Protection against radiation-induced mutagenesis in V79 cells by 2-[(aminopropyl)amino] ethanethiol under conditions of acute hypoxia

The effects of the radioprotector 2-[(aminopropyl)amino] ethanethiol (WR-1065) on radiation-induced cell killing and mutagenesis at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 Chinese hamster cells under hypoxic or aerobic conditions were examined. Conditions of acute hypoxia were attained by gassing 10(6) cells in 1-ml volumes in individual glass ampoules for 2 min with nitrogen. Ampoules were then sealed and incubated at 37 degrees C for 60 min. Following this treatment, cell survival after irradiation as expected was significantly enhanced. The effect of acute hypoxia on the formation of HGPRT mutants by irradiation was also investigated. Mutation frequencies were determined with a 6-day expression time and corrected for the number of spontaneous background mutants. Although mutation induction was approximately linear as a function of radiation dose under most conditions tested, it was significantly reduced in cell populations made acutely hypoxic prior to irradiation. Protection against mutation induction was apparent and similar when cells were irradiated in the presence of the radioprotector, regardless of whether they were also hypoxic or aerated. If cells were irradiated in air and then made hypoxic, no significant protection was still observed. These results suggest that the antimutagenic effect of WR-1065 is not due solely to its ability to scavenge radiation-induced oxygen-free radicals, but rather that it may also modulate these effects through the scavenging of metabolically induced free radicals and/or the chemical repair of radiation-induced DNA lesions.     

Association of WR-1065 with CHO AA8 cells, nuclei, and nucleoids

The radioprotector WR-1065 (N-(2-mercaptoethyl)-1,3-diaminopropane) has been shown to be the active moiety involved in protecting mammalian cells from the cytotoxic and mutagenic effects of ionizing radiation after administration of WR-1065 or the phosphorylated form, WR-2721. Initial experiments demonstrated that, in our hands, WR-1065 protects Chinese hamster AA8 cells from killing by (a) mechanism(s) other than induction of hypoxia. AA8 cells were then incubated in the presence of [14C]WR-1065 to determine whether association of WR-1065 in vivo was random or targeted to the nucleus or the nuclear matrix. The kinetics of incorporation of labeled material showed rapid incorporation for the first 30 min and little, if any, additional incorporation over the next 2.5 h. Examination of nuclei and nucleoids generated from the AA8 cells indicated that approximately 10% of the drug was localized in the nucleus and the drug that remained was not dislodged with repeated washes of the filters. Association kinetics of the drug with nuclei and nucleoids indicated that there was little increase in drug association with time, suggesting that there may be a limited number of strong association sites in the nucleus, but these sites are either with DNA or with matrix proteins. Exposure of the AA8 cells to 6 Gy of 60Co gamma rays did not significantly alter the association of the drug with AA8 cells. Incubating AA8 cells in [14C]WR-1065 for 30 min and then incubating in drug-free medium indicated that nearly all of the drug was lost from cells within the first 5 min of incubation in drug-free medium. The low level of tightly bound matrix-associated label may be important in generating alterations in matrix organization that have been observed previously in this laboratory.     

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

Serosurvey of Brucella spp. infection in the Kafue lechwe (Kobus leche kafuensis) of the Kafue flats in Zambia

One of the diseases of veterinary and public health importance affecting the Kafue lechwe (Kobus leche kafuensis) on the Kafue flats is brucellosis, for which only scant information is available. During the 2003 (October), 2004 (December), and 2008 (July-December) hunting seasons in the Kafue flats, we conducted a study to determine the seroprevalence of Brucella spp. in the Kafue lechwe and to evaluate serologic tests for detection of Brucella spp. antibodies in lechwe. The Rose Bengal Test (RBT), competitive enzyme-linked immunosorbent assay (cELISA), and fluorescence polarization assay (FPA) were used. A total of 121 Kafue lechwe were hunted for disease investigations in 2003, 2004, and 2008 in the Kafue Flat Game Management Area. Of these, 21.6%, (95% confidence interval [CI]: 14.2-29.1%) had detectable antibodies to Brucella spp. The Kafue lechwe in Lochnivar National Park had higher antibody results than those in Blue Lagoon National Park (odds ratio=3.0; 95% CI: 0.94-9.4). Infection levels were similar in females (21.6%) and males (21.7%). Results were similar among RBT, FPA, cELISA tests, suggesting that these could effectively be used in diagnosing brucellosis in the Kafue lechwe. Our study demonstrates the presence of Brucella infections in the Kafue lechwe in two national parks located in the Kafue flats and further highlights the suitability of serologic assays for testing the Kafue lechwe. Because the Kafue lechwe is the most hunted wildlife species in Zambia, hunters need to be informed of the public health risk of Brucella spp. infection.     

