Compared Protein Expression Levels Research Articles

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

Identification and drug metabolic characterization of four new CYP2C9 variants CYP2C9*72-*75 in the Chinese Han population.

Cytochrome 2C9 (CYP2C9), one of the most important drug metabolic enzymes in the human hepatic P450 superfamily, is required for the metabolism of 15% of clinical drugs. Similar to other CYP2C family members, CYP2C9 gene has a high genetic polymorphism which can cause significant racial and inter-individual differences in drug metabolic activity. To better understand the genetic distribution pattern of CYP2C9 in the Chinese Han population, 931 individuals were recruited and used for the genotyping in this study. As a result, seven synonymous and 14 non-synonymous variations were identified, of which 4 missense variants were designated as new alleles CYP2C9*72, *73, *74 and *75, resulting in the amino acid substitutions of A149V, R150C, Q214H and N418T, respectively. When expressed in insect cell microsomes, all four variants exhibited comparable protein expression levels to that of the wild-type CYP2C9 enzyme. However, drug metabolic activity analysis revealed that these variants exhibited significantly decreased catalytic activities toward three CYP2C9 specific probe drugs, as compared with that of the wild-type enzyme. These data indicate that the amino acid substitution in newly designated variants can cause reduced function of the enzyme and its clinical significance still needs further investigation in the future.

Comparative proteome analysis of the ligamentum flavum of patients with lumbar spinal canal stenosis.

Thickening of the ligamentum flavum is considered to be the main factor for lumbar spinal canal stenosis (LSCS). Although some mechanisms have been speculated in the thickening of the ligamentum flavum, there are only a few comprehensive approaches to investigate its pathology. The objective of this study was to investigate the pathology of thickened ligamentum flavum in patients with LSCS based on protein expression levels using shotgun proteome analysis. Ligamentum flavum samples were collected from four patients with LSCS (LSCS group) and four patients with lumbar disc herniation (LDH) as controls (LDH group). Protein mixtures were digested and analyzed by liquid chromatography/mass spectrometry analysis. To compare protein expression levels between the LSCS and LDH groups, the mean Mascot score was compared. Biological processes were assessed using Gene Ontology analysis. A total of 1151 proteins were identified in some samples of ligamentum flavum. Among these, 145 proteins were detected only in the LSCS group, 315 in the LDH group, and 691 in both groups. The demonstrated biological processes occurring in the LSCS group included: extracellular matrix organization, regulation of peptidase activity, extracellular matrix disassembly, and negative regulation of cell growth. Proteins related to fibrosis, chondrometaplasia, and amyloid deposition were found highly expressed in the LSCS group compared with those in the LDH group. Tissue repair via fibrosis, chondrometaplasia, and amyloid deposits may be important pathologies that occur in the thickened ligamentum flavum of patients with LSCS.

Prostaglandin D2 contributes to cisplatin-induced neuropathic pain in rats via DP2 receptor in the spinal cord.

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) is a major reason for stopping or changing anticancer therapy. Among the proposed pathomechanisms underlying CIPN, proinflammatory processes have attracted increasing attention. Here we assessed the role of prostaglandin D2 (PGD2) signaling in cisplatin-induced neuropathic pain.MethodsCIPN was induced by intraperitoneal administration of cisplatin 2 mg/kg for 4 consecutive days using adult male Sprague-Dawley rats. PGD2 receptor DP1 and/or DP2 antagonists were administered intrathecally and the paw withdrawal thresholds were measured using von Frey filaments. Spinal expression of DP1, DP2, hematopoietic PGD synthase (H-PGDS), and lipocalin PGD synthase (L-PGDS) proteins were analyzed by western blotting.ResultsThe DP1 and DP2 antagonist AMG 853 and the selective DP2 antagonist CAY10471, but not the DP1 antagonist MK0524, significantly increased the paw withdrawal threshold compared to vehicle controls (P = 0.004 and P < 0.001, respectively). Western blotting analyses revealed comparable protein expression levels in DP1 and DP2 in the spinal cord. In the CIPN group the protein expression level of L-PGDS, but not of H-PGDS, was significantly increased compared to the control group (P < 0.001).ConclusionsThe findings presented here indicate that enhanced PGD2 signaling, via upregulation of L-PGDS in the spinal cord, contributes to mechanical allodynia via DP2 receptors in a cisplatin-induced neuropathic pain model in rats, and that a blockade of DP2 receptor activation may present a novel therapeutic target for managing CIPN.

