Force spectroscopy with a small dithering of AFM tip: a method of direct and continuous measurement of the spring constant of single molecules and molecular complexes

A new method of direct and continuous measurement of the spring constant of single molecule or molecular complex is elaborated. To that end the standard force spectroscopy technique with functionalized tips and samples is combined with a small dithering of the tip. The change of the dithering amplitude as a function of the pulling force is measured to extract the spring constant of the complex. The potentialities of this method are illustrated for the experiments with single bovine serum albumin-its polyclonal antibody (Ab-BSA) and fibrinogen-fibrinogen complexes.     

Copper(II) complexes based on a new chelating 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine ligand: Synthesis and crystal structures. Lone pair-π, C-H···π, π-π and C-H···A (A = N, Cl) non-covalent interactions

A new bidentate chelating pyrazolylpyrimidine ligand bearing a strong electron-donating substituent, i.e. 4-(3,5-diphenyl-1H-pyrazol-1-yl)-6-(piperidin-1-yl)pyrimidine (L) (Scheme 1), has been synthesized and used to obtain the copper(II) complexes by reaction with CuCl₂. The molar ratio Cu:L = 1:2 leads to isolation of a complex having CuL₂Cl₂ empirical formula, while the molar ratio Cu:L = 1:1 gives a complex with CuLCl₂ empirical formula. The crystal structure of L as well as the structures of both complexes were studied by single crystal X-ray diffraction. The crystal structure of CuL₂Cl₂ compound is formed by trans-[CuL₂Cl₂] mononuclear molecules. Surprisingly, in contrast to the previous compound having molecular structure, the crystal structure of CuLCl₂ consists of mononuclear [CuL₂Cl]⁺ complex cations and dinuclear [Cu₂Cl₆]²⁻ anions. Thus, formula of CuLCl₂ complex can be represented as [CuL₂Cl]₂[Cu₂Cl₆]. In both complexes molecules of L adopt bidentate chelating coordination mode through N² atom of pyrazole and N³ atom of pyrimidine rings forming five-membered CuN₃C metallocycles. Owing to C-H···N interactions and π-π-stacking L molecules form 2D network. In the structure of trans-[CuL₂Cl₂] there exist double lone pair(N(piperidine))-π(pyrimidine) interactions and C-H···Cl contacts resulting in the formation of 1D chains. Layered 2D structure of [CuL₂Cl]₂[Cu₂Cl₆] results from C-H···Cl, C-H···π and double lone pair(Cl([CuL₂Cl]⁺ complex cation)-π(pyrimidine) interactions.     

Male-Induced Sociosexual Behavior by Vaginal Secretions in Macaca arctoides

Odor communication in Old World monkeys and apes is controversial, because most females have evolved visual and behavioral cues to signal fertility, e.g., sexual swellings. Female stump-tailed macaques (Macaca arctoides) do not have swellings, and mediation of chemical communication likely occurs because males engage in sexual behavior mostly throughout the periovulatory phase of the menstrual cycle. We tested whether vaginal secretions from different cycle phases, with saline solution as a control, promote changes in the frequency of male genital exploration, copulation, and coercive behavior toward females different from the donors, while female donors were apart from the group. Males explored more female genitals when exposed to follicular, periovulatory, and early luteal secretions in comparison to saline or menstrual or late luteal secretions. The increase in coercive behavior after exposure to follicular and periovulatory secretions most likely was a male response to the lack of cooperation of target females in engaging in copulation, as the latter were not receptive during the tests. The strength of male response to vaginal secretions varied significantly as a result of individual variability between donor females, yet the variability does not correlate either to dominance rank or to female age. Exploratory behavior of males correlates significantly with their social rank. Our results suggest that vaginal secretions are among the cues that male Macaca arctoides use to acknowledge the reproductive status of females in the absence of visual signals.     

