American Journal of Physiology-lung Cellular and Molecular Physiology, Jun 1, 1991
Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of supero... more Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of superoxide dismutase, which, although identical in enzymic activity, are widely dissimilar in structure. Manganese (Mn) superoxide dismutase located in the mitochondrial matrix and copper/zinc (Cu/Zn) superoxide dismutase in the cytosol are different in molecular weight, sub-unit number and amino acid composition (Weisiger & Fridovich, 1973; Johansen et al., 1979; Harris et al., 1980). The synthesis of both enzymes is controlled by nuclear DNA. Little else is known about the regulation of these enzymes, although recently the synthesis of Cu/Zn superoxide dismutase was reported in a cell-free rabbit reticulocyte lysate system programmed with human placental mRNA (Bannister et al., 1980).
The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both a... more The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.
In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of r... more In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of regulatory molecules in the TATA proximal portion of the maize alcohol dehydrogenase-1 gene. In several tissues and organs, Adh1 is transcriptionally induced by anaerobic stress, and the various alleles of Adh1 can show a quantitative differential response to induction. Two regulatory molecules were found to be bound to the promoter only when the gene was induced. The binding sites for these factors mapped to regions located at -100 to -108 and -186 to -190 relative to the site of transcript initiation. Two additional regulatory molecules were bound to sites located at positions -117 to -120 and -138 to -145 regardless of the state of transcription. However, the factor bound at the -138 to -145 region was found to alter its binding characteristics when the gene was induced.
Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD)... more Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.
Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of supero... more Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of superoxide dismutase, which, although identical in enzymic activity, are widely dissimilar in structure. Manganese (Mn) superoxide dismutase located in the mitochondrial matrix and copper/zinc (Cu/Zn) superoxide dismutase in the cytosol are different in molecular weight, sub-unit number and amino acid composition (Weisiger & Fridovich, 1973; Johansen et al., 1979; Harris et al., 1980). The synthesis of both enzymes is controlled by nuclear DNA. Little else is known about the regulation of these enzymes, although recently the synthesis of Cu/Zn superoxide dismutase was reported in a cell-free rabbit reticulocyte lysate system programmed with human placental mRNA (Bannister et al., 1980).
American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 1997
We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuro... more We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuronal, and smooth muscle cells (SMC) after acetic acid-induced colitis. To examine the regulation of MnSOD in the SMC more specifically, primary cultures of colonic SMC were developed by enzymatic digestion of the circular muscle layer from an adult rat. SMC were treated for 2-72 h with 0.5 microgram/ml Escherichia coli endotoxin [lipopolysaccharide (LPS)], 10 ng/ml tumor necrosis factor (TNF)-alpha, or 2 ng/ml interleukin-1 beta (IL-1 beta). Cotreatments were performed with IL-1 beta and 4 microM actinomycin or 50 microM cycloheximide. Northern analysis demonstrated 23-fold, 8-fold, and 6-fold inductions of MnSOD mRNA by IL-1 beta, LPS, and TNF-alpha, respectively. Induction of MnSOD by IL-1 beta was eliminated by actinomycin but not by cycloheximide, implicating a requirement for de novo transcription. Western analysis resulted in a 23.7-fold induction of MnSOD protein after 48-h treatment with IL-1 beta. Induction of MnSOD by IL-1 beta and other inflammatory mediators may serve as a protective mechanism to reduce oxygen free radical- and nitric oxide-mediated cell damage during inflammation.
