Urinary and plasma proteomics to discover biomarkers for diagnosing between diabetic nephropathy and minimal change nephrotic syndrome or membranous nephropathy

The choice of treatment for primary nephrotic syndrome depends on the pathologic type of the disorder. Renal biopsy is necessary for a definitive diagnosis, but it is burdensome for the patients, and can be avoided if tests could be performed using urine or plasma. In this study, we analyzed 100 urinary proteins, 141 plasma proteins, and 57 urine/plasma ratios in cases of diabetic nephropathy (DN; n = 11), minimal change nephrotic syndrome (MCNS; n = 14), and membranous nephropathy (MN; n = 23). We found that the combination of urinary retinol-binding protein 4 and SH3 domain-binding glutamic acid-rich-like protein 3 could distinguish between MCNS and DN, with an area under the curve (AUC) of 0.9740. On the other hand, a selectivity index (SI) based on serotransferrin and immunoglobulin G, which is often used in clinical practice, distinguished them with an AUC of 0.9091. Similarly, the combination of urinary afamin and complement C3 urine/plasma ratio could distinguish between MN and DN with an AUC of 0.9842, while SI distinguished them with an AUC of 0.8538. Evidently, the candidates identified in this study were superior to the SI method. Thus, the aim was to test these biomarkers for accurate diagnosis and to greatly reduce the burden on patients.     

Therapeutic Efficacy of C-Kit-Targeted Radioimmunotherapy Using 90Y-Labeled Anti-C-Kit Antibodies in a Mouse Model of Small Cell Lung Cancer

Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the ⁹⁰Y isotope.

Methods

¹¹¹In- or ¹²⁵I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of ⁹⁰Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7.

Results

[¹¹¹In]12A8 and [¹¹¹In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [¹¹¹In]12A8 and [¹¹¹In]67A2 accumulated in tumors, but not in major organs. [¹¹¹In]67A2 uptake by the tumor was 1.7 times higher than for [¹¹¹In]12A8. [⁹⁰Y]12A8, but not [⁹⁰Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [⁹⁰Y]12A8, and 1.85 and 3.7 MBq of [⁹⁰Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [⁹⁰Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [⁹⁰Y]12A8, whereas no dose-dependent increase was observed following [⁹⁰Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well.

Conclusion

Treatment with [⁹⁰Y]12A8 and [⁹⁰Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.     

Kinetic Studies on Xylanase Induction by β-Xyloside in Streptomyces sp.

Xylanase induction by β-xyloside was investigated in non-growing conditions using non-induced mycelia of Streptomyces sp. No. 3137 harvested from glucose medium. The mycelia started to produce xylanase without lag time when β-xyloside was added. The rate of xylanase synthesis was dependent on the concentration of β-xyloside added to the inducing culture medium. The induction constants of various β-xylosides were calculated from the Lineweaver-Burk plots; those of methyl-, isopropyl-, butyl- and ethylencyanohydrin-β-d-xylosides were 10.53 mm, 3.83 mm, 0.55mm and 0.25 mm, respectively. Some α-xylosides repressed xylanase synthesis. The rate of xylanase synthesis decreased suddenly after the addition of α-xyloside. The inhibition constants of methyl-, ethyl- and isopropyl-α-d-xylosides were 8.80 mm, 12.50 mm and 33.33 mm, respectively. The xylanase induction was also repressed by glucose. However, this repression was completely restored after consuming additional glucose.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Microbial Oxidation of Cephalosporins to Cephalosporin Sulfoxides

The highest inhibition rate of conidial germination of Pyricularia oryzae was shown by extracts of rice plant leaves inoculated by a pathogen after treatment with probenazole, a rice blast controlling agent. Four anti-conidial germination substances were isolated from these extracts. Substances A, C and D inhibited the germination of the conidia at concentrations between 100 and 200 mcg/ml, and substance B caused morphological changes in the germination tubes of the conidia with a little inhibition of germination. These substances were differentiated from momilactone A, B and the degraded or metabolized products of probenazole. Besides anti-conidial germination activity, they showed antimicrobial activities against several kinds of phytopathogenic bacteria of fungi on agar plates by diffusion method.     