TCL-302: Immunohistochemical Characterization of Anaplastic Large Cell Lymphoma Using Tissue Microarray

Context: Anaplastic Large Cell Lymphoma (ALCL) is a type of mature T-cell lymphomas with systemic and primary cutaneous (PCALCL) subtypes. Systemic ALCL includes anaplastic lymphoma kinase (ALK)+ and ALK-. ALK+ usually has the NPM/ALK gene, which has favorable prognosis compared to ALK-. PCALCL is usually indolent. DUSP22 rearrangement in ALK-ALCL has a favorable outcome like ALK+, indicating alternate pathways of ALCL. Objective: Our primary aim was to compare protein expression levels of selected signaling molecules in oncogenic pathways of ALCL (JAK1, STAT3, DUSP22, ERB4, BLIMP1, SOCS3) among the three subtypes. We also correlated expression levels to ORR, PFS, DFS, and OS. Design: This is a retrospective study correlating protein expression levels of various antibodies across 27 patient tissue samples. Setting: The study was set at H. Lee Moffitt Cancer Center and Research Institute, an academic referral center. Patients or other participants: We identified 50 ALCL patients from 2000–2019. Twenty-seven tissue samples (6 ALK+, 10 ALK-, 8 PCALCL, 3 controls) met eligibility criteria, which included pathologist-reviewed diagnosis of ALCL subtypes (ALK+, ALK-, PCALCL) and consent to the Tissue Core database. Interventions: Replicate cores were taken and TMAs were constructed from formalin-fixed, paraffin-embedded lymph node or skin biopsies. IHC was performed with Abs to selected proteins. Stained TMA was scanned using Aperio™ ScanScope XT2 with a 200×/0.8NA objective lens at 5 min/slide via Basler tri-linear array detection. TMAs were analyzed using Aperio Nuclear V9 Default algorithm with thresholds for stain intensity: Strong = 130, Moderate = 188, Weak = 162 to determine the % positive biomarker stain within each core. Main outcome measures: The primary study outcome measurement was comparison of protein expression levels based on specific pathways known or hypothesized to be involved in ALCL pathogenesis. We hypothesized that these protein expression levels would correlate to time to event outcomes Results: PCALCL, ALK+, and ALK- subgroups demonstrated a significant difference in SOCS3 expression with mean and std respectively: 42.4% (0.153), 17.4% (0.089), and 21.0% (0.155);p=0.008. Among all pairs, ALK+ and PCALCL were statistically different (p=0.011). Patients with high BLIMP1 expression (% larger than median, 28.5%) regardless of ALCL subtype were associated with better mOS (not reached) compared to patients with low ( Conclusions: We identified a significant difference in expression of two Abs among three subtypes of ALCL. Increased BLIMP1 was associated with favorable prognosis and could potentially be a positive prognostic marker. Increased SOCS3 appears more prevalent in PCALCL than systemic ALCL, suggesting a potential utility in DDx. We plan to validate our results in a larger sample. Adding secondary markers like Bcl-2 can be used in the future to correlate expression levels and response to targeted agents, such as venetoclax. Funding: University of South Florida Graduate Medical Education Grant ($7500).

Immunohistochemical characterization of anaplastic large cell lymphoma using tissue microarray.

