Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(I)-carbonyl complex.

We have developed a technetium labeling technology based on a new organometallic chemistry, which involves simple mixing of the novel reagent, a 99m Tc(I)-carbonyl compound, with a His-tagged recombinant protein. This method obviates the labeling of unpaired engineered cysteines, which frequently create problems in large-scale expression and storage of disulfide-containing proteins. In this study, we labeled antibody single-chain Fv fragments to high specific activities (90 mCi/mg), and the label was very stable to serum and all other challenges tested. The pharmacokinetic characteristics were indistinguishable from iodinated scFv fragments, and thus scFV fragments labeled by the new method will be suitable for biodistribution studies. This novel labeling method should be applicable not only to diagnostic imaging with 99mTc, but also to radioimmunotherapy approaches with 186/188 Re, and its use can be easily extended to almost any recombinant protein or synthetic peptide.

Exploring the space of histidine containing dipeptides in search of novel efficient RCS sequestering agents.

The study reports a set of forty proteinogenic histidine-containing dipeptides as potential carbonyl quenchers. The peptides were chosen to cover as exhaustively as possible the accessible chemical space, and their quenching activities toward 4-hydroxy-2-nonenal (HNE) and pyridoxal were evaluated by HPLC analyses. The peptides were capped at the C-terminus as methyl esters or amides to favor their resistance to proteolysis and diastereoisomeric pairs were considered to reveal the influence of configuration on quenching. On average, the examined dipeptides are less active than the parent compound carnosine (βAla + His) thus emphasizing the unfavorable effect of the shortening of the βAla residue as confirmed by the control dipeptide Gly-His. Nevertheless, some peptides show promising activities toward HNE combined with a remarkable selectivity. The results emphasize the beneficial role of aromatic and positively charged residues, while negatively charged and H-bonding side chains show a detrimental effect on quenching. As a trend, ester derivatives are slightly more active than amides while heterochiral peptides are more active than their homochiral diastereoisomer. Overall, the results reveal that quenching activity strongly depends on conformational effects and vicinal residues (as evidenced by the reported QSAR analysis), offering insightful clues for the design of improved carbonyl quenchers and to rationalize the specific reactivity of histidine residues within proteins.

Burkholderia sartisoli sp. nov., isolated from a polycyclic aromatic hydrocarbon-contaminated soil.

A Gram-negative, rod-shaped, aerobic bacterium, designated strain RP007(T), was isolated from a polycyclic aromatic hydrocarbon-contaminated soil in New Zealand. Two additional strains were recovered from a compost heap in Belgium (LMG 18808) and from the rhizosphere of maize in the Netherlands (LMG 24204). The three strains had virtually identical 16S rRNA gene sequences and whole-cell protein profiles, and they were identified as members of the genus Burkholderia, with Burkholderia phenazinium as their closest relative. Strain RP007(T) had a DNA G+C content of 63.5 mol% and could be distinguished from B. phenazinium based on a range of biochemical characteristics. Strain RP007(T) showed levels of DNA-DNA relatedness towards the type strain of B. phenazinium and those of other recognized Burkholderia species of less than 30 %. The results of 16S rRNA gene sequence analysis, DNA-DNA hybridization experiments and physiological and biochemical tests allowed the differentiation of strain RP007(T) from all recognized species of the genus Burkholderia. Strains RP007(T), LMG 18808 and LMG 24204 are therefore considered to represent a single novel species of the genus Burkholderia, for which the name Burkholderia sartisoli sp. nov. is proposed. The type strain is RP007(T) (=LMG 24000(T) =CCUG 53604(T) =ICMP 13529(T)).

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.

