Cytotaxonomic study of Cephalota hispanica (Gory, 1833) and Spiralia maura (Linnaeus, 1758), two cicindelids from Portugal (Coleoptera)

Cephalota hispanica and Spiralia maura have similar numbers of autosomes but distinct chromosome morphology and sex chromosome systems, (9II+X₁X₂Y/9II+X₁X₂ and 9II+X₁X₂X₃Y/9II+X₁X₂X₃, respectively). The former sex chromosome system follows the pattern of Nearctic and Indian groups and the latter the Palearctic type. The chromosomal polymorphism found suggests the occurrence of other structural rearrangements in the process of differentiation of cicindelids than those usually referred to robertsonian translocations. Moreover, the polyploidy observed in males of both species suggests pre-meiotic endoreduplication phenomena.     

Cytogenetic characterization of ten araneomorph spiders (Araneae): Karyotypes and meiotic features

Karyotypes, sex chromosome systems and meiotic characteristics are reported for ten spider species belonging to the families Gnaphosidae, Philodromidae, Salticidae, Oxyopidae and Sicariidae by using standard Giemsa staining. The male diploid numbers (2n) and sex chromosome systems are as follows: Berinda hakani 2n = 22 (X₁X₂), Berinda ensigera 2n = 22 (X₁X₂), Trachyzelotes lyonneti 2n = 22 (X₁X₂), Trachyzelotes malkini 2n = 22 (X₁X₂), Zelotes caucasius 2n = 22 (X₁X₂) (Gnaphosidae); Thanatus pictus 2n = 28 (X₁ X₂), Tibellus macellus 2n = 24 (X₁ X₂) (Philodromidae); Neon reticulatus 2n = 21 (X₀) (Salticidae); Peucetia virescens 2n = 28 (X₁X₂) (Oxyopidae) and Loxosceles rufescens 2n = 21 (X₁ X₂Y) (Sicariidae). All species have monoarmed chromosomes with the exception of L. rufescens that has biarmed (metacentric and submetacentric) chromosomes. The obtained data are the first results for the genera Berinda, Trachyzelotes and Neon. Additionally, with the exception of L. rufescens, all species are being chromosomally analyzed for the first time.     

Gintonin, a ginseng-derived lysophosphatidic acid receptor ligand, potentiates ATP-gated P2X1 receptor channel currents

Ginseng, the root of Panax ginseng C.A. Meyer, is used as a general tonic. Recently, we isolated a novel ginsengderived lysophosphatidic acid (LPA) receptor ligand, gintonin. Gintonin activates G protein-coupled LPA receptors with high affinity in cells endogenously expressing LPA receptors, e.g., Xenopus oocytes. P2X receptors are ligandgated ion channels activated by extracellular ATP, and 7 receptor subtypes (P2X₁–P2X₇) have been identified. Most of the P2X₁ receptors are expressed in the smooth muscles of genitourinary organs involved in reproduction. A main characteristic of the P2X₁ receptor is rapid desensitization after repeated ATP treatment of cells or tissues expressing P2X₁ receptors. In the present study, we examined the effect of gintonin on P2X₁ receptor channel activity. P2X₁ receptors were heterologously expressed in Xenopus oocytes. ATP treatment of oocytes expressing P2X₁ receptors induced large inward currents (I _ATP ), but repetitive ATP treatments induced a rapid desensitization of I _ATP . Gintonin treatment after P2X₁ receptor desensitization potentiated I _ATP in a concentration-dependent manner. We further examined the signaling transduction pathways involved in gintonin-mediated potentiation of I _ATP . Gintoninmediated I _ATP potentiation was blocked by Ki16425, an LPA1/3 receptor antagonist, a PKC inhibitor, a PLC inhibitor, and a PI4-Kinase inhibitor but not by a calcium chelator. In addition, mutations of the phosphoinositide binding site of the P2X₁ receptor greatly attenuated the gintonin-mediated I _ATP potentiation. These results indicate that G protein-coupled LPA receptor activation by gintonin is coupled to the potentiation of the desensitized P2X₁ receptor through a phosphoinositide-dependent pathway.     

