A novel image analysis method combined with recently developed powerful computing hardware is proposed, in which the time course for the image data of silver staining gel taken during the developmental process was used for the analysis of protein spots in two-dimensional polyacrylamide gel electrophoresis. The alignment procedure of the time course gel images using the landmark spots of plastic fragments was a critical step in the precise analysis of protein spots. The merged, or adjacent, protein spots that inhibit the quantification and identification of the proteins were successfully segmented from regions that were automatically determined from the previous time gel image, which effectively removed operator subjectivity. Furthermore, the effect of termination time of the developing process on the gel image analysis was not a concern, because the present analysis used the whole-time course of the gel image.

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Due to the negative charges on their surface membrane, erythrocytes usually do not agglutinate each other in vivo. However, when mixed with 5% glucose solution in a test tube, some feline erythrocytes exhibit agglutination. To investigate the reasons for this phenomenon, we extracted erythrocyte membrane proteins from these cells and subjected them to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In samples in which agglutination did not occur, a band was seen at the 36-kDa position, whereas in samples showing agglutination, this band disappeared or has lower intensity. The 36-kDa position corresponds to glycophorin A in the human erythrocyte membrane, but no immunochemical cross-reaction with this band was seen. We therefore conducted mass spectrometry in order to investigate the composition, and found a partial amino acid sequence, His-Ile-Thr-Ser-Tyr-Pro-Glu-Thr-His-Glu-Gly. Furthermore, although no protein showing this sequence was found in any database, this protein was confirmed to be an acidic glycoprotein, therefore it is thought to be a glycophorin-like molecule in the feline erythrocyte membrane that could contribute to inhibition of agglutination.

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The purpose of this study was to develop and evaluate a semiautomatic analyzer for total urinary protein assay and to develop a software program for identifying the affected part of the kidney by using data obtained by cellulose acetate electrophoresis (CAE) with silver staining.
 The limit of detection of this semiautomatic analyzer was 2.5 mg/L, and the assay precision was good, with a coefficient of variation (CV) less than 10%. Moreover, there was a good correlation between the results obtained by using this semiautomatic analyzer and the manual method.
 The urinary protein fractions of patients who were diagnosed with renal disease on the basis of renal biopsy were analyzed by performing CAE with silver staining. The relative mobility (RM) of several bands in the β fraction was calculated. The algorithm of the method used for calculating RM was programmed, and the RM ranges detected in several major protein bands were incorporated in the programmed software to classify the types of renal disorders. The programmed software could be used to classify the types of nephropathy in the case of diabetic patients with albumin concentrations of 30 mg/gCre or less.
 This semiautomatic analyzer can help detect nephropathy in early stages, and the urinary protein fraction analysis software can help determine the type of renal disorder in diabetic patients before microalbuminuria is detected, thus facilitating early treatment.

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Patients with Dent disease excrete high amounts of urinary α₁-microglobulin (α₁-m), β₂-microglobulin (β₂-m), and retinol-binding protein (RBP). We found that the levels of α₁-m, β₂-m, and RBP in patients with Dent disease were higher than those in healthy subjects by 20 times, 130 times, and 200 times, respectively. These results confirmed the results of previous studies.
 The cellulose acetate membrane electrophoresis (CAE) showed that the characteristics of the urinary protein fraction in patients with Dent disease included appearance of pre-albumin, low albumin value, equal α₁-globulin (α₁-G) and α₂-globulin (α₂-G) values, disappearance of β-globulin (β-G), and appearance of slow α₂-G (RBP) and slow β-G (β₂-m).
 Urinary albumin (ALB), α₁-m, β₂-m, and RBP of patients with Dent disease 1 (mutation of CLCN 5) and Dent disease 2 (mutation of OCRL 1) and of healthy subjects were identified using western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six bands from 84 kDa to 130 kDa were detected in addition to a main band of ALB (67 kDa). Seven bands from 51 kDa to 92 kDa were detected in addition to a main band of α₁-m (34 kDa). A 17-kDa band was detected in addition to a main band of β₂-m (14 kDa). RBP was detected only in the main band (17 kDa). The ALB and α₁-m bands, which were in the high molecular weight range in addition to the main band, were densely stained in Dent disease 2.

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Previously, we discovered that the rat liver showed enhanced expression of intestinal-type alkaline phosphatase (ALP) on high fat-feeding. Here we tested the hypothesis that intestinal-type ALP (Akp3 or Akp6) might also be regulated in the liver of Akp3^-/- mice upon high fat-feeding. C57Bl/6J male Akp3^-/-, Akp3^+/-, or wild-type mice were fed a normal control or high-fat diet and the effects on intestinal-type ALP mRNA in liver were investigated. We found that Akp3 mRNA is only expressed in the intestine and not in the liver, while Akp6 mRNA is expressed in both the upper intestine and liver of wild-type mice. The intensity of Akp6 mRNA expression in the liver was not enhanced in the Akp3^-/- mice compared with the wild-type mice fed on a high-fat diet. The nucleotide sequence of the PCR product of Akp6 from the liver was identified as being the same as that of Akp6 in the intestine. This is the first report concerning IAP mRNA expression in the mouse liver, but further studies will be needed to determine if this ectopic expression of intestinal-type ALP is associated with any unique function.

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