity of human parotid secretory protein (PSP)
Authors: Geetha C. , Venkatesh S.G. , Fasciotto Dunn B.H. , Gorr S.-U.
Published: June 2004

Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands. A related sequence (C20orf70) was identified on human chromosome 20. The goal of this study was to determine if PSP is expressed in the human parotid gland. The cDNA for human PSP was amplified from a human parotid cDNA sample. A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy. Immunoaffinity chromatography suggested that PSP was expressed in human saliva. PSP is related to bactericidal/permeability-increasing protein (BPI). To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells. The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP. These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.
Insulin secretion by ‘kiss-and-run’ exocytosis in clonal pancreatic islet β-cells
Authors: Tsuboi T. , Rutter G.A.
Published: June 2004

Exocytotic release of neuropeptides and hormones is generally believed to involve the complete merger of the secretory vesicle with the plasma membrane. However, recent data have suggested that ‘kiss-and-run’ mechanisms may also play a role. To analyse secretory events in neuroendocrine β-cells, we imaged chimaeric reporters targeted to either the vesicle membrane [chimaeras of synaptobrevin-2 and pH-sensitive green fluorescent protein (synapto·pHluorin) or of phogrin (phosphatase on the granule of insulinoma) and enhanced green fluorescent protein (EGFP) (phogrin·EGFP)] or the lumen [neuropeptide Y (NPY)·pH-insensitive yellow fluorescent protein (Venus)] by evanescent wave microscopy. Unexpectedly, the frequency of NPY·Venus release events was only 17–27% of that of vesicle fusion reported with synapto·pHluorin, but not phogrin·EGFP, indicating that exocytosis of cargo peptides that is likely to require complete collapse of the vesicle into the plasma membrane is relatively rare. However, both the frequency and the kinetics of NPY·Venus release were modulated by stimulus strength or by overexpression of synaptotagmin IV, demonstrating the plasticity of ‘kiss-and-run’ fusion.
Relationship between fusion pore opening and release during mast cell exocytosis studied with patch amperometry
Authors: Tabares L. , Lindau M. , Alvarez de Toledo G.
Published: June 2004

We have studied exocytosis in rat peritoneal mast cells by cell-attached patch amperometry. Step increases in capacitance were accompanied by typical amperometric spikes due to the release of 5-hydroxytryptamine (serotonin), indicating exocytosis of typical mast cell granules. We have measured the time course of fusion pore expansion, and correlated it with release from the granule matrix. The fusion pore of mast cell granules grows in three stages. The initial expansion of the pore occurred at a rate of 5 nS/s, and in many cases an observable amperometric foot was detected. A second, rapid expansion phase occurred with a rate as high as 1000 nS/s, coinciding with the upstroke of the amperometric spike. A third, slower phase, with a rate of 5 nS/s, completed the final expansion of the fusion pore. These data reveal the very late stages in the exocytotic process, and demonstrate that the size of the fusion pore does not limit release during the upstroke of the amperometric spike or during the final, slow expansion that occurs during for the decay of the amperometric spike.
Red, yellow, green go! – a novel tool for microscopic segregation of secretory vesicle pools according to their age
Authors: Wiegand U.K. , Duncan R.R. , Greaves J. , Chow R.H. , Shipston M.J. , Apps D.K.
Published: June 2004

Large dense-core vesicles (LDCVs) were labelled in cultured bovine adrenal chromaffin cells expressing fluorescent chimaeric ‘cargo’ proteins that were targeted to these secretory vesicles. When the cells were stimulated with nicotine 48 h after transduction, the fractional loss of fluorescent LDCVs was much greater than the fractional catecholamine secretion, implying selective release of newly assembled vesicles. This was confirmed using a fluorescent ‘timer’ construct that changes its fluorescence emission from green to red over several hours, and by measurement of the location and mobility of LDCVs in live cells by confocal fluorescence microscopy. Newly assembled (green) LDCVs were located mostly in peripheral regions of the cells, were virtually immobile and could be released by nicotine, but not by Ba2+; in contrast, older (red) LDCVs were centrally located and relatively mobile, and their exocytotic release was triggered by Ba2+, but not by nicotine. We describe the image restoration procedure that is necessary in order to analyse the behaviour of LDCVs labelled with this construct.
Kinases in clathrin-mediated endocytosis
Authors: Korolchuk V. , Banting G.
Published: June 2004

