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D R Galloway

Publications and source records attributed to D R Galloway.

36 records · Page 2Linked to original sources
PubMed1990

Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: enhancement of elastase activity.

A 22-kilodalton protein purified from the culture supernatant fraction of Pseudomonas aeruginosa (strains PA220 and PAO1) was found to enhance the elastolytic activity of purified P. aeruginosa elastase. N-terminal sequence analysis identified the protein as a fragment of the lasA gene product (P.A. Schad and B.H. Iglewski, J. Bacteriol. 170:2784-2789, 1988). However, comparative analysis with the reported LasA sequence indicated that the purified LasA fragment is longer than the deduced sequence reported. The purified LasA fragment had minimal elastolytic and proteolytic activity and did not enhance the proteolytic activity of purified elastase, yet enhanced the elastolytic activity more than 25-fold. The LasA fragment was found to also enhance the elastolytic activities of thermolysin, human neutrophil elastase, and proteinase K. The results presented here suggest that the LasA protein interacts with the elastin substrate rather than modifying elastase.

Bacterial Proteins↗
PubMed1989

Biochemical analysis of CRM 66. A nonfunctional Pseudomonas aeruginosa exotoxin A.

Pseudomonas aeruginosa exotoxin A (ETA) is an ADP-ribosyltransferase which inactivates protein synthesis by covalently attaching the ADP-ribose portion of NAD+ onto eucaryotic elongation factor 2 (EF-2). A direct biochemical comparison has been made between ETA and a nonenzymatically active mutant toxin (CRM 66) using highly purified preparations of each protein. The loss of ADP-ribosyltransferase activity and subsequent cytotoxicity have been correlated with the presence of a tyrosine residue in place of a histidine at position 426 in CRM 66. In the native conformation, CRM 66 demonstrated a limited ability (by a factor or at least 100,000) to modify EF-2 covalently and lacked in vitro and in vivo cytotoxicity, yet CRM 66 appeared to be normal with respect to NAD+ binding. Upon activation with urea and dithiothreitol, CRM 66 lost ADP-ribosyltransferase activity entirely yet CRM 66 retained the ability to bind NAD+. Replacement of Tyr-426 with histidine in CRM 66 completely restored cytotoxicity and ADP-ribosyltransferase activity. These results support previous findings from this laboratory (Wozniak, D. J., Hsu, L.-Y., and Galloway, D. R. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8880-8884) which suggest that the His-426 residue of ETA is not involved in NAD+ binding but appears to be associated with the interaction between ETA and EF-2.

ADP Ribose Transferases↗
PubMed1988

His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II.

Exotoxin A (ETA) is recognized as the most toxic product associated with the opportunistic pathogen Pseudomonas aeruginosa. Identification of the amino acids in the polypeptide sequence that are required for toxin activity is critical for vaccine development. By defining the nucleotide sequence of the structural gene of a mutant that encodes an enzymatically inactive ETA (CRM 66), we identified an essential amino acid (His-426), which is involved in the ADP-ribosyltransferase activity associated with functional ETA. A monoclonal antibody that inhibits ETA enzymatic activity in vitro fails to react with ETA variants that have a His 426----Tyr substitution. Several mono-ADP-ribosylating toxins, including diphtheria and pertussis toxins, within the primary amino acid sequences carry a histidine residue that is conserved in spacing and in location with respect to other critical residues. Analysis of the three-dimensional structure of ETA revealed that His-426 is not associated with the proposed NAD+ binding site. These findings should be useful for the design and construction of toxin vaccines.

ADP Ribose Transferases↗
PubMed1987

Nucleotide sequence and characterization of toxR: a gene involved in exotoxin A regulation in Pseudomonas aeruginosa.

We have previously reported the discovery and subsequent cloning of a regulatory gene, designated toxR, which appears to regulate the expression of the exotoxin A (ETA) structural gene toxA. Subsequent work by this laboratory has resulted in the subcloning of the toxR gene and its transfer to a high copy number plasmid (pGW28). Functional analysis of the toxR gene using a Tn5 insertion along with toxR deletions indicates that inactivation of toxR results in a dramatic reduction of ETA production. Nucleotide sequence analysis of pGW28 has revealed a 675 bp major open reading frame (225 codons) which could encode for a protein of 24,626 daltons. Using S1 nuclease mapping, the toxR RNA transcript has been shown to originate 20 bp upstream of the presumptive translation initiation codon. Experiments using a toxA specific probe have revealed the the toxR gene product appears to regulate the expression of ETA at the transcriptional level.

