Nucleotide sequence of a gene cluster involved in entry of E colicins and single-stranded DNA of infecting filamentous bacteriophages into Escherichia coli.

Mutations in fii or tolA of the fii-tolA-tolB gene cluster at 17 min on the Escherichia coli map render cells tolerant to high concentrations of the E colicins and do not allow the DNA of infecting single-stranded filamentous bacteriophages to enter the bacterial cytoplasm. The nucleotide sequence of a 1,854-base-pair DNA fragment carrying the fii region was determined. This sequence predicts three open reading frames sequentially coding for proteins of 134, 230, and 142 amino acids, followed by the potential start of the tolA gene. Oligonucleotide mutagenesis of each open reading frame and maxicell analysis demonstrated that all open reading frames are expressed in vivo. Sequence analysis of mutant fii genes identified the 230-amino acid protein as the fii gene product. Chromosomal insertion mutations were constructed in each of the two remaining open reading frames. The phenotype resulting from an insertion of the chloramphenicol gene into the gene coding for the 142-amino acid protein is identical to that of mutations in fii and tolA. This gene is located between fii and tolA, and we propose the designation of tolQRA for this cluster in which tolQ is the former fii gene and tolR is the new open reading frame. The protein products of this gene cluster play an important role in the transport of large molecules such as the E colicins and filamentous phage DNA into the bacterium.     

NADH diferric transferrin reductase in liver plasma membrane

Evidence is presented that rat liver plasma membranes contain a distinct NADH diferric transferrin reductase. Three different assay procedures for demonstration of the activity are described. The enzyme activity is highest in isolated plasma membrane, and activity in other internal membranes is one-eighth or less than in plasma membrane. The activity is inhibited by apotransferrin and antitransferrin antibodies. Trypsin treatment of the membranes leads to rapid loss of the transferrin reductase activity as compared with NADH ferricyanide reductase activity. Erythrocyte plasma membranes, which lack transferrin receptors, show no diferric transferrin reductase activity, although NADH ferricyanide reductase is present. The transferrin reductase is inhibited by agents that inhibit diferric transferrin reduction by intact cells and is activated by CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfate) detergent. Inhibitors of mitochondrial electron transport have no effect on the activity. We propose that the NADH diferric transferrin reductase in plasma membranes measures the activity of the enzyme that causes the reduction of diferric transferrin by intact cells. This transmembrane electron transport system requires the transferrin receptor for diferric transferrin reduction. Because the transmembrane electron transport has been shown to stimulate cell growth, the reduction of diferric transferrin at the cell surface may be an important function for diferric transferrin in stimulation of cell growth, in addition to its role in iron transport.     

Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive proton pore of the clathrin-coated vesicle proton translocating complex

The clathrin-coated vesicle proton translocating complex is composed of a maximum of eight polypeptides. The function of the components of this system have not been defined. Proton pumping catalyzed by the reconstituted, 200-fold purified proton translocating complex of clathrin-coated vesicles is inhibited 50% at a dicyclohexylcarbodiimide (DCCD)/protein ratio of 0.66 mumol of DCCD/mg of protein. At an identical DCCD/protein ratio, the 17-kDa component of the proton pump is labeled by [14C]DCCD. Through toluene extraction, the 17-kDa subunit has been isolated from the holoenzyme. The 17-kDa polypeptide diminished proteoliposome acidification when coreconstituted with either bacteriorhodopsin or the intact clathrin-coated vesicle proton translocating ATPase. In both instances, treatment of the 17-kDa polypeptide with DCCD restored proteoliposome acidification. Moreover, the proton-conducting activity of the 17-kDa polypeptide is abolished by trypsin digestion. These results demonstrate that the 17-kDa polypeptide present in the isolated proton ATPase of clathrin-coated vesicles is a subunit which functions as a transmembranous proton pore.     

墨红鲜花香气(头香)成分分析

利用 XAD-4憎水性吸附树脂采集墨红头香,以毛细管气相色谱双柱保留指数和 GC/MS/DS 联用方法鉴定头香的化学成份。共分离鉴定或初步鉴定了45种组份,其中含量较大的有乙酸芳樟酯(14.98%),柠檬烯(12.07%),甲基苯甲醚(9.88%),香茅醇(4.82%),乙酸巳酯(3.98%),β-石竹烯(4.55%),芳樟醇(3.18%),正巳醇(3.17%)等.     

Pregnancy diagnosis in zoo animals by estrogen determination in feces

Estrogen concentration in feces was investigated in five different herbivorous species of zoo animals. Using a nonspecific estrogen radioimmunoassay, in four species (red buffalo, yak, Grevy's zebra, and Nubian ibex) pregnancy was revealed by measuring estrogen concentration in feces. In hippopotamus, the levels of fecal estrogens were not different between pregnant and nonpregnant animals.     

EGF receptors on TEA3A1 endocrine thymic epithelial cells

Thymic endocrine epithelial cell line TEA3A1 can be maintained and passaged in a serum-free WAJC404A medium supplemented with insulin, transferrin, dexamethasone and EGF. EGF not only promotes the growth of these cells but also regulates the activation of phospholipase A2 enzyme activity. The binding of [125I]EGF to the TEA3A1 cells is temperature and time dependent, saturable and can be blocked by excess unlabelled EGF. Two classes of EGF receptors are found on these cells. One with Kd of 5 X 10(-11)M (approximately 3000 sites/cell) and the other with Kd of 5 X 10(-9)M (approximately 30,000 sites/cell). The resynthesis of EGF receptor in TEA3A1 cells after down-regulation requires about 24 hrs and can be blocked by both actinomycin D and cycloheximide.     

The conformation of gramicidin A in dimethylsulphoxide solution. A full analysis of the one- and two-dimensional 1H, 13C, and 15N nuclear-magnetic-resonance spectra

The combined application of one- and two-dimensional high-field NMR techniques has led to the first assignment of the 1H, 13C, and 15N spectra of the pentadecapeptide gramicidin A in dimethylsulphoxide solution. The 62.9-MHz and 100.6-MHz 13C spin-lattice relaxation times and 13C-[1H] NOE factors for the backbone alpha carbons have been analysed in the 'model-free' approach to give a single correlation time (tau m) for isotropic overall molecular motion and an order parameter and internal correlation time for each C alpha H group in the backbone. The relatively high and constant values for the order parameter along the backbone indicate a degree of ordering of the structure, while the internal correlation times show that internal motions are progressively more rapid towards the N terminus. The average values of the vicinal HNC alpha H couplings are 7.4 Hz and 8.4 Hz respectively for the alternate L- and D-amino acid residues. The values are not consistent with either a ribbon conformation for the backbone or a right-handed beta 6.3 helix; they are consistent with the model proposed by Glickson et al. [Glickson, J. D., Mayers, D. F., Settine, J. M. & Urry, D. W. (1972) Biochemistry 11, 477-486] in which there is a rapid conformational order in equilibrium disorder equilibrium, the ordered structure being the left-handed beta 6.3 helix and the disordered state having local random-coil character.