The correlation between bismuth and uranyl staining and phosphorus content of intracellular structures as determined by electron spectroscopic imaging

Four groups of intracellular structures can be recognized according to bismuth and uranyl staining and phosphorus content. (1) Those which contain phosphorus and stain strongly with uranyl acetate but not with bismuth (ribosomes, heterochromatin and mature ribosomal precursor granules), presumably because of their nucleic acid content. (2) Those which contain phosphorus and stain with uranyl acetate and bismuth (interchromatin granules, immature ribosomal precursor granules and mitochondrial granules), presumably because at least some of their phosphate is available to react with bismuth. (3) Those which contain little phosphorus but which stain strongly with bismuth and weakly with uranyl acetate (Golgi complex beads), perhaps because some ligand in addition to phosphate reacts with bismuth, and (4) those which do not contain phosphorus and stain with neither uranyl acetate nor bismuth (portasomes). Uranyl staining correlates strongly with the phosphorus content of nucleic acids, proteins and inorganic deposits. Bismuth will stain some phosphorylated molecules but not all. Thus only some phosphates stain with bismuth.     

A vibrational study of the CD2 stretching bands of selectively deuterated palmitic and stearic acids

The C-D stretching regions of the infrared and Raman spectra of 14 different palmitic and stearic acids containing isolated CD₂ groups are reported. Anomalous behaviour is observed when substitution occurs near the terminal methyl group, which behaviour can not be explained in terms of crystal field effects.     

Multiple mechanisms of cytochrome P450-catalyzed substrate hydroxylations

We have examined the 5-$\text{exo}$-hydroxylation of camphor by cytochrome P450 in [¹⁸O] water/buffer solution. In the $\text{NADH}\text{O}{}_2\text{-dependent}$ reaction of the reconstituted multienzyme system, no ¹⁸O-label is observed in the product alcohol. Similarly, in the m-chloroperbenzoic acid or cumene hydroperoxide supported reactions with ferric P450, solvent oxygen is not incorporated into hydroxycamphor. When the analagous reaction is carried out using iodosobenzene as the exogenous oxidant, however, the alcoholic oxygen of the product is derived entirely from the solvent. These results cannot be explained by equilibration of the iodosobenzene oxygen with solvent water before reacting with P450, and suggest a unique mechanism for iodosobenzene-supported P450 oxygenations. We propose two distinct mechanistic activities for cytochrome P450: a hydroxylase, and an oxene transferase, with the former encompassing the classic oxygenase as well as “peroxygenase” reactions.     

A general synthesis of long chain ω- and (ω-1)-hydroxy fatty acids

A method for the synthesis of long chain fatty acids substituted at the ω and ω-1 positions has been developed. The key step is the isomerization of the triple bond of an alkyn-1-ol from an internal position in the chain to the free terminus with a new, convenient reagent, sodium aminopropylamide (NaAPA). Standard functional group manipulations i.e., Jones oxidation, esterification and hydroboration of the triple bond are used to prepare ω-hydroxy fatty esters. The generality of the method is illustrated with syntheses of ω-hydroxy fatty esters with 24, 26, 28 and 30 carbon chains.In the 24 carbon series, hydration of the terminal triple bond of alkynoic ester 4a followed by reduction gave the (ω-1)-hydroxy ester.     

Studies on the origin of the near-infrared (800–900 nm) absorption of cytochrome c oxidase

Data are presented which were collected in the course of the past ten years and bear on the correlation of absorbance at 800 nm and the EPR signal at g = 2 (‘copper signal’) of cytochrome c oxidase in various states of oxidation and ligation. Both EPR and optical reflectance spectra were obtained at low temperature (?170 to ?190°C). For some sets of samples spectra were recorded in the range 500–1100 nm. A particular effort was made to study this correlation with what are called ‘mixed valence’ states (Greenwood, C., Wilson, M.T. and Brunori, M. (1974) Biochem. J. 137, 205–215), when cytochrome a and the EPR-detectable copper are thought to be oxidized and the other components reduced and vice versa. These data show no evidence that the copper component of cytochrome oxidase which has so far not been detected by EPR makes a contribution to the absorption between 800 and 900 nm exceeding 10–15% of the total, which is close to or within the error of the respective measurements. For the various states of the oxidase examined in this work the 700–800 nm region did not appear to be more useful than the 800–900 nm region for determining the state of the EPR-undetectable copper in a reliable way. These conclusions are in agreement with results presented previously from other laboratories concerning the relationship of optical (approx. 800 nm) and EPR spectroscopic (g = 2) data obtained with the enzyme.     

