Amphiphilic cationic peptides mediate cell adhesion to plastic surfaces

Four amphiphilic peptides, each with net charges of +2 or more at neutrality and molecular weights under 4 kilodaltons, were found to mediate the adhesion of normal rat kidney fibroblasts to polystyrene surfaces. Two of these peptides, a model for calcitonin (peptide 1, MCT) and melittin (peptide 2, MEL), form amphiphilic alpha-helical structures at aqueous/nonpolar interfaces. The other two, a luteinizing hormone-releasing hormone model (peptide 3, LHM) and a platelet factor model (peptide 4, MPF) form beta-strand structures in amphiphilic environments. Although it contains only 10 residues, LHM mediated adhesion to surfaces coated with solutions containing as little as 10 pmoles/ml of peptide. All four of these peptides were capable of forming monolayers at air-buffer interfaces with collapse pressures greater than 20 dynes/cm. None of these four peptides contains the tetrapeptide sequence Arg-Gly-Asp-Ser, which has been associated with fibronectin-mediated cell adhesion. Ten polypeptides that also lacked the sequence Arg-Gly-Asp-Ser but were nonamphiphilic and/or had net charges less than +2 at neutrality were all incapable of mediating cell adhesion (Pierschbacher and Ruoslahti, 1984). The morphologies of NRK cells spread on polystyrene coated with peptide LHM resemble the morphologies on fibronectin-coated surfaces, whereas cells spread on surfaces coated with MCT or MEL exhibit strikingly different morphologies. The adhesiveness of MCT, MEL, LHM, and MPF implies that many amphiphilic cationic peptides could prove useful as well defined adhesive substrata for cell culture and for studies of the mechanism of cell adhesion.     

Sexual advertisement and immune function in an arachnid species (Lycosidae)

A simple version of the immunocompetence handicap hypothesizesthat through condition-dependence, the size of the sexual traitmay be positively related to immune function at the populationlevel. In the present study, we investigated the relationshipbetween sexual advertisement and immune function in a naturalpopulation of male wolf spiders, Hygrolycosa rubrofasciata (Araneae:Lycosidae). Males of H. rubrofasciata have a costly and condition-dependentacoustic signal, courtship drumming. In the mating season, malesdrum against dry leaves while wandering around the habitat searchingfor receptive females. Males increase their mating success byincreasing their drumming rate and mobility. We used drummingrate and mobility measured without female proximity as estimatesof sexual advertisement. As estimates of male immune function,we used encapsulation rate and lytic activity. Encapsulationrate is a common challenging technique, which measures immuneresponse against multicellular parasites. Lytic activity isa monitoring technique, which measures immune response againstpathogens. Our results show that males with higher drummingrate had higher encapsulation rate. This suggests that femalesmight use drumming rate as a signal for choosing males withgood immunocompetence. Moreover, our results show that maleswith higher mobility had higher lytic activity. As females aremore likely to encounter those males that have higher mobility,this might also select males with better immune function. Ourresults suggest that the immunocompetence handicap might workalso among spiders, although we could not assess the causalityof the relationship between sexual selection and immune functionin this correlational study.     

