Stable expression of blood group H determinants and GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase in mouse cells after transfection with human DNA

We report here the application of a genetic approach to identify and isolate human DNA sequences controlling the expression of a GDP-L-fucose: beta-D-galactoside 2-alpha-L-fucosyltransferase [alpha-1,2)fucosyltransferase). Mouse L cells were chosen as host cells for this scheme since they express the necessary substrate and acceptor molecules for surface display of blood group H Fuc alpha 1----2 G al linkages constructed by (alpha-1,2) fucosyltransferases. However, they do not express cell surface blood group H structures nor detectable (alpha-1,2)fucosyltransferase activity. We therefore asked if (alpha-1,2)fucosyltransferase activity could be expressed and detected in these cells after transfection with human DNA sequences. These cells were transfected with genomic DNA isolated from a human cell line (A431) that expresses (alpha-1,2)fucosyltransferase. A panning procedure and fluorescence-activated cell sorting were used to isolate a mouse transfectant cell line that expresses cell surface H Fuc alpha 1----2 Gal linkages and a cognate (alpha-1,2)fucosyltransferase. Southern blot analysis showed that the genome of this cell line contains several hundred kilobase pairs of human DNA. Genomic DNA from this primary transfectant was used to transfect mouse L cells, and several independent, H-expressing secondary transfectants were isolated by immunological selection. Each expresses an (alpha-1,2)fucosyltransferase. Southern blot analysis demonstrated that the genome of each secondary transfectant contains common, characteristic human DNA restriction fragments. These results show that transfected human DNA sequences determine expression of the (alpha-1,2)fucosyltransferases in the mouse transfectants, that these sequences represent a single locus, and that they are within or linked to specific human restriction fragments identifiable in each secondary transfectant. These sequences may represent a human (alpha-1,2)fucosyltransferase gene.     

Resistance of heparinase-derived heparin fragments to biotransformation

The biotransformation of heparinase-derived heparin fragments was examined via a combined approach using 35S-labeled heparin fragments as well as unlabeled chemically defined heparin fragments. Rats dosed with either [35S]di-, tetra-, hexa-, or octasaccharide fragments (2 mg/kg body weight, intravenously) excreted 63-69% of the injected radioactivity into the urine within 24 h with two-thirds being excreted during the first 6 h. Gel permeation chromatography of the urinary material shows that the tetra- and octasaccharides have undergone minor (approximately 5%) depolymerization whereas no change was observed for the di- and hexasaccharides. No N-desulfation was demonstrated for any of the substances. The hexa- and octasaccharide metabolites present in the urine 24 h after dosing exhibited the same antifactor Xa activity as that of the injected material. A chemically defined trisulfated disaccharide and a hexasulfated tetrasaccharide were prepared and dosed in a similar manner. Only one metabolite was recovered from animals dosed with disaccharide. This compound was characterized by anion exchange chromatography, proton nuclear magnetic resonance spectroscopy, Fourier transform infrared spectrometry, and mass spectrometry and shown to be identical to the injected disaccharide. Five metabolites were isolated from the urine of rats dosed with the hexasulfated tetrasaccharide. The major metabolite, consisting of at least 65% of the total, was characterized as described for the disaccharide and shown to be identical to the injected compound. The remaining material appeared to be disaccharides and, possibly, a tetrasaccharide conjugate. Taken together, our results show that the heparinase-derived heparin fragments are very resistant to biotransformation compared with heparin and endogenous heparin fragments. These fragments may therefore be useful in defining structure activity relationships in vivo.     

In vitro studies of prolactin inhibition of luteinizing hormone action on Leydig cells of rats and mice

