Isolation and characterization of a glycosylated form of human insulin-like growth factor I produced in Saccharomyces cerevisiae

Expression and secretion of human insulin-like growth factor-I (IGF-I) in Saccharomyces cerevisiae was achieved by linking an actin (ACT) promoter to an MF alpha 1 prepro leader peptide/IGF-I gene fusion. Purified human IGF-I from yeast culture media was found to contain, in addition to the native form, also a glycosylated variant. Structural studies showed that both IGF-I forms were processed identically, resulting in 70-amino-acid long polypeptides, with intact N-terminal and C-terminal residues of glycine and alanine, respectively. The glycosylation site was determined to threonine-29 (Thr29), by 1H NMR spectroscopy and protein sequence analysis of an isolated tryptic peptide(22-36). No other glycosylation sites were found. Only mannose was detected in the sugar analysis, with an estimated content of 4.5% w/w corresponding to 2 mannose residues per molecule of IGF-I. The carbohydrate structure, determined by 1H and 13C NMR spectroscopy, was found to be alpha-D-Manp(1----2)alpha-D-Manp(1----3)Thr corresponding to an O-linked glycoprotein structure. No other post-translational modifications could be identified in the glycosylated IGF-I form. Furthermore, this form was highly active, comparable to native IGF-I, exhibiting a specific activity of 20,500 units/mg, as determined by a radio-receptor assay.     

Aint/Tacc3 is highly expressed in proliferating mouse tissues during development, spermatogenesis, and oogenesis.

Aint was originally identified on the basis of its interaction in vitro with the aryl hydrocarbon nuclear receptor translocator (Arnt). Arnt is a common heterodimerization partner in the basic helix-loop-helix (bHLH)-PER-ARNT-SIM (PAS) protein family and is involved in diverse biological functions. These include xenobiotic metabolism, hypoxic response, and circadian rhythm. In addition, Arnt has a crucial role during development. Aint is a member of a growing family of transforming acidic coiled-coil (TACC) proteins and is the murine homologue of human TACC3. Here we report the spatiotemporal expression of Tacc3 mRNA and protein in embryonic, postnatally developing, and adult mouse tissues using in situ hybridization and immunocytochemistry. Tacc3 mRNA was highly expressed in proliferating cells of several organs during murine development. However, the only adult tissues expressing high levels were testis and ovary. Immunocytochemistry revealed that Tacc3 is a nuclear protein. Our results suggest that Tacc3 has an important role in murine development, spermatogenesis, and oogenesis.     

Endophytic fungi in wild and cultivated grasses in Finland

We examined the occurrence of vertically via host seeds transmitted endophyte infections of 14 grass species in natural populations in Finland and totally 97 agricultural cultivars of 13 grass species. Although endophyte infections were widespread in native grass species, overall endophyte occurrence and frequencies were lower than published reports have suggested. In natural populations, 10 out of 14 grass species examined harbor fungal endophytes in their seeds. The highest species-specific mean incidences of endophyte infected plants in infected populations were found in Agrostis capillaris. Festuca arundinacea. F. ovina. F. Pratensis. F. rubra and Phleum pratense (67%, 98%, 29%., 42%. 32% and 33%, respectively). Mean incidences were < 20% in Dactylis glomerata. Deschampsia flexuosa. D. cespitosa and Elymus repens. and no infections were detected in Calamagrostis lapponica. C. epigejos. Alopecurus pratensis and Phalaris arundinacea. However, we detected a very high variation in infection incidences among natural populations and a large proportion of populations was, indeed, endophyte-free. This supports the ideas that 1) endophytic fungi provide selective advantage of infected grasses to their uninfected eonspecifics in some habitats, and/or 2) fungi are occasionally transmitted horizontally by spores. In grass cultivars, endophyte infected seeds were detected only in F. Pratensis and Lolium perenne. and endophyte frequencies were either very high or very low. Cultivars of 11 other grass species were endophyte-free.     

TGF-β induces the expression of SAP30L, a novel nuclear protein

Background  

We have previously set up an in vitro mesenchymal-epithelial cell co-culture model which mimics the intestinal crypt villus axis biology in terms of epithelial cell differentiation. In this model the fibroblast-induced epithelial cell differentiation from secretory crypt cells to absorptive enterocytes is mediated via transforming growth factor-β (TGF-β), the major inhibitory regulator of epithelial cell proliferation known to induce differentiation in intestinal epithelial cells. The aim of this study was to identify novel genes whose products would play a role in this TGF-β-induced differentiation.     

