The cDNA for casein kinase 1 (CK1) of Plasmodium falciparum was cloned, sequenced, and expressed in bacteria. The single major open reading frame of the 1.2-kilobase pair cDNA coded for a 324-amino acid polypeptide of ϳ37 kDa, the predicted sequence of which showed strong identity with known CK1 isoforms. The purified recombinant enzyme exhibited properties characteristic of CK1, such as inhibition by CK1-7, the ability to phosphorylate a highly specific peptide substrate, and a strong preference for ATP over GTP. A casein kinase activity, partially purified from soluble extracts of P. falciparum by affinity chromatography through CK1-7 columns displayed identical properties. The activity showed a stage-specific expression in the parasite, in the order trophozoite > ring > > schizont. Northern analysis indicated the existence of two major CK1 mRNAs, 2.4 and 3.2 kilobase pairs long, the levels of which were in the order ring > schizont > trophozoite. Mutagenesis of recombinant CK1 defined important amino acid residues and their potential role in the conformation of the enzyme. The malarial CK1 appeared to be the one of the smallest and perhaps the most primitive CK1 enzymes known, containing little sequence information beyond the minimal catalytic domain.The parasitic protozoan, Plasmodium falciparum, is the causative agent of malaria throughout the world and is responsible for an annual death toll of nearly 3 million, the majority of which are children and pregnant mothers (1). However, tools available to control malaria are inadequate, and drug-resistant strains are widespread; moreover, the immediate prospect of a useful vaccine is uncertain. Thus, there is an urgent need to obtain fundamental knowledge about the various cellular processes of P. falciparum at the molecular level, so that susceptible targets can be identified. With this long-term goal in mind, we have initiated studies of the signal transduction system in P. falciparum. Since reversible protein phosphorylation and dephosphorylation constitute a major mechanism of signal transduction (2), one of our immediate goals has been to characterize the various protein kinases in P. falciparum. In this paper, we report the characterization, expression, stagespecific regulation, and mutational analysis of P. falciparum casein kinase 1 (PfCK1). 1 Casein kinase-1 and -2 (CK1 and CK2) are multipotential Ser/Thr protein kinases, originally purified from rabbit reticulocyte lysates using casein as substrate (reviewed in Refs. 3 and 4). In the subsequent years, both enzymes were shown to phosphorylate, and thus regulate, a wide variety of cellular proteins. The sequence alignment of CK1 genes of various organisms and deletion analysis of recombinant yeast CK1 have recently resulted in the delineation of the following domains in the prototype 45-kDa yeast enzyme (summarized in Refs. 5 and 6): an N-terminal catalytic domain of about 300 amino acids followed by a 12-residue stretch conserved among some forms but not in others; a hydrophilic 85-residue segment predi...

No abstract

Background Measuring anti-spike protein antibodies in human plasma or serum is commonly used to determine prior exposure to SARS-CoV-2 infection and to assess the anti-viral protection capacity. According to the mass-action law, a lesser concentration of tightly binding antibody can produce the same quantity of antibody-antigen complexes as higher concentrations of lower affinity antibody. Thus, measurements of antibody levels reflect both affinity and concentration. These two fundamental parameters cannot be disentangled in clinical immunoassays, and so produce a bias which depends on the assay format. Methods To determine the apparent affinity of anti-spike protein antibodies, a small number of antigen-coated magnetic microparticles were imaged by fluorescence microscopy after probing antigen-antibody equilibria directly in patient plasma. Direct and indirect anti-SARS-CoV-2 immunoassays were used to measure antibody levels in the blood of infected and immunised individuals. Findings We observed affinity maturation of antibodies in convalescent and vaccinated individuals, showing that higher affinities are achieved much faster by vaccination. We demonstrate that direct and indirect immunoassays for measuring anti-spike protein antibodies depend differently on antibody affinity which, in turn, affects accurate interpretation of the results. Interpretation Direct immunoassays show substantial antibody affinity dependence. This makes them useful for identifying past SARS-CoV-2 exposure. Indirect immunoassays provide more accurate quantifications of anti-viral antibody levels. Funding The authors are all full-time employees of Abbott Laboratories. Abbott Laboratories provided all operating funds. No external funding sources were used in this study.

No abstract

Rod photoreceptor cGMP phosphodiesterase (PDE6) is a three-subunit (a, b, g2) enzyme that functions to reduce intracellular cytoplasmic cGMP levels, an integral feature of the phototransduction cascade of vision. To allow assessment of the potential for defects in the gene encoding the alpha-subunit (PDE6A) to cause visual dysfunction, and to begin to dissect the basis for photoreceptor-specific expression of this gene, we have characterized the structural gene and upstream region. The human PDE6A gene consists of 22 exons spanning about 60 kb with the intron/exon junctions highly conserved in comparison to the mouse and human PDE6B genes. Using ribonuclease protection and primer extension assays, a predominant transcription start point (tsp) was identified 120 bp upstream of the initiator ATG. To begin functional analysis of the PDE6A promoter, approx 4 kb of sequence were determined upstream of the tsp. Comparison of this upstream sequence with an approximately 500 bp sequence upstream of the mouse Pde6a gene revealed five distinct segments of identity all within 100 bp upstream of the human PDE6A tsp. A TATA box adjacent to a photoreceptor-specific RET1-like binding site, an SP1 site, and two novel putative cis-element sequences were found. A consensus initiator element sequence is present at the tsp. Additionally, within a 2.5-kb segment beginning 900 bp upstream of the tsp two Alu, a MIR, an L1, and two MER repetitive elements were found. Electrophoretic mobility shift assays generate a retina-specific bandshift using a 322-bp fragment containing the putative promoter region or a multimer of the RET1-like site. DNA footprinting assays revealed footprints over the primary transcription startpoint and the RET1-like and TATA box regions. These results indicate that a 220-bp segment of the PDE6A gene upstream region is important for tissue-specific expression.

No abstract

No abstract

No abstract