Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein ß-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.

Ticks in the ecotone: the impact of agri-environment field margins on the presence and intensity of Ixodes ricinus ticks (Acari: Ixodidae) in farmland in southern England.

The present study aimed to assess whether agri-environment field margins provide a habitat for the sheep/deer tick Ixodes ricinus. Field studies were conducted in arable farmland in southern England in both extant and newly constructed field margins. The presence and intensity (i.e. the mean number of nymphs per transect, excluding zeros) of questing nymphs and adult I. ricinus were compared between field margins with three adjacent habitats: woodland, hedgerow and arable land. The presence and intensity of ticks within a field margin was also compared between three ecozones: the ecotone, the margin and the crop. It was found that field margins do support I. ricinus, although the intensity of ticks was associated with field margins with adjacent woodland, with a higher tick intensity along the ecotonal ecozone, compared with the rest of the margin or the crop edge. The presence of a hedge also increased the likelihood of finding questing nymphs in a field margin compared with a margin adjacent to arable land. This effect, however, was less pronounced than in field margins with adjacent woodland. The provision of footpaths within the margin (at least 1-2 m from the ecotone), or on the edge of the crop where paths run next to woodland known to be an important tick habitat, could be promoted to minimize tick exposure. In addition, based on the results of the present study, raising awareness that walking alongside woodlands also constitutes a tick risk could be promoted.

Has the red sheep tick, Haemaphysalis punctata, recently expanded its range in England?

The red sheep tick, Haemaphysalis punctata (Ixodida: Ixodidae), has been reported as present in the U.K. for more than a century; however, very little has been written about its distribution. In recent years, numbers of detections of this tick species reported to the Public Health England (PHE) Tick Surveillance Scheme have increased. This rise in the number of records may be attributable to increased tick surveillance activities or to the increased distribution of this species of tick in parts of England. This paper reviews published reports of H. punctata and all data held by the Biological Records Centre and PHE, and summarizes a number of field studies conducted by PHE and the Animal and Plant Health Agency over the past 8 years. It would appear from the evidence presented here that H. punctata may be expanding its range across the eastern part of the South Downs National Park, where there have also been reports of this tick species biting humans. It is possible that the movement of sheep between grassland sites is facilitating this spread. Further studies that better elucidate the ecology of this tick and its possible role as a vector of human and veterinary diseases are now warranted.

Distribution of the tick Dermacentor reticulatus in the United Kingdom.

The recent implication of Dermacentor reticulatus (Ixodida: Ixodidae) in the transmission of canine babesiosis in the U.K. has highlighted the lack of accurate published data on its distribution in this country. This paper aims to collate and appraise historical data for D. reticulatus, to supplement such data with more recent surveillance data and to report on field sampling conducted during 2009-2016. These updated data facilitate better understanding of the current distribution of this tick in the U.K., which will better inform disease risk assessments. There appear to be four known regions of the U.K. in which D. reticulatus currently occurs, including western Wales, North and South Devon, and Essex. The majority of foci are located in coastal sand dunes and maritime grasslands, including grazing marsh. However, more recently the tick has been detected in urban greenspace in Essex. The emergence of this tick as a vector of babesiosis in the U.K. and its recent apparent spread in Essex into urban greenspace highlight the need for continued surveillance and for further research into its status as a vector of human and veterinary pathogens.

The use of syndromic surveillance to monitor the incidence of arthropod bites requiring healthcare in England, 2000-2013: a retrospective ecological study.

