Volume 383, Issue 10

Cellular membranes contain many types and species of lipids. One of the most important functional consequences of this heterogeneity is the existence of microdomains within the plane of the membrane. Sphingolipid acyl chains have the ability of forming tightly packed platforms together with sterols. These platforms or lipid rafts constitute segregation and sorting devices into which proteins specifically associate. In budding yeast, Saccharomyces cerevisiae, lipid rafts serve as sorting platforms for proteins destined to the cell surface. The segregation capacity of rafts also provides the basis for the polarization of proteins at the cell surface during mating. Here we discuss some recent findings that stress the role of lipid rafts as key players in yeast protein sorting and cell polarity.

In the RNA world hypothesis it is postulated that RNA was the first genetic molecule. Recent discoveries in gene silencing research on plants, fungi and animals show that RNA indeed plays a key role not only in controlling invading nucleic acids, like viruses and transposable elements, but also in regulating the expression of transgenes and endogenous genes. Doublestranded RNAs were identified to be the triggering structures for the induction of a specific and highly efficient RNA silencing system, in which enzyme complexes, like Dicer and RISC, facilitate as molecular machines the processing of dsRNA into characteristic small RNA species. RNA silencing can be transmitted rapidly from silenced to nonsilenced cells by short and long distance signaling. There is evidence that at least one component of the signal is a specific, degradationresistant RNA.

Recognition of danger of infection by innate immune cells is a prerequisite to combat infections and to activate T and B cells. Pathogenassociated molecular patterns (PAMP) play a fundamental role in this process. PAMPs are sensed by at least ten different Tolllike receptors (TLR). Within the realm of PAMPs, CpG DNA that is recognized by TLR-9 has an outstanding propensity to induce a milieu that favors activation of T lymphocytes and biases Th1-dominated immune responses. Therefore CpG DNA has become a promising immunotherapeutical candidate to assist and to direct immune responses such as in vaccination or modulation of allergic responses. As opposed to other PAMPs, CpG DNA can be synthesized with defined purity and base composition. Moreover, chemical substitutions can confer new qualities to synthetic CpG DNA.

Granulocyte colonystimulating factor (GCSF) is one of the most prominent endogenous proteins in broad clinical use. While its biological and clinical effects are relatively well studied, little is known about its endogenous formation in health and disease. However, such knowledge is crucial to decide in which situations GCSF should be applied efficiently in the clinic, i.e. when endogenous production does not suffice. The dramatic changes induced by GCSF in the differential blood cell count are directly immunomodulatory, strengthening the innate defence by multiplying neutrophilic granulocytes. A multitude of further immunomodulatory effects contribute to the regulation of the concerted host defence. In this review, following a short introduction into the biology of GCSF, the available data on endogenous formation in a number of animal models and human diseases is compiled. The cellular sources and inducers of GCSF formation are reviewed and the regulation of GCSF expression on both the transcriptional and translational level are discussed. The emerging understanding of the role and regulation of endogenous GCSF formation opens up possibilities to define therapeutic windows as well as targets for diagnostics or drug development. Lastly, the modulation of GCSF formation by various pharmacological agents alerts to putative side effects of these drug treatments.

Mitogenactivated protein kinases (MAPKs) are evolutionarily conserved enzymes which connect cellsurface receptors to regulatory targets within cells and convert receptor signals into various outputs. In mammalian cells, four distinct MAPKs have been identified: the extracellular signalrelated kinases (ERK)-1/2, the cjun Nterminal kinases or stressactivated protein kinases 1 (JNK1/2/3, or SAPK1s), the p38 MAPKs (p38 α/β/γ/δ, or SAPK2s), and the ERK5 or big MAP kinase 1 (BMK1). The p38 MAPK cascade is activated by stress or cytokines and leads to phosphorylation of its central elements, the p38 MAPKs. Downstream of p38 MAPKs there is a diversification and extensive branching of signalling pathways. For that reason, we will focus in this review on the different signalling events that are triggered by p38 activity, and analyse how these events contribute to specific gene expression and cellular responses.

Major advances have been made during the last decade in our understanding of adrenal steroid hormone biosynthesis. Two key players in these pathways are the human mitochondrial cytochrome P450 enzymes CYP11B1 and CYP11B2, which catalyze the final steps in the biosynthesis of cortisol and aldosterone. Using data from mutations found in patients suffering from steroid hormonerelated diseases, from mutagenesis studies and from the construction of threedimensional models of these enzymes, structural information could be deduced that provide a clue to the stereo and regiospecific steroid hydroxylation reactions carried out by these enzymes. In this review, we summarize the current knowledge on the physiological function and the biochemistry of these enzymes. Furthermore, the pharmacological and toxicological importance of these steroid hydroxylases, the means for the identification of their potential inhibitors and possible biotechnological applications are discussed.

