Altered response to viral infection by tobacco plants perturbed in ubiquitin system.

Tobacco plants can be perturbed in their ubiquitin system by expression of a ubiquitin variant with a lys to arg change at position 48. Plants expressing this ubiquitin variant have a tendency to form necrotic lesions resembling those occurring as defense reactions against some pathogens. Using the interaction between tobacco and tobacco mosaic virus (TMV), it was investigated whether the ubiquitin variant influences plant responses to pathogen infection. Expression of the ubiquitin variant leads to changes in the response of both resistant and susceptible tobacco varieties. Resistant plants form fewer, but darker lesions. In sensitive plants, TMV replication is inhibited, but not completely abolished. Moreover, susceptible plants expressing the ubiquitin variant induce PR-1 mRNA in response to virus, whereas controls do not express PR-1 under these conditions.     

Multiple translational products from a Mycoplasma hyorhinis gene expressed in Escherichia coli.

We analyzed protein expression from a cloned Mycoplasma hyorhinis genomic fragment that produces in Escherichia coli a set of related polypeptides of 110, 100, 65, and 55 kilodaltons from a coding region of just over 3.0 kilobases. Expression of these multiple products resulted from a mechanism operating at the translational level but not from truncation at UGA termination codons, which are known to encode tryptophan in several mycoplasma species. The structural relatedness of the proteins was demonstrated by two-dimensional tryptic peptic mapping, but their generation by posttranslational processing was ruled out by pulse-chase labeling analysis. Examination of proteins expressed from plasmid constructs and tryptic peptide analysis of these polypeptides and the original set of proteins revealed that they share carboxy-terminal regions, an observation inconsistent with truncation at UGA codons. Expression of proteins from this cloned fragment was not dependent on vector sequences and was observed when the coding region was placed under control of a T7 promoter, suggesting that all products were translated from a single message. Expression of related products in mycoplasmas was examined by immunoblot analysis of M. hyorhinis proteins with antiserum against overexpressed recombinant proteins. A single 115-kilodalton mycoplasma protein was detected, which is larger than any of the related proteins expressed in E. coli. Our analysis indicated that translation initiation sites are used in E. coli that are not active in mycoplasmas, thereby defining differences between the translational regulatory signals of mycoplasmas and eubacteria.     

Using viral genomics to develop viral gene products as a novel class of drugs to treat human ailments

A novel strategy for drug discovery and advancement has been developed by exploiting viral genomics. By manipulating cell function through the therapeutic administration of specific gene products from viruses, an important new class of therapeutics could be developed for the treatments of major human diseases. As it may turn out in the human genome arena, the groups which can establish a stronghold in the field would be in the best position to exploit this new drug development strategy.     

Detection and quantification of gene amplification products by a nonisotopic automated system.

We describe in this report the ability to determine human immunodeficiency virus proviral copy number by an automated nonisotopic method. Our system utilizes a FACStarPLUS cell sorter, the GeneAmp PCR System 9600 and a Biomek 1000 robotic workstation. Linking these three machines allows cell populations to be sorted and the DNA amplified and quantitated with minimal technical effort. We have developed this system to quantitate proviral DNA copy number in sorted subpopulations of peripheral blood cells in one day.     

Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene.

A transient chloramphenicol acetyltransferase (CAT) expression system was developed for Borrelia burgdorferi. An Escherichia coli vector containing a promoterless Streptococcus agalactiae cat gene was constructed. Promoters for ospA, ospC, and flaB were placed upstream of this cat gene, and CAT assays were performed in E. coli from these stably maintained plasmids. The plasmids with putative promoters ospA and flaB were found to be approximately 20-fold more active than were the plasmids with ospC or no promoter. The level of activity correlated well with the resistance to chloramphenicol that each plasmid provided. Next, the nonreplicative plasmid constructs were transformed by electroporation into B. burgdorferi. CAT assays were performed by both thin-layer chromatography and the fluor diffusion method. Measurement of CAT activity demonstrated that the ospA promoter was again about 20-fold more active than the promoterless cat gene. The flaB and ospC promoters increased the activity seven- and threefold, respectively, over that with the promoterless construct. This simple transient-expression assay was shown to be an effective method to study promoter function in B. burgdorferi in the absence of a well-developed genetic system.     

