Array-based ELISAs for high-throughput analysis of human cytokines.

In this report, we describe the development of a mini-array system suitable for high-throughput quantification of proteins. This mini-array is a multiplexed, sandwich-type ELISA that measures the concentration of seven different human cytokines--TNF-alpha, IFN alpha, IFN gamma, IL-1 alpha, IL-1 beta, IL-6, and IL-10--from a single sample in each well of a 96-well plate. The mini-array is produced by spotting monoclonal antibodies (mAbs) in a 3 x 3 pattern in the bottom of the wells of 96-well polystyrene plates. Cytokines that are captured by the arrayed mAbs are detected by using biotinylated mAbs, followed by the addition of a streptavidin-horseradish peroxidase (HRP) conjugate and a chemiluminescent substrate. The light produced from the HRP-catalyzed oxidation of the substrate is measured at each spot in the array by imaging the entire plate with a commercially available CCD camera. Here, we demonstrate that these 96-well-plate format mini-arrays have performance characteristics that make them suitable for the high-throughput screening of anti-inflammatory compounds.

Xplore mRNA assays for the quantification of IL-1 beta and TNF-alpha mRNA in lipopolysaccharide-induced mouse macrophages.

Because the accurate measurement of a number of cytokine mRNA transcripts provides valuable knowledge about cytokine gene regulation, we have developed the Xplore assay for the quantification of cytokine mRNA. This microplate-based assay is rapid (under four hours), quantitative over three orders of magnitude and carries no risk of false-positive values from contamination with amplified target. Here, we describe the use of Xplore assays to measure the steady-state mRNA levels of TNF-alpha and IL-1 beta produced by mouse WEHI and J774 macrophage-like cell lines.

In vitro and in vivo bioactivity of single-chain interleukin-12.

Interleukin-12 is a heterodimeric cytokine with potent immunoregulatory properties, making it a potential vaccine adjuvant and an immune response modulator. The study of its function is confounded by its heterodimeric structure. In order to facilitate the study of interleukin-12 in both in vitro and in vivo models, we constructed a single-chain porcine interleukin-12 gene and expressed the recombinant protein in Pichia pastoris. Single-chain porcine interleukin-12 was bioactive in vitro on both human and porcine cells as measured by its ability to induce proliferation of lymphoblasts and interferon-gamma secretion by lymph node cells. In contrast, the p40 subunit of porcine interleukin-12 alone did not induce proliferation or inhibit the activity of the single-chain porcine interkeukin-12. The in vivo bioactivity of single-chain porcine interleukin-12 was demonstrated in an oral immunization model where it increased antigen-specific IgA and IgG in jejunal mucus. These results indicate that binding of interleukin-12 to its receptor and transduction of intracellular signals requires both p40 and p35 subunits. The bioactivity of interleukin-12 expressed as a single polypeptide will facilitate its in vivo delivery and study of its structure and function.

Expression of human interleukin-17 in Pichia pastoris: purification and characterization.

A Pichia pastoris expression clone has been developed to produce the human cytokine interleukin-17 (hIL-17). Characterization of purified recombinant hIL-17 made with this clone demonstrated that it shared many characteristics with hIL-17 produced in mammalian cells. The hIL-17 produced in Pichia had the correct N-terminus of natural mature hIL-17 and a glycosylation pattern similar to hIL-17 produced in mammalian cells; both Pichia and human cells add approximately 5 kDa of sugars via N-linked glycosylation and both express a mixture of the glycosylated and nonglycosylated forms. Gel filtration provides evidence that the Pichia produced hIL-17 exists as a dimer in solution. A combination of cation-exchange and gel-filtration chromatography yielded 3.5 mg of highly purified and biologically active hIL-17 from a 10-liter fermentation. These results show that P. pastoris is a useful system to produce recombinant hIL-17 in structure/function studies of this molecule.

A side chain at position 48 of the human immunodeficiency virus type-1 protease flap provides an additional specificity determinant.

Substitution of glycine with glutamic acid at position 48 of the human immunodeficiency virus protease resulted in an enzyme with reduced activity on one of the protease processing sites in the viral Pol polyprotein precursor. Cleavage at this site was restored by a second-site substitution in the substrate replacing an aspartic acid with either glycine or asparagine. These results suggest that the glutamic acid side chain in the mutant protease has an unfavorable charge-charge interaction with this position in the substrate. Cleavage of a processing site in the viral Gag polyprotein precursor with the mutant enzyme was enhanced, and this enhancement was dependent on the presence of an arginine residue in the substrate, again suggesting a charge-charge interaction. The potential for such interactions was confirmed using molecular modeling. The effect of the position 48 substitution was attributed to a 10-fold increase in Km for the processing site in Pol. These results indicate that the addition of a side chain at position 48 can alter the specificity of the HIV-1 protease to substrate in a sequence specific manner and that compensatory changes can be made in the substrate.

