Characterization of cytostatically active glycosphingolipids isolated from thioglycollate-elicited murine macrophages

Two acidic glycosphingolipids (gangliosides) derived from mouse macrophage membranes and separated by thin-layer chromatography have a strong cytostatic effect on human and mouse tumor cells. The structure of the two gangliosides, named M phi G1 and M phi G2, was elucidated by application of physicochemical and immunochemical methods. Gas chromatography and mass spectrometry of M phi G1 and M phi G2 classified them as isomeric monosialogangliosides with ceramide moieties composed of sphingosine as the long-chain base, C16 and C18 fatty acids, respectively, and a lacto-tetraose backbone. For M phi G1, additional immunochemical findings led to the proposed structure IV3NeuAc-nLcOse4Cer. The immunochemical reactions of M phi G2 suggest a branched structure for the oligosaccharide moiety.     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

Two WXXF-based motifs in NECAPs define the specificity of accessory protein binding to AP-1 and AP-2

The adaptor proteins AP-2 and AP-1/GGAs are essential components of clathrin coats at the plasma membrane and trans-Golgi network, respectively. The adaptors recruit accessory proteins to clathrin-coated pits, which is dependent on the adaptor ear domains engaging short peptide motifs in the accessory proteins. Here, we perform an extensive mutational analysis of a novel WXXF-based motif that functions to mediate the binding of an array of accessory proteins to the alpha-adaptin ear domain of AP-2. Using nuclear magnetic resonance and mutational studies, we identified WXXF-based motifs as major ligands for a site on the alpha-ear previously shown to bind the DPW-bearing proteins epsin 1/2. We also defined the determinants that allow for specific binding of the alpha-ear motif to AP-2 as compared to those that allow a highly related WXXF-based motif to bind to the ear domains of AP-1/GGAs. Intriguingly, placement of acidic residues around the WXXF cores is critical for binding specificity. These studies provide a structural basis for the specific recruitment of accessory proteins to appropriate sites of clathrin-coated vesicle formation.     

Factor Xa inhibitors based on a 2-carboxyindole scaffold: SAR of neutral P1 substituents

A series of novel, highly potent 2-carboxyindole-based factor Xa inhibitors is described. Structural requirements for neutral ligands, which bind in the S1 pocket of factor Xa were investigated with the 2-carboxyindole scaffold. This privileged fragment assembly approach yielded a set of equipotent, selective inhibitors with structurally diverse neutral P1 substituents.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

Cold sweetening in diploid potato: mapping quantitative trait loci and candidate genes

A candidate gene approach has been used as a first step to identify the molecular basis of quantitative trait variation in potato. Sugar content of tubers upon cold storage was the model trait chosen because the metabolic pathways involved in starch and sugar metabolism are well known and many of the genes have been cloned. Tubers of two F(1) populations of diploid potato grown in six environments were evaluated for sugar content after cold storage. The populations were genotyped with RFLP, AFLP, and candidate gene markers. QTL analysis revealed that QTL for glucose, fructose, and sucrose content were located on all potato chromosomes. Most QTL for glucose content mapped to the same positions as QTL for fructose content. QTL explaining >10% of the variability for reducing sugars were located on linkage groups I, III, VII, VIII, IX, and XI. QTL consistent across populations and/or environments were identified. QTL were linked to genes encoding invertase, sucrose synthase 3, sucrose phosphate synthase, ADP-glucose pyrophosphorylase, sucrose transporter 1, and a putative sucrose sensor. The results suggest that allelic variants of enzymes operating in carbohydrate metabolic pathways contribute to the genetic variation in cold sweetening.     

