Conservation of gene linkage in dispersed vertebrate NK homeobox clusters

Nk homeobox genes are important regulators of many different developmental processes including muscle, heart, central nervous system and sensory organ development. They are thought to have arisen as part of the ANTP megacluster, which also gave rise to Hox and ParaHox genes, and at least some NK genes remain tightly linked in all animals examined so far. The protostome–deuterostome ancestor probably contained a cluster of nine Nk genes: (Msx)–(Nk4/tinman)–(Nk3/bagpipe)–(Lbx/ladybird)–(Tlx/c15)–(Nk7)–(Nk6/hgtx)–(Nk1/slouch)–(Nk5/Hmx). Of these genes, only NKX2.6–NKX3.1, LBX1–TLX1 and LBX2–TLX2 remain tightly linked in humans. However, it is currently unclear whether this is unique to the human genome as we do not know which of these Nk genes are clustered in other vertebrates. This makes it difficult to assess whether the remaining linkages are due to selective pressures or because chance rearrangements have “missed” certain genes. In this paper, we identify all of the paralogs of these ancestrally clustered NK genes in several distinct vertebrates. We demonstrate that tight linkages of Lbx1–Tlx1, Lbx2–Tlx2 and Nkx3.1–Nkx2.6 have been widely maintained in both the ray-finned and lobe-finned fish lineages. Moreover, the recently duplicated Hmx2–Hmx3 genes are also tightly linked. Finally, we show that Lbx1–Tlx1 and Hmx2–Hmx3 are flanked by highly conserved noncoding elements, suggesting that shared regulatory regions may have resulted in evolutionary pressure to maintain these linkages. Consistent with this, these pairs of genes have overlapping expression domains. In contrast, Lbx2–Tlx2 and Nkx3.1–Nkx2.6, which do not seem to be coexpressed, are also not associated with conserved noncoding sequences, suggesting that an alternative mechanism may be responsible for the continued clustering of these genes.     

Mapping the extracellular topography of the α-chain in free and in membrane-bound acetylcholine receptor by antibodies against overlapping peptides spanning the entire extracellular parts of the chain

The extracellular surface of theα-chain ofTorpedo california acetylcholine receptor (AChR) was mapped for regions that are accessible to binding with antibodies against a panel of synthetic overlapping peptides which encompassed the entire extracellular parts of the chain. The binding of the antipeptide antibodies to membrane-bound AChR (mbAChR) and to isolated, soluble AChR. was determined. The specificity of each antiserum was narrowed down by determining the extent of its cross-reaction with the two adjacent peptides that overlap the immunizing peptide. With mbAChR, high antibody reactivity was obtained with antisera against peptidesα1–16,α89–104,α158–174,α262–276, andα388–408. Lower, but significant, levels of reactivity were obtained with antibodies against peptidesα67–82,α78–93,α100–115, andα111–126. On the other hand, free AChR bound high levels of antibodies against peptidesα34–49,α78–93,α134–150,α170–186, andα194–210. It also bound moderate levels of antibodies against peptidesα262–276 andα388–408. Low, yet significant, levels of binding were exhibited by antibodies against peptidesα45–60,α111–126, andα122–138. These binding studies, which enabled a comparison of the accessible regions in mbAChR and free AChR, revealed that the receptor undergoes considerable changes in conformation upon removal from the cell membrane. The exposed regions found here are discussed in relation to the functional sites of AChR (i.e., the acetylcholine binding site, the regions that are recognized by anti-AChR antibodies, T-cells and autoimmune responses and the regions that bind short and long neurotoxins).     

