The JC and BK human polyoma viruses appear to be recent introductions to some South American Indian tribes: There is no serological evidence of cross-reactivity with the simian polyoma virus SV40

In an effort to understand the unusual cytogenetic damage earlier encountered in the Yanomama Indians, plasma samples from 425 Amerindians representing 14 tribes have been tested for hemagglutination inhibition antibodies to the human JC polyoma virus and from 369 Amerinds from 13 tribes for hemagglutination inhibition antibodies to the human BK polyoma virus. There is for both viruses highly significant heterogeneity between tribes for the prevalence of serum antibody titers ≥1/40, the pattern of infection suggesting that these two viruses only relatively recently have been introduced into some of these tribes. Some of these samples, from populations with no known exposure to the simian polyoma virus SV40, also were tested for antibodies to this virus by using an immunospot assay. In contrast to the findings of Brown et al. (Brown, P., Tsai, T. & Gajdusek, D. C. (1975) Am. J. Epidemiol. 102, 331–340), none of the samples was found to possess antibodies to SV40. In addition, no significant titers to SV40 were found in a sample of 97 Japanese adults, many of whom had been found to exhibit elevated titers to the JC and BK viruses. This study thus suggests that these human sera contain significant antibody titers to the human polyoma viruses JC and BK but do not appear to contain either cross-reactive antibodies to SV40 or primary antibodies resulting from SV40 infection.

Antibody C219 recognizes an α-helical epitope on P-glycoprotein

The ABC transporter, P-glycoprotein, is an integral membrane protein that mediates the ATP-driven efflux of drugs from multidrug-resistant cancer and HIV-infected cells. Anti-P-glycoprotein antibody C219 binds to both of the ATP-binding regions of P-glycoprotein and has been shown to inhibit its ATPase activity and drug binding capacity. C219 has been widely used in a clinical setting as a tumor marker, but recent observations of cross-reactivity with other proteins, including the c-erbB2 protein in breast cancer cells, impose potential limitations in detecting P-glycoprotein. We have determined the crystal structure at a resolution of 2.4 Å of the variable fragment of C219 in complex with an epitope peptide derived from the nucleotide binding domain of P-glycoprotein. The 14-residue peptide adopts an amphipathic α-helical conformation, a secondary structure not previously observed in structures of antibody–peptide complexes. Together with available biochemical data, the crystal structure of the C219-peptide complex indicates the molecular basis of the cross-reactivity of C219 with non-multidrug resistance-associated proteins. Alignment of the C219 epitope with the recent crystal structure of the ATP-binding subunit of histidine permease suggests a structural basis for the inhibition of the ATP and drug binding capacity of P-glycoprotein by C219. The results provide a rationale for the development of C219 mutants with improved specificity and affinity that could be useful in antibody-based P-glycoprotein detection and therapy in multidrug resistant cancers.

Isolation and characterization of neutral-lipid-containing organelles and globuli-filled plastids from Brassica napus tapetum

The monolayer tapetum cells of the maturing flowers of Brassica napus contain abundant subcellular globuli-filled plastids and special lipid particles, both enriched with lipids that are supposed to be discharged and deposited onto the surface of adjacent maturing pollen. We separated the two organelles by flotation density gradient centrifugation and identified them by electron microscopy. The globuli-filled plastids had a morphology similar to those described in other plant species and tissues. They had an equilibrium density of 1.02 g/cm3 and contained neutral esters and unique polypeptides. The lipid particles contained patches of osmiophilic materials situated among densely packed vesicles and did not have an enclosing membrane. They exhibited osmotic properties, presumably exerted by the individual vesicles. They had an equilibrium density of 1.05 g/cm3 and possessed triacylglycerols and unique polypeptides. Several of these polypeptides were identified, by their N-terminal sequences or antibody cross-reactivity, as oleosins, proteins known to be associated with seed storage oil bodies. The morphological and biochemical characteristics of the lipid particles indicate that they are novel organelles in eukaryotes that have not been previously isolated and studied. After lysis of the tapetum cells at a late stage of floral development, only the major plastid neutral ester was recovered, whereas the other abundant lipids and proteins of the two tapetum organelles were present in fragmented forms or absent on the pollen surface.

