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L P Fernando

Publications and source records attributed to L P Fernando.

At least 19 recordsLinked to original sources
PubMed2003

Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells.

The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.

Antibodies↗
PubMed2001

HIV transmissions from a window-period platelet donation.

Recently, blood centers began investigational testing for HIV RNA by pooled nucleic acid testing (NAT). A 35-year-old frequent platelet donor tested HIV p24 antigen positive, antibody negative before implementation of NAT. He made 2 platelet donations (day -4 and -11) immediately before testing positive for HIV. The donor's HIV seroconversion was monitored, and stored samples were tested retrospectively for HIV RNA. Platelet recipients were tested for HIV infection. The day -4 sample tested positive for HIV RNA by pooled and individual sample NAT. The day -11 sample tested negative for HIV RNA by both NAT tests. The 2 recipients of the day -4 platelets tested HIV RNA and p24 antigen positive. The recipient of the day -11 platelets could not be tested because he had died. HIV NAT would have prevented transmission of HIV had it been available at the time of this donor's HIV seroconversion.

Adult↗
PubMed2000

Suppression of Cox-2 and TNF-alpha mRNA in endotoxin tolerance: effect of cycloheximide, antinomycin D, and okadaic acid.

Lipopolysaccharide (LPS)-tolerant human promonocytic THP-1 cells produce decreased levels of inflammatory mediators such as eicosanoids and tumor necrosis factor alpha (TNFalpha) in response to LPS. We hypothesized that transcriptional repression by newly synthesized proteins may be a mechanism for the reduced cellular response to a secondary challenge with LPS. THP-1 cells were desensitized after a 3.5 h or 20 h pre-exposure to LPS (1 microg/mL) and subsequently challenged with LPS (10 microg/mL). In cells rendered tolerant by exposure to LPS for 20 h, LPS-induced expression of cyclooxygenase (Cox)-2 and TNFalpha mRNA was suppressed. Cycloheximide (10 microM) prevented the transcriptional down-regulation of Cox-2 mRNA and to a lesser extent, TNFalpha mRNA, in LPS-tolerant cells. Transcriptional arrest with actinomycin D stabilized steady-state expression of Cox-2 mRNA in naive and tolerant cells but destabilized TNFalpha mRNA expression in LPS-tolerant cells. The observation that in naive cells Cox-2 and TNFalpha mRNA levels subside at 3 to 4 h after LPS (10 microg/mL or 1 microg/mL) suggested that LPS tolerance may occur earlier. Therefore, in subsequent experiments, the effect of LPS pretreatment for only 3.5 h was examined. This abbreviated tolerance regimen diminished secondary LPS-induced Cox-2 mRNA expression but had a lesser effect on TNFalpha mRNA expression. However, cycloheximide augmented both Cox-2 and TNFalpha mRNA expression in this group. Also, the serine/threonine phosphatase inhibitor okadaic acid augmented Cox-2 and TNFalpha mRNA expression in the LPS-tolerant cells. Although LPS-induced TNFalpha production in LPS-tolerant cells was suppressed relative to the naive cells, okadaic acid induced comparable levels of TNFalpha in tolerant and naive cells. These findings support the concept that LPS tolerance is associated with induction of proteins that alter expression of certain genes. Expression of Cox-2 mRNA appears to be particularly sensitive to down-regulation and, to a lesser extent, TNFalpha mRNA. However, this seems to vary depending on the LPS pretreatment regimen. The ability of a phosphatase inhibitor to induce TNFalpha and expression of Cox-2 and TNFalpha mRNA in LPS tolerance suggests that there may be alterations in phosphorylation status of signaling pathways, transcriptional mechanisms, or post-transcriptional mRNA stability.

Cycloheximide↗
PubMed1999

Tolerance to LPS decreases macrophage G protein content.

