Property and application of novel ferrocenyl-azobenzene labeled peptide nucleic acid monomers with the dual stimulus-response characteristics

In this work, three novel types of peptide nucleic acid (PNA) monomers labeled by ferrocenyl-azobenzene units (Fc-Azo-PNAs, 1a, 1b, and 1c) have been synthesized and they all exhibited good reversible photoswitch and redox properties. In the different solvents of ethanol (EtOH) and acetonitrile (ACN), photoisomerization rate constants (K) of three PNA monomers under UV light irradiation have also been analyzed and compared, discovering that the values of K are closely correlative to their molecular structures. The electrochemical properties of the synthesized PNA monomers are also investigated and the typical redox properties are displayed, demonstrating that they can be regarded as the anchor to be grafted at the end of PNA oligomers for detecting themselves. Finally, according to the analyses of UV-Vis absorption spectrum and differential pulse voltammetry (DPV), the synthesized PNA monomers afford the best detection limit down to 10⁻⁸ M in EtOH. The results also illuminate that PNA monomers with new photo-redox dual stimulus-response characteristics can be realized to detect themselves by the metal complex unit together with azo moiety; moreover, the hybridization with DNA also can be tuned by the isomerization of azo moiety.     

Synthesis of novel ribavirin hydrazone derivatives and anti-proliferative activity against A549 lung cancer cells

A series of novel ribavirin hydrazone derivatives were synthesized by the reaction of ribavirin hydrazone with benzaldehyde or acetophenone derivatives. The structures of the compounds were determined by IR, ¹H NMR, and HRESIMS. Preliminary biological evaluation showed that one compound (7h) inhibits the growth of A549 cells at 20 μM.     

Sperm cryopreservation in guppies and black mollies—A generalized freezing protocol for livebearers in Poeciliidae

In this study, we evaluated various parameters of sperm cryopreservation in two livebearers, guppies (Poecilia reticulata) and black mollies (P. latipinna). Our results suggested a common freezing protocol for the guppies and mollies: suspend sperm in Hanks’ balanced salt solution (HBSS) at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 °C/min, and thaw at 40 °C in a water bath for 7 s. Live young were produced from females inseminated with frozen-thawed sperm in both species. In guppies, percent fertilization (F) and the number of embryos (N) produced with cryopreserved sperm (F = 50%, N = 74, from 26 females) were similar to those of fresh controls (F = 54%, N = 61, from 22 females). Interestingly, this same freezing protocol has been used successfully for sperm cryopreservation in green swordtails Xiphophorus helleri, and platyfish of X. couchianus with post-thaw motility as high as 80%. All these species belong to the family of Poeciliidae, and their sperm are similar in morphology exhibiting the absence of acrosome, elongate sperm head, and the long mitochondrial sheaths. Besides their internal fertilization reproduction mode, these fish are also small in size (2–4 cm) and live in a freshwater environment. Sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. However, results presented in this study suggested otherwise. Thus, sperm cryopreservation methods optimized for one species may be applicable to others if they are taxonomical closely related species with similar sperm morphology and reproduction mode. Considering the enormous number of fish species on the planet, development of generalized sperm freezing protocols for species in groups could have additional advantages for genetic conservation.     

Validation of eight names of Chinese taxa in Ranunculaceae, Rosaceae and Scrophulariaceae

Eight names in five genera Delphinium (Ranunculaceae), Lindernia and Mimulicalyx (Scrophulariaceae), Sorbus and Spiraea (Rosaceae) from China were not validly published because two gatherings were simultaneously designated as types in the protologues, or were simultaneously cited, without indicating the type in the protologues. These eight names are validated here, with one specimen designated as the holotype.     

A genetic linkage map construction for sesame (Sesamum indicum L.)