The preparation of a novel radioiodination reagent, the (aminooxy)acetyl derivative of (p-[125]-iodophenyl)ethylamine, is described. Conventional radioiodination of proteins involves the formation of iodotyrosine residues, but for in vivo applications such as thyroid or stomach immunoscintigraphy, the susceptibility of these residues to tissue dehalogenases constitutes a serious disadvantage. Using our new compound, which has a particularly nonreactive aromatic ring, we confirm and extend studies published by other workers indicating the much greater in vivo stability of iodophenyl compounds compared to the more conventional iodophenolic ones. In addition, the aminooxy group of our reagent gives a stable and specific linkage to aldehyde groups formed by periodate oxidation on the sugar moiety of antibody molecules. In vitro, favorable binding activity and high stability was obtained with a (([125I]iodoaryl)amino)oxy labeled monoclonal antibody directed against carcinoembryonic antigen. In vivo, using paired labeling experiments in nude mice bearing colon carcinoma xenografts, the (([125I]iodoaryl)amino)oxy-MAb (MAb = monoclonal antibody) was compared with the same MAb 131I-labeled by conventional chloramine-T method. Tumor 125I concentration of (arylamino)oxy MAb (measured as percent injected dose per gram) was significantly higher as compared to values obtained with a conventionally labeled 131I antibody. Additionally, thyroid uptake, an indicator of iodine release from the antibody, was up to 25 times lower after injection of 125I-MAb obtained by the new method as compared to the conventionally iodinated 131I-MAb.

Physiology and transcriptome of the polycyclic aromatic hydrocarbon-degrading Sphingomonas sp. LH128 after long-term starvation.

The survival, physiology and gene expression profile of the phenanthrene-degrading Sphingomonas sp. LH128 was examined after an extended period of complete nutrient starvation and compared with a non-starved population that had been harvested in exponential phase. After 6 months of starvation in an isotonic solution, only 5 % of the initial population formed culturable cells. Microscopic observation of GFP fluorescent cells, however, suggested that a larger fraction of cells (up to 80 %) were still alive and apparently had entered a viable but non-culturable (VBNC) state. The strain displayed several cellular and genetic adaptive strategies to survive long-term starvation. Flow cytometry, microscopic observation and fatty acid methyl ester (FAME) analysis showed a reduction in cell size, a change in cell shape and an increase in the degree of membrane fatty acid saturation. Transcriptome analysis showed decreased expression of genes involved in ribosomal protein biosynthesis, chromosomal replication, cell division and aromatic catabolism, increased expression of genes involved in regulation of gene expression and efflux systems, genetic translocations, and degradation of rRNA and fatty acids. Those phenotypic and transcriptomic changes were not observed after 4 h of starvation. Despite the starvation situation, the polycyclic aromatic hydrocarbon (PAH) catabolic activity was immediate upon exposure to phenanthrene. We conclude that a large fraction of cells maintain viability after an extended period of starvation apparently due to tuning the expression of a wide variety of cellular processes. Due to these survival attributes, bacteria of the genus Sphingomonas, like strain LH128, could be considered as suitable targets for use in remediation of nutrient-poor PAH-contaminated environments.

Generation of polycyclic aromatic hydrocarbons (PAHs) during woodworking operations.

Occupational exposures to wood dust have been associated with an elevated risk of sinonasal cancer (SNC). Wood dust is recognized as a human carcinogen but the specific cancer causative agent remains unknown. One possible explanation is a co-exposure to; wood dust and polycyclic aromatic hydrocarbons (PAHs). PAHs could be generated during incomplete combustion of wood due to heat created by use of power tools. To determine if PAHs are generated from wood during common wood working operations, PAH concentrations in wood dust samples collected in an experimental chamber operated under controlled conditions were analyzed. In addition, personal air samples from workers exposed to wood dust (n = 30) were collected. Wood dust was generated using three different power tools: vibrating sander, belt sander, and saw; and six wood materials: fir, Medium Density Fiberboard (MDF), beech, mahogany, oak and wood melamine. Monitoring of wood workers was carried out by means of personal sampler device during wood working operations. We measured 21 PAH concentrations in wood dust samples by capillary gas chromatography-ion trap mass spectrometry (GC-MS). Total PAH concentrations in wood dust varied greatly (0.24-7.95 ppm) with the lowest being in MDF dust and the highest in wood melamine dust. Personal PAH exposures were between 37.5-119.8 ng m(-3) during wood working operations. Our results suggest that PAH exposures are present during woodworking operations and hence could play a role in the mechanism of cancer induction related to wood dust exposure.

Sawhorse-type diruthenium tetracarbonyl complexes containing porphyrin-derived ligands as highly selective photosensitizers for female reproductive cancer cells.

