Juan Medina - Academia.edu (original) (raw)
Papers by Juan Medina
Gastroenterology, 2008
Background & Aims: Cl ؊ /HCO 3 ؊ anion exchanger 2 (AE2) is involved in intracellular pH (pH i ) ... more Background & Aims: Cl ؊ /HCO 3 ؊ anion exchanger 2 (AE2) is involved in intracellular pH (pH i ) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2 a.b -deficient mice. Methods: In this murine model, splenocyte pH i and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Results: Ae2 a,b ؊/؊ mice exhibit splenomegaly, elevated pH i in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8 ؉ T-cell population, and under represented CD4 ؉ FoxP3 ؉ /regulatory T cells. Most Ae2 a,b ؊/؊ mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2 a,b ؊/؊ mice had extensive portal inflammation with CD8 ؉ and CD4 ؉ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2 a,b ؊/؊ mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Conclusions: Ae2 deficiency alters pH i homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. Abbreviations used in this paper: AE2, Cl ؊ /HCO 3 ؊ anion exchanger 2; AMA, antimitochondrial antibodies; FACS, fluorescence-activated cell sorter; hALP, hepatic alkaline phosphatase; IL, interleukin; PBC, primary biliary cirrhosis; PCR, polymerase chain reaction; PDC-E2, E2 component of the pyruvate dehydrogenase complex; pH i , intracellular pH; Q-TOF/MS, quadrupole/time-of-flight mass spectrometry; Treg, regulatory T cells.
Gastroenterology, 2003
Abbreviations used in this paper: ICP, intrahepatic cholestasis of pregnancy; MDR3, multidrug res... more Abbreviations used in this paper: ICP, intrahepatic cholestasis of pregnancy; MDR3, multidrug resistance 3 glycoprotein; PFIC, progressive familial intrahepatic cholestasis.
Hepatology, 1994
Sodium-independent Cl−/HCO3− exchange activity has been observed in isolated rat hepatocytes and ... more Sodium-independent Cl−/HCO3− exchange activity has been observed in isolated rat hepatocytes and intrahepatic bile duct epithelial cells, where it is involved in intracellular pH regulation and, possibly, biliary bicarbonate secretion. Monoclonal antibodies to the membrane domain of human chloride/bicarbonate anion exchanger proteins, AE1 and AE2, were prepared so that we might determine by immunohistochemical methods the presence and location of these antiporters in the human liver. To obtain the antibody against AE1, we immunized mice with injections of washed human erythrocytes. The selected monoclonal antibody was found to be specific for the 17-kD proteolytic membrane fragment of AE1 protein. The antibody to AE2 was produced with a 14-mer synthetic peptide, whose sequence corresponds specifically to amino acid residues 871 to 884 in the deduced primary structure of human kidney AE2 protein. When the monoclonal antibody to AE2 peptide was employed for the immunohistochemical study of liver specimens (by both immunofluorescence and immunoperoxidase), a clearly defined staining was present at the canalicular membrane of hepatocytes, as well as the luminal side of the membrane of bile duct epithelial cells from small and medium-sized bile ducts. No staining was observed in the liver parenchyma with the monoclonal antibody to AE1, which instead strongly decorated the erythrocytes in liver blood vessels. We conclude that AE2 immunoreactivity is present in human liver, where it localizes very specifically to the membrane regions, which appear most probably involved in the transport of bicarbonate to bile (i.e., the canalicular membrane of hepatocytes and the apical side of epithelial cells of small and medium bile ducts). (HEPATOLOGY 1994;19:1400–1406.)
Transgenic Research, 2002
Complementary DNA expression of mature human serum albumin was engineered into potato plants unde... more Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.
Biochemical and Biophysical Research Communications, 2000
Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exc... more Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b1 corresponds to the rat variant “b” and encodes three initial residues (MTQ) in AE2b1 isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b2 encodes eight initial residues (MDFLLRPQ) in AE2b2 isoform. The relative abundance of AE2b1 and AE2b2 mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b1 and AE2b2 transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.
Gastroenterology, 2008
Background & Aims: Cl ؊ /HCO 3 ؊ anion exchanger 2 (AE2) is involved in intracellular pH (pH i ) ... more Background & Aims: Cl ؊ /HCO 3 ؊ anion exchanger 2 (AE2) is involved in intracellular pH (pH i ) regulation and transepithelial acid-base transport, including secretin-stimulated biliary bicarbonate excretion. AE2 gene expression was found to be reduced in liver biopsy specimens and blood mononuclear cells from patients with primary biliary cirrhosis (PBC), a disease characterized by chronic nonsuppurative cholangitis associated with antimitochondrial antibodies (AMA) and other autoimmune phenomena. In mice with widespread Ae2 gene disruption, we previously reported altered spermiogenesis and reduced gastric acid secretion. We now describe the hepatobiliary and immunologic changes observed in these Ae2 a.b -deficient mice. Methods: In this murine model, splenocyte pH i and T-cell populations were studied by flow cytometry. CD3-stimulated cytokine secretion was estimated using cytokine arrays. AMA were evaluated by immunoblotting and proteomics. Hepatobiliary changes were assessed by immunohistopathology, flow cytometry, and serum biochemistry. Cholangiocyte gene expression was analyzed by real-time polymerase chain reaction. Results: Ae2 a,b ؊/؊ mice exhibit splenomegaly, elevated pH i in splenocytes, increased production of interleukin-12p70 and interferon gamma, expanded CD8 ؉ T-cell population, and under represented CD4 ؉ FoxP3 ؉ /regulatory T cells. Most Ae2 a,b ؊/؊ mice tested positively for AMA, showing increased serum levels of immunoglobulin M and G, and liver-specific alkaline phosphatase. About one third of Ae2 a,b ؊/؊ mice had extensive portal inflammation with CD8 ؉ and CD4 ؉ T lymphocytes surrounding damaged bile ducts. Cholangiocytes isolated from Ae2 a,b ؊/؊ mice showed gene expression changes compatible with oxidative stress and increased antigen presentation. Conclusions: Ae2 deficiency alters pH i homeostasis in immunocytes and gene expression profile in cholangiocytes, leading to immunologic and hepatobiliary changes that resemble PBC. Abbreviations used in this paper: AE2, Cl ؊ /HCO 3 ؊ anion exchanger 2; AMA, antimitochondrial antibodies; FACS, fluorescence-activated cell sorter; hALP, hepatic alkaline phosphatase; IL, interleukin; PBC, primary biliary cirrhosis; PCR, polymerase chain reaction; PDC-E2, E2 component of the pyruvate dehydrogenase complex; pH i , intracellular pH; Q-TOF/MS, quadrupole/time-of-flight mass spectrometry; Treg, regulatory T cells.