Sesame (Sesamum indicum L.) is one of the oldest oilseed crops with high seed oil quality. The first sesame genetic linkage map based on F2 segregating population of an intraspecific cross between two cultivars was constructed. Using three types of PCR-based markers, 284 polymorphic loci including 10 EST-SSR marker, 30 AFLP marker and 244 RSAMPL marker, respectively, had been screened. Subsequently, a total of 220 molecular markers were mapped in 30 linkage groups covering a genetic length of 936.72 cM, and the average distance between markers was 4.93 cM. In this map, the linkage groups contained from 2 to 33 loci each and ranged in distance from 6.44 cM to 74.52 cM. Based on map information, sesame genome length was estimated to be approximately 1,232.53 cM, and genome coverage of this map was about 76.0%. As a starting point of sesame genome study, the genetic linkage map will be hopeful to tag traits of breeding interest and further aid in the sesame molecular breeding. Furthermore, RSAMPL marker had been also appreciated in this paper, for its first usage in genetic map construction and higher utilization potential in some crop species lacking much genome information.     

Native Cuscuta campestris restrains exotic Mikania micrantha and enhances soil resources beneficial to natives in the invaded communities

Nutrients in exotic species and invaded communities play a key role in determining the dynamics of invaders and the invasibility of a receipt community. This study focused on the effects of the native holoparasite Cuscuta campestris (for short Cuscuta) on nutrients in the exotic invasive Mikania micrantha (for short Mikania) and stands invaded by Mikania. We conducted a set of field investigations on Mikania with Cuscuta parasitism for 1–4 years, and measured soil properties, community composition, and the growth and nutrient content of Mikania and Cuscuta in two types of sub-communities (i.e. with Mikania only, or with Mikania and Cuscuta). Cuscuta dramatically reduced the cover, biomass, and nutrients (i.e. N, P, and K content) of Mikania, significantly enhanced soil water, pH and nutrient content (i.e. organic matter, total N and P, available P and K), and greatly increased the cover and species richness of native plants. In addition, N and K of Cuscuta were positively correlated with N of Mikania, which was negatively associated with soil total N, available P and K. These findings suggest that Cuscuta may be an effective measure against Mikania and be beneficial to the restoration of invaded communities.     

Independent Transport and Sorting of Functionally Distinct Protein Families in Tetrahymena thermophila Dense Core Secretory Granules

