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D C Lee

Publications and source records attributed to D C Lee.

At least 127 records · Page 7Linked to original sources
PubMed1989

Regulation of transforming growth factor alpha messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol ester and hormones.

Transforming growth factor alpha (TGF-alpha) is produced by many transformed cells, but little is known about the regulation of its expression. We examined TGF-alpha mRNA levels in a set of cloned neoplastic cell lines derived by chemical transformation of a normal rat liver epithelial cell. The untransformed parental cell line, WB-344, did not express a detectable level of TGF-alpha mRNA, whereas GP6ac, a transformed line capable of autonomous growth in soft agar, expressed TGF-alpha. When GP6ac cells were treated with agents thought to regulate protein kinase C activity, e.g., the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF-alpha mRNA levels increased by 8- to 11-fold. The induction of TGF-alpha mRNA was detectable at 2 h, was maximal at 8-12 h, and declined by 24 h. Angiotensin, bradykinin, epinephrine, and epidermal growth factor also increased TGF-alpha mRNA by 2- to 5-fold. In contrast, parental WB cells neither expressed TGF-alpha mRNA, nor responded to TPA. TPA also increased epidermal growth factor receptor mRNA in GP6ac cells but the effect was less prolonged; maximal levels were seen at 4 h after TPA exposure and returned to control levels by 12 h. TPA increased TGF-alpha mRNA in GP6ac cells, in part, by increasing transcription of the TGF-alpha gene as measured by run-on transcription rates in isolated nuclei. In addition, the induction of TGF-alpha by TPA was blocked by concurrent incubation with agents that inhibit protein synthesis. However, if TPA was present for at least 2 h, subsequent addition of cycloheximide enhanced the effect of TPA. This indicates that the induction of TGF-alpha in GP6ac cells is comprised of at least two phases demarcated by the requirement for protein synthesis. The time course of induction and the sensitivity to inhibition of protein synthesis distinguish the effect of TPA on TGF-alpha mRNA from that of other genes regulated by TPA, e.g., c-myc and c-fos. These data also suggest that chemical transformation of rat liver epithelial cells leads to expression of TGF-alpha mRNA, and that once expressed, TGF-alpha mRNA can be modulated in a protein kinase C-dependent manner.

Animals↗
PubMed1989

The TGF-alpha precursor expressed on the cell surface binds to the EGF receptor on adjacent cells, leading to signal transduction.

The 50 amino acid form of TGF-alpha is cleaved from a conserved integral membrane glycoprotein by a protease that, in many tumor cells, appears to be limiting. To test whether the membrane-bound precursor has biological activity in the absence of processing, we introduced amino acid substitutions at the proteolytic cleavage sites. BHK cells transfected with expression vectors containing these altered sequences do not secrete detectable levels of mature TGF-alpha into the medium, but express high levels of proTGF-alpha at the cell surface. Coincubation of these BHK cells with A431 cells demonstrates that membrane-bound proTGF-alpha may bind to EGF receptors on the surface of contiguous cells, induce receptor autophosphorylation, and thereby produce a rapid rise in A431 intracellular calcium levels. Thus, proTGF-alpha can be biologically active in the absence of processing, a fact that may have implications for the integral membrane precursors of related growth factors.

Amino Acid Sequence↗
PubMed1988

Characterization of high molecular weight transforming growth factor alpha produced by rat hepatocellular carcinoma cells.

In addition to the mature 50 amino acid transforming growth factor alpha (TGF alpha), some transformed cells appear to produce multiple higher molecular weight forms. The structure and derivation of most of these larger soluble TGF alpha species remain to be established. We previously reported that a chemically induced rat hepatocellular carcinoma cell line, JM1, secreted acid-stable proteins which bind to epidermal growth factor receptors and stimulate DNA synthesis in primary cultures of normal adult rat hepatocytes. Purification and characterization of these hepatoma-derived growth factors have indicated their relationship to TGF alpha. Two EGF-competing activities of apparent Mr 30K and 10K were separated by gel filtration of concentrated JM1-conditioned medium and further purified by ion-exchange chromatography and reverse-phase HPLC. Both growth factors were detected by a radioimmunoassay specific for TGF alpha. Western blotting with antibodies to the 50 amino acid TGF alpha revealed that the lower molecular weight factor comigrated with the synthetic 6-kDa rat TGF alpha. The higher molecular weight TGF alpha appeared on immunoblots as a diffuse band of 18-21 kDa, which converted to the mature 6-kDa form upon digestion with elastase, confirming a precursor-product relationship. However, the 18-21-kDa proteins did not react with antibodies directed against the carboxy-terminal cytoplasmic segment of the transmembrane TGF alpha precursor. Enzymatic deglycosylation of the 18-21-kDa TGF alpha species by sequential removal of sialic acids and O- and N-linked carbohydrate reduced the molecular weight to 11K. The size and soluble nature of this polypeptide suggest that it represents the extracellular domain of the transmembrane TGF alpha precursor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Lipid-protein interaction. The incorporation of myelin proteolipid apoprotein into phosphatidylcholine bilayers.

