Selection and characterization of Affibody ligands binding to Alzheimer amyloid beta peptides

Affibody (Affibody) ligands specific for human amyloid beta (Abeta) peptides (40 or 42 amino acid residues in size), involved in the progress of Alzheimer's disease, were selected by phage display technology from a combinatorial protein library based on the 58-amino acid residue staphylococcal protein A-derived Z domain. Post-selection screening of 384 randomly picked clones, out of which 192 clones were subjected to DNA sequencing and clustering, resulted in the identification of 16 Affibody variants that were produced and affinity purified for ranking of their binding properties. The two most promising Affibody variants were shown to selectively and efficiently bind to Abeta peptides, but not to the control proteins. These two Affibody ligands were in dimeric form (to gain avidity effects) coupled to affinity resins for evaluation as affinity devices for capture of Abeta peptides from human plasma and serum. It was found that both ligands could efficiently capture Abeta that were spiked (100 microgml(-1)) to plasma and serum samples. A ligand multimerization problem that would yield suboptimal affinity resins, caused by a cysteine residue present at the binding surface of the Affibody ligands, could be circumvented by the generation of second-generation Affibody ligands (having cysteine to serine substitutions). In an epitope mapping effort, the preferred binding site of selected Affibody ligands was mapped to amino acids 30-36 of Abeta, which fortunately would indicate that the Affibody molecules should not bind the amyloid precursor protein (APP). In addition, a significant effort was made to analyze which form of Abeta (monomer, dimer or higher aggregates) that was most efficiently captured by the selected Affibody ligand. By using Western blotting and a dot blot assay in combination with size exclusion chromatography, it could be concluded that selected Affibody ligands predominantly bound a non-aggregated form of analyzed Abeta peptide, which we speculate to be dimeric Abeta. In conclusion, we have successfully selected Affibody ligands that efficiently capture Abeta peptides from human plasma and serum. The potential therapeutic use of these optimized ligands for extracorporeal capture of Abeta peptides in order to slow down or reduce amyloid plaque formation, is discussed.     

Parallel evolution of facial stripe patterns in the Neolamprologus brichardi/pulcher species complex endemic to Lake Tanganyika

Colour pattern diversity can be due to random processes or to natural or sexual selection. Consequently, similarities in colour patterns are not always correlated with common ancestry, but may result from convergent evolution under shared selection pressures or drift. Neolamprologus brichardi and Neolamprologus pulcher have been described as two distinct species based on differences in the arrangement of two dark bars on the operculum. Our study uses DNA sequences of the mitochondrial control region to show that relatedness of haplotypes disagrees with species assignment based on head colour pattern. This suggests repeated parallel evolution of particular stripe patterns. The complete lack of shared haplotypes between populations of the same or different phenotypes reflects strong philopatric behaviour, possibly induced by the cooperative breeding mode in which offspring remain in their natal territory and serve as helpers until they disperse to nearby territories or take over a breeding position. Concordant phylogeographic patterns between N. brichardi/N. pulcher populations and other rock-dwelling cichlids suggest that the same colonization routes have been taken by sympatric species and that these routes were affected by lake level fluctuations in the past.     

C-terminal truncation of alpha-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha

In eukaryotic cells, COPI vesicles retrieve resident proteins to the endoplasmic reticulum and mediate intra-Golgi transport. Here, we studied the Hansenula polymorpha homologue of the Saccharomyces cerevisiae RET1 gene, encoding α-COP, a subunit of the COPI protein complex. H. polymorpha ret1 mutants, which expressed truncated α-COP lacking more than 300 C-terminal amino acids, manifested an enhanced ability to secrete human urokinase-type plasminogen activator (uPA) and an inability to grow with a shortage of Ca²⁺ ions, whereas a lack of α-COP expression was lethal. The α-COP defect also caused alteration of intracellular transport of the glycosylphosphatidylinositol-anchored protein Gas1p, secretion of abnormal uPA forms, and reductions in the levels of Pmr1p, a Golgi Ca²⁺-ATPase. Overexpression of Pmr1p suppressed some ret1 mutant phenotypes, namely, Ca²⁺ dependence and enhanced uPA secretion. The role of COPI-dependent vesicular transport in cellular Ca²⁺ homeostasis is discussed.     

