Effects of L-Carnitine Treatment on Kidney Mitochondria and Macrophages in Mice with Diabetic Nephropathy.

INTRODUCTION: In diabetic nephropathy (DN), mitochondrial dysfunction and leakage of mitochondrial DNA (mtDNA) are caused by the downregulation of superoxide dismutase 2 (SOD2). mtDNA induces the activation of Toll-like receptor (TLR) 9, which is present in macrophages (Mφs), and triggers their activation. METHODS: We orally administered L-carnitine, which exerts protective effects on the mitochondria, to obesity-induced DN (db/db) mice for 8 weeks. We then investigated the effects of L-carnitine on kidney mitochondrial reactive oxygen species (mtROS) production, circulating mtDNA content, and kidney CD11bhigh/CD11blow Mφ functions. RESULTS: In db/db mice, mtROS production increased in proximal tubular cells and kidney CD11blow Mφs; both Mφ types showed enhanced TLR9 expression. L-Carnitine treatment suppressed mtROS production in both proximal tubular cells and CD11blow Mφs (p < 0.01), with improved SOD2 expression in the kidney (p < 0.01), decreased circulating mtDNA content, and reduced albuminuria. Moreover, it suppressed Mφ infiltration into kidneys and reduced TLR9 expression in Mφs (p < 0.01), thereby lowering tumor necrosis factor-α production in CD11bhigh Mφs (p < 0.05) and ROS production by CD11blow Mφs (p < 0.01). Collectively, these changes alleviated DN symptoms. CONCLUSION: The positive effects of L-carnitine on DN suggest its potential as a novel therapeutic agent against obesity-linked DN.

Effects of a CCR2 antagonist on macrophages and Toll-like receptor 9 expression in a mouse model of diabetic nephropathy.

The pathogenesis of diabetic nephropathy (DN) is related to macrophage (Mφ) recruitment to the kidneys, tumor necrosis factor-α (TNF-α) production, and oxidative stress. Toll-like receptor 9 (TLR9) activation is reportedly involved in systemic inflammation, and it exacerbates this condition in metabolic syndrome. Therefore, we hypothesized that TLR9 plays a role in the pathogenesis of DN. Two subsets of kidney Mφs in DN model (db/db) mice were analyzed using flow cytometry to evaluate their distribution and TLR9 expression and function. Mice were administered the CCR2 antagonist INCB3344 for 8 wk; changes in Mφ distribution and function and its therapeutic effects on DN pathology were examined. Bone marrow-derived CD11bhigh (BM-Mφ) and tissue-resident CD11blow Mφs (Res-Mφ) were identified in the mouse kidneys. As DN progressed, the BM-Mφ number, TLR9 expression, and TNF-α production increased significantly. In Res-Mφs, reactive oxygen species (ROS) production and phagocytic activity were enhanced. INCB3344 decreased albuminuria, serum creatinine level, BM-Mφ abundance, TLR9 expression, and TNF-α production by BM-Mφs and ROS production by Res-Mφs. Both increased activation of BM-Mφ via TLR9 and TNF-α production and increased ROS production by Res-Mφs were involved in DN progression. Thus, inactivating Mφs and their TLR9 expression by INCB3344 is a potential therapeutic strategy for DN.NEW & NOTEWORTHY We classified kidney macrophages (Mφs) into bone marrow-derived Mφs (BM-Mφs) expressing high CD11b and tissue-specific resident Mφ (Res-Mφs) expressing low CD11b. In diabetic nephropathy (DN) model mice, Toll-like receptor 9 (TLR9) expression and TNF-α production via TLR9 activation in BM-Mφs and ROS production in Res-Mφs were enhanced. Furthermore, CCR2 antagonist suppressed the kidney infiltration of BM-Mφs and their function and the ROS production by Res-Mφs, with concomitant TLR9 suppression. Our study presents a new therapeutic strategy for DN.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Alectinib re-challenge in small cell lung cancer transformation after chemotherapy failure in a patient with ALK-positive lung cancer: A case report.

