Effect of myeloperoxidase oxidation and N-homocysteinylation of high-density lipoprotein on endothelial repair function.

Endothelial cell (EC) migration is essential for healing vascular injuries. Previous studies suggest that high-density lipoprotein (HDL) and apolipoprotein A-I (apoA-I), the major protein constituent of HDL, have endothelial healing functions. In cardiovascular disease, HDL is modified by myeloperoxidase (MPO) and N-homocysteine, resulting in apoA-I/apoA-II heterodimer and N-homocysteinylated (N-Hcy) apoA-I formation. This study investigated whether these modifications attenuate HDL-mediated endothelial healing. Wound healing assays were performed to analyze the effect of MPO-oxidized HDL and N-Hcy HDL in vitro. HDL obtained from patients with varying troponin I levels were also examined. MPO-oxidized HDL reduces EC migration compared to normal HDL in vitro, and N-Hcy HDL showed a decreasing trend toward EC migration. EC migration after treatment with HDL from patients was decreased compared to HDL isolated from healthy controls. Increased apoA-I/apoA-II heterodimer and N-Hcy apoA-I levels were also detected in HDL from patients. Wound healing cell migration was significantly negatively correlated with the ratio of apoA-I/apoA-II heterodimer to total apoA-II and N-Hcy apoA-I to total apoA-I. MPO-oxidized HDL containing apoA-I/apoA-II heterodimers had a weaker endothelial healing function than did normal HDL. These results indicate that MPO-oxidized HDL and N-Hcy HDL play a key role in the pathogenesis of cardiovascular disease.

[Multi-course web-learning system for supporting students of medical technology].

Web-Learning system was developed to support the self-learning for national qualification examination and medical engineering practice by students. The results from small tests in various situations suggest that the unit-learning systems are more effective, especially for the early stage of their self learning. In addition, the answers of some questionnaire suggest that the students' motivation has a certain relation with the number of the questions in the system. That is, the less number of the questions, the easier they are worked out with a higher learning motivation by students. Thus, the system was extended to enable students to study various subjects and/or units by themselves. The system enables them to have learning effects more easily by the exercise during lectures. The effectiveness of the system was investigated on medical associated subjects installed in the system. The concerning questions of Medical engineering and Pathological histology are adequately divided into several groups, of which sixteen Web-Learning subsystems were well composed for their practical application. Our concerning various unit-learning systems were confirmed much useful for most students comparing with the case of the overall Web-Learning system.

Identification of sites in apolipoprotein A-I susceptible to chymase and carboxypeptidase A digestion.

MCs (mast cells) adversely affect atherosclerosis by promoting the progression of lesions and plaque destabilization. MC chymase cleaves apoA-I (apolipoprotein A-I), the main protein component of HDL (high-density lipoprotein). We previously showed that C-terminally truncated apoA-I (cleaved at the carboxyl side of Phe(225)) is present in normal human serum using a newly developed specific mAb (monoclonal antibody). In the present study, we aimed to identify chymase-induced cleavage sites in both lipid-free and lipid-bound (HDL(3)) forms of apoA-I. Lipid-free apoA-I was preferentially digested by chymase, at the C-terminus rather than the N-terminus. Phe(229) and Tyr(192) residues were the main cleavage sites. Interestingly, the Phe(225) residue was a minor cleavage site. In contrast, the same concentration of chymase failed to digest apoA-I in HDL(3); however, a 100-fold higher concentration of chymase modestly digested apoA-I in HDL(3) at only the N-terminus, especially at Phe(33). CPA (carboxypeptidase A) is another MC protease, co-localized with chymase in severe atherosclerotic lesions. CPA, in vitro, further cleaved C-terminal Phe(225) and Phe(229) residues newly exposed by chymase, but did not cleave Tyr(192). These results indicate that several forms of C-terminally and N-terminally truncated apoA-I could exist in the circulation. They may be useful as new biomarkers to assess the risk of CVD (cardiovascular disease).

Determination of myeloperoxidase-induced apoAI-apoAII heterodimers in high-density lipoprotein.

