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Plants have a penchant for perfuming the atmosphere around them. Since antiquity it has been known that both floral and vegetative parts of many species emit substances with distinctive smells. The discovery of the gaseous hormone ethylene 70 years ago brought the realization that at least some of the compounds emitted may have physiological significance without any distinctive smell to humans. At present, more than 1,000 low M r organic compounds have been reported to be emitted from plants, although a comprehensive list is available only for floral volatiles (Knudsen et al., 1993). Our knowledge of the occurrence and distribution of plant volatiles has been significantly extended in the last 15 years thanks to the adoption of simple, sensitive methods for headspace sampling and the availability of relatively inexpensive bench-top instruments for gas chromatography-mass spectrometry. The substances reported are largely lipophilic products with molecular masses under 300. Most can be assigned to the following classes (in order of decreasing size): terpenoids, fatty acid derivatives including lipoxygenase pathway products, benzenoids and phenylpropanoids, C5-branched compounds, and various nitrogen and sulfur containing compounds. Nearly all of these classes are emitted from vegetative parts as well as flowers (Knudsen et al., 1993), and some are even emitted from roots (Steeghs et al., 2004). A major discovery of the last decade is that plants commonly emit much greater amounts and varieties of volatiles after herbivore damage, and not just from the site of injury (Pare and Tumlinson, 1999). Major progress in plant volatile research, as in other areas of plant biology, has come from the use of molecular and biochemical techniques. A large number of genes encoding enzymes of volatile biosynthesis have recently been reported. In vitro characterization of the heterologously expressed enzymes, especially determination of their substrate and product specificity, has helped clarify the pathways of volatile formation. In addition, investigation of the spatial and temporal patterns of gene expression has provided new information on the factors regulating the emission of plant volatile compounds. In this update, we survey the latest advances on the biosynthesis and regulation of plant volatiles, beginning with a brief review of the function of these substances. Perhaps the greatest mysteries surrounding volatiles concern their function in the life of the plant. While it is generally assumed that compounds emitted from flowers serve to attract and guide pollinators (Reinhard et al., 2004), only scattered attempts have been made to demonstrate the ability of individual substances to attract specific pollinators. Many floral volatiles have anti-microbial or anti-herbivore activity (DeMoraes et al., 2001; Friedman et al., 2002; Hammer et al., 2003), and so could also act to protect valuable reproductive parts of plants from enemies. Among vegetative volatiles, the most intensively studied substance is isoprene, a simple five-carbon terpene emitted from the foliage of many woody species (Sharkey and Yeh, 2001b). The function of isoprene is still controversial, and this compound may act to increase the tolerance of photosynthesis to high temperatures by stabilizing the thylakoid membranes (Sharkey et al., 2001a) or by quenching reactive oxygen species (Loreto and Velikova, 2001). The release of volatiles from vegetative organs following herbivore damage seems to be a general property of plant species. Contributions to this special issue cover herbivore-induced volatiles from cabbage (Brassica oleracea; Vuorinen et al., 2004b), cucumber (Cucumis sativus; Mercke et al., 2004), Lotus japonicus (Arimura et al., 2004b), and maize (Zea mays; Degen et al., 2004). These substances have been demonstrated to serve as indirect plant defenses. That is, they attract arthropods that prey upon or parasitize herbivores, thus minimizing further damage to plant tissue (Pare and Tumlinson, 1999; Dicke and Van Loon, 2000). In some cases, herbivore-induced volatiles may also act as direct defenses, repelling (DeMoraes et al., 2001; Kessler and Baldwin, 2001) or intoxicating (Vancanneyt et al., 2001) herbivores and pathogens (Andersen et al., 1994). The possibility that these substances also act in plant-plant communication has been discussed (Arimura et al., 2000; Dicke and Bruin, 2001; Engelberth et al., 2004). Herbivore-induced volatiles could additionally have physiological roles within the plant, with their release being a consequence of their volatility and membrane solubility. Like isoprene, some herbivore-induced monoterpenes and sesquiterpenes have the potential to combine with various reactive oxygen species (Hoffmann et al., 1997; Bonn and Moortgat, 2003), and so could protect against internal oxidative damage (Delfine et al., 2000; Loreto et al., 2004b). In fact, ozone fumigation has recently been reported to promote the emission of herbivore-induced volatiles (Vuorinen et al., 2004a). Yet, it is still unclear why oxidative stress is likely to be significantly higher after herbivore damage. Further studies are needed to help elucidate the roles of these and other plant volatiles. The growing number of reports on genes involved in volatile formation, as described in the following sections, should enable investigators to manipulate volatile emission and test its function in plants. Terpenes, as the largest class of plant secondary metabolites, have many volatile representatives. The majority of hemiterpenes (C5), monoterpenes (C10), sesquiterpenes (C15), and even some diterpenes (C20) have high enough vapor pressures at normal atmospheric conditions to allow significant release into the air. The basic pathway of volatile terpenoid biosynthesis is conveniently treated in three phases: (1) formation of the basic C5 units, (2) condensation of two or three C5 units to form C10, C15, or