Serine/arginine-rich splicing factors belong to a class of intrinsically disordered proteins

Serine/arginine-rich (SR) splicing factors play an important role in constitutive and alternative splicing as well as during several steps of RNA metabolism. Despite the wealth of functional information about SR proteins accumulated to-date, structural knowledge about the members of this family is very limited. To gain a better insight into structure-function relationships of SR proteins, we performed extensive sequence analysis of SR protein family members and combined it with ordered/disordered structure predictions. We found that SR proteins have properties characteristic of intrinsically disordered (ID) proteins. The amino acid composition and sequence complexity of SR proteins were very similar to those of the disordered protein regions. More detailed analysis showed that the SR proteins, and their RS domains in particular, are enriched in the disorder-promoting residues and are depleted in the order-promoting residues as compared to the entire human proteome. Moreover, disorder predictions indicated that RS domains of SR proteins were completely unstructured. Two different classification methods, the charge-hydropathy measure and the cumulative distribution function (CDF) of the disorder scores, were in agreement with each other, and they both strongly predicted members of the SR protein family to be disordered. This study emphasizes the importance of the disordered structure for several functions of SR proteins, such as for spliceosome assembly and for interaction with multiple partners. In addition, it demonstrates the usefulness of order/disorder predictions for inferring protein structure from sequence.     

Sic1 is phosphorylated by CK2 on Ser201 in budding yeast cells

We have previously identified Ser201 of Sic1, a yeast cyclin-dependent kinase inhibitor, as an in vitro target of protein kinase CK2. Here we present new evidence, by using specific anti-P-Ser201 antibodies and 2-D gel electrophoresis coupled to MALDI mass spectrometry analysis, that Sic1 is phosphorylated in vivo on Ser201 shortly after its de novo synthesis, during late anaphase in glucose-grown cells. This phosphorylation is also detected in Sic1 immunopurified from G1 cells. In agreement with these data we also show that the catalytic alpha' subunit of CK2, whose function is required for cell cycle progression, is detected in Sic1 immunopurified complexes, and that phosphorylation on Ser201 is reduced after CK2 inactivation at the non-permissive temperature in a cka1delta cka2(ts) yeast strain. These data strongly support the notion that CK2 phosphorylates Sic1 in vivo.     

Novel RasGRF1-derived Tat-fused peptides inhibiting Ras-dependent proliferation and migration in mouse and human cancer cells

Mutations of RAS genes are critical events in the pathogenesis of different human tumors and Ras proteins represent a major clinical target for the development of specific inhibitors to use as anticancer agents. Here we present RasGRF1-derived peptides displaying both in vitro and in vivo Ras inhibitory properties. These peptides were designed on the basis of the down-sizing of dominant negative full-length RasGRF1 mutants. The over-expression of these peptides can revert the phenotype of K-RAS transformed mouse fibroblasts to wild type, as monitored by several independent biological readouts, including Ras-GTP intracellular levels, ERK activity, morphology, proliferative potential and anchorage independent growth. Fusion of the RasGRF1-derived peptides with the Tat protein transduction domain allows their uptake into mammalian cells. Chemically synthesized Tat-fused peptides, reduced to as small as 30 residues on the basis of structural constraints, retain Ras inhibitory activity. These small peptides interfere in vitro with the GEF catalyzed nucleotide dissociation and exchange on Ras, reduce cell proliferation of K-RAS transformed mouse fibroblasts, and strongly reduce Ras-dependent IGF-I-induced migration and invasion of human bladder cancer cells. These results support the use of RasGRF1-derived peptides as model compounds for the development of Ras inhibitory anticancer agents.     

Functionality of Varroa-resistant honey bees (Hymenoptera: Apidae) when used in migratory beekeeping for crop pollination

Two types of honey bees, Apis mellifera L. (Hymenoptera: Apidae), bred for resistance to Varroa destructor Anderson & Trueman were evaluated for performance when used in migratory crop pollination. Colonies of Russian honey bees (RHB) and outcrossed bees with Varroa-sensitive hygiene (VSH) were managed without miticide treatments and compared with colonies of Italian honey bees that served as controls. Control colonies were managed as groups which either were treated twice each year against V. destructor (CT) or kept untreated (CU). Totals of 240 and 247 colonies were established initially for trials in 2008 and 2009, respectively. RHB and VSH colonies generally had adult and brood populations similar to those of the standard CT group regarding pollination requirements. For pollination of almonds [Prunus dulcis (Mill.) D.A.Webb] in February, percentages of colonies meeting the required six or more frames of adult bees were 57% (VSH), 56% (CT), 39% (RHB), and 34% (CU). RHB are known to have small colonies in early spring, but this can be overcome with appropriate feeding. For later pollination requirements in May to July, 94-100% of colonies in the four groups met pollination size requirements for apples (Malus domestica Borkh.), cranberries (Vaccinium macrocarpon Aiton), and lowbush blueberries (Vaccinium angustifolium Aiton). Infestations with V. destructor usually were lowest in CT colonies and tended to be lower in VSH colonies than in RHB and CU colonies. This study demonstrates that bees with the VSH trait and pure RHB offer alternatives for beekeepers to use for commercial crop pollination while reducing reliance on miticides. The high frequency of queen loss (only approximately one fourth of original queens survived each year) suggests that frequent requeening is necessary to maintain desired genetics.     

