CHO cells expressing the human P₅-ATPase ATP13A2 are more sensitive to the toxic effects of herbicide paraquat

P-ATPases are membrane transporters energized by ATP. The subfamily of P₅-ATPases is the least studied P-ATPases and the ion substrate specificity of the P₅ subfamily is not known. Mutations of the human P₅ATPase gene ATP13A2 has been shown to underlie a form of Parkinson disease (PD). We investigated the link between ATP13A2 and environmental factors related to PD development. Increasing concentrations of the synthetic polyamine analog paraquat induced a greater cytotoxic effect over CHO cells expressing ATP13A2. Paraquat-toxicity was associated with increased production of cellular reactive oxygen species and this increment was reversed by the natural polyamine spermidine. Acridine orange fluorescence intensity suggested that ATP13A2 induced the expansion of acidic vesicles that become more alkaline upon external addition of spermidine. Polyamine uptake is proposed to be initiated by a plasma membrane carrier followed by sequestration into acidic vesicles of the late endocytic compartment through an unidentified active mechanism; because ATP13A2 is located in lysosomes and late endosomes, our results open the possibility that ATP13A2 could be one of those active transporters capable of transporting polyamines like spermidine as well as its toxic analog paraquat.     

Mechanisms of kin discrimination inferred from pedigrees and the spatial distribution of mates

Where animals avoid inbreeding, different mechanisms of kin discrimination can leave different 'signatures' in the patterns of observed mate relationship. For example, consider a species with no paternal care. If a female avoids mating with familiar individuals, one would expect a deficit of offspring whose parents are maternal half-siblings, but paternal half-siblings would be unfamiliar with each other and thus have offspring at the frequency expected by chance. If spatial cues are used to avoid inbreeding, a female would be expected to produce few offspring with males (even unrelated males) living near her birth site. We searched for these and other signatures with data from a long-term study of banner-tailed kangaroo rats, Dipodomys spectabilis, in Arizona, USA, using a combination of intensive censusing, mapping of available dens, microsatellite-based parentage determination, and a randomization routine that determines the numbers of offspring expected if females in the population mate indiscriminately among the males available to them. The data are consistent with the hypothesis that kangaroo rats discriminate kin by familiarity developed via association early in life, rather than by using spatial cues or self-referential phenotype matching. Our approach should be widely applicable as a means of assessing the degree to which kin discrimination exists (in contexts like nepotism as well as inbreeding avoidance) and in inferring what cues animals use to assess categories of relationship.     

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.     

Pleiotrophin is expressed in avian somites and tendon anlagen

Pleiotrophin (Ptn) is a secreted, developmentally regulated growth factor associated with the extracellular matrix. During mammalian embryogenesis, Ptn has been suggested to play a role in the development of various embryonic structures including nervous system and skeleton. In the avian embryo, Ptn has been proposed to be involved in limb cartilage development, but embryonic Ptn expression has not been comprehensively studied. We isolated a cDNA fragment containing the full-length coding sequence of chick Ptn and studied the expression of Ptn in detail until embryonic day 10. We, furthermore, isolated a 6,385-bp phage clone containing the Ptn cDNA of 2,551 bp and additional 3,787 bp downstream of the published Ptn cDNA sequence classifying a yet Ptn-unrelated chEST clone as the 3′ untranslated region of Ptn. Our studies revealed novel expression domains in developing somites and during limb formation. We found prominent expression in the somitocoel cells of epithelial somites, and in a sclerotomal subcompartment, the syndetome, which gives rise to the axial tendons in the vertebral motion segment. In the limbs, Ptn was markedly expressed in tendon anlagen and in phalangeal joints. Our results introduce Ptn as a novel marker gene in avian somite and tendon development.     