Bovine myelin proteolipid apoprotein (PLA), obtained in high yield and purity by a novel ultrafiltration procedure, has been used to study the perturbations produced by this protein on phosphatidylcholine bilayers, using infrared spectroscopy, nuclear magnetic resonance and fluorescence polarisation. PLA interacts with phospholipids in a similar manner to other intrinsic proteins. For bilayers in the fluid state, the fatty-acyl chain static order, as measured by deuterium NMR, is slightly increased in the presence of the protein, except at very high PLA concentrations. Phosphorus NMR reveals some perturbation of the phospholipid polar group by PLA, but to a smaller degree than occurs with other intrinsic proteins. An increase in static order above tc (the onset temperature for gel-to-fluid transition) is also detected by infrared spectroscopy. Studies using steady-state polarisation of diphenylhexatriene fluorescence indicate that the microviscosity of the bilayer increases as a function of the protein mole fraction. From these data an estimation of the average number of lipids perturbed per protein monomer has been made, and a figure of 37 phospholipid molecules determined. The data are compatible with a picture of a hydrophobic polypeptide, perturbing the phospholipids close to it, but allowing rapid (greater than 10(4) s-1) exchange with all the lipid molecules in the system.

Apoproteins↗
PubMed1988

Extraction of polyurethane block copolymers: effects on bulk and surface properties and biocompatibility.

In order to study changes occurring in polyurethane block copolymers upon solvent extraction, a base polymer containing approximately 50% polyurethane hard segment based on 4,4'-bis(p-phenyl isocyanate), 1,4-butanediol, and poly(tetramethylene oxide) of MW 1000 was synthesized. Portions of this polymer were extracted using methanol, toluene, and acetone. Multidetector gel permeation chromatography was used to characterize the effect of extraction on molecular weight and molecular weight distribution. Extraction also affected bulk and surface properties and the blood compatibility as assessed using a canine ex vivo blood-contacting experiment. Extracted materials possessed a higher molecular weight than the base polymer and had narrower molecular weight distributions. Acetone extraction resulted in the polymer with the highest ultimate tensile strength. Contrary to expectations, the surface properties and blood compatibility of the material studied were affected minimally by extraction.

Biocompatible Materials↗
PubMed1988

Expression of transforming growth factor alpha during development.

Transforming growth factors (TGFs) are polypeptides that are produced by transformed and tumour cells, and that can confer phenotypic properties associated with transformation on normal cells in culture. One of these growth-regulating molecules, transforming growth factor alpha (TGF-alpha), is a 50 amino acid polypeptide that is related to epidermal growth factor (EGF) and binds to the EGF receptor. Previous studies have shown that TGF-alpha is expressed during rodent embryogenesis between 7 and 14 days gestation. To investigate the cellular sites of TGF-alpha mRNA expression during development, we have performed Northern analyses and in situ hybridization histochemistry on the conceptus and maternal tissues at various gestational ages. Contrary to previous reports, both Northern analyses and in situ hybridization histochemistry indicate that TGF-alpha mRNA is predominantly expressed in the maternal decidua and not in the embryo. Decidual expression is induced following implantation, peaks at day 8, and declines through day 15 when the decidua is being resorbed. In situ hybridization revealed that expression of TGF-alpha mRNA is highest in the region of decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and embryo. In addition, we could not detect TGF-alpha mRNA expression in other maternal tissues, indicating that the induction of TGF-alpha transcripts in the decidua is tissue specific, and not a pleiotropic response to changes in hormonal milieu that occur during pregnancy. The developmentally regulated expression of TGF-alpha mRNA in the decidua, together with the presence of EGF receptors in this tissue, suggests that this peptide may stimulate mitosis and angiogenesis locally by an autocrine mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Expression of somatomedin/insulin-like growth factor messenger ribonucleic acids in the human fetus: identification, characterization, and tissue distribution.

