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Biomedical subjects

M E Lee

Publications and source records attributed to M E Lee.

At least 91 records · Page 5Linked to original sources
PubMed1996

Recovery of olfactory function in thirteen-day-old rats after olfactory bulb transplantation but not after olfactory bulb ablation.

Previous studies have shown recovery of olfactory ability along with reconnectivity of olfactory nerve (ON) following both olfactory bulb (OB) lesions and OB transplants (TX) when performed in newborn rats. The purpose of the present study is to correlate functional recovery with patterns of anatomical reconnectivity in older, postnatal (PN) 13-day-old rats (a possible critical period for plasticity in the system). Reinnervation of olfactory areas was seen in all OB TX animals regardless of the extent of functional recovery. Eight of nineteen animals with OB TXs demonstrated some degree of behavioral recovery. No reinnervation or behavioral recovery of OB lesion animals was observed. At this age, behavioral recovery is dependent upon reconnectivity within the system and transplantation may be required to facilitate this process.

Age Factors↗
PubMed1996

Suppression of interleukin-1beta-induced nitric-oxide synthase promoter/enhancer activity by transforming growth factor-beta1 in vascular smooth muscle cells. Evidence for mechanisms other than NF-kappaB.

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-beta1 down-regulates inducible NOS after its induction by interleukin (IL)-1beta by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5'-flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1beta or vehicle. IL-1beta increased the activity of the plasmid containing -1485 to +31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1beta-responsive elements. Further deletion analysis revealed that a construct containing -234 to +31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1beta stimulation. Mutation of the NF-kappaB site within this region partially reduced IL-1beta-inducible activity; however, a large portion of activity remained independent of the NF-kappaB site. TGF-beta1 suppressed promoter/enhancer activity after IL-1beta stimulation, and this suppression was complete in the construct with a mutated NF-kappaB site. In addition, TGF-beta1 did not decrease the binding of nuclear proteins to the NF-kappaB site. These data suggest that the ability of TGF-beta1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-kappaB motif in vascular smooth muscle cells.

Animals↗
PubMed1996

Molecular cloning and characterization of SmLIM, a developmentally regulated LIM protein preferentially expressed in aortic smooth muscle cells.

Differentiated, quiescent vascular smooth muscle cells assume a dedifferentiated, proliferative phenotype in response to injury, one of the hallmarks of arteriosclerosis. Members of the LIM family of zinc-finger proteins are important in the differentiation of various cells including striated muscle. We describe here the molecular cloning and characterization of a developmentally regulated smooth muscle LIM protein, SmLIM, that is expressed preferentially in the rat aorta. This 194-amino acid protein has two LIM domains, and comparisons of rat SmLIM with its mouse and human homologues reveal high levels of amino acid sequence conservation (100 and 99%, respectively). SmLIM is a nuclear protein and maps to human chromosome 3. SmLIM mRNA expression was high in aorta but not in striated muscle and low in other smooth muscle tissues such as intestine and uterus. In contrast with arterial tissue, SmLIM mRNA was barely detectable in venous tissue. The presence of SmLIM expression within aortic smooth muscle cells was confirmed by in situ hybridization. In vitro, SmLIM mRNA levels decreased by 80% in response to platelet-derived growth factor-BB in rat aortic smooth muscle cells. In vivo, SmLIM mRNA decreased by 60% in response to vessel wall injury during periods of maximal smooth muscle cell proliferation. The down-regulation of SmLIM by phenotypic change in vascular smooth muscle cells suggests that it may be involved in their growth and differentiation.

Amino Acid Sequence↗
PubMed1996

Increased proteolytic processing of protein tyrosine phosphatase mu in confluent vascular endothelial cells: the role of PC5, a member of the subtilisin family.