Influence of Developmental Conditions on Immune Function and Dispersal-Related Traits in the Glanville Fritillary (Melitaea cinxia) Butterfly

Organisms in the wild are constantly faced with a wide range of environmental variability, such as fluctuation in food availability. Poor nutritional conditions influence life-histories via individual resource allocation patterns, and trade-offs between competing traits. In this study, we assessed the influence of food restriction during development on the energetically expensive traits flight metabolic rate (proxy of dispersal ability), encapsulation rate (proxy of immune defence), and lifespan using the Glanville fritillary butterfly, Melitaea cinxia, as a model organism. Additionally, we examined the direct costs of flight on individual immune function, and whether those costs increase under restricted environmental conditions. We found that nutritional restriction during development enhanced adult encapsulations rate, but reduced both resting and flight metabolic rates. However, at the individual level metabolic rates were not associated with encapsulation rate. Interestingly, individuals that were forced to fly prior to the immune assays had higher encapsulation rates than individuals that had not flown, suggesting that flying itself enhances immune response. Finally, in the control group encapsulation rate correlated positively with lifespan, whereas in the nutritional restriction group there was no relationship between these traits, suggesting that the association between encapsulation rate on adult lifespan was condition-dependent. Thus stressful events during both larval development (food limitation) and adulthood (forced flight) induce increased immune response in the adult butterflies, which may allow individuals to cope with stressful events later on in life.     

Correction to: Genetic characterization of Addison’s disease in Bearded Collies

Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.     

Insect oviposition preference between Epichloë‐symbiotic and Epichloë‐free grasses does not necessarily reflect larval performance

Variation in plant communities is likely to modulate the feeding and oviposition behavior of herbivorous insects, and plant‐associated microbes are largely ignored in this context. Here, we take into account that insects feeding on grasses commonly encounter systemic and vertically transmitted (via seeds) fungal Epichloë endophytes, which are regarded as defensive grass mutualists. Defensive mutualism is primarily attributable to alkaloids of fungal origin. To study the effects of Epichloë on insect behavior and performance, we selected wild tall fescue (Festuca arundinacea) and red fescue (Festuca rubra) as grass–endophyte models. The plants used either harbored the systemic endophyte (E+) or were endophyte‐free (E?). As a model herbivore, we selected the Coenonympha hero butterfly feeding on grasses as larvae. We examined both oviposition and feeding preferences of the herbivore as well as larval performance in relation to the presence of Epichloë endophytes in the plants. Our findings did not clearly support the female's oviposition preference to reflect the performance of her offspring. First, the preference responses depended greatly on the grass–endophyte symbiotum. In F. arundinacea, C. hero females preferred E+ individuals in oviposition‐choice tests, whereas in F. rubra, the endophytes may decrease exploitation, as both C. hero adults and larvae preferred E? grasses. Second, the endophytes had no effect on larval performance. Overall, F. arundinacea was an inferior host for C. hero larvae. However, the attraction of C. hero females to E+ may not be maladaptive if these plants constitute a favorable oviposition substrate for reasons other than the plants' nutritional quality. For example, rougher surface of E+ plant may physically facilitate the attachment of eggs, or the plants offer greater protection from natural enemies. Our results highlight the importance of considering the preference of herbivorous insects in studies involving the endophyte‐symbiotic grasses as host plants.     

A multi-component LC–MS/MS method for detection of ten plant-derived psychoactive substances in urine

A sensitive and specific LC–MS/MS method for simultaneous detection of 10 plant-derived psychoactive substances (atropine, N,N-dimethyltryptamine, ephedrine, harmaline, harmine, ibogaine, lysergic acid amide, psilocin, scopolamine and yohimbine) in urine was developed. Direct injection of urine diluted with 3 deuterated internal standards allowed for a readily accessible method suitable for application in clinical intoxication cases. Separation was achieved using reversed phase chromatography and gradient elution with a total analysis time of 14 min. Electrospray ionization was used and ions were monitored in the positive selected reaction monitoring mode. The calibration curves were linear (r² > 0.999) and the total imprecision at high (1000 μg/L) and low (50 μg/L) substance concentrations were 4.9–13.8% and 8.3–26%, respectively. Infusing the analytes post column and injecting matrix samples showed limited influence by ion suppression. The multi-component method proved to be useful for investigation of authentic cases of intoxication with plant-derived psychoactive drugs and was indicated to cover the clinically relevant concentration ranges.