Cytochrome P450 (P450) reactions are of interest because of their relevance to the oxidative metabolism of drugs, steroids, carcinogens, and other chemicals. One of the considerations about functional characterization is which steps of the catalytic cycle are ratelimiting. Detailed analysis indicates that several different steps can be ratelimiting with individual P450 reactions. NDealkylation of parasubstituted N,Ndimethylanilines is a function of the electron withdrawing/donating properties of the substituent and the oxidationreduction potential of the substrate, supporting a role in ratelimiting electron transfer from substrate to the high valent P450. In the oxidations of ethanol and acetaldehyde by human P450 2E1, a step following product formation must be the slow step (but not product release per se). Several oxidations catalyzed by human P450s 1A2 and 2D6 show slow CH bond breaking, and apparent highvalent iron complexes accumulate in the reaction steadystate. Kinetic simulations were used to test the suitability of potential schemes and to probe the effects of changes in individual reaction steps.

Membranes contain proteins that catalyze a variety of reactions, which lead to the selective permeability of the membrane. For membrane proteins to function as receptors, transporters, channels, and ATPases, they must be targeted to their correct membrane and inserted into the lipid bilayer. Recently, a new membrane component called YidC was discovered that mediates the insertion of proteins into membranes in bacteria. YidC homologs also exist in mitochondria and chloroplasts. Depletion of YidC from the cell interferes with the insertion of membrane proteins that insert both dependent and independent of the Sec YEG/SecDFYajC machinery. YidC directly interacts with membrane proteins during the membrane protein insertion process and assists in the folding of the hydrophobic regions into the membrane bilayer. The chloroplast and bacterial YidC homologs are truly functional homologs because the chloroplast homolog Alb3 functionally complements the bacterial YidC depletion strain. The role of YidC in the membrane insertion pathway will be reviewed.

The vascular endothelial growth factor (VEGF) receptor family in mammals contains three members, VEGFR1(Flt-1), VEGFR2(KDR/Flk-1) and VEGFR3 (Flt-4), which directly regulate the formation of blood vessels and lymphatic vessels. These two circular systems are essential for the supply of O2 and nutrients to all tissues of the body as well as the drainage of excess fluids with waste metabolites from peripheral tissues. VEGF receptors have a characteristic structure with 7 Iglike domains in the extracellular domain and a cytoplasmic tyrosine kinase domain with a long kinase insert region. Recently, some novel findings on the phylogenetical conservation of VEGF receptor genes in animals were reported: the conservation of the VEGFR1/solubleVEGFR1 gene in birds, and the conservation of the VEGFRPDGFRlike receptor gene in nonvertebrates. Based on this new information as well as established observations, here the possibility is discussed that the three VEGFR genes phylogenetically segregated not at once when the vertebrates established, but in a stepwise manner: two genes first (the VEGFR1/R2 progenitor and the VEGFR3 gene), and subsequently the three genes VEGFR1, R2 and R3.

Different forms of tumor necrosis factor (TNF) interact with two specific receptors for TNF (TNFR) on the cell membrane to induce a variety of effects. While sharing structural similarities in their extracellular domains, the two TNFRs differ in their intracellular domain, their signal transduction, and consequently their function. In addition, one of the two TNFRs can be expressed in two differently located isoforms. This makes the TNFTNFR system very complex. The dual TNF function for either cell death or survival upon interaction of members of the TNF ligand family with members of the TNF receptor family will be discussed.

In contrast to the wellknown regulation of cytochrome P450 (CYP) activity by enzyme induction, which represents a process with slow onset and slow offset, more recent studies revealed phosphorylation as a fast (within observation instantaneous) and isoenzymeselective regulation. The phosphorylated enzyme (investigated isozyme: CYP2B1) was fully inactive. The phosphorylation is mediated by PKA and hence under control of hormones and drugs that alter cellular cAMP levels. The consequences for the metabolic control of toxic species derived from drugs and environmental carcinogens are discussed. This information will help to improve therapy with drugs metabolized by CYPs which are phosphorylated by PKA, especially if these drugs possess a narrow window between required effectiveness and unacceptable toxicity.

