Retinyl palmitate is non-genotoxic in Chinese hamster ovary cells in the dark or after pre-irradiation or simultaneous irradiation with UV light

Retinyl palmitate (RP), an ingredient of cosmetic and medical skin-care preparations, has been reported to be photo-genotoxic/photo-clastogenic in mouse lymphoma cells (Tk locus) as well as in human Jurkat T-cells, as measured by use of the comet assay. Given that these results were obtained under exploratory conditions, we re-investigated the photo-genotoxicity of RP following a protocol consistent with current recommendations for photo-genotoxicity testing of drugs and chemicals. We tested RP in Chinese hamster ovary (CHO) cells in the dark (standard chromosome aberration test), under pre-irradiation (UVA irradiation of cells and subsequent treatment with RP) or simultaneous irradiation (irradiation of cells and RP together, standard photo-genotoxicity protocol) conditions. UVA irradiation was applied at 350 and 700 mJ/cm2 with the high UV dose targeted to produce a small increase in the incidence of structural chromosome aberrations (CA) in cells not treated with RP. RP was tested up to and above its limit of solubility in the culture medium (20-40 μg/mL). There was no overt cytotoxicity under dark or different irradiation conditions. Treatment of cells with RP in the dark, as well as treatment under pre- or simultaneous irradiation conditions failed to produce biologically significant increases in the incidence of CA, whereas the positive control substances 4-nitroquinolone and 8-methoxypsoralene produced significantly positive effects in the dark or under simultaneous irradiation, respectively. Overall, our results failed to confirm the reported positive photo-genotoxic effects, and suggest that they may have been due to the test conditions, i.e. high irradiation doses, high cytotoxicity or re-irradiation of photo-products. In conclusion, our data suggest that, under standard conditions for testing photo-genotoxicity, RP had no in vitro genotoxic or photo-genotoxic potential and is therefore unlikely to pose a local or systemic genotoxic or photo-genotoxic risk.     

Comparative sequence analysis of the complete set of 40S ribosomal proteins in the Senegalese sole (Solea senegalensis Kaup) and Atlantic halibut (Hippoglossus hippoglossus L.) (Teleostei: Pleuronectiformes): phylogeny and tissue- and development-specific expression

Background  

Ribosomal proteins (RPs) are key components of ribosomes, the cellular organelle responsible for protein biosynthesis in cells. Their levels can vary as a function of organism growth and development; however, some RPs have been associated with other cellular processes or extraribosomal functions. Their high representation in cDNA libraries has resulted in the increase of RP sequences available from different organisms and their proposal as appropriate molecular markers for phylogenetic analysis.     

Electrophoretic study of cationic cobalt(III) complexes with [N2O] and [N2O2] Schiff base ligands

The electrophoretic migration behavior of acid-sensitive cationic alkylcobalt(III) complexes with tridentate Schiff bases and amines as well as that of related ‘inorganic’ cobalt(III) chelates with tridentate and tetradentate Schiff bases was studied. A correlation of the electrophoretic mobility of the organocobalt complexes in question with their structure was established. An attempt to optimize the analytical procedures for capillary electrophoretic separation of these rather labile complex cations is outlined. Their decomposition in solutions under ambient conditions was surveyed using this technique.     

The pyruvate, orthophosphate dikinase regulatory proteins of Arabidopsis are both bifunctional and interact with the catalytic and nucleotide-binding domains of pyruvate, orthophosphate dikinase

Pyruvate orthophosphate dikinase (PPDK) is a key enzyme in C(4) photosynthesis and is also found in C(3) plants. It is post-translationally modified by the PPDK regulatory protein (RP) that possesses both kinase and phosphotransferase activities. Phosphorylation and dephosphorylation of PPDK lead to inactivation and activation respectively. Arabidopsis thaliana contains two genes that encode chloroplastic (RP1) and cytosolic (RP2) isoforms of RP, and although RP1 has both kinase and phosphotransferase activities, to date RP2 has only been shown to act as a kinase. Here we demonstrate that RP2 is able to catalyse the dephosphorylation of PPDK, although at a slower rate than RP1 under the conditions of our assay. From yeast two-hybrid analysis we propose that RP1 binds to the central catalytic domain of PPDK, and that additional regions towards the carboxy and amino termini are required for a stable interaction between RP2 and PPDK. For 21 highly conserved amino acids in RP1, mutation of 15 of these reduced kinase and phosphotransferase activity, while mutation of six residues had no impact on either activity. We found no mutant in which only one activity was abolished. However, in some chimaeric fusions that comprised the amino and carboxy termini of RP1 and RP2 respectively, the kinase reaction was severely compromised but phosphotransferase activity remained unaffected. These findings are consistent with the findings that both RP1 and RP2 modulate reversibly the activity of PPDK, and possess one bifunctional active site or two separate sites in close proximity.     

