Glucose kinase of Streptomyces coelicolor A3(2): large-scale purification and biochemical analysis

Glucose kinase of Streptomyces coelicolor A3(2) is essential for glucose utilisation and is required for carbon catabolite repression (CCR) exerted through glucose and other carbon sources. The protein belongs to the ROK-family, which comprises bacterial sugar kinases and regulators. To better understand glucose kinase function, we have monitored the cellular activity and demonstrated that the choice of carbon sources did not significantly change the synthesis and activity of the enzyme. The DNA sequence of the Streptomyces lividans glucose kinase gene glkA was determined. The predicted gene product of 317 amino acids was found to be identical to S. coelicolor glucose kinase, suggesting a similar role for this protein in both organisms. A procedure was developed to produce pure histidine-tagged glucose kinase with a yield of approximately 10 mg/l culture. The protein was stable for several weeks and was used to raise polyclonal antibodies. Purified glucose kinase was used to explore protein-protein interaction by surface plasmon resonance. The experiments revealed the existence of a binding activity present in S. coelicolor cell extracts. This indicated that glucose kinase may interact with (an)other factor(s), most likely of protein nature. A possible cross-talk with proteins of the phosphotransferase system, which are involved in carbon catabolite repression in other bacteria, was investigated.     

Experimental excursions on adaptive landscapes: density-dependent selection on egg size

Abstract. Theories of density-dependent natural selection suggest that intraspecific competition will favor juveniles of high competitive ability. Empirical evidence has been provided from laboratory selection experiments, but field studies are lacking due to the logistical difficulties of experimentally manipulating population densities in natural settings. Here, we present data from a decade-long experimental field study of side-blotched lizards, Uta stansburiana that overcomes these difficulties. We tested the hypothesis that density-dependent natural selection causes egg size to increase from early to late clutches in this and many other species. Using a novel combination of environmental manipulations of hatchling density and phenotypic manipulations of egg size, we demonstrate that the nature of selection on egg size changes dramatically in the absence of older competitors. The strength of selection on egg size among later-clutch hatchlings released in areas without competitors from early clutches became almost doubled in magnitude, compared to that among hatchlings released in the presence of older competitors. These experimental findings demonstrate density-dependent natural selection on egg size; however, they contradict the classical idea that egg size increases during the reproductive season because of competition between early and late hatchlings. The results indicate that competitive age or size asymmetries between early and late hatchlings can override within-cohort asymmetries due to egg size. We suggest that competition could be an important mediator of oscillating selection pressures in this and other systems. Finally, we discuss the utility of "double-level," simultaneous experimental manipulation of both phenotypic traits that are targets of selection (e.g., egg size) as well the environmental agents of selection (e.g., population density).     

Characterization of SPACR, a sialoprotein associated with cones and rods present in the interphotoreceptor matrix of the human retina: immunological and lectin binding analysis

Rod and cone photoreceptors project from the outer retinal surface into a carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM glycoconjugates are distributed around rods and cones. Wheat germ agglutinin (WGA) strongly decorates the rod matrix domains and weakly decorates the cone matrix domains. This study characterizes the major WGA-binding glycoprotein in the human IPM, which we refer to as SPACR (sialoprotein associated with cones and rods). SPACR, which has a molecular weight of 147 kDa, was isolated and purified from the IPM by lectin affinity chromatography. A polyclonal antibody to SPACR was prepared that colocalizes in tissue preparations with WGA-binding domains in the IPM. Sequential digestion of SPACR with N- and O- glycosidases results in a systematic increase in electrophorectic mobility, indicating the presence of both N- and O-linked glycoconjugates. Complete deglycosylation results in a reduction in the relative molecular mass of SPACR by about 30%. Analysis of lectin binding allowed us to identify some of the structural characteristics of SPACR glycoconjugates. Treatment with neuraminidase exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA), specific for alpha2-6 linked sialic acid, does not, indicating that the dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate, NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR suggests that this glycoprotein may contribute substantially to the polyanionic nature of the IPM. The carbohydrate chains present on SPACR could also provide sites for extensive crosslinking and participate in the formation of the ordered IPM lattice that surrounds the elongate photoreceptors projecting from the outer retinal surface.      