The process of clathrin-coated vesicle (CCV) formation/disassembly has been studied intensively, and numerous proteins have been identified which aid this process. We have learnt a great deal about individual components of the CCV machinery, and now the ultimate aim is to elucidate the mechanisms regulating clathrin-mediated trafficking. One of the fundamental processes governing the complicated network of interactions is phosphorylation. It has been known for some time that several proteins associated with clathrin-coated vesicles are substrates for protein kinases. These proteins include clathrin, three of the four adaptor complex subunits, dynamin 1, synaptojanin 1 and the amphiphysins. However, the identities of the kinases involved in this process remained largely unknown until recently. This short review discusses advances in our knowledge of how CCV formation/disassembly is regulated by the phosphorylation/dephosphorylation cycle and the role played by specific protein kinases in that process.
Imaging SNAREs at work in ‘unroofed’ cells – approaches that may be of general interest for functional studies on membrane proteins
Authors: Lang T.
Published: June 2004

When cultured cells are subjected to a brief ultrasound pulse, their upper parts burst, but the basal plasma membranes with their embedded membrane–protein complexes remain intact. Such two-dimensional, paraformaldehyde-fixed plasma membrane sheets have been used in the past to visualize the morphology of the inner plasmalemmal leaflet by electron or light microscopy. More recently, fluorescence microscopy of unfixed native membranes has been applied to study SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) function. For instance, biochemical reactions of the plasmalemmal SNAREs with soluble fluorescent SNAREs, patching of SNARE and raft domains, and online monitoring of SNARE-mediated membrane fusion has been performed. The results obtained with the membrane sheet system have added some novel aspects to our understanding of the regulation of neuronal exocytosis. Surprisingly, SNAREs are concentrated in cholesterol-dependent microdomains that are different from membrane rafts. SNAREs in such domains are highly reactive, and define sites for vesicle exocytosis. Secretory granules that fuse on the membrane sheets are retrieved intact in a dynamin-dependent process, suggesting that the ‘kiss-and-run’ mechanism is not a reversed SNARE reaction, but is driven by a biochemically different mechanism. So far, studies of this type have focused on neuronal exocytosis; however, the method might be widely applicable. Data obtained with this system are derived from a 100% pure plasma membrane preparation that is only several seconds old, and membrane proteins are studied in their natural microenvironment that is defined by local lipid composition and putative bound proteins. Hence this approach yields results that most probably reflect the situation in a live cell.
Assembly and subunit diversity of nicotinic acetylcholine receptors
Authors: Millar N.S.
Published: June 2004

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of neurotransmitter-gated ion channels which contain five transmembrane subunits arranged around a central pore. Distinct receptor subtypes are expressed at the vertebrate skeletal neuromuscular junction, in mechanosensory cells and within the central and peripheral nervous systems. A total of 17 nAChR subunits (α1–α10, β1–β4, γ, δ and ∊) have been identified in vertebrate species, which can co-assemble to generate a wide variety of nAChRs. Nicotinic receptors also constitute an abundant and diverse family of receptors in invertebrates. As a consequence of studies which have been conducted with both native and recombinant nAChRs, the subunit composition of nAChRs and the rules governing subunit co-assembly are becoming clearer. In this paper the extent of nAChR subunit diversity and evidence for receptor subunit composition is reviewed.
Multiple assembly signals in γ-aminobutyric acid (type A) receptor subunits combine to drive receptor construction and composition
Authors: Bollan K. , Robertson L.A. , Tang H. , Connolly C.N.
Published: June 2004

