From transformation to chronification of migraine: pathophysiological and clinical aspects.

Chronic migraine is a neurological disorder characterized by 15 or more headache days per month of which at least 8 days show typical migraine features. The process that describes the development from episodic migraine into chronic migraine is commonly referred to as migraine transformation or chronification. Ample studies have attempted to identify factors associated with migraine transformation from different perspectives. Understanding CM as a pathological brain state with trigeminovascular participation where biological changes occur, we have completed a comprehensive review on the clinical, epidemiological, genetic, molecular, structural, functional, physiological and preclinical evidence available.

The role of Trichoderma spp. and silica gel in plant defence mechanisms and insect response in vineyard.

Several elicitors, stimulating induced resistance mechanisms, have potential in preventing or mitigating pathogen infections. Some of these compounds, triggering the production of jasmonic acid (JA), a precursor of herbivore-induced plant volatiles, could also play a central role in indirect resistance to pest species, by improving beneficial arthropod performance, and necrotrophic pathogens. In the current work, Trichoderma gamsii/T. asperellum and silica gel treatments - alone and in combination - were studied to evaluate the plant defence mechanism on grapevines (Vitis vinifera L.) by laboratory and field trials. JA production level was measured before and after Plasmopara viticola infection on potted vines. JA production induced by silica gel was higher than that caused by Trichoderma before infection. In Trichoderma-treated plants, JA production increased after P. viticola inoculation. In vineyard field trials, Mymaridae (Hymenoptera: Chalcidoidea) showed higher captures in transparent sticky traps on silica gel-treated plants, in comparison with control. On the other hand, no significant attraction was detected for Ichneumonoidea and other Chalcidoidea in silica gel and T. gamsii/T. asperellum-treated plants. The potential effects of elicitors are discussed, in the frame of attract and reward strategy.

Inner retinal inhibition shapes the receptive field of retinal ganglion cells in primate.

The centre-surround organisation of receptive fields is a feature of most retinal ganglion cells (RGCs) and is critical for spatial discrimination and contrast detection. Although lateral inhibitory processes are known to be important in generating the receptive field surround, the contribution of each of the two synaptic layers in the primate retina remains unclear. Here we studied the spatial organisation of excitatory and inhibitory synaptic inputs onto ON and OFF ganglion cells in the primate retina. All RGCs showed an increase in excitation in response to stimulus of preferred polarity. Inhibition onto RGCs comprised two types of responses to preferred polarity: some RGCs showed an increase in inhibition whilst others showed removal of tonic inhibition. Excitatory inputs were strongly spatially tuned but inhibitory inputs showed more variable organisation: in some neurons they were as strongly tuned as excitation, and in others inhibitory inputs showed no spatial tuning. We targeted one source of inner retinal inhibition by functionally ablating spiking amacrine cells with bath application of tetrodotoxin (TTX). TTX significantly reduced the spatial tuning of excitatory inputs. In addition, TTX reduced inhibition onto those RGCs where a stimulus of preferred polarity increased inhibition. Reconstruction of the spatial tuning properties by somatic injection of excitatory and inhibitory synaptic conductances verified that TTX-mediated inhibition onto bipolar cells increases the strength of the surround in RGC spiking output. These results indicate that in the primate retina inhibitory mechanisms in the inner plexiform layer sharpen the spatial tuning of ganglion cells.

Subclinical pulmonary edema in endurance athletes.

Strenuous exercise may cause progressive and proportional haemodynamic overload damage to the alveolar membrane, even in athletes. Despite the high incidence of arterial desaturation reported in endurance athletes has been attributed, into other factors, also to the damage of the alveolar-capillary membrane this evidence is equivocal. Some studies demonstrated flood of the interstitial space and consequent increase in pulmonary water content, but most of them were able to show this through indirect signs of interstitial oedema. The present review illustrates the literature's data in favour or against pulmonary interstitial edema due to intense exercise in athletes.

