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J E Shively

Publications and source records attributed to J E Shively.

At least 91 records · Page 5Linked to original sources
PubMed1991

Microsequence analysis of peptides and proteins. IX. An improved, compact, automated instrument.

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w x 38 x 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5-10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.

Amino Acid Sequence↗
PubMed1991

Structural analysis of proteins by laser desorption and electrospray mass spectrometry.

The development of electrospray and matrix-assisted laser desorption mass spectrometry has provided protein chemists with tools for peptide and protein structure analysis with unprecedented sensitivity and molecular weight range. The two technologies can be viewed as competitive with respect to their molecular mass determinations, but complementary with respect to their differences in instrumentation, sample preparation methods, and nature of spectra produced.

Animals↗
PubMed1991

Binding of nonspecific cross-reacting antigen, a granulocyte membrane glycoprotein, to Escherichia coli expressing type 1 fimbriae.

Nonspecific cross-reacting antigen (NCA) is a well-characterized membrane glycoprotein on granulocytes, macrophages, and lung epithelium. Structural studies at the protein and genomic levels have revealed that NCA is a member of the immunoglobulin supergene family, and hybridization studies showed that the transcript level of NCA is induced by treatment with gamma interferon. These studies, as well as the expression of NCA on granulocytes, suggest a role for NCA in immune response. For a first step in studying this possible role, we have examined the binding of two glycoforms of NCA designated NCA-50 (Mr, 50,000) and TEX-75 (Mr, 75,000). Here we report the results from binding assays which demonstrate carbohydrate-mediated binding of Escherichia coli expressing type 1 fimbriae and of isolated type 1 fimbriae to NCA-50. TEX-75 did not bind to the purified fimbriae but bound slightly to the bacterial strain. Inhibition studies showed that the binding to NCA-50 involved interaction of mannose moieties on NCA-50 and lectins on the fimbriae. The binding of NCA-50 to bacterial fimbriae was confirmed by electron microscopy studies, using immunolabeling techniques. In addition, we show that the surface expression of NCA-50 (and presumably of other NCA species) on isolated polymorphonuclear leukocytes is increased following activation with the bacterial peptide formylmethionyl-leucyl-phenylalanine, consistent with a role for NCA in immune response.

Antigens, Neoplasm↗
PubMed1991

Human proopiomelanocortin-(79-96), a proposed androgen stimulatory hormone, does not affect steroidogenesis in cultured human fetal adrenal cells.

Whether the production of human adrenal androgens is stimulated by a hormone other than ACTH or angiotensin-II has long been controversial. A candidate for such a cortical androgen stimulatory hormone (CASH) is the proximal 18-amino acid hinge region of proopiomelanocortin (POMC). This region of POMC lies to the amino-terminal side of the ACTH region, displays considerable amino acid sequence variation among mammals, and has been reported to stimulate the secretion of dehydroepiandrosterone (DHEA) from cultured adult adrenal cells. We cultured human fetal adrenal cells with 10(-8) M CASH-18, 10(-13) M ACTH, 10(-8) M CASH-18 plus 10(-13) M ACTH, 10(-8) M ACTH, or vehicle alone. Culture media were assayed for cortisol, DHEA, and DHEA sulfate, and cellular RNA was assayed for P450scc and P450c17 mRNAs. ACTH (10(-8 M) increased steroid secretion and the mRNAs for P450scc and P450c17, but the other treatments were indistinguishable from the control. Thus, CASH-18 does not affect steroidogenesis in primary cultures of human fetal adrenal cells. These data could be consistent with a model in which adrenarche, the onset of adrenal androgen synthesis at 8-10 yr of age, is mediated by developmentally programmed synthesis of a receptor for CASH. Alternatively, they are also consistent with this region of POMC having no CASH activity.

Adrenal Glands↗
PubMed1991

cDNA cloning of carboxyl ester lipase from human pancreas reveals a unique proline-rich repeat unit.

