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Volume 54 (2010)
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Volume 54, Issue 1
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Full Paper
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Hiroto Maeda, Waka Horie, Toshifumi Watanabe, Kazuko Sakaguchi, Aiko T ...2010 Volume 54 Issue 1 Pages 1-6
Published: 2010
Released on J-STAGE: March 29, 2010
JOURNAL FREE ACCESSIn the present study, we performed micro two-dimensional polyacrylamide gel electrophoresis (M2D-PAGE) analysis of primary cat urine samples (0.25 μL) collected by non-invasive catheterization. Urine albumin levels were measured after identifying albumin spots by Western Blotting and mass spectrometry by using chicken ovalbumin as a protein standard. The levels determined by M2D-PAGE method were confirmed to be higher than those obtained with the turbidimetric immunoassay that has been been used in veterinary clinical tests employing anti-human albumin antibody and human Alb as a standard sample. These findings suggest that this assay is useful for the early diagnosis of chronic renal failure, including cases classified as stage I according to the International Renal Interest Society (IRIS). We also suggest a new cut off value of 20 mg/dL, which is lower than the previously used 30 mg/dL assumed to be the threshold for no admission.
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Technical Note
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Ryo Kubota, Shigeru Kogami, Takehide Matsuda, Kohei Fukushima, Toshiak ...2010 Volume 54 Issue 1 Pages 7-11
Published: 2010
Released on J-STAGE: March 29, 2010
JOURNAL FREE ACCESSOptical densitometer is widely used for electrophoretic pattern analysis on serum proteins, isoenzymes and lipoproteins separated by electrophoresis with cellulose acetate membrane or polyacrylamide gel-support.
In this report, we evaluated an electrophoretic pattern analysis system of “Fingerprinter” (Jokoh Co., Ltd.) based on its image processing technology on the digital image captured by CCD camera (CCD method).
In the first place, to establish the compatibility between the optical density and the pixel values measured by densitometry and CCD camera, respectively, we obtained a conversion formula between those values. Then, the CCD method was compared with the optical densitometry in the measurement of serum protein fractions, of urine samples and alkaline phosphatase isozyme in serum samples.
The CCD method exhibited good correlation (r=>0.95) with the densitometry and similar reproducibility to the results of the densitometry. Furthermore, by overlaying separation patterns on the display of a PC, the CCD method allowed us to check easily the protein fractionation and the ALP isozyme analysis.
These results demonstrate the practical utility of the CCD method using “Fingerprinter” in the data processing of clinical laboratory tests. This CCD method should facilitate the management of patient information including electrophoresis charts.
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Hironobu Naiki2010 Volume 54 Issue 1 Pages 13-17
Published: 2010
Released on J-STAGE: September 29, 2010
JOURNAL FREE ACCESSIt has been proposed that a nucleation-dependent polymerization model may explain the general mechanisms of amyloid fibril formation both in vitro and in vivo, in various types of human as well as murine amyloidosis. This model consists of two phases, i.e., energetically unfavorable nucleation phase and energetically favorable extension phase. β2-Microglobulin (β2-m)-related amyloidosis is a common and serious complication in long-term hemodialysis patients. We previously reported that various types of glycosaminoglycans and proteoglycans, as well as apolipoprotein E, a representative amyloid-associated protein stabilizes β2-m amyloid fibrils and inhibit their depolymerization at neutral pH. Lysophospholipids (LPLs) and non-esterified fatty acids (NEFAs) are candidate biological molecules that induce β2-m amyloid fibril formation at neutral pH. Recently, we found that some LPLs, especially lysophosphatidic acid (LPA), as well as NEFAs induce the extension of β2-m amyloid fibrils at neutral pH, by partially unfolding the compact structure of β2-m to an amyloidogenic conformer, as well as by stabilizing the extended fibrils. We also observed that hemodialysis patients have significantly higher plasma concentrations of LPA than healthy subjects and that patient plasma samples with the highest LPA concentrations stabilize β2-m amyloid fibrils more potently than normal plasma samples. Moreover, in patients receiving hemodialysis regularly, administration of heparin results in an acute increase in plasma NEFA, largely by the activation of lipoprotein lipase. These results suggest possible roles of LPLs and NEFAs in the development of β2-m-related amyloidosis.
