Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or... more Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic ...
High-mobility group box 1 (HMGB1) is a chromatin-associated protein that in response to stress or... more High-mobility group box 1 (HMGB1) is a chromatin-associated protein that in response to stress or injury, translocates from the nucleus to the extracellular milieu where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and to a much lesser extent in healthy subjects. Differential HMGB1c levels also were detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link-site mapping and MS analysis revealed...
Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet... more Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet-unknown mechanism. Here, we show that application of cyclic tensile stress (CTS) in vitro abrogates the catabolic effects of IL-1 on chondrocytes. The effects of CTS are mediated by down-regulation of IL-1-dependent inducible NO production, and are directly attributed to the inhibition of inducible NO synthase (iNOS) mRNA expression and protein synthesis. The inhibition of iNOS induction by CTS is paralleled by abrogation of IL-1-induced down-regulation of proteoglycan synthesis. Furthermore, CTS inhibits iNOS expression and up-regulates proteoglycan synthesis at concentrations of IL-1 frequently observed in inflamed arthritic joints, suggesting that the actions of CTS may be clinically relevant in suppressing the sustained effects of pathological levels of IL-1 in vivo. These results are the first to demonstrate that mechanisms of the intracellular actions of CTS in IL-1-activated chondrocytes are mediated through inhibition of a key molecule in the signal transduction pathway that leads to iNOS expression.
Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplast... more Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplastic cells removed from murine hosts at progressive stages of growth. Cells from three ascites cancers and one fibrosarcoma were tested and showed declining ODC production with progressive growth. The cells were incubated with serum or malignant effusion fluid taken from the murine hosts at progressive stages of growth. For 2 to 3 weeks after tumor implantation, sera and, in particular, ascites fluids increasingly stimulated ODC production in cells at all stages of growth. With advancing disease, without the malignant growth having reached a stationary phase, the collected fluids decreasingly stimulated ODC production in the cells. The stimulating factor(s) in host serum and malignant effusion fluid were not tumor specific in the one combination tested.
Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ri... more Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adeniylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
Purpose: The temporomandibular joint is a place of motion where release of proinflammatory cytoki... more Purpose: The temporomandibular joint is a place of motion where release of proinflammatory cytokines like interleukin-1β (IL-1β) induces cartilage destruction via production of nitric oxide (NO). The purpose of this study was to evaluate the effects of ...
patient, the injured knee and the unharmed knee showed no difference in varus angle. In 5 patient... more patient, the injured knee and the unharmed knee showed no difference in varus angle. In 5 patients the measurement of telomere lengths showed that telomere length of lateral femoral condyle >ACL >medial femoral condyle; in one patient the measure showed telomere length of medial femoral condyle >ACL > lateral femoral condyle. Conclusions: From test 1, we found expression of asporin in ACL increased in the OA patients, it suggested degeneration of ACL in the OA patients. From test 2, we found that ACL injury would increase the varus angles of the knee. From test 3, we found that senescent degree in osteoarthritis knee is medial femoral condyle >ACL > lateral femoral condyle. We infered that ACL might degenerate or get injured at first, and then led to knee varus, and led to osteoarthritis at last.
The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. T... more The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. The enzyme is a single polypeptide of about 100,000 in molecular weight. No apparent separation of the nicking and sealing activities could be ob- tained in attempts to separate the two activities by a variety of methods, including limited protease digestion, thermal dena- turation, and differential inhibition. Monoclonal antibodies obtained from hybridomas likewise did not preferentially inhibit one of the two activities. These results suggest that the two catalytic functions are carried by the same essential residues of the active enzyme site.
The canonical Wnt/b-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathwa... more The canonical Wnt/b-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.
Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or... more Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic ...
High-mobility group box 1 (HMGB1) is a chromatin-associated protein that in response to stress or... more High-mobility group box 1 (HMGB1) is a chromatin-associated protein that in response to stress or injury, translocates from the nucleus to the extracellular milieu where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and to a much lesser extent in healthy subjects. Differential HMGB1c levels also were detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link-site mapping and MS analysis revealed...
Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet... more Continuous passive motion manifests therapeutic effects on inflamed articular joints by an as-yet-unknown mechanism. Here, we show that application of cyclic tensile stress (CTS) in vitro abrogates the catabolic effects of IL-1 on chondrocytes. The effects of CTS are mediated by down-regulation of IL-1-dependent inducible NO production, and are directly attributed to the inhibition of inducible NO synthase (iNOS) mRNA expression and protein synthesis. The inhibition of iNOS induction by CTS is paralleled by abrogation of IL-1-induced down-regulation of proteoglycan synthesis. Furthermore, CTS inhibits iNOS expression and up-regulates proteoglycan synthesis at concentrations of IL-1 frequently observed in inflamed arthritic joints, suggesting that the actions of CTS may be clinically relevant in suppressing the sustained effects of pathological levels of IL-1 in vivo. These results are the first to demonstrate that mechanisms of the intracellular actions of CTS in IL-1-activated chondrocytes are mediated through inhibition of a key molecule in the signal transduction pathway that leads to iNOS expression.
Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplast... more Ornithine decarboxylase (ODC) production was used as an indicator of mitotic activity in neoplastic cells removed from murine hosts at progressive stages of growth. Cells from three ascites cancers and one fibrosarcoma were tested and showed declining ODC production with progressive growth. The cells were incubated with serum or malignant effusion fluid taken from the murine hosts at progressive stages of growth. For 2 to 3 weeks after tumor implantation, sera and, in particular, ascites fluids increasingly stimulated ODC production in cells at all stages of growth. With advancing disease, without the malignant growth having reached a stationary phase, the collected fluids decreasingly stimulated ODC production in the cells. The stimulating factor(s) in host serum and malignant effusion fluid were not tumor specific in the one combination tested.
Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ri... more Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adeniylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
Purpose: The temporomandibular joint is a place of motion where release of proinflammatory cytoki... more Purpose: The temporomandibular joint is a place of motion where release of proinflammatory cytokines like interleukin-1β (IL-1β) induces cartilage destruction via production of nitric oxide (NO). The purpose of this study was to evaluate the effects of ...
patient, the injured knee and the unharmed knee showed no difference in varus angle. In 5 patient... more patient, the injured knee and the unharmed knee showed no difference in varus angle. In 5 patients the measurement of telomere lengths showed that telomere length of lateral femoral condyle >ACL >medial femoral condyle; in one patient the measure showed telomere length of medial femoral condyle >ACL > lateral femoral condyle. Conclusions: From test 1, we found expression of asporin in ACL increased in the OA patients, it suggested degeneration of ACL in the OA patients. From test 2, we found that ACL injury would increase the varus angles of the knee. From test 3, we found that senescent degree in osteoarthritis knee is medial femoral condyle >ACL > lateral femoral condyle. We infered that ACL might degenerate or get injured at first, and then led to knee varus, and led to osteoarthritis at last.
The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. T... more The DNA topoisomerase from Agrobacterium tumefaciens has been purified to apparent homogeneity. The enzyme is a single polypeptide of about 100,000 in molecular weight. No apparent separation of the nicking and sealing activities could be ob- tained in attempts to separate the two activities by a variety of methods, including limited protease digestion, thermal dena- turation, and differential inhibition. Monoclonal antibodies obtained from hybridomas likewise did not preferentially inhibit one of the two activities. These results suggest that the two catalytic functions are carried by the same essential residues of the active enzyme site.
The canonical Wnt/b-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathwa... more The canonical Wnt/b-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.
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Papers by Sudha Agarwal