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Paul C Kuo

Publications and source records attributed to Paul C Kuo.

At least 37 records · Page 2Linked to original sources
PubMed2004

Identification of S-nitrosylated proteins in endotoxin-stimulated RAW264.7 murine macrophages.

Nitric oxide (NO) is an omnipresent regulator of cell function in a variety of physiologic and pathophysiologic states. In part, NO exerts its actions by S-nitrosylation of target thiols, primarily in cysteine residues. Delineating the functional correlates of S-nitrosylation can begin with identification of the entire population of S-nitrososylated proteins. Recently, the biotin switch technique was developed to allow a proteomic approach to identification of the "universe" of S-nitrsoylated proteins. In this study using endotoxin-stimulated RAW264.7 murine macrophages, we have utilized the biotin-switch technique and protein sequencing to identify S-nitrosylated proteins in this setting. In contrast to other studies utilizing exogenous sources of NO, our approach utilizes endogenous NO synthesis as the basis for S-nitrosylation. Our results indicate multiple unique proteins not previously identified as S-nitrosylation targets: enolase, pyruvate kinase, elongation factor-1 and -2, plastin-2, FRAG-6, CEM-16, and SMC-6. While the ubiquitous nature of NO argues for some degrees of commonality, S-nitrosylation of unique proteins specific to endotoxin stimulated macrophages suggests regulatory mechanisms for which NO is necessary, but not sufficient.

Amidines↗
PubMed2004

A transcriptional repressor of osteopontin expression in the 4T1 murine breast cancer cell line.

Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids which is an important mediator of tumor metastatic behavior. We have previously demonstrated that endotoxin-dependent OPN gene transcription is regulated by a constitutive transcriptional repressor protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNP-A/B). However, in the context of cancer, the role of hnRNP-A/B in the transcriptional regulation of OPN and its metastasis-promoting functions has not been previously studied. We examined hnRNP-A/B in the 4T1 murine mammary epithelial tumor cell line, a thioguanine resistant subline which closely mimics stage IV breast cancer in humans. Our data indicate that hnRNP-A/B p37 binds to the OPN promoter, significantly decreases OPN promoter activity and mRNA levels, ablates OPN protein expression, and inhibits OPN dependent in vitro correlates of metastatic behavior, motility, and invasion. These results are unique and may suggest new therapies to re-establish loco-regional control of cancers.

Animals↗
PubMed2004

Differential osteopontin expression in phenotypically distinct subclones of murine breast cancer cells mediates metastatic behavior.

Cancer progression depends on an accumulation of metastasis-supporting cell signaling molecules, which target signal transduction pathways and, ultimately, gene expression. One such molecule, osteopontin (OPN), represents a key molecular signaling event in tumor progression and metastasis. However, the transcriptional regulatory mechanisms that underlie OPN expression in the setting of breast cancer have not been well studied. In this regard, we have examined the differential transcriptional regulation of OPN in the murine mammary epithelial tumor cell lines, 4T1 and 4T07, which are sublines derived from the parental population of 410.4 cells from Balb/cfC3H mice. These lines are phenotypically heterogeneous in their metastatic behavior. 4T1 hematogenously metastasizes to the lung, liver, bone, and brain, whereas 4T07 is highly tumorigenic but fails to metastasize. The tumor growth and metastatic spread of 4T1 cells closely mimics stage IV breast cancer. We demonstrate that a Ras-independent, phosphoinositide-3 kinase-dependent, c-Jun N-terminal kinase-dependent phosphorylation of c-Jun results in binding of an AP-1 c-Jun homodimer to the OPN promoter in 4T1 cells. This differential up-regulation of OPN gene transcription and protein expression in 4T1 cells conveys in vitro correlates of a metastatic phenotype. These results provide new insight into the transcriptional regulation of OPN as a key mediator of metastatic behavior in malignancy.

Animals↗
PubMed2004

Osteopontin increases CD44 expression and cell adhesion in RAW 264.7 murine leukemia cells.

