65 results on '"Sven Ulrik Gorr"'
Search Results
2. The salivary protein BPIFA2 differentially regulates sodium preference and blood pressure in male and female mice
- Author
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Sven-Ulrik Gorr and Michael Nevels
- Subjects
Saliva ,SPLUNC2 ,hypertension ,sodium intake ,Technology ,Medicine ,Science - Abstract
BPIFA2 (PSP, SPLUNC2, C20orf70) is a major salivary protein of uncertain physiological function. BPIFA2 is downregulated in salivary glands of spontaneously hypertensive rats, pointing to a role in blood pressure regulation. This study used a novel Bpifa2 knockout mouse model to test the role of BPIFA2 in sodium preference and blood pressure. Blood pressure did not differ between wild-type male and female mice but was significantly lower in male knockout mice compared to male wild-type mice. In contrast, blood pressure was increased in female knockout mice compared to female wild-type mice. Female wild-type mice showed a significant preference for 0.9% saline compared to male mice. This difference was reduced in the knockout mice. BPIFA2 is an LPS-binding protein but it remains to be determined if the reported effects are mediated by the LPS-binding activity of BPIFA2.
- Published
- 2020
- Full Text
- View/download PDF
3. In vivo activity and low toxicity of the second-generation antimicrobial peptide DGL13K.
- Author
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Sven-Ulrik Gorr, Craig M Flory, and Robert J Schumacher
- Subjects
Medicine ,Science - Abstract
Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect.
- Published
- 2019
- Full Text
- View/download PDF
4. A D-enantiomer of the antimicrobial peptide GL13K evades antimicrobial resistance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii.
- Author
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Helmut Hirt, Jeffrey W Hall, Elliot Larson, and Sven-Ulrik Gorr
- Subjects
Medicine ,Science - Abstract
Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.
- Published
- 2018
- Full Text
- View/download PDF
5. Antimicrobial GL13K peptide coatings killed and ruptured the wall of Streptococcus gordonii and prevented formation and growth of biofilms.
- Author
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Xi Chen, Helmut Hirt, Yuping Li, Sven-Ulrik Gorr, and Conrado Aparicio
- Subjects
Medicine ,Science - Abstract
Infection is one of the most prevalent causes for dental implant failure. We have developed a novel antimicrobial peptide coating on titanium by immobilizing the antimicrobial peptide GL13K. GL13K was developed from the human salivary protein BPIFA2. The peptide exhibited MIC of 8 µg/ml against planktonic Pseudonomas aeruginosa and their biofilms were reduced by three orders of magnitude with 100 µg/ml GL13K. This peptide concentration also killed 100% of Streptococcus gordonii. At 1 mg/ml, GL13K caused less than 10% lysis of human red blood cells, suggesting low toxicity to mammalian cells. Our GL13K coating has also previously showed bactericidal effect and inhibition of biofilm growth against peri-implantitis related pathogens, such as Porphyromonas gingivalis. The GL13K coating was cytocompatible with human fibroblasts and osteoblasts. However, the bioactivity of antimicrobial coatings has been commonly tested under (quasi)static culture conditions that are far from simulating conditions for biofilm formation and growth in the oral cavity. Oral salivary flow over a coating is persistent, applies continuous shear forces, and supplies sustained nutrition to bacteria. This accelerates bacteria metabolism and biofilm growth. In this work, the antimicrobial effect of the coating was tested against Streptococcus gordonii, a primary colonizer that provides attachment for the biofilm accretion by P. gingivalis, using a drip-flow biofilm bioreactor with media flow rates simulating salivary flow. The GL13K peptide coatings killed bacteria and prevented formation and growth of S. gordonii biofilms in the drip-flow bioreactor and under regular mild-agitation conditions. Surprisingly the interaction of the bacteria with the GL13K peptide coatings ruptured the cell wall at their septum or polar areas leaving empty shell-like structures or exposed protoplasts. The cell wall rupture was not detected under regular culture conditions, suggesting that cell wall rupture induced by GL13K peptides also requires media flow and possible attendant biological sequelae of the conditions in the bioreactor.
- Published
- 2014
- Full Text
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6. Re-probing of Immunoblots after Storage for More than a Decade
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Sven-Ulrik Gorr
- Subjects
Biology (General) ,QH301-705.5 - Published
- 1997
- Full Text
- View/download PDF
7. The antimicrobial peptide DGL13K is active against resistant gram-negative bacteria and subinhibitory concentrations stimulate bacterial growth without causing resistance
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Jadyn C. Anderson, Sven Ulrik Gorr, Hunter V Brigman, and Elizabeth B. Hirsch
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Gram-negative bacteria ,biology ,Chemistry ,Pseudomonas aeruginosa ,medicine.drug_class ,Klebsiella pneumoniae ,Antibiotics ,Antimicrobial peptides ,biology.organism_classification ,medicine.disease_cause ,Antimicrobial ,Acinetobacter baumannii ,Microbiology ,Antibiotic resistance ,medicine - Abstract
Antimicrobial peptides may be alternatives to traditional antibiotics with reduced bacterial resistance. The antimicrobial peptide GL13K was derived from the salivary protein BPIFA2. This study determined the relative activity of the L-and D-enantiomers of GL13K to wild-type and drug-resistant strains of three gram-negative species and against Pseudomonas aeruginosa biofilms. DGL13K displayed in vitro activity against ESBL and KPC-producing Klebsiella pneumoniae (MICs 16-32 μg/ml), MDR and XDR P. aeruginosa, and XDR Acinetobacter baumannii carrying metallo-beta-lactamases (MICs 8-32 μg/ml). P. aeruginosa showed low inherent resistance to DGL13K and the increased metabolic activity and growth caused by sub-MIC concentrations of GL13K peptides did not result in acquired bacterial resistance. Daily dosing for approximately two weeks did not increase the MIC of DGL13K or cause cross-resistance between LGL13K and DGL13K. These data suggest that DGL13K is a promising candidate antimicrobial peptide for further development.
- Published
- 2020
8. The parotid secretory protein BPIFA2 is a salivary surfactant that affects lipopolysaccharide action
- Author
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Thomas T. Wheeler, Seshagiri Rao Nandula, Ian Huxford, Sven Ulrik Gorr, and Conrado Aparicio
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Lipopolysaccharides ,medicine.medical_specialty ,Saliva ,Lipopolysaccharide ,Physiology ,medicine.medical_treatment ,030204 cardiovascular system & hematology ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Surface-Active Agents ,0302 clinical medicine ,Pulmonary surfactant ,Physiology (medical) ,Internal medicine ,medicine ,Metabolome ,Animals ,Metabolomics ,Oral mucosa ,Salivary Proteins and Peptides ,Mastication ,Mice, Knockout ,Nutrition and Dietetics ,Insulin ,General Medicine ,Mice, Inbred C57BL ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Knockout mouse ,030217 neurology & neurosurgery - Abstract
New findings What is the central question of this study? The salivary protein BPIFA2 binds lipopolysaccharide, but its physiological function is not known. This study uses a new knockout mouse model to explore the physiological role of BPIFA2 in the oral cavity and systemic physiology. What is the main finding and its importance? BPIFA2 is a crucial surfactant in mouse saliva. In its absence, saliva exhibits the surface tension of water. Depletion of BPIFA2 affects salivary and ingested lipopolysaccharide and leads to systemic sequelae that include increased insulin secretion and metabolomic changes. These results suggest that the lipopolysaccharide-binding activity of BPIFA2 affects the activity of ingested lipopolysaccharide in the intestine and that BPIFA2 depletion causes mild metabolic endotoxaemia. Abstract Saliva plays important roles in the mastication, swallowing and digestion of food, speech and lubrication of the oral mucosa, antimicrobial and anti-inflammatory activities, and the control of body temperature in grooming animals. The salivary protein BPIFA [BPI fold containing family A member 2; former names: parotid secretory protein (PSP), SPLUN2 and C20orf70] is related to lipid-binding and lipopolysaccharide (LPS)-binding proteins expressed in the mucosa. Indeed, BPIFA2 binds LPS, but the physiological role of BPIFA2 remains to be determined. To address this question, Bpifa2 knockout (Bpifa2tm1(KOMP)Vlcg ) (KO) mice were phenotyped, with emphasis on the saliva and salivary glands. Stimulated whole saliva collected from KO mice was less able to spread on a hydrophobic surface than wild-type saliva, and the surface tension of KO saliva was close to that of water. These data suggest that BPIFA2 is a salivary surfactant that is mainly responsible for the low surface tension of mouse saliva. The reduced surfactant activity of KO saliva did not affect consumption of dry food or grooming, but saliva from KO mice contained less LPS than wild-type saliva. Indeed, mice lacking BPIFA2 responded to ingested LPS with an increased stool frequency, suggesting that BPIFA2 plays a role in the solubilization and activity of ingested LPS. Consistent with these findings, BPIFA2-depleted mice also showed increased insulin secretion and metabolomic changes that were consistent with a mild endotoxaemia. These results support the distal physiological function of a salivary protein and reinforce the connection between oral biology and systemic disease.
- Published
- 2020
9. The parotid secretory protein BPIFA2 is a salivary surfactant that affects LPS action
- Author
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Sven Ulrik Gorr, Thomas T. Wheeler, Conrado Aparicio, Seshagiri Rao Nandula, and Ian Huxford
- Subjects
0303 health sciences ,Saliva ,medicine.medical_specialty ,Physiological function ,Chemistry ,Parotid secretory protein ,030206 dentistry ,Surfactant activity ,3. Good health ,03 medical and health sciences ,stomatognathic diseases ,0302 clinical medicine ,medicine.anatomical_structure ,Endocrinology ,Pulmonary surfactant ,stomatognathic system ,Internal medicine ,medicine ,Salivary Proteins ,Oral mucosa ,Mastication ,030304 developmental biology - Abstract
Saliva plays important roles in the mastication, swallowing and digestion of food, speech and lubrication of oral mucosa, antimicrobial and anti-inflammatory activity and control of body temperature in grooming animals. The salivary protein BPIFA2 (BPI fold containing family A member 2; former names: Parotid Secretory Protein, PSP, SPLUNC2, C20orf70) is related to lipid-binding and LPS-binding proteins expressed in mucosa. Indeed, BPIFA2 binds LPS but the physiological role of BPIFA2 remains to be determined. To address this question,Bpifa2knockout (Bpifa2tm1(KOMP)Vlcg) (KO) mice were phenotyped with a special emphasis on saliva and salivary glands. Saliva collected from KO mice was less able to spread on a hydrophobic surface than wild-type saliva and the surface tension of KO saliva was close to that of water. These data suggest that BPIFA2 is a salivary surfactant that is mainly responsible for the low surface tension of mouse saliva. The reduced surfactant activity of KO saliva did not affect consumption of dry food or grooming, but saliva from KO mice contained less LPS than wild-type saliva. Indeed, mice lacking BPIFA2 responded to ingested LPS with an increased stool frequency, suggesting that BPIFA2 plays a role in the solubilization and activity of ingested LPS. Consistent with these findings, BPIFA2-depleted mice also showed increased insulin secretion and metabolomic changes that were consistent with a mild endotoxemia. These results support the distal physiological function of a salivary protein and reinforce the connection between oral biology and systemic disease.
