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E H Holmes

Publications and source records attributed to E H Holmes.

At least 37 records · Page 2Linked to original sources
PubMed1998

Measurement of serum prostate-specific membrane antigen, a new prognostic marker for prostate cancer.

OBJECTIVES: To describe current results with Western blot assay for prostate specific membrane antigen (PSMA) using 7E11.C5 antibody and the development of an additional antibody measurement for PSMA by a new sandwich immunoassay. METHODS: A population of patients from a screening group, from a difficult diagnostic group, from a pre- and postoperative radical prostatectomy group, and from a group with metastatic disease followed for a serial period, provided the serum values for a prospective assessment of PSMA by Western blot assay. A new monoclonal antibody was sought, reacting to the C-terminal region of PSMA in order to develop a sandwich radioimmunoassay. RESULTS: PSMA values in screened patients correlate with the more advanced stage of the cancers determined. In postprostatectomy patients, the PSMA value corresponds more with preoperative values and with the values of those with a poor clinical course. In difficult diagnostic cases, the PSMA value is increased, specifically in hormone-refractory cases and particularly in those cases judged by other criteria, such as the National Prostatic Cancer Project, to be in clinical progression compared with those judged to be in clinical remission. The level of PSMA value appears to be independent of homogeneous tumor volume and to be more related to that of prior hormone treatment, or to where prostate cancer cells can be documented to be outside the prostate. A new monoclonal antibody, 3F5.4G6, reacts with the extracellular domain of PSMA near the C-terminal region. This is in contrast to the previously measured antibody 7E11.C5, which reacts with an N-terminal epitope. 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5. The epitopes are essentially at opposite ends of the molecule. The 3F5.4G6 antibody reacts with the LNCaP line but not with DU145, or PC3. These two antibodies to PSMA are well suited for use in a new sandwich immunoassay. CONCLUSIONS: PSMA provides a prostatic cancer serum test by using Western blot, which suggests a clinical prognostic value not seen with other markers. New antibodies, such as 3F5.4G6, reacting with the extracellular domain of PSMA combined with 7E11.C5, appear to offer an opportunity for a new sandwich immunoassay.

Antibodies, Monoclonal↗
PubMed1998

Isolation and characterization of monoclonal antibodies specific for the extracellular domain of prostate specific membrane antigen.

PURPOSE: Monoclonal antibodies specific for protein epitopes of prostate specific membrane antigen (PSMA) expressed on the external surface of prostatic epithelial cells were prepared to provide material for use in the diagnosis or treatment of prostatic cancer. MATERIALS AND METHODS: Mice were immunized with LNCaP cell membranes followed by purified PSMA before fusion. Hybridomas were screened by reactivity with purified PSMA. Resulting antibodies were characterized by enzyme-linked immunosorbent assay, Western blot and fluorescence-activated cell sorter analyses. RESULTS: Monoclonal antibody producing hybridomas designated 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 were obtained which displayed specificities for differing regions of the extracellular domain of the PSMA protein. These antibodies reacted strongly with PSMA from multiple sources and specifically stained unfixed PSMA expressing cells by flow cytometric analysis. CONCLUSIONS: The antibodies obtained displayed strong reactivity and specificity for extracellular epitopes of PSMA. These antibodies will have value in future diagnostic and therapeutic applications focusing on PSMA as a target antigen.

Animals↗
PubMed1998

Prostate-specific membrane antigen: current and future utility.

Prostate cancer is the most common cancer and the second leading cause of cancer-related death among men. New prostatic markers are needed to increase diagnostic and prognostic effectiveness. One such new marker is prostate-specific membrane antigen (PSMA). PSMA is a highly prostate-restricted membrane glycoprotein that is expressed in normal prostatic epithelial cells and elevated in prostate cancers, especially in poorly differentiated, metastatic, and hormone refractory carcinomas. It has been measured in serum with immunocompetitive and Western blot assays, and its levels have been found to be correlated with the prediction of treatment failure and disease prognosis. Reverse transcriptase-polymerase chain reaction (RT-PCR) assays with primers specific for PSMA have been shown to be more effective than PSA-specific primers in detecting hematogenous circulating prostate cancer cells; however, no clear benefit in patient staging or utility as a predictor of clinical outcome or response to treatment has so far been obtained using RT-PCR methods. PSMA is currently utilized as an immunoscintigraphic target using the antibody conjugate CYT-356 (ProstaScint; Cytogen, Princeton, NJ) and has been shown to have clinical value, particularly in detecting occult prostate cancer. Another current application of PSMA is in immunotherapy of prostate cancer, in which promising results have been obtained in a phase I trial, and a phase II trial is underway. The research summarized in this article indicates that PSMA is an excellent target for diagnostic and therapeutic applications in prostate cancer.