We report the isolation and nucleotide sequence of the cDNA for carboxyl ester lipase (CEL) from human pancreas. CEL was purified from human pancreas and microsequence analysis was performed on the amino-terminal and internal peptides. Peptide sequence was used to design oligonucleotide probes for screening a human pancreas cDNA library. Partial length cDNAs for CEL were isolated from the library, and the 5' portion of the cDNA was obtained using the anchored polymerase chain reaction. The deduced amino acid sequence indicates that mature CEL contains 722 amino acids and is synthesized with a 20 amino acid leader peptide. The amino acid sequence is rich in proline (12.2%), with 68% of the proline residues occurring within the final 25% of protein length. This is due to the occurrence of a series of proline-rich tandem repeat units near the carboxyl terminus, and accounts for the previously observed species variation in CEL size and amino acid composition. The primary sequence of CEL shows strong similarity to members of the serine esterase family, including the identical G-E-S-A-G motif at the putative active site. A striking homology also occurs between CEL and acetylcholinesterase and cholinesterase, essential enzymes of the nervous system. Proteins with cholesteryl esterase activity have been detected in extra-pancreatic tissues including liver, intestine, kidney, aorta, macrophage, and in the milk of some species (human, gorilla, cat, dog), but not others (rat, cow). To clarify the structural relationships between these various esterases and CEL, we used the CEL cDNA to study expression in pancreas and liver. CEL mRNA was abundant in pancreas of human and rat, with the human CEL mRNA approximately 300 nucleotides larger than that from rat. CEL mRNA was not detected in human adult or fetal liver, nor in rat liver. These results indicate that CEL is not synthesized in significant amounts in liver, and suggest that the cholesterol esterase activity that has been described in liver may be due to a distinct enzyme, or may be derived from pancreas, as has been proposed for the cholesterol esterase activity in intestine.

Amino Acid Sequence↗
PubMed1990

Sequence and glycosylation site identity of two distinct glycoforms of nonspecific cross-reacting antigen as demonstrated by sequence analysis and fast atom bombardment mass spectrometry.

Nonspecific cross-reacting antigen (NCA) is a highly glycosylated membrane protein which is immunologically and structurally related to carcinoembryonic antigen, an important tumor-associated antigen. Two glycoforms of NCA were purified from a single liver metastasis of a colonic carcinoma and characterized with respect to their primary sequence and position of glycosylation sites. The two glycoforms (designated TEX (tumor-extracted antigen), Mr 75,000, and NCA, Mr 45,000) each showed a deglycosylated Mr of 35,000 and yielded identical peptide maps. The structural characterization of TEX and NCA and the assignment of glycosylation sites was performed by fast atom bombardment mass spectrometry and microsequence analysis of the resulting peptides. This approach showed that TEX and NCA were identical with respect to primary sequence and provided direct evidence that 11 of the 12 predicted asparagine-linked glycosylation sites were glycosylated in both TEX and NCA. Indirect evidence was obtained for glycosylation at the other site. Both glycoforms also contain ethanolamine linked to Gly-286, a finding consistent with the conclusion that these proteins are anchored to the plasma membrane through a glycosyl-phosphatidylinositol tail. The large difference in the molecular weights of glycosylated TEX and NCA suggests significant variations in their oligosaccharide structures.

Amino Acid Sequence↗
PubMed1990

Transcription of biliary glycoprotein I gene in malignant and non-malignant human liver tissues.

Biliary glycoprotein I (BGP I) is a member of carcinoembryonic antigen (CEA) gene family consisting of at least 11 related genes. The transcription of BGP I gene was analysed in malignant and non-malignant human liver tissues with a 396-bp 3'-untranslated region probe from a cDNA clone 4-13 which was newly isolated from an adult human colon cDNA library. Among 21 tissue samples from 14 patients with hepatocellular carcinoma, 16 samples were clearly shown to express a single 3.9-kb message. This message was also found in the hepatoma cell line HuH-7. When the malignant tissues were compared to the non-malignant ones for the intensity of the band, no significant difference was observed. mRNAs of CEA and non-specific cross-reacting antigen (NCA) were not detected in 5 samples which were shown to have the message of the BGP I gene. These data suggest that the human hepatocyte and its malignant transformant produce BGP I, and that this could correspond to the cross-reacting antigen previously detected in the liver.

Antigens, CD↗
PubMed1990

Expression of complementary DNA and genomic clones for carcinoembryonic antigen and nonspecific cross-reacting antigen in Chinese hamster ovary and mouse fibroblast cells and characterization of the membrane-expressed products.

