Search PubMed⌕ Search

Biomedical subjects

J E Shively

Publications and source records attributed to J E Shively.

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

Solid-phase synthesis and characterization of carcinoembryonic antigen (CEA) domains.

Carcinoembryonic antigen (CEA), a 180,000 dalton cell surface glycoprotein expressed on tumors of the colon, breast, ovary, and lung, has seven predicted immunoglobulin-like domains (N-A1-B1-A2-B2-A3-B3), most of which are recognized by distinct monoclonal antibodies. To study the individual domains, we have prepared several of the domains (N, A3, B3, and A3-B3) by solid-phase peptide synthesis. The syntheses were performed by the Fmoc method using single couplings, elevated temperatures for both the coupling and deblocking reactions, and a flexible solvent system for the coupling reactions. The syntheses were accomplished on an in-house built synthesizer which allowed for temperature control and flexible solvent control during the course of the coupling reactions. Due to the large size of the peptides (84-184 residues), it was anticipated that the overall purity of the final product would not exceed 60% even for an average coupling yield of 99.5%. Therefore, several of the peptides were synthesized with a His6 "tail" at the amino terminus, allowing for purification on a Ni-NTA chelate column. For the most part, the purified peptides exhibited single sharp peaks by RP-HPLC, migrated at their expected molecular weights by gel permeation chromatography, gave correct masses by electrospray ionization or matrix-assisted laser desorption ionization time of flight mass spectrometry, gave the expected amino acid analyses, N-terminal sequences, and tryptic maps, and bound their appropriate monoclonal antibodies. The N-domain was extremely hydrophobic, requiring 6M guanidinium hydrochloride for solubilization, the A3 domain was soluble in dilute acid, and the B3 domain had an intermediate solubility. The affinity constants of the A3 domain and several mutants (also made by peptide synthesis) are reported, along with characterization of the 178 amino acid two-domain peptide, A3-B3. Although there is no evidence for proper folding of these domains by NMR, their ability to bind monoclonal antibodies with high affinity suggests that this is a plausible approach for producing individual domains of CEA.

Amino Acid Sequence↗
PubMed1998

Dose escalation trial of indium-111-labeled anti-carcinoembryonic antigen chimeric monoclonal antibody (chimeric T84.66) in presurgical colorectal cancer patients.

UNLABELLED: Chimeric T84.66 (cT84.66) is a high-affinity (1.16x10(11) M(-1)) IgG1 monoclonal antibody against carcinoembryonic antigen (CEA). The purpose of this pilot trial was to evaluate the tumor-targeting properties, biodistribution, pharmacokinetics and immunogenicity of 111In-labeled cT84.66 as a function of administered antibody protein dose. METHODS: Patients with CEA-producing colorectal cancers with localized disease or limited metastatic disease who were scheduled to undergo definitive surgical resection were each administered a single intravenous dose of 5 mg of isothiocyanatobenzyl diethylenetriaminepentaacetic acid-cT84.66, labeled with 5 mCi of 111In. Before receiving the radiolabeled antibody, patients received unlabeled diethylenetriaminepentaacetic acid-cT84.66. The amount of unlabeled antibody was 0, 20 or 100 mg, with five patients at each level. Serial blood samples, 24-hr urine collections and nuclear images were collected until 7 days postinfusion. Human antichimeric antibody response was assessed up to 6 mo postinfusion. RESULTS: Imaging of at least one known tumor site was performed in all 15 patients. Fifty-two lesions were analyzed, with an imaging sensitivity rate of 50.0% and a positive predictive value of 76.9%. The antibody detected tumors that were not detected by conventional means in three patients, resulting in a modification of surgical management. Interpatient variations in serum clearance rates were observed and were secondary to differences in clearance and metabolic rates of antibody and antibody:antigen complexes by the liver. Antibody uptake in primary tumors, metastatic sites and regional metastatic lymph nodes ranged from 0.4% to 134% injected dose/kg, resulting in estimated 90Y-cT84.66 radiation doses ranging from 0.3 to 193 cGy/mCi. Thirteen patients were evaluated 1-6 mo after infusion for human antichimeric antibody, and none developed a response. No major differences in tumor imaging, tumor uptake, pharmacokinetics or organ biodistribution were observed with increasing protein doses, although a trend toward increasing blood uptake and decreasing liver uptake was observed with increasing protein dose. CONCLUSION: Chimeric T84.66 demonstrated tumor targeting comparable to other radiolabeled intact anti-CEA monoclonal antibodies. Its immunogenicity after single administration was lower than murine monoclonal antibodies. These properties make 111In-cT84.66, or a lower molecular weight derivative, attractive for further evaluation as an imaging agent. Yttrium-90 dosimetry estimates predict potentially cytotoxic radiation doses to select tumor sites, which makes 90Y-cT84.66 also appropriate for further evaluation in Phase I radioimmunotherapy trials. Although clinically important changes in biodistribution, pharmacokinetics and tumor targeting with increasing protein doses of 111In-cT84.66 were not demonstrated, the results do suggest that antibody clearance from the blood is driven by hepatic uptake and metabolism, with more rapid blood clearance seen in patients with liver metastases. These patients with rapid clearance and potentially unfavorable biodistribution for imaging and therapy may, therefore, be a more appropriate subset in which to evaluate the role of administering higher protein doses. This underscores the need to further identify, characterize and understand those factors that influence the biodistribution and clearance of radiolabeled anti-CEA antibodies, to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Adult↗
PubMed1997