C20 prenyl diphosphates, and (3) conversion of the resulting prenyl diphosphates to end products. The formation of basic C5 units, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) proceeds via two alternative pathways: the long known mevalonate pathway from acetyl-CoA and the methylerythritol phosphate pathway from pyruvate and glyceraldehyde-3-phosphate, discovered only in the last 10 years (for review, see Rodriguez-Concepcion and Boronat, 2002). The methylerythritol phosphate pathway, localized in the plastids, is thought to provide IPP and DMAPP for hemiterpene, monoterpene, and diterpene biosynthesis, while the cytosol-localized mevalonate pathway provides C5 units for sesquiterpene biosynthesis. However, metabolic “cross-talk” between the two pathways is prevalent (Schuhr et al., 2003), particularly in the direction from plastids to cytosol (Laule et al., 2003). This issue contains two contributions concerning the regulation of the basic pathways in relation to isoprene formation. Although produced largely by the plastidial pathway, isoprene also seems to arise from extra-plastidial sources, but there is apparently no cross-talk between the two pathways in its formation (Loreto et al., 2004a). The plastidial pathway is controlled by tight feedback regulation on its first step, deoxyxylulose-5-phosphate synthase (Wolfertz et al., 2004). In the second phase of terpene biosynthesis, IPP and DMAPP condense to form geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate, the precursors of monoterpenes, sesquiterpenes, and diterpenes, respectively. These reactions are catalyzed by short-chain prenyltransferases (Koyama and Ogura, 1999; Liang et al., 2002). FPP is synthesized by a large family of homodimeric prenyltransferases called FPP synthases. However, the situation regarding GPP formation is more complex. While the GPP synthases of Arabidopsis (Bouvier et al., 2000) and grand fir (Abies grandis; Burke and Croteau, 2002) are homodimers, like other short-chain prenyltransferases, those reported from peppermint (Mentha × piperita) leaves (Burke et al., 1999) and the flowers of snapdragon (Antirrhinum majus) and Clarkia breweri (Tholl et al., 2004) are unusual heterodimeric enzymes, with each subunit being a member of the prenyltransferase protein family. The third phase of terpene volatile biosynthesis involves the conversion of the various prenyl diphosphates, DMAPP (C5), GPP (C10), FPP (C15), and geranylgeranyl diphosphate (C20), to hemiterpenes (isoprene and 2-methyl-3-buten-2-ol), monoterpenes, sesquiterpenes, and diterpenes, respectively. These reactions, carried out by a large family of enzymes known as terpene synthases (Cane, 1999; Wise and Croteau, 1999), produce the primary representatives of each skeletal type. The investigation of terpene synthases is a very active area of plant volatile research and this issue contains four contributions describing the isolation of genes of this type from Norway spruce (Picea abies; Martin et al., 2004), Arabidopsis (Chen et al., 2004), cucumber (Mercke et al., 2004), and L. japonicus (Arimura et al., 2004b). These gene sequences give new insights into the evolutionary origin and genetic regulation of terpene synthases. One of the most outstanding properties of these enzymes is their proclivity for making multiple products from a single substrate. Hence, there has been much curiosity about the carbocationic reaction mechanism. The elucidation of the first crystal structures of plant terpene synthases (Starks et al., 1997; Whittington et al., 2002) now puts this work on a much stronger experimental footing. Many terpene volatiles are direct products of terpene synthases, but others are formed through transformation of the initial products by oxidation, dehydrogenation, acylation, and other reaction types. These are discussed in the following section. The terpene pathways are essentially biosynthetic, building up a carbon skeleton, and the immediate products formed by the large family of terpene synthases discussed above are mostly hydrocarbons, although sometimes they contain a hydroxyl group (e.g. linalool synthase produces linalool, a tertiary alcohol). Such compounds are already fairly volatile. In contrast, most other volatile compounds are produced through the shortening of a carbon skeleton, often followed by further modification, or simply by modification of the existing carbon skeleton. Compounds that are already somewhat volatile may also be modified, resulting in enhanced volatility or changed olfactory properties. The majority of these modifications involve the reduction or removal of carboxyl groups, the addition of hydroxyl groups, and the formation of esters and ethers. Each type of modification is catalyzed by a group (or several groups) of related enzymes constituting protein families. Some of these protein families had been previously recognized from biochemical research into nonvolatile compounds but some were only recently identified as part of the research into the biosynthesis of plant volatiles. Modifications for which enzymatic reactions and enzymes have been identified in plants are described below. Representative modification reactions leading to the biosynthesis of compounds with enhanced or changed volatility and olfactory properties. SAM, S-adenosyl-l-Met; CVOMT, chavicol O-methytransferase; SAMT, S-adenosyl-l-Met:salicylic acid carboxyl methytransferase; and BEAT, acetyl-coenzyme A:benzyl alcohol acetyltransferase. Cytochrome P450 enzymes are also very important in the biosynethsis of volatile phenylpropenes such as eugenol and the benzenoid vanillin. Both of these compounds, found in a wide variety of species both in flowers and in leaves, are derived from Phe and share with nonvolatile phenylpropanoids the earlier steps of 4-hydroxylation of cinnamate by 4CH, a P450 enzyme (Frank et al., 1996), and 3-hydroxylation by the newly discovered P450 enzyme that utilizes the shikimic or quinic ester of coumarate rather than coumaric