Dynamic and quantitative Ca2+ measurements using improved cameleons

Cameleons are genetically-encoded fluorescent indicators for Ca2+ based on green fluorescent protein variants and calmodulin (CaM). Because cameleons can be targeted genetically and imaged by one- or two-photon excitation microscopy, they offer great promise for monitoring Ca2+ in whole organisms, tissues, organelles, and submicroscopic environments in which measurements were previously impossible. However, the original cameleons suffered from significant pH interference, and their Ca2+-buffering and cross-reactivity with endogenous CaM signaling pathways was uncharacterized. We have now greatly reduced the pH-sensitivity of the cameleons by introducing mutations V68L and Q69K into the acceptor yellow green fluorescent protein. The resulting new cameleons permit Ca2+ measurements despite significant cytosolic acidification. When Ca2+ is elevated, the CaM and CaM-binding peptide fused together in a cameleon predominantly interact with each other rather than with free CaM and CaM-dependent enzymes. Therefore, if cameleons are overexpressed, the primary effect is likely to be the unavoidable increase in Ca2+ buffering rather than specific perturbation of CaM-dependent signaling.

Isolation and Characterization of Glutathione S-Transferase Isozymes from Sorghum1

Two glutathione S-transferase (GST) isozymes, A1/A1 and B1/B2, were purified from etiolated, O-1,3-dioxolan-2-yl-methyl-2,2,2,-trifluoro-4′-chloroacetophenone-oxime-treated sorghum (Sorghum bicolor L. Moench) shoots. GST A1/A1, a constitutively expressed homodimer, had a subunit molecular mass of 26 kD and an isoelectric point of 4.9. GST A1/A1 exhibited high activity with 1-chloro-2, 4,dinitrobenzene (CDNB) but low activity with the chloroacetanilide herbicide metolachlor. For GST A1/A1, the random, rapid-equilibrium bireactant kinetic model provided a good description of the kinetic data for the substrates CDNB and glutathione (GSH). GST B1/B2 was a heterodimer with subunit molecular masses of 26 kD (designated the B1 subunit) and 28 kD (designated the B2 subunit) and a native isoelectric point of 4.8. GST B1/B2 exhibited low activity with CDNB and high activity with metolachlor as the substrate. The kinetics of GST B1/B2 activity with GSH and metolachlor fit a model describing a multisite enzyme having two binding sites with different affinities for these substrates. Both GST A1/A1 and GST B1/B2 exhibited GSH-conjugating activity with ethacrynic acid and GSH peroxidase activity with cumene hydroperoxide, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid and 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid. Both GST A1/A1 and GST B1/B2 are glycoproteins, as indicated by their binding of concanavalin A. Polyclonal antibodies raised against GST A1/A1 exhibited cross-reactivity with the B1 subunit of GST B1/B2. Comparisons of the N-terminal amino acid sequences of the GST A1, B1, and B2 subunits with other type I θ-GSTs indicated a high degree of homology with the maize GST I subunit and a sugarcane GST.

NADH-Monodehydroascorbate Oxidoreductase Is One of the Redox Enzymes in Spinach Leaf Plasma Membranes1

