Long-Term Toxicity of 213Bi-Labelled BSA in Mice

Background

Short-term toxicological evaluations of alpha-radioimmunotherapy have been reported in preclinical assays, particularly using bismuth-213 (²¹³Bi). Toxicity is greatly influenced not only by the pharmacokinetics and binding specificity of the vector but also by non-specific irradiation due to the circulating radiopharmaceutical in the blood. To assess this, an acute and chronic toxicity study was carried out in mice injected with ²¹³Bi-labelled Bovine Serum Albumin (²¹³Bi-BSA) as an example of a long-term circulating vector.

Method

Biodistribution of ²¹³Bi-BSA and ¹²⁵I-BSA were compared in order to evaluate ²¹³Bi uptake by healthy organs. The doses to organs for injected ²¹³Bi-BSA were calculated. Groups of nude mice were injected with 3.7, 7.4 and 11.1 MBq of ²¹³Bi-BSA and monitored for 385 days. Plasma parameters, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN) and creatinine, were measured and blood cell counts (white blood cells, platelets and red blood cells) were performed. Mouse organs were examined histologically at different time points.

Results

Haematological toxicity was transient and non-limiting for all evaluated injected activities. At the highest injected activity (11.1 MBq), mice died from liver and kidney failure (median survival of 189 days). This liver toxicity was identified by an increase in both ALT and AST and by histological examination. Mice injected with 7.4 MBq of ²¹³Bi-BSA (median survival of 324 days) had an increase in plasma BUN and creatinine due to impaired kidney function, confirmed by histological examination. Injection of 3.7 MBq of ²¹³Bi-BSA was safe, with no plasma enzyme modifications or histological abnormalities.

Conclusion

Haematological toxicity was not limiting in this study. Liver failure was observed at the highest injected activity (11.1 MBq), consistent with liver damage observed in human clinical trials. Intermediate injected activity (7.4 MBq) should be used with caution because of the risk of long-term toxicity to kidneys.     

Growth of hemopoietic cells after incubation with VP 16-213

We examined the effect of various concentrations of VP 16-213 (25-125 microM/l, 2-h incubation on normal and complete remission bone marrow from patients with acute leukaemia and on leukaemic blasts. The maximal tolerated dose of the drug for normal bone marrow GM-CFC was between 75 and 100 microM/l whereas that for complete remission bone marrow was distinctly lower. More early stem cells measured by aid of LTBMC were more resistant in normal, but not in every remission bone marrow. We have to examine if these LTBMC results are influenced by a damaged microenvironment by using 2 stage LTBMC. Spontaneous leukaemic cells showed a different, sometimes lower sensitivity to VP 16-213 doses maximally tolerated by normal hemopoietic cells so that the VP 16-213 incubation must not be effective for every leukaemia.     

Russian Article pp. 213–225

The kinetics of assimilation of hydrocarbons having a different structure by Candida yeasts was studied. The rates of oxidation of various carbon atoms in the molecules of isoalkanes, alkylaromatic hydrocarbons and n-alkanes were evaluated in the range of C₁₁ to C₂₈. Metabolic inhomogeneity of carbon atoms in the molecules of isolakanes and alkylaromatic hydrocarbons was observed. A competition in the assimilation of the called hydrocarbons and n-alkanes and also a competition in the assimilation of n-alkanes of a different molecular weight, i.e. metabolic inhomogeneity of carbon atoms of n-dodecane and n-tetracosane was found out for yeasts.     

Characterization of the naphthalene-degrading bacterium, Rhodococcus opacus M213

Bacterial strain M213 was isolated from a fuel oil-contaminated soil in Idaho, USA, by growth on naphthalene as a sole source of carbon, and was identified as Rhodococcus opacus M213 by 16S rDNA sequence analysis and growth on substrates characteristic of this species. M213 was screened for growth on a variety of aromatic hydrocarbons, and growth was observed only on simple 1 and 2 ring compounds. No growth or poor growth was observed with chlorinated aromatic compounds such as 2,4-dichlorophenol and chlorobenzoates. No growth was observed by M213 on salicylate, and M213 resting cells grown on naphthalene did not attack salicylate. In addition, no salicylate hydroxylase activity was detected in cell free lysates, suggesting a pathway for naphthalene catabolism that does not pass through salicylate. Enzyme assays indicated induction of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase on different substrates. Total DNA from M213 was screened for hybridization with a variety of genes encoding catechol dioxygenases, but hybridization was observed only with catA (encoding catechol 1,2-dioxygenase) from R. opacus 1CP and edoD (encoding catechol 2,3-dioxygenase) from Rhodococcus sp. I1. Plasmid analysis indicated the presence of two plasmids (pNUO1 and pNUO2). edoD hybridized to pNUO1, a very large (approximately 750 kb) linear plasmid.     

