Fibulin-2 is involved in early extracellular matrix development of the outgrowing mouse mammary epithelium

Cell–matrix interactions control outgrowth of mammary epithelium during puberty and pregnancy. We demonstrate here that the glycoprotein fibulin-2 (FBLN2) is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth. FBLN2 was specifically localized to the cap cells of the terminal end buds during puberty and to myoepithelial cells during very early pregnancy (days 2–3) even before morphological changes to the epithelium become microscopically visible, but was down-regulated thereafter. Exposure to exogenous oestrogen (E2) or E2 plus progesterone (P) increased Fbln2 mRNA expression in the pubertal gland, indicating hormonal control. FBLN2 was co-expressed and co-localised with the proteoglycan versican (VCAN) and co-localised with laminin (LN), while over-expression of FBLN2 in HC-11 cells increased cell adhesion to several extracellular matrix proteins including LN and fibronectin, but not collagens. Mammary glands from Fbln2 knockout mice showed no obvious phenotype but increased fibulin-1 (FBLN1) staining was detected, suggesting a compensatory mechanism by other fibulin family members. We hypothesise that similar to embryonic aortic smooth muscle development, FBLN2 and VCAN expression alters the cell–matrix interaction to allow mammary ductal outgrowth and development during puberty and to enable epithelial budding during pregnancy.     

Central nervous system stem cells in the embryo and adult

The central nervous system is generated from neural stem cells during embryonic development. These cells are multipotent and generate neurons, astrocytes and oligodendrocytes. The last few years it has been found that there are populations of stem cells also in the adult mammalian brain and spinal cord. In this paper, we review the recent development in the field of embryonic and adult neural stem cells. Received 26 March 1998; received after revision 27 April 1998; accepted 27 April 1998     

Lecticans: organizers of the brain extracellular matrix

Lecticans are a family of chondroitin sulfate proteoglycans, encompassing aggrecan, versican, neurocan and brevican. These proteoglycans are characterized by the presence of ahyaluronan-binding domain and a C-type lectin domain in their core proteins. Through these domains, lecticans interact with carbohydrate and protein ligands in the extracellular matrix and act as linkers of these extracellular matrix molecules. In adult brain, lecticans are thought to interact with hyaluronan and tenascin-R to form a ternary complex. We propose that the hyaluronan-lectican-tenascin-R complex constitutes the core assembly of the adult brain extracellular matrix, which is found mainly in pericellular spaces of neurons as ‘perineuronal nets’. Received 27 September 1999; accepted 26 October 1999     

Unveiling molecular mechanisms of bacterial surface proteins: <Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> as a model organism for structural studies

Bacteria present a variety of molecules either on their surface or in a cell-free form. These molecules take part in numerous processes in the interactions with their host, with its tissues and other molecules. These molecules are essential to bacterial pathogenesis either during colonization or the spread/invasion stages, and most are virulence factors. This review is focused on such molecules using Streptococcus pneumoniae, a Gram-positive bacterium, as an example. Selected surface proteins are introduced, their structure described, and, whenever available, their mechanisms of function on an atomic level are explained. Such mechanisms for hyaluronate lyase, pneumococcal surface protein A, pneumolysin, histidine-triad and fibronectin-binding proteins are discussed. Elucidation of molecular mechanisms of virulence factors is essential for the understanding of bacteria and their functional properties. Structural biology appears pivotal for these studies, as structural and mechanistic insights facilitate rational approach to the development of new treatments. Received 12 March 2007; received after revision 28 June 2007; accepted 18 July 2007     

Affinity capillary electrophoresis in biomolecular recognition

Affinity capillary electrophoresis is a new method for studies of biomolecular recognition. Applications reported in the literature include chiral separation of racemic biomolecules, measurement of binding constants, estimation of kinetic on- and off-rate constants, determination of binding stoichiometries (a useful tool in examining electrostatic interactions), estimation of effective charges and molecular weights of proteins, characterization of enzymatic activities and library screening for tight-binding drug candidates in solution. This technique demands only small amounts of sample (nanolitre injection volumes, picograms of proteins), involves no radiolabelled materials or chemically immobilized ligands, and does not require changes in spectroscopic characteristics upon binding. This paper reviews the most recent applications of affinity capillary electrophoresis and its use in the analysis of biomolecules. Received 9 January 1998; received after revision 27 February 1998; accepted 3 March 1998     

Differential leaf resistance to insects of transgenic sweetgum (Liquidambar styraciflua) expressing tobacco anionic peroxidase

Leaves of transgenic sweetgum (Liquidambar styraciflua) trees that expressed tobacco anionic peroxidase were compared with leaves of L. styraciflua trees that did not express the tobacco enzyme. Leaves of the transgenic trees were generally more resistant to feeding by caterpillars and beetles than wild-type leaves. However, as for past studies with transgenic tobacco and tomato expressing the tobacco anionic peroxidase, the degree of relative resistance depended on the size of insect used and the maturity of the leaf. Decreased growth of gypsy moth larvae appeared mainly due to decreased consumption, and not changes in the nutritional quality of the foliage. Transgenic leaves were more susceptible to feeding by the corn earworm, Helicoverpa zea. Thus, it appears the tobacco anionic peroxidase can contribute to insect resistance, but its effects are more predictable when it is expressed in plant species more closely related to the original gene source. Received 4 March 1998; received after revision 27 April 1998; accepted 30 April 1998     

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin light-chain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed.     

