Biomedical subjects
J P Brown
Publications and source records attributed to J P Brown.
Isolation of two novel sialyl-Lewis X-active oligosaccharides by high-performance liquid affinity chromatography using monoclonal antibody Onc-M26.
A monoclonal antibody, Onc-M26, that recognizes a cancer-associated antigen expressed by most human adenocarcinomas of the breast was shown previously to recognize a carbohydrate epitope carried on a hexaglycosyl ganglioside carrying the sialyl-Lewis X (SLex) antigen (P.S. Linsley et al., 1988, Cancer Res. 48, 2138-2148). Evidence that the antibody binds even more avidly to minor gangliosides containing more complex carbohydrate chains prompted us to search for a higher affinity epitope among sialylated oligosaccharides from pooled human milk. Affinity chromatography of a partially purified fraction of monosialylated milk oligosaccharides on a column containing monoclonal antibody Onc-M26 bound to a macroporous silica matrix gave a peak with a retention volume significantly greater than that of a standard SLex-active hexasaccharide. The retained material consisted of two nonasaccharides, each containing the SLex tetrasaccharide sequence, Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3) GlcNAc, linked beta 1-6 to a 3,6-disubstituted galactosyl residue.
On the carcinogenicity of cadmium by the oral route.
Cadmium and cadmium compounds are carcinogenic both by inhalation and by injection. For purposes of risk assessment, a prudent public health approach has been that, if a chemical has been demonstrated to be carcinogenic by one route, it should be considered carcinogenic by all routes. This policy has been questioned for several toxic metals including cadmium. After reviewing the literature on cadmium carcinogenicity and genotoxicity, we think that cadmium should be considered noncarcinogenic by the oral route. The bases for this decision included: (1) a database for genotoxicity of cadmium with more negative test results than positive results and with most positive results in in vitro tests, indicating that cadmium has limited genotoxicity; (2) some epidemiologic evidence of respiratory tract cancer and prostatic cancer in people occupationally exposed to airborne cadmium but no reliable evidence of gastrointestinal tract cancers in workers; and (3) a large dietary oncogenicity study in rats of cadmium chloride at several dose levels, including a maximally tolerated dose (50 ppm) in males, which showed no increase of tumors due to cadmium ingestion in all of the 19 tissues examined. The conclusion that an agent, which has been shown to be carcinogenic by one route of exposure, is not carcinogenic by a second route should be made only in the presence of robust data which indicate the lack of effect via the second route of exposure.
The future of prevention in dental schools.
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Risk assessment for benzo[a]pyrene.
Benzo[a]pyrene is the most studied carcinogenic polycyclic aromatic hydrocarbon and one of the most potent, and it is often used as a toxicological prototype or surrogate for all carcinogenic polycyclic aromatic hydrocarbons. Quantitative risk assessment of benzo[a]pyrene has been hampered by the quality of the data sets available for quantitation. The EPA has calculated both potency slopes and unit risks using the linearized multistage model. We have confirmed and refined that analysis. Risks based on feeding, inhalation, and intratracheal administration of benzo[a]pyrene show fairly good concordance. Others have applied the Moolgavkar two-stage model to the data and estimated a lower potency for benzo[a]pyrene. We think that such conclusion is premature. Longer term exposures of animals to benzo[a]pyrene are necessary to provide the data needed for more refined carcinogenic risk estimations.
Expression of a yeast glycolytic gene is subject to dosage limitation.
The Saccharomyces cerevisiae pyruvate kinase-encoding gene (PYK1) has been transformed back into yeast using a derivative of the multicopy vector, pJDB207. High levels of PYK1 expression in these transformants are limited by at least two separate mechanisms. Pyruvate kinase assays and polysome analyses demonstrate that the translation of the PYK1 mRNA is inhibited as its abundance increases. The abundance of the PYK1 mRNA per gene copy also decreases as the copy number of the PYK1 gene increases. This is the first report which demonstrates that a eukaryotic glycolytic gene is subject to dosage limitation at the translational level.
Finding protein similarities with nucleotide sequence databases.
In this chapter we describe strategies for the searching of translated nucleotide sequence databases. By applying standard searching techniques developed for protein databases, we have found that previously unrecognized homologies can be detected. In addition, we have shown that extremely high sensitivity can be obtained using the scoring matrix strategy for short regions of similarity. The latter approach is particularly effective for detecting homologs found at the ends of sequences and within data of poor quality. These individual methods are demonstrated for the LysR family of bacterial activator proteins. Successive applications of these methods allow for sensitive detection of complex relationships, as demonstrated for the AraC family and for the complex LuxR-OmpR-NtrC families of bacterial activator proteins. Although our examples are drawn from bacterial sequences, these methods are likewise effective for higher eukaryotic genomic sequences, where protein-coding sequences are usually interrupted by introns. This should be particularly important in the future, since much of the expected increase in nucleotide sequence databases is likely to come from eukaryotic genomic sequencing projects.
