Urinary albumin (ALB) and transferrin (TF) are useful markers for impaired renal function. Compared to ALB, TF is more readily excreted into urine from an early stage of renal failure because of its smaller number of negative charges. However, few evidence of a change in isoelectric point (pI) value of urinary TF in nephropathy patients has been reported. We analyzed pI values of urinary TF by an isoelectric focusing, and compared the results in normal subjects with those in diabetic patients at various stages of nephropathy. Urine samples of diabetics were randomly collected from 24 patients and 7 healthy volunteers. An apotransferrin with a high pI value was detected in samples of healthy subjects. However, the pI value of TF bands derived from diabetic patients shifted to the lower side, and the more nephropathy stages progressed, greater change of pI value was detected. In addition, TF with low iron-binding capacity was detected in some samples from diabetics. These results suggest that determination of the pI value of urinary TF in diabetic patients may be useful diagnostic markers for an early detection of diabetic nephropathy.

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Virulent strains of Acidovorax avenae subsp. citrulli (Aac) and avenae cause bacterial fruit blotch in cucurbits and bacterial stripe in rice, respectively. Here, we describe a rapid (1 h) method to identify virulent strains of Aac using a combination of short PCR and matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Single-stranded DNA of the PCR products was analyzed by MALDI-TOF MS after digestion by an enzyme and alkali denaturation. The mass spectra differed among the strains as a result of the differences in their nucleotide sequences. This new method allows rapid identification of virulent strains of Aac.

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Background: Urinary exosomal proteins have recently emerged as important candidates for elucidating the mechanisms underlying physiological events and disease-related metabolism in the kidney. Here, we evaluated standard sample preparation methods for two-dimensional gel electrophoresis (2DE) to determine which one yielded the maximum protein recovery from urinary exosomes for protein identification.
Materials and Methods: Urinary exosomes were purified from a healthy subject by using ultracentrifugation. The final pellets were dissolved with PBS or RIPA buffer. After being desalted, these exosomal protein solutions were each treated with 1 of 4 rehydration buffers (Rbs) containing detergents in the following formulations: CHAPS (Rb1), CHAPS and Triton X-100 (Rb2), dodecyl maltoside (Rb3), and ASB-14 (Rb4).
Results: For all Rbs, a much greater number of protein spots was detected in the samples isolated with RIPA than with PBS. Only minor differences were observed in the number of protein spots for Rb1-3. The largest protein spots were detected using the combination of RIPA buffer and Rb4; however, the background on the 2DE gel was high in the region of >66 kD and at the lower pH values. For all combinations, the co-precipitation of the urinary Tamm-Horsfall protein masked the protein spots in the 66-100 kD region.
Conclusion: For extracting a large number of proteins with a relatively clear background on silver-stained 2DE gels, the optimal exosomal protein-dissolving buffer is RIPA buffer. All of the evaluated Rbs, except for the one containing ASB-14 as a detergent, is suitable for solubilizing exosomal proteins on 2DE.

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The purpose of the present study was to find the possibility of a glucose-protein interaction using p-hydroxyacetophenone (p-HAP)-Sepharose resin. Additionally, two proteins were identified as p-HAP-Sepharose binding proteins. In this study, seven polypeptides were found to have an affinity with p-HAP in the liver cytosolic fraction. More specifically, this study demonstrated that the four polypeptides from the seven also have an affinity with glucose. Moreover, to confirm the results from this experiment using a p-HAP affinity column, a commercially available horse liver alcohol dehydrogenase (HLADH) was examined. HLADH bounded to a p-HAP affinity column was eluted by both glucose and p-HAP. The in vitro enzymatic activity of the HLADH, in the presence of ethanol as a substrate, was significantly decreased by incubation with glucose (up to 250 mM). These results suggested that glucose has an affinity with HLADH and decreases its activity in vitro.

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