- Published
- 2020
- Full Text
- View/download PDF
10. The salivary protein BPIFA2 differentially regulates sodium preference and blood pressure in male and female mice
- Author
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Michael Nevels and Sven-Ulrik Gorr
- Subjects
medicine.medical_specialty ,Physiological function ,Saliva ,medicine.medical_treatment ,Sodium ,Male mice ,chemistry.chemical_element ,Biology ,Endocrinology ,Blood pressure ,chemistry ,Internal medicine ,Knockout mouse ,medicine ,Salivary Proteins ,Saline - Abstract
BPIFA2 (PSP, SPLUNC2, C20orf70) is a major salivary protein of uncertain physiological function. BPIFA2 is downregulated in salivary glands of spontaneously hypertensive rats, pointing to a role in blood pressure regulation. This study used a novel Bpifa2 knockout mouse model to test the role of BPIFA2 in sodium preference and blood pressure. Blood pressure did not differ between wild-type male and female mice but was significantly lower in male knockout mice compared to male wild-type mice. In contrast, blood pressure was increased in female knockout mice compared to female wild-type mice. Female wild-type mice showed a significant preference for 0.9% saline compared to male mice. This difference was reduced in the knockout mice. BPIFA2 is an LPS-binding protein but it remains to be determined if the reported effects are mediated by the LPS-binding activity of BPIFA2.
- Published
- 2020
11. In vivo activity and low toxicity of the second-generation antimicrobial peptide DGL13K
- Author
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Craig M. Flory, Robert J. Schumacher, and Sven Ulrik Gorr
- Subjects
0301 basic medicine ,Life Cycles ,Luminescence ,Erythrocytes ,Physiology ,Staphylococcus ,Peptide ,Moths ,medicine.disease_cause ,Toxicology ,Pathology and Laboratory Medicine ,Median lethal dose ,Mice ,Larvae ,Antibiotics ,Medicine and Health Sciences ,Staphylococcus Aureus ,Skin ,chemistry.chemical_classification ,Mice, Inbred BALB C ,Multidisciplinary ,biology ,Antimicrobials ,Physics ,Electromagnetic Radiation ,Pseudomonas Aeruginosa ,Drugs ,Animal Models ,Antimicrobial ,Bacterial Pathogens ,Anti-Bacterial Agents ,Galleria mellonella ,Experimental Organism Systems ,Staphylococcus aureus ,Medical Microbiology ,Toxicity ,Physical Sciences ,Medicine ,Female ,Pathogens ,Research Article ,Methicillin-Resistant Staphylococcus aureus ,Lysis (Medicine) ,Science ,030106 microbiology ,Antimicrobial peptides ,Mouse Models ,Microbial Sensitivity Tests ,Research and Analysis Methods ,Microbiology ,Lethal Dose 50 ,03 medical and health sciences ,Model Organisms ,In vivo ,Pseudomonas ,Microbial Control ,Tissue Repair ,medicine ,Animals ,Humans ,Pseudomonas Infections ,Amino Acid Sequence ,Microbial Pathogens ,Pharmacology ,Bacteria ,fungi ,Organisms ,Biology and Life Sciences ,biology.organism_classification ,030104 developmental biology ,HEK293 Cells ,chemistry ,Animal Studies ,Wound Infection ,Physiological Processes ,Developmental Biology ,Antimicrobial Cationic Peptides - Abstract
Antimicrobial peptides have been evaluated as possible alternatives to traditional antibiotics. The translational potential of the antimicrobial peptide DGL13K was tested with focus on peptide toxicity and in vivo activity in two animal models. DGL13K was effective against Pseudomonas aeruginosa, Staphylococcus aureus and methicillin-resistant S. aureus with minimal bactericidal concentrations similar to the minimal inhibitory concentration. The peptide showed low toxicity to human red blood cells and HEK cells with median lethal dose around 1 mg/ml. The median lethal dose in greater wax moth larvae (Galleria mellonella) was about 125mg/kg while the peptide caused no skin toxicity in a mouse model. A novel high-throughput luminescence assay was used to test peptide activity in infected G. mellonella, thus reducing vertebrate animal use. DGL13K killed P. aeruginosa in both the G. mellonella model and a mouse burn wound infection model, with bacterial viability 3-10-fold lower than in untreated controls. Future experiments will focus on optimizing peptide delivery, dose and frequency to further improve the antibacterial effect.
- Published
- 2019
12. Antimicrobial Peptide GL13K Is Effective in Reducing Biofilms of Pseudomonas aeruginosa
- Author
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Sven Ulrik Gorr and Helmut Hirt
- Subjects
Pharmacology ,Alanine ,chemistry.chemical_classification ,Pseudomonas aeruginosa ,Antimicrobial peptides ,Biofilm ,Peptide ,Biology ,medicine.disease_cause ,biology.organism_classification ,Antimicrobial ,Anti-Bacterial Agents ,Microbiology ,Infectious Diseases ,Anti-Infective Agents ,chemistry ,Biochemistry ,Biofilms ,medicine ,Pharmacology (medical) ,Peptides ,Mechanisms of Action: Physiological Effects ,Peptide sequence ,Bacteria - Abstract
Human parotid secretory protein (PSP; BPIF2A) is predicted to be structurally similar to bactericidal/permeability-increasing protein and lipopolysaccharide (LPS)-binding protein. Based on the locations of known antimicrobial peptides in the latter two proteins, potential active peptides in the PSP sequence were identified. One such peptide, GL13NH 2 (PSP residues 141 to 153) was shown previously to interfere with LPS binding and agglutinate bacteria without bactericidal activity. By introducing three additional positively charged lysine residues, the peptide was converted to the novel bactericidal cationic peptide GL13K (MIC for Pseudomonas aeruginosa , 8 μg/ml [5.6 μM]). We investigated the antibiofilm activity of GL13K against static, monospecies biofilms of P. aeruginosa PAO1. Two-hour exposure of a 24-h biofilm to 64 μg/ml (44.8 μM) GL13K reduced biofilm bacteria by 10 2 , and 100 μg/ml (70 μM) GL13K reduced bacteria by 10 3 . Similar results could be achieved on 48-h-old biofilms. Lower concentrations of GL13K (32 μg/ml [22.4 μM]) were successful in reducing biofilm cell numbers in combination with tobramycin. This combination treatment also achieved total eradication of the biofilm in a majority (67.5%) of tested samples. An alanine scan of GL13K revealed the importance of the leucine residue in position six of the peptide sequence, where replacement led to a loss of antibiofilm activity, whereas the impact of replacing charged residues was less pronounced. Bacterial metalloproteases were found to partially inactivate GL13K but not a d amino acid version of the peptide.
- Published
- 2013
13. Membrane selectivity and biophysical studies of the antimicrobial peptide GL13K
- Author
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Rolf Schmidt, Christine E. DeWolf, Vinod Balhara, and Sven Ulrik Gorr
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Circular dichroism ,Model membrane ,Lipid Bilayers ,Antimicrobial peptides ,Biophysics ,Peptide ,02 engineering and technology ,Microscopy, Atomic Force ,Biochemistry ,Protein Structure, Secondary ,03 medical and health sciences ,Lipid bilayer ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Liposome ,Circular Dichroism ,technology, industry, and agriculture ,Phosphatidylglycerols ,Isothermal titration calorimetry ,Cell Biology ,021001 nanoscience & nanotechnology ,Random coil ,Membrane disruption ,Crystallography ,Membrane ,chemistry ,Dual polarization interferometry ,Liposomes ,Phosphatidylcholines ,Antimicrobial peptide ,0210 nano-technology ,Supported lipid bilayer: atomic force microscopy ,Antimicrobial Cationic Peptides - Abstract
GL13K is a short (13 amino acid) antimicrobial peptide derived from the parotid secretory protein. GL13K has been found to exhibit anti-inflammatory and antibacterial activities in physiological salt conditions. We investigated the mechanism of interaction of GL13K, with model membranes comprising 1, 2-dioleoylphosphatidylcholine (DOPC) and 1, 2-dioleoylphosphatidylglycerol (DOPG) using various biophysical and imaging techniques. Circular dichroism studies showed that GL13K adopts a β-sheet structure in the presence of negatively charged DOPG liposomes while it retains its random coil structure with zwitterionic DOPC liposomes. GL13K did not cause any fusion of these liposomes but was able to selectively disrupt the negatively charged membranes of DOPG leading to vesicular leakage. There was no or minimal evidence of GL13K interaction with DOPC liposomes, however an analysis of supported lipid bilayers (SLBs) using atomic force microscopic (AFM) imaging and dual polarization interferometry (DPI) suggested that GL13K can interact with the surface of a DOPC planar bilayer. In the case of DOPG bilayers, AFM and DPI clearly showed membrane thinned regions where a portion of lipid molecules has been removed. These results suggest that the mechanism of GL13K action on bacterial membranes involves localized removal of lipid from the membrane via peptide-induced micellization.
