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D C Lee

Publications and source records attributed to D C Lee.

At least 91 records · Page 5Linked to original sources
PubMed1994

Expression of EGF receptor mRNA in rat nigrostriatal system.

In situ hybridization with a cRNA probe was used to determine the cellular expression of mRNA for the epidermal growth factor receptor (EGF-R) within the rat dopaminergic nigrostriatal system. Hybridization for EGF-R mRNA was distributed throughout the ventral mesencephalon within cells of the substantia nigra pars compacta and ventral tegmental area (VTA). Unilateral lesion of the nigrostriatal pathway with the neurotoxin 6-hydroxydopamine resulted in substantial elimination of EGF-R cRNA hybridization in the ipsilateral substantia nigra and VTA. EGF-R mRNA was also demonstrated within a population of striatal somata, with highest levels of expression detected neonatally. These results indicate that a subpopulation of ventral midbrain dopaminergic neurons, as well as striatal perikarya, express the EGF-R and, thus, may be directly responsive to endogenous EGF, transforming growth factor-alpha, or another EGF-R ligand in vivo.

Animals↗
PubMed1994

Purification and secondary structural analysis of tissue inhibitor of metalloproteinases-1.

In connective tissue diseases such as rheumatoid arthritis, the matrix metalloproteinases are the primary enzymes involved in tissue degradation. Tissue inhibitor metalloproteinases-1 (TIMP-1) is a specific inhibitor of these enzymes, which is thought to regulate their action in vivo. The structure and function of TIMP-1 may therefore be important as the basis for the rational design of therapeutic agents. This paper describes a simple and effective method for the purification of sufficient quantities of TIMP-1 for spectroscopic studies. Circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy have, together, showed TIMP-1 to be mostly in a beta-sheet conformation, with significant amounts of alpha-helix and beta-turn. Two-dimensional nuclear magnetic resonance spectroscopy indicated a correspondingly high proportion of beta-sheet. CD and FTIR have also shown TIMP-1 to have high thermostability.

Circular Dichroism↗
PubMed1994

Zeta-crystallin: a lens-specific promoter and the gene recruitment of an enzyme as a crystallin.

A novel enzyme with quinone oxidoreductase activity has undergone gene recruitment in certain mammals, acquiring a second function as the lens structural protein zeta-crystallin. Here we show that recruitment of this enzyme crystallin can be explained by the lens specificity of an alternative promoter which does not require host-specific factors. The strong lens preference of this promoter is apparent in both cultured cell transfections and in transgenic mice. While proximal regions of the promoter have some activity in the brain of transgenic mice this is abolished by the addition of more distal regions. The minimal active promoter is differentially footprinted by extracts from lens and non-lens cells. Deletion within the major region footprinted in lens, ZPE (zeta protected element), abolishes promoter function. This is the first example of a lens-specific promoter in an enzyme crystallin gene and the first demonstration of gene recruitment by this mechanism.

Animals↗
PubMed1994

The mouse waved-2 phenotype results from a point mutation in the EGF receptor tyrosine kinase.

Mice harboring the waved-1 (wa-1) and waved-2 (wa-2) mutations exhibit skin and eye abnormalities that are strikingly similar to those of TGF-alpha-deficient mice, and wa-1 and TGF-alpha were recently shown to be allelic. Because the wa-2 mutation was mapped previously to the vicinity of the EGF/TGF-alpha receptor (EGFR) gene on mouse chromosome 11, we hypothesized that the wa-2 phenotype might result from a defect in either the expression or activity of EGFR, or both. In the present report, we show that EGFR mRNA and protein of normal size are expressed in wa-2 liver and skin at levels that are comparable to those in the corresponding normal tissues, and that the ability of wa-2 EGFR to bind ligand is unaltered. However, ligand-dependent autophosphorylation of wa-2 EGFR is diminished 5- to 10-fold in vitro, and the ability of wa-2 EGFR to phosphorylate an exogenous substrate is reduced by > 90% compared with that of the control receptor. EGF-induced tyrosine phosphorylation, including that of EGFR itself, is also diminished in skin, particularly at lower dose of exogenous EGF. To establish the nature of the wa-2 mutation, we determined the nucleotide sequence of the coding region of normal and wa-2 murine EGFR cDNAs. A comparison of these sequences revealed a single-nucleotide transversion resulting in the substitution of a glycine for a conserved valine residue near the amino terminus of the tyrosine kinase domain. The importance of this mutation was confirmed by showing that its introduction into an otherwise normal EGFR markedly reduced the receptor's tyrosine kinase activity in transfected Chinese hamster ovary cells. Finally, in situ hybridization analysis demonstrated expression of EGFR predominantly in the outer root sheath of active hair follicles in neonatal mice. As we previously localized TGF-alpha mRNA to the inner root sheath, this pattern of EGFR expression is consistent with the effect of the wa-2 mutation on hair structure, and together with our previous characterization of TGF-alpha-deficient mice, reveals a critical role for signaling by this ligand/receptor system in skin.

