Adherence of diarrhea-associated Escherichia coli was studied by scanning electron microscopy. Enteropathogenic E. coli (EPEC) adherence factor-positive (EAF+) E. coli of EPEC serotypes (class I EPEC) adhered to plastic and human jejunal and ileal mucosa, similar to case and HeLa cells. Localized adherence, elongation of cell microvilli, and "locking" of the bacterial aggregates by the elongated microvilli were evident after incubation for 20 min. EAF+ E. coli adhered strikingly to mucus but rarely to M cells in Peyer's patch-associated epithelium. Most enteroaggregative E. coli (EAggEC) strains adhered to plastic, similar to HeLa cells. Some diffuse-adhering E. coli (DAEC) strains displayed no adherence to plastic but formed "dimples" on HeLa cells. Both EAggEC and DAEC adhered at lower levels to human small intestines (except M cells) than did EAF+ E. coli. In all cases of EAF+ E. coli, EAggEC, and DAEC, strains were found with atypical characteristics. The data demonstrate the unique adherence characteristics of EAF+ E. coli, EAggEC, and DAEC.

Complex II (succinate-ubiquinone oxidoreductase) from Escherichia coli is composed of four nonidentical subunits encoded by the sdhCDAB operon. Gene products of sdhC and sdhD are small hydrophobic subunits that anchor the hydrophilic catalytic subunits (flavoprotein and iron-sulfur protein) to the cytoplasmic membrane and are believed to be the components of cytochrome b 556 in E. coli complex II. In the present study, to elucidate the role of two hydrophobic subunits in the heme b ligation and functional assembly of complex II, plasmids carrying portions of the sdh gene were constructed and introduced into E. coli MK3, which lacks succinate dehydrogenase and fumarate reductase activities. The expression of polypeptides with molecular masses of about 19 and 17 kDa was observed when sdhC and sdhD were introduced into MK3, respectively, indicating that sdhC encodes the large subunit (cybL) and sdhD the small subunit (cybS) of cytochrome b 556 . An increase in cytochrome b content was found in the membrane when sdhD was introduced, while the cytochrome b content did not change when sdhC was introduced. However, the cytochrome b expressed by the plasmid carrying sdhD differed from cytochrome b 556 in its CO reactivity and red shift of the ␣ absorption peak to 557.5 nm at 77 K. Neither hydrophobic subunit was able to bind the catalytic portion to the membrane, and only succinate dehydrogenase activity, not succinateubiquinone oxidoreductase activity, was found in the cytoplasmic fractions of the cells. In contrast, significantly higher amounts of cytochrome b 556 were expressed in the membrane when sdhC and sdhD genes were both present, and the catalytic portion was found to be localized in the membrane with succinate-ubiquinone oxidoreductase and succinate oxidase activities. These results strongly suggest that both hydrophobic subunits are required for heme insertion into cytochrome b 556 and are essential for the functional assembly of E. coli complex II in the membrane. Accumulation of the catalytic portion in the cytoplasm was found when sdhCDAB was introduced into a heme synthesis mutant, suggesting the importance of heme in the assembly of E. coli complex II.Complex II (succinate-ubiquinone oxidoreductase) is a tricarboxylic acid cycle enzyme associated with the inner membrane of mitochondria or the cytoplasmic membrane of bacteria (1, 2). Complex II in Escherichia coli is encoded by the sdhCDAB operon (3, 4) and contains four nonidentical subunits and five prosthetic groups (5, 6). The largest hydrophilic subunit (flavoprotein; Fp), 1 encoded by sdhA, has covalently bound FAD, and the second largest subunit (iron-sulfur protein; Ip), encoded by sdhB, contains three different types of iron-sulfur clusters termed S-1[2Fe-2S], S-2[4Fe-4S], and S-3[3Fe-4S]. Two hydrophobic heme b-containing subunits are encoded by sdhC and sdhD, and at least the sdhC product is a component of cytochrome b 556 (7).Generally, the Fp and Ip subunits comprise the catalytic portion of complex II and catalyze electron transfer from suc...

The ability of the novel water-soluble provitamin E, alpha-tocopherol-6-O-phosphate, to protect against ultraviolet B-induced damage in cultured mouse skin was investigated and compared with the protectiveness of alpha-tocopherol acetate in cultured mouse skin. Pretreatment of skin with 0.5% (9.4 mM) alpha-tocopherol-6-O-phosphate in medium for 3 h significantly prevented such photodamage as sunburn cell formation, DNA degradation, and lipid peroxidation, which were induced in control cultured skin by a single dose of ultraviolet B irradiation at 0 to 40 kJ per m2 (290-380 nm, maximum 312 nm). This protection was greater than that seen with alpha-tocopherol acetate, the most common provitamin E that is used in commercial human skin care products. The concentration of alpha-tocopherol in cultured skin pretreated with 0.5% alpha-tocopherol-6-O-phosphate rose to approximately two to three times that found in the control skin and the reduction in cutaneous alpha-tocopherol that was induced by ultraviolet irradiation was significantly inhibited. In the group pretreated with 0.5% alpha-tocopherol acetate, however, conversion of alpha-tocopherol acetate to alpha-tocopherol was not observed, although the level of provitamin incorporated into the cultured skin was the same as that for alpha-tocopherol-6-O-phosphate. These findings indicated that the enhanced ability of alpha-tocopherol-6-O-phosphate to protect against ultraviolet B-induced skin damage compared with alpha-tocopherol acetate may have been due to alpha-tocopherol-6-O-phosphate's conversion to alpha-tocopherol. Moreover, following pretreatment with a 0.5% alpha-tocopherol-6-O-phosphate, alpha-tocopherol-6-O-phosphate was incorporated into the human skin in a three-dimensional model and 5% of the incorporated alpha-tocopherol-6-O-phosphate was converted to alpha-tocopherol. These results suggest that treatment with the novel provitamin E, alpha-tocopherol-6-O-phosphate may be useful in preventing ultraviolet-induced human skin damage.