The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoin... more The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoi...
Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan tha... more Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membran...
Acetyl-coenzyme A carboxylase (ACC) catalyzes the rate-limiting step in the biosynthesis of long-... more Acetyl-coenzyme A carboxylase (ACC) catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. Transcription of the single-copy ACC gene from two independent promoters, together with the differential splicing of the transcripts, gives rise to mature ACC mRNA having the same open reading frame (ORF), but exhibiting heterogeneity in their 5' untranslated region (5'-UTR). Class 1 ACC mRNA are transcribed from the inducible promoter 1 and their 5'-end leading sequences are provided by exon 1. Class 2 ACC mRNA are transcribed from the constitutively expressing promoter 2 and their leading sequences are derived from exon 2. In order to understand the role of different 5' UTR of ACC mRNA we have synthesized in vitro transcripts with defined ACC mRNA 5' UTR and examined their relative translational efficiencies in rabbit reticulocyte lysates. The major translation product of both forms of ACC mRNA was initiated at the first AUG of the ORF. Class 1 transcripts had a 6-9-fold better translational efficiency than class 2 transcripts, based on the quantity of major peptide produced by a given amount of transcript. The poor translational efficiency of class 2 transcripts can be improved by the removal of sequences contributed by exon 2, suggesting that they play an inhibitory role in the translation of class 2 types of ACC mRNA. In addition to their higher translational efficiency, the class 1 transcripts can also initiate translation at in-frame non-AUG codons, located in exon 1, i.e. upstream to the starting AUG of the common ACC mRNA ORF. This results in novel ACC peptides with extended N termini. These observations are consistent with the hypothesis that the 5' UTR heterogeneity in the ACC mRNA may be involved in post-transcriptional control, at the level of translation, of the ACC gene expression.
Proceedings of the National Academy of Sciences, 1989
Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-li... more Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. We have previously characterized five acetyl-CoA carboxylase mRNA species that differ in their 5' untranslated regions but not in the coding region. We have now characterized the exon-intron structure of the genomic DNA that encodes the 5' untranslated region of the mRNA. Generation of different forms of the mRNA is the result of the selective use of two promoters and differential splicing of five different exons. These five exons contain a total of 645 nucleotides and they are scattered over a 50-kilobase-pair genomic DNA region that we have characterized.
The purpose of this study was to evaluate DNA vaccination in cysticercosis prevention by using a ... more The purpose of this study was to evaluate DNA vaccination in cysticercosis prevention by using a Taenia crassiceps cDNA of a recombinant antigen (KETc7) that has been reported as protective against murine cysticercosis. The KETc7 cDNA was cloned into the pcDNA3 plasmid alone or with the betaglycan signal peptide sequence (pTc-7 and pTc-sp7, respectively). Positive expression of the pTc-sp7 product was confirmed by transfection of C33 cells and immunofluorescence using sera of mice infected with T. crassiceps. Immunization of mice with 3 injections of pTc-sp7 DNA at the higher dose (200 microg) was the most effective to induce antibody with or without bupivacaine. Immunization with pTc-sp7 induced protection against challenge with T. crassiceps cysticerci as successfully as previously observed with the KETc7 recombinant protein. Antibodies elicited by DNA immunization with pTc-sp7 specifically reacted with the native protein of 56 kDa previously reported, which is immunolocalized in the tegument of T. crassiceps cysticerci. The 56-kDa antigen is also present in Taenia solium oncospheres, cysticerci, and adult tissue. The protection induced in DNA-immunized mice and the observation that the injected plasmid remains as an episomic form within muscle cells, encouraged us to continue testing this procedure to prevent T. solium cysticercosis.
Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model wit... more Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model with which to test the efficacy of new vaccine candidates and delivery systems against pig cysticercosis. A high level of protection against murine cysticercosis was previously observed by intramuscular or intradermal DNA immunization with the use of the sequence of the recombinant KETc7 antigen cloned in pcDNA3 (pTc-sp7). To determine the effect of KETc7 differential expression in DNA vaccination, KETc7 was cloned in pGEM 11Zf(+) under the control of the tissue-specific regulatory promoter phosphoenolpyruvate carboxykinase (pPc-sp7). A high level of protection was induced by intrahepatic immunization with pPc-sp7, pTc-sp7 and the empty vector in the absence of any specific immunity. The empty vector pGEM 11Zf(+), the plasmid with the highest content of CpG sequences, provided to the most efficient protection. This protection was related to an increased number of splenocytes, enhanced nonspecific splenocyte proliferation, and intensified intrahepatic INF-gamma production. Overall, intrahepatic plasmid CpG-DNA immunization provokes an exacerbated nonspecific immune response that can effectively control Taenia crassiceps cysticercosis.