Antigens, Neoplasm↗
PubMed1997

A family of human beta4-galactosyltransferases. Cloning and expression of two novel UDP-galactose:beta-n-acetylglucosamine beta1, 4-galactosyltransferases, beta4Gal-T2 and beta4Gal-T3.

BLAST analysis of expressed sequence tags (ESTs) using the coding sequence of the human UDP-galactose:beta-N-acetylglucosamine beta1, 4-galactosyltransferase, designated beta4Gal-T1, revealed a large number of ESTs with identical as well as similar sequences. ESTs with sequences similar to that of beta4Gal-T1 could be grouped into at least two non-identical sequence sets. Analysis of the predicted amino acid sequence of the novel ESTs with beta4Gal-T1 revealed conservation of short sequence motifs as well as cysteine residues previously shown to be important for the function of beta4Gal-T1. The likelihood that the identified ESTs represented novel galactosyltransferase genes was tested by cloning and sequencing of the full coding region of two distinct genes, followed by expression. Expression of soluble secreted constructs in the baculovirus system showed that these genes represented genuine UDP-galactose:beta-N-acetylglucosamine beta1, 4-galactosyltransferases, thus designated beta4Gal-T2 and beta4Gal-T3. Genomic cloning of the genes revealed that they have identical genomic organizations compared with beta4Gal-T1. The two novel genes were located on 1p32-33 and 1q23. The results demonstrate the existence of a family of homologous galactosyltransferases with related functions. The existence of multiple beta4-galactosyltransferases with the same or overlapping functions may be relevant for interpretation of biological functions previously assigned to beta4Gal-T1.

Amino Acid Sequence↗
PubMed1997

Comparison of serum PSMA, PSA levels with results of Cytogen-356 ProstaScint scanning in prostatic cancer patients.

BACKGROUND: Stored serum from clinical trial cases undergoing ProstaScint (CYT-356) scanning were available for Prostate Specific Membrane Antigen (PSMA) assay. Prostate Specific Antigen (PSA) levels had already been determined. This provided an opportunity to see what correlations existed between the serum markers and the ProstaScint scan. A group of patients had the studies preprostatectomy, whereas another group had the studies postprostatectomy. METHODS: The scan results, serum PSA, serum PSMA, and clinical data were separately analyzed. PSMA serum levels were determined by Western blot. RESULTS: Preoperatively, radical prostatectomy patients showed a correlation between serum PSA or PSMA levels and the ProstaScint scan in the total group (n = 86), or in an untreated group (n = 38). Preoperatively, PSMA correlated with the pathological stage, whereas PSA correlated with the scan. Postoperatively, only PSMA serum levels correlated with the scan in an untreated group (n = 40). CONCLUSIONS: Preoperatively or postoperatively, Western blot PSMA serum levels predict the stage of disease or local, regional, or distant metastases, as shown by ProstaScint scan. Both the scan and the serum tests provide prognostic information and evaluate the extent of disease to a more significant degree than previously possible.

Adult↗
PubMed1997

Evaluation of PSA, free PSA, PSMA, and total and bone alkaline phosphatase levels compared to bone scans in the management of patients with metastatic prostate cancer.

BACKGROUND: Metastatic prostate cancer clinical evaluation is difficult. A revaluation of new prostate markers with regard to bone scans was performed. METHODS: Serial markers, including bone alkaline phosphatase (BAP), total alkaline phosphatase (TAP), prostate-specific antigen, total (PSA) and free (fPSA), and prostate-specific membrane antigen (PSMA), were obtained in patients under evaluation and treatment for possible or known metastatic prostate cancer. These were correlated with bone scan results (BSR). RESULTS: Seventy patients were observed from mid-October 1996-January 1997, during which time 171 serum samples were obtained and correlated with semiquantitative bone scan status. PSA and fPSA provided some correlation with BAP and BSR, but only at high levels (> 16-50 ng/ml). Receiver-operating curve (ROC) analysis demonstrated that BAP and TAP had a significant discriminating ability for positive and negative bone scans (> .78), compared to PSMA, PSA, and fPSA. However, percent BAP and TAP only correlated with BSR at a level above six lesions. As the lesions detected by BSR increased, the correlation increased. CONCLUSIONS: BAP is a valuable marker for clinical response evaluations to use in the serial follow-up of patients with metastatic prostate cancer, and correlates well with the bone scan as the number of lesions increase to > 6. PSA or fPSA show comparable results, but only at high levels (> 16-50 ng/ml).

