Oxidized lipoproteins inhibit surfactant phosphatidylcholine synthesis via calpain-mediated cleavage of CTP:phosphocholine cytidylyltransferase

We investigated effects of pro-atherogenic oxidized lipoproteins on phosphatidylcholine (PtdCho) biosynthesis in murine lung epithelial cells (MLE-12). Cells surface-bound, internalized, and degraded oxidized low density lipoproteins (Ox-LDL). Ox-LDL significantly reduced [3H]choline incorporation into PtdCho in cells by selectively inhibiting the activity of the rate-regulatory enzyme, CTP:phosphocholine cytdylyltransferase (CCT). Ox-LDL coordinately increased the cellular turnover of CCTalpha protein as determined by [35S]methionine pulse-chase studies by inducing the calcium-activated proteinase, calpain. Forced expression of calpain or exposure of cells to the calcium ionophore, A23187, increased CCTalpha degradation, whereas overexpression of the endogenous calpain inhibitor, calpastatin, attenuated Ox-LDL-induced CCTalpha degradation. The effects of Ox-LDL on CCTalpha breakdown were attenuated in calpain-deficient cells. In vitro calpain digestion of CCTalpha isolated from cells transfected with truncated or internal deletion mutants indicated multiple cleavage sites within the CCTalpha primary structure, leading to the generation of a 26-kDa (p26) fragment. Calpain hydrolysis of purified CCTalpha generated p26, which upon NH2-terminal sequencing localized a calpain attack site within the CCTalpha amino terminus. Expression of a CCTalpha mutant where the amino-terminal cleavage site and a putative carboxyl-terminal hydrolysis region were modified resulted in an enzyme that was significantly less sensitive to proteolytic cleavage and restored the ability of cells to synthesize surfactant PtdCho after Ox-LDL treatment. Thus, these results provide a critical link between proatherogenic lipoproteins and their metabolic target, CCTalpha, resulting in impaired surfactant metabolism.     

Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction

Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg2+ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.     

Effect of imidacloprid tree treatments on the occurrence of Formosan subterranean termites,Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), in independent monitors

Periodic sampling of 87 independent monitors, initially active with the Formosan subterranean termite, Coptotermes formosanus Shiraki, was conducted. Monitors, located in eight sectors adjacent to seven buildings, were various distances (1-46 m) from 57 trees treated with 0.1% imidacloprid foam. Termites collected from six of the eight sectors showed latent mortality attributed to imidacloprid intoxication at all monitor-tree distances. Approximately 6 mo after treatment, termite populations had recovered in these sectors. Another sector showed termite population suppression for approximately 15 mo, followed by recovery. Imidacloprid tree treatments did not control C. formosanus populations in independent monitors adjacent to the treatments.     

The potential role of ectomycorrhizal fungi in determining Douglas-fir resistance to defoliation by the western spruce budworm (Lepidoptera: Tortricidae)

There is phenotypic variation among individual trees of interior Douglas-fir (Pseudotsuga menziesii var. glauca [Beissn.] Franco) in their resistance to defoliation by the western spruce budworm (Choristoneura occidentalis Freeman). We evaluated the potential role of ectomycorrhizal fungi in determining this resistance using half-sib seedlings derived from parent trees that are resistant versus susceptible to budworm defoliation in the field. The seedlings were inoculated with Laccaria bicolor ectomycorrhizal fungi, fertilized, or untreated. Approximately 48 d after treatment, late-instar larvae from a nondiapausing laboratory colony of C. occidentalis were allowed to feed on pairs of resistant versus susceptible seedlings for 1 wk. Chemical analyses of current-year shoots for nitrogen (N), phosphorus (P), magnesium (Mg), and zinc (Zn) indicated that the fungus increased foliar concentrations of P and Mg in resistant seedlings, but it did not increase their growth rate. However, L. bicolor had no effect on foliar concentrations of P or Mg in susceptible seedlings, even though seedling growth rates increased slightly in response to the inoculation. L. bicolor had no effect on foliar levels of N or Zn in any of the seedlings. As expected, fertilization increased levels of N and P in the foliage of both resistant and susceptible seedlings, but it did not affect levels of Mg and Zn. Surprisingly, the fertilizer treatment had no effect on seedling growth rates. Despite these differences, late-instar budworms showed no feeding preference among untreated, mycorrhizal, or fertilized seedlings. The fact that seedlings from resistant versus susceptible Douglas-firs responded differently to the L. bicolor treatment lends preliminary support to the hypothesis that ecotmycorrhizae might play a role in Douglas-fir resistance to damage from the western spruce budworm. Finally, differences in foliar concentrations of N and P among untreated seedlings from different maternal trees suggested that foliar nutritional chemistry is influenced by the tree's genotype.     

