The expression of Plasmodium falciparum bloodstage antigens in Escherichia coli

A library of cDNA clones expressing proteins of the asexual blood stages of a Papua New Guinean isolate of Plasmodium falciparum (isolate FCQ27/PNG (FC27] was constructed in the bacteriophage vector lambda gt11-Amp3. In an in situ colony immunoassay, human serum was used to identify colonies producing natural immunogens. Sera from donors of defined clinical status, or reactive to a defined subset of natural immunogens were used to identify clones of particular interest (for example, clones reacting with convalescent but not with acute serum or clones expressing the isolate specific S-antigen of FC27). Antisera raised by immunizing mice and rabbits with cloned antigens were used to characterize the P. falciparum proteins corresponding to the antigen-positive clones. Nucleotide sequence analysis of an antigen found on the surface of cells infected with ring stage parasites revealed an unusual sequence coding for eight, four and three amino acid repeats rich in acidic amino acids. The discussion centres on the use of cloned antigens as tools for the analysis of the host-protective immune response and selection of candidate vaccine molecules.     

Multiphasic activation of smooth muscle adenylate cyclase by pretreatment with guanyl-5′-yl imidodiphosphate (Gpp(NH)p) suggests multiple enzyme populations

The activation of uterine smooth muscle adenylate cyclase was studied by pretreating the particulate form of the enzyme with the GTP analog guanyl-5′-yl imidodiphosphate (Gpp(NH)p). Pretreatment with Gpp(NH)p left the enzyme in an irreversibly activated state which survived subsequent washing in guanyl nucleotide-free buffer. Activation under these conditions was multiphasic with rapid and slow components. At 23 °C slow activation proceeded at about $\text{1}\text{10}\text{th}$ the rate of rapid activation. The onset of the slow phase took longer at lower temperatures. Routine adenylate cyclase assay conditions (conversion of [³²P]ATP to cyclic [³²P]AMP) carried out without pretreatment probably characterized the rapidly activated component. The simplest kinetic model suggests not only the generally accepted two-step association reaction, but also implies the existence of more than one enzyme form, each of which is characterized by a separate activation rate. The complex kinetics of activation might be explained by a heterogeneous mixture of unassociated and preassociated nucleotide binding and catalytic subunits.     

Comparison of yeast mitochondrial Phe-tRNA synthetase subunits to their cytoplasmic counterparts: Isolation and determination of amino acid compositions

The α and β subunits of yeast mitochondrial Phe-tRNA synthetase are separated and isolated by means of chromatography on DEAE-cellulose, after enzyme alkylation with iodoacetate. The comparison of amino acid compositions of yeast mitochondrial and cytoplasmic native Phe-tRNA synthetases and their components shows significant differences. Results indicate that the two enzymes are coded for by different nuclear genes.     

Fusion of newly formed phagosomes with endosomes in intact cells and in a cell-free system

Phagosomes are membrane-bound vesicles, formed by the receptor-mediated internalization of particulate ligands, which exchange soluble and membrane proteins with other endocytic compartments as a part of their maturation process. This exchange of material is undoubtedly mediated by fusion of phagosomes with other membrane-bound compartments of the endocytic pathway. By using a particulate probe (fixed Staphylococcus aureus coated with mouse anti-dinitrophenol monoclonal antibody) localized in phagosomes and a soluble probe (dinitrophenol-derivitized beta-glucuronidase) internalized by receptor-mediated endocytosis, we have studied phagosome-endosome and phagosome-lysosome fusion in intact cells and in a cell-free system. Vesicle fusion was assessed by measuring beta-glucuronidase activity associated with S. aureus particles after lysis of the membranes. In intact macrophages, newly formed phagosomes fused with early endosomes and with lysosomes. Fusion with lysosomes was observed to commence after a short lag period of about 5 min. In broken-cell preparations, phagosomes were able to fuse with early endosomes. It was not possible to reconstitute phagosome-lysosome fusion in vitro. In vitro phagosome-endosome fusion required energy and cytosolic- and membrane-associated proteins. A nonhydrolyzable analog of GTP stimulated fusion at low cytosol concentrations and inhibited fusion at high cytosol concentrations. These observations indicate that the mechanisms mediating phagosome-endosome fusion are similar to those described for endosome-endosome fusion. Our results suggest that exchange of material with endosomes is an important step in the process of phagosome maturation.     

Comparison of an electrochemiluminescent homogenous immunoassay and an ELISA for monitoring productivity in mammalian cell bioreactors

Summary An Enzyme Linked Immuno Sorbent Assays (ELISA) and an Electro-chemiluminescent Immuno Assay (CIA) are compared for the purpose of monitoring product formation in mammalian cell bioreactors. The ELISA had a relative standard deviation of 10%, compared to 8% for the CIA . The CIA was found to be a fast and accurate alternative to the ELISA. The use of more than one immuno assay format was also shown to provide additional insight into process performance.     

Yeast mitochondrial and cytoplasmic valyl-tRNA synthetases

Yeast mitochondrial tRNA synthetase has been partially purified and chromatographic, catalytic and antigenic properties have been compared to the cytoplasmic homologous enzyme from yeast. No significant differences could be observed between the two enzymes with respect to their behaviour during ammonium sulfate precipitation or in chromatographic separation on DEAE cellulose, hydroxylapatite and Sephadex G 200. The Km of the two enzymes toward tRNAs from yeast mitochondria, yeast cytoplasm or E. coli are pratically identical. The antigenic properties of the two enzymes are very similar; antisera against either the mitochondria or the cytoplasmic enzyme lead to the inhibition of their catalytic properties. The mitochondrial ValRS is formed by a single polypeptide chain whose molecular weight is 125,000 daltons, a value very close to that of the yeast cytoplasmic enzyme.