8048 Background: Anaplastic Large Cell Lymphoma (ALCL) is a subtype of mature T-cell lymphoma comprised of systemic (ALK+ or ALK-) and primary cutaneous (PCALCL) forms. ALK+, usually associated with NPM1-ALK gene, has a favorable prognosis. PCALCL is usually indolent. Despite worse prognosis for ALK-, pts with DUSP22 rearrangement have favorable outcomes similar to ALK+ while those with p63 aberration have poor outcomes. We hypothesize that other genetic alterations in ALCL could aid in diagnosis and prognosis. We compared protein expression levels of selected signaling molecules (JAK1, STAT3, DUSP22, ERBB4/HER4, PRDM1/BLIMP1 and SOCS3) among the subtypes and correlated to outcomes. Methods: Of 50 pts with ALCL at Moffitt (MCC) from 2000-2019, 27 tissue samples (6 ALK+, 10 ALK-, 8 PCALCL, 3 controls) met eligibility criteria. Tissue microarrays (TMAs) were constructed from formalin-fixed, paraffin-embedded lymph node biopsies. Up to 3 replicate cores were taken from each block. Immunohistochemistry was performed with antibodies to the selected proteins. Stained TMA slides were scanned using the Aperio™ ScanScope XT2 to determine % positive biomarker stain within each core per established algorithm. Statistics: ANOVA was used to compared protein expression levels, with pairwise tests when significant. T-test compared PCALCL to systemic ALCL. Boxplots were created for differences in protein expression levels and time to event outcomes (OS and RFS) using Log rank test and Cox proportional hazards models. Kaplan-Meier curves were used for clinical time-to-event outcomes and quartiles of the protein expression levels. Results: The 3 subgroups had a significant difference in SOCS3 expression with mean and std respectively: 42.4% (0.153), 17.4% (0.089), and 21.0% (0.155), p = 0.008. Among all pairs, ALK+ and PCALCL groups were statistically different (P = 0.011). Patients with high BLIMP1 (≥28.5%) across 3 ALCL subgroups were associated with better median OS (not reached) in comparison to pts with lower expression (27.3 mos) (Log rank test p = 0.014, HR = 0.17, 95% CI 0.034-0.829). The same was seen for median RFS (81.1 vs. 12.5 mos). (Log rank test p = 0.009, HR 0.23, 95% CI 0.069-0.755). Conclusions: High BLIMP1 suggests favorable prognosis in ALCL and could potentially be a positive prognostic marker. High SOCS3 appears more prevalent in PCALCL than systemic and could aid in differential. Larger samples should be used to validate results. Secondary markers like Bcl-2 can be considered in the future to correlate expression levels to targeted treatments like venetoclax.

Calcium Homeostasis Modulator (CALHM1/2) and Pulmonary Arterial Hypertension

RATIONALEConcentric pulmonary vascular remodeling is a major cause for the elevated pulmonary vascular resistance (PVR) and pulmonary arterial pressure (PAP) in patients with pulmonary arterial hypertension (PAH). Excessive PASMC proliferation and inhibited PASMC apoptosis have been implicated in the development and progression of pulmonary vascular wall thickening in PAH. Transition from the contractile phenotype to the synthetic or proliferative phenotype of PASMC is thus an important pathogenic process that promotes vascular remodeling. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMC is not only a trigger for PASMC contraction (and pulmonary vasoconstriction), but also an important stimulus for PASMC proliferation. Calcium homeostasis modulators (CALHM), CALHM1/2, are membrane Ca2+ channels that are allosterically regulated by voltage and extracellular Ca2+. CALHM1/2 are closed at the resting membrane potential but can be opened by strong membrane depolarization. Reduction of extracellular [Ca2+] increases CALHM open probability, which allows the channel to be activated at negative potential. We aimed to study whether CALHM1/2 are involved in the phenotypical transition of PASMC and whether CALHM1/2 are upregulated in highly proliferative PASMC from patients with PAH.METHODSFreshly isolated pulmonary artery with the endothelium removed (as contractile PASMC) and primary cultured PASMC (as synthetic or proliferative PASMC) from rats were used for this study. Western blot analysis was used to compare protein expression levels of CALHM1 and CALHM2 in fresh PA tissue and cultured PASMC derived from the same rats. In addition, we compared protein expression levels of CALHM1/2 in PASMC from normal subjects and PAH patients.RESULTSThe protein expression level of CALHM2 in proliferative PASMC (the primary cultured PASMC in growth factor‐containing medium) was significantly higher than in contractile PASMC (the freshly isolated PA tissue). The increased CALHM2 in proliferative PASMC was indeed associated with a significantly increased expression of PCNA, a cell proliferation marker, and a significantly decreased expression of MHC and TGLN4, markers for contractile phenotype of SMC. Furthermore, we also observed that mRNA expression level of CALHM2 was significantly higher in PASMC isolated from PAH patients than in PASMC from normal subjects.CONCLUSIONUpregulation of CALHM2 may play an important role in the phenotypical transition of PASMC from the contractile phenotype to the synthetic or proliferative phenotype. Ca2+ influx through voltage‐ and extracellular Ca2+‐regulated CALHM2 in the plasma membrane of PASMC is an important trigger for PASMC proliferation and may play an important role in stimulating PASMC proliferation in patients with PAH.Support or Funding InformationNIH