Persistent efficacy of doramectin against experimental challenge with Ostertagia ostertagi in cattle.

Two studies were conducted in North America to evaluate the persistent efficacy of doramectin injectable solution against experimental challenge with infective larvae of Ostertagia ostertagi. In both studies, four groups of 10 randomly-assigned calves, negative for trichostrongyle-type eggs on fecal examination, were treated subcutaneously in the midline of the neck with saline (1 ml 50 kg-1) on Day 0 or doramectin (200 micrograms kg-1 = 1 ml 50 kg-1) on Day 0, 7, or 14. Two additional calves from the same pool of animals were randomly assigned as larval-viability monitors and received no treatment. Beginning on Day 14 and continuing through Day 28, the 40 treated calves each were given approximately 1000 infective larvae of O. ostertagi by gavage daily; the two larval-viability monitors were inoculated in a similar manner with approximately 30,000 larvae as a single dose on Day 28. Animals were slaughtered on Day 42 in one study and on Days 42, 43, or 46 in the second. The abomasum from each calf was harvested and processed for worm recovery. A 2% aliquot of abomasal contents plus wash was examined for worm quantification and identification. Geometric mean O. ostertagi burdens were calculated from the log (O. ostertagi count + 1) and were used to estimate percentage reduction. In both studies, doramectin injectable solution was > or = 99.6% efficacious in reducing infection resulting from challenge with infective larvae of O. ostertagi for at least 21 days posttreatment; by 28 days posttreatment, efficacy was 87.3% in one study and 99.7% in the other.

Animals↗
PubMed1997

Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors.

Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.

Amino Acid Sequence↗
PubMed1997

Clinical evaluation of indium-111-labeled chimeric anti-CEA monoclonal antibody.