Mammalian γ-aminobutyric acid type A (GABAA) receptors are constructed from a large repertoire of subunits (α1–α6, β1–β3, γ1–γ3, δ, ∊, θ and π) into a pentameric ion channel. GABAA receptor assembly occurs within the endoplasmic reticulum (ER) and involves interactions with chaperone molecules. Only specific subunit combinations can produce functional surface receptors (with a fixed stoichiometry); other subunit combinations are retained within the ER and degraded. Thus, receptor assembly occurs by defined pathways to limit the diversity of GABAA receptors. The key to understanding how receptor diversity is achieved and controlled is the identification of assembly signals capable of distinguishing between other subunit partners. Analysis of an assembly box in α1 (residues 57–68) has revealed an absolute requirement for this region in the assembly of αβ receptors. Furthermore, a selective requirement for a single amino acid (R66) is observed for the assembly of α1β2, but not α1β1 or α1β3, receptors. In addition, we have characterized an assembly signal in the β3 subunit that is capable of driving the assembly of β3, γ2β3 and α1β3 receptors. Interestingly, this signal does not appear to utilize the α1 assembly box, suggesting the presence of alternative assembly signals within the α1 subunit. Although this β3 signal is sufficient to permit the formation of βγ receptors it is not necessary, suggesting that alternative assembly signals also exist within the β3 subunit. These findings support the belief that GABAA receptor assembly occurs via multiple defined pathways that may be determined by subunit availability.
Real-time imaging of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor) movements in neurons
Authors: Perestenko P. , Ashby M.C. , Henley J.M.
Published: June 2004

The mechanisms that regulate α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) synthesis, transport, targeting and surface expression are of fundamental importance for fast excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system. It has become apparent that these control processes involve complex sets of protein–protein interactions and many of the proteins responsible have been identified. We have been working to visualize AMPAR movement in living neurons in order to investigate the effects of blocking protein interactions. Here we outline the approaches used and the results obtained thus far.
Pre- and post-synaptic mechanisms regulating the clustering of type A γ-aminobutyric acid receptors (GABAA receptors)
Authors: Fritschy J.-M. , Schweizer C. , Brünig I. , Lüscher B.
Published: June 2004

Postsynaptic clustering of GABAA (type A γ-aminobutyric acid) receptors is essential to ensure proper function of GABAergic synapses. This process is initiated during synapse formation and is maintained throughout life. The tubulin-associated protein gephyrin is required for clustering of GABAA receptors, but its specific role in this process is not understood. A second protein associated selectively with GABAA receptors at postsynaptic sites is dystrophin. It is present in a subset of GABAergic synapses along with several partners, forming the dystrophin-associated protein complex. In this review, we discuss recent advances in the role of neuronal activity and trans-synaptic signaling for the clustering of gephyrin and dystrophin during synaptogenesis and on the role of these proteins for plasticity and maintenance of mature synapses.
Linking endocytic cargo to clathrin: structural and functional insights into coated vesicle formation
Authors: Owen D.J.
Published: June 2004

Clathrin-mediated endocytosis is the major process by which transmembrane proteins are internalized from the cell's limiting membrane into the first compartment of the endosomal system, the early endosome. From here, these transmembrane cargo proteins, which are of widely varying type and function, are trafficked to their required destination. Endocytosis plays, therefore, an important role in cell signalling, nutrient uptake, cellular homoeostasis and the interaction of the cell with its external environment. The formation of clathrin-coated endocytic vesicles requires the complex interplay of many proteins with each other and with the membrane itself. Their formation has served as a paradigm for formation of all types of transport vesicle, which move cargo between the various membrane-bound compartments of the cell. Clathrin-coated vesicles (CCVs) possess three layers: the inner membrane layer, in which the transmembrane cargo is embedded, linked to the outer clathrin lattice by a layer of cargo-binding adaptors and proteins that aid and regulate vesicle formation. Protein X-ray crystallography in combination with biochemical, biophysical and cell biological assays has been used to investigate the structure and function of some of the proteins that make up the middle layer of CCVs. These proteins are diverse in their functions, but are all modular in nature, consisting of folded domains joined by long unstructured linkers. Within these linkers are short motifs that interact with the folded domains of other components of the CCV formation machinery. Many of these folded domains also bind directly to the membrane. These interactions, whose molecular basis we have studied, have affinities in the low micromolar range, making them readily reversible and easily regulated. The mechanism of CCV formation is discussed in the light of this structural and biochemical data.
Proteases of the complement system
Authors: Sim R.B. , Tsiftsoglou S.A.
Published: June 2004