BACKGROUND: Osteopontin (OPN) is an inducible cell attachment protein which binds alphavbeta3-integrin and CD44 receptors to promote tumor metastasis. We hypothesized that OPN alters expression of its CD44 receptor to promote neoplastic cell migration. METHODS: RAW264.7 cells were stimulated with OPN (0-10 nM) for 0-12 hours to determine the time- and concentration-dependence of CD44 protein and mRNA expression. In selected instances, a competitive ligand for the alphavbeta3-integrin, GRGDSP (50 nM), or an inhibitor of protein synthesis, anisomycin (10 microg/ml), was added. Cell adhesion to hyaluronan was assayed with the crystal violet assay. RESULTS: OPN upregulates plasma membrane total CD44 protein in a concentration-(ANOVA P = 0.001) and time-dependent (ANOVA P = 0.001) fashion. CD44v6 is not altered. Cell adhesion to hyaluronate increases in parallel with CD44 expression. Steady state mRNA levels for CD44 are not altered by OPN. 5 nM OPN increases CD44 protein half-life from 105 +/- 11 minutes to 278 +/- 15 minutes. (P < 0.03) Blockade of either alphavbeta3-integrin ablates the OPN-dependent increase in CD44. CONCLUSIONS: These data indicate that OPN increases plasma membrane CD44 expression and cell adhesion by binding to its alphavbeta3-integrin receptor. We conclude that OPN may promote tumor metastatic behavior by CD44 expression.

Animals↗
PubMed2004

Peroxide-mediated chromatin remodelling of a nuclear factor kappa B site in the mouse inducible nitric oxide synthase promoter.

Hepatocyte expression of iNOS (inducible nitric oxide synthase) and synthesis of nitric oxide convey protective antioxidant functions in models of sepsis, shock and reperfusion. However, the underlying redox-sensitive mechanisms that regulate hepatocyte expression of iNOS and its antioxidant functions are largely unknown. Activity of the transcription factor NF-kappaB (nuclear factor kappaB) is known to be redox-modulated. In this regard, the mouse hepatocyte iNOS promoter has NF-kappaB-binding sites at nt -1044 to -1034 and at nt -114 to -104, which are considered to be critical for iNOS expression in response to pro-inflammatory cytokine stimulation. The relative contribution of these two NF-kappaB-binding sites in the mouse iNOS promoter to hepatocyte iNOS promoter activity in the context of oxidative stress has not been characterized previously. In addition, although the cis - and trans -regulatory factors controlling mouse hepatocyte iNOS expression have been well-characterized, the local changes in chromatin structure that accompany activation of iNOS gene transcription have not been considered. In the present study, we demonstrate that (1) in the absence of exogenous oxidative stress, the NF-kappaB site at nt -114 is inactive and (2) peroxide-mediated oxidative stress induces hyperacetylation and enhanced accessibility of the restriction enzyme to this NF-kappaB region. Our results suggest that chromatin structural changes activate this NF-kappaB site and increase interleukin-1beta-stimulated iNOS expression in the presence of oxidative stress.

Animals↗
PubMed2004

S-nitrosylation of heterogeneous nuclear ribonucleoprotein A/B regulates osteopontin transcription in endotoxin-stimulated murine macrophages.

Osteopontin (OPN) is a highly hydrophilic and negatively charged sialoprotein of approximately 298 amino acids that contains a Gly-Arg-Gly-Asp-Ser sequence. It is a secreted protein with diverse regulatory functions, including cell adhesion and migration, tumor growth and metastasis, atherosclerosis, aortic valve calcification, and repair of myocardial injury. Despite the many recognized functions of OPN, very little is known of the transcriptional regulation of OPN. In this regard, we have previously demonstrated that OPN transcription and promoter activity are significantly up-regulated in response to NO in a system of endotoxin-stimulated murine macrophages. However, the specific cis- and trans-regulatory elements that determine the extent of endotoxin- and NO-mediated induction of OPN synthesis are unknown. In this follow-up study, we demonstrate that: 1) OPN gene transcription is regulated by a constitutive transcriptional repressor protein, heterogeneous nuclear ribonucleoprotein A/B (hnRNP A/B); 2) inhibition of in vivo hnRNP DNA binding activity is accompanied by increased S-nitrosylation of hnRNP A/B in the setting of lipopolysaccharide (LPS)-mediated NO synthesis; 3) inhibition of LPS mediated NO synthesis restores hnRNP DNA binding and decreases the extent of S-nitrosylation; and 4) S-nitrosylation of hnRNP at cysteine 104 inhibits in vitro DNA binding activity, which is reversed by dithiothreitol. Our findings suggest that LPS induced S-nitrosylation of hnRNP inhibits its activity as a constitutive repressor of the OPN promoter and results in enhanced OPN expression.