Studying Genetic Variability of Pomegranate (Punica granatum L.) Based on Chloroplast DNA and Barcode Genes

Chloroplast DNA has been used extensively to analyze plant phylogenies at different taxonomic levels because of its size, organization and sequence conservation. In the present research, two chloroplastic regions, petA–psaJ, trnC–trnD and four DNA barcodes (trnH–psbA, ITS, rbcL, matK), were used to introduce suitable regions for the assessment of genetic diversity among P. granatum L. genotypes. Analysis of psbE–petL in petA–psaJ region revealed 1,300 nucleotides with 4.29 % genetic diversity among genotypes, while trnC–petN in trnC–trnD region showed 1.8 % genetic diversity. Therefore, despite the results obtained from the study of other plants, the trnC–trnD region had a low potential for the evaluation of diversity among pomegranate genotypes. Analysis of DNA barcodes in pomegranate showed that trnH–psbA (genetic diversity 2.91 %) provides the highest intra-species variation, followed by ITS (genetic diversity 0.44 %). Eighteen genotypes from different geographical origins of Iran were used to investigate psbE–petL and trnH–psbA potential as novel barcodes to determine genetic polymorphism and characterize pomegranate genotypes. The results suggested that two regions, psbE–petL and trnH–psbA, were more suitable for determining intra-species relationships of pomegranate.     

Three New Species of Predatory Nematodes (Dorylaimida: Actinolaimoidea) from Korea

Three new species of predatory nematodes belonging to family Actinolaimidae, superfamily Actinolaimoidea, order Dorylaimida are described and illustrated. Westindicus sanaensis sp. n. has a body 1.9–2.4mm long; b = 3.7–4.3; c = 8.1–8.5; odontostyle 26.5–28.0μm long; spicules 71–74μm long; ventromedian supplements 15 and comes close to Westindicus cheongsongensis Choi et al, 1998 and Westindicus keralaensis Khan et al., 1994. Egtitus koriensis sp. n. is 1.8–2.0 mm long; b = 3.4–4.1; c = 14–18; odontostyle 20–21 μm, and comes close to Egtitus elaboratus (Cobb, 1906) Thorne, 1967; Egtitus itanagrus Khan et al., 1994 and Egtitus adhricus Khan & Jairajpuri, 1994. Stopractinca glandulus sp. n. is 2.3–2.4 mm long; b = 4.0–4.1; c = 11–12; odontostyle 26–28μm and differs from other two known species viz., Stopractincta orientalis Khan et al., 1994 and Stopractincta malnadensis Dhanam et al., 1994 in having very thick body cuticle and longer cardia, and in the presence of a gland at the base of oesophagus.     

Ecological notes on the species of Phacus Dujardin (Euglenophyta) from the central region of Portugal

Twenty-one species, belonging to the genus Phacus, were identified during the study of samples from the central region of Portugal collected in lentic systems. The abundance of each taxon was determined. Water samples were taken for determination, by means of standard methods, of physicochemical parameters (water temperature, pH, organic matter (K₂Cr₂O₇), conductivity, alkalinity, nitrogen as N(NH₄⁺), N(NO₂^–) and N(NO₃^–), orthophosphate P(PO₄^3–) and metals in a total of 35 parameters). Some species were found more frequently, namely Phacus agilis Skuja, Ph. aenigmaticus Drez., Ph. caudatus Hübn., Ph. gigas Da Cunha, Ph. triqueter (Ehr.) Duj., Ph. longicauda (Ehr.) Duj. and Ph. tortus (Lemm.) Skv. Eighteen taxa were found in the sampling sites characterized by the following variation intervals of the environmental parameters: water temperature: 11.4–21.6 °C; pH: 6.2–7.5; dichromate oxidability: 10–59 mg l^–1; conductivity: 145–779 μS cm^–1; nitrogen as NO₃^–: n.d.–2.852 mg l^–1; orthophosphate: n.d.–0.892 mg l^–1; chloride: 14.2–109.3 mg l^–1; sodium: 10.3–47.5 mg l^–1 and total iron: 135–6446 μg l^–1. In this work, information concerning the environmental conditions that preceded the occurrence of these species as well as results of the cytological and morphologic studies (with bright field microscopy as a resource) is presented and discussed.     