Small cell lung cancer (SCLC) transformation is a rare resistance mechanism to anaplastic lymphoma kinase-tyrosine kinase inhibitors (ALK-TKIs), for which cytotoxic chemotherapy is often initiated. However, no case has been reported so far in which the SCLC component disappeared after chemotherapy and the tumor responded to ALK-TKI treatment again. A 41-year-old, never-smoker man was diagnosed with multiple metastatic lung adenocarcinoma harboring ALK gene rearrangements. After tumor re-growth was treated with alectinib, histological analysis of re-biopsy of the primary lesion showed combined small cell carcinoma, and cytotoxic chemotherapy was administered. After resistance to chemotherapy developed, the third biopsy of the primary lesion showed the original ALK gene rearrangements without the SCLC component. Alectinib was re-administered, and partial response was obtained. Biopsy for ALK-positive lung cancer that progressed after chemotherapy for SCLC transformation might be useful for decision-making regarding the therapeutic strategy.

Impact of bronchoalveolar lavage lymphocytosis on the effects of anti-inflammatory therapy in idiopathic non-specific interstitial pneumonia, idiopathic pleuroparenchymal fibroelastosis, and unclassifiable idiopathic interstitial pneumonia.

BACKGROUND: Idiopathic non-specific interstitial pneumonia (iNSIP), idiopathic pleuroparenchymal fibroelastosis (iPPFE), and unclassifiable idiopathic interstitial pneumonia (IIP) are IIPs with chronic fibrotic phenotypes, and unlike idiopathic pulmonary fibrosis, they have often been treated with anti-inflammatory drugs, including corticosteroids and immunosuppressants. However, the impact of bronchoalveolar lavage (BAL) lymphocytosis on the effects of anti-inflammatory therapy has never been evaluated. This study aimed to elucidate whether BAL lymphocytosis can be used to predict the efficacy of anti-inflammatory drugs for iNSIP, iPPFE, and unclassifiable IIP. METHODS: Japanese patients diagnosed with iNSIP, iPPFE, and unclassifiable IIP by multidisciplinary discussion were identified using the nationwide registry. Eligible patients were stratified into four groups with and without BAL lymphocytosis and anti-inflammatory therapy to compare overall survival (OS) and changes in lung function. BAL lymphocytosis was defined as a lymphocyte differential count > 15%, and the cut-off was corroborated by survival classification and regression tree analysis. RESULTS: Overall, 186 patients (37 iNSIP, 16 iPPFE, and 133 unclassifiable IIP) were analyzed. Limited to patients treated with anti-inflammatory drugs (n = 123), patients with BAL lymphocytosis had a better prognosis [hazard ratio (HR), 0.26; 95% confidence interval (CI), 0.11-0.63; P = 0.003], higher slope of forced vital capacity (FVC) % predicted for 2 years, and longer OS (log-rank test, P = 0.012) than those without BAL lymphocytosis. On multivariate analysis, BAL lymphocytosis (HR 0.31; 95% CI 0.13-0.75; P = 0.009) was a prognostic factor for OS, along with age and FVC % predicted. Conversely, for patients managed without anti-inflammatory therapy (n = 63), the presence or absence of BAL lymphocytosis had no prognostic value. CONCLUSIONS: BAL lymphocytosis is associated with good outcomes in patients treated with anti-inflammatory drugs, but has no prognostic value when anti-inflammatory drugs are not used. BAL lymphocytosis may provide a predictive biomarker for identifying patients with iNSIP, iPPFE and unclassifiable IIP who are likely to benefit from anti-inflammatory drugs.

Impact of interstitial lung disease associated with immune checkpoint inhibitors on prognosis in patients with non-small-cell lung cancer.