UNLABELLED: Chimeric T84.66 (cT84.66) is a high-affinity (1.16 x 10[11] M[-1]) IgG1 monoclonal antibody (MAb) against carcinoembryonic antigen (CEA). This pilot trial evaluated the tumor-targeting properties, biodistribution, pharmacokinetics and immunogenicity of 111In-labeled cT84.66. METHODS: Patients with CEA-producing metastatic malignancies were administered a single intravenous dose of 5 mCi 111In-diethylenetriaminepentaacetic acid-cT84.66. Serial blood samples, 24-hr urine collections and nuclear images were collected up to 7 days postinfusion. Human antichimeric antibody response was assessed up to 6 mo postinfusion. RESULTS: Imaging of at least one known tumor site was observed in 14 of 15 (93%) patients. Seventy-four lesions were analyzed with an imaging sensitivity rate of 45.1% and a positive predictive value of 94.1%. In one patient, two additional bone metastases developed within 6 mo of antibody administration at sites initially felt to be falsely positive on scan. One patient developed a human antichimeric antibody response predominantly to the murine portion of the antibody. The antibody cleared serum with a median T(1/2alpha) of 6.53 hr and a T(1/2beta) of 90.87 hr. Interpatient variations in serum clearance rates were observed and were secondary to differences in clearance and metabolic rates of antibody-antigen complexes by the liver. One patient demonstrated very rapid clearance of antibody by the liver, which compromised antibody localization to the primary tumor. Antibody uptake in primary and metastatic tumors ranged from 0.5% to 10.5% injected dose/kg, resulting in estimated radiation doses ranging from 0.97 to 21.3 cGy/mCi 90Y. Antibody uptake in regional lymph nodes ranged from 1.3% to 377% injected dose/kg, resulting in estimated radiation doses ranging from 2.0 to 617 cGy/mCi 90Y. CONCLUSION: Chimeric T84.66 demonstrated tumor targeting that was comparable to that of other radiolabeled intact anti-CEA Mabs. Its immunogenicity after single administration was lower than murine Mabs. These properties make cT84.66 or a lower molecular weight derivative attractive for further evaluation as an imaging agent. These same properties also make it appropriate for future evaluation in Phase I therapy trials. Finally, a wide variation in the rate of antibody clearance was observed, with one patient demonstrating very slow clearance, resulting in the highest estimated marrow dose of the group, and one patient demonstrating unusually rapid clearance, resulting in poor antibody localization to tumor. Data from this study suggest that serum CEA levels, antibody-antigen complex clearance and, therefore, antibody clearance are influenced by both the production and clearance rates of CEA. This underscores the need to further identify, characterize and understand those factors that influence the biodistribution and clearance of radiolabeled anti-CEA antibodies to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Adult↗
PubMed1996

Role of interferon regulatory factor-1 in the induction of biliary glycoprotein (cell CAM-1) by interferon-gamma.

Biliary glycoprotein (BGP), also known as C-CAM-1, has been shown to be down-regulated in colon and prostate tumors. Previously, we demonstrated that BGP mRNA is up-regulated by interferon-gamma (IFN-gamma) in colon cancer cell lines (Takahashi, H., Okai, Y., Paxton, R. J., Hefta, L. J. F., and Shively, J. E. (1993) Cancer Res. 53, 1612-1619). We now show that the BGP promoter contains an interferon-sensitive response element (ISRE) that is specifically protected in in vivo footprints. Interferon regulatory factor-1 (IRF-1) was identified as the ISRE-binding factor by electrophoretic mobility shift assays. The induction of IRF-1 mRNA by IFN-gamma in HT-29 cells reaches a maximum at 6 h and is superinduced by cycloheximide. Four mRNA species for BGP are induced by IFN-gamma, the major band of which is inhibited by cycloheximide. Transfection of HT-29 cells with an IRF-1 expression plasmid (pAct-1) transactivates a BGP promoter reporter gene containing wild-type (but not mutant) ISRE. Electrophoretic mobility shift assay analysis of a second footprint reveals the binding of Sp1, an Sp1-like protein, and upstream stimulatory factor. The Sp1-like complex was also induced by IFN-gamma treatment of HT-29 cells and may be a second point of transcriptional control for the BGP gene.

Adenosine Triphosphatases↗
PubMed1996

Minibody: A novel engineered anti-carcinoembryonic antigen antibody fragment (single-chain Fv-CH3) which exhibits rapid, high-level targeting of xenografts.

