Kiyoko Shiba

Transforming growth factor beta type II receptor gene mutations in adenomas from hereditary nonpolyposis colorectal cancer

BACKGROUND & AIMS Germline mutations of DNA mismatch repair genes are responsible for cancer susceptibility in hereditary nonpolyposis colorectal cancer (HNPCC) kindreds. Transforming growth factor beta type II receptor (TGF-beta RII) has been found to be somatically altered in HNPCC. The aim of this study was to clarify further the role of TGF-beta RII alterations in HNPCC tumorigenesis, particularly in adenomas. METHODS Fourteen adenoma specimens and 13 cancer specimens from 10 patients with HNPCC were screened for mutations in the short repeated sequences of the TGF-beta RII gene by polymerase chain reaction-single-strand conformation polymorphism. Mismatch repair genes, replication errors, and c-K-ras 2 were also analyzed in HNPCC tumors. RESULTS Alterations of the TGF-beta RII gene at the short poly(A) repeat were found in 8 (57%) adenoma specimens and 11 (85%) cancer specimens. They were found at an earlier stage of adenomas. Two adenoma specimens showed two-hit inactivation of mismatch repair genes. Replication errors were detectable in 13 (93%) adenoma specimens. Mutations in c-K-ras 2 codon 12 were detected at a 50% frequency in adenoma specimens. CONCLUSIONS These data indicate a strong association between TGF-beta RII gene alterations and adenoma-carcinoma progression in HNPCC.

Prevalence of Microalbuminuria and Relationship to the Risk of Cardiovascular Disease in the Japanese Population

The prevalence of microalbuminuria and its relationship to cardiovascular disease risk factors were examined in subjects participating in an annual physical and laboratory examination program. The urinary albumin concentration and the urinary albumin/creatinine ratio were determined in morning urine specimens. A turbidimetric immunoassay was used for the measurement of urinary albumin. Of the 731 subjects, 41 (5.6%) who were weakly positive or positive on a routine dipstick test for protein were excluded from the final analysis of data. Microalbuminuria was present in 14.5% of the men, in 12.4% of the women, and in 13.2% of the entire subject population when defined as a urinary albumin concentration of 30–299 μg/ml. The prevalence of microalbuminuria was significantly higher in subjects with a high normal blood pressure (15.0%) or hypertension (26.2%) as compared with normotensive subjects (6.5%). Subjects with impaired glucose tolerance (24.3%) or hyperglycemic subjects (50.0%) had a significantly higher prevalence of microalbuminuria than normoglycemic subjects (11.3%). The prevalence of microalbuminuria was significantly higher in subjects with left ventricular hypertrophy (47.1%) as compared with those with normal electrocardiograms (11.3%). A good correlation was observed between urinary albumin concentration and albumin/creatinine ratio, and both showed a significant positive correlation with age, systolic and diastolic blood pressures, and fasting plasma glucose, total serum protein, albumin, and triglyceride levels, but not with angiotensin-converting enzyme activity. Multiple regression analysis demonstrated that both the urinary albumin concentration and the albumin/creatinine ratio show a significant positive correlation with systolic blood pressure and fasting plasma glucose. The prevalence of microalbuminuria was about 13% in this Japanese cohort, and the systolic blood pressure and the fasting plasma glucose level were demonstrated as independent risk indicators for both urinary microalbumin level and urinary microalbumin/creatinine ratio.

