Cell density-induced changes in lipid composition and intracellular trafficking

Cell density is one of the extrinsic factors to which cells adapt their physiology when grown in culture. However, little is known about the molecular changes which occur during cell growth and how cellular responses are then modulated. In many cases, inhibitors, drugs or growth factors used for in vitro studies change the rate of cell proliferation, resulting in different cell densities in control and treated samples. Therefore, for a comprehensive data analysis, it is essential to understand the implications of cell density on the molecular level. In this study, we have investigated how lipid composition changes during cell growth, and the consequences it has for transport of Shiga toxin. By quantifying 308 individual lipid species from 17 different lipid classes, we have found that the levels and species distribution of several lipids change during cell growth, with the major changes observed for diacylglycerols, phosphatidic acids, cholesterol esters, and lysophosphatidylethanolamines. In addition, there is a reduced binding and retrograde transport of Shiga toxin in high density cells which lead to reduced intoxication by the toxin. In conclusion, our data provide novel information on how lipid composition changes during cell growth in culture, and how these changes can modulate intracellular trafficking.     

Molecular chaperones in the processing and presentation of antigen to helper T cells

Helper T lymphocytes recognize peptide fragments of antigen bound to Major Histocompatibility Complex (MHC) class II molecules on the surfaces of antigen presenting cells (APC). Antigen processing involves internalization of the antigen into an acidic compartment where the antigen is degraded and the resulting peptide fragments of the antigen are bound to MHC class II molecules and the complexes subsequently displayed at the APC surface. Thus, antigen processing represents a complex, intracellular assembly process which may, like many intracellular protein folding and assembly processes, require the function of molecular chaperones. This contribution focuses on the evidence which suggests that members of the heat shock protein family of molecular chaperones play a role in this pathway.     

The role of the insulin-like growth factor (IGF) axis in osteogenic and odontogenic differentiation

The insulin-like growth factor (IGF) axis is a multicomponent molecular network which has important biological functions in the development and maintenance of differentiated tissue function(s). One of the most important functions of the IGF axis is the control of skeletal tissue metabolism by the finely tuned regulation of the process of osteogenesis. To achieve this, the IGF axis controls the activity of several cell types—osteoprogenitor cells, osteoblasts, osteocytes and osteoclasts to achieve the co-ordinated development of appropriate hard tissue structure and associated matrix deposition. In addition, there is an increasing awareness that the IGF axis also plays a role in the process of odontogenesis (tooth formation). In this review, we highlight some of the key findings in both of these areas. A further understanding of the role of the IGF axis in hard tissue biology may contribute to tissue regeneration strategies in cases of skeletal tissue trauma.     

Bacterial phytotoxins: Mechanisms of action

Many species of phytopathogenic procaryotes produce toxins that appear to function in disease development. The affect the plant in different ways, the end result of which is the elicitation of chlorosis, necrosis, watersoaking, growth abnormalities or wilting. The most extensively studied toxins cause chlorosis. They specifically inhibit diverse enzymes, all critical to the plant cell. This inhibition results in a complex series of metabolic dysfunctions ultimately resulting in symptom expression. Substances causing growth abnormalities consist of known phytohormones and other compounds with plant hormone-like activities, but which have no structural relationship to the known hormones. The former act in the usual manner but, because of their elevated levels and imbalances, the host's regulatory mechanisms are overwhelmed and abnormal growth results (hyperplasia, shoot or root formation); the mechanisms of action of the latter group are unknown. High molecular weight, carbohydrate-containing substances, also acting in unknown ways, cause tissue watersoaking or wilting. Likewise, we know little about toxins causing necrosis except for syringomycin which affects ion transport across the plasmalemma.     

The response of human skeletal muscle tissue to hypoxia

Hypoxia refers to environmental or clinical settings that potentially threaten tissue oxygen homeostasis. One unique aspect of skeletal muscle is that, in addition to hypoxia, oxygen balance in this tissue may be further compromised when exercise is superimposed on hypoxia. This review focuses on the cellular and molecular responses of human skeletal muscle to acute and chronic hypoxia, with emphasis on physical exercise and training. Based on published work, it is suggested that hypoxia does not appear to promote angiogenesis or to greatly alter oxidative enzymes in skeletal muscle at rest. Although the HIF-1 pathway in skeletal muscle is still poorly documented, emerging evidence suggests that muscle HIF-1 signaling is only activated to a minor degree by hypoxia. On the other hand, combining hypoxia with exercise appears to improve some aspects of muscle O₂ transport and/or metabolism.     

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K_V) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type K_V channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.     

