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ABSTRACT: Three-dimensional gel electrophoresis (3-DE), which combines agarose gel electrophoresis and IEF/SDS-PAGE, was developed to characterize monoclonal proteins (M-proteins). However, original 3-DE method has not been optimized and its specificity has not been demonstrated. The main goal of this study was to optimize the procedure of 3-DE and then compare it with 2-DE. We developed a high-sensitive 3DE method in which M-proteins extracted from first dimension, agarose gel, by diffusing into 150mM NaCl and the recovery of M-proteins was 90.6%. To validate the utility of the high-sensitive 3DE, we compared it with the original 3-DE method. We found that high-sensitive 3DE provided for greater M-proteins recovery and was more effective in terms of detecting spots on SDS-PAGE gels than the original 3-DE. Moreover, high-sensitive 3-DE separates residual normal IgG from M-proteins which could not be done by 2-DE. Applying the high-sensitive 3-DE to clinical samples, we found that the characteristics of M-proteins vary tremendously between individuals. We believe that our high-sensitive 3-DE method described here will prove useful in further studies of the heterogeneity of M-proteins.
Analytical Biochemistry 03/2013; · 3.00 Impact Factor
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ABSTRACT: To determine whether cystatin C accurately reflects renal function in asthma, we investigated serum cystatin C concentrations in a large number of asthmatic patients by adjusting for several confounding factors that might affect serum cystatin C concentrations.
A total of 126 asthmatic patients and 126 healthy volunteers, matched for age and gender, were studied.
Serum cystatin C concentrations in symptomatic subjects with asthma were significantly higher than in healthy controls (p < 0.001) and asymptomatic subjects with asthma (p = 0.007), whereas no significant difference was observed between healthy controls and asymptomatic subjects. In asthmatic subjects, serum cystatin C concentrations were not influenced by inhaled corticosteroid (ICS). However, serum cystatin C concentrations were significantly higher in subjects who were regularly treated by oral corticosteroid (OCS) (p = 0.001).
Serum cystatin C concentrations are elevated in asthmatic patients; particularly while symptomatic and/or taking OCS but not ICS. Serum cystatin C concentrations may not accurately reflect renal function in those patients.
Clinical Chemistry and Laboratory Medicine 08/2012; 50(8):1367-71. · 2.15 Impact Factor
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ABSTRACT: Serum Krebs von den Lungen-6 (KL-6), which is classified as human mucin-1 (MUC1), is used as a marker of sarcoidosis and other interstitial lung diseases. However, there remain some limitations due to a lack of information on the factors contributing to increased levels of serum KL-6. This study was designed to investigate the factors contributing to increased levels of serum KL-6 by molecular analysis.
Western blot analysis using anti-KL-6 antibody was performed simultaneously on the bronchoalveolar lavage fluid (BALF) and serum obtained from 128 subjects with sarcoidosis.
KL-6/MUC1 in BALF showed three bands and five band patterns. These band patterns were associated with the MUC1 genotype and the KL-6 levels. KL-6/MUC1 band patterns in serum were dependent on molecular size class in BALF. Significantly increased levels of serum KL-6, serum/BALF KL-6 ratio and serum soluble interleukin 2 receptor were observed in the subjects with influx of high molecular size KL-6/MUC1 from the alveoli to blood circulation. The multivariate linear regression analysis involving potentially relevant variables such as age, gender, smoking status, lung parenchymal involvement based on radiographical stage and molecular size of KL-6/MUC1 in serum showed that the molecular size of KL-6/MUC1 in serum was significant independent determinant of serum KL-6 levels.
The molecular structural variants of KL-6/MUC1 and its leakage behavior affect serum levels of KL-6 in sarcoidosis. This information may assist in the interpretation of serum KL-6 levels in sarcoidosis.
Journal of Translational Medicine 05/2012; 10:111. · 3.41 Impact Factor
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ABSTRACT: To date, two studies have reported lower total serum immunoglobulin E (IgE) levels and lower prevalence of atopy in patients with sarcoidosis compared with healthy subjects. However, those reports did not consider age or gender differences between cases and controls. In addition, the association between total serum IgE levels and clinical manifestations of sarcoidosis has not been clarified. This study assessed total serum IgE levels and prevalence of atopy in patients with sarcoidosis after taking age and sex differences into account and evaluated associations between total serum IgE levels and clinical manifestations of sarcoidosis. Total serum IgE levels and prevalence of atopy on initial visits were compared between 189 patients with sarcoidosis and 378 age- and sex-matched controls. Associations between total serum IgE levels and involvement of each affected organ were evaluated. Changes in total serum IgE levels during the clinical course of sarcoidosis were also evaluated. Total serum IgE levels were significantly lower in patients with sarcoidosis than in controls, independent of atopic status (atopic subjects, p = 0.025; nonatopic subjects, p < 0.001). Total serum IgE levels did not differ according to the involvement of different organs. Total serum IgE levels decreased further, albeit only slightly, after disease remission (p < 0.001). Increased susceptibility to sarcoidosis may be attributable to several underlying genetic or environmental factors that result in lower total serum IgE levels.
