Stephen Brand | Babson College (original) (raw)

Papers by Stephen Brand

Research paper thumbnail of Role of the Proteasome and NF-kappa B in Streptococcal Cell Wall-Induced Polyarthritis

Pnas, 1998

The transcription factor NF-kappaB activates a number of genes whose protein products are proinfl... more The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Research paper thumbnail of Prevention of Chronic Renal Allograft Rejection by Soluble CD83

Transplantation, Nov 1, 2010

Background. Recombinant human soluble CD83 had previously exhibited significant immunosuppressive... more Background. Recombinant human soluble CD83 had previously exhibited significant immunosuppressive properties that involved interference with dendritic cell maturation in both mouse and humans, inhibition of autoimmunity in mice, and induction of antigen-specific mouse cardiac allograft tolerance when used in combination with other immunosuppressive drugs. Our current research focus turned to examining the effects of peritransplant soluble CD83 (sCD83) administration on prevention of chronic renal allograft rejection. Methods. Fisher344-to-Lewis orthotopic rat renal transplants were performed with sequential recipient killing on postoperative days (PODs) 2, 14, and 140 to examine both the acute and chronic effects of peritransplant sCD83 treatment in rat recipients. Results. Recipients treated with sCD83 exhibited a marked decrease in IgM and IgG deposition in the graft and antidonor antibody levels in the circulation, as early as POD14 and persisting until POD140. sCD83 treatment also reduced the infiltration of T cells and monocytes into the graft tissue and inhibited intragraft expression of MyD88 and inflammatory cytokine levels during the observation period. sCD83-treated grafts demonstrated normal histology beyond POD140, including dramatic reductions in tubular atrophy and interstitial fibrosis compared with untreated recipients. Conclusion. We have demonstrated that peritransplant treatment with recombinant sCD83 attenuates both innate and adaptive immune responses and leads to prevention of chronic rejection in a rat renal transplant model. Because sCD83 is of human origin, the therapeutic approach used in our rodent transplant model holds significant promise for clinical transplantation.

Research paper thumbnail of Method for monitoring proteasome inhibitor drug action

Research paper thumbnail of Combination Therapy With Epidermal Growth Factor and Gastrin Increases -Cell Mass and Reverses Hyperglycemia in Diabetic NOD Mice

Diabetes, 2005

Combination therapy with epidermal growth factor (EGF) and gastrin induces ␤-cell regeneration in... more Combination therapy with epidermal growth factor (EGF) and gastrin induces ␤-cell regeneration in rodents with chemically induced diabetes. We investigated whether EGF plus gastrin could correct hyperglycemia in NOD mice with autoimmune diabetes. Combined treatment with EGF (1 g/kg) and gastrin (3 g/kg) for 2 weeks restored normoglycemia after diabetes onset in NOD mice, whereas EGF or gastrin alone did not. Fasting blood glucose remained normal (3.5-6.5 mmol/l) or mildly elevated (<11 mmol/l) in five of six mice (83%) for 10 weeks after EGF plus gastrin treatment was stopped, whereas all mice treated with vehicle or EGF or gastrin alone became severely hyperglycemic (12-35 mmol/l). Pancreatic ␤-cell mass was increased threefold and insulin content was increased eightfold in mice treated with EGF plus gastrin compared with pretreatment values. The correction of hyperglycemia correlated significantly with increases in pancreatic ␤-cell mass and insulin content. In addition, splenic cells from mice treated with EGF plus gastrin delayed diabetes induction by adoptive transfer of diabetogenic cells into immunodeficient NOD-scid mice, suggesting the induction of immunoregulatory cells in NOD mice treated with EGF plus gastrin. We conclude that a short course of combined EGF and gastrin therapy increases pancreatic ␤-cell mass and reverses hyperglycemia in acutely diabetic NOD mice; the impact of this combined therapy may result from the effects of EGF and gastrin on ␤-cells, immune cells, or both.

Research paper thumbnail of Autoreactive epitope profiles of the proliferating cell nuclear antigen define two classes of autoantibodies

The Journal of Immunology, Apr 15, 1994

Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic functi... more Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic function and autoantibody recognition.