Advances in tenascin-C biology

Tenascin-C is an extracellular matrix glycoprotein that is specifically and transiently expressed upon tissue injury. Upon tissue damage, tenascin-C plays a multitude of different roles that mediate both inflammatory and fibrotic processes to enable effective tissue repair. In the last decade, emerging evidence has demonstrated a vital role for tenascin-C in cardiac and arterial injury, tumor angiogenesis and metastasis, as well as in modulating stem cell behavior. Here we highlight the molecular mechanisms by which tenascin-C mediates these effects and discuss the implications of mis-regulated tenascin-C expression in driving disease pathology.     

What’s new in the renin-angiotensin system?

Virtually all existing evidence on the function of angiotensin II (Ang II) in the regulation of tissue homeostasis and blood pressure regulation bears on the more restricted question of what other mechanisms or systems may amplify or inhibit the actions of this important peptide. Whereas there is evidence that Ang II may potentiate the effects of catecholamines, various cytokines and also growth factors, the repertoire of substances which may inhibit the actions of Ang II is more limited and has been restricted primarily to prostacyclin, bradykinin and nitric oxide. Advances in receptor pharmacology and introduction of selective antagonists to two of the receptor subtypes at which Ang II binds permitted a more critical examination of the functions of the renin angiotensin system in physiological and pathophysiological conditions, as well as uncovering the previously unsuspected possibility that within the biochemical pathways leading to the formation of the peptide the renin angiotensin system could process either its immediate precursor (angiotensin I) or the actual Ang II peptide into an alternative form, angiotensin-(1-7) [Ang-(1-7)], the function of which was to antagonize the effects of Ang II. We review here the biological actions of Ang-(1-7) and discuss how this discovery may change altogether the perception of how the renin angiotensin system functions in the regulation of tissue perfusion pressure and the regulation of salt and water metabolism.     

Mammalian iron transport

Iron is essential for basic cellular processes but is toxic when present in excess. Consequently, iron transport into and out of cells is tightly regulated. Most iron is delivered to cells bound to plasma transferrin via a process that involves transferrin receptor 1, divalent metal-ion transporter 1 and several other proteins. Non-transferrin-bound iron can also be taken up efficiently by cells, although the mechanism is poorly understood. Cells can divest themselves of iron via the iron export protein ferroportin in conjunction with an iron oxidase. The linking of an oxidoreductase to a membrane permease is a common theme in membrane iron transport. At the systemic level, iron transport is regulated by the liver-derived peptide hepcidin which acts on ferroportin to control iron release to the plasma.     

Peptide aptamers: exchange of the thioredoxin-A scaffold by alternative platform proteins and its influence on target protein binding

Peptide aptamers have emerged as powerful new tools for molecular medicine. They can specifically bind to and functionally inactivate a given target molecule under intracellular conditions. Typically, peptide aptamers are generated by screening a randomized peptide expression library, displayed from the Escherichia coli thioredoxin A (TrxA) protein. Here, we transferred peptide moieties from defined TrxA-based peptide aptamers to alternative scaffold proteins, such as the green fluorescent protein and staphylococcal nuclease. Yeast and mammalian two-hybrid assays as well as in vitro binding analyses show that the TrxA scaffold can be a major determinant for the binding of peptide aptamers. In addition, we demonstrate that TrxA can correctly display peptide sequences that correspond to the binding domains of natural interaction partners. Therefore, sequence analyses of TrxA-based peptide aptamers, isolated by two-hybrid screening from randomized expression libraries, should also be useful to find cellular binding partners for a given target protein, by homology. Received 1 August 2002; received after revision 17 September 2002; accepted 19 September 2002 RID="*" ID="*"Corresponding author.     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

Cellular copper distribution: a mechanistic systems biology approach

Copper is an essential but potentially harmful trace element required in many enzymatic processes involving redox chemistry. Cellular copper homeostasis in mammals is predominantly maintained by regulating copper transport through the copper import CTR proteins and the copper exporters ATP7A and ATP7B. Once copper is imported into the cell, several pathways involving a number of copper proteins are responsible for trafficking it specifically where it is required for cellular life, thus avoiding the release of harmful free copper ions. In this study we review recent progress made in understanding the molecular mechanisms of copper transport in cells by analyzing structural features of copper proteins, their mode of interaction, and their thermodynamic and kinetic parameters, thus contributing to systems biology of copper within the cell.     

