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Biomedical subjects

J E Shively

Publications and source records attributed to J E Shively.

At least 145 records · Page 8Linked to original sources
PubMed1987

Isolation of a cDNA for adrenodoxin reductase (ferredoxin-NADP+ reductase). Implications for mitochondrial cytochrome P-450 systems.

Using specific antibodies against adrenodoxin reductase (AR), we screened lambda gt11 cDNA expression libraries constructed from bovine adrenal cortex mRNA, and isolated several putative clones coding for this enzyme. Concurrently we determined the amino acid sequences of fragments from it. A deoxyinosine-containing oligonucleotide probe, generated for one of the sequences, reacted specifically with one of the cloned cDNAs of about 1600 base pairs. The codon sequence of this cDNA matched the peptide sequences, further confirming its identity as a copy of AR mRNA. RNA blot analysis indicates that in the adrenal cortex and corpus luteum there is only one major mRNA (approximately 2000 bp) for AR. The levels of this mRNA are at least 40-fold lower in the liver and kidney which are also known to contain in homologue of AR. As compared to adrenodoxin and cytochrome P-450scc mRNAs, AR mRNA levels in the adrenal cortex appear to be about 10-fold lower. Southern blot analysis of bovine and human genomic DNAs reveals that in both of these species there is only one gene for AR. These results indicate that only a single reductase serves the different mitochondrial P-450 systems in steroidogenic tissues.

Adrenal Cortex↗
PubMed1987

Microsequence analysis of peptides and proteins: trimethylsilylisothiocyanate as a reagent for COOH-terminal sequence analysis.

A reinvestigation of the isothiocyanate-based chemistry for cyclic degradations of peptides and proteins revealed that the reagent trimethylsilylisothiocyanate (TMS-ITC) gives superior results in terms of coupling efficiency and lack of complicating side reactions. Acetic anhydride (10 min at various temperatures) was used to activate the carboxyl terminus, and 6 N HCl (30 min at room temperature) was used for cleavage as originally described by G.R. Stark (Biochemistry 8, 4735, 1968). Reaction conditions for efficient coupling were explored using subtractive chemistry on bradykinin, a nonapeptide, and separation of the reaction products by reverse-phase high-performance liquid chromatography. The products were analyzed by fast atom bombardment-mass spectrometry and shown to be the N-acetylated starting material and the N-acetylated des-Arg9 derivative of bradykinin. The pseudo-first-order rate constants measured at 50, 70, and 90 degrees C were 5.6 X 10(-5), 5.1 X 10(-4), and 8.6 X 10(-4) s-1, respectively. In order to obtain complete couplings within 30-40 min at 50 degrees C, the effect of pyridine catalysis was studied. The addition of 0.225 M pyridine resulted in roughly doubling the rates at 50 and 70 degrees C. In the case of bradykinin, the reaction with TMS-ITC in the presence of the pyridine catalyst at 50 degrees C was complete in 15 min. In order to apply this methodology to the analysis of proteins, the thiohydantoin derivatives of amino acids were synthesized and separated by reverse-phase HPLC. The derivatives were also characterized by mass spectrometry. The above reaction conditions were tested on 3 nmol of sperm whale apomyoglobin for three cycles of degradation. The sample was first coupled to p-phenylene diisothiocyanate-derivatized aminopropyl glass with a 90% yield. The approximate initial yield of glycine at cycle one was 30%. The first three cycles corresponded exactly to the predicted carboxy-terminal sequence of myoglobin. These results demonstrate the feasibility of a new Stark reagent for automated carboxy-terminal chemistry.

Amino Acids↗
PubMed1987

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2).

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.

Amino Acid Sequence↗
PubMed1987

Carcinoembryonic antigen (CEA) and two crossreacting antigens of 165 kD and 105 kD isolated from meconium exhibit identical amino terminal sequences.

Two antigens of 165 kD and 105 kD crossreacting with the carcinoembryonic antigen (CEA) were isolated from human meconium by perchloric acid extraction, differential immunoaffinity chromatography, and size exclusion HPLC. The sequences of the 30 amino terminal amino acids of both antigens are identical with the corresponding sequence of CEA.

Amino Acid Sequence↗
PubMed1987

Neurotensin-like immunoreactivity generated by pepsin from human plasma and gastric tissue.