bioRxiv (Cold Spring Harbor Laboratory), Oct 21, 2021
As secondary lymphoid organs, the spleen and lymph node represent important hubs for both innate ... more As secondary lymphoid organs, the spleen and lymph node represent important hubs for both innate and adaptive immunity 1,2. Neuroanatomical and tracing data, largely derived from rodents, suggest that lymph nodes contain sensory and sympathetic innervation 3,4 , whereas the spleen contains postganglionic sympathetic innervation 5 , with conflicting views regarding the
OBJECTIVE: To test the hypothesis that a mutation in the voltage-gated potassium channel Kv3.3 ca... more OBJECTIVE: To test the hypothesis that a mutation in the voltage-gated potassium channel Kv3.3 causes disordered binary hearing and processing of sound localization cues. BACKGROUND: We studied an SCA13 kindred carrying an autosomal dominant mutation in the gene coding for Kv3.3, which is expressed at high levels in binaural auditory brainstem pathways. DESIGN/METHODS: Genotype status of affected and unaffected family members was confirmed following auditory testing. Clinical severity was determined utilizing the Scale for the Assessment and Rating of Ataxia (SARA) with a range from 0 (asymptomatic) to 40 (severe disability). We conducted hearing tests with 13 heterozygous family members (affected listeners), 6 homozygous family members (familial controls), and an age-matched control group of 16 unrelated normal-hearing individuals (non-familial controls). All listeners demonstrated age-appropriate pure-tone audiograms. We used an adaptive psychophysical procedure measuring threshol...
Highlights d ACE2 mRNA and protein are expressed in human pancreatic ducts and microvasculature d... more Highlights d ACE2 mRNA and protein are expressed in human pancreatic ducts and microvasculature d ACE2 mRNA was rarely detected and at low levels in human pancreatic endocrine cells d Pancreatic ACE2 protein expression changes across the lifespan and correlates with BMI d SARS-CoV-2 NP was detected in ducts, but not endocrine cells, of COVID-19 pancreata
Current evidence suggests that alteration in cellular metabolism leads to pancreatic β-cell dysfu... more Current evidence suggests that alteration in cellular metabolism leads to pancreatic β-cell dysfunction and loss in type 1 diabetes (T1D). The mammalian target of rapamycin, mTOR is a key regulator of cell survival and growth in response to intracellular energy levels, nutrients, and growth factor signaling. Dysregulated mTOR complex 1 (mTORC1) has been implicated in β-cell loss and dysfunction in T2D but has not been well studied in T1D. We hypothesized that genes associated with three pathways involved in the regulation of mTORC1 would display altered expression in T1D organ donor pancreata compared to nondiabetic controls. We isolated total RNA from 30 T1D and 30 unaffected control human organ donor pancreata obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD) program and performed real-time qPCR on 26 genes related to these pathways, all of which demonstrated significant (p<0.05) induction. In relation to cellular energy balance, we observed induction o...
American Journal of Physiology-Lung Cellular and Molecular Physiology, 1996
Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observ... more Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observed is related to injury of the pulmonary microvasculature. In this study, we evaluated the response of the pulmonary microvascular endothelial cell to high oxygen concentrations, using two-dimensional protein gel electrophoresis as a direct molecular assay of differences between cells exposed to room air or hyperoxia. We observed a differential expression of five specific proteins within 24 h of a hyperoxic insult that we termed hyperoxia-responsive proteins. After 4 h of hyperoxia there was a decrease in two of the proteins. From 8 to 24 h we observed a repression of a third and an induction of the other two proteins. One of the induced proteins was also increased by heat shock and hydrogen peroxide and has characteristics similar to heat shock protein (HSP) 32 (heme oxygenase 1). Western analysis using an antibody specific to rat heme oxygenase 1 verified that this oxygen-responsive pr...
American Journal of Physiology-Cell Physiology, 1992
The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-m... more The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-methyl-3-isobutylxanthine (MIX), insulin, and dexamethasone to the culture medium. During differentiation, we have demonstrated that the level of both annexin I mRNA and protein decreases. The half-times for this reduction were 2 h and 10 h for annexin I mRNA and protein, respectively. Of the added agents in the differentiation medium, only MIX caused a decline in annexin I expression in 3T3-L1 fibroblasts. The MIX effect in fibroblasts was reversible and required de novo transcription but not protein synthesis. Although MIX could be replaced by high levels of theophylline, neither agonists of the beta-adrenergic receptor nor intracellular second messengers, cAMP and cGMP, were able to reduce annexin I. The potential role of annexin I in cellular differentiation is discussed.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 1994
Even though endothelial cells from different locations have similarities, there are potential mor... more Even though endothelial cells from different locations have similarities, there are potential morphological and functional differences between cells from different vascular regions, as well as between species. Our laboratory is interested in studying the molecular regulation of vasoactive substances in pulmonary vasculature. Therefore, we have developed reproducible methodology to isolate and maintain cultures of human pulmonary artery endothelial cells. The major innovation has been the employment of sections of pulmonary artery from heart transplant donors, from which endothelial cells are isolated. Cell monolayers were identified as endothelial cells by phase-contrast microscopy. Representative dishes of cells were further characterized by indirect immunofluorescent staining for factor VIII antigen, uptake of acetylated low-density lipoprotein, and electron microscopy. These cells were also evaluated for the expression of endothelin-1 (ET-1), a vasoactive 21-amino acid peptide de...