Preparation and Enzymatic Hydrolysis of Maltosyl-α-cyclodextrin

Maltosyl-α-cyclodextrin (6-α-maltosylcyclomaltohexaose, M-CD) was prepared from maltose and α-cyclodextrin by the reverse action of Bacillus pullulanase, and the action of α-amylases on this dextrin was examined. Among α-amylases tested, Thermoactinomyces vulgaris α-amylase (TVA) and Taka-amylase A (TAA) were found to attack the M-CD. Their action pattern on M-CD was studied. These α-amylases cleaved, first the cyclodextrin ring of M-CD, and the branched octasaccharides formed were immediately degraded to form glucose, branched tetraose, or pentaose, though the action pattern was different for TVA and TAA. In addition, TAA also split M-CD into glucose and glucosyl-α-cyclodextrin. Fission products at various stages of the reaction were separated and analyzed by paper chromatography and high performance liquid chromatography, their structures were analyzed, and the degradation pattern of M-CD was found.     

Purification and Some Properties of β-Xylosidase from Emericella nidulans

β-Xylosidase was purified 662 fold from a culture filtrate by ammonium sulfate fractionation, gel filtration on Biogel P-100, DEAE-Sephadex chromatography, and gel filtration on Sephadex G-200. With isoelectric focusing, the purified β-xylosidase found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis. The molecular weight was estimated by gel filtration to be 240,000, and 116,000 by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 3.25, and contained 4% carbohydrate residue. The optimum pH was found to be in the range of 4.5 ~ 5, and the optimum temperature was 55°C. The enzyme activity was inhibited by Hg^{2 +}, SDS, and N-bromosuccinimide at a concentration of 1 × 10^?3 m, and also p-chloromercuribenzoate at a concentration of 1 × 10^?4m. The purified enzyme hydrolyzed phenyl β-d-xyloside (k_o = 302.6 sec^?1),β-nitrophenyl β-d-xyloside (k_o = 438.9 sec^?1), o-nitrophenyl β-d-xyloside (k_o = 431.0 sec^?1), p-chlorophenyl β-d-xyloside (k_o = 207.9 sec^?1), o-chlorophenyl β-d-xyloside (k_o = 211.8 sec^?1), β-methylphenyl β-d-xyloside k_o = 96.5 sec^?1), o-methylphenyl β-d-xyloside (k_o = 83.1 sec^?1), p-methoxyphenyl β-d-xyloside (k_o = 99.3 sec^?1), o-methoxyphenyl β-d-xyloside (k_o= 100.0 sec^?1), xylobiose (k_o = 992A sec^?1), xylotriose (k_o = 1321.9 sec^?1), xylotetraose (k_o = 7S9.1 sec^?1) and xylopentaose (k_o = 508.0 sec^?1). On enzymic hydrolysis of phenyl β-d-xyloside, the reaction product was found to be β-d-xylose with retention of the configuration. The purified β-xylosidase was practically free of a-xylosidase and β-glucosidase activities.     

Extracellular Accumulation of Phospholipids,UDP-N-Acetylhexosamine Derivatives and L-Glutamic Acid by Penicillin-treated Corynebacterium alkanolyticum

The addition of penicillin to cells of Corynebacterium alkanolyticum No. 314 growing on n-paraffins medium caused the simultaneous excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid.

Among many antibiotics which inhibit cell wall synthesis, only the inhibitors of peptideglycan transpeptidase such as penicillin G and cephaloridine were effective for inducing the excretion of phospholipids, UDP-N-acetylhexosamine derivatives and L-glutamic acid, while the others promoted only the excretion of UDP-N-acetylhexosamine derivatives.

From the close relationship between the excretion of L-glutamic acid and the excretion of phospholipids, it was suggested that the action of penicillins and cephalosporins on the cell membrane resulted in the excretion of L-glutamic acid.