Biology and distribution of hemichordates (Enteropneusta) with emphasis on Harrimaniidae and description of Protoglossus bocki sp. nov. from Scandinavia

A new species of the genus Protoglossus is described from the west coast of the Scandinavian Peninsula. It lives buried in clay bottoms below the halocline where the salinity is at least 33–34 psu. Body small, estimated maximal length 1.5 cm. Collar broader than long, with a forward inclination. The thickest part is the collar region where it can be up to 1 mm in diameter. Proboscis colouration light pink to golden yellow; collar white with transversal yellow bands; branchial, hepatic and intestinal regions translucent pale yellow to golden yellow; brown intestine visible through the body wall. Proboscis groove extends through posterior half of proboscis. Nine to 17 pairs of gill openings, the size of the posteriormost successively smaller. It differs from the other European species, Protoglossus koehleri, in colouration, smaller size, fewer gill openings, body shape and proportions. It was sequenced (18S rRNA gene) and clustered within the family Harrimaniidae, with Saxipendium as its closest relative.     

Dynamic Changes in Mucus Thickness and Ion Secretion during Citrobacter rodentium Infection and Clearance

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.     

Field-based video observations of wild barnacle cyprid behaviour in response to textural and chemical settlement cues

Many marine invertebrate larvae respond behaviourally to environmental settlement cues, yet behaviours are often only inferred from settlement patterns or are limited to laboratory studies. The behaviour of wild cypris larvae of Semibalanus balanoides L. was filmed on settlement tiles in the field. Tiles were of five different textures with a nested treatment of crude conspecific adult extract (AE). The effects of texture and AE on eleven defined behaviours were analysed. Texture affected the gross and net exploratory distances, velocity, acceleration and time spent exploring. AE attracted more cyprids during the first minute of immersion and increased the time spent on surfaces. Relatively few arrivals that either travel far and fast, or exit the surface rapidly, may indicate a lower chance of settlement. An increase in time spent on a surface may increase the probability of being in contact with the surface when the sign stimulus to settle occurs.     

Structural,Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the −7 to −4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.     

Catalytic Turnover Triggers Exchange of Subunits of the Magnesium Chelatase AAA+ Motor Unit

The ATP-dependent insertion of Mg²⁺ into protoporphyrin IX is the first committed step in the chlorophyll biosynthetic pathway. The reaction is catalyzed by magnesium chelatase, which consists of three gene products: BchI, BchD, and BchH. The BchI and BchD subunits belong to the family of AAA+ proteins (ATPases associated with various cellular activities) and form a two-ring complex with six BchI subunits in one layer and six BchD subunits in the other layer. This BchID complex is a two-layered trimer of dimers with the ATP binding site located at the interface between two neighboring BchI subunits. ATP hydrolysis by the BchID motor unit fuels the insertion of Mg²⁺ into the porphyrin by the BchH subunit. In the present study, we explored mutations that were originally identified in semidominant barley (Hordeum vulgare L.) mutants. The resulting recombinant BchI proteins have marginal ATPase activity and cannot contribute to magnesium chelatase activity although they apparently form structurally correct complexes with BchD. Mixing experiments with modified and wild-type BchI in various combinations showed that an exchange of BchI subunits in magnesium chelatase occurs during the catalytic cycle, which indicates that dissociation of the complex may be part of the reaction mechanism related to product release. Mixing experiments also showed that more than three functional interfaces in the BchI ring structure are required for magnesium chelatase activity.     

Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum

The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass subunit of photosystem I (PSI) containing one transmembrane helix. Homoplastomic transformants with an inactivated psaJ gene were devoid of PSI-J protein. The mutant plants were slightly smaller and paler than wild-type because of a 13% reduction in chlorophyll content per leaf area caused by an approximately 20% reduction in PSI. The amount of the peripheral antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the core subunits, but Lhca1 and Lhca4 were present in relative excess. The functional size of the PSI antenna was not affected, suggesting that PSI-J is not involved in binding of light-harvesting complex I. The specific PSI activity, measured as NADP(+) photoreduction in vitro, revealed a 55% reduction in electron transport through PSI in the mutant. No significant difference in the second-order rate constant for electron transfer from reduced plastocyanin to oxidized P700 was observed in the absence of PSI-J. Instead, a large fraction of PSI was found to be inactive. Immunoblotting analysis revealed a secondary loss of the luminal PSI-N subunit in PSI particles devoid of PSI-J. Presumably PSI-J affects the conformation of PSI-F, which in turn affects the binding of PSI-N. This together renders a fraction of the PSI particles inactive. Thus, PSI-J is an important subunit that, together with PSI-F and PSI-N, is required for formation of the plastocyanin-binding domain of PSI. PSI-J is furthermore important for stability or assembly of the PSI complex.     

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Effects of four trichothecene mycotoxins on activation marker expression and cell proliferation of human lymphocytes in culture

Four trichothecene mycotoxins – the type A trichothecenes T2-toxin and diacetoxyscirpenol and the type B trichothecenes nivalenol and deoxynivalenol – were studied. The effects of these mycotoxins on the expression of the sequentially expressed activation markers CD69, CD25, and CD71 and on proliferation of human lymphocytes were studied in culture with a duration of up to 72 h.All the examined toxins affected activation marker expression in a similar way. After 6 h, the CD69 expression was lower in exposed cultures compared to controls. After 24 and 48 h of exposure, an increased frequency of cells expressing CD69 was found in exposed cultures, indicating a delay in downregulation of CD69 expression. Stimulation of CD25 expression was observed for doses below the IC₅₀ value, while suppression was found for higher doses. The pattern was different from that detected for CD69 expression, in that an increased expression of CD25 never occurred after exposure to the highest concentration of the toxin, and in that no stimulatory effects were found after 48 h of exposure, indicating that the response was inhibited and not delayed. The effects of toxin exposure on CD71 expression were in many respects similar to the effects on CD25 expression.We conclude that the trichothecene mycotoxins investigated in this study inhibited the cell cycle in a similar way and exert their main antiproliferative action rather early in the cell cycle, before or in conjunction with CD25 expression.     

Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay

It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.