In vivo footprinting experiments have been used to analyze the binding of transacting regulatory ... more In vivo footprinting experiments have been used to analyze the binding of transacting regulatory factors in the 5' flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding transacting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adhl) gene. One binding site is similar in sequence_to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a transacting factor. Several of the remaining binding sites apparently represent a class' of elements sharing the sequence 5'-GTGG-3' within their footprint. Comparisons with maize Adhl in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.
Biochemical and Biophysical Research Communications, 1993
Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepa... more Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepatocytes or hepatoma cells. One of these proteins, MP-73, was synthesized at a rate 2- to 3-fold greater when cells were incubated for 3-9 h under conditions of amino acid deprivation versus amino acid supplementation. Immunoblotting with polyclonal antibodies prepared against MP-73 localized it to the inner mitochondrial membrane. MP-73 appears to be a hydrophobic, integral membrane protein. MP-73 antibody was used to identify a partial cDNA (NS3.2) of approximately 2 kb. A probe prepared from pNS3.2 identified a transcript in rat Fao hepatoma cells of approximately 4.4 kb that was increased in abundance by more than 20-fold following amino acid starvation of the cells.
American Journal of Respiratory Cell and Molecular Biology, 1995
Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especial... more Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.
American Journal of Physiology-lung Cellular and Molecular Physiology, Jun 1, 1991
Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of supero... more Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of superoxide dismutase, which, although identical in enzymic activity, are widely dissimilar in structure. Manganese (Mn) superoxide dismutase located in the mitochondrial matrix and copper/zinc (Cu/Zn) superoxide dismutase in the cytosol are different in molecular weight, sub-unit number and amino acid composition (Weisiger & Fridovich, 1973; Johansen et al., 1979; Harris et al., 1980). The synthesis of both enzymes is controlled by nuclear DNA. Little else is known about the regulation of these enzymes, although recently the synthesis of Cu/Zn superoxide dismutase was reported in a cell-free rabbit reticulocyte lysate system programmed with human placental mRNA (Bannister et al., 1980).
The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both a... more The gene for the amino acid biosynthetic activity asparagine synthetase (AS) is induced by both amino acid and glucose deprivation of cells. The data reported here document that the human AS gene is induced following activation of the Unfolded Response Pathway (UPR), also known as the Endoplasmic Reticulum Stress Response (ERSR) in mammals. Increased AS transcription occurs in response to glucose deprivation, tunicamycin, or azetidine-2-carboxylate, all known to activate the UPR/ERSR pathway. Previously identified ERSR target genes contain multiple copies of a single highly conserved cis-element. In contrast, the human AS gene does not contain the ERSR element, as it has been described for other responsive genes. Instead, AS induction requires an Sp1-like sequence, a sequence previously shown to be associated with amino acid control of transcription, and possibly, a third region containing no consensus sequences for known transcription factors. Oligonucleotides covering each of these regions form DNA-protein complexes in vitro, and for some the amount of these complexes is greater when nuclear extracts from glucose-starved cells are tested. These results document that a wider range of metabolic activities are activated by the UPR/ERSR pathway than previously recognized and that genomic elements other than those already described can serve to enhance transcription of specific target genes.