Structure, function and evolution of antifreeze proteins

Antifreeze proteins bind to ice crystals and modify their growth. These proteins show great diversity in structure, and they have been found in a variety of organisms. The ice-binding mechanisms of antifreeze proteins are not completely understood. Recent findings on the evolution of antifreeze proteins and on their structures and mechanisms of action have provided new understanding of these proteins in different contexts. The purpose of this review is to present the developments in contrasting research areas and unite them in order to gain further insight into the structure and function of the antifreeze proteins. Received 2 September 1998; received after revision 21 October 1998; accepted 2 November 1998     

Molecular cloning and analysis of the protein modules of aggrecans

The large aggregating chondroitin sulfate proteoglycan of cartilage, aggrecan, has served as a prototype of proteoglycan structure. Molecular cloning has elucidated its primary structure and revealed both known and unknown domains. To date the complete structures of chicken, rat and human aggrecans have been deduced, while partial sequences have been reported for bovine aggrecan. A related proteoglycan, human versican, has also been cloned and sequenced. Both aggrecan and versican have two lectin domains, one at the amino-terminus which binds hyaluronic acid and one at the carboxyl-terminus whose physiological ligand is unknown. Both lectins have homologous counterparts in other types of proteins. Within the aggrecans the keratan sulfate domain may be variably present and also has a prominent repeat in some species. The chondroitin sulfate domain has three distinct regions which vary in their prominence in different species. The complex molecular structure of aggrecans is consistent with the concept of exon shuffling and aggrecans serve as suitable prototypes for comprehending the evolution of multi-domain proteins.     

Tenascins,a growing family of extracellular matrix proteins

The tenascins are a family of large multimeric extracellular matrix proteins consisting of repeated structural modules including heptad repeats, epidermal growth factor (EGF)-like repeats, fibronectin type III repeats, and a globular domain shared with the fibrinogens. The tenascins are believed to be involved in the morphogenesis of many organs and tissues. To date three members of the tenascin family have been described, tenascin-C, tenascin-R, and tenascin-X. Tenascin-R seems to be specific for the central and peripheral nervous system, tenascin-X is most prominent in skeletal and heart muscle, while tenascin-C is present in a large number of developing tissues including the nervous system, but is absent in skeletal and heart muscles. Tenascin-C was the original tenascin discovered, partly because of its overexpression in tumors. Inferring from cell biological studies, it has been proposed that tenascin-C is an adhesion-modulating protein.     

Long-term changes in tyrosine phosphorylation of the abundant nuclear proteins during granulocytic differentiation of HL-60 cells

The two-dimensional electrophoretic patterns of nuclear proteins and their tyrosine phosphorylation were compared for HL-60 cells before and after differentiation induction to granulocytes by dimethyl sulfoxide, all-trans retinoic acid and N ⁶,O ²-dibutyryl adenosine 3′5′-cyclic monophosphate. Regardless of the inducer used, some nuclear proteins, which are tyrosine-phosphorylated in proliferating HL-60 cells, undergo gradual dephosphorylation 12–72 h after induction of differentiation, followed by drastic dephosphorylation during maturation to granulocytes. At least 13 nuclear proteins with a molecular mass of 35–110 kDa are dephosphorylated, and 6 nuclear proteins undergo tyrosine phosphorylation. Analysis of the nuclear proteins differentially extracted by salt and detergents indicates that changes in their tyrosine phosphorylation during the maturation stage of differentiating granulocytes occur mainly in proteins which are abundant in nucleoplasm, chromatin and residual nuclear structures. The abundance of these proteins, residing in the nuclear structures, and their long-term modification in phosphorylation during the maturation stages of differentiation strongly suggest that tyrosine phosphorylation of these proteins is involved in reorganization of the differentiating cell nucleus. Received 21 September 1998; received after revision 24 November 1998; accepted 3 December 1998     