Aged↗
PubMed1997

Fucosylation of complex glycosphingolipids by recombinant fucosyltransferase-VII.

Fucosyltransferase VII (FucT-VII) is one of five known alpha 1-->3fucosyltransferases capable of transferring fucose to the C-3 position of N-acetylglucosamine residues found in lactosamine based glycans. Previous studies have indicated that FucT-VII has a very restricted specificity, capable of fucosylating only terminally alpha 2-->3sialylated carbohydrate substrates, resulting in the synthesis of the sialyl Lewis x (sLe(x)) epitope. Although FucT-VII is expressed in cells of myeloid origin, the monosialylganglioside fraction of HL60 cells contains only internally and/or multiply fucosylated polylactosamine structures; no monofucosylated sLe(x) derivatives are detected. We now report that the structure of the final product formed by the action of FucT-VII on sialynorhexaosylceramide (a glycosphingolipid substrate having multiple fucosylation sites) is extended monofucosyl sLe(x) and fucosylation is restricted to the terminal GlcNAc-V. This indicates that the biosynthesis of all fucosylated monosialylated gangliosides found in HL60 cells (including the E-selectin binding fractions) involves at least one additional alpha 1-->3fucosyltransferase.

Carbohydrate Sequence↗
PubMed1997

Monoclonal antibody 7E11.C5 staining of viable LNCaP cells.

BACKGROUND: Prostate-specific membrane antigen (PSMA) is a transmembrane glycoprotein defined by the monoclonal antibody 7E11.C5. The 7E11.C5 antibody forms the basis of an in vivo diagnostic imaging agent (ProstaScint, Cyt-356) for identification of metastatic prostate cancer. The epitope on PSMA recognized by 7E11.C5 has been determined to be the first 6 amino acids from the N-terminal, expressed on the cytoplasmic side of the plasma membrane. Thus, the basis for 7E11.C5 specificity in imaging studies remains unclear. METHODS: Fluorescence-activated cell sorter (FACS) analysis of fixed and viable cultured cells was used to determine the staining intensity with FITC-labeled antibodies. RESULTS: The results indicate that FITC-labeled 7E11.C5 antibody is taken up and specifically labels viable LNCaP cells in vitro. Labeling intensity of viable cells after 2 hr of antibody incubation was similar to that of fixed cells. No labeling of cells that do not express PSMA was observed, nor was labeling observed with LNCaP cells treated with an isotype-matched irrelevant antibody. CONCLUSIONS: Uptake and labeling of PSMA by FITC-labeled 7E11.C5 in viable cells in vitro strongly suggest that this is a major basis for effectiveness of the 7E11.C5 antibody during in vivo imaging applications with 111In-labeled antibody (ProstaScint, Cyt-356).

Antibodies, Monoclonal↗
PubMed1996

Evaluation and comparison of two new prostate carcinoma markers. Free-prostate specific antigen and prostate specific membrane antigen.

BACKGROUND: Two new prostate cancer markers, free-prostate specific antigen (f-PSA) and prostate specific membrane antigen (PSMA) were recently introduced. This report summarizes a prospective two-year multicenter test of their diagnostic or prognostic capabilities. Total PSA was also measured. METHODS: There were four clinical groups studied: (1) 226 individuals from a screening project undergoing ultrasound and biopsy evaluation had markers obtained: (2) 68 patients suspected of having prostate cancer and undergoing 2 or more biopsies had the markers obtained on multiple occasions: (3) 100 patients undergoing radical prostatectomy had markers obtained pre- and post-operatively: and (4) 31 patients with metastatic prostate cancer each had multiple samples for marker assay obtained over a 2-year period. In all, 465 patients had one or more samples obtained and studied. RESULTS: Free-PSA affords little additional diagnostic advantage compared with total PSA in the screening population. The receiver operating characteristic curves for diagnostic accuracy were ranked: (1) PSA density; (2) total PSA; (3) f-PSA; and (4) PSMA, PSMA showed the best correlation with stage of the primary tumor in the screened group. In the multiple negative biopsy group, f-PSA varied from 12 to 21%. PSMA values were evaluated in all histologic categories. PSA density was > or = 0.15 in all categories. In the prostatectomy cases PSA values postoperatively were quite low in Stage II; f-PSA was of no value. Later, f-PSA was increased in association with elevated total PSA values. Mean PSMA values were above normal in all postoperative time periods except in Stage III patients at 6 months to 1 year postoperatively. PSA densities were all > or = 0.15. In patients with metastatic carcinoma, elevated PSMA values correlated best with a poor prognosis (clinical progression), as has been described. CONCLUSIONS: These data suggest that f-PSA values do not provide additional diagnostic benefit compared with total PSA in screening populations, in the presence of suspected cancer, postprostatectomy, or in metastatic disease. PSMA is of prognostic significance, especially in the presence of metastatic disease, and correlates well with the stage of disease in cancers detected in a screened population.