- Published
- 2013
14. Hydrophobic Oxime Ethers: A Versatile Class of pDNA and siRNA Transfection Lipids
- Author
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Ralph J. Knipp, Seshagiri Rao Nandula, Laura E. Gordon, Michael H. Nantz, Sven Ulrik Gorr, Geoffrey J. Clark, and Souvik Biswas
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Lung Neoplasms ,Cell Survival ,Genetic Vectors ,Green Fluorescent Proteins ,Breast Neoplasms ,Transfection ,Biochemistry ,Article ,chemistry.chemical_compound ,Cell Line, Tumor ,Oximes ,Drug Discovery ,Humans ,Organic chemistry ,Luciferase ,RNA, Small Interfering ,General Pharmacology, Toxicology and Pharmaceutics ,Luciferases ,Cytotoxicity ,Pharmacology ,Liposome ,Chemistry ,Organic Chemistry ,Gene Transfer Techniques ,Cationic polymerization ,DNA ,Oxime ,Lipids ,Combinatorial chemistry ,Polynucleotide ,Click chemistry ,Molecular Medicine ,Click Chemistry ,Hydrophobic and Hydrophilic Interactions ,Ethers - Abstract
The manipulation of the cationic lipid structures to increase polynucleotide binding and delivery properties, while also minimizing associated cytotoxicity, has been a principal strategy for developing next-generation transfection agents. The polar (DNA binding) and hydrophobic domains of transfection lipids have been extensively studied; however, the linking domain comprising the substructure used to tether the polar and hydrophobic domains has attracted considerably less attention as an optimization variable. Here, we examine the use of an oxime ether as the linking domain. Hydrophobic oxime ethers were readily assembled via click chemistry by oximation of hydrophobic aldehydes using an aminooxy salt. A facile ligation reaction delivered the desired compounds with hydrophobic domain asymmetry. Using the MCF-7 breast cancer, H1792 lung cancer and PAR C10 salivary epithelial cell lines, our findings show that lipoplexes derived from oxime ether lipids transfect in the presence of serum at higher levels than commonly used liposome formulations, based on both luciferase and green fluorescent protein (GFP) assays. Given the biological compatibility of oxime ethers and their ease of formation, this functional group should find significant application as a linking domain in future designs of transfection vectors.
- Published
- 2011
15. Antimicrobial peptides and periodontal disease
- Author
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Sven Ulrik Gorr and Mahsa Abdolhosseini
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chemistry.chemical_classification ,Saliva ,medicine.drug_class ,medicine.medical_treatment ,Antibiotics ,Antimicrobial peptides ,Peptide ,Biology ,Antimicrobial ,Microbiology ,Cathelicidin ,Immune system ,chemistry ,Immunology ,medicine ,Periodontics ,Defensin - Abstract
Gorr S-U, Abdolhosseini M. Antimicrobial peptides and periodontal disease. J Clin Periodontol 2011; 38 (Suppl. 11): 126–141. doi: 10.1111/j.1600-051X.2010.01664.x. Abstract Aims: The goal of this review is to identify the antimicrobial proteins in the oral fluids, saliva and gingival crevicular fluid and identify functional families and candidates for antibacterial treatment. Results: Periodontal biofilms initiate a cascade of inflammatory and immune processes that lead to the destruction of gingival tissues and ultimately alveolar bone loss and tooth loss. Treatment of periodontal disease with conventional antibiotics does not appear to be effective in the absence of mechanical debridement. An alternative treatment may be found in antimicrobial peptides and proteins, which can be bactericidal and anti-inflammatory and block the inflammatory effects of bacterial toxins. The peptides have co-evolved with oral bacteria, which have not developed significant peptide resistance. Over 45 antibacterial proteins are found in human saliva and gingival crevicular fluid. The proteins and peptides belong to several different functional families and offer broad protection from invading microbes. Several antimicrobial peptides and proteins (AMPs) serve as templates for the development of therapeutic peptides and peptide mimetics, although to date none have demonstrated efficacy in human trials. Conclusions: Existing and newly identified AMPs may be developed for therapeutic use in periodontal disease or can serve as templates for peptide and peptide mimetics with improved therapeutic indices.
- Published
- 2011
16. A D-enantiomer of the antimicrobial peptide GL13K evades antimicrobial resistance in the Gram positive bacteria Enterococcus faecalis and Streptococcus gordonii
- Author
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Elliot Larson, Helmut Hirt, Jeffrey W. Hall, and Sven Ulrik Gorr
- Subjects
0301 basic medicine ,Autolysis (biology) ,Cell Membranes ,lcsh:Medicine ,Pathology and Laboratory Medicine ,Biochemistry ,Bacterial cell structure ,Isomers ,Anti-Infective Agents ,Stereochemistry ,Medicine and Health Sciences ,Stereoisomers ,lcsh:Science ,Multidisciplinary ,biology ,Chemistry ,Gram Positive Bacteria ,Streptococcus gordonii ,Proteases ,Antimicrobial ,Bacterial Pathogens ,Enzymes ,Medical Microbiology ,Cell Processes ,Physical Sciences ,Pathogens ,Cellular Structures and Organelles ,Research Article ,Gram-positive bacteria ,030106 microbiology ,Antimicrobial peptides ,Enterococcus Faecalis ,Microbial Sensitivity Tests ,Gram-Positive Bacteria ,Microbiology ,Enterococcus faecalis ,03 medical and health sciences ,Isomerism ,Microbial Control ,Drug Resistance, Bacterial ,Microbial Pathogens ,Gram-Positive Bacterial Infections ,Pharmacology ,Bacteria ,lcsh:R ,Organisms ,Chemical Compounds ,Biology and Life Sciences ,Proteins ,Bacteriology ,Cell Biology ,biology.organism_classification ,Peptide Fragments ,Enantiomers ,Biofilms ,Enzymology ,lcsh:Q ,Antimicrobial Resistance ,Autolysis ,Enterococcus ,Antimicrobial Cationic Peptides - Abstract
Antimicrobial peptides represent an alternative to traditional antibiotics that may be less susceptible to bacterial resistance mechanisms by directly attacking the bacterial cell membrane. However, bacteria have a variety of defense mechanisms that can prevent cationic antimicrobial peptides from reaching the cell membrane. The L- and D-enantiomers of the antimicrobial peptide GL13K were tested against the Gram-positive bacteria Enterococcus faecalis and Streptococcus gordonii to understand the role of bacterial proteases and cell wall modifications in bacterial resistance. GL13K was derived from the human salivary protein BPIFA2. Minimal inhibitory concentrations were determined by broth dilution and a serial assay used to determine bacterial resistance. Peptide degradation was determined in a bioassay utilizing a luminescent strain of Pseudomonas aeruginosa to detect peptide activity. Autolysis and D-alanylation-deficient strains of E. faecalis and S. gordonii were tested in autolysis assays and peptide activity assays. E. faecalis protease inactivated L-GL13K but not D-GL13K, whereas autolysis did not affect peptide activity. Indeed, the D-enantiomer appeared to kill the bacteria prior to initiation of autolysis. D-alanylation mutants were killed by L-GL13K whereas this modification did not affect killing by D-GL13K. The mutants regained resistance to L-GL13K whereas bacteria did not gain resistance to D-GL13K after repeated treatment with the peptides. D-alanylation affected the hydrophobicity of bacterial cells but hydrophobicity alone did not affect GL13K activity. D-GL13K evades two resistance mechanisms in Gram-positive bacteria without giving rise to substantial new resistance. D-GL13K exhibits attractive properties for further antibiotic development.
- Published
- 2018
17. Antimicrobial peptides of the oral cavity
- Author
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Sven Ulrik Gorr
- Subjects
Mouth ,business.industry ,medicine.drug_class ,Extramural ,Antibiotics ,Antimicrobial peptides ,MEDLINE ,Gingival Crevicular Fluid ,Pharmacology ,Oral cavity ,Pharmacotherapy ,Anti-Infective Agents ,Humans ,Periodontics ,Medicine ,Mouth Diseases ,Saliva ,business ,Periodontal Diseases ,Antimicrobial Cationic Peptides - Published
- 2009
18. Secretory cargo composition affects polarized secretion in MDCK epithelial cells
- Author
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Sven Ulrik Gorr, David V. Cohn, Ulrike Kühn, and Brigitte H. Fasciotto
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Molecular Sequence Data ,Clinical Biochemistry ,Cell ,Parathyroid hormone ,Biology ,Cell Line ,Mice ,Dogs ,medicine ,Animals ,Humans ,Secretion ,Amino Acid Sequence ,Molecular Biology ,Furin ,Secretory pathway ,Cell Polarity ,Chromogranin A ,Biological Transport ,Epithelial Cells ,Cell Biology ,General Medicine ,Cell biology ,Cholesterol ,Secretory protein ,medicine.anatomical_structure ,Parathyroid Hormone ,biology.protein ,Cattle ,Mutant Proteins ,Parathyroid gland - Abstract
Polarized epithelial cells secrete proteins at either the apical or basolateral cell surface. A number of non-epithelial secretory proteins also exhibit polarized secretion when they are expressed in polarized epithelial cells but it is difficult to predict where foreign proteins will be secreted in epithelial cells. The question is of interest since secretory epithelia are considered as target tissues for gene therapy protocols that aim to express therapeutic secretory proteins. In the parathyroid gland, parathyroid hormone is processed by furin and co-stored with chromogranin A in secretory granules. To test the secretion of these proteins in epithelial cells, they were expressed in MDCK cells. Chromogranin A and a secreted form of furin were secreted apically while parathyroid hormone was secreted 60% basolaterally. However, in the presence of chromogranin A, the secretion of parathyroid hormone was 65% apical, suggesting that chromogranin can act as a "sorting escort" (sorting chaperone) for parathyroid hormone. Conversely, apically secreted furin did not affect the sorting of parathyroid hormone. The apical secretion of chromogranin A was dependent on cholesterol, suggesting that this protein uses an established cellular sorting mechanism for apical secretion. However, this sorting does not involve the N-terminal membrane-binding domain of chromogranin A. These results suggest that foreign secretory proteins can be used as "sorting escorts" to direct secretory proteins to the apical secretory pathway without altering the primary structure of the secreted protein. Such a system may be of use in the targeted expression of secretory proteins from epithelial cells.