A chloroplast-derived C4V3 polypeptide from the human immunodeficiency virus (HIV) is orally immunogenic in mice

Although the human immunodeficiency virus (HIV) causes one of the most important infectious diseases worldwide, attempts to develop an effective vaccine remain elusive. Designing recombinant proteins capable of eliciting significant and protective mammalian immune responses remain a priority. Moreover, large-scale production of proteins of interest at affordable cost remains a challenge for modern biotechnology. In this study, a synthetic gene encoding a C4V3 recombinant protein, known to induce systemic and mucosal immune responses in mammalian systems, has been introduced into tobacco chloroplasts to yield high levels of expression. Integration of the transgene into the tobacco plastome has been verified by Southern blot hybridization. The recombinant C4V3 protein is also detected in tobacco chloroplasts by confocal microscopy. Reactivity of the heterologous protein with both an anti-C4V3 rabbit serum as well as sera from HIV positive patients have been assayed using Western blots. When administered by the oral route in a four-weekly dose immunization scheme, the plant-derived C4V3 has elicited both systemic and mucosal antibody responses in BALB/c mice, as well as CD4+ T cell proliferation responses. These findings support the viability of using plant chloroplasts as biofactories for HIV candidate vaccines, and could serve as important vehicles for the development of a plant-based candidate vaccine against HIV.     

Short-term and long-term leptin exposure differentially affect human natural killer cell immune functions

Epidemiological studies have indicated that obesity is associated with a higher risk for certain cancers caused by elevated levels of adipocyte-derived hormones. Leptin, one such hormone produced by adipocytes, is a major regulator of metabolism and has also been shown to modulate immunity. However, its role in regulating human natural killer (NK) cell functions is largely unknown. Here, we show that the leptin receptor (Ob-R) is expressed on 5% of NK cells isolated from blood donors, as measured with flow cytometry, and expression of the signal-transducing long form of the leptin receptor Ob-Rb was confirmed with quantitative PCR. The Ob-R+ subpopulation displayed a lower expression of CD16, a cell surface receptor mediating antibody-dependent activation. Short-term stimulation with leptin increased IFNγ secretion, CD69 activation marker expression, and cytotoxic lysis of tumor cells; this was mediated by an improved conjugate forming between NK cells and tumor cells as well as higher expression of tumor necrosis factor-related apoptosis-inducing ligand. On the contrary, long-term incubation with leptin significantly impaired these NK cell immune functions and decreased cell proliferation. In addition, phosphorylation of Jak-2 after leptin stimulation was reduced in peripheral mononuclear blood cells from obese humans compared with normal-weight controls. NK cells represent an immune cell population that is crucial for an effective antitumor response. Here, we show that long-term exposure to leptin, similarly to the situation in obese individuals with elevated serum leptin levels, significantly impairs integral parts of NK cell immune functions, possibly linking leptin to increased cancer susceptibility in obesity.     

Development of microsatellite markers for Anadenanthera colubrina (Leguminosae), a neotropical tree species

? Premise of the study: We developed and characterized nuclear microsatellite markers for Anadenanthera colubrina, a tropical tree species widely distributed in South America. ? Methods and Results: Leaf samples of mature A. colubrina trees, popularly called "angico," were collected from an area that is greatly impacted by agricultural practices in the region of Ribeir?o Preto in S?o Paulo State in southeastern Brazil. Twenty simple sequence repeat (SSR) markers were developed, 14 of which had polymorphic loci. A total of 96 alleles were detected with an average of 6.86 alleles per polymorphic locus. The expected heterozygosity, calculated at polymorphic loci, ranged from 0.18 to 0.83. Finally, we demonstrated that 18 loci were cross-amplified in A. peregrina. ? Conclusions: A total of 14 polymorphic markers suggest a high potential for genetic diversity, gene flow, and mating system analyses in A. colubrina.