Erwinia carotovora as a recipient system for the natural hybrid plasmid RP4::Mu cts62

The plasmid RP4::Mu cts62 is transferred from Escherichia coli cells into a recipient strain Erwinia carotovora 268 by conjugation with the frequency 1.5-5 x 10(-7) per donor cell. The maximal frequencies of transfer are obtained by cultivation of donor and recipient cells for 3-5 h on the filters. Structural and functional validity of the plasmid in transconjugants is expressed in preservation of all antibiotic-resistant markers of RP4, thermosensitivity to growth at 42 degrees C as well as spontaneous and thermally-induced production and zygotic induction of bacteriophage determined by the genome of Mu cts62, total length of the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid restricts. Location and orientation of Mu cts62 genome in the plasmid RP4::Mu cts62 in Erwinia carotovora transconjugant cells has been determined. A single bacteriophage genome has been shown to transpose into the chromosome of the cell with the elimination of RP4 fragment under the conditions of thermal induction.     

Equilibrium and structure of thallium(III)–ethylenediamine complexes in pyridine solution and in solid

The formation of three [Tl(en)_n]³⁺ complexes (n=1–3) in a pyridine solvent has been established by means of ²⁰⁵Tl and ¹H NMR. Their stepwise stability constants based on concentrations, K_n=[Tl(en)_n ³⁺]/{[Tl(en)_n−1 ³⁺]·[en]}, at 298 K in 0.5 M NaClO₄ ionic medium in pyridine, were calculated from ²⁰⁵Tl NMR integrals: log K₁=7.6±0.7; log K₂=5.2±0.5 and log K₃=2.64±0.05. Linear correlation between both the ²⁰⁵Tl NMR shifts and spin–spin coupling ²⁰⁵Tl–¹H versus the stability constants has been found and discussed. A single crystal with the composition [Tl(en)₃](ClO₄)₃ was synthesized and its structure determined by X-ray diffraction. The Tl³⁺ ion is coordinated by three ethylenediamine ligands via six N-donor atoms in a distorted octahedral fashion.     

The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA₂ and lyso-PLA₂ activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA₁ activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.     

SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

The axial channel of the proteasome core particle is gated by the Rpt2 ATPase and controls both substrate entry and product release.

Substrates enter the proteasome core particle (CP) through a channel that opens upon association with the regulatory particle (RP). Using yeast mutants, we show that channel opening is mediated by the ATPase domain of Rpt2, one of six ATPases in the RP. To test whether degradation products exit through this channel, we analyzed their size distribution. Their median length from an open-channel CP mutant was 40% greater than that from the wild-type. Thus, channel opening may enhance the yield of peptides long enough to function in antigen presentation. These experiments demonstrate that gating of the RP channel controls both substrate entry and product release, and is specifically regulated by an ATPase in the base of the RP.     

Cutting edge: regulation of TLR4-driven B cell proliferation by RP105 is not B cell autonomous

Mechanistic understanding of RP105 has been confounded by the fact that this TLR homolog has appeared to have opposing, cell type-specific effects on TLR4 signaling. Although RP105 inhibits TLR4-driven signaling in cell lines and myeloid cells, impaired LPS-driven proliferation by B cells from RP105(-/-) mice has suggested that RP105 facilitates TLR4 signaling in B cells. In this article, we show that modulation of B cell proliferation by RP105 is not a function of B cell-intrinsic expression of RP105, and identify a mechanistic role for dysregulated BAFF expression in the proliferative abnormalities of B cells from RP105(-/-) mice: serum BAFF levels are elevated in RP105(-/-) mice, and partial BAFF neutralization rescues aberrant B cell proliferative responses in such mice. These data indicate that RP105 does not have dichotomous effects on TLR4 signaling and emphasize the need for caution in interpreting the results of global genetic deletion.     