PURPOSE: Immune checkpoint inhibitor-associated interstitial lung disease (ICI-ILD) is a serious immune-related adverse event. We aimed to evaluate the impact of ICI-ILD severity and imaging patterns or post-ILD cancer therapy on prognosis in patients with non-small-cell lung cancer (NSCLC). METHODS: We retrospectively analysed NSCLC patients who developed ICI-ILD in our institution between January 2016 and March 2019. The primary objective was to report prognosis following onset of ICI-ILD, stratified by severity grade or imaging pattern. The secondary objective was the analysis of cancer therapy after ICI-ILD. RESULTS: Among 222 patients treated with ICI, 27 (12.2%) developed ICI-ILD. No trend for different prognosis depending on severity grade was seen unless ICI-ILD was fatal. Most patients (91.3%) with organising pneumonia (OP) or nonspecific interstitial pneumonia pattern on imaging showed grade 1 or 2, while all patients with a diffuse alveolar damage (DAD) pattern showed grade 3 or higher, and one reached grade 5. Among patients who overcame ICI-ILD, eight patients (30.8%) have been followed up without chemotherapy because of long-term disease control and seven had shown an OP pattern on imaging at onset of ICI-ILD. Three patients underwent ICI rechallenge, but two showed ICI-ILD recurrence and no patient achieved response to rechallenge treatment. CONCLUSION: The DAD pattern may predict short-term adverse prognosis for ICI-ILD. Once ICI-ILD is overcome, severity grade is not associated with prognosis. Even if initial immunotherapy proves effective, ICI rechallenge requires careful consideration.

Therapeutic plasma exchange for clinically amyopathic dermatomyositis (CADM) associated with rapidly progressive interstitial pneumonia.

BACKGROUND: Patients with clinically amyopathic dermatomyositis (CADM) with anti-melanoma differentiation-associated gene 5 antibody (anti-MDA5 Ab) frequently develop rapidly progressive interstitial pneumonia (RPIP), often with fatal outcomes. Therapeutic plasma exchange (TPE) has been reported as effective against CADM-RPIP refractory to conventional immunosuppressive therapy. However, the detailed mechanisms by which TPE improves disease activity of CADM-RPIP remain unclear. AIM: To elucidate the clinical and demographic characteristics of patients with anti-MDA5 Ab-positive CADM-RPIP treated with TPE and to analyze changes in laboratory findings before, during, and after TPE. MATERIALS & METHODS: Patients hospitalized for CADM-RPIP and treated with TPE in 2017 and 2018 were analyzed retrospectively. RESULTS: Three patients were successfully treated with TPE, with good tolerance. Anti-MDA5 Ab titers decreased significantly over the course of TPE. CONCLUSION: We emphasize that TPE could represent an effective treatment option for CADM-RPIP refractory to traditional therapy. Removal of anti-MDA5 Ab and other pathogenic factors may facilitate favorable outcomes.

PEPITEM/Cadherin 15 Axis Inhibits T Lymphocyte Infiltration and Glomerulonephritis in a Mouse Model of Systemic Lupus Erythematosus.

Control of lymphocyte infiltration in kidney is a potential therapeutic strategy for lupus nephritis, considering that control of lymphocyte migration by sphingosine 1 phosphate has been implicated in inflammation-related pathology. The peptide inhibitor of the transendothelial migration (PEPITEM)/cadherin (CDH) 15 axis was recently reported to promote sphingosine 1 phosphate secretion. In this study, we investigated whether CDH15 is expressed in the kidney of MRL/lpr mice and whether lymphocyte infiltration is suppressed by exogenously administered PEPITEM. Mice (18 wk old) were randomized into 4-wk treatment groups that received PEPITEM or PBS encapsulated in dipalmitoylphosphatidylcholine liposomes. Enlargement of the kidney, spleen, and axillary lymph nodes was suppressed by PEPITEM treatment, which also blocked infiltration of double-negative T lymphocytes into the kidney and glomerular IgG/C3 deposition, reduced proteinuria, and increased podocyte density. Immunohistochemical analysis revealed that the PEPITEM receptor CDH15 was expressed on vascular endothelial cells of glomeruli and kidney arterioles, skin, and peritoneum in lupus mice at 22 wk of age but not in 4-wk-old mice. These results suggest that PEPITEM inhibits lymphocyte migration and infiltration into the kidney, thereby preserving the kidney structure and reducing proteinuria. Thus, PEPITEM administration may be considered as a potential therapeutic tool for systemic lupus erythematosus.

Proteogenomic analysis of granulocyte macrophage colony- stimulating factor autoantibodies in the blood of a patient with autoimmune pulmonary alveolar proteinosis.