A novel engineered antibody fragment (VL-VH-CH3, or "minibody") with bivalent binding to carcinoembryonic antigen (CEA) was produced by genetic fusion of a T84.66 (anti-CEA) single-chain antibody (scFv) to the human IgG1 CH3 domain. Two designs for the connecting peptide were evaluated. In the T84.66/212 LD minibody, a two-amino acid linker (generated by fusion of restriction sites) was used to join VH and CH3. In the T84.66/212 Flex minibody, the human IgG1 hinge plus an additional 10 residues were used as the connecting peptide. Size exclusion chromatography of purified minibodies demonstrated that both proteins had assembled into Mr80,000 dimers as expected. Furthermore, analysis by SDS-PAGE under nonreducing conditions was consistent with disulfide bond formation in the hinge of the T84.66 Flex minibody. Purified minibodies retained high affinity for CEA (KA, 2 x 10(9) M(-1)) and demonstrated bivalent binding to antigen. Tumor targeting properties were evaluated in vivo using athymic mice bearing LS174T human colon carcinoma xenografts. 123I-labeled T84.66 minibodies demonstrated rapid, high tumor uptake, reaching 17% injected dose/gram (%ID/g) for the LD minibody and 33%ID/g for the Flex minibody at 6 h following injection. Radioiodinated minibody also cleared rapidly from the circulation, yielding high tumor:blood uptake ratios: 44.5 at 24 h for the LD minibody and 64.9 at 48 h for the Flex minibody. Rapid localization by the T84.66/212 Flex minibody allowed imaging of xenografts at 4 and 19 h after administration.

Animals↗
PubMed1996

Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers.

BACKGROUND: Genetic engineering can produce novel antibody fragments with improved properties for applications such as tumor targeting in vivo. OBJECTIVES: To produce stable monomeric (27 kDa) and dimeric (55 kDa) forms of a single-chain Fv (scFv) from the anti-carcinoembryonic antigen (anti-CEA) antibody T84.66, and assess the targeting and biodistribution properties in an animal model. STUDY DESIGN: ScFv were constructed with either a 28 or 14 amino acid connecting peptide and expressed by secretion from E. coli. Following affinity purification, proteins were characterized by gel electrophoresis and mass spectrometry. Binding properties were assessed by size exclusion HPLC after incubation with antigen, and affinities determined by surface plasmon resonance. The shorter linker favored formation of dimers (and higher multimers) which showed unusual stability. ScFv were radiolabeled with 125I for tumor targeting and biodistribution studies of monomeric or dimeric forms were conducted in athymic mice bearing LS174T human colorectal carcinoma xenografts. RESULTS: 125I-scFv monomers and dimers targeted exhibited rapid clearance kinetics in tumor-bearing mice. Nevertheless, the anti-CEA scFvs targeted very well to xenografts, leading to high tumor: normal organ ratios (greater than 20:1 at 24 h) for both forms. Tumor localization of the non-covalent dimers was much higher than monomers, reaching 10-15% injected dose per gram at 1 h. CONCLUSION: Non-covalent dimers of scFv (also known as diabodies) are stable, easy to produce and show excellent targeting as compared to monomeric scFv, probably due to increased mass and valency.

Animals↗
PubMed1996

Evidence that CCK-58 has structure that influences its biological activity.

Many biologically active peptides exist in multiple molecular forms, but the functional significance of regions outside the region of bioactivity is unknown. The biological and immunological data presented in this study indicate that cholecystokinin-58 (CCK-58), unlike other forms of cholecystokinin, has structure that influences its bioactivity. CCK-58 was purified from acid extracts of canine intestinal mucosa until a single absorbance peak was obtained during reverse-phase chromatography. Amino acid analysis precisely determined the peptide concentrations of purified CCK-58 and synthetic CCK-8. Our hypothesis was that if the amino terminus of CCK-58 influences its bioactivity then its activity would be modified when this region was removed from the peptide. To evaluate the importance of the amino terminus of CCK-58 to influence its biological activity, the abilities of CCK-58 and CCK-8 to release amylase from pancreatic acini were compared before and after tryptic digestion. Tryptic digestion of CCK-58 decreased the half-maximal stimulation (EC50) for amylase release from 96 to 28 pM. The EC50 for digested CCK-58 was similar to that for CCK-8 (17 pM). These results suggest that CCK-58 has a structure that shields its bioactive carboxyl terminus. This is further supported by the finding that carboxyl fragments generated from CCK-58 by trypsin or by partial acid hydrolysis were greater than twofold more immunoreactive than the intact CCK-58. The diminished activity of CCK-58 SK shields the carboxyl terminus, which is important to its biological and immunological activities.