Cleavage and subsequent release of the extracellular domains of receptor protein tyrosine phosphatases (RPTP) occur at high cell density and may have an important role in regulating their activity. Because cleavage of RPTP occurs at a target motif (RXK/RR) recognized by a family of subtilisin/kexin-like endoproteases, we postulated that members of the subtilisin family may have an important role in this cleavage. We show in this report that the membrane-associated RPTPmu--both in its full 200-kDa form and as a 100-kDa cleavage product--is upregulated 4- and 7-fold, respectively, as human umbilical vein endothelial cells (HUVEC) approach confluence. To determine whether RPTPmu cleavage depended on PC5 (a subtilisin/kexin like endoprotease present in endothelial cells), we transfected COS cells with expression plasmids coding for RPTPmu and PC5 or the closely related protease PACE4. PC5, but not PACE4, cleaved RPTPmu, and RPTPmu cleavage was absent in COS cells transfected with an expression plasmid encoding a mutant PC5 whose active-site serine had been mutated to alanine. We also performed RNA blot analysis to determine whether PC5 expression was affected by confluence in HUVEC. PC5 mRNA levels were upregulated by more than 30-fold when confluence in HUVEC increased from 25% to 100%. These results indicate that PC5 may have an important role in mediating the cleavage of RPTPmu in response to contact inhibition in HUVEC.

Amino Acid Sequence↗
PubMed1996

Overexpression and purification of human immunodeficiency virus type 1 env derived epitopes in Escherichia coli.

In order to develop a reliable and inexpensive serodiagnostic method, a part of envelope gene of HIV-1, gp120' and gp41' (HIV-1 env a.a. 295-474 and a.a. 556-647) was cloned into a T7 expression vector (pET3d). The fusion protein (gp120'-gp41') was overexpressed in Escherichia coli, then purified to homogeneity by a simple gel filtration chromatography. Western blot analysis and enzyme-linked immunosorbent assay (ELISA) using the purified fusion protein showed a high sensitivity and a specificity for the detection of anti HIV-1 antibodies in testing human plasma. These results suggest that the expression scheme employing a direct expression vector and the rapid purification method are reliable and applicable for obtaining a large quantity of HIV-1 env protein for diagnoses of HIV-1 infections.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

Animals↗
PubMed1996

Induction of apoptosis by pyrrolidinedithiocarbamate and N-acetylcysteine in vascular smooth muscle cells.

Pyrrolidinedithiocarbamate (PDTC) and N-acetylcysteine (NAC) have been used as antioxidants to prevent apoptosis in lymphocytes, neurons, and vascular endothelial cells. We report here that PDTC and NAC induce apoptosis in rat and human smooth muscle cells. In rat aortic smooth muscle cells, PDTC induced cell shrinkage, chromatin condensation, and DNA strand breaks consistent with apoptosis. In addition, overexpression of Bcl-2 suppressed vascular smooth muscle cell death caused by PDTC and NAC. The viability of rat aortic smooth muscle cells decreased within 3 h of treatment with PDTC and was reduced to 30% at 12 h. The effect of PDTC and NAC on smooth muscle cells was not species specific because PDTC and NAC both caused dose-dependent reductions in viability in rat and human aortic smooth muscle cells. In contrast, neither PDTC nor NAC reduced viability in human aortic endothelial cells. The use of antioxidants to induce apoptosis in vascular smooth muscle cells may help prevent their proliferation in arteriosclerotic lesions.

Acetylcysteine↗
PubMed1996

Role of CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

CD44, the principal receptor for hyaluronic acid, is a widely distributed cell surface proteoglycan involved in cellular activation, proliferation, and migration. These processes are also central to the vascular smooth muscle cell's response to arterial wall injury. We evaluated the expression of CD44 and its isoform, CD44-V6, on vascular smooth muscle cells in vitro and in vivo and assessed the role of CD44 in DNA synthesis. Cultured vascular smooth muscle cells expressed CD44 and CD44-V6 at levels equal to or higher than those of the beta 1 and beta 2 integrins. In a rat carotid artery balloon injury model, CD44 and CD44-V6 mRNAs were unregulated in vascular smooth muscle cells after injury, and CD44 protein expression was greatest at the luminal edge of the growing neointima. CD44-expressing smooth muscle cells proliferated actively, and hyaluronic acid expression increased after injury in a temporal pattern similar to that of CD44. Through binding to hyaluronic acid, CD44 augmented DNA synthesis in cultured human and rat smooth muscle cells by 48 +/- 7.8 and 100 +/- 12.5%, respectively, an effect inhibited by an anti-CD44 antibody that blocked hyaluronate binding. These observations support a role for CD44 in the reaction of vascular smooth muscle cells to arterial wall injury.

