Depleted uranium-catalyzed oxidative DNA damage: absence of significant alpha particle decay

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalized human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha particle) and a chemical (metal) component. Since DU has a low-specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. In the current study we demonstrate that DU can generate oxidative DNA damage and can also catalyze reactions that induce hydroxyl radicals in the absence of significant alpha particle decay. Experiments were conducted under conditions in which chemical generation of hydroxyl radicals was calculated to exceed the radiolytic generation by one million-fold. The data showed that markers of oxidative DNA base damage, thymine glycol and 8-deoxyguanosine could be induced from DU-catalyzed reactions of hydrogen peroxide and ascorbate similarly to those occurring in the presence of iron catalysts. DU was 6-fold more efficient than iron at catalyzing the oxidation of ascorbate at pH 7. These data not only demonstrate that DU at pH 7 can induced oxidative DNA damage in the absence of significant alpha particle decay, but also suggest that DU can induce carcinogenic lesions, e.g. oxidative DNA lesions, through interaction with a cellular oxygen species.     

Selective binding of specific rat epididymal secretory proteins to spermatozoa and erythrocytes

Tissue pieces from the caput epididymidis of the rat were incubated in vitro with (³⁵S) methionine to produce radioactive secretory proteins. The radioactive secretory proteins so formed were tested for their ability to bind to washed rat spermatozoa collected from the rete testis and cauda epididymidis, and to rat erythrocytes. The sperm and erythrocytes bound approximately 5% of the total radioactive protein. Binding was protein-specific in that only selected proteins became associated with the cells. Binding was not cell-specific, however, since testicular spermatozoa, caudal spermatozoa, and erythrocytes all bound the same proteins to a similar degree.     

Immunoquantification and enzyme kinetics of alpha-L-iduronidase in cultured fibroblasts from normal controls and mucopolysaccharidosis type I patients.

alpha-L-Iduronidase activity is deficient in mucopolysaccharidosis type I (MPS I; Hurler syndrome, Scheie syndrome) patients and results in the disruption of the sequential degradation of the glycosaminoglycans dermatan sulfate and heparan sulfate. A monoclonal antibody-based immunoquantification assay has been developed for alpha-L-iduronidase, which enables the detection of at least 16 pg alpha-L-iduronidase protein. Cultured human skin fibroblasts from 12 normal controls contained 17-54 ng alpha-L-iduronidase protein/mg extracted cell protein. Fibroblasts from 23 MPS I patients were assayed for alpha-L-iduronidase protein content. Fibroblast extracts from one MPS I patient contained at least six times the level of alpha-L-iduronidase protein for normal controls--but contained no associated enzyme activity--and is proposed to represent a mutation affecting the active site of the enzyme. Fibroblast extracts from 11 MPS I patients contained 0.05-2.03 ng alpha-L-iduronidase protein/mg extracted cell protein, whereas immunodetectable protein could not be detected in the other 11 patients. Four fibroblast extracts with no immunodetectable alpha-L-iduronidase protein had residual alpha-L-iduronidase activity, suggesting that the mutant alpha-L-iduronidase in cultured cells from these MPS I patients has been modified to mask or remove the epitopes detected by two monoclonal antibodies used in the quantification assay. Both the absence of immunoreactivity in a mild MPS I patient and high protein level in a severe MPS I patient present limitations to the use of immunoquantification analysis as a sole measure of patient phenotype. Enzyme kinetic analysis of alpha-L-iduronidase from MPS I fibroblasts revealed a number of patients with either abnormal substrate binding or catalytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of a noncanonical disulfide bond in the stability,affinity, and flexibility of a VHH specific for the Listeria virulence factor InlB

A distinguishing feature of camel (Camelus dromedarius) VHH domains are noncanonical disulfide bonds between CDR1 and CDR3. The disulfide bond may provide an evolutionary advantage, as one of the cysteines in the bond is germline encoded. It has been hypothesized that this additional disulfide bond may play a role in binding affinity by reducing the entropic penalty associated with immobilization of a long CDR3 loop upon antigen binding. To examine the role of a noncanonical disulfide bond on antigen binding and the biophysical properties of a VHH domain, we have used the VHH R303, which binds the Listeria virulence factor InlB as a model. Using site directed mutagenesis, we produced a double mutant of R303 (C33A/C102A) to remove the extra disulfide bond of the VHH R303. Antigen binding was not affected by loss of the disulfide bond, however the mutant VHH displayed reduced thermal stability (T_m = 12°C lower than wild‐type), and a loss of the ability to fold reversibly due to heat induced aggregation. X‐ray structures of the mutant alone and in complex with InlB showed no major changes in the structure. B‐factor analysis of the structures suggested that the loss of the disulfide bond elicited no major change on the flexibility of the CDR loops, and revealed no evidence of loop immobilization upon antigen binding. These results suggest that the noncanonical disulfide bond found in camel VHH may have evolved to stabilize the biophysical properties of the domain, rather than playing a significant role in antigen binding.     

