Human Hepatic Transporter Signature Peptides for Quantitative Targeted Absolute Proteomics: Selection, Digestion Efficiency, and Peptide Stability.

PURPOSE: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. METHODS: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. RESULTS: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. CONCLUSIONS: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.

Antiplatelet Effect of Mirtazapine via Co-blocking of the 5-HT2A and α2-Adrenergic Receptors on Platelets.

Mirtazapine (MTZ) is a noradrenergic and specific serotonergic antidepressant. MTZ is reportedly associated with an increased risk of bleeding. However, the underlying mechanism remains unclear. In this study, we investigated the antiplatelet effect of MTZ in mice via light transmission aggregometry to elucidate the mechanism of MTZ-induced bleeding. The results of the ex vivo study showed that the oral administration of MTZ (20 or 100 mg/kg) significantly suppressed platelet aggregation mediated by the synergic interaction of 5-hydroxytryptamine (5-HT) and adrenaline. Additionally, MTZ significantly suppressed platelet aggregation, mediated by the synergic interaction of ADP and 5-HT or adrenaline. Similar results were obtained in vitro, under the condition of 5-HT- and adrenaline-induced platelet aggregation. Overall, the results suggest that MTZ exerts antiplatelet effect by co-blocking 5-HT2A and α2-adrenergic receptors on platelets and suppresses platelet aggregation mediated by ADP, increased by either 5-HT or adrenaline. Thus, a detailed monitoring of bleeding is recommended for patients taking MTZ.

Proteomic Evaluation of Plasma Membrane Fraction Prepared from a Mouse Liver and Kidney Using a Bead Homogenizer: Enrichment of Drug-Related Transporter Proteins.

Quantifying the protein levels of drug transporters in plasma membrane fraction helps elucidate the function of these transporters. In this study, we conducted a proteomic evaluation of enriched drug-related transporter proteins in plasma membrane fraction prepared from mouse liver and kidney tissues using the membrane protein extraction kit and a bead homogenizer. Crude and plasma membrane fractions were prepared using either the Dounce or bead homogenizer, and protein levels were determined using quantitative proteomics. In liver tissues, the plasma membrane fractions were more enriched in transporter proteins than the crude membrane fractions; the average enrichment ratios of plasma-to-crude membrane fractions were 3.31 and 6.93 using the Dounce and bead homogenizers, respectively. The concentrations of transporter proteins in plasma membrane fractions determined using the bead homogenizer were higher than those determined using the Dounce homogenizer. Meanwhile, in kidney tissues, the plasma membrane fractions were enriched in transporters localized in the brush-border membrane to the same degree for both the homogenizers; however, the membrane fractions obtained using either homogenizer were not enriched in Na+/K+-ATPase and transporters localized in the basolateral membrane. These results indicate that fractionation, using the bead homogenizer, yielded transporter-enriched plasma membrane fractions from mouse liver and kidney tissues; however, no enrichment of basolateral transporters was observed in plasma membrane fractions prepared from kidney tissues.

Pericardial Effusion in Association With Periodontitis: Case Report and Review of 8 Patients in Literature.

Periodontal diseases are well-known background for infective endocarditis. Here, we show that pericardial effusion or pericarditis might have origin also in periodontal diseases. An 86-year-old man with well-controlled hypertension and diabetes mellitus developed asymptomatic increase in pericardial effusion. Two weeks previously, he took oral new quinolone antibiotics for a week because he had painful periodontitis along a dental bridge in the mandibular teeth on the right side and presented cheek swelling. The sputum was positive for Streptococcus species. He was healthy and had a small volume of pericardial effusion for the previous 5 years after drug-eluting coronary stents were inserted at the left anterior descending branch 10 years previously. The differential diagnoses listed for pericardial effusion were infection including tuberculosis, autoimmune diseases, and metastatic malignancy. Thoracic to pelvic computed tomographic scan demonstrated no mass lesions, except for pericardial effusion and a small volume of pleural effusion on the left side. Fluorodeoxyglucose positron emission tomography disclosed many spotty uptakes in the pericardial effusion. The patient denied pericardiocentesis, based on his evaluation of the risk of the procedure. He was thus discharged in several days and followed at outpatient clinic. He underwent dental treatment and pericardial effusion resolved completely in a month. He was healthy in 6 years until the last follow-up at the age of 92 years. We also reviewed 8 patients with pericarditis in association with periodontal diseases in the literature to reveal that periodontal diseases would be the background for developing infective pericarditis and also mediastinitis on some occasions.