Differences of protein expression profiles, KRAS and BRAF mutation, and prognosis in right-sided colon, left-sided colon and rectal cancer

To compare protein expression levels, gene mutation and survival among Right-Sided Colon Cancer (RSCC), Left-Sided Colon Cancer (LSCC) and rectal cancer patients, 57 cases of RSCC, 87 LSCC and 145 rectal cancer patients were included retrospectively. Our results demonstrated significant differences existed among RSCC, LSCC and rectal cancer regarding tumor diameter, differentiation, invasion depth and TNM stage. No significant difference was identified in expression levels of MLH1, MSH2, MSH6, PMS2, β-Tubulin III, P53, Ki67 and TOPIIα, and gene mutation of KRAS and BRAF among three groups. Progression Free Survival (PFS) of RSCC was significantly lower than that of LRCC and rectal cancer. In univariate analyses, RSCC, preoperative chemoradiotherapy, poor differentiation, advanced TNM stage, elevated serum CEA and CA19-9 level, tumor deposit, perineural and vascular invasion were found to be predictive factors of shorter PFS. In multivariate analyses, only differentiation and TNM stages were found to be independent predictors of PFS. In conclusion, compared with LSCC and rectal cancer, RSCC has larger tumor size, poor differentiation, advanced TNM stage and shorter survival. The shorter survival in RSCC might be attributed to the advanced tumor stage caused by its inherent position feature of proximal colon rather than genetic difference.

Abstract 3320: Human bone marrow- and adipose-mesenchymal stem cells secrete inflammatory cytokines and growth factors in the presence of breast tumor cells

Abstract Introduction: Stem cell and adipocyte-epithelial interactions are mediated by tumor-enhancing factors such as cytokines, chemokines, and growth factors. The objective of our study was to identify novel secreted cytokines and growth factors common to mesenchymal stem cells and adipocytes and breast tumor cells using protein array analysis. Methods: Bone marrow (BM-MSC) or adipose (ASC) mesenchymal stem cells or their preadipocytes were co-cultured indirectly with MDA-MB-231 invasive breast cancer cells. Conditioned media was collected to compare protein expression levels using a cytokine antibody array. Signals were visualized, scanned, and quantitated by densitometry. Ingenuity Pathway Analysis mapped direct and indirect relationships between highly expressed proteins involved in obesity and the inflammation. Results: Within a week, stem cells derived from either bone marrow or adipose tissue differentiated into preadipocytes, as evident by staining with oil red O solution. Greater upregulation of cytokine and growth factor expression was observed when mesenchymal stem cells or preadipocytes were cultured in the presence of breast cancer cells. In BM-MSCs, IL-6, IL-6R, FGF9, ADIPOQ, and CXCL1 were the top five upregulated proteins, and IL13, IL2, CCL8, CXCL9, and TNF were the top five downregulated proteins when cultured with breast cancer cells. IL13, IL2, TNF, IL6, IL6R, and ADIPOQ are obesity-related while CXCL1, IL6, IL6R, ADIPOQ, CCL8, CXCL9, TNF, IL2, and IL13 are involved in inflammation. In ASCs, CXCL5, CCL5, IFNG, FGF7, and PDGF BB were the top five upregulated proteins, and CXCL11, ICAM3, IL6R, CCL16, and EGF were the top five downregulated proteins when cultured with breast cancer cells. CXCL11, IL6R, and CCL5 are obesity-related while IFNG, EGF, CCL16, IL6R, and CXCL11 are involved in the inflammatory response. IL7, CXCL3, ANG, CXCL11, and IGFBP1 were the top five upregulated proteins, and TNF, CCL18, IGFBP2, CCL15, and CCL20 were the top five downregulated proteins when BM-MSCs were differentiated into preadipocytes. CCL16, IL17, ICAM3, AGRP, and TNFSF18 were the top five upregulated proteins, and CCL3, IL6, FGF9, NAP1L4, and CSF1 were the top five downregulated proteins when ASCs were differentiated into preadipocytes. Conclusions: We identified a number of metabolic and inflammatory cytokines and growth factors common to bone and adipose tissue that could be involved in reciprocal communication between stem cells, their preadipocytes, and invasive breast cancer cells. A number of unique cytokines and growth factors were differentially expressed and under investigation for their roles in breast cancer. Citation Format: Brandi Talkington, Leah Figurski, Yuya Kudo, Kelsey Sadlek, Linda Vona-Davis. Human bone marrow- and adipose-mesenchymal stem cells secrete inflammatory cytokines and growth factors in the presence of breast tumor cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3320.