Changes in the human mitochondrial genome after treatment of malignant disease

Mitochondrial DNA (mtDNA) is the only extrachromosomal DNA in human cells. The mitochondrial genome encodes essential information for the synthesis of the mitochondrial respiratory chain. Inherited defects of this genome are an important cause of human disease. In addition, the mitochondrial genome seems to be particularly prone to DNA damage and acquired mutations may have a role in ageing, cancer and neurodegeneration. We wished to determine if radiotherapy and chemotherapy used in the treatment of cancer could induce changes in the mitochondrial genome. Such changes would be an important genetic marker of DNA damage and may explain some of the adverse effects of treatment. We studied samples from patients who had received radiotherapy and chemotherapy for point mutations within the mtDNA control region, and for large-scale deletions. In blood samples from patients, we found a significantly increased number of point mutations compared to the control subjects. In muscle biopsies from 7 of 8 patients whom had received whole body irradiation as well as chemotherapy, the level of a specific mtDNA deletion was significantly greater than in control subjects. Our studies have shown that in patients who have been treated for cancer there is an increased level of mtDNA damage.     

A multiple in silico program approach for the prediction of mutagenicity from chemical structure

We have conducted an evaluation of three of the most widely used commercial toxicity prediction programs, Toxicity Prediction by Komputer Assisted Technology (TOPKAT), Deductive Estimation of Risk from Existing Knowledge (DEREK) for Windows (DfW) and CASETOX. The three programs were evaluated for their ability to predict Ames test mutagenicity using 520 proprietary drug candidate (Test set 1) and 94 commercial (Test set 2) compounds. The study demonstrates that these three commercially available programs are useful, with limitations in their ability to predict mutagenicity over a wide range of chemical space, i.e. global predictivity. Individually, each of the programs performed at an acceptable level for overall accuracy, i.e. the ability to predict the correct outcome. However, analysis of the predictions indicates that the overall accuracy figure is heavily weighted by the ability of the programs to correctly predict non-mutagens, whereas none of the programs individually performed well in the prediction of novel mutagenic structures, i.e. Ames positive compounds. The performance of these programs' in predicting Ames positive mutagens appeared to be independent of the chemical utility of the compound, i.e. industrial, agricultural or pharmaceutical. The combination of program predictions provided some improvement in overall accuracy, sensitivity and specificity.     

Distribution patterns of postmortem damage in human mitochondrial DNA

The distribution of postmortem damage in mitochondrial DNA retrieved from 37 ancient human DNA samples was analyzed by cloning and was compared with a selection of published animal data. A relative rate of damage (rho(v)) was calculated for nucleotide positions within the human hypervariable region 1 (HVR1) and cytochrome oxidase subunit III genes. A comparison of damaged sites within and between the regions reveals that damage hotspots exist and that, in the HVR1, these correlate with sites known to have high in vivo mutation rates. Conversely, HVR1 subregions with known structural function, such as MT5, have lower in vivo mutation rates and lower postmortem-damage rates. The postmortem data also identify a possible functional subregion of the HVR1, termed "low-diversity 1," through the lack of sequence damage. The amount of postmortem damage observed in mitochondrial coding regions was significantly lower than in the HVR1, and, although hotspots were noted, these did not correlate with codon position. Finally, a simple method for the identification of incorrect archaeological haplogroup designations is introduced, on the basis of the observed spectrum of postmortem damage.     