Biotransformation of 6,6-Dimethylfulvene by Pseudomonas putida RE213

The biotransformation of 6,6-dimethylfulvene [5-(1-methylethylidene)-1,3-cyclopentadiene], a nonaromatic C(inf5) carbocyclic analog of isopropylbenzene, was examined by using Pseudomonas putida RE213, a Tn5-generated dihydrodiol-accumulating mutant of the isopropylbenzene-degrading strain P. putida RE204. 6,6-Dimethylfulvene was converted to a single chiral product identified as (+)-(1R,2S)-cis-1,2-dihydroxy-5-(1-methylethylidene)-3-cyclopentene. This isopropylbenzene 2,3-dioxygenase-catalyzed transformation demonstrates the potential of bacterial arene dioxygenases for the direct conversion of cyclopentadienylidene compounds to homochiral C(inf5) carbocyclic cis-diols for use in enantiocontrolled organic syntheses.     

RNF213, a new nuclear marker for acanthomorph phylogeny

We show that RNF213 is a nuclear gene suitable for investigating large scale acanthomorph teleosteans interrelationships. The gene recovers many clades already found by several independent studies of acanthomorph molecular phylogenetics and considered as reliable. Moreover, we performed phylogenetic analyses of three other independent nuclear markers, first separately and then of all possible combinations (Dettaï, A., Lecointre, G., 2004. In search of nothothenioid (Teleostei) relatives. Antarct. Sci. 16 (1), 71–85. URL http://dx.doi.org/10.1017/S0954102004) of the four genes. This was coupled with an assessment of the reliability of clades using the repetition index of Li and Lecointre (Li, B., Lecointre, G., 2008. Formalizing reliability in the taxonomic congruence approach. Article accepted by Zoologica Scripta. URL http://dx.doi.org/10.1111/j.1463-6409.2008.00361.x). This index was improved here to handle the incomplete taxonomic overlap among datasets. The results lead to the identification of new reliable clades within the ‘acanthomorph bush’. Within a clade containing the Atherinomorpha, the Mugiloidei, the Plesiopidae, the Blennioidei, the Gobiesocoidei, the Cichlidae and the Pomacentridae, the Plesiopidae is the sister-group of the Mugiloidei. The Apogonidae are closely related to the Gobioidei. A clade named ‘H’ grouping a number of families close to stromateids and scombrids (Stromateidae, Scombridae, Trichiuridae, Chiasmodontidae, Nomeidae, Bramidae, Centrolophidae) is related to another clade named ‘E’ (Aulostomidae, Macrorhamphosidae, Dactylopteridae). The Sciaenidae is closely related to the Haemulidae. Within clade ‘X’ (Dettaï, A., Lecointre, G., 2004. In search of nothothenioid (Teleostei) relatives. Antarct. Sci. 16 (1), 71–85. URL http://dx.doi.org/10.1017/S0954102004), the Cottoidei, the Zoarcoidei, the Gasterosteidae, the Triglidae, the Scorpaenidae, the Sebastidae, the Synanceiidae, and the Congiopodidae form a clade. Within clade ‘L’ (Chen, W.-J., Bonillo, C., Lecointre, G., 2003. Repeatability of clades as a criterion of reliability: a case study for molecular phylogeny of Acanthomorpha (Teleostei) with larger number of taxa. Mol. Phylogenet. Evol. 26, 262–288; Dettaï, A., Lecointre, G., 2004. In search of nothothenioid (Teleostei) relatives. Antarct. Sci. 16 (1), 71–85. URL http://dx.doi.org/10.1017/S0954102004) grouping carangoids with flatfishes and other families (Centropomidae, Menidae, Sphyraenidae, Polynemidae, Echeneidae, Toxotidae, Xiphiidae), carangids are the stem-group of echeneids and coryphaenids, and sphyraenids are the sister-group to the Carangoidei. The Howellidae, the Epigonidae and the Lateolabracidae are closely related. We propose names for most of the clades repeatedly found in acanthomorph phylogenetic studies of various teams of the past decade.     

