Colicine Typing of Shigella sonnei

The colicine typing method of Shigella sonnei is described with experimental evidence supporting it as well as the manner of selection of eight indicator strains. The disparity of principle between this method and that of Abbott and Shannon's method is (1) selection of the indicators only from wild strains existing in this country, (2) employment of heart infusion broth for colicine production, (3) performance of the typing within 48 hours, and (4) determination of types and subtypes of test-strains by combining their colicinogenic activity against the indicators and their sensitivity to colicines produced by the indicators. A modification of the method is advocated which requires three days to extract colicine by cultivation and one day for sensitivity tests and which uses peptone as the sole nutriment in media. The efficiency of the technique of Abbott and Shannon, McGeachie and McCormick, and the authors' two methods was compared using the selected indicators. Only the technique of McGeachie and McCormick showed some discrepancies.     

Site-targeted non-viral gene delivery by direct DNA injection into the pancreatic parenchyma and subsequent in vivo electroporation in mice

The pancreas is considered an important gene therapy target because the organ is the site of several high burden diseases, including diabetes mellitus, cystic fibrosis, and pancreatic cancer. We aimed to develop an efficient in vivo gene delivery system using non-viral DNA. Direct intra-parenchymal injection of a solution containing circular plasmid pmaxGFP DNA was performed on adult anesthetized ICR female mice. The injection site was sandwiched with a pair of tweezer-type electrode disks, and electroporated using a square-pulse generator. Green fluorescent protein (GFP) expression within the injected pancreatic portion was observed one day after gene delivery. GFP expression reduced to baseline within a week of transfection. Application of voltages over 40 V resulted in tissue damage during electroporation. We demonstrate that electroporation is effective for safe and efficient transfection of pancreatic cells. This novel gene delivery method to the pancreatic parenchyma may find application in gene therapy strategies for pancreatic diseases and in investigation of specific gene function in situ.     

Vitronectin secretion by hepatic and non-hepatic human cancer cells

Summary Thirty-eight human cancer cell lines and subclones derived from 12 different organs were screened for vitronectin secretion in their culture media. By immunoblotting analysis we detected high secretion by three out of five hepatoma cell lines tested but no secretion by the others. In addition, significant secretion was observed in seven non-hepatic cancer cell lines and subclones derived from the cervix, lung, and pancreas. These vitronectin-secreting cells included PLC/PRF/5, HuH-6 #5, HuH-7, HeLa S3, HeLa · P3 #2, #3, #6, #8, A549, and MIAPaCa-2. The results were further confirmed by quantitative analysis using sandwich enzyme-linked immunoassay, and activity analysis of cell attachment promotion on Western blotted filters.     

Bioactive jatrophane diterpenes from Euphorbia guyoniana

Chromatographic investigation of the methylenechloride/methanol extract of the aerial parts of Euphorbia guyoniana afforded two jatrophane diterpenes, designated guyonianins E and F, in addition to a known jatrophane diterpene. The structures of the compounds were determined by comprehensive NMR analyses, including DEPT, COSY, HMQC, HMBC, NOESY and HRMS. These compounds exhibited cytotoxicity against human embryonic kidney 293 (HEK293) cells with IC₅₀ values of 35–100 μM.     

Fish otolith contains a unique structural protein, otolin-1.

A collagen-like protein was identified from the otoliths of the chum salmon, Oncorhynchus keta. The otolith, composed mainly of calcium carbonate with small amount of organic matrices, is formed in the inner ear and serves as a part of the hearing and balance systems. Although the organic matrices may play important roles in the growth of otolith, little is known about their chemical nature and physiological function. In this study, a major organic component of the otolith, designated otolin-1, which may serve as a template for calcification, was purified. The sequences of two tryptic peptides from otolin-1 revealed high homology with parts of a saccular collagen which had been described previously [Davis, J.G., Oberholtzer, J.C., Burns, F.R. & Greene, M.I. (1995) Science 267, 1031-1034]. Cloning of a cDNA coding for otolin-1 revealed that the deduced amino-acid sequence contained a collagenous domain in the central part of the protein. Although collagen is the most abundant structural protein in the animal body, otolin-1 mRNA was expressed specifically in the sacculus. Immunohistochemical studies showed that otolin-1 is synthesized in the transitional epithelium and transferred to the otolith and otolithic membrane. This is the first report concerning characterization of a structural protein containing many tandem repeats of the sequence, Gly-Xaa-Yaa, typical for collagen from the biomineral composed of calcium carbonate.     

The synthesis and biodistribution of [11C]metformin as a PET probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1) in vivo

In order to develop a new positron emission tomography (PET) probe to study hepatobiliary transport mediated by the multi-drug and toxin extrusion transporter 1 (MATE1), ¹¹C-labelled metformin was synthesized and then evaluated as a PET probe. [¹¹C]Metformin ([¹¹C]4) was synthesized in three steps, from [¹¹C]methyl iodide. Evaluation by small animal PET of [¹¹C]4 showed that there was increased concentrations of [¹¹C]4 in the livers of mice pre-treated with pyrimethamine, a potential inhibitor of MATEs, inhibiting the hepatobiliary excretion of metformin. Radiometabolite analysis showed that [¹¹C]4 was not degraded in vivo during the PET scan. Biodistribution studies were undertaken and the organ distributions were extrapolated into a standard human model. In conclusion, [¹¹C]4 may be useful as a PET probe to non-invasively study the in vivo function of hepatobiliary transport and drug–drug interactions, mediated by MATE1 in future clinical investigations.     

Heat denaturation increases the sensitivity of the cytomegalovirus loop‐mediated isothermal amplification method

A new protocol for CMV LAMP with an additional heat denaturation step was developed. While the sensitivity of the original CMV LAMP method was 500 copies/tube, sensitivity was increased by up to 100 copies/tube by additional heat denaturation. CMV DNA was detected in 103 of 350 samples (29.4%) by the original CMV LAMP procedure and 148 of 350 samples (42.3%) by the new CMV LAMP protocol. When the pp65 antigenemia assay was used as the standard method, the sensitivity, specificity, PPV, and NPV of the new protocol were 92.9%, 77.7%, 62.2%, and 96.5%, respectively.