The complement system is a group of about 35 soluble and cell-surface proteins which interact to recognize, opsonize and clear or kill invading micro-organisms or altered host cells (e.g. apoptotic or necrotic cells). Complement is a major part of the innate immune system. Recognition proteins such as C1q, MBL (mannan-binding lectin) and ficolins bind to targets via charge or sugar arrays. Binding causes activation of a series of serine protease proenzymes, such as C1r, C1s and MASP2 (MBL-associated serine protease 2), which in turn activate the atypical serine proteases factor B and C2, which then activate the major opsonin of the system, C3. Activated C3 binds covalently to targets, and is recognized by receptors on phagocytic cells. Two of the complement proteases, factors D and I, circulate not as proenzymes, but in activated form, and they have no natural inhibitors; their substrates are transient protein complexes (e.g. C3bB and C3bH) which form during complement activation. Factor B and C2 also have no natural inhibitor; they are active only when proteolytically cleaved and bound in an unstable, short-lived complex with C3b or C4b. C1r, C1s and the MASPs, in contrast, are regulated more conventionally by the natural serpin, C1-inhibitor. Complement proteases in general have very narrow specificity, and low substrate turnover with both natural and synthetic substrates. Excessive activation of complement is inflammatory, and causes tissue damage (e.g. in rheumatoid arthritis, or in ischaemia/reperfusion injury). Substances that regulate complement activation are likely to be useful in the regulation of inflammation. Complement activation might potentially be controlled at many different steps. Much attention has been focused on controlling the formation or activity of the protease complexes C3bBb and C4b2a (containing activated factor B and C2 respectively), as these generate the inflammatory peptides C3a and C5a.
Improvement of the anti-C3 activity of compstatin using rational and combinatorial approaches
Authors: Morikis D. , Soulika A.M. , Mallik B. , Klepeis J.L. , Floudas C.A. , Lambris J.D.
Published: June 2004

Compstatin is a 13-residue cyclic peptide that has the ability to inhibit the cleavage of C3 to C3a and C3b. The effects of targeting C3 cleavage are threefold, and result in hindrance of: (i) the generation of the pro-inflammatory peptide C3a, (ii) the generation of opsonin C3b (or its fragment C3d), and (iii) further complement activation of the common pathway (beyond C3) with the end result of the generation of the membrane attack complex. We will report on our progress on: (i) rational design of more active compstatin analogues based on the three-dimensional structure of compstatin, (ii) experimental combinatorial design based on the generation of a phage-displayed peptide library partially randomized with the implementation of structure-induced restraints, and (iii) theoretical combinatorial design based on a novel computational optimization method, structure-induced restraints and flexible structural templates. All three approaches have resulted in analogues with improved activities. Currently, the lead analogue has the sequence acetyl-I[CVYQDWGAHRC]T-NH2 (where the brackets denote cyclization), and is 16-fold more active than the parent peptide. We will also report on our progress towards understanding the dynamic character of compstatin using molecular dynamics simulations. The identification of an ensemble of interconverting conformers of compstatin with variable populations is a first step towards the incorporation of dynamic elements in the design of new analogues using dynamics–activity relationships in addition to structure–activity relationships.
Proteolytic cascade in the amyloidogenesis of Alzheimer's disease
Authors: Sidera C. , Parsons R. , Austen B.
Published: June 2004

