Characterization and function of the T-box 1 gene in Chinese giant salamander Andrias davidianus

We characterized the Andrias davidianus T-box 1 (Tbx1) gene. Tbx1 expression was high in testis and low in other examined tissues. Immunohistochemistry detected tbx1 expression in somatic and germ cells 62 days post-hatching (dph), prior to gonad differentiation. At 210 dph, after gonad differentiation, tbx1 was expressed in spermatogonia and testis somatic cells and in granulosa cells in ovary. Tbx1 expression was up-regulated in ovary after high temperature treatment. In the neomale, tbx1 expression showed a similar profile to normal males, and vice-versa for genetic male. Over-expression of tbx1 in females after injection of TBX1 protein down-regulated the female-biased genes cyp19a and foxl2 and up-regulated the male-biased amh gene. When tbx1 was knocked down by tbx1/siRNA, cyp19a and foxl2 expression was up-regulated, and expression of amh, cyp26a, dmrt1, and wt1 was down-regulated. Results suggest that tbx1 influenced sex-related gene expression and participates in regulation of A. davidianus testis development.     

Analysis of Expression,Cellular Localization,and Function of Three Inhibitors of Apoptosis (IAPs) from Litopenaeus vannamei during WSSV Infection and in Regulation of Antimicrobial Peptide Genes (AMPs)

Inhibitors of apoptosis (IAPs) play important roles in apoptosis and NF-κB activation. In this study, we cloned and characterized three IAPs (LvIAP1-3) from the Pacific white shrimp, Litopenaeusvannamei . LvIAP1-3 proteins shared signature domains and exhibited significant similarities with other IAP family proteins. The tissue distributions of LvIAP1-3 were studied. The expression of LvIAP1-3 was induced in the muscle after white spot syndrome virus (WSSV) infection. LvIAP1 expression in the gill, hemocytes, hepatopancreas, and intestine was responsive to WSSV and Vibrio alginolyticus infections. LvIAP2 expression in the gill, hemocytes, and hepatopancreas was also responsive to WSSV infection. The expression of LvIAP3 in the gill, hemocytes, and intestine was reduced after V . alginolyticus infection. When overexpressed in Drosophila S2 cells, GFP labeled-LvIAP2 was distributed in the cytoplasm and appeared as speck-like aggregates in the nucleus. Both LvIAP1 and LvIAP3 were widely distributed throughout the cytoplasm and nucleus. The expression of LvIAP1, LvIAP2, and LvIAP3 was significantly knocked down by dsRNA-mediated gene silencing. In the gill of LvIAP1- or LvIAP3-silenced shrimp, the expression of WSSV VP28 was significantly higher than that of the dsGFP control group, suggesting that LvIAP1 and LvIAP3 may play protective roles in host defense against WSSV infection. Intriguingly, the LvIAP2-silenced shrimp all died within 48 hours after dsLvIAP2 injection. In the hemocytes of LvIAP2-silenced shrimps, the expression of antimicrobial peptide genes (AMPs), including Penaeidins, lysozyme, crustins, Vibrio penaeicidae-induced cysteine and proline-rich peptides (VICPs), was significantly downregulated, while the expression of anti-lipopolysaccharide factors (ALFs) was upregulated. Moreover, LvIAP2 activated the promoters of the NF-κB pathway-controlled AMPs, such as shrimp Penaeidins and Drosophila drosomycin and attacin A, in Drosophila S2 cells. Taken together, these results reveal that LvIAP1 and LvIAP3 might participate in the host defense against WSSV infection, and LvIAP2 might be involved in the regulation of shrimp AMPs.     

Interactions of Insecticidal Toxin Gene Products from Xenorhabdus nematophilus PMFI296