Animals↗
PubMed1996

Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

Activating Transcription Factors↗
PubMed1996

Staurosporine resistance accompanies DNA tumor virus-induced immortalization and is independent of the expression and activities of ERK1, ERK2, cyclin A, cyclin-dependent kinase (cdk) 2, and cdk4.

Staurosporine, a potent protein kinase inhibitor, has been shown to arrest the growth of a number of normal cell types in the G1 phase of the cell cycle, while having little effect on several transformed lines. We wished to determine whether increased resistance to staurosporine was a common feature of virus-immortalized human cells and whether this phenotype was an early event following the expression of SV40 tumor antigens. Human foreskin keratinocytes immortalized by the SV40 DNA tumor virus displayed an increased resistance to staurosporine-induced growth arrest when compared with normal parental cells, as has been seen in human diploid fibroblasts. Keratinocytes immortalized by human papillomaviruses, or by just the human papillomavirus E6 and E7 oncogenes were also staurosporine resistant, suggesting that this phenotype often accompanies the immortalization of human cells by DNA tumor viruses. Acquisition of staurosporine resistance was a late event during immortalization, because precrisis human diploid fibroblasts that expressed the SV40 large T and small t antigens were not resistant to staurosporine. The same parental cells that were fully immortalized by SV40 were resistant. Staurosporine resistance was not the result of increased activities and/or expression of cyclin A, cyclin-dependent kinase (cdk) 2, cdk4, or the mitogen-activated kinases ERK1 and ERK2. Although increased activities and/or expression of cyclin A and cdk2 and cdk4 proteins, but not ERK1 or ERK2, were associated with immortalization, similar increases were found in staurosporine-sensitive precrisis cells expressing SV40 tumor antigens.

Antigens, Polyomavirus Transforming↗
PubMed1995

Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial growth factor.

KDR/flk-1 is one of two receptors for vascular endothelial growth factor, a potent angiogenic peptide. KDR/flk-1 is an early marker for endothelial cell progenitors, and its expression is restricted to endothelial cells in vivo. To investigate the molecular mechanisms regulating expression of KDR/flk-1, we cloned and characterized the promoter of the human KDR/flk-1 gene. The transcription start site was localized by primer extension and ribonuclease protection to a nucleotide 303 base pairs (bp) 5' of the initiation methionine codon. The 5'-flanking sequence is rich in G and C residues and contains five Sp1 elements but no TATA consensus sequence. By reporter gene transfection experiments, we found that approximately 4 kilobases of KDR/flk-1 5'-flanking sequence directed high level luciferase activity in bovine aortic endothelial cells; further deletion analysis revealed positive regulatory elements between bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. Mutation of an atypical GATA sequence between bp +105 and +127 did not affect promoter activity, suggesting that GATA elements are not essential for the high level promoter activity of this gene. Consistent with endothelial cell-restricted expression of KDR/flk-1 mRNA, we found that the 4-kilobase flanking sequence directed high level promoter activity in endothelial cells but not in other cell types. To our knowledge this is the first report characterizing the KDR/flk-1 promoter. Understanding the KDR/flk-1 promoter will allow us to investigate endothelial cell-specific gene regulation and to uncover methods for targeting gene delivery specifically to endothelial cells.

Animals↗
PubMed1995

Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP.

