Effects of the number of markers per haplotype and clustering of haplotypes on the accuracy of QTL mapping and prediction of genomic breeding values

The aim of this paper was to compare the effect of haplotype definition on the precision of QTL-mapping and on the accuracy of predicted genomic breeding values. In a multiple QTL model using identity-by-descent (IBD) probabilities between haplotypes, various haplotype definitions were tested i.e. including 2, 6, 12 or 20 marker alleles and clustering base haplotypes related with an IBD probability of > 0.55, 0.75 or 0.95. Simulated data contained 1100 animals with known genotypes and phenotypes and 1000 animals with known genotypes and unknown phenotypes. Genomes comprising 3 Morgan were simulated and contained 74 polymorphic QTL and 383 polymorphic SNP markers with an average r² value of 0.14 between adjacent markers. The total number of haplotypes decreased up to 50% when the window size was increased from two to 20 markers and decreased by at least 50% when haplotypes related with an IBD probability of > 0.55 instead of > 0.95 were clustered. An intermediate window size led to more precise QTL mapping. Window size and clustering had a limited effect on the accuracy of predicted total breeding values, ranging from 0.79 to 0.81. Our conclusion is that different optimal window sizes should be used in QTL-mapping versus genome-wide breeding value prediction.     

Rapid plasma membrane-endosomal trafficking of the lymph node sinus and high endothelial venule scavenger receptor/homing receptor stabilin-1 (FEEL-1/CLEVER-1)

The sinusoidal endothelia of liver, spleen, and lymph node are major sites for uptake and recycling of waste macromolecules through promiscuous binding to a disparate family of scavenger receptors. Among the most complex is stabilin-1, a large multidomain protein containing tandem fasciclin domains, epidermal growth factor-like repeats, and a C-type lectin-like hyaluronan-binding Link module, which functions as an endocytic receptor for acetylated low density lipoprotein and advanced glycation end products. Intriguingly, stabilin-1 has also been reported to mediate both homing of leukocytes across lymph node high endothelial venules and adhesion of metastatic tumor cells to peritumoral lymphatic vessels. Currently, however, it is not clear how stabilin-1 mediates these distinct functions. To address the issue, we have investigated the tissue and subcellular localization of stabilin-1 in detail and assessed the functional status of its Link module. We show that stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and EEA-1+ve (early endosome antigen 1) early endosomes. In addition, we show using full-length stabilin-1 cDNA and a stabilin-1/CD44 chimera in HeLa cells that intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. Finally, we provide evidence that the stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing.     

Direct immobilization of gangliosides onto gold-carboxymethyldextran sensor surfaces by hydrophobic interaction: applications to antibody characterization

We describe a novel immobilization technique to investigate interactions between immobilized gangliosides (GD3, GM1, and GM2) and their respective antibodies, antibody fragments, or binding partners using an optical biosensor. Immobilization was performed by direct injection onto a carboxymethyldextran sensor chip and did not require derivatization of the sensor surface or the ganglioside. The ganglioside appeared to bind to the sensor surface by hydrophobic interaction, leaving the carbohydrate epitope available for antibody or, in the case of GM1, cholera toxin binding. The carboxyl group of the dextran chains on the sensor surface did not appear to be involved in the immobilization as evidenced by equivalent levels of immobilization following conversion of the carboxyl groups into acyl amino esters, but rather the dextran layer provided a hydrophilic coverage of the sensor chip which was essential to prevent nonspecific binding. This technique gave better reactivity and specificity for anti- ganglioside monoclonal antibodies (anti-GD3: KM871, KM641, R24; and anti-GM2: KM966) than immobilization by hydrophobic interaction onto a gold sensor surface or photoactivated cross-linking onto carboxymethydextran. This rapid immobilization procedure has facilitated detailed kinetic analysis of ganglioside/antibody interactions, with the surface remaining viable for a large number of cycles (>125). Kinetic constants were determined from the biosensor data using linear regression, nonlinear least squares and equilibrium analysis. The values of kd, ka, and KAobtained by nonlinear analysis (KAKM871 = 1.05, KM641 = 1.66, R24 = 0.14, and KM966 = 0.65 x 10(7) M- 1) were essentially independent of concentration and showed good agreement with data obtained by other analytical methods.      

The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis>

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.     

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.     

Delivery of antibodies to the cytosol: Debunking the myths

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed. Numerous strategies have been proposed, but the most robust and widely applicable procedure still remains to be identified, since a quantitative ranking of the efficiencies has not yet been done. To achieve this, we developed a novel efficiency evaluation method for antibody delivery based on a fusion protein consisting of a human IgG₁ Fc and the recombination enzyme Cre (Fc-Cre). Applied to suitable GFP reporter cells, it allows the important distinction between proteins trapped in endosomes and those delivered to the cytosol. Further, it ensures viability of positive cells and is unsusceptible to fixation artifacts and misinterpretation of cellular localization in microscopy and flow cytometry. Very low cytoplasmic delivery efficiencies were found for various profection reagents and membrane penetrating peptides, leaving electroporation as the only practically useful delivery method for antibodies. This was further verified by the successful application of this method to bind antibodies to cytosolic components in living cells.     