Complementary DNA clones coding for both carcinoembryonic antigen (CEA), a well characterized colonic tumor marker, and nonspecific cross-reacting antigen (NCA), a related antigen, were expressed in Chinese hamster ovary (CHO) cells and L-cells (mouse fibroblasts). A genomic clone coding for CEA was also expressed in CHO cells. Positive clones were identified by fluorescence flow cytometry and enzyme-linked immunosorbent assay. Membrane location of the recombinant CEA and NCA was confirmed by indirect immunofluorescence labeling of the transfectants, followed by visualization under a fluorescence microscope. The apparent molecular weight of the expressed CEA and NCA were 180,000 and 96,000, respectively, for both cell lines, as determined by immunoblot analysis. The CEA and NCA expressed on CHO cells were sensitive to treatment with phosphatidylinositol-specific phospholipase C (PI-PLC), whereas the CEA and NCA proteins on L-cells were resistant to removal by PI-PLC. Unlike NCA, which contains three methionine residues, the only methionine in CEA is in the C-terminal hydrophobic domain. This domain in CEA was shown to be removed and replaced by a phosphatidylinositol glycan (PI-G) anchor (Hefta et al., Proc. Natl. Acad. Sci. USA, 85: 4648-4652, 1988). The recombinant CEA from both CHO cells and L-cells could be labeled with [3H]-ethanolamine (a component of the PI-G anchor) but not with [35S] methionine, whereas the recombinant NCA could be labeled with both [3H]ethanolamine and [35S]methionine. The labeling studies and PI-PLC treatment results are consistent with the CEA and NCA expressed on CHO cells possessing a PI-G anchor. The CEA expressed on the L-cell transfectants may contain a PI-G anchor which is resistant to cleavage by PI-PLC. In addition, the membrane-bound and secreted levels of CEA from the CHO and L-cell transfectants were determined.

Animals↗
PubMed1990

Cloning of the genes for T84.66, an antibody that has a high specificity and affinity for carcinoembryonic antigen, and expression of chimeric human/mouse T84.66 genes in myeloma and Chinese hamster ovary cells.

Carcinoembryonic antigen (CEA) is one of the best characterized tumor-associated antigens and is extensively used in the in vitro immunodiagnosis of human colon adenocarcinomas. Among a number of anti-CEA monoclonal antibodies, the murine monoclonal antibody T84.66 shows the highest specificity and affinity for CEA and has been used successfully for in vivo tumor imaging in mice and humans. We report here the cloning and sequencing of the genes coding for monoclonal antibody T84.66 and the amino acid sequence of the variable regions for the heavy and light chains. We also report the construction of mouse/human chimeric IgG1 antibody genes using T84.66 variable region genes and human constant region genes. The resulting chimeric gene constructs were transfected into murine myeloma cells (Sp2/0) by electroporation and into Chinese hamster ovary cells by lipofection. The chimeric antibodies obtained exhibited the same specificity and affinity for CEA as that of the T84.66 immunoglobulin produced by the murine hybridoma cell line. Antibody concentrations in culture medium supernatants were clonally variable but similar (15-480 ng/ml) for both Sp2/0 and Chinese hamster ovary transfectants; the average production by Chinese hamster ovary transfectants was only 3-5-fold less than Sp2/0 transfectants. Ascites production of Sp2/0 transfectants is sufficiently high (900 micrograms/ml) for initial in vivo studies with humanized T84.66.

Amino Acid Sequence↗
PubMed1990

Carboxy-terminal sequencing: formation and hydrolysis of C-terminal peptidylthiohydantoins.

Proteins and peptides can be sequenced from the carboxy terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory indicated that the use of trimethylsilyl isothiocyanate (TMS-ITC) as a coupling reagent significantly improved the yields and reaction conditions and reduced the number of complicating side products [Hawke et al. (1987) Anal. Biochem. 166, 298]. The present study further explores the conditions for formation of the peptidylthiohydantoins by TMS-ITC and examines the cleavage of these peptidylthiohydantoin derivatives into a shortened peptide and thiohydantoin amino acid derivative. Schizophrenia-related peptide (Thr-Val-Leu) was used as a model peptide and was treated with acetic anhydride and TMS-ITC at 50 degrees C for 30 min, and the peptidylthiohydantoin derivatives were isolated by reverse-phase HPLC and characterized by FAB-MS. The purified derivatives were subjected to a variety of cleavage conditions, and rate constants for hydrolysis were determined. Hydrolysis with acetohydroxamate as reported originally by Stark [(1968) Biochemistry 7, 1796] was found to give excellent cleavage of the terminal thiohydantoin amino acid, but also led to the formation of stable hydroxamate esters of the shortened peptide which are poorly suited for subsequent rounds of degradation. Hydrolysis with 2% aqueous triethylamine under mild conditions (1-5 min at 50 degrees C) was found to be more suitable for carboxy-terminal sequence analysis by the thiocyanate method. The shortened peptide, which could be isolated and subjected to a second round of degradation, and the released thiohydantoin amino acid are formed in good yield (90-100%). Several other small peptides containing 15 different C-terminal amino acid side chains were also investigated in order to examine any interfering reactions that might occur when these side chains are encountered in a stepwise degradation using the thiocyanate chemistry. Quantitative yields of peptidylthiohydantoins were obtained for all the amino acids examined with the following exceptions: low yields were obtained for C-terminal Glu or Thr, and no peptidylthiohydantoins were obtained for C-terminal Pro or Asp. Asparagine was found to form cyclic imides (64%) at the penultimate position (C-2) during hydrolysis of the peptidylthiohydantoins by 2% aqueous triethylamine. Cleavage of C-terminal Asn under these conditions led to the formation of the expected shortened peptide (69%), but also to the formation of a shortened peptide (31%) with a C-terminal amide. Problems with Glu and Thr could be solved by minimizing the reaction time with acetic anhydride.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1990