Allergy and Asthma Proceedings 01/2012; 33(1):90-4. · 2.17 Impact Factor
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ABSTRACT: There has been only one report showing high levels of transferrin (Tf) in bronchoalveolar lavage fluid (BALF) in patients with sarcoidosis. This study was designed to assess the levels of Tf in both BALF and serum and to examine the relationship between the levels of Tf and other disease markers in sarcoidosis. Subjects were 64 sarcoidosis and 10 healthy controls. Tf in BALF and serum was measured by nephelometric assay. Median Tf levels in BALF from sarcoidosis was 0.70 (range, 0.00-3.97) mg/dl, which was significantly higher compared with controls (0.36 (range, 0.00-1.02) mg/dl; p = 0.005). In contrast, median Tf levels in serum from sarcoidosis was 258 (range, 171- 383) mg/dl, which was significantly lower compared with controls (322 (range, 234-356) mg/dl; p = 0.003). Tf levels in BALF were significantly correlated with both the percentage of lymphocytes (r = 0.617, p = 0.001) and serum angiotensin-converting enzyme activity (r = 0.363, p = 0.003) and serum soluble interleukin-2 receptor (r = 0.450, p = 0.001) in sarcoidosis. Levels of Tf in BALF from patients with sarcoidosis were not influenced by smoking status. The levels of Tf in sarcoidosis are high in BALF, but low in serum. Increased levels of Tf in BALF may reflect the disease activity.
Beiträge zur Klinik der Tuberkulose 04/2010; 188(2):151-7. · 1.90 Impact Factor
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ABSTRACT: The androgen receptor (AR) is a transcription factor belonging to the family of nuclear receptors that mediate the action of androgen. AR plays an important role in normal development of the prostate, as well as in the progression of prostate cancer. AR is regulated by several posttranslational modifications, including phosphorylation, acetylation, and ubiquitination. In this study, we found that the putative E3 ubiquitin ligase TRIM68, which is preferentially expressed in prostate cancer cells, interacts with AR and enhances transcriptional activity of the AR in the presence of dihydrotestosterone. We also found that TRIM68 functionally interacts with TIP60 and p300, which act as coactivators of AR, and synergizes in the transactivation of AR. Overexpression of TRIM68 in prostate cancer cells caused an increase in secretion of prostate-specific antigen (PSA), one of the most reliable diagnostic markers for prostate cancer, whereas knockdown of TRIM68 attenuated the secretion of PSA and inhibited cell growth and colony-forming ability. Moreover, we showed that TRIM68 expression is significantly up-regulated in human prostate cancers compared with the expression in adjacent normal tissues. These results indicate that TRIM68 functions as a cofactor for AR-mediated transcription and is likely to be a novel diagnostic tool and a potentially therapeutic target for prostate cancer.
Cancer Research 06/2008; 68(9):3486-94. · 7.86 Impact Factor
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ABSTRACT: Ubiquitylation appears to be involved in the membrane trafficking system including endocytosis, exocytosis, and ER-to-Golgi transport. We found that PIRH2, which was identified as an interacting protein for androgen receptor or p53, interacts with and ubiquitylates the epsilon-subunit of coatmer complex, epsilon-COP. PIRH2 promotes the ubiquitylation of epsilon-COP in vitro and in vivo and consequently promotes the degradation of epsilon-COP. The interaction between PIRH2 and epsilon-COP is affected by the presence of androgen, and PIRH2 in the presence of androgen promotes ubiquitylation of epsilon-COP in vivo. Furthermore, overexpression of the wild type of PIRH2 in prostate cancer cells causes downregulation of the secretion of prostate-specific antigen (PSA), a secretory protein in prostate epithelial cells and one of diagnostic markers for prostate cancer. Our results indicate that PIRH2 functions as a regulator for COP I complex.
