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Biomedical subjects

T A Rado

Publications and source records attributed to T A Rado.

At least 19 recordsLinked to original sources
PubMed1998

PU.1 regulates the expression of the human neutrophil elastase gene.

PU.1 is a transcription factor present in B-cells and macrophages. Here, we report our studies on the role of PU.1 in myelopoiesis using human neutrophil elastase (HNE) as a model. HNE, a component of the primary granules of mature granulocytes, is a serine protease which is transcriptionally restricted to the late promyelocytic stage of granulocytic maturation. The first 200 bp of the HNE promoter directs myeloid specific expression of a reporter gene and a 30-bp element within this region was been identified as the major determinant of myeloid specific expression [S. Srikanth, T. Rado, A 30-bp element is responsible for the myeloid specific activity of the human neutrophil elastase promoter, J. Biol. Chem. 269 (1994) 32626-32632.]. We now show that the B-cell and macrophage specific transcription factor, PU.1, binds to the PU.1 consensus site within the 30-bp element to activate transcription. Substitution mutations within this recognition sequence results in the loss of PU.1 binding and in a 90% decrease in promoter activity in myeloid cells. Cotransfection of PU.1 and a reporter gene controlled by the HNE promoter into non-myeloid HeLa cells resulted in activation of reporter gene transcription.

Amino Acid Substitution↗
PubMed1994

A 30-base pair element is responsible for the myeloid-specific activity of the human neutrophil elastase promoter.

Human neutrophil elastase (HNE), a serine protease, is expressed only in the promyelocytic stages of granulocyte maturation. We examined several regions of the promoter for transcriptional activity and report that a 30-base pair (bp) element located between -76 and -106 in the 5'-flanking region of HNE is sufficient for myeloid-specific expression of HNE. Gel shift assays using nuclear extracts from myeloid and non-myeloid cells reveal several myeloid-specific complexes binding to the 30-bp element. Examination of DNA-protein interactions shows that at least two myeloid-specific proteins of 38 and 55 kDa bind to this element. DNase I protection analysis reveals two distinct footprints between -80 to -91 and -94 to -104 within this element. Transient expression studies using deletion constructs of the HNE 5'-flanking region show that the 30-bp element is active in myeloid cells K 562 and U 937 but not in HeLa cells. Internal deletion of this element results in a 60-85% loss of promoter activity in myeloid cells. Additional functional studies also show that a 19-bp region between -112 and -131 contributes to transcriptional activity of the elastase promoter as well.

Base Composition↗
PubMed1994

Mutation in the negative regulatory element of the erythropoietin receptor gene in a case of sporadic primary polycythemia.

A 42-year-old Caucasian male with sporadic primary polycythemia has been followed by us for 13 years. During the time of observation, his hemoglobin had been stable, and he has never had an elevated white count or platelet count or any other stigmata of polycythemia vera (PV). Both of his parents, his three children, and all siblings have been hematologically normal. The in vitro culture of erythroid progenitors revealed an absence of autonomous erythropoietin (Epo)-independent erythroid colonies but demonstrated a marked increase in the sensitivity of erythroid progenitors to Epo. We have undertaken a study designed to determine whether a mutation in the Epo receptor (Epo-R) gene could cause the polycythemia phenotype seen in either dominant or recessive primary polycythemia described by us and others, or in polycythemia vera. We have sequenced the cytoplasmic positive and negative regulatory domains of the Epo-R genomic DNA, and a transversion of C to T in nucleotide 6148 was found in one of the patient's chromosomes. This mutation is located in the negative regulatory domain and results in a change from proline to serine (P488S). We have subsequently analyzed more than 40 chromosomes from unrelated normal subjects, as well as autosomal dominant, recessive, and sporadic primary polycythemia and polycythemia vera subjects. In no instance was the same or any other mutation in the Epo-R found. To determine if this Epo-R mutation is a cause of increased sensitivity of erythroid progenitors to erythropoietin, Ba/F3 cells (interleukin-3-dependent murine lymphoid line) were transfected with normal and mutated Epo-R cDNA, rendering the transfected cells viable and able to proliferate in Epo. Transfectants with wild-type and mutant Epo-R cDNA exhibited no difference in the presence of Epo. More recently, we were able to obtain DNA from the seven family members of the propositus and found that the nonpolycythemic mother and one of the siblings have the same Epo-R mutation. We conclude that this first described mutation of Epo-R encountered in humans does not appear on its own to explain the polycythemia phenotype; however, the possibility that it may interact with some other acquired or congenital abnormality in generating the polycythemia phenotype cannot be excluded.