The present study demonstrates precursors of neurotensin-like immunoreactivity (NTLI) endogenous to human gastric tissue and plasma, and the existence of a gastric NTLI-generating enzyme system. The molecular size of the NTLI-precursors in plasma and gastric tissue were estimated by gel permeation chromatography to be ca 50,000-60,000 and 60,000-70,000 Da, respectively. The neurotensin-like peptide generated from the precursor was detected with a carboxyl-terminally directed antiserum but did not cross-react with an amino-terminally directed antiserum. A neurotensin-like peptide isolated from pepsin-treated human plasma was characterized by mass spectrometry and its amino acid sequence determined. This novel nonapeptide, referred to as kinetensin, failed to affect pentagastrin-stimulated acid secretion or blood pressure in the rat. Sequence homologies between neurotensin, kinetensin and proteins of the serum albumin family suggest a common evolutionary origin and raise questions regarding albumin-like proteins as precursors of regulatory peptides.

Amino Acid Sequence↗
PubMed1987

Hepatic lipase: a member of a family of structurally related lipases.

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.

Amino Acid Sequence↗
PubMed1987

Affinity alkylation of the active site of C21 steroid side-chain cleavage cytochrome P-450 from neonatal porcine testis: a unique cysteine residue alkylated by 17-(bromoacetoxy)progesterone.

The affinity alkylating progesterone analogue 17-(bromoacetoxy)progesterone has been used to label the active site of a microsomal cytochrome P-450 enzyme from neonatal pig testis. The enzyme causes removal of the C20 and C21 side chains from the substrates progesterone and pregnenolone by catalyzing both 17-hydroxylase and C17,20-lyase reactions, which produce the corresponding C19 steroidal precursors of testosterone. The progesterone analogue causes simultaneous inactivation of the two catalytic activities of the enzyme by a first-order kinetic process that obeys saturation kinetics. Progesterone and 17-hydroxyprogesterone each protect the enzyme against inactivation. The progesterone and analogue is a competitive inhibitor of the enzyme with Ki values of 8.4 microM and 7.8 microM for progesterone and 17-hydroxyprogesterone, respectively. The enzyme inactivation and kinetic data are consistent with a theory proposing that the analogue and the two substrates compete for the same active site. The radioactive analogue 17-[( 14C]bromoacetoxy)progesterone causes inactivation of the enzyme with incorporation of 1.5-2.2 mol of the analogue per mole of inactivated enzyme. When this experiment is carried out in the presence of a substrate, then 0.9-1.2 mol of radioactive analogue is incorporated per mole of inactivated enzyme. The data suggest that the analogue can bind to two different sites, one of which is related to the catalytic site. Radiolabeled enzyme samples, from reactions of the 14C-labeled analogue with the enzyme alone or with enzyme in the presence of a substrate, were subjected to amino acid analysis and also to tryptic digestion and peptide mapping.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1987

Demonstration of structural differences between the two subunits of human-plasma fibronectin in the carboxy-terminal heparin-binding domain.

Structural differences between the two subunits of human plasma fibronectin were studied by analyzing the carboxy-terminal heparin-binding domain (Hep-2). Two fragments (29 kDa and 38 kDa) derived from the Hep-2 domain were purified from thermolysin-digested human plasma fibronectin. Identical NH2-terminal sequences were obtained for both fragments through 16 Edman cycles. Neither domain contained the 90-amino-acid extra domain which is predicted by cDNA analysis of the cellular form of fibronectin. We have examined the primary structures of the 29-kDa and 38-kDa Hep-2 domains produced from the two chains of plasma fibronectin by analyzing the tryptic peptides by fast atom bombardment/mass spectrometry and comparison with the predicted fragments deduced from the corresponding cDNA-derived peptide sequences. Peptides that were unique to each domain were further characterized by microsequence analysis. The two domains showed identical amino acid sequences through 274 residues, followed by a region of variability. The 29-kDa domain contains 279 amino acids with an estimated relative molecular mass (Mr) of 30,460. This domain is located in the heavy chain of plasma fibronectin and contains three repeats of type III sequences plus a portion of the connecting segment (IIICS) region. The 38-kDa domain contains 359 amino acids and one O-linked glycosyl unit for an estimated Mr of 39,263. This domain is from the light chain of plasma fibronectin and contains four repeats of type III sequences with the deletion of the entire 120-amino-acid IIICS area. Secondary structure analysis by Chou/Fasman and circular dichroism reveals extensive beta-sheet structure for these domains. Key sulfhydryl and glycosylation sites are located near the mRNA splice junctions for the two chains. It is postulated that the splice junctions are adjacent to a flexible domain joining two regions of extensive beta-sheet structure.

Amino Acid Sequence↗
PubMed1987

Imaging of colon carcinoma with 111-indium-labeled anti-CEA monoclonal antibodies (Indacea) prior to surgery.

