An essential tryptophan residue in alkaline phosphatase from pearl oyster (<Emphasis Type="Italic">Pinctada fucata</Emphasis>)

Alkaline phosphatases are ubiquitous enzymes found in most species including the pearl oyster, Pinctada fucata, where it is presumably involved in nacreous biomineralization processes. In the present study, we have purified alkaline phosphatases from the pearl oyster and modified the tryptophan residues using N-bromosuccinimide (NBS). We show that the resulting inactivation of purified alkaline phosphatase by NBS is dependent on modification of only one of five tryptophan residues in the enzyme. Substrate protection experiments showed that the tryptophan residue was not located at the substrate-binding site but was involved in the catalytic activity. Published in Russian in Biokhimiya, 2008, Vol. 73, No. 1, pp. 107–112.     

<Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and<Emphasis Type="Italic">Vibrio alginolyticus</Emphasis>: Short generation-time marine bacteria

The growth rates of 30 different strains ofVibrio parahaemolyticus andVibrio alginolyticus at 37°C was determined. Each species consists of two major groups, one having a short generation time (12–14 min) and one with a longer generation time (20–25 min). The diversity in generation times of different strains belonging to the same species is discussed. The effect of temperature, salt, and nutrient concentrations on the growth rate of oneV. alginolyticus strain (NCMB 1803) was studied. The most striking is the effect of the temperature; at 39°C the generation time is 10–11 min, while at 21°C it is 60 min. The heat of activation for growth calculating from such data is 22,580 kcal/mole/hr⁻¹. The ecological significance of these results is discussed.     

Isolation of <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio splendidus</Emphasis> from captive-bred seahorses with disease symptoms

Vibrio species isolated from diseased seahorses were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The results demonstrated that Vibrio alginolyticus and Vibrio splendidus were predominant in the lesions of these seahorses. To our knowledge, this is the first time that these bacterial species have been associated with disease symptoms in captive-bred seahorses.     

Identification and comparative analysis of microRNAs in <Emphasis Type="Italic">Pinus massoniana</Emphasis> infected by <Emphasis Type="Italic">Bursaphelenchus xylophilus</Emphasis>

Pine wilt disease seriously endangers pine tree growth and can lead to wilting and death. In this study, Masson pine (Pinus massoniana) was used as the donor plant to study miRNA expression by high-throughput sequencing in needle leaves of trees during the first 3 days after infection by Bursaphelenchus xylophilus. The results showed that the number of differentially expressed miRNAs first increased and then decreased in samples within 3 days of infection, with the highest expression in samples collected on day 2. Analysis of miRNA target genes in needles on different days after infection revealed enrichment in two pathways: plant hormone signal transduction and RNA transport. Further comparison of the samples indicated differential expression of 10 miRNAs on different days after infection, and the corresponding target genes, such as those participating in plant hormone signal transduction, were also significantly enriched. In addition, the concentrations of zeatin and indole acetic acid in needle tissues were significantly lower after infection than in the control sample. The above results indicated that B. xylophilus infection influenced the expression of miRNAs related to plant hormone signal transduction in P. massoniana, decreasing plant hormone synthesis and ultimately affecting the growth of Masson pine.     

Occurrence of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in the German Bight over a seasonal cycle

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10³ N l⁻¹ and MPNs up to 240 N g⁻¹ were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.     

Comparative morphometric study of the invasive pearl oyster <Emphasis Type="Italic">Pinctada radiata</Emphasis> along the Tunisian coastline

In order to study the relative growth of the pearl oyster Pinctada radiata in Tunisia, a total of 330 individuals of this species were collected from six sites along the Tunisian coastline. Quantitative measurements of collected oysters were conducted for shell height, shell length, shell width, hinge length, height and width of the nacreous part and wet weight. The size structure of the sampled populations was described and the relative growth between different morphometric characteristics was estimated as allometric growth lines for the six P. radiata samples. It appeared that the majority of examined samples were dominated by large individuals that exceed a shell height of 42 mm. The maximum size (100.5 mm), recorded in Bizerta lagoon, is bigger than that recorded elsewhere in particular in the Red Sea. Size distribution analysis also showed that the majority of P. radiata samples were dominated by two or more size groups. Differences of allometric regression were found between the examined samples for the tested relationships. Moreover, the Factorial Discriminant Analysis, coupled with Ascending Hierarchic Classification, classified the sub-populations according to geographic locations.     

Isolation and Characterization of the N-linked Oligosaccharides in Nacrein from <Emphasis Type="Italic">Pinctada fucata</Emphasis>

We analyzed the structure of the N-linked oligosaccharides enzymatically liberated from the organic matrix (OM) component in the nacreous layer of Japanese pearl oyster: Pinctada fucata. The lectin-blot analysis of the soluble OM after separation by SDS-PAGE, four components, with sizes of approximately 55 kDa, 35 kDa, 25 kDa, and 21 kDa were detected with GNA lectin, which recognized terminal mannose of high mannose and hybrid types of N-glycan. The 55-kDa component of the soluble OM detected by lectin blotting was identified as nacrein by using liquid chromatography/mass spectrometry (LC/MS). LC/MS analysis of the N-glycan liberated from nacrein detected a hybrid-type N-glycan, which contained sulfite and sialic acid at its terminus. The data strongly imply that nacrein, a sulfated OM glycoprotein, participates in molluscan biomineralization by creating a favorable environment for calcium ion uptake through sulfonic acid and sialic acid.     