Group A Rotavirus as a Cause of Neonatal Calf Enteritis in Sweden

Faeces samples were collected during outbreaks of neonatal calf diarrhoea in 14 beef and dairy herds. Samples from 33 calves were taken at the onset of diarrhoea as well as from 30 calves with no signs of enteritis. No vaccines or medical treatment had previously been given. The mean age of the calves was 16.8 days (SD 8.2). The clinical evaluation of faeces consistency corresponded well to the dry matter content of the faeces (p<0.001). The samples were analyzed for rotavirus, Cryptosporidium species and Escherichia coli K99+. Group A rotavirus was detected by ELISA and RNA Polyacrylamide gel electrophoresis (RNA-PAGE) in 14/33 (43.8%) of the samples from scouring calves and 1/30 (3.7%) of the samples from non-scouring calves. The correlation between group A rotavirus and diarrhoea was statistically significant (p<0.001). No non-group A rotaviruses were found by RNA-PAGE. Cryptosporidium species were detected through demonstration of oocysts in smears from 12/63 (19.0%) of the faecal samples, but no statistically significant correlation between diarrhoea and detection of oocysts was demonstrated. Escherichia coli K99+ was not detected in any faeces sample. The clear association between group A rotavirus and diarrhoea is suggested to be due to low pathogenic load in the herds.     

Clutch detachment in a copepod after capture by a predator

When the egg-carrying copepod, Eudiaptomus gracilis, is capturedby larvae of predatory Chaoborus species (C.obscuripes or C.flavicans)in laboratory experiments, the external egg clutch detachesin 16-29% of caw, thereby allowing the eggs to escape from ingestion.Clutch detachment may be due to active removal by the femalebefore she is eaten or the result of accidental loss by thepredator during handling. Detached clutches were significantlylarger than thase that were ingested along with the female.Clutch detachment is advantageous to the female since ingestedeggs are lost, whereas detached eggs hatch normally and maypotentially propagate the genotype. Chaoborus predation maytherefore influence the evolution of clutch size and the degreeof iteroparity in Eudiaptomus.     

The non-competitive NMDA antagonists MK-801 and PCP, as well as the competitive NMDA antagonist SDZ EAA494 (D-CPPene), interact synergistically with clonidine to promote locomotion in monoamine-depleted mice

The present study shows that high doses of the non-competitive NMDA antagonist phencyclidine (PCP) as well as of the competitive NMDA antagonist SDZ EAA494 (D-CPPene) increase locomotion in monoamine-depleted mice. The pattern of movement produced following treatment with these agents is very similar to that previously observed following MK-801 administration to monamine-depleted mice. When subthreshold doses of MK-801, PCP and SDZ EAA494 were combined with the alpha-adrenergic agonist clonidine, a dramatic stimulation of locomotion was observed in monoamine-depleted mice; the gross appearance of the animals was similar with the three drug combinations. These results support our previous conclusion that suppression of glutamatergic neurotransmission promotes the locomotor stimulatory potential of other (e.g. adrenergic) transmitter systems. The present findings may be of relevance for future treatment strategies in (L-DOPA-resistant) Parkinson's disease.     

Enantiomeric conservation of the male‐produced sex pheromone facilitates monitoring of threatened European hermit beetles (Osmoderma spp.)

Hermit beetles of the genus Osmoderma (Coleoptera: Scarabaeidae: Cetoniinae) are known for their fruity odour, which is released in large amounts by males. Two species of the genus occur in Europe, the eastern Osmoderma barnabita (Motschulsky) and the western Osmoderma eremita (Scopoli). Previous studies on Swedish populations of O. eremita showed that the compound responsible for the characteristic scent, γ‐decalactone, functions as a sex pheromone for the attraction of conspecific females. Male O. eremita only release the (R)‐enantiomer of the lactone, and both sexes are anosmic to the opposite enantiomer. As the distribution areas of the two hermit beetle species partly overlap, it may be expected that they use different enantiomeric compositions of γ‐decalactone as pheromones to promote species discrimination. This paper reports on the identification of the sex pheromone of O. barnabita. Surprisingly, males from a Polish population produce only the (R)‐enantiomer of γ‐decalactone, and conspecific females show equal attraction to the (R)‐enantiomer and a racemic mixture of the compound, indicating that O. barnabita is anosmic to the (S)‐enantiomer, similarly to what was observed for O. eremita. A mtDNA sequence analysis of the cytochrome oxidase subunit I gene of Polish and Swedish beetles confirmed their taxonomical status as O. barnabita and O. eremita, respectively, with an average sequence divergence of 10.5% between beetles from the two studied areas. Although genetic data suggest that these species diverged several million years ago, they still rely on the same enantiomer of γ‐decalactone for mate finding. Thus, the male‐produced pheromone in Osmoderma spp. may be regarded as a territorial signal being exploited by females, rather than a cue for determining species identity. Our data show that the same compound can be used to facilitate monitoring of both beetle species, which are considered indicator species of the species‐rich fauna of saproxylic insects in Europe.     