acid or coumaroyl-CoA (Schoch et al., 2001; Gang et al., 2002a). Cytochrome P450 enzymes are crucial in the biosynthesis of volatiles derived from fatty acids, and in particular, in the octodecanoic pathway. Two different P450 enzymes, 9-LOX and 13-LOX, can introduce a peroxide into linoleic acid (18:3) at the respective positions (Howe and Schilmiller, 2002). Subsequent cleavage of the hydrocarbon chain by produces and respectively. The is important of volatiles, the of compounds that are emitted the is and 2002). In addition to the cleavage of linoleic acid at the to also produces a compound that can be to acid (Howe and Schilmiller, 2002). While acid is not by its is are large and family of with representatives found to be involved in the biosynthesis of volatiles. Such enzymes have been in the of volatile and apparently alcohol can short-chain such as and to and that are also found in leaves et al., This of tight substrate to the of by genetic et al., 1999). Some terpene such as and are to by et al., et al., and are by the of the is have and are found in many plants, and its a major floral in many species et al., and is also likely derived from in a reaction catalyzed by a member of the family et al., 2004). A large of plant volatiles contain a hydroxyl group a The group is in a reaction catalyzed by a in which as the plant to share a they into families that share primary et al., 2003). A large family of with involved in the of both volatile and nonvolatile to or has been identified and as the family et al., 2003). of this family have been to the of eugenol to form in flowers of breweri and in the of and also the of chavicol to in the et al., 1997; et al., 2000; Gang et al., a major compound in many is produced from in two reactions catalyzed by two very and et al., 2002; et al., 2002). Both enzymes can out both is more with while is more with et 2002). flowers a compound with three groups, which is synthesized from and can the of the second and third and but not the of also known as et al., 2002; et al., 2002). The enzyme that this also to the but is only related to and et al., 2004). important × to be produced by the of This to be to a wide of including of the pathway such as and alcohol et al., 2002). above as the substrate of a eugenol and important volatile on its and important also contain a group on their The pathways of these compounds share the first steps with the pathway. Gang et demonstrated that eugenol is derived from a the and reactions and the which is catalyzed by This is also likely to be the for although this has not been demonstrated is a member of the family of plants et al., 2003). Some esters are wide in the plant has been reported in floral (Knudsen and 1993), and it is also commonly emitted from vegetative under by or et al., 2001; et al., 2003). enzyme of acid acid carboxyl first reported from breweri flowers et al., 1999). has been identified from several other plant species et al., 2002; et al., et al., et al., 2003). This which as the a new type of plant known as or the first two of the of the first three enzymes identified in this Some enzymes have been to be to also acid a compound to for the group in (for et al., 2004). the other acid carboxyl the enzyme for the snapdragon floral volatile et al., 2000; et al., 2000). are by the Arabidopsis including enzyme that both and (Chen et al., 2003). is not known all of these are involved in the biosynthesis of volatile compounds, but to the family have been to be for the three reactions in the biosynthesis of a nonvolatile in et al., 2003). However, Arabidopsis to acid to form et al., 2001). While this may act as internal in Arabidopsis and other plant it is also emitted from plants (Howe and Schilmiller, and has also been reported in the floral of several plant species (Knudsen et al., it is likely to be formed by most often with but also with such or to volatile compounds is also In all known such plant volatile esters are synthesized by a recently discovered family of plant called after the first of the first four enzymes identified and 2000). The basic reaction catalyzed by these enzymes is the of group from to the hydroxyl group of alcohol Many enzymes are involved in the of nonvolatile compounds such as or derivatives et al., and Croteau, or in steps of pathways that may to the of volatiles such as eugenol et al., 2002a). involved in volatile alcohol acetyl-CoA from breweri which produced et al., alcohol which produces in flowers of Clarkia et al., 2002) and et al., 2004), and in leaves of et al., and which produces and is in leaves of Arabidopsis et al., 2002). The enzymes often wide substrate for both the and the alcohol the alcohol enzyme can also to the alcohol et al., 2004). a enzyme from can use a of such as and and to various such as and et al., 2000; et al., 2004). from has wide substrate et al., 2004), and a enzyme can both and et al., 2003). In such cases, the type of volatile formed in a tissue more on the internal of the than on the and of the enzyme for these et al., et al., 2004). The shortening by two of the chain to the of phenylpropanoids to the formation of benzenoid compounds. The by which this is is not In and metabolic described in this that both the and pathways are involved in the formation of benzenoid compounds in et al., 2004). However, a discovery also that in the of acid a third pathway, via the pathway, may also in plants, as it in et al., 2001). of a volatile compound into the atmosphere on both the of its biosynthesis and the of its progress in the last decade in the isolation and characterization of genes for the formation of volatile compounds has the investigation of the regulation of the biosynthesis of plant volatiles. has been found that volatiles are synthesized in the from which they are in the of plant from which they can into the atmosphere or after being synthesized et al., and 2000; et al., et al., 2004) or in the structures or for found in and et al., Gang et al., 2001; et al., 