Molecular and Cellular Biochemistry 02/2008; 307(1-2):73-82. · 2.06 Impact Factor
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Masahiko Shigemura,
Takanori Moriyama,
Hitoshi Shibuya,
Masato Obara,
Tomoyuki Endo,
Satoshi Hashino,
Hiroshi Yokouchi,
Masahiro Asaka,
Chikara Shimizu,
Hitoshi Chiba,
Masaharu Nishimura
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ABSTRACT: There have been several reports describing a notable hyperamylasaemia in patients with multiple myeloma. Such amylase-producing myelomas have been mainly described in the context of concomitant salivary-type hyperamylasaemia, with sialyl salivary-type amylase identified in a portion of those cases. We investigated the incidence of the production of sialyl salivary-type amylase in serum of multiple myeloma patients.
Eleven patients (6 male and 5 female) who had been diagnosed as having multiple myeloma were enrolled in this study. Sialyl salivary-type amylase was detected by isoamylase electrophoresis and HPLC analysis, and identified by detecting either abnormal neuraminidase-sensitive band through isoamylase electrophoresis or abnormal extra-elution peak of amylase by means of HPLC analysis.
Sialyl salivary-type amylase was detected in 7 out of 11 (63.6%) patients. Median total amylase activity was 154 U/l (range 109-43020). Isoamylase electrophoretic patterns of patients' serum were normal in 5 patients (71.4%) out of 7 patients and salivary-dominant in 2 (50.0%) out of 4 patients.
We consider that there is no significant relationship between total serum amylase level and amylase isoenzyme pattern in the incidence of production of sialyl salivary-type amylase with multiple myeloma.
Clinica Chimica Acta 03/2007; 376(1-2):121-5. · 2.54 Impact Factor
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Clinical Chemistry and Laboratory Medicine 02/2004; 42(6):677-80. · 2.15 Impact Factor
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Hiroshi Yokouchi,
Koichi Yamazaki,
Hajime Asahina, Masahiko Shigemura,
Takanori Moriyama,
Kazuo Takaoka,
Jun Moriya,
Tomoo Itoh,
Ichiro Kinoshita,
Hirotoshi Dosaka-Akita,
Yutaka Tsutsumi,
Masaharu Nishimura
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ABSTRACT: Few studies have successfully established an amylase-producing lung cancer cell line or have examined its cytological, biochemical and biological features.
Cancer cells, isolated from pleural effusion using a gradient method, were cultivated.
Amylase production from the newly established cell line was confirmed by positive staining for alpha-amylase and increased amylase levels in the culture supernatant. Electron microscopy revealed zymogen granule-like structures. Sialylation of salivary-type amylase was confirmed directly from the cell line by examining the neuraminidase sensitivity and amylase elution profile under high-performance liquid chromatography. Neither EGFR or KRAS mutation were found.
This cell line offers a useful tool for analyzing the pathogenesis and pathophysiology of amylase-producing lung cancers. Moreover, it might be useful for probing the metastasis and invasiveness of lung cancer cells and for developing an early diagnostic method based on sialylated salivary-amylase production.
Anticancer research 26(4B):2821-7. · 1.73 Impact Factor
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ABSTRACT: Sarcoidosis is a multisystem disorder characterized by a T-helper 1 (Th1)-mediated immune response. Conversely, atopy is characterized by the presence of a specific immunoglobulin E (IgE) E response in association with a Th2-type immune response. Several epidemiological studies have shown that atopic status influences disease activity and clinical course for several Th1-mediated diseases. The aim of this study was to evaluate associations between atopic status and clinical findings of sarcoidosis. We further evaluated the impact of atopic status on the clinical course of pulmonary sarcoidosis. We defined atopy as a positive specific IgE response to at least one common inhaled allergen (multiple antigen simultaneous test scores, lumicount of >1.01). Subjects comprised 134 patients given a diagnosis of sarcoidosis between 2000 and 2006, divided into atopic and nonatopic groups. Several clinical findings were compared between the two groups. Furthermore, 100 subjects observed 2 years after diagnosis were divided into resolving and persistent clinical course groups according to chest radiography and associations with atopic status were evaluated. Atopy was more prevalent among men than women (p = 0.009) and subjects with atopy were younger (p = 0.002) and showed less frequent lung parenchymal lesions (stages II and III; p = 0.018) compared with subjects without atopy. The prevalence of atopy was higher in the resolving clinical course group than in the persistent clinical course group (p = 0.002) and this association was independent of sex, age, presence of lung parenchymal lesions, and presence of extrapulmonary lesions (p = 0.037). Classification of sarcoidosis based on atopic status might be useful for predicting the clinical course of pulmonary sarcoidosis.
Allergy and Asthma Proceedings 31(3):238-43. · 2.17 Impact Factor