Chiral recognition of cyclic alpha-hydroxyketones by CD-sensitive zinc tetraphenylporphyrin tweezer

A combined chemical/chiroptical microscale protocol for the determination of absolute configurations of cyclic alpha-hydroxyketones is described. The hydroxyl group in cyclic alpha-hydroxyketones is converted into (3-aminopropylamino)acetate (NH2CH2CH2CH2NHCH2COOR), or more generally, according to a newly developed protocol, into (3-hydroxypropylamino)acetate group (HOCH2CH2CH2NHCH2COOR). The resultant conjugated compound forms a 1:1 host-guest complex with a dimeric zinc porphyrin tweezer, which exhibits exciton-coupled bisignate CD spectrum centered around the 420-nm porphyrin Soret band due to induced helicity between the two porphyrins in the complex. The absolute configurations of the alpha-stereogenic center is then determined by comparison of the sign of the observed CD exciton couplet of the complex with that of the preferred porphyrin twist predicted by the Merck Molecular Force Field (MMFFs) method.     

Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum

A genetic approach was used to increase phosphatidylinositol(4,5)bisphosphate [PtdIns(4,5)P2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKIalpha in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P2 100-fold. In vivo studies revealed that the rate of 32Pi incorporation into whole-cell PtdIns(4,5)P2 increased >12-fold, and the ratio of [3H]PtdInsP2 to [3H]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [3H]inositol trisphosphate and [3H]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] binding assay showed that total cellular Ins(1,4,5)P3/g fresh weight was >40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKIalpha increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.     

The effect of rearing history and aphid density on volatile‐mediated foraging behaviour of Diaeretiella rapae

1. Parasitoid females foraging for hosts rely on cues derived from the insect host, the host plant and/or their interaction, and all of these can be learned during the immature and adult stages. 2. The present study investigated the importance of rearing history on foraging behaviour of Diaeretiella rapae, an endoparasitoid often associated with aphids feeding on brassicaceous plant species. 3. Parasitoids were reared on one of the four possible combinations, comprising two brassicaceous host plant species, Brassica nigra or Raphanus sativus, and two aphid species Brevicoryne brassicae or Myzus persicae. These parasitoids were tested in a Y‐tube olfactometer and given the choice between volatiles emitted by an aphid‐infested plant (25 or 100 aphids per plant) and an uninfested control plant. The parasitoid's responses were compared when offered: (i) the same plant–aphid combination as the one on which it had been reared; (ii) the same host plant infested with the alternative aphid species; or (iii) an alternative plant with the alternative aphid species. 4. Aphid density did affect the behavioural responses to the various odour sources, but rearing history did not. Diaeretiella rapae only preferred aphid‐induced to non‐induced plant volatiles at low aphid infestation level, whereas they did not discriminate between volatiles at high aphid infestation level. 5. It is concluded that aphid‐induced volatiles of brassicaceous plants play an important role during host habitat location, but seem less important for parasitoids to locate the aphid host itself. The data are discussed in the light of manipulation of host plant defences by aphids.     

Gender differences in the modulation of cardiac gene expression by dietary conjugated linoleic acid isomers

In an earlier study, we showed that dietary conjugated linoleic acid (CLA) isomers can exert differential effects on heart function in male and female rats, but the underlying mechanisms for these actions are not known. Cardiomyocyte Ca2+ cycling is a key event in normal cardiac contractile function and defects in Ca2+ cycling are associated with cardiac dysfunction and heart disease. We therefore hypothesized that abnormalities in the sarcolemmal (SL) and sarcoplasmic reticulum (SR)-mediated regulation of intracellular Ca2+ contribute to altered cardiac contractile function of male and female rats owing to dietary CLA isomers. Healthy male and female Sprague-Dawley rats were fed different CLA isomers, (cis-9, trans-11 (c9,t11) and trans-10, cis-12 (t10,c12)) individually and in combination (50:50 mix as triglyceride or fatty acids) from 4 to 20 weeks of age. We determined the mRNA levels of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 2a, ryanodine receptor, phospholamban, calsequestrin, Na+-Ca2+-exchanger (NCX), and L-type Ca2+ channel in the left ventricle (LV) by RT-PCR. The SR function was assessed by measurement of Ca2+-uptake and -release. Significant gender differences were seen in the LV NCX, L-type Ca2+ channel, and ryanodine receptor mRNA expression levels in control male and female rats. Dietary CLA isomers in the various forms induced changes in the mRNA levels of SERCA 2a, NCX, and L-type Ca2+ channel in the LV of both male and female hearts. Whereas protein contents of the Ca2+ cycling proteins were altered, changes in SR Ca2+-uptake and -release were also detected in both male and female rats in response to dietary CLA. The results of this study demonstrate that long-term dietary supplementation can modulate cardiac gene expression and SR function in a gender-related manner and may, in part, contribute to altered cardiac contractility.     

Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658-724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2-1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1-657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1-657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.