Apolipoprotein A-I mimetic peptide helix number and helix linker influence potentially anti-atherogenic properties

We hypothesize that apolipoprotein A-I (apoA-I) mimetic peptides better mimicking the punctuated alpha-helical repeats of full-length apoA-I are more anti-inflammatory and anti-atherogenic. This study compares a monomeric apoA-I mimetic helix to three different tandem helix peptides in vitro: 4F (18 mer), 4F-proline-4F (37 mer, Pro), 4F-alanine-4F (37 mer, Ala), and 4F-KVEPLRA-4F [the human apoA-I 4/5 interhelical sequence (IHS), 43 mer]. All peptides cleared turbid lipid suspensions, with 4F being most effective. In contrast to lipid clearance, tandem peptides were more effective at remodeling mouse HDL. All four peptides displaced apoA-I and apoE from the HDL, leaving a larger particle containing apoA-II and peptide. Peptide-remodeled HDL particles show no deficit in ABCG1 cholesterol efflux despite the loss of the majority of apoA-I. Tandem peptides show greater ability to efflux cholesterol from lipid-loaded murine macrophages, compared with 4F. Although 4F inhibited oxidation of purified mouse LDL, the Ala tandem peptide increased oxidation. We compared several tandem 4F-based peptides with monomeric 4F in assays that correlated with suggested anti-inflammatory/anti-atherogenic pathways. Tandem 4F-based peptides, which better mimic full-length apoA-I, exceed monomeric 4F in HDL remodeling and cholesterol efflux but not LDL oxidation protection. In addition, apoA-I mimetic peptides may increase reverse cholesterol transport through both ABCA1 as well as ABCG1 pathways.     

Left-handed helix of polyproline ii type in linker regions of DNA-binding proteins

Linker segments assuming the polyproline II type conformation within DNA-protein complexes were sought in protein and linker databases. Seventy-three linker-DNA complexes were found. The mean length of polyproline II type segments was six residues, and prolines were not predominant there. It was shown that the symmetrical position of prolines in these segments prevented the formation of the cooperative water network involving amide groups. An example of specific proline location in some motility apparatus proteins is presented.     

The site of binding of linker histone to the nucleosome does not depend upon the amino termini of core histones.

Using nucleosomes reconstituted on a defined sequence of DNA, we have investigated the question as to whether the N-terminal tails of core histones play a role in determining the site of binding of a linker histone. Reconstitutes used histone cores of three types: intact, lacking the N-terminal H3 tails, or lacking all tails. In each case the same, single defined position for the histone core was observed, using high-resolution mapping. The affinity for binding of linker histone H1(o) was highest for the intact cores, lowest for the tailless cores. However, the location of the linker histone, as judged by micrococcal nuclease protection, was exactly the same in each case, an asymmetric site of about 17 bp to one side of the core particle DNA.     

CD spectra of indolicidin antimicrobial peptides suggest turns, not polyproline helix.

Indolicidin is a 13-residue antimicrobial peptide-amide isolated from the cytoplasmic granules of bovine neutrophils that contains five Trp and three Pro residues. Falla et al. [(1996) J. Biol. Chem. 271, 19298] suggested that indolicidin forms a poly-L-proline II helix based upon the circular dichroism (CD) spectra of a closely related peptide (indolicidin methyl ester). In contrast, we found no evidence of poly-L-proline II helix formation in the CD spectra of native indolicidin in various solvents or when bound to micelles and membranes [Ladokhin et al. (1997) Biophys. J. 72, 794]. We interpreted the spectra as arising from unordered and/or beta-turn structures, but noted a sharp negative band at 227 nm arising from the tryptophan residues that would mask spectral features characteristic of poly-L-proline II helix. We have reexamined this issue by means of CD measurements of native indolicidin and several of its analogues. None of the features characteristic of a poly-L-proline helix (or alpha- or 3(10)-helix) were observed for any of the peptides studied. To eliminate artifacts associated with tryptophan, we synthesized indolicidin-L and indolicidin-F in which all five tryptophans were replaced with leucines or phenylalanines, respectively. The changes in CD spectra of these Trp-free peptides upon transfer into membrane-like environments were found to be consistent with the formation of beta-turns. For the native indolicidin in SDS micelles, temperature increases resulted in a coupled diminution of two sharp bands, a negative one at 227 nm and a positive one at 217 nm. This phenomenon, which is absent in indolicidin-L variants with single Leu-->Trp substitutions, is consistent with exciton splitting produced by the stacking of indole rings. Type VI turns in model peptides in aqueous solution are known to be promoted by stacking interactions between cis-proline and neighboring aromatic residues [Yao et al. (1994) J. Mol. Biol. 243, 754]. Molecular modeling of indolicidin with a -Trp(6)-cis-Pro(7)-Trp(8)- type VIa turn demonstrated the feasibility of this turn conformation and revealed the possibility of an accompanying amphipathic structure. We therefore suggest that turn conformations are the principal structural motif of indolicidin and that these turns greatly enhance membrane activity.     