Recently, attempts to reveal the structures of autoantibodies comprehensively using improved proteogenomics technology, have become popular. This technology identifies peptides in highly purified antibodies by using an Orbitrap device to compare spectra from liquid chromatography-tandem mass spectrometry against a cDNA database obtained through next-generation sequencing. In this study, we first analyzed granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies in a patient with autoimmune pulmonary alveolar proteinosis, using the trapped ion mobility spectrometry coupled with quadrupole time-of-flight (TIMS-TOF) instrument. The TIMS-TOF instrument identified peptides that partially matched sequences in up to 156 out of 162 cDNA clones. Complementarity-determining region 3 (CDR3) was fully and partially detected in nine and 132 clones, respectively. Moreover, we confirmed one unique framework region 4 (FR4) and at least three unique across CDR3 to FR4 peptides via de novo peptide sequencing. This new technology may thus permit the comprehensive identification of autoantibody structure.

Prognostic factors in nursing and healthcare-associated pneumonia.

BACKGROUND AND OBJECTIVE: Nursing and healthcare-associated pneumonia (NHCAP) is a category of healthcare-associated pneumonia modified for the healthcare system in Japan. To date, only a few studies have examined the prognostic factors of NHCAP in a prospective cohort. This study aimed to investigate the prognostic factors related to 30-day mortality in patients with NHCAP by analyzing prospective data. METHODS: We analyzed patients hospitalized for NHCAP who were enrolled between October 2010 and February 2017. Age, sex, comorbidities, vital signs and laboratory findings were used as prognostic variables. The primary outcome was 30-day mortality. RESULTS: Of 817 NHCAP patients identified, the mean age was 78.0 ± 11.1 years, 580 (71.0%) were men and 30-day mortality was 13.1% (107/817). On multivariate analysis, male sex (odds ratio [OR]: 2.07, 95% confidence interval [CI]: 1.18-3.63), malignancy (OR: 2.35, 95%CI: 1.38-4.01), performance status (PS) (OR: 1.55, 95%CI: 1.23-1.96), body temperature (OR: 0.77, 95%CI: 0.61-0.97), heart rate (OR: 1.02, 95%CI: 1.01-1.03), respiratory rate (OR: 1.04, 95%CI: 1.01-1.08), serum albumin (Alb) (OR: 0.45, 95%CI: 0.30-0.66) and blood urea nitrogen (BUN) (OR: 1.02, 95%CI: 1.01-1.03) were significantly related to 30-day mortality. On the other hand, the risk factors for involvement by drug-resistant pathogens predicted a better prognosis (OR: 0.39, 95%CI: 0.19-0.82). CONCLUSIONS: Male sex, malignancy, poor PS, hypothermia, tachycardia, tachypnea, low serum Alb and high BUN are worse prognostic factors. Thus, the risk of drug-resistant pathogens is not necessarily related to poor prognosis.

Distinct methylation levels of mature microRNAs in gastrointestinal cancers.

The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.

Serial Manifestation of Acute Kidney Injury and Nephrotic Syndrome in a Patient with TAFRO Syndrome.

A 76-year-old woman suddenly developed anasarca and a fever, and an examination revealed thrombocytopenia, reticulin fibrosis, and acute kidney injury, yielding the diagnosis of thrombocytopenia, anasarca, fever, reticulin fibrosis, organomegaly (TAFRO) syndrome. Renal replacement therapy and steroid treatment were soon started. Her proteinuria was minor at first; however, once the kidney function improved, nephrotic syndrome occurred. A kidney biopsy showed membranoproliferative glomerulonephritis-like glomerulopathy with massive macrophage infiltration. Although kidney dysfunction is often observed in TAFRO syndrome patients, its detailed mechanism is unclear. This case suggests that TAFRO syndrome involves both acute kidney injury with minor proteinuria and nephrotic syndrome, and these disorders can develop serially in the same patient.

Rapid clinical improvement of amyloid A amyloidosis following treatment with tocilizumab despite persisting amyloid deposition: a case report.