Distribution of P2Y<Subscript>2</Subscript> receptors in the guinea pig enteric nervous system and its coexistence with P2X<Subscript>2</Subscript> and P2X<Subscript>3</Subscript> receptors,neuropeptide Y,nitric oxide synthase and calretinin

The distribution of P2Y₂ receptor-immunoreactive (ir) neurons and fibers and coexistence of P2Y₂ with P2X₂ and P2X₃ receptors, neuropeptide Y (NPY), calretinin (CR), calbindin (CB) and nitric oxide synthase (NOS) was investigated with immunostaining methods. The results showed that P2Y₂-ir neurons and fibers were distributed widely in myenteric and submucous plexuses of the guinea pig stomach corpus, jejunum, ileum and colon. The typical morphology of P2Y₂-ir neurons was a long process with strong positive staining on the same side of the cell body. The P2Y₂-ir neurons could be Dogiel type 1. About 40–60% P2X₃-ir neurons were immunoreactive for P2Y₂ in the myenteric plexus and all the P2X₃-ir neurons expressed the P2Y₂ receptor in the submucosal plexus; almost all the NPY-ir neurons and the majority of CR-ir neurons were also immunoreactive for P2Y₂, especially in the myenteric plexus of the small intestine; no P2Y₂-ir neurons were immunoreactive for P2X₂ receptors, CB and NOS. It is shown for the first time that S type/Dogiel type 1 neurons with fast P2X and slow P2Y receptor-mediated depolarizations could be those neurons expressing both P2Y₂-ir and P2X₃-ir and that they are widely distributed in myenteric and submucosal plexuses of guinea pig gut.     

Ceramide-1-Phosphate, in Contrast to Ceramide, Is Not Segregated into Lateral Lipid Domains in Phosphatidylcholine Bilayers

Sphingolipids are key lipid regulators of cell viability: ceramide is one of the key molecules in inducing programmed cell death (apoptosis), whereas other sphingolipids, such as ceramide 1-phosphate, are mitogenic. The thermotropic and structural behavior of binary systems of N-hexadecanoyl-D-erythro-ceramide (C₁₆-ceramide) or N-hexadecanoyl-D-erythro-ceramide-1-phosphate (C₁₆-ceramide-1-phosphate; C₁₆-C1P) with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was studied with DSC and deuterium nuclear magnetic resonance (²H-NMR). Partial-phase diagrams (up to a mole fraction of sphingolipids X = 0.40) for both mixtures were constructed based on DSC and ²H-NMR observations. For C₁₆-ceramide-containing bilayers DSC heating scans showed already at X_cer = 0.025 a complex structure of the main-phase transition peak suggestive of lateral-phase separation. The transition width increased significantly upon increasing X_cer, and the upper-phase boundary temperature of the mixture shifted to ∼65°C at X_cer = 0.40. The temperature range over which ²H-NMR spectra of C₁₆-ceramide/DPPC-d₆₂ mixtures displayed coexistence of gel and liquid crystalline domains increased from ∼10° for X_cer = 0.1 to ∼21° for X_cer = 0.4. For C16-C1P/DPPC mixtures, DSC and ²H-NMR observations indicated that two-phase coexistence was limited to significantly narrower temperature ranges for corresponding C1P concentrations. To complement these findings, C₁₆-ceramide/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and C16-C1P/POPC mixtures were also studied by ²H-NMR and fluorescence techniques. These observations indicate that DPPC and POPC bilayers are significantly less perturbed by C₁₆-C1P than by C₁₆-ceramide and that C₁₆-C1P is miscible within DPPC bilayers at least up to X_C1P = 0.30.     

Evolutionary breakpoint regions and chromosomal remodeling in Harttia (Siluriformes: Loricariidae) species diversification

The Neotropical armored catfish genus Harttia presents a wide variation of chromosomal rearrangements among its representatives. Studies indicate that translocation and Robertsonian rearrangements have triggered the karyotype evolution in the genus, including differentiation of sex chromosome systems. However, few studies used powerful tools, such as comparative whole chromosome painting, to clarify this highly diversified scenario. Here, we isolated probes from the X₁ (a 5S rDNA carrier) and the X₂ (a 45S rDNA carrier) chromosomes of Harttia punctata, which displays an X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system. Those probes were applied in other Harttia species to evidence homeologous chromosome blocks. The resulting data reinforce that translocation events played a role in the origin of the X₁X₂Y sex chromosome system in H. punctata. The repositioning of homologous chromosomal blocks carrying rDNA sites among ten Harttia species has also been demonstrated. Anchored to phylogenetic data it was possible to evidence some events of the karyotype diversification of the studied species and to prove an independent origin for the two types of multiple sex chromosomes, XX/XY₁Y₂ and X₁X₁X₂X₂/X₁X₂Y, that occur in Harttia species. The results point to evolutionary breakpoint regions in the genomes within or adjacent to rDNA sites that were widely reused in Harttia chromosome remodeling.     

