Site‐specific inhibition of integrin αvβ3‐vitronectin association by a ser‐asp‐val sequence through an Arg‐Gly‐Asp‐binding site of the integrin

A functional proteomic technology using protein chip and molecular simulation was used to demonstrate a novel biomolecular interaction between P11, a peptide containing the Ser‐Asp‐Val (SDV) sequence and integrin αvβ3. P11 (HSDVHK) is a novel antagonistic peptide of integrin αvβ3 screened from hexapeptide library through protein chip system. An in silico docking study and competitive protein chip assay revealed that the SDV sequence of P11 is able to create a stable inhibitory complex onto the vitronectin‐binding site of integrin αvβ3. The Arg‐Gly‐Asp (RGD)‐binding site recognition by P11 was site specific because the P11 was inactive for the complex formation of a denatured form of integrin–vitronectin. P11 showed a strong antagonism against αvβ3‐GRGDSP interaction with an IC₅₀ value of 25.72±3.34 nM, whereas the value of GRGDSP peptide was 1968.73±444.32 nM. The binding‐free energies calculated from the docking simulations for each P11 and RGD peptide were ?3.99 and ?3.10 kcal/mol, respectively. The free energy difference between P11 and RGD corresponds to approximately a 4.5‐fold lower K_i value for the P11 than the RGD peptide. The binding orientation of the docked P11 was similar to the crystal structure of the RGD in αvβ3. The analyzed docked poses suggest that a divalent metal–ion coordination was a common driving force for the formation of both SDV/αvβ3 and RGD/αvβ3 complexes. This is the first report on the specific recognition of the RGD‐binding site of αvβ3 by a non‐RGD containing peptide using a computer‐assisted proteomic approach.     

Differential CCR1-mediated chemotaxis signaling induced by human CC chemokine HCC-4/CCL16 in HOS cells

Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.     

Pathogenicity, virulence factors, and strategies to fight against Burkholderia cepacia complex pathogens and related species

The Burkholderia cepacia complex (Bcc) is a group of 17 closely related species of the β-proteobacteria subdivision that emerged in the 1980s as important human pathogens, especially to patients suffering from cystic fibrosis. Since then, a remarkable progress has been achieved on the taxonomy and molecular identification of these bacteria. Although some progress have been achieved on the knowledge of the pathogenesis traits and virulence factors used by these bacteria, further work envisaging the identification of potential targets for the scientifically based design of new therapeutic strategies is urgently needed, due to the very difficult eradication of these bacteria with available therapies. An overview of these aspects of Bcc pathogenesis and opportunities for the design of future therapies is presented and discussed in this work.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.     

Identification and Characterization of a Dual-Acting Antinematodal Agent against the Pinewood Nematode,Bursaphelenchus xylophilus

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is a mycophagous and phytophagous pathogen responsible for the current widespread epidemic of the pine wilt disease, which has become a major threat to pine forests throughout the world. Despite the availability of several preventive trunk-injection agents, no therapeutic trunk-injection agent for eradication of PWN currently exists. In the characterization of basic physiological properties of B. xylophilus YB-1 isolates, we established a high-throughput screening (HTS) method that identifies potential hits within approximately 7 h. Using this HTS method, we screened 206 compounds with known activities, mostly antifungal, for antinematodal activities and identified HWY-4213 (1-n-undecyl-2-[2-fluorphenyl] methyl-3,4-dihydro-6,7-dimethoxy-isoquinolinium chloride), a highly water-soluble protoberberine derivative, as a potent nematicidal and antifungal agent. When tested on 4 year-old pinewood seedlings that were infected with YB-1 isolates, HWY-4213 exhibited a potent therapeutic nematicidal activity. Further tests of screening 39 Caenorhabditis elegans mutants deficient in channel proteins and B. xylophilus sensitivity to Ca²⁺ channel blockers suggested that HWY-4213 targets the calcium channel proteins. Our study marks a technical breakthrough by developing a novel HTS method that leads to the discovery HWY-4213 as a dual-acting antinematodal and antifungal compound.     

Resetting and regulation of FLOWERING LOCUS C expression during Arabidopsis reproductive development

The epigenetic regulation of the floral repressor FLOWERING LOCUS C ( FLC ) is one of the critical factors that determine flowering time in Arabidopsis thaliana . Although many FLC regulators, and their effects on FLC chromatin, have been extensively studied, the epigenetic resetting of FLC has not yet been thoroughly characterized. Here, we investigate the FLC expression during gametogenesis and embryogenesis using FLC::GUS transgenic plants and RNA analysis. Regardless of the epigenetic state in adult plants, FLC expression disappeared in gametophytes. Subsequently, FLC expression was reactivated after fertilization in embryos, but not in the endosperm. Both parental alleles contributed equally to the expression of FLC in embryos. Surprisingly, the reactivation of FLC in early embryos was independent of FRIGIDA (FRI) and SUPPRESSOR OF FRIGIDA 4 (SUF4) activities. Instead, FRI , SUF4 and autonomous-pathway genes determined the level of FLC expression only in late embryogenesis. Many FLC regulators exhibited expression patterns similar to that of FLC , indicating potential roles in FLC reprogramming. An FVE mutation caused ectopic expression of FLC in the endosperm. A mutation in PHOTOPERIOD-INDEPENDENT EARLY FLOWERING 1 caused defects in FLC reactivation in early embryogenesis, and maintenance of full FLC expression in late embryogenesis. We also show that the polycomb group complex components, Fertilization-Independent endosperm and MEDEA, which mediate epigenetic regulation in seeds, are not relevant for FLC reprogramming. Based on our results, we propose that FLC reprogramming is composed of three phases: (i) repression in gametogenesis, (ii) reactivation in early embryogenesis and (iii) maintenance in late embryogenesis.     