Abnormalities, Multiple↗
PubMed1994

Conserved linkage of early growth response 4, annexin 4, and transforming growth factor alpha on mouse chromosome 6.

The mouse genes encoding early growth response 4 (Egr4), annexin IV (Anx4), and transforming growth factor alpha (Tgfa) have been mapped to a linkage group on mouse chromosome 6 that is conserved on human chromosome 2p11-p13. The genes are closely linked, with 0/215 recombinants between Anx4 and Tgfa and 1/215 recombinants between these genes and Egr4. The genes are located approximately 2 cM distal to mnd2, a mouse mutation causing neuromuscular disease. The results demonstrate that mnd2 is located at an internal position within this conserved linkage group.

Animals↗
PubMed1994

Protein interactions at Sp1-like sites in the TGF alpha promoter as visualized by in vivo genomic footprinting.

Transcription from the rat TGF alpha promoter initiates at two predominant sites (-188 and -58) in a G+C-rich region that does not contain TATA or CAAT motifs. Previous studies using transfected reporter constructs implicated the transcription factor Sp1 in active expression from the promoter, particularly from the -58 site (Chen et al., 1992; Shin et al., 1992). In the present report we have examined the functionality of two adjacent clusters of Sp1-like recognition sites that are located in the upstream portion of the promoter from -300 to -273. A double-stranded oligonucleotide, which spanned this region and contained the putative Sp1 elements, demonstrated similar gel-mobility shifts in the presence of both crude HeLa cells nuclear extract and pure Sp1 protein. Mutations that simultaneously altered several of the overlapping Sp1 elements significantly reduced the gel-mobility shift activity of this oligonucleotide probe and, when introduced into the promoter templates, inhibited transcription in vitro from the proximal -188 start site. To confirm the binding of protein to these sites in cells, we carried out an in vivo genomic footprinting analysis of this portion of the TGF alpha promoter in normal and transformed rat liver epithelial cell lines that express the endogenous gene at varying levels. This analysis revealed clear evidence of protein/DNA interaction at Sp1-like sites in the -300 and -273 region in cells actively expressing the gene but not in a normal, parental cell line that expressed very low levels of TGF alpha mRNA. Collectively, these results corroborate the functional importance of Sp1 binding elements in the -300 to -273 region, and together with previous findings, indicate that two clusters of Sp1 binding sites respectively determine levels of transcription from the -188 and -58 start sites. Our additional finding that Sp1 mRNA and protein were present at similar levels in normal and transformed cells that expressed the endogenous TGF alpha gene at markedly different levels, suggests that the activity of the TGF alpha promoter could be regulated via the accessibility of Sp1 protein.

Amino Acid Sequence↗
PubMed1994

Lens expression of TGF alpha in transgenic mice produces two distinct eye pathologies in the absence of tumors.