213 Structures and interactions of proteins involved in ER-associated protein degradation

Proteins are translocated into the endoplasmic reticulum (ER) of cells in an unfolded state, and acquire their native conformation in the ER lumen after signal peptide cleavage. ER-associated degradation (ERAD) of folding-incompetent protein chains is mediated by the protein complexes residing in the ER membrane. We study the architecture and function of one of these, the HRD complex assembled around the E3 ubiquitin ligase Hrd1. The recognition of ERAD substrates is linked to the maturation of their carbohydrate structures. The HRD complex-associated lectin Yos9 is involved in ERAD substrate recognition by binding carbohydrates through its mannose-6-phosphate receptor homology (MRH) domain. We have determined the crystal structure of a central domain of Yos9, adjacent to the MRH domain, which was previously annotated as interaction region with the HRD subunit Hrd3 (Hanna et al., 2012). We find that this domain does not support Hrd3 association which we map to the N-terminal half of Yos9 instead. In contrast, the domain has a function in Yos9 dimerization as seen in the crystal structure, in various solution experiments and as supported by mutagenesis of dimer interface residues. The dimerization of the ER-luminal Yos9, in conjunction with studies of the cytosolic domain of the HRD component Usa1 (Horn et al., 2009) and other biochemical data thus supports a model of a HRD complex that exists and functions as a dimer or a higher multimer. The delivery of ubiquitinated ERAD substrates to the proteasome is mediated by the cytosolic AAA ATPase Cdc48 (p97 in mammalian cells). The p97 (VCP) serves a wide variety of cellular functions in addition to its role in ERAD, including organelle membrane fusion, mitosis, DNA repair, and apoptosis. These different functions are linked to the binding of adaptor proteins to p97, many of which contain ubiquitin regulatory X (UBX) domains. One of these adaptors, ASPL (alveolar soft part sarcoma locus), uses a substantially extended UBX domain for binding to the N domain of p97 where a lariat-like, mostly α-helical extension wraps around one subunit of p97. By this binding ASPL triggers the dissociation of functional p97 hexamers leading to partial inactivation of the AAA ATPase. To the best of our knowledge, this is the first time that the structural basis for adaptor protein-induced inactivation by hexamer dissociation of p97 and, indeed, any AAA ATPase has been demonstrated. This observation has far reaching implications for AAA ATPase-regulated processes.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Draft Genome Sequence of Serratia marcescens Strain LCT-SM213

Serratia marcescens is a species of Gram-negative, rod-shaped bacterium of the family Enterobacteriaceae. S. marcescens can cause nosocomial infections, particularly catheter-associated bacteremia, urinary tract infections, and wound infections. Here, we present the draft genome sequence of Serratia marcescens strain LCT-SM213, which was isolated from CGMCC 1.1857.     

Characterization of pES213, a small mobilizable plasmid from Vibrio fischeri

Most Vibrio fischeri strains isolated from the Euprymna scolopes light organ carry plasmids, often including both a large (>40kb) plasmid, and one or more small (<12kb) plasmids. The large plasmids share homology with pES100, which is the lone plasmid in V. fischeri type strain ES114. pES100 appears to encode a conjugative system similar to that on plasmid R721. The small plasmids lack extensive similarity to pES100, but they almost always occur in cells that also harbor a large plasmid resembling pES100. We found that many or all of these small plasmids share homology with pES213, a plasmid in strain ES213. We determined the 5501-bp pES213 sequence and generated selectable antibiotic resistance encoding pES213 derivatives, which enabled us to examine replication, retention, and transfer in V. fischeri. An 863-bp fragment of pES213 with features characteristic of theta-type replicons conferred replication without requiring any pES213 open reading frame (ORF). We estimated that pES213 derivatives were maintained at 9.4 copies per genome, which corresponds well with a model of random plasmid segregation to daughter cells and the approximately 10(-4) per generation frequency of plasmid loss. pES213 derivatives mobilized between V. fischeri strains at frequencies up to approximately 10(-4) in culture and in the host, apparently by employing the pES100 conjugative apparatus. pES213 carries two homologs of the putative pES100 origin of transfer (oriT), and V. fischeri strains lacking the pES100 conjugative relaxase, including a relaxase mutant, failed to serve as donors for transmission of pES213 derivatives. In other systems, genes directing conjugative transfer can function in trans to oriT, so it was noteworthy that ORFs adjacent to oriT, VFB51 in pES100 and traYZ in pES213, enhanced transfer 100- to 1000-fold when provided in cis. We also identified and disrupted the V. fischeri recA gene. RecA was not required for stable pES213 replication but surprisingly was required in donors for efficient transfer of pES213 derivatives. These studies provide an explanation for the prevalence and co-occurrence of pES100- and pES213-type plasmids, illuminate novel elements of pES213 mobilization, and provide the foundation for new genetic tools in V. fischeri.