Adult↗
PubMed1992

Differential expression of two sodium channel subtypes in human brain.

Two partial human brain sodium channel cDNA sequences (designated HBSC I and II) have been cloned and mapped to chromosome 2q23-2q24 by chromosome microdissection-PCR (CMPCR). The distribution of HBSC I and II mRNA in human brain was studied by means of a novel approach based on the ligase detection reaction. These studies demonstrate that HBSC I and II mRNA is heterogeneously distributed in brain, and that the relative ratio of the two forms can vary as much as 7-fold between different regions.

Amino Acid Sequence↗
PubMed1992

Recombinant synthesis, purification, and nucleotide binding characteristics of the first nucleotide binding domain of the cystic fibrosis gene product.

The majority of mutations which lead to clinical cystic fibrosis are located within the two predicted nucleotide binding domains of the cystic fibrosis gene product. We have used a prokaryotic expression system to synthesize and purify the first nucleotide binding domain (NBD-1, amino acids 426-588) with and without the most common mutation associated with the disease (the deletion of phenylalanine at position 508, delta F508). Both wild type and delta F508 NBD-1 bind ATP-agarose in a quantitatively comparable manner; this binding was inhibited by excess Na2ATP, trinitrophenol-ATP, or 8-azido-ATP. Irreversible NBD-1 labeling by an ATP analog was demonstrated using [32P]8-azido-ATP. This covalent labeling was inhibited by preincubation with Na2ATP, with half-maximal inhibition for Na2ATP occurring at approximately 5 mM for both the wild type and delta F508 nucleotide binding domain. These experiments are among the first to confirm the expectation that the cystic fibrosis transmembrane conductance regulator NBD-1 binds nucleotide. Since, under the conditions used in our study, NBD-1 without phenylalanine 508 displays very similar nucleotide binding characteristics to the wild type protein, our results support previous structural models which predict that the delta F508 mutation should not cause an alteration in ATP binding.

Adenosine Triphosphate↗
PubMed1992

Transfection of wild-type CFTR into cystic fibrosis lymphocytes restores chloride conductance at G1 of the cell cycle.

We complemented the Cl- conductance defect in cystic fibrosis lymphocytes by transfection with wild-type cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR). Stable transfectants were selected and subjected to molecular and functional analyses. We detected expression of endogenous CFTR mRNA in several CF and non-CF lymphoid cell lines by PCR. Expression from cDNA in the transfectants was demonstrated by amplifying vector-specific sequences. Both fluorescence and patch-clamp assays showed that transfectants expressing wild-type CFTR acquired properties previously associated with Cl- conductance (GCl) regulation in non-CF lymphocytes: (i) GCl was elevated in the G1 phase of the cell cycle, (ii) cells fixed at G1 increase GCl in response to increased cellular cAMP or Ca2+, (iii) agonist-induced increases in GCl were lost as the cells progressed to the S phase of the cell cycle. The cell cycle and agonist dependent regulation of GCl was not observed in CF lymphocytes transfected with CFTR cDNA containing stop codons in all reading frames at exon 6. Our findings indicate that lymphocytes express functional CFTR since wild-type CFTR corrects the defects in Cl- conductance regulation found in CF lymphocytes. Evaluation of the mechanism of this novel, CFTR-mediated regulation of GCl during cell cycling should provide further insights into the function of CFTR.

B-Lymphocytes↗
PubMed1992

Expression and regulation of the cystic fibrosis gene during rat liver regeneration.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are responsible for cystic fibrosis. The CFTR gene has recently been identified and encodes a 6.5-kb mRNA transcript. Recent observations showing that CFTR expression increases during differentiation of epithelial cells suggested that CFTR may also be regulated in the liver in response to partial hepatectomy (PH). We studied the expression of CFTR in rat regenerating liver and investigated the mechanisms that regulate CFTR RNA levels during a 120-h period after PH. Northern and slot-blot analysis revealed a liver-specific biphasic increase of CFTR mRNA levels, which peaks at 2 and 24 h post-PH. In contrast to these findings, the mode of regulation of the homologous gene MDR-1 showed a clearly different pattern. Nuclear run-on analysis demonstrated increased levels of CFTR transcription corresponding to the time points where an increase in CFTR message was observed. Similarly, the beta-actin gene, which increases transiently during liver regeneration, showed increased nuclear run-on activity 4 h posthepatectomy, indicating that the nuclei were functional. No increase of MDR-1 gene transcription was detected, confirming the previous finding that the increase in MDR-1 mRNA level in regenerating liver results from a post-transcriptional event such as message stabilization. This study indicates that expression of the CFTR gene is regulated during the regenerative process of the liver. The data also suggest that the increase in CFTR and MDR expression levels result from two distinct regulatory mechanisms.

