Urinary albumin and transferrin as early diagnostic markers of chronic kidney disease.

Feline renal diseases are increasingly noted in veterinary practice. It is important to diagnose and identify the pathological basis of renal dysfunction accurately at an early stage, but there are only a few reports on this area in clinical veterinary medicine. We investigated the efficacy of measurement of urinary albumin (u-Alb) and urinary transferrin (u-Tf) for early diagnosis using 5-µl urine samples collected noninvasively by catheterization from normal (IRIS stage I) cats and cats with stage I chronic kidney disease (CKD). The u-Alb levels in normal and stage I CKD cats were 6.0 ± 4.5 and 11.2 ± 8.4 mg/dl, respectively, and the u-Tf levels were 0.09 ± 0.42 and 0.52 ± 0.79 mg/dl, respectively. Based on ROC curve analysis, the sensitivity and specificity of u-Alb and u-Tf were higher than those of the currently used biomarker, the plasma creatinine level. The sensitivity of u-Alb was higher than that of u-Tf, whereas the specificity of u-Tf was higher than that of u-Alb. The validity of the threshold albumin level (20 mg/dl) was confirmed by measurements using SDS-PAGE. Since leakage of u-Tf in urine precedes leakage of u-Alb, inclusion of u-Tf in biochemistry tests may be appropriate for IRIS staging as a diagnostic marker of early diagnosis of renal disorder in cats.

Time course of virulence factors produced by group A streptococcus during a food-borne epidemic.

We studied the protein amount and activity of the major virulence factors hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase), which are produced by Streptococcus pyogenes type T-25, with a food poisoning outbreak. The three virulence factors were analyzed by activity and amount of protein using supernatants at 2-30 h of culture. All these virulence factors were confirmed by their activity. Streptolysin O (SLO), SpeB, and NADase were immunochemically confirmed at protein level by Western blot analysis. Two hemolytic forms (70 and 60 kDa) of SLO were identified. SpeB was detected as a 44-kDa precursor form and a 30-kDa mature form. NADase was 50 kDa. SLO protein peaked at 8 h of culture, which corresponded with the hemolytic activity peak. Conversion from precursor to SpeB protein peaked at 14 h of culture. The conversion peak corresponded to the activity expression time. Also, mature SpeB protein peaked at 24 h of culture and corresponded to SpeB activity peak. Electrophoretic analysis clarified the relationship between SLO protein and SpeB protein, although amounts of SLO and SpeB have been reported to be inversely proportional to activity. NADase protein peaked at 12 h of culture, but protein level did not correspond to the peak. Because the NADase protein peak was closer to SpeB activity than SLO protein, our results suggested NADase protein was degraded at 12 h of culture. The time course production of these virulence factors is discussed.

Properties of metabolic substances produced by group A streptococcus from a food-borne epidemic.

Here we report a large food poisoning outbreak by Streptococcus pyogenes that occurred in Kanagawa, Japan, in July 2005. To compare cases of type T-B3264 (Chiba) and type T-28 (Tokyo) reported to date, we studied the properties and activity of the major virulence factors produced by Streptococcus pyogenes type T-25 (Kanagawa): hemolysin, cysteine protease streptococcal pyrogenic exotoxin B (SpeB), and NAD glycohydrolase (NADase). These virulence factors were also analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The titer of hemolysin was 9 50% hemolytic dose (HD(50)) per milliliter (HD(50)/ml) for T-25, 173 HD(50)/ml for T-28, and 147 HD(50)/ml for T-B3264. The hemolytic titer of T-25 was very low compared with those of T-28 and T-B3264. Each hemolysin produced by the three strains was dependent on its reductant, and its properties differed among strains. The major hemolysin of T-25 was identified as streptolysin O (SLO), because cholesterol or γ-globulin, but not phospholipids, inhibited its hemolysis. In contrast, the major hemolysin of T-28 and T-B3264 was streptolysin S (SLS). Although the SpeB activity of T-25 (4.8 U/ml) was lower than that of T-B3264, its NADase activity (19.1 U) was the largest of the three strains. The conversion from the SpeB precursor to mature SpeB was confirmed by SDS-PAGE analysis of T-25 at 6 h of culture; no conversion was identified for T-28 and T-B3264 at 6 h. SpeB of T-25 was converted quickly, most likely because of the degradation of SLO by SpeB, thereby resulting in the very low hemolytic titer of T-25. These results suggest that the three strains have diverse properties and activities of major virulence factors. The specific interactions of these virulence factors are thought to be involved in the pathosis of these strains.