The MURFI linker for multiple reading frame insertion of a sense or nonsense codon into DNA.

Blunt-end palindromic DNA linkers with a central restriction site have been designed for the multiple reading frame insertion (abbreviated MURFI) of a sense or nonsense codon into DNA. We have utilized an amber MURFI linker, 5'CTAG TCTAGA CTAG3' to disrupt the lacZ gene, yielding truncated beta-galactosidase proteins. Conditional disruption of the tetr gene in E. coli has also been demonstrated. Nonsense codon MURFI linkers permit conditional fusion of multiple gene products while sense codon linkers can add structural elements (e.g. beta-turn, cationic segment, hydrophobic segment) or a desired amino acid to a protein (e.g. methionine, cysteine). Shotgun or alternatively site-directed insertion of the symmetric linkers is possible. The over-all length of the linker may be adjusted to retain the original reading frame, matching nucleotide additions or subtractions at recipient DNA sites. If a linker restriction site occurs elsewhere in the target DNA, single linker copies may still be inserted using non-phosphorylated linkers.     

The Relationship of Carbohydrates to Cold Acclimation of Hedera helix L. cv. Thorndale

While both the total sugar content and cold hardiness greatly increased during artificial cold acclimation, no direct parallelism was demonstrated. In fact, plants hardened in the dark exhibited an increase in hardiness during the period when the total sugar content declined, furthermore, while there was evidence for an accumulation of sugars (especially sucrose) incorporation of ¹⁴C indicated that all of the isolated fractions were in a dynamic state. Dehardening, paralleled by a large increase in the starch content, was more rapid in the light than in the dark. Furthermore, in stems the sugar content increased after 3 days of dehardening but declined after 7 days. This increase may represent the release of sugars from a previously unextractable form such as a glycoprotein complex. Starvation experiments indicate that photosynthates produced during the cold acclimation period are preferentially used during cold acclimation rather than reserve carbohydrates. This was also indicated by the smaller amount of starch hydrolysis in plants hardened in the light. Thus, while there appears to be a role for carbohydrates in the cold acclimation process, the lack of parallelism between sugar content and hardiness may be interpreted as indicating 1) cold acclimation is not merely an accumulation of sugars or an osmotic effect per se, and 2) under normal conditions, the level of carbohydrates is not limiting the rate or degree of cold acclimation.     

Small-angle X-ray scattering studies of calmodulin mutants with deletions in the linker region of the central helix indicate that the linker region retains a predominantly alpha-helical conformation

Two mutant forms of calmodulin were examined by small-angle X-ray scattering in solution and compared with the wild-type protein. Each mutant has deletions in the linker region of the central helix: one lacks residues Glu-83 and Glu-84 (Des2) and the other lacks residues Ser-81 through Glu-84 (Des4). The deletions change both the radii of gyration and the maximum dimensions of the molecules. In the presence of Ca2+, the observed radii of gyration are 22.4 A for wild-type bacterially expressed calmodulin, 19.5 A for Des2 calmodulin, and 20.3 A for Des4 calmodulin. A reduction in the radius of gyration by 1-2 A on removal of calcium, previously observed in the native protein, was also found in the wild type and the Des4 mutant; however, no significant size change was observed in the Des2 mutant. The large calcium-dependent conformational change in calmodulin induced by the binding of melittin [Kataoka, M., Head, J.F., Seaton, B.A., & Engelman, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6944-6948] was observed in all the bacterially expressed proteins. Each protein appears to undergo a transition from a dumbbell shape to a more globular conformation on binding melittin in the presence of calcium, although quantitatively the changes in the wild-type and Des4 proteins greatly exceed those in Des2. Modeling shows the central linker region of the molecule. Thus, the structure of the linker region is stable enough to maintain the average orientation and separation of the lobes yet flexible enough to permit the lobes to approach each other upon binding a peptide.     

