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.

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.

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.

Generation of minisatellite variant repeat codes on an automated DNA sequencer using fluorescent dye-labeled primers.

We demonstrate the potential to generate unambiguous minisatellite variant repeat PCR profiles on an automated DNA sequencer using fluorescent dye-labeled primers. The products of a 24-cycle PCR amplification using 100 ng of template DNA were analyzed by conventional blot hybridization and also on an Applied Biosystems 373A DNA Sequencer. The results from the sequencer were clear to the 20th repeat when using a primer labeled with 6-FAM, whereas conventional analysis allowed codes to be defined to in excess of the 60th repeat. Use of a modified Tag primer sequence, which eliminates the potential for self-annealing, did not improve the clarity of the results under the PCR cycling conditions used.

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.

Identification of a volatile attractant for Diabrotica and Acalymma spp. from blossoms of Cucurbita maxima duchesne.

Fractionation of headspace volatiles from Cucurbita maxima blossoms by high-performance liquid chromatography resulted in the isolation of a single component which was highly active in an electroantennogram bioassay on Diabrotica undecimpunctata howardi antennae. This compound was identified as indole by gas chromatography-mass spectrometry. Field-trapping bioassays were conducted which indicated that indole is a potent attractant of the western corn rootworm,D. virgifera virgifera, and the striped cucumber beetle,Acalymma vittatum. The southern corn rootworm,D. u. howardi, did not respond, despite its strong EAG response. The sex ratio ofD. v. virgifera found in indole-baited traps varied seasonally. Males were trapped in abundance in late July and later September, 1983, while females were more abundant August and early September. The effectiveness of indole as aD. v. virgifera attractant also varied seasonally. A prolonged period of depressed trap catches occurred in early August 1983, during the silking and tasseling period of the corn in the field where trapping was carried out.

Report of the European DNA profiling group (EDNAP)--towards standardisation of short tandem repeat (STR) loci.

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using short tandem repeat (STR) loci. Two different STRs were chosen--HUMTH01 and the AT-rich HUMACTBP2 (SE33). The former locus has only five common alleles, whereas the latter is complex and has at least 30 alleles. Laboratories were asked to test seven blood stains and to report the results to the coordinating laboratory. The exercise demonstrated that the simple STR systems such as HUMTH01 are more amenable to adoption as standard loci than complex AT-rich systems.

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.

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.

Perioperative analgesia by 3-in-one block in total hip arthroplasty. Prospective randomized blind study.

In a prospective randomized blind study of 182 patients undergoing total hip arthroplasty, the patients per- and postoperative need for analgesics was assessed in two groups, one receiving general anesthesia and a 3-in-one block, the other group only general anesthesia. Although we found a significant reduction in analgesics per- and postoperatively in the 3-in-one block group, the difference in analgesics was small and has no clinical relevance in the patient with a healthy cardiovascular status. We conclude that 3-in-one block in combination with light general anesthesia is not the anesthetic of choice in total hip arthroplasty using the posterior approach.

[Maternal hemodynamic monitoring in severe pre-eclampsia]. / Maternel haemodynamisk monitorering ved svaer praeeklampsi.

Based on a review of the literature, hemodynamic signs and measurements in severe preeclampsia are described. Advantages and disadvantages of various hemodynamic methods and resulting indications for therapy are discussed. We found that in respect of hemodynamic monitoring in severe preeclampsia there are no indications for central venous pressure and conclude that at present there is no alternative to right heart catheterization (Swan-Ganz catheter).

[Treatment of women with severe pre-eclampsia in an intensive care unit during a 3-year period]. / Behandling af kvinder med svaer praeeklampsi på intensiv afdeling over en treårig periode.

During the period 1.1.1986-31.12.1988, the number of patients with severe preeclampsia referred to the intensive care unit in Sønderborg Hospital increased. On the basis of a retrospective study of the hospital records, possible reasons and certain characteristics of this increase are pointed out. Failure of treatment of severe hypertension and oliguria were the most frequent reasons for referral. Respiratory complications predominated: four women had pulmonary oedema and eight required ventilatory support. Despite difficulties in assessing the haemodynamic status, these nineteen cases appear to illustrate the unpredictable variation in severe preeclampsia per se. Insufficient volume expansion may explain a few episodes of oliguria, but neither excessive volume expansion nor unacceptable delay in observation appear to offer plausible explanations for the increase observed. The authors conclude that the frequency of haemodynamic complications in this analysis illustrated the potentially disastrous effect of preeclampsia in itself. Early delivery should always be considered in patients with even minor symptoms of treatment failure, regardless of the age of gestation. Should haemodynamic complications occur despite this, referral to an intensive care unit should be considered.