Changes of α1‐acid glycoprotein microheterogeneity in acute inflammation stages analyzed by isoelectric focusing using serum obtained postoperatively

The relationship between variations of α1‐acid glycoprotein (orosomucoid, AGP) microheterogeneity detected from isoelectric focusing (IEF) patterns and clinical stage of acute inflammation based on serum C‐reactive protein (CRP) levels and interleukin‐6 (IL‐6) levels was investigated. Serum samples were obtained from healthy subjects, and from patients with esophageal or stomach carcinoma before and after operation. Samples without neuraminidase treatment were used for AGP microheterogeneity analysis, and samples with neuraminidase treatment for AGP heterogeneity analysis. In AGP microheterogeneity, nine bands were detected in the range of pI 3.18—3.57 in sera obtained from healthy subjects. In patients, AGP microheterogeneity changed the first day after operation; the percentage of bands surrounding pI 3.5 increased, and the highest value appeared in sera taken the first or second day after operation and then decreased quickly. These bands showed reactivity for concanavalin A (Con A). The increase in Con A‐reactive AGP occurred later than the increase in IL‐6, and occurred earlier than the increase in CRP. On the seventh day after operation, the percentage of bands around pI 3.2 increased. These bands showed the reactivity for Datura stramonium agglutinin. On the other hand, in samples with neuraminidase treatment, little change of AGP heterogeneity was observed in most samples, which did not reflect the stage of inflammation. These findings suggested that AGP microheterogeneity detection was a useful marker for the clinical stage of inflammation.

Rapid and sensitive electrophoresis of urinary protein clearly reveals the pathophysiological feature of renal diseases

Aim:  The diagnostic approach for renal diseases with the electrophoretic pattern of urinary protein on cellulose acetate (CA) membrane differentiates the causes of proteinuria. However, this method has not been used routinely because of its difficulty in obtaining a clear image. This study was performed in order to re‐evaluate this method with an improved system.

Analysis of urinary albumin, transferrin, N-acetylβ-D-glucosaminidase and β2-microglobulin in patients with impaired glucose tolerance

We investigated the changes in urinary albumin and urinary transferrin as glomerular proteins, and in urinary N‐acetyl‐β‐D‐glucosaminidase and urinary β2‐microglobulin as tubular proteins, in patients with impaired glucose tolerance. We attempted to compare the proteins of normal subjects to those of diabetics with pre‐nephropathy. Transferrin and N‐acetyl‐β‐D‐glucosaminidase levels were significantly increased in patients with impaired glucose tolerance, while albumin and β2‐microglobulin levels were only slightly increased. In addition, there was no significant difference in transferrin levels between patients with impaired glucose tolerance and type 2 diabetics with pre‐nephropathy.

Rapid and highly sensitive colloidal silver staining on cellulose acetate membrane for analysis of urinary proteins

In order to establish a rapid staining method sensitive enough to stain the protein bands on cellulose acetate membrane and suitable for the analysis of urinary proteins in healthy subjects, we conducted a detailed examination of Kovariks method designed for the analysis of proteins in cerebrospinal fluid in detail. For purpose of our study, we changed the pH of the staining solution used in Kovariks method to pH 4.4 by adding acetic acid. With this solution, our staining method revealed a degree of sensitivity about three times higher than that of the original method, but specific sensitivity for albumin and γ‐globulin remained almost unchanged. The solution preparation we used was much more simple and the reagent much more stable than in the original method. Our method also showed better reproducibility and linearity than the original method. These results point to the suitability of this method for the analysis of low‐concentration proteins, such as urinary proteins, in healthy subjects.

Re-evaluation of normal human urinary proteins fractionated by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis

Proteins in normal human urine were clearly fractionated into 26 bands with molecular weights from 14,000 to 230,000 by means of one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) coupled with silver staining. The main band contained uromucoid, and the second main band had albumin. However, when urine samples from healthy persons were electrophoresed in the absence of SDS using polyacrylamide gel or agarose gel, or a cellulose acetate membrane, albumin but not uromucoid, frequently formed the main protein band. It is suggested that this is due to the complexing of uromucoid subunits to form a large molecule which cannot penetrate into the gel. In order to correctly fractionate all the proteins contained in normal human urine, it was concluded that it was best to treat a urine sample with SDS with pre-condensation, fractionate it by SDS-PAGE and stain fractionated proteins by a highly sensitive method such as silver staining.