Synthesis of novel acetylene-containing amino acids

Summary.  Novel synthetic procedures for the modification of non-proteinogenic acetylene-containing amino acids have been developed. The functionalization either proceeds via zinc/copper-mediated introduction of alkyl substituents, or via tungsten-catalyzed ring-closing alkyne metathesis reactions. Received March 28, 2002 Accepted October 3, 2002 Published online December 18, 2002 Acknowledgements These investigations are supported (in part) by the Netherlands Research Council for Chemical Sciences (CW) with financial aid from the Netherlands Technology Foundation (STW). Authors' address: Floris P. J. T. Rutjes, Prof. Dr., Department of Organic Chemistry, University of Nijmegen, Toernooiveld 1, NL-6525 ED Nijmegen, The Netherlands, E-mail: rutjes@sci.kun.nl  2, selected data: ¹H NMR (300 MHz, CDCl₃) δ 5.32 (d, J = 7.7 Hz, 1H), 4.44–4.40 (m, 1H), 3.76 (s, 3H), 2.75–2.73 (d, J = 5.0 Hz, 2H), 1.44 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.0, 155.0, 80.3, 74.6, 52.6, 51.9, 41.7, 28.3, 24.0; mp = 55°C.  Typical procedure for 5: zinc dust (116 mg, 1.408 mmol) was weighed into a 20 mL flask, which was repeatedly evacuated (with heating using a heat gun) and flushed with argon. Dry DMF (0.5 mL, distilled from CaH₂) and 1,2-dibromoethane (9.2 μL, 0.106 mmol) were added and the flask was heated at 80°C for 40 min. The reaction mixture was allowed to cool to room temperature, trimethylsilyl chloride (4 μL, 0.035 mmol) was added and the resulting mixture was stirred vigorously for a further 30 min under argon. Iodocyclohexane (69 μl, 0.528 mmol) was added and stirred at room temperature for 3 h more after which stirring was ceased to settle the zinc. CuCN (41 mg, 0.458 mmol) and LiCl (40 mg, 0.915 mmol) were heated to 150°C for 2 h and cooled to room temperature. Addition of DMF (1 mL) formed a soluble CuCN·2LiCl complex within 5 min. After cooling the Cu-complex to −15°C, the organozinc reagent was added dropwise followed by the bromoacetylene 2 (116 mg, 0.352 mmol). The mixture was allowed to stir overnight at room temperature. Water was added and the suspension was extracted using heptane, washed with brine, dried (MgSO₄) and concentrated. Purification using flash column chromatography (10% EtOAc in heptane) yielded 5 (100 mg, 81%) as a colorless oil. 5: IR ν 3355, 2929, 2852, 2359, 2337, 1749, 1717, 1498, 1447, 1365, 1251, 1181, 1060; ¹H NMR (300 MHz, CDCl₃) δ 5.28 (d, J = 7.7 Hz, 1H), 4.43–4.38 (m, 1H), 3.73 (s, 3H), 2.69–2.63 (m, 2H), 2.13 (m, 1H), 1.73–1.22 (m, 10H), 1.43 (s, 9H); ¹³C NMR (75 MHz, CDCl₃) δ 171.4, 155.0, 88.1, 79.9, 73.8, 52.3, 32.7, 32.7, 28.8, 28.2, 25.8, 24.6, 23.1; HRMS (EI): calculated for C₁₇H₂₇NO₄ 309.1940, found 309.1937.  A solution of the tungsten catalyst (7 mg, 10 mol%) in C₆H₅Cl (2 mL) was treated with a solution of 14 (49.0 mg, 0.120 mmol) in C₆H₅Cl (5.0 mL) under an argon atmosphere and the resulting mixture was heated at 80°C for 3 h. Evaporation followed by flash column chromatography (80% EtOAc in heptane) afforded 15 (21.0 mg, 50%; 64% after correction for starting material) and 14 (16 mg, 33%) as colorless oils. 15: [α]_D =–14.6 (c = 1, CH₂Cl₂); IR ν 3313, 2931, 2865, 2249, 1744, 1667, 1520, 1366, 1170; ¹H NMR (400 MHz, CDCl₃) δ 7.14 (d, J = 8.7 Hz, 1H), 6.08 (d, J = 8.3 Hz, 1H), 4.78 (q, J = 6.8 Hz, 1H), 4.27 (q, J = 7.9 Hz, 1H), 3.73 (s, 3H), 2.17–2.15 (m, 4H), 2.07–1.96 (m, 2H), 1.79–1.52 (m, 4H), 1.45 (s, 9H), 0.89–0.83 (m, 2H); ¹³C NMR (100 MHz, CDCl₃) δ 173.2, 171.8, 155.8, 80.4, 80.2, 79.3, 53.8, 52.5, 51.2, 32.8 (2×), 28.1, 24.6, 24.2, 18.3 (2×); HRMS (EI): calculated for C₁₈H₂₈N₂O₅     

Circadian and Ultradian Rhythms in Heart Rate and Motor Activity of Smokers, Abstinent Smokers, and Nonsmokers

In a field study, heart rate and motor activity were assessed continuously in 12 male smokers during 2 smoking and 2 abstinence days and in 12 male nonsmokers during 4 days. A circadian analysis revealed earlier activity acrophases in smokers than nonsmokers and earlier heart rate acrophases in abstinent than smoking smokers. Furthermore, heart rate acrophases of smoking smokers significantly anticipated activity acrophases;, whereas in abstinent smokers and nonsmokers the two parameters oscillated in phase. With the use, in smoking smokers, of the individual average smoking interval as a hypothetical ultradian period length, significant periodicities were found for heart rates in 16 and for activity in 15 of 24 observation days. These rhythms were nicotine independent and based on heart rate and activity increases prior to lighting up the cigarettes. Individual frequency spectra for the 16 h after getting up and the 7 h after going to bed did not reveal single dominant frequencies but rather complex frequency distributions. Power spectra of the daytime data revealed no group differences for activity and no heart rate differences between smoking smokers and nonsmokers. In abstinent smokers, however, a significant reduction of heart rate frequencies slower than 1 cycle/135 min and a significant increase of heart rate frequencies faster than 1 cycle/20 min were observed as compared with all other groups. This effect persisted over the 2 abstinence days, suggesting an activity-independent change in the frequency distribution of heart rates after quitting smoking.     