Diruthenium tetracarbonyl complexes of the type [Ru2(CO)4(l2-g2-O2CR)2L2] containing a Ru-Ru backbone with four equatorial carbonyl ligands, two carboxylato bridges, and two axial two-electron ligands in a sawhorse-like geometry have been synthesized with porphyrin-derived substituents in the axial ligands [1: R is CH3, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin], in the bridging carboxylato ligands [2: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is PPh3; 3: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 1,3,5-triaza-7-phosphatricyclo [3.3.1.1]decane], or in both positions [4: RCO2H is 5-(4-carboxyphenyl)-10,15,20-triphenyl-21,23H-porphyrin, L is 5-(4-pyridyl)-10,15,20-triphenyl-21,23H-porphyrin]. Compounds 1-3 were assessed on different types of human cancer cells and normal cells. Their uptake by cells was quantified by fluorescence and checked by fluorescence microscopy. These compounds were taken up by human HeLa cervix and A2780 and Ovcar ovarian carcinoma cells but not by normal cells and other cancer cell lines (A549 pulmonary, Me300 melanoma, PC3 and LnCap prostate, KB head and neck, MDAMB231 and MCF7 breast, or HT29 colon cancer cells). The compounds demonstrated no cytotoxicity in the absence of laser irradiation but exhibited good phototoxicities in HeLa and A2780 cells when exposed to laser light at 652 nm, displaying an LD50 between 1.5 and 6.5 J/cm2 in these two cell lines and more than 15 J/cm2 for the others. Thus, these types of porphyric compound present specificity for cancer cell lines of the female reproductive system and not for normal cells; thus being promising new organometallic photosensitizers.

Occupational exposure of truck drivers to dust and polynuclear aromatic hydrocarbons: a pilot study in Geneva, Switzerland.

The exposure to dust and polynuclear aromatic hydrocarbons (PAH) of 15 truck drivers from Geneva, Switzerland, was measured. The drivers were divided between "long-distance" drivers and "local" drivers and between smokers and nonsmokers and were compared with a control group of 6 office workers who were also divided into smokers and nonsmokers. Dust was measured on 1 workday both by a direct-reading instrument and by sampling. The local drivers showed higher exposure to dust (0.3 mg/m3) and PAH than the long-distance drivers (0.1 mg/m3), who showed no difference with the control group. This observation may be due to the fact that the local drivers spend more time in more polluted areas, such as streets with heavy traffic and construction sites, than do the long-distance drivers. Smoking does not influence exposure to dust and PAH of professional truck drivers, as measured in this study, probably because the ventilation rate of the truck cabins is relatively high even during cold days (11-15 r/h). The distribution of dust concentrations was shown in some cases to be quite different from the expected log-normal distribution. The contribution of diesel exhaust to these exposures could not be estimated since no specific tracer was used. However, the relatively low level of dust exposure dose not support the hypothesis that present day levels of diesel exhaust particulates play a significant role in the excess occurrence of lung cancer observed in professional truck drivers.

Approaches to identifying and quantifying polycyclic aromatic hydrocarbons of molecular weight 302 in diesel particulates

Among the PAH class of compounds, high molecular weight PAH are now considered as relevant cancer inducers, but not all of them have the same biological activity. However, their analysis is difficult, mainly due to the presence of numerous isomers and due to their low volatility. Retention indices (Ri) for 13 dibenzopyrenes and homologues were determined by high-resolution capillary gas chromatography (GC) with four different stationary phases: a 5% phenyl-substituted methylpolysiloxane column (DB-5 ms), a 35% phenyl-substituted methylpolysiloxane column (BPX-35), a 50% phenyl-substituted methylpolysiloxane column (BPX-50), and a 35% trifluoropropylmethyl polysiloxane stationary phase (Rtx-200). Correlations for retention on each phase were investigated by using 8 independent molecular descriptors. Ri has been shown to be linearly correlated to PAH volume, polarisability alpha, Hückel-pi energy on the four examined columns. Ionisation potential Ip is a fourth variable which improves the regression model for DB-5ms, BPX-35, and BPX-50 column. Correlation coefficients ranging from r2 = 0.935 to r2 = 0.952 are then observed. Application of these indices to the identification and quantification of PAH with MW 302 in certified diesel particulate matter SRM 1650a is presented and discussed. [Authors]