Selection of ovine PrP high-producer subclones from a transfected epithelial cell line

The hallmarks of prion diseases are the conversion of the normal prion into an abnormal protease resistant isoform and its brain accumulation. Purification of the native abnormal prion isoform for biochemical and biophysical studies has been hampered by poor recovery from brain tissue. An epithelial cell transfected with the ovine VRQ allele prion, called Rov9, has been used to select prion high-producer cells by flow cytometry. The representative clone 4 described here produced 6.2 microg of cellular prion protein per mg of total protein extract, representing 8- to 10-fold the amount produced by the Rov9 parental cells. After exposure to the scrapie agent (PG128/98), clone 4 produced 2.6 microg of abnormal isoform per mg of total protein. When infected clone 4 cell cultures were treated with tunicamycin, 80% of the abnormal isoform was deglycosylated. The infectivity of the prions produced in clone 4 cultures was confirmed in a mouse bioassay. Such high-producer clones represent new tools for producing large amounts of glycosylated and/or non-glycosylated PrP(Sc) and for a powerful screening of clinical samples' infectivity.     

FKBP12 Modulation of the Binding of the Skeletal Ryanodine Receptor onto the II-III Loop of the Dihydropyridine Receptor

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ of the II-III loop of the skeletal DHPR α₁-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu⁷²⁴-Pro⁷⁶⁰ antagonizes this effect. Two peptides, covering these sequences (peptide A_Sk and C_Sk, respectively) were immobilized on polystyrene beads. We demonstrate that peptide A_Sk binds to the skeletal isoform of RyR (RyR1) whereas peptide C_Sk does not. Using surface plasmon resonance detection, we show that 1) domain Thr⁶⁷¹-Leu⁶⁹⁰ is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A_Sk with RyR1 is not modulated by Ca²⁺ (pCa 9-2) nor by Mg²⁺ (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC₅₀ = 10 nM) and inhibited by both rapamycin (IC₅₀ = 5 nM) and FK506. Peptide A_Sk induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A_Sk on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR α₁-subunit corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ and show that the association of FKBP12 with RyR1 specifically modulates this interaction.     

A new translational repression element and unusual transcriptional control regulate expression of don juan during Drosophila spermatogenesis.

The Drosophila don juan (dj) gene encodes a basic protein that is expressed solely in the male germline and shows structural similarities to the linker histone H1. Don Juan is located in two different subcellular structures: in the nucleus during the phase of chromatin condensation and later in the mitochondrial derivatives starting with spermatid individualization. The don juan gene is transcribed in primary spermatocytes under the control of 23 bp upstream in combination with downstream sequences. During meiotic stages and in early spermatid stages don juan mRNA is translationally repressed for several days. Analysis of male sterile mutants which fail to undergo meiosis shows that release of dj mRNA from translational repression is independent of meiosis. In gel retardation assays 60 nucleotides at the end of the dj leader form four major complexes with proteins that were extracted from testes but not with protein extracts from ovaries. Transformation studies prove that in vivo 35 bp within that region of the dj mRNA is essential to confer translational repression. UV cross-linking studies show that a 62 kDa protein specifically binds to the same region within the 5′ untranslated region. The dj translational repression element, TRE, is distinct from the translational control element, TCE, described earlier for all members of the Mst(3)CGP gene family. Moreover, expression studies in several male sterile mutants reveal that don juan mRNA is translated in earlier developmental stages during sperm morphogenesis than the Mst(3)CGP mRNAs. This proves that translational activation of dormant mRNAs in spermatogenesis occurs at different time-points which are characteristic for each gene, an essential feature for coordinated sperm morphogenesis.     

Netherlands Twin Register: a focus on longitudinal research.

In 1986 we began The Netherlands Twin Register (NTR) by recruiting young twins and multiples a few weeks or months after birth. Currently we register around 50% of all newborn multiples in The Netherlands. Their parents receive a questionnaire at registration and afterwards when the children are 2, 3, 5, 7, 10 and 12 years of age. Teachers are asked to rate the behavior of the children at ages 7, 10 and 12 years. Adolescent and young-adult twins were recruited through City Councils in the early 1990s. These twins, their parents and siblings participate in longitudinal survey studies that include items about health, fertility, lifestyle, addiction, personality and psychopathology, religion, socioeconomic status, and educational attainment. The total number of twins and multiples registered with the NTR is currently over 60,000. Subgroups of twins and siblings take part in studies of cognitive development, brain function and neuropsychological indices of attention processes, and molecular genetic studies of classical and behavioral cardiovascular risk factors. DNA samples are currently collected in selected twin families for two large linkage studies, which aim to find QTLs for anxious depression and for nicotine addiction. Sisters who are mothers of DZ twins contribute DNA samples for a linkage study of DZ twinning. Large cohorts of phenotyped family members from the general population are very valuable for genetic epidemiological studies and permit selection of informative families for gene finding studies.     