Comparative proteomic analysis of fibrosarcoma and skin fibroblast cell lines.

Comparative proteomic analysis of normal and cancer cell lines provides for a better understanding of the molecular mechanism of cancer development and is essential for developing more effective strategies for new biomarker or drug target discovery. The purpose of this study is to compare protein expression levels between fibrosarcoma and fibroblast cell lines. In our study, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) techniques were carried out to compare the protein profile between fibrosarcoma and fibroblast cell lines. We prepared cell lysate samples to analyze intracellular proteins and secretome samples to analyze extracellular proteins in both cell lines. Our results revealed 13 upregulated proteins and 1 downregulated protein of which all of them identified in fibrosarcoma cell line after the comparison with fibroblast cell line cell lysates. When comparing secretome profiles of both cell lines, we found and identified 13 proteins only expressed in fibrosarcoma cell line. These identified proteins have common functions such as cell proliferation, cell differentiation, invasion, metastasis, and apoptosis in cancer. The data obtained from this study indicates that these proteins have importance on understanding the molecular mechanism of fibrosarcoma. These proteins may serve as candidate biomarkers and drug targets for future clinical studies.

Protease- and Acid-catalyzed Labeling Workflows Employing &lt;sup&gt;18&lt;/sup&gt;O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing &lt;sup&gt;18&lt;/sup&gt;O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, (18)O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes(1-3). With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of (18)O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao(4), Miyagi and Rao(5) and Ye et al.(6)). (18)O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques(7). Depending on the protease utilized, (18)O-labeling can result in the incorporation of up to two (18)O-atoms in the C-terminal carboxyl group of the cleavage product(3). The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction(8). In our PALeO (protease-assisted labeling employing (18)O-enriched water) adaptation of enzymatic (18)O-labeling, we utilized 50% (18)O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases(9) and monitor proteolytic reactions(10-11). Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second (18)O-atom. Such "double-labeling" enzymes can be used for postdigestion (18)O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing (18)O-enriched water beyond enzymes and uses acidic pH conditions to introduce (18)O-stable isotope signatures into peptides.

Oncogene‐ and Oxidative Stress‐Induced Cellular Senescence Shows Distinct Expression Patterns of Proinflammatory Cytokines in Vascular Endothelial Cells

Senescent cells are metabolically active and produce a variety of proinflammatory cytokines. It was previously reported that atherosclerotic plaques contain senescent cells, suggesting that senescence may contribute to the progression of atherosclerosis. In this study, we induced cellular senescence in vascular endothelial cells (VECs) using hydrogen peroxide (H2O2) or an adenovirus that expresses a constitutively active mutant of Ras (AdRas12V) and studied the expression of cytokines. Both H2O2 treatment and AdRas12V infection induced senescence in VECs, as assessed by senescence-associated β-Gal activity and the expression of proteins such as p53 and p21CIP1. In addition, both treatments induced the expression of a variety of cytokines, including interleukin-1β (IL-1β) and nerve growth factor (NGF). AdRas12V infection induced IL-1β expression more significantly than H2O2 treatment, whereas both treatments induced comparable mRNA and protein expression levels of NGF. These results suggest that senescent cells express different patterns of proinflammatory cytokines, depending on the trigger that induced senescence. It is therefore possible that senescent cells can differentially induce inflammation in the surrounding tissues, depending on the cause of senescence.