Cytogenetic,cross-mating and molecular evidence of four cytological races of Anopheles crawfordi (Diptera: Culicidae) in Thailand and Cambodia

Twenty-nine isolines of Anopheles crawfordi were established from wild-caught females collected from cow-baited traps in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and four types of Y (Y₁, Y₂, Y₃, and Y₄) chromosomes were identified, according to differing amounts of extra heterochromatin. These sex chromosomes represent four metaphase karyotypes, i.e., Forms A (X₁, X₂, X₃, Y₁), B (X₁, X₂, X₃, Y₂), C (X₂, Y₃) and D (X₂, Y₄). Forms C and D are novel metaphase karyotypes confined to Thailand, whereas forms A and B appear to be common in both Thailand and Cambodia. Cross-mating experiments between the four karyotypic forms indicated genetic compatibility in yielding viable progenies and synaptic salivary gland polytene chromosomes. The results suggest that the forms are conspecific and A. crawfordi comprises four cytological races, which is further supported by very low intraspecific variation (mean genetic distance = 0.000–0.018) of the nucleotide sequences in ribosomal DNA (ITS2) and mitochondrial DNA sequences (COI, COII).     

X1X1X2X2/X1X2Y sex chromosome systems in the Neotropical Gymnotiformes electric fish of the genus Brachyhypopomus

Several types of sex chromosome systems have been recorded among Gymnotiformes, including male and female heterogamety, simple and multiple sex chromosomes, and different mechanisms of origin and evolution. The X₁X₁X₂X₂/X₁X₂Y systems identified in three species of this order are considered homoplasic for the group. In the genus Brachyhypopomus, only B. gauderio presented this type of system. Herein we describe the karyotypes of Brachyhypopomus pinnicaudatus and B. n. sp. FLAV, which have an X₁X₁X₂X₂/X₁X₂Y sex chromosome system that evolved via fusion between an autosome and the Y chromosome. The morphology of the chromosomes and the meiotic pairing suggest that the sex chromosomes of B. gauderio and B. pinnicaudatus have a common origin, whereas in B . n. sp. FLAV the sex chromosome system evolved independently. However, we cannot discard the possibility of common origin followed by distinct processes of differentiation. The identification of two new karyotypes with an X₁X₁X₂X₂/X₁X₂Y sex chromosome system in Gymnotiformes makes it the most common among the karyotyped species of the group. Comparisons of these karyotypes and the evolutionary history of the taxa indicate independent origins for their sex chromosomes systems. The recurrent emergence of the X₁X₁X₂X₂/X₁X₂Y system may represent sex chromosomes turnover events in Gymnotiformes.     

Synthesis and in vitro activity of N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine P2X(7) antagonists

Synthesis and biological evaluation of a novel class of substituted N-benzyl-1-(2,3-dichlorophenyl)-1H-tetrazol-5-amine derivatives resulted in the identification of potent P2X₇ antagonists. These compounds were assayed for activity at both the human and rat P2X₇ receptors. On the benzyl moiety, a variety of functional groups were tolerated, including both electron-withdrawing and electron-donating substituents. Ortho-substitution on the benzyl group provided the greatest potency. The ortho-substituted analogs showed approximately 2.5-fold greater potency at human compared to rat P2X₇ receptors. Compounds 12 and 38 displayed hP2X₇pIC₅₀s >7.8 with less than 2-fold difference in potency at the rP2X₇.     

Die Cytogenetik zweier norddeutscher Populationen von Nosopsyllus fasciatus Bosc (Aphaniptera)