Amino acid analysis of internal sequences of purified NADH-hexacyanoferrate(III) oxidoreductase (NFORase), obtained from highly purified plasma membranes (PM) of spinach (Spinacia oleracea L.) leaves, showed 90 to 100% homology to internal amino acid sequences of monodehydroascorbate (MDA) reductases (EC 1.6.5.4) from three different plant species. Specificity, kinetics, inhibitor sensitivity, and cross-reactivity with anti-MDA reductase antibodies were all consistent with this identification. The right-side-out PM vesicles were subjected to consecutive salt washing and detergent (polyoxyethylene 20 dodecylether and 3-[(3-cholamido-propyl)-dimethylammonio]-1-propane sulfonate [CHAPS]) treatments, and the fractions were analyzed for NFORase and MDA reductase activities. Similar results were obtained when the 300 mm sucrose in the homogenization buffer and in all steps of the salt-washing and detergent treatments had been replaced by 150 mm KCl to mimic the conditions in the cytoplasm. We conclude that (a) MDA reductase is strongly associated with the inner (cytoplasmic) surface of the PM under in vivo conditions and requires washing with 1.0 m KCl or CHAPS treatment for removal, (b) the PM-bound MDA reductase activity is responsible for the majority of PM NFORase activity, and (c) there is another redox enzyme(s) in the spinach leaf PM that cannot be released from the PM by salt-washing and/or CHAPS treatment. The PM-associated MDA reductase may have a role in reduction of ascorbate in both the cytosol and the apoplast.

Heat-Stress Response of Maize Mitochondria1

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42°C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13°C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.

Selenocysteine, identified as the penultimate C-terminal residue in human T-cell thioredoxin reductase, corresponds to TGA in the human placental gene.

The possible relationship of selenium to immunological function which has been suggested for decades was investigated in studies on selenium metabolism in human T cells. One of the major 75Se-labeled selenoproteins detected was purified to homogeneity and shown to be a homodimer of 55-kDa subunits. Each subunit contained about 1 FAD and at least 0.74 Se. This protein proved to be thioredoxin reductase (TR) on the basis of its catalytic activities, cross-reactivity with anti-rat liver TR antibodies, and sequence identities of several tryptic peptides with the published deduced sequence of human placental TR. Physicochemical characteristics of T-cell TR were similar to those of a selenocysteine (Secys)-containing TR recently isolated from human lung adenocarcinoma cells. The sequence of a 12-residue 75Se-labeled tryptic peptide from T-cell TR was identical with a C-terminal-deduced sequence of human placental TR except that Secys was present in the position corresponding to TGA, previously thought to be the termination codon, and this was followed by Gly-499, the actual C-terminal amino acid. The presence of the unusual conserved Cys-Secys-Gly sequence at the C terminus of TR in addition to the redox active cysteines of the Cys-Val-Asn-Val-Gly-Cys motif in the FAD-binding region may account for the peroxidase activity and the relatively low substrate specificity of mammalian TRs. The finding that T-cell TR is a selenoenzyme that contains Se in a conserved C-terminal region provides another example of the role of selenium in a major antioxidant enzyme system (i.e., thioredoxin-thioredoxin reductase), in addition to the well-known glutathione peroxidase enzyme system.

Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.

Inositol 1,4,5-trisphosphate receptor subtype 3 in pancreatic islet cell secretory granules revisited.

It has been reported that the inositol 1,4,5-trisphosphate receptor subtype 3 is expressed in islet cells and is localized to both insulin and somatostatin granules [Blondel, O., Moody, M. M., Depaoli, A. M., Sharp, A. H., Ross, C. A., Swift, H. & Bell, G. I. (1994) Proc. Natl. Acad. Sci. USA 91, 7777-7781]. This subcellular localization was based on electron microscope immunocytochemistry using antibodies (affinity-purified polyclonal antiserum AB3) directed to a 15-residue peptide of rat inositol trisphosphate receptor subtype 3. We now show that these antibodies cross-react with rat, but not human, insulin. Accordingly, the anti-inositol trisphosphate receptor subtype 3 (AB3) antibodies label electron dense cores of mature (insulin-rich) granules of rat pancreatic beta cells, and rat granule labeling was blocked by preabsorption of the AB3 antibodies with rat insulin. The immunostaining of immature, Golgi-associated proinsulin-rich granules with AB3 antibodies was very weak, indicating that cross-reactivity is limited to the hormone and not its precursor. Also, the AB3 antibodies labeled pure rat insulin crystals grown in vitro but failed to stain crystals grown from pure human insulin. By immunoprecipitation, the antibodies similarly displayed a higher affinity for rat than for human insulin. We could not confirm the labeling of somatostatin granules using AB3 antibodies.

