Cholesterol attenuates the interaction of the antimicrobial peptide gramicidin S with phospholipid bilayer membranes

We have investigated the effect of the presence of 25 mol percent cholesterol on the interactions of the antimicrobial peptide gramicidin S (GS) with phosphatidylcholine and phosphatidylethanolamine model membrane systems using a variety of methods. Our circular dichroism spectroscopic measurements indicate that the incorporation of cholesterol into egg phosphatidylcholine vesicles has no significant effect on the conformation of the GS molecule but that this peptide resides in a range of intermediate polarity as compared to aqueous solution or an organic solvent. Our Fourier transform infrared spectroscopic measurements confirm these findings and demonstrate that in both cholesterol-containing and cholesterol-free dimyristoylphosphatidylcholine liquid-crystalline bilayers, GS is located in a region of intermediate polarity at the polar--nonpolar interfacial region of the lipid bilayer. However, GS appears to be located in a more polar environment nearer the bilayer surface when cholesterol is present. Our (31)P-nuclear magnetic resonance studies demonstrate that the presence of cholesterol markedly reduces the tendency of GS to induce the formation of inverted nonlamellar phases in model membranes composed of an unsaturated phosphatidylethanolamine. Finally, fluorescence dye leakage experiments indicate that cholesterol inhibits the GS-induced permeabilization of phosphatidylcholine vesicles. Thus in all respects the presence of cholesterol attenuates but does not abolish the interactions of GS with, and the characteristic effects of GS on, phospholipid bilayers. These findings may explain why it is more potent at disrupting cholesterol-free bacterial than cholesterol-containing eukaryotic membranes while nevertheless disrupting the integrity of the latter at higher peptide concentrations. This additional example of the lipid specificity of GS may aid in the rational design of GS analogs with increased antibacterial but reduced hemolytic activities.     

A differential scanning calorimetric and 31P NMR spectroscopic study of the effect of transmembrane alpha-helical peptides on the lamellar-reversed hexagonal phase transition of phosphatidylethanolamine model membranes

We have investigated the effects of the model alpha-helical transmembrane peptide Ac-K(2)L(24)K(2)-amide (L(24)) on the thermotropic phase behavior of aqueous dispersions of 1,2-dielaidoylphosphatidylethanolamine (DEPE) to understand better the interactions between lipid bilayers and the membrane-spanning segments of integral membrane proteins. We studied in particular the effect of L(24) and three derivatives thereof on the liquid-crystalline lamellar (L(alpha))-reversed hexagonal (H(II)) phase transition of DEPE model membranes by differential scanning calorimetry and (31)P nuclear magnetic resonance spectroscopy. We found that the incorporation of L(24) progressively decreases the temperature, enthalpy, and cooperativity of the L(alpha)-H(II) phase transition, as well as induces the formation of an inverted cubic phase, indicating that this transmembrane peptide promotes the formation of inverted nonlamellar phases, despite the fact that the hydrophobic length of this peptide exceeds the hydrophobic thickness of the host lipid bilayer. These characteristic effects are not altered by truncation of the side chains of the terminal lysine residues or by replacing each of the leucine residues at the end of the polyleucine core of L(24) with a tryptophan residue. Thus, the characteristic effects of these transmembrane peptides on DEPE thermotropic phase behavior are independent of their detailed chemical structure. Importantly, significantly shortening the polyleucine core of L(24) results in a smaller decrease in the L(alpha)-H(II) phase transition temperature of the DEPE matrix into which it is incorporated, and reducing the thickness of the host phosphatidylethanolamine bilayer results in a larger reduction in the L(alpha)-H(II) phase transition temperature. These results are not those predicted by hydrophobic mismatch considerations or reported in previous studies of other transmembrane alpha-helical peptides containing a core of an alternating sequence of leucine and alanine residues. We thus conclude that the hydrophobicity and conformational flexibility of transmembrane peptides can affect their propensity to induce the formation of inverted nonlamellar phases by mechanisms not primarily dependent on lipid-peptide hydrophobic mismatch.     