Male mating preference of two cryptic species of the herbivorous insect Eccritotarsus catarinensis

When using biological control against pest populations, more than one biocontrol agent might be introduced simultaneously. This could be counterproductive in the event of negative interactions between the biocontrol agents. Within and between species interactions have a strong impact on mating behaviour and reproduction, and can have an impact on the effectiveness of biological control. We studied the reproductive compatibility between two geographically isolated strains (Brazil and Peru) of Eccritotarsus catarinensis (Heteroptera: Miridae), a biocontrol agent against the invasive aquatic weed, Eichhornia crassipes. By performing inter- and intra-species mating experiments, we investigated whether or not males from each of the cryptic species would be able to distinguish between partners from either species, and if they would mate with partners from the opposite species. Our results showed the decrease in lifetime fecundity, and most importantly, the lack of production of offspring from eggs resulting from forced hybridisation. We showed that Peruvian males mated only with females from their own species, and did not mate with females from the Brazilian species. In contrast, Brazilian males mated equally with females from both species, but needed significantly more time in order to commence a mating, and no offspring were produced from eggs resulting from hybridisation. Although future studies demand more rigorous controls, our results indicate asymmetrical sexual isolation between the two species. We speculate on mechanisms involved in reproductive isolation in the two cryptic species, and the possible implications for effective biocontrol, and include some morphological measurements that might support our assumptions.     

A Pathogenic Mosaic TP53 Mutation in Two Germ Layers Detected by Next Generation Sequencing

Background

Li-Fraumeni syndrome is caused by germline TP53 mutations and is clinically characterized by a predisposition to a range of cancers, most commonly sarcoma, brain tumours and leukemia. Pathogenic mosaic TP53 mutations have only rarely been described.

Methods and Findings

We describe a 2 years old child presenting with three separate cancers over a 6 month period; two soft tissue mesenchymal tumors and an aggressive metastatic neuroblastoma. As conventional testing of blood DNA by Sanger sequencing for mutations in TP53, ALK, and SDH was negative, whole exome sequencing of the blood DNA of the patient and both parents was performed to screen more widely for cancer predisposing mutations. In the patient''s but not the parents'' DNA we found a c.743 G>A, p.Arg248Gln (CCDS11118.1) TP53 mutation in 3–20% of sequencing reads, a level that would not generally be detectable by Sanger sequencing. Homozygosity for this mutation was detected in all tumor samples analyzed, and germline mosaicism was demonstrated by analysis of the child''s newborn blood spot DNA. The occurrence of separate tumors derived from different germ layers suggests that this de novo mutation occurred early in embryogenesis, prior to gastrulation.

Conclusion

The case demonstrates pathogenic mosaicim, detected by next generation deep sequencing, that arose in the early stages of embryogenesis.     

Measurement of Free Intracellular Calcium in the Brain by 19F-Nuclear Magnetic Resonance Spectroscopy

We report the first measurement of the free intracellular calcium level in an actively metabolising intact cerebral tissue preparation. To this end, we applied the recently developed 19F-nuclear magnetic resonance calcium chelator, 5,5'-F2-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBAPTA), in superfused cerebral cortical slices to give values for the intracellular Ca2+ concentration of 350 and 480 nM, at external calcium concentrations of 1.2 and 2.4 mM, respectively. Under both conditions, the intracellular Ca2+ concentration was increased by depolarisation using a high external K+ concentration. Interleaved 31P spectra showed that the presence of the 5FBAPTA had a deleterious effect on the metabolic state of the tissue with an external Ca2+ concentration of 1.2 mM, but normal viability was maintained using 2.4 mM.     

The metabolism of synthetic leukotriene B4 in synovial fluid and whole human blood

The metabolism in vitro of synthetic leukotriene B4 (LTB4) in synovial fluid from rheumatoid arthritis and osteoarthritis patients and in whole blood from these same patient groups and from normal volunteers has been studied. A linear relationship existed between a plot of the time of incubation of samples with LTB4 and the percentage of the initial concentration of LTB4 at each time point. The slope of this line, the rate constant for metabolism, has been used to compare different samples. LTB4 was metabolised more rapidly in the synovial fluid of rheumatoid arthritis patients than osteoarthritis patients. Furthermore, LTB4 was metabolised more rapidly in the blood of rheumatoid arthritis patients than either osteoarthritis patients or normal volunteers. These differences in metabolism correlate with the polymorphonuclear leukocyte (PMN) and albumin content of samples. It is suggested that binding of LTB4 to albumin in vivo will in part determine the available concentration of LTB4 in inflammatory lesions.     

Molecular characterization of PoGT8D and PoGT43B, two secondary wall-associated glycosyltransferases in poplar

Dicot wood is mainly composed of cellulose, lignin and glucuronoxylan (GX). Although the biosynthetic genes for cellulose and lignin have been studied intensively, little is known about the genes involved in the biosynthesis of GX during wood formation. Here, we report the molecular characterization of two genes, PoGT8D and PoGT43B, which encode putative glycosyltransferases, in the hybrid poplar Populus alba x tremula. The predicted amino acid sequences of PoGT8D and PoGT43B exhibit 89 and 75% similarity to the Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9, respectively, both of which have been shown to be required for GX biosynthesis. The PoGT8D and PoGT43B genes were found to be expressed in cells undergoing secondary wall thickening, including the primary xylem, secondary xylem and phloem fibers in stems, and the secondary xylem in roots. Both PoGT8D and PoGT43B are predicted to be type II membrane proteins and shown to be targeted to Golgi. Overexpression of PoGT43B in the irx9 mutant was able to rescue the defects in plant size and secondary wall thickness and partially restore the xylose content. Taken together, our results demonstrate that PoGT8D and PoGT43B are Golgi-localized, secondary wall-associated proteins, and PoGT43B is a functional ortholog of IRX9 involved in GX biosynthesis during wood formation.