The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci prese... more The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the β-glycan receptor (pTc-sp7), has been shown to be effective in ...
Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of p... more Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in...
The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter... more The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter II (PII). Depending upon which promoter is active, two classes of ACC mRNA are formed. The physiological significance of the presence of two promoters in the gene is not clear at this time. However, this question can be indirectly approached by examination of their expression patterns under different physiological conditions. We have examined the activities of these two promoters under different physiological conditions by means of primer extension analysis. Under normal conditions, the Wistar rat, fed standard chow ad libitum, expresses only basal levels of PI in white adipose tissue and PII in the liver. Starvation leads to the virtual disappearance of transcriptional products from these promoters. When fatty acid synthesis is stimulated by refeeding a fat-free diet to starved rats, both PI and PII are activated in the liver; however, in white adipose tissue, only PI, not PII, is responsive to this nutritional induction. On the other hand, in streptozotocin-diabetic rats, in which the activity of both promoters in both tissues is depressed, the administration of insulin quickly induces PI in adipose tissues, but has no significant effect on either of the promoters in the liver. During the weaning transition, the increase in hepatic lipogenesis is accompanied by activation of PI and PII when the pups are weaned onto a fat-free diet. Weaning onto a standard chow causes only a slight increase in PII. During the lactation period, profound alterations occur in the metabolism of the lipogenic tissues. In the lactating rat mammary gland only PII is active, and its activity is increased throughout lactation, reaching a plateau by day 7. Concomitantly, all ACC gene activity is completely shut off in the adipose tissue, while in the liver, PII, the only promoter active, is affected only minimally. The fat accumulation of the genetically obese Zucker rats is largely due to an abnormally high hepatic lipogenesis. In the obese Zucker rat (fa/fa), the level of expression of PII is similar to that in its lean siblings; however, PI is constitutively expressed at high levels, comparable to those in the Wistar rat that has been subjected to the starvation/refeeding induction. These studies demonstrate that the in vivo transcriptional control of the dual promoter rat ACC gene is a highly regulated and tissue-specific process.
Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may co... more Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels ∼4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c in...
The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoin... more The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoi...
Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan tha... more Betaglycan, also known as the TGF-beta type III receptor, is a membrane-anchored proteoglycan that presents TGF-beta to the type II signaling receptor, a transmembrane serine/threonine kinase. The betaglycan extracellular region, which can be shed by cells into the medium, contains a NH2-terminal domain related to endoglin and a COOH-terminal domain related to uromodulin, sperm receptors Zp2 and 3, and pancreatic secretory granule GP-2 protein. We identified residues Ser535 and Ser546 in the uromodulin-related region as the glycosaminoglycan (GAG) attachment sites. Their mutation to alanine prevents GAG attachment but does not interfere with betaglycan stability or ability to bind and present TGF-beta to receptor II. Using a panel of deletion mutants, we found that TGF-beta binds to the NH2-terminal endoglin-related region of betaglycan. The remainder of the extracellular domain and the cytoplasmic domain are not required for presentation of TGF-beta to receptor II; however, membran...