Light-induced charge generation in polymeric nanoparticles restores vision in advanced-stage retinitis pigmentosa rats.

Retinal dystrophies such as Retinitis pigmentosa are among the most prevalent causes of inherited legal blindness, for which treatments are in demand. Retinal prostheses have been developed to stimulate the inner retinal network that, initially spared by degeneration, deteriorates in the late stages of the disease. We recently reported that conjugated polymer nanoparticles persistently rescue visual activities after a single subretinal injection in the Royal College of Surgeons rat model of Retinitis pigmentosa. Here we demonstrate that conjugated polymer nanoparticles can reinstate physiological signals at the cortical level and visually driven activities when microinjected in 10-months-old Royal College of Surgeons rats bearing fully light-insensitive retinas. The extent of visual restoration positively correlates with the nanoparticle density and hybrid contacts with second-order retinal neurons. The results establish the functional role of organic photovoltaic nanoparticles in restoring visual activities in fully degenerate retinas with intense inner retina rewiring, a stage of the disease in which patients are subjected to prosthetic interventions.

Chromosome painting in two genera of South American monkeys: species identification, conservation, and management.

Cytogenetic studies showed that a number of New World primate taxa, particularly the genera Alouatta, Aotus, and Callicebus, have highly derived karyotypes. Cytogenetics in these primates, at every level of analysis, has contributed to the recognition of species and revealed that their number was certainly underestimated by researchers relying solely on traditional morphological data. Further attention was drawn to Alouatta and Aotus because they are characterized by translocations of the Y chromosome to autosomes, generating multiple sex chromosome systems. Here we present a report on the hybridization of human chromosome-specific paints on metaphases from 4 individuals originally assigned to Alouatta caraya and 1 individual of Aotuslemurinus. This is only the third karyotype studied with chromosome painting out of more than 10 known karyomorphs in Aotus. The banded chromosomes matched those of karyotype II as defined by Ma et al. [1976a], and we were able to more precisely assign the origin of the sample to A. l. griseimembra. Our results on the Argentinean Alouatta caraya samples were generally comparable to the banding and hybridization pattern of previous studies of A. caraya including the presence of an X(1)X(1)X(2)X(2)/X(1)X(2)Y(1)Y(2) sex chromosome system. The karyotype of the Brazilian Alouatta sample labeled as A. caraya differs from the 3 Argentinean samples by at least 10 chromosome rearrangements. The diploid number, G banding, and hybridization pattern of this female cell line was almost identical to previous painting results on Alouatta guariba guariba. Therefore we must conclude that this cell line is actually from an A. guariba guariba individual. The contribution of cytogenetic tools in identifying species or in this case assigning individuals or cell lines to their precise taxonomic allocation is stressed. Gathering further molecular cytogenetic data on New World primates should be conservation and management priorities.

Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA.

Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence. In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production. Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha. In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein. We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR. One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs. Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE.

Structure of the complex of leech-derived tryptase inhibitor (LDTI) with trypsin and modeling of the LDTI-tryptase system.

BACKGROUND: Tryptase is a trypsin-like serine proteinase stored in the cytoplasmic granules of mast cells, which has been implicated in a number of mast cell related disorders such as asthma and rheumatoid arthritis. Unlike almost all other serine proteinases, tryptase is fully active in plasma and in the extracellular space, as there are no known natural inhibitors of tryptase in humans. Leech-derived tryptase inhibitor (LDTI), a protein of 46 amino acids, is the first molecule found to bind tightly to and specifically inhibit human tryptase in the nanomolar range. LDTI also inhibits trypsin and chymotrypsin with similar affinities. The structure of LDTI in complex with an inhibited proteinase could be used as a template for the development of low molecular weight tryptase inhibitors. RESULTS: The crystal structure of the complex between trypsin and LDTI was solved at 2.0 A resolution and a model of the LDTI-tryptase complex was created, based on this X-ray structure. LDTI has a very similar fold to the third domain of the turkey ovomucoid inhibitor. LDTI interacts with trypsin almost exclusively through its binding loop (residues 3-10) and especially through the sidechain of the specificity residue Lys8. Our modeling studies indicate that these interactions are maintained in the LDTI-tryptase complex. CONCLUSIONS: The insertion of nine residues after residue 174 in tryptase, relative to trypsin and chymotrypsin, prevents inhibition by other trypsin inhibitors and is certainly responsible for the higher specificity of tryptase relative to trypsin. In LDTI, the disulfide bond between residues 4 and 25 causes a sharp turn from the binding loop towards the N terminus, holding the N terminus away from the 174 loop of tryptase.

