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.     

Electronic Cigarette and Traditional Cigarette Use among Middle and High School Students in Florida, 2011–2014

Recent youth trends in the prevalence of e-cigarette and traditional cigarette use in Florida were examined in a cross-sectional, representative state sample from 2011 to 2014. Traditional cigarette use among youth declined during the study period. Experimentation with and past 30-day use of e-cigarettes among Florida youth tripled over 4 years. Past 30-day e-cigarette use exceeded traditional cigarette use in 2014; 10.8% of high school and 4.0% of middle school students reported recent e-cigarette use, compared with 8.7% of high school and 2.9% of middle school students for traditional cigarettes (P<0.001). By 2014, 20.5% of high school and 8.5% of middle school students reported ever use of e-cigarettes. Among ever e-cigarette users in 2014, 30.3% of high school and 42.2% of middle school students had never smoked traditional cigarettes. Given the concern that significant rates of e-cigarette use by U.S. adolescents may have a negative effect on public health, further review of e-cigarette advertising, marketing, sales, and use among U.S. youth is warranted.     

A method to study zhizosphere processes in thin soil layers of different proximity to roots

Frankia is the diverse bacterial genus that fixes nitrogen within root nodules of actinorhizal trees and shrubs. Systematic and ecological studies of Frankia have been hindered by the lack of morphological, biochemical, or other markers to readily distinguish strains. Recently, nucleotide sequence of 16 S RNA from the small ribosomal subunit has been used to classify and identify a variety of microorganisms. We report nucleotide sequences from portions of the 16 S ribosomal RNA from Frankia strains AcnI1 isolated from Alnus viridis ssp. crispa (Ait.) Turrill and PtI1 isolated from Purshia tridentata (Pursh) DC. The number of nucleotide base substitutions and gaps we find more than doubles the previously reported sequence diversity for the same variable regions within other strains of Frankia.     

Development of mating disruption for control of pine sawfly populations

Mating disruption of the pine sawflyNeodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) was strongly indicated by reduced male trap catches in pine plantations permeated with the sex phermone, (2S, 3S, 7S)-diprionly acetate. The trap catch reduction was 95 to near 100% when dispensers every 10 m were used, giving a total release of about 3 mg per hectare and day. Two mg of pheromone per cotton roll dispenser maintained low catches for the whole season (about 2 months) without any renewal of disruption dispensers. Anerythro-mixture was as effective as the pure pheromone isomer. The effects of the experiments on population density and sex ratio were not possible to investigate, due to a general collapse of the population, also outside experimental plots, the year after the experiments.     

A Novel Method for Rapid Hybridization of DNA to a Solid Support

Here we present a novel approach entitled Magnetic Forced Hybridization (MFH) that provides the means for efficient and direct hybridization of target nucleic acids to complementary probes immobilized on a glass surface in less than 15 seconds at ambient temperature. In addition, detection is carried out instantly since the beads become visible on the surface. The concept of MFH was tested for quality control of array manufacturing, and was combined with a multiplex competitive hybridization (MUCH) approach for typing of Human Papilloma Virus (HPV). Magnetic Forced Hybridization of bead-DNA constructs to a surface achieves a significant reduction in diagnostic testing time. In addition, readout of results by visual inspection of the unassisted eye eliminates the need for additional expensive instrumentation. The method uses the same set of beads throughout the whole process of manipulating and washing DNA constructs prior to detection, as in the actual detection step itself.     

The transthyretin-related protein family.

A number of proteins related to the homotetrameric transport protein transthyretin (TTR) forms a highly conserved protein family, which we present in an integrated analysis of data from different sources combined with an initial biochemical characterization. Homologues of the transthyretin-related protein (TRP) can be found in a wide range of species including bacteria, plants and animals, whereas transthyretins have so far only been identified in vertebrates. A multiple sequence alignment of 49 TRP sequences from 47 species to TTR suggests that the tertiary and quaternary features of the three-dimensional structure are most likely preserved. Interestingly, while some of the TRP orthologues show as little as 30% identity, the residues at the putative ligand-binding site are almost entirely conserved. RT/PCR analysis in Caenorhabditis elegans confirms that one TRP gene is transcribed, spliced and predominantly expressed in the worm, which suggests that at least one of the two C. elegans TRP genes encodes a functional protein. We used double-stranded RNA-mediated interference techniques in order to determine the loss-of-function phenotype for the two TRP genes in C. elegans but detected no apparent phenotype. The cloning and initial characterization of purified TRP from Escherichia coli reveals that, while still forming a homotetramer, this protein does not recognize thyroid hormones that are the natural ligands of TTR. The ligand for TRP is not known; however, genomic data support a functional role involving purine catabolism especially linked to urate oxidase (uricase) activity.     

Relationship between fish kairomone concentration in a lake and phototactic swimming by Daphnia

Light-induced swimming behaviour of Daphnia can lead to dielvertical migration. When this occurs, Daphnia may escape frompredation by juvenile 0+ fish. For this to happen, swimmingin response to the change in light intensity at dawn and duskmust be enhanced. This enhanced swimming reaction can be elicitedby fish-associated kairomone in the laboratory. We studied theeffect of these fish-associated kairomones in Lake Maarsseveen,The Netherlands. A bioassay was conducted in which light-inducedswimming reactions of Daphnia were used to quantify the kairomonesignal strength. In two successive years, 1998 and 1999, waterfrom Lake Maarsseveen was tested weekly in early summer. Sampleswere taken from the epilimnion at 3 m depth and from the hypolimnionat 15 m depth. Simultaneously, we caught 0+ perch (Perca fluviatilis)with bongo nets to determine its abundance. The length and weightof individuals caught in 1998 were determined to establish alength–weight relationship. Using this relationship andthe information on perch density from the trawls, the relativefish biomass was calculated for these two years. It was shownthat water from the epilimnion layer increasingly enhanced light-inducedswimming reactions until the second week of June, then the effectgradually disappeared. Water from the hypolimnion had no sucheffect. In 1998, these changes in signal strength correlatedwith the relative biomass of the 0+ perch, but in 1999, themaximum of the enhancement lagged 2 weeks behind the maximumof the biomass of the 0+ perch. This lag may be due to a differentdevelopment of the thermocline. We conclude that kairomone concentrationmay well correlate with 0+ perch biomass and thereby might informDaphnia not only about the presence, but also about the abundance,of juvenile perch.