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Masahide Yazaki, Ayako Suzuki, Shu-ichi Ikeda, Fuyuki Kametani2010 Volume 54 Issue 1 Pages 19-22
Published: 2010
Released on J-STAGE: September 29, 2010
JOURNAL FREE ACCESSAmyloidosis is characterized by deposition of amyloid fibrils onto various visceral organs and so far, over 20 types of amyloidoses have been identified. The type of amyloidosis is decided from that of amyloid precursor proteins. Because the treatment of amyloidosis is different in each type of amyloidosis, it is quite important to make a definite diagnosis of amyloidosis. Here we report availability of the biochemical analysis of amyloid fibrils extracted from small biopsy samples. Two systemic amyloidosis patients were studied, who could not be diagnosed by immunohistochemical examinations. One patient presented with nephropathy due to renal amyloidosis and another patient suffered from amyloid polyneuropathy. The biochemical studies revealed that amyloid fibrils were derived from immunoglobulin heavy chain in the former (AH amyloidosis) and λ light chain in the latter (AL amyloidosis). Thus, the correct diagnosis was made by our methods. Our result indicates that biochemical analysis of amyloid fibrils extracted from small biopsy samples may be quite useful in making a diagnosis of amyloidosis.
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Konen Obayashi, Yukio Ando2010 Volume 54 Issue 1 Pages 23-25
Published: 2010
Released on J-STAGE: September 29, 2010
JOURNAL FREE ACCESSMutated forms of transthyretin (TTR) are the precursor protein of familial amyloidotic polyneuropathy (FAP). Since plasma TTR is predominantly synthesized by the liver, liver transplantation has been performed as an effective therapy for FAP. However, such surgery has several problems, so we must develop novel essential therapies for FAP. We have tried several other approaches. In mutated TTR, an unstable form of tetrameric form of TTR occurs, resulting in misfolding of the TTR molecule, which leads to amyloid fibril formation. Since mutated TTR exposes cryptic epitopes on the surface of TTR molecule, the induction of an antibody for the epitopes was thought to be effective. We synthesized ATTR Y78P, a spontaneously misfolded TTR, and injected it into amyloid laden transgenic rats having human ATTR V30M to induce the antibody for amyloid fibrils. On the other hand, β-cyclodextrin (β-CyD) may be useful for preventing amyloid formation. Moreover, single stranded oligonucleotides (SSOs) or short interference RNA (siRNA) is a promising tool for gene therapy for FAP. These therapies may become novel strategies for essential FAP therapy instead of liver transplantation.
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Toshiyuki Yamada2010 Volume 54 Issue 1 Pages 27-29
Published: 2010
Released on J-STAGE: September 29, 2010
JOURNAL FREE ACCESSAlthough amyloidosis is diagnosed pathologically, there are several biochemical approaches. Detection of Bence Jones protein is critical for AL type. Gene polymorphism of serum amyloid A can suggest susceptibility for AA type. Detection of variant transthyretin for ATTR type is getting accelerated using mass spectrometry. β2-microglobulin is measured to evaluate the efficiency of dialysis in Aβ2m type.
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Takashi Manabe, Ya Jin2010 Volume 54 Issue 1 Pages 31-34
Published: 2010
Released on J-STAGE: December 10, 2010
JOURNAL FREE ACCESSThe advances in mass spectrometric techniques greatly facilitated the assignment of polypeptides separated by two-dimensional gel electrophoresis (2-DE). The features of mass spectrometric techniques, high-sensitivity and the ability to assign multiple polypeptides simultaneously, were more suited for the assignment of proteins separated by gel electrophoresis under non-denaturing conditions. The results of mass spectrometric assignment of human plasma proteins, separated on non-denaturing micro 2-DE gels, were summarized.
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Masamichi Oh-Ishi, Yoshio Kodera, Tadakazu Maeda, Takeshi Tomonaga, Ta ...2010 Volume 54 Issue 1 Pages 35-39
Published: 2010
Released on J-STAGE: December 10, 2010
JOURNAL FREE ACCESSThere is substantial interest in applying disease proteomics to the identification of diagnostic markers. However, a 2-DE with immobilized pH gradients is not particularly suitable in analyzing high-molecular-mass (HMM) proteins larger than 100 kDa. A 2-DE with agarose IEF gels in the first dimension (agarose 2-DE), which is sufficiently good at separating HMM proteins as large as 600 kDa, has been used for separation of proteins from human primary colorectal cancer, esophageal squamous cell carcinoma and hepatocellular carcinoma. We could find and identify many diagnostic marker candidates in these cancer tissues with LC-MS/MS. Interestingly, a 195-kDa HMM protein, periplakin, was significantly downregulated in esophageal cancer. In hepatocellular carcinoma, a 190-kDa HMM protein, clathrin heavy chain (CHC), was significantly upregulated. These results suggest that the agarose 2-DE based proteomics is a useful strategy for finding new diagnostic markers with HMM.
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