Myeloperoxidase secreted by macrophages and neutrophils in atherosclerotic lesions generates a tyrosyl radical in apolipoprotein (apo) AI, a major protein component of high-density lipoprotein (HDL), thus inducing the formation of apoAI-apoAII heterodimers. It can also cause nitration and chlorination of tyrosine residues. Determining the apoAI-apoAII heterodimer could provide useful information as to functional changes in HDL and/or the progression of atherosclerotic lesions. To this end, the apoAI-apoAII heterodimer was identified in normal human serum by immunoblotting; the band intensity was increased by treatment with myeloperoxidase. This apparent increase in heterodimer formation was quantitatively confirmed by ELISA. In normal human serum, a significant correlation between the concentrations of apoAI-apoAII heterodimer and free apoAII (r=0.763), but not free apoAI (r=0.093), was observed, indicating that heterodimer formation is likely induced on HDL particles carrying both apoAI and apoAII (Lp-AI/AII). In preliminary studies, the levels of apoAI-apoAII heterodimer were statistically higher in plasma from subjects with acute myocardial infarction (AMI) as compared to controls. These findings indicate the possibility that the apoAI-apoAII heterodimer, including nitration and chlorination modifications, may serve as an indicator of atherosclerotic lesions.

The oral administration of thermophile-fermented compost extract and its influence on stillbirths and growth rate of pre-weaning piglets.

Food produced via fermentation with mesophilic bacteria has been used to confer health benefits. In contrast, mammalian physiological responses to the intake of thermophile-fermented products have not been thoroughly investigated. We examined the effects of administering a compost extract consisting of fermented marine animals with thermophiles, including Bacillaceae, to pregnant sows and piglets. Retrospective studies were performed on two different swine farms (n=330-1050 sows). The rate of stillbirth was markedly lower in all parities of the compost extract-fed group compared to those of the control group (p≦0.001). Additionally, the birth to weaning period of newborns was significantly shorter (p<0.0001), while the ratio of weanlings per liveborn piglets was increased by more than 6.5% in the compost extract-fed group. Thus thermophiles and their products in the compost extract might promote growth and reduce stillbirths of piglets during the birth to weaning period.

Oral administration of multispecies microbial supplements to sows influences the composition of gut microbiota and fecal organic acids in their post-weaned piglets.

The timings of the administration of microbial supplements to control the populations of gut microbiota of piglets have been poorly understood. Here the effects of temporal administering multispecies microbial supplements to sows on the composition of gut microbiota and on the bacteria-mediated fecal metabolites in their offsprings were investigated. During gestation and lactation, pregnant sows were fed either a normal diet (group A) or a diet with multispecies supplements comprised of nine microbial species such as Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium bifidum, Enterococcus faecium, Candida pintolopesii, and Aspergillus oryzae etc. (group B). All of the sows' piglets were temporarily fed with the same supplements around weaning in accordance with the guideline of the farm. This regimen was followed by a normal diet in both groups over one month thereafter. Under such conditions, the concentration of short-chain fatty acids (SCFAs) in fecal samples remarkably increased in group B compared to group A. When 16S rDNA sequences of the fecal bacteria were analyzed, the microbial structure of bacteria was different between both goups. Especially the Clostridium cluster IV and subcluster XIVa were particularly increased in group B, although the administered microbes were undetectable. Thus, temporal administration of multispecies-microbial supplements to pregnant sows changes the composition of SCFAs and gut microbiota in their offsprings.

Detection of chymase-digested C-terminally truncated apolipoprotein A-I in normal human serum.

In atherosclerotic artery walls, mast cells, an inflammatory cell, are activated and secrete some proteases including chymase. Chymase, a chymotrypsin-like protease, cleaves the C-terminus of apolipoprotein A-I (apoA-I) at Phe225. This cleavage reduces the ability of apoA-I to promote the efflux of cellular cholesterol. The aim of this study is to detect C-terminally truncated apoA-I in normal human serum. For this purpose, we generated a monoclonal antibody that specifically recognizes C-terminally truncated apoA-I by immunizing mice with a peptide that corresponds to human apoA-I amino acid residues 216-225. The monoclonal antibody, termed 16-4 mAb, selectively reacted with recombinant C-terminally truncated apoA-I, but not recombinant full-length apoA-I. A two-dimensional electrophoresis analysis also indicated that only two out of six spots that contained apoA-I fragments and had a molecular mass of 26 kDa after chymase digestion reacted with the 16-4 mAb. We detected an extremely small amount of C-terminally truncated apoA-I in normal human serum by concentrating the serum through affinity chromatography using a 16-4 mAb-conjugated resin, and then performing Western blot analysis. The 16-4 mAb could be useful to examine whether C-terminally truncated apoA-I is associated with the progression of atherosclerosis.

[Urinary albumin fragmentation and immunoreactivity].

