Drug Resistance of Enteric Bacteria XIII. Distribution of R Factors in Escherichia coli Strains Isolated from Livestock

Escherichia coli strains isolated from 151 swine and 108 fowl, which were kept at the Animal Health Center, Maebashi, Japan, were surveyed for drug resistance and distribution of R factors. All of the swine and 38% of the fowl excreted E. coli strains resistant to tetracycline, chloramphenicol, streptomycin, and sulfanilamide, or certain combinations thereof. Among 278 resistant cultures isolated from swine, 13% were found to be resistant to one antibiotic, whereas 87% were resistant to more than one antibiotic. Among these resistant strains, 40% carried R factors which were transferable by the usual conjugal process. The resistance patterns of these R factors included 36% which were singly resistant and 64% which were multiply resistant. Among 54 resistant cultures isolated from fowl, 24% were singly resistant and 76% were multiply resistant. Of the resistant strains from fowl, 22% carried R factors. The resistance patterns of R factors included 50% of the singly resistant type and 50% which were multiply resistant. In spite of feeding with dairy products containing only tetracycline, a high incidence of multiple resistance was observed in the E. coli strains and the R factors isolated from these animals.     

Biochemical basis for the resistance of guinea pigs to carcinogenesis by 2-acetylaminofluorene

Studies were made on why guinea pigs are resistant to carcinogenesis by 2-acetylaminofluorene. Cytochrome P-448 and arylhydrocarbon hydroxylase were not induced in either the microsomes and nuclei of guinea pigs by 3-methylcholanthrene treatment. 3-Methylcholanthrene treatment caused only 2-fold increase in the binding of 2-acetylaminofluorene to DNA in nuclei isolated from guinea pigs, while it caused 17-fold increase in the binding in rat nuclei. Microsomes from 3-methylcholanthrene treated rats had 5 times more effect than Microsomes from 3-methylcholanthrene treated guinea pigs on the binding of 2-acetylaminofluorene to DNA of nuclei from untreated guinea pigs. N-Hydroxy-2-acetylaminofluorene combined equally well with the DNA of rats and guinea pigs. In guinea pigs, there was a good correlation between the low inducibility of cytochrome P-448 and the low binding of 2-acetylaminofluorene to DNA. Our results clearly showed that guinea pigs are resistant to tumor induction by 2-acetylaminofluorene through inability to carry out the first step of activation of 2-acetylaminofluorene.     

Comparison of ES cell fate in sandwiched aggregates and co-cultured aggregates during blastocyst formation by monitored GFP expression

Markers and the means to detect them are required to monitor the fate of living cells. However, few suitable markers for living cells were known until a green fluorescent protein (GFP) was discovered. We have established mouse embryonic stem (ES) cell lines that express mutant GFP under the chicken beta-actin (CAG) promoter. Using these cell lines, we were able to follow the migration of ES cells during blastocyst formation both in sandwiching and coculture methods, even if only a single ES cell was used. Furthermore, the contribution of ES cells to the inner cell mass (ICM) was easily estimated at the blastocyst stage. We compared sandwiching with coculture aggregation relative to the contribution of the ES cell in the ICM, and the results indicated that there was no difference in the ratios of chimeric embryos having ICM contributed from cultured ES cells. Furthermore, an aggregated single ES cell was able to contribute three or four cells to the ICM at the blastocyst stage. Thus we conclude that one, instead of two, embryos is enough to make aggregation with ES cells, and a single ES cell attached to an embryo is enough to produce germline chimeras. Moreover, we could clearly observe single cell fate during blastocyst formation. This suggests that our established cell line can be used for monitoring single cell fate in vivo. In addition, we have shown that up to five doses of 30 sec of UV irradiation using GFP filters have no effect on the embryonic development.     

Orally active anti-proliferation agents: novel diphenylamine derivatives as FGF-R2 autophosphorylation inhibitors

(6,7-Disubstituted-quinolin-4-yloxy-phenyl)(4-substituted-phenyl)amine derivatives were synthesized and evaluated by a cellular autophosphorylation assay for FGF-R2 in the human scirrhous gastric carcinoma cell line, OCUM-2MD3. We also performed metabolic stability studies showing that substitutions at the 7-position of quinoline affect its biological stability. In this study, we achieved a remarkable improvement in the solubility and metabolic stability of the diphenylamine derivative. The most promising compound 15e showed a significant decrease in tumor volume when orally administered.     