2002). of volatile compounds is the plant organs in flowers produce the most and the of volatile compounds, which the flowers are for tissue also of volatile organic compounds, which could be by damage or by or et al., and Tumlinson, 1997; et al., 2004a). In such as significant amounts of volatile compounds in the and the emitted volatiles only a of the produced et al., 2000). and emission of volatile compounds is also a emission in flowers and in leaves and the of leaves are and not is not or flowers are for and relatively or decreasing the et al., and 2000; et al., 2000). The temporal in of enzymes for the steps of volatile formation, enzyme protein and the expression of genes that the biosynthesis of volatiles is largely at the of gene expression et al., et al., 2000). is still unclear to and other to this In more than biochemical pathway is for a of volatile compounds from different plant A of the regulation of benzenoid and emission in snapdragon flowers that the emission of and compounds is of individual metabolic pathways and the expression of genes that enzymes involved in the steps of biosynthesis et al., 2003). However, factors that multiple pathways leading to the formation of have not been The of the enzyme for the of the biosynthesis of a volatile is not the only The for the regulation of of volatile compounds also the of substrate in the et al., 2000). In the of enzymes that are to use several such as and the of the type of produced product et al., et al., et al., 2004). The of substrate in the regulation of the biosynthesis of volatile compounds also recently by metabolic the linalool synthase gene under the of the into the between organs in the of the synthesized linalool or its more on the availability of the substrate GPP in the tissue than on the expression of the linalool synthase gene et al., 2001). In peppermint the of which the conversion of to methylerythritol the to GPP and to about a increase in the and Croteau, 2001). regulation of GPP formation can at the of GPP as in snapdragon the subunit can a in GPP biosynthesis (Tholl et al., 2004). regulation of GPP synthase by product and substrate could also to the of the to GPP and to (Tholl et al., 2004). enzymes for the substrate as in the of three synthases and into plants, the of emission in leaves to that on the of the enzymes for while the emission in flowers were that the GPP not et al., 2004). These that while the investigation of the regulation of the steps of volatile biosynthesis is important a of the regulation of the through the biochemical pathway is for the of and emission of secondary volatile compounds. of volatile compounds from flowers and leaves of some plant as well as herbivore-induced volatiles, the The release of floral volatiles in these species a with emission the or which generally with the of potential and is controlled by a or by and et al., et al., as well as volatiles emitted from and leaves, a emission et al., Loreto et al., et al., 2001; and Martin et al., et al., with some emission also controlled by a for in et al., 2002). In flowers the and emission of some volatile compounds, such as the volatile ester is by the of substrate availability which in could be at the of expression of genes encoding the enzymes of its biosynthesis et al., While regulation of isoprene emission is well (Wolfertz et al., 2003), is known to about the molecular for emission of vegetative volatiles. there is a of several between the beginning of herbivore damage and the release of volatile compounds, which could be in by the of the expression of genes for their biosynthesis (Arimura et al., 2004a). volatile compounds are after herbivore damage, they arise from (Pare and Tumlinson, factors such as and can the emission of volatiles and the and of and et al., 2000). In addition, the in emission of floral volatiles which after the et al., 2003). very is known about the release of synthesized volatile compounds from plant In the of release is a function of the properties of the compound and the properties of and membranes (in of monoterpenes which are synthesized in the through which the compound has to of volatile compounds emitted and within the plant tissue that the emission of volatiles is not a function of their volatility but could also involve a and et al., 2000). The membranes of the it or be more to some volatile compounds or the emitted substances may be with different than the volatiles. is known about between various the of the release and these to the regulation of volatile Plants produce a of volatile compounds for both general and advances in with ability to and genes and the enzymes they from many plant have enhanced of plants such compounds and their The of should come from the significance of these volatiles for plant and their on the of plants with their

With the ageing population in most countries, disorders of bone and mineral metabolism are becoming increasingly relevant to every day clinical practice. Consequently, the interest in, and the need for effective measures to be used in the screening, diagnosis and follow-up of such pathologies has markedly grown. Together with clinical and imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease. In recent years, the isolation and characterisation of cellular and extracellular components of the skeletal matrix have resulted in the development of molecular markers that are considered to reflect either bone formation or bone resorption. These biochemical indices are non-invasive, comparatively inexpensive and, when applied and interpreted correctly, helpful tools in the diagnostic and therapeutic assessment of metabolic bone disease. Part I of this article provides an overview of the basic biochemistry of bone markers, and sources of non-specific variability. Part II (to be published in a subsequent issue of this journal) will review the current evidence regarding the clinical use of biochemical markers of bone remodelling in metabolic and metastatic bone disease.