β-Amyloid, a neurotoxic peptide deposited in the brains of Alzheimer's disease patients, is released by a series of membrane-limited proteolytic events. β-Secretase activity is enhanced by cellular targeting into intracellular cholesterol-rich microdomains, which are dispersed by statins.
In vitro and in vivo effects of oxidative damage to deoxyguanosine
Authors: Greenberg M.M.
Published: June 2004

Biochemical, biophysical and biological studies of oligonucleotides containing lesions at defined sites provide a molecular basis for the effects of DNA lesions. dG (deoxyguanosine) is the most easily oxidized of the four native nucleotides. The chemical reactivity of dG correlates with compilations of mutations, which reveal that a significant fraction of transitions or transversions involve dG. OxodG (7,8-dihydro-8-hydroxy-2´-deoxyguanosine) is widely recognized as an important lesion derived from the oxidation of dG, and significant effort has been expended in studies of its effects on DNA structure and function. Recently, the properties of other lesions derived from dG and/or the oxidation of OxodG have been uncovered. Studies on these lesions reveal that they too are biologically significant.
Synthesis and function of hepatic very-low-density lipoprotein
Authors: Gibbons G.F. , Wiggins D. , Brown A.-M. , Hebbachi A.-M.
Published: June 2004

Most of the triacylglycerol (TAG) utilized for the assembly of very-low-density lipoprotein (VLDL) in the secretory apparatus of the hepatocyte is mobilized by lipolysis of the cytosolic TAG pool, followed by re-esterification. The lipases involved include arylacetamide deacetylase and/or triacylglycerol hydrolase. Some of the re-esterified products of lipolysis gain access to an apolipoprotein-B-rich VLDL precursor to form mature VLDL. Some, however, are returned to the cytosolic pool in a process that is stimulated by insulin and inhibited by microsomal triacylglycerol transfer protein (MTP). Phospholipids also contribute to VLDL TAG in a process which involves ADP-ribosylation factor-1 (ARF-1)-mediated activation of phospholipase D. The temporary storage of TAG in the liver, followed by its mobilization and secretion as VLDL, form part of a process by which the liver protects vulnerable body tissues from excess lipotoxic non-esterified (‘free’) fatty acids in the plasma.
Enterocyte fatty acid uptake and intestinal fatty acid-binding protein
Authors: Tso P. , Nauli A. , Lo C.-M.
Published: June 2004

This article reviews our current understanding of the uptake of fatty acids by the enterocytes of the intestine. The micellar solubilization of fatty acids by bile salts and the factors regulating that process are discussed. The mechanism of how micellar solubilization of fatty acids promotes the uptake of fatty acids by enterocytes and their relative importance is reviewed. Additionally, discussion of the various fatty acid transporters located at the brush border membrane of the enterocytes is included. Finally, a summary of our current understanding of the function of fatty-acid-binding proteins inside enterocytes is provided.
Increased FAT (fatty acid translocase)/CD36-mediated long-chain fatty acid uptake in cardiac myocytes from obese Zucker rats
Authors: Coort S.L.M. , Luiken J.J.F.P. , van der Vusse G.J. , Bonen A. , Glatz J.F.C.
Published: June 2004

Disturbed cardiac lipid homoeostasis in obesity is regarded as a key player in the development of cardiovascular diseases. In this study, we show that FAT (fatty acid translocase)/CD36-mediated LCFA (long-chain fatty acid) uptake in cardiac myocytes from young adult obese Zucker rats is markedly increased, but insensitive to insulin. Basal and insulin-induced glucose uptake rates in these myocytes are not changed, suggesting that during the development from obesity to hyperglycaemic Type II diabetes, alterations in cardiac LCFA uptake precede alterations in cardiac glucose uptake.
The surprising diversity of Δ6-desaturase substrates
Authors: Guillou H. , D'Andrea S. , Rioux V. , Jan S. , Legrand P.
Published: June 2004

