Biomedical subjects
Publications and source records attributed to J E Shively.
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The complete amino acid sequence of human liver NADPH-cytochrome P-450 reductase has been determined by microsequence analysis and mass spectrometry. The total sequence consists of 676 amino acids initiated by an amino-terminal acetyl group. There is no evidence for posttranslational modifications, including Asn-linked glycosylation. The human enzyme exhibits sequence homology in the range of 92-95% with other mammalian enzymes. Sequence differences were mainly confined to several hydrophilic regions in the NH2-terminal and COOH-terminal domains. Since the human enzyme is immunochemically distinct from the rabbit enzyme despite similar enzymatic properties, it is likely that these variable hydrophilic regions are potential antigenic determinants. The NADPH-depleted enzyme is inactivated by either fluorescein isothiocyanate, a lysine-specific reagent, or 5-(iodoacetamido)fluorescein, a cysteine-specific reagent. In both cases, protection by NADP(H) prevents enzyme inactivation by the reagents. Isolation of fluorescent peptide from 5-(iodoacetamido)fluorescein-inactivated enzyme identified Cys 565 as the specifically NADPH-protected residue.
The epitope reactivities of 52 well-characterized monoclonal antibodies (Mabs) against carcinoembryonic antigen from 11 different research groups were studied using competitive solid-phase immunoassays. About 60% of all possible combinations of Mabs as inhibitors and as the primary binding antibody were investigated. The inhibition data were analyzed by a specially developed computer program "EPITOPES" which measures concordance and discordance in inhibition patterns between Mabs. The analysis showed that 43 of the 52 Mabs (83%) could be classified into one of five essentially noninteracting epitope groups (GOLD 1-5) containing between four and 15 Mabs each. The epitopes recognized by the Mabs belonging to groups 1 to 5 were peptide in nature. With one or two possible exceptions non-classifiable Mabs were either directed against carbohydrate epitopes (4 Mabs) or were inactive in the tests used. Within epitope groups GOLD 1, 4, and 5 two partially overlapping subgroups were distinguished. Mabs with a high degree of carcinoembryonic antigen specificity generally belonged to epitope groups GOLD 1 and 3.
Sodium butyrate induces morphological and biochemical changes consistent with a more differentiated phenotype in some colon cancer cell lines. These changes include increased expression of carcinoembryonic antigen (CEA) and other oncodevelopmental markers. We utilized domain-specific probes and polyclonal antibodies against CEA-related antigens to study sodium butyrate-induced expression of the CEA gene family in a villous adenoma-derived cell line, which is nontumorigenic in nude mice (VACO 235), and two colonic carcinoma cell lines known to respond to sodium butyrate exposure by phenotypic differentiation (HT-29 and LS 174T). The induction begins as quickly as 24 h after exposure and occurs primarily at a transcriptional level, although some translational control is also evident. No evidence was found for gene amplification, rearrangement, or methylation to account for the mechanism of this transcriptional control. [35S]Cysteine pulse-labeled cell lysate immunoblots and polyadenylated RNA blot hybridization suggest that increases in mRNA transcript and CEA-related glycoprotein levels are primarily due to increased synthesis rather than decreased degradation. A considerable amount of heterogeneity is seen in the biosynthesis of the CEA-related glycoproteins, with each cell line showing a distinct pattern of CEA-related antigen expression from a limited number of mRNA transcripts.
A peptide identical in structure to the carboxyl-terminal flanking nonapeptide of rat progastrin, predicted by cDNA sequence, was synthesized. The synthetic peptide was used for production of a rabbit antiserum. This antiserum was used to develop a radioimmunoassay specific for rat carboxyl terminal flanking peptide. This assay was used to monitor the purification of immunoreactivity from rat antral extracts. Gel permeation, anion exchange and reverse phase chromatography steps resulted in a single absorbance peak associated with the carboxyl terminal flanking peptide immunoreactivity. The purified peptide eluted in the same position as the synthetic peptide during all three types of chromatography. This material was shown to be identical in mass to Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn, the predicted sequence of the carboxyl terminal nonapeptide of rat progastrin.