Animals↗
PubMed2004

Determining benchmarks for evaluation and management coding in an academic division of general surgery.

BACKGROUND: Academic divisions of general surgery are facing ever-increasing financial pressures. Cost-cutting is a common approach to maintaining profitability, but strategies to increase revenue should not be ignored. One specific avenue for enhanced revenue generation in general surgery is that of coding for evaluation and management (E&M). Although this is the financial life-blood for many of the consultative services in departments of medicine, E&M coding is an often neglected and misunderstood component of surgical care. STUDY DESIGN: The financial records for the Division of General Surgery were reviewed for the period of January 2001 to June 2003. Specifically, charges and receipts for inpatient procedures and hospital visits (CPT codes 99231, 99232, and 99233) were determined. The analysis was limited to surgeons with a primary clinical focus based at the University hospital rather than the neighboring community or Veteran's Affairs hospitals. In addition, ICD-9 and All Patient Refined Diagnosis Related Groups (APR-DRG) data were analyzed to determine the surgeon-specific number of inpatients and inpatient-days with more than one ICD-9 code or secondary ICD-9 codes, or both, or an APR-DRG severity of illness score of 2, 3, or 4. These categories were defined to determine the number of inpatient-days for which E&M coding could be billed for management of secondary medical diagnoses. RESULTS: Analysis demonstrates that actual E&M charges were 40% to 47% of predicted minimums for E&M charges for the period under study. In theory, this result translates into an annual gain in receipts of 400,000 dollars to 600,000 dollars. CONCLUSIONS: We conclude that the ICD-9 and APR-DRG models may serve as benchmarks to determine the limits for E&M revenue stream, and E&M coding may represent an underutilized source of revenue among academic departments of surgery.

Academic Medical Centers↗
PubMed2004

The role of Osteopontin in tumor metastasis.

Osteopontin (OPN) is a glyco-phosphoprotein that is expressed and secreted by numerous human cancers. OPN functions in cell adhesion, chemotaxis, macrophage-directed interleukin-10 (IL-10) suppression, stress-dependent angiogenesis, prevention of apoptosis, and anchorage-independent growth of tumor cells by regulating cell-matrix interactions and cellular signaling through binding with integrin and CD44 receptors. While constitutive expression of OPN exists in several cell types, induced expression has been detected in T-lymphocytes, epidermal cells, bone cells, macrophages, and tumor cells in remodeling processes such as inflammation, ischemia-reperfusion, bone resorption, and tumor progression. Recently, substantial evidence has linked OPN with the regulation of metastatic spread by tumor cells. However, the molecular mechanisms that define the role of OPN in tumor metastasis are incompletely understood. Transcriptional regulators that contribute to the induction of OPN expression have received significant attention as potential modulators of the OPN-mediated metastatic phenotype. The following review will discuss the molecular structure of OPN, the evidence for its functional role in tumor cell metastasis, the downstream signals that activate invasive mechanisms, and the recent reports concerning regulation of OPN transcription.

Extracellular Matrix↗
PubMed2004

Intraoperative fluid management during orthotopic liver transplantation.