A single gene encoding a δ6-desaturase (FADS2) has been isolated and characterized in mammalian species. This δ6-desaturase plays a major role in the biosynthesis of PUFAs (polyunsaturated fatty acids). It catalyses the rate-limiting desaturation of linoleic acid (C18:2n−6) and α-linolenic acid (C18:3n−3) required for the biosynthesis of long-chain PUFAs. Moreover, recent studies have provided strong evidence that this δ6-desaturase also acts on 24-carbon PUFAs of both the n−6 and n−3 series. Another substrate of this δ6-desaturase has been identified through complementary works from different investigators. This δ6-desaturase acts on a saturated fatty acid, palmitic acid (C16:0), leading to the newly characterized biosynthesis of hexadecenoic acid (C16:1n−10) or sapienate.
Transcriptional regulation of human SREBP-1c (sterol-regulatory-element-binding protein-1c): a key regulator of lipogenesis
Authors: Tarling E. , Salter A. , Bennett A.
Published: June 2004

Sterol-regulatory-element-binding protein 1c (SREBP-1c) is one member of the family of transcription factors that stimulate sterol and fatty-acid biosynthesis in animal cells. Human SREBP-1c, mapped to chromosome 17p11.2, is expressed in liver, intestine, skeletal muscle and adipocytes. A section of genomic sequence from a chromosome 17 library, thought to contain the SREBP-1c promoter, was cloned. Putative transcription-factor-binding sites and a potential transcriptional start site were identified using the Genomatix Suite of sequence analysis tools (MatInspector®). Sequence analysis showed the human promoter to be 42% identical with the previously published mouse sequence. Two novel transcription-factor-binding sites were identified: those for PDX-1 (pancreatic–duodenal homoeobox-1) and HNF-4 (hepatic nuclear factor-4). Co-transfection experiments with overexpression plasmids for PDX-1 and HNF-4 suggested that both factors stimulate SREBP-1c gene expression, although further work is required to ascertain their mechanisms of action.
Scavenger receptor class B type I in high-density lipoprotein metabolism, atherosclerosis and heart disease: lessons from gene-targeted mice
Authors: Trigatti B. , Covey S. , Rizvi A.
Published: June 2004

The scavenger receptor class B type I (SR-BI) is a multi-ligand receptor that can mediate the binding and bi-directional lipid transfer between high-density lipoproteins (HDLs) and cells. It is expressed in a variety of tissues, including the liver, and in macrophages in atherosclerotic plaques. The physiological role of SR-BI has been tested in vivo by the genetic manipulation of SR-BI levels in mice. Mice lacking SR-BI exhibit impaired hepatic-selective HDL cholesterol uptake and increased atherosclerosis, suggesting that SR-BI is required for hepatic reverse cholesterol transport and normally protects against atherosclerosis. Surprisingly, elimination of SR-BI in apolipoprotein E knockout mice results in rapid development of occlusive coronary artery disease, accompanied by spontaneous myocardial infarction, reduced heart function and early death, which points to a role for SR-BI in protection against coronary heart disease. The in vivo role of macrophage SR-BI has been less clear. We have used bone-marrow transplantation to demonstrate that bone-marrow-derived SR-BI also normally protects against atherosclerosis in low-density lipoprotein receptor knockout mice. These results suggest that SR-BI may have multiple protective effects against atherosclerosis in different tissues.
ATP-binding cassette transporter A1: regulation of cholesterol efflux
Authors: Knight B.L.
Published: June 2004

