Highly sensitive quantification of vancomycin in plasma samples using liquid chromatography-tandem mass spectrometry and oral bioavailability in rats

We developed a highly sensitive liquid chromatography-tandem mass spectrometry assay (LC-MS-MS) for a glycopeptide antibacterial drug, vancomycin (VCM), in rat plasma. After precipitating 100 micro l of plasma with 300 micro l of 10% trifluoroacetic acid-methanol (2:1, v/v), the supernatant was diluted with 300 micro l of distilled water and was passed through a filter. LC-MS-MS equipped with electrospray ionization in the positive ion mode used a pair of ions at 725/144 m/z for VCM in the multiple reaction-monitoring mode with a sample injection volume of 20 micro l. The calibration curve had a linear range from 0.01 to 20 micro g/ml when linear least square regression was applied to the concentration versus peak area plot. The drug in the sample was detected within 5 min. Precision, accuracy and limit of quantitation indicated that this method was suitable for the quantitative determination of VCM in rat plasma. Using this method, we defined for the first time that the oral bioavailability of VCM in rats was 0.069%. This method can be applied to basic pharmacokinetic and pharmaceutical studies in rats.     

Gangliosides that associate with lipid rafts mediate transport of cholera and related toxins from the plasma membrane to endoplasmic reticulm

Cholera toxin (CT) travels from the plasma membrane of intestinal cells to the endoplasmic reticulum (ER) where a portion of the A-subunit, the A1 chain, crosses the membrane into the cytosol to cause disease. A related toxin, LTIIb, binds to intestinal cells but does not cause toxicity. Here, we show that the B-subunit of CT serves as a carrier for the A-subunit to the ER where disassembly occurs. The B-subunit binds to gangliosides in lipid rafts and travels with the ganglioside to the ER. In many cells, LTIIb follows a similar pathway, but in human intestinal cells it binds to a ganglioside that fails to associate with lipid rafts and it is sorted away from the retrograde pathway to the ER. Our results explain why LTIIb does not cause disease in humans and suggest that gangliosides with high affinity for lipid rafts may provide a general vehicle for the transport of toxins to the ER.     

Effects of cyclohexenonic long-chain fatty alcohol on diabetic rat trachea

In order to investigate the diabetes-associated neuropathy and prevent effects of cyclohexenonic long-chain fatty alcohol, a neurotrophic substance, in trachea, we studied its effect on streptozotocin-diabetic hyper-reactivity in the rat trachea. Diabetes was induced in 8-week-old male Sprague-Dawley rats by administering an intraperitoneal injection of streptozotocin (50 mg/kg). The rats were divided randomly into four groups and were maintained for four weeks: age-matched control rats, diabetic rats without treatment with cyclohexenonic long-chain fatty alcohol, and diabetic rats treated with cyclohexenonic long-chain fatty alcohol (2 and 8 mg/kg, i.p. every day). The serum glucose and insulin levels were determined, and the contractile responses of the trachea induced by carbachol and KCl were investigated. Treatment with cyclohexenonic long-chain fatty alcohol did not alter the rats' diabetic status, i.e., body weight, thickness of the trachea, serum glucose levels, and serum insulin levels, but significantly improved the diabetic-induced hyper-reactivity of the rat trachea in a dose-dependent manner. There was no significant difference in either the carbachol- or KCl-induced contractile forces between groups with or without mucosa in the functional studies. In histological examinations, thinning of cricoid cartilage, thickness of basal membrane, and degeneration, fragmentation of elastic fibers in the submucosal layer, and hypertrophy of smooth muscle bundle in the membranous wall of trachea were observed in the diabetic rat trachea, which were improved by treatment with cyclohexenonic long-chain fatty alcohol. Our data indicate that this drug can prevent hyper-reactivity in the diabetic trachea.     

Direct Detection by PCR of Escherichia coli O157 and Enteropathogens in Patients with Bloody Diarrhea

Direct detection of Escherichia coli O157 and foodborne pathogens associated with bloody diarrhea were achieved using polymerase chain reaction (PCR) after the preparation of DNA from stool specimens using the microspin technique. PCR was compared with cultivation and toxin production tests with respect to the efficiency of detection of each pathogen; E. coli O157, Vibrio parahaemolyticus, Salmonella serovar Enteritidis and Campylobacter jejuni. Detection of some or all of the above pathogens in clinical stool specimens was achieved using PCR. The minimum number of cells required for the detection of the above pathogens by PCR was 10¹ CFUs/0.5 g of stool sample. PCR was completed within 6 hr. The above pathogens were also detected in cultivation and toxin production tests. Partial purification of the template DNA using the microspin technique was essential for the elimination of PCR inhibitors from the DNA samples. This PCR method is an accurate, easy-to-read screening method for the detection of Shiga-like toxin producing E. coli O157 and enteropathogens associated with bloody diarrhea in stool specimens.     

The mechanism of the degradation of psaB gene product,one of the photosynthetic reaction center subunits of Photosystem I,upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala⁵⁰⁰ and Val⁵⁰¹ on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.     

Subcellular Localization and Polymorphism of Bovine FABP4 in Bovine Intramuscular Adipocytes

Fatty acid binding protein 4 (FABP4) I74 V, a gene polymorphism associated with unsaturated fatty acid contents, was discovered in Japanese Black cattle. Individuals with FABP4 I/I genotype contain a significantly high level of palmitoleic acid compared to those with FABP4 V/V genotype. It remains unknown how the FABP4 polymorphism leads to different palmitoleic acid contents.

We overexpressed FABP4 of different genotypes in bovine intramuscular preadipocytes and examined whether the intracellular localization of FABP4 and the expression levels of lipid metabolism-related genes were different among cells expressing different genotypes. Nuclear localization was observed for the FABP4 V/V, while the FABP4 I/I almost did not. The cells expressing FABP4 of different genotypes were comparable in terms of the expression levels of genes involved in lipid metabolism. FABP4 I/I was localized in most of the lipid droplets 4 days after differentiation induction, whereas approximately 25% lipid droplet co-localized with FABP4 in cells expressing FABP4 V/V. The lipid droplet size increased when palmitoleic acid was added compared to the size observed when palmitic acid was added. These results suggest that lipid droplet enlargement caused by palmitoleic acid and genotype-dependent differences in the fatty acid transporting capacity underlie variations in palmitoleic acid content among FABP4 polymorphisms.     

Molecular Composition of the 16S Toxin Produced by a Clostridium botulinum Type D Strain, 1873

The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873). Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined. It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin. The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases. However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity. The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.