- Published
- 2007
19. Human variability in innate immunity
- Author
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George Hajishengallis, Donald R. Demuth, Michael Martin, Denis F. Kinane, and Sven Ulrik Gorr
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Innate immune system ,Virulence Factors ,Extramural ,business.industry ,Toll-Like Receptors ,MEDLINE ,Genetic Variation ,Immunity, Innate ,Human variability ,Disease susceptibility ,Cytokines metabolism ,Immunity ,Immunology ,Animals ,Cytokines ,Humans ,Periodontics ,Medicine ,Disease Susceptibility ,Periodontitis ,business ,Porphyromonas gingivalis ,Antimicrobial Cationic Peptides ,Signal Transduction - Published
- 2007
20. Isoproterenol increases sorting of parotid gland cargo proteins to the basolateral pathway
- Author
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Sven Ulrik Gorr, Jinlian Tan, S. G. Venkatesh, and Douglas S. Darling
- Subjects
Male ,Physiology ,Endogeny ,Protein Sorting Signals ,Biology ,Article ,Rats, Sprague-Dawley ,medicine ,Animals ,Parotid Gland ,Secretion ,Salivary Proteins and Peptides ,Secretory pathway ,Salivary gland ,Secretory Vesicles ,Isoproterenol ,Cell Biology ,Adrenergic beta-Agonists ,Hydrogen-Ion Concentration ,Secretory Vesicle ,Rats ,Transport protein ,Protein Transport ,medicine.anatomical_structure ,Secretory protein ,Biochemistry ,Amylases ,Proline-Rich Protein Domains ,Peptides - Abstract
Exocrine cells have an essential function of sorting secreted proteins into the correct secretory pathway. A clear understanding of sorting in salivary glands would contribute to the correct targeting of therapeutic transgenes. The present work investigated whether there is a change in the relative proportions of basic proline-rich protein (PRP) and acidic PRPs in secretory granules in response to chronic isoproterenol treatment, and whether this alters the sorting of endogenous cargo proteins. Immunoblot analysis of secretory granules from rat parotids found a large increase of basic PRP over acidic PRPs in response to chronic isoproterenol treatment. Pulse chase experiments demonstrated that isoproterenol also decreased regulated secretion of newly synthesized secretory proteins, including PRPs, amylase and parotid secretory protein. This decreased efficiency of the apical regulated pathway may be mediated by alkalization of the secretory granules since it was reversed by treatment with mild acid. We also investigated changes in secretion through the basolateral (endocrine) pathways. A significant increase in parotid secretory protein and salivary amylase was detected in sera of isoproterenol-treated animals, suggesting increased routing of the regulated secretory proteins to the basolateral pathway. These studies demonstrate that shifts of endogenous proteins can modulate regulated secretion and sorting of cargo proteins.
- Published
- 2007
21. The Salivary Protein PSP/Bpifa2 Protects Against Intestinal Inflammation
- Author
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Helmut Hirt, Seshagiri Rao Nandula, and Sven Ulrik Gorr
- Subjects
business.industry ,digestive, oral, and skin physiology ,Inflammatory Bowel Diseases ,medicine.disease ,digestive system ,Biochemistry ,digestive system diseases ,Malnutrition ,Diarrhea ,Immune system ,Weight loss ,Intestinal inflammation ,Immunology ,Genetics ,Salivary Proteins ,Medicine ,sense organs ,medicine.symptom ,business ,Molecular Biology ,Biotechnology - Abstract
Inflammatory bowel diseases are associated with intestinal inflammation, diarrhea, malnutrition and weight loss. IBD appears to result from immune system dysregulation, diet and changes to the gut ...
- Published
- 2015
22. Parotid Secretory Granules: Crossroads of Secretory Pathways and Protein Storage
- Author
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S. G. Venkatesh, Douglas S. Darling, and Sven Ulrik Gorr
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Secretory granule maturation ,Protein Sorting Signals ,Article ,03 medical and health sciences ,0302 clinical medicine ,stomatognathic system ,Internal medicine ,Major Salivary Gland ,medicine ,Humans ,Parotid Gland ,Secretion ,Salivary Proteins and Peptides ,General Dentistry ,Secretory pathway ,Glycoproteins ,Salivary gland ,Chemistry ,Secretory Vesicles ,030206 dentistry ,Secretory Vesicle ,Cell biology ,030104 developmental biology ,Secretory protein ,Endocrinology ,medicine.anatomical_structure ,Adenomere - Abstract
Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins—including amylase, proline-rich proteins, and parotid secretory protein (PSP)—to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.
- Published
- 2005
23. Parotid secretory protein is expressed and inducible in human gingival keratinocytes
- Author
-
Hideki Shiba, Germán Barbieri, Hidemi Kurihara, Sven Ulrik Gorr, Denis F. Kinane, and S. G. Venkatesh
- Subjects
Keratinocytes ,Cell Culture Techniques ,Gingiva ,Biology ,Immunofluorescence ,Microbiology ,Downregulation and upregulation ,medicine ,Humans ,RNA, Messenger ,Salivary Proteins and Peptides ,Porphyromonas gingivalis ,Periodontitis ,Messenger RNA ,Estradiol ,medicine.diagnostic_test ,Reverse Transcriptase Polymerase Chain Reaction ,Tumor Necrosis Factor-alpha ,medicine.disease ,biology.organism_classification ,eye diseases ,Up-Regulation ,medicine.anatomical_structure ,Humoral immunity ,Periodontics ,Tumor necrosis factor alpha ,Keratinocyte - Abstract
Background: Parotid secretory protein (PSP) is a major salivary protein that is thought to possess both antibacterial and anti-inflammatory activity. A major question is whether PSP expression can be regulated by humoral factors and bacteria. Periodontitis is an inflammatory lesion initiated by interaction between gingival keratinocytes and periodontopathogenic microorganisms such as the Gram-negative anaerobe Porphyromonas gingivalis. Cytokines and sex hormones have been implicated in the progression of various forms of periodontal diseases. Materials and methods: We investigated the expression of PSP and its regulation in primary cultures of human gingival keratinocytes (HGK). HGK at the third or fourth passage were exposed to heat-killed P. gingivalis, tumor necrosis factor-α (TNF-α) and 17β-estradiol. The PSP mRNA levels were examined by real-time polymerase chain reaction (PCR). The protein expression of PSP was confirmed by immunofluorescence. Results: Heat-killed P. gingivalis, TNF-α and 17β-estradiol all resulted in increased HGK levels of mRNA for PSP as determined by real-time PCR analysis. Immunofluorescence demonstrated increased PSP localized within the cytoplasm of HGK following exposure to killed P. gingivalis. Conclusion: The present study has demonstrated for the first time that PSP is expressed in keratinocytes and that it can be up-regulated by bacteria and humoral factors. Thus PSP may have a role in the innate defense system at the gingival epithelial surface.
- Published
- 2005
24. Design and Validation of Anti-inflammatory Peptides from Human Parotid Secretory Protein
- Author
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Sven Ulrik Gorr, Lynne Bingle, S. G. Venkatesh, C. Geetha, and Colin D. Bingle
- Subjects
0301 basic medicine ,Lipopolysaccharide ,medicine.drug_class ,Polymyxin ,Molecular Sequence Data ,Peptide ,Plunc ,Biology ,Protein Engineering ,Cell Line ,Gene product ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,Sequence Analysis, Protein ,medicine ,Humans ,Secretion ,Amino Acid Sequence ,Cloning, Molecular ,Salivary Proteins and Peptides ,General Dentistry ,Peptide sequence ,Glycoproteins ,chemistry.chemical_classification ,Analysis of Variance ,Tumor Necrosis Factor-alpha ,Macrophages ,Membrane Proteins ,Blood Proteins ,030206 dentistry ,Phosphoproteins ,Molecular biology ,eye diseases ,Anti-Bacterial Agents ,030104 developmental biology ,Secretory protein ,Biochemistry ,chemistry ,RNA ,Sequence Alignment ,Antimicrobial Cationic Peptides - Abstract
Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNFα from RAW 264.7 macrophage cells. At 200 μg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 μg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.
- Published
- 2005
25. Differential aggregation properties of secretory proteins that are stored in exocrine secretory granules of the pancreas and parotid glands
- Author
-
Sven Ulrik Gorr, Darrin J. Cowley, and S. G. Venkatesh
- Subjects
Male ,Physiology ,Permeability ,Rats, Sprague-Dawley ,Exocrine secretion ,stomatognathic system ,medicine ,Animals ,Parotid Gland ,Amylase ,Salivary Proteins and Peptides ,Pancreas ,chemistry.chemical_classification ,Salivary gland ,biology ,Secretory Vesicles ,Cell Biology ,Hydrogen-Ion Concentration ,Saponins ,Rats ,Parotid gland ,Enzyme ,medicine.anatomical_structure ,Secretory protein ,chemistry ,Biochemistry ,Proteoglycan ,Amylases ,biology.protein ,Calcium - Abstract
Low-pH- and calcium-induced aggregation of regulated secretory proteins has been proposed to play a role in their retention and storage in secretory granules. However, this has not been tested for secretory proteins that are stored in the exocrine parotid secretory granules. Parotid granule matrix proteins were analyzed for aggregation in the presence or absence of calcium and in the pH range of 5.5 to 7.5. Amylase did not aggregate under these conditions, although
- Published
- 2004
26. Expression and anti-bacterial activity of human parotid secretory protein (PSP)
- Author
-
B. H Fasciotto Dunn, C. Geetha, Sven Ulrik Gorr, and S. G. Venkatesh
- Subjects
Saliva ,Peptide ,PC12 Cells ,Biochemistry ,Cell Line ,stomatognathic system ,Affinity chromatography ,Complementary DNA ,medicine ,Animals ,Humans ,Parotid Gland ,Secretion ,Salivary Proteins and Peptides ,chemistry.chemical_classification ,biology ,Molecular biology ,Recombinant Proteins ,eye diseases ,Anti-Bacterial Agents ,Rats ,Parotid gland ,stomatognathic diseases ,medicine.anatomical_structure ,Microscopy, Fluorescence ,chemistry ,Pituitary Gland ,Pseudomonas aeruginosa ,biology.protein ,Chromosome 20 ,Antibody - Abstract
Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands. A related sequence (C20orf70) was identified on human chromosome 20. The goal of this study was to determine if PSP is expressed in the human parotid gland. The cDNA for human PSP was amplified from a human parotid cDNA sample. A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy. Immunoaffinity chromatography suggested that PSP was expressed in human saliva. PSP is related to bactericidal/permeability-increasing protein (BPI). To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells. The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP. These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.
