Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations

Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column.     

Synthesis and evaluation of novel 2-pyridone derivatives as inhibitors of phosphodiesterase3 (PDE3): A target for heart failure and platelet aggregation

Twenty-six 2-pyridone derivatives (8a-8z), which are structurally analogous to amrinone and milrinone two important cardiotonic drugs, are synthesized and characterized. The synthesis of 2-pyridone derivatives involves addition, followed by cyclization between Baylis-Hillman acetates (7a-7k) and enamino esters or nitriles (3a-3e). Thus synthesized pyridones were subjected to PDE3 inhibitory activity, 14 pyridones were found to be hits out of 26 pyridones synthesized and out of 14 hits, there are 5 pyridones found to be lead compounds having excellent PDE3 inhibitory activity. Further we have carried out computational analysis to understand protein/enzyme and 2-pyridone derivative interactions to identify amino acid residues involved in the vicinity of binding and compared with milrinone drug.     

Sequence Capture and Next Generation Resequencing of the MHC Region Highlights Potential Transplantation Determinants in HLA Identical Haematopoietic Stem Cell Transplantation

How cells coordinate the immune system activities is important for potentially life-saving organ or stem cell transplantations. Polymorphic immunoregulatory genes, many of them located in the human major histocompatibility complex, impact the process and assure the proper execution of tolerance-versus-activity mechanisms. In haematopoietic stem cell transplantation, on the basis of fully human leukocyte antigen (HLA)-matched donor–recipient pairs, adverse effects like graft versus leukaemia and graft versus host are observed and difficult to handle. So far, high-resolution HLA typing was performed with Sanger sequencing, but for methodological reasons information on additional immunocompetent major histocompatibility complex loci has not been revealed. Now, we have used microarray sequence capture and targeted enrichment combined with next generation pyrosequencing for 3.5 million base pair human major histocompatibility complex resequencing in a clinical transplant setting and describe 3025 variant single nucleotide polymorphisms, insertions and deletions among recipient and donor in a single sequencing experiment. Taken together, the presented data show that sequence capture and massively parallel pyrosequencing can be used as a new tool for risk assessment in the setting of allogeneic stem cell transplantation.     

An electrostatic/hydrogen bond switch as the basis for the specific interaction of phosphatidic acid with proteins

Phosphatidic acid (PA) is a minor but important phospholipid that, through specific interactions with proteins, plays a central role in several key cellular processes. The simple yet unique structure of PA, carrying just a phosphomonoester head group, suggests an important role for interactions with the positively charged essential residues in these proteins. We analyzed by solid-state magic angle spinning 31P NMR and molecular dynamics simulations the interaction of low concentrations of PA in model membranes with positively charged side chains of membrane-interacting peptides. Surprisingly, lysine and arginine residues increase the charge of PA, predominantly by forming hydrogen bonds with the phosphate of PA, thereby stabilizing the protein-lipid interaction. Our results demonstrate that this electrostatic/hydrogen bond switch turns the phosphate of PA into an effective and preferred docking site for lysine and arginine residues. In combination with the special packing properties of PA, PA may well be nature's preferred membrane lipid for interfacial insertion of positively charged membrane protein domains.     

Deletion 17q12 is a recurrent copy number variant that confers high risk of autism and schizophrenia

Autism spectrum disorders (ASD) and schizophrenia are neurodevelopmental disorders for which recent evidence indicates an important etiologic role for rare copy number variants (CNVs) and suggests common genetic mechanisms. We performed cytogenomic array analysis in a discovery sample of patients with neurodevelopmental disorders referred for clinical testing. We detected a recurrent 1.4 Mb deletion at 17q12, which harbors HNF1B, the gene responsible for renal cysts and diabetes syndrome (RCAD), in 18/15,749 patients, including several with ASD, but 0/4,519 controls. We identified additional shared phenotypic features among nine patients available for clinical assessment, including macrocephaly, characteristic facial features, renal anomalies, and neurocognitive impairments. In a large follow-up sample, the same deletion was identified in 2/1,182 ASD/neurocognitive impairment and in 4/6,340 schizophrenia patients, but in 0/47,929 controls (corrected p = 7.37 × 10⁻⁵). These data demonstrate that deletion 17q12 is a recurrent, pathogenic CNV that confers a very high risk for ASD and schizophrenia and show that one or more of the 15 genes in the deleted interval is dosage sensitive and essential for normal brain development and function. In addition, the phenotypic features of patients with this CNV are consistent with a contiguous gene syndrome that extends beyond RCAD, which is caused by HNF1B mutations only.     