Animals↗
PubMed1992

Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes.

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.

B-Lymphocytes↗
PubMed1991

Expression of the human neutrophil elastase gene: positive and negative transcriptional elements in the 5' flanking region.

The structure and position of cis-acting DNA sequences which regulate tissue specific expression of the human neutrophil elastase (HNE) gene have been investigated. We have identified a positive and a negative regulatory element upstream from the promoter region. The ability of these sequences to regulate transcription in myeloid and non-myeloid cells was studied by inserting varying lengths of HNE 5'-flanking sequence into a reporter plasmid containing the bacterial chloramphenicol acetyltransferase (CAT) gene. CAT activity in U937 was minimal in the absence of promoter and in the presence of HNE sequence to -102 bp. Inclusion of sequence up to -153 bp resulted in a 5.6-fold increase in CAT activity that was not observed in non-myeloid transfectants. Extension of the insert to include additional HNE sequence to -196 bp resulted in a decrease in CAT activity to control levels.

Base Sequence↗
PubMed1991

Expression of cloned human lactoferrin in baby-hamster kidney cells.

Human lactoferrin was expressed from a cloned cDNA introduced into mammalian cells in tissue culture. Total RNA was extracted from human bone marrow, and lactoferrin cDNA was synthesized by primer-specific polymerase chain reaction after oligo(dT)-primed first-strand synthesis. The cDNA was sequenced to confirm its identity with previously published human lactoferrin sequences and cloned into the eukaryotic expression vector pNUT. Recombinant vector DNA containing the human lactoferrin sequence was introduced into baby-hamster kidney (BHK) cells in culture, and stable transfectants were produced by dominant marker selection. Human lactoferrin was expressed from the metallothionein promoter of pNUT by Zn2+ induction. The protein was secreted into the tissue-culture medium and was subsequently purified to homogeneity in a single step. Initial characterization suggests that the protein expressed by BHK cells is identical with native human lactoferrin.

Amino Acid Sequence↗
PubMed1991

Regulation of human bone marrow lactoferrin and myeloperoxidase gene expression by tumor necrosis factor-alpha.

Lactoferrin (LF) and myeloperoxidase (MPO) are glycoproteins synthesized in early myeloid cells (promyelocytes, myelocytes) and stored in granules of polymorphonuclear neutrophilic granulocytes. Both proteins are involved in the host inflammatory response, and LF has been found to have myelosuppressive activity in vivo and in vitro. Little is known, however, about the regulation of their production. We investigated the stability of LF and MPO mRNA and the effects of purified recombinant human TNF-alpha on LF and MPO levels in normal human bone marrow. Low density human bone marrow cells were cultured in the presence or absence of actinomycin D (10 micrograms/ml) or TNF-alpha (200 U/ml). LF and MPO RNA levels were analyzed by Northern blots using, respectively, a 650-bp insert from the plasmid pHL41, and a 2.3-kb insert from the plasmid pMPO2 as probes. It was found that: 1) LF mRNA is a fairly stable molecule, with a half-life of between 8 and 9 h, whereas MPO is less stable, with a half-life of between 4 and 5 h; 2) TNF-alpha decreases both LF and MPO mRNA levels, an effect seen by 24 h with MPO mRNA and 48 h with LF mRNA; 3) nuclear run-on assays revealed that TNF decreases transcription of the LF gene by 70% and the MPO gene by 50%; and 4) the suppressive effect of TNF-alpha on LF and MPO mRNA levels is not due to cell killing or selective differentiation and is reversible.

Bone Marrow↗
PubMed1991

Direct amplification of a single dissected chromosomal segment by polymerase chain reaction: a human brain sodium channel gene is on chromosome 2q22-q23.