- Published
- 2003
27. Sorting of an exocrine secretory protein to the regulated secretory pathway in endocrine cells
- Author
-
Maylene Chu, Darrin J. Cowley, and Sven Ulrik Gorr
- Subjects
Cell type ,DNA, Complementary ,Octoxynol ,Detergents ,Immunoblotting ,Biophysics ,Enteroendocrine cell ,Biology ,PC12 Cells ,Biochemistry ,Cell Line ,Endocrine Glands ,Chromogranins ,Animals ,Endocrine system ,Secretion ,Salivary Proteins and Peptides ,Molecular Biology ,Cells, Cultured ,Constitutive secretory pathway ,Chromogranin A ,Cell Biology ,Alkaline Phosphatase ,Rats ,Protein Transport ,Secretory protein ,biology.protein ,Electrophoresis, Polyacrylamide Gel ,Cell fractionation ,Protein Binding ,Subcellular Fractions - Abstract
Regulated secretory proteins are stored in secretory granules. While the sorting and storage process appears similar in endocrine and exocrine cells, the extent of overlap of sorting between endocrine and exocrine cell types is not clear. It is predicted that exocrine regulated secretory proteins that are stored with high efficiency in exocrine granules would also be stored efficiently in endocrine granules. To test this hypothesis, parotid secretory protein (PSP), which is stored efficiently in parotid acinar cells, was expressed in the endocrine cell lines GH4C1 and PC12. PSP undergoes stimulated secretion in both cell types. Secretion is similar to that of the endocrine regulated secretory protein chromogranin A but distinct from secreted alkaline phosphatase, a marker for the constitutive secretory pathway in endocrine cells. Subcellular fractionation of GH4C1 cells revealed that PSP co-fractionates with chromogranin A but not with secreted alkaline phosphatase.
- Published
- 2002
28. A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland
- Author
-
S. G. Venkatesh and Sven Ulrik Gorr
- Subjects
Male ,Exocrine gland ,Physiology ,Biology ,Rats, Sprague-Dawley ,Sulfation ,Acinus ,Culture Techniques ,medicine ,Protein biosynthesis ,Animals ,Parotid Gland ,Glycosides ,Salivary Proteins and Peptides ,Salivary gland ,Sulfates ,Cell Biology ,Rats ,Parotid gland ,Protein Transport ,medicine.anatomical_structure ,Secretory protein ,Biochemistry ,Proteoglycan ,Amylases ,Chlorates ,biology.protein ,Proteoglycans ,Acids ,Hymecromone - Abstract
Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.
- Published
- 2002
29. N-terminal proteolytic processing of porcine chromogranin A in parathyroid tissue
- Author
-
Sven Ulrik Gorr, Brigitte H. Fasciotto, and David V. Cohn
- Subjects
endocrine system ,Sucrose ,Swine ,Physiology ,Sequence analysis ,Acid Phosphatase ,Immunoblotting ,Molecular Sequence Data ,Clinical Biochemistry ,In Vitro Techniques ,Biology ,Cytoplasmic Granules ,Biochemistry ,Parathyroid Glands ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Endocrinology ,Sequence Analysis, Protein ,Chromogranins ,Animals ,Endocrine system ,Amino Acid Sequence ,Protein Precursors ,Peptide sequence ,chemistry.chemical_classification ,L-Lactate Dehydrogenase ,Edman degradation ,Chromogranin A ,Parathyroid chief cell ,Peptide Fragments ,chemistry ,biology.protein ,Glycoprotein ,Protein Processing, Post-Translational - Abstract
Chromogranin A (CgA) is a glycoprotein stored in secretory granules of many endocrine and neuroendocrine cells. CgA undergoes tissue specific processing to release regulatory peptides. In the parathyroid, although processing is limited and variable, several CgA-derived peptides have been characterized including parastatin and betagranin. An early stage of CgA processing is the generation of a 64-kDa fragment (CgA64). In this study, we have purified CgA64 from porcine parathyroid glands by chromatographic separations. Edman degradation of this CgA64 yielded the N-terminal sequence NDQAELKEGTEEASSKEAAEKRGDXAVEKND corresponding to pCgA(94-125). Amino acid composition suggests that CgA64 corresponds to CgA(94-430) (i.e. the entire CgA molecule, less the N-terminal residues 1-93). To determine the origin of CgA64, we fractionated parathyroid membrane vesicles by sucrose gradient centrifugation. Intact CgA is predominantly located in dense sucrose fractions (secretory granules), whereas CgA64 is located near the top of the gradient (soluble protein fraction). In vitro incubation of these fractions revealed that the conversion of CgA did not occur in intact granules. These results indicate that CgA64 is not present in intact granules suggesting that it is not a naturally occurring secretory product in parathyroid cells.
- Published
- 2002
30. Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells
- Author
-
Renu K. JAIN, Paul B. M. JOYCE, Miguel MOLINETE, Philippe A. HALBAN, and Sven-Ulrik GORR
- Subjects
fungi ,Cell Biology ,Molecular Biology ,Biochemistry - Abstract
Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys49 and Cys71), which could play a role in this oligomerization. Site-directed mutagenesis of Cys49 and Cys71 showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.
- Published
- 2001
31. Aggregation Chaperones Enhance Aggregation and Storage of Secretory Proteins in Endocrine Cells
- Author
-
Renu K. Jain, Paul B.M. Joyce, and Sven-Ulrik Gorr
- Subjects
Cell Biology ,Molecular Biology ,Biochemistry - Published
- 2000
32. Polarized Secretion of the Regulated Secretory Protein Chromogranin A
- Author
-
David V. Cohn, Sven Ulrik Gorr, and Ulrike Kühn
- Subjects
endocrine system ,Glycosylation ,Biophysics ,Endogeny ,Biology ,Kidney ,Biochemistry ,Cell Line ,chemistry.chemical_compound ,Dogs ,N-linked glycosylation ,Chromogranins ,Animals ,Secretion ,Molecular Biology ,Polarity (international relations) ,Chondroitin Sulfates ,Chromogranin A ,Cell Biology ,Tunicamycin ,Hydrogen-Ion Concentration ,Endocytosis ,Cell biology ,carbohydrates (lipids) ,Secretory protein ,chemistry ,biology.protein ,Carbohydrate Metabolism ,Cattle ,Ammonium chloride - Abstract
Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75–80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.
- Published
- 2000
33. Sorting of a Constitutive Secretory Protein to the Regulated Secretory Pathway of Exocrine Cells
- Author
-
Sven Ulrik Gorr and Yancy R. Moore
- Subjects
Barium Compounds ,Biophysics ,Substance P ,Endogeny ,Cycloheximide ,Cytoplasmic Granules ,Transfection ,Biochemistry ,Dexamethasone ,Cell Line ,Potassium Chloride ,chemistry.chemical_compound ,Chlorides ,Animals ,Secretion ,Amylase ,Pancreas ,Molecular Biology ,biology ,Granule (cell biology) ,Sodium butyrate ,Cell Biology ,3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester ,Alkaline Phosphatase ,Rats ,Cell biology ,Butyrates ,Secretory protein ,chemistry ,Amylases ,biology.protein ,Tetradecanoylphorbol Acetate ,Cholecystokinin - Abstract
Exocrine cells secrete granule proteins by regulated or constitutive-like secretory pathways. It is thought that all secretory proteins can enter immature secretory granules in exocrine cells. To test this hypothesis, we expressed the constitutive secretory protein secreted alkaline phosphatase (SEAP) in the exocrine cell line AR42J and compared its secretion to that of amylase, an endogenous regulated secretory protein. Secretion of SEAP and amylase were stimulated about 1.5-fold by substance P and 2-fold by barium chloride. In dexamethasone-treated cells, SEAP and amylase secretion were stimulated about 1.8-fold by substance P, 5-fold by barium chloride, and 4-fold by cholecystokinin-8. Cycloheximide reduced basal secretion of SEAP and amylase by 50%, increasing cholecystokinin-stimulated secretion to about 10-fold. Sodium butyrate induced expression of SEAP 2-fold but had no effect on stimulated secretion. These results suggest that SEAP is stored in secretory granules in AR42J cells.
- Published
- 1999
34. Bio-inspired stable antimicrobial peptide coatings for dental applications
- Author
-
Xi Chen, Yuping Li, Conrado Aparicio, Mahsa Abdolhosseini, Sven Ulrik Gorr, and Kyle V. Holmberg
- Subjects
Materials science ,Cell Survival ,Biomedical Engineering ,chemistry.chemical_element ,Peptide ,engineering.material ,Biochemistry ,Article ,Biomaterials ,Coating ,Coated Materials, Biocompatible ,Biomimetic Materials ,Materials Testing ,Molecular Biology ,Porphyromonas gingivalis ,chemistry.chemical_classification ,Dental Implants ,biology ,General Medicine ,biology.organism_classification ,Antimicrobial ,In vitro ,Anti-Bacterial Agents ,chemistry ,engineering ,Implant ,Bacteria ,Biotechnology ,Biomedical engineering ,Titanium ,Antimicrobial Cationic Peptides - Abstract
We developed a novel titanium coating that has applications for preventing infection-related implant failures in dentistry and orthopedics. The coating incorporates an antimicrobial peptide, GL13K, derived from parotid secretory protein, which has been previously shown to be bactericidal and bacteriostatic in solution. We characterized the resulting physicochemical properties, resistance to degradation, activity against Porphyromonas gingivalis and in vitro cytocompatibility. Porphyromonas gingivalis is a pathogen associated with dental peri-implantitis, an inflammatory response to bacteria resulting in bone loss and implant failure. Our surface modifications obtained a homogeneous, highly hydrophobic and strongly anchored GL13K coating that was resistant to mechanical, thermochemical and enzymatic degradation. The GL13K coatings had a bactericidal effect and thus significantly reduced the number of viable bacteria compared to control surfaces. Finally, adequate proliferation of osteoblasts and human gingival fibroblasts demonstrated the GL13K coating's cytocompatibility. The robustness, antimicrobial activity and cytocompatibility of GL13K-biofunctionalized titanium make it a promising candidate for sustained inhibition of bacterial biofilm growth. This surface chemistry provides a basis for development of multifunctional bioactive surfaces to reduce patient morbidities and improve long-term clinical efficacy of metallic dental and orthopedic implants.