Biological activity and pathological implications of misfolded proteins

The physiological metabolism of proteins guarantees that different cellular compartments contain the appropriate concentration of proteins to perform their biological functions and, after a variable period of wear and tear, mediates their natural catabolism. The equilibrium between protein synthesis and catabolism ensures an effective turnover, but hereditary or acquired abnormalities of protein structure can provoke a premature loss of biological function, an accelerated catabolism and diseases caused by the loss of an irreplaceable function. In certain proteins, abnormal structure and metabolism are associated with a strong tendency to self-aggregation into a polymeric fibrillar structure, and in these cases the disease is not principally caused by the loss of an irreplaceable function but by the action of this new biological entity. Amyloid fibrils are an apparently inert, insoluble, mainly extracellular protein polymer that kills the cell without tissue necrosis but by activation of the apoptotic mechanism. We analyzed the data reported so far on the structural and functional properties of four prototypic proteins with well-known biological functions (lysozyme, transthyretin, β2-microglobulin and apolipoprotein AI) that are able to create amyloid fibrils under certain conditions, with the perspective of evaluating whether the achievement of biological function favors or inhibits the process of fibril formation. Furthermore, studying the biological functions carried out by amyloid fibrils reveals new types of protein-protein interactions in the transmission of messages to cells and may provide new ideas for effective therapeutic strategies. Received 9 November 1998; received after revision 15 January 1999; accepted 15 January 1999     

SNAREs and SNARE regulators in membrane fusion and exocytosis

Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or ‘protein export’) pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. ‘SNARE-masters’) in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented. Received 8 October 1998; received after revision 26 November 1998; accepted 26 November 1998     

Roles of the DNA mismatch repair and nucleotide excision repair proteins during meiosis

Numerous proteins are involved in the nucleotide excision repair (NER) and DNA mismatch repair (MMR) pathways. The function and specificity of these proteins during the mitotic cell cycle has been actively investigated, in large part due to the involvement of these systems in human diseases. In contrast, comparatively little is known about their functioning during meiosis. At least three repair pathways operate during meiosis in the yeast Saccharomyces cerevisiae to repair mismatches that occur as a consequence of heteroduplex formation in recombination. The first pathway is similar to the one acting during postreplicative mismatch repair in mitotically dividing cells, while two pathways are responsible for the repair of large loops during meiosis, using proteins from MMR and NER systems. Some MMR proteins also help prevent recombination between diverged sequences during meiosis, and act late in recombination to affect the resolution of crossovers. This review will discuss the current status of DNA mismatch repair and nucleotide excision repair proteins during meiosis, especially in the yeast S. cerevisiae. Received 21 September 1998; received after revision 23 November 1998; accepted 23 November 1998     

Identifying the conformational state of bi-liganded haemoglobin

While most researchers agree on the global features of cooperative ligand binding to haemoglobin (Hb), the internal mechanisms remain open to debate. This is not due to inaccurate measurements, but is rather a consequence of the cooperative ligand binding that decreases the equilibrium populations of the partially liganded states and makes observation of the transitions between these substates more difficult. For example, the equilibrium population of the doubly liganded tetramers is typically less than 5% of the total Hb. As a result many models with widely varying mechanisms may fit the oxygen equilibrium curve, but may not be consistent with observations of other parameters, such as ligand-binding kinetics or subunit association equilibria. The wide range of methods and models has led to divergent conclusions about the properties of specific substates. One notable debate concerns the properties of the doubly liganded forms. The simple two-state model predicts a shift in the allosteric equilibrium based on the number of ligands bound, but not on their distribution within the tetramer. From studies of dimer-tetramer equilibria of various pure and hybrid forms, it was concluded that a tetramer with two ligands bound on the same α β dimer (species 21, an asymmetric hybrid) shows an enhanced tetramer stability, similar to singly liganded Hb, relative to the other three types of doubly liganded tetramers which resemble the triply liganded forms [Ackers et al. (1992), Science 255: 54–63]. The implications of this model and the relevant experiments will be reviewed here. Received 27 April 1998; received after revision 17 July 1998; accepted 10 August 1998     

Some cellular and molecular properties of abscisic acid: its particular involvement in growing plant roots

Several characteristics of the plant hormone abscisic acid (ABA) are critically discussed, more or less directly, in relation to the extension of root cells. A few topics have been selected some biochemical characteristics of ABA (chemical structure, metabolism), inhibiting-β complex, inhibiting regulators from root caps, endogenous ABA in growing roots (ABA gradients, microsurgical experiments, light effects), applied ABA on elongating roots, ABA and indol-3yl acetic acid (IAA) interactions (root growth, proton extrusion, hormone transport, auxin herbicides), ABA effect on the root cell cycle, ABA and drought cells of elongating roots [water deficit conditions, IAA and jasmonic acid (JA) as ‘stress hormones’ other than ABA, gene expression]. Received 28 January 1998; received after revision 20 April 1998; accepted 21 April 1998