Automated multi-dimensional purification of tagged proteins

The capacity for high throughput purification (HTP) is essential in fields such as structural genomics where large numbers of protein samples are routinely characterized in, for example, studies of structural determination, functionality and drug development. Proteins required for such analysis must be pure and homogenous and available in relatively large amounts. ÄKTAT 3D system is a powerful automated protein purification system, which minimizes preparation, run-time and repetitive manual tasks. It has the capacity to purify up to 6 different His₆- or GST-tagged proteins per day and can produce 1–50 mg protein per run at >90% purity. The success of automated protein purification increases with careful experimental planning. Protocol, columns and buffers need to be chosen with the final application area for the purified protein in mind.     

Identification of a common risk haplotype for canine idiopathic epilepsy in the ADAM23 gene

Background

Idiopathic epilepsy is a common neurological disease in human and domestic dogs but relatively few risk genes have been identified to date. The seizure characteristics, including focal and generalised seizures, are similar between the two species, with gene discovery facilitated by the reduced genetic heterogeneity of purebred dogs. We have recently identified a risk locus for idiopathic epilepsy in the Belgian Shepherd breed on a 4.4 megabase region on CFA37.

Results

We have expanded a previous study replicating the association with a combined analysis of 157 cases and 179 controls in three additional breeds: Schipperke, Finnish Spitz and Beagle (p_c = 2.9e–07, p_GWAS = 1.74E-02). A targeted resequencing of the 4.4 megabase region in twelve Belgian Shepherd cases and twelve controls with opposite haplotypes identified 37 case-specific variants within the ADAM23 gene. Twenty-seven variants were validated in 285 cases and 355 controls from four breeds, resulting in a strong replication of the ADAM23 locus (p_raw = 2.76e–15) and the identification of a common 28 kb-risk haplotype in all four breeds. Risk haplotype was present in frequencies of 0.49–0.7 in the breeds, suggesting that ADAM23 is a low penetrance risk gene for canine epilepsy.

Conclusions

These results implicate ADAM23 in common canine idiopathic epilepsy, although the causative variant remains yet to be identified. ADAM23 plays a role in synaptic transmission and interacts with known epilepsy genes, LGI1 and LGI2, and should be considered as a candidate gene for human epilepsies.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1651-9) contains supplementary material, which is available to authorized users.     

Primers targeting mitochondrial genes of avian haemosporidians: PCR detection and differential DNA amplification of parasites belonging to different genera

Haemosporida is a diverse group of vector-borne parasitic protozoa, ubiquitous in terrestrial vertebrates worldwide. The renewed interest in their diversity has been driven by the extensive use of molecular methods targeting mitochondrial genes. Unfortunately, most studies target a 478?bp fragment of the cytochrome b (cytb) gene, which often cannot be used to separate lineages from different genera found in mixed infections that are common in wildlife. In this investigation, an alignment constructed with 114 mitochondrial genome sequences belonging to four genera (Leucocytozoon, Haemoproteus, Plasmodium and Hepatocystis) was used to design two different sets of primers targeting the cytb gene as well as the other two mitochondrial DNA genes: cytochrome c oxidase subunit 1 and cytochrome c oxidase subunit 3. The design of each pair of primers required consideration of different criteria, including a set for detection and another for differential amplification of DNA from parasites belonging to different avian haemosporidians. All pairs of primers were tested in three laboratories to assess their sensitivity and specificity under diverse practices and across isolates from different genera including single and natural mixed infections as well as experimental mixed infections. Overall, these primers exhibited high sensitivity regardless of the differences in laboratory practices, parasite species, and parasitemias. Furthermore, those primers designed to separate parasite genera showed high specificity, as confirmed by sequencing. In the case of cytb, a nested multiplex (single tube PCR) test was designed and successfully tested to differentially detect lineages of Plasmodium and Haemoproteus parasites by yielding amplicons with different sizes detectable in a standard agarose gel. To our knowledge, the designed assay is the first test for detection and differentiation of species belonging to these two genera in a single PCR. The experiments across laboratories provided recommendations that can be of use to those researchers seeking to standardise these or other primers to the specific needs of their field investigations.     

Characterisation of Age-Dependent Beta Cell Dynamics in the Male db/db Mice

Aim

To characterise changes in pancreatic beta cell mass during the development of diabetes in untreated male C57BLKS/J db/db mice.

Methods

Blood samples were collected from a total of 72 untreated male db/db mice aged 5, 6, 8, 10, 12, 14, 18, 24 and 34 weeks, for measurement of terminal blood glucose, HbA_1c, plasma insulin, and C-peptide. Pancreata were removed for quantification of beta cell mass, islet numbers as well as proliferation and apoptosis by immunohistochemistry and stereology.

Results

Total pancreatic beta cell mass increased significantly from 2.1 ± 0.3 mg in mice aged 5 weeks to a peak value of 4.84 ± 0.26 mg (P < 0.05) in 12-week-old mice, then gradually decreased to 3.27 ± 0.44 mg in mice aged 34 weeks. Analysis of islets in the 5-, 10-, and 24-week age groups showed increased beta cell proliferation in the 10-week-old animals whereas a low proliferation is seen in older animals. The expansion in beta cell mass was driven by an increase in mean islet mass as the total number of islets was unchanged in the three groups.