Specimens of the populations Hamburg and Wilhelmshaven of the ratflea N. fasciatus exhibit variation of the chromosome number in the range of 2n=20–23 and 2n=20–27 respectively, resulting from individual differences in the number of supernumerary chromosomes beyond the basic chromosome complement of 2n=20. The supernumerary chromosomes are mostly euchromatic and partly or completely homologous to each other and to the 10. pair of the basic complement. The numerical variation in the population Wilhelmshaven is produced by recurrent mitotic non-disjunction of the supernumerary chromosomes in anaphase II of spermatogenesis. Constant mitotic non-disjunction and preferential segregation of the supernumerary chromosomes towards the pronucleus leads to their accumulation in the population.—A multiple sex-chromosome mechanism of the type X₁ X₂ Y₁ Y₂ (male): X₁ X₁ X₂ X₂ (female) has been demonstrated for the population Wilhelmshaven of N. fasciatus. The X₁ X₂ Y₁ Y₂-chain of four is restricted to the male meiosis, in oogenesis two sex bivalents (X₁ X₁ and X₂ X₂) are formed. — The cytogenetic data presented do not support the concept of a closer phylogenetic relationship between the Aphaniptera and Nematocera, but do not preclude the possibility of a kinship of Aphaniptera and Neomecoptera.     

Dodonaea viscosa var. angustifolia leaf: New source of polysaccharide and its anti-oxidant activity

Ultrasonic technology was applied for polysaccharide extraction from the leaves of Dodonaea viscosa and response surface methodology (RSM) was used to optimize the effects of processing parameters on polysaccharide extraction yield. Three independent variables were extraction time (X₁), extraction temperature (X₂) and ultrasonic power (X₃), respectively. The statistical analysis indicated the independent variables (X₁, X₂, X₃), the quadratic terms (X₁₁ and X₃₃) and the interaction terms (X₁X₂, X₁X₃, X₂X₃) had significant effects on the yield of polysaccharides (P < 0.05). The optimal extraction conditions of D. viscosa leaf were determined as follows: extraction time 50.54 min, extraction temperature 85 °C and ultrasonic power 400 W. Under these conditions, the experimental yield of polysaccharides was 9.455 ± 0.24%, which was agreed closely with the predicted value (9.398%). The evaluation of anti-oxidant activity suggested that the polysaccharide exhibited significant protection against DPPH and hydroxyl radicals and could be explored as a nutraceutical agent.     

Karyological analysis of Cylindera trisignata (Latreille & Dejean, 1822) from Portugal (Coleoptera,Cicindelidae)

Cellular suspensions of germinal tissues of Cylindera trisignata provided the definition of its karyotype: 2n=23 and 2n=26 for the males and females respectively. This Palearctic species has a sex chromosome system of the X₁X₂X₃X₄Y / X₁X₁X₂X₂X₃X₃X₄X₄ type, only found until now in Cicindela maroccana pseudomaroccana. The heterosomes are not well-differentiated from the general morphological standpoint. To explain the origin of the 4X condition, a mechanism of dissociation of the X chromosomes rather than an incorporation of autosomal segments is proposed. However, based on the occurrence of distinct ploidy levels, both in male and female cells, with a relatively high incidence for the tetraploid condition with regular autosomal bivalents, the hypothesis of a possible evolutionary role of polyploidy is suggested.     

Origin of the X1X1X2X2/X1X2Y sex chromosome system of Harttia punctata (Siluriformes,Loricariidae) inferred from chromosome painting and FISH with ribosomal DNA markers

Harttia is a genus in the subfamily Loricariinae that accommodates fishes popularly known as armored catfishes. They show extensive karyotypic diversity regarding interspecific numerical/structural variation of the karyotypes, with the presence of the XX/XY₁Y₂ multiple sex chromosome system, as found in H. carvalhoi. In this context, this study aimed to characterize Harttia punctata chromosomally, for the first time, and to infer the rearrangements that originated the X₁X₁X₂X₂/X₁X₂Y multiple sex chromosome system present in this species. The data obtained in this study, with classical (Giemsa, C-banding and AgNORs) and molecular methodologies (fluorescence in situ hybridization) and chromosome microdissection, indicated that a translocation between distinct acrocentric chromosomes bearing rRNA genes, accompanied by deletions in both chromosomes, might have originated the neo-Y chromosome in this species. The data also suggest that the multiple sex chromosome systems present in H. carvalhoi and H. punctata had an independent origin, evidencing the recurrence of chromosome alterations in species from this genus.     

