Chemical and spectroscopic evidence for the formation of a ferryl Fea3 intermediate during turnover of cytochrome c oxidase

When partially reduced cytochrome c oxidase samples are reoxidized with dioxygen, an EPR-silent dioxygen intermediate, which is at the three-electron level of dioxygen reduction, is trapped at the dioxygen reduction site. The intermediate has novel spectral features at 580 and 537 nm. Combined optical and EPR results reveal that this intermediate reacts rapidly with CO at 277-298 K causing the abolition of the 580/537 mm features and the appearance of a rhombic CuB EPR signal. A ferryl Fea3, or an intermediate at the same formal level of oxidation, is proposed to oxidize CO to CO2 producing an EPR-detectable CuB adjacent to a low-spin ferrous Fea3-dioxygen (or carbon monoxide) adduct.     

Carbon isotopic fractionation in heterotrophic microbial metabolism.

Differences in the natural-abundance carbon stable isotopic compositions between products from aerobic cultures of Escherichia coli K-12 were measured. Respired CO2 was 3.4% depleted in 13C relative to the glucose used as the carbon source, whereas the acetate was 12.3% enriched in 13C. The acetate 13C enrichment was solely in the carboxyl group. Even though the total cellular carbon was only 0.6% depleted in 13C, intracellular components exhibited a significant isotopic heterogeneity. The protein and lipid fractions were -1.1 and -2.7%, respectively. Aspartic and glutamic acids were -1.6 and +2.7%, respectively, yet citrate was isotopically identical to the glucose. Probable sites of carbon isotopic fractionation include the enzyme, phosphotransacetylase, and the Krebs cycle.     

A three-dimensional computational simulation of some sources of measurement artifact in microvascular pulsed ultrasound Doppler velocimetry

Recent applications of 20 MHz pulsed ultrasound Doppler velocimetry (PUDVM) in microsurgical research have necessarily employed piezoelectric crystals whose diameter is not negligible compared to the lumen size (1-2 mm) of many vessels of interest. A three-dimensional numerical model was developed to explore relationships between actual and detected flow field parameters, for (steady) Poiseuille flow, when appreciable velocity gradients exist within the PUDVM sample volume. Validation studies showed that highly accurate velocity profiles could be obtained in the limiting case of a very small sample volume (0.1 mm radius), but that for currently employed crystals (approximately equal to 0.5 mm radius) there was appreciable underestimation of the centersteam velocity, and appreciable overestimation of the flow stream diameter. Errors in perceived velocity and flow rate were found to be relatively insensitive to perturbations in the sample volume thickness, in the size of the sampling range increment, or in the angle of insonation beam divergence. By contrast, these apparent flow parameters were found to be very sensitive to perturbations of sample volume diameter or of the Doppler angle. Small variations in the degree of partial sample volume overlap of the flowstream periphery were shown to be capable of causing large fluctuations in apparent flow stream diameter.     

Deurithitrema gingae n.g., n.sp. (Digenea: Plagiorchioidea) from the kidneys of a salt-water crocodile (Crocodylus porosus Schneider) from Australia

Summary The name Deurithitrema gingae n. g., n. sp. is proposed for worms from the kidneys of one Crocodylus porosus from Darwin, Northern Territory, Australia. The following combination of characters distinguishes the new genus from all others: prepharynx very short; oesophagus absent; caeca reaching to posterior end of body; testes tandem in middle third of body, contiguous or amost so; cirrus-sac large, over-reaches ventral sucker, contains bipartite seminal vesicle, prostatic regions and ejaculatory duct with spines around its opening into genital atrium; genital pore immediately in front of ventral sucker; uterus coiled to posterior end of body; vitellaria lateral from ventral sucker to slightly behind testes; excretory vesicle small, receiving a single ventro-lateral duct from each side behind end of uterine loops. Deurithitrema belongs to the superfamily Plagiorchioidea Dollfus, 1930. It superficially resembles a number of genera, but its relationships within the superfamily are obscure.     

Electron spin relaxation of CuA and cytochrome a in cytochrome c oxidase. Comparison to heme, copper, and sulfur radical complexes

The method of continuous saturation has been used to measure the electron spin relaxation parameter T1T2 at temperatures between 10 and 50 K for a variety of S = 1/2 species including: CuA and cytochrome a of cytochrome c oxidase, the type 1 copper in several blue copper proteins, the type 2 copper in laccase, inorganic Cu(II) complexes, sulfur radicals, and low spin heme proteins. The temperature dependence and the magnitude of T1T2 for all of the species examined are accounted for by assuming that the Van Vleck Raman process dominates the electron spin-lattice relaxation. Over the entire temperature range examined, the relaxation of the type 1 coppers in six to seven times faster than that of type 2 copper, inorganic copper, and sulfur radicals, in spite of the similar g-anisotropies of these species. This result may indicate that the coupling of the phonon bath to the spin center is more effective in type 1 coppers than in the other complexes studied. The relaxation of CuA of cytochrome oxidase exhibits an unusual temperature dependence relative to the other copper complexes studied, suggesting that the protein environment of this center is different from that of the other copper centers studied and/or that CuA is influenced by a magnetic dipolar interaction with another, faster-relaxing paramagnetic site in the enzyme. A comparison of the saturation characteristics of the CuA EPR signal in native and partially reduced CO complexes of the enzyme also suggests the existence of such an interaction. The implications of these results with respect to the disposition of the metal centers in cytochrome oxidase are discussed.     