Estimation of monoclonal antibody-associated 90Y activity needed to achieve certain tumor radiation doses in colorectal cancer patients.

In these measurements, we quantitated, via surgical samples, human primary tumor uptake of the anti-carcinoembryonic antigen monoclonal antibody T84.66. Uptake was measured in units of percentage of injected dose/kg with 111In as the radiolabel. All 11 colorectal lesions were nonnecrotic and were visualized upon scanning. Tumor volume was calculated using the three orthogonal dimensions as described by pathology. Uptake mean +/- SD was 6.55 +/- 3.55% injected dose/kg with a range of 1.2 to 10.4% injected dose/kg. Lesion mean volume was 36 cm3 with a range of 1.5 to 304 cm3. Using mean values, assuming no biological clearance and that the biodistribution of the monoclonal is independent of its radiolabel, the predicted human tumor 90Y beta dose was 0.12 Gy/mCi. Therefore a 10-Gy tumor dose would require 83 mCi of i.v. activity. Using least and highest uptake results, requisite activity values were 3-fold larger and smaller respectively. Thus, there was approximately an order of magnitude variation in the amount of 90Y predicted to achieve a given tumor dose in colorectal cancer patients. Murine and human uptake values were consistent if lesion mass and carcinoembryonic antigen content were taken into account.

Antibodies, Monoclonal↗
PubMed1990

Presurgical imaging with indium-labeled anti-carcinoembryonic antigen for colon cancer staging.

Over a 4-year period, 108 patients with known or suspected colorectal cancer were studied by radioimmunoconjugate scintigraphy prior to operative procedures. Study subjects received 0.2 to 40 mg i.v. of murine anti-carcinoembryonic antigen monoclonal antibody labeled with 2-5 mCi of 111In (Indacea). Resected tissues were analyzed for 111In and carcinoembryonic antigen content. Tumor, liver, and draining lymph nodes had over 10% injected dose/kg compared to less than 2.5% injected dose/kg for other normal tissues. Primary tumors that were successfully imaged were significantly larger and had higher 111In and carcinoembryonic antigen content. In 54 patients, primary tumors were visualized with a sensitivity of 78%. Hepatic metastases (58 patients) were visualized as negative filling defects (sensitivity, 45%). Extrahepatic (intraabdominal) metastases (25 patients) were visualized (sensitivity, 48%) as areas of increased uptake. Extraabdominal metastases were uncommon (10 patients; sensitivity, 80%). Of 56 patients with known or suspected hepatic metastases who presented with no evidence of extrahepatic disease by conventional tests (X-ray, computerized tomographic scan), 20 (36%) were documented to have extrahepatic metastases at exploratory surgery and 10 of these (50%) had the extrahepatic disease localized by the Indacea scan. The management of these 10 patients was, or could have been, modified by the scan findings and unnecessary surgery eliminated.

Adult↗
PubMed1990

Localization of intraperitoneal xenografts of human colon cancer by radiolabeled anti-CEA antibodies.

An intraperitoneal xenograft tumor model was developed in the nude mouse to represent advanced colorectal disease in the human patient. Intraperitoneal (ip) and subcutaneous (sc) tumor xenografts were successfully localized by indium 111-labeled anti-carcinoembryonic antigen monoclonal antibodies (Indacea) administered intravenously (IV) or intraperitoneally (IP). In the sc model, tumor uptake (% injected dose per g of tumor tissue--%ID/g) was significantly improved at 72 hours postinjection when Indacea was administered IP (44.01 +/- 1.94 vs. 33.74 +/- 0.87, P less than 0.005). In the ip model, tumor uptake at 72 hours was improved with IP Indacea (28.23 +/- 5.42 vs. 19.36 +/- 5.50, P less than 0.01) and the tumor to blood ratio was significantly improved for IP Indacea (17.83 +/- 2.95 vs. 8.45 +/- 1.38, P less than 0.01). Tumor mass was not a contributing factor in these differences. This study demonstrates that ip xenografts can be successfully imaged using radiolabeled antibodies and that the IP route of administration results in better tumor uptake of the antibody in ip lesions.