Dense core granules (DCGs) in Tetrahymena thermophila contain two protein classes. Proteins in the first class, called granule lattice (Grl), coassemble to form a crystalline lattice within the granule lumen. Lattice expansion acts as a propulsive mechanism during DCG release, and Grl proteins are essential for efficient exocytosis. The second protein class, defined by a C-terminal β/γ-crystallin domain, is poorly understood. Here, we have analyzed the function and sorting of Grt1p (granule tip), which was previously identified as an abundant protein in this family. Cells lacking all copies of GRT1, together with the closely related GRT2, accumulate wild-type levels of docked DCGs. Unlike cells disrupted in any of the major GRL genes, ΔGRT1 ΔGRT2 cells show no defect in secretion, indicating that neither exocytic fusion nor core expansion depends on GRT1. These results suggest that Grl protein sorting to DCGs is independent of Grt proteins. Consistent with this, the granule core lattice in ΔGRT1 ΔGRT2 cells appears identical to that in wild-type cells by electron microscopy, and the only biochemical component visibly absent is Grt1p itself. Moreover, gel filtration showed that Grl and Grt proteins in cell homogenates exist in nonoverlapping complexes, and affinity-isolated Grt1p complexes do not contain Grl proteins. These data demonstrate that two major classes of proteins in Tetrahymena DCGs are likely to be independently transported during DCG biosynthesis and play distinct roles in granule function. The role of Grt1p may primarily be postexocytic; consistent with this idea, DCG contents from ΔGRT1 ΔGRT2 cells appear less adhesive than those from the wild type.In eukaryotes, the directional transport of lumenal proteins throughout the network of membrane-bound organelles depends on reversible assembly of multisubunit protein complexes in the cytoplasm. For example, the assembly of a localized clathrin coat at a cell''s surface facilitates both the concentration of specific transmembrane receptors together with their bound ligands at that site and the invagination and budding of the plasma membrane, resulting in endocytosis (18). Similarly, other cytosolic coats assemble and direct traffic at the endoplasmic reticulum (ER) and Golgi apparatus (4). For one protein trafficking pathway in eukaryotic cells, however, the determinative protein self-assembly occurs not in the cytoplasm but within the lumen of the secretory pathway itself. Dense core granules (DCGs) are secretory vesicles whose lumenal cargo consists of a condensed polypeptide aggregate. This cargo is secreted when the vesicles fuse with the plasma membrane in response to a specific extracellular stimulus, an event called regulated exocytosis. The aggregation of the cargo occurs progressively within the secretory pathway, beginning in the trans-Golgi network (TGN), and may be promoted by multiple factors including compartment-specific proton and calcium levels (23). Aggregation facilitates the vesicular storage of concentrated secretory proteins but also serves as a sorting mechanism to segregate DCG proteins from proteins that are secreted via other pathways. Evidence for this mechanism includes in vitro experiments showing that some proteins released via constitutive exocytosis remain soluble under TGN-like conditions that promote DCG protein aggregation (10). In vivo, sorting would result if aggregated and soluble proteins exit the TGN in different carriers. Importantly, there is no evidence that sorting of DCG proteins at the TGN requires assembly of cytosolic coat complexes.While aggregative sorting represents an attractively simple mechanism, relatively little is known about the structure or dynamic properties of the aggregates themselves. This is an interesting issue, as illustrated by several phenomena. First, aggregates in some cell types, like those formed by proinsulin in pancreatic β cells, can become reordered as protein crystals during a multistage process called granule maturation (13). Second, Aplysia bag cells can sort different subsets of DCG proteins into distinct granules, suggesting that aggregation can be finely regulated and that different aggregates have different properties in vivo (20). Both of these phenomena have also been observed within the DCGs of unicellular ciliates (3, 14). In addition, ciliate DCGs demonstrate another degree of subtlety in DCG formation because the granule cores in many of these organisms are divided into distinct domains (25). The domain organization indicates that DCG proteins in these cells can segregate from one another even as they are sorted to the same vesicular destination. While the structures of DCGs in many ciliates have been captured by electron microscopy, molecular studies have advanced in two species, Tetrahymena thermophila and Paramecium tetraurelia (30, 33).In many ciliates, the individual DCGs are organized in at least two distinct domains within the lumen. First, the bulk of the cargo is organized as a core crystal that expands, spring-like, upon exocytosis (28). This expansion can drive rapid extrusion of the DCG contents, which may be essential for hunting or defensive behaviors (17). In addition, many ciliate DCGs possess a single polarized tip structure that is involved in DCG docking to the plasma membrane and exocytic fusion (25). These tip structures are also filled with condensed, highly organized proteins, which appear by both genetic and morphological criteria to be different from proteins making up the expansible core (1, 21). The proteins that form the distinct domains are beginning to be identified and analyzed. Those that constitute the expansible springs are encoded by homologous families of genes named GRL (granule lattice) in Tetrahymena and tmp (trichocyst matrix) in Paramecium (11, 12, 15). Assembly of Grl proteins begins in the ER with formation of heterooligomers. This is an obligatory step, as shown by the fact that deletion of individual Grl proteins by targeted gene disruption resulted in the ER retention of remaining Grl proteins (12). Further assembly of Grl proteins to form a crystal occurs during DCG maturation and is accompanied by site-specific proprotein processing (34). Upon exocytosis, the expansion of the crystalline core is controlled by calcium binding to the fully processed Grl proteins (34).In addition to the GRL family-encoded proteins, 13 other lumenal DCG proteins have been putatively or definitively identified in Tetrahymena, and homologous proteins are predicted in the Paramecium genome (6). The entire set belongs to a gene family that is defined by a carboxy-terminal β/γ-crystallin domain, which may function as a DCG-targeting motif (16). Studies of two different members of this family in Tetrahymena, IGR1 (induced during granule regeneration 1) and GRT1 (granule tip 1), suggested that these proteins are functionally distinct from the spring-forming Grl proteins. First, whereas gene disruption of any of the highly transcribed GRL genes resulted in grossly aberrant spring formation, no such defect was seen upon disruption of IGR1 (16). However, this could be explained by the fact that IGR1 encodes a relatively low-abundance protein in DCGs, and furthermore its function could be redundant with that of the highly related gene, IGR2.The second protein in the β/γ-crystallin domain family that has been investigated is the 80-kDa product of the GRT1 gene. Grt1p was first detected as one of the most abundant DCG components released during exocytosis (32). Biochemical analysis showed that Grt1p differs in its solubility from the Grl proteins and also that it is packaged intact in DCGs rather than undergoing proteolytic processing (31). Since processing is essential for Grl protein assembly and function, this difference appears highly significant. Second, Grt1p accumulates at a single pole of each DCG, corresponding to the tip of the organelle that docks and then fuses with the plasma membrane (5). Two Mendelian mutants with defects in DCG maturation show delocalized Grt1p, and these mutant DCGs can dock but do not appear to undergo exocytosis (5). These results suggested that Grt1p might be involved in forming a DCG tip domain that interacted with the plasma membrane.We have now investigated the trafficking and function of Grt1p. Our data provide both direct biochemical and cell-biological evidence that Grt1p and Grl proteins form distinct complexes during DCG biogenesis in Tetrahymena. Together with earlier results, our experiments provide genetic evidence that Grl and Grt complexes can be independently trafficked to DCGs. Cells lacking GRT1, together with the closely related GRT2, still show rapid and efficient release of DCG contents upon stimulation with secretagogues, but the released DCG contents are subtly different from those of the wild type, suggesting that Grt1p may primarily serve a postexocytic function.     