Recombinant human interferons have recently been shown to enhance tumor antigen expression, including carcinoembryonic antigen (CEA), on the surface of human carcinoma cells, which results in an increase in the targeting of antitumor monoclonal antibodies (MAb) in vivo. We report here the effect of recombinant human gamma-interferon (HuIFN-gamma) on the expression of human CEA and its related transcripts in several human colon carcinoma and normal human fibroblast cell lines. The colon tumor cell lines HT-29, WiDr, and LS-174T were each shown to express different constitutive levels of CEA glycopeptide, as measured by the binding of the CEA-specific MAb COL-4. Treatment with HuIFN-gamma enhanced the level of binding of COL-4 in total cell extracts of HT-29 and WiDr cells 2.5- and 6.5-fold, respectively. Using a CEA complementary DNA probe, this increase in MAb binding was shown to be accompanied by a 6- to 11-fold increase in the steady state levels of three CEA transcripts with sizes of 4.2, 3.5, and 2.8 kilobases. On the other hand, HuIFN-gamma treatment had no effect on the level of COL-4 binding or expression of CEA transcripts in LS-174T colon carcinoma cells, which are high constitutive expressors of CEA glycoprotein. Normal human fibroblast cell lines MRC-5 and WI38 had no detectable cytoplasmic CEA glycopeptide levels nor did they contain detectable levels of CEA mRNA, either before or after treatment with HuIFN-gamma. In contrast, HuIFN-gamma induced the de novo expression of the normal major histocompatibility complex class II antigen, HLA-DR, on HT-29 and WiDr colon cancer cells as well as the two fibroblast cell lines. Treatment of the LS-174T cell line with HuIFN-gamma did not result in the induction of class II HLA-DR antigen. These observations suggest that some common factors may be involved in the regulation of the CEA and class II histocompatibility genes. In addition, the demonstration that HuIFN-gamma enhances CEA expression in some carcinoma cell lines but fails to induce de novo expression of CEA transcripts in fibroblasts supports the potential application of HuIFN-gamma in enhancement of tumor targeting of antitumor MAbs and adds to our understanding of the mechanism of gamma-interferon-mediated up-regulation of some tumor antigens.
Administration of a large dose (0.2 mg) of unlabeled specific anticarcinoembryonic antigen (anti-CEA) monoclonal antibody (MAB) to nude mice bearing LS174T human colon cancer xenografts significantly decreased normal liver uptake of 111In-labeled anti-CEA MAB (Indacea). Mice bearing tumors of approximately 1 g showed liver accumulation of indium-111 at 48 h following injection of 2 micrograms/10 microCi Indacea of 33.8 +/- 1.5% injected dose per gram (%ID/g) (N = 25). Treatment with 0.2 mg unlabeled anti-CEA MAB reduced this to 8.9 +/- 0.5% ID/g (N = 22; P less than 0.001). The dose of pretreatment was found to be critical. Increasing the amount of unlabeled MAB to 2.0 mg did not significantly improve the liver level of indium-111, but did compromise the tumor uptake of Indacea (15.9 +/- 1.3 versus 12.4 +/- 0.4% ID/g; P less than 0.05). Lowering the dose of pretreatment 10-fold resulted in increased (P less than 0.001) liver uptake of the label (26.5 +/- 2.8% ID/g). The unlabeled anti-CEA MAB treatment given as a single dose or fractionated over several days gave the same results. The decrease in liver uptake was the same for i.v. administration of the unlabeled MAB given 1 week prior to Indacea injection or mixed together with Indacea. With i.p. administration, simultaneous injection of the unlabeled MAB with Indacea was not as effective as pretreatment (20 min to 7 days) in decreasing the liver uptake of 111In (P less than 0.05). Epitope specificity and affinity were shown to be important considerations in the choice of MAB combinations used for pretreatment and imaging. Pretreatment with nonspecific MAB was ineffective in decreasing liver uptake of Indacea.
Five members of the human CEA gene family [human pregnancy-specific beta 1-glycoprotein (PS beta G); hsCGM1, 2, 3 and 4] have been isolated and identified through sequencing the exons containing their N-terminal domains. Sequence comparisons with published data for CEA and related molecules reveal the existence of highly-conserved gene subgroups within the CEA family. Together with published data eleven CEA family members have so far been determined. Apart from the highly conserved coding sequences, these genes also show strong sequence conservation in their introns, indicating a duplication of whole gene units during the evolution of the CEA gene family.