Acids↗
PubMed1995

Initial experience evaluating 90yttrium-radiolabeled anti-carcinoembryonic antigen chimeric T84.66 in a phase I radioimmunotherapy trial.

Chimeric T84.66 (cT84.66) is a high-affinity (5 x 10(10) M-1) anti-carcino-embryonic antigen (CEA) IgG1. In a recently completed pretherapy imaging trial, 111In-labeled cT84.66 demonstrated targeting of CEA-producing metastatic sites and low immunogenicity, with human antichimeric antibody (HACA) response in only 1 of 15 patients after a single administration. The purpose of the present study was to evaluate cT84.66-diethylenetriaminepentaacetic acid labeled with 90Y in a dose-escalation Phase I trial. Patients with metastatic CEA-producing malignancies received imaging doses of 5 mCi 111In-labeled cT84.66 first, followed 1-2 weeks later by 5 mg cT84.66 labeled with the therapeutic dose of 90Y. Immediately following the therapeutic infusion, diethylenetriaminepentaacetic acid was administered by continuous i.v. infusion over 3 days at 250 mg/m2 body surface area/24 h. Biodistribution, tumor targeting, absorbed radiation dose estimates, antibody clearance, and HACA response were evaluated through blood samples, 24-h urine collections, and nuclear images performed at serial time points after infusion. To date, three patients with metastatic colorectal cancer have been evaluated at the first dose level of 5 mCi/m2. No side effects were associated with antibody administration. Localization of the antibody to nonhepatic metastatic sites was observed. Size-exclusion high-performance liquid chromatography demonstrated the formation of CEA:antibody complexes in serum in all three patients. A significant variation among patients in the clearance rate of the antibody and complexes from blood to liver was seen, which resulted in a reciprocal relationship between estimated liver dose and red marrow dose. Patients who demonstrated faster clearance to liver demonstrated greater excretion of a low-molecular-weight metabolite through the urine. Two patients developed HACA response, which persisted at 4 months after therapy. At this first dose level, no tumor responses were seen and reversible grade 1 thrombocytopenia was observed in 2 patients. cT84.66 demonstrated effective localization in CEA-producing tumors. Its low immunogenicity after a single administration makes it attractive for further evaluation as a radioimmunotherapeutic agent. However, further evaluation is needed to determine whether its immunogenicity will remain low after multiple administrations. Additionally, in two of the three patients, we identified rapid clearance of the antibody to the liver. This underscores the need to identify, characterize, and understand further those factors that influence the biodistribution and clearance of anti-CEA antibodies to allow for better selection of patients for therapy and rational planning of radioimmunotherapy.

Carcinoembryonic Antigen↗
PubMed1995

In vitro and in vivo footprint analysis of the promoter of carcinoembryonic antigen in colon carcinoma cells: effects of interferon gamma treatment.

