Biomedical subjects
Publications and source records attributed to H J Yeh.
Alkaloids of a new class are present in skin extracts of the dendrobatid poison frog, Minyobates bombetes, of Colombia. The structure of the major alkaloid of this class, 251F, has been determined as a trimethylcyclopenta[b]quinolizidinemethanol 1 by nmr, gc-Ft-ir, and ms studies including ms-ms. At least nine congeners of 251F were detected in these extracts.
Platelet-derived growth factor (PDGF) may be a critical factor in the temporal differentiation of glial elements in the mammalian central nervous system. We have used in situ hybridization and immunoperoxidase staining to investigate the localization of PDGF A and have observed high levels of PDGF A-chain mRNA and immunoreactive PDGF A in neurons of embryonic and adult mice. PDGF A-chain expression was shown to be developmentally regulated and tissue specific. Every neuronal population examined in the central and peripheral nervous systems expresses PDGF A transcripts. Variable, significantly weaker signals are observed in glial cells. In contrast to known neurotrophic factors, the PDGF A transcripts are widely distributed among neurons. This generalized distribution of PDGF A transcripts, together with the known effects of PDGF on glial cells in vitro, suggests a unique role of neurons in regulating the proliferation and differentiation of glial cells in vivo.
The aS,7S-absolute configuration of (-)-colchicine (1) and (-)-N-acetylcolchinol methyl ether (3, NCME) suggested on the basis of 1H NMR data and negative Cotton effects at about 260 nm (EtOH) is firmly established by an X-ray analysis of urea 5, a compound derived from 3. Binding of these compounds to tubulin requires an aS-configuration of the biaryl system.
CHO cells were transfected with plasmid pSV2-PDGF-A (containing human PDGF-A cDNA) by calcium phosphate method. Twenty transfected cell lines were obtained after G418 selection. The selected 2 cell lines At1 and Aot7), with prominent changes in morphology and growth behaviour, showed transcription of PDGF-A chain mRNA much higher than CHO cells, strong fluorescent PDGF-specific reaction, appearing that PDGF-like proteins were synthesized in cytoplasm of these cells. At1 and Aot7 cells not only had increased growth rate, but also formed large colonies in soft agar and grew into fibrosarcomas in nude mice. These results suggested that the expression of exogenous PDGF-A gene might cause the uncontrolled growth and malignant transformation of CHO cells.
During the past four years in our hospital, real-time sonographic examinations were performed prior to barium enema reductions in 48 proven intussusception cases. Four major sonographic findings were noted. First, a length of target configuration consisting of two rings of low echogenicity separated by an intermediate hyperechoic ring was seen on the cross-sectional image of the intussuscepted bowel. Second, a doughnut configuration consisting of a hypoechoic rim and a dense central echogenic core was noted on the cross section near the apex of the intussusceptum. Third, no demonstrable movement or change was observed in the target or doughnut configuration. Fourth, all of the exterior sonolucent rims of the target were thicker than 0.6 cm. Operative reductions were necessary in all 13 cases whose exterior rims were thicker than 1.6 cm. On the contrary, only 15 of the remaining 35 cases whose exterior rims were between 0.6 and 1.5 cm needed surgical management (p = 0.0033, Fisher's exact test).
CHO cells were transfected with plasmid pSM-1 (containing human c-sis cDNA) singly or co-transfected with pSV 2 neo DNA by calcium phosphate method. After low serum or G418 selection several cell lines with expression of platelet-derived growth factor (PDGF) were obtained. One among them, FB5, was of the highest PDGF expression and showed the following biological characteristics when compared with CHO cells: (1) a prominent change in morphology from spindle to round in shape: (2) increase of growth rate; (3) growth in low serum (2%) medium as a semisuspension culture; (4) growth on soft agar to larger colonies; (5) synthesis of PDGF in cytoplasm identified by immunofluorescent method; (6) the conditioned medium stimulated DNA synthesis of NRK cells; (7) RNA dot hybridization showing high transcription of PDGF mRNA; (8) southern blot showing integration of human c-sis gene was still stable after 7 months. These results indicated that intergration of exogenous c-sis gene and its high expression might cause CHO cells to high growth rate and even transformation. The establishment of this stable transformed cell line, FB5 is thought to be a good model for further study on the function of PDGF in cell growth control and cell transformation.
Measuring ellipticities of (+/-)-colchicine and (+/-)-deacetamidocolchicine in the presence of tubulin afforded net positive CD bands with maxima at 340 nm resulting from reduction of the negative ellipticities upon binding of (-) enantiomers to the protein. Results of optical studies together with earlier NMR conformational analysis of these molecules substantiate the hypothesis that colchicinoids bind to tubulin with the phenyl-tropolone moiety in the 'aS' configuration. Natural colchicine which binds to tubulin, therefore, should be referred to as (-)-(aS,7S)-colchicine.