The ATP-binding cassette transporter A1 (ABCA1) is involved in the regulation of cholesterol efflux from cells. Mutations in ABCA1 give rise to familial high-density lipoprotein (HDL) deficiency and Tangier disease, which is characterized by very low levels of HDL in plasma and cholesteryl ester accumulation in tonsils and other reticuloendothelial cells. The mechanism of action of ABCA1 is still unclear, but requires the transfer of phospholipid and cholesterol to apolipoprotein A1 bound by or close to the transporter. An important factor in the regulation of ABCA1 is cholesterol itself, which provides oxysterol ligands for liver X receptors that stimulate ABCA1 transcription. ABCA1-deficient mice show increased cholesterol absorption, suggesting that ABCA1 could also help to transport dietary cholesterol back out of intestinal absorptive cells into the lumen. Thus ABCA1 is intimately connected to various aspects of the regulation of whole-body cholesterol metabolism and probably plays an important role in protecting against the development of cardiovascular disease.
Circulating levels of the chemokines JE and KC in female C3H apolipoprotein-E-deficient and C57BL apolipoprotein-E-deficient mice as potential markers of atherosclerosis development
Authors: Parkin S.L. , Pritchett J.P. , Grimsditch D.C. , Bruckdorfer K.R. , Sahota P.K. , Lloyd A. , Overend P. , Benson G.M.
Published: June 2004

We have investigated serum chemokines for their suitability as markers of atherosclerosis development in apoE (apolipoprotein E)-deficient (−/−) mice. Female C3H apoE−/− and C57BL apoE−/− mice were fed on either diet W (Western diet; 6 weeks) or normal rodent diet (12 weeks). Serum lipids (0, 6 and 12 weeks) and terminal chemokine levels were measured using commercially available assays, whereas the lesion area was determined using Oil-Red O-stained aortic sections. Serum lipids were higher in C3H apoE−/− mice for both diets throughout the study; however, lesions were significantly larger in C57BL apoE−/− mice fed on either diet. Chemokine levels were significantly lower in C3H apoE−/− mice fed on the normal diet, but no difference was observed between the two groups fed on diet W. We conclude that serum chemokine levels are potential markers for atherosclerosis susceptibility in C3H and C57BL apoE−/− mice fed on a normal rodent diet.
Faecal sterol output is increased by arachidyl amido cholanoic acid (Aramchol) in rats
Authors: Leikin-Frenkel A. , Weinbroum A.A , Leikin-Gobbi D. , Krupitzky L. , Goldiner I. , Shafat L. , Gilat T. , Konikoff F.M.
Published: June 2004

Fatty acid–bile acid conjugates (FABACs) were shown recently to have important and multiple effects on cholesterol metabolism. In human fibroblasts, they were found to markedly enhance cholesterol efflux by an ATP-binding cassette transporter A1-dependent pathway. In C57L/J mice, they increased CYP7A1 activity and RNA expression, while decreasing moderately 3-hydroxy-3-methylglutaryl-CoA reductase activity. In C57L/J mice and in hamsters, they also decreased serum cholesterol levels, whereas in other animals, this effect was not seen in short-term experiments. In the present study, we investigated potential mechanisms of action of arachidyl amido cholanoic acid (Aramchol), with particular reference to biliary and faecal sterol outputs in rats. Supplementation with Aramchol at a dose of 150 mg·kg−1·day−1 increased neutral sterol output by approx. 50%, while the faecal outputs of bile salts and total sterols increased by almost 2-fold. Biliary lipid outputs were not significantly different between the control and FABAC-supplemented animals. These findings indicate an overall catabolic effect of FABACs on body cholesterol.
Specific markers of lipid peroxidation issued from n−3 and n−6 fatty acids
Authors: Guichardant M. , Chantegrel B. , Deshayes C. , Doutheau A. , Moliere P. , Lagarde M.
Published: June 2004