Mental retardation and abnormal skeletal development (Dyggve-Melchior-Clausen dysplasia) due to mutations in a novel,evolutionarily conserved gene

Dyggve-Melchior-Clausen dysplasia (DMC) and Smith-McCort dysplasia (SMC) are similar, rare autosomal recessive osteochondrodysplasias. The radiographic features and cartilage histology in DMC and SMC are identical. However, patients with DMC exhibit significant developmental delay and mental retardation, the major features that distinguish the two conditions. Linkage studies localized the SMC and DMC disease genes to chromosome 18q12-21.1, providing evidence suggesting that they are allelic disorders. Sequence analysis of the coding exons of the FLJ90130 gene, a highly evolutionarily conserved gene within the recombination interval defined in the linkage study, identified mutations in SMC and DMC patients. The affected individuals in two consanguinous DMC families were homozygous for a stop codon mutation and a frameshift mutation, respectively, demonstrating that DMC represents the FLJ90130-null phenotype. The data confirm the hypothesis that SMC and DMC are allelic disorders and identify a gene necessary for normal skeletal development and brain function.     

The genetic origins of the Andaman Islanders

Mitochondrial sequences were retrieved from museum specimens of the enigmatic Andaman Islanders to analyze their evolutionary history. D-loop and protein-coding data reveal that phenotypic similarities with African pygmoid groups are convergent. Genetic and epigenetic data are interpreted as favoring the long-term isolation of the Andamanese, extensive population substructure, and/or two temporally distinct settlements. An early colonization featured populations bearing mtDNA lineage M2, and this lineage is hypothesized to represent the phylogenetic signal of an early southern movement of humans through Asia. The results demonstrate that Victorian anthropological collections can be used to study extinct, or seriously admixed populations, to provide new data about early human origins.     

C3 shrub expansion in a C4 grassland: positive post-fire responses in resources and shoot growth

Changes in land management and reductions in fire frequency have enabled woody species to increase in grasslands worldwide. Nevertheless, fire is rarely eliminated from grasslands, and for shrubs to survive, they must be able cope with fire and replace aboveground structures. Because new shoots may have more available solar radiation, greater root?:?shoot ratios, and thus more resources available belowground after fire compared to undisturbed shrub communities, we hypothesized that carbon, nutrient, and water relations may be enhanced in stems compared to those in an undisturbed grassland. However, this same post-fire resource pulse stimulates the grasses and may intensify competitive interactions between shrubs and grasses. To test these predictions, we measured seasonal patterns in net photosynthesis (A), predawn xylem pressure potentials (XPP), leaf nitrogen (N) content, and productivity of Cornus drummondii shoots from shrub patches (islands) of different sizes in mesic grasslands burned annually, burned infrequently, and protected from fire. Seasonal average A was 20% higher (P = 0.016) in burned than in unburned shrubs, regardless of island size. Shrubs in burned sites also produced shoots with higher leaf N than unburned shrubs, and N content was higher in leaves from small islands compared to large islands (P < 0.0001). Burning caused a decrease in late summer predawn XPP in small islands (-3.1 MPa), whereas burned large islands did not differ from unburned shrubs. Post-fire productivity of new shoots was significantly greater compared to shoots in unburned sites. These results indicate that a transient period of high resource availability after fire allows for increased growth and rapid recovery of grassland shrubs. Thus, although fire has a negative effect on aboveground biomass of shrubs, the post-fire increases in resource availability, which enhance growth in the dominant grasses, are also important for recovery of woody species.