cDNA cloning and structure of mouse putative Ah receptor.

Mouse cDNA clones for a putative Ah receptor have been isolated from a cDNA library of mRNA from Hepa-1 cells by an oligonucleotide probe produced by PCR with a pair of primers which was synthesized according to the reported N-terminal sequence of 26 amino acids. The cDNA clones encode a polypeptide of 805 amino acids with a helix-loop-helix motif and with some similarity to a certain region designated PAS of Drosophila Per and Sim, and human Arnt protein. Cotransfection of an expression vector of the Ah receptor with a reporter plasmid pMC6.3k consisting of CYP1A1 promoter and CAT structural gene into CV-1 cells enhanced the CAT expression in response to added 3-methylcholanthrene.     

A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.

Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.     

Three-dimensional solution structure of the B domain of staphylococcal protein A: comparisons of the solution and crystal structures.

The three-dimensional solution structure of the recombinant B domain (FB) of staphylococcal protein A, which specifically binds to the Fc portion of immunoglobulin G, was determined by NMR spectroscopy and hybrid distance geometry-dynamical simulated annealing calculations. On the basis of 692 experimental constraints including 587 distance constraints obtained from the nuclear Overhauser effect (NOE), 57 torsion angle (phi, chi 1) constraints, and 48 constraints associated with 24 hydrogen bonds, a total of 10 converged structures of FB were obtained. The atomic root mean square difference among the 10 converged structures is 0.52 +/- 0.10 A for the backbone atoms and 0.98 +/- 0.08 A for all heavy atoms (excluding the N-terminal segment from Thr1 to Glu9 and the C-terminal segment from Gln56 to Ala60, which are partially disordered). FB is composed of a bundle of three alpha-helices, i.e., helix I (Gln10-His19), helix II (Glu25-Asp37), and helix III (Ser42-Ala55). Helix II and helix III are antiparallel to each other, whereas the long axis of helix I is tilted at an angle of about 30 degrees with respect to those of helix II and helix III. Most of the hydrophobic residues of FB are buried in the interior of the bundle of the three helices. It is suggested that the buried hydrophobic residues form a hydrophobic core, contributing to the stability of FB.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of a ricin mutant showing rescue of activity by a noncatalytic residue.

Ricin A chain is an N-glycosidase which removes a single adenine base from a conservative loop of 28S rRNA, thereby inactivating eukaryotic ribosomes. The mechanism of action has been proposed to include transition-state stabilization of an oxycarbonium ion on the substrate ribose by interaction with Glu 177. Conversion of Glu 177 to Gln reduces activity nearly 200-fold [Ready, M. P., Kim, Y., & Robertus, J. D. (1991) Proteins: Struct., Funct., Genet. 10, 270-278] while conversion to Ala (E177A) reduces activity only 20-fold [Schlossman, D., Withers, D., Welsh, P., Alexander, A., Robertus, J., & Frankel, A. (1989) Mol. Cell. Biol. 9, 5012-5021]. X-ray analysis of the latter mutant protein shows that a residue at the edge of the active site, Glu 208, rotates into the space left vacant by the mutation. Its rearranged carboxylate partially substitutes for that of Glu 177. This is equivalent to the rescue of enzyme activity by a second-site reversion. Kinetic analysis shows the E177A mutation affects kcat and not Km, consistent with the notion that the carboxylate serves in transition-state stabilization.     

Kinetic studies on CO binding to reconstituted myoglobins with four synthetic hemes; structural control in ligand binding to myoglobin.

Examination was made of CO binding reactions to four kinds of modified sperm whale myoglobin (Mb), whose heme was reconstituted by iron complexes of synthetic porphyrins such as porphine (Por), meso-tetramethylporphyrin (TMeP), meso-tetraethylporphyrin (TEtP) and meso-tetra(n-propyl)porphyrin (TnPrP), using flash photolysis and stopped-flow methods. The CO association rate was found to be 5- to 20-times and dissociation rate 10- to 36-times accelerated by replacement with synthetic hemes. These features could be explained based on characteristic structures of modified Mbs indicated by X-ray crystallography. The side chain of Arg-45 protruded from the heme vicinity into the solvent region and heme was tilted by interactions of meso-alkyl side chains with surrounding peptides, resulting in the formation of widely opened channels and pockets for ligand passage. These structural features indicate the CO ligand to more easily enter or exit from heme pockets of reconstituted myoglobins, compared to native Mb.     

Simultaneous convective and diffusive mass transfers in a hemodialyser

The mass transfer in a hemodialyser in the presence of combined dialysis and ultrafiltration has been calculated by integration of mass fluxes across the boundary layers in blood and dialysate phase taking into account the partial rejection of solute as well as changes in local blood flow and ultrafiltration flux along the membrane. Clearances of creatinin, vitamin B12, and myoglobin have been calculated as a function of blood and ultrafiltrate flow rate and were found to be in good agreement with in vitro measurements. The data suggest the following empirical correlation for the hemodiafiltration clearance.     

The primary structure of human Rieske iron-sulfur protein of mitochondrial cytochrome bc1 complex deduced from cDNA analysis

We isolated a cDNA encoding human Rieske Fe-S protein of mitochondrial cytochrome bc1 complex from a fibroblast cDNA library by colony hybridization. The cDNA contains the nucleotide sequence encoding all of the amino acids (274 residues) comprising the putative precursor to the protein. Based on the known amino acid sequence of bovine Rieske Fe-S protein, the N-terminal extension sequence is presumed to be composed of 78 amino acids with a molecular weight of 8053. The mature protein consists of the same number of amino acid residues as that of its rat and bovine counterparts, having a homology of about 92% with the latter.     

Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface.

Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.     

Aconitine-induced modification of single sodium channels in neuroblastoma cell membrane

Aconitine-modified sodium channels in the neuroblastoma cell membrane were investigated with patch-clamp technique in outside-out configuration. When aconitine (0.1 mmol/l) was present in the pipette solution two types of modified single sodium channels were observed. The first type showed openings with normal amplitude (slope conductance 15.5 pS) and bursting behaviour. The second type of modified channel openings was characterized with low amplitude (slope conductance 2.8 pS) and longer open time as comparing to unmodified channels. The low-amplitude channels were shown to have altered ion selectivity: they were permeable to NH4+. Both populations of aconitine-modified channels could be blocked by tetrodotoxin. In contrast to macroscopic current experiments (Mozhayeva et al. 1977) the development of aconitine modification was not affected by repetitive stimulation and external application of the agent had no effect on single sodium channels in outside-out membrane patch.