Gastroenterology, 2003
Abbreviations used in this paper: ICP, intrahepatic cholestasis of pregnancy; MDR3, multidrug res... more Abbreviations used in this paper: ICP, intrahepatic cholestasis of pregnancy; MDR3, multidrug resistance 3 glycoprotein; PFIC, progressive familial intrahepatic cholestasis.
Hepatology, 1994
Sodium-independent Cl−/HCO3− exchange activity has been observed in isolated rat hepatocytes and ... more Sodium-independent Cl−/HCO3− exchange activity has been observed in isolated rat hepatocytes and intrahepatic bile duct epithelial cells, where it is involved in intracellular pH regulation and, possibly, biliary bicarbonate secretion. Monoclonal antibodies to the membrane domain of human chloride/bicarbonate anion exchanger proteins, AE1 and AE2, were prepared so that we might determine by immunohistochemical methods the presence and location of these antiporters in the human liver. To obtain the antibody against AE1, we immunized mice with injections of washed human erythrocytes. The selected monoclonal antibody was found to be specific for the 17-kD proteolytic membrane fragment of AE1 protein. The antibody to AE2 was produced with a 14-mer synthetic peptide, whose sequence corresponds specifically to amino acid residues 871 to 884 in the deduced primary structure of human kidney AE2 protein. When the monoclonal antibody to AE2 peptide was employed for the immunohistochemical study of liver specimens (by both immunofluorescence and immunoperoxidase), a clearly defined staining was present at the canalicular membrane of hepatocytes, as well as the luminal side of the membrane of bile duct epithelial cells from small and medium-sized bile ducts. No staining was observed in the liver parenchyma with the monoclonal antibody to AE1, which instead strongly decorated the erythrocytes in liver blood vessels. We conclude that AE2 immunoreactivity is present in human liver, where it localizes very specifically to the membrane regions, which appear most probably involved in the transport of bicarbonate to bile (i.e., the canalicular membrane of hepatocytes and the apical side of epithelial cells of small and medium bile ducts). (HEPATOLOGY 1994;19:1400–1406.)
Transgenic Research, 2002
Complementary DNA expression of mature human serum albumin was engineered into potato plants unde... more Complementary DNA expression of mature human serum albumin was engineered into potato plants under the transcriptional control of patatin B33 promoter and potato proteinase inhibitor II terminator. Protein secretion was achieved by using the signal sequence from potato proteinase inhibitor II. Recombinant albumin accumulated up to 0.2% of total soluble tuber protein in single transformant lines, regardless of the potato cultivar used. Electrophoretic mobility and N-terminal amino acid sequence analysis of partially purified recombinant albumin confirmed proper processing of an immune responsive recombinant albumin, and revealed that the proteinase inhibitor II signal sequence was correctly removed. No further optimisation of these yields was obtained by HSA expression in patatin antisense plants (line Pas58). Subcellular localisation showed that recombinant protein was successfully targeted to the apoplast. Potato tubers may be used, by applying this technology, to produce other heterologous proteins of interest in the biopharmaceutical industry.
Biochemical and Biophysical Research Communications, 2000
Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exc... more Previously, we isolated the human AE2 (SLC4A2) gene, a member of the sodium-independent anion exchanger family. Rat ortholog of this gene was reported to drive alternative transcription yielding N-terminal variants of the AE2a message. We thus analyzed the human AE2 gene in this regard. Using HepG2 cells, two alternative first exons, each splicing to exon 3 in alternative transcripts, were found to be transcribed from overlapping sequences of intron 2. Exon 1b1 corresponds to the rat variant “b” and encodes three initial residues (MTQ) in AE2b1 isoform that replace the first 17 amino acids of AE2a protein, while the novel exon 1b2 encodes eight initial residues (MDFLLRPQ) in AE2b2 isoform. The relative abundance of AE2b1 and AE2b2 mRNAs was about 10% of AE2a mRNA each. Alternate promoter sequences have multiple potential binding motifs for liver-enriched factors, and dual-luciferase assays indicated that they possess the ability for driving transcription in transiently transfected HepG2 cells. Tissue survey showed that expression of human AE2b1 and AE2b2 transcripts is restricted to liver and kidney, while AE2a mRNA was encountered in all examined tissues. Our findings reveal a characteristic tissue-specific expression of two N-terminal variants of human AE2 from overlapping sequences within intron 2, one of which is a novel isoform.