We have devised a general strategy for gene mapping based upon the direct amplification of a target sequence within a single microdissected Giemsa-banded chromosomal segment using the polymerase chain reaction. The usefulness of this approach was demonstrated by mapping a cloned human brain sodium channel (alpha subunit) gene sequence to chromosome 2q22-q23. When DNA from single, dissected chromosome segments 2q21-qter and 2q24-pter were used as templates, a sodium channel-specific 172-base-pair polymerase chain reaction product was obtained. This product was not synthesized when segments 2q21-pter and 2q24-qter were used. Chromosome microdissection-polymerase chain reaction is not only a simple, fast, and accurate method for gene mapping but also may offer significant advantages for other applications, such as cancer cytogenetics and linkage analysis.

Animals↗
PubMed1990

Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

B-Lymphocytes↗
PubMed1990

A cystic fibrosis pancreatic adenocarcinoma cell line.

We established a pancreatic adenocarcinoma cell line (CFPAC-1) from a patient with cystic fibrosis (CF) and assessed some of its properties. The cells show epithelial morphology and express cytokeratin and oncofetal antigens characteristic of pancreatic duct cells. Basal and stimulated levels of cAMP and cAMP-dependent protein kinase and the biophysical properties of single Cl- channels in CFPAC-1 are similar to those of airway and sweat gland primary cultures and Cl(-)-secreting epithelial cell lines. Anion transport and single Cl- channel activity was stimulated by Ca2+ ionophores but not by forskolin, cAMP analogs, or phosphodiesterase inhibitors. The cells express the CF gene and manifest the most common CF mutation, deletion of three nucleotides resulting in a phenylalanine-508 deletion. These properties have been stable through greater than 80 passages (24 months), suggesting that CFPAC-1 can serve as a continuous cell line that displays the CF defect.

Adenocarcinoma↗
PubMed1989

Current approaches to the molecular and physiological basis of cystic fibrosis.

Cystic fibrosis (CF) is the most common disease caused by a single gene abnormality within the caucasian population. Its severity of expression in homozygotes varies widely, and the disease involves multiple organ systems. During the past few years, major advances in CF research have been made. These advances have occurred primarily in the fields of physiology and molecular genetics. As a result of these advances, it is now generally accepted that the basic defect in CF is the inability of an epithelial chloride channel to respond to adrenergic stimulation in affected organs. The recent major breakthrough in CF research is the localization of the CF gene and identification of the mutation responsible for the majority of cases of CF. In this article, the evidence which has led to this conclusion, as well as possible mechanisms by which a mutation in a single codon can produce the CF defects are reviewed. Finally, new approaches to the characterization of the CF gene by complementation of the defect in immortal cell lines displaying the transport phenotype associated with CF are discussed.

Blood Proteins↗
PubMed1987

Expression of granulocyte colony-stimulating factor by human cell lines.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.

Base Sequence↗
PubMed1987

Characterization of a new human diploid myeloid leukemia cell line (PLB-985) with granulocytic and monocytic differentiating capacity.

A new human diploid cell line, designated PLB-985, has been established from the peripheral blood of a patient with acute nonlymphocytic leukemia (ANLL). Cells of this line are capable of granulocytic and monocytic maturation in the presence of inducing agents. By morphology, the analysis of surface antigens, and cytochemical staining PLB-985 cells are myelomonoblasts. Transmission electron microscopy reveals them to be devoid of neutrophilic primary or secondary granules and to have an open chromatin pattern with frequent nucleoli. The modal karyotype of the line is 46,XX, with no consistent marker chromosomes or recognizable translocations. Myelomonoblasts of this line form colonies in soft agar and induce tumors (chloromas) in nude mice. Growth of the cells in the presence of dimethyl sulfoxide, cis-retinoic acid, or dibutyryl cyclic adenosine monophosphate results in granulocytic maturation as determined by morphology, histochemical staining characteristics, and incorporation of 35S-methionine into the neutrophil primary granule proteinases elastase and cathepsin G. The tumor-promoting phorbol ester phorbol myristate acetate induces PLB-985 cells to differentiate as monocytes. Cells grown in the presence of this agent rapidly become adherent to plastic, display markedly increased phagocytosis of latex particles, stain positively for alpha-naphthyl acetate esterase, and lose the ability to synthesize the neutrophilic proteinases. Induction of differentiation along either pathway is accompanied by a marked decrease in myc oncogene transcription.

Antigens, Neoplasm↗