Effect of centrifuge-induced artificial gravity and ergometric exercise on cardiovascular deconditioning, myatrophy, and osteoporosis induced by a -6 degrees head-down bedrest

We have reported that centrifuge-induced artificial gravity with ergometric exercise could reduce developing cardiovascular deconditioning in humans. In the present study, we examined this load could prevent the myatrophy and osteoporosis induced by head-down bedrest for 20 days. Subjects were ten healthy male volunteers with informed consent. They were requested to lie down at -6 degrees for 20 days, and evaluation for cardiovascular deconditioning, myatrophy, and osteoporosis. As the result, high G-load with low intensity exercise suppressed the orthostatic intolerance and increase in serum osteoporotic marker, whereas low G-load with high intensity ergometric exercise maintained the maximal oxygen intake, heart dimension, and prevented myatrophy. The combination of high/low G-load with low/high intensity exercise will determine the optimal protocol for prevention of cardiovascular deconditioning, myatrophy, and osteoporosis.     

Dipeptidyl peptidase with strict substrate specificity of an anaerobic periodontopathogen Porphyromonas gingivalis

A dipeptidyl peptidase which hydrolyzed Xaa-Ala-p-nitroanilide was purified to homogeneity by sequential procedures including ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic interaction chromatography, gel filtration and isoelectric focusing from the cell extract of Porphyromonas gingivalis. The purified enzyme hydrolyzed p-nitroanilide derivatives of Lys-Ala, Ala-Ala, and Val-Ala, but not Xaa-Pro. Enzyme activity was maximum at neutral pHs. Its molecular mass was 64 kDa with an isoelectric point of 5.7. The enzyme belonged to the family of serine peptidases.     

Regulation of proton-to-electron stoichiometry in photosynthetic electron transport: physiological function in photoprotection

The primary stable products of photosynthetic electron flow are NADPH and ATP. Stoichiometry of their production depends on the ratio of protons pumped across the thylakoid membrane to electrons passed through the electron transport pathway (H(+)/e(-) ratio). Flexible requirements of the ATP/NADPH ratio by various assimilatory reactions in chloroplasts must be fulfilled by the H(+)/e(-) ratio during the electron flow. In addition to the well-known role of Delta pH during ATP synthesis, Delta pH also functions as a trigger of the down-regulation of photosystem II (PSII) photochemistry. Excessive light energy is safely dissipated as heat by this regulatory process to suppress the generation of toxic reactive oxygen species. Thus, regulation of the H(+)/e(-) ratio may function in the photoprotection, as well as in the regulation of the ATP/NADPH production ratio. It has long been the consensus that the H(+)/e(-) ratio can be controlled by regulating the proton-transporting Q-cycle in the cytochrome b(6)f complex and by the cyclic electron flow around photosystem I (PSI). Despite the possible physiological importance and the long history of interest, the molecular identity of Q-cycle regulation and the cyclic electron flow around PSI have been remained unclear. The recent improvements in research tools, including the genetic approach using chlorophyll fluorescence imaging and establishment of the chloroplast transformation technique, are providing new insights into classical topics. In this review, we focus on regulation of the H(+)/e(-) ratio especially from the view of photosynthetic regulation.     

Analytical method for lipoperoxidation relevant reactive aldehydes in human sera by high-performance liquid chromatography–fluorescence detection

A validated, simple and sensitive HPLC method was developed for the simultaneous determination of lipoperoxidation relevant reactive aldehydes: glyoxal (GO), acrolein (ACR), malondialdehyde (MDA), and 4-hydroxy-2-nonenal (HNE) in human serum. The studied aldehydes were reacted with 2,2′-furil to form fluorescent difurylimidazole derivatives that were separated on a C₁₈ column using gradient elution and fluorescence detection at excitation and emission wavelengths of 250 and 355 nm, respectively. The method showed good linearity over the concentration ranges of 0.100–5.00, 0.200–10.0, 0.200–40.0, and 0.400–10.0 nmol/mL for GO, ACR, HNE, and MDA, respectively, with detection limits ranging from 0.030 to 0.11 nmol/mL. The percentage RSD of intraday and interday precision did not exceed 5.0 and 6.2%, respectively, and the accuracy (%found) ranged from 95.5 to 103%. The proposed method was applied for monitoring the four aldehydes in sera of healthy, diabetic, and rheumatic human subjects with simple pretreatment steps and without interference from endogenous components. By virtue of its high sensitivity and accuracy, our method enabled detection of differences between analytes concentrations in sera of human subjects under different clinical conditions.     

High doses of ultraviolet-C irradiation increases vasoactive intestinal contractor/endothelin-2 expression in keratinocytes of the newborn mouse epidermis

We examined the expression profiles of vasoactive intestinal contractor/endothelin-2 (VIC/ET-2) at both gene and peptide level in skin irradiated with different ultraviolet wavelengths. We found that VIC/ET-2 gene expression is sensitive only to ultraviolet-C (UVC) irradiation and has an immediate response. These results provide direct evidence that high doses of UVC irradiation induce an increase in gene expression and protein production of VIC/ET-2 and endothelin (ET) receptors in a dose-dependent manner in epidermal keratinocytes. We suggest that VIC/ET-2 can play an essential role in the maintenance, protection and hyperpigmentation of the epidermis exposed to UVC irradiation from artificial or natural sources.