Conclusions/Interpretation

The age-dependent beta cell dynamics in male db/db mice has been described from 5-34 weeks of age and at the same time alterations in insulin/glucose homeostasis were assessed. High beta cell proliferation and increased beta cell mass occur in young animals followed by a gradual decline characterised by a low beta cell proliferation in older animals. The expansion of beta cell mass was caused by an increase in mean islet mass and not islet number.     

Atypical metachromatic leukodystrophy?

Summary A 3/12-year-old slightly retarded boy with marked deficiency of arylsulfatase A (ASA) activity in leucocytes and fibroblasts and almost no cerebroside sulfatase (CS) activity in fibroblasts was tested with the sulfatide-loading test. On this test his fibroblasts showed impaired degradation. A pathological excretion of sulfatides was seen in his urine. Nerve conduction velocity, visual evoked potential, auditory brain stem evoked response, and somatosensory evoked potential were all normal.His father and older brother had similarly low levels of ASA in leucocytes and fibroblasts and 1.7–2% residual CS activity in fibroblasts. Although both were clinically normal, their fibroblasts accumulated increased amounts of sulfatides when challenged in the sulfatide-loading test.In this family, this test thus will be of no value in prenatal diagnosis to discriminate among low ASA fetuses with pseudoarylsulfatase A deficiency and fetuses with this unusual ASA deficiency variant.     

Arabidopsis PDK1: identification of sites important for activity and downstream phosphorylation of S6 kinase

The Arabidopsis thaliana protein kinase AtPDK1 was identified as a homologue of the mammalian 3-phosphoinositide-dependent protein kinase-1 (PDK1), which is involved in a number of physiological processes including cell growth and proliferation. We now show that AtPDK1, expressed in E. coli as a recombinant protein, undergoes autophosphorylation at several sites. Using mass spectrometry, three phosphorylated amino acid residues, Ser-177, Ser-276 and Ser-382, were identified, followed by mutational analyses to reveal their roles. These residues are not conserved in mammalian PDK1s. Mutation of Ser-276 in AtPDK1 to alanine resulted in an enzyme with no detectable autophosphorylation. Autophosphorylation was significantly reduced in the Ser177Ala mutant but was only slightly reduced in the Ser382Ala mutant. Other identified sites of importance for autophosphorylation and/or activity of AtPDK1 were Asp-167, Thr-176, and Thr-211. Sites in the mammalian PDK1 corresponding to Asp-167 and Thr-211 are essential for PDK1 autophosphorylation and activity. Autophosphorylation was absent in the Asp167Ala mutant while the Thr176Ala and The211Ala mutants exhibited very low but detectable autophosphorylation, pointing to both similarity and difference between mammalian and plant enzymes. We also demonstrate that AtS6k2, an A. thaliana homologue to the mammalian S6 kinases, is an in vitro target of AtPDK1. Our data clearly show that Asp-167, Thr-176, Ser-177, Thr-211, and Ser-276 in AtPDK1 are important for the downstream phosphorylation of AtS6k2. The results confirm that AtPDK1, like mammalian PDK1, needs phosphorylation at several sites for full downstream phosphorylation activity. Finally, we investigated A. thaliana 14-3-3 proteins as potential AtPDK1 regulatory proteins and the effect of phospholipids on the AtPDK1 activity. Nine of the 12 14-3-3 isoforms tested enhanced AtPDK1 activity whereas one isoform suppressed the activity. No significant effects on AtPDK1 activity by the various phospholipids (including phosphoinositides) were evident.     

Uptake and intracellular transportation of a bacterial surface protein in lymphoid cells

Some strains of the human pathogen Streptococcus pyogenes express a surface protein called protein H, which is released from the streptococcal surface by a cysteine proteinase produced by the bacteria. Here, we find that soluble protein H binds to the surface of lymphocytes and granulocytes, and that the molecule is taken up by lymphocytes and transported to the perinuclear region. The translocation over the cell membrane is rapid, and the uptake and intracellular transportation is not dependent on actin polymerization. Protein H could be immunoprecipitated from cell extracts and nuclear preparations of lymphocytes, and analysis of molecular interactions between protein H and proteins of different cellular compartments demonstrated a binding to nucleophosmin/ B23, a protein known to shuttle between the cytoplasm and the nucleus, and to the nuclear proteins SET and hnRNP A2/B1. Nucleophosmin/B23 was co-immunoprecipitated with protein H from cell and nuclear extracts, and binding experiments, including kinetic analyses, suggest that protein H dissociating from nucleophosmin/B23 complexes in the perinuclear region or in the nucleus binds to proteins SET and hnRNP A2/B1. Finally, the uptake and intracellular transportation of protein H was found to result in a cytostatic effect on B and T lymphocytes.