Tissue regeneration in humans is limited and excludes vitals organs like heart and brain. Transformation experiments with oncogenes like T antigen have shown that retrodifferentiation of the respective cells is possible but hard to control. To bypass the risk of cancer formation a protein therapy approach has been developed. The transient delivery of proteins rather than genes could still induce terminallydifferentiated cells to reenter the cell cycle. This approach takes advantage of proteintransducing domains that mediate the transfer of cargo proteins into cells. The goal of this brief review is to outline the basics of protein transduction and to discuss potential applications for tissue regeneration.

From clinical studies the proinflammatory cytokine TNFα was proposed to play a key role in human cytomegalovirus (HCMV) reactivation from latency. In vitro experiments confirmed that TNFα stimulates the activity of the HCMV IE1/2 enhancer/promoter, which controls immediate early protein IE1 and IE2 gene expression via activation of the transcription factor NFκB and its binding to putative binding sites in the IE1/2 enhancer. NFκB was also proposed to be involved in IE1-mediated autostimulation of this promoter. The IE1/2 enhancer of HCMV contains four putative NFκB binding sites which differ in their distance to the transcription start site as well as in their sequence. Construction and testing of a series of promoter mutants demonstrated that NFκB is essential for both TNFα and IE1 stimulation. Furthermore, we were able to show that although all four NFκB sites bind NFκB with similar affinity in vitro, the contribution to TNFα and IE1 stimulation differs in correlation with the distance to the transcription start site and the sequence. Site 1 and 3 play the most dominant role and site 2 an intermediate, while site 4, which is conserved in sequence but far distant from the transcription start site, had no influence on NFκBmediated regulation of the IE1/2 promoter. Specific inhibition of NFκB signalling by coexpression of a dominantnegative IκB variant reduced TNFα stimulation of the IE1/2 enhancer/promoter by up to 80%. From this data, inhibitors of NFκB activation are suggested to be an alternative therapeutical strategy to interfere with HCMV (re)activation in undifferentiated monocyte/granulocyte progenitor cells in patients with a high risk of inflammationrelated HCMV (re)activation.

Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2 3 fold molar excess over ribosomes and a fraction of it is not ribosomeassociated after cell lysis. Here we show that TF follows a threestate equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent Kd of 18 M. Dimerization is mediated by the Nterminal ribosome binding domain and the Cterminal domain of TF, whereas the central peptidyl prolyl isomerase (PPIase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosomeassociated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TFribosome complex of approximately 1.2 M. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomerdimer equilibrium with approximately two thirds of TF existing in a dimeric state.

The vitamin Dbinding protein (DBP) binds to monomeric actin with high affinity. The variation in DBP isoforms is due to genetic polymorphism and varying glycosylation. To obtain a homogeneous preparation, the cDNA for human DBP and truncations thereof were cloned and various systems were applied for heterologous bacterial and yeast expression. The fulllength protein and the N and Cterminal halves of DBP remained insoluble probably because the protein did not fold to its native threedimensional structure due to formation of accidental intra and intermolecular disulfide bonds during expression in bacteria or yeast. This problem was overcome by cloning of a Cterminal fragment comprising residues 369 to 435 that did not contain disulfide bonds and was completely soluble. Binding of the Cterminal fragment to monomeric actin was demonstrated by comigration with actin during native polyacrylamide gel electrophoresis and surface plasmon resonance, however, at considerably lower affinity than fulllength DBP. This suggests that in addition to the Cterminal amino acid sequence other parts (amino acid residues or sugar moieties) of DBP participate in actin binding. The Cterminal fragment was found to inhibit denaturation of actin and to decrease the rate of actin polymerisation both at the barbed and at the pointed end in a concentrationdependent manner. According to a quantitative analysis of the polymerisation kinetics, association of actin monomers to nucleate filaments was not prevented by binding of the Cterminal fragment to actin. These data suggest that the sites on the surface of actin that are involved in actin nucleation and elongation are different.