Products of the gastrin-releasing peptide gene were isolated from culture medium supernatant of a small cell lung cancer line, NCI-H345, by several (high performance liquid chromatography) HPLC steps. The column eluates were monitored by immunoassay and absorbance profiles. Gastrin-releasing peptide was identified in HPLC eluates by a specific radioimmunoassay. Two carboxyl-terminal gastrin-releasing peptide gene-associated peptides were identified by a radioimmunoassay specific for their predicted carboxyl terminus. The amino termini of these two peptides were determined by microsequence analysis. The shorter peptide was revealed to be a fragment of the larger peptide. Expression of an alternate mRNA was shown by isolation and characterization of a novel tetradecapeptide. Amino acid analysis, microsequence analysis, and mass spectral analysis confirmed that the structure was Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser. This peptide represents the carboxyl terminus of a peptide resulting from alternate processing of gastrin releasing peptide mRNA. This mRNA contains a 19-base deletion, creating a frame shift. A radioiodinated synthetic analog of this peptide (Tyr-Leu-Val-Asp-Ser-Leu-Leu-Gln-Val-Leu-Asn-Val-Lys-Glu-Gly-Thr-Pro-Ser ) bound specifically to a small cell cancer line with high affinity, suggesting possible biological activity of the isolated peptide.
One hundred patients with known or suspected colorectal cancer were studied by radioimmunoconjugate scintigraphy prior to operation. Study subjects received murine monoclonal anticarcinoembryonic antigen labeled with indium 111 (Indacea). Sensitivity of imaging was 76 percent for primary tumors, 44 percent for hepatic metastases, 38 percent for extrahepatic abdominal metastases, and 78 percent for extraabdominal metastases. Seventeen of 46 patients (37 percent) with known or suspected hepatic metastases and no evidence of extrahepatic disease by conventional imaging methods had extrahepatic metastases at exploratory surgery. Nine of the 17 patients had disease accurately predicted by the Indacea scanning. The management of each of these nine patients was, or could have been, modified by the scan findings and unnecessary surgery eliminated. A number of patients without post-operative disease had an unexplained increase in plasma carcinoembryonic antigen level due to production of human antimouse antibody. The addition of excess mouse immunoglobulin to the plasma prior to assay blocked this artifactual increase.
The effects of hyperthermia on carcinoembryonic antigen (CEA) expression were investigated. The human colon adenocarcinoma cell line, LS174T, was heated in vitro for 42 degrees C/1 hr, 43 degrees C/1 hr, or 45 degrees C/30 min. Carcinoembryonic antigen membrane expression was assayed by live cell radioimmunoassay 0-6 days after heating. A heat exposure of 45 degrees C/30 min resulted in an initial decrease in carcinoembryonic antigen membrane expression 1 day post-heating followed by a 2-3 fold increase which peaked 3 days post-heating. Carcinoembryonic antigen expression began returning to normal by the sixth day. Heat exposures of 42 degrees C/1 hr and 43 degrees C/1 hr also resulted in increased carcinoembryonic antigen expression but this increase was of lesser magnitude and of shorter duration. Carcinoembryonic antigen shed into the medium, as measured by enzyme immunoassay, also increased after heating in a temperature-dependent fashion. Flow cytometry analysis demonstrated that cells in all phases of the cell cycle expressed this increase. We conclude that hyperthermia results in significant changes in carcinoembryonic antigen membrane expression and shedding. The implications that these findings have with regards to clinical hyperthermia and radioimmunotherapy are discussed.
The effects of external beam radiation on tumor uptake of radiolabeled monoclonal antibody were investigated. Nude mice bearing carcinoembryonic antigen (CEA)-producing subcutaneous human tumor xenografts (LS174T) were irradiated (60Co) with a single fraction of 0, 2 or 20 Gy, 6 or 11 days after tumor inoculation. An Indium-111 labeled anti-CEA monoclonal antibody (T84.66) was injected 1-2 hrs after irradiation. Biodistribution studies performed at 48 hrs showed a statistically significant (p less than 0.01) inverse correlation between tumor mass and tumor antibody uptake. Tumor age was also a significant factor with 11 day old tumors having significantly less uptake (p less than 0.0001) compared to 6 day old tumors for a given mass. Radiation increased tumor antibody uptake only in those tumors where growth inhibition also occurred. Multiple regression analysis showed that this inverse correlation between tumor mass and antibody uptake was the same for irradiated and nonirradiated tumors. We conclude that, in this model system, radiation does not act independently to enhance tumor antibody deposition. Radiation's primary effect is to reduce tumor mass, with mass reduction then resulting in an increase in antibody uptake. The clinical implications of this study are discussed.
This article describes highlights of the state of the art in protein structural analysis, and comments on the current trends toward increased sensitivity and integrated isolation-structure methodologies.