- Published
- 2013
35. Pancreastatin stimulates secretion from neonatal rat atrial cells
- Author
-
Sven Ulrik Gorr and Stefanie S. Pence
- Subjects
medicine.medical_specialty ,Cell type ,Heart Ventricles ,Stimulation ,Biology ,Pancreastatin ,chemistry.chemical_compound ,Internal medicine ,medicine ,Animals ,Secretion ,Heart Atria ,cardiovascular diseases ,Atrium (heart) ,Molecular Biology ,Cells, Cultured ,Chromogranin A ,Pancreatic Hormones ,Rats ,Endocrinology ,medicine.anatomical_structure ,chemistry ,Chondroitin sulfate proteoglycan ,cardiovascular system ,biology.protein ,Proteoglycans ,Signal transduction ,Cardiology and Cardiovascular Medicine ,Signal Transduction - Abstract
S.-U. Gorr and S. S. Pence . Pancreastatin Stimulates Secretion from Neonatal Rat Atrial Cells. Journal of Molecular and Cellular Cardiology (1995) 27, 767–771. Atrial myocardial cells store atrial natriuretic factor and secrete the peptide in response to extracellular stimulation, Ventricular myocardial cells also exhibit stimulated secretion of atrial natriuretic factor but with little or no intracellular storage. The stimulated secretion of other proteins from these cells is poorly characterized. In the present study the secretion of sulfated macromolecules from Na235SO4 labeled atrial and ventricular myocardial cells was quantitated. Both cell types secreted a chondroitin sulfate proteoglycan, as evidenced by glycanase digestion. Endothelin-1, a known stimulator of ANF secretion, stimulated secretion from atrial cells about 70% but had no effect on ventricular cell secretion. The differential stimulation of atrial and ventricular cell secretion was not due to the absence of a regulated secretory pathway in the latter cell type since phorbol ester stimulated secretion from both cell types. This result indicates that differences exist in the early steps of signal transduction between atrial and ventricular cells. Pancreastatin, a regulatory peptide derived from chromogranin A, stimulated atrial cell secretion 90%. This result suggests that chromogranin A, which has been identified in atrial secretory granules, may play an autoregulatory role in atrial secretion.
- Published
- 1995
36. Lysine substitutions convert a bacterial-agglutinating peptide into a bactericidal peptide that retains anti-lipopolysaccharide activity and low hemolytic activity
- Author
-
Seshagiri Rao Nandula, Sven Ulrik Gorr, Mahsa Abdolhosseini, Jonathan Song, and Helmut Hirt
- Subjects
Lipopolysaccharides ,Male ,Agglutination ,Physiology ,Gram-positive bacteria ,Peptide ,medicine.disease_cause ,Biochemistry ,Hemolysis ,Article ,Microbiology ,Cellular and Molecular Neuroscience ,Mice ,Endocrinology ,Anti-Infective Agents ,medicine ,Escherichia coli ,Animals ,chemistry.chemical_classification ,Alanine ,biology ,Lysine ,Streptococcus gordonii ,medicine.disease ,biology.organism_classification ,Peptide Fragments ,Amino acid ,Anti-Bacterial Agents ,Mice, Inbred C57BL ,chemistry ,Amino Acid Substitution ,Pseudomonas aeruginosa ,Porphyromonas gingivalis ,Bacteria ,Antimicrobial Cationic Peptides - Abstract
GL13NH2 is a bacteria-agglutinating peptide derived from the sequence of the salivary protein parotid secretory protein (PSP, BPIFA2, SPLUNC2, C20orf70). The peptide agglutinates both Gram negative and Gram positive bacteria, and shows anti-lipopolysaccharide activity in vitro and in vivo. However, GL13NH2 does not exhibit bactericidal activity. To generate a more cationic peptide with potential bactericidal activity, three amino acid residues were replaced with lysine residues to generate the peptide GL13K. In this report, the antibacterial and anti-inflammatory activities of GL13K were characterized. GL13K had lost the ability to agglutinate bacteria but gained bactericidal activity. Substitution of individual amino acids in GL13K with alanine did not restore bacterial agglutination. GL13K was bactericidal against Pseudomonas aeruginosa, Streptococcus gordonii and Escherichia coli but not Porphyromonas gingivalis. Unlike the agglutinating activity of GL13NH2, the bactericidal activity of GL13K against Pseudomonas aeruginosa was retained in the presence of saliva. Both GL13NH2 and GL13K exhibited anti-lipopolysaccharide activity. In GL13K, this activity appeared to depend on a serine hydroxyl group. GL13K protected mice from lipopolysaccharide- induced sepsis and the peptide exhibited a low level of hemolysis, suggesting that it may be suitable for in vivo application.
- Published
- 2012
37. Characterization of Constitutive Protein Secretion in PC12 Cells That Lack the Regulated Secretory Pathway
- Author
-
Renu K. Jain and Sven Ulrik Gorr
- Subjects
Time Factors ,biology ,Chemistry ,General Neuroscience ,Chromogranin A ,Regulated secretory pathway ,Alkaline Phosphatase ,PC12 Cells ,General Biochemistry, Genetics and Molecular Biology ,Rats ,Norepinephrine (medication) ,Norepinephrine ,Sulfation ,Secretory protein ,History and Philosophy of Science ,Biochemistry ,Chromogranins ,Regulated secretion ,biology.protein ,medicine ,Animals ,Constitutive secretion ,Cells, Cultured ,medicine.drug - Published
- 2002
38. Antimicrobial peptides in periodontal innate defense
- Author
-
Sven Ulrik Gorr
- Subjects
Proteases ,Saliva ,Neutrophils ,Antimicrobial peptides ,Bacterial Toxins ,Proteinase Inhibitory Proteins, Secretory ,Antigen-Presenting Cells ,Biology ,Article ,Microbiology ,Immune system ,Immunity ,Humans ,Secretion ,Salivary Proteins and Peptides ,Periodontal Diseases ,Gene Expression Profiling ,Biofilm ,Mouth Mucosa ,Epithelial Cells ,Gingival Crevicular Fluid ,Immunity, Innate ,Oral microbiology ,Biofilms ,Immunology ,Biomarkers ,Antimicrobial Cationic Peptides - Abstract
The development of oral biofilms and the host response to biofilm bacteria and their toxins are important factors in the development of periodontal disease. An early component of the host response is the secretion of antimicrobial proteins and peptides (AMPs) by salivary glands, oral epithelial cells and neutrophils. Over 45 AMPs have been identified in the oral cavity. All are found in saliva and several are also present in gingival crevicular fluid. Of these, 13 are up regulated in periodontal disease while 11 are downregulated. However, the concentrations of most AMPs found in oral fluids are below the effective in vitro concentrations, suggesting that local concentrations must be higher for effect or that additional biological functions are important in the oral cavity. Thus, in addition to direct antibacterial activity (e.g. bactericidal activity, bacterial agglutination), AMPs may affect the course of periodontal disease by inactivating bacterial or host proteases (e.g. secretory leukoprotease inhibitor) or bind bacterial toxins, including lipopolysaccharides (e.g. LL-37). Several AMPs (e.g. defensins) also act as immune system alarmins, i.e. endogenous mediators that recruit and activate antigen-presenting cells to enhance innate and adaptive immune responses. The differential regulation of AMP expression in periodontal disease suggests that AMP panels, including up- and downregulated proteins, can be used in oral fluid diagnosis of periodontal disease and to monitor treatment outcome.
- Published
- 2011
39. Dual host-defence functions of SPLUNC2/PSP and synthetic peptides derived from the protein
- Author
-
Julie Sotsky, Mahsa Abdolhosseini, Sven Ulrik Gorr, and Anuradha P. Shelar
- Subjects
Lipopolysaccharides ,Saliva ,Hemagglutination ,Lipopolysaccharide ,Peptide ,Plasma protein binding ,Biochemistry ,Article ,Microbiology ,chemistry.chemical_compound ,Animals ,Humans ,Salivary Proteins and Peptides ,chemistry.chemical_classification ,biology ,Biological activity ,Bacterial Infections ,Haemolysis ,biology.organism_classification ,Immunity, Innate ,Peptide Fragments ,Anti-Bacterial Agents ,chemistry ,Bacteria ,Protein Binding - Abstract
PSP (parotid secretory protein)/SPLUNC2 (short palate, lung and nasal epithelium clone 2) is expressed in human salivary glands and saliva. The protein exists as an N-glycosylated and non-glycosylated form and both appear to induce agglutination of bacteria, a major antibacterial function for salivary proteins. Both forms of PSP/SPLUNC2 bind LPS (lipopolysaccharide), suggesting that the protein may also play an anti-inflammatory role. Based on the predicted structure of PSP/SPLUNC2 and the location of known antibacterial and anti-inflammatory peptides in BPI (bactericidal/permeability-increasing protein) and LBP (LPS-binding protein), we designed GL13NH2 and GL13K, synthetic peptides that capture these proposed functions of PSP/SPLUNC2. GL13NH3 agglutinates bacteria, leading to increased clearance by macrophages and reduced spread of infection in a plant model. GL13K kills bacteria with a minimal inhibitory concentration of 5–10 μg/ml, kills bacteria in biofilm and retains activity in 150 mM NaCl and 50% saliva. Both peptides block endotoxin action, but only GL13K appears to bind endotoxin. The peptides do not cause haemolysis, haemagglutination in serum, inhibit mammalian cell proliferation or induce an inflammatory response in macrophages. These results suggest that the GL13NH2 and the modified peptide GL13K capture the biological activity of PSP/SPLUNC2 and can serve as lead compounds for the development of novel antimicrobial and anti-inflammatory peptides.