The effects of tolerizing doses of LPS on mRNA and protein levels of three different G protein subunits were investigated to understand the mechanism(s) responsible for the reduction in Gialpha protein content in LPS tolerance. Tolerance was induced in rats using Salmonella enteritidis LPS (intraperitoneal route) with a single dose of 100 microg/kg. Peritoneal macrophages were harvested 6 and 24 h later. In some studies, a second dose of LPS 500 microg/kg was given on the following day, and peritoneal macrophages were harvested 5 days after the first injection. Macrophage RNA or a crude membrane fraction was prepared from macrophages, and the mRNA level or the protein content for Gialpha3, Gialpha2, and Gsalpha was analyzed using Northern or Western blots, respectively. Compared with the control levels, the message for Gialpha3 was reduced (p < .025) at 6 and 24 h and 5 day time periods after LPS treatment. The Gialpha2 mRNA was increased relative to the control levels (p < .05) at 6 h and 5 days after LPS treatment, respectively, and Gsalpha message was not significantly changed. The half-life of Gialpha3 mRNA was not significantly different in control versus tolerant macrophages. The Gialpha3 mRNA and membrane protein were not significantly changed by incubation with LPS for intervals up to 6 h in vitro. Macrophage membrane Gialpha3 and Gialpha1 and 2 protein content from tolerant rats were reduced compared with the controls at 6 and 24 h, respectively (p < .05). These studies are consistent with our previous observations of selective changes in macrophage Gialpha protein content in LPS tolerance and raise the possibility that this may affect signal transduction events in these cells.

Animals↗
PubMed1999

Protective effect of tyrphostin AG-556 on shock induced by endotoxin or gram positive bacteria.

The effects of tyrphostin AG-556 (TYR), a tyrosine kinase inhibitor, were evaluated on shock induced by lipopolysaccharide (LPS) or group B streptococcus (GBS) in rats. Mortality and mean survival time were monitored. Plasma 6-keto prostaglandin F1alpha (6-keto PGF1alpha) was also measured at four hours after LPS injection. The effects of TYR on the production of 6-keto PGF1alpha thromboxane B2(TXB2) and nitrite (NO) from LPS or GBS stimulated in vitro peritoneal rat macrophage were also examined. Salmonella enteritidis LPS (12 mg/kg, i.v. ) (n=6) produced severe shock (100% mortality). Simultaneous treatment with TYR (n=6) significantly (p < 0.01) extended mean survival time and 33% of rats survived. Plasma 6-keto PGF1alpha concentrations were increased in LPS controls, whereas TYR (5 mg/kg) significantly (p < 0.05) decreased the production. Animals treated with GBS/D-galactosamine (n=9) also exhibited shock with 100% lethality and TYR again prolonged survival time (p < 0.05) with 55% of the animals surviving. To evaluate direct effects of TYR on mediator production induced by LPS or GBS, rat macrophages were stimulated with heat-killed GBS or LPS with or without TYR. Supernatants were collected at 24 h for determination of TXB2, 6-keto PGF1alpha and NO. All mediators measured were significantly increased (p < 0.05) with LPS or GBS. TYR inhibited (p < 0.05) the production of all mediators from macrophages induced by LPS or GBS. The decrease in eicosanoids was associated with a reduction of the content of cyclooxygenase-2 (COX-2) as determined by western blotting. Collectively, these results suggest that TYR ameliorates toxic shock induced by LPS or gram positive bacteria. This protection is associated with suppression of macrophage mediator production.

6-Ketoprostaglandin F1 alpha↗
PubMed1998

Increased prostacyclin and PGE2 stimulated cAMP production by macrophages from endotoxin-tolerant rats.