Transgenic mice carrying the rat TGF alpha minigene linked to the alpha A crystallin promoter were generated to investigate the effects of expression of this growth factor in the lens of the eye. All transgenic mice exhibited eye abnormalities in the absence of overt tumors, and two distinct and heritable phenotypes were observed. Five lineages produced 'squinting' transgenic mice characterized by microphthalmic eyes with severe lens and retinal dysplasia, and four lineages produced 'bulged' transgenic mice with eyes that exhibited enlarged globes, lens epithelial hyperplasia, anterior segment dysgenesis, and in some cases retinal dysplasia. The eye perturbations of both phenotypes were evident histologically by 1 week of age, and the eyes of squinting mice were abnormal during embryonic development. The squinting phenotype was dominant over the bulged phenotype in intercrosses, suggesting that position effects from the transgene integration site resulted in differences in TGF alpha expression between the two phenotypes. In situ hybridization showed that TGF alpha transgene expression was confined to the lens or lens rudiment of all transgenic eyes despite the involvement of non-lenticular tissues in the pathology. These results show that inappropriate expression of TGF alpha in the eye can disrupt the communication required to coordinate normal eye development.

Animals↗
PubMed1993

An avian alpha B-crystallin. Non-lens expression and sequence similarities with both small (HSP27) and large (HSP70) heat shock proteins.

alpha B-crystallin is multifunctional, serving as both a major structural protein in the lens and a small heat-shock protein (shsp) in other tissues in mammals. Cloning and Northern analysis show similarly that alpha B-crystallin mRNA is present in all mature tissues examined in a bird (Anas platyrhynchos), although there are some differences in the pattern of transcripts seen. Interestingly, sequence analysis not only shows that duck alpha B-crystallin is a member of the shsp family, as expected, but that this family shares more distant similarity with another heat shock protein family, the highly conserved HSP70s of both eukaryotes and prokaryotes. This raises the possibility that large and small hsps may share structural and perhaps functional features.

Amino Acid Sequence↗
PubMed1993

TGF alpha deficiency results in hair follicle and eye abnormalities in targeted and waved-1 mice.

To explore the physiological roles of transforming growth factor alpha (TGF alpha), we disrupted the mouse gene by homologous recombination in embryonic stem cells. Homozygous mutant mice were viable and fertile, but displayed pronounced waviness of the whiskers and fur, accompanied by abnormal curvature, disorientation, and misalignment of the hair follicles. Homozygous and, to a lesser extent, heterozygous mice displayed eye abnormalities of variable incidence and severity, including open eyelids at birth, reduced eyeball size, and superficial opacity. Histological examination revealed eyelid and anterior segment dysgenesis, corneal inflammation and scarring, and lens and retinal defects. Although TGF alpha deficiency affected skin and eyes, wound healing in these tissues was not impaired. Similar hair and eye defects have been previously associated with the recessive mutation waved-1 (wa-1), and Northern analysis revealed reduced expression of TGF alpha in wa-1 mice. Crosses between wa-1 homozygotes and TGF alpha-targeted mice confirmed that wa-1 and TGF alpha are allelic.

Alleles↗
PubMed1993

A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens.

A discrete 10-kDa polypeptide (10K) is expressed from early stages in the embryonic chicken lens. Since this has potential as a marker for lens cell development, chicken 10K and its homologues from mouse and human lenses were identified by protein sequencing and cloning. Surprisingly, lens 10K proteins appear to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), originally identified in activated human T cells. Using microdissection and PCR techniques, we find that expression of 10K/MIF is strongly correlated with cell differentiation in the developing chicken lens. Northern blot analysis shows that 10K/MIF is widely expressed in mouse tissues. These results suggest that proteins with MIF activity may have roles beyond the immune system, perhaps as intercellular messengers or part of the machinery of differentiation itself. Indeed, partial sequence of other small lens proteins identifies another MIF-related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations that may follow damage to the lens.

Amino Acid Sequence↗
PubMed1993

Changes in structure of alpha 2-macroglobulin upon reaction with trypsin as assessed by light scattering and differential scanning calorimetry.

Employing a combination of static and dynamic light scattering, as well as differential scanning calorimetry (DSC), the structural changes which appear in alpha 2-macroglobulin (alpha 2M) upon trypsin binding have been further characterized. Light-scattering measurements suggest that a 15% reduction in both the hydrodynamic radius and radius of gyration occurs when two molecules of trypsin complex to alpha 2M. Approx. 85% of this trypsin-induced compaction results from the binding of the first proteinase. A complementary result was obtained from DSC measurements in which the major fraction of the trypsin-induced conversion of alpha 2M to a single more thermally stable form results from interaction with the first proteinase molecule. These observations support a functionally asymmetric model of trypsin binding to alpha 2M in which the significant reduction in size of the complex is primarily due to the initial interaction of alpha 2M with a single proteinase molecule.