Four genes on a genomic fragment from Xenorhabdus nematophilus PMFI296 were shown to be involved in insecticidal activity towards three commercially important insect species. Each gene was expressed individually and in combinations in Escherichia coli, and the insecticidal activity of the lysates was determined. The combined four genes (xptA1, xptA2, xptB1, and xptC1), in E. coli, showed activity towards Pieris brassicae, Pieris rapae, and Heliothis virescens. The genes xptA1, xptB1, and xptC1 were involved in expressing activity towards P. rapae and P. brassicae, while the genes xptA2, xptB1, and xptC1 were needed for activity towards H. virescens. When each of these three genes was expressed individually in E. coli and the cell lysates were used in insect assays or mixed and then used, insecticidal activity was detected at a very low level. If the genes xptB1 and xptC1 were expressed in the same E. coli cell and this cell lysate was mixed with cells expressing xptA1, activity was restored to P. rapae and P. brassicae. Similarly mixing XptB1/C1 lysate with XptA2 lysate restored activity towards H. virescens. Individual gene disruptions in X. nematophilus PMFI296 reduced activity to insects; this activity was restored by complementation with cells expressing either xptA1 or xptA2 for their respective disruptions or E. coli expressing both xptB1 and xptC1 for individual disruptions of either of these genes. The genes xptA2, xptC1, and xptB1 were expressed as an operon in PMFI296 and inactivation of xptA2 or xptC1 resulted in silencing of downstream gene(s), while xptA1 was expressed as a single gene. Therefore, the two three gene product combinations interact with each other to produce good insecticidal activity.     

Pro-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from Phaseolus aureus was photoinactivated in the presence of methylene blue or rose bengal. Pro and several imino acid analogues protected the enzyme against dye-mediated photoinactivation but ATP was ineffective. Together with kinetic data, this evidence suggested that a His-residue near the Pro-binding site was involved in the enzyme reaction. In the absence of methylene blue, Phaseolus enzyme was stable to light whilst that from Delonix was rapidly and reversibly photoinactivated. ATP as well as Pro, protected the Delonix enzyme against dye-independent photoinactivation. In the presence of methylene blue, the Delonix enzyme was more rapidly photoinactivated than in the absence of the dye. p-Chloromercuribenzoate (pCMB)-inhibited enzyme from both Phaseolus and Delonix was reactivated by sulphydryl reducing reagents. Reactivation of Delonix enzyme was markedly temperature-dependent whilst Phaseolus enzyme was reactivated equally efficiently at all temperatures tested. ATP, tRNA, Pro and several analogues of Pro, protected both the Phaseolus and Delonix enzymes against pCMB inhibition. The possible roles of the His-residue and SH group are discussed in relation to the known differences in substrate specificity between the Phaseolus and Delonix enzymes.     

Axial and Radial Hydraulic Resistance to Roots of Maize (Zea mays L.)

A root pressure probe was employed to measure hydraulic properties of primary roots of maize (Zea mays L.). The hydraulic conductivity (Lp_r) of intact root segments was determined by applying gradients of hydrostatic and osmotic pressure across the root cylinder. In hydrostatic experiments, Lp_r was constant along the segment except for an apical zone of approximately 20 millimeters in length which was hydraulically isolated due to a high axial resistance. In osmotic experiments, Lp_r decreased toward the base of the roots. Lp_r (osmotic) was significantly smaller than Lp_r (hydrostatic). At various distances from the root tip, the axial hydraulic resistance per unit root length (R_x) was measured either by perfusing excised root segments or was estimated according to Poiseuille's law from cross-sections. The calculated R_x was smaller than the measured R_x by a factor of 2 to 5. Axial resistance varied with the distance from the apex due to the differentiation of early metaxylem vessels. Except for the apical 20 millimeters, radial water movement was limiting water uptake into the root. This is important for the evaluation of Lp_r of roots from root pressure relaxations. Stationary water uptake into the roots was modeled using measured values of axial and radial hydraulic resistances in order to work out profiles of axial water flow and xylem water potentials.     

Scorpion venom complexity fractal analysis. Its relevance for comparing venoms

We analyzed the venom elution pattern of 15 scorpions species. Data were scanned at 1 Hz and stored digitally. Approximate fractal dimension (D) [Sevcik (1998)] was calculated for minutes 0-60 of the elutions. D was calculated for either the whole time range, or calculated using a window of 500 points, which was displaced by one time increment recursively, and stored [(t_i,D_i) sets]. We avoid the term complexity as much as possible since defining complexity is difficult; instead we propose the term contortedness and represent it by the variable Q=D−1. To compare venom contortednesses of different species, a phase plot with their (t_i,Q_i) sets was constructed and determination coefficient (d_s) were calculated squaring the Spearman rank correlation coefficient. (t_i,Q_i) sets of several elutions of the same specie were averaged and compared with other species finding that some were amazingly similar (Tityus clathratus vs Tityus caripitensis, d_s = 0.813). Tityus discrepans was similar to 6 of 8 species of the same genus (d_s ranging from 0.23 to 0.49), and also similar to Centruroides gracilis and Chactas laevipes (d_s 0.54 and 0.49, respectively). Serendipitously,T. discrepans was chosen many years ago to produce anti-Tityus antivenom in Venezuela; perhaps the clinical success in neutralizing the venom of the other known Venezuelan Tityus, stems from the mimetism of this venom with the remaining species’ venom.     