BACKGROUND: Amyloid A amyloidosis is one of the most common forms of amyloidosis. It is secondary to rheumatoid arthritis, which is difficult to manage and has a poor prognosis. We present a patient with rheumatoid arthritis and amyloid A amyloidosis who was treated with tocilizumab, a humanized monoclonal antibody against interleukin 6 receptor, resulting in improvement in both proteinuria and gastrointestinal symptoms; however, amyloid deposition remained. CASE PRESENTATION: A 67-year-old woman who had previously been treated for rheumatoid arthritis presented with abdominal pain and diarrhea. Right renal cell carcinoma was found, and amyloid A amyloidosis was diagnosed concomitantly based on colon biopsy. The renal cell carcinoma was resected, and the non-cancerous part of the renal tissue also showed amyloid A deposition. Following surgery, protein levels in the urine increased to the nephrotic range, and administration of tocilizumab was initiated, which resulted in resolution of the proteinuria. The patient's gastrointestinal symptoms were also alleviated. However, repeat colon biopsy showed amyloid deposition. CONCLUSIONS: This case of amyloid A amyloidosis suggests that amyloid deposition indicates only structural change of the affected tissue, and that it is not amyloid deposition per se that causes the clinical symptoms of amyloidosis.

Low concentrations of recombinant granulocyte macrophage-colony stimulating factor derived from Chinese hamster ovary cells augments long-term bioactivity with delayed clearance in vitro.

To date, the biological activity of granulocyte macrophage-colony stimulating factor (GM-CSF) has been investigated by using mostly Escherichia coli- or yeast cell-derived recombinant human GM-CSF (erhGM-CSF and yrhGM-CSF, respectively). However, Chinese hamster ovary cell-derived recombinant human GM-CSF (crhGM-CSF), as well as natural human GM-CSF, is a distinct molecule that includes modifications by complicated oligosaccharide moieties. In the present study, we reevaluated the bioactivity of crhGM-CSF by comparing it with those of erhGM-CSF and yrhGM-CSF. The effect of short-term stimulation (0.5h) on the activation of neutrophils/monocytes or peripheral blood mononuclear cells (PBMCs) by crhGM-CSF was lower than those with erhGM-CSF or yrhGM-CSF at low concentrations (under 60pM). Intermediate-term stimulation (24h) among the different rhGM-CSFs with respect to its effect on the activation of TF-1 cells, a GM-CSF-dependent cell line, or PBMCs was not significantly different. In contrast, the proliferation/survival of TF-1 cells or PBMCs after long-term stimulation (72-168h) was higher at low concentrations of crhGM-CSF (15-30pM) than that of cells treated with other GM-CSFs. The proportion of apoptotic TF-1 cells after incubation with crhGM-CSF for 72h was lower than that of cells incubated with other rhGM-CSFs. These effects were attenuated by desialylation of crhGM-CSF. Clearance of crhGM-CSF but not desialylated-crhGM-CSF by both TF-1 cells and PBMCs was delayed compared with that of erhGM-CSF or yrhGM-CSF. These results suggest that sialylation of oligosaccharide moieties delayed the clearance of GM-CSF, thus eliciting increased long-term bioactivity in vitro.

Periodontal and serum protein profiles in patients with rheumatoid arthritis treated with tumor necrosis factor inhibitor adalimumab.

BACKGROUND: Tumor necrosis factor (TNF)-α inhibitor has been shown to affect the periodontal condition of patients with rheumatoid arthritis (RA). The aim of the present study is to assess the effect of a fully humanized anti-TNF-α monoclonal antibody, adalimumab (ADA), on the periodontal condition of patients with RA and to compare serum protein profiles before and after ADA therapy. METHODS: The study participants consisted of 20 patients with RA treated with ADA. Clinical periodontal and rheumatologic parameters and serum cytokine levels were evaluated at baseline and 3 months later. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant difference in abundance before and after ADA therapy were found and identified using mass spectrometry and protein databases. RESULTS: The patients showed a significant decrease in gingival index (P = 0.002), bleeding on probing (P = 0.003), probing depth (P = 0.002), disease activity score including 28 joints using C-reactive protein (P <0.001), and serum levels of TNF-α (P <0.001) and interleukin-6 (P <0.001) after ADA medication, although plaque levels were comparable. Among a total of 495 protein spots obtained, nine spots were significantly decreased in abundance at reassessment, corresponding to complement factor H, phospholipase D, serum amyloid A, complement component 4, and α-1-acid glycoprotein (P <0.01). CONCLUSION: These results suggest a beneficial effect of ADA therapy on the periodontal condition of patients with RA, which might be related to differences in serum protein profiles before and after ADA therapy.