OBJECTIVE: To assess clinical safety of a low central venous pressure (CVP) fluid management strategy in patients undergoing liver transplantation. DESIGN: Retrospective record review comparing 2 transplant centers, one using the low CVP method and the other using the normal CVP method. SETTING: University-based, academic, tertiary care centers. PARTICIPANTS: Patients undergoing orthotopic cadaveric liver transplantation. INTERVENTIONS: Each center practiced according to its own standard of care. Center 1 maintained an intraoperative CVP <5 mmHg using fluid restriction, nitroglycerin, forced diuresis, and morphine. If pressors were required to maintain systolic arterial pressure >90 mmHg, phenylephrine or norepinephrine was used. At center 2, CVP was kept 7 to 10 mmHg and mean arterial pressure >75 mmHg with minimal use of vasoactive drugs. MEASUREMENTS AND MAIN RESULTS: Data collected included United Network for Organ Sharing status, surgical technique, intraoperative transfusion rate, preoperative and peak postoperative creatinine, time spent in intensive care unit and hospital, incidence of death, and postoperative need for hemodialysis. Principal findings include an increased rate of transfusion in the normal CVP group but increased rates of postoperative renal failure (elevated creatinine and more frequent need for dialysis) and 30-day mortality in the low CVP group. CONCLUSIONS: Despite success in lowering blood transfusion requirements in liver resection patients, a low CVP should be avoided in patients undergoing liver transplantation.

Adrenergic alpha-Agonists↗
PubMed2003

Osteopontin inhibits expression of cytochrome c oxidase in RAW 264.7 murine macrophages.

Osteopontin (OPN) functions as both a cell attachment protein and a cytokine that signals through two CAM molecules: alpha(v)beta(3)-integrin and CD44. OPN initiates a number of signal transduction pathways that control cell survival, proliferation, and migration. In this study, utilizing RAW 264.7 murine macrophages, we demonstrate that expression of the mitochondrial protein, CCOI, is significantly decreased in the setting of OPN stimulation. This effect is blocked by the CD44 competitive ligand, hylauronate; GRGDSP, a hexapeptide that blocks OPN-integrin binding, had no effect. CCOI mRNA and transcription were significantly decreased in the presence of OPN; CCOI mRNA half-life was unaltered by OPN. Additional mitochondrial run-on studies, which included genes from L-strand and H-strand, suggest that OPN terminates transcription of the distal H-strand. CCO enzyme activity was also significantly decreased by OPN. Our results indicate that OPN inhibits CCOI expression as the result of a novel CD44-dependent transcriptional regulatory mechanism of the mitochondrial H strand.

Animals↗
PubMed2003

Osteopontin-dependent CD44v6 expression and cell adhesion in HepG2 cells.

The interaction of osteopontin (OPN) with CD44 and alphavbeta3-integrin has been implicated in numerous signal transduction pathways that may promote cancer metastasis. CD44v6 is a splice variant of CD44 which has been identified as a marker of cancer progression. In this study, immortalized liver carcinoma cells (HepG2) were used to examine the effect of OPN on two isoforms of CD44: CD44 standard (CD44 s) and CD44v6. Western blots demonstrated that OPN up-regulated plasma membrane CD44v6 protein expression in a concentration- and time-dependent fashion. CD44v6 levels returned to control levels when OPN-alphavbeta3-integrin binding was blocked by an RGD peptide or tyrosine kinase activity was inhibited. OPN significantly increased CD44v6 protein synthesis, while simultaneously decreasing protein degradation. Steady-state mRNA levels of both CD44s and CD44v6 were unaltered in the presence of OPN stimulation. OPN increased HepG2 in vitro adhesion to hyaluronate (HA); excess soluble HA extinguished OPN-mediated HepG2 adhesion, indicating CD44 dependence. In conclusion, OPN binds to the alphavbeta3-integrin to increase plasma membrane CD44v6 expression and augment in vitro adhesion to HA. This may contribute to the mechanism by which OPN enhances metastatic behavior in hepatocellular cancer cells.

Blotting, Northern↗
PubMed2003

Beneficial effect of plasmapheresis and intravenous immunoglobulin on renal allograft survival of patients with acute humoral rejection.

