Plants express a lipid transfer protein with high similarity to mammalian sterol carrier protein-2

This is the first report describing the cloning and characterization of sterol carrier protein-2 (SCP-2) from plants. Arabidopsis thaliana SCP-2 (AtSCP-2) consists of 123 amino acids with a molecular mass of 13.6 kDa. AtSCP-2 shows 35% identity and 56% similarity to the human SCP-2-like domain present in the human D-bifunctional protein (DBP) and 30% identity and 54% similarity to the human SCP-2 encoded by SCP-X. The presented structural models of apo-AtSCP-2 and the ligand-bound conformation of AtSCP-2 reveal remarkable similarity with two of the structurally known SCP-2s, the SCP-2-like domain of human DBP and the rabbit SCP-2, correspondingly. The AtSCP-2 models in both forms have a similar hydrophobic ligand-binding tunnel, which is extremely suitable for lipid binding. AtSCP-2 showed in vitro transfer activity of BODIPY-phosphatidylcholine (BODIPY-PC) from donor membranes to acceptor membranes. The transfer of BODIPY-PC was almost completely inhibited after addition of 1-palmitoyl 2-oleoyl phosphatidylcholine or ergosterol. Dimyristoyl phosphatidic acid, stigmasterol, steryl glucoside, and cholesterol showed a moderate to marginal ability to lower the BODIPY-PC transfer rate, and the single chain palmitic acid and stearoyl-coenzyme A did not affect transfer at all. Expression analysis showed that AtSCP-2 mRNA is accumulating in most plant tissues. Plasmids carrying fusion genes between green fluorescent protein and AtSCP-2 were transformed with particle bombardment to onion epidermal cells. The results from analyzing the transformants indicate that AtSCP-2 is localized to peroxisomes.     

Theoretical and empirical power of regression and maximum-likelihood methods to map quantitative trait loci in general pedigrees

Both theoretical calculations and simulation studies have been used to compare and contrast the statistical power of methods for mapping quantitative trait loci (QTLs) in simple and complex pedigrees. A widely used approach in such studies is to derive or simulate the expected mean test statistic under the alternative hypothesis of a segregating QTL and to equate a larger mean test statistic with larger power. In the present study, we show that, even when the test statistic under the null hypothesis of no linkage follows a known asymptotic distribution (the standard being chi(2)), it cannot be assumed that the distribution under the alternative hypothesis is noncentral chi(2). Hence, mean test statistics cannot be used to indicate power differences, and a comparison between methods that are based on simulated average test statistics may lead to the wrong conclusion. We illustrate this important finding, through simulations and analytical derivations, for a recently proposed new regression method for the analysis of general pedigrees to map quantitative trait loci. We show that this regression method is not necessarily more powerful nor computationally more efficient than a maximum-likelihood variance-component approach. We advocate the use of empirical power to compare trait-mapping methods.     

Early population differentiation in extinct aborigines from Tierra del Fuego-Patagonia: ancient mtDNA sequences and Y-chromosome STR characterization

Ancient mtDNA was successfully recovered from 24 skeletal samples of a total of 60 ancient individuals from Patagonia-Tierra del Fuego, dated to 100-400 years BP, for which consistent amplifications and two-strand sequences were obtained. Y-chromosome STRs (DYS434, DYS437, DYS439, DYS393, DYS391, DYS390, DYS19, DYS389I, DYS389II, and DYS388) and the biallelic system DYS199 were also amplified, Y-STR alleles could be characterized in nine cases, with an average of 4.1 loci per sample correctly typed. In two samples of the same ethnic group (Aonikenk), an identical and complete eight-loci haplotype was recovered. The DYS199 biallelic system was used as a control of contamination by modern DNA and, along with DYS19, as a marker of American origin. The analysis of both mtDNA and Y-STRs revealed DNA from Amerindian ancestry. The observed polymorphisms are consistent with the hypothesis that the ancient Fuegians are close to populations from south-central Chile and Argentina, but their high nucleotide diversity and the frequency of single lineages strongly support early genetic differentiation of the Fuegians through combined processes of population bottleneck, isolation, and/or migration, followed by strong genetic drift. This suggests an early genetic diversification of the Fuegians right after their arrival at the southernmost extreme of South America.     

Nuclear overexpression of NAD(P)H:quinone oxidoreductase 1 in Chinese hamster ovary cells increases the cytotoxicity of mitomycin C under aerobic and hypoxic conditions

The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.     

Long-term modulation of mitochondrial Ca2+ signals by protein kinase C isozymes

The modulation of Ca2+ signaling patterns during repetitive stimulations represents an important mechanism for integrating through time the inputs received by a cell. By either overexpressing the isoforms of protein kinase C (PKC) or inhibiting them with specific blockers, we investigated the role of this family of proteins in regulating the dynamic interplay of the intracellular Ca2+ pools. The effects of the different isoforms spanned from the reduction of ER Ca2+ release (PKCalpha) to the increase or reduction of mitochondrial Ca2+ uptake (PKCzeta and PKCbeta/PKCdelta, respectively). This PKC-dependent regulatory mechanism underlies the process of mitochondrial Ca2+ desensitization, which in turn modulates cellular responses (e.g., insulin secretion). These results demonstrate that organelle Ca2+ homeostasis (and in particular mitochondrial processing of Ca2+ signals) is tuned through the wide molecular repertoire of intracellular Ca2+ transducers.     

Chondrocyte protein with a poly-proline region (CHPPR) is a novel mitochondrial protein and promotes mitochondrial fission

We have recently identified a chondrocyte protein with a poly-proline region, referred to as CHPPR, and showed that this protein is expressed intracellularly in chick embryo chondrocytes. Conventional fluorescence and confocal localization of CHPPR shows that CHPPR is sorted to mitochondria. Furthermore, immunoelectron microscopy of CHPPR transfected cells demonstrates that this protein is mostly associated with the mitochondrial inner membranes. Careful analysis of CHPPR expressing cells reveals, instead of the regular mitochondrial tubular network, the presence of a number of small spheroid mitochondria. Here we show that the domain responsible for network-spheroid transition spans amino acid residues 182-309 including the poly-proline region. Functional analyses of mitochondrial activity rule out the possibility of mitochondrial damage in CHPPR transfected cells. Since cartilage expresses high levels of CHPPR mRNA when compared to other tissues and because CHPPR is associated with late stages of chondrocyte differentiation, we have investigated mitochondrial morphology in hypertrophic chondrocytes by MitoTracker Orange labeling. Confocal microscopy shows that these cells have spheroid mitochondria. Our data demonstrate that CHPPR is able to promote mitochondrial fission with a sequence specific mechanism suggesting that this event may be relevant to late stage of chondrocyte differentiation.     

Optimal design of experiments with anticipated pattern of missing observations

We propose a general method of designing an experiment when there are potentially failing trials. We use polynomial models and the Michaelis-Menten model as examples and construct different types of optimal designs under a broad class of response probability functions. We show that the usual optimal designs, that assume all observations are available at the end of the experiment, can be quite inefficient if the anticipated missingness pattern is not accounted for at the design stage. We also investigate robustness properties of the proposed designs to specification of their nominal values and the response probability functions.