A new structural class of serine protease inhibitors revealed by the structure of the hirustasin-kallikrein complex.

BACKGROUND: Hirustasin belongs to a class of serine protease inhibitors characterized by a well conserved pattern of cysteine residues. Unlike the closely related inhibitors, antistasin/ghilanten and guamerin, which are selective for coagulation factor Xa or neutrophil elastase, hirustasin binds specifically to tissue kallikrein. The conservation of the pattern of cysteine residues and the significant sequence homology suggest that these related inhibitors possess a similar three-dimensional structure to hirustasin. RESULTS: The crystal structure of the complex between tissue kallikrein and hirustasin was analyzed at 2.4 resolution. Hirustasin folds into a brick-like structure that is dominated by five disulfide bridges and is sparse in secondary structural elements. The cysteine residues are connected in an abab cdecde pattern that causes the polypeptide chain to fold into two similar motifs. As a hydrophobic core is absent from hirustasin the disulfide bridges maintain the tertiary structure and present the primary binding loop to the active site of the protease. The general structural topography and disulfide connectivity of hirustasin has not previously been described. CONCLUSIONS: The crystal structure of the kallikrein-hirustasin complex reveals that hirustasin differs from other serine protease inhibitors in its conformation and its disulfide bond connectivity, making it the prototype for a new class of inhibitor. The disulfide pattern shows that the structure consists of two domains, but only the C-terminal domain interacts with the protease. The disulfide pattern of the N-terminal domain is related to the pattern found in other proteins. Kallikrein recognizes hirustasin by the formation of an antiparallel beta sheet between the protease and the inhibitor. The P1 arginine binds in a deep negatively charged pocket of the enzyme. An additional pocket at the periphery of the active site accommodates the sidechain of the P4 valine.

The 1.2 A crystal structure of hirustasin reveals the intrinsic flexibility of a family of highly disulphide-bridged inhibitors.

BACKGROUND: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 A structure of the hirustasin-kallikrein complex is known, classical methods such as molecular replacement were not successful in solving the structure of free hirustasin. RESULTS: Ab initio real/reciprocal space iteration has been used to solve the structure of free hirustasin using either 1.4 A room temperature data or 1.2 A low temperature diffraction data. The structure was also solved independently from a single pseudo-symmetric gold derivative using maximum likelihood methods. A comparison of the free and complexed structures reveals that binding to kallikrein causes a hinge-bending motion between the two hirustasin subdomains. This movement is accompanied by the isomerisation of a cis proline to the trans conformation and a movement of the P3, P4 and P5 residues so that they can interact with the cognate protease. CONCLUSIONS: The inhibitors from this protein family are fairly flexible despite being highly cross-linked by disulphide bridges. This intrinsic flexibility is necessary to adopt a conformation that is recognised by the protease and to achieve an optimal fit, such observations illustrate the pitfalls of designing inhibitors based on static lock-and-key models. This work illustrates the potential of new methods of structure solution that require less or even no prior phase information.

Interferon-alpha hybrids.

The alpha-interferons (IFN-alpha) belong to a family of polypeptides comprising several subtypes. Using recombinant DNA technology, it has been possible to create IFN hybrids that provide novel combinations of the amino acid residues from the parental protein sequences. They have been used to study structure-activity relationships of IFN-alpha and interactions with the IFN-alpha receptor, and to create analogs of natural IFNs with novel properties for potential therapeutic application. The biological data obtained with these hybrids are now evaluated in terms of the published structural and homology models of IFN-beta and -alpha.