The effects of cisplatinum and vincristine on peripheral nerve regeneration

Current treatment modalities for extremity sarcoma often include tumor extirpation plus neoadjuvant therapy. Limb-sparing surgery may require reconstruction of critical nerve defects. Neurotoxic side effects from adjuvant chemotherapy have been reported and raise concerns regarding the effects of chemotherapy on nerve regeneration. In an attempt to define the effects of adjuvant chemotherapy on peripheral nerve regeneration, cisplatin and vincristine were administered to rats following isografting of the posterior tibial nerve. Parameters used to assess peripheral nerve regeneration included walking track analysis and histomorphology. Sixty 250-g Sprague-Dawley rats were randomly allocated into one of three treatment groups. Each animal underwent a 15-mm reversed interposition nerve isograft from 30 donor rats into the right posterior tibial nerve. Ten animals served as control. The remaining animals were divided into two groups of 25 animals each. One group received cisplatin (75 mg/m2) and the other group received vincristine (1 mg/m2). Chemotherapy was administered at 4-week cycles for a total of six cycles (24 weeks). Walking track analysis was performed monthly. Nerve specimens were harvested from the grafted segment and the distal posterior tibial nerve for histomorphology. Walking track analysis demonstrated no statistical difference in print length between the control and chemotherapeutic groups at the conclusion of the study. The number of axons per square millimeter and nerve fiber density were not statistically different between control and chemotherapeutic groups. In the rodent posterior tibial nerve model, postoperative adjuvant therapy does not significantly alter functional outcome in peripheral nerve regeneration. The practice of immediate nerve grafting after tumor extirpation, despite planned postoperative chemotherapy, is supported.     

Alternative roles for putative ice-binding residues in type I antifreeze protein

Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.     

Penile vibratory stimulation in the marmoset monkey: a practical alternative to electro-ejaculation, yielding ejaculates of enhanced quality

The availability of sufficient amounts of spermatozoa of high quality is one of the main limiting factors in reproductive research and development of reproductive technologies in marmoset monkeys (Callithrix jacchus). Penile vibrostimulation (PVS) has been successfully used in semen collection in the squirrel monkey but with poor success rate in the marmoset. We report here on an improved protocol for PVS with a success rate of almost 90%. Ejaculates obtained by PVS were of enhanced quality compared with those obtained by rectal probe electro-ejaculation (RPE). PVS ejaculates contained on average three to fourfold higher numbers of total and motile spermatozoa. Assessment of sperm kinematics using computer-assisted sperm analysis indicated that there are also functional differences between spermatozoa collected by PVS and RPE. Marmoset spermatozoa in samples obtained by RPE swim in a more convoluted manner compared with those obtained by PVS.     

Isolation of Pea Thioredoxin f Precursor Protein and Characterization of its Biochemical Properties

Like many other soluble chloroplastic enzymes, thioredoxin f is nuclear-encoded and expressed as a precursor protein. After synthesis in the cytosol, it is imported into the chloroplast with subsequent cleavage of the transit sequence in the stroma. We report the expression and the partial purification of the recombinant precursor thioredoxin f protein. The prethioredoxin f was found to be located essentially in the insoluble Echerichia coli fraction, but could be renatured after urea treatment followed by dialysis. The renatured protein was active in the dithiothreitol- and thioredoxin-dependent activation of NADP malate dehydrogenase and also of fructose bisphosphatase and in the ferredoxin-thioredoxin-dependent fructose bisphosphatase activation. These data are discussed in relation with the known properties of mature thioredoxin f.     

Defining the minimum size of a hydrophobic cluster in two-stranded alpha-helical coiled-coils: effects on protein stability