Antigens, Neoplasm↗
PubMed1996

Measurement of prostate-specific membrane antigen in the serum with a new antibody.

Work to date has identified prostate-specific membrane antigen (PSMA) as a membrane-bound glycoprotein with high specificity for prostatic epithelial cells. PSMA reacts with the monoclonal antibody 7E11.C5, which is present in serum, seminal fluid, and prostatic epithelial cells, and is increased in its expression in the presence of a hormone refractory state associated with prostatic cancer. This report confirms these results and further documents the presence of the monoclonal antibody 3F5.4G6, which reacts with the extracellular domain of PSMA. This region of PSMA is also an element present in a truncated version of the protein, so-called PSM'. Immune precipitation with either 7E11.C5 or 3F5.4G6 yields an isolated protein species that are reactive with the reciprocal antibody in Western blot analysis. Thus, 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5, but at different epitopes on essentially opposite ends of the molecule. These two antibodies are well suited for use in a sandwich immunoassay, either one as a capture or detection antibody. Current work on this is underway. This report also confirms that 7E11.C5 Western blots for PSMA are negative with normal human brain tissue. The monoclonal antibody 9H10 does not react with 3F5.4G6 or with 7E11.C5 in studies conducted herein. Moreover, 3F5.4G6 reacts with PSMA found in the LNCaP cell line, but not DU-145 or PC3, which lack PSMA.

Amino Acid Sequence↗
PubMed1996

Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.

BACKGROUND: Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates. METHODS: In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments. RESULTS: The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells. CONCLUSIONS: Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.

Amino Acid Sequence↗
PubMed1995

Structure-function analysis of human alpha 1-->3fucosyltransferases. A GDP-fucose-protected, N-ethylmaleimide-sensitive site in FucT-III and FucT-V corresponds to Ser178 in FucT-IV.

Human alpha 1-->3fucosyltransferases constitute a family of closely related membrane-bound enzymes distinguished by differences in acceptor specificities and inherent protein biochemical properties. One such biochemical property is sensitivity to enzyme inactivation by sulfhydral-group modifying reagents such as N-ethylmaleimide. The basis for this property has been studied using a fusion protein of FucT-III and FucT-V composed of Protein A coupled to the catalytic domain of the enzyme. The results indicate that modification of FucT-V by 5,5'-dithiobis(2-nitrobenzoic acid) resulted in efficient enzyme inactivation that could be reversed by excess thiol reagent suggesting that the free sulfhydral group on the enzyme was required for activity. Recombinant forms of both FucT-III and FucT-V were irreversibly inactivated by N-ethylmaleimide and could be effectively protected from inactivation by GDP-fucose and GDP but not by UDP-galactose, fucose, or N-acetyllactosamine. Analysis of the distribution of Cys residues in aligned sequences of cloned human alpha 1-->3fucosyltransferases indicated one site, Cys143 of FucT-III and Cys156 of FucT-V, corresponded to the highly conservative replacement of Ser178 in FucT-IV, an enzyme insensitive to N-ethylmaleimide. A site-directed mutagenesis experiment was performed to replace Ser178 of FucT-IV with a Cys residue. The mutant FucT-IV enzyme was active; however, the Km for GDP-fucose was increased about 3-fold compared to the native enzyme to 28 +/- 3 microM. This enzyme was N-ethylmaleimide sensitive and could be partially protected by GDP-fucose but not N-acetyllactosamine. These results support the importance of Ser178 of FucT-IV in donor substrate binding and strongly suggest analogous Cys residues are the GDP-fucose protectable, N-ethylmaleimide-sensitive sites present in FucT-III and -V.