Research paper thumbnail of Epidermal growth factor protein and gene, and methods of use therefor

Research paper thumbnail of Method for Monitoring Proteasome Inhibitor Drug Action

Research paper thumbnail of Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet β-cells from pancreatic duct cells and an increase in functional β-cell mass

The Journal of Clinical Endocrinology and Metabolism, 2005

Human pancreases were obtained, with informed consent of relatives, from brain-dead organ donors.... more Human pancreases were obtained, with informed consent of relatives, from brain-dead organ donors. Tissue procurement and experimental protocols were approved by the human ethics committee of University of Alberta Hospitals. Pancreases were removed from donors

Research paper thumbnail of Inhibition of NF-?B activation in vitro and in vivo: Role of 26S proteasome

Meth Enzymology, 1999

The results observed with PS-273 have also been demonstrated with other boronic acid proteasome i... more The results observed with PS-273 have also been demonstrated with other boronic acid proteasome inhibitors, as well as the peptide aldehyde and lactacystin class of proteasome inhibitors. The relative potencies for inhibition in the above mentioned assays correlates generally with Ki values for proteasome inhibition. This is evidence that the intracellular mode of action of the inhibitors is indeed through proteasome inhibition.

Research paper thumbnail of Treatment of Infarcts Through Inhibition of NF-Kappab

Research paper thumbnail of Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein

Protein Expression and Purification, Oct 1, 2010

The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of ... more The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tended to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutics is currently under exploration.

Research paper thumbnail of Novel Epidermal Growth Factor Protein and Gene, and Methods of Use Therefor

Research paper thumbnail of Treatment for Diabetes

Research paper thumbnail of Treatment of infarcts

Research paper thumbnail of A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway

The American journal of physiology, 1999

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-... more A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathw...

Research paper thumbnail of Spasmolytic polypeptide: a trefoil peptide secreted by rat gastric mucous cells

Gastroenterology, 1994

Spasmolytic polypeptide (SP) is a trefoil peptide expressed in the digestive tract. This study ai... more Spasmolytic polypeptide (SP) is a trefoil peptide expressed in the digestive tract. This study aimed to determine the structure and distribution of SP expression in the rat gastrointestinal tract. The structure of rat SP was determined from the sequence of complementary DNAs isolated from antral RNA. SP gene expression was localized by Northern blotting and in situ hybridization in the adult and fetal rat digestive tract. Expression of the SP peptide was localized by immunocytochemistry and Western blot analysis. SP messenger (m)RNA was found predominantly in the stomach with highest expression in the antrum. High levels of SP mRNA were expressed in the fetal stomach before gastrin and somatostatin expression. Surprisingly, SP mRNA and peptide did not colocalize in the gastric mucosa, SP mRNA being superficial to SP peptide immunoreactivity throughout the gastric mucosa. Abundant SP immunoreactivity was seen in the lumen of the gastric glands and the mucus layer adherent to the gast...

Research paper thumbnail of Inhibition of NF-κB activation in vitro and in vivo: Role of 26S proteasome

Methods in Enzymology, 1999

The results observed with PS-273 have also been demonstrated with other boronic acid proteasome i... more The results observed with PS-273 have also been demonstrated with other boronic acid proteasome inhibitors, as well as the peptide aldehyde and lactacystin class of proteasome inhibitors. The relative potencies for inhibition in the above mentioned assays correlates generally with Ki values for proteasome inhibition. This is evidence that the intracellular mode of action of the inhibitors is indeed through

Research paper thumbnail of Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein

Protein Expression and Purification, 2009

An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human... more An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (1) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10% of total cellular protein, with a cell density of 8 OD 600 was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed.

Research paper thumbnail of Role of the proteasome and NF- B in streptococcal cell wall-induced polyarthritis

Proceedings of the National Academy of Sciences, 1998

The transcription factor NF-B activates a number of genes whose protein products are proinf lamma... more The transcription factor NF-B activates a number of genes whose protein products are proinf lammatory. In quiescent cells, NF-B exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IB is degraded by the ubiquitinproteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-B. This suppression should therefore decrease transcription of many genes encoding proinf lammatory proteins and should ultimately have an anti-inf lammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway.