In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of r... more In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of regulatory molecules in the TATA proximal portion of the maize alcohol dehydrogenase-1 gene. In several tissues and organs, Adh1 is transcriptionally induced by anaerobic stress, and the various alleles of Adh1 can show a quantitative differential response to induction. Two regulatory molecules were found to be bound to the promoter only when the gene was induced. The binding sites for these factors mapped to regions located at -100 to -108 and -186 to -190 relative to the site of transcript initiation. Two additional regulatory molecules were bound to sites located at positions -117 to -120 and -138 to -145 regardless of the state of transcription. However, the factor bound at the -138 to -145 region was found to alter its binding characteristics when the gene was induced.
Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD)... more Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.
Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of supero... more Saccharomyces cereuisiae, in common with all other eukaryotic cells, contains two forms of superoxide dismutase, which, although identical in enzymic activity, are widely dissimilar in structure. Manganese (Mn) superoxide dismutase located in the mitochondrial matrix and copper/zinc (Cu/Zn) superoxide dismutase in the cytosol are different in molecular weight, sub-unit number and amino acid composition (Weisiger & Fridovich, 1973; Johansen et al., 1979; Harris et al., 1980). The synthesis of both enzymes is controlled by nuclear DNA. Little else is known about the regulation of these enzymes, although recently the synthesis of Cu/Zn superoxide dismutase was reported in a cell-free rabbit reticulocyte lysate system programmed with human placental mRNA (Bannister et al., 1980).
American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 1997
We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuro... more We have observed a rapid induction of manganese superoxide dismutase (MnSOD) in epithelial, neuronal, and smooth muscle cells (SMC) after acetic acid-induced colitis. To examine the regulation of MnSOD in the SMC more specifically, primary cultures of colonic SMC were developed by enzymatic digestion of the circular muscle layer from an adult rat. SMC were treated for 2-72 h with 0.5 microgram/ml Escherichia coli endotoxin [lipopolysaccharide (LPS)], 10 ng/ml tumor necrosis factor (TNF)-alpha, or 2 ng/ml interleukin-1 beta (IL-1 beta). Cotreatments were performed with IL-1 beta and 4 microM actinomycin or 50 microM cycloheximide. Northern analysis demonstrated 23-fold, 8-fold, and 6-fold inductions of MnSOD mRNA by IL-1 beta, LPS, and TNF-alpha, respectively. Induction of MnSOD by IL-1 beta was eliminated by actinomycin but not by cycloheximide, implicating a requirement for de novo transcription. Western analysis resulted in a 23.7-fold induction of MnSOD protein after 48-h treatment with IL-1 beta. Induction of MnSOD by IL-1 beta and other inflammatory mediators may serve as a protective mechanism to reduce oxygen free radical- and nitric oxide-mediated cell damage during inflammation.
bioRxiv (Cold Spring Harbor Laboratory), Oct 21, 2021
As secondary lymphoid organs, the spleen and lymph node represent important hubs for both innate ... more As secondary lymphoid organs, the spleen and lymph node represent important hubs for both innate and adaptive immunity 1,2. Neuroanatomical and tracing data, largely derived from rodents, suggest that lymph nodes contain sensory and sympathetic innervation 3,4 , whereas the spleen contains postganglionic sympathetic innervation 5 , with conflicting views regarding the
OBJECTIVE: To test the hypothesis that a mutation in the voltage-gated potassium channel Kv3.3 ca... more OBJECTIVE: To test the hypothesis that a mutation in the voltage-gated potassium channel Kv3.3 causes disordered binary hearing and processing of sound localization cues. BACKGROUND: We studied an SCA13 kindred carrying an autosomal dominant mutation in the gene coding for Kv3.3, which is expressed at high levels in binaural auditory brainstem pathways. DESIGN/METHODS: Genotype status of affected and unaffected family members was confirmed following auditory testing. Clinical severity was determined utilizing the Scale for the Assessment and Rating of Ataxia (SARA) with a range from 0 (asymptomatic) to 40 (severe disability). We conducted hearing tests with 13 heterozygous family members (affected listeners), 6 homozygous family members (familial controls), and an age-matched control group of 16 unrelated normal-hearing individuals (non-familial controls). All listeners demonstrated age-appropriate pure-tone audiograms. We used an adaptive psychophysical procedure measuring threshol...