Base Sequence↗
PubMed1995

The etiology and prediction of breast cancer. Fourier transform-infrared spectroscopy reveals progressive alterations in breast DNA leading to a cancer-like phenotype in a high proportion of normal women.

BACKGROUND: The authors previously have shown by gas chromatography-mass spectrometry that the hydroxyl radical (.OH) induces alterations in the DNA base structure of the female breast, which are premalignant markers of breast cancer. Fourier transform-infrared (FT-IR)-spectroscopy also has a high potential for revealing a broad array of structural changes in DNA that may provide important new insight into breast cancer etiology and prediction. METHODS: DNA from normal reduction mammoplasty tissue, invasive ductal carcinoma, and nearby microscopically normal tissue was analyzed by FT-IR spectroscopy. Statistical models based on DNA spectral properties were developed and compared with a statistical model previously used with base modifications. RESULTS: Substantial differences were found in the spectral properties of DNA from women with normal and cancerous breast tissue, indicating an ability to discriminate cancerous tissue from noncancerous tissue with a sensitivity and specificity of 83%. Most importantly, the normal population was divided into subgroups in which a nonrandom progression was identified and a cancer-like DNA phenotype that was highly correlated (r > or = 0.90) with that of the patients with cancer was exhibited in 59% of the women. The spectral data, which also were highly correlated with the base-model data, were used to establish a model for predicting the probability of breast cancer. Consistent with the high cancer reoccurrence rate in the ipsilateral breast, 8 of 10 of the microscopically normal tissue specimens remaining after tumor excision were classified as cancerous using this model. CONCLUSIONS: Progressive structural changes in the DNA of the normal female breast, leading to a premalignant cancer-like phenotype in a high proportion of women, are the basis for a new paradigm for understanding the etiology of breast cancer and predicting its occurrence at early stages of oncogenesis. The results also suggest therapeutic strategies for potentially reversing the extent of DNA damage, which may be useful in disease prevention and treatment.

Breast↗
PubMed1995

Comparison of prostate specific antigen, prostate specific membrane antigen, and LNCaP-based enzyme-linked immunosorbent assays in prostatic cancer patients and patients with benign prostatic enlargement.

Serum assays for prostate specific antigen (PSA; monoclonal), for prostate specific membrane antigen (PSM; Western blot), and a LNCaP/7E11.C5-based competitive enzyme-linked immunosorbent assay (ELISA) were evaluated in a small number of prostate cancer patients with localized or disseminated disease, and judged to be in clinical progression or remission based on National Prostate Cancer Project (NPCP) criteria. PSA values recognized the presence of clinical progression in localized disease (B1-C) and to a lesser degree disseminated disease (D1-D2). In contrast, to a limited degree the ELISA test recognized clinical progression mainly in disseminated disease and chiefly in stage D2. PSM values were elevated in both D1 and D2 but not in a linear fashion as observed with PSA. The ELISA and PSM results may be assessing a different clinical response to prostatic cancer than that recognized by PSA. This could reflect a developing clone of resistant prostatic cells as previously postulated. To further pursue this possibility a secondary generation of monoclonal antibodies to PSM is being developed. The ELISA levels for benign prostatic enlargement were not elevated above normal. In contrast both with PSA and PSM the assays reflected levels significantly above the normal range in benign prostatic hypertrophy (BPH).

Antibodies, Monoclonal↗
PubMed1994

Blood group ABO-related glycosylation of urothelial cell lines: immunocytological, enzymatic, and genetic characterization.

Three immortalized, human urothelial cell lines were characterized with respect to their ABO-related carbohydrate phenotypes using a panel of monoclonal antibodies directed to a series of carbohydrate epitopes (Lac, sialylated Lac, Le(a), sialylated Le(a), Le(x), sialylated Le(x), H types I and II, Ley, Leb, A monofucosylated types I and II, ALey, Aleb, and A type III). The glycosyltransferases forming some of these epitopes (beta 1-3/4 galactosyltransferase, alpha 1-2 fucosyltransferase, alpha 1-3 galactosyltransferase, and alpha 1-3-N-acetyl-galactosaminyltransferase) were determined by enzyme assays. The ABO gene complex was analyzed by Southern blotting, Northern blotting, and polymerase chain reaction across the O deletion and across base differences between the A and B alleles. The immunocytochemical stainings showed marked differences between the three cell lines; the high grade (tumorigenic, metastatic) cell line showed difucosylated types I and II structures, and the low grade (nontumorigenic, nonmetastatic) cell lines showed monofucosylated types I and II structures. Polymerase chain reaction genotyping of the cell lines indicated that one was OO, one was AA, and one was A plus a mutated allele. Northern blotting showed RNA encoding the A transferase. However, even though both of the A cell lines seemed to have an intact gene, which could produce A transferase and transcribed RNA, none of them showed any activity of the A gene encoded enzyme or any A-structures at the cell surface. In contrast, the three other examined glycosyltransferases were active. The three urothelial cell lines reflect in vivo findings in humans. They represent a competent system for in vitro studies of the different carbohydrate transferase genes responsible for the carbohydrate structures expressed on the cell surface in bladder tumors.