Sublethal administration of lipopolysaccharide (LPS) renders rats tolerant to multiple lethal stimuli. Tolerant macrophages exhibit differential alterations in LPS-stimulated cytokine and inflammatory mediator release. Increased cAMP levels stimulated by PGE2 or prostacyclin (PGI2) result in differential effects on LPS-induced cytokine release and protect against the pathophysiological changes of endotoxemia. In the present studies, we sought to determine whether PGE2- and PGI2-stimulated cAMP levels are altered in tolerant macrophages. Incubation of macrophages with cicaprost or 11-deoxy-PGE1 in the presence of phosphodiesterase inhibitors resulted in significantly higher (2.5- to 6.5-fold) cAMP concentrations in tolerant macrophages compared with control. In contrast, isoproterenol-stimulated cAMP levels were not significantly different between control and tolerant cells. Also, incubation of tolerant macrophages with LPS did not result in significantly elevated cAMP levels. Prostacyclin (IP) receptor mRNA levels were significantly increased in tolerant cells compared with controls, whereas [3H]PGE2 binding and PGE2 EP4 receptor mRNA levels were not significantly changed. These studies suggest that LPS tolerance induces selective alterations in eicosanoid regulation of cAMP formation.

Adenylyl Cyclases↗
PubMed1997

Activation of the chicken metallothionein promoter by metals and oxidative stress in cultured cells and transgenic mice.

Cis-acting elements in the chicken metallothionein promoter were tested for their ability to direct responses of reporter genes to metal ions and oxidative stress in transfected mouse cells and in transgenic mice. In addition, protein interactions with the promoter were analyzed by the electrophoretic mobility shift assay. In transient transfection assays and in transgenic mice, 107-bp of the chicken MT promoter was sufficient to direct responses to Zn. This promoter region also directed response to oxidative stress in transfected cells and transgenic mice, but in transgenic mice, maximal responsiveness to oxidative stress apparently involved other elements in the proximal promoter region (307-bp). The proximal 200-bp of the promoter contains sequences homologous to a metal response element (-47-bp), Sp1 binding sites (-70-bp and -161-bp), and an antioxidant response element (-189-bp). Electrophoretic mobility shift assay demonstrated that metal response element binding activity was low in control Hepa cell nuclear extracts, but was induced 6-fold after 45 min of H2O2 treatment. In contrast, Sp1 binding remained unchanged, and no evidence for specific binding to the core antioxidant response element consensus sequence was obtained. These studies demonstrate that cis-acting elements mediating induction of metallothionein gene expression by metals and oxidative stress are present in the chicken metallothionein promoter and suggest a role for increased binding of the transcription factor MTF-1 to the metal response element(s).

Animals↗
PubMed1996

Avian metallothioneins: structure, regulation and evolution.

This article reviews studies of the molecular biology of the avian metallothionein (MT) genes. Analysis of cloned genes and/or cDNAs from chicken, turkey, pheasant and quail suggests that each of these species possesses a very simple MT gene family. The MT from these birds is a cysteine-rich protein of 63 amino acids that shares extensive structural homology with the mammalian MTs, and, remarkably, the deduced amino acid sequence of the major metallothionein is identical in each of these birds. The chicken MT gene is inducible by dietary or injected metal ions [i.e., zinc (Zn) and copper (Cu)], bacterial lipopolysaccharide and oxidative stress. Furthermore, it is expressed during liver development. The turkey and chicken MT genes are identical in gross structure to other functional MT genes. They consist of three exons separated by two intervening sequences. Comparisons of the nucleotide sequences of the turkey and chicken MT genes revealed regions of exceptionally high sequence conservation, suggesting important functions such as splicing, polyadenylation and transcriptional activation. Structure-function studies of the chicken MT promoter using transient transfection assays and transgenic mice have revealed cis-acting promoter sequences involved in the induction of chicken MT gene expression by metals ions. A metal-responsive enhancer was located in the proximal 107-bp of the chicken MT promoter in a region highly conserved in both the turkey and chicken MT genes. Function of this enhancer element apparently requires cooperation of transcription factors interacting with an Sp 1 binding site and a single palindromic metal-responsive element. In this regard, the structure of the proximal region of the chicken and turkey MT promoters is unique. Our current studies suggest that this enhancer regulates gene expression in a position-independent manner in transgenic mice.

Amino Acid Sequence↗
PubMed1995

Antibodies to the rat beta 3 subunit of the gamma-aminobutyric acid A receptors.

