Identification and characterization of a histamine-binding lipocalin-like molecule from the relapsing fever tick Ornithodoros turicata.

Lipocalins are low molecular weight membrane transporters that are abundantly expressed in the salivary glands and other tissues of ticks. In this study, we identified a lipocalin-like molecule, designated as otlip, from the soft ticks Ornithodoros turicata, the vector for the relapsing fever causing spirochete Borrelia turicatae. We noted that the expression of otlip was developmentally regulated, with adult ticks expressing significantly higher levels in comparison to the larvae or nymphal ticks. Expression of otlip was evident in both fed and unfed O. turicata ticks, with significantly increased expression in the salivary glands in comparison to the midgut or ovary tissues. High conservation of the biogenic amine-binding motif was evident in the deduced primary amino acid sequence of Otlip. Protein modelling of Otlip revealed conservation of most of the residues involved in binding histamine or serotonin ligand. In vitro assays demonstrated binding of recombinant Otlip with histamine. Furthermore, prediction of post-translational modifications revealed that Otlip contained phosphorylation and myristoylation sites. Taken together, our study not only provides evidence for the presence of a lipocalin-like molecule in O. turicata ticks but also suggests a role for this molecule in the salivary glands of this medically important vector.

A novel family of RGD-containing disintegrins (Tablysin-15) from the salivary gland of the horsefly Tabanus yao targets αIIbß3 or αVß3 and inhibits platelet aggregation and angiogenesis.

A novel family of RGD-containing molecules (Tablysin-15) has been molecularly characterised from the salivary gland of the haematophagous horsefly Tabanus yao. Tablysin-15 does not share primary sequence homology to any disintegrin discovered so far, and displays an RGD motif in the N-terminus of the molecule. It is also distinct from disintegrins from Viperidae since its mature form is not released from a metalloproteinase precursor. Tablysin-15 exhibits high affinity binding for platelet αIIbß3 and endothelial cell αVß3 integrins, but not for α5ß1 or α2ß1. Accordingly, it blocks endothelial cell adhesion to vitronectin (IC50 ~1 nM) and marginally to fibronectin (IC50 ~1 µM), but not to collagen. It also inhibits fibroblast growth factor (FGF)-induced endothelial cell proliferation, and attenuates tube formation in vitro. In platelets, Tablysin-15 inhibits aggregation induced by collagen, ADP and convulxin, and prevents static platelet adhesion to immobilised fibrinogen. In addition, solid-phase assays and flow cytometry demonstrates that αIIbß3 binds to Tablysin-15. Moreover, immobilised Tablysin-15 supports platelet adhesion by a mechanism which was blocked by anti-integrin αIIbß3 monoclonal antibody (e.g. abciximab) or by EDTA. Furthermore, Tablysin-15 dose-dependently attenuates thrombus formation to collagen under flow. Consistent with these findings, Tablysin-15 displays antithrombotic properties in vivo suggesting that it is a useful tool to block αIIbß3, or as a prototype to develop antithrombotics. The RGD motif in the unique sequence of Tablysin-15 represents a novel template for studying the structure-function relationship of the disintegrin family of inhibitors.

Nitrophorins and related antihemostatic lipocalins from Rhodnius prolixus and other blood-sucking arthropods.

Recent gene sequence and crystal structure determinations of salivary proteins from several blood-sucking arthropods have revealed an unusual evolutionary relationship: many such proteins derive their functions from lipocalin protein folds. Many blood-sucking arthropods have independently evolved the ability to overcome a host organism's means of preventing blood loss (called hemostasis). Most blood feeders have proteins that induce vasodilation, inhibit blood coagulation, and reduce inflammation, but do so by distinctly different mechanisms. Despite this diversity, in many cases the antihemostatic activities in such organisms reside in proteins with lipocalin folds. Thirteen such lipocalins are described in this review, with a particular focus on the heme-containing nitrophorins from Rhodnius prolixus, which transport nitric oxide, sequester histamine, and disrupt blood coagulation. Also described are the antiplatelet compounds RPAI, moubatin, and pallidipin from R. prolixus, Ornithodoros moubata, and Triatoma pallidipennis; the antithrombin protein triabin from T. pallidipennis; and the tick histamine binding proteins from Rhipicephalus appendiculatus.

Kinetics and equilibria in ligand binding by nitrophorins 1-4: evidence for stabilization of a nitric oxide-ferriheme complex through a ligand-induced conformational trap.