Several lines of evidence indicate that multiple human fetal tissues synthesize somatomedins/insulin-like growth factors (Sm/IGFs). To investigate the synthesis of Sm/IGFs in vivo, we isolated polyadenylated RNAs from multiple human fetal tissues of 16-20 weeks gestation and performed Northern and dot-blot analyses using 32P-labeled cDNAs and oligodeoxyribonucleotide (oligomer) probes complementary to human Sm-C/IGF-I and IGF-II mRNAs. Sm-C/IGF-I mRNAs were present in all tissues studied. IGF-II mRNAs were detectable in all tissues except the cerebral cortex and hypothalamus. IGF-II mRNA levels were consistently higher than Sm-C/IGF-I mRNAs in all tissues where IGF-II mRNAs were detectable (varying from 2-fold higher in spleen and thymus to 650-fold higher in liver), suggesting that IGF-II is synthesized in greater quantities than Sm-C/IGF-I in most tissues during early fetal life. Liver, adrenal, and skeletal muscle had the highest levels of IGF-II mRNAs, while placenta and stomach had the highest level of Sm-C/IGF-I mRNAs. Multiple Sm-C/IGF-I and IGF-II transcripts were identified with estimated sizes 0.7, 5.3, and 8.0 kilobases (kb) for Sm-C/IGF-I and 1.8, 2.0, 2.8, 3.0, 4.0, 4.8, and 6.2 kb for IGF-II. The 5.3-kb species was the most abundant Sm-C/IGF-I mRNA. The largest Sm-C/IGF-I transcript (8.0 kb) was identified in the intestine, muscle, kidney, placenta, stomach, heart, skin, pancreas, hypothalamus, and brain stem and was most abundant in the hypothalamus and muscle. The smallest transcript (0.7 kb) was detectable only in spleen, adrenal, placenta, and stomach. On the other hand, nearly all species of IGF-II mRNAs were found in tissues with detectable mRNAs, with the 6.2-kb mRNA being the most abundant. The variation in abundance and species of Sm-C/IGF-I and IGF-II mRNAs among different human fetal tissues suggests tissue-specific differences in Sm-C/IGF-I and IGF-II gene expression, mRNA precursor processing, and/or mRNA stability. Such differences may have significance for the roles of Sm-C/IGF-I and IGF-II during human fetal development. The finding of Sm/IGF mRNAs in many human fetal tissues also supports a local role for Sm/IGFs in human fetal development.

Electrophoresis, Polyacrylamide Gel↗
PubMed1987

Changes in the secondary structure of apolipoprotein B-100 after Cu2+-catalysed oxidation of human low-density lipoproteins monitored by Fourier transform infrared spectroscopy.

Fourier transform infrared (FTIR) spectra have been obtained of human low-density lipoproteins (LDL) in H2O and 2H2O buffers. The absorption bands are assigned to vibrations of the lipid and apolipoprotein B-100 components. The analysis of second-derivative spectra allowed an assignment of individual protein bands to alpha-helical, random, coil or beta-structure and beta-turn conformations. Changes in the FTIR spectra after Cu2+-catalysed oxidation of the LDL particles indicate that the structure of apolipoprotein B-100 becomes less ordered, with some alterations of alpha-helical and beta-turn conformation. The main beta-structure absorption at 1620 cm-1 is unaffected by oxidation. Taking into account the resistance to oxidation and the slow H-2H exchange it is suggested that the beta-structure is hidden from external factors whereas other structures are mostly present on the surface of the LDL particle. Oxidation affects mainly the surface region of apolipoprotein B-100 and leads to a structural rearrangement which consequently changes the receptor specificity of the LDL.

Apolipoprotein B-100↗
PubMed1987

Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy.

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.

Calcium↗
PubMed1987

Fourier transform infrared spectroscopic study of the structure and conformational changes of the human erythrocyte glucose transporter.