Enzymatic glycosylation of reducing oligosaccharides linked to a solid phase or a lipid via a cleavable squarate linker.

Reducing oligosaccharides were converted into their corresponding glycosylamines, and these were reacted with 3,4-diethoxy-3-cyclobuten-1,2-dione (squaric acid diethyl ester). The resulting derivatives could be linked to amino-functionalized lipids, solids, or proteins. Treatment of the obtained lipid or solid conjugates with aqueous bromine or, alternatively, with ammonia-ammonium borate cleaved the linkage and regenerated the oligosaccharide glycosylamines, which were in turn rapidly hydrolyzed to the reducing oligosaccharides. To demonstrate the usefulness of this linkage in enzymatic oligosaccharide synthesis, lactose was linked to a lipid or a solid phase, the obtained conjugates were then subjected to two enzymatic glycosylations (either consecutively or 'one-pot'). The resulting materials were then cleaved to give, in both cases, the expected reducing tetrasaccharide (lacto-N-neotetraose) in good yield.     

To adhere or not to adhere

The modulation of cell adhesion is fundamental to the morphogenesis that accompanies proper embryonic development. Cadherins are a large family of calcium-dependent cell adhesion molecules whose spatial and temporal expression is critical to the formation of the neural crest, a unique, multipotent cell type that contributes to the patterning of the vertebrate body plan. Neural crest cells arise from the embryonic ectoderm through inductive interactions and reside in the dorsal aspect of the neural tube. These cells under go an epithelial-to-mesenchymal transition and migrate to precise destinations in the embryo, where they go on to differentiate into such diverse structures as melanocytes, elements of the peripheral nervous system, and the craniofacial skeleton. Distinct cadherins are expressed during the induction, migration and differentiation of the neural crest. With the advent of genomic sequencing, assembly and annotation for various model organisms, it has become possible to elucidate the molecular mechanisms underlying cadherin expression, and how these cadherins function, during neural crest development. This review explores the known roles of cadherins and details, where relevant, how different cadherins are regulated during the formation of the neural crest.     

To age or not to age

According to the antagonistic pleiotropy theory of ageing, natural selection has favoured genes conferring short-term benefits to the organism at the cost of deterioration in later life. The 'disposable soma' theory expresses this as a life-history strategy in which somatic maintenance is below the level required to prevent ageing, thus enabling higher immediate fertility. It has been argued that a non-ageing strategy will always be bettered by a low but non-zero rate of ageing, because the costs of such ageing will be felt only in the distant future when they are of negligible importance. Here, we examine this argument critically. We find that a non-ageing strategy will be locally optimal if, in the presence of ageing, the onset of deterioration is sufficiently rapid or early. Conversely, ageing will be optimal if deterioration is sufficiently slow or late. As the temporal profile of ageing changes from one of steady deterioration to one involving a sudden loss of vitality after a period of little or no decline, the conditions for a non-ageing strategy to be locally optimal become progressively more stringent. But for all forms of profile considered, conditions can be found for which a strategy involving no ageing is locally optimal.     

Binding of netropsin to a DNA triple helix.

The interaction of netropsin, a minor groove binding drug, with T-A-T triple helix and A-T double helix was studied using circular dichroism spectroscopy and thermal denaturation. The triple helix was made by an oligonucleotide (dA)12-x-(dT)12-x-(dT)12, where x is a hexaethylene glycol chain bridged between the 3' phosphate of one strand and the 5' phosphate of the following strand. This oligonucleotide is able to fold back on itself to form a very stable triplex. Changing the conditions allows the same oligonucleotide in a duplex form with a (dT)12 dangling arm. Circular dichroism spectroscopy demonstrates that netropsin can bind to the triple helical structure. Spectral analysis shows that the bound drug exhibits a conformation and an environment similar in double-stranded and in triple-stranded structure. However, the binding constant to the triple-stranded structure is found smaller than the binding constant to the double-stranded one. Thermal denaturation experiments demonstrate that netropsin destabilizes the triplex whereas it stabilizes the duplex.