Climate change experts predict the number of nuisance-biting arthropods in England will increase but there is currently no known surveillance system in place to monitor or assess the public health impact of arthropod bites. This retrospective ecological study utilized arthropod bites requiring healthcare from five national real-time syndromic surveillance systems monitoring general practitioner (GP) consultations (in-hours and out-of-hours), emergency department (ED) attendances and telephone calls to remote advice services to determine baseline incidence in England between 2000 and 2013 and to assess the association between arthropod bites and temperature. During summer months (weeks 20-40) we estimated that arthropod bites contribute a weekly median of ~4000 GP consultations, 750 calls to remote advice services, 700 ED and 1300 GP out-of-hours attendances. In all systems, incidence was highest during summer months compared to the rest of the year. Arthropod bites were positively associated with temperature with incidence rate ratios (IRRs) that ranged between systems from 1·03 [95% confidence interval (CI) 1·01-1·06] to 1·14 (95% CI 1·11-1·16). Using syndromic surveillance systems we have established and described baseline incidence of arthropod bites and this can now be monitored routinely in real time to assess the impact of extreme weather events and climate change.

Transglutaminase: new insights into gelatin nanoparticle cross-linking.

Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in vivo and in vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25°C using an ion-free solvent at a neutral pH and have been terminated after 12 h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production.

Investigation of ecological and environmental determinants for the presence of questing Ixodes ricinus (Acari: Ixodidae) on Gower, South Wales.

The spatial heterogeneity of questing Ixodes ricinus (L.) (Acari: Ixodidae) within endemic areas in Great Britain is well established. Their presence is acutely responsive to blood host availability and their ability to maintain water balance, which are in turn governed by a variety of ecological and environmental factors. This article details the findings of a 3-yr study on the Gower peninsula, south Wales, which investigated the contribution of such factors (both ground- and geographic information systems [GIS] -derived) for predicting the presence of questing I. ricinus (Q(P)), at a local scale. Statistically significant univariate associations were found between Q(P) and calcareous/ neutral grassland and heathland habitats, particularly those grazed by livestock, and various factors that intuitively promote tick survival. For example, topographical features, such as certain aspects, that reduce exposure to cold northerly winds and the hot midday sun, favored Q(P). Similarly, positive associations were found with substrata composed of less permeable soil types and less permeable superficial/bedrock geologies that promote a moist microhabitat and reduce the likelihood of desiccation. Q(P) was also higher in areas of high soil moisture. This study highlighted a number of GIS-derived data sets that could be applied in the development of local and national predictive maps for I. ricinus in Great Britain. An understanding of the influence of these factors on questing I. ricinus can aid targeted tick control programs and help to educate the public, and those occupationally exposed, in understanding likely I. ricinus prolific areas within an I. ricinus endemic region.

Preliminary evaluation of exotic tick species and exotic pathogens imported on migratory birds into the British Isles.

Field studies were carried out to determine whether ticks are being imported into the British Isles on migratory birds. During spring and autumn migration 2004, ticks were collected from ringed birds at 11 bird observatories and 3 inland Riparia riparia colonies. A total of 38 ticks of 4 species (Ixodes ricinus, I. frontalis, I. lividus, I. arboricola) were collected from 12 species of bird. Ticks were tested for viruses in the Flavivirus and Nairovirus genera, with no positives found. This data demonstrates that ticks are being imported into the British Isles on migratory birds with future work recommended to determine the quantity of ticks imported and to detect low prevalence pathogens.

Rhipicephalus sanguineus importation into the UK: surveillance, risk, public health awareness and One Health response.

As part of Public Health England's assessment of vectorborne disease risk to public health in the UK, tick specimens are regularly submitted by veterinarians for identification via the Tick Surveillance Scheme. Recently, a number of these specimens have been identified as the brown dog tick, Rhipicephalus sanguineus This species is non-endemic to the UK and presents a risk to both human and animal health due to its role in the transmission of various tickborne pathogens. Although current climatic conditions in the UK are unlikely to permit the survival of this species outdoors, indoor infestations can occur and this can present a risk of disease transmission within an infested property. This paper documents 40 importation events involving R sanguineus on recently travelled or imported dogs into the UK since 2012. It also provides details of the response following these detections in line with the One Health concept. With the increasing number of dogs travelling or being imported, it is likely that importation and infestation events in the UK will continue and may result in pathogen transmission. It is therefore important to raise awareness of this risk and share lessons learned to improve our prevention and response strategies to this emerging issue.