The influence of the hyaluronanbinding protease (PHBSP), a plasma enzyme with FVII and prourokinase activating potency, on components of the contact phase (kallikrein/kinin) system was investigated. No activation or cleavage of the proenzymes involved in the contact phase system was observed. The procofactor high molecular weight kininogen (HK), however, was cleaved in vitro by PHBSP in the absence of any charged surface, releasing the activated cofactor and the vasoactive nonapeptide bradykinin. Glycosoaminoglycans strongly enhanced the reaction. The cleavage was comparable to that of plasma kallikrein, but clearly different from that of coagulation factor FXIa. Upon extended incubation with PHBSP, the light chain was further processed, partially removing about 60 amino acid residues from the Nterminus of domain D5 of the light chain. These cleavage site(s) were distinct from plasma kallikrein or FXIa cleavage sites. PHBSP and, more interestingly, also plasma kallikrein could cleave low molecular weight kininogen in vitro, indicating that domains D5H and D6H are no prerequisite for kininogen cleavage. PHBSP was also able to release bradykinin from HK in plasma where the procofactor circulates predominantly in complex with plasma kallikrein or FXI. In conclusion, PHBSP represents a novel kininogencleaving and bradykininreleasing enzyme in plasma that shares significant catalytic similarities with plasma kallikrein. Since they are structurally unrelated in their heavy chains (propeptide), their similar in vivo catalytic activities might be directed at distinct sites where PHBSP could induce processes that are related to the kallikrein/kinin system.

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [Vörös et al., Eur. J. Biochem. 251 (1998), 36 44], two fulllength cDNAs (LOX2:Hv:2, LOX2:Hv:3) were isolated from barley leaves (Hordeum vulgare cv. Salome) and characterized. Both of them encode 13-lipoxygenases with putative target sequences for chloroplast import. Immunogold labeling revealed preferential, if not exclusive, localization of lipoxygenase proteins in the stroma. The ultrastructure of the chloroplast was dramatically altered following methyl jasmonate treatment, indicated by a loss of thylakoid membranes, decreased number of stacks and appearance of numerous osmiophilic globuli. The three 13-lipoxygenases are differentially expressed during treatment with jasmonate, salicylate, glucose or sorbitol. Metabolite profiling of free linolenic acid and free linoleic acid, the substrates of lipoxygenases, in water floated or jasmonatetreated leaves revealed preferential accumulation of linolenic acid. Remarkable amounts of free 9- as well as 13-hydroperoxy linolenic acid were found. In addition, metabolites of these hydroperoxides, such as the hydroxy derivatives and the respective aldehydes, appeared following methyl jasmonate treatment. These findings were substantiated by metabolite profiling of isolated chloroplasts, and subfractions including the envelope, the stroma and the thylakoids, indicating a preferential occurrence of lipoxygenasederived products in the stroma and in the envelope. These data revealed jasmonateinduced activation of the hydroperoxide lyase and reductase branch within the lipoxygenase pathway and suggest differential activity of the three 13-lipoxygenases under different stress conditions.

A biosensoric approach has been developed to determine the activity of telomerase in tumor cell lysates. An optical sensor, the grating coupler, was used to monitor the association and dissociation of unlabeled compounds on the sensor surface in real time, by virtue of an evanescent field. An oligonucleotide was immobilized on the surface of the optical biosensor and linked with two other oligonucleotides by complementary sequences in an overlapping manner. The 3end of the last one carried the sequence of the telomeric substrate (TS) primer used for elongation by telomerase in the telomeric repeat amplification protocol (TRAP) assay. This primer sequence was phosphorothioate (PS) modified, which is known to strongly increase the affinity to the primer binding site of telomerase protein and consequently the velocity of the telomerase reaction. We show that the PS primer binds to the modified biosensor and is elongated effectively by the telomerase from HL-60 cell lysates. A synthesis rate of 1 nucleotide/min was determined. The inhibitory effect of peptide nucleic acid (PNA) was shown by using immobilized TS. The velocity of the telomerase reaction was slowed down and the signal intensity was below the signaltonoise ratio. Most nucleic acid detection systems use amplification steps such as polymerase chain reaction (PCR) to increase the amount of the probe. Since telomerase is a polymerase itself amplification of DNA by PCR is not required. Furthermore, no purification steps were required since all measurements were performed with crude cell extract.

With respect to the mechanism of chaperonelike activity, we examined the behavior of haptoglobin under heat shock conditions. Secondary structure changes during heat treatment were followed by circular dichroism, Raman and infrared spectroscopy. A model of the haptoglobin tetramer, based on its sequence homology with serine proteases and the CCP modules, has been proposed. Sequence regions responsible for the chaperonelike activity were not fully identical with the region that takes part in formation of the hemoglobinhaptoglobin complex. We can postulate the presence of at least two different chaperonebinding sites on each haptoglobin heavy chain.