Nitrophorins 1-4 (NP1-4) are ferriheme proteins from the blood-sucking insect Rhodnius prolixus that transport nitric oxide (NO) to the victim, sequester histamine, and inhibit blood coagulation. Here, we report kinetic and thermodynamic analyses for ligand binding by all four proteins and their reduction potentials. All four undergo biphasic association and dissociation reactions with NO. The initial association is fast (1.5-33 microM(-)(1) s(-)(1)) and similar to that of elephant metmyoglobin. However, unlike in metmyoglobin, a slower second phase follows ( approximately 50 s(-)(1)), and the stabilized final complexes are resistant to autoreduction (E degrees = +3 to +154 mV vs normal hydrogen electrode). NO dissociation begins with a slow, pH-dependent step (0.02-1.4 s(-)(1)), followed by a faster phase that is again similar to that of metmyoglobin (3-52 s(-)(1)). The equilibrium dissociation constants are quite small (1-850 nM). NP1 and NP4 display larger release rate constants and smaller association rate constants than NP2 and NP3, leading to values for K(d) that are about 10-fold greater. The results are discussed in light of the recent crystal structures of NP1, NP2, and NP4, which display open, polar distal pockets, and of NP4-NO, which displays an NO-induced conformational change that leads to expulsion of solvent and complete burial of the NO ligand in a now nonpolar distal pocket. Taken together, the results suggest that tighter NO binding in the nitrophorins is due to the trapping of the molecule in a nonpolar distal pocket rather than through formation of particularly strong Fe-NO or hydrogen bonds.

The crystal structure of nitrophorin 2. A trifunctional antihemostatic protein from the saliva of Rhodnius prolixus.

Nitrophorin 2 (NP2) (also known as prolixin-S) is a salivary protein that transports nitric oxide, binds histamine, and acts as an anticoagulant during blood feeding by the insect Rhodnius prolixus. The 2.0-A crystal structure of NP2 reveals an eight-stranded antiparallel beta-barrel containing a ferric heme coordinated through His(57), similar to the structures of NP1 and NP4. All four Rhodnius nitrophorins transport NO and sequester histamine through heme binding, but only NP2 acts as an anticoagulant. Here, we demonstrate that recombinant NP2, but not recombinant NP1 or NP4, is a potent anticoagulant; recombinant NP3 also displays minor activity. Comparison of the nitrophorin structures suggests that a surface region near the C terminus and the loops between beta strands B-C and E-F is responsible for the anticoagulant activity. NP2 also displays larger NO association rates and smaller dissociation rates than NP1 and NP4, which may result from a more open and more hydrophobic distal pocket, allowing more rapid solvent reorganization on ligand binding. The NP2 protein core differs from NP1 and NP4 in that buried Glu(53), which allows for larger NO release rates when deprotonated, hydrogen bonds to invariant Tyr(81). Surprisingly, this tyrosine lies on the protein surface in NP1 and NP4.

Nitric oxide binding to nitrophorin 4 induces complete distal pocket burial.

The nitrophorins comprise an unusual family of proteins that use ferric (Fe(III)) heme to transport highly reactive nitric oxide (NO) from the salivary gland of a blood sucking bug to the victim, resulting in vasodilation and reduced blood coagulation. We have determined structures of nitrophorin 4 in complexes with H2O, cyanide and nitric oxide. These structures reveal a remarkable feature: the nitrophorins have a broadly open distal pocket in the absence of NO, but upon NO binding, three or more water molecules are expelled and two loops fold into the distal pocket, resulting in the packing of hydrophobic groups around the NO molecule and increased distortion of the heme. In this way, the protein apparently forms a 'hydrophobic trap' for the NO molecule. The structures are very accurate, ranging between 1.6 and 1.4 A resolutions.

The crystal structure of nitrophorin 4 at 1.5 A resolution: transport of nitric oxide by a lipocalin-based heme protein.