Development and evaluation of monoclonal antibody-based immunoassays: breast carcinoma-associated mucins as tumor markers.
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Georgia's public health experience with diabetes control.
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Monoclonal antibodies reactive with mucin glycoproteins found in sera from breast cancer patients.
Monoclonal antibodies to mucin glycoproteins have previously been shown to detect elevated antigen levels in sera from breast cancer patients. To determine whether different mucin epitopes represent better targets for serum assays, we have produced and characterized 14 new monoclonal antibodies directed against the mucin glycoproteins detected by antibody W1. Many of the new antibodies differed from each other in their ability to bind to mucins from various sources. Cross-competition analyses of antibody binding indicated that while epitopes for some antibodies were distinct, most epitopes showed complex structural or steric relationships with those for other antibodies. Antibody M26 bound glycolipids from meconium and kidney, indicating that it recognized a carbohydrate epitope. Antibodies M15, M22, M23, and M27 bound to structurally or sterically related epitopes on deglycosylated milk-derived mucin, suggesting that they recognized core protein epitopes. Enzyme immunoassays were developed with the new antibodies and evaluated for their ability to discriminate between sera from breast cancer patients and from controls with benign breast disease. The best single test in terms of sensitivity and specificity used a combination of two antibodies, antibody M29 for antigen capture and antibody M38 for antigen detection. A second test using antibody M26 for antigen capture and antibody M38 for antigen detection detected elevated antigen levels in sera from some patients which were in the control range of the M29/M38 test. By combining results from these tests, significantly more cancer patients were detected than with the W1 and CA 15.3 tests.
Molecular cloning of the human B cell CD20 receptor predicts a hydrophobic protein with multiple transmembrane domains.
CD20 is an antigen expressed on normal and malignant human B cells that is thought to function as a receptor during B cell activation. Here we report the isolation of a CD20-specific cDNA clone from a lambda gt11 library using a polyclonal antiserum raised against purified CD20 antigen. Additional cDNA clones were then isolated from a lambda gt10 library. Alignment of the sequences of overlapping lambda clones reveal a single consensus sequence except for a divergence that preceded the first methionine within the open reading frame. Normal B cells and B cell lines contain a prominent 2.6 kb mRNA and a lower level of a 3.3 kb mRNA. An oligonucleotide derived from one of the divergent sequences hybridized to the 3.3 kb mRNA only, indicating that the two mRNA species are derived from an alternative splicing mechanism. The predicted amino acid sequence of CD20 reveals three major hydrophobic regions of approximately 53, 25 and 20 amino acids. CD20 lacks an NH2-terminal signal peptide and contains a highly charged COOH-terminal domain. Although CD20 is immunoprecipitated as a doublet of 33 and 35 kd proteins from B cells, in vitro translation of CD20 cDNA produced a single 33 kd protein that was specifically immunoprecipitated with monoclonal CD20 antibodies. CD20 was strongly phosphorylated on resting B cells after CDw40 stimulation, suggesting that CD20 may be functionally regulated by a protein kinase(s).
Anti-tumor effects of antibody-alkaline phosphatase conjugates in combination with etoposide phosphate.
Two anti-tumor monoclonal antibodies, L6 (anticarcinoma) and 1F5 (anti-B lymphoma), were covalently linked to alkaline phosphatase (AP), forming conjugates that could bind to the surface of antigen-positive tumor cells. The conjugates were capable of converting a relatively noncytotoxic prodrug, etoposide phosphate (EP), into etoposide--a drug with significant antitumor activity. In vitro studies with a human colon carcinoma cell line, H3347, demonstrated that while EP was less toxic than etoposide by a factor of greater than 100, it was equally toxic when the cells were pretreated with L6-AP, a conjugate that bound to the surface of H3347 cells. The L6-AP conjugate localized in H3347 tumor xenografts in nude mice and histological evaluation indicated that the targeted enzyme (AP) was distributed throughout the tumor mass. A strong antitumor response was observed in H3347-bearing mice that were treated with L6-AP followed 18-24 hr later by EP. This response, which included the rejection of established tumors, was superior to that of EP (P less than 0.005) or etoposide (P less than 0.001) given alone. The IF5-AP conjugate did not bind to H3347 cells and did not enhance the toxicity of EP on these cells in vitro. In addition, IF5-AP did not localize to H3347 tumors in nude mice and did not demonstrate enhanced antitumor activity in combination with the prodrug.
Recombinant vaccinia virus vaccine against the human melanoma antigen p97 for use in immunotherapy.