Immobilization of the hydantoin cleaving enzymes from Arthrobacter aurescens DSM 3747.

The immobilization procedure of the two industrially important hydantoin cleaving enzymes--hydantoinase and L-N-carbamoylase from Arthrobacter aurescens DSM 3747--was optimized. Using different methods (carbodiimide, epoxy activated carriers) it was possible to immobilize the crude hydantoinase from A. aurescens DSM 3747 to supports containing primary amino groups with a yield of up to 60%. Immobilization on more hydrophobic supports such as Eupergit C and C 250 L resulted in lower yields of activity, whereas the total protein coupled remained constant. All attempts to immobilize the crude L-N-carbamoylase resulted in only low activity yields. Therefore, the enzyme was highly purified and used in immobilization experiments. The pure enzyme could easily be obtained in large amounts by cultivation of a recombinant Escherichia coli strain following a three step purification protocol consisting of cell disruption, chromatography on Streamline diethylaminoethyl and Mono Q. The immobilization of the L-N-carbamoylase was optimized with respect to the coupling yield by varying the coupling method as well as the concentrations of protein, carrier and carbodiimide. Using 60 mM of water-soluble carbodiimide, nearly 100% of the enzyme activity and protein could be immobilized to EAH Sepharose 4B.

Fast purification and kinetic studies of the glycerol-3-phosphate dehydrogenase from the yeast Saccharomyces cerevisiae.

The glycerol-3-phosphate dehydrogenase has been purified from Saccharomyces cerevisiae 140-fold to electrophoretic homogeneity by a simple procedure involving affinity and ion exchange chromatography. The purified enzyme was most active at pH 6.8 and 51 degrees C. Its molecular mass was determined to be 45000 +/- 2000 Da by SDS-polyacrylamide gel electrophoresis. At physiological pH values the thermodynamic equilibrium constant was determined to be 3.5 x 10(-3) (M-1). Product inhibition as well as competitive inhibition patterns were found which clearly indicate that the kinetic mechanism of the glycerol-3-phosphate dehydrogenase is random bi-bi with the formation of dead-end complexes. In vivo concentrations of selected metabolites and kinetic expression for G3P-DH were used to explain regulatory properties of this enzyme under conditions of short-term glucose effect in Saccharomyces cerevisiae.

Purification and characterization of a highly selective epoxide hydrolase from Nocardia sp. EH1.

A highly enantioselective, soluble epoxide from Nocardia sp. EH1 was purified to homogeneity via a four-step procedure: (i) hydrophobic interaction chromatography on Phenyl Sepharose CL-4B, (ii) anion exchange chromatography on SOURCE 30Q, followed by (iii) a second hydrophobic interaction chromatography on Phenyl Sepharose HP, and finally (iv) gel-filtration on Superdex 75 HR 10/30. The pure protein was shown to be a monomer of integral of 34 kDa possessing an optimum pH of 8-9. Neither UV-absorbing cofactors nor metal ions were required for activity. In contrast to whole-cell activity, the partially purified enzyme proved to be considerably less stable. Stabilization was achieved by addition of non-ionic detergents such as Tween 80 or Triton X-100, causing a shift of the temperature optimum from 35 to 40 degrees C. Both effects combined led to an enhancement of the relative activity of up to approximately 150% of that of the native enzyme.

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization.

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5'-monosubstituted D,L-hydantoins to L-amino acids, a stereoselective hydantoinase, a stereospecific L-N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described L-N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate L-5-methylthioethylhydantoin. Enzymatic racemization of D-5-indolylmethylenehydantoin in D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.

Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as new member of cyclic amidases.