Efficient detection of eukaryotic calcium-sensing receptor (CaSR) by polyclonal antibody against prokaryotic expressed truncated CaSR

Calcium-sensing receptor (CaSR), which is better known for its action as regulating calcium homeostasis, can bind various ligands. To facilitate research on CaSR and understand the receptor's function further, an in silico designed truncated protein was developed. The resulting protein folding indicated that 99% of predicted three dimensional (3D) structure residues are located in favored and allowed Ramachandran plots. However, it was found that such protein does not fold properly when expressed in prokaryotic host cells. Thioredoxin (Trx) tag was conjugated to increase the final protein's solubility, which could help obtain the soluble antigen with better immunogenic properties. The truncated recombinant proteins were expressed and purified in two forms (Trx-CaSR: RR19 and CaSR: RRJ19). The polyclonal antibody was induced by the rabbit immunization with the form of RR19. Western blot on mouse kidney lysates evidenced the proper immune recognition of the receptor by the produced antibody. The specificity and sensitivity of antibodies were also assayed by immunohistofluorescence. These experiments affirmed antibody's ability to indicate the receptor on the cell surface in native form and the possibility of applying such antibodies in further cellular and tissue assays.

     

Identification of gene products programmed by restriction endonuclease DNA fragments using an E. coli in vitro system.

DNA restriction enzyme fragments have been used to programme the synthesis of polypeptides in an in vitro system without apparent loss in fidelity compared with supercoiled templates. The system is extremely sensitive, less than 1 microgram of DNA can be used to direct the synthesis of 35S-labelled polypeptides of sufficiently high specific activity such that products can be identified by SDS-PAGE after a few hours autoradiography. The ability to analyse fragments can be used to readily assign specific proteins to small regions of the coding template, to identify cloned gene products distinct from those of the vector, and to identify cloned genes expressed from their own promoters. The in vitro system can be used successfully with bacterial DNA from other species and efficient extracts can be prepared from any E. coli K-12 strain, which should greatly facilitate the purification of factors controlling the expression of specific genes by complementation assay.     

Sheep antibody response to cell wall antigens expressed in vivo by Pasteurella haemolytica serotype A2

Abstract Cells of Pasteurella haemolytica A2 were harvested directly from the pleural fluid of sheep with pneumonic pasteurellosis and the protein composition of their envelopes was examined by SDS-PAGE. Several high molecular-weight proteins expressed in pleural fluid were absent when the same isolate was grown in nutrient broth, but were produced during culture in iron restricted media. When the cell wall envelopes were examined by immunoblotting with sera and lung washes of lambs recovering from pneumonic pasteurellosis, antibodies to the major outer membrane proteins, the iron-regulated proteins and one other 'in vivo'-expressed antigen were present. These results have implications for the formulation of effective vaccines against pasteurellosis in sheep.     

Control of central nervous system viral persistence by neutralizing antibody

Replication of the neurotropic JHM strain of mouse hepatitis virus within the central nervous system is controlled by cellular immunity. However, following initial clearance, virus reactivates in the absence of humoral immunity. Viral recrudescence is prevented by the transfer of antiviral antibody (Ab). To characterize the specificity and biological functions of Ab critical for maintaining viral persistence, monoclonal Abs specific for the viral spike, matrix, and nucleocapsid proteins were transferred into infected B-cell-deficient mice following initial virus clearance. Neutralizing immunoglobulin G (IgG) but not IgA anti-spike Ab suppressed virus recrudescence, reduced viral antigen in most cell types except oligodendroglia, and was associated with reduced demyelination. Nonneutralizing monoclonal Abs specific for the spike, matrix, and nucleocapsid proteins did not prevent recrudescence, demonstrating that neutralization is critical for maintaining JHM mouse hepatitis virus persistence within the central nervous system. Ab-mediated protection was not associated with alterations in virus-specific T-cell function or inflammation. Furthermore, neutralizing Ab delayed but did not prevent virus recrudescence. These data indicate that following acute viral clearance cellular immunity is ineffective in controlling virus recrudescence and suggest that the continued presence of neutralizing Ab is the essential effector in maintaining viral persistence within the central nervous system.     