extracellular matrix reactive oxygen intermediates transforming growth factor-β1 Kruppel-like factor receptor tyrosine kinase discoidin domain receptor platelet-derived growth factor extracellular signal-regulated kinase endothelin-1 matrix metalloproteinase tissue inhibitor of metalloproteinase monocyte chemotactic protein-1 retinoic acid interleukin-10 Encapsulation of injury with fibrosis is a highly evolved response of adult tissues. In liver, the components of the process have been greatly clarified, leading to a coherent view of how wound healing occurs in response to injury. Hepatic fibrogenesis provides an important biological and clinical context for emerging concepts in molecular biochemistry that is relevant to many other tissues. Chronic injury leading to fibrosis in liver occurs in response to a variety of insults, including viral hepatitis (especially hepatitis B and C), alcohol abuse, drugs, metabolic diseases due to overload of iron or copper, autoimmune attack of hepatocytes or bile duct epithelium, or congenital abnormalities (1.Friedman S.L. Schiff E. Sorrell M. Maddrey W. Diseases of the Liver. 8th Ed. Lippincott-Raven, Philadelphia1998: 371-386Google Scholar). Typically, injury is present for months to years before significant scar accumulates, although the time course may be accelerated in congenital liver disease. Liver fibrosis is reversible, whereas cirrhosis, the end-stage consequence of fibrosis, is generally irreversible. Thus, efforts to understand fibrosis focus primarily on events that lead to the early accumulation of scar in hopes of identifying therapeutic targets to slow its progression. Like other parenchyma, the normal liver contains an epithelial component (hepatocytes), an endothelial lining (which in liver is distinguished by fenestrae or pores), tissue macrophages (Kupffer cells), and a perivascular mesenchymal cell called the stellate cell (previously called Ito cell, lipocyte, perisinusoidal cell, or fat-storing cell) (Fig. 1); stellate cells are the key fibrogenic cell (see next section). The cellular elements of liver are organized within the sinusoid, or microvascular unit, with the subendothelial space of Disse separating the epithelium (hepatocytes) from the sinusoidal endothelium. In normal liver this space contains a basement membrane-like matrix, although it is not electron-dense like a typical basement membrane. The normal subendothelial extracellular matrix (ECM)1 is essential for maintaining the differentiated function of all resident liver cells. Establishing the importance of the normal ECM in liver has illuminated recent attempts to develop artificial liver support by recognizing that all cellular elements and supporting structures (not just the hepatocyte compartment) must be reconstituted to preserve differentiated function of liver ex vivo (2.Mitaka T. Sato F. Mizuguchi T. Yokono T. Mochizuki Y. Hepatology. 1999; 29: 111-125Crossref PubMed Scopus (206) Google Scholar). As the liver becomes fibrotic, there are both quantitative and qualitative changes in composition of the hepatic ECM. The total content of collagens and noncollagenous components increases 3–5-fold, accompanied by the shift in the type of ECM in subendothelial space from the normal low density basement membrane-like matrix to interstitial type matrix containing fibril-forming collagens. Hepatic stellate cells comprise 15% of the total number of resident liver cells. In normal liver they are the principal storage site for retinoids (3.Wang X.D. Nutr. Rev. 1999; 57: 51-59Crossref PubMed Scopus (57) Google Scholar). Stellate cells constitute a heterogeneous group of cells that are functionally and anatomically similar but different in their expression of cytoskeletal filaments, their retinoid content, and their potential for ECM production (4.Knittel T. Kobold D. Saile B. Grundmann A. Neubauer K. Piscaglia F. Ramadori G. Gastroenterology. 1999; 117: 1205-1221Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Stellate cells have an intriguing embryologic origin, with recent evidence suggesting that they are neural crest-derived because they express glial fibrillary acidic protein and nestin (Ref. 5.Niki T. Pekny M. Hellemans K. Bleser P.D. Berg K.V. Vaeyens F. Quartier E. Schuit F. Geerts A. Hepatology. 1999; 29: 520-527Crossref PubMed Scopus (242) Google Scholar and references therein). A neural crest origin is further supported by studies in rat neural crest stem cells, which differentiate into myofibroblasts that express smooth muscle α-actin (6.Morrison S.J. White P.M. Zock C. Anderson D.J. Cell. 1999; 96: 737-749Abstract Full Text Full Text PDF PubMed Scopus (637) Google Scholar), a marker of activated stellate cells. These observations raise the possibility of using neural crest-specific promoters to drive transgene expression selectively in stellate cells in vivo and the prospect of reconstituting stellate cells from a neural crest precursor as part of efforts to repopulate liver. The perivascular orientation and long cytoplasmic processes of stellate cells facilitate their interactions with neighboring cell types. These processes are adjacent to hepatic nerves, which can respond to α-adrenergic stimulation with an influx of cytosolic calcium and release of osmolytes (7.vom Dahl S. Bode J.G. Reinehr R.M. Monnighoff I. Kubitz R. Haussinger D. Hepatology. 