BACKGROUND: Nitrophorins are nitric oxide (NO) transport proteins from the saliva of blood-feeding insects, which act as vasodilators and anti-platelet agents. Rhodnius prolixus, an insect that carries the trypanosome that causes Chagas' disease, releases four NO-loaded nitrophorins during blood feeding, whereupon the ligand is released into the bloodstream or surrounding tissue of the host. Histamine, a signaling molecule released by the host upon tissue damage, is tightly bound by the nitrophorins; this may facilitate the release of NO and reduce inflammation in the host. RESULTS: Recombinant nitrophorin 4 (NP4) was expressed in Escherichia coli, reconstituted with heme, and found to bind NO and histamine in a manner similar to that of the natural protein. The crystal structure of NP4 revealed a lipocalin-like eight-stranded beta barrel, with heme inserted into one end of the barrel. His59 ligates the proximal site on the heme, a solvent molecule (NH3) ligates the distal site, and three additional solvent molecules occupy the distal pocket. Buried in the protein interior are Glu55 and three solvent molecules. A detailed comparison with other lipocalins suggests that NP4 is closely related to the biliverdin-binding proteins from insects. CONCLUSIONS: The nitrophorins have a unique hemoprotein structure and are completely unlike the globins, the only other hemoproteins designed to transport dissolved gases. Compared with the recently described structure of NP1, the NP4 structure is considerably higher resolution, confirms the unusual placement of ionizable groups in the protein interior, and clarifies the solvent arrangement in the distal pocket. It also provides a striking example of structural homology where sequence homology is minimal.

A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis.

A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This omega-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

Crystal structures of a nitric oxide transport protein from a blood-sucking insect.

The nitrophorins are heme-based proteins from the salivary glands of the blood-sucking insect Rhodnius prolixus that deliver nitric oxide gas (NO) to the victim while feeding, resulting in vasodilation and inhibition of platelet aggregation. The nitrophorins also bind tightly to histamine, which is released by the host to induce wound healing. Here we present three crystal structures of nitrophorin 1 (NP1): bound to cyanide, which binds in a manner similar to NO (2.3 A resolution); bound to histamine (2.0 A resolution); and bound to what appears to be NH3 from the crystallization solution (2.0 A resolution). The NP1 structures reveal heme to be sandwiched between strands of a lipocalin-like beta-barrel, and in an arrangement unlike any other gas-transport protein discovered to date. The heme is six-coordinate with a histidine (His 59) on the proximal side, and ligand in a spacious pocket on the distal side. The structures confirm that NO and histamine compete for the same binding pocket and become buried on binding. The dissociation constant for histamine binding was found to be 19 nM, approximately 100-fold lower than that for NO.

Human thioredoxin homodimers: regulation by pH, role of aspartate 60, and crystal structure of the aspartate 60 --> asparagine mutant.

Thioredoxins are a group of ca. 12 kDa redox proteins that mediate numerous cytosolic processes in all cells. Human thioredoxin can be exported out of the cell where it has additional functions including the ability to stimulate cell growth. A recent crystal structure determination of human thioredoxin revealed an inactive dimeric form of the protein covalently linked through a disulfide bond involving Cys 73 from each monomer [Weichsel et al. (1996) Structure 4, 735-751]. In the present study, apparent dissociation constants (Kapp) for the noncovalently linked dimers were determined at various pHs using a novel assay in which preformed dimers, but not monomers, were rapidly linked through oxidation (with diamide) of the Cys 73 disulfide bond, and the relative amounts of monomer and dimer were detected by gel filtration. The values obtained were pH-dependent, varying between 6.1 and 166 microM for the pH range of 3.8-8.0, and were consistent with the titration of a single ionizable group having a pKa of 6.5. A similar value was obtained using gel filtration at pH 3.8 (Kapp = 164 microM), and the crystal structure of the diamide-oxidized protein was determined to be nearly identical to that obtained in the absence of diamide. Asp 60 lies in the dimer interface and was found to be responsible for the pH dependence for dimer formation, and therefore must have a pKa elevated by approximately 2.5 pH units. Mutation of Asp 60 to asparagine abolished nearly all of the pH dependence for dimer formation. The crystal structure of the D60N mutant revealed a dimer nearly identical to the wild type, but, surprisingly, it had the Asn 60 side chain rotated out of the dimer interface and replaced with two water molecules. The values obtained for Kapp suggest human thioredoxin may dimerize in vivo and possible roles for such dimers are discussed.

Nitric oxide binding and crystallization of recombinant nitrophorin I, a nitric oxide transport protein from the blood-sucking bug Rhodnius prolixus.