The analysis of the carcinoembryonic antigen (CEA) promoter in the colon carcinoma lines HT-29 and SW403, using HeLa as a control, was performed using gel mobility shift assays and in vitro and in vivo footprinting before and after IFN-gamma treatment. Using a 332-bp probe extending from the start of translation (+1 to -331), we detected 4-5 specific complexes that increased with intensity with IFN-gamma treatment as measured by a gel mobility shift assay. In contrast, no complexes were observed for probes covering the regions -500 to -1000 and -1000 to -1500. DNase I in vitro footprinting with the 332-bp probe revealed three footprints, FP-I to FP-III, none of which changed during the course of IFN-gamma treatment. Using probes corresponding to each footprint, 6-7 specific complexes were observed by gel mobility shift assays. Although minor changes were observed on IFN-gamma treatment, no consistent pattern was observed for all cell lines tested. Several of the proteins involved in the promoter complexes were identified by antibody super shifts, UV cross-linking, and Southwestern blotting. FP-I bound an Sp1-like protein, binding to a GT box sequence, and USF (upstream regulatory factor). FP-II and FP-III bound Sp1, binding through the consensus sequence for a GC box. Lower molecular weight complexes of an unknown nature were observed with sequence specificity for both single- and double-stranded DNA. DNase I in vivo footprints confirmed the boundaries of FP-I to FP-III and revealed a fourth but weaker footprint, FP-IV. The strongest in vivo footprints were observed for SW403 cells, with weaker and no footprints observed for HeLa cells, thus correlating with the degree of CEA transcriptional activity (HeLa cells make no CEA mRNA). DNase I hypersensitive sites correlated well with the boundaries of the footprints and also revealed activity around the start of transcription (-110). The specific pattern for DNase I hypersensitivity for Sp1 in the CEA promoter was the same as observed for the SV40 early promoter. In vivo footprinting with dimethyl sulfate revealed protein binding at the Sp1 consensus sequences in FP-II and FP-III and at the USF consensus sequence in FP-I. We conclude that in vivo footprinting is the most accurate predictor of the state of transcriptional activity of the CEA gene. It is also likely that Sp1 and USF play a major role in CEA transcriptional activation and that the majority of IFN-gamma effects are at the posttranscriptional level.

Base Sequence↗
PubMed1995

Benzodiazepines and peptides stimulate pregnenolone synthesis in brain mitochondria.

Mitochondria isolated from rat brain were found to cleave cholesterol to produce pregnenolone, the precursor for hormonal steroids, at a mean rate of 21.0 pmol pregnenolone.mg protein-1.min-1. This rate-limiting step in steroidogenesis was significantly stimulated by PK 11195 (1-(2-chlorophenyl)-N-methyl-(1-methylpropyl)-3-isoquinoline carboxamide) and Ro5 4864 (4'-chlorodiazepam), ligands which bind to peripheral benzodiazepine receptors with high affinity. Low-affinity ligands for the peripheral benzodiazepine receptor such as Ro15 1788 (ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5 alpha][1,4] benzo-3-carboxylate) and clonazepam had no significant effect on the rate of pregnenolone synthesis. Furthermore, the rank order of potency of these compounds as inhibitors of [3H]Ro5 4864 binding was identical to the rank order for steroid production. Since the 86-amino acid peptide diazepam binding inhibitor is also thought to bind to the peripheral benzodiazepine receptor, four fragments of this peptide, a random sequence and steroidogenesis activator peptide were also evaluated for their ability to interact with peripheral benzodiazepine receptors and to stimulate steroidogenesis in rat brain mitochondria. Steroidogenesis activator peptide and two fragments of diazepam binding inhibitor significantly stimulated pregnenolone biosynthesis. In contrast to the peripheral benzodiazepine receptor ligands, no correlation between peptide potency in displacing [3H]Ro5 4864 binding and steroidogenesis was observed.

Amino Acid Sequence↗
PubMed1995

Automated carboxy-terminal sequence analysis of polypeptides containing C-terminal proline.