Comparative analysis of serum proteins in relation to rheumatoid arthritis and chronic periodontitis.

BACKGROUND: Rheumatoid arthritis (RA) and chronic periodontitis (CP) are chronic inflammatory conditions and share many pathologic features. The common molecular pathogenesis of the two inflammatory diseases is unclear. The aim of the present study is to evaluate serum protein profiles specific for patients with RA and CP by a comprehensive proteomic analysis. METHODS: The study participants were: 10 patients with RA, 10 patients with CP, 10 patients with RA and CP, and 10 healthy controls. All groups were balanced for age, sex, and smoking status. Serum protein spot volume was examined with two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins with significant differences in abundance among the four groups were determined with computer image analysis and identified with mass spectrometry and protein databases. RESULTS: A total of 1,694 protein spots were obtained in sera of the four groups. Seven spots were significantly different in abundance among the four groups. Of these, three spots (complement component 3, complement factor H, and ceruloplasmin) were significantly different in the RA+CP group compared with the other three groups (P <0.05). The similar profiles of complement component 3, complement factor H, and ceruloplasmin were observed by enzyme-linked immunosorbent assay. CONCLUSION: These results suggest that patients with RA and CP may exhibit three serum proteins with different abundance compared with healthy controls and patients with RA only or CP only.

Proteome analysis of proliferative response of bystander cells adjacent to cells exposed to ionizing radiation.

Recently (Cytometry 2003, 56A, 71-80), we reported that direct cell-to-cell contact is required for stimulating proliferation of bystander rat liver cells (WB-F344) cocultured with irradiated cells, and neither functional gap junction intercellular communication nor long-range extracellular factors appear to be involved in this proliferative bystander response (PBR). The molecular basis for this response is unknown. Confluent monolayers of WB-F344 cells were exposed to 5-Gray (Gy) of gamma-rays. Irradiated cells were mixed with unirradiated cells and co-cultured for 24 h. Cells were harvested and protein expression was examined using 2-DE. Protein expression was also determined in cultures of unirradiated and 5-Gy irradiated cells. Proteins were identified by MS. Nucleophosmin (NPM)-1, a multifunctional nucleolar protein, was more highly expressed in bystander cells than in either unirradiated or 5-Gy irradiated cells. Enolase-alpha, a glycolytic enzyme, was present in acidic and basic variants in unirradiated cells. In bystander and 5-Gy irradiated cells, the basic variant was weakly expressed, whereas the acidic variant was overwhelmingly present. These data indicate that the presence of irradiated cells can affect NPM-1 and enolase-alpha in adjacent bystander cells. These proteins appear to participate in molecular events related to the PBR and suggest that this response may involve cellular defense, proliferation, and metabolism.

Mapping of phosphorylated proteins on two-dimensional polyacrylamide gels using protein phosphatase.

This study developed an enzymatic method for high-throughput mapping of phosphoproteins on two-dimensional (2-D) polyacrylamide gels. Proteins of cultured rat skin fibroblasts were divided into two aliquots, one of which was dephosphorylated using recombinant lambda protein phosphatase and the other was not treated with the enzyme. The two aliquots were then subjected to 2-D electrophoresis. Phosphoproteins could be mapped on the 2-D gel of the nontreated aliquot by comparing the gels of the two aliquots, because the phosphoproteins in the treated aliquot shifted to more basic positions on the gel. This technique revealed that approximately 5% of the detectable proteins were phosphorylated. Fourteen phosphoproteins were identified by mass spectrometry, including proteasome component C8 and small glutamine-rich tetratricopeptide repeat-containing protein. Furthermore, the extent of phosphorylation of two actin modulating proteins, destrin and cofilin, was found to be significantly reduced when the cells were chemically or enzymatically detached from the culture dishes. The method developed by this study can generally be applied to all biological materials and is useful for high-throughput mapping of phosphoproteins in proteome research.