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

Recombinant hirustasin: production in yeast, crystallization, and interaction with serine proteases.

A synthetic gene coding for the 55-amino acid protein hirustasin, a novel tissue kallikrein inhibitor from the leech Hirudo medicinalis, was generated by polymerase chain reaction using overlapping oligonucleotides, fused to the yeast alpha-factor leader sequence and expressed in Saccharomyces cerevisiae. Recombinant hirustasin was secreted mainly as incompletely processed fusion protein, but could be processed in vitro using a soluble variant of the yeast yscF protease. The processed hirustasin was purified to better than 97% purity. N-terminal sequence analysis and electrospray ionization mass spectrometry confirmed a correctly processed N-terminus and the expected amino acid sequence and molecular mass. The biological activity of recombinant hirustasin was identical to that of the authentic leech protein. Crystallized hirustasin alone and in complex with tissue kallikrein diffracted beyond 1.4 A and 2.4 A, respectively. In order to define the reactive site of the inhibitor, the interaction of hirustasin with kallikrein, chymotrypsin, and trypsin was investigated by monitoring complex formation in solution as well as proteolytic cleavage of the inhibitor. During incubation with high, nearly equimolar concentration of tissue kallikrein, hirustasin was cleaved mainly at the peptide bond between Arg 30 and Ile 31, the putative reactive site, to yield a modified inhibitor. In the corresponding complex with chymotrypsin, mainly uncleaved hirustasin was found and cleaved hirustasin species accumulated only slowly. Incubation with trypsin led to several proteolytic cleavages in hirustasin with the primary scissile peptide bond located between Arg 30 and Ile 31. Hirustasin appears to fall into the class of protease inhibitors displaying temporary inhibition.

Antitumor activity of recombinant adenoviral vectors expressing murine IFN-alpha in mice injected with metastatic IFN-resistant tumor cells.

Recent studies have shown that gene therapy with type I interferon (IFN) in an adenovirus vector is a powerful tool to suppress the growth of human tumors transplanted in immune-deficient mice. However, in these studies the host immune-mediated effects, which may be important in mediating the long-term control of tumor growth by these cytokines, was not studied. In this paper, we evaluate the antitumor efficacy of different adenoviral vectors containing mouse IFN-alpha genes (i.e., a first-generation replication-defective vector containing IFN-alpha1 and two different second-generation vectors containing IFN-alpha2) in immunocompetent DBA/2 mice transplanted with highly metastatic Friend leukemic cells resistant in vitro to type I IFN. We found that injection of all the different adenovirus vectors containing mouse IFN-alpha( genes resulted in a marked antitumor response in mice transplanted either subcutaneously or intravenously with IFN-resistant Friend leukemic cells compared to tumor-bearing animals inoculated with a control vector. Tumor growth inhibition after injection of IFN-adenovirus vectors was associated with a prolonged presence of high IFN levels in the sera of the injected mice. Suppression of metastatic tumor growth was also observed after a single injection of the IFN--adenovirus recombinant vectors, whereas a comparable antitumor response generally required several injections of high doses of IFN. Altogether, these results demonstrate that IFN--adenoviral vectors can efficiently inhibit metastatic tumor growth by host-mediated mechanisms and suggest that adenovirus-mediated IFN-alpha gene therapy may represent an attractive alternative to the conventional clinical use of this cytokine, which generally requires multiple injections of high IFN doses for a prolonged period of time.

Purification, analysis, and enzymatic activity of recombinant human synovial fluid phospholipase A2 and N-terminal variants.