Safrole oxide induces apoptosis by up-regulating Fas and FasL instead of integrin beta4 in A549 human lung cancer cells

Previously, we found that 3,4-(methylenedioxy)-1-(2',3'-epoxypropyl)-benzene (safrole oxide) induced a typical apoptosis in A549 human lung cancer cells by activating caspase-3, -8, and -9. In this study, we further investigated which upstream pathways were activated by safrole oxide during the apoptosis. Immunofluorescence assay combined with laser scanning confocal microscopy revealed that both Fas and Fas ligand (FasL) were up-regulated by the small molecule. In addition, Fas protein distribution was altered, showing a clustering distribution instead of a homogeneous one. Subsequently, Western blot analysis confirmed the up-regulations of Fas and its membrane-binding form of FasL (m-FasL), as well as P53 protein. Conversely, safrole oxide hardly affected integrin beta4 subunit expression or distribution, which was reflected from the data obtained by immunofluorescence assay combined with laser scanning confocal microscopy. The results suggested that Fas/FasL pathway might be involved in safrole oxide-induced apoptosis of A549 cells, while integrin beta4 might be irrelevant to the apoptosis. Nevertheless, we first found the strong expression of integrin beta4 in A549 cells. The study first suggested that safrole oxide might be used as a small molecular promoter of Fas/FasL pathway to elicit apoptosis in A549 cells, which would lay the foundation for us to insight into the new strategies for lung cancer therapy.     

Composition and variability of airborne fungi in an enclosed rabbit house in China

Sampling was conducted from June 2007 to May 2008 in an enclosed rabbit house to investigate composition and variability of airborne fungi. Samples were collected using an Andersen-6 sampler, with Sabouraud culture medium as sampling medium. The results showed that monthly mean concentration was 2.79–5.46 × 10³ colony forming unit/m³ air (CFU/m³ air), with the maximum level in October, and the minimum level in January. Within a day, the maximum level occurred at 09:00, followed by 17:00 and then 13:00. A total of 6,523 fungal colonies, belonging to 17 genera and 36 species, were obtained. The predominant genera included Cladosporium, Penicillium, Aspergillus and Altemaria, comprising 71.45% of the colony count. The obtained fungi of the year were mainly centralized in the stage D of the sampler (2.0–3.0 μm), accounting for 37.8% of the colonies. The minimum value occurred at stage F (<0.65 μm), accounting for 1.10% of the colonies.     

Bovine prolactin promotes the expression of human transferrin in the milk of transgenic mice

The bovine prolactin vector was injected directly into the mammary glands of mice carrying the human transferrin transgene to investigate its effect on the production of human transferrin in milk. The mean levels of human transferrin in two experimental groups were increased by approx. 60% compared with the control group: 1143 ± 196 ng/ml (experimental group 1; two injections) and 1160 ± 189 ng/ml (experimental group 2; three injections) versus 714 ± 75 ng/ml (control group). These findings suggest the potential utility of the prolactin vector for efficient expression of valuable pharmaceutical proteins in transgenic animal mammary glands.