Biomedical subjects
Publications and source records attributed to M E Baker.
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Although glycyrrhizic acid, a major constituent of licorice root, has important pharmacological effects in humans, the biological activity of glycyrrhizic acid and its aglycone glycyrrhetinic acid in plants is unknown. Here we report that these licorice-derived compounds and the analog carbenoxolone inhibit desaturation of linoleic acid (C18:2 omega 6) in soybean chloroplasts using monogalactosyldiacylglycerol and phosphatidylcholine substrates in an in vitro assay for desaturase activity. At 10 nM glycyrrhetinic acid, there is significant inhibition of desaturation of linoleic acid suggesting that licorice-derived compounds could prove useful in investigating biochemical pathways of linoleic acid desaturation in plant chloroplasts and plant desaturase regulation, which has application in modification of plant response to environmental stress, as well as optimization of oil seed composition.
We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.
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Although plants have long been known to have important pharmacological effects in humans, the mechanism by which plant-derived compounds act in humans is still being elucidated. Two important pathways for the biological actions of plant-derived compounds involved binding either to hormone receptors or to enzymes that metabolize hormones. What are the origins of this interaction between plant-derived compounds and animals? And what insights can we gain from investigating this question? Some answers come from recent sequence analyses, revealing that 17 beta-hydroxysteroid dehydrogenase, which regulates estrogen and androgen levels in humans, and 15-hydroxyprostaglandin dehydrogenase, which regulates prostaglandin E2 and F2 alpha levels in humans, have a common ancestor with proteins in rhizobia that are important in forming nitrogen-fixing nodules in legume roots, and 3 beta-hydroxysteroid dehydrogenase, which regulates progestin and androgen levels in humans, has a common ancestor with enzymes important in the synthesis of anthocyanins. This evolutionary kinship, when combined with the structural similarities between flavonoids, licorice-derived compounds, and steroid hormones, provides another perspective on the hormone-like activity of flavonoids and other plant-derived compounds in humans: some of the hormone-like activity of plant-derived compounds is due to binding to steroid and prostaglandin dehydrogenases.
Significant technical advances have occurred in both computed tomography (CT) and magnetic resonance (MR) imaging that have resulted in improved visualization of the pancreas by both techniques. In this article we will consider the advances in both modalities and consider the relative roles of each in imaging the spectrum of pancreatic pathology.
We previously reported that the D39N mutant of Drosophila alcohol dehydrogenase (ADH), in which Asp-39 is replaced with asparagine, has a 60-fold increase in affinity for NADP+ and a 1.5-fold increase in kcat compared to wild-type ADH [Chen et al. (1991) Eur. J. Biochem. 202, 263-267] and proposed that this part of ADH is close to the 2'-phosphate on the ribose moiety of NADP+. Here we report the effect of replacing Ala-46 with an argine residue, and A46R mutant, on binding of NADP+ to ADH and its catalytic efficiency with the NADP+ cofactor, and a modeling of the three-dimensional structure of the NAD(+)-binding region of ADH. The A46R mutant has a 2.5-fold lower Km(app)NADP+ and a 3-fold higher kcat with NADP+ compared to wild-type ADH; binding of NAD+ to the mutant was unchanged and kcat with NAD+ was lowered by about 30%. For the A46R mutant, the ratio of kcat/Km of NAD+ to NADP+ is 85, over ten-fold lower than that for wild-type ADH. Our model of the 3D structure of the NAD(+)-binding region of ADH shows that Ala-46 is over 10 A from the ribose moiety of NAD+, which would suggest that there is little interaction between this residue and NAD+ and explain why its mutation to arginine has little effect on NAD+ binding. However, the positive charge at residue 46 can neutralize some of the coulombic repulsion between Asp-39 and the 2'-phosphate on the ribose moiety of NADP+, which would increase its affinity for the A46R mutant. We also constructed a double mutant, D39N/A46R mutant, which we find has a 30-fold lower Km(app)NADP+ and 8-fold higher kcat with NADP+ as a cofactor compared to wild-type ADH; binding of NAD+ to this double mutant was lowered by 5-fold and kcat was increased by 1.5-fold. As a result, kcat/Km for the double mutant was the same for NAD+ and NADP+. The principle effect of the two mutations in ADH is to alter its affinity for the nucleotide cofactor; kcat decreases slightly in A46R with NAD+ and remains unchanged or increases in the other mutants.