Ontogeny of tick hemocytes: a comparative analysis of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> and <Emphasis Type="Italic">Ornithodoros moubata</Emphasis>

Hemocytes of two tick species, Ixodes ricinus and Ornithodoros moubata, were investigated with the aim to determine their ultrastructural characteristics and developmental relationships. Only a limited number of ultrastructural features was shown to be unequivocally homological across all hemocyte types. The two species, representing distant groups of ticks, differ in the composition of their circular cell populations. In I. ricinus, three groups of distinct morphological types of hemocytes could be determined according to well-defined ultrastructural features: a typical non-phagocytic granular cell with electron-dense granula and homogeneous cytoplasm (Gr II), and two different types of phagocytic hemocytes, namely plasmatocytes with a low number of granula and phagocytic granolocytes, designated as Gr I. In contrast, an additional cell type resembling insect spherulocytes was determined in O. moubata. This cell type does not seem to be homologous to any I. ricinus hemocyte and may represent a cell type typical of soft ticks only. Possible ontogenetic lineages of the hemocytes of both tick-species were inferred.     

Macrophage response to <Emphasis Type="Italic">Mycobacteriumtuberculosis</Emphasis> infection

The immune response to Mycobacterium tuberculosis (Mtb) infection is the formation of multicellular lesions, or granolomas, in the lung of the individual. However, the structure of the granulomas and the spatial distribution of the immune cells within is not well understood. In this paper we develop a mathematical model investigating the early and initial immune response to Mtb. The model consists of coupled reaction-diffusion-advection partial differential equations governing the dynamics of the relevant macrophage and bacteria populations and a bacteria-produced chemokine. Our novel application of mathematical concepts of internal states and internal velocity allows us to begin to study this unique immunological structure. Volume changes resulting from proliferation and death terms generate a velocity field by which all cells are transported within the forming granuloma. We present numerical results for two distinct infection outcomes: controlled and uncontrolled granuloma growth. Using a simplified model we are able to analytically determine conditions under which the bacteria population decreases, representing early clearance of infection, or grows, representing the initial stages of granuloma formation.     

<Emphasis Type="Italic">Galleria</Emphasis><Emphasis Type="Italic">mellonella</Emphasis> are Resistant to <Emphasis Type="Italic">Pneumocystis</Emphasis><Emphasis Type="Italic">murina</Emphasis> Infection

Studying Pneumocystis has proven to be a challenge from the perspective of propagating a significant amount of the pathogen in a facile manner. The study of several fungal pathogens has been aided by the use of invertebrate model hosts. Our efforts to infect the invertebrate larvae Galleria mellonella with Pneumocystis proved futile since P. murina neither caused disease nor was able to proliferate within G. mellonella. It did, however, show that the pathogen could be rapidly cleared from the host.     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Revision of the <Emphasis Type="Italic">Serrulati</Emphasis> species of <Emphasis Type="Italic">Penstemon</Emphasis> section <Emphasis Type="Italic">Saccanthera</Emphasis> subsection <Emphasis Type="Italic">Serrulati</Emphasis>

A revision of Penstemon sect. Saccanthera subsect. Serrulati includes a new species (P. salmonensis), a new variety (P. triphyllus var. infernalis), and the elevation of a subspecies to species (P. curtiflorus), bringing the total number of species to eight, which are keyed and described, complete with nomenclature and type citations.     

Gel shift analysis of the <Emphasis Type="Italic">empA</Emphasis> promoter region in <Emphasis Type="Italic">Vibrio anguillarum</Emphasis>

Background  

The induction of metalloprotease encoded by empA in Vibrio anguillarum occurs at high cell density in salmon intestinal mucus. Previously we have shown that there are significant differences in empA expression in two strains of V. anguillarum, M93Sm and NB10. It is hypothesized that differences in empA regulation are due to differences in binding of regulatory elements.     

<Emphasis Type="Italic">Agrobacterium</Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of callus cells of <Emphasis Type="Italic">Crataegus</Emphasis><Emphasis Type="Italic">aronia</Emphasis>

A genetic transformation system has been developed for callus cells of Crataegus aronia using Agrobacterium tumefaciens. Callus culture was established from internodal stem segments incubated on Murashige and Skoog (MS) medium supplemented with 5 mg l⁻¹ Indole-3-butyric acid (IBA) and 0.5 mg l⁻¹ 6-benzyladenine (BA). In order to optimize the callus culture system with respect to callus growth and coloration, different types and concentrations of plant growth regulators were tested. Results indicated that the best average fresh weight of red colored callus was obtained on MS medium supplemented with 2 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg l⁻¹ kinetin (Kin) (callus maintenance medium). Callus cells were co-cultivated with Agrobacterium harboring the binary plasmid pCAMBIA1302 carrying the mgfp5 and hygromycin phosphotransferase (hptII) genes conferring green fluorescent protein (GFP) activity and hygromycin resistance, respectively. Putative transgenic calli were obtained 4 weeks after incubation of the co-cultivated explants onto maintenance medium supplemented with 50 mg l⁻¹ hygromycin. Molecular analysis confirmed the integration of the transgenes in transformed callus. To our knowledge, this is the first time to report an Agrobacterium-mediated transformation system in Crataegus aronia.     

Identification of <Emphasis Type="Italic">S</Emphasis> haplotypes in <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis of <Emphasis Type="Italic">SP11</Emphasis> alleles

A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.     

Genome-wide comparative analysis of the <Emphasis Type="Italic">IQD</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Background  

Calcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.