Proteins and peptides can be sequenced from the carboxy-terminus with isothiocyanate reagents to produce amino acid thiohydantoin derivatives. Previous studies in our laboratory have focused on automation of the thiocyanate chemistry using diphenyl phosphoroisothiocyanatidate (DPP-ITC) and pyridine to derivatize the C-terminal amino acid to a thiohydantoin and sodium trimethylsilanolate for specific hydrolysis of the derivatized C-terminal amino acid (Bailey, J. M., Nikfarjam, F., Shenoy, N. S., and Shively, J. E. (1992) Protein Sci. 1, 1622-1633). A major limitation of this approach was the inability to derivatize C-terminal proline. We now describe chemistry based on the DPP-ITC/pyridine reaction which is capable of derivatizing C-terminal proline to a thiohydantoin. The reaction of DPP-ITC/pyridine with C-terminal proline rapidly forms an acyl isothiocyanate which is capable of forming a quaternary amine containing thiohydantoin. Unlike formation of peptidylthiohydantoins with the other 19 commonly occurring amino acids in which cyclization to a thiohydantoin is concomitant with loss of a proton from the amide nitrogen, proline has no amide proton and as a result the newly formed proline thiohydantoin contains an unprotonated ring nitrogen. This cyclic structure if left unprotonated will regenerate C-terminal proline during the cleavage reaction. However, if protonated by the addition of acid, the proline thiohydantoin ring is stabilized and can be readily hydrolyzed to proline thiohydantoin and a shortened peptide by the addition of water vapor or alternatively by sodium or potassium trimethylsilanolate, the reagent normally used for the cleavage reaction. By introducing vaporphase trifluoroacetic acid (TFA) for the protonation reaction and water vapor for the hydrolysis reaction we have been able to automate the chemistry required for derivatization of C-terminal proline.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetic Anhydrides↗
PubMed1994

Purification and sequence of rat oxyntomodulin.

Structural information about rat enteroglucagon, intestinal peptides containing the pancreatic glucagon sequence, has been based previously on cDNA, immunologic, and chromatographic data. Our interests in testing the physiological actions of synthetic enteroglucagon peptides in rats required that we identify precisely the forms present in vivo. From knowledge of the proglucagon gene sequence, we synthesized an enteroglucagon C-terminal octapeptide common to both proposed enteroglucagon forms, glicentin and oxyntomodulin, but sharing no sequence overlap with glucagon. We then developed a radioimmunoassay using antibodies raised against the octapeptide that was specific for enteroglucagon peptides without cross-reacting with glucagon. Rat intestine was extracted, and one presumptive enteroglucagon form was purified by following the enteroglucagon C-terminal octapeptide-like immunoreactivity through several HPLC purification steps. Structural characterization of the material by amino acid composition, microsequence, and mass spectral analyses identified the peptide as rat oxyntomodulin. The 37-residue peptide consists of pancreatic glucagon plus the C-terminal extension, Lys-Arg-Asn-Arg-Asn-Asn-Ile-Ala. This now permits synthesis of an unambiguous duplicate of endogenous rat oxyntomodulin for physiological studies.

Amino Acid Sequence↗
PubMed1994

Prognostic relevance of carcinoembryonic antigen and estrogen receptor status in breast cancer patients.

BACKGROUND: Expression of carcinoembryonic antigen (CEA) has been reported in 10-95% of breast cancer. Its value as a predictor of disease progression is controversial. METHODS: The expression of CEA in 202 Stages I and II breast carcinomas was assessed by immunohistochemistry, and the results were correlated with various histologic and clinical parameters to establish CEA's biologic relevance. The mean follow-up of the patients was 6.5 years. The monoclonal antibody used does not cross-react with other molecules in the CEA gene family. RESULTS: One hundred, thirteen (56%) tumors expressed CEA in more than 15% of the cells. Expression of CEA was associated with positive estrogen receptor (ER) status (P = 0.003). Univariate Cox regression analysis showed that, whereas disease free survival (DFS) and overall survival (OS) were not associated significantly with CEA expression, tumor size, nuclear grade, ER status, lymph node metastases, and stage were. When ER status was stratified to CEA expression, patients who were ER negative and had CEA-negative tumors had a 3.9 times higher risk (P = 0.032) of death than did the patients with CEA-positive tumors. Cox regression analysis revealed that ER was the only parameter with significant interacting effect with CEA. Multivariate, stepwise Cox regression analysis showed that CEA expression, tumor size, and nuclear grade were the only significant independent predictors of DFS, and nuclear grade and lymph node metastasis the only significant predictors of OS in the ER-positive group. The only significant independent predictor of DFS and OS in the ER-negative group was CEA. When CEA expression was stratified to ER status, patients whose tumors lacked CEA and ER had threefold higher risk of disease relapse (P = 0.002) and a 5.3-fold higher risk of death (P = 0.0001) than those with ER-positive and CEA-negative tumors. Multivariate analysis showed that the association between CEA and ER was enhanced further after compensating for other parameters with independent predictive value. CONCLUSIONS: The association between CEA and ER was the most important independent predictor of a subgroup of patients (CEA-negative, ER-positive) with the most favorable prognosis. The results imply that the association of several tumor markers may provide tumor profiles with superior predictive value than a single parameter.