Recombinant human synovial fluid phospholipase A2 (rPLA2) and several variants with N-terminal sequences modified by addition or deletion of one or two amino acid residues (ala or Met; Des-Asn1, Leu2) have been expressed in mammalian cells and in Escherichia coli, respectively, purified to homogeneity, and characterized. The observed values for the molecular mass of rPLA2 and variants are in complete agreement with the predicted values for a correctly folded structure containing seven disulfide bridges. Moreover, the relative proportions of the various types of secondary structures of the variants of rPLA2, as measured by CD spectroscopy, are similar to that found for native porcine pancreatic PLA2, indicating that the recombinant proteins are correctly folded. Enzymatic activities of rPLA2 with modified N-termini decreased to 1.3-0.005% of the activity of the mature rPLA2, emphasizing a key role of the N-terminus for catalytic activity.

Refolding, isolation and characterization of crystallizable human interferon-alpha 8 expression in Saccharomyces cerevisiae.

Human interferon-alpha 8 was expressed in Saccharomyces cerevisiae and found to accumulate intracellularly in an insoluble form. The protein could be solubilized and converted to a biologically active form with high yield by a denaturation-refolding procedure. The interferon-alpha 8 was further purified to apparent homogeneity by copper-chelate affinity chromatography and anion-exchange chromatography and fully characterized by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, mass spectrometry, circular-dichroism (CD) spectroscopy and specific activity. Secondary-structure predictions from CD spectroscopy indicate that the molecule is correctly folded. Peptide mapping supported the correct sequence and the expected disulfide-bridge connectivity. The purified protein elutes on reversed-phase high-pressure liquid chromatography (RP-HPLC) as two peaks. Electrospray mass spectrometry and N-terminal sequence analysis of the minor component indicated the existence of an N-terminal acetyl group for the later eluting HPLC-component. In anti-viral assays, the two IFN forms were equally active. Hexagonal crystals of this interferon preparation could be obtained. On the basis of the electrophoretic mobility, HPLC profile, and biological activity assay, the crystalline material was judged to be identical to the uncrystallized interferon. Interferon in crystallized form was found to be stable for up to 24 months and, therefore, could be used for long-term storage, particularly for material intended for clinical use.

Effects of heavy ions on visual function and electrophysiology of rodents: the ALTEA-MICE project.

ALTEA-MICE will supplement the ALTEA project on astronauts and provide information on the functional visual impairment possibly induced by heavy ions during prolonged operations in microgravity. Goals of ALTEA-MICE are: (1) to investigate the effects of heavy ions on the visual system of normal and mutant mice with retinal defects; (2) to define reliable experimental conditions for space research; and (3) to develop animal models to study the physiological consequences of space travels on humans. Remotely controlled mouse setup, applied electrophysiological recording methods, remote particle monitoring, and experimental procedures were developed and tested. The project has proved feasible under laboratory-controlled conditions comparable in important aspects to those of astronauts' exposure to particle in space. Experiments are performed at the Brookhaven National Laboratories [BNL] (Upton, NY, USA) and the Gesellschaft für Schwerionenforschung mbH [GSI]/Biophysik (Darmstadt, FRG) to identify possible electrophysiological changes and/or activation of protective mechanisms in response to pulsed radiation. Offline data analyses are in progress and observations are still anecdotal. Electrophysiological changes after pulsed radiation are within the limits of spontaneous variability under anesthesia, with only indirect evidence of possible retinal/cortical responses. Immunostaining showed changes (e.g. increased expression of FGF2 protein in the outer nuclear layer) suggesting a retinal stress reaction to high-energy particles of potential relevance in space.

Craniofacial morphometric variation and the biological history of the peopling of Sardinia.