The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions.

The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p2 site, however, was cleaved approximately 20-fold faster when the adjacent downstream p2/NC site was blocked from cleavage or when the p2 domain of Gag was deleted. These results suggest that the presence of a C-terminal p2 tail on processing intermediates slows cleavage at the upstream CA/p2 site. We also found that lower pH selectively accelerated cleavage of the CA/p2 processing site in the full-length precursor and as a peptide primarily by a sequence-based mechanism rather than by a change in protein conformation. Deletion of the p2 domain of Gag results in released virions that are less infectious despite the presence of the processed final products of Gag. These findings suggest that the p2 domain of HIV-1 Gag regulates the rate of cleavage at the CA/p2 processing site during sequential processing in vitro and in infected cells and that p2 may function in the proper assembly of virions.

Evolution of host cell RNA into efficient template RNA by Q beta replicase: the origin of RNA in untemplated reactions.

Q beta replicase can replicate a single molecule of certain species of RNA to 10(14) copies in minutes. This replication ability has been used for in vitro studies of molecular evolution and is currently being utilized as a method of amplifying RNAs that contain probe sequences. It has been observed that Q beta replicase can produce replicatable RNA even in the absence of exogenously added template RNA. The origin of this RNA has been ascribed either to contamination with replicatable RNA or to an ability of Q beta replicase to synthesize RNA de novo from the nucleotides present in the reaction. Technologies that employ Q beta replicase require a thorough understanding of the conditions that lead to this so-called spontaneous RNA production. We have created an expression system and purification method with which we produce gram quantities of highly purified Q beta replicase, and we have identified reaction conditions that prevent the amplification of RNA in assays that do not contain added RNA. However, when these reaction conditions are relaxed, spontaneous RNA replication is seen in up to 100% of the assays. To understand the origin of this RNA, we have cloned several spontaneously produced RNAs. Sequence analysis of one of these RNAs shows that it arose by the evolution of Escherichia coli tRNA into a replicatable template and not by de novo synthesis from nucleoside triphosphates in the reaction.

The function of reovirus proteins during the reovirus multiplication cycle: analysis using monoreassortants.

When cultured cells are injected with mixtures of cores of two reovirus strains, a high proportion of reassortants are monoreassortants, that is, virus particles that contain one genome segment of 1 parent and 9 genome segments of the other. We have isolated two complete sets of monoreassortants, those that contain a single serotype 2 genome segment and 9 serotype 3 genome segments, and those that contain 1 serotype 3 genome segment and 9 serotype 1 genome segments. We have used the former set of monoreassortants (because reovirus serotypes 2 and 3 are less closely related than serotypes 1 and 3) to assess the effect of all 10 genome segments, or rather of the proteins that they encode, in controlling parameters of the reovirus multiplication cycle such as yield size, extent of viral ssRNA, dsRNA and protein synthesis, plaque size, and cytopathogenicity. Among the major findings are: proteins lambda 2, mu 1/mu 1C, and sigma 3 control yield size and extent of RNA and protein synthesis; proteins mu 2 and sigma 1 control severity of cytopathic effects; and proteins sigma 1, mu 1/mu 1C, and mu 2 control plaque size. Identification of monoreassortant phenotypes is useful for identifying which viral proteins are functionally involved at the various stages of the reovirus multiplication cycle.

Virulence genes of poxviruses and reoviruses.

Identification of viral genes that specify virulence, however defined, is of critical importance for the design of viral vaccines. In particular, the targeted development not only of avirulent vaccine strains but also of viruses to be used as carriers for foreign genes depends on the inactivation or deletion of genes that harm the host. This paper illustrates approaches to identifying viral pathogenesis genes in two model systems involving cowpox virus and reovirus, respectively.

Ferric iron reductase of Rhodopseudomonas sphaeroides.

Ferric iron reductase activity was examined in the facultative photosynthetic bacterium Rhodopseudomonas sphaeroides. The specific activities of extracts from cells grown under phototrophic and aerobic conditions were similar and not affected by the concentration of iron in the growth media. The activity was resolved by ion-exchange column chromatography into two fractions, designated iron reductase A and iron reductase B, with molecular weights of 41,000 and 32,000, respectively. Both of these soluble cytoplasmic enzymes required the presence of flavin mononucleotide for activity and utilized NADH to reduce iron supplied as ferric citrate. Iron reductase B was responsible for the majority of activity in crude extracts and was purified 556-fold by conventional protein purification techniques. The apparent Km values of iron reductase B for NADH, Fe3+, and flavin mononucleotide were determined to be 18.2, 8.3, and 3.2 microM, respectively.

