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B K Ray

Publications and source records attributed to B K Ray.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lipopolysaccharide-mediated induction of the bovine interleukin-6 gene in monocytes requires both NF-kappa B and C/EBP binding sites.

The interleukin-6 (IL-6) gene expression in bovine monocytes is highly induced following bacterial lipopolysaccharide (LPS) stimulation. To identify the promoter element(s) involved in the inducible transcription of IL-6, a 5'-flanking region containing 230 bp of the bovine IL-6 gene was linked to a reporter gene coding for bacterial chloramphenicol acetyltransferase (CAT) and analyzed for its ability to confer LPS-responsiveness to the reporter CAT gene in monocytic cells. Using mutant reporter genes, we demonstrate that although mutation in the NF-kappa B element produces the major loss of induction, both NF-kappa B and C/EBP elements are necessary for maximal transcriptional activation of the bovine IL-6 gene. Gel electrophoretic mobility-shift assays have detected induced DNA-binding activities in the LPS-stimulated monocytes. Further characterization has revealed the activation and interaction of C/EBP-alpha, C/EBP-beta (NF-IL6), NFKB1 (p50), and RelA (p65) to their specific binding elements present in the bovine IL-6 gene. These results suggest a model in which induction of C/EBP-alpha in differentiating monocytes contributes and synergizes with induced C/EBP-beta and NF-kappa B, which are activated following LPS stimulation, to mediate a high rate of IL-6 transcription under inflammatory conditions.

Animals↗
PubMed1994

Sp1-mediated transactivation of the rabbit alpha 1 acid glycoprotein-encoding gene involves a cis-acting element in the 5'-proximal promoter region.

Analysis of the regulatory promoter region of the rabbit alpha 1-acid glycoprotein (alpha 1-AGP)-encoding gene revealed the presence of a G + C-rich region that is a potential binding site for the transcription factor Sp1. DNase I footprinting and competition with Sp1-specific wild-type oligodeoxyribonucleotides showed that Sp1 interacts with a tandem array of GGGCGG motifs within the alpha 1-AGP promoter. Transfection assays using both liver and nonliver cells have demonstrated that these Sp1-binding elements are transcriptionally active and overproduction of Sp1 can significantly induce the expression of this gene. Previously, we have identified two adjacent C/EBP-binding elements just upstream from these Sp1-binding regions. We now demonstrate by both in vivo and in vitro analyses that C/EBP and Sp1 bind to the alpha 1-AGP promoter and transactivate the expression of this gene in an independent manner.

Animals↗
PubMed1994

Expression and structural analysis of a novel highly inducible gene encoding alpha 1-antitrypsin in rabbit.

alpha 1-Antitrypsin is a major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of neutrophil elastase that hydrolyzes structural proteins. This protein, in rabbit is expressed as three isoforms designated as F, S-1, and S-2. An inducible form of this protein has been cloned from an acute-phase cDNA library of rabbit liver. Structural study has revealed that the cloned cDNA is the S-2 isoform previously identified by partial peptide sequence analysis. The amino acid sequence of the reactive center of the S-2 form is not conserved and thus can be categorized as of unorthodox type. mRNA analysis has indicated that the transcription of the S-2 isoform which is normally present at a very low concentration increases more than 100-fold under inflammatory conditions while the expression of the F and S-1 isoforms changes about 1.5-fold under similar conditions. The S-2 isoform is biologically active and capable of inhibiting both elastase and chymotrypsin. The high level of induction of this active isoform of alpha 1-antitrypsin under inflammatory conditions and its apparent resistance to oxidation-mediated inactivation, common to the orthodox forms of alpha 1-antitrypsin, make the S-2 isoform suitable for gene therapy in diseases associated with alpha 1-antitrypsin deficiency.

Acute-Phase Reaction↗
PubMed1994

Expression of the gene encoding alpha 1-acid glycoprotein in rabbit liver under acute-phase conditions involves induction and activation of beta and delta CCAAT-enhancer-binding proteins.