ADP Ribose Transferases↗
PubMed1986

Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa.

We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.

ADP Ribose Transferases↗
PubMed1984

Antibody response of infected mice to outer membrane proteins of Pseudomonas aeruginosa.

The antibody response to outer membrane proteins of Pseudomonas aeruginosa was studied in mice experimentally infected with P. aeruginosa 220. The infection consisted of an abscess established by subcutaneous injection of bacteria. Sera from these mice were analyzed by indirect radioimmunoprecipitation and immunoblot methods for the presence of antibodies to proteins of the isolated outer membrane. Sera from mice 14 days postinfection were shown to contain antibodies directed against proteins that comigrated with the major outer membrane proteins F (porin), H2, and I (lipoprotein). A 16,000-dalton protein that did not appear to be a major outer membrane protein also elicited a significant antibody response in some instances. It is concluded that mice, in response to infection, elicit an immunological response to outer membrane proteins of P. aeruginosa.

Animals↗
PubMed1984

Toxoids of Pseudomonas aeruginosa exotoxin-A: photoaffinity inactivation of purified toxin and purified toxin derivatives.

For the preparation of greatly detoxified but highly immunogenic toxoids, two enzymatically active, low-toxicity derivatives of Pseudomonas aeruginosa exotoxin-A were further inactivated by photoaffinity labeling. These derivatives were formed during toxin purification, when a relatively crude toxin preparation was concentrated by ammonium sulfate precipitation and subsequently dialyzed. These derivatives, designated peak-1 protein (PK-1) and peak-2 protein (PK-2) were antigenically indistinguishable from native toxin, but had isoelectric points (5.00 and 4.90, respectively) that were different from that of the native toxin (4.95). Although the enzymatic activities and molecular weights of PK-1 and PK-2 were similar to those of native toxin, their toxicities were greatly reduced (ca. 500-fold). Photoaffinity labeling of fully active toxin-A, purified by a process which limits the formation of these derivatives, decreased its enzymatic activity (ca. 30-fold) and toxicity (ca. 100-fold). Likewise, photoaffinity labeling of purified PK-1 and PK-2 decreased their enzymatic activities and toxicities (ca. 30-fold and 100-fold, respectively) and, thus, yielded toxoids that were ca. 50,000-fold less toxic than unpurified native toxin. These toxoids were irreversibly detoxified and highly immunogenic during 9 months of storage at 4 degrees C.

ADP Ribose Transferases↗
PubMed1984

Production and characterization of monoclonal antibodies to exotoxin A from Pseudomonas aeruginosa.

Hybridomas secreting monoclonal antibodies specific for exotoxin A from Pseudomonas aeruginosa strain PA103 were derived from the fusion of spleen cells from mice immunized with: (i) purified exotoxin A, (ii) Formalin-treated exotoxin A, (iii) exotoxin A covalently coupled to Sepharose 4B, or (iv) P. aeruginosa-infected mice. All hybridomas were screened and selected by using an enzyme-linked immuno-adsorbent assay. All antibody isotypes were represented (immunoglobulins G, A, and M) as determined by enzyme-linked immunoadsorbent assay. The most productive fusions resulted from immunization with antigens coupled to an insoluble matrix, such as Sepharose 4B, or by infection of mice. Several hybridomas were selected and cloned by limiting dilution. The specificity of the monoclonal antibodies for exotoxin A was demonstrated by indirect immunoprecipitation of 125I-labeled exotoxin A followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and by the immunoblotting technique. The protective ability of certain monoclonal antibodies was demonstrated in vitro by toxin neutralization in tissue culture and in vivo by prolonged survival time in the burned mouse infection model, after passive immunization. One monoclonal antitoxin displayed specificity for PA103-derived exotoxin yet failed to react with exotoxin purified from PAO-PR1 or PAO1, suggesting that structural differences exist between these exotoxins.