Fourier transform infrared spectroscopy has been used to study the secondary structure of the human erythrocyte glucose transporter after purification and reconstitution in erythrocyte lipids. The spectra indicate that the glucose transporter contains, in addition to the predominant alpha-helical structure, an appreciable amount of beta-structure and random coil conformation. A study of the time dependency of H-2H exchange revealed that more than 80% of the polypeptide backbone is readily accessible to the solvent. This result indicates that a portion of the intramembrane-spanning region of the membrane protein is exposed to the solvent, suggesting the existence of an intraprotein aqueous channel. The residual (10-20%) portion of the protein which exchanges slowly includes some alpha-helical structure, probably situated in a hydrophobic environment inside the membrane. The infrared spectra of transporter preparations were also examined after incubation with substrate and substrate analogues. Compared with the spectra recorded under conditions in which the "inward-facing" form predominates, a small but reproducible shift in the bands assigned to alpha-helical and beta-strand structures is observed after incubation with 4,6-O-ethylidene-D-glucose, which largely fixes the transporter in the "outward-facing" conformation. An increase of temperature, which is known to increase the proportion of transporter in the outward-facing conformation, results in a similar shift in this alpha-helical absorption band.

Erythrocyte Membrane↗
PubMed1987

Bovine and human cDNA sequences encoding a putative benzodiazepine receptor ligand.

cDNAs containing the entire coding sequence of endozepine, a putative ligand of the benzodiazepine receptor, were isolated from bovine and human cDNA libraries. These libraries were constructed using a novel oligonucleotide adapter molecule that allowed us to combine the use of G/C tailing with the preservation of the unique Eco RI site in the vector, lambda gt10. The amino acid sequences derived from these cDNA clones are identical to those previously determined for the purified proteins and are homologous to a related rat protein termed diazepam-binding inhibitor. The endozepine proteins are highly conserved, as illustrated by the finding that the nucleotide sequences of the coding regions are 93% conserved between the bovine and human forms. Analysis of these sequences indicates that endozepine is not, as expected, derived from a precursor molecule containing a transient signal peptide. Moreover, Northern analyses using the cloned cDNAs as hybridization probes indicate that the 650-nucleotide endozepine mRNA is expressed in a number of peripheral tissues in addition to brain. These observations may be consistent with a recent report describing the presence in peripheral tissues of benzodiazepine receptors on the outer mitochondrial membrane (Anholt et al., 1986). In addition to the endozepine cDNAs, we also isolated a bovine cDNA clone which encodes a larger protein, a portion of which is homologous to endozepine. This related protein may be synthesized in a precursor form containing putative signal peptide and membrane-spanning domains.

Amino Acid Sequence↗
PubMed1987

Expression and characterization of transforming growth factor alpha precursor protein in transfected mammalian cells.

Analysis of a cDNA clone derived from retrovirus-transformed rat fibroblasts has recently suggested that the mature 50-amino-acid form of transforming growth factor alpha (TGF alpha) is derived from a 159-amino-acid transmembrane precursor by proteolytic cleavage. To understand the processing of the TGF alpha precursor molecule in more detail, we have expressed this protein in baby hamster kidney (BHK) fibroblasts under control of the metal-ion-inducible metallothionein promoter and characterized the expressed precursor with site-specific antipeptide antibodies. One of the BHK transfectants, termed 5:2, expressed the TGF alpha mRNA in a cadmium- and zinc-inducible manner. The TGF alpha precursor protein was detected by immunoprecipitation analysis of radiolabeled cell cultures. In the induced 5:2 cells, a polypeptide of Mr 13,000 to 17,000 was readily identified by peptide antisera made to three different regions of the TGF alpha precursor protein. No such protein species were observed in BHK cells treated with cadmium and zinc or in uninduced 5:2 cells. However, two cell lines known to produce TGF alpha naturally, Leydig testicular tumor cells and Snyder-Theilan feline sarcoma virus-transformed Fisher rat embryo fibroblasts, possessed detectable levels of immunologically related Mr 13,000 to 17,000 proteins. Cell fractionation studies indicate that the Mr 13,000 to 17,000 species expressed in induced 5:2 cells is membrane associated, consistent with predictions based on the cDNA sequence of the TGF alpha precursor. Media conditioned by induced 5:2 cells contained epidermal growth factor receptor-competing activity, which, upon size fractionation, was similar in size to the mature processed form of TGF alpha. These data show that these nontransformed BHK cells possess the ability to process the TGF alpha precursor molecule into its native form.

Animals↗
PubMed1987

Expression of rat transforming growth factor alpha mRNA during development occurs predominantly in the maternal decidua.