Research paper thumbnail of Epithelial glycoprotein is a member of a family of epithelial cell surface antigens homologous to nidogen, a matrix adhesion protein

Proceedings of the National Academy of Sciences, 1990

The cell surface antigen, epithelial glycoprotein, defined by the monoclonal antibody HEA 125, is... more The cell surface antigen, epithelial glycoprotein, defined by the monoclonal antibody HEA 125, is expressed on virtually all epithelial cell membranes but not on mesodermal or neural cell membranes. The cDNA encoding epithelial glycoprotein was isolated by HEA 125 antibody enrichment of colon tumor cDNA expressed transiently in COS cells. The sequence of the epithelial glycoprotein antigen is identical to the cell membrane protein recognized by the monoclonal antibody KS 1/4 and is homologous to the tumor-associated antigen GA733. These proteins share sequence homology to nidogen, an extracellular matrix component that appears to participate in cell-matrix adhesion. These proteins also share a homologous domain found in the B1 chain of laminin, a matrix adhesion protein, and placental protein 12, an insulin-like growth factor I binding protein secreted during pregnancy that has been implicated in regulation of fetal growth. This common domain is also repeated multiple times within t...

Research paper thumbnail of Role of the Proteasome and NF-kappa B in Streptococcal Cell Wall-Induced Polyarthritis

Pnas, 1998

The transcription factor NF-kappaB activates a number of genes whose protein products are proinfl... more The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Research paper thumbnail of Prevention of Chronic Renal Allograft Rejection by Soluble CD83

Transplantation, Nov 1, 2010

Background. Recombinant human soluble CD83 had previously exhibited significant immunosuppressive... more Background. Recombinant human soluble CD83 had previously exhibited significant immunosuppressive properties that involved interference with dendritic cell maturation in both mouse and humans, inhibition of autoimmunity in mice, and induction of antigen-specific mouse cardiac allograft tolerance when used in combination with other immunosuppressive drugs. Our current research focus turned to examining the effects of peritransplant soluble CD83 (sCD83) administration on prevention of chronic renal allograft rejection. Methods. Fisher344-to-Lewis orthotopic rat renal transplants were performed with sequential recipient killing on postoperative days (PODs) 2, 14, and 140 to examine both the acute and chronic effects of peritransplant sCD83 treatment in rat recipients. Results. Recipients treated with sCD83 exhibited a marked decrease in IgM and IgG deposition in the graft and antidonor antibody levels in the circulation, as early as POD14 and persisting until POD140. sCD83 treatment also reduced the infiltration of T cells and monocytes into the graft tissue and inhibited intragraft expression of MyD88 and inflammatory cytokine levels during the observation period. sCD83-treated grafts demonstrated normal histology beyond POD140, including dramatic reductions in tubular atrophy and interstitial fibrosis compared with untreated recipients. Conclusion. We have demonstrated that peritransplant treatment with recombinant sCD83 attenuates both innate and adaptive immune responses and leads to prevention of chronic rejection in a rat renal transplant model. Because sCD83 is of human origin, the therapeutic approach used in our rodent transplant model holds significant promise for clinical transplantation.

Research paper thumbnail of Method for monitoring proteasome inhibitor drug action

Research paper thumbnail of Combination Therapy With Epidermal Growth Factor and Gastrin Increases -Cell Mass and Reverses Hyperglycemia in Diabetic NOD Mice

Diabetes, 2005

Combination therapy with epidermal growth factor (EGF) and gastrin induces ␤-cell regeneration in... more Combination therapy with epidermal growth factor (EGF) and gastrin induces ␤-cell regeneration in rodents with chemically induced diabetes. We investigated whether EGF plus gastrin could correct hyperglycemia in NOD mice with autoimmune diabetes. Combined treatment with EGF (1 g/kg) and gastrin (3 g/kg) for 2 weeks restored normoglycemia after diabetes onset in NOD mice, whereas EGF or gastrin alone did not. Fasting blood glucose remained normal (3.5-6.5 mmol/l) or mildly elevated (<11 mmol/l) in five of six mice (83%) for 10 weeks after EGF plus gastrin treatment was stopped, whereas all mice treated with vehicle or EGF or gastrin alone became severely hyperglycemic (12-35 mmol/l). Pancreatic ␤-cell mass was increased threefold and insulin content was increased eightfold in mice treated with EGF plus gastrin compared with pretreatment values. The correction of hyperglycemia correlated significantly with increases in pancreatic ␤-cell mass and insulin content. In addition, splenic cells from mice treated with EGF plus gastrin delayed diabetes induction by adoptive transfer of diabetogenic cells into immunodeficient NOD-scid mice, suggesting the induction of immunoregulatory cells in NOD mice treated with EGF plus gastrin. We conclude that a short course of combined EGF and gastrin therapy increases pancreatic ␤-cell mass and reverses hyperglycemia in acutely diabetic NOD mice; the impact of this combined therapy may result from the effects of EGF and gastrin on ␤-cells, immune cells, or both.