Balancing food and density‐dependence in the spatial distribution of an interference‐prone forager

Foraging distributions are thought to be density‐dependent, because animals not only select for a high availability and quality of resources, but also avoid conspecific interference. Since these processes are confounded, their relative importance in shaping foraging distributions remains poorly understood. Here we aimed to rank the contribution of density‐dependent and density‐independent effects on the spatio‐temporal foraging patterns of eurasian oystercatchers. In our intertidal study area, tides caused continuous variation in oystercatcher density, providing an opportunity to disentangle conspecific interference and density‐independent interactions with the food landscape. Spatial distributions were quantified using high‐resolution individual tracking of foraging activity and location. In a model environment that included a realistic reconstruction of both the tides and the benthic food, we tested a family of behaviour‐based optimality models against these tracking data. Density‐independent interactions affected spatial distributions much more strongly than conspecific interference, even in an interference‐prone species like oystercatchers. Spatial distributions were governed by avoidance of bill injury costs, selection for high interference‐free intake rates and a decreasing availability of benthic bivalve prey after their exposure. These density‐independent interactions outweighed interference competition in terms of effect size. We suggest that the bottleneck in our mechanistic understanding of foraging distributions may be primarily the role of density‐independent prey attributes unrelated to intake rates, like damage costs in the case of oystercatchers foraging on perilous prey. At a landscape scale, above the finest inter‐individual distances, effects of conspecific interaction on spatial distributions may have been overemphasised.     

Transfer of dysbiotic gut microbiota has beneficial effects on host liver metabolism

Gut microbiota dysbiosis has been implicated in a variety of systemic disorders, notably metabolic diseases including obesity and impaired liver function, but the underlying mechanisms are uncertain. To investigate this question, we transferred caecal microbiota from either obese or lean mice to antibiotic‐free, conventional wild‐type mice. We found that transferring obese‐mouse gut microbiota to mice on normal chow (NC) acutely reduces markers of hepatic gluconeogenesis with decreased hepatic PEPCK activity, compared to non‐inoculated mice, a phenotypic trait blunted in conventional NOD2 KO mice. Furthermore, transferring of obese‐mouse microbiota changes both the gut microbiota and the microbiome of recipient mice. We also found that transferring obese gut microbiota to NC‐fed mice then fed with a high‐fat diet (HFD) acutely impacts hepatic metabolism and prevents HFD‐increased hepatic gluconeogenesis compared to non‐inoculated mice. Moreover, the recipient mice exhibit reduced hepatic PEPCK and G6Pase activity, fed glycaemia and adiposity. Conversely, transfer of lean‐mouse microbiota does not affect markers of hepatic gluconeogenesis. Our findings provide a new perspective on gut microbiota dysbiosis, potentially useful to better understand the aetiology of metabolic diseases.     

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Improved mass spectrometry compatibility is afforded by ammoniacal silver staining

Sequence coverage in MS analysis of protein digestion-derived peptides is a key issue for detailed characterization of proteins or identification at low quantities. In gel-based proteomics studies, the sequence coverage greatly depends on the protein detection method. It is shown here that ammoniacal silver detection methods offer improved sequence coverage over standard silver nitrate methods, while keeping the high sensitivity of silver staining. With the development of 2D-PAGE-based proteomics, another burden is placed on the detection methods used for protein detection on 2-D-gels. Besides the classical requirements of linearity, sensitivity, and homogeneity from one protein to another, detection methods must now take into account another aspect, namely their compatibility with MS. This compatibility is evidenced by two different and complementary aspects, which are (i) the absence of adducts and artefactual modifications on the peptides obtained after protease digestion of a protein detected and digested in - gel, and (ii) the quantitative yield of peptides recovered after digestion and analyzed by the mass spectrometer. While this quantitative yield is not very important per se, it is however a crucial parameter as it strongly influences the S/N of the mass spectrum and thus the number of peptides that can be detected from a given protein input, especially at low protein amounts. This influences in turn the sequence coverage and thus the detail of the analysis provided by the mass spectrometer.     

N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells

In plants, N -linked glycans are processed in the Golgi apparatus to complex-type N -glycans of limited size containing a β(1,2)-xylose and/or an α(1,3)-fucose residue. Larger mono- and bi-antennary N -linked complex glycans have not often been described. This study has re-examined the structure of such plant N -linked glycans, and, through both immunological and structural data, it is shown that the antennae are composed of Lewis a (Le^a) antigens, comprising the carbohydrate sequence Galβ1-3[Fucα1-4]GlcNAc. Furthermore, a fucosyltransferase activity involved in the biosynthesis of this antigen was detected in sycamore cells. This is the first characterization in plants of a Lewis antigen that is usually found on cell-surface glycoconjugates in mammals and involved in recognition and adhesion processes. Le^a-containing N -linked glycans are widely distributed in plants and highly expressed at the cell surface, which may suggest a putative function in cell/cell communication.