Relationship between an increase of juvenile hormone titer in early instars and the induction of diapause in fully grown larvae of Sesamia nonagrioides

The larvae of Sesamia nonagrioides (Lepidoptera: Noctuidae) grown at 25 degrees C and long photoperiod (16:8h light:dark) pupate in the 5th or 6th (mostly) larval instar, while the larvae reared under a short photoperiod (12:12h) enter diapause during which they consume some food and undergo up to 12 (usually 3-4) stationary larval molts. Diapause programming includes an increase of juvenile hormone (JH) titer in the hemolymph from about 20 to 50 nM in the 4th and 5th instar larvae (titer in earlier instars was not measured). JH I, II, and III are present in approximate ratio 1-2:10:1. The JH titer drops to zero before pupation but remains around 20 nM during diapause. Perfect extra larval molts associated with a body weight increase can be induced in the non-diapausing larvae with a JH analogue (JHA). The weight rise is due to accumulation of reserves and not to a general body growth. The timing of extra molts is similar to the molting pattern of the diapausing larvae only when JHA is present since early larval instars. In the diapausing larvae, JHA application affects neither molting periodicity nor the body weight. It is concluded that (1) Increased JH titer in early larval instars is a part of diapause programming; (2) The extension of larval stage in the diapausing larvae, but not the timing pattern of extra molts, is due to continuously high JH titer; (3) The diapause program includes low food intake, maintenance of a certain body weight, and periodic larval molts.     

Diadenosine tetraphosphate (Ap4A) is compartmentalized in nuclei of mammalian cells

The intracellular compartmentation of Ap4A in various growth and cell-cycle stages in mammalian cells was studied by applying a non-aqueous extraction procedure for cell nuclei. In both slowly and in exponentially growing Ehrlich ascites tumour cells from random cultures, more than 75% of the whole cellular Ap4A content is localized in the nuclei. In G1 and early S-phase cells of synchronized baby hamster kidney (BHK) fibroblast cultures, approx. 90% of the intracellular Ap4A pool is confined to the nuclear compartment. In contrast, Ap4A is distributed to nearly equal amounts between cytoplasm and nuclei during mid-S phase. After transition through the S-phase, increasing proportions of Ap4A (78% 18 h and 96% 22 h after serum replenishing, respectively) are again localized in the nuclear compartment.     

Why boys will be boys: two pathways of fetal testicular androgen biosynthesis are needed for male sexual differentiation

Human sexual determination is initiated by a cascade of genes that lead to the development of the fetal gonad. Whereas development of the female external genitalia does not require fetal ovarian hormones, male genital development requires the action of testicular testosterone and its more potent derivative dihydrotestosterone (DHT). The "classic" biosynthetic pathway from cholesterol to testosterone in the testis and the subsequent conversion of testosterone to DHT in genital skin is well established. Recently, an alternative pathway leading to DHT has been described in marsupials, but its potential importance to human development is unclear. AKR1C2 is an enzyme that participates in the alternative but not the classic pathway. Using a candidate gene approach, we identified AKR1C2 mutations with sex-limited recessive inheritance in four 46,XY individuals with disordered sexual development (DSD). Analysis of the inheritance of microsatellite markers excluded other candidate loci. Affected individuals had moderate to severe undervirilization at birth; when recreated by site-directed mutagenesis and expressed in bacteria, the mutant AKR1C2 had diminished but not absent catalytic activities. The 46,XY DSD individuals also carry a mutation causing aberrant splicing in AKR1C4, which encodes an enzyme with similar activity. This suggests a mode of inheritance where the severity of the developmental defect depends on the number of mutations in the two genes. An unrelated 46,XY DSD patient carried AKR1C2 mutations on both alleles, confirming the essential role of AKR1C2 and corroborating the hypothesis that both the classic and alternative pathways of testicular androgen biosynthesis are needed for normal human male sexual differentiation.     

Targeted Next Generation Sequencing as a Reliable Diagnostic Assay for the Detection of Somatic Mutations in Tumours Using Minimal DNA Amounts from Formalin Fixed Paraffin Embedded Material

Background

Targeted Next Generation Sequencing (NGS) offers a way to implement testing of multiple genetic aberrations in diagnostic pathology practice, which is necessary for personalized cancer treatment. However, no standards regarding input material have been defined. This study therefore aimed to determine the effect of the type of input material (e.g. formalin fixed paraffin embedded (FFPE) versus fresh frozen (FF) tissue) on NGS derived results. Moreover, this study aimed to explore a standardized analysis pipeline to support consistent clinical decision-making.

Method

We used the Ion Torrent PGM sequencing platform in combination with the Ion AmpliSeq Cancer Hotspot Panel v2 to sequence frequently mutated regions in 50 cancer related genes, and validated the NGS detected variants in 250 FFPE samples using standard diagnostic assays. Next, 386 tumour samples were sequenced to explore the effect of input material on variant detection variables. For variant calling, Ion Torrent analysis software was supplemented with additional variant annotation and filtering.

Results

Both FFPE and FF tissue could be sequenced reliably with a sensitivity of 99.1%. Validation showed a 98.5% concordance between NGS and conventional sequencing techniques, where NGS provided both the advantage of low input DNA concentration and the detection of low-frequency variants. The reliability of mutation analysis could be further improved with manual inspection of sequence data.

Conclusion

Targeted NGS can be reliably implemented in cancer diagnostics using both FFPE and FF tissue when using appropriate analysis settings, even with low input DNA.