Lsamp–/– mice display lower sensitivity to amphetamine and have elevated 5-HT turnover

In mice, the limbic system-associated membrane protein (Lsamp) gene has been implicated in locomotion, anxiety, fear reaction, learning, social behaviour and adaptation. Human data links the LSAMP gene to several psychiatric disorders and completed suicide. Here, we investigated changes in major monoamine systems in mice lacking the Lsamp gene. First, the locomotor and rewarding effects of amphetamine were studied in Lsamp^–/– mice and Lsamp^+/+ mice. Second, monoamine levels in major brain regions in response to saline and amphetamine injections were measured and, third, the expression levels of dopamine system-related genes in the brain were studied in these mice. Lsamp^–/– mice displayed lower sensitivity to amphetamine in the motility box. Likewise, in the place preference test, the rewarding effect of amphetamine was absent in Lsamp^–/– mice. In all brain regions studied, Lsamp^–/– mice displayed lower serotonin (5-HT) baseline levels, but a greater 5-HT turnover rate, and amphetamine increased the level of 5-HT and lowered 5-HT turnover to a greater extent in Lsamp^–/– mice. Finally, Lsamp^–/– mice had lower level of dopamine transporter (DAT) mRNA in the mesencephalon. In conclusion, Lsamp-deficiency leads to increased endogenous 5-HT-ergic tone and enhanced 5-HT release in response to amphetamine. Elevated 5-HT function and reduced activity of DAT are the probable reasons for the blunted effects of amphetamine in these mice. Lsamp^–/– mice are a promising model to study the neurobiological mechanisms of deviant social behaviour and adaptation impairment observed in many psychiatric disorders.     

A novel recombinant baculovirus expressing insect neurotoxin and producing occlusion bodies that contain Bacillus thuringiensis Cry toxin

A novel recombinant baculovirus, designated AcB5A, was constructed to develop an improved baculovirus insecticide with additional beneficial properties. Bacillus thuringiensis crystal protein gene (cry1–5) and an insect-specific neurotoxin gene (AaIT) were introduced into the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) genome by fusion of polyhedrin–cry1–5–polyhedrin under the control of polyhedrin gene promoter, and by insertion of AaIT under the control of early promoter of ORF3004 from Cotesia plutellae bracovirus. RT-PCR analysis with total RNA from AcB5A-infected cells indicated that cry1–5 and AaIT genes were normally transcribed. The 150 kDa of polyhedrin–Cry1–5–polyhedrin fusion protein was produced by AcB5A and occluded into polyhedra produced by the recombinant virus. This protein was activated when treated with trypsin to form a crystal protein of approximately 65 kDa. The AcB5A showed a high level of insecticidal activity against Plutella xylostella larvae and a significant reduction in the lethal time against Spodoptera exigua larvae compared to those infected with wild-type AcMNPV. The expression level of the fusion protein decreased after in vivo passage as a result of homologous recombination between the two polyhedrin genes.     

Cytotoxic withanolides from the aerial parts of Tubocapsicum anomalum

Ten new withanolides (1–10) and three artificial withanolides (11–13) were isolated from the aerial parts of Tubocapsicum anomalum, together with five known analogues (14–18). Their structures were determined on the basis of extensive spectroscopic and chemical methods. They include seven acnistin–type (1–4, 11, 14 and 15), three withajardin–type (5–7), and eight normal–type (8–10, 12, 13 and 16–18) withanolides. Of normal–type withanolides, a chemical conversion from the 16α,17α–epoxywithanolide (16) to Δ^13,14–16α–hydroxywithanolide (18) was achieved by Wagner–Meerwein rearrangement. All isolates were evaluated for their cytotoxicity against four human tumor cell lines (HCT–116, HepG2, MCF–7 and A375). Among them, compounds 1–3, 6–8, 14, 16–18 showed cytotoxic activity with IC₅₀ values of 0.24–8.71 μM.     