ABO Blood-Group System↗
PubMed1994

Photoidentification of mannosyltransferases of dolichol cycle in the mammary gland. Purification and characterization of GDP-Man:Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-dolichol mannosyltransferase.

Glc3Man9GlcNAc2-P-P-Dol serves as the major precursor for the biosynthesis of asparagine-linked glycoproteins in eukaryotes. The first 5 of the 9 mannosyl residues during the assembly of the oligosaccharide moiety within the dolichol cycle in the endoplasmic reticulum are incorporated directly by the action of GDP-Man-requiring mannosyltransferases while the remaining last 4 mannosyl residues are transferred by Man-P-Dol-requiring enzymes. In an earlier study (Shailubhai, K., Illeperuma, C., Tayal, M., and Vijay, I. K. (1990) J. Biol. Chem. 265, 14105-14108), we identified the enzyme UDP-Glc:Dol-P glucosyltransferase by photolabeling rat mammary microsomes with 5-N3-[beta-32P]UDP-Glc. Applying a similar strategy, GDP-hexanolamine-125I-azidosalicylic acid, an analog of GDP-Man, was found to photolabel two polypeptides of 37 and 69 kDa among the microsomal proteins of the rat mammary gland. A differential ammonium sulfate saturation (60-80%) of the detergent-solubilized microsomal proteins enriched the 69-kDa polypeptide. Photolabeling of this polypeptide was specifically inhibited by guanine-containing nucleotides and nucleotide-sugars and was associated with a GDP-Man-requiring mannosyltransferase. The mannosyltransferase was purified nearly 16,000-fold and shown to contain the 69-kDa polypeptide. The purified enzyme catalyzes the transfer of [14C]Man from GDP-[14C]Man to Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol in alpha 1,3-linkage to give [14C]Man alpha 1-->3Man beta 1-->4GlcNAc beta 1-->4GlcNAc-P-P-Dol as the product. Antibodies raised against the 69-kDa polypeptide removed the enzymatic activity from the detergent extract of the rat mammary microsomes and reacted specifically with a polypeptide band of the same size on immunoblots. The purified enzyme showed a pH optima of 7.4-7.8, Km approximately 4 microM for GDP-Man, approximately 2-fold activation by phosphatidylcholine, and a strong inhibition by sulfhydryl-selective reagents, N-ethylmaleimide and p-chloromercuribenzoate. The availability of the highly purified enzyme and a monospecific antibody should allow its molecular cloning for investigating the regulation of the machinery for protein N-glycosylation upon hormonally modulated growth and differentiation of the mammary gland during its ontogeny.

Animals↗
PubMed1994

Western blot assay for prostate-specific membrane antigen in serum of prostate cancer patients.

There is a need for the development of new diagnostic tools for the early detection of prostate cancer. A candidate molecule for a new screening test is a prostate-specific membrane antigen (PSM) recognized by the monoclonal antibody 7E11.C5. We carried out studies aimed at identifying PSM in the serum of normal and benign prostatic hyperplasia (BPH) donors and patients with adenocarcinoma of the prostate, in order to judge whether the development of a serum assay using this marker was feasible. By Western blotting, we found significant levels of PSM in serum samples from prostatic cancer patients, in the seminal fluid of pooled normal donors, in BPH patients, and in normal male sera. Similar to prostate-specific antigen (PSA), PSM was present in seminal plasma in higher concentrations than in serum, and PSM levels in prostatic cancer patients were significantly higher than in normal controls. These data suggest that the development of an assay utilizing the PSM and new monoclonal antibodies directed against the antigen, could provide a feasible test for prostatic cancers.

Adenocarcinoma↗