Fine mapping of the chicken salmonellosis resistance locus (SAL1)

Salmonella enterica serovar Typhimurium is a Gram-negative bacterium that has a significant impact on both human and animal health. It is one of the most common food-borne pathogens responsible for a self-limiting gastroenteritis in humans and a similar disease in pigs, cattle and chickens. In contrast, intravenous challenge with S. Typhimurium provides a valuable model for systemic infection, often causing a typhoid-like infection, with bacterial replication resulting in the destruction of the spleen and liver of infected animals. Resistance to systemic salmonellosis in chickens is partly genetically determined, with bacterial numbers at systemic sites in resistant lines being up to 1000-fold fewer than in susceptible lines. Identification of genes contributing to disease resistance will enable genetic selection of resistant lines that will reduce Salmonella levels in poultry flocks. We previously identified a novel resistance locus on Chromosome 5, designated SAL1 . Through the availability of high-density SNP panels in the chicken, combined with advanced back-crossing of the resistant and susceptible lines, we sought to refine the SAL1 locus and identify potential positional candidate genes. Using a 6^th generation backcross mapping population, we have confirmed and refined the SAL1 locus as lying between 54.0 and 54.8 Mb on the long arm of Chromosome 5 ( F  = 8.72, P  = 0.00475). This region spans 14 genes, including two very striking functional candidates; CD27-binding protein ( Siva ) and the RAC -alpha serine/threonine protein kinase homolog , AKT1 ( protein kinase B , PKB ).     

Dna stability and survival of bacillus subtilis spores in extreme dryness

The inactivation of Bacillus subtilis spores during long-term exposure (up to several months) to extreme dryness (especially vacuum) is strain-dependent, through only to a small degree. During a first phase (lasting about four days) monolayers of spores lose about 20% of their viability, regardless of the strain studied. During this phase loss in viability can be equally attributed both to damages of hydrophobic structures (membranes and proteins) and DNA. During a second phase lasting for the remaining time of experimental observation (weeks, months and years) the loss in viability is slowed. A viability of 55% to 75% (depending on the strain) is attained after a total exposure of 36 days. The loss in viability during the second phase can be correlated with the occurrence of DNA double strand breaks. Also covalent DNA-protein cross-links are formed by vacuum exposure. If the protein moiety of these cross-links is degraded by proteinase K-treatment in vitro additional DNA double strand breaks result. The data are also discussed with respect to survival on Mars and in near Earth orbits.     

Interaction of calcium and vanadate with fluorescein isothiocyanate labeled Ca2+-ATPase from sarcoplasmic reticulum: kinetics and equilibria

The interaction of Ca2+ and vanadate with fluorescein isothiocyanate (FITC) labeled sarcoplasmic reticulum (SR) Ca2+-ATPase has been studied by following the kinetics of changes in the reporter group fluorescence and equilibrium fluorescence levels. The vanadate species bound to the enzyme is clearly monomeric orthovanadate, probably H2VO4-. Vanadate binding is noncooperative, suggesting an absence of interactions between the Ca2+-ATPase subunits. The fluorescence experiments confirm the existence of a calcium-enzyme-vanadate complex (in the presence of magnesium). On the basis of the fluorescence properties of this complex, it is similar in its conformation to the calcium-enzyme complex, i.e., "E1-like" rather than "E2-like". However, Ca2+ binds to the enzyme-vanadate complex via sites that are only accessible from the interior of the SR vesicles. The complex Ca2E*Van, which is rapidly formed, isomerizes very slowly (t1/2 approximately 1 min) to the stable ternary complex. The mutual destabilization between bound vanadate and two bound Ca2+ ions is only 1.6 kcal/mol, much smaller than that produced by the interaction of calcium and phosphate.     

Susceptibility of autologous target cells to lysis by lymphokine-activated effectors from interferon-α-treated chronic myelogenous leukaemia patients

Summary Chronic myelogenous leukemia (CML) patients in chronic phase display compromised lymphokine-activated killer (LAK) cell induction, which is partly restored after therapy with interferon . However, the relative resistance of the leukemic cells from these patients to autologous or allogeneic LAK lysis is not affected by this treatment. In an attempt to render CML cells more susceptible to lysis or cytostasis, they were precultured in serum-free medium with or without recombinant growth factors. In eight patients studied, interleukin-3 (IL-3) significantly enhanced the spontaneous short-term (6-day) proliferation of CML cells, with retention of ability to form colonies in methylcellulose. Culture in either medium alone or IL-3 led to a significant enrichment of CD14⁺ and CD33⁺ cells but to a reduction in CD34⁺ cells. In contrast, culture of the same cells in IL-2 (to generate autologous LAK activity) resulted in a loss of CD14⁺ and CD33⁺ as well as CD34⁺ cells but in a significant increase in CD3⁺ and CD56⁺ cells. Despite similarities in their phenotypes, IL-3 cultured cells but not those cultured in medium alone acquired susceptibility to lysis by the IL-2-cultured autologous LAK cells. These results may have significance for the design of novel combination immunotherapy in CML.This work was supported in part by the Deutsche Forschungsgemeinschaft (SFB 120)