Polymerase chain reaction was used to amplify the cDNA region that codes for the large intracellular loop of the beta 3 subunit of the gamma-aminobutyric acidA/benzodiazepine receptors (GABAAR/BZDR) from rat brain. The amplified cDNA was inserted into the prokaryotic expression vector pGEX-3X and a fusion protein containing glutathione-S-transferase and beta 3 intracellular loop moieties was expressed in bacteria. The fusion protein was affinity-purified and it was used to raise a rabbit anti-beta 3 antiserum. The anti-beta 3 antiserum immunoprecipitated the gamma-aminobutyric acidA receptor from rat and bovine brain. Immunoblots of the affinity-purified GABAAR/BZDR from bovine brain revealed that the anti-beta 3 antiserum reacted with a 57 kDa peptide, whereas the monoclonal antibody 62-3G1 that recognized both beta 2 and beta 3 reacted with 55 and 57 kDa peptides. The anti-beta 3 antiserum showed specificity for the beta 3 subunit vs beta 2 and beta 1.

Animals↗
PubMed1995

Monoclonal antibodies to the human gamma 2 subunit of the GABAA/benzodiazepine receptors.

The large intracellular loop (IL) of the gamma 2 subunit of the cloned human gamma-aminobutyric acidA (GABAA) receptor (gamma 2 IL) was expressed in bacteria as glutathione-S-transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the gamma 2 IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABAA/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000-49,000-M(r) peptide band in immunoblots of affinity-purified GABAA receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABAA receptors in various brain regions.

Amino Acid Sequence↗
PubMed1993

Evidence of hepatitis in patients receiving transfusions of blood components containing antibody to hepatitis C.

When hepatitis C virus antibody (anti-HCV) enzyme immunoassay (EIA1) testing became available in 1990, we tested samples from previously transfused blood units, traced the recipients of reactive units, and evaluated the recipients for HCV infection during the 12 months after transfusion. Ten of 42 recipients of EIA1-reactive blood were anti-HCV reactive on follow-up by EIA1 and 12 were reactive by a second-generation assay (EIA2). Reverse transcriptase-polymerase chain reaction (RT-PCR) detected HCV RNA in 5 seronegative recipients. In all, 17 of 42 recipients (40%) of EIA1-reactive blood had evidence of HCV infection. In comparison, 54 surgery patients, who received either no transfusion or autologous EIA1-nonreactive blood, remained EIA1 nonreactive and RT-PCR negative for 1 year; 1 patient (1.8%) became EIA2 reactive (P < or = .01). Of the recipients of anti-HVC reactive blood transfusions (reactive by both EIA1 and a supplemental 4-antigen strip immunoblot assay [RIBA2]), 14 (93%) of the recipients had evidence of HCV infection compared with only 3 of 27 recipients (11%) of EIA1-reactive, RIBA2-nonreactive blood (P < or = .01). Thus, blood components reactive for anti-HCV EIA1 may have transmitted HCV up to 40% of the time, but blood components found reactive by both EIA1 and RIBA2 may transmit HCV with a frequency of greater than 90%.

Adult↗
PubMed1993

Antibodies to the human gamma 2 subunit of the gamma-aminobutyric acidA/benzodiazepine receptor.

A gamma-aminobutyric acidA (GABAA) receptor (GABAAR) gamma 2 subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage lambda gt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors lambda gt11 and pGEX-3X. Both beta-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the gamma 2IL were purified, and a rabbit antibody to the LacZ-gamma 2IL was made. The antibody reacted with the gamma 2IL of both LacZ and GST fusion proteins and immunoprecipitated the GABAAR/benzodiazepine receptor (GABAAR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABAAR/BZDR immunoblots with a wide peptide band of 44,000-49,000 M(r). Immunoprecipitation studies with the anti-gamma 2IL antibody suggest that in the cerebral cortex, 87% of the GABAARs with high affinity for benzodiazepines and 70% of the GABAARs with high affinity for muscimol contain at least a gamma subunit, probably a gamma 2. These results indicate that there are [3H]muscimol binding GABAARs that do not bind [3H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABAAR/BZDR antibodies indicate that the most abundant combination of GABAAR subunits in the cerebral cortex involves alpha 1, gamma 2 (or other gamma), and beta 2 and/or beta 3 subunits. These subunits coexist in > 60% of the GABAAR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20-25%) of the cerebellar GABAAR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain alpha 6, also contain gamma 2 (or other gamma) subunit(s). The alpha 1 and beta 2 or beta 3 subunits are also frequently associated with gamma 2 (or other gamma) and alpha 6 in these cerebellar receptors.