[Use of the APACHE II scoring method for registering patients receiving intensive care at a central hospital]. / Anvendelse af APACHE II scoring til registrering af intensivpatienter på et centralsygehus.

The APACHE II scoring method was employed prospectively for registration of patients in intensive care in a central hospital. Scoring was performed in a total of 390 patients. Seventy-three patients (20.2%) of the patients died in hospital. A significant difference in the APACHE II score was found in the surviving patients (9 points) as compared with those who died (19 points). A significant linear connection was found between the frequency of death observed and the estimated risk of death in hospital. The APACHE II score could not be correlated to the number of bed-days in the intensive care unit but to the resources employed measured as the number of therapeutic procedures carried out. The APACHE II scoring method was found to be employable in the prognosis for survival as a control of quality, as a tool for description of the degree of severity of illness and as an indirect measurement of the resources required.

[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.

[Massive theophylline poisoning. A case with survival without hemoperfusion]. / Massiv teofyllinforgiftning. Et tilfoelde med overlevelse uden hoemoperfusion.

We describe a case of intentional acute theophylline intoxication with a very high theophylline level managed successfully without haemoperfusion. Treatment included aggressive support of the cardiovascular system and control of seizure activity.

[Hemolysis, elevated liver enzymes and thrombocytopenia: the HELLP syndrome--a manifestation of severe pre-eclampsia]. / Haemolyse, et forhøjet leverenzymtal og trombocytopeni: HELLP-syndromet--en manifestation af svaer praeeklampsi?

On the basis of a review of the literature and five case histories, the pathophysiological and clinical features in pregnant women with haemolysis, elevated liver enzymes and thrombocytopenia which together constitute the HELLP syndrome (haemolysis, elevated liver enzymes and low platelet count) are illustrated. This syndrome describes a complicated obstetric course with greatly increased neonatal and maternal morbidities. Hypertension/proteinuria are common but are not obligatory. The condition should be suspected in pregnant women with pain under the right rib margin and unexplained jaundice. The diagnosis is verified by the blood picture, liver enzyme count and a blood smear. Women with verified HELLP syndrome and gestational age greater than 34 weeks should be delivered immediately. Women with the same syndrome and gestational age less than 32 weeks should be delivered if the condition cannot be rapidly controlled. In exceptional cases cesarean section may be necessary. On the basis of the coagulation status, the defective plasma components may be supplied (e.g. fresh frozen plasma and antithrombin-III). Plasmapheresis and specific pharmacological intervention must be considered as experimental at present, although these present promising therapeutic possibilities.

[Pneumonia among patients admitted to intensive care units. An epidemiological multicenter study of APACHE II score, incidence and course]. / Pneumoni blandt patienter indlagt på intensiv terapiafsnit. Epidemiologisk multicenterstudie of APACHE II-score, incidens og forløb.

Pneumonia in patients in intensive care units (ICU) is associated with several diagnostic difficulties and high mortality. This study was conducted to describe the diagnostic procedures and clinical characteristics of the pneumonic and critically ill patient in relation to APACHE II score. The material consisted of 193 patients admitted to seven Danish ICUs and is also included in a European epidemiologic survey (EURO.NIS). Twenty-eight (14.5%) developed pneumonia and 18 (9.3%) of these were nosocomial (> 48h after admission). Patients with pneumonia had a significantly higher APACHE II score, duration of stay and mortality. The techniques used to diagnose pneumonia were mainly conventional and did not or only seldom include protected brush, bronchoalveolar lavage or pulmonary biosy. The predominant pathogens isolated in tracheal aspirate were Gram-negative bacilli (50%). The APACHE II scoring system was found to stratify patients with respect to mortality, duration of stay and pneumonia diagnosed in ICU.

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.