Cloning and characterization of a binding subunit of the interleukin 13 receptor that is also a component of the interleukin 4 receptor.

Interleukins 4 (IL-4) and 13 (IL-13) have been found previously to share receptor components on some cells, as revealed by receptor cross-competition studies. In the present study, the cloning is described of murine NR4, a previously unrecognized receptor identified on the basis of sequence similarity with members of the hemopoietin receptor family. mRNA encoding NR4 was found in a wide range of murine cells and tissues. By using transient expression in COS-7 cells, NR4 was found to encode the IL-13 receptor alpha chain, a low-affinity receptor capable of binding IL-13 but not IL-4 or interleukins 2, -7, -9, or -15. Stable expression of the IL-13 receptor alpha chain (NR4) in CTLL-2 cells resulted in the generation of high-affinity IL-13 receptors capable of transducing a proliferative signal in response to IL-13 and, moreover, led to competitive cross-reactivity in the binding of IL-4 and IL-13. These results suggest that the IL-13 receptor alpha chain (NR4) is the primary binding subunit of the IL-13 receptor and may also be a component of IL-4 receptors.

Presence of antibodies to different subunits of replication protein A in autoimmune sera.

A human cDNA expression library was used to investigate the nature of molecules recognized by serum from a patient with Sjögren syndrome that exhibits a mixed immunofluorescence pattern and reacts with multiple components on an immunoblot. The data demonstrated that this serum contains IgG antibodies specific for the 70- and 32-kDa subunits of replication protein A (RPA; RPA-70 and RPA-32, respectively), a highly conserved multisubunit DNA binding protein. Affinity purification of serum autoantibodies demonstrated a complete lack of cross-reactivity between RPA-70 and RPA-32, suggesting a direct participation of the native protein complex in the autoimmune response in this patient. Purified anti-RPA-70 and anti-RPA-32 antibodies labeled nuclear and cytoplasmic components in an immunofluorescence assay, suggesting that RPA is present in both cellular compartments. Additional sera from 55 patients with different autoimmune conditions were screened against purified RPA-70 and RPA-32 recombinant proteins. One of these 55 sera was positive and reacted with only RPA-32. Twenty sera from healthy control individuals did not react with RPA. These results show that RPA is a target for autoantibodies in human autoimmune diseases, although its precise frequency, occurrence in other autoimmune diseases, and pathological significance remain to be fully elucidated.

Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: Relevance to diseases of the nervous system

Many proteins contain reiterated glutamine residues, but polyglutamine of excessive length may result in human disease by conferring new properties on the protein containing it. One established property of a glutamine residue, depending on the nature of the flanking residues, is its ability to act as an amine acceptor in a transglutaminase-catalyzed reaction and to make a glutamyl–lysine cross-link with a neighboring polypeptide. To learn whether glutamine repeats can act as amine acceptors, we have made peptides with variable lengths of polyglutamine flanked by the adjacent amino acid residues in the proteins associated with spinocerebellar ataxia type 1 (SCA1), Machado–Joseph disease (SCA3), or dentato-rubral pallido-luysian atrophy (DRPLA) or those residues adjacent to the preferred cross-linking site of involucrin, or solely by arginine residues. The polyglutamine was found to confer excellent substrate properties on any soluble peptide; under optimal conditions, virtually all the glutamine residues acted as amine acceptors in the reaction with glycine ethyl-ester, and lengthening the sequence of polyglutamine increased the reactivity of each glutamine residue. In the presence of transglutaminase, peptides containing polyglutamine formed insoluble aggregates with the proteins of brain extracts and these aggregates contained glutamyl–lysine cross-links. Repeated glutamine residues exposed on the surface of a neuronal protein should form cross-linked aggregates in the presence of any transglutaminase activated by the presence of Ca2+.