Laser spectroscopy for measuring the parameters of a plasma containing helium and argon

Laser spectroscopy diagnostics used in experiments on the PNX-U facility are described. The working gas was argon with an additive of helium. The 2³ P → 3³ D transition was excited by means of optical pumping, and helium fluorescence at wavelengths of 388 and 706.5 nm was observed. The Doppler temperature of helium atoms was determined by scanning the profile of the absorption line with the help of a tunable laser. The sum of the signals so obtained provides information on the local density of helium atoms. It was proposed to determine the local value of the electron density N _e (R) from the ratio between the fluorescence intensities at wavelengths of 388 and 706.5 nm. The ratio of these intensities as a function of N _e for He I was calculated in the collisional-radiative model, and relevant measurements of N _e in the PNX-U facility were performed. When diagnosing the argon component, the main attention was paid to measurements of the ion temperature T _i (R, t). In the course these measurements, anomalous heating of Ar II ions was revealed. The concentration of singly charged argon ions was estimated.     

Effects of newly synthesised platinum(ii) complexes on mitochondrial functions

Protease deficient recA431 mutants of Escherichia coli are defective in their capacity for induction of SOS responses and were intermediate in their sensitivities to ultraviolet light (UV) and cis-platinum(II)diamminodichloride (cis-PDD). Survival after treatment determined as colony forming ability was greater in rec⁺ strains and decreased in recA13 mutants which are defective in both recA proteolytic and recombination capabilites. In contrast, recA431 mutants were as sensitive to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) as the recA13 cells. When cells carried either the pKM101 or N3 plasmid, survival after treatment with the three mutagens was increased. Presence of these plasmids in cells also resulted in hypermutagenicity as indicated by reversion of the argE3 mutation using a modified Ames test. Mutagenesis by NTG and cis-PDD was increased, as was survival of cells treated with UV light, cis-PDD and NTG in both recA⁺ and recA431 (protease deficient) strains. No plasmid mediated enhancement of mutagenesis or cell survival was observed in recA13 mutants. Thus, the ability of the plasmids to enhance cell survival and mutagenesis was dependent on recombination proficiency of the recA gene product and not its regulatory proteolytic activity. Unlike UV or NTG, presence of one of these plasmids was needed to detect reversion of the argE3 mutation by cis-PDD.     

Regulation and function of Arabidopsis AtGALK2 gene in abscisic acid response signaling

AtGALK2 belongs to galactokinase of GHMP family in Arabidopsis thaliana. Two homozygous T-DNA insertion mutants (Atgalk2-1 and Atgalk2-2) of the AtGALK2 gene were identified. The AtGALK2 gene was highly expressed in flowers and roots, but less in stems, leaves and petioles. It was found that the expression of AtGALK2 gene was induced by NaCl and ABA. The two Atgalk2 mutants showed higher germination activity when treated with ABA and NaCl than wild type (Col-0). Through comparing the results of seed germination, root growth, stomatal aperture, water loss, and proline accumulation between the Atgalk2 mutants and Col-0, it was found that Atgalk2 mutants showed less sensitive to ABA than Col-0. The expression levels of ABI1, ABI2, RAB18, ABF3, RD22, RD29A, and RD29B in the Atgalk2 mutants were higher than in Col-0. However, the expression level of OST1 in the Atgalk2 mutants was lower than in Col-0. Taken together, these results suggested AtGALK2 was required for abscisic acid regulation of seed germination, root growth and gene expression, and was involved in salt and osmotic stress response in the early development stage. This study provides important clues to galactokinase activities of GHMP family in ABA signaling and plant development.     