Research paper thumbnail of Autoreactive epitope profiles of the proliferating cell nuclear antigen define two classes of autoantibodies

The Journal of Immunology, Apr 15, 1994

Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic functi... more Trimeric structure of human proliferating cell nuclear antigen. Implications for enzymatic function and autoantibody recognition.

Research paper thumbnail of Epidermal growth factor protein and gene, and methods of use therefor

Research paper thumbnail of Method for Monitoring Proteasome Inhibitor Drug Action

Research paper thumbnail of Combination therapy with epidermal growth factor and gastrin induces neogenesis of human islet β-cells from pancreatic duct cells and an increase in functional β-cell mass

The Journal of Clinical Endocrinology and Metabolism, 2005

Human pancreases were obtained, with informed consent of relatives, from brain-dead organ donors.... more Human pancreases were obtained, with informed consent of relatives, from brain-dead organ donors. Tissue procurement and experimental protocols were approved by the human ethics committee of University of Alberta Hospitals. Pancreases were removed from donors

Research paper thumbnail of Inhibition of NF-?B activation in vitro and in vivo: Role of 26S proteasome

Meth Enzymology, 1999

The results observed with PS-273 have also been demonstrated with other boronic acid proteasome i... more The results observed with PS-273 have also been demonstrated with other boronic acid proteasome inhibitors, as well as the peptide aldehyde and lactacystin class of proteasome inhibitors. The relative potencies for inhibition in the above mentioned assays correlates generally with Ki values for proteasome inhibition. This is evidence that the intracellular mode of action of the inhibitors is indeed through proteasome inhibition.

Research paper thumbnail of Treatment of Infarcts Through Inhibition of NF-Kappab

Research paper thumbnail of Structural identification of recombinant human CD83 mutant variant as a potent therapeutic protein

Protein Expression and Purification, Oct 1, 2010

The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of ... more The formation of aberrant disulfide bonds is a structural consideration for the manufacturing of the extracellular domain of human CD83 (hCD83ext), a potential therapeutic protein. In certain instances, hCD83ext protein products, even when stored frozen, tended to dimerize or even multimerize through the formation of aberrant intermolecular disulfide bonds. Herein, we discovered an analytical inconsistency and applied a modified sample preparation protocol for proper structural analysis of hCD83ext products which are heterologously expressed in Escherichia coli and subsequently purified. In addition, a mutant derivative with the Cys100Ser mutation was identified as an improved version which did not form dimers or multimers. The identification of this mutant variant as a more potent therapeutic protein than other hCD83ext species demonstrated that the structural variation associated with disulfide bond formation can be a critical issue for rigorous control of the quality and bioactivity of therapeutic proteins. The application of this mutant variant for protein therapeutics is currently under exploration.

Research paper thumbnail of Novel Epidermal Growth Factor Protein and Gene, and Methods of Use Therefor

Research paper thumbnail of Treatment for Diabetes

Research paper thumbnail of Treatment of infarcts

Research paper thumbnail of A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway

The American journal of physiology, 1999

A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-... more A new model of cachexia is described in which muscle protein metabolism related to the ubiquitin-proteasome pathway was investigated. Cloning of the colon-26 tumor produced a cell line, termed R-1, which induced cytokine (noninterleukin-1beta, interleukin-6 and tumor necrosis factor-alpha)-independent cachexia. Implantation of R-1 cells in mice elicited significant (20-30%) weight loss and decreased blood glucose by 70%, and adipose tissue levels declined by 95% and muscle weights decreased by 20-25%. Food intake was unaffected. The decrease in muscle weight reflected a decline in insoluble, but not soluble, muscle protein that was associated with a significant increase in net protein degradation. The rate of ubiquitin conjugation of proteins was significantly elevated in muscles of cachectic mice. Furthermore, the proteasome inhibitor lactacystin blocked the increase in protein breakdown but had no significant effect on proteolysis. Several markers of the ubiquitin-proteasome pathw...