[A case of intragastric wall abscess formation during bevacizumab combined chemotherapy].

A 38-year-old man was given a diagnosis of as sigmoid colon cancer and underwent sigmoid colectomy. Post-operative pathological staging was stage IIIb. He then underwent adjuvant chemotherapy. One year and 4 months after the surgery, CT scans revealed multiple liver and lung metastases. He was given mFOLFOX6+bevacizumab, which was changed later to FOLFIRI+bevacizumab. After these chemotherapies, he was admitted to the hospital due to sudden abdominal pain and high grade fever. Obstructive jaundice was initially diagnosed, but detailed study of initial CT revealed intragastric wall abscess. After the drainage of the abscess, his conditions improved. We speculated that the abscess formation was caused by mucosal damage due to bevacizumab.

Quantitative and targeted proteomics-based identification and validation of drug efficacy biomarkers.

Proteomics refers to the large-scale study of proteins, providing comprehensive and quantitative information on proteins in tissue, blood, and cell samples. In many studies, proteomics utilizes liquid chromatography-mass spectrometry. Proteomics has developed from a qualitative methodology of protein identification to a quantitative methodology for comparing protein expression, and it is currently classified into two distinct methodologies: quantitative and targeted proteomics. Quantitative proteomics comprehensively identifies proteins in samples, providing quantitative information on large-scale comparative profiles of protein expression. Targeted proteomics simultaneously quantifies only target proteins with high sensitivity and specificity. Therefore, in biomarker research, quantitative proteomics is used for the identification of biomarker candidates, and targeted proteomics is used for the validation of biomarkers. Understanding the specific characteristics of each method is important for conducting appropriate proteomics studies. In this review, we introduced the different characteristics and applications of quantitative and targeted proteomics, and then discussed the results of our recent proteomics studies that focused on the identification and validation of biomarkers of drug efficacy. These findings may enable us to predict the outcomes of cancer therapy and drug-drug interactions with antibiotics through changes in the intestinal microbiome.

Transient, Tunable Expression of NTCP and BSEP in MDCKII Cells for Kinetic Delineation of the Rate-Determining Process and Inhibitory Effects of Rifampicin in Hepatobiliary Transport of Taurocholate.

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.

Presence of anti-nuclear antibodies is a risk factor for the appearance of anti-drug antibodies during infliximab or adalimumab therapy in patients with rheumatoid arthritis.

This study aimed to directly analyze the potential relationship of anti-nuclear antibodies (ANA) before and after the administration of TNF-α inhibitors (TNFi) with the appearance of anti-drug antibodies (ADrA) in patients with rheumatoid arthritis (RA). A total of 121 cases, viz., 38, 53, and 30 cases treated with infliximab (IFX), adalimumab (ADA), and etanercept (ETN), respectively, were enrolled. The ANA titers were measured using indirect immunefluorescence assay (IF-ANA) and multiplex flow immunoassay (ANA Screen) before and serially during the therapy. The anti-IFX antibodies (HACA) and anti-ADA antibodies (AAA) were measured with a radioimmunoassay. ADrA turned positive in 14 (36.8%) among 38 patients treated with IFX, and 16 (30.2%) among 53 treated with ADA. All of them were positive for IF-ANA before TNFi administration, while ADrA never appeared in any of the 15 patients negative for IF-ANA (< 40). IF-ANA of high titers (≥ 320 and ≥ 640) before IFX treatment showed a significant association with the appearance of HACA 52 weeks after IFX (P = 0.040 and 0.017, respectively), whereas AAA appearance was not related to IF-ANA titers before treatment. Moreover, IF-ANA of high titers before IFX treatment was significantly associated with inefficacy and discontinuation of the treatment. The positivity of anti-SS-A antibodies before therapy might be a risk factor for ADrA appearance in patients treated with IFX or ADA. The percentage of patients whose IF-ANA titers increased was significantly higher with IFX than with ADA or ETN treatments (P = 0.026 and 0.022, respectively). High ANA titers and positive ANA Screen after IFX therapy showed a significant association with HACA appearance and possibly led to treatment failure. Among the three TNFi, only IFX showed a close relationship with IF-ANA and ADrA appearance, suggesting the interaction of immunogenicity with autoimmunity as well as the advantage of ANA measurement before TNFi therapy.