The most abundant protein of the brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is a protein that migrates at a molecular weight of approximately 46 kDa on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bovine brain capillary 46 kDa protein was purified by SDS-PAGE and Sephadex G-25 gel filtration. The purified protein migrated as a single band of molecular weight of approximately 42,000 Da on subsequent SDS-PAGE followed by silver staining. The protein was digested by trypsin and tryptic peptides were analyzed by reverse phase high-performance liquid chromatography (HPLC). Two of these peptides, 11 and 18 amino acids in length, were sequenced and found to be identical to amino acid sequences corresponding to portions of cytoplasmic actin. The SDS-PAGE gel-purified 46 kDa protein was also subjected to limited proteolysis using S. aureus V8 protease, and this resulted in the formation of a prominent 31 kDa doublet as well as smaller proteolytic fragments, and these fragments were of identical molecular weight to those generated from limited proteolysis of bovine actin. Electron microscopic immunoperoxidase studies with primary cultures of bovine brain capillary endothelium showed that immunoreactive actin is intimately associated with the plasma membranes. In conclusion, the brain capillary 46 kDa protein is cytoplasmic actin and is localized to the endothelial plasma membrane. Modulations of brain capillary endothelial actin may play a role in the regulation of BBB permeability.
Delivery of cholesterol to inner mitochondrial membranes is rate-limiting for steroidogenesis in the zona fasciculata of adrenal cortex. A protein that stimulates this process was isolated to homogeneity from bovine adrenal tissue. This protein's primary structure has been determined in its entirety by a combination of automated Edman microsequencing, fast-atom bombardment mass spectrometry (FAB-MS). The sequence was identical to that previously reported for bovine brain endozepine, except that it lacks the last two residues, -Gly-Ile, at the C terminus. To our knowledge, isolation of an endozepine-related protein from a tissue other than brain has not been reported previously. Endozepine competes with benzodiazepines for saturable binding sites in synaptosomes and in mitochondria of specific peripheral tissues. Previous reports have localized the adrenal benzodiazepine receptor to the outer mitochondrial membrane. In this report, we show that the prototypic benzodiazepine, diazepam, effects a stimulation of adrenal mitochondrial cholesterol delivery similar to that observed for endozepine. The effective diazepam concentration was consistent with that previously shown to displace a high-affinity ligand of the mitochondrial benzodiazepine receptor. The action of diazepam in adrenal mitochondria suggests that the mediation of corticotropin-induced steroidogenesis may be the physiological function of the peripheral-type benzodiazepine receptor. These studies provide new insights into the previously unknown function of peripheral benzodiazepine receptors and should allow new investigations into the stimulation of steroidogenesis by endozepines and benzodiazepines in the brain and in certain peripheral tissues.
Blood-brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB-specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16-18K triplet of low-molecular-weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary-free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16-18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and C4 reverse-phase HPLC. Amino acid sequencing of the N-terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are alpha-globin, histone 2B, histone 3, and histone 2A, respectively. SDS-PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16-18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14-18K proteins seen on SDS-PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS-PAGE.
A full-length human placental aromatase cDNA clone, Aro 2, was isolated upon screening a human placental cDNA library with an aromatase cDNA probe and an oligonucleotide probe whose sequence was derived from a human aromatase genomic clone. Nucleotide sequence microheterogeneity was found in the 3'-untranslated region among Aro 2 and in two previously described human aromatase cDNA clones. Both the minor sequence differences and the expression of a single protein species in placental tissue suggest the presence of different alleles for aromatase. Northern blot analyses using one cDNA and two oligonucleotide probes are consistent with the two mRNA messages of 2.9 and 2.5 kilobases arising in human placenta as a consequence of differential processing. Several yeast expression plasmids containing the aromatase cDNA we cloned were constructed. The enzyme was expressed in Saccharomyces cerevisiae. The expressed activity was inhibited by the known aromatase inhibitor, 4-hydroxyandrostenedione. A level of 2 micrograms aromatase/mg partially purified yeast microsomes was estimated by analyses of carbon monoxide difference spectra on microsomal fractions from yeast carrying plasmid pHARK/VGAL. Using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, an apparent Michaels-Menken constant (Km) of 34 nM and a maximum velocity (Vmax) of 23 pmol [3H]water formed per min/mg protein were obtained for the yeast synthesized aromatase by transformation with plasmid pHARK/VGAL. The kinetic results are similar to those determined for human placental aromatase, and suggest that the yeast synthesized aromatase will be useful for further structure-function studies.