Transcription of the gene encoding alpha 1-acid glycoprotein is highly induced during acute inflammation which has been previously shown to be mediated by some inducible members of the CCAAT-enhancer-binding (C/EBP) transcription-factor family. In this study, we demonstrate that the involved inducible C/EBP isoforms are C/EBP-beta and C/EBP-delta, and together they control the high-level induction of the alpha 1-acid glycoprotein gene in response to inflammatory signals. We observed that dephosphorylation severely inhibits the DNA-binding ability of C/EBP-delta and its transactivating potential increases in the presence of cellular phosphatase inhibitors, such as okadaic acid and sodium orthovanadate. These results suggest that C/EBP-delta is regulated by phosphorylation. Transient transfections using expression vectors of C/EBP-alpha, C/EBP-beta and C/EBP-delta have shown that while individually all three isoforms can transactivate the alpha 1-acid glycoprotein-chloramphenicol-acetyltransferase gene transcription, co-expression of C/EBP-alpha and C/EBP-beta isoforms results in lower levels of reporter gene expression than the levels predicted from their additive transactivation level. In vitro DNA-binding studies have shown that C/EBP-alpha and C/EBP-beta isoforms both interact and form complexes with the alpha 1-acid glycoprotein gene C/EBP-binding element under normal noninduced conditions during which alpha 1-acid glycoprotein is expressed at a very low level. Higher than additive levels of reporter gene expression are observed when combinations of C/EBP-delta and C/EBP-beta or C/EBP-delta and C/EBP-alpha are used. Together, these data demonstrate that C/EBP-beta and C/EBP-delta are the major proteins responsible for the acute-phase induction of alpha 1-acid-glycoprotein gene expression and they require phosphorylation for transactivation potential.

Acute-Phase Reaction↗
PubMed1994

Serum amyloid A gene expression under acute-phase conditions involves participation of inducible C/EBP-beta and C/EBP-delta and their activation by phosphorylation.

Serum amyloid A (SAA) is a plasma protein whose synthesis is markedly increased in the liver during the inflammatory process. Previous analysis of SAA promoter function implicated the involvement of the CCAAT/enhancer-binding protein (C/EBP) in controlling this process. In this study, using antibodies against three C/EBP isoforms in DNA-binding and Western blot (immunoblot) assays, we found that in response to inflammatory signals, both C/EBP-delta and C/EBP-beta are induced and that their interactions with the SAA promoter element are necessary for the increased SAA gene expression. Cotransfections of liver cells with an SAA promoter-linked reporter chloramphenicol acetyltransferase gene and murine sarcoma virus-expressed C/EBP-delta or C/EBP-beta confirm such phenomena. The increased transactivating ability in the presence of the cellular phosphatase inhibitors okadaic acid and sodium orthovanadate, coupled with the observation that dephosphorylation severely inhibits the DNA-binding ability in vitro, implicates a role of phosphorylation in the regulation of the activities of the C/EBP-delta isoform. Consistent with these findings, we have detected higher levels of DNA-binding activity of C/EBP-delta prepared from cells treated with phosphatase inhibitors. We also present evidence that C/EBP-delta is a phosphoprotein. These results suggest that C/EBP-delta is regulated by phosphorylation and, in conjunction with C/EBP-beta, is one of the major proteins responsible for the increased transcription of the SAA gene in response to inflammatory stimuli.

Animals↗
PubMed1993

Regulation of rabbit alpha 1-acid glycoprotein gene expression in acute-phase liver. Identification of inducible and constitutive proteins like CCAAT-enhancer binding protein that interact with the 5'-proximal promoter elements.

To identify the cis-acting DNA sequences responsible for inducible transcription of rabbit alpha 1-acid glycoprotein gene, 5'-flanking region containing 529 bp of this gene and its various 5'-deletions were linked to the reporter gene coding for the bacterial chloramphenicol acetyltransferase (CAT) and analyzed for their ability to confer cytokine-mediated inducibility to the reporter CAT gene in liver cells. Deletion analysis has identified a 151-bp region from the sequence -186 to -35, that contains the regulatory promoter element(s) responsible for stimulation mediated by cytokines present in the conditioned-medium. Using mobility shift assays, we have identified highly inducible nuclear factors in acute liver nuclear extract that interact with this regulatory promoter region. DNase I footprint analysis has revealed two adjacent nuclear factor binding sites and competition of DNA-binding activity has indicated that the distal element of these two sites has higher affinity for nuclear factors than the proximal one. Both of these two regions have been found to be capable of directing conditioned-medium-induced transcription. Studies on the characterization of nuclear factors binding to these elements have shown that they belong to a class of transcription factors called CCAAT-enhancer binding protein (C/EBP). Our results indicate that binding of C/EBP-like factors to the inducible promoter elements of rabbit alpha 1-acid glycoprotein gene is highly specific and the induction of this gene under acute-phase conditions may involve their participation.

Animals↗
PubMed1993

Functional NF-kappa B element in rabbit serum amyloid A gene and its role in acute phase induction.