BACKGROUND: Acute humoral rejection (AHR) has been associated with enhanced graft loss. Our study compared the renal allograft survival of patients with AHR treated with plasmapheresis (PP) and intravenous immunoglobulin (IVIG) with allograft survival in patients with acute cellular rejection (ACR). METHODS: We retrospectively analyzed all kidney transplants performed at our institution between January 1999 and August 2001 (n=286). Recipients were classified into three groups according to biopsy reports: AHR, ACR, or no rejection. The ACR group was further divided into early and late rejection (<90 and >90 days posttransplant, respectively). RESULTS: After a mean follow-up of 569+/-19 days, the incidence of AHR was 5.6% (n=16). Recipient presensitization, delayed graft function, early rejection, and higher creatinine at diagnosis were characteristic of AHR. Most AHR patients (14/16) were treated with PP and IVIG. One patient received only IVIG, whereas another received only PP. All AHR patients were given steroid pulses, but only four received antilymphocyte therapy because of concomitant severe ACR. The ACR group comprised 43 patients (15%). One patient with mild rejection received no therapy, 20 improved with steroids alone, and 22 required additional antilymphocyte therapy. One-year graft survival by Kaplan Meier analysis was 81% and 84% in the AHR and ACR groups, respectively (P=NS). Outcomes remained similar when AHR patients were compared with those with early ACR. CONCLUSIONS: We conclude that AHR, when diagnosed early and treated aggressively with PP and IVIG, carries a short-term prognosis that is similar to ACR.

Acute Disease↗
PubMed2003

Tolerance and the "Holy Grail" of transplantation.

Advances in transplantation biology have greatly improved patient outcomes following transplant surgery. However, generalized immunosuppression remains the Achilles heel of modern transplantation surgery with its associated infectious and neoplastic morbidities. Tolerance remains the ultimate goal for the entire field. Although recent advances in transplant immunology suggest that tolerance may be achievable in the near future, the complex and redundant nature of the human immune system may not allow us to circumvent such a basic function as the recognition of nonself. In this paper, advances in transplant immunology are reviewed and their potential relevance to achieving the "Holy Grail" of transplantation are discussed.

Cytokines↗
PubMed2003

Optimization of operating room allocation using linear programming techniques.

BACKGROUND: New and innovative approaches must be used to rationally allocate scarce resources such as operating room time while simultaneously optimizing the associated financial return. In this article we use the technique of linear programming to optimize allocation of OR time among a group of surgeons based on professional fee generation. STUDY DESIGN: For the period of December 1, 2000, to July 31, 2002, the following individualized data were obtained for the Division of General Surgery at Duke University Medical Center: allocated OR time (hours), case mix as determined by CPT codes, total OR time used, and normalized professional charges and receipts. Inpatient, outpatient, and emergency cases were included. The Solver linear programming routine in Microsoft Excel (Microsoft Corp.) was used to determine the optimal mix of surgical OR time allocation to maximize professional receipts. RESULTS: Our model of optimized OR allocation would maximize weekly professional revenues at 237,523 US dollars, a potential increase of 15% over the historical value of 207,700 US dollars or an annualized increase of approximately 1.5 million US dollars. CONCLUSIONS: Our results suggest that mathematical modeling techniques used in operations research, management science, or decision science may rationally optimize OR allocation to maximize revenue or to minimize costs. These techniques may optimize allocation of scarce resources in the context of the goals specific to individual academic departments of surgery.

Fees, Medical↗
PubMed2003

De-novo cholangiocarcinoma in the setting of recurrent primary sclerosing cholangitis following liver transplant.

Orthotopic liver transplantation is the only definitive therapeutic option in patients with primary sclerosing cholangitis (PSC) and end-stage liver disease. However, PSC recurs in up to 20% of patients transplanted for this indication. To date, no patient has been reported to develop cholangiocarcinoma (CCA) post-transplant, without biliary tract cancer having been present pretransplant. Here, we report recurrent PSC complicated by de-novo CCA in a 31-year-old man transplanted for PSC 8 years earlier. Cholangiocarcinoma was confirmed using a combination of computed tomography, cholangiography, positron emission tomography and histological examination of biliary cytology. He has since been successfully re-transplanted following preoperative chemo-radiotherapy. No viable tumor was identified in the explanted liver. This case establishes that long-term complications associated with PSC and biliary-enteric surgery such as CCA may become apparent in new grafts post-transplant.

Adult↗