Acetyl-coenzyme A carboxylase (ACC) catalyzes the rate-limiting step in the biosynthesis of long-... more Acetyl-coenzyme A carboxylase (ACC) catalyzes the rate-limiting step in the biosynthesis of long-chain fatty acids. Transcription of the single-copy ACC gene from two independent promoters, together with the differential splicing of the transcripts, gives rise to mature ACC mRNA having the same open reading frame (ORF), but exhibiting heterogeneity in their 5' untranslated region (5'-UTR). Class 1 ACC mRNA are transcribed from the inducible promoter 1 and their 5'-end leading sequences are provided by exon 1. Class 2 ACC mRNA are transcribed from the constitutively expressing promoter 2 and their leading sequences are derived from exon 2. In order to understand the role of different 5' UTR of ACC mRNA we have synthesized in vitro transcripts with defined ACC mRNA 5' UTR and examined their relative translational efficiencies in rabbit reticulocyte lysates. The major translation product of both forms of ACC mRNA was initiated at the first AUG of the ORF. Class 1 transcripts had a 6-9-fold better translational efficiency than class 2 transcripts, based on the quantity of major peptide produced by a given amount of transcript. The poor translational efficiency of class 2 transcripts can be improved by the removal of sequences contributed by exon 2, suggesting that they play an inhibitory role in the translation of class 2 types of ACC mRNA. In addition to their higher translational efficiency, the class 1 transcripts can also initiate translation at in-frame non-AUG codons, located in exon 1, i.e. upstream to the starting AUG of the common ACC mRNA ORF. This results in novel ACC peptides with extended N termini. These observations are consistent with the hypothesis that the 5' UTR heterogeneity in the ACC mRNA may be involved in post-transcriptional control, at the level of translation, of the ACC gene expression.
Proceedings of the National Academy of Sciences, 1989
Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-li... more Acetyl-CoA carboxylase [acetyl-CoA:carbondioxide ligase (ADP-forming), EC 6.4.1.2] is the rate-limiting enzyme in the biogenesis of long-chain fatty acids. We have previously characterized five acetyl-CoA carboxylase mRNA species that differ in their 5' untranslated regions but not in the coding region. We have now characterized the exon-intron structure of the genomic DNA that encodes the 5' untranslated region of the mRNA. Generation of different forms of the mRNA is the result of the selective use of two promoters and differential splicing of five different exons. These five exons contain a total of 645 nucleotides and they are scattered over a 50-kilobase-pair genomic DNA region that we have characterized.
The purpose of this study was to evaluate DNA vaccination in cysticercosis prevention by using a ... more The purpose of this study was to evaluate DNA vaccination in cysticercosis prevention by using a Taenia crassiceps cDNA of a recombinant antigen (KETc7) that has been reported as protective against murine cysticercosis. The KETc7 cDNA was cloned into the pcDNA3 plasmid alone or with the betaglycan signal peptide sequence (pTc-7 and pTc-sp7, respectively). Positive expression of the pTc-sp7 product was confirmed by transfection of C33 cells and immunofluorescence using sera of mice infected with T. crassiceps. Immunization of mice with 3 injections of pTc-sp7 DNA at the higher dose (200 microg) was the most effective to induce antibody with or without bupivacaine. Immunization with pTc-sp7 induced protection against challenge with T. crassiceps cysticerci as successfully as previously observed with the KETc7 recombinant protein. Antibodies elicited by DNA immunization with pTc-sp7 specifically reacted with the native protein of 56 kDa previously reported, which is immunolocalized in the tegument of T. crassiceps cysticerci. The 56-kDa antigen is also present in Taenia solium oncospheres, cysticerci, and adult tissue. The protection induced in DNA-immunized mice and the observation that the injected plasmid remains as an episomic form within muscle cells, encouraged us to continue testing this procedure to prevent T. solium cysticercosis.
Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model wit... more Experimental murine cysticercosis caused by Taenia crassiceps has proved to be a useful model with which to test the efficacy of new vaccine candidates and delivery systems against pig cysticercosis. A high level of protection against murine cysticercosis was previously observed by intramuscular or intradermal DNA immunization with the use of the sequence of the recombinant KETc7 antigen cloned in pcDNA3 (pTc-sp7). To determine the effect of KETc7 differential expression in DNA vaccination, KETc7 was cloned in pGEM 11Zf(+) under the control of the tissue-specific regulatory promoter phosphoenolpyruvate carboxykinase (pPc-sp7). A high level of protection was induced by intrahepatic immunization with pPc-sp7, pTc-sp7 and the empty vector in the absence of any specific immunity. The empty vector pGEM 11Zf(+), the plasmid with the highest content of CpG sequences, provided to the most efficient protection. This protection was related to an increased number of splenocytes, enhanced nonspecific splenocyte proliferation, and intensified intrahepatic INF-gamma production. Overall, intrahepatic plasmid CpG-DNA immunization provokes an exacerbated nonspecific immune response that can effectively control Taenia crassiceps cysticercosis.