Carbon monoxide (CO) is a product of the enzyme heme oxygenase (HO; EC 1.14.99.3). In vascular smooth muscle cells, exogenously administered CO increases cyclic guanosine 3',5'-monophosphate (cGMP), which is an important regulator of vessel tone. We report here that smooth muscle cells produce CO via HO and that it regulates cGMP levels in these cells. Hypoxia, which has profound effects on vessel tone, significantly increased the transcriptional rate of the HO-1 gene resulting in corresponding increases of its mRNA and HO enzymatic activity. In addition, under the same conditions, rat aortic and pulmonary artery smooth muscle cells accumulated high levels of cGMP following a similar time course to that of HO-1 production. The increased accumulation of cGMP in smooth muscle cells required the enzymatic activity of HO, since it was abolished by a specific HO inhibitor, tin protoporphyrin. In contrast, N omega-nitro-L-arginine, a potent inhibitor of nitric oxide (NO) synthesis, had no effect on cGMP produced by smooth muscle cells, indicating that NO is not responsible for the activation of guanylyl cyclase in this setting. Furthermore, conditioned medium from hypoxic smooth muscle cells stimulated cGMP production in recipient cells and this stimulation was completely inhibited by tin protoporphyrin or hemoglobin, an inhibitor of CO production and a scavenger of CO, respectively. This report shows that HO-1 is expressed by vascular smooth muscle cells and that its product, CO, may regulate vascular tone under physiologic and pathophysiologic (such as hypoxic) conditions.

Animals↗
PubMed1995

Mutational analysis of the catalytic residues lysine 230 and tyrosine 160 in the NADP(+)-dependent isocitrate dehydrogenase from Escherichia coli.

Two site-directed mutants of isocitrate dehydrogenase (IDH) of Escherichia coli have been studied by site-directed mutagenesis kinetic and structural studies. Substitution of phenylalanine for tyrosine at position 160 (Y160F) showed 0.4% of the kcat of wild-type with isocitrate as substrate, while the Km for isocitrate remained unchanged. When the postulated intermediate, oxalosuccinate, was enzymatically decarboxylated, Y160F showed a higher kcat and a similar Km to the wild type values. The rate of reduction of oxalosuccinate to isocitrate by the Y160F mutant was greatly decreased relative to the wild-type. Substitution of methionine for lysine at position 230 decreased kcat to 1.1% of that of the wild-type and Km increased by a factor of 500-600. The decarboxylation of oxalosuccinate was undetectable for the K230M mutant. The structure of the site-directed mutants of IDH with and without a bound complex of isocitrate and Mg2+ was solved at 2.5 A resolution and compared by difference mapping against previously determined enzyme structures. The structural studies show that (i) the overall protein-folding side chain conformations and active sites of both mutants are isomorphous with wild-type enzyme, (ii) isocitrate and magnesium bind to both enzyme mutants with the same relative conformation and binding interactions as wild-type enzyme, and (iii) the mutated side chains (Phe 160 and Met 230) are positioned for catalysis in a similar conformation as that observed for the wild-type enzyme. Hence, the alteration of the side chain functional groups is directly related to the loss of enzyme activity. Possible roles of the active site tyrosine and lysine are discussed.

Catalysis↗
PubMed1995

Induction of vascular endothelial growth factor gene expression by interleukin-1 beta in rat aortic smooth muscle cells.

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen for vascular endothelial cells and promotes neovascularization in vivo. To determine whether interleukin-1 beta (IL-1 beta), which is present in atherosclerotic lesions, induces VEGF gene expression in vascular smooth muscle cells, we performed RNA blot analysis on rat aortic smooth muscle cells (RASMC) with a rat VEGF cDNA probe. IL-1 beta increased VEGF mRNA levels in RASMC in a time- and dose-dependent manner. As little as 0.1 ng/ml IL-1 beta increased VEGF mRNA levels by 2-fold and 10 ng/ml IL-1 beta increased VEGF mRNA by 4-fold. We also measured the half-life of VEGF mRNA and performed nuclear run-on experiments before and after addition of IL-1 beta to see if IL-1 beta increased VEGF mRNA levels by stabilizing the mRNA or by increasing its rate of transcription. The normal, 2-h half-life of VEGF mRNA in RASMC was lengthened to 3.2 h (60%) by IL-1 beta, and IL-1 beta increased the rate of VEGF gene transcription by 2.1-fold. In immunoblot experiments with an antibody specific for VEGF, we found that IL-1 beta increased VEGF protein levels in RASMC by 3.3-fold. Together these data indicate that IL-1 beta induces VEGF gene expression in smooth muscle cells. This IL-1 beta-induced expression of VEGF may accelerate the progression of atherosclerotic lesions by promoting the development of new blood vessels.