Research paper thumbnail of Spasmolytic polypeptide: a trefoil peptide secreted by rat gastric mucous cells

Gastroenterology, 1994

Spasmolytic polypeptide (SP) is a trefoil peptide expressed in the digestive tract. This study ai... more Spasmolytic polypeptide (SP) is a trefoil peptide expressed in the digestive tract. This study aimed to determine the structure and distribution of SP expression in the rat gastrointestinal tract. The structure of rat SP was determined from the sequence of complementary DNAs isolated from antral RNA. SP gene expression was localized by Northern blotting and in situ hybridization in the adult and fetal rat digestive tract. Expression of the SP peptide was localized by immunocytochemistry and Western blot analysis. SP messenger (m)RNA was found predominantly in the stomach with highest expression in the antrum. High levels of SP mRNA were expressed in the fetal stomach before gastrin and somatostatin expression. Surprisingly, SP mRNA and peptide did not colocalize in the gastric mucosa, SP mRNA being superficial to SP peptide immunoreactivity throughout the gastric mucosa. Abundant SP immunoreactivity was seen in the lumen of the gastric glands and the mucus layer adherent to the gast...

Research paper thumbnail of Inhibition of NF-κB activation in vitro and in vivo: Role of 26S proteasome

Methods in Enzymology, 1999

The results observed with PS-273 have also been demonstrated with other boronic acid proteasome i... more The results observed with PS-273 have also been demonstrated with other boronic acid proteasome inhibitors, as well as the peptide aldehyde and lactacystin class of proteasome inhibitors. The relative potencies for inhibition in the above mentioned assays correlates generally with Ki values for proteasome inhibition. This is evidence that the intracellular mode of action of the inhibitors is indeed through

Research paper thumbnail of Bioprocess development for production, purification, and structural characterization of recombinant hCD83ext as a potential therapeutic protein

Protein Expression and Purification, 2009

An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human... more An effective bioprocess for the production of hCD83ext (i.e. the extracytoplasmic domain of human CD83) as a potential therapeutic protein was developed. It primarily consists of (1) cell cultivation for the production of recombinant glutathione-S-transferase-hCD83ext (GST-hCD83ext) fusion protein and (2) downstream processing for purification of hCD83ext. The developed bioprocess is robust, reproducible, easy to operate, and, most importantly, can generate hCD83ext with a high yield and purity. For cell cultivation, a high GST-hCD83ext expression level, estimated to be more than 10% of total cellular protein, with a cell density of 8 OD 600 was obtained by tuning several culture parameters, including medium recipe, host/vector system, induction condition, temperature, and aeration. For downstream processing, milligrams of very pure and low-endotoxin hCD83ext was obtained through simultaneous binding and cleavage of GST-hCD83ext in a GST affinity chromatographic column followed by a polishing step using anion exchange chromatography. To identify potential factors associated with bioactivity consistency, structural changes for the final product of hCD83ext were characterized and monitored. Formation of various hCD83ext multimeric forms, including dimer, trimer, and tetramer, via intermolecular disulfide bonds was observed.

Research paper thumbnail of Role of the proteasome and NF- B in streptococcal cell wall-induced polyarthritis

Proceedings of the National Academy of Sciences, 1998

The transcription factor NF-B activates a number of genes whose protein products are proinf lamma... more The transcription factor NF-B activates a number of genes whose protein products are proinf lammatory. In quiescent cells, NF-B exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IB is degraded by the ubiquitinproteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-B. This suppression should therefore decrease transcription of many genes encoding proinf lammatory proteins and should ultimately have an anti-inf lammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway.

Research paper thumbnail of Epithelial glycoprotein is a member of a family of epithelial cell surface antigens homologous to nidogen, a matrix adhesion protein

Proceedings of the National Academy of Sciences, 1990

The cell surface antigen, epithelial glycoprotein, defined by the monoclonal antibody HEA 125, is... more The cell surface antigen, epithelial glycoprotein, defined by the monoclonal antibody HEA 125, is expressed on virtually all epithelial cell membranes but not on mesodermal or neural cell membranes. The cDNA encoding epithelial glycoprotein was isolated by HEA 125 antibody enrichment of colon tumor cDNA expressed transiently in COS cells. The sequence of the epithelial glycoprotein antigen is identical to the cell membrane protein recognized by the monoclonal antibody KS 1/4 and is homologous to the tumor-associated antigen GA733. These proteins share sequence homology to nidogen, an extracellular matrix component that appears to participate in cell-matrix adhesion. These proteins also share a homologous domain found in the B1 chain of laminin, a matrix adhesion protein, and placental protein 12, an insulin-like growth factor I binding protein secreted during pregnancy that has been implicated in regulation of fetal growth. This common domain is also repeated multiple times within t...