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Licorice has long been known to promote the healing of ulcers. In the 1950s, studies with licorice-derived compounds revealed that the anti-ulcer effects of licorice are due to inhibition of 15-hydroxyprostaglandin dehydrogenase and delta 13-prostaglandin reductase. 15-Hydroxyprostaglandin dehydrogenase converts prostaglandins E2 and F2 alpha to 15-ketoprostaglandins, which are inactive. delta 13-Prostaglandin reductase metabolizes the inactive delta 13-prostaglandin to 13,14-dihydro,15-ketoprostaglandin, which is further metabolized and excreted in urine. Thus, licorice-derived compounds have the effect of raising the local concentration of prostaglandins that promote mucous secretion and cell proliferation in the stomach, leading to healing of ulcers. 11 beta-Hydroxysteroid dehydrogenase, which also is inhibited by licorice-derived compounds, shares a common ancestor with 15-hydroxyprostaglandin dehydrogenase. Both enzymes are homologous to Streptomyces hydrogenans 3 alpha,20 beta-hydroxysteroid dehydrogenase, which also is inhibited by licorice. Thus, licorice inhibits enzymes that diverged at least 2 billion years ago from a common ancestor. Other oxidoreductases in bacteria, plants, and animals that are inhibited by licorice-derived compounds are likely to be discovered in the future.
Amino acid sequence comparisons have revealed that mammalian 11 beta-hydroxysteroid and 17 beta-hydroxysteroid dehydrogenases and bacterial 3 alpha, 20 beta- and 3 beta-hydroxysteroid dehydrogenases are homologs; that is, these enzymes are descended from a common ancestor. These steroid dehydrogenases are also homologous to human 15-hydroxyprostaglandin dehydrogenase and to proteins found in Rhizobia, bacteria that form nitrogen-fixing nodules in the roots of legumes. We constructed a multiple sequence alignment of these proteins, which, when combined with the recently determined tertiary structure of Streptomyces hydrogenans 3 alpha, 20 beta-hydroxysteroid dehydrogenase and a homologous enzyme, rat dihydropteridine reductase, identifies segments and residues that are likely to be structurally important in the functioning of these enzymes especially regarding specificity for NADPH and NADH.
The colorimetric indicator pararosaniline has been immobilized onto the cation-exchange resins Amberlite IRC-50, Dowex 50W-X8 and cellulose phosphate by electrostatic bonding. The reflectance of each reagent phase was measured using a bifurcated fibre-optic system and a flow cell. Pararosaniline immobilized on cellulose phosphate was found to respond to formaldehyde without requiring the addition of sulfite to develop the purple chromogen. This immobilized system demonstrated a linear response to 50-2500 micrograms of formaldehyde and had a correlation coefficient of 0.9979. Acetaldehyde and butyraldehyde did not produce any interference. However, exposure to the unsaturated aldehydes, acrolein and crotonaldehyde, gave rise to responses that were much greater than that observed with formaldehyde.
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OBJECTIVE: The purpose of this study was to describe the MR appearance of hepatic adenomas and correlate the MR imaging features with pathologic findings. MATERIALS AND METHODS: MR examinations were performed in 14 patients with 66 hepatic adenomas. The diagnosis of hepatic adenoma was proved pathologically in nine patients (22 lesions). In five other patients (44 lesions), who had type I glycogen storage disease and were known to be at risk for hepatic adenomas, the diagnosis was established by repeated sonographic examinations that showed stability, reduction, or resolution of hepatic tumors. T1- and T2-weighted spin-echo MR images obtained at 1.5 T were retrospectively reviewed for the signal intensity of the lesions relative to liver, the signal pattern, the presence of a capsule, and the presence of hemorrhage. Histopathologic specimens (22 lesions) were reviewed for fat content (graded 0-3), the presence of a capsule, and the presence of hemorrhage. RESULTS: On T1-weighted images, 51 (77%) of 66 lesions were hyperintense, 11 (17%) were hypointense, and four (6%) were isointense with respect to liver. On T2-weighted images, 49 (74%) of 66 lesions were hyperintense, 12 (18%) were isointense, and five (8%) were hypointense. Sixty-one (92%) of 66 lesions were heterogeneous. Eleven (17%) of 66 lesions were hemorrhagic. Of the 22 lesions reviewed histopathologically, 17 were hyperintense on T1-weighted images; 15 of these had a fat content of grade 2 or 3 and two had hemorrhage. All 15 lesions that had a fat content of grade 2 or 3 were hyperintense on T1-weighted images. CONCLUSION: Hepatic adenomas have a variable MR appearance but most often are hyperintense with respect to liver on T1- and T2-weighted images. The high signal intensity often relates to the increased fat content of these lesions.