Cholecystokinin activates both A- and C-type vagal afferent neurons

Patch-clamp electrophysiological methods were used on dissociated rat nodose neurons maintained in culture to determine whether responses to cholecystokinin (CCK) were associated with capsaicin-resistant (A type) or capsaicin-sensitive (C type) neurons. Nodose neurons were classified as A or C type on the basis of the characteristics of the Na+ current, a hyperpolarization-activated current, and sensitivity to a low concentration of capsaicin to ascertain the presence of vanilloid receptor 1 that has been associated with C-type neurons in sensory ganglia. It was expected that only capsaicin-sensitive C-type neurons would respond to CCK, because most vagally mediated actions of CCK are blocked by capsaicin treatment. However, we found that subpopulations of both A- and C-type neurons responded to CCK (24 and 38%, respectively). Thus some vagally mediated actions of CCK may be mediated by capsaicin insensitive A-type neurons.     

Pyrithiones as antifoulants: Environmental chemistry and preliminary risk assessment

Pyrithiones are widely used as bactericides, fungicides, or algicides in a variety of products such as shampoos, metal working fluids, adhesives, sealants, and coatings. This broad antimicrobial activity, along with low water solubility and favorable environmental chemistry, makes zinc pyrithione and copper pyrithione potentially ideal replacements for TBT in marine antifouling paints. Several studies on the toxicity and environ- ? mental fate of these pyrithiones have been conducted in freshwater and saltwater systems. Environmental fate studies show that pyrithiones rapidly degrade in the water column to less toxic compounds. Sediment accumulation is also prevented by the facile reduction of a critical functional group under anaerobic conditions. Modeling programs were used to calculate the predicted environmental concentration (PEC) for pyrithione. Comparison of PECs calculated for more persistent antifoulants with actual measured concentrations provided a measure of the bias inherent to the models. The results indicate a pyrithione risk quotient (PEC/PNEC) < 1. The findings are consistent with the absence of ecological effects during the long history of the use of zinc pyrithione as an antidandruff agent.     

Adaptation to high-fat diet reduces inhibition of gastric emptying by CCK and intestinal oleate

Rats maintained on low-fat (LF) or high-fat (HF) diets were fitted with gastric cannulas and duodenal catheters. Intraperitoneal injection of 0.250-2.0 microg/kg cholecystokinin (CCK) significantly inhibited gastric emptying of a 5-ml NaCl load in LF rats by 26.2-55. 1% compared with emptying after vehicle injection. By contrast, CCK-induced inhibition of gastric emptying was significantly less in HF rats given the same CCK doses (10.0-31.7% inhibition over the same CCK dose range). A 20-min intraduodenal infusion of oleate (0.03 or 0.06 kcal/ml) also resulted in significant inhibition of gastric emptying in LF rats (24 and 89%, respectively). Oleate-induced inhibition of gastric emptying was significantly attenuated in rats maintained on the HF diet (2 and 56%, respectively). Unlike CCK injections or oleate infusion, intraduodenal maltotriose infusion inhibited gastric emptying to a similar degree in LF and HF rats (77 and 78%, respectively). These results indicate that feeding HF diets diminishes the enterogastric inhibition of gastric emptying by intestinal oleate and diminishes the ability of CCK to inhibit gastric emptying.     

Caffeine-induced Ca(2+) sparks in mouse ventricular myocytes

Ca(2+) sparks are spatially localized intracellular Ca(2+) release events that were first described in 1993. Sparks have been ascribed to sarcoplasmic reticulum Ca(2+) release channel (ryanodine receptor, RyR) opening induced by Ca(2+) influx via L-type Ca(2+) channels or by spontaneous RyR openings and have been thought to reflect Ca(2+) release from a cluster of RyR. Here we describe a pharmacological approach to study sparks by exposing ventricular myocytes to caffeine with a rapid solution-switcher device. Sparks under these conditions have properties similar to naturally occurring sparks in terms of size and intracellular Ca(2+) concentration ([Ca(2+)](i)) amplitude. However, after the diffusion of caffeine, sparks first appear close to the cell surface membrane before coalescing to produce a whole cell transient. Our results support the idea that a whole cell [Ca(2+)](i) transient consists of the summation of sparks and that Ca(2+) sparks consist of the opening of a cluster of RyR and confirm that characteristics of the cluster rather than the L-type Ca(2+) channel-RyR relation determine spark properties.