A nitric oxide transport protein (nitrophorin I) from the salivary glands of the blood-sucking bug Rhodnius prolixus has been expressed as an insoluble form in Escherichia coli, reconstituted with heme, and characterized with respect to NO binding kinetics and equilibria. NO binding and absorption spectra for recombinant nitrophorin I were indistinguishable from those of the insect-derived protein. The degree of NO binding, the rate of NO release, and the Soret absorption maxima for nitrophorin I were all pH dependent. The NO dissociation constant rose 9-fold over the pH range 5.0-8.3, from 0.19 x 10(-6) to 1.71 x 10(-6). The NO dissociation rate rose 2500-fold between pH 5.0 and pH 8.3, from 1.2 x 10(-3) to 3.0 s(-1). Thus, the NO association rate must also be pH dependent and reduced at pH 5.0 by approximately 280-fold. These factors are consistent with nitrophorin function: NO storage in the apparent low pH of insect salivary glands and NO release into the tissue of the insect's host, where vasodilation is induced. The reversible nature of NO binding, which does not occur with most other heme proteins, and the apparent kinetic control of NO release are discussed. We also report crystals of nitrophorin I that are suitable for structure determination by X-ray crystallography. The most promising crystal form contains two protein molecules in the asymmetric unit and diffracts beyond 2.0 A resolution.

Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1.

Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize alpha-pinene, limonene, of the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.

[Fatal traffic accidents in Denmark. Prehospital changes in consciousness and loss of life-years due to traffic accidents]. / Trafikdrab i Danmark. AEndringer i den cerebrale tilstand i den proehospitale fase for trafikdraebte samt tabte leveår ved trafikulykker.

The aim of the study was to investigate the level of consciousness during the prehospital period of 299 persons killed in traffic accidents, and to determine the amount of lost years of life, during the period from January 1st 1986 to December 31st 1991. The study was a retrospective investigation based on police reports, medical records, death certificates and autopsy reports. The investigation was done in an area of Denmark without medically staffed prehospital care. The study demonstrates that the patients' cerebral condition worsened dramatically during the prehospital period. For these 299 killed persons a total of 10,368 years of life were lost, an average of 34.7 years per victim. One hundred and eighty-five persons between 18 and 65 years of age had a loss of 5,390 years of productivity. Medically staffed prehospital care at a high level ought to be built up in the whole country.

[Fatal traffic accidents in Denmark. Survival time and factors of importance for the prehospital phase]. / Trafikdrab i Danmark. Overlevelsestiden samt faktorer af betydning for den praehospitale tidsfase og det terapifrie intervals laengde for trafikdraebte.

The aim of the study was to determine the length of time of survival, and which factors affected the length of the prehospital phase/treatment-free period for persons who died as a result of a traffic accident in the County of Southern Jutland during the period from 1 January 1986 to 31 December 1991. The study was conducted as a retrospective investigation based on police reports and medical records. Two hundred and ninety-nine traffic victims were included. One hundred and nineteen were still alive when the ambulance reached the scene of the accident. Thirty-five of these died within one hour of the accident occurring, and 24 of the 35 died before reaching the hospital. The number of patients who died days to weeks after the trauma was lower than expected. In 80.2% of the traffic accidents (comprising 229 people killed and 193 wounded) there were other factors than distance from accident site to hospital that had a negative effect on the length of the prehospital phase and treatment-free period. It is concluded that the time that elapses between the accident occurring and the patient arriving at hospital must be better utilized, and that when evaluating the length of the prehospital phase/treatment-free period there are other factors than distance to hospital that must be taken into account.

Further validation of a multiplex STR system for use in routine forensic identity testing.

A polymerase chain reaction- (PCR) based short tandem repeat (STR) system has recently been developed for use in routine forensic identity testing. The methodology involves the simultaneous amplification of alleles at four loci on different chromosomes, followed by the fluorescent detection of products using an automated DNA sequencer. The adoption of this technology into operational casework offers several advantages over systems currently in use, particularly the ability to obtain results from very old or small samples, reduced operator time when compared with conventional DNA (single locus probe) analysis and the potential for automation. Validation studies were incorporated into the development work on this system. The scope of these studies has been extended by further investigation carried out in this laboratory to test the reliability of the system under normal operational procedures. It was demonstrated that the precision of size determination was sufficient for the discrimination of alleles and size windows for allelic designation were established. A collaborative exercise carried out in conjunction with two independent laboratories demonstrated the robustness of allelic designation. Having tested both the DNA quantification and amplification techniques against DNA samples from a wide range of animal and microbial species, it was confirmed that results are only obtained from higher primate DNA. The PCR methodology was tested with both simulated and real casework samples (over 250 in total). Reportable results were obtained from most items yielding extracted DNA. Approximately 20% of the casework items from which no grouping (ABO, PGM) nor SLP results were obtained, gave reportable STR results. A method for the routine purification of DNA extracts which failed to amplify was established and validated for use in forensic casework. The STR multiplex system developed by Kimpton et al. proved robust and reliable when tested under the operational procedures in place in this laboratory.