1999; 29: 195-204Crossref PubMed Scopus (17) Google Scholar). Following liver injury of any etiology, hepatic stellate cells undergo a response known as “activation,” which is the transition of quiescent cells into proliferative, fibrogenic, and contractile myofibroblasts. Stellate cell activation is a remarkably pleiotropic yet tightly programmed response occurring in a reproducible sequence (Fig.2). The organization of stellate cell activation into a defined temporal sequence provides a framework in which cellular events can be placed into a discrete biologic context. Early events have been termed initiation (also referred to as the “preinflammatory” stage). Initiation encompasses rapid changes in gene expression and phenotype that render the cells responsive to cytokines and other local stimuli. Initiationis associated with transcriptional events and induction of immediate early genes. It results from paracrine stimulation due to rapid, disruptive effects of liver injury on the homeostasis of neighboring cells and from early changes in ECM composition.Perpetuation incorporates those cellular events that amplify the activated phenotype through enhanced cytokine expression and responsiveness; this component of activation results from autocrine and paracrine stimulation, as well as from accelerated ECM remodeling. Stimuli initiating stellate cell activation derive from injured hepatocytes and neighboring endothelial and Kupffer cells in addition to rapid, subtle changes in ECM composition. Hepatocytes and Kupffer cells are a potent source of reactive oxygen intermediates (ROI) (8.Maher J.J. Alcohol Clin. Exp. Res. 1999; 23: 917-921Crossref PubMed Scopus (33) Google Scholar). These compounds exert paracrine stimulation of stellate cells. Moreover, their activity is amplified in vivo by depletion of antioxidants as typically occurs in diseased liver. In cultured stellate cells, conditioned medium from hepatocytes undergoing oxidative stress increases proliferation and collagen synthesis (9.Svegliati Baroni G. D'Ambrosio L. Ferretti G. Casini A. Di Sario A. Salzano R. Ridolfi F. Saccomanno S. Jezequel A.M. Benedetti A. Hepatology. 1998; 27: 720-726Crossref PubMed Scopus (263) Google Scholar). Overexpression in stellate cells of the enzyme cytochrome P4502E1, which generates ROI, stimulates collagen I gene expression; this effect is attenuated by antioxidants (10.Nieto N. Friedman S.L. Greenwel P. Cederbaum A. Hepatology. 1999; 30: 987-996Crossref PubMed Scopus (161) Google Scholar). Endothelial cells play a dual role in early stellate cell activation. Injury to sinusoidal endothelial cells stimulates production of a splice variant of cellular fibronectin (EIIIA isoform), which has an activating effect on stellate cells (11.Jarnagin W.R. Rockey D.C. Koteliansky V.E. Wang S.S. Bissell D.M. J. 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An advantage to the stellate cell system in these studies has been the opportunity to analyze “in vivo” gene expression in freshly purified, homogenous cell isolates. These efforts have yielded a transcription factor (13.Ratziu V. Lalazar A. Wong L. Dang Q. Collins C. Shaulian E. Jensen S. Friedman S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9500-9505Crossref PubMed Scopus (238) Google Scholar), an adhesion molecule (ICAM-1) (14.Hellerbrand C. Wang S.C. Tsukamoto H. Brenner D.A. Rippe R.A. Hepatology. 1996; 24: 670-676Crossref PubMed Google Scholar), and interestingly, the prion protein (15.Ikeda K. Kawada N. Wang Y.Q. Kadoya H. Nakatani K. Sato M. Kaneda K. Am. J. Pathol. 1998; 153: 1695-1700Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), among others. One representative effort to identify regulatory genes during early stellate cell activation has resulted in the cloning of a Kruppel-like factor (KLF) zinc finger gene, Zf9/COPEB/GBF(recently renamed “KLF6”). KLF6 mRNA is rapidly induced in liver injury in vivo and in culture (13.Ratziu V. Lalazar A. Wong L. Dang Q. Collins C. Shaulian E. Jensen S. Friedman S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9500-9505Crossref PubMed Scopus (238) Google Scholar) and can transactivate genes regulating ECM accumulation (16.Kim Y. Ratziu V. Choi S.G. Lalazar A. Theiss G. Dang Q. Kim S.J. Friedman S.L. J. Biol. Chem. 1998; 273: 33750-33758Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). At least two other KLF proteins also regulate stellate cell activation. Sp1, the first member of the KLF family, binds more actively to its consensus motif in activated versus quiescent stellate cells (17.Philipsen S. Suske G. Nucleic Acids Res. 1999; 27: 2991-3000Crossref PubMed Scopus (537) Google Scholar, 18.Rippe R.A. Almounajed G. Brenner D.A. Hepatology. 1995; 22: 241-251PubMed Google Scholar). Basic transcription element binding protein 1 (BTEB1) mediates the increase in collagen gene expression, which occurs in response to UV radiation or expression of the transcription factor Jun (19.Chen A. Davis B.H. J. Biol. Chem. 1999; 274: 158-164Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Perpetuation of stellate cell activation involves key phenotypic responses mediated by increased cytokine effects and remodeling of ECM (12.Friedman S.L. Semin. Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google Scholar). Enhanced cytokine responses occurs through multiple mechanisms (12.Friedman S.L. Semin. Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google Scholar); among these, increased expression of cell membrane receptors and enhanced signaling are especially important (see Ref. 20.Pinzani M. Marra F. Carloni V. 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J. 1999; 30: Full Text Full Text PDF PubMed Scopus Google Scholar). is increased in and hepatic are many of this autocrine expression is important (see Ref. A.M. J. 1995; Scholar for of the gene has been in stellate cells (16.Kim Y. Ratziu V. Choi S.G. Lalazar A. Theiss G. Dang Q. Kim S.J. Friedman S.L. J. Biol. Chem. 1998; 273: 33750-33758Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). activity is also enhanced in activated stellate cells through of latent into the cytokine by a (12.Friedman S.L. Semin. Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google Scholar). and activity of are by a number of binding proteins W. A.M. K. S. Gastroenterology. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). A splice variant of latent binding which a has been identified in stellate cells K. S. C. W. P. A.M. Hepatology. 1998; 27: PubMed Scopus Google Scholar); this the biologic of during Enhanced signaling also the response to injury in stellate cells. binding of to its signaling I and receptors yet type receptor mRNA is during stellate cell activation (12.Friedman S.L. Semin. Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google D. A.M. T. S. Hepatology. 1999; 29: PubMed Scopus Google Scholar). of collagen synthesis during activation is among the molecular responses of stellate cells to injury and is mediated by both transcriptional and not all of which can be to activation of the type I collagen has been (13.Ratziu V. Lalazar A. Wong L. Dang Q. Collins C. Shaulian E. Jensen S. Friedman S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9500-9505Crossref PubMed Scopus (238) Google Scholar, D.A. J. M. Am. J. Google Scholar, Y. S. Greenwel P. M. M. K. F. Hepatology. 1995; 22: Google Scholar). In the of collagen mRNA increases in activated with quiescent stellate cells B. C. M. M. S. Brenner D.A. Cell. Biol. PubMed Google Scholar). A the of the collagen mRNA mediates this enhanced mRNA through an with the B. C. Brenner D.A. Cell. Biol. 1999; 19: PubMed Google Scholar). in matrix activity lead to remodeling of the hepatic ECM during liver which both and stellate cell activation. Stellate cells express all the key components for matrix (see T. Kobold D. Saile B. Grundmann A. Neubauer K. Piscaglia F. Ramadori G. Gastroenterology. 1999; 117: 1205-1221Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar, J. 1995; Scholar, and G. M. C. G. Salzano R. Casini A. S. M. Hepatology. 1999; 29: PubMed Scopus Google Scholar for In they are a key source of matrix as well as J. 1995; Scholar), of which the normal subendothelial ECM. of the normal subendothelial ECM its by fibril-forming which further stellate cell growth and production in a N. K. B. Hepatology. 1999; 30: PubMed Scopus Google Scholar, M. 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Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google Scholar). ECM in liver is an important of growth (12.Friedman S.L. Semin. Liver Dis. 1999; 19: 129-140Crossref PubMed Scopus (351) Google Scholar). Stellate cells can also amplify through the release of and monocyte are factor and F. R. C. S. S. M. G. P. Am. J. Pathol. 1998; Google Scholar, M. L. S.L. Am. J. PubMed Google Scholar). The of is through stimulation M. Marra F. Carloni V. Liver. 1998; 18: PubMed Scopus Google Scholar). of adhesion stellate cell activation further during liver injury T. C. Kobold D. Neubauer K. M. S. Ramadori G. Am. J. Pathol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). from and liver injury the number of activated stellate cells as tissue is an intriguing to activated stellate cells during they to quiescent cells or are they have to A key is an activated stellate cell can to a quiescent One that may this response is interleukin-10 and increases interstitial activity S.C. 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Steroidogenesis entails processes by which cholesterol is converted to biologically active steroid hormones. Whereas most endocrine texts discuss adrenal, ovarian, testicular, placental, and other steroidogenic processes in a gland-specific fashion, steroidogenesis is better understood as a single process that is repeated in each gland with cell-type-specific variations on a single theme. Thus, understanding steroidogenesis is rooted in an understanding of the biochemistry of the various steroidogenic enzymes and cofactors and the genes that encode them. The first and rate-limiting step in steroidogenesis is the conversion of cholesterol to pregnenolone by a single enzyme, P450scc (CYP11A1), but this enzymatically complex step is subject to multiple regulatory mechanisms, yielding finely tuned quantitative regulation. Qualitative regulation determining the type of steroid to be produced is mediated by many enzymes and cofactors. Steroidogenic enzymes fall into two groups: cytochrome P450 enzymes and hydroxysteroid dehydrogenases. A cytochrome P450 may be either type 1 (in mitochondria) or type 2 (in endoplasmic reticulum), and a hydroxysteroid dehydrogenase may belong to either the aldo-keto reductase or short-chain dehydrogenase/reductase families. The activities of these enzymes are modulated by posttranslational modifications and by cofactors, especially electron-donating redox partners. The elucidation of the precise roles of these various enzymes and cofactors has been greatly facilitated by identifying the genetic bases of rare disorders of steroidogenesis. Some enzymes not principally involved in steroidogenesis may also catalyze extraglandular steroidogenesis, modulating the phenotype expected to result from some mutations. Understanding steroidogenesis is of fundamental importance to understanding disorders of sexual differentiation, reproduction, fertility, hypertension, obesity, and physiological homeostasis.

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner-Doudoroff pathway and Calvin cycle and divides into an oxidative and non-oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6-phosphate into carbon dioxide, ribulose 5-phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the 'Warburg effect' of cancer cells. The non-oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6-phosphate and glyceraldehyde 3-phosphate as well as sedoheptulose sugars, yielding ribose 5-phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non-oxidative branch can supply glycolysis with intermediates derived from ribose 5-phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.