Relationship between phosphorylated histone H2AX formation and cell survival in human microvascular endothelial cells (HMEC) as a function of ionizing radiation exposure in the presence or absence of thiol-containing drugs

Human microvascular endothelial cells (HMEC) were exposed to ionizing radiation at doses ranging from 0 to 16 Gy in either the presence or absence of the active thiol forms of amifostine (WR1065), phosphonol (WR255591), N-acetyl-l-cysteine (NAC), captopril or mesna. Each of these clinically relevant thiols, administered to HMEC at a dose of 4 mM for 30 min prior to irradiation, is known to exhibit antioxidant properties. The purpose of this investigation was to determine the relationship(s), if any, between the frequency of radiation-induced histone H2AX phosphorylation at serine 139 (gamma-H2AX) in cells and subsequent survival, as assessed by colony-forming ability, in exposed cell populations as a function of the presence or absence of each of the five thiol compounds during irradiation. gamma-H2AX formation in irradiated cells, as a function of relative DNA content, was quantified by bivariant flow cytometry analysis with FITC-conjugated gamma-H2AX antibody and nuclear DAPI staining. gamma-H2AX formation in cells was measured as the relative fold increase as a function of the treatment conditions. The frequency of gamma-H2AX-positive cells increased with increasing dose of radiation followed by a dose- and time-dependent decay. The most robust response for gamma-H2AX formation occurred 1 h after irradiation with their relative frequencies decreasing as a function of time 4 and 24 h later. To assess the effects of the various thiols on gamma-H2AX formation, all measurements were made 1 h after irradiation. WR1065 was not only effective in protecting HMEC against gamma-H2AX formation across the entire dose range of radiation exposures used, but it was also significantly more cytoprotective than either its prodrug (WR2721) or disulfide (WR33278) analogue. WR1065 had no significant effect on gamma-H2AX formation when administered immediately or up to 30 min after radiation exposure. An inhibitory effect against gamma-H2AX formation induced by 8 Gy of radiation was expressed by each of the thiols tested. NAC, captopril and mesna were equally effective in reducing the frequency of gamma-H2AX formation, with both WR1065 and WR255591 exhibiting a slightly more robust protective effect. Each of the five thiols was effective in reducing the frequency of gamma-H2AX-positive cells across all phases of the cell cycle. In contrast to the relative ability of each of these thiols to inhibit gamma-H2AX formation after irradiation, NAC, captopril and mesna afforded no protection to HMEC as determined using a colony-forming survival assay. Only WR1065 and WR255591 were effective in reducing the frequencies of radiation-induced gamma-H2AX-positive cells as well as protecting against cell death. These results suggest that the use of gamma-H2AX as a biomarker for screening the efficacy of novel antioxidant radioprotective compounds is highly problematic since their formation and disappearance may be linked to processes beyond simply the formation and repair of radiation-induced DSBs.     

Multiplexed expression and screening for recombinant protein production in mammalian cells

Background  

A variety of approaches to understanding protein structure and function require production of recombinant protein. Mammalian based expression systems have advantages over bacterial systems for certain classes of protein but can be slower and more laborious. Thus the availability of a simple system for production and rapid screening of constructs or conditions for mammalian expression would be of great benefit. To this end we have coupled an efficient recombinant protein production system based on transient transfection in HEK-293 EBNA1 (HEK-293E) suspension cells with a dot blot method allowing pre-screening of proteins expressed in cells in a high throughput manner.     

A new brace treatment similar for adolescent scoliosis and kyphosis based on restoration of thoracolumbar lordosis. Radiological and subjective clinical results after at least one year of treatment

Study design

A prospective treatment study with a new brace was conducted Objective. To evaluate radiological and subjective clinical results after one year conservative brace treatment with pressure onto lordosis at the thoracolumbar joint in children with scoliosis and kyphosis.

Summary of background data

Conservative brace treatment of adolescent scoliosis is not proven to be effective in terms of lasting correction. Conservative treatment in kyphotic deformities may lead to satisfactory correction. None of the brace or casting techniques is based on sagittal forces only applied at the thoracolumbar spine (TLI= thoracolumbar lordotic intervention). Previously we showed in patients with scoliosis after forced lordosis at the thoracolumbar spine a radiological instantaneous reduction in both coronal curves of double major scoliosis.

Methods

A consecutive series of 91 children with adolescent scoliosis and kyphosis were treated with a modified symmetric 30 degrees Boston brace to ensure only forced lordosis at the thoracolumbar spine. Scoliosis was defined with a Cobb angle of at least one of the curves [greater than or equal to] 25 degrees and kyphosis with or without a curve <25 degrees in the coronal plane. Standing radiographs were made i) at start, ii) in brace at beginning and iii) after one year treatment without brace.

Results

Before treatment start ??in brace?? radiographs showed a strong reduction of the Cobb angles in different curves in kyphosis and scoliosis groups (sagittal n = 5 all p < 0.001, pelvic obliquity p < 0.001). After one year of brace treatment in scoliosis and kyphosis group the measurements on radiographs made without brace revealed an improvement in 3 Cobb angles each.

Conclusion

Conservative treatment using thoracolumbar lordotic intervention in scoliotic and kyphotic deformities in adolescence demonstrates a marked improvement after one year also in clinical and postural criteria. An effect not obtained with current brace techniques.