We have constructed a recombinant vaccinia virus, v-p97NY, which expresses the human melanoma-associated glycoprotein p97. Immunization with v-p97NY could induce humoral and cell-mediated immunity to p97, including delayed-type hypersensitivity, in mice and in two of two monkeys (Macaca fascicularis). The fact that an immune response was induced also in monkeys is important because normal cells from monkeys, but not from mice, express a low level of cross-reactive p97. Mice immunized with v-p97NY rejected transplants of syngeneic mouse melanoma expressing p97. A rejection response could be detected also when immunization was started 2 days after tumor transplantation, irrespective of whether the transplanted cells grew subcutaneously or as lung metastases. Evidence was obtained that melanoma cells lacking p97 may be killed as "bystanders" at the site of an immune response to melanoma cells expressing p97.
Characterization of a recombinant vaccinia virus expressing human melanoma-associated antigen p97.
p97 is a cell surface glycoprotein expressed at high levels in most human melanomas but present only in trace amounts in normal adult tissues. We are interested in exploring the possibility of using recombinant vaccinia virus to express a specific tumor-associated antigen as a vaccine against human cancer. To this end, we constructed a recombinant virus, v-p97NY, which contains the entire coding sequence for p97 under the control of the vaccinia virus 7.5K promoter. Upon infection of tissue culture cells, v-p97NY expressed high levels of a membrane-bound glycoprotein immunoreactive with a p97-specific monoclonal antibody. Immunization of mice with this recombinant elicited high-titered antibodies against p97. Spleen cells isolated from these mice proliferated in vitro when stimulated either with purified p97 protein or with syngeneic cells expressing p97 antigen. Delayed-type hypersensitivity was also observed in immunized mice after challenge with p97-expressing cells. These findings indicate the potential usefulness of v-p97NY and similar recombinants in tumor immunotherapy.
Difference in cell binding patterns of two monoclonal antibodies recognizing distinct epitopes on a human melanoma-associated oncofetal antigen.
Two monoclonal antibodies (MAbs), 140.240 and 96.5, generated independently in different laboratories, have been shown to detect the target structures of 87,000 (gp87) and 97,000 (p97) glycoproteins, respectively, both strongly expressed by melanoma cells and fetal small intestine. To determine whether MAb 140.240 and MAb 696.5 recognized a same target structure, they were tested in immunoprecipitation/SDS-PAGE using NP-40 lysates of melanoma cells labelled with [35S]methionine for 18 hr. Both antibodies precipitated a single band with Mr = 87,000. Reciprocal immunodepletion studies showed that neither of the two antibodies detected the 87,000 band in the lysate immuno depleted by either antibody, suggesting that these two antibodies recognize the same or extremely similar molecules. Two-dimensional tryptic peptide mapping analysis showed that the two identified molecules shared the same finger-printing pattern. A 40,000 fragment of the 87,000 molecule produced by protease digestion was precipitated by MAb 96.5 but not MAb 140.240, indicating that the epitopes recognized by the two antibodies are localized at discrete sites on the molecule. Serological studies on these two antibodies revealed slightly different binding patterns in the MAb 140.240 exhibited a more melanoma-restricted specificity, while MAb 96.5 had a specificity to melanoma and to some other cell types. The observed difference in epitope specificity may be important in the clinical applications of these antibodies.
Psychological distress in medical patients referred for psychiatric consultation.
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Active bone turnover of the cortico-endosteal envelope in postmenopausal osteoporosis.
We developed a computerized semiautomatic method allowing accurate measurement of bone remodeling activity of the cortico-endosteal envelope of iliac crest biopsies. Measurement of bone turnover of the cortico-endosteal and of the trabecular envelopes were performed in 33 untreated women with postmenopausal vertebral osteoporosis and in 8 age-matched normal women. In normal women, bone remodeling was not different in the trabecular and the cortico-endosteal envelopes. In contrast, osteoporotic women had a marked, significant increase of bone formation and resorption at the cortico-endosteal envelope when compared to trabecular bone. Despite this difference, bone turnover activities of both envelopes were related, as indicated by significant correlations for most parameters between both envelopes. Serum bone gla-protein, previously reported to reflect trabecular bone formation, was also highly correlated with static and dynamic assessment of bone formation of the cortico-endosteal envelope of osteoporotic women but did not reflect bone resorption. When trabecular and endosteal measurements were pooled, correlations between serum bone gla-protein and bone formation were similar or even improved. In conclusion, our data suggest that patients with postmenopausal osteoporosis have active bone turnover at the cortico-endosteal envelope which may contribute significantly to bone loss in this disease. Because the different bone envelopes react differently to various therapeutic regimens, bone turnover of the cortico-endosteal envelope should be considered when assessing the effect of new treatments.