Polymorphisms in the promoter region of ESR2 gene and breast cancer susceptibility

Genetic variations like single nucleotide polymorphisms (SNPs) in genes involved in estrogen biosynthesis, metabolism and signal transduction have been suggested to affect breast cancer susceptibility. In this study we tested the hypothesis that polymorphisms in the promoter of ESR2 gene may be associated with increased risk for breast cancer. We analyzed three SNPs in the promoter region of human ESR2 gene by means of allele-specific tetra-primer PCR. A total of 318 sporadic breast cancer cases and 318 age-matched controls were included in the study. With regard to homozygous genotypes, women with sporadic breast cancer more frequently carried the CC genotype of ESR2 promoter SNP rs2987983 (OR 1.99, p = 0.005). Calculation of allele positivity demonstrated that presence of T allele of this SNP was more frequent in healthy women. Our data suggest that a SNP in the promoter region of ESR2 gene might be able to affect breast cancer risk. These results further support the emerging hypothesis that ERβ is an important factor in breast cancer development.     

Synthetic LXR agonist attenuates plaque formation in apoE-/- mice without inducing liver steatosis and hypertriglyceridemia

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism. LXR agonists have been shown to limit the cellular cholesterol content by inducing reverse cholesterol transport, increasing bile acid production, and inhibiting intestinal cholesterol absorption. Most of them, however, also increase lipogenesis via sterol regulatory element-binding protein-1c (SREBP1c) and carbohydrate response element-binding protein activation resulting in hypertriglyceridemia and liver steatosis. We report on the antiatherogenic properties of the steroidal liver X receptor agonist N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA) in apolipoprotein E (apoE)-deficient mice. Long-term administration of DMHCA (11 weeks) significantly reduced lesion formation in male and female apoE-null mice. Notably, DMHCA neither increased hepatic triglyceride (TG) levels in male nor female apoE-deficient mice. ATP binding cassette transporter A1 and G1 and cholesterol 7alpha-hydroxylase mRNA abundances were increased, whereas SREBP1c mRNA expression was unchanged in liver, and even decreased in macrophages and intestine. Short-term treatment revealed even higher changes on mRNA regulation. Our data provide evidence that DMHCA is a strong candidate as therapeutic agent for the treatment or prevention of atherosclerosis, circumventing the negative side effects of other LXR agonists.     

Erythrocytes serve as a reservoir for cellular and extracellular sphingosine 1‐phosphate

Sphingosine 1‐phosphate (S1P) in blood is phosphorylated, stored, and transported by red blood cells (RBC). Release of S1P from RBC into plasma is a regulated process that does not occur in plasma‐ or serum‐free media. Plasma fractionation and incubations with isolated and recombinant proteins identified high density lipoprotein (HDL) and serum albumin (SA) as non‐redundant endogenous triggers for S1P release from RBC. S1P bound to SA and HDL was able to stimulate the S1P₁ receptor in calcium flux experiments. The binding capability of acceptor molecules triggers S1P release, as demonstrated with the anti‐S1P antibody Sphingomab?. More S1P was extracted from RBC membranes by HDL than by SA. Blood samples from anemic patients confirmed a reduced capacity for S1P release in plasma. In co‐cultures of RBC and endothelial cells (EC), we observed transcellular transportation of S1P as a second function of RBC‐associated S1P in the absence of SA and HDL and during tight RBC‐EC contact, mimicking conditions in tissue interstitium and capillaries. In contrast to S1P bound to SA and HDL, RBC‐associated S1P was significantly incorporated by EC after S1P lyase (SGPL1) inhibition. RBC‐associated S1P, therefore, has two functions: (1) It contributes to the cellular pool of SGPL1‐sensitive S1P in tissues after transcellular transportation and (2) it helps maintain extracellular S1P levels via SA and HDL independently from SGPL1 activity. J. Cell. Biochem. 109: 1232–1243, 2010. © 2010 Wiley‐Liss, Inc.     

Novel small molecule bradykinin B1 receptor antagonists. Part 1: Benzamides and semicarbazides

The synthesis and SAR of two series of bradykinin B₁ receptor antagonists is described. The benzamide moiety proved to be a suitable replacement for the aryl ester functionality of biaryl based antagonists. In addition, it was found that semicarbazides can effectively replace cyclopropyl amino acids. The compounds with the best overall profile were biaryl semicarbazides which display high antagonistic activity, low Caco-2 efflux and high oral bioavailability in the rat.