Patients with primary and/or metastatic colorectal cancer who had been scheduled for operative intervention were injected intravenously with 200 micrograms of a high-affinity anti-carcinoembryonic antigen (CEA) monoclonal antibody labeled with 2 mCi of 111-indium (Indacea). Patients were imaged by gamma camera at 24 and 48 hours. Primary tumors were identified in 3/10 cases and were not visualized in 3/10 cases. Four scans were considered equivocal. Hepatic metastases were identified as image defects in 5/13 cases and were not visualized in 8/13 cases. All tumors contained CEA by immunoperoxidase staining. In all cases, the primary tumor uptake (5.44 +/- 1.07% ID/kg) was much higher than the uptake of the adjacent fat (0.18 +/- 0.04% ID/kg). There was a direct correlation between tumor CEA content, tumor radioactivity, and the imaging of primary tumor by Indacea. High liver uptake (30.3 +/- 3.0% ID/kg), seen when scanning all patients, was the main limitation of imaging and led to photopenic visualization of hepatic metastases. These results suggest that selection of patients with colorectal carcinoma on the basis of tumor CEA content will lead to improved rates of tumor imaging by Indacea in post-surgical scanning.

Antibodies, Monoclonal↗
PubMed1987

A monoclonal antibody to the heavy chain of human IgM with a particular reaction pattern in human B-cell malignancies.

We produced a monoclonal antibody to the human mu-chain. Its specificity was proved by amino acid-sequence analysis of immunoabsorbent purified cell surface antigen. In binding studies an affinity constant (K-aff) of 5.7 X 10(9) M-1 was determined. When compared with a polyclonal anti-IgM antiserum it was found that this antibody may react with a determinant of the mu-chain differently exposed in clonal B-cell disorders rendering it to a reagent of particular diagnostic interest.

Amino Acid Sequence↗
PubMed1987

Microsequence analysis of peptides and proteins. VI. A continuous flow reactor for sample concentration and sequence analysis.

We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2-3 cm) containing 5-10 mg of Polybrene-coated, spherical, porous silica (100-200-micron particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0-ml samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95-100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes.

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.

Adrenal Glands↗
PubMed1987

Cytochrome P450c17 (steroid 17 alpha-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues.

P450c17 is the single enzyme mediating both 17 alpha-hydroxylase (steroid 17 alpha-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. We sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, lambda hac17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17. Comparison of the amino acid sequence of P450c17 with two other human steroidogenic cytochromes P450 show much greater homology with P450c21 (28.9%), another microsomal enzyme, than with P450scc (12.3%), a mitochondrial enzyme.

Adrenal Glands↗
PubMed1987

Sequence analysis of carcinoembryonic antigen: identification of glycosylation sites and homology with the immunoglobulin supergene family.

A direct method for the determination of N-linked glycosylation sites in highly glycosylated proteins is described. Carcinoembryonic antigen (CEA) and a nonspecific crossreacting antigen (NCA) were chemically deglycosylated, and peptide maps were prepared by reverse-phase HPLC. The peptides were sequenced on a gas-phase microsequencer, and glycosylation sites were identified as the phenylthiohydantoin derivative of N-acetylglucosaminylasparagine. The sequences were confirmed by fast atom bombardment mass spectrometry. Highly homologous, extended amino-terminal sequences were determined for CEA and two NCAs, NCA-95 and NCA-55. Cysteine-containing sequences for CEA and NCA-95 show up to 95% sequence homology, and the CEA sequences also show internal sequence homologies. A comparison of the CEA sequences with known protein sequences suggests that CEA may be a member of the immunoglobulin supergene family. The protein sequence data have been used to identify a genomic DNA clone for one of the NCA antigens [Thompson, J., Pande, H., Paxton, R. J., Shively, L., Padma, A., Simmer, R. L., Todd, C. W., Riggs, A. D. & Shively, J. E. (1987) Proc. Natl. Acad. Sci. USA, in press] and a cDNA clone for CEA [Zimmermann, W., Ortlieb, B., Friedrich, R. & von Kleist, S. (1987) Proc. Natl. Acad. Sci. USA, in press].

Amino Acids↗
PubMed1987

Cloning and tissue-specific expression of mouse macrophage colony-stimulating factor mRNA.