Serum amyloid A (SAA) protein synthesis is highly induced in acute inflammatory conditions. Such induction process is primarily regulated at the transcriptional level and large amount of SAA mRNA has been found to accumulate in rabbit liver during acute inflammation. To identify the promoter element(s) involved in inducible transcription of SAA, a 5' flanking region of this gene has been fused to a reporter chloramphenicol acetyl transferase (CAT) gene and transfected into BNL liver cells. This DNA is capable of inducing synthesis of the reporter gene in response to lipopolysaccharide-stimulated monocyte-derived conditioned medium. Deletion analyses have shown that a region from -135 to -78 that contains a putative NF-kappa B element is responsible for the inducible function of the promoter. Gel shift assay has detected DNA-binding activity in the induced cells that appear to interact with the potential NF-kappa B element located within -112 to -78 of the SAA gene. Similar factor(s) have also been detected in the lipopolysaccharide-treated rabbit liver which is highly active in transcribing SAA mRNA. Appearance of these factors in acute-phase induced animals and their binding to the NF-kappa B-like element in the SAA proximal promoter region correlated with SAA mRNA synthesis suggests functional role of this promoter element in SAA gene expression under LPS-induced acute-phase condition.

Acute-Phase Proteins↗
PubMed1993

Analysis of the promoter element of the serum amyloid A gene and its interaction with constitutive and inducible nuclear factors from rabbit liver.

In an effort to identify regulatory elements of the serum amyloid A (SAA) gene that play a major role in its expression under acute-phase conditions, we studied the expression of a set of chimeric SAA-chloramphenicol acetyltransferase (CAT) plasmids containing a progressively deleted upstream 5' sequence of the SAA gene. Two regulatory regions (-314 to -135 and -135 to -31) capable of driving cytokine-induced transcription have been identified. Gel retardation assays revealed that the regulatory region located between positions -314 and -135 is a major site of interaction for highly inducible and constitutive nuclear proteins in acute-phase rabbit liver. DNase I footprint and competition analyses showed that this region contains two adjacent nuclear protein binding sites (between -191 and -140) with varying affinity for protein binding. Both of these binding sites are capable of driving cytokine-induced transcription of a reporter gene containing a minimal promoter. Detailed analyses of the inducible nuclear proteins that bind to this promoter element showed that they are homologues of the CCAAT/enhancer binding protein (C/EBP) family. Accumulation of the inducible nuclear factors under acute conditions, when maximal transcription activity has been reported, suggests a critical role for these proteins in the expression of the SAA gene.

Animals↗
PubMed1992

Identification of novel inducible nuclear factors that interact with the acute phase responsive promoter element of rabbit alpha 1-acid glycoprotein gene.

Alpha 1-acid glycoprotein (alpha 1-AGP) is a major acute phase protein that is highly inducible during hepatic acute-phase response. To identify the promoter element(s) required for increased gene transcription under acute condition a 529 bp 5'-flanking region of alpha 1-AGP gene is fused to the chloramphenicol acetyl transferase (CAT) reporter gene and introduced into BNL liver cells. This DNA fragment is capable of inducing synthesis of the reporter gene-product about ten-fold when transfected cells are exposed to conditioned medium from lipopolysaccharide-stimulated peripheral monocytes. Deletion analyses have shown that sequences located between -224 to -22 are capable of eliciting this inducible promoter function. Using electromobility shift assay we have identified two novel inducible nuclear factors from turpentine-induced acute liver that can interact with this regulatory promoter region. Our results indicate that binding of these two factors to the promoter region of alpha 1-AGP gene is highly specific in nature and the induction of this gene under acute condition may involve their participation.

Animals↗
PubMed1992

Cloning and structural characterization of a rabbit genomic DNA for alpha 1 acid glycoprotein.

The gene for rabbit alpha 1 acid glycoprotein (AGP) has been isolated from a lambda EMBL3 genomic DNA library. Isolated clone contains a 12 Kbp fragment of rabbit genomic DNA. Restriction endonuclease mapping has localized the gene within a 4.2 Kbp fragment spanning two EcoRI sites. Southern blot analysis of the rabbit genomic DNA and its comparison with the cloned gene indicates that there is only one gene for AGP present per genome. DNA sequence analysis of the cloned gene indicates that the entire gene, TATA box to the polyadenylation signal, is located within the 4.2 Kbp region and contains six exons representing the full-length cDNA described earlier (1). The 5'-end of alpha 1-AGP gene sequences from rabbit, human, rat and mouse have been compared. Such analysis reveals two conserved regions located between -63 bp and -36 bp and -29 bp and -1 bp of putative transcription start site, which may play a role in transcriptional induction of this gene during acute response. In addition to this conserved domain, DNA sequence upstream of the major transcription start site contains a potential element for Sp1 binding and a 18 bp long palindrome sequence followed by a short repeating dinucleotide sequence, which may be important in the regulation of AGP gene induction.