Urinary albumin (ALB) has been measured as a marker for the early detection of diabetic nephropathy. In 2004, Comper et al. developed a gel-filtration high-performance liquid chromatography (HPLC) procedure for the determination of urinary ALB. They demonstrated the presence in its albumin fraction of non immunoreactive ALB with the total molecular weight of a monomeric ALB that was non-reactive with the existing anti-ALB antibody, and reported that the level of urinary non-immunoreactive ALB was higher in diabetic patients than in normal subjects. In this study, we isolated urinary ALB from diabetic patients using an anti-ALB antibody-coupled affinity column to test its immunoreactivity. In some diabetic patients, the results of HPLC and turbidimetric immunoassay for urinary ALB were discrepant. Western blot analysis showed that ALB samples from such patients were contaminated with proteins other than ALB, and contained ALB, whose molecular weight became lower using a reductive procedure. In addition, the reactivity of ALB with anti-ALB antibody differed depending on whether it was in a reduced or non-reduced state. These results indicate that ALB in such patients is susceptible to structural changes due to disease-induced urinary factors and, thus, their urine contains ALB with an altered reactivity to antibody.

Urinary protein analysis in pre- and postoperative cancer patients.

Urinary proteins from six patients with esophageal cancer and two with stomach cancer were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analyses were performed on days-1 to 3, 5, 7, 10, 14, and 21 (or 22) after surgery. The protein patterns were scanned by densitometry and divided into nine fractions. The main proteins in the fractions (Fr.) were identified as follows: immunoglobulin G in Fr. A, Tamm-Horsfall glycoprotein (THP) in Fr. B, transferrin in Fr. C, albumin in Fr. D, alpha(1)-acid glycoprotein in Fr. E, alpha(1)-microglobulin in Fr. F, retinol binding protein in Fr. G, and beta(2)-microglobulin in Fr. I. The protein in Fr. H was not identified. The percentage of each fraction was calculated from the densitometry pattern of each lane. The percentage values were averaged among all the patients, and pre- and postoperative data were compared. The percentage of Frs. E, F, and G increased on days 1-7, and the changes in these three proteins were similar to changes in serum C-reactive protein (CRP). In particular, the percentage of Fr. G peaked within 1 day of operation, which was faster than for CRP. Conversely, other fractions decreased. These results suggest that urinary protein analysis is useful for monitoring the response to surgical stress.

Urinary protein fraction in healthy subjects using cellulose acetate membrane electrophoresis followed by colloidal silver staining.

We previously reported a rapid and highly sensitive colloidal silver staining solution suitable for the cellulose acetate membrane. This method was useful for detecting even very small amounts of urinary protein. In the present study, we examined urinary protein fractions in healthy subjects, using cellulose acetate membrane electrophoresis (CAE) with a highly sensitive colloidal silver staining, in an attempt to determine the clinical relevance of urinary protein fractions. Sixty unconcentrated spot urine specimens were analyzed by CAE and calculated by densitometry. All of the samples were separated into five fractions by CAE. The mean +/- 1 SD of the percentage of five fractions was 28.37 +/- 8.51 in albumin, 4.30 +/- 4.19 in alpha1-globulin, 14.41 +/- 6.14 in alpha2-globulin, 19.45 +/- 7.10 in beta-globulin, and 33.46 +/- 8.24 in gamma-globulin. The albumin/globulin (A/G) ratio was 0.41 +/- 0.17. These six items and the concentrations of total protein, albumin, and beta-N-acetyl-D-glucosaminidase (NAG) did not significantly differ between males and females. NAG is the marker of tubulointerstitial nephropathy. The results suggest that there are no gender-dependent differences in the urinary protein fractions of healthy subjects.

Change in relative mobility of M protein following neuraminidase treatment in patients with multiple myeloma and monoclonal gammopathy of undetermined significance.

The aim of this study was to clarify the relationship between the relative mobility of M protein in various disease states using cellulose acetate membrane electrophoresis. To examine the carbohydrate chain of the M protein, sera from patients with multiple myeloma (MM), various cancers, and benign disease were treated with neuraminidase. The relative mobility in benign disease and MM patient sera following neuraminidase treatment varied among individuals, while that of IgG M protein in sera from cancer patients was from 0.2 to 0.3. Thus, the relative mobility in cancer patients was narrower than in those with MM or benign disease.However, after neuraminidase treatment, there was no significant difference between relative mobility in cancer patient's sera and those in other disease patients.