Nonlinear Dependences of Optical Properties of Spherical Core–Shell Silver–Gold and Gold–Silver Nanoparticles on Their Parameters

Modeling of nonlinear optical properties of spherical core–shell gold–silver and silver–gold nanoparticles (NPs) placed in water was carried out on the base of extended Mie theory. Efficiency cross sections of absorption σ _abs, scattering σ _sca, and extinction σ _ext of radiation with wavelengths λ?=?400 and 532 nm for core–shell NPs with constant core radii r ₀₀?=?5, 10, 20, and 40 nm and in the range of relative radii r ₁/r ₀₀?=?1–8 were calculated (r ₁ is the radius of shell). Dependences of optical properties of gold–silver and silver–gold NPs on increasing of core radius r ₀ in the range 0???r ₁ under condition r ₁?=?const and increasing of r ₀ under r ₁???r ₀?=?const were investigated. Results show the nonlinear behavior of optical properties of core–shell gold–silver and silver–gold NPs on radiation wavelengths (optical indexes of metals), different core and shell radii, and their correlation, on relative NP radii r ₁/r ₀₀. An increase and decrease of absorption, scattering, and extinction efficiency cross sections of core–shell NPs with changing of wavelengths, core and shell radii, and relative NP radii r ₁/r ₀₀ are established. These dependences can be used for experimental investigation of the interesting first stages of shell formation on core and optical determination of core–shell NP parameters.     

A theoretical investigation into the cooperativity effect between the H∙∙∙O and H∙∙∙F– interactions and electrostatic potential upon 1:2 (F–:N-(Hydroxymethyl)acetamide) ternary-system formation

The cooperativity effects between the O/N–H???F^– anionic hydrogen-bonding and O/N–H???O hydrogen-bonding interactions and electrostatic potentials in the 1:2 (F^–:N-(Hydroxymethyl)acetamide (signed as “ha”)) ternary systems are investigated at the B3LYP/6-311++G** and MP2/6-311++G** levels. A comparison of the cooperativity effect in the “F^–???ha???ha” and “FH???ha^–???ha” systems is also carried out. The result shows that the increase of the H???O interaction energy in the O–H???O–H, N–H???O–H or N–H???O?=?C link is more notable than that in the O–H???O?=?C contact upon ternary-system formation. The cooperativity effect is found in the complex formed by the O/N–H???F^– and O/N–H???O interactions, while the anti-cooperativity effect is present in the system with only the O/N–H???F^– H-bond or the “FH???ha^–???ha” complex by the N^–???H–F contact. Atoms in molecules (AIM) analysis and shift of electron density confirm the existence of cooperativity. The most negative surface electrostatic potential (V _S,min ) correlates well with the interaction energy E′ _{int.(ha???F–)} and synergetic energy E _syn., respectively. The relationship between the change of V _S,min (i.e., ΔV _S,min ) and E _syn. is also found.

Establishment of Elymus natans improves soil quality of a heavily degraded alpine meadow in Qinghai-Tibetan Plateau, China

Elymus natans is a dominant native species widely planted to restore the heavily degraded alpine meadows in Qinghai-Tibetan plateau. The objective of this study was to determine how E. natans establishment affected the quality and fertility of a heavily degraded soil. Soil samples (at depths of 0–10, 10–20 and 20–30 cm) were collected from the 3- and 7-year-old E. natans re-vegetated grasslands, and in the heavily degraded alpine meadow (control). The establishment of E. natans promoted plant cover and aboveground biomass. Compared to the non-reseeded meadow, the concentration of total organic C increased by 13% in the soil under 3-year-old reseeded E. natans grassland at 0–10 cm, and by 7–33% in the soil under 7-year-old reseeded E. natans grassland at 0–10, 10–20 and 20–30 cm depths. Rapid increases in total and available N were also observed in two E. natans re-vegetated grasslands, especially in the 0–10 cm soil layer. Across three sampling depths, total P concentration was increased by 17–35% and 18–54% in 3- and 7-year-old reseeded soil respectively, compared to the soil of control. After 3 years of E. natans growth, microbial biomass C increased by 13–58% at 0–10 and 10–20 cm layers; while it increased by 43–87% in 7-year-old reseeded treatment at 0–10, 10–20 and 20–30 cm depths relative to control. A similar increasing trend was observed for microbial biomass N and P generally. Significant increase in neutral phosphatase, urease, catalase and dehydrogenase was also found in 3- and 7-year-old re-vegetated grasslands compared with heavily degraded meadow. Our results suggest a significant positive impact of E. natans establishment on soil quality. Thus, E. natans establishment could be an effective and applicable measure in restoring heavily degraded alpine meadow in the region of Qinghai-Tibetan Plateau.     