Rapid estimation of numbers of whiteflies (Hemiptera: Aleurodidae) and thrips (Thysanoptera: Thripidae) on sticky traps

The presence or absence of greenhouse whiteflies, Trialeurodes vaporariorum Westwood, and thrips, primarily western flower thrips, Frankliniella occidentalis (Pergande), in cells of a grid laid over 7.6 cm by 12.7 cm sticky traps was used to estimate the population density of these pests on the trap. The method accurately predicted trap population densities of between 15 and 192 individuals per side for thrips on blue and yellow traps and between 15 and 168 whiteflies per side on yellow traps. The distribution of both whiteflies and thrips tended to be clustered on the sides and upper edge of the traps. The method is useful in giving a far more rapid estimate than counting individuals, particularly at high population densities.     

The control of ionized calcium in squid axons

Measurements of the Ca content, [Ca](T), of freshly isolated squid axons show a value of 60 μmol/kg axoplasm. Axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 3 mM Ca(Na) seawater show little change in Ca content over 4 h, while axons in 10 mM Ca(Na) seawater show gains of 18 μmol/Ca/kgxh. In 10 Ca (Choline) seawater the gain is 2,400 μmol/kgxh. Using aequorin confined to a dialysis capillary in the center of an axon, one finds that [Ca](i) is in a steady state with 3 Ca (Na) seawater, and that both 10 Ca (Na) and 3 Ca (choline) seawater cause increases in [Ca](i). In 3 Ca (Na) seawater-3 Ca (choline) seawater mixtures, 180 mM [Na](0) (40 perecent Na) is as effective as 450 mM [Na](0) (100 percent Na) in maintaining a normal [Ca](1); lower [Na] causes an increase in [Ca](i). If axons are injected with the ATP-splitting enzyme apyrase, the resulting [Ca](1) is not loading with high [Ca](0) or low [Na](0) solutions. Depolarization of an axon with 100 mM K (Na) seawater leads to an increase in the steady-state level of [Ca](1) that is reversed upon returning the axon to normal seawater. Freshly isolated axons treated with either CN or FCCP to inhibit mitochondrial Ca buffering can still maintain a normal [Ca](i) in 1 Ca (Na) seawater.     

CDK1 inhibition sensitizes normal cells to DNA damage in a cell cycle dependent manner

Cyclin-dependent kinase 1 (CDK1) orchestrates the transition from the G2 phase into mitosis and as cancer cells often display enhanced CDK1 activity, it has been proposed as a tumor specific anti-cancer target. Here we show that the effects of CDK1 inhibition are not restricted to tumor cells but can also reduce viability in non-cancer cells and sensitize them to radiation in a cell cycle dependent manner.

Radiosensitization by the specific CDK1 inhibitor, RO-3306, was determined by colony formation assays in three tumor lines (HeLa, T24, SQ20B) and three non-cancer lines (HFL1, MRC-5, RPE). Initial results showed that CDK1 inhibition radiosensitized tumor cells, but did not sensitize normal fibroblasts and epithelial cells in colony formation assays despite effective inhibition of CDK1 signaling. Further investigation showed that normal cells were less sensitive to CDK1 inhibition because they remained predominantly in G1 for a prolonged period when plated in colony formation assays. In contrast, inhibiting CDK1 a day after plating, when the cells were going through G2/M phase, reduced their clonogenic survival both with and without radiation. Our finding that inhibition of CDK1 can damage normal cells in a cell cycle dependent manner indicates that targeting CDK1 in cancer patients may lead to toxicity in normal proliferating cells. Furthermore, our finding that cell cycle progression becomes easily stalled in non-cancer cells under normal culture conditions has general implications for testing anti-cancer agents in these cells.     

Relaxin-3 and RXFP3 expression, and steroidogenic actions in the ovary of teleost fish

Relaxins are peptides similar in secondary structure to insulins. In teleost genomes, five or six relaxin genes have been identified. Two relaxins group closely with mammalian relaxin-3 on phylogenetic analysis and are named relaxin-3a and b. We refer to the remainder as relaxins c to f. Ovarian expression of relaxin-3a, d and f genes, and the relaxin-3 receptor gene Rxfp3, was studied in Danio rerio using RT-PCR. Immunohistochemistry was used to determine the distribution of relaxin-3 peptides and RXFP3 in the ovary of Fundulus heteroclitus (killifish). Thirdly, enzyme immunoassays and ovarian follicular culture were used to determine the effect of treatment with human recombinant relaxin-3 on the production of 17beta-estradiol and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one in killifish ovarian follicles. Relaxin-3a, d, f, and Rxfp3 genes were expressed regardless of sex or reproductive condition. Relaxin-3 immunostaining was present in mid to late follicular stages within cortical alveoli of the oocyte cytopasm, whereas receptor staining was localized to follicular cells. Treatment with relaxin-3 enhanced 17beta-estradiol production in early and late maturing follicles, but did not have an effect in vitellogenic follicles. Relaxin-3 appeared to suppress the release of MIS production. This suggests that relaxin peptides may be involved with estradiol-dependant events in follicular development.