Animals↗
PubMed1995

Olfactory bulb transplants establish afferent and efferent connections with host forebrain in rat.

We are using wheat germ agglutinin-conjugated horseradish peroxidase (WHRP) to study reconnectivity in the transplanted (TX) olfactory bulb (OB) in Sprague-Dawley rats. Tritium-labeled OBs from fetal rat donors of Embryonic Days 14-15 were immediately grafted into neonatal rats in the site from which the host OB had been removed. Following survival times of 7 weeks and longer, WHRP solution was injected into the TX OB, and subjects were perfused after 24 h. The WHRP transport is seen in fibers from the TX OB into layer I of the host olfactory peduncle (OP) and olfactory cortex (OC) and in cell bodies in layers II and III of the OP and OC, the lateral hypothalamus, and the contralateral anterior olfactory nucleus (AON). These findings reaffirm that the axons from a TX OB make connections with some appropriate areas of the host brain and also indicate that axons from cells in the target areas of the host brain, including contralateral AON, reinnervate the TX OB.

Afferent Pathways↗
PubMed1995

The ATF site mediates downregulation of the cyclin A gene during contact inhibition in vascular endothelial cells.

Contact inhibition mediates monolayer formation and withdrawal from the cell cycle in vascular endothelial cells. In studying the cyclins--key regulators of the cell cycle--in bovine aortic endothelial cells (BAEC), we found that levels of cyclin A mRNA decreased in confluent BAEC despite the presence of 10% fetal calf serum. We then transfected into BAEC a series of plasmids containing various lengths of the human cyclin A 5' flanking sequence and the luciferase gene. Plasmids containing 3,200, 516, 406, 266, or 133 bp of the human cyclin A promoter directed high luciferase activity in growing but not confluent BAEC. In contrast, a plasmid containing 23 bp of the cyclin A promoter was associated with a 65-fold reduction in activity in growing BAEC, and the promoter activities of this plasmid were identical in both growing and confluent BAEC. Mutation of the activating transcription factor (ATF) consensus sequence at bp -80 to -73 of the cyclin A promoter decreased its activity, indicating the critical role of the ATF site. We identified by gel mobility shift analysis protein complexes that bound to the ATF site in nuclear extracts from growing but not confluent BAEC and identified (with antibodies) ATF-1 as a binding protein in nuclear extracts from growing cells. Also, ATF-1 mRNA levels decreased in confluent BAEC. Taken together, these data suggest that the ATF site and its cognate binding proteins play an important role in the downregulation of cyclin A gene expression during contact inhibition.

Activating Transcription Factor 1↗
PubMed1995

Cooperative interaction of GATA-2 and AP1 regulates transcription of the endothelin-1 gene.

Endothelin-1 (ET-1) is a 21-amino-acid vasoactive peptide initially characterized as a product of endothelial cells. Reporter gene transfection experiments have indicated that a GATA site and an AP1 site are essential for ET-1 promoter function in endothelial cells, and GATA-2 appears to be the active GATA factor which regulates ET-1 expression. To look for interactions between AP1 and GATA-2, transactivation experiments were performed with expression vectors encoding c-Jun, c-Fos, and GATA-2. Cooperativity between the AP1 complex and GATA-2 was observed as a synergistic increase in transcriptional activity of the ET-1 reporter plasmid. In addition, AP1 was able to potentiate the action of GATA-2 on reporter constructs lacking a functional AP1 site. In a similar fashion, GATA-2 was able to potentiate the action of AP1 despite deletion of the GATA site. Experiments with GATA-1 and GATA-3 expression vectors provided evidence that this capacity to interact with AP1 may be a characteristic of all GATA family members. Biochemical evidence for AP1-GATA interaction was provided by immunoprecipitation experiments. A GATA-2-specific antiserum was shown to immunoprecipitate in vitro-synthesized Jun and Fos protein from reticulocyte lysate. Also, antisera directed against Jun and Fos were able to immunoprecipitate from nuclear extracts a GATA-binding protein, indicating the association of AP1 and GATA proteins in vivo.

Base Sequence↗