Enhanced antibacterial activity of an attacin-coleoptericin hybrid protein fused with a helical linker

Previously, we isolated and characterized attacin from Spodoptera exigua and a coleoptericin-like protein from Protaetia brevitarsis seulensis. In this study, we fused these two genes encoding antimicrobial proteins to obtain a hybrid protein with enhanced antimicrobial activity. To fuse the two antimicrobial proteins, we employed helical and non-helical linker sequences that function as inter-domain linkers in proteins. We used the Gly–Gly–Gly–Gly–Ser peptide as a non-helical linker. The hybrid protein produced using this linker showed less antimicrobial activity against Escherichia coli, Bacillus subtilis, Burkholderia glumae, Pseudomonas corrugate, and Erwinia rhapontici than either of the two parental antimicrobial proteins. In addition, the MIC value of the hybrid protein was 23.1 μM, which indicates poor activity against E. coli. When we used three Glu–Ala–Ala–Ala–Lys (EAAAK) peptide sequences as a helical linker to fuse the two proteins, the resultant hybrid protein had much higher antimicrobial activity than the parental antimicrobial proteins. In particular, this hybrid protein had strong antimicrobial activity against P. corrugate. These results indicate that the EAAAK motif can be used to effectively separate two antimicrobial proteins and produce a hybrid protein with more antimicrobial activity than either of the parent proteins.     

Reidentification of pheromone composition of Sparganothis sulfureana (Clemens) and evidence of geographic variation in male responses from two US states

GC-EAD analyses of pheromone gland extracts of calling female Sparganothis sulfureana revealed at least 6 compounds that consistently elicited antennal responses from male antennae. In addition to the major pheromone compound, (E)-11-tetradecenyl acetate (E11–14:OAc), which was previously reported, the other compounds were found to be (E)-9-dodecenyl acetate (E9–12:OAc), (Z)-9-dodecenyl acetate (Z9–12:OAc), (Z)-9-tetradecenyl acetate (Z9–14:OAc), (Z)-11-tetradecenyl acetate (Z11–14:OAc), and (E)-11-tetradecenol (E11–14:OH). Tetradecyl acetate, hexadecyl acetate and hexadecenyl acetates were also present in the extracts, but elicited no EAG response from male antennae. Wind tunnel tests demonstrated that males from New Jersey responded equally well to a blend containing five pheromone components in relative to the pheromone glands of calling females. Different male-response profiles from field-trapping tests conducted in the states of Wisconsin and New Jersey were observed, respectively. Significantly higher numbers of male S. sulfureana were caught in New Jersey in traps baited with the binary blend of E11–14:OAc (30 μg) with 1% of Z11–14:OAc, but males from Wisconsin responded equally well to traps containing blends of E11–14:OAc with 0–10% of Z11–14:OAc. The addition of more than 10% of Z11–14:OAc to the primary pheromone compound reduced male captures significantly in both states. Male catches were doubled by adding E9–12:OAc and E11–14:OH to the most attractive binary blend in both states. The trapping test with caged live virgin female moths showed that males in Wisconsin preferred females from the local population than those from New Jersey. The differences in male responses observed may indicate the existence of pheromone polymorphism in this species.     

Comparison of the hydrodynamics and mass transfer characteristics in internal-loop airlift bioreactors utilizing either a novel membrane-tube sparger or perforated plate sparger

Two gas spargers, a novel membrane-tube sparger and a perforated plate sparger, were compared in terms of hydrodynamics and mass transfer (or oxygen transfer) performance in an internal-loop airlift bioreactor. The overall gas holdup ε _T, downcomer liquid velocity V _d, and volumetric mass transfer coefficient K _L a were examined depending on superficial gas velocity U _G increased in Newtonian and non-Newtonian fluids for the both spargers. Compared with the perforated plate sparger, the bioreactor with the membrane-tube sparger increased the values of ε _T by 4.9–48.8 % in air–water system when the U _G was from 0.004 to 0.04 m/s, and by 65.1–512.6 % in air–CMC solution system. The V _d value for the membrane-tube sparger was improved by 40.0–86.3 %. The value of K _L a was increased by 52.8–84.4 % in air–water system, and by 63.3–836.3 % in air–CMC solution system. Empirical correlations of ε _T, V _d, and K _L a were proposed, and well corresponding with the experimental data with the deviation of 10 %.     