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OBJECTIVE: Although the hepatotoxic effects of systemic chemotherapy are well known, CT findings in the liver after systemic chemotherapy have received little attention in the literature. In some patients with breast carcinoma metastatic to the liver who have received chemotherapy, a morphologic pattern develops similar to that associated with cirrhosis. This pattern is characterized by a lobular hepatic contour, segmental volume loss, and enlargement of the caudate lobe. The purpose of this study was to describe the CT appearance of this pseudocirrhosis and to correlate it with pathologic findings. MATERIALS AND METHODS: We reviewed 65 CT examinations of 22 patients with stage IV breast carcinoma with hepatic metastases, who were receiving systemic chemotherapy and for whom abdominal CT scans showed pseudocirrhosis. Nineteen of 22 patients had follow-up CT scans at intervals ranging from 1 to 15 months. Criteria for the diagnosis of pseudocirrhosis included a lobular hepatic contour, segmental volume loss, and enlargement of the caudate lobe. CT findings were correlated with pathologic findings in seven patients. RESULTS: In all patients, CT scans showed retraction of the capsular surface of the liver (15 diffuse, seven focal) with a lobular margin, a finding also seen in advanced cirrhosis. The retraction occurred at the site of subjacent metastases. Findings evolved over 1-3 months. Six of seven patients had pathologic findings suggestive of nodular regenerative hyperplasia. No patients had pathologic evidence of cirrhosis. CONCLUSION: In patients undergoing systemic chemotherapy for breast cancer metastatic to the liver, a pattern may develop that mimics the CT appearance of hepatic cirrhosis. Pathologic findings suggest nodular regenerative hyperplasia as a possible cause.
The chicken is a useful model for studies of lipid biochemistry because of the profound changes in lipid metabolism during development and the marked effects of estrogen on its lipids. Here we report the cloning of a cDNA encoding chicken sterol carrier protein 2 (SCP2)/sterol carrier protein x (SCPx), proteins that are believed to play key roles in intracellular lipid movement and metabolism. The 2.4-kb clone, isolated from a liver cDNA library by homology screening with a rat SCP2 cDNA, encodes a 58-kDa polypeptide, SCPx, which encompasses the sequence for SCP2 at its C-terminus. Comparison of the deduced amino acid sequence of the chicken protein with those of mammals revealed conservation of structure from an evolutionary standpoint. Like the other vertebrate SCPx proteins, the chicken protein contains a conserved Arg-Gly-Asp sequence and a cysteine residue in the N-terminus that aligns with the active site cysteine of Escherichia coli 3-ketoacyl-CoA thiolase, a protein that was previously shown to be homologous to vertebrate SCPx. Northern blot analysis of poly(A)+ RNA from different chicken tissues revealed two mRNA species, one of 2.5 kb, encoding SCPx, and one of 1.4 kb, presumably encoding SCP2. High levels of the 2.5 and 1.4-kb mRNAs were found in liver, intestine, and ovarian granulosa cells, consistent with a role for these proteins in lipid metabolism. There was no change in the 2.5-kb mRNA in chicken liver with development (Day 20 embryo to 4 weeks posthatch), but there was a 10-fold increase in the 1.4-kb mRNA by 1 week posthatch. Treatment of roosters with a single injection of estradiol (25 mg/kg) caused a twofold increase in the 2.5-kb mRNA in liver at 6 h after estradiol administration, reaching a maximum fourfold increase at 48 h, while the 1.4-kb mRNA increased twofold at 48 h. The two SCP2/SCPx mRNAs were most abundant in granulosa cells from small follicles. A reduction in SCP2/SCPx gene expression was observed with follicular maturation, correlating with falling estrogen production. In summary, we have documented (i) marked conservation of SCP2/SCPx structure across species and (ii) developmental and hormonal regulation of the mRNAs which encode proteins thought to be involved in lipid metabolism.
Various protease inhibitors (e.g. phenylmethanesulfonyl fluoride (PMSF), tosyl-phenylalanine chloromethyl ketone (TosPheCH2Cl)) and substrates (e.g., tosyl-arginine methyl ester (TosArgOMe), tryptophan methyl ester (TrpOMe)) inhibit the binding of adrenal and sex steroids to their cognate receptors (Hubbard and Kalimi (1985) Mol. Cell. Biochem. 66, 101-109). Here we extend this finding to the receptor for 3,5,3'-triiodothyronine (T3) in rat liver nuclei. We find that PMSF, TosPheCH2Cl and other protease inhibitors as well as TosArgOMe, TrpOMe, tyrosine methyl ester (TyrOMe) and tyrosine ethyl ester (TyrOEt) inhibit binding of 125I-T3 to its receptor in rat liver nuclei. Inhibition by protease substrates appears to be at or close to the hormone binding domain. By analogy with the known mechanism of binding of protease inhibitors and substrates to enzymes, we suggest that the T3 receptor contains a nucleophilic site at or close to the hormone binding domain.