Prediction of the articular eminence shape in a patient with unilateral hypoplasia of the right mandibular ramus before and after distraction osteogenesis—A simulation study

The aim of this work was to predict the shape of the articular eminence in a patient with unilateral hypoplasia of the right mandibular ramus before and after distraction osteogenesis (DO). Using a patient-specific musculoskeletal model of the mandible the hypothesis that the observed differences in this patient in the left and right articular eminence inclinations were consistent with minimisation of joint loads was tested. Moreover, a prediction was made of the final shape of the articular eminence after DO when the expected remodelling has reached a steady state. The individual muscle forces and the average TMJ loading were computed for each combination of articular eminence angles both before and after DO. This exhaustive parameter study provides a full overview of average TMJ loading depending on the angles of the articular eminences. Before DO the parameter study resulted in different articular eminence inclinations between left and right sides consistent with patient data obtained from CT scans, indicating that in this patient the articular eminence shapes result from minimisation of joint loads. The simulation model predicts development of almost equal articular eminence shapes after DO. The same tendency was observed in cone beam CT scans (NewTom) of the patient taken 6.5 years after surgery.     

Comparison of Extended-Spectrum β-Lactamase (ESBL) CTX-M Genotypes in Franklin Gulls from Canada and Chile

Migratory birds have been suggested to contribute to long-distance dispersal of antimicrobial resistant bacteria, but tests of this hypothesis are lacking. In this study we determined resistance profiles and genotypes of ESBL-producing bacteria in randomly selected Escherichia coli from Franklin´s gulls (Leucophaeus pipixcan) at breeding sites in Canada and compared with similar data from the gulls'' wintering grounds in Chile. Resistant E. coli phenotypes were common, most notably to ampicillin (30.1%) and cefadroxil (15.1%). Furthermore, 17.0% of the gulls in Canada carried ESBL producing bacteria, which is higher than reported from human datasets from the same country. However, compared to gulls sampled in Chile (30.1%) the prevalence of ESBL was much lower. The dominant ESBL variants in Canada were bla _CTX-M-14 and bla _CTX-M-15 and differed in proportions to the data from Chile. We hypothesize that the observed differences in ESBL variants are more likely linked to recent exposure to bacteria from anthropogenic sources, suggesting high local dissemination of resistant bacteria both at breeding and non-breeding times rather than a significant trans-hemispheric exchange through migrating birds.     

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us understand the disease mechanisms and advance the discoveries of new treatment options. In vitro cell cultures of monolayers or co-cultures lack the three-dimensional (3D) environment and tissue responses. Herein, we describe an innovative in vitro model of a human lung tissue, which holds promise to be an effective tool for studying the complex events that occur during infection with Mycobacterium tuberculosis (M. tuberculosis). The 3D tissue model consists of tissue-specific epithelial cells and fibroblasts, which are cultured in a matrix of collagen on top of a porous membrane. Upon air exposure, the epithelial cells stratify and secrete mucus at the apical side. By introducing human primary macrophages infected with M. tuberculosis to the tissue model, we have shown that immune cells migrate into the infected-tissue and form early stages of TB granuloma. These structures recapitulate the distinct feature of human TB, the granuloma, which is fundamentally different or not commonly observed in widely used experimental animal models. This organotypic culture method enables the 3D visualization and robust quantitative analysis that provides pivotal information on spatial and temporal features of host cell-pathogen interactions. Taken together, the lung tissue model provides a physiologically relevant tissue micro-environment for studies on TB. Thus, the lung tissue model has potential implications for both basic mechanistic and applied studies. Importantly, the model allows addition or manipulation of individual cell types, which thereby widens its use for modelling a variety of infectious diseases that affect the lungs.