Genetic studies to re-affiliate Edwardsiella tarda fish isolates to Edwardsiella piscicida and Edwardsiella anguillarum species

Until 2012, the genus Edwardsiella was composed by three species Edwardsiella tarda, Edwardsiella hoshinae and Edwardsiella ictaluri. In 2013, Edwardsiella piscicida, compiling fish pathogenic strains previously identified as E. tarda was described, and more recently a new species isolated from diseased eel was reported, namely Edwardsiella anguillarum.The incorporation of these species into the genus makes necessary a revision of the taxonomic position of the isolates previously identified as E. tarda. Using AFLP technique, MLSA studies and in silico DNA–DNA hybridization, 46 of 49 E. tarda isolates were re-assigned as E. piscicida and 2 as E. anguillarum, whereas it was confirmed previous classification of the Edwardsiella types and reference strains used. The study of the taxonomic resolution of the genes 16S rRNA, adk, atpD, dnaJ, glnA, hsp60, tuf as well as the possible combinations among housekeeping genes, showed that the gene dnaJ was the more resolutive. In conclusion, the use of molecular techniques is necessary to accurately identify Edwardsiella isolates, especially when differentiating new species from E. tarda.     

Pyridine salvage and nicotinic acid conjugate synthesis in leaves of mangrove species

The metabolic fate of [carbonyl-¹⁴C]nicotinamide was surveyed in leaf disks of seven mangrove species, Bruguiera gymnorrhiza, Rhizophora stylosa, Kandeliaobovata, Sonneratia alba, Pemphis acidula, Lumnitzera racemosa and Avicennia marina, with and without 250 mM NaCl. Uptake of [¹⁴C]nicotinamide by leaf disks was stimulated by 250 mM NaCl in K. candel, R. stylosa, A. marina and L. racemosa. [Carbonyl-¹⁴C]nicotinamide was converted to nicotinic acid and was utilised for the synthesis of nucleotides and nicotinic acid conjugates. Formation of nicotinic acid by the deaminase reaction was rapid; there was little accumulation of nicotinamide in the disks 3 h after administration. Radioactivity from [carbonyl-¹⁴C]nicotinamide was incorporated into pyridine nucleotides (mainly NAD and NADP) in all mangrove leaves, and the rates varied from 2% (in L. racemosa) to 15% (S. alba) of the total radioactivity taken up. NaCl generally reduced nicotinic acid salvage for NAD and NADP. In all mangrove leaf disks, the most heavily labelled compounds (up to 70% of total radioactivity) were trigonelline (N-methylnicotinic acid) and/or nicotinic acid N-glucoside. Trigonelline was formed in all mangrove plants, but N-glucoside synthesis was found only in leaves of A. marina and K. obovata. In A. marina, incorporation of radioactivity into N-glucoside (51%) was much greater than incorporation into trigonelline (2%). In general, NaCl stimulates the synthesis of these pyridine conjugates. The rate of decarboxylation of nicotinic acid in roots of A. marina seedlings was much greater than for the corresponding reaction observed in leaves.     

Kinetics of Formate Metabolism in Methanobacterium formicicum and Methanospirillum hungatei

The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K_m and V_max values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 μM. The K_m and V_max values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h⁻¹ g⁻¹ (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 μM. The apparent K_m for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K_m for H₂ uptake by cultures of Methanobacterium formicicum was 6 μM dissolved H₂. Formate and H₂ were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H₂ uptake was severely depressed when formate was above saturation and the dissolved H₂ was below 6 μM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation.     

Heterogenous expression of poly-γ-glutamic acid synthetase complex gene of Bacillus licheniformis WBL-3

Bacillus licheniformis WBL-3, one of poly-γ-glutamic acid (γ-PGA) producers, depends on the existence of glutamate in the medium. In this paper, γ-PGA synthetase complex gene (pgsBCA) was cloned from Bacillus licheniformis WBL-3. pgsBCA gene of B. licheniformis WBL-3 was highly homologous with pgs-BCA gene of B. licheniformis 14580. The similarity was 97%, but the similarity of pgsBCA gene between B. licheniformis WBL-3 and Bacillus subtilis IFO3336 was only 74%. However, when pgsBCA was expressed in Escherichia coli, the E. coli clone produced γ-PGA extracellularly. The yield of γ-PGA was 8.624 g/l. This result infers that B. licheniformis and B. subtilis has the similar γ-PGA biosynthesis mechanism, namely, glutamic acid is catalyzed by an ATP-dependent amide ligase to synthesize γ-PGA.     