Lasp1 is down-regulated in NMDA receptor antagonist-treated mice and implicated in human schizophrenia susceptibility

Mice treated with MK-801, a non-competitive antagonist of the N-methyl-d-aspartic (NMDA) acid receptor, are important animal models for schizophrenia studies. In the present study, we compared protein expression levels in the hippocampus of mice treated with MK-801 (0.6 mg/kg) or saline once daily for 7 days. Changes in the proteome were detected by two-dimensional electrophoresis, and the six proteins exhibiting differential expression were identified by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry. Down-regulation of one of these proteins, Lasp1 (LIM and SH3 protein 1), in MK-801-treated mice was confirmed by western blotting and immunohistochemical analyses. Lasp1 is a multidomain protein that may recruit signaling molecules to the actin-based cytoskeleton and is known to concentrate in synaptic sites of hippocampal neurons. We next investigated whether polymorphisms in the human LASP1 gene were associated with schizophrenia in the Korean population. A single-nucleotide polymorphism in the LASP1 gene promoter region was associated with schizophrenia susceptibility. Our results suggest that LASP1 might be associated with NMDA receptor antagonism and schizophrenia susceptibility and, thus, might be involved in the pathophysiology of schizophrenia.

Identifying TRKA and TRKB specific pathways in neuroblastoma through phosphoproteomic analysis.

e20008 Background: Neuroblastoma is the most common solid tumor found in children and is difficult to treat given its genetic and clinical heterogeneity. The tyrosine kinase receptor, TrkB, is often co-expressed with the MYCN oncogene in high-risk tumors, whereas the TrkA receptor is most often found expressed in low-risk, non-MYCN amplified samples. There are differences in the gene expression profiles of TrkB- and TrkA-over-expressing cell lines, but they do not explain the phenotypic variation. We hypothesize that differences in protein translation and post-translational modifications have profound downstream effects on cellular signaling and disease phenotype. Methods: We performed quantitative proteomics using stable isotope labeling, phosphopeptide enrichment, and tandem mass spectrometry on parental SY5Y neuroblastoma cells and cell lines stably transfected with either TrkA or TrkB. Receptors were activated with NGF or BDNF and activation was inhibited with CEP-701 (Lestaurtinib), a selective tyrosine kinase inhibitor, currently in clinical trials. Samples were separated by gel electrophoresis, digested with trypsin, and applied to the mass spectrometer for protein identification. Results: We have performed quantitative phosphoproteomic analysis and compared protein expression levels and patterns in TrkA overexpressing, TrkB overexpressing, and parental SY5Y cells. The TrkA and TrkB receptors were activated with NGF and BDNF ligands, respectively, as evidenced by increased phosphorylation of ERK and AKT, with inhibition by CEP-701. Changes in protein abundance and pathway activation following both ligand binding and inhibition are being determined by quantitative phosphoproteomic analysis, and TrkA-specific and TrkB-specific differences will be presented. Conclusions: As current genomic techniques may underestimate the differences in protein expression, proteomic profiling holds great promise for describing how post-translational modifications such as phosphorylation can affect tumor phenotype. Our work may reveal key elements of TRK signaling pathways important in neuroblastoma tumorigenesis, and may lead to the identification of novel targets for therapy development.

Antibody microarray analysis of the serum proteome in primary breast cancer patients

Noninvasive biomarkers are urgently needed for detecting breast cancer as early as possible since the risk of recurrence, morbidity, and mortality is closely related to disease stage at the time of primary surgery. There are currently no such biomarkers in clinical use as a diagnostic tool. Proteomic analysis of protein expression patterns in body fluids has potential for use in identifying biomarkers of breast cancer. The aim of this study was to compare protein expression levels in the sera of primary breast cancer patients and healthy controls. An antibody microarray tool with 23 antibodies immobilized on nitrocellulose slides was used to determine the levels of acute phase proteins, interleukins, and complement factors in the sera of 101 study participants (49 women with primary breast cancer and 52 healthy age-matched controls). Statistical analysis of reaction intensities identified 6 proteins that showed significantly (p < 0.05) different levels in breast cancer patients vs. healthy subjects. The neural network distinguished cancer patients from controls with a sensitivity of 69% and a specificity of 76%. Thus, antibody microarray analysis could be used as a tool for the development of improved diagnostics and biomarker discovery for breast cancer patients. Further validation of the results and de novo screening of new biomarkers could facilitate the early diagnosis of breast cancer.