Animals↗
PubMed1990

Size and subdomain architecture of the glucan-binding domain of sucrose:3-alpha-D-glucosyltransferase from Streptococcus sobrinus.

Mild trypsin proteolysis of Streptococcus sobrinus sucrose:3-alpha-D-glucosyltransferase (GTF-I) reduced most of the enzyme to small products but left a few large fragments undigested. The digest had no glucosyl transfer activity, but several digestion products had an affinity for glucan equivalent to that of the native enzyme. The glucan-binding fragments ranged in size from 17 to 60 kilodaltons (kDa), with a particularly prominent 42-kDa fragment. The largest of these (60 kDa) appears to be the full extent of the domain since it increases in abundance when the enzyme is protected with glucan during proteolysis. The presence of smaller fragments with glucan-binding function and intact tertiary structure indicates that the full domain must be built on glucan-binding subdomains. Among the range of glucan-binding fragments, only the 42-kDa segment could be satisfactorily purified. It was subjected to N-terminal sequence analysis and, because of some ambiguity, was also subjected to chymotrypsin digestion and sequence of several chymotryptic peptides. The sequence data established that the 42-kDa domain fragment is initiated approximately two-thirds into the 170-kDa enzyme in a region previously identified as a segment of the gene that includes the glucan-binding domain (J. J. Ferretti, M. L. Gilpin, and R. R. B. Russell, J. Bacteriol. 169:4271-4278, 1987). The site is approximately 60 kDa from the C terminus and covers a region characterized by extensive amino acid sequence repeats. The data are discussed in the context of the size range of the glucan-binding fragments and subdomain architecture of the full glucan-binding domain.

Amino Acid Sequence↗
PubMed1990

Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression.

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.

Amino Acid Sequence↗
PubMed1990

The 25-kilodalton insulin-like growth factor (IGF)-binding protein inhibits both basal and IGF-I-mediated growth of chick embryo pelvic cartilage in vitro.

Insulin-like growth factor (IGF)-I stimulates the growth of many tissues, including growth plate cartilage. However, the role of IGF-binding proteins in the growth process is controversial. We purified a 25-kDa IGF-binding protein (BP-25) from amniotic fluid. We tested the effect of this BP-25 preparation on both basal and IGF-I-stimulated growth of chick embryo pelvic cartilages maintained in serum-free organ culture. Cartilage wet weight was 4.1 +/- 0.3 mg/cartilage initially; after 3 days, BP-25 inhibited both basal and IGF-I-stimulated growth. Control cartilages weighed 7.4 +/- 0.7 mg/cartilage, while those incubated with 100 nM BP-25 weighed 5.8 +/- 0.5 mg/cartilage (P less than 0.001 vs. control); BP-25 concentrations as low as 0.2 nM significantly inhibited basal cartilage growth. Cartilages incubated with 1.25 nM IGF-I weighed 10.4 +/- 0.8 mg/cartilage (P less than 0.001 vs. control), while those incubated with both 100 nM BP-25 and 1.25 nM IGF-I weighed 8.1 +/- 0.5 mg/cartilage (P less than 0.001 vs. cartilage incubated with IGF-I alone); BP-25 concentrations as low as 0.4 nM significantly inhibited IGF-I-stimulated cartilage growth. BP-25 also inhibited basal and IGF-I-stimulated increases in cartilage dry weight, [3H]thymidine incorporation into DNA, and 35SO4 incorporation into proteoglycan. A second BP-25 preparation, which in the presence of 1% platelet-poor plasma acts synergistically with IGF-I to stimulate DNA synthesis and cell replication of fibroblasts and smooth muscle cells in tissue culture, inhibited IGF-I-stimulated cartilage growth to the same degree as did our BP-25 preparation. In separate experiments, proteins present in serum-free medium conditioned for 3 days by chick cartilages were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and incubated with [125I]IGF-I. This medium was found to contain two IGF-binding proteins; one appeared to be the chick equivalent of BP-25, while the other had a molecular mass similar to that of a poorly characterized human 34-kDa IGF-binding protein. We conclude that purified BP-25 inhibits the growth of chick embryo pelvic cartilage in our serum-free organ culture system. Since conditioned medium from these cartilages contains both IGF-I-like peptides and IGF-binding proteins such as BP-25, we suggest that the IGF-binding proteins present may act to down-regulate the growth-promoting effects of the local IGF peptides.

Animals↗