IgA-producing lymphoplasmacytic lymphoma carrying the chromosomal abnormality t(8;14).

IgA-producing lymphoplasmacytic lymphoma (LPL) is rare and IgH/c-myc translocation is rare in LPL. This is the first report of a case of IgA-producing LPL carrying t(8;14). An 86-year-old woman presented inguinal and intra-abdominal lymph node swelling, and lytic bone lesions in the lumbar vertebrae. A diagnosis of IgA-producing LPL was immunohistochemically made by inguinal lymph node biopsy. The serum IgA level was 1,180 mg/dL, which was revealed to be composed of IgA-λ monoclonal protein. Bone marrow chromosomal analysis demonstrated a complex abnormal karyotype, including t(8;14)(q24;q32), which was confirmed by FISH analysis. Abnormal lymphocytes positive for CD19, CD20, cyIgA, and cyλ were detected on flow cytometry analysis of marrow cells. Best supportive care was selected because of dementia and refractory urinary tract infection. Circulating lymphoplasmacytic cells with the same phenotype and karyotype were observed, and increased in number. The aggressive clinical course, including lytic bone lesions, may have been due to IgH/c-myc translocation or the nature of IgA-producing LPL.

Acute monocytic leukemia diagnosed by flow cytometry includes acute myeloid leukemias with weakly or faintly positive non-specific esterase staining.

A diagnosis of acute monocytic leukemia (AML-M5) based on α-naphthyl butyrate esterase (α-NB) staining has some problems, because AML-M5 leukemic cells often show weak or faint positivity on α-NB staining. In these situations, some cases of AML-M5 tend to be misdiagnosed as AML-M0. Therefore, we evaluated the significance of weak or faint α-NB staining in AML-M5 diagnosed by flow cytometry (FCM). Nineteen AML cases in which leukemic cells were negative for naphthol AS-D chloroacetate esterase staining were studied. For FCM, we defined leukemic cells as having a monocytic nature when more than 10% of the leukemic cells were positive for at least one of the following antigens: CD4, CD11c, CD14, and CD64. The monocytic nature determined by FCM was consistent with positive or weak positivity on α-NB staining. Five of 6 cases in which leukemic cells exhibited faint positivity for α-NB staining could be diagnosed as AML-M5 by FCM, while negative α-NB staining was consistent with a diagnosis of AML-M0. These results suggest that AML-M5 should be taken into consideration even when leukemic cells are faintly positive for α-NB staining.

Liquid imidazole-borane complex.

Physical properties of liquid imidazole-borane complex were investigated to demonstrate their utility as aprotic polar solvents or liquid electrolytes appropriate for selective ion transport.

A case of uveal, palpebral, and orbital invasions in adult T-Cell leukemia.

BACKGROUND: Patients with adult T-cell leukemia (ATL) may have eyelid lymphoma, uveitis, or cytomegalovirus retinitis due to being immunocompromised. However, there have been few reports on the invasion of multiple ocular lesions. We treated 1 unusual ATL patient with uveitis in whom multiple ocular invasions were suspected. CASE: A woman in whom ATL was diagnosed 10 years previously complained of blurred vision and decreased visual acuity in the right eye. Anterior uveitis of the right eye was suspected. One week later the cells increased in the anterior chamber, and fibrin exudates and hyphema appeared. She was admitted to our hospital. OBSERVATIONS: The visual acuity was 0.04 in the right eye and finger-counting from 30 cm in the left. She was treated with systemic steroid therapy. Inflammation disappeared, but both eyelids became swollen and multiple ocular lesions appeared. She was given carcinostatic therapy once more and the mass lesions decreased. Mass lesions appeared in the iris and in the bulbar conjunctiva. Computed tomography and magnetic resonance imagining (MRI) showed that the mass lesions extended to the right orbit and both nasal cavities. MRI also demonstrated choroidal thickening in the left eye. CONCLUSION: This case documents that ATL cells may cause severe uveitis and invade multiple ocular tissues such as the iris, eyelid, choroids, and orbit.