Serendipitous syntheses of Rh(H)2Cl(PRPh2)3 complexes, and their crystal structures, where R = Me, Cy (cyclohexyl)

Reactions of RhCl(cod)(THP) (cod = 1,5-cyclooctadiene; THP = P(CH₂OH)₃) with PMePh₂ or PCyPh₂ (Cy = cyclohexyl) in acetone/MeOH solution under H₂ surprisingly form the complexes cis, mer-Rh(H)₂Cl(PRPh₂)₃ (R = Me or Cy); both complexes are characterized by crystallography (the first structures in which the hydride ligands of such dihydrido-chloro-trisphosphine complexes have been located), and by detailed ¹H and ³¹P NMR spectroscopy. The key role of the THP in the observed chemistry is discussed.     

Dielectric Spectroscopy: a Rapid Method for the Determination of Solvent Biocompatibility During Biotransformations

Dielectric spectroscopy provides a convenient means of determining the degree of intactness of biological cells. 4-terminal dielectric measurements of suspensions of Saccharomyces cerevisiae at 0.4 MHz show that, as with all other biological cells, these organisms possess a substantial β-dispersion. The additional of octanol to such suspensions causes a rapid decrease in the electrical capacitance of the suspension, which parallels the cellular viability as determined by methylene blue staining. The kinetics of cell death are determined in part by the rate of dissolution of the organic solvent in the aqueous phase. The toxicity of several organic solvents to S. cerevisiae is studied using this technique, and is found to be dependent upon the polarity of the solvent. The present method provides a simple and rapid means for assessing the biocompatibility of solvents used in biotransformations.     

Curium(III) complexation with pyoverdins secreted by a groundwater strain of Pseudomonas fluorescens

Pyoverdins, bacterial siderophores produced by ubiquitous fluorescent Pseudomonas species, have great potential to bind and thus transport actinides in the environment. Therefore, the influence of pyoverdins secreted by microbes on the migration processes of actinides must be taken into account in strategies for the risk assessment of potential nuclear waste disposal sites. The unknown interaction between curium(III) and the pyoverdins released by Pseudomonas fluorescens (CCUG 32456) isolated from the granitic rock aquifers at the Äspö Hard Rock Laboratory (Äspö HRL), Sweden, is the subject of this paper. The interaction between soluble species of curium(III) and pyoverdins was studied at trace curium(III) concentrations (3 × 10^?7 M) using time-resolved laser-induced fluorescence spectroscopy (TRLFS). Three Cm³⁺—P. fluorescens (CCUG 32456) pyoverdin species, M_pH_qL_r, could be identified from the fluorescence emission spectra, CmH₂L⁺, CmHL, and CmL^?, having peak maxima at 601, 607, and 611 nm, respectively. The large formation constants, log β₁₂₁ = 32.50 ± 0.06, log β₁₁₁ = 27.40 ± 0.11, and log β₁₀₁ = 19.30 ± 0.17, compared to those of other chelating agents illustrate the unique complexation properties of pyoverdin-type siderophores. An indirect excitation mechanism for the curium(III) fluorescence was observed in the presence of the pyoverdin molecules.     

Copper induced oxidation of serotonin: analysis of products and toxicity

Serotonin is a major neurotransmitter that controls many functions, ranging from mood and behaviour through to sleep and motor functions. The non-enzymatic oxidation of serotonin is of significant importance as some oxidation products are considered to be neurotoxic. An interaction between copper and serotonin has been suggested by symptoms observed in a number of neurodegenerative diseases such as Wilson's and Prion diseases. Using PC12 cells as a model of neuronal cells, we show that the interaction between copper and serotonin is toxic to undifferentiated cells. The toxicity is largely due to reactive oxygen species as cell death is significantly reduced in the presence of the antioxidant mannitol. Differentiation of the PC12 cells also confers resistance to the oxidative process. In vitro oxidation of serotonin by copper results in the eventual formation of a coloured pigment, thought to be a melanin-like polymeric species. Using spectroscopic methods we provide evidence for the formation of a single intermediate product. This dimeric intermediate was identified and characterized as 5,5'-dihydroxy-4,4'-bitryptamine. These results indicate that copper structurally alters serotonin and this process may play a role in copper related neurodegenerative diseases.