The alpha-helical coiled-coil motif is characterized by a heptad repeat pattern (abcdefg)(n) in which residues a and d form the hydrophobic core. Long coiled-coils (e.g., tropomyosin, 284 residues per polypeptide chain) typically do not have a continuous hydrophobic core of stabilizing residues, but rather one that consists of alternating clusters of stabilizing and destabilizing residues. We have arbitrarily defined a cluster as a minimum of three consecutive stabilizing or destabilizing residues in the hydrophobic core. We report here on a series of two-stranded, disulfide-bridged parallel alpha-helical coiled-coils that contain a central cassette of three consecutive hydrophobic core positions (d, a, and d) with a destabilizing cluster of three consecutive Ala residues in the hydrophobic core on each side of the cassette. The effect of adding one to three stabilizing hydrophobes in these positions (Leu or Ile; denoted as [see text]) was investigated. Alanine residues (denoted as [see text]) are used to represent destabilizing residues. The peptide with three Ala residues in the d a d cassette positions ([see text]) was among the least stable coiled-coil (T(m) = 39.3 degrees C and Urea(1/2) = 1.9 M). Surprisingly, the addition of one stabilizing hydrophobe (Leu) to the cassette or two stabilizing hydrophobes (Leu), still interspersed by an Ala in the cassette ([see text]), also did not lead to any gain in stability. However, peptides with two adjacent hydrophobes in the cassette ([see text])([see text]) did show a gain in stability of 0.9 kcal/mole over the peptide with two interspersed hydrophobes ([see text]). Because the latter three peptides have the same inherent hydrophobicity, the juxtaposition of stabilizing hydrophobes leads to a synergistic effect, and thus a clustering effect. The addition of a third stabilizing hydrophobe to the cassette ([see text]) resulted in a further synergistic gain in stability of 1.7 kcal/mole (T(m) = 54.1 degrees C and Urea(1/2) = 3.3M). Therefore, the role of hydrophobicity in the hydrophobic core of coiled-coils is extremely context dependent and clustering is an important aspect of protein folding and stability.     

Horned lizard (Phrynosoma) phylogeny inferred from mitochondrial genes and morphological characters: understanding conflicts using multiple approaches

The genus Phrynosoma includes 13 species of North American lizards characterized by unique and highly derived morphologies and ecologies. Understanding interspecific relationships within this genus is essential for testing hypotheses about character evolution in this group. We analyzed mitochondrial ND4 and cytochrome b gene sequence data from all species of Phrynosoma in conjunction with a previously published dataset including 12S and 16S rRNA gene sequences and morphological characters. We used multiple phylogenetic methods and diagnostic tests for data combinability and taxonomic congruence to investigate the data in separate and combined analyses. Separate data partitions resulted in several well-supported lineages, but taxonomic congruence was lacking between topologies from separate and combined analyses. Partitioned Bremer support analyses also reveals conflict between data partitions in certain tree regions. When taxa associated with well-supported clades were removed from analyses, phylogenetic signal was lost. Combined, our results initially suggest conflict between data partitions, but further tests show the data are only appropriate for phylogenetic reconstruction of those parts of the topology that were well resolved. Nonetheless, our data analyses reveal five well-supported clades: (1) Phrynosoma ditmarsi and Phrynosoma hernandesi, (2) P. ditmarsi, P. hernandesi, and Phrynosoma douglasii, (3) P. ditmarsi, P. hernandesi, P. douglasii, and Phrynosoma orbiculare, (4) Phrynosoma mcallii and Phrynosoma platyrhinos, and (5) Phrynosoma braconnieri and Phrynosoma taurus.     

Alpha 1-adrenergic and cholinergic agonists activate MAPK by separate mechanisms to inhibit secretion in lacrimal gland

Thepurpose of this study was to determine the role of p42/p44mitogen-activated protein kinase (MAPK) in₁-adrenergically and cholinergically stimulated proteinsecretion in rat lacrimal gland acinar cells and the pathways used bythese agonists to activate MAPK. Acini were isolated by collagenasedigestion and incubated with the ₁-adrenergic agonistphenylephrine or the cholinergic agonist carbachol, and activation ofMAPK and protein secretion were then measured. Phenylephrine andcarbachol activated MAPK in a time- and concentration-dependent manner.Inhibition of MAPK significantly increased phenylephrine- andcarbachol-induced protein secretion. Inhibition of EGF receptor (EGFR)with AG1478, an inhibitor of the EGFR tyrosine kinase activity,significantly increased phenylephrine- but not carbachol-inducedprotein secretion. Whereas phenylephrine-induced activation of MAPK wascompletely inhibited by AG1478, activation of MAPK by carbachol wasnot. Phenylephrine stimulated tyrosine phosphorylation of the EGFR, whereas carbachol stimulated p60^Src, and possibly Pyk2, toactivate MAPK. We conclude that, in the lacrimal gland, activation ofMAPK plays an inhibitory role in ₁-adrenergically andcholinergically stimulated protein secretion and that these agonistsuse different signaling mechanisms to activate MAPK.