Previous in vivo studies have shown that in male rabbits prolactin inhibits the testosterone production stimulated by luteinizing hormone (LH) or human chorionic gonadotropin (hCG). This inhibition has now been studied in vitro using both mouse and rat testicular interstitial cells. First, the dose response of human LH (hLH) stimulation of testosterone was studied in detail using testicular interstitial cells from both species. Next, a small but stimulatory dose of hLH was selected and extensive prolactin doses were studied in vitro. NIH B-6 (bovine) prolactin in varying doses was added to the interstitial cells 30 min prior to the addition of a constant dose of hLH. Under these circumstances prolactin inhibited LH action over a wide range of doses. In both species a biphasic dose-response curve existed: large doses of 100 to 1000 ng/ml produced less inhibition or augmented LH action, compared to smaller doses. Next, entire hLH dose-response curves were produced in the presence of three doses of prolactin (0.33, 33, and 1000 ng/ml) as well as in the absence of prolactin. The addition of prolactin shifted the hLH dose-response curve to the right and depressed the maximal response in comparison to the curve without prolactin. Finally, inhibitory doses of prolactin resulted in no detectable change in LH receptor number as estimated from Scatchard plots. It is concluded that prolactin inhibits LH action on interstitial cells as determined by rate of testosterone production except at very large doses of prolactin where LH action is less inhibited or augmented. The inhibitory action of prolactin in this in vitro interstitial cell assay was not accompanied by a decrease in LH receptor number. Thus, a postreceptor action is likely to be involved.     

Analysis of mortality metrics associated with a comprehensive range of disorders in Denmark, 2000 to 2018: A population-based cohort study

BackgroundThe provision of different types of mortality metrics (e.g., mortality rate ratios [MRRs] and life expectancy) allows the research community to access a more informative set of health metrics. The aim of this study was to provide a panel of mortality metrics associated with a comprehensive range of disorders and to design a web page to visualize all results.Methods and findingsIn a population-based cohort of all 7,378,598 persons living in Denmark at some point between 2000 and 2018, we identified individuals diagnosed at hospitals with 1,803 specific categories of disorders through the International Classification of Diseases-10th Revision (ICD-10) in the National Patient Register. Information on date and cause of death was obtained from the Registry of Causes of Death. For each of the disorders, a panel of epidemiological and mortality metrics was estimated, including incidence rates, age-of-onset distributions, MRRs, and differences in life expectancy (estimated as life years lost [LYLs]). Additionally, we examined models that adjusted for measures of air pollution to explore potential associations with MRRs. We focus on 39 general medical conditions to simplify the presentation of results, which cover 10 broad categories: circulatory, endocrine, pulmonary, gastrointestinal, urogenital, musculoskeletal, hematologic, mental, and neurologic conditions and cancer. A total of 3,676,694 males and 3,701,904 females were followed up for 101.7 million person-years. During the 19-year follow-up period, 1,034,273 persons (14.0%) died. For 37 of the 39 selected medical conditions, mortality rates were larger and life expectancy shorter compared to the Danish general population. For these 37 disorders, MRRs ranged from 1.09 (95% confidence interval [CI]: 1.09 to 1.10) for vision problems to 7.85 (7.77 to 7.93) for chronic liver disease, while LYLs ranged from 0.31 (0.14 to 0.47) years (approximately 16 weeks) for allergy to 17.05 (16.95 to 17.15) years for chronic liver disease. Adjustment for air pollution had very little impact on the estimates; however, a limitation of the study is the possibility that the association between the different disorders and mortality could be explained by other underlying factors associated with both the disorder and mortality.ConclusionsIn this study, we show estimates of incidence, age of onset, age of death, and mortality metrics (both MRRs and LYLs) for a comprehensive range of disorders. The interactive data visualization site (https://nbepi.com/atlas) allows more fine-grained analysis of the link between a range of disorders and key mortality estimates.

In a population-based study, Oleguer Plana-Ripoll and colleagues report on and develop an online resource to study mortality metrics and life expectancy associated with different health conditions among individuals living in Denmark.     

Optimal and freely chosen paddling rate during moderate kayak ergometry

Moderate paddling, as in long distance kayaking, constitutes an endurance activity, which shares energetic aspects with activities such as long distance running and road cycling. The aim of the present study was to investigate whether in moderate paddling there is a U-shaped relationship between oxygen uptake and stroke rate, and also whether elite kayakers apply a freely chosen stroke rate, which is energetically optimal. Eleven young male elite kayakers performed moderate kayak ergometry at preset target stroke rates of 65, 75, and 90 strokes min^-1, and at a freely chosen stroke rate, while physiological responses including oxygen uptake were measured. The results showed that considering average values calculated across all participants, there was an approximately U-shaped relationship between oxygen uptake and target stroke rate with a minimum at 75 strokes min^-1. The freely chosen stroke rate was 67.0 ± 6.1 strokes min^-1. Thus, the freely chosen stroke rate, for the group in total, appeared to be lower and require higher oxygen uptake as compared to the energetically optimal preset target stroke rate. Eight out of 11 participants had a higher oxygen uptake (5.1% ± 6.7%, p = 0.028, across all participants) at their freely chosen stroke rate than at the preset target stroke rate, which resulted in the lowest oxygen uptake. In conclusion, an approximately U-shaped relationship between oxygen uptake and stroke rate for young elite kayakers during moderate ergometer kayaking was found. Additionally, the freely chosen stroke rate was systematically lower and, consequently, required higher oxygen uptake than the preset stroke rate, which resulted in the lowest oxygen uptake.     