Molecular diagnosis of parasites

Summary New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene clonign and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Molecular diagnosis of parasites

New developments in molecular biology have generated exciting possibilities for improved diagnosis of parasitic diseases. Through gene cloning and expression and peptide synthesis, defined parasite antigens can be produced in vitro for use in serodiagnosis, while nuclear hybridization techniques offer a vastly improved approach to identification of parasites in the tissue specimens of infected hosts as a means of diagnosis. Furthermore, the advent of the polymerase chain reaction technique has made it possible to increase the sensitivity of nuclear hybridization techniques, through amplification of target DNA sequences of the parasites in test material, by in situ synthesis of these sequences prior to hybridization with the diagnostic probe. Finally, through the use of monoclonal antibody technology, it is possible to design highly specific and sensitive serological assays, as well as assays for parasite antigen detection in tissue fluids and in the excreta of infected hosts, as a means of diagnosis.     

Regulatory peptide immunocytochemistry at light- and electron microscopical levels

Immunocytochemical techniques applied at both light- and electron microscopical levels are valuable in the study of regulatory peptide distribution in normal and diseased tissue, whether in the form of sections or whole cell preparations. Successful immunolocalization depends on adequate preservation of the peptide antigen and the tissue structure in which it resides; a suitably specific and sensitive labelled antibody detecting system. In general, peptides are stable molecules, most of which retain their antigenicity after conventional cross-linking fixation and tissue processing, allowing standard immunocytochemical methods to be used for light- and electron microscopy. Regulatory peptides are derived from precursor molecules and several 'families' of structurally similar peptides are now generally recognised. Region-specific antibodies may be needed to overcome problems of cross-reactivity or to identify a bioactive form in the presence of its precursor. Multiple co-localisation of different related and unrelated peptides in the same cell or even storage granule is now recognised and can be identified by immunocytochemistry.     

The plasma membrane proton-translocating ATPase

Living cells require membranes and membrane transporters for the maintenance of life. After decades of biochemical scrutiny, the structures and molecular mechanisms by which membrane transporters catalyze transmembrane solute movements are beginning to be understood. The plasma membrane proton-translocating adenosine triphosphatase (ATPase) is an archetype of the P-type ATPase family of membrane transporters, which are important in a wide variety of cellular processes. The H+-ATPase has been crystallized and its structure determined to a resolution of 8 angstrom in the membrane plane. When considered together with the large body of biochemical information that has been accumulated for this transporter, and for enzymes in general, this new structural information is providing tantalizing insights regarding the molecular mechanism of active ion transport catalyzed by this enzyme.     

Molecular aspects of membrane fission in the secretory pathway

Membrane fission is essential in various intracellular dissociative transport steps. The molecular mechanisms by which endocytic vesicles detach from the plasma membrane are being rapidly elucidated. Much less is known about the fission mechanisms operating at Golgi tubular networks; these include the Golgi transport and sorting stations, the trans-Golgi and cis-Golgi networks, where the geometry and physical properties of the membranes differ from those at the cell surface. Here we discuss the lipid and protein machineries that have so far been related to the fission process, with emphasis on those acting in the Golgi complex. Received 10 May 2002; received after revision 20 June 2002; accepted 26 June 2002 RID="*" ID="*"Corresponding author.     

Bridging the molecular and biological functions of the oxysterol-binding protein family

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large eukaryotic gene family that transports and regulates the metabolism of sterols and phospholipids. The original classification of the family based on oxysterol-binding activity belies the complex dual lipid-binding specificity of the conserved OSBP homology domain (OHD). Additional protein- and membrane-interacting modules mediate the targeting of select OSBP/ORPs to membrane contact sites between organelles, thus positioning the OHD between opposing membranes for lipid transfer and metabolic regulation. This unique subcellular location, coupled with diverse ligand preferences and tissue distribution, has identified OSBP/ORPs as key arbiters of membrane composition and function. Here, we will review how molecular models of OSBP/ORP-mediated intracellular lipid transport and regulation at membrane contact sites relate to their emerging roles in cellular and organismal functions.     

Studies of bone matrix molecules give us insights into bone remodelling

Bone is one of the most dynamic tissues, with constant remodelling and repair of minor defects during the whole life cycle. These processes have key roles in replacing fatigued, non-functional tissue with fresh tissue which has the proper mechanical properties. In this process, a balance between breakdown of less functional tissue and new tissue production is a prerequisite for tissue integrity and function. One of the major health problems in the elderly, i.e. osteoporosis and ensuing bone fracture, is caused by an imbalance between breakdown and synthesis in this continuous bone remodelling. Thus the study of cellular and molecular events governing the relevant processes is a prerequisite for understanding mechanisms and for future efficient therapeutical intervention.