Molecular Detection of Rickettsia typhi in Cats and Fleas

Background

Rickettsia typhi is the etiological agent of murine typhus (MT), a disease transmitted by two cycles: rat-flea-rat, and peridomestic cycle. Murine typhus is often misdiagnosed and underreported. A correct diagnosis is important because MT can cause severe illness and death. Our previous seroprevalence results pointed to presence of human R . typhi infection in our region; however, no clinical case has been reported. Although cats have been related to MT, no naturally infected cat has been described. The aim of the study is to confirm the existence of R . typhi in our location analyzing its presence in cats and fleas.

Methodology/Principal Findings

221 cats and 80 fleas were collected from Veterinary clinics, shelters, and the street (2001-2009). Variables surveyed were: date of collection, age, sex, municipality, living place, outdoor activities, demographic area, healthy status, contact with animals, and ectoparasite infestation. IgG against R . typhi were evaluated by indirect immunofluorescence assay. Molecular detection in cats and fleas was performed by real-time PCR. Cultures were performed in those cats with positive molecular detection. Statistical analysis was carried out using SPSS. A p < 0.05 was considered significant.Thirty-five (15.8%) cats were seropositive. There were no significant associations among seropositivity and any variables. R . typhi was detected in 5 blood and 2 cultures. High titres and molecular detection were observed in stray cats and pets, as well as in spring and winter. All fleas were Ctenocephalides felis. R . typhi was detected in 44 fleas (55%), from shelters and pets. Co-infection with R . felis was observed.

Conclusions

Although no clinical case has been described in this area, the presence of R . typhi in cats and fleas is demonstrated. Moreover, a considerable percentage of those animals lived in households. To our knowledge, this is the first time R . typhi is detected in naturally infected cats.     

Cloning,Sequencing and Functional Expression in Escherichia coli of dmc Gene Encoding Periplasmic Tetraheme Cytochrome c3from Desulphovibrio desulphuricans M6

A small soluble protein, periplasmic tetraheme cytochrome c₃, was purified fromDesulphovibrio desulfuricans M6 and its gene, dmc, was cloned and the complete nucleotide sequence determined. The purity index of purified cytochrome c₃was 3.3 and the molecular weight was determined as 14.5 kDa by SDS-PAGE. It was found that the 387 bp of dmc gene encoded 21 amino acids of hydrophobic signal peptide and 107 residues of apoprotein. The nucleotide sequence and the predicted amino acid sequence of dmc showed 76% and 83% identities to those of 13 kDa cytochrome c₃from D. desulphuricans ATCC 27774, respectively.dmc gene was functionally expressed in aerobically grown Escherichia coli BL-21(DE3) by co-expressing eightccm genes which were reported to be involved in cytochrome c maturation. The molecular weight of overexpressed holocytochrome c₃was identical to that of the original protein. Visible spectrum of dithionite-reduced form exhibited typical characteristics of c -type cytochromes. In addition, the redox potential was measured to −340 mV by cyclic voltammetry.     

Influence of photoperiods on the growth rate and biomass productivity of green microalgae

The effect of different photoperiods: 24 h illumination and a 12:12-h light/dark (12L:12D) cycle on the growth rate and biomass productivity was studied in five algal species: Neochloris conjuncta, Neochloris terrestris, Neochloris texensis, Botryococcus braunii and Scenedesmus obliquus. The green microalgae examined differ in the reproduction mode. Continuous illumination stimulated the growth of B. braunii and S. obliquus more effectively than the growth of the microalgal species from the genus Neochloris. However, under shorter duration of light of the same intensity (12L:12D cycle), the growth of all the three species of Neochloris was stimulated. Under continuous illumination, the specific growth rate in the first phase of B. braunii and S. obliquus cultures was higher than the growth rate of Neochloris, whereas under the 12L:12D cycle, the specific growth rate of all the three Neochloris species was generally higher than that in B. braunii and S. obliquus. As a result, the light regime influenced algal biomass productivity differently. The maximum biomass productivity was obtained in B. braunii and S. obliquus cultures carried out at continuous illumination. All the Neochloris species produced biomass more efficiently at the 12L:12D cycle, which was two–threefold higher than that of B. braunii and S. obliquus. The unicellular species of the green microalgae from the genus Neochloris, examined for the first time in this study, are promising prospective objects for algal biotechnology.