Locked nucleic acid: a potent nucleic acid analog in therapeutics and biotechnology

Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic physiochemical properties of LNA and some of the difficulties that may be encountered when applying LNA technology. The central part of the review focuses on the use of LNA molecules in regulation of gene expression, including delivery to cells, stability, unspecific effects, toxicity, pharmacokinetics, and design of LNA oligonucleotides. The last part evaluates LNA as a diagnostic tool in genotyping.     

Protein Evolution via Amino Acid and Codon Elimination

Background

Global residue-specific amino acid mutagenesis can provide important biological insight and generate proteins with altered properties, but at the risk of protein misfolding. Further, targeted libraries are usually restricted to a handful of amino acids because there is an exponential correlation between the number of residues randomized and the size of the resulting ensemble. Using GFP as the model protein, we present a strategy, termed protein evolution via amino acid and codon elimination, through which simplified, native-like polypeptides encoded by a reduced genetic code were obtained via screening of reduced-size ensembles.

Methodology/Principal Findings

The strategy involves combining a sequential mutagenesis scheme to reduce library size with structurally stabilizing mutations, chaperone complementation, and reduced temperature of gene expression. In six steps, we eliminated a common buried residue, Phe, from the green fluorescent protein (GFP), while retaining activity. A GFP variant containing 11 Phe residues was used as starting scaffold to generate 10 separate variants in which each Phe was replaced individually (in one construct two adjacent Phe residues were changed simultaneously), while retaining varying levels of activity. Combination of these substitutions to generate a Phe-free variant of GFP abolished fluorescence. Combinatorial re-introduction of five Phe residues, based on the activities of the respective single amino acid replacements, was sufficient to restore GFP activity. Successive rounds of mutagenesis generated active GFP variants containing, three, two, and zero Phe residues. These GFPs all displayed progenitor-like fluorescence spectra, temperature-sensitive folding, a reduced structural stability and, for the least stable variants, a reduced steady state abundance.

Conclusions/Significance

The results provide strategies for the design of novel GFP reporters. The described approach offers a means to enable engineering of active proteins that lack certain amino acids, a key step towards expanding the functional repertoire of uniquely labeled proteins in synthetic biology.     

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.     

The RDP (Ribosomal Database Project).

The Ribosomal Database Project (RDP) is a curated database that offers ribosome-related data, analysis services and associated computer programs. The offerings include phylogenetically ordered alignments of ribosomal RNA (rRNA) sequences, derived phylogenetic trees, rRNA secondary structure diagrams, and various software for handling, analyzing and displaying alignments and trees. The data are available via anonymous FTP (rdp.life.uiuc.edu), electronic mail (server@rdp.life.uiuc.edu), gopher (rdpgopher.life.uiuc.edu) and WWW (http://rdpwww.life.uiuc.edu/ ). The electronic mail and WWW servers provide ribosomal probe checking, approximate phylogenetic placement of user-submitted sequences, screening for possible chimeric rRNA sequences, automated alignment, and a suggested placement of an unknown sequence on an existing phylogenetic tree.     

Epigenome dysregulation in cholangiocarcinoma

Epigenomics is a fast-evolving field of research that has lately attracted considerable interest, mainly due to the reversibility of epigenetic marks. Clinically, among solid tumors, the field is still limited. In cholangiocarcinoma (CCA) it is well known that the epigenetic landscape is deregulated both during carcinogenesis and disease progression as a consequence of aberrant mechanisms leading to genome instability. In this article, we will briefly review the molecular alterations that have been described in the transformation of normal cholangiocytes into malignant derivatives, focusing on the role of non-coding RNA (ncRNA) interactions, DNA methylation, post-translational modifications (PTMs) of histones and chromatin remodeling complexes.