[Properties of metabolic substance produced by group A Streptococcus from two food-borne epidemic].

We report the finding for two food-poisonins outbreaks occurring in Tokyo and Chiba in September 2003. Patients in the Tokyo outbreak suffered from fever varying widely from 35.9 degrees C to 39.4 degrees C. Throat pain was predominant, accompanied by headache, cough, and joint pain. Patients in the Chiba outbreak suffered from malaise in addition to the above symptoms. To clarify the relationship between pathology and virulence factors, we studied the properties of hemolysins and proteases produced by the causative bacteria, Streptococcus pyogenes, specifically type T-28 in the Tokyo outbreak and type T-B3264 in the Chiba outbreak. The main S. pyogenes T serotypes isolated in 2003 were types T12, T1, T4, and T3, followed types T-28 and T-B3264. The hemolytic titer of hemolysins, which are metabolic, was 173HD50/mL for T-28 and 147HD50/mL for T-B3264. Hemolysins produced by both strains did not depend on reducing agents and were not inhibited by gamma-globulin or cholesterol, indicating the streptolysin S (SLS) rather than hemolysis inhibition by phospholipids. The fact that the titer increased slightly in the presence of reducing agents indicates that some amount of streptolysin O may also have been present. Protease production was four times greater for T-B3264 than for T-28. Proteases produced by both strains were similarly inhibited by sodium tetrathionate, iodoacetate, and normal serum. The outbreak infection was caused by infiltration of food-borne Streptococcus bacteria via the upper airway during eating. The primary cause of predominant throat pain was thought to be SLS cytotoxicity in the upper respiratory mucous membrane. This toxin was also thought to assist in Streptococcus bacteria infiltration and proliferation. Proteases produced by pathogenic bacteria were thought to have acted on the body as potent virulence factors.

Exoenzymes of Trametes versicolor can metabolize coplanar PCB congeners and hydroxy PCB.

Two fractions containing the oxidase activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) were obtained using ion-exchange DEAE-Sepharose column chromatography of the culture fluid of white-rot fungus, Trametes versicolor. These two fractions can reduce the level of coplanar PCB congeners (Co-PCBs). The ABTS oxidase in the first fraction passed through the DEAE-Sepharose column. The ABTS oxidase in the second fraction was adsorbed to the column at

Novel and extensive aspects of paraquat-induced pulmonary fibrogenesis: comparative and time-course microarray analyses in fibrogenic and non-fibrogenic rats.

Although paraquat (PQ) is widely known to induce pulmonary fibrosis, the molecular mechanisms are poorly understood. Therefore, to bring a new dimension to the elucidation of the mechanisms, we conducted microarray experiments to investigate the expression profiles of 1,090 genes in the lungs during the progressive phase of PQ-induced pulmonary fibrosis in rats. After several s.c. injections of PQ, rats were divided into a fibrogenic group and a non-fibrogenic group. Time-course gene expression analysis of the fibrogenic group showed altered gene regulation throughout the experimental period. The expression levels of many cell membrane channel, transporter, and receptor genes were substantially altered. These genes were classified into two categories: polyamine transporter- and electrolyte/fluid balance-related genes. Moreover, comparative analysis of the fibrogenic and the non-fibrogenic group revealed 36 genes with significantly different patterns of expression, including the pro-apoptotic gene Bad. This indicates that Bad is a key factor in apoptosis and that apoptosis provides a major turning point in PQ-induced pulmonary fibrosis. Notably, subtypes of transforming growth factor (TGF)-beta genes that are considered to play a pivotal role in fibrogenesis showed no differences in expression between the two groups, though TGF-beta3 was markedly induced in both groups. These results provide novel and extensive insights into the molecular mechanisms that lead to pulmonary fibrosis after exposure to PQ.