Direct analysis of airborne mite allergen (Der f1) in the residential atmosphere by chemifluorescent immunoassay using bioaerosol sampler.

Dermatophagoides farinae allergen (Der f1) is one of the most important indoor allergens associated with allergic diseases in humans. Mite allergen Der f1 is usually associated with particles of high molecular weight; thus, Der f1 is generally present in settled dust. However, a small quantity of Der f1 can be aerosolized and become an airborne component. Until now, a reliable method of detecting airborne Der f1 has not been developed. The aim of this study was to develop a fiber-optic chemifluorescent immunoassay for the detection of airborne Der f1. In this method, the Der f1 concentration measured on the basis of the intensity of fluorescence amplified by an enzymatic reaction between the labeled enzyme by a detection antibody and a fluorescent substrate. The measured Der f1 concentration was in the range from 0.49 to 250 ng/ml and a similar range was found by enzyme-linked immunosorbent assay (ELISA). This method was proved to be highly sensitive to Der f1 compared with other airborne allergens. For the implementation of airborne allergen measurement in a residential environment, a bioaerosol sampler was constructed. The airborne allergen generated by a nebulizer was conveyed to a newly sampler we developed for collecting airborne Der f1. The sampler was composed of polymethyl methacrylate (PMMA) cells for gas/liquid phases and some porous membranes which were sandwiched in between the two phases. Der f1 in air was collected by the sampler and measured using the fiber-optic immunoassay system. The concentration of Der f1 in aerosolized standards was in the range from 0.125 to 2.0 mg/m(3) and the collection rate of the device was approximately 0.2%.

Molecular heterogeneity of urinary albumin in glomerulonephritis: Comparison of cardiovascular disease with albuminuria

BACKGROUND Despite the unstable structure of urinary albumin in kidney diseases, urinary albumin fragments have been identified by denaturing methods such as two-dimensional electrophoresis. This study examined the relationship between the structural heterogeneity of urinary albumin and protease effects. METHODS Urine samples from patients with glomerulonephritis (GN), cardiovascular diseases (CVD), and healthy subjects were analyzed by non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), Western blot, diagonal 2-dimensional non-reducing/reducing (d2D) SDS PAGE, and albumin zymography. RESULTS The major band was monomer albumin in CVD and healthy subjects; however, 13 urinary albumin bands ranging from 55 to 172 kDa were identified by non-reducing SDS PAGE in GN. The results from d2D SDS PAGE showed urinary albumin polymerization between disulfide bridges, interactions with other proteins, and reduction induced degradation in GN patients. The results from albumin zymography showed that low-molecular mass forms of albumin did not necessarily correspond to high protease activity. Furthermore, concentrated healthy urine showed similar protease digestion as in GN without low-molecular mass of albumin. CONCLUSIONS The molecular alterations observed cannot be explained only by urinary proteases. The specific alteration of urinary albumin molecules in GN can be attributed to different mechanisms to CVD.

Occurrence of serum M-protein species in Japanese patients older than 50 years based on relative mobility in cellulose acetate membrane electrophoresis.

We investigated the occurrence of serum M‐protein species in 2,007 Japanese patients older than 50 years of age. All sera samples were analyzed by cellulose acetate membrane electrophoresis. The relative mobility of an M‐protein band was calculated by dividing the migration distance of M protein by that of albumin. M proteins were found to be present in 71 of 2,007 cases (3.5%). Men 80–89 years old showed the highest occurrence of M proteins, 11.0%. The relative mobility of M‐protein bands, especially the band of the IgA‐type M protein, increased as the patients age advanced. The patients had higher levels of the IgG‐type M protein than healthy Japanese subjects. We found that the occurrence of M‐protein species in Japanese patients increases with their age. The IgG‐type M protein was most frequently expressed among other types. The mobility of the M protein was greater in older patients probably because of aging‐related changes in the carbohydrate chain of immunoglobulins composing an M‐protein molecule. J. Clin. Lab. Anal. 14:64–69, 2000.