Highlights d ACE2 mRNA and protein are expressed in human pancreatic ducts and microvasculature d... more Highlights d ACE2 mRNA and protein are expressed in human pancreatic ducts and microvasculature d ACE2 mRNA was rarely detected and at low levels in human pancreatic endocrine cells d Pancreatic ACE2 protein expression changes across the lifespan and correlates with BMI d SARS-CoV-2 NP was detected in ducts, but not endocrine cells, of COVID-19 pancreata
Current evidence suggests that alteration in cellular metabolism leads to pancreatic β-cell dysfu... more Current evidence suggests that alteration in cellular metabolism leads to pancreatic β-cell dysfunction and loss in type 1 diabetes (T1D). The mammalian target of rapamycin, mTOR is a key regulator of cell survival and growth in response to intracellular energy levels, nutrients, and growth factor signaling. Dysregulated mTOR complex 1 (mTORC1) has been implicated in β-cell loss and dysfunction in T2D but has not been well studied in T1D. We hypothesized that genes associated with three pathways involved in the regulation of mTORC1 would display altered expression in T1D organ donor pancreata compared to nondiabetic controls. We isolated total RNA from 30 T1D and 30 unaffected control human organ donor pancreata obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD) program and performed real-time qPCR on 26 genes related to these pathways, all of which demonstrated significant (p<0.05) induction. In relation to cellular energy balance, we observed induction o...
American Journal of Physiology-Lung Cellular and Molecular Physiology, 1996
Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observ... more Exposure to high partial pressures of oxygen are toxic to the lung, and much of the damage observed is related to injury of the pulmonary microvasculature. In this study, we evaluated the response of the pulmonary microvascular endothelial cell to high oxygen concentrations, using two-dimensional protein gel electrophoresis as a direct molecular assay of differences between cells exposed to room air or hyperoxia. We observed a differential expression of five specific proteins within 24 h of a hyperoxic insult that we termed hyperoxia-responsive proteins. After 4 h of hyperoxia there was a decrease in two of the proteins. From 8 to 24 h we observed a repression of a third and an induction of the other two proteins. One of the induced proteins was also increased by heat shock and hydrogen peroxide and has characteristics similar to heat shock protein (HSP) 32 (heme oxygenase 1). Western analysis using an antibody specific to rat heme oxygenase 1 verified that this oxygen-responsive pr...
American Journal of Physiology-Cell Physiology, 1992
The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-m... more The differentiation of 3T3-L1 fibroblasts to adipocytes can be accelerated by the addition of 1-methyl-3-isobutylxanthine (MIX), insulin, and dexamethasone to the culture medium. During differentiation, we have demonstrated that the level of both annexin I mRNA and protein decreases. The half-times for this reduction were 2 h and 10 h for annexin I mRNA and protein, respectively. Of the added agents in the differentiation medium, only MIX caused a decline in annexin I expression in 3T3-L1 fibroblasts. The MIX effect in fibroblasts was reversible and required de novo transcription but not protein synthesis. Although MIX could be replaced by high levels of theophylline, neither agonists of the beta-adrenergic receptor nor intracellular second messengers, cAMP and cGMP, were able to reduce annexin I. The potential role of annexin I in cellular differentiation is discussed.
American Journal of Physiology-Lung Cellular and Molecular Physiology, 1994
Even though endothelial cells from different locations have similarities, there are potential mor... more Even though endothelial cells from different locations have similarities, there are potential morphological and functional differences between cells from different vascular regions, as well as between species. Our laboratory is interested in studying the molecular regulation of vasoactive substances in pulmonary vasculature. Therefore, we have developed reproducible methodology to isolate and maintain cultures of human pulmonary artery endothelial cells. The major innovation has been the employment of sections of pulmonary artery from heart transplant donors, from which endothelial cells are isolated. Cell monolayers were identified as endothelial cells by phase-contrast microscopy. Representative dishes of cells were further characterized by indirect immunofluorescent staining for factor VIII antigen, uptake of acetylated low-density lipoprotein, and electron microscopy. These cells were also evaluated for the expression of endothelin-1 (ET-1), a vasoactive 21-amino acid peptide de...