Physiology of F-Pilin Synthesis and Utilization

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to study the synthesis and turnover of F-pilin in membrane preparations of Escherichia coli K-12 under conditions which have been reported to affect the production of F-pili. Incorporation of [³⁵S]methionine into membrane F-pilin by cells in log phase was barely detectable at 25°C, but increased with temperature. The labeled pilin band was prominent in membranes from 37°C cultures and even more prominent if the growth temperature was raised to 42°C. The appearance of other tra products in the membranes was similarly temperature dependent. In cultures grown in glucose minimal medium at 37°C, the relative amount of membrane pilin and traT product synthesis remained unchanged from early log phase through early stationary phase; provision of glycerol or arabinose as a substitute carbon source had no obvious effect. Turnover of traT product and membrane F-pilin, as assessed in an Flac tra mutant strain which is incapable of elaborating pili, was not rapid. Both traT product and pilin subunits labeled in mid-log phase cells were still apparent in the membranes after growth of the cells to stationary phase. The relative amount of labeled pilin decreased with prolonged incubation in stationary phase, but the relative amount of traT product did not decrease even after the culture was incubated for 24 h. When wild-type Flac piliated cells were used, a similar result was obtained, but in this case, loss of F-pilin from the preparations could be acclerated by blending the cells. Although intermittent blending during culture growth caused a slow depletion of the labeled pilin pool, continuous blending resulted in the rapid disappearance of this pool from our preparations. Loss of other membrane polypeptides was not accelerated by our blending procedure, and blending did not affect the turnover of the pilin pool of the Flac tra mutant. Our data are consistent with a model in which pilin subunits are assembled transiently into pili, conserved by retraction, and made available for subsequent reassembly. Growth in 0.01% sodium dodecyl sulfate did not accelerate loss of pilin from the Flac strain compared with the Flac tra strain, and we suggest that in the presence of sodium dodecyl sulfate at this concentration, F-pili are not elaborated from cell surfaces.     

Some characteristics of monolayers of 1-palmitoyl-2-oleoyl-phosphatidylglycerol with and without dipalmitoylphosphatidylcholine during dynamic compression and expansion

Some properties of monolayers of $\text{1-}\text{palmitoyl}\text{-2-}\text{oleoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phospho}\text{-rac-}\text{glycerol}$ (POPG) alone or of POPG in mixtures with $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) have been measured near 35°C during dynamic compression and expansion at 3.6 cm²·s^?1. (2) The mean values of minimum surface tension (corresponding to maximum surface pressure) which could be obtained with pure POPG monolayers at high compression ranged from 15 to 18 mN·m^?1 in the presence of Na⁺, Ca²⁺ or low pH (2.0) in the subphase. (3) The presence of Ca²⁺ or low pH in the subphase increased the collapse plateau ratios obtained on cyclic compression. This might represent enhanced respreading into the monolayer of pure POPG from a collapsed form during reexpansion of the surface. (4) Monolayers containing 10% or 30% POPG and 90% or 70% DPPC could be compressed to surface tensions approaching zero. (5) In such mixed monolayers, 10% or 30% POPG did not appear to enhance respreading, as measured by collapse plateau ratios, in the presence of Na⁺ or Ca²⁺ in the subphase.     

Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

Blastomere ablation and the developmental origin of identified monoamine-containing neurons in the leech

Ablation of different identifiable blastomeres of the early embryo of the leech Helobdella triserialis was found to lead to the absence of different sets of segmentally iterated monoamine-containing neurons in subsequent development. Thus the ablation of one of the paired N ectoteloblasts leads to the absence of one member of each of the three bilateral pairs of serotonin-containing neurons (one of which is the Retzius cell) from each segmental ganglion. The ablation of one of the paired OP blastomeres (precursors of the paired O and P ectoteloblasts) leads to the absence of one member of each of the two bilateral pairs of lateral dopamine-containing neurons that lie in the body wall of each segment. And the ablation of one of the paired Q ectoteloblast leads to the absence of one member of the bilateral pair of medial dopamine-containing neurons that lie in the body wall of each segment. These results suggest that each of these sets of monoamine-containing neurons is derived from a particular blastomere. Upon ablation of that blastomere the set does not develop from any other source.