ADP Ribose Transferases↗
PubMed1981

Immunochemical delineation of an oncofetal antigen on normal and simian virus 40-transformed human fetal melanocytes.

Human melanoma cells of uveal origin shed 94,000- and 240,000-dalton glycoproteins in common with most melanoma cell lines of dermal origin. Normal human melanocytes derived from fetal uvea shed a 90,000-dalton glycoprotein that was found to be immunologically identical with the 94,000-dalton glycoprotein of melanoma cells. Expression of this 90,000-dalton molecule was confined to fetal cells of ectodermal origin. After simian virus 40 (SV40) transformation of human fetal melanocytes, there was an apparent increase in molecular size of this component to 94,000 daltons. In contrast, the 240,000-dalton glycoprotein was not synthesized or shed by uninfected or SV40-transformed fetal melanocytes. These data suggest that the 94,000-dalton glycoprotein is an oncofetal antigen of ectodermal origin.

Antigens, Neoplasm↗
PubMed1981

Production and characterization of monoclonal antibody to a melanoma specific glycoprotein.

An immunogen consisting of a 4M urea extract derived from human melanoma cells (M14), that was devoid of HLA-A,B,C, HLA-DR antigens and fibronectin was adsorbed to lens culinaris lectin-Sepharose 4B and used to immunize mice for production of monoclonal antibody to a melanoma-specific glycoprotein. Screening for hybridomas secreting antibodies to melanoma associated antigens was facilitated by use of a solid phase target antigen of chemically defined medium of melanoma cells (CDM). Use of these procedures allowed us to select 40 hybridomas secreting antibody which recognized determinants on melanoma cells not found on lymphoid cells. Further characterization of one of these hybridomas, 9.2.27, indicated that the antibody it secreted recognized a 240K dalton glycoprotein found on all melanoma cell lines tested but not on carcinoma, lymphoid, or fibroblastoid cultures. These results demonstrate the utility of soluble antigen preparations devoid of strongly immunogenic non tumor-specific molecules in the elicitation of tumor specific antibody. Preliminary results suggest that immunogens of this kind are superior to intact melanoma cells for production of tumor specific hybridomas.

Animals↗
PubMed1981

Serological and immunochemical analysis of the specificity of xenoantiserum 8986 elicited with hybrids between human melanoma cells and murine fibroblasts.

Antisera were elicited in rabbits with hybrids derived from the fusion of human melanoma cells with murine fibroblasts. Following absorption with cultured human lymphoid cells, Xenoantiserum 8986 reacts with cultured human melanoma cells and other tumors of nonlymphoid origin. Rosette inhibition assays showed that the xenoantiserum reacts with structures which carry the determinants recognized by the monoclonal antibodies 165.28T and 653.25N and which are recognized by a xenoantiserum elicited with cultured human melanoma cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the immune complexes formed by reacting spent medium of cultured melanoma cells with Xenoantiserum 8986 showed that the antiserum contains antibodies reacting with a M.W. 240,000 melanoma-associated antigen and a M.W. 94,000 melanoma-associated antigen.

Animals↗
PubMed1981

Human melanoma-associated antigens: role of carbohydrate in shedding and cell surface expression.

The shedding of 2 tumor-specific glycoprotein antigens from human melanoma cells into spent culture medium was selectively inhibited by nontoxic doses (0.5 microgram/ml) of tunicamycin, an inhibitor of N-asparagine-linked glycosylation. The inhibition of shedding of these 2 antigens with m.w. of 240K and 94K is complete within 24 hr after addition of tunicamycin. During this time interval, our data suggest that these glycosylated cell surface antigens are replaced by their nonglycosylated forms. Removal of tunicamycin or addition of N-acetyl glucosamine restores shedding of these melanoma-associated antigens with initially reduced glycosylation. This same selective inhibition of shedding was observed with cultures adapted to grow in high doses (2.5 microgram/ml) of tunicamycin that otherwise killed > 98% of the cells upon first exposure. In contrast to other glycoproteins found in spent culture medium of melanoma cells, the shedding of melanoma-associated antigens is strictly dependent on glycosylation.

Animals↗
PubMed1981

Tumor-associated antigens in spent medium of human melanoma cells: immunochemical characterization with xenoantisera.

