Peter N. Graves

Preparation of influenza virus subviral particles lacking the HA1 subunit of hemagglutinin: unmasking of cross-reactive HA2 determinants.

Acid treatment of influenza A and B virus preparations followed by addition of dithiothreitol (DTT) and centrifugation through a sucrose cushion removes the HA1 subunit of hemagglutinin from virus. Rabbit sera made against these subviral particles and untreated virus were tested in a radioimmune precipitation assay using [35S]cysteine-labeled virus. Conditions of the assay permitted discrimination of discrete HA1- and HA2-specific antibody populations. It was found that (a) sera raised to intact influenza A virus preparations contained both HA1- and HA-2 specific antibodies, (b) sera made to subviral particles of influenza A virus contained HA2-specific antibody but had little or no detectable HA1-specific antibody. (c) the HA2-specific antibodies were partially cross-reactive with the HA2 of an influenza A virus of a different subtype, and (d) sera raised against two strains of untreated influenza B viruses contained antibodies which were cross-reactive with the HA2 as well as the NP of influenza A viruses.

Heterologous Expression and Functional Characterization of a Mouse Renal Organic Anion Transporter in Mammalian Cells

Organic anion transporters play an essential role in eliminating a wide range of organic anions including endogenous compounds, xenobiotics, and their metabolites from kidney, thereby preventing their potentially toxic effects within the body. The goal of this study was to extend our previous study on the functional characterization and post-translational modification of a mouse kidney organic anion transporter (mOAT), in a mammalian cell system, COS-7 cells. The transporter-mediated p-aminohippurate (PAH) uptake was saturable, probenecid-sensitive, and inhibited by a wide range of organic anions including vitamins, anti-hypertensive drugs, anti-tumor drugs, and anti-inflammatory drugs. Tunicamycin, an inhibitor of asparagine-linked glycosylation, significantly inhibited the transport activity. Immunofluorescence provided evidence that most of the protein remained in the intracellular compartment in tunicamycin-treated cells. Diethyl pyrocarbonate (DEPC), a histidine residue-specific reagent, completely blocked PAH transport. The inhibitory effect by DEPC was significantly protected (90%) by pretreating the cells with excess unlabeled PAH, suggesting that the histidine residues may be close to the PAH binding sites. Finally,in situ mRNA localization was studied in postnatal mouse kidney. The expression was observed in proximal tubules throughout development. We conclude that COS-7 cells may be useful in pharmacological and molecular biological studies of this carrier. The carbohydrate moieties are necessary for the proper trafficking of mOAT to the plasma membrane, and histidine residues appear to be important for the transport function.

CTLA-4 and autoimmune thyroid disease: lack of influence of the A49G signal peptide polymorphism on functional recombinant human CTLA-4☆

A single nucleotide A49G polymorphism (SNP) of CTLA-4 has been linked and associated with the autoimmune thyroid diseases (AITD) and thyroid autoantibody secretion. We have explored the functional mechanisms of CTLA-4 by means of recombinant human CTLA-4 expressed on transfected Jurkat T cells. Analysis of CTLA-4 transcripts with quantitative real-time PCR demonstrated similar baseline and PHA-stimulated levels for both the A49 and G49 alleles, which were markedly enhanced by anti-CTLA-4 engagement. Both alleles also coded for proteins which were expressed on the cell membrane, as measured by FACS analysis using anti-CTLA-4 (G: 34.4+/-11.9% cells, A: 27.6+/-8.6% cells) (p=ns). Baseline and PHA-stimulated IL-2 production were also similar among control and CTLA-4 clones expressing both alleles. After anti-CTLA-4 engagement, IL-2 production was markedly inhibited in a dose- and time-dependent manner but this also appeared to be similar in the A and G allele expressing cells (95.7+/-1.2% inhibition and 94.9+/-1.1% inhibition, respectively). In conclusion, both the extrinsic and intrinsic actions of human CTLA-4 were not affected by the signal peptide A49G polymorphism. Therefore, the linkage of the CTLA-4 A49G SNP to AITD is most likely secondary to linkage disequilibrium.

Absence of lutropin(LH) receptor mRNA in the rat thyroid : further evidence for specificity cross-over at the thyroid-stimulating hormone receptor level

Chorionic gonadotropin (CG) and purified lutropin (LH) activate intact thyroid tissue and isolated thyroid cells. A recent report has suggested that the presence of aberrant LH/CG receptors in human and rat thyroid tissue may interact with gonadotropin thus explaining the mechanisms of thyroid cell stimulation. To detect putative thyroidal LH receptor mRNA, a segment of the transmembrane region containing domains 3 through 6 of the rat (r) LH receptor was targeted for amplification using the polymerase chain reaction (PCR). cDNA prepared from a rLH receptor-positive control tissue (testis) was efficiently amplified under stringent annealing conditions giving a 486 bp product as predicted. However, cDNAs from thyroidal tissue and from the thyroid-stimulating hormone (TSH)- and hCG-responsive 1B-6 subclone of Fisher rat thyroid cells (FRTL-5) yielded no detectable 486 bp product. A smaller (non-LH) fragment amplified to similar extents from both testis and thyroidal cDNAs provided a useful internal control for amplification. This allowed the conclusion that specificity cross-over between LH/CG and TSH occurs at the TSH receptor and that the LH/CG receptor gene is transcriptionally silent in rat thyroidal cells.

GAUCHER DISEASE: ENZYMATIC AND MOLECULAR STUDIES

Human acid β-glucosidase (β-Glc, D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) is a membrane associated lysosomal enzyme which cleaves the βglucosidic linkage of its natural substrate, glucosylceramide (GC), and synthetic β-glucosides1,2. The defective activity of this enzyme leads to the subtypes and variants of Gaucher disease3. The normal mature polypeptide has a predicted molecular mass of 55,834 Da4,5. The normal enzyme also contains 7- 15% carbohydrate6,7 and extensive glycosidic remodelling occurs post-translationally in cultured fibroblasts8,9. Except for signal sequence clipping, no other proteolytic processing has been detected with the human enzyme from several sources7,9. A catalytically essential carboxylate, Asp443, has been provisionally assigned near the carboxy terminus of this 497 amino acid enzyme by using the affinity label. Bromo[3H]conduritol B epoxide, an active sitedirected covalent inhibitor10. In addition, kinetic studies have suggested the presence of three subsites within the active site with specificities for the glycon head group and the acyl or alkyl moieties of substrates and/or inhibitors11,12. These latter subsites influence apparent kcat values or binding constants, respectively11. Similar studies have also suggested a relatively hydrophilic region within the active site which is located at the junction of the three subsites and has been proposed to have specificity for the hydroxyl and amine group of sphingosine11. The glycon binding site contains residues for binding of properly configured hydroxyl groups at C-2, C-3 and C-4 of glucose as well as a residue with pKaapp=6.7 which is necessary for the formation of binary (EI) complexes12. Continued study of these subsites should provide insight into structure/function correlations of normal β-Glc as well as into the nature of the enzymatic defects in the subtypes and variants of Gaucher disease.