Calorimetry, Differential Scanning↗
PubMed1993

Carbonyl-metabolizing enzymes and their relatives recruited as structural proteins in the eye lens.

The refractive properties of the eye lens are determined by abundant soluble structural proteins known as crystallins. While some crystallins are common to most vertebrates, others are abundant only in groups of related species. These taxon-specific crystallins all turn out to be enzymes, apparently recruited by modification of gene expression without prior gene duplication. They include eta-crystallin, accounting for up to 25% of protein in elephant shrew lenses and apparently identical to cytoplasmic aldehyde dehydrogenase; rho-crystallin from frog lenses, a member of the same superfamily as aldose and aldehyde reductases; and zeta-crystallin, found in guinea pig and camel lenses, which is structurally related to alcohol dehydrogenase (ADH). Unlike ADH, zeta-crystallin requires NADPH rather than NAD+/NADH as cofactor. Molecular modelling of zeta-crystallin shows that amino-acid changes around the co-factor binding site are responsible for this change in affinity. Purified guinea pig lens zeta-crystallin has a substrate preference for orthoquinones which are reduced by a single electron transfer mechanism. cDNA sequencing of zeta-crystallin suggests that the expression in lens as a crystallin depends on a different gene promoter from that used predominantly in liver. The putative guinea pig zeta-crystallin lens promoter has now been assayed for function in transfection studies. Elements with positive and negative effects on transcription, at least one of which has tissue preferred function, have been defined. When introduced into transgenic mice this promoter exhibits tissue-specific expression in the lens. This is the first identification of a lens-specific, alternative promoter in an enzyme crystallin gene.

Aldehyde Dehydrogenase↗
PubMed1993

Transcription termination/polyadenylation occurs at multiple sites in the human type I interferon receptor gene.

Based on the previously reported sequence, we isolated an independent cDNA clone encoding a binding component of the human type I interferon receptor (IFN-R). This cDNA is identical to the published sequence except that it lacks 62 bases of 5' untranslated sequence and terminates at the first of two potential polyadenylation sites. In Northern blot analyses of poly(A)+RNAs from both IFN-sensitive and IFN-resistant Daudi cells, this cloned cDNA hybridized to a predominant mRNA of 2.4 kb, as well as to mRNAs of 1.8, 4.8, and 5.6 kb, and occasionally 6.9 kb. These various transcripts, which were also observed at similar levels in Raji B cells and two T-cell lines, Jurkat and MOLT-4, were detected after high-stringency washes, and by alternate probes corresponding to subfragments of the cDNA. In contrast, only the 4.8- and 5.6-kb transcripts hybridized to a polymerase chain reaction (PCR)-derived probe that corresponded to genomic sequences immediately down-stream from the second polyadenylation site. These results indicate that the latter transcripts arise from the same gene as the predominant 2.4-kb mRNA due to incomplete transcription termination at either of the known polyadenylation sites. Finally, Northern blot analysis of total RNAs revealed the presence of the predominant 2.4-kb type I IFN-R transcript in numerous tissues from second trimester human fetuses, suggesting that the type I IFN-R gene is constitutively expressed in multiple cell types.

Cell Line↗
PubMed1993

Cellular localization of transforming growth factor-alpha mRNA in rat forebrain.

The cellular localization of transforming growth factor-alpha (TGF alpha) mRNA in juvenile and adult rat forebrain was examined using in situ hybridization with a 35S-labeled cRNA probe. TGF alpha cRNA-labeled neuronal perikarya were distributed across many forebrain regions including the olfactory bulb, caudate-putamen, nucleus accumbens, olfactory tubercle, ventral pallidum, amygdala, hippocampal stratum granulosum and CA3 stratum pyramidale, and piriform, entorhinal, and retrosplenial cortices. TGF alpha cRNA-hybridizing cells were also localized to several thalamic nuclei and to the suprachiasmatic, dorsomedial, and ventromedial nuclei of the hypothalamus. In addition, labeled cells were present in regions of white matter including the corpus callosum, anterior commissure, internal and external capsules, optic tract, and lateral olfactory tract. Thus, both neurons and glia appear to synthesize TGF alpha in normal brain. Hybridization densities were greater in neuronal fields at 2 weeks of age compared with the adult, suggesting a role for TGF alpha in the development of several forebrain systems. Our results demonstrating the prominent and wide-spread expression of TGF alpha mRNA in forebrain, combined with the extremely low abundance of epidermal growth factor mRNA in brain, support the argument that TGF alpha is the principal endogenous ligand for the epidermal growth factor receptor in normal brain.