Antibodies, Monoclonal↗
PubMed1993

Adhesion to carcinoembryonic antigen by human colorectal carcinoma cells involves at least two epitopes.

Carcinoembryonic antigen (CEA) may be involved in both cell-cell and cell-substrate adhesion. Our purpose was to determine whether epitopes involved in the homophilic binding of human colorectal carcinoma cells to CEA participated in adhesion to basement membrane proteins. Three human colorectal adenocarcinoma cell lines and one CHO cell line transfected with CEA cDNA were tested in a solid-phase adhesion assay. The 2 CEA-expressing carcinoma cell lines (KM-12c and CCL 188) and the transfectant, but not the parental CHO line, bound to CEA. The CEA-non-producing carcinoma line (Clone A) did not bind to CEA. All colorectal carcinoma cell lines, the transfectant and the parental CHO line bound to laminin, while the colorectal carcinoma lines bound to type-IV collagen. MAbs to epitopes on CEA that cross-react with non-specific cross-reacting antigen (NCA) inhibited adhesion of CEA-expressing cells to CEA. MAbs to non-cross-reactive epitopes of CEA did not block adhesion to CEA. When the inhibitory anti-CEA antibodies were compared in a competitive radioimmunoassay, 2 distinct epitopes were identified. Epitope I is in the N-terminal domain and defined by MAbs MN3, T84.1 and C110, whereas epitope II is located in the repeating loop domains and is recognized by antibodies MN15, PR3B10 and NP1. None of the antibodies to epitope I or II blocked adhesion by KM-12c or CCL 188 cells to laminin or type-IV collagen. Thus, at least 2 different regions on CEA participate in adhesion to CEA but not to collagen or laminin by CEA-expressing human colorectal carcinoma cells.

Adenocarcinoma↗
PubMed1993

Compact protein sequencer for the C-terminal sequence analysis of peptides and proteins.

We describe the construction of a compact protein sequencer designed specifically for the C-terminal sequence analysis of peptides and proteins. This sequencer has a vertical flow path and is equipped with a continuous flow reactor (CFR). The flow paths for the various reagents and solvents have been minimized. A unique feature of this instrument is the design of a quadrate valve (quad valve) which permits the delivery of four solvents or reagents to the conversion flask (CF). Combination of two of these quad valves in series permits the delivery of eight solvents and reagents to the CFR. The CF contains three inputs from the top, one for transfer of the contents of the CFR, one which is used as a vent, and one for input of solvents or reagents from the CF quad valve. The CF drains from the bottom, connecting to a switching valve which allows delivery either to a waste bottle or to an on-line HPLC. Another unique feature of this instrument is the design of an optical flow detector which permits injection of approximately 90% of the contents of the CF for HPLC analysis. The overall size of the instrument (11 w x 16.5 h x 23.5 d in.) is smaller than commercially available instruments for protein sequencing and represents the first time an instrument has been constructed specifically for C-terminal sequence analysis. The utility of this instrument is demonstrated with the C-terminal sequence analysis of protein samples noncovalently applied to Zitex strips and with a peptide covalently attached to carboxylic acid-modified polyethylene film.

Amino Acids↗