Silver staining of proteins in polyacrylamide gels

Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram range) with the use of very simple and cheap equipment and chemicals. It is compatible with downstream processing, such as mass spectrometry analysis after protein digestion. The sequential phases of silver staining are protein fixation, then sensitization, then silver impregnation and finally image development. Several variants of silver staining are described here, which can be completed in a time range from 2 h to 1 d after the end of the electrophoretic separation. Once completed, the stain is stable for several weeks.     

Recombinant kallikrein expression: site-specific integration for hK6 production in human cells

Kallikreins have been implicated in carcinogenesis and are promising biomarkers for the diagnosis and follow-up of various cancers. To evaluate the functions and clinical interest of kallikreins, it is important to be able to produce them as recombinant proteins. Here we summarize the various strategies used to produce kallikreins, emphasizing their advantages and limitations. We also describe an approach to achieve high-level production of kallikreins, such as hK6, with correct folding and activity, combining an expression system with targeted transgene integration and an efficient cultivation device to increase yield, the CELLine bioreactor. This novel method for recombinant kallikrein production will be useful to study their bio-pathological functions and to develop anti-bodies.     

Apc tumor suppressor gene is the "zonation-keeper" of mouse liver

The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. We provide here compelling evidence that the Wnt/beta-catenin pathway plays a key role in liver zonation. First, we show the complementary localization of activated beta-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes. We then analyzed the immediate consequences of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1, and we show that Wnt/beta-catenin signaling inversely controls the perivenous and periportal genetic programs. Finally, we show that genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of beta-catenin signaling, and that perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From our results, we propose that Apc is the liver "zonation-keeper" gene.     

Immunoproteomic analysis of the murine antibody response to successful and failed immunization with live anti-Francisella vaccines

Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA\2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice.     

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.     

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.