Animals↗
PubMed1993

Transforming growth factor alpha dramatically enhances oncogene-induced carcinogenesis in transgenic mouse pancreas and liver.

To characterize the effect(s) of transforming growth factor alpha (TGF alpha) during multistage carcinogenesis, we examined tumor development in pancreas and liver of transgenic mice that coexpressed TGF alpha with either viral (simian virus 40 T antigens [TAg]) or cellular (c-myc) oncogenes. In pancreas, TGF alpha itself was not oncogenic, but it nevertheless dramatically accelerated growth of tumors induced by either oncogene alone, thereby reducing the host life span up to 60%. Coexpression of TGF alpha and TAg produced an early synergistic growth response in the entire pancreas together with the more rapid appearance of preneoplastic foci. Coexpression of TGF alpha and c-myc also accelerated tumor growth in situ and produced transplantable acinar cell carcinomas whose rate of growth was TGF alpha dependent. In liver, expression of TGF alpha alone increased the incidence of hepatic cancer in aged mice. However, coexpression of TGF alpha with c-myc or TAg markedly reduced tumor latency and accelerated tumor growth. Significantly, expression of the TGF alpha and myc transgenes in hepatic tumors was induced up to 20-fold relative to expression in surrounding nonneoplastic liver, suggesting that high-level overexpression of these proteins acts as a major stimulus for tumor development. Finally, in both pancreas and liver, combined expression of TGF alpha and c-myc produced tumors with a more malignant (less differentiated) appearance than did expression of c-myc alone, consistent with an influence of TGF alpha upon the morphological character of c-myc-induced tumor progression. These findings demonstrate the importance of TGF alpha expression during multistage carcinogenesis in vivo and point to a major role for this growth factor as a potent stimulator of tumor growth.

Animals↗
PubMed1993

Regulation of fat and muscle development by transforming growth factor alpha in transgenic mice and in cultured cells.

Transgenic mice overexpressing transforming growth factor alpha (TGF-alpha) under control of the metallothionein promoter had, on average, 20% reductions in body and carcass weights compared to nontransgenic littermates. This loss resulted from significant decreases in the comparative weights of bone, muscle, and especially fat. Transgenic epididymal fat pads were reduced by 40-80%, and total body fat content by 50%, relative to control animals. Distal hindlimb muscle weights were 20% below normal, and other skeletal muscles were visibly smaller in size. Weight reductions were accompanied by decreases in the cellularity of transgenic fat pads and muscles and by decreases in the number and area of striated muscle fibers. These findings were not obviously attributable to differences in metabolic rates since transgenic and control mice displayed similar levels of energy expenditure per unit lean body mass. The effects of TGF-alpha on the development of these tissues could be mimicked in culture for fat but not muscle. Thus, TGF-alpha did not inhibit the differentiation of the mouse skeletal myoblast cell line C2C12 as evidenced by the expression of muscle-specific actin and fusion to form multinucleated myotubes. However, TGF-alpha repressed the differentiation of the preadipocyte cell line 3T3-F442A in a dose-dependent and reversible manner as judged by morphological conversion and diminished expression of mRNAs encoding the adipocyte-specific markers adipsin and glycerophosphate dehydrogenase. This repression, which occurred without marked stimulation of proliferation, was incomplete even in the presence of high concentrations of growth factor. Despite its effects on adipose development, introduction of the metallothionein-TGF-alpha transgene into the ob/ob genetic background did not suppress the marked obesity characteristic of this mutation. Finally, endogenous TGF-alpha epidermal growth factor receptor mRNAs were detected in normal adipose tissue, suggesting that regulation of adipogenesis by this growth factor may be physiological.

Adipose Tissue↗