Iron transport and its relation to heme biosynthesis in Rhodopseudomonas sphaeroides.

The uptake of iron supplied as ferric citrate or ferric parabactin was examined in aerobically grown whole cells and vesicles of Rhodopseudomonas sphaeroides. Inner and outer membrane fractions from R. sphaeroides contained no membrane proteins which were inducible by growth in low-iron medium. Vesicles composed of the inner membrane and devoid of outer membrane and periplasmic proteins were able to transport iron supplied as ferric citrate and ferric parabactin. This uptake required the presence of NADH. When the electrical component of the proton motive force was depleted in whole cells, the uptake of iron supplied as ferric parabactin was completely inhibited. The uptake of iron supplied as ferric citrate was inhibited by gallium citrate; however, Ga3+ was not transported. The relationship between iron uptake and heme synthesis was examined by treating whole cells with N-methylprotoporphyrin which inhibits ferrochelatase, the enzyme which inserts ferrous iron into protoporphyrin to form heme. This treatment reduced ferrochelatase activity by 82% but had no effect on iron uptake, indicating that iron uptake and heme synthesis are not directly coupled. The fate of transported iron was investigated by measuring intracellular concentrations of heme and nonheme iron. It was determined that newly transported iron exists primarily as nonheme iron.

Siderophore utilization and iron uptake by Rhodopseudomonas sphaeroides.

The growth of Rhodopseudomonas sphaeroides in iron-deficient medium did not result in the production of detectable levels of siderophores of either the catechol or hydroxamate type. Iron-limited cultures of R. sphaeroides were not able to remove iron from ferric transferrin unless supplemented with 2,3-dihydroxybenzoic acid. R. sphaeroides was shown to take up 59Fe+3 when it was supplied as ferric chloride, ferric citrate, or ferric parabactin, but not when supplied as ferric rhodotorulate or ferric Desferal. When iron was supplied as ferric citrate, citrate was not taken up by the cells. The growth rate of R. sphaeroides under iron-limiting conditions was decreased by the addition of either Desferal or rhodotorulic acid, while the addition of citrate or parabactin did not affect growth.

Clinical evaluation of the improved streptex method for grouping streptococci.

The improved Streptex method for serogrouping streptococci incorporates a new extraction enzyme and a simplified procedure requiring no centrifugation. A total of 114 clinical isolates of beta-hemolytic streptococci were serogrouped from primary plates, isolation plates, and Todd-Hewitt broth cultures using this system. Results were compared to those of the heat extraction Lancefield precipitin method. An additional 33 stock culture isolates of related streptococcal species and 5 strains of Listeria were serogrouped to assess the specificity of the test. Agreement between the two methods was 82.5% with primary plates and 96.5% with both isolation plates and broth cultures. Four isolates from three different serogroups were nongroupable by the Lancefield method, but did agglutinate in specific Streptex antisera; therefore, the enzyme extraction procedure appeared more sensitive than the heat extraction method. Streptex accurately grouped five isolates of gamma-hemolytic group B streptococci, but failed to detect antigen in 33% of the group D streptococcal extracts tested. In addition, cross-reactions were observed with strains of alpha-hemolytic streptococci. Streptex produced fewer ambiguous results and required fewer repeat tests. When used with isolation plates or broth cultures, Streptex is both sensitive and specific for the grouping of beta-hemolytic streptococci of groups A, B, C, F, and G.

Aerobic ferrisiderophore reductase assay and activity stain for native polyacrylamide gels.

The reduction of ferric iron from microbial iron-binding compounds (siderophores) releases the iron from the siderophore so that it may be utilized by the microorganism. A method to detect aerobic ferrisiderophore reductase activity using ferrozine as a ferrous iron trap is shown to be applicable to cytoplasmic fractions from Rhodopseudomonas sphaeroides and four other different species of bacteria. The ferrisiderophore reductase uses reduced nicotinamide cofactors as reducing agents, and activity is stimulated by flavins. This assay has been adapted as a staining method to locate ferrisiderophore reductase activity in native polyacrylamide gels.

Diagnosis of streptococcal pharyngitis and rheumatic fever.

Eradication of streptococci from the throat has been shown to prevent rheumatic fever, so that an accurate diagnosis of streptococcal pharyngitis is desirable. Throat cultures could be better utilized than they have been for this purpose.