Elucidating Interactions and Conductivity of Newly Synthesised Low Bandgap Polymer with Protic and Aprotic Ionic Liquids

In this paper, we have examined the conductivity and interaction studies of ammonium and imidazolium based ionic liquids (ILs) with the newly synthesised low bandgap polymer (Poly(2-heptadecyl-4-vinylthieno[3,4-d]thiazole) (PHVTT)). Use of low bandgap polymers is the most suitable way to harvest a broader spectrum of solar radiations for solar cells. But, still there is lack of most efficient low bandgap polymer. In order to solve this problem, we have synthesised a new low bandgap polymer and investigated its interaction with the ILs to enhance its conductivity. ILs may undergo almost unlimited structural variations; these structural variations have attracted extensive attention in polymer studies. The aim of present work is to illustrate the state of art progress of implementing the interaction of ILs (protic and aprotic ILs) with newly synthesised low bandgap polymer. In addition to this, our UV-Vis spectroscopy, confocal Raman spectroscopy and FT-IR spectroscopy results have revealed that all studied ILs (tributylmethylammonium methyl sulfate ([N₁₄₄₄][MeSO₄] from ammonium family) and 1-methylimidazolium chloride ([Mim]Cl, and 1-butyl-3-methylimidazolium chloride ([Bmim]Cl from imidazolium family) have potential to interact with polymer. Our semi empirical calculation with help of Hyperchem 7 shows that protic IL ([Mim]Cl) interacts strongly with the low bandgap polymer through the H-bonding. Further, protic ILs shows enhanced conductivity than aprotic ILs in association with low bandgap polymer. This study provides the combined effect of low bandgap polymer and ILs that may generate many theoretical and experimental opportunities.     

Cleavage Events and Sperm Dynamics in Chick Intrauterine Embryos

This study was undertaken to elucidate detailed event of early embryogenesis in chicken embryos using a noninvasive egg retrieval technique before oviposition. White Leghorn intrauterine eggs were retrieved from 95 cyclic hens aged up to 54-56 weeks and morphogenetic observation was made under both bright field and fluorescent image in a time course manner. Differing from mammals, asymmetric cleavage to yield preblastodermal cells was observed throughout early embryogenesis. The first two divisions occurred synchronously and four polarized preblastodermal cells resulted after cruciform cleavage. Then, asynchronous cleavage continued in a radial manner and overall cell size in the initial cleavage region was smaller than that in the distal area. Numerous sperms were visible, regardless of zygotic nuclei formation. Condensed sperm heads were present mainly in the perivitelline space and cytoplasm, and rarely in the yolk region, while decondensed sperm heads were only visible in the yolk. In conclusion, apparent differences in sperm dynamics and early cleavage events compared with mammalian embryos were detected in chick embryo development, which demonstrated polarized cleavage with penetrating supernumerary sperm into multiple regions.     

Postnatal fate of the ultimobranchial remnants in the rat thyroid gland

The ultimobranchial follicles (UBFs) are considered embryonic remnants from the ultimobranchial body (UBB). They are follicular structures that vary in size and appearance depending on the age of the rat. The main objective of this article was to study the progressive changes in shape, size, and frequency of the UBFs in the postnatal rat, from birth to old‐age. To accomplish that objective, a systematic morphometric and incidental study of the UBF has been carried out in 110 Wistar rats of different ages and both sexes, divided into three groups: 1) young rats (5–90‐day‐old); 2) adult rats (6–15‐month‐old), and 3) old rats (18–24‐month‐old). The glands were serially sectioned and immunostained for calcitonin at five equidistant levels. According to our results, UBFs were observed in all thyroid glands but a more exhaustive sampling was occasionally necessary in male rats. In young rats, immature UBFs predominantly appeared whereas in adult rats, mature UBFs with cystic appearance and variable luminal content prevailed. We frequently found spontaneous anomalous UBFs in old rats, which we have termed as “ultimobranchial cystadenomata.” Additionally, in young rats, UBF areas significantly increased with age and they were larger when compared to that of normal thyroid follicles. Likewise, in adult rats, UBFs were significantly larger than normal thyroid follicles but only in female rats. In general, UBFs in females were also significantly larger than those found in male rats. Finally, all these differences related to UBFs together with a higher incidence in females of UB cystadenomata suggest a sexual dimorphism in regard to the destiny of these embryonic remnants during postnatal thyroid development. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.