Effect of retinyl palmitate and 13-cis retinoic acid on immune functions in immunodeficient, nude mice

Nude mice are deficient in thymus gland development and hence lacking functional, mature T-lymphocytes. Weanling nude mice were given various deficient and high retinyl palmitate (RP) or 13-cis retinoic acid (CRA) diets. The high RP (vitamin A) diets stimulated phagocytosis in the absence of mature T-helper cells. However, T-cell dependent mitogens did not cause significant mitogenesis in any group, while LPS, a B-cell mitogen, did. RP had no effect on mitogenesis. NK cell activity was increased only at a very high level of RP, as has been reported with conventional mice. Macrophage production of cytotoxic factors was unaffected by high levels of RP or CRA. Direct cytotoxicity in vitro of tumor cells was increased only at very high RP levels. Thus, mature T cells may be needed for RP to produce normal activation of macrophage, except at very high RP levels.     

Lipid peroxidation rate and the antioxidant protection system during the readaptation period after long-term space flights on board the International Space Station

The content of lipid peroxidation (LPO) products (diene conjugates (DC), malondialdehyde (MDA), Schiff bases (SB), and tocopherol (TP, a main lipid antioxidant) were measured in blood serum of 17 astronauts taking part in long-term (125–217 days) missions on board the International Space Station (ISS) during the preflight period, on the day of the landing, and on the 7th and 14th days after landing (the rehabilitation period, RP). A decrease in the DC and MDA levels against a background of an increase in TP has been found in a group of eight astronauts after landing on board the Space Shuttle spacecraft and a group of eight astronauts after a space flight on board the Soyuz TM in the course of RP. The changes in measured indices were more pronounced in the group of astronauts after the space flight on board the Space Shuttle spacecraft. Inhibition of LPO during RP was regarded as an adequate response to readaptation stress to the conditions on earth. The possible mechanisms of differences in the efficiency of LPO inhibition between groups are discussed: the changes in the biomembrane phase state under the conditions of deceleration load during disorbiting and the stressful reaction to landing on board different spacecrafts.     

Comparison in halide binding ability between the unsaturated clusters [Pd3(μ-dppm)3(CO)] and [PdPtCo(μ-dppm)2(CO)3(CNBu)] (dppm = Ph2PCH2PPh2)

The trinuclear clusters [Pd₃(μ-dppm)₃(CO)]²⁺ and [PtPdCo(μ-dppm)₂(CO)₃(CN^tBu)]⁺ exhibit a large and a small cavity, respectively, formed by the phenyl rings of the bridging diphosphine ligands. Their binding constants (K₁₁) with halide ions (X⁻) were obtained by UV-Vis spectroscopy. The binding ability varies as I⁻ > Br⁻ > Cl⁻, and [Pd₃(μ-dppm)₃(CO)]²⁺ > [ptPdCo(μ-dppm)₂-(CO)₃(CN^tBu)]⁺. The MO diagram for the related cluster [Pd₂Co(μ-dppm)₂(CO)₄]⁺ has been addressed theoretically in order to predict the nature of the lowest energy electronic bands. For this class of compounds, the lowest energy bands are assigned to charge transfers from the Co center to the Pd₂ centers.     

Polynuclear chromium(III) carboxlylates: Part 2. Chromium(III) acetate — what's in it?

Chromium(III) acetate has been widely used in industry for decades. The commercial material is an ill-defined substance, which represents a large number of compounds having different compositions, physical properties and appearances. Several samples of Cr(III) acetate, from various commercial sources were examined by ion-exchange chromatography. All the samples were found to contain several species such as [Cr₃O(O₂CCH₃)₆(H₂O)₃]⁺ and other positively charged purple complexes. They also contain various amounts of the neutral violet complex [Cr₈(OH)₈(O₂CCH₃)₁₆] (1) which crystallizes upon slow evaporation of its aqueous solution. 1 is a cyclic octanuclear complex with hydroxo and acetate ligands bridging the adjacent Cr(III) ions. The structure of a well-defined Cr(III) acetate, namely, [Cr(H₂O)₆](O₂CCH₃)₃ (2) has been determined crystallographically and its decomposition products were examined by ion-exchange chromatography. Compound 2 decomposes under ambient conditions, releasing acetic acid and water producing neutral and charged polynuclear Cr(III) complexes.     