A hydantoinase from Arthrobacter aurescens DSM 3745 has been purified to homogeneity with a yield of 77% using a three-step purification procedure. The active enzyme is a tetramer consisting of four identical subunits, each with a molecular mass of 49670 Da as determined by mass spectrometry. The N-terminal amino acid sequence of the enzyme indicates sequence identities to cyclic amidases involved in the nucleotide metabolism as the D-hydantoinase from Agrobacterium radiobacter (53%), the D-selective dihydropyrimidinase from Bacillus stearothermophilus (38%), the allantoinase from Rana catesbeiana (26%), as well as to the catalytic subunit of the urease from Helicobacter pylori (50%). However, all studies based on substrate-dependent growth, induction and catalytic behavior documented the novelty of the bacterial hydantoinase and that its physiological role is not related to any of these enzymes or known metabolic pathways. Its substrate specificity differs from hydantoinases listed in Enzyme Nomenclature and is rather more predominant for the cleavage of aryl- than for alkyl-hydantoin derivatives. It is shown that the stereoselectivity of this enzyme depends on the substrate used for bioconversion: although it is strictly L-selective for the cleavage of D,L-5-indolylmethylhydantoin, it appears to be D-selective for the hydrolysis of D,L-methylthioethylhydantoin. Due to these findings we conclude that this novel bacterial hydantoinase should be classified as a new member of the EC-group 3.5.2 of cyclic amidases.

Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli.

An L-N-carbamoyl amino acid amidohydrolase (L-N-carbamoylase) from Arthrobacter aurescens DSM 3747 was cloned in E. coli and the nucleotide sequence was determined. After expression of the gene in E. coli the enzyme was purified to homogeneity and characterized. The enzyme was shown to be strictly L-specific and exhibited the highest activity in the hydrolysis of beta-aryl substituted N alpha-carbamoyl-alanines as e.g. N-carbamoyl-tryptophan. Carbamoyl derivatives of beta-alanine and charged aliphatic amino acids were not accepted as substrates. The N-carbamoylase of A. aurescens DSM 3747 differs from all known enzymes with respect to its substrate specificity although amino acid sequence identity scores of 35-38% to other N-carbamoylases have been detected. The enzyme consists of two subunits of 44,000 Da, and has an isoelectric point of 4.3. The optima of temperature and pH were determined to be 50 degrees C and pH 8.5 respectively. At 37 degrees C the enzyme was completely stable for several days.

Results of a systematic adverse drug reaction (ADR)-screening concerning bradycardia caused by drug interactions in departments of internal medicine in Rostock.

OBJECTIVES: The concomitant intake of drugs, which is frequently needed, may be associated with drug interactions. We report results on the screening of ADRs responsible for hospital admissions involving bradycardia. This investigation was part of a BfArM pilot project with the objective of monitoring and reporting ADRs. METHOD: Beginning in 1997, a trained medical staff member of the Pharmacovigilance Center, Rostock, prospectively screened all hospital admissions to the Departments of Internal Medicine of the 2 hospitals in Rostock (40,000 hospital admissions). ADRs leading to hospital admission were registered, evaluated and reported. RESULTS: A total of 1,441 ADRs were recorded by the Pharmacovigilance Center Rostock in the period up to December 2000. 12% (n = 173) of all ADRs involve the cardiovascular system; 83 patients (5.7% of all ADRs) suffered from bradycardia. Bradycardia was the most frequent cardiovascular ADR observed. Of these patients, 88% were receiving 3 - 10 different drugs. Drugs suspected of causing bradycardia were: digitalis (n = 62), beta-blockers (n = 47), calcium channel blockers with negative chronotropic effect (n = 45), and antiarrhythmic drugs (n = 3). 54 patients had received more than I of these drugs concomitantly as outpatients, increasing the risk of drug interactions: 18 patients received digitalis + calcium channel blocker; 14 patients digitalis + beta-blocker; 7 patients beta-blocker + calcium channel blocker; 12 cases digitalis + beta-blocker + calcium channel blocker. CONCLUSION: The results show that special attention should be given to patients who receive more than 1 drug when there is a high risk of bradycardia. Drug combinations which may cause drug interactions should be avoided, especially when other equivalent therapeutic options are available.

Results of systematic screening for serious gastrointestinal bleeding associated with NSAIDs in Rostock hospitals.

