The nuclear-coded subunits of yeast cytochrome c oxidase. II. The amino acid sequence of subunit VIII and a model for its disposition in the inner mitochondrial membrane

The amino acid sequence of subunit VIII from yeast cytochrome c oxidase is reported. This 47-residue (Mr = 5364) amphiphilic polypeptide has a polar NH2 terminus, a hydrophobic central section, and a dilysine COOH terminus. An analysis of local hydrophobicity and predicted secondary structure along the peptide chain predicts that the hydrophobic central region is likely to be transmembranous. Subunit VIII from yeast cytochrome c oxidase exhibits 40.4% homology to bovine heart cytochrome c oxidase subunit VIIc , at the level of primary structure. Secondary structures and hydrophobic domains predicted from the sequences of both polypeptides are also highly conserved. From the location of hydrophobic domains and the positions of charged amino acid residues we have formulated a topological model for subunit VIII in the inner mitochondrial membrane.     

Deductive analysis of a protein-synthesis mutant of Escherichia coli

A mutant of E. coli, T ^s68b, selected as unable to grow at 43 C, is unable to synthesize proteins at 43 C, though it can carry out this function at 30 C. This defect is shown to be in the protein-synthetic rather than the RNA-synthetic machinery by an analysis of the response of the strain to infection with the RNA bacteriophage f2. An analysis of the capacity for RNA synthesis and the polyribosome content of these cells at 44 C indicates that the defect resides in the elongation step of protein synthesis. No defects could be detected in vitro. The results are discussed in light of similar data on other mutants and in relation to the general approach of analyzing complex mutants selected with ill-defined phenotypes.This work was supported by Grant GM14368 from the National Institute of Health.     

Microbial biomass in a shallow, urban aquifer contaminated with aromatic hydrocarbons: analysis by phospholipid fatty acid content and composition

The city of Perth contains a number of sites that have been contaminated with hydrocarbons due to leakage from petroleum underground storage tanks. Microbial biomass in groundwater and sediment cores from above and below the water table, and from within and outside a plume of hydrocarbon contamination, was examined using phospholipid fatty acid methyl ester analysis. Microbial numbers, calculated from the phospholipid content, ranged from 0·9 × 10⁶ to 7·8 × 10⁶' Escherichia coli equivalent cells' g⁻¹ dry wt of sediment. Over 96% of the microbial biomass was attached to the sediment and the proportion of attached cells did not decrease within the plume of contaminants. The amount of biomass within aquifer samples seemed to be related more to the proximity of the rhizosphere to the shallow aquifer, and other unknown urban inputs, rather than to the effects of the plume of contaminants. Fatty acids common to many bacterial groups dominated within the plume, and as such the analyses gave limited insight into microbial community structure. For site assessment of intrinsic remediation of shallow aquifers in urban areas, estimates of microbial biomass may not provide information that is readily applicable to plume management.     

Ascorbic acid uptake in guinea pig intestinal mucosa

Cellular accumulation of ascorbic acid was investigated in vitro in distal intestinal mucosa of guinea pig. With ¹⁴C-ascorbic acid present at 8 μM/L in the bathing media, tissue/media (T/M) concentration ratios of at least 5 were routinely achieved. Recently absorbed ascorbic acid appeared to be free in solution in the cellular fluid in that it diffused from tissue exposed to poisons with a disappearance half-time of approximately 10 minutes. Ascorbic acid uptake was highly dependent on the presence of sodium in the bathing media; total Tris substitution resulted in a 97% decrease in uptake. Also, metabolically depleted tissue did not accumulate ascorbic acid against a concentration gradient. Uptake of ¹⁴C-ascorbic acid from a bathing solution concentration of 8 μM/L was reduced 67% in the presence of 0.8 mM/L nonlabeled ascorbic acid. Recently absorbed ¹⁴C-ascorbic acid moved more rapidly back into the lumen when the luminal solution contained nonlabeled ascorbic acid (5 mM) than when it contained mannitol (5mM). This demonstration of counter transport substantiates a carrier mechanism in the brush border.     

Toxicity of abamectin to cockroaches (Dictyoptera: Blattellidae, Blattidae).

Abamectin was fed to German cockroaches, Blattella germanica (L.), in non-choice tests. LT50s and LC50s were estimated by probit analysis. The LT50s for the German cockroach ranged from 4.4 to 1.7 d for males, from 9.0 to 2.4 d for females, and from 4.4 to 1.6 d for nymphs for bait concentrations of abamectin between 0.0025 and 0.0500%. The LC50s of abamectin were 0.0110 and 0.0040% from males, 0.0240 and 0.0090% for females, and 0.0200 and 0.0080% for nymphs at 3 and 6 d, respectively. The LT50 values of 0.0550% abamectin bait were 3.4, 3.4, 2.4, 7.5, 2.9, and 4.5 d for Periplaneta americana (L.), P. fuliginosa (Serville), P. brunnea Burmeister, P. australasiae (F.), Blatta orientalis L., and Supella longipalpa (Serville). Although the bait was effective against various cockroach species, time to death for the larger species was longer than for the German cockroach. In preference tests in which male German cockroaches were allowed to feed on rat chow or abamectin bait, all died within 5 d of exposure to abamectin bait. Abamectin bait consumption was not significantly lower than that of untreated rat chow. Arena tests with 0.0550% abamectin bait resulted in 31-75% mortality of German cockroaches after 9 d, with most control being achieved by treating harborages with the bait. The hydramethylnon standard resulted in 65% mortality after 9 d.     

Spectra and structure of alpha-tocopherol radicals produced in anoxic media.

Electron spin resonance and flash photolysis studies have been combined to determine, amid conflicting reports from radiolysis studies, the spectrum for the phenoxyl radical of alpha-tocopherol. Triplet state ketones were used to abstract the alpha-tocopherol phenolic hydrogen in order to obtain both the transient ESR spectrum and optical spectrum. Analysis of the ESR data yields g = 2.00469, a(H, 5-CCH3) = 6.04G, a(H,7-CCH3) = 4.51G, a(H, CH2) = 1.38G, a(H', CH2) = 1.49G, and a(H,8-CCH3) = 0.89G. The g factor shows large spin population on oxygen in this radical and demonstrates conclusively involvement of the phenoxyl radical. Both spectra were in agreement with those produced by radiolysis of alpha-tocopherol in N2 saturated EtOH. While the spectral characteristics of the phenoxyl radical now appear to be clarified, uncertainties remain concerning optical spectra for radiolysis of alpha-tocopherol in air- and oxygen-containing systems.     

Plasminogen activator: The major secreted neutral protease of cultured skeletal muscle cells

Clonal mouse skeletal muscle cells which differentiate in culture and from synpases with neuronal cells were found to secrete high levels of protease activity as measured with an ¹²⁵I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean typsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occured with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromusclar contacts in experimental and pathologic denervation.