Animals↗
PubMed1991

Rabbit serum amyloid a gene: cloning, characterization and sequence analysis.

A genomic DNA clone that encodes serum amyloid A protein has been isolated from a rabbit liver lambda EMBL3 genomic library. Southern blot analysis indicates that the cloned gene is a member of multiple SAA gene family in rabbit. The nucleotide sequence of this DNA shows that the cloned gene corresponds to a SAA cDNA recently isolated from an acute phase rabbit liver cDNA library (1). This rabbit SAA gene contains four exons which are located within a 4500-base pair region spanning a putative TATA box and an AATAAA polyadenylation signal. DNA sequence upstream of the major transcription start site also contains other structural elements such as the sequences for NF-kappa B, IL-6 receptor and Sp1 binding. Significance of these regulatory elements in SAA gene expression is discussed.

Animals↗
PubMed1991

Molecular cloning and nucleotide sequence of complementary DNA encoding rabbit alpha 1-acid glycoprotein.

Alpha 1-acid glycoprotein (AGP) complementary DNA clone has been isolated from an acute phase rabbit liver library. Complete and full-length nucleotide sequence of this cDNA has been determined. The cDNA contains 35 nucleotides 5' untranslated region followed by 606 nucleotides of coding region and a 112 nucleotides 3' untranslated region. Comparison of this sequence with that of human, rat and mouse reveals a high degree of homology with human alpha 1-AGP. Northern blot analysis of messenger RNA for alpha 1-AGP in normal and acute livers demonstrates remarkable induction of transcription of this gene in rabbit in response to acute inflammation by the administration of turpentine. Such a response of AGP gene expression makes it a major member of the acute phase responsive genes in rabbit liver.

Amino Acid Sequence↗
PubMed1991

Complementary DNA cloning and nucleotide sequence of rabbit serum amyloid A protein.

A complementary DNA clone encoding serum amyloid A protein has been isolated from an acute rabbit liver cDNA library. Complete nucleotide sequence analysis reveals that the cloned gene contains a 24 bases 5' untranslated region, 369 bases coding region and a 106 bases 3' untranslated region. Primer extension analysis indicates that the full-length 5' untranslated region contains 80 nucleotides. Northern blot analysis of mRNA from normal and acute rabbit livers demonstrates that this gene is expressed constitutively at a low level and undergoes induction of transcription in response to acute inflammation by the administration of turpentine.

Amino Acid Sequence↗
PubMed1990

Calcium-dependent protein phosphorylation in Babesia bovis and its role in growth regulation.

Intracellular growth of protozoan parasite Babesia bovis has been followed to study the effect of some chemical agents on growth regulation. Using an in vitro parasite culture system we present evidence that the normal growth of the parasite is dependent upon available calcium and a Ca2(+)-binding protein, calmodulin, because sequestration of either of these 2 components from the culture medium causes inhibition of parasitic growth. Further studies demonstrate that the parasite contains a protein kinase that can phosphorylate a 40-kDa parasitic protein and its activity is regulated by calcium and calmodulin. Both the enzyme and its substrate are present in the membrane of the parasite. In addition, the parasite also contains a highly active protein kinase C activity that is documented by phosphorylating histone, a known substrate for protein kinase C. These findings suggest a possible correlation between the growth of parasite and calcium/calmodulin-dependent protein phosphorylation activity.

Animals↗
PubMed1988

Induction of eIF-4E phosphorylation by the addition of L-pyrroline-5-carboxylic acid to rabbit reticulocyte lysate.

Addition of L-pyrroline-5-carboxylic acid to reticulocyte lysates inhibits protein synthesis and induced phosphoproteins of 25 and 14 kDa. The 25 kDa phosphoprotein had the same Mr and pI as phosphorylated eIF-4E. Incubation of lysates with L-pyrroline-5-carboxylic acid did not alter the crosslinking of eIF-4E to reovirus mRNA caps. These results suggest that modifications of the translational apparatus other than eIF-4E phosphorylation may mediate the inhibitory effect seen with L-pyrroline-5-carboxylic acid and/or that phosphorylation of eIF-4E may effect functions subsequent to its interaction with the mRNA cap such as protein-protein interactions with other cap-specific translation factors.

Animals↗