Expression of cytochrome P450 genes of the CYP4 family in midgut and fat body of the tobacco hornworm, Manduca sexta.

Two conserved regions in the alignment of cytochrome P450 family 4 (CYP4) proteins served as guide to the synthesis of degenerate oligonucleotide primers. The primers were used in PCR from a midgut cDNA library and RT-PCR from fat body mRNA, both from last instar larvae of the tobacco hornworm, Manduca sexta. The PCR products of 443-449 bp were cloned and sequenced. Nine P450 clones representing four new genes were obtained from the midgut. Fifteen P450 clones representing three new genes were obtained from the fat body. Two genes were expressed in both tissues. A number of putative allelic variants were also observed for three of the P450 genes. The resulting sequences of 130-132 amino acids were aligned to generate a parsimony analysis of CYP4 P450 proteins. Two new subfamilies of CYP4 were designated from M. sexta by these procedures, CYP4L and CYP4M. The sequence of a full-length cDNA clone for CYP4M2 (41.2% identity to CYP4C1) confirmed that the PCR products obtained by this method were P450s belonging to the CYP4 family. The developmental expression of the CYP4 genes appeared to be coordinately regulated in both fat body and midgut. In the fat body, CYP4 mRNA levels declined after the first day of the final larval instar, peaked during the wandering stage, and fell again until the prepupal molt. Midgut CYP4 mRNA levels were higher during the active feeding, midwandering, prepupal, and pupal stages. Addition of 2-tridecanone or 2-undecanone to the diet induced several P450s in the midgut and in the fat body. Phenobarbital induced CYP4M1 in the fat body and dietary clofibrate induced the mRNA levels of CYP4M1 and CYP4M3 in the midgut. The results indicate that at least four CYP4 genes are expressed in single tissues of a Lepidopteran insect. Several of these P450 may be involved in tissue responses to xenobiotics.

Photoaffinity labeling of methyl farnesoate epoxidase in cockroach corpora allata.

The last enzyme in the biosynthetic pathway to juvenile hormone III in the corpora allata of hemimetabolous insects is methyl farnesoate epoxidase, a cytochrome P450 monooxygenase. Assays with intact glands incubated in vitro and with gland homogenates have identified a series of 1,5-disubstituted imidazoles as potent inhibitors of the enzyme. We have designed, synthesized and tested two imidazoles, diazirine-Ice T and benzophenone-Ice T, in which a radiolabeled and photoactivatable diazirine or benzophenone group was introduced to label the hydrophobic substrate binding site of the enzyme. Our results show that these bifunctional compounds inhibit JH III synthesis by intact glands as well as methyl farnesoate epoxidation by gland homogenates. Moreover both compounds selectively label a protein of ca. 55 kDa in corpora allata of the cockroach, Diploptera punctata. These photoaffinity labels, which use an imidazole to coordinate to the heme iron and a photoreactive group to modify the hydrophobic substrate binding pocket, are specific and effective probes for the molecular analysis of methyl farnesoate epoxidase.

Report on the second EDNAP collaborative STR exercise. European DNA Profiling Group.

The European DNA Profiling Group (EDNAP) has previously carried out collaborative exercises to determine which STR systems will produce results that can be reproduced by different laboratories. The first EDNAP exercise involving STR systems focused on different types of loci: a simple locus with six common alleles (HUMTH01) and a complex locus with > 35 alleles (ACTBP2). Generally the simpler STR system was found to be readily amenable for use across a wide range of different technologies, whereas a more complex locus presented difficulties. The second EDNAP STR exercise was intended to take the process of investigation a stage further. Some laboratories are developing automation, coupled with fluorescent methods of detection and multiplex applications, whereas others use manual methods involving visual detection techniques such as silver staining. The purpose of this exercise was to determine whether loci amenable to multiplexing with automation (as a quadruplex reaction) could also be successfully used with manual methods, either by multiplexing in duplex reactions or alternatively by using just a single pair of PCR primers.