Folding and dimerization of tick-borne encephalitis virus envelope proteins prM and E in the endoplasmic reticulum

Flavivirus envelope proteins are synthesized as part of large polyproteins that are co- and posttranslationally cleaved into their individual chains. To investigate whether the interaction of neighboring proteins within the precursor protein is required to ensure proper maturation of the individual components, we have analyzed the folding of the flavivirus tick-borne encephalitis (TBE) virus envelope glycoproteins prM and E by using a recombinant plasmid expression system and virus-infected cells. When expressed in their polyprotein context, prM and E achieved their native folded structures with half-times of approximately 4 min for prM and about 15 min for E. They formed heterodimeric complexes within a few minutes after synthesis that were required for the final folding of E but not for that of prM. Heterodimers could also be formed in trans when these proteins were coexpressed from separate constructs. When expressed without prM, E could form disulfide bonds but did not express a specific conformational epitope and remained sensitive to reduction by dithiothreitol. This is consistent with a chaperone-like role for prM in the folding of E. PrM was able to achieve its native folded structure without coexpression of E, but signal sequence cleavage at the N terminus was delayed. Our results show that prM is an especially rapidly folding viral glycoprotein, that polyprotein cleavage and folding of the TBE virus envelope proteins occurs in a coordinated sequence of processing steps, and that proper and efficient maturation of prM and E can only be achieved by cosynthesis of these two proteins.     

Biochemical and functional characterization of recombinant Rhodnius prolixus platelet aggregation inhibitor 1 as a novel lipocalin with high affinity for adenosine diphosphate and other adenine nucleotides

The Rhodnius prolixus aggregation inhibitor 1 (RPAI-1) is a novel blood-sucking salivary molecule that binds to ADP and attenuates platelet aggregation. In this report, we determine the binding constants and specificity of RPAI-1 for adenine nucleotides and its functional significance. By the Hummel-Dreyer method of equilibrium gel filtration, we show that RPAI-1 binds ADP with a K(0.5) of 48.6 plus minus 12.2 nM. RPAI-1 also displays high-affinity binding to ATP, AMP, Ado, AP4A, and alpha,beta Met ADP; however, RPAI-1 does not bind to inosine, guanosine, uridine, or cytidine. Binding is not modified by EDTA, indicating that Ado structure but not phosphate groups or Ca(2+) is necessary for binding. By computer simulation, we show that RPAI-1 is more effective in scavenging low but not high concentrations of ADP, in contrast to R. prolixus apyrase. RPAI-1 inhibits in vitro the ADP-dependent platelet-rich plasma aggregation by collagen (COLL), TRAP, PAF, and A23187 but did not block platelet aggregation by ristocetin or phorbol myristate acetate (PMA) and only slightly attenuated that by convulxin. RPAI-1 prolongs the closure time as assessed with PFA-100, when COLL-Epi but not COLL-ADP cartridges are employed. RPAI-1 also affects platelet-mediated hemostasis time and COLL-induced thrombus formation at high shear as assessed with the Clot Signature Analyzer. We conclude that RPAI-1 exerts an antiplatelet effect due to scavenging of low concentrations of ADP in vitro and in vivo. RPAI-1 is the first lipocalin described so far with unique specificity for adenine nucleotides.     

Jasmonate-induced lipid peroxidation in barley leaves initiated by distinct 13-LOX forms of chloroplasts

In addition to a previously characterized 13-lipoxygenase of 100 kDa encoded by LOX2:Hv:1 [V?r?s et al., Eur. J. Biochem. 251 (1998), 36-44], two full-length 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 jasmonate-treated 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 lipoxygenase-derived products in the stroma and in the envelope. These data revealed jasmonate-induced 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.     

Cellular junctions of myelinated nerves (Review)

Myelinated nerves are specifically designed to allow the efficient and rapid propagation of action potentials. Myelinating glial cells contain several types of cellular junctions that are found between the myelin lamellas themselves in specialized regions of non-compact myelin and between the myelin membrane and the underlying axon. These include most of the junctional specializations found in epithelial cells, including tight, gap and adherens junctions. However, whereas in epithelial cells these junctions are formed between different cells, in myelinating glia these so called autotypic junctions are found between membrane lamellae of the same cell. In addition, myelinating glial cells form a heterotypic septate-like junction with the axon around the nodes of Ranvier and, in the peripheral nerve system, contact the basal lamina, which surrounds myelinating Schwann cells. This short review discusses the structure, molecular composition and function of the junctions present in myelinating cells, concentrating on the axo-glial junction.     

Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A

Progression through S phase of the eukaryotic cell cycle is regulated by the action of the cyclin dependent protein kinase 2 (CDK2) in association with cyclin A. CDK2/cyclin A phosphorylates numerous substrates. Substrate specificity often employs a dual recognition strategy in which the sequence flanking the phospho-acceptor site (Ser.Pro.X.Arg/Lys) is recognized by CDK2, while the cyclin A component of the complex contains a hydrophobic site that binds Arg/Lys.X.Leu ("RXL" or "KXL") substrate recruitment motifs. To determine additional sequence specificity motifs around the RXL sequence, we have performed X-ray crystallographic studies at 2.3 A resolution and isothermal calorimetry measurements on complexes of phospho-CDK2/cyclin A with a recruitment peptide derived from E2F1 and with shorter 11-mer peptides from p53, pRb, p27, E2F1, and p107. The results show that the cyclin recruitment site accommodates a second hydrophobic residue either immediately C-terminal or next adjacent to the leucine of the "RXL" motif and that this site makes important contributions to the recruitment peptide recognition. The arginine of the RXL motif contacts a glutamate, Glu220, on the cyclin. In those substrates that contain a KXL motif, no ionic interactions are observed with the lysine. The sequences N-terminal to the "RXL" motif of the individual peptides show no conservation, but nevertheless make common contacts to the cyclin through main chain interactions. Thus, the recruitment site is able to recognize diverse but conformationally constrained target sequences. The observations have implications for the further identification of physiological substrates of CDK2/cyclin A and the design of specific inhibitors.     

TOR and PKA signaling pathways converge on the protein kinase Rim15 to control entry into G0

The highly conserved Tor kinases (TOR) and the protein kinase A (PKA) pathway regulate cell proliferation in response to growth factors and/or nutrients. In Saccharomyces cerevisiae, loss of either TOR or PKA causes cells to arrest growth early in G(1) and to enter G(0) by mechanisms that are poorly understood. Here we demonstrate that the protein kinase Rim15 is required for entry into G(0) following inactivation of TOR and/or PKA. Induction of Rim15-dependent G(0) traits requires two discrete processes, i.e., nuclear accumulation of Rim15, which is negatively regulated both by a Sit4-independent TOR effector branch and the protein kinase B (PKB/Akt) homolog Sch9, and release from PKA-mediated inhibition of its protein kinase activity. Thus, Rim15 integrates signals from at least three nutrient-sensory kinases (TOR, PKA, and Sch9) to properly control entry into G(0), a key developmental process in eukaryotic cells.     

Development of Agrobacterium tumefaciens C58-induced plant tumors and impact on host shoots are controlled by a cascade of jasmonic acid,auxin, cytokinin,ethylene and abscisic acid

The development of Agrobacterium tumefaciens-induced plant tumors primarily depends on the excessive production of auxin and cytokinin by enzymes encoded on T-DNA genes integrated into the plant genome. The aim of the present study was to investigate the involvement of additional phytohormone signals in the vascularization required for rapid tumor proliferation. In stem tumors of Ricinus communis L., free auxin and zeatin riboside concentrations increased within 2 weeks to 15-fold the concentrations in control stem tissue. Auxin and cytokinin immunolocalization revealed the highest concentrations within and around tumor vascular bundles with concentration gradients. The time-course of changes in free auxin concentration in roots was inversely correlated with that in the tumors. The high ethylene emission induced by increased auxin- and cytokinin correlated with a 36-fold accumulation of abscisic acid in tumors. Ethylene emitted from tumors and exogenously applied ethylene caused an increase in abscisic acid concentrations also in the host leaves, with a diminution in leaf water vapor conductance. Jasmonic acid concentration reached a maximum already within the first week of bacterial infection. A wound effect could be excluded. The results demonstrate the concerted interaction of a cascade of transiently induced, non-T-DNA-encoded phytohormones jasmonic acid, ethylene and abscisic acid with T-DNA-encoded auxin and zeatin riboside plus trans-zeatin, all of which are required for successful plant tumor vascularization and development together with inhibition of host plant growth.     

Biological and environmental monitoring of hospital personnel exposed to antineoplastic agents: a review of analytical methods

In order to assess occupational exposure of hospital personnel involved in the preparation and administration of antineoplastic drugs, biological and environmental monitoring are essential to identify the main exposure routes and to quantify potential health risks. If workplace contamination cannot be completely avoided, it is of utmost importance to reduce exposure to the lowest possible levels. To this aim, not only do education and training of the exposed subjects play an important role, but accurate standardized sampling techniques and analytical methods are also required. A critical overview of the most significant methods available in the literature is presented and their value is discussed, especially with respect to their sensitivity and specificity. In addition, attention is given to validation procedures and, consequently, to their reliability. The results from the most important surveys carried out at hospital departments are also discussed, with a view to improving both monitoring strategies and moreover working conditions.