OBJECTIVES: Gastrointestinal bleeding and ulcers may lead to life-threatening complications. One of the causes is use of non-steroidal anti-inflammatory drugs (NSAIDs). METHODS: All hospital admissions in 1998 to the Departments of Internal Medicine, including the Intensive Care Unit and the Department of Surgery, in 2 hospitals in Rostock were prospectively screened for gastrointestinal bleeding. Whether the bleeding was due to an adverse drug reaction ADR or not was assessed using the rating scale of Begaud et al. [1985] for each drug taken. The risk profile and the drug history of all patients with gastrointestinal bleeding were registered. RESULTS: A total of 58 patients with gastrointestinal bleeding due to NSAIDs were documented. Risk factors for bleeding were cardiac diseases, hypertension, diabetes, age over 60 years, history of ulcer, a Helicobacter pylori infection, smoking and consumption of alcohol together with drugs known to have a risk of causing gastrointestinal bleeding and ulcers (antiplatelet drugs, anticoagulants, corticosteroids). About 70% of these patients had 3 or more risk factors, but only 20% had been receiving effective prophylaxis with a proton pump inhibitor. CONCLUSION: Gastrointestinal problems resulting from the use of NSAIDs are clinically important. It is concluded, that individual risk profiles, as a criterion for the prophylactic use of effective protective drugs, would be helpful in patients management.

[Challenges in inpatient ophthalmology for elderly patients]. / Herausforderungen in der stationären Augenheilkunde bei hoch- und höchstbetagten Patienten.

PURPOSE: The demographic changes will lead to an increase in elderly patients in the field of medicine and in particular ophthalmology. The purpose of this study was to assess ocular problems of inpatients in the clinical setting with a focus on patients over 60 years old, especially elderly patients over 80 years and very old patients over 90 years. METHODS: All consecutive inpatients were included in the study. Medical data were analyzed concerning the diagnosis, operative indications and comorbidities and the presence of dementia disorders was also considered. The effects of age or diseases on the inpatient course were investigated. RESULTS: At the eye clinic in Frankfurt (Oder) the mean age of patients was 69 years and was the highest of all medical and surgical disciplines. The age of patients of internal medicine was similarly with 67 years but orthopedic and urological patients were on average 64 years old. In the other departments the mean age of patients was clearly less than 60 years old. Of the patients 26% were aged 80 years or older. A total of 31 patients over 90 years old were treated as inpatients (2.0%) during the year 2012. The treatment-relevant diagnosis increased with age. Nearly 40% had a reduced mobility at an age of 80-89 years and 60% at an age over 90 years. In the age group over 90 years approximately 25% had dementia and 20% a visual disorder. CONCLUSION: Old and very old patients with ocular problems and more than 80-90 years of age had an increased comorbidity concerning the relevant treatment diagnostics. This has relevant implications and challenges for nursing and medical care in the clinical setting.

[Bilateral panuveitis with papillary swelling]. / Beidseitige Panuveitis mit Papillenschwellung.

Lues (syphilis) is a chronic cyclic infectious disease which can continue for decades if untreated. A simultaneous HIV infection can result in false negative results in serological tests for lues. The occurrence of neurolues has frequently been described in HIV positive patients. In the differential diagnosis an early ocular manifestion of lues should be considered. A 40-year-old homosexual patient presented in our hospital with bilateral pain-free increasing loss of vision. The ophthalmological examination revealed an intermediately expressed panuveitis with streaky opacity of the vitreous body and pronounced bilateral papillary swelling. Following systemic anti-inflammatory therapy with cortisone the situation worsened after initial improvement. The serological investigations revealed both HIV and lues infections. Intravenous therapy with mega units of penicillin led to a slow improvement of clinical symptoms and also vision. In cases of uveitis of unclear origin together with a HIV infection and suspected lues, regular serological testing should be carried out because the occurrence of late complications of lues can be avoided by the diagnosis of lues and adequate treatment.