Mitochondria are membrane bound organelles present in almost all eukaryotic cells. Responsible for orchestrating cellular energy production, they are central to the maintenance of life and the gatekeepers of cell death. Thought to have originated from symbiotic ancestors, they carry a residual genome as mtDNA encoding 13 proteins essential for respiratory chain function. Mitochondria comprise an inner and outer membrane that separate and maintain the aqueous regions, the intermembrane space and the matrix. Mitochondria contribute to many processes central to cellular function and dysfunction including calcium signalling, cell growth and differentiation, cell cycle control and cell death. Mitochondrial shape and positioning in cells is crucial and is tightly regulated by processes of fission and fusion, biogenesis and autophagy, ensuring a relatively constant mitochondrial population. Mitochondrial dysfunction is implicated in metabolic and age related disorders, neurodegenerative diseases and ischemic injury in heart and brain.

The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.

Malignant cells are known to have accelerated metabolism, high glucose requirements, and increased glucose uptake. Transport of glucose across the plasma membrane of mammalian cells is the first rate-limiting step for glucose metabolism and is mediated by facilitative glucose transporter (GLUT) proteins. Increased glucose transport in malignant cells has been associated with increased and deregulated expression of glucose transporter proteins, with overexpression of GLUT1 and/or GLUT3 a characteristic feature. Oncogenic transformation of cultured mammalian cells causes a rapid increase of glucose transport and GLUT1 expression via interaction with GLUT1 promoter enhancer elements. In human studies, high levels of GLUT1 expression in tumors have been associated with poor survival. Studies indicate that glucose transport in breast cancer is not fully explained by GLUT1 or GLUT3 expression, suggesting involvement of another glucose transporter. Recently, a novel glucose transporter protein, GLUT12, has been found in breast and prostate cancers. In human breast and prostate tumors and cultured cells, GLUT12 is located intracellularly and at the cell surface. Trafficking of GLUT12 to the plasma membrane could therefore contribute to glucose uptake. Several factors have been implicated in the regulation of glucose transporter expression in breast cancer. Hypoxia can increase GLUT1 levels and glucose uptake. Estradiol and epidermal growth factor, both of which can play a role in breast cancer cell growth, increase glucose consumption. Estradiol and epidermal growth factor also increase GLUT12 protein levels in cultured breast cancer cells. Targeting GLUT12 could provide novel methods for detection and treatment of breast and prostate cancer.

The matrix metalloproteinases (MMPs) form an enzyme family of which gelatinase B (MMP-9) represents the largest and most complex member. We focus here on the biochemical properties, regulation, and functions of gelatinase B. The tight regulation of gelatinase B activity is highly complex and is established at five different levels. The transcription of the gelatinase B-gene depends on various cis-elements in its gene promotor and is induced or repressed by a large variety of soluble factors, including cytokines, growth factors, and hormones and by cellular contacts acting through specific signaling pathways. The specific regulation of its secretion occurs in cells storing gelatinase B in granules. After secretion, progelatinase B must be activated through an activation network. The enzyme activity is further regulated by inhibition and by other mechanisms, such as fine-tuning and stabilization by glycosylation. The ability of gelatinase B to degrade components of the extracellular matrix and to regulate the activity of a number of soluble proteins confers an important role in various physiological and pathological processes. These include reproduction, growth, development, inflammation, and vascular and proliferative diseases.

Many types of nanoparticles (NPs) are tested for use in medical products, particularly in imaging and gene and drug delivery. For these applications, cellular uptake is usually a prerequisite and is governed in addition to size by surface characteristics such as hydrophobicity and charge. Although positive charge appears to improve the efficacy of imaging, gene transfer, and drug delivery, a higher cytotoxicity of such constructs has been reported. This review summarizes findings on the role of surface charge on cytotoxicity in general, action on specific cellular targets, modes of toxic action, cellular uptake, and intracellular localization of NPs. Effects of serum and intercell type differences are addressed. Cationic NPs cause more pronounced disruption of plasma-membrane integrity, stronger mitochondrial and lysosomal damage, and a higher number of autophagosomes than anionic NPs. In general, nonphagocytic cells ingest cationic NPs to a higher extent, but charge density and hydrophobicity are equally important; phagocytic cells preferentially take up anionic NPs. Cells do not use different uptake routes for cationic and anionic NPs, but high uptake rates are usually linked to greater biological effects. The different uptake preferences of phagocytic and nonphagocytic cells for cationic and anionic NPs may influence the efficacy and selectivity of NPs for drug delivery and imaging.

Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.

Alzheimer's disease (AD) is the most common neurodegenerative disorder seen in age-dependent dementia. There is currently no effective treatment for AD, which may be attributed in part to lack of a clear underlying mechanism. Studies within the last few decades provide growing evidence for a central role of amyloid β (Aβ) and tau, as well as glial contributions to various molecular and cellular pathways in AD pathogenesis. Herein, we review recent progress with respect to Aβ- and tau-associated mechanisms, and discuss glial dysfunction in AD with emphasis on neuronal and glial receptors that mediate Aβ-induced toxicity. We also discuss other critical factors that may affect AD pathogenesis, including genetics, aging, variables related to environment, lifestyle habits, and describe the potential role of apolipoprotein E (APOE), viral and bacterial infection, sleep, and microbiota. Although we have gained much towards understanding various aspects underlying this devastating neurodegenerative disorder, greater commitment towards research in molecular mechanism, diagnostics and treatment will be needed in future AD research.