New insights into sex chromosome evolution in anole lizards (Reptilia,Dactyloidae)

Anoles are a clade of iguanian lizards that underwent an extensive radiation between 125 and 65 million years ago. Their karyotypes show wide variation in diploid number spanning from 26 (Anolis evermanni) to 44 (A. insolitus). This chromosomal variation involves their sex chromosomes, ranging from simple systems (XX/XY), with heterochromosomes represented by either micro- or macrochromosomes, to multiple systems (X₁X₁X₂X₂/X₁X₂Y). Here, for the first time, the homology relationships of sex chromosomes have been investigated in nine anole lizards at the whole chromosome level. Cross-species chromosome painting using sex chromosome paints from A. carolinensis, Ctenonotus pogus and Norops sagrei and gene mapping of X-linked genes demonstrated that the anole ancestral sex chromosome system constituted by microchromosomes is retained in all the species with the ancestral karyotype (2n?=?36, 12 macro- and 24 microchromosomes). On the contrary, species with a derived karyotype, namely those belonging to genera Ctenonotus and Norops, show a series of rearrangements (fusions/fissions) involving autosomes/microchromosomes that led to the formation of their current sex chromosome systems. These results demonstrate that different autosomes were involved in translocations with sex chromosomes in closely related lineages of anole lizards and that several sequential microautosome/sex chromosome fusions lead to a remarkable increase in size of Norops sagrei sex chromosomes.     

Differentiation and evolutionary relationships in Erythrinus erythrinus (Characiformes, Erythrinidae): comparative chromosome mapping of repetitive sequences

Erythrinus erythrinus presents extensive karyotypic diversity, with four karyomorphs (A–D) differing in the number of chromosomes, karyotype structure or sex chromosomes systems. Karyomorph A has 2n = 54 chromosomes in males and females without heteromorphic sex chromosomes, while karyomorph C has 2n = 52 chromosomes in females and 2n = 51 chromosomes in males, due a X₁X₁X₂X₂/X₁X₂Y sex chromosome system. Three allopatric populations of the karyomorph A and one population of the karyomorph C were now in deep investigated by molecular cytogenetic analyses, using repetitive DNAs as probes. The results reinforced the relatedness among populations of the karyomorph A, despite their large geographic distribution. Karyomorph C, however, showed a remarkably difference in the genomic constitution, especially concerning the amount and distribution of the 5S rDNA and Rex3 sequences on chromosomes. In addition, although karyomorphs C and D share several features, exclusive chromosomal markers show the derivative evolutionary pathway between them. Thus, besides the classical chromosomal rearrangements, the repetitive DNAs were useful tools to reveal the biodiversity, relatedness and differentiation of this fish group. The chromosomal set strongly corroborates that E. erythrinus corresponds to a species complex instead of a single biological entity.     

Cleavage Specificity of Mycobacterium tuberculosis ClpP1P2 Protease and Identification of Novel Peptide Substrates and Boronate Inhibitors with Anti-bacterial Activity

The ClpP1P2 protease complex is essential for viability in Mycobacteria tuberculosis and is an attractive drug target. Using a fluorogenic tripeptide library (Ac-X₃X₂X₁-aminomethylcoumarin) and by determining specificity constants (k_cat/K_m), we show that ClpP1P2 prefers Met ≫ Leu > Phe > Ala in the X₁ position, basic residues or Trp in the X₂ position, and Pro ≫ Ala > Trp in the X₃ position. We identified peptide substrates that are hydrolyzed up to 1000 times faster than the standard ClpP substrate. These positional preferences were consistent with cleavage sites in the protein GFPssrA by ClpXP1P2. Studies of ClpP1P2 with inactive ClpP1 or ClpP2 indicated that ClpP1 was responsible for nearly all the peptidase activity, whereas both ClpP1 and ClpP2 contributed to protein degradation. Substrate-based peptide boronates were synthesized that inhibit ClpP1P2 peptidase activity in the submicromolar range. Some of them inhibited the growth of Mtb cells in the low micromolar range indicating that cleavage specificity of Mtb ClpP1P2 can be used to design novel anti-bacterial agents.     

Achiasmate male meiosis in two Cymatia species (Hemiptera,Heteroptera, Corixidae)

The karyotype and male meiosis, with a particular focus on the presence or absence of chiasmata between the homologs, were studied in the water boatman species Cymatia rogenhoferi (Fieber) and Cymatia coleoptrata (Fabricius) (Corixidae, Cymatiainae). It is shown that the species have 2n = 33 (28A+2m+X₁X₂Y) and 2n = 24 (20A+2m+XY) respectively, post-reduction of sex chromosomes, and achiasmate meiosis of an alignment type in males. Cytogenetic and some morphological diagnostic characters separating Cymatia Flor from the rest of Corixidae are discussed.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.