Macrophage colony-stimulating factor (CSF-1) stimulates the production of macrophages from bone marrow progenitor cells. We have identified a cDNA clone for murine CSF-1 by antibody screening of a mouse L-cell cDNA library in the expression vector lambda gt11. A screen of about 150,000 recombinant plaques yielded 6 clones that reacted well with an antibody raised against denatured and reduced mouse L-cell CSF-1. These clones were further screened with synthetic oligonucleotides based on the amino-terminal amino acid sequence of CSF-1. One clone, which hybridized to the oligonucleotides, was sequenced and found to contain a single open reading frame. This encompassed 68 amino acids of the mature protein, including the entire amino-terminal sequence we previously reported. This is preceded by what appears to be a 31 amino acid signal peptide. Blot analysis showed that this cDNA hybridizes to a major mRNA species of about 4.5 kilobases (kb) as well as several smaller, less abundant mRNA species (3.8, 2.3, and 1.4 kb) present in mouse L cells. A similar pattern of hybridization was observed with mRNA from a human pancreatic carcinoma cell line that produces CSF-1. Striking differences in the qualitative and quantitative expression of mRNA species for CSF-1 were observed in various mouse tissues. Liver expressed primarily a 1.4-kb species, heart and lung expressed primarily a 4.5-kb species, brain expressed high levels of both the 4.5-kb and 1.4-kb species, and intestine lacked detectable CSF-1 transcripts. Southern blot analysis suggests that the CSF-1 gene is present as a single copy in the mouse haploid genome and that it is not rearranged or amplified in L cells.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of a gene belonging to the carcinoembryonic antigen gene family and discussion of a domain model.

Carcinoembryonic antigen (CEA) is a glycoprotein important as a tumor marker for colonic cancer. Immunological and biochemical studies have shown it to be closely related to a number of other glycoproteins, which together make up a gene family. We have cloned a member of this gene family by using long oligonucleotide probes (42-54 nucleotides) based on our protein sequence data for CEA and NCA (nonspecific cross-reacting antigen) and on human codon usage. The clone obtained (lambda 39.2) hybridizes with six probes and has a 15-kilobase insert. The 5' end of the gene is contained within a 2700-base-pair EcoRI fragment, which hybridizes with five of the six synthetic probes. Sequencing of the 5' end region revealed the location and structure of one exon and two putative intron boundaries. The exon encodes part of the leader sequence and the NH2-terminal 107 amino acids of NCA. Southern blot analysis of human normal and tumor DNA, using as probes two lambda 39.2 fragments that contain coding sequences, suggests the existence of 9-11 genes for the CEA family. One of the restriction fragments described here has been used by Zimmermann et al. [Zimmermann, W., Ortlieb, B., Friedrich, R. & von Kleist, S. (1987) Proc. Natl. Acad. Sci. USA 84, 2960-2964] to isolate partial cDNA clones for CEA. The identity of this clone was verified with our protein sequence data [Paxton, R., Mooser, G., Pande, H., Lee, T.D. & Shively, J.E. (1987) Proc. Natl. Acad. Sci. USA 84, 920-924]. We discuss a domain structure for CEA based on the CEA sequence data and the NCA exon sequence data. It is likely that this gene family evolved from a common ancestor shared with neural cell adhesion molecule and alpha 1 B-glycoprotein and is perhaps a family within the immunoglobulin superfamily.

Amino Acid Sequence↗
PubMed1987

Amyloid angiopathy of Alzheimer's disease: amino acid composition and partial sequence of a 4,200-dalton peptide isolated from cortical microvessels.

The cardinal lesions of Alzheimer's disease are neurofibrillary tangles, senile neuritic plaques, and vascular amyloid, the latter generally involving cortical arteries and small arterioles. All three lesions are composed of amyloid-like, beta-pleated sheet fibrils. Recently, a 4,200-dalton peptide has been isolated from extraparenchymal meningeal vessels, neuritic plaques, and neurofibrillary tangles. The assumption of N-terminal homogeneity in vascular amyloid has been used as an argument for a neuronal (versus blood) origin of the peptide. However, intracortical microvessels from Alzheimer's disease have not been previously isolated. The present studies describe the isolation of a microvessel fraction from Alzheimer's disease and control fresh autopsy human brain. Alzheimer's disease isolated brain microvessels that were extensively laden with amyloid and control microvessels were solubilized in 90% formic acid and analyzed by urea sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The arteriole fraction from the Alzheimer's subject with extensive amyloid angiopathy contained a unique 4,200-dalton peptide, whereas the arterioles or capillaries isolated from two controls and two Alzheimer's disease subjects without angiopathy did not. This peptide was purified by HPLC and amino acid composition analysis showed the peptide is nearly identical to the 4,200-dalton peptide recently isolated from neuritic plaques or from neurofibrillary tangles. Sequence analysis revealed N-terminal heterogeneity. The N-terminal sequence was: Asp-Ala-Glu-Phe-Arg-His-Asp-Ser-Gly-Tyr, which is identical to the N-terminal sequence of the 4,200-dalton peptide isolated previously from extraparenchymal meningeal vessels and neuritic plaques.(ABSTRACT TRUNCATED AT 250 WORDS)

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