Evidence for a major retinitis pigmentosa locus on 19q13.4 (RP11) and association with a unique bimodal expressivity phenotype.

Retinitis pigmentosa (RP) is the name given to a heterogeneous group of retinal degenerations mapping to at least 16 loci. The autosomal dominant form (ARP), accounting for approximately 25% of cases, can be caused by mutations in two genes, rhodopsin and peripherin/RDS, and by at least six other loci identified by linkage analysis. The RP11 locus for adRP has previously been mapped to chromosome 19q13.4 in a large English family. This linkage has been independently confirmed in a Japanese family, and we now report three additional unrelated linked U.K. families, suggesting that this is a major locus for RP. Linkage analysis in the U.K. families refines the RP11 interval to 5 cM between markers D19S180 and AFMc001yb1. All linked families exhibit incomplete penetrance; some obligate gene carriers remain asymptomatic throughout their lives, whereas symptomatic individuals experience night blindness and visual field loss in their teens and are generally registered as blind by their 30s. This "bimodal expressivity" contrasts with the variable-expressivity RP mapping to chromosome 7p (RP9) in another family, which has implications for diagnosis and counseling of RP11 families. These results may also imply that a proportion of sporadic RP, previously assumed to be recessive, might result from mutations at this locus.     

Effect of controlled redox potential and dissolved oxygen on the in vitro refolding of an E. coli alkaline phosphatase and chicken lysozyme

The development of efficient purification strategies of recombinant active protein derived from inclusion bodies requires the knowledge of the effect of environmental variables, such as redox potential (RP) and dissolved oxygen tension (DOT), in order to control the protein folding process. However, that information is scarce and only few in vitro studies of the impact of such variables have been reported under constant controlled conditions. In this work, the effect of controlled RP and DOT on the refolding of E. coli alkaline phosphatase (AP) and chicken lysozyme (CL) enzymes were studied. Disulphide bonds of both enzymes were reduced in an instrumented vessel using 2-mercaptoethanol and nitrogen. In the latter case, guanidine hydrochloride was also used to denature the protein. Such conditions caused protein conformational changes, as determined by the intrinsic fluorescence spectra that correlated with a decrease on the activity in both cases. Reduced enzymes were then oxidized, under different constant and predetermined RP or DOT, by manipulating the gas composition in the vessel. Folding kinetics were followed as the recovery of enzyme activity. Results showed that the percentage of recovery and rate of increase of enzymatic activity directly depended on the RP and DOT. A higher folding efficiency was found under controlled DOT compared to controlled RP conditions. These results are useful for establishing protein folding strategies to improve the recovery of active protein from inclusion bodies.     

Simultaneous high-performance liquid chromatographic determination of quinupristin, dalfopristin and their main metabolites in human plasma

Quinupristin–dalfopristin (30:70, w/w) is a new streptogramin, which has been developed for intravenous use. A specific and sensitive HPLC method was developed to measure simultaneously quinupristin (RP 57669) and dalfopristin (RP 54476) and their main metabolites in human plasma. The metabolites measured by this method were RP 69012 (glutathione-conjugated) and RPR 100391 (cysteine-conjugated) from quinupristin and RP 12536 (natural pristinamycin IIA), from dalfopristin. Solid-phase extraction with disposable cartridges was combined with reversed-phase HPLC and fluorimetric detection for RP 57669, RP 69012 and RPR 100391 and UV detection for RP 54476 and RP 12536. The method provided good recovery and low limits of quantitation (0.025 mg l⁻¹ for RP 57669, RP 54476 and RP 12536, and of 0.010 mg l⁻¹ for RP 69012 and RPR 100391). The validated range of concentrations of the method was: 0.025–5000 mg l⁻¹ for RP 57669, RP 54476 and RP 12536 and 0.010–0.750 mg l⁻¹ for RP 69012 and RPR 100391.