Natural products as sources of new fungicides (V): Design and synthesis of acetophenone derivatives against phytopathogenic fungi in vitro and in vivo

A series of acetophenone derivatives (10a–10i, 11, 12a–12g, 13a–13g, 14a–14d and 15a–15l) were designed, synthesized and evaluated for antifungal activities in vitro and in vivo. The antifungal activities of 53 compounds were tested against several plant pathogens, and their structure–activity relationship was summarized. Compounds 10a–10f displayed better antifungal effects than two reference fungicides. Interestingly, the most potent compound 10d exhibited antifungal properties against Cytospora sp., Botrytis cinerea, Magnaporthe grisea, with IC₅₀ values of 6.0–22.6?µg/mL, especially Cytospora sp. (IC₅₀?=?6.0?µg/mL). In the in vivo antifungal assays, 10d displayed the significant protective efficacy of 55.3% to Botrytis cinerea and 73.1% to Cytospora sp. The findings indicated that 10d may act as a potential pesticide lead compound that merits further investigation.     

Comparative analysis of DNA alkylation by conjugates between pyrrole–imidazole hairpin polyamides and chlorambucil or seco-CBI

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide–chlorambucil conjugates 1–4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py–Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1–4 alkylated DNA at the sequences recognized by the Py–Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide–seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py–Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide–seco-CBI conjugate 5 was similar to that of polyamide–chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1–4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents.     

The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB–dnaE2 gene cassette and generation of superior feather degrading bacteria

The lexA–imuB–dnaE2 gene cassette contributing to the TLS (translesion synthesis) polymerase activity and can easily cause mutation after DNA damage in many bacteria. But it was previously thought that TLS polymerase activity was unlikely to exist in the radio-resistant genus Deinococcus. In our preliminary studies, the lexA–imuB–dnaE2 gene cassette was found in a newly isolated feather-degrading Deinococcus ficus. Here we have attempted to determine the imuB gene sequence from another Deinococcus species namely D. grandis, by using the newly designed primers. The destroying of either imuB or dnaE2 gene in D. ficus leads to the increase in UV sensitivity and decrease in UV-induced mutations, which demonstrated the existence of TLS polymerase activity in D. ficus. In the presence of lexA–imuB–dnaE2, it is possible to obtain mutants with various keratinolytic activities after UV exposure. The keratinolytic activity of mutant strain CC-ZG207 increased by approximately twofold during growth in liquid feather medium. In contrast, the mutant strain CC-ZG227 showed only half of the keratinolytic activity compared with the wild type strain. By utilizing SDS–PAGE and zymogram profile analysis, the change in the protease activity was observed. We have proposed that the superior mutants of D. ficus can be created under UV stress, which is mediated by the lexA–imuB–dnaE2 gene cassette.     

Conodonts of the genus Lochriea near the Visean–Serpukhovian boundary (Mississippian) at the Naqing section,Guizhou Province,South China

The First Appearance Datum (FAD) of the conodont Lochriea ziegleri in the lineage Lochriea nodosa–L. ziegleri has been considered the most suitable definition and global correlation-level for a revised base of the Serpukhovian Stage. Abundant specimens of Lochriea are recorded throughout the Late Visean–early Serpukhovian interval in the Naqing section, South China. Among them, the P1 elements, with wide morphological variability, enable confirmation and refinement of main lineages within the genus. Two lineages are proposed: 1) noded Lochriea species, such as L. mononodosa–L. nodosa–L. ziegleri, L. senckenbergica and L. multinodosa, and 2) ridged Lochriea species such as L. monocostata–L. costata–L. cruciformis. The possibility for their phylogenetic relationships is evaluated in this paper.