Dimeric porin from Paracoccus denitrificans.

Paracoccus denitrificans was shown to contain a 33,000-dalton porin, which produced pores of large (1.6 to 1.8 nm) diameter. Cross-linking studies showed that the porin existed as dimers in the outer membrane.     

Stimulation of glutamine transport by osmotic stress in Escherichia coli K-12.

Osmotic stress produced by high concentrations of sucrose stimulated the high-affinity transport of glutamine in Escherichia coli cells. Glutamine transport via a low-affinity system was not affected. Osmotic stress produced by NaCl, in contrast, inhibited the transport of glutamine and some other amino acids. Maltose transport was strongly inhibited by osmotic stress.     

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups.

Recently, beta-lactam agents containing iron-chelating moieties, such as E0702, which contains catechol, and pirazmonam and U-78,608, which contain 3-hydroxypyridone, have been developed. By determining the susceptibility to these agents of Escherichia coli mutants lacking various iron-repressible outer membrane proteins, we showed that two of these proteins with hitherto unknown functions, Fiu and Cir, were apparently involved in the transport of monomeric catechol and its analogs. These results confirm the conclusion of Curtis and co-workers, which was obtained by using a different set of catechol-containing antibiotics (N. A. C. Curtis, R. L. Eisenstadt, S. J. East, R. J. Cornford, L. A. Walker, and A. J. White, Antimicrob. Agents Chemother. 32:1879-1886, 1988). E0702 was shown to enhance the uptake of radioactive ferric iron into wild-type cells but not into cir fiu double mutants. By combining the influx of E0702 with its hydrolysis by a periplasmic beta-lactamase, we showed that the wild-type cells transported unliganded E0702 at a rate comparable to or even higher than the rate of transport of the E0702-Fe3+ complex. We postulate that the main function of Cir and Fiu may be to recapture the hydrolytic products of enterobactin, such as 2,3-dihydroxybenzoic acid and 2,3-dihydroxybenzoylserine.     

Blood ammonia concentration in mice: normal reference values and changes during growth

To establish normal reference values of blood ammonia concentration in mice, possible factors which may affect blood ammonia determination such as age, sex, strain and sampling site of blood, were investigated by the microdiffusion method. The ammonia concentration in blood collected from the tail (mixture of venous and arterial blood) and left ventricle was significantly higher than that in venous blood collected from the orbital sinus and right auricle. Neither strain nor sex differences were observed. Blood ammonia concentration in newborn pups was 92 +/- 19 micrograms/dl immediately after birth and increased to 184 +/- 22 micrograms/dl 24 hours after birth. This increased level was maintained for a week. By 15 days, it decreased to the level at birth. Thereafter, no significant variation with age was observed. Blood ammonia concentration in mice after weaning, irrespective of age, sex and strain, varied from 38 to 123 micrograms/dl, with a mean +/- SD of 79 +/- 18 micrograms/dl.     

Establishment of a monoclonal antibody recognizing ultraviolet light-induced (6-4) photoproducts

We obtained a monoclonal antibody directed against UV-induced DNA damage. Analysis of the antigenic determinant in UV-irradiated DNA recognized by this antibody, 64M-1, revealed that it bound UV-irradiated oligo- or poly-nucleotides containing thymine-thymine or thymine-cytosine sequences. The antibody failed to bind DNA irradiated with 313 nm UV in the presence of acetophenone, which contained predominantly thymine dimers as DNA damage. The binding activity of this antibody to 254-nm UV-irradiated DNA decreased with 313-nm UV irradiation, and the decrease of this binding activity correlated with the decrease of fluorescence corresponding to (6-4) photoproducts. These results suggest that the antigenic determinant recognized by this monoclonal antibody is a (6-4) photoproduct. Using autoradiography with 3H-antibody, we could detect the formation of the (6-4) photoproduct in individual human cells irradiated with 254-nm UV doses as low as 20 J/m2.     

The acrAB homolog of Haemophilus influenzae codes for a functional multidrug efflux pump.

Disruption of gene HI0894 or HI0895 in Haemophilus influenzae Rd, homologs of Escherichia coli acrAB multidrug efflux genes, caused hypersusceptibility to erythromycin, rifampin, novobiocin, and dyes such as ethidium bromide and crystal violet and increased accumulation of radioactive erythromycin, showing that these genes are expressed and contribute to the baseline level resistance of this organism through active drug efflux. The gene disruption did not produce detectable changes in susceptibility to several other antibiotics, possibly because rapid influx of small antibiotic molecules through the large H. influenzae porin channels counterbalances their efflux.     

Antigen structural requirements for recognition by a cyclobutane thymine dimer-specific monoclonal antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine dimer,and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.