Antibodies to immune response gene products inhibit the IgM and IgG antibody response but not the development of resistance to Toxoplasma gondii

We have examined the effects of a monoclonal antibody directed against immune response gene products on the appearance of antibodies and development of resistance to Toxoplasma gondii. In vivo administration of a single dose of anti-I-Ak antibody to C3H/He (H-2k) and not BALB/c (H-2d) mice suppressed both the IgM and IgG response to two different strains of Toxoplasma. Administration of anti-I-Ak antibody to mice 5 days before and 10 days after infection resulted in complete inhibition of IgM and a more pronounced inhibition of IgG response to Toxoplasma. Under these experimental conditions, development of resistance against a subsequent challenge with a virulent strain of Toxoplasma was not affected. The microbicidal and tumoricidal activities of macrophages obtained from anti-I-Ak-treated, Toxoplasma-infected mice and mice infected with Toxoplasma alone were equivalent. Mice treated with anti-I-Ak antibody demonstrated a decreased proliferative response to lipopolysaccharide, a B-cell mitogen. Enumeration of B-cell numbers in anti-I-Ak-treated mice revealed a pronounced decrease in B-cell counts.     

Distinctive patterns in prokaryotic community composition in response to viral lysis and flagellate grazing in freshwater microcosms

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Expression of a biologically active ovine trophoblastic interferon using a baculovirus expression system.

Ovine trophoblast protein (oTP) an embryonic interferon, which plays a key role in maternal recognition of pregnancy, has been expressed in insect cells using a baculovirus expression system. A cDNA coding for oTP was inserted downstream of the strong polyhedrin promoter. Cells infected with recombinant virus produced biologically active oTP and greater than 90% was secreted into the culture medium during infection. High amount of antiviral activity were produced (up to 5 x 10(5) IU per ml of culture medium). Recombinant oTP (roTP) was purified by immunoaffinity chromatography and found to be identical to authentic oTP with respect to molecular mass and N-terminal amino acid sequence.     

Purification and characterization of a single-chain chimeric anti-p185 antibody expressed by CHO-GS system

Monoclonal antibody A21 reacts specifically with the extracellular domain of p185c-erbB-2 oncoprotein, a member of the epidermal growth factor receptor family. In a previous study, we constructed a single-chain chimeric antibody, assembled using an A21 single-chain Fv antibody and a human IgG1 Fc fragment. In this study, we expressed this chimeric antibody using a CHO-GS system, and developed a simple and efficient method for its purification. After only one step using affinity purification, the recovery rate and purity of the antibody attained was 60 and 91%, respectively. After a second step, using reverse phase HPLC purification, the purity was above 99%. The high purity of the recombinant antibody allowed us to identify a number of its intrinsic molecular properties, including antigen binding activity, measurement of affinity constant, N-terminal sequencing, and mass spectrometer analysis. These results further augment the potential of this recombinant antibody to be a drug candidate for cancer therapy.     

Two of the three influenza viral polymerase proteins expressed by using baculovirus vectors form a complex in insect cells.

Each of the influenza virus polymerase (P) genes PB1, PB2, and PA was inserted into a baculovirus vector under the control of the polyhedrin promoter. In insect (Spodoptera frugiperda) cells infected by each baculovirus recombinant containing a P gene insert, a large amount of the encoded P protein was synthesized. Gel electrophoretic analysis of the total proteins in infected cells revealed the presence of a new protein band corresponding to the encoded P protein that was abundant enough to be stained with Coomassie blue. In cells infected simultaneously with both the PB1 and PB2 baculovirus recombinants, a PB1-PB2 complex was formed that was immunoprecipitated with an antiserum specific for either PB1 or PB2. In cells infected simultaneously with all three P baculovirus recombinants, a PB1-PB2 complex lacking the PA protein was formed. Formation of this PB1-PB2 complex partially mimics events that occur in influenza virus-infected cells, where all three P proteins form a complex with each other (B. M. Detjen, C. St. Angelo, M. G. Katze, and R. M. Krug, J. Virol. 61:16-22, 1987). These results indicate that the ability of PB1 and PB2 to form a complex is an intrinsic property of these two proteins that does not require the participation of other influenza viral gene products. Possible reasons for the absence of the PA protein from the immunoprecipitable P protein complex in insect cells infected by the three P baculovirus recombinants are discussed.