In vivo footprinting experiments have been used to analyze the binding of transacting regulatory ... more In vivo footprinting experiments have been used to analyze the binding of transacting regulatory factors in the 5' flanking region upstream of the alcohol dehydrogenase (Adh) gene from Arabidopsis thaliana. Protein-DNA interactions were detected by dimethyl sulfate footprinting and genomic sequencing, using an A. thaliana cell suspension culture that constitutively expressed the Adh gene. Several distinct footprinting domains have been characterized, and the potential effects of the corresponding transacting factors have been inferred from a comparison with data from the maize alcohol dehydrogenase-1 (Adhl) gene. One binding site is similar in sequence_to one of the anaerobic response elements (ARE) of the maize gene, which has also been shown to bind to a transacting factor. Several of the remaining binding sites apparently represent a class' of elements sharing the sequence 5'-GTGG-3' within their footprint. Comparisons with maize Adhl in vivo protein interactions reveal that the elements of Adh promoter structure are highly conserved, but the relative and absolute positions of the elements are variable.
Biochemical and Biophysical Research Communications, 1993
Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepa... more Biosynthesis of several rat liver proteins is enhanced by amino acid deprivation of cultured hepatocytes or hepatoma cells. One of these proteins, MP-73, was synthesized at a rate 2- to 3-fold greater when cells were incubated for 3-9 h under conditions of amino acid deprivation versus amino acid supplementation. Immunoblotting with polyclonal antibodies prepared against MP-73 localized it to the inner mitochondrial membrane. MP-73 appears to be a hydrophobic, integral membrane protein. MP-73 antibody was used to identify a partial cDNA (NS3.2) of approximately 2 kb. A probe prepared from pNS3.2 identified a transcript in rat Fao hepatoma cells of approximately 4.4 kb that was increased in abundance by more than 20-fold following amino acid starvation of the cells.
American Journal of Respiratory Cell and Molecular Biology, 1995
Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especial... more Endothelin-1 (ET-1) is known to be involved in a variety of pathophysiologic conditions, especially of the pulmonary vasculature. The aim of this study was to investigate physiologic mediators potentially involved in the pathogenesis of pulmonary hypertension, for their effects on ET-1 gene expression at both the transcriptional and translational level. Rat microvascular and pulmonary artery endothelial cells grown in culture were exposed to vasoactive mediators (thrombin or an anoxic gas mixture) and inflammatory mediators (lipopolysaccharide, interleukin 1 alpha, interleukin 1 beta, or tumor necrosis factor alpha) for various time periods. The change in prepro-ET-1 (ppET-1) mRNA levels in these cells in response to stimuli was a time-dependent phenomenon. The inflammatory mediators caused an acute rise in ppET-1 mRNA levels whereby peak induction occurred after 1 h with a rapid decline to control levels by 4 h. The vasoactive mediators elicited a more sustained response whereby a significant elevation in ppET-1 mRNA expression occurred quickly and remained elevated through 4 h. The pattern of induction was more rapid for thrombin than for anoxic gas exposure. Radioimmunoassay analysis demonstrated a similar response for thrombin and the inflammatory mediators in ET-1 mature peptide release, whereas the effect of anoxic gas exposure was divergent. Significant elevations were noted after 6 h for thrombin as well as each of the inflammatory mediators except IL-1 alpha. In response to the anoxic gas exposure, however, a significant rise in ET-1 peptide release was not evident until after 24 h. To determine the level at which ppET-1 mRNA induction is regulated, cells were cotreated with each of the stimuli and actinomycin D or cycloheximide. Results indicate that the induction of ppET-1 mRNA levels is likely due to de novo transcription, as well as mRNA stabilization. In summary, inflammatory and vasoactive agents are important regulators of ET-1 gene expression in rat pulmonary endothelial cells; most important, we observed a differential response at the mRNA or peptide level depending on the mediator involved.
Uploads
Papers by Harry Nick