Xenoantisera to human melanoma cells and to partially purified melanoma-associated antigens were coupled to protein A-bearing Staphylococcus Aureus or protein A-Sepharose and used as immunoadsorbents for the indirect immunoprecipitation of intrinsically radiolabeled proteins released into culture medium from various cultured human tumor and nontumor cell lines. These radiolabeled immunoprecipitates when analyzed by SDS-PAGE revealed highly reproducible molecular profiles of proteins and glycoproteins released by various cultured tumor lines and control cells into their spent culture media. A comparison of molecular profiles together with data indicating the binding specificity of known xenoantisera produced against human melanoma cells or their extracts led to the discovery of 2 macromolecules that are associated with human melanoma cells; a glycoprotein with a subunit m.w. of 240,000 (240K) and a single-chain glycoprotein of 94,000 (94K) m.w. also found in association with human carcinoma cells are described in this report.

Animals↗
PubMed1981

Molecular profiles of human melanoma-associated antigens.

Melanoma-associated antigens (MAA) shed into spent culture medium of intrinsically radiolabeled melanoma cells react specifically with monoclonal and polyclonal anti-melanoma xenoantiserums and are represented by two glycoproteins with molecular weights of 240,000 (240K) and 94,000 (94K): 240K is present only on melanoma cells whereas 94k is also found on carcinoma cells and on fetal melanocytes. Both 240K and 94K have been obtained radiochemically pure by utilizing cellulose ion-exchange and antibody affinity chromatography. The two antigens have different charge properties, as 240K binds to CM-cellulose while 94K does not. A difference in carbohydrate moieties is also indicated since 240K binds selectively to lentil lectin and 94K to ricin lectin. Two-dimensional gel electrophoresis an tryptic peptide maps of the two antigens reveal distinct and characteristics profiles. Subunit structure determination of both antigens suggests 94K to be a single chain whereas 240K appears to be a subunit of a larger molecular structure.

Antibodies, Neoplasm↗
PubMed1980

Human melanoma associated antigens: a solid-phase assay for detection of specific antibody.

A solid-phase radioimmunometric binding assay is described utilizing 125I-labeled protein A for the detection of antibody to human melanoma associated antigens. The novel aspect of this assay is the use of chemically defined spent culture medium of melanoma cells at target antigens previously depleted of fibronectin by affinity chromatography. This makes it possible to screen for antibody in unabsorbed antiserum. Sensitivity, reproducibility and ease of performance of the assay are optimized by conjugating target antigens to a background of bovine serum albumin dried onto polyvinyl 96-well microtiter plates and cross-linked with glutaraldehyde. The use of an immobilized soluble antigen target derived from a large pool of spent culture medium facilitates direct interassay comparisons and permits extensive absorption analysis of antisera. The assay has considerable potential in screening for alloantibody and both poly- and monoclonal xeonoantibody to human melanoma associated antigens.

Animals↗
PubMed1980

Lack of association of serologically detectable human melanoma-associated antigens with beta 2 microglobulin: serologic and immunochemical evidence.

Serologic and immunochemical assays showed that human melanoma-associated antigens (MAA) identified with operationally specific xenoantisera were neither spatially nor structurally associated with beta 2-microglobulin (beta 2-mu), the light chain of the HLA-A,B antigen molecular complex; i.e., cultured melanoma cells coated with a specific anti-beta 2-mu xenoantiserum maintained their reactivity with anti-MAA xenoantisera. Furthermore, soluble MAA were not bound by a beta 2-mu immunoadsorbent. Finally, MAA were shed into the culture medium of melanoma cells and then were immunoprecipitated with specific anti-MAA xenoantisera, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they appeared as two distinct structures with molecular weights of 240,000 and 94,000 but comprised no structure with the characteristic 12,000 molecular weight of beta 2-mu. Conversely immunoprecipitates obtained by the reaction of spent culture medium of [3H]valine-labeled melanoma cells with anti-beta 2-mu xenoantiserum had the 12,000-molecular-weight component but no structures with the molecular weights established for MAA. Thus the data refute the contention that serologically detectable MAA have a molecular structure similar to that of HLA antigens.

Antibodies, Neoplasm↗