Arteether (6) has been prepared from dihydroquinghaosu (3) by etherification with ethanol in the presence of Lewis acid and separated from its chromatographically slower moving alpha-dihydroqinghaosu ethyl ether (7). The absolute stereochemistry at C-12 has been determined by 1H NMR data (J11,12, NOESY). Ethyl ethers 6 and 7 showed potent in vitro inhibition of Plasmodium falciparum, and both compounds were highly potent antimalarials in mice infected with a drug-sensitive strain of Plasmodium berghei. Crystalline arteether (6) and its oily epimer 7 were 2-3 times more potent schizontocides than quinghaosu (1), but deoxy compounds 8, 9, and 11 were 100-300 times less potent in vitro than their corresponding peroxy precursors. Pharmacological studies have shown arteether(6) to have antimalarial activity in animals comparable to artesunate (2) and artemether (4), both of which are fast-acting blood schizontocides in humans. Arteether (6) has now been chosen for a clinical evaluation in high-risk malaria patients.
The platelet-derived growth factor (PDGF) is a potent mitogen for cells derived from mesenchyme and is highly related to the transforming protein of the simian sarcoma virus (SSV), p28v-sis. Both PDGF and p28v-sis appear to initiate DNA synthesis and cell proliferation through an interaction with PDGF cell surface receptors but the identity of the PDGF induced signals which are recognized within the nucleus is unknown. It has been suggested that PDGF is directly transported to the nucleus although conflicting data have been obtained when nuclear fractions have been analysed for PDGF-like proteins. Specific antisera recognizing PDGF and p28v-sis were used to isolate and partially characterize proteins from nuclei of SSV-transformed NRK fibroblasts. Nuclear proteins of 66, 65, and 44 kD were identified in immunoprecipitates. These proteins were displaced competitively from binding to anti-PDGF antisera by either PDGF or by mitogenically active recombinant v-sis protein homodimers and the proteins were not recognized by non-immune sera. Proteins of 66, 65, and 44 kD also were partially purified from nuclear extracts with anti-PDGF immunoaffinity chromatography and were identified in silver stained PAGE gels. The data establish the presence of proteins antigenically related to PDGF within the nuclei of SSV-transformed cells and suggest possible roles of nuclear proteins related antigenically to PDGF and p28v-sis in cell growth and transformation.
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Methyl 3-deoxy-3-fluoro-alpha- and beta-D-glucopyranosides and alpha- and beta-D-glucofuranosides were prepared by methanolysis of 3-deoxy-3-fluoro-1,2:5,6-di-O-isopropylidene-alpha-D-glucofuranose. Crystalline 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-alpha-D-glucopyranosyl chloride (2) and the corresponding glycosyl bromide (3) were prepared from 1,3,4,6-tetra-O-acetyl-2-deoxy-2-fluoro-beta-D-glucopyranose (1). Reaction of 2 with methanol under the conditions of both silver triflate- and silver perchlorate-catalyzed glycosylation showed remarkable lack of stereoselectivity for the formation of the corresponding methyl alpha-glycoside, despite the presence at C-2 of the fluorine functionality presumably not capable of neighboring-group participation. Pure methyl 2-deoxy-2-fluoro-alpha- and beta-D-glucopyranosides were obtained by fractional crystallization from the mixture formed by methanolysis of 1. The structure of these substances as well as of several other derivatives of 2-deoxy-2-fluoro- and 3-deoxy-3-fluoro-D-glucose were verified by n.m.r. (1H, 13C, and 19F) spectroscopy.
13C, 15N, and 1H nuclear magnetic resonance measurements indicate that chloroform-soluble threonine-containing tripeptide derivatives, such as t-Boc-Thr-Gly-Gly-OBz, form three strong hydrogen bonds to the cytosine moiety of 2',3'-O-isopropylidene-5'-O-t-butyldimethylsilylcytidine. The C = O and NH of the central peptide residue plus the OH of the threonine side chain appear to form bonds to the N(4')H2, N(3), and C(2) = O, respectively, of the pyrimidine. An association constant calculated from the cytidine 15N(4') nuclear magnetic resonance response to added peptide is four times larger than the corresponding cytosine-guanine constant. It is suggested that cytosine-peptide bonding was part of the primitive genetic coding mechanism early in evolution and accounts for the origin of the cytosine-centered codons for the hydroxy amino acids, serine and threonine, in the present code.
Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.
Platelet-derived growth factor (PDGF) is the major growth factor in serum and a potent mitogen for cells mesenchymal origin. It is a highly basic heterodimeric protein with a molecular mass of approximately 30 kDa and binds to a cell surface receptor with high affinity. The amino acid sequence of PDGF revealed sequence homology to the v-sis gene product of simian sarcoma virus (SSV), a transforming retrovirus. Characterization of cells transformed by SSV has revealed PDGF-related proteins in subcellular organelles and in conditioned media consistent with the autocrine stimulation of cell growth through cell surface receptors and perhaps through an internal autocrine mechanism as the growth factor and its receptor are processed. PDGF is also a potent chemotactic agent for inflammatory and other mesenchymal cells and has been implicated in normal tissue repair processes such as wound healing, as well as in aberrant proliferative processes like atherogenesis.