- Published
- 2011
40. Human Parotid Secretory Protein is a lipopolysaccharide-binding protein: identification of an anti-inflammatory peptide domain
- Author
-
Mahsa Abdolhosseini, Paul B.M. Joyce, Julie Sotsky, Anuradha P. Shelar, and Sven Ulrik Gorr
- Subjects
Clinical Biochemistry ,Anti-Inflammatory Agents ,Peptide ,Plunc ,Article ,Protein structure ,Humans ,Secretion ,Salivary Proteins and Peptides ,Molecular Biology ,chemistry.chemical_classification ,Membrane Glycoproteins ,biology ,Macrophages ,Acute-phase protein ,Cell Biology ,General Medicine ,Molecular biology ,eye diseases ,Peptide Fragments ,Protein Structure, Tertiary ,Secretory protein ,chemistry ,Biochemistry ,biology.protein ,Tumor necrosis factor alpha ,Carrier Proteins ,Lipopolysaccharide binding protein ,Acute-Phase Proteins - Abstract
Parotid secretory protein (PSP) (C20orf70) is a salivary protein of unknown function. The protein belongs to the palate, lung, and nasal epithelium clone (PLUNC) family of mucosal secretory proteins that are predicted to be structurally similar to lipid-binding and host-defense proteins including bactericidal/permeability-increasing protein and lipopolysaccharide-binding protein. However, the PLUNC proteins exhibit significant sequence variation and different biological functions have been proposed for different family members. This study tested the functional implications of the proposed similarity of PSP to the acute phase protein lipopolysaccharide-binding protein (LBP). PSP was identified in human saliva and was soluble in 70% ethanol, as shown for other PLUNC proteins. PSP binds lipopolysaccharide and can be eluted by non-ionic detergent, but not by urea or high salt. A synthetic PSP peptide, GL13NH2, which corresponds to a lipopolysaccharide-inhibiting peptide from LBP, inhibited the binding of lipopolysaccharide to both PSP and lipopolysaccharide-binding protein. Peptides from other regions of PSP and the control peptide polymyxin B showed no effect on the binding of PSP to lipopolysaccharide. GL13NH2 also inhibited lipopolysaccharide-stimulated secretion of tumor necrosis factor from macrophages. The other PSP peptides had no effect in this assay. PSP peptides had no or only minor effect on macrophage cell viability. These results indicate that PSP is a lipopolysaccharide-binding protein that is functionally related to LBP, as suggested by their predicted structural similarities.
- Published
- 2011
41. Antimicrobial-peptide coating that ruptures the wall of Gram positive bacteria
- Author
-
Xi Chen, Helmut Hirt, Yuping Li, Conrado Aparicio, and Sven Ulrik Gorr
- Subjects
chemistry.chemical_classification ,Materials science ,biology ,Gram-positive bacteria ,Peptide ,engineering.material ,biology.organism_classification ,Antimicrobial ,Microbiology ,Coating ,chemistry ,Mechanics of Materials ,engineering ,General Materials Science ,General Dentistry - Published
- 2014
42. Autocrine inhibition of parathyroid cell secretion requires proteolytic processing of chromogranin A
- Author
-
Brigitte H. Fasciotto, David V. Cohn, and Sven Ulrik Gorr
- Subjects
endocrine system ,medicine.medical_specialty ,Swine ,medicine.medical_treatment ,Enteroendocrine cell ,Biology ,Biochemistry ,Pancreastatin ,Parathyroid Glands ,Endocrinology ,Internal medicine ,Chromogranins ,medicine ,Animals ,Protease Inhibitors ,Secretion ,Autocrine signalling ,Cells, Cultured ,Antiserum ,Protease ,Chromogranin A ,Parathyroid chief cell ,Precipitin Tests ,biology.protein ,Surgery ,Protein Processing, Post-Translational ,Peptide Hydrolases - Abstract
Summary Chromogranin A (CgA, Secretory Protein-I) is a protein of about 450 amino acids representing a major soluble component of the secretory granules of parathyroid and other endocrine and neuroendocrine cells. In the parathyroid, CgA is costored and cosecreted with parathormone (PTH). We earlier found that CgA and the derived peptide, pancreastatin, inhibited secretion of PTH and CgA by parathyroid cells in culture and that CgA antiserum stimulated secretion above the maximum achieved at low (0.5 mM) Ca2+. In the present study, porcine parathyroid cells were incubated at different cell concentrations at low Ca2+. The amount of secreted CgA increased over the 6-h incubation period at 1 × 106 to 4 × 106 cells/ml, but plateaued after 3 h at 6 × 106 cells/ml. Secretion did not plateau when antiserum to CgA and pancreastatin were present at the start of incubation or resumed when the antisera were added at 3 h. Conditioned medium contained a factor or factors that blocked secretion by fresh parathyroid cells at 0.5 mM Ca2+. Pulse-chase studies revealed that 40% of the secreted CgA was processed after 6 h of chase, α-2-macroglobulin, an inhibitor of proteolytic processing, increased the amount of CgA in the medium by 30% at 1 h of chase and decreased the amount processed to 20% by 6 h. Other protease inhibitors similarly enhanced the amount of CgA in the medium. These data indicate that proteolytic processing of intact CgA is requisite for its autocrine inhibitory activity.
- Published
- 1992
43. Sulfated secreted forms of bovine and porcine parathyroid chromogranin A (secretory protein-I)
- Author
-
David V. Cohn, J.W. Hamilton, and Sven Ulrik Gorr
- Subjects
chemistry.chemical_classification ,endocrine system ,Glycosylation ,endocrine system diseases ,biology ,Chromogranin A ,Cell Biology ,Oligosaccharide ,Biochemistry ,chemistry.chemical_compound ,Proteoglycan ,chemistry ,biology.protein ,Endocrine system ,Secretion ,Tyrosine ,Glycoprotein ,Molecular Biology - Abstract
Chromogranin A (secretory protein-I) is an acidic sulfated glycoprotein found in secretory granules of most endocrine and neuroendocrine cells. In the parathyroid it is co-stored and secreted with parathormone in response to hypocalcemia. Differences in post-translational modifications have been reported between chromogranin A from the bovine adrenal and porcine parathyroid glands. The former has been reported to be sulfated mainly on oligosaccharide residues and apparently includes a proteoglycan form, whereas the latter was previously reported to be tyrosine sulfated with little of the proteoglycan form present. Here we have directly compared 35SO4-labeled parathyroid chromogranin A from the pig and the cow to determine if these reported differences were tissue or species specific. We find that the chromogranin A secreted by the bovine gland contains a proteoglycan form, whereas that from the porcine gland does not. Moreover, chromogranin A of both species is primarily sulfated on oligosaccharide residues with little if any tyrosine sulfate detected. Differences were detected in the structure of sulfated O-linked oligosaccharides in bovine and porcine parathyroid chromogranin A.
- Published
- 1991
44. Autocrine Regulation of Parathyroid Secretion: Inhibition of Secretion by Chromogranin-A (Secretory Protein-I) and Potentiation of Secretion by Chromogranin-A and Pancreastatin Antibodies*
- Author
-
Sven Ulrik Gorr, Brigitte H. Fasciotto, David V. Cohn, and Agnes M. Bourdeau
- Subjects
endocrine system ,medicine.medical_specialty ,Swine ,Parathyroid hormone ,Nerve Tissue Proteins ,Enteroendocrine cell ,Pancreastatin ,Parathyroid Glands ,Endocrinology ,Internal medicine ,Chromogranins ,medicine ,Animals ,Secretion ,Autocrine signalling ,Cells, Cultured ,biology ,Immune Sera ,Chromogranin A ,Parathyroid chief cell ,Pancreatic Hormones ,Parathyroid Hormone ,biology.protein ,Calcium ,Endocrine gland - Abstract
Chromogranin-A, also referred to as secretory protein-I, is a 50-kDa protein present in and secreted by most endocrine cells together with the native hormone. Porcine chro- mogranin-A contains a sequence identical to pancreastatin, sug- gesting that it is the precursor of pancreastatin. Pancreastatin is a potent inhibitor of parathyroid gland secretion, and it and chromogranin-A inhibit glucose-stimulated insulin release by the pancreas. It is possible that chromogranin-A, pancreastatin, or a related peptide is a physiological inhibitor of secretion by the parathyroid as well as other endocrine glands. As a test of this hypothesis, parathyroid cells in culture were incubated with purified porcine chromogranin-A or antisera to chromogranin- A and pancreastatin. In the absence of exogenous chromogranin- A or antisera, secretion of chromogranin-A and PTH at 0.5 mM Ca 2+ was about twice that at 3.0 mM Ca 2+ . When intact chro- mogranin-A was added to the incubation medium at 0.5 mM Ca 2+ , secretion was reduced to the basal level obtained at 3.0 mM Ca 2+ . Chromogranin-A did not affect the secretion of cells incubated at 3.0 mM Ca 2+ . At 1 h of incubation, 100 nM chro- mogranin-A was equivalent in potency to 1 nM pancreastatin, but after 3 h the two agents were equipotent. This suggests that chromogranin-A was processed into biologically active peptide(s) during incubation. Antisera directed against chromogranin-A or pancreastatin potentiated the secretion of both chromogranin- A and PTH at 0.5 mM, but not 3.0 mM, Ca 2+ . This stimulatory action of the antisera was dose dependent from 1:3200 to 1:400 final dilution, was effective within 2 h, and did not shift the Ca 2+ set-point for glandular secretion. These results are consonant with chromogranin-A-derived peptides serving as an autocrine inhibitor of parathyroid gland secretion. (Endocrinology 127: 1329-1335, 1990)
- Published
- 1990
45. Secretion of sulfated and nonsulfated forms of parathyroid chromogranin A (secretory protein-I)
- Author
-
Sven Ulrik Gorr and David V. Cohn
- Subjects
Tyrosine sulfation ,chemistry.chemical_classification ,endocrine system ,biology ,Chemistry ,Chromogranin A ,Radioimmunoassay ,Cell Biology ,Biochemistry ,Secretory protein ,Sulfation ,biology.protein ,Secretion ,Tyrosine ,Glycoprotein ,Molecular Biology - Abstract
Chromogranin A (secretory protein-I) is an acidic, sulfated glycoprotein found in secretory granules of most endocrine cells but not in exocrine or epithelial cells. Parathyroid chromogranin A is sulfated on tyrosine residues, whereas adrenal chromogranin A appears to be sulfated mainly on oligosaccharide residues. Chromogranin B, on the other hand, is tyrosine-sulfated in the bovine adrenal whereas this protein is absent from the parathyroid. The role of this tissue- or species-specific sulfation of chromogranin is not known. Tyrosine sulfation is a common post-translational modification of proteins in the exocytotic pathway and has been suggested to play a role in the sorting or intracellular transport of secretory proteins. To test this, porcine parathyroid tissue slices were metabolically labeled with 35SO4 and [3H]Lys, and the tissue and incubation medium analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoprecipitation with chromogranin A-specific antiserum or by radioimmunoassay for parathormone. Secretion of total and 3H-labeled chromogranin A was about 3- and 7-fold higher, respectively, at 0.5 mM than at 3.0 mM Ca2+, and secretion of 35SO4-labeled chromogranin A was 67-fold higher. This indicates that either sulfated chromogranin A is directed primarily to the Ca2+-regulated pathway or that sulfation occurs following sorting to this pathway. Sodium chlorate (1-10 mM) inhibited sulfation in a dose-dependent manner by up to 95% but it had no effect on the onset or rate of chromogranin A secretion. These data indicate that regulated secretion of parathyroid chromogranin A does not require sulfation of tyrosine residues.