The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci prese... more The nucleotide sequence of a protective recombinant antigen of Taenia crassiceps cysticerci present in all stages of Taenia solium (KETc7), cloned into pcDNA3 plasmid with the signal peptide sequence of the β-glycan receptor (pTc-sp7), has been shown to be effective in ...
Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of p... more Taenia crassiceps recombinant antigens KETc1 and KETc12 have been shown to induce high level of protection against experimental murine T. crassiceps cysticercosis, an experimental model successfully used to test candidate antigens for use in vaccination against porcine Taenia solium cysticercosis. Based on the deduced amino acid sequence, KETc1 and KETc12 were chemically synthesized in linear form. Immunization with KETc1 induced 66.7 to 100% protection against murine cysticercosis, and immunization with KETc12 induced 52.7 to 88.1% protection. The elicited immune response indicated that both peptides contain at least one B-cell epitope (as demonstrated by their ability to induce specific antibodies) and one T-cell epitope that strongly stimulated the proliferation of T cells primed with either the free peptide or total cysticercal T. crassiceps antigens. The high percentage of spleen cells expressing inflammatory cytokines points to the likelihood of a T1 response being involved in...
The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter... more The acetyl-coenzyme-A carboxylase (ACC) gene contains two promoters: promoter I (PI) and promoter II (PII). Depending upon which promoter is active, two classes of ACC mRNA are formed. The physiological significance of the presence of two promoters in the gene is not clear at this time. However, this question can be indirectly approached by examination of their expression patterns under different physiological conditions. We have examined the activities of these two promoters under different physiological conditions by means of primer extension analysis. Under normal conditions, the Wistar rat, fed standard chow ad libitum, expresses only basal levels of PI in white adipose tissue and PII in the liver. Starvation leads to the virtual disappearance of transcriptional products from these promoters. When fatty acid synthesis is stimulated by refeeding a fat-free diet to starved rats, both PI and PII are activated in the liver; however, in white adipose tissue, only PI, not PII, is responsive to this nutritional induction. On the other hand, in streptozotocin-diabetic rats, in which the activity of both promoters in both tissues is depressed, the administration of insulin quickly induces PI in adipose tissues, but has no significant effect on either of the promoters in the liver. During the weaning transition, the increase in hepatic lipogenesis is accompanied by activation of PI and PII when the pups are weaned onto a fat-free diet. Weaning onto a standard chow causes only a slight increase in PII. During the lactation period, profound alterations occur in the metabolism of the lipogenic tissues. In the lactating rat mammary gland only PII is active, and its activity is increased throughout lactation, reaching a plateau by day 7. Concomitantly, all ACC gene activity is completely shut off in the adipose tissue, while in the liver, PII, the only promoter active, is affected only minimally. The fat accumulation of the genetically obese Zucker rats is largely due to an abnormally high hepatic lipogenesis. In the obese Zucker rat (fa/fa), the level of expression of PII is similar to that in its lean siblings; however, PI is constitutively expressed at high levels, comparable to those in the Wistar rat that has been subjected to the starvation/refeeding induction. These studies demonstrate that the in vivo transcriptional control of the dual promoter rat ACC gene is a highly regulated and tissue-specific process.
Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may co... more Acetyl-CoA carboxylase I (ACCI) is a key lipogenic enzyme whose induction in islet β-cells may contribute to glucolipotoxicity. Here, we provide evidence that enhanced insulin release plays an important role in the activation of this gene by glucose. Glucose (30 vs. 3 mmol/l) increased ACCI mRNA levels ∼4-fold and stimulated ACCI (pII) promoter activity >30-fold in MIN6 cells. The latter effect was completely suppressed by blockade of insulin release or of insulin receptor signaling. However, added insulin substantially, but not completely, mimicked the effects of glucose, suggesting that intracellular metabolites of glucose may also contribute to transcriptional stimulation. Mutational analysis of the ACCI promoter, and antibody microinjection, revealed that the effect of glucose required sterol response element binding protein (SREBP)-1c. Moreover, adenoviral transduction with dominant-negative-acting SREBP1c blocked ACCI gene induction, whereas constitutively active SREBP1c in...
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