The subcellular distribution of v-sis-related protein(s) was analyzed in three simian sarcoma virus (SSV)-transformed cell lines with immunofluorescence and protein A-gold labeling techniques using rabbit polyclonal anti-platelet-derived-growth factor (PDGF) antisera. Antigenically reactive proteins were recognized in subcellular organelles related to protein synthesis and processing, including polyribosomes, endoplasmic reticulum, and the Golgi apparatus, as well as on the cytoplasmic surface of plasma membranes. Prominent immunoreactive proteins were also shown in association with nuclear chromatin in intact cells and in isolated nuclei using Lowicryl K4M resin embedding techniques. Protein A-gold labeling was markedly reduced in sections of non-SSV-transformed fibroblasts incubated with anti-PDGF and absent from SSV-transformed cells if Epon resin was substituted for Lowicryl in the embedding process or if sections were with irrelevant antisera. Nuclear localization of v-sis-related antigens was confirmed in a nitrocellulose-based immunoassay using nuclei isolated from SSV-transformed fibroblasts. Thus, polypeptides recognized antigenically as related to the v-sis gene product not only may be found in subcellular organelles associated with protein synthesis and packaging but also may be found in the nucleus of SSV-transformed cells. These results raise the possibility that v-sis- or PDGF-like proteins may function within the nucleus of SSV-transformed cells.
We are exploring the active site and the mechanism of the pyridoxal phosphate dependent reactions of the bacterial tryptophan synthase alpha 2 beta 2 complex by use of substrate analogues and of reaction intermediate analogues. Fluorine-19 nuclear magnetic resonance studies and absorption spectroscopy are used to study the binding and reactions of the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)- and (3R)-2,3-dihydro-5-fluorotryptophan. Tryptophan synthase specifically and tightly binds the 3S diastereoisomer of both 2,3-dihydro-5-fluoro-D-tryptophan and 2,3-dihydro-5-fluoro-L-tryptophan, whereas it binds 5-fluoro-D-tryptophan more tightly than 5-fluoro-L-tryptophan. Unexpectedly, we find that the D and L isomers of 5-fluorotryptophan, of tryptophan, and of (3S)-2,3-dihydro-5-fluorotryptophan are slowly interconverted by isomerization reactions. Since these isomerization reactions are 10(3)-10(5) times slower than the beta-replacement and beta-elimination reactions catalyzed by tryptophan synthase, they have no biochemical significance in vivo. However, the occurrence of these slow reactions does throw some light on the nature of the active site of tryptophan synthase and its requirements for substrate binding. Our results raise the interesting question of whether tryptophan synthase itself serves a catalytic role in these slow reactions or whether the enzyme simply binds the substrate and pyridoxal phosphate stereospecifically and thus promotes the intrinsic catalytic activity of pyridoxal phosphate.
Metabolism of (+)-, (-)-, and (+/-)-trans-3,4-dihydroxy-3, 4-dihydrobenzo[c]phenanthrenes by liver microsomes from rats and mice and by a purified monooxygenase system reconstituted with cytochrome P-450c has been examined. Bay-region 3,4-diol 1,2-epoxides are minor metabolites of both enantiomers of the 3,4-dihydrodiol with liver microsomes from 3-methylcholanthrene-treated rats or with the reconstituted system (less than 10% of total metabolites). Microsomes from control and phenobarbital-treated rats and from control mice form higher percentages of these diol epoxides (13-36% of total metabolites). Microsomes from 3-methylcholanthrene-treated rats and cytochrome P-450c in the reconstituted system form exclusively the diol expoxide-1 diastereomer, in which the benzylic hydroxyl group and oxirane oxygen are cis to each other, from the (+)-(3S,4S)-dihydrodiol. The same enzymes selectively form the diol expoxide-2 diastereomer, with its oxirane oxygen and benzylic hydroxyl groups trans to each other, from the (-)-(3R,4R)-dihydrodiol (77% of the total diol epoxides). Liver microsomes from control rats show similar stereoselectivity whereas liver microsomes from phenobarbital-treated rats and from control mice are less stereoselective. Three bis-dihydrodiols and three phenolic dihydrodiols are also formed from the enantiomeric 3,4-dihydrodiols of benzo[c]phenanthrene. A single diastereomer of one of these bis-dihydrodiols with the newly introduced dihydrodiol group at the 7,8-position accounts for 79-88% of the total metabolites of the (-)-(3R,4R)-dihydrodiol formed by liver microsomes from 3-methylcholanthrene-treated rats or by the reconstituted system containing epoxide hydrolase. In contrast, the (+)-(3S,4S)-dihydrodiol is metabolized to two diastereomers of this bis-dihydrodiol, a third bis-dihydrodiol, and two phenolic dihydrodiols.