Previous studies have shown that transforming growth factor alpha is expressed during rodent development. To establish the site(s) of transforming growth factor alpha mRNA expression during rat embryogensis, we performed in situ hybridization and Northern blot analyses on samples of embryonic and maternal tissues at various gestational ages. Our results indicate that the high levels of transforming growth factor alpha mRNA that are observed during early development are the result of expression in the maternal decidua and not in the embryo. Decidual expression appears to be induced after implantation, peaks at day 8, and then slowly declines through day 15 at which time the decidua is being resorbed. Expression of transforming growth factor alpha mRNA is highest in that region of the decidua adjacent to the embryo and is low or nondetectable in the uterus, placenta, and other maternal tissues. The developmentally regulated expression of transforming growth factor alpha mRNA in the decidua, together with the presence of epidermal growth factor receptors in this tissue, suggests that transforming growth factor alpha stimulates proliferation locally through an autocrine mechanism. Since epidermal growth factor receptors are present in the embryo and placenta, transforming growth factor alpha produced in the decidua may also act on these tissues through paracrine or endocrine mechanisms.

Animals↗
PubMed1987

Transferrin messenger ribonucleic acid: molecular cloning and hormonal regulation in rat Sertoli cells.

Transferrin-specific cDNA clones were isolated from a rat liver cDNA library prepared from transferrin-enriched mRNA. Hybrid selection and sequence analysis confirmed that the selected clone contained the carboxy-terminal coding region of the transferrin mRNA. Northern blot analysis was used to demonstrate the presence of transferrin mRNA in liver and Sertoli cells. Transferrin mRNA levels were measured in total RNA isolated from cultured rat Sertoli cells after treatment with FSH, insulin, retinol, and testosterone. The results showed a 2- to 4-fold increase in the level of transferrin mRNA, which peaked on the fourth day of culture after initiation of treatment, with FSH, insulin, retinol, and testosterone. This induction is gene specific, since no change in the mRNA levels for either the catalytic or regulatory subunits of cAMP-dependent protein kinase was observed. The effects of hormones, vitamin A (retinol), and Bu2 cAMP on transferrin mRNA and transferrin secretion (measured by RIA) in cultured Sertoli cells were compared. In general, a direct relationship between the amount of transferrin mRNA present in the cells and the amount of transferrin secreted into the culture medium was observed. These results demonstrate the important role that vitamin A, testosterone, and peptide hormones play in modulating transferrin gene expression in Sertoli cells.

Animals↗
PubMed1987

The effects of temperature on biological membranes and their models.

The physical effects of temperature on biological membranes are reviewed. Our current understanding of membrane structure is based on a model in which proteins are inserted in or are attached to a lipid bilayer structure. The lipid composition varies between cell types. Phospholipid bilayers undergo phase transitions at temperatures which are dependent on chain length and the structure of the headgroup. These factors also affect the miscibility of phospholipid types. Cholesterol orders the phospholipid chains above their phase transition temperature but disorders them below this temperature. Cerebrosides, which are important constituents of the myelin membrane, form metastable states under certain conditions. The activities of membrane-bound enzymes may be affected by the physical state of the lipid bilayer which, in turn, is affected by temperature. The interactions between the lipid and protein components of membranes have been investigated by a variety of techniques.

Animals↗
PubMed1986

A Fourier transform infrared investigation of the structural differences between ribonuclease A and ribonuclease S.

Fourier transform infrared spectroscopy was used to investigate the small conformational differences which exist between ribonuclease A and ribonuclease S in aqueous systems. Deconvolution and derivative methods were used to observe the overlapping components of the amide I and II bands. These proteins give identical spectra in H2O and after complete exchange in 2H2O. However structural differences are revealed by monitoring the rate of 1H-2H exchange by Fourier transform infrared spectroscopy. At equivalent times of exposure in 2H2O buffer ribonuclease S undergoes greater isotopic exchange than ribonuclease A. Thus complete exchange takes place for ribonuclease S but not ribonuclease A after incubation at room temperature for 8 days. Complete 1H-2H exchange of ribonuclease A was achieved by incubation at 62 degrees C for 30 min. The available X-ray data and comparison with the infrared spectra of other soluble proteins was used to assign the components of the amide I and II bands to various secondary structures. In particular, band shifts observed during the later stages of exchange are associated with slowly exchanging residues in beta-strand and alpha-helical regions. The higher rate of exchange for ribonuclease S is associated with a greater conformational flexibility and a more open structure. The results show that it is necessary to be cautious in making band assignments based on exchange methods unless the extent of exchange is known. Furthermore, it is seen that the combination of Fourier transform infrared spectroscopy and hydrogen-deuterium exchange is a powerful technique for revealing small differences in protein secondary structure.

Fourier Analysis↗