Antibodies↗
PubMed1991

Serological discrimination between HTLV-I and HTLV-II antibodies by ELISA using synthetic peptides as antigens.

Using the peptides from amino acids 100-130 of the HTLV-I gag protein, 175-199 of the HTLV-I env protein and the corresponding peptides of HTLV-II (amino acids 106 to 135 of the gag protein and 171 to 196 of the env protein), we tested for reactivity against antibodies by enzyme immunoassay in sera from HTLV-I and HTLV-II carriers. The peptides derived from the env proteins have high specificity for antibody binding. The peptide based on amino acids 175-199 of HTLV-I reacted with antibodies in sera from all HTLV-I carriers, and the peptide composed of amino acids 171-196 of HTLV-II reacted with antibodies in sera from all HTLV-II carriers. For the peptides derived from the gag proteins, we observed some cross-reactivity in sera from persons with anti-HTLV-I and anti-HTLV-II, due to antibody binding to the peptide corresponding to 12 amino acids from the C-terminal end of the gag protein. Separate enzyme immunoassays that used the four synthetic peptides as antigens clearly distinguished between serum with antibodies to HTLV-I or HTLV-II in various individuals and excluded false positive results using the particle agglutination assay that used a whole-virus lysate of HTLV-I as antigen.

Amino Acid Sequence↗
PubMed1991

Stable expression of a secreted form of the mouse IFN-gamma receptor by rat cells.

The biologic effects of IFN-gamma are mediated through a receptor that is expressed in relatively low abundance on normal mammalian cells. As a consequence, investigations of the physicochemical and ligand-binding properties of the purified receptor have been limited. The work reported here characterizes a secreted form of the receptor for mouse IFN-gamma, made by deletion of the nucleotides that code for the anchoring domain from a cDNA that encodes the receptor binding protein and its related signal peptide. When transfected into rat XC cells, this construct produced up to approximately 1 mg/liter of a secreted protein that had the characteristics of the native receptor. Both the secreted protein and its mRNA were of sizes that were consistent with loss of the transmembrane region. The protein was detectable by a mAb that is specific for an epitope that is found in the ligand binding site of the receptor for mouse IFN-gamma, as well as by a goat polyclonal IgG that is monospecific for the mouse IFN-gamma R. Supernates that contained the secreted protein blocked binding of IFN-gamma to mouse IFN-gamma R and inhibited in a dose-dependent manner the IFN-gamma-mediated priming of mouse bone marrow culture-derived macrophages for tumor cell killing. Availability of relatively large amounts of a secreted protein that retains ligand-binding activity should facilitate purification and basic studies of the receptor binding protein and could provide new approaches to the treatment/prevention of diseases that arise due to inappropriate response of cells to IFN-gamma. In addition, because this secreted receptor, unlike others, consists of both the extracellular and intracellular domains, it is likely that it will be useful in determining how the cytoplasmic portion of the receptor is involved in receptor function.

Amino Acid Sequence↗
PubMed1991

Relative prevalence and risk factors of HTLV-I and HTLV-II infection in US blood donors.