Several markers of lipid peroxidation are available with different degrees of specificity, from malondialdehyde as a global marker, to F2-isoprostane, which is specifically produced from arachidonic acid. Among these, 4-hydroxynonenal is recognized as a breakdown product of fatty acid hydroperoxides, such as 15-hydroperoxy-eicosatetraenoic acid and 13-hydroperoxy-octade cadienoic acid from the n−6 fatty acids. Furthermore, 4-hydroxyhexenal (4-HHE) derives from n−3 fatty acid hydroperoxides. We have recently described the occurrence of 4-hydroxydodecadienal (4-HDDE) from the 12-lipoxygenase product of arachidonic acid 12-hydroperoxy-eicosatetraenoic acid. These three hydroxy-alkenals may be measured in human plasma by GC–MS, but they may partly be generated in the course of sampling, and the relative volatility of 4-HHE makes its measurement quite unreliable. We have successfully characterized and measured the stable oxidized carboxylic acid products from the hydroxy-alkenals 4-HNA, 4-HHA and 4-HDDA in urine. The ratio between 4-HHA and 4-HNA found in the same urinary sample might provide useful information on the location of lipid peroxidation, accounting for the high enrichment of the cerebrovascular system with docosahexaenoic acid, the main n−3 fatty acid in humans.
A novel antioxidant action of ethanolamine plasmalogens in lowering the oxidizability of membranes
Authors: Maeba R. , Ueta N.
Published: June 2004

We have demonstrated a novel antioxidant action of ethanolamine plasmalogens both in protecting cholesterol from oxidation by free radicals and in lowering the oxidizability of membranes, along with the action of scavenging radicals, by the oxygen-uptake method using large unilamellar vesicles and the water-soluble azo-radical initiator, AAPH [2,2´-azobis-(2-amidino-propane) dihydrochloride].
Plasmalogens: targets for oxidants and major lipophilic antioxidants
Authors: Engelmann B.
Published: June 2004

Cellular membranes and plasma lipoproteins are less efficiently protected against oxidative stress than the various aqueous compartments of mammalian organisms. Here, previous results on the role of plasmalogens in lipid oxidation are evaluated on the basis of criteria required for an antioxidant. The plasmalogen-specific enol ether double bond is targeted by a vast variety of oxidants, including peroxyl radicals, metal ions, singlet oxygen and halogenating species. Oxidation of the vinyl ether markedly prevents the oxidation of highly polyunsaturated fatty acids, and products of plasmalogen degradation do not propagate lipid oxidation. This protection is also demonstrated intramolecularly, thus ascertaining the function of plasmalogens as a major storage pool for polyunsaturated fatty acids. Although cells rapidly incorporate and synthesize plasmalogens de novo, their plasmalogen contents can be deliberately increased by supplementation with precursors. Thus plasmalogens terminate lipid-oxidation processes, are present in adequate locations at sufficient concentrations, and are rapidly regenerated, classifying them as efficient antioxidants in vitro. Future work should address the in vivo role of plasmalogens in lipid oxidation and the biological function of plasmalogen interactions with oxidants.
Antioxidants and atherosclerosis
Authors: Niki E.
Published: June 2004

The oxidative modification of low-density lipoprotein (LDL) can be induced by various active species by different mechanisms. Vitamin E and other radical-scavenging antioxidants can inhibit the free radical-mediated oxidation of LDL, but they are not effective against LDL oxidation induced by non-radical mechanisms.
What does the lipoprotein oxidation phenomenon mean?
Authors: Ferns G.A.A. , Lamb D.J.
Published: June 2004

The evidence that oxidative lipid modification may be involved in the genesis of common diseases, such as atherosclerosis, is persuasive, but it was, until recently, conjecture based on in vitro findings, or investigation using experimental animal models. Recent clinical intervention studies in patients at high risk of cardiovascular events have been, at best, inconclusive. This has led to a general consensus that antioxidant supplements are of no value in the prevention of cardiovascular disease in subjects at high risk. However, epidemiological studies have demonstrated that the protective effects of antioxidant supplements, specifically vitamin E, were particularly evident amongst healthy subjects taking supplements. The picture is further clouded by the uncertain mechanism of lipoprotein modification within the artery wall, the possibility that some antioxidants may, under certain conditions, become pro-oxidants, the complex interactions between lipid- and water-soluble antioxidants, and the fact that free-radical-mediated events may only be important in the early stages of atherogenesis. Recent results also suggest that the biological efficacy of antioxidants, such as α-tocopherol, may be compromised by the conditions extant within the plaque. It is evidently important that the position on the benefits of antioxidants, whether in food or as supplements, in disease prevention is clarified.