Identification of Soat1 as a Quantitative Trait Locus Gene on Mouse Chromosome 1 Contributing to Hyperlipidemia

We previously identified two closely linked quantitative trait loci (QTL) on distal chromosome 1 contributing to major variations in plasma cholesterol and triglyceride levels in an intercross derived from C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE−/−) mice. Soat1, encoding sterol o-acyltransferase 1, is a functional candidate gene located underneath the proximal linkage peak. We sequenced the coding region of Soat1 and identified four single nucleotide polymorphisms (SNPs) between B6 and C3H mice. Two of the SNPs resulted in amino-acid substitutions (Ile147Val and His205Tyr). Functional assay revealed an increased enzyme activity of Soat1 in peritoneal macrophages of C3H mice relative to those of B6 mice despite comparable protein expression levels. Allelic variants of Soat1 were associated with variations in plasma cholesterol and triglyceride levels in an intercross between B6.apoE−/− and C3H.apoE−/− mice. Inheritance of the C3H allele resulted in significantly higher plasma lipid levels than inheritance of the B6 allele. Soat1 variants were also significantly linked to major variations in plasma esterified cholesterol levels but not with free cholesterol levels. Trangenic expression of C3H Soat1 in B6.apoE−/− mice resulted in elevations of plasma cholesterol and triglyceride levels. These results indicate that Soat1 is a QTL gene contributing to hyperlipidemia.

Identification of ETFB as a candidate protein that participates in the mechanoregulation of fibroblast cell number in collagen gel culture

Fibroblast activation is strongly influenced by mechanical environment in the wound-healing process, especially in fibrosis. Mechanically stressed three-dimensional collagen embedded culture is a useful model representing fibroblasts in morphological as well as biochemical situations encountered during fibrosis. To find key proteins involved in reducing the number of fibroblasts during mechanical stress, we performed two-dimensional gel electrophoresis (2DE)-based differential display and siRNA-based functional screening with collagen gel culture focusing on the differences between attached and detached culture environments. Membrane extracts of fibroblasts from 1 day of attached or detached cultures were subjected to 2DE. We compared protein expression levels and identified the attached-culture-dominant proteins by MALDI-TOF-MS. Next, fibroblasts were transfected with siRNA and embedded in collagen gel. Cell number was counted after 3 days in culture. Eight attached culture dominant proteins were identified with MALDI-TOF-MS. Transfection of siRNA against these proteins demonstrated that electron transfer flavoprotein β subunit (ETFB)-specific siRNA reduced the cell number in the attached culture without a decrease in the detached culture. ETFB participates in the mechanoregulation of fibroblast cell number in collagen gel culture.

Tissue Factor and Tissue Factor Pathway Inhibitors TFPI and TFPI2 in Human Secretory Endometrium—Possible Link to Female Infertility

The aim of this study was to investigate tissue factor (TF) and its inhibitors TFPI and TFPI2 in secretory endometrium of fertile women and in women with unexplained infertility in relation to endometrial receptivity. In addition, common variation in the regulatory area of TF and TFPI genes was studied. Immunostaining of TF and TFPI, together with the appearance of pinopodes, revealed similar expression pattern in fertile endometrium throughout the secretory phase, being highest at the time of implantation. When compared protein expression levels at the time of implantation, infertile women demonstrated significantly higher TFPI expression in luminal epithelium. Furthermore, polymorphism TF -603 A/G was associated with the endometrial protein level in infertile women, being highest in women with GG genotype, and variation TFPI -287 T/C was associated with unexplained infertility, where infertile women presented more frequently T allele than fertile women. Contrary to TF and TFPI, TFPI2 showed different mRNA and protein expression patterns in fertile endometrium, and no differences between fertile and infertile women were detected. We conclude that the TF pathway is involved in normal endometrial maturation, where TF and TFPI seem to have important roles at the time of embryo implantation. Higher TFPI expression level during the time of embryo implantation and TFPI -287 T allele could be risk factors for unexplained infertility. No distinct involvement of TFPI2 in the regulation of endometrial receptivity and unexplained infertility was found.

RPPanalyzer: Analysis of reverse-phase protein array data

RPPanalyzer is a statistical tool developed to read reverse-phase protein array data, to perform the basic data analysis and to visualize the resulting biological information. The R-package provides different functions to compare protein expression levels of different samples and to normalize the data. Implemented plotting functions permit a quality control by monitoring data distribution and signal validity. Finally, the data can be visualized in heatmaps, boxplots, time course plots and correlation plots. RPPanalyzer is a flexible tool and tolerates a huge variety of different experimental designs. The RPPAanalyzer is open source and freely available as an R-Package on the CRAN platform http://cran.r-project.org/.