The clinical significance of human T-cell lymphotropic virus type II (HTLV-II) infection, unlike that of HTLV-I, is unknown, and the major known association of HTLV-II seropositivity is with intravenous drug abuse. Screening of blood donors for HTLV-I, now routine in North America, does not distinguish this retrovirus from HTLV-II. To find out more about the seroepidemiology of and risk factors for HTLV I and II, blood from 480,000 volunteer donors in five geographically separate US urban centres was tested for antibodies to HTLV-I/II and HIV-1. Confirmed HTLV-I/II seropositive donors were then followed up by DNA amplification to distinguish type I from type II and by interviews focusing on possible risk factors. HTLV seroprevalence was 3.3 times greater than that for HIV-1 (0.043% vs 0.013%). DNA amplification on 65 of the 207 HTLV-I/II seropositive donors revealed that 34 (52%) had HTLV-II infection and 28 (43% had HTLV-I; 3 samples were uninformative. Interviews of 49 donors showed that whereas HTLV-I was principally associated with donor origin from endemic regions, the major risk factor for HTLV-II infection was intravenous drug use. The surprisingly high rate of HTLV-II infection in US blood donors raises important public health and donor counselling issues since HTLV-I infection is associated with adult T-cell leukaemia and a neurological disorder while the pathogenicity of HTLV-II is as yet unclear.

Adult↗
PubMed1989

The natural history of transfusion-associated infection with human immunodeficiency virus. Factors influencing the rate of progression to disease.

Patients infected by the human immunodeficiency virus (HIV) as a result of blood transfusions are unique in that their dates of infection are well defined and their medical conditions before infection are known. To characterize the natural history of transfusion-associated HIV infection, we studied 694 recipients of blood from 112 donors in whom AIDS later developed and from 31 donors later found to be positive for HIV antibody. Of the recipients tested, 85 were seronegative, 116 were seropositive, and 19 had AIDS. Of 101 HIV-seropositive recipients followed for a median of 55 months after infection, 54 had Centers for Disease Control Class IV disease, including 43 with AIDS. Life-table analysis suggested that AIDS will develop in 49 percent of infected recipients (95 percent confidence limits, 36 to 62 percent) within seven years after infection. As compared with recipients without AIDS, the 43 recipients with AIDS had received more transfusions at the time of infection (median, 21 vs. 7; P = 0.01). HIV-infected blood donors in whom AIDS developed were grouped according to whether AIDS developed within 29 months (the median) after donation (Group 1) or 29 or more months after donation (Group 2). As compared with the 31 recipients of blood from Group 2 blood donors, the 31 recipients of blood from Group 1 donors were more likely to have AIDS four years after infection (49 percent vs. 4 percent; P = 0.005) and illnesses resembling acute retroviral syndrome (14 of 24 vs. 5 of 22; P = 0.03). We conclude that most recipients of HIV-infected blood become seropositive, that AIDS develops in about half these recipients within seven years, and that the risk may be higher when AIDS develops in the blood donor soon after donation.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Cloning and expression of an avian metallothionein-encoding gene.

The chicken metallothionein gene (cMT), isolated from a chicken genomic DNA phage lambda library, was found to be approximately 1.5 kb in length and to consist of three exons, separated by two intervening sequences. The number and placement of the introns in the cMT gene is precisely the same as that in the mammalian metallothionein-coding genes. S1 nuclease mapping indicated a prominent transcription start point (tsp) 62 bp 5' to the translation start codon. The promoter region analyzed (623 bp) contained three regions of homology (at -47, -488, and -577 bp relative to the tsp) with the metal regulatory element (MRE) consensus sequence, and three potential Sp1 binding sites. Two of the putative MREs (-47, -577) were 12 to 14-bp palindromes, which suggests that they are binding sites for trans-acting proteins. The intact cMT gene was functional in mammalian cells, and the cMT promoter could confer metal responsiveness on the firefly luciferase cDNA (Luc) in transient expression assays. Deletion mutagenesis established that 107 bp of 5'-flanking sequence, containing the proximal MRE and a putative Sp1-binding site, were sufficient for transient expression and metal induction of the cMT promoter-Luc fusion gene.

Amino Acid Sequence↗