The aim of this work is to explore the pattern of craniofacial morphometric variation and the relationships among five prehistoric Sardinian groups dated from Late Neolithic to the Nuragic Period (Middle and Late Bronze Age), in order to formulate hypotheses on the peopling history of Sardinia. Biological relationships with coeval populations of central peninsular Italy were also analysed to detect influences from and towards extra-Sardinian sources. Furthermore, comparison with samples of contemporary populations from Sardinia and from continental Italy provided an indication of the trend leading to the final part of the peopling history. Finally, Upper Palaeolithic and Mesolithic samples were included in the analyses to compare the prehistoric Sardinians with some of their potential continental ancestors. The analysis is based on multivariate techniques including Mahalanobis D(2) distance, non-parametric multidimensional scaling (MDS) and principal component analysis (PCA). The results showed the tendency to progressive differentiation between Sardinian groups and peninsular Italian groups, with the possible exception of a discontinuity showed by the Bonnànaro (Early Bronze Age) Sardinian sample. Several aspects of the morphological results were found to agree with the current genetic evidence available for the present-day Sardinian population and a Nuragic sample: (1) biological divergence between the Sardinian and peninsular Italian populations; (2) similarity/continuity among Neolithic, Bronze Age and recent Sardinians; (3) biological separation between the Nuragic and Etruscan populations; (4) contribution of a Palaeo-Mesolithic gene pool to the genetic structure of current Sardinians.

Randomized, double-blind, phase III, controlled trial comparing levobupivacaine 0.25%, ropivacaine 0.25% and bupivacaine 0.25% by the caudal route in children.

BACKGROUND: The rationale for replacing racemic bupivacaine with the s-enantiomers levobupivacaine and ropivacaine is to provide a wider margin of safety with the same analgesic efficacy and less postoperative motor block. In a randomized, double-blind, phase III, controlled trial we compared the caudal administration of levobupivacaine 0.25% and ropivacaine 0.25% with bupivacaine 0.25% in children. METHODS: Ninety-nine ASA I-II children less than 10 yr old scheduled for elective sub-umbilical surgery were randomized to receive caudal block with bupivacaine 0.25%, ropivacaine 0.25% or levobupivacaine 0.25%. The primary outcome of the study was the clinical efficacy of the caudal block during the operation. Secondary outcome measures were analgesic onset time, pain relief after the operation and residual motor blockade. RESULTS: The proportion of children with effective analgesia during the operation was similar among groups. There were no significant differences in the analgesic onset time of the caudal block. Bupivacaine produced a significant incidence of residual motor block compared with levobupivacaine or ropivacaine at wake-up (P<0.01). There were no significant differences in the number of patients receiving rescue analgesia after surgery. However, analgesic block lasted significantly longer in patients receiving bupivacaine (P=0.03). CONCLUSION: During sub-umbilical surgery, caudal levobupivacaine, ropivacaine and bupivacaine provided comparable analgesic efficacy. Bupivacaine produced a higher incidence of residual motor blockade and a longer analgesic block than ropivacaine and levobupivacaine.

Characterization of the RNA binding proteins forming complexes with a novel putative regulatory region in the 3'-UTR of TNF-alpha mRNA.

Tumor necrosis factor-alpha (TNF-alpha) is a key cytokine regulator of an early immune response and the central mediator of deleterious effects of systemic inflammatory response syndrome. High production of TNF-alpha by macrophages requires two signals: the first signal induces transcription, while the second signal releases the translational repression of TNF-alpha mRNA. The translational control of TNF-alpha expression is conferred by sequences in the 3'-untranslated region (3'-UTR) of its mRNA. Previously, we have characterized protein complexes binding to the main AU-rich region in the 3'-UTR of murine TNF-alpha mRNA. Here we describe a second protein binding region which is located 147 bases downstream of the first region and interacts with at least seven distinct protein species present in murine macrophages. The second protein binding motif contains a single AUAUUUAU sequence motif; a mutation of this sequence to AUAGGUAU abrogates the binding of proteins. Some of the macrophage proteins mutually compete for the binding to both regions, while others seem to be region specific. The existence of the two protein binding domains explains the previously published data addressing the translatibility of a reporter gene linked to various deletion mutants of the TNF-alpha 3'-UTR. Both the sequence and position of the two putative protein binding regions are highly conserved across species, indicating their important role in the regulation of translational repression and inducibility of TNF-alpha synthesis.