- Published
- 1990
46. Secretory granule biogenesis and chromogranin A: master gene, on/off switch or assembly factor?
- Author
-
Robert Day and Sven Ulrik Gorr
- Subjects
endocrine system ,Cell type ,Endocrinology, Diabetes and Metabolism ,Secretory Vesicles ,Granule (cell biology) ,Chromogranin A ,Enteroendocrine cell ,Biology ,Exocytosis ,Cell biology ,Endocrinology ,Gene expression ,biology.protein ,Chromogranins ,Animals ,Gene ,Biogenesis - Abstract
Secretory granules are found in specialized cell types, including endocrine cells, suggesting that a coordinated programme of gene expression is involved in their biogenesis. Indeed, it has been proposed that chromogranin A (CgA) acts as an on/off switch for secretory granule biogenesis. However, this proposed function is difficult to reconcile with the large body of evidence suggesting that secretory granules exist in the absence of CgA and that cells can synthesize CgA in the absence of secretory granules. Indeed, recent evidence suggests that, rather than a master gene or universal on/off switch, a series of on/off switches combines to induce expression of subsets of secretory granule-associated genes. The assembly of newly synthesized proteins and the inclusion of existing granule proteins would produce functional secretory granules. CgA and related proteins might act as assembly factors in this process.
- Published
- 2002
47. In vitro aggregation of the regulated secretory protein chromogranin A
- Author
-
Sven Ulrik Gorr, Paul B.M. Joyce, Wen Tzu Chang, Renu K. Jain, and Chitta Geetha
- Subjects
Octoxynol ,chemistry.chemical_element ,Protein aggregation ,Calcium ,Biochemistry ,Divalent ,Hydrophobic effect ,Epitopes ,Cations ,Chromogranins ,Animals ,Histidine ,Magnesium ,Molecular Biology ,Edetic Acid ,chemistry.chemical_classification ,biology ,Chromogranin A ,Cell Biology ,Alkaline Phosphatase ,In vitro ,Recombinant Proteins ,Zinc ,Secretory protein ,Spectrometry, Fluorescence ,chemistry ,Chaperone (protein) ,biology.protein ,Cattle ,Research Article ,Molecular Chaperones - Abstract
Aggregation chaperones, consisting of secretory proteins that contain a hexa-histidine epitope tag, enhance the calcium-induced aggregation of regulated secretory proteins and their sorting to secretory granules. The goal of this study was to gain a better understanding of this unusual aggregation mechanism. Hexa-histidine-epitope-tagged secreted alkaline phosphatase, an aggregation chaperone, enhanced the in vitro aggregation of chromogranin A in the presence of calcium, but not in the presence of magnesium or other divalent cations. As an exception, chromogranin was completely aggregated by zinc, even in the absence of the aggregation chaperone. In addition, fluorescence spectroscopy of the aggregation reaction mixture showed an increase in fluorescence intensity consistent with the formation of protein aggregates. The calcium-induced aggregation of chromogranin A was completely inhibited by 0.2% Triton X-100, suggesting that it involves hydrophobic interactions. In contrast, the detergent did not affect chaperone-enhanced aggregation, suggesting that this aggregation does not depend on hydrophobic interactions. EDTA-treated chaperone did not enhance chromogranin A aggregation, indicating that divalent cations are necessary for chaperone action. Although the structure of the aggregation chaperone was not important, the size of the chaperone was. Thus the free His-hexapeptide could not substitute for the aggregation chaperone. Based on these results, we propose that the hexa-histidine tag, in the context of a polypeptide, acts as a divalent cation-dependent nucleation site for chromogranin A aggregation.
- Published
- 2002
48. Comparative sorting of neuroendocrine secretory proteins: a search for common ground in a mosaic of sorting models and mechanisms
- Author
-
Paul B.M. Joyce, Darrin J. Cowley, Renu K. Jain, Ulrike Kuehn, and Sven Ulrik Gorr
- Subjects
Cell type ,biology ,Sorting ,Chromogranin A ,Proteins ,Protein Sorting Signals ,Biochemistry ,Neurosecretory Systems ,Cell biology ,Transport protein ,Protein Transport ,Endocrinology ,Secretory protein ,Models, Chemical ,Proteins metabolism ,biology.protein ,Animals ,Humans ,Molecular Biology - Abstract
Endocrine, neuroendocrine and exocrine cells store regulated secretory proteins in secretory granules, while constitutive and constitutive-like secretory proteins are secreted directly without storage. Sorting of secretory proteins takes place in the trans-Golgi network (sorting for entry) or immature secretory granules (sorting by retention). The relative contribution of these sorting steps and the sorting signals and mechanisms involved in each step has been the subject of intense studies and debate in recent years. New evidence now suggests that: (1) two proteins with structurally similar sorting signals can use different sorting mechanisms; (2) one protein with multiple sorting signals can be sorted differently in different cell types; and (3) one cell type can recognize different sorting signals and use different sorting mechanisms. The latter finding suggests that sorting must be a regulated event. While the current image of sorting is complex, recent findings are pointing to common features that form a mosaic of related sorting mechanisms.
- Published
- 2001
49. Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells
- Author
-
Paul B.M. Joyce, V. Lilla, Mariella Ravazzola, Renu K. Jain, Sven Ulrik Gorr, Philippe A. Halban, and Miguel Molinete
- Subjects
Islets of Langerhans/ physiology/secretion ,Signal peptide ,Endocrinology, Diabetes and Metabolism ,Genetic Vectors ,Green Fluorescent Proteins ,Biology ,Protein Sorting Signals ,Cytoplasmic Granules ,Transfection ,Green fluorescent protein ,Adenoviridae ,Cell Line ,Islets of Langerhans ,Alkaline Phosphatase/genetics/metabolism ,Cytosol ,Insulin Secretion ,Internal Medicine ,Animals ,Insulin ,Secretion ,Cytoplasmic Granules/metabolism/ultrastructure ,Cytosol/metabolism/ultrastructure ,Proinsulin ,ddc:616 ,Recombinant Proteins/metabolism ,Protein Sorting Signals/genetics ,Luminescent Proteins/analysis/genetics ,Alkaline Phosphatase ,Fusion protein ,Molecular biology ,Recombinant Proteins ,Kinetics ,Luminescent Proteins ,Secretory protein ,Cell culture ,Insulin/ secretion - Abstract
Aims/hypothesis. Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. Methods. SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. Results. Secreted alkaline phosphatase showed high basal secretion (6.6 % total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8 % total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. Conclusion/interpretation. These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both. [Diabetologia (2000) 43: 1157–1164]
- Published
- 2000
50. N- and C-terminal domains direct cell type-specific sorting of chromogranin A to secretory granules
- Author
-
Sven Ulrik Gorr, Yancy R. Moore, Darrin J. Cowley, Paul B.M. Joyce, and Douglas S. Darling
- Subjects
endocrine system ,endocrine system diseases ,Cell ,Mutant ,Enteroendocrine cell ,Protein Sorting Signals ,Cytoplasmic Granules ,Biochemistry ,PC12 Cells ,medicine ,Chromogranins ,Animals ,Molecular Biology ,Cells, Cultured ,chemistry.chemical_classification ,biology ,Constitutive secretory pathway ,Chromogranin A ,Biological Transport ,Cell Biology ,Intracellular Membranes ,digestive system diseases ,Recombinant Proteins ,Amino acid ,Cell biology ,Cell Compartmentation ,Protein Structure, Tertiary ,Rats ,medicine.anatomical_structure ,Secretory protein ,chemistry ,Pituitary Gland ,biology.protein ,Protein Binding - Abstract
Chromogranins are a family of regulated secretory proteins that are stored in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation (regulated secretion). A conserved N-terminal disulfide bond is necessary for sorting of chromogranins in neuroendocrine PC12 cells. Surprisingly, this disulfide bond is not necessary for sorting of chromogranins in endocrine GH4C1 cells. To investigate the sorting mechanism in GH4C1 cells, we made several mutant forms removing highly conserved N- and C-terminal regions of bovine chromogranin A. Removing the conserved N-terminal disulfide bond and the conserved C-terminal dimerization and tetramerization domain did not affect the sorting of chromogranin A to the regulated secretory pathway. In contrast, removing the C-terminal 90 amino acids of chromogranin A caused rerouting to the constitutive secretory pathway and impaired aggregation properties as compared with wild-type chromogranin A. Since this mutant was sorted to the regulated secretory pathway in PC12 cells, these results demonstrate that chromogranins contain independent N- and C-terminal sorting domains that function in a cell type-specific manner. Moreover, this is the first evidence that low pH/calcium-induced aggregation is necessary for sorting of a chromogranin to the regulated secretory pathway of endocrine cells.
- Published
- 2000
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