Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Probiotic <Emphasis Type=Italic>Lactobacillus</Emphasis> strains: <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> studies

Three Lactobacillus strains (LOCK 0900, LOCK 0908, LOCK 0919) out of twenty-four isolates were selected according to their antagonistic activity against pathogenic bacteria, resistance to low pH and milieu of bile salts. Intragastric administration of a mixture of these strains to Balb/c mice affected cytokine T_H1-T_H2 balance toward nonallergic T_H1 response. Spleen cells, isolated from lactobacilli-treated mice and re-stimulated in vitro with the mixture of heat-inactivated tested strains, produced significantly higher amounts of anti-allergic tumor necrosis factor- and interferon-γ than control animals whereas the level of pro-allergic interleukin-5 was significantly lower. Lactobacillus cells did not translocate through the intestinal barrier into blood, liver and spleen; a few Lactobacillus cells found in mesenteric lymph nodes could create antigenic reservoir activating the immune system. The mixture of Lactobacillus LOCK 0900, LOCK 0908 and LOCK 0919 strains represents a probiotic bacterial preparation with possible use in prophylaxis and/or therapy of allergic diseases.     

Marker assisted pea breeding: <Emphasis Type=Italic>eIF4E</Emphasis> allele specific markers to <Emphasis Type=Italic>pea seed-borne mosaic virus</Emphasis> (PSbMV) resistance

Traditional plant breeding relies upon crosses and subsequent selection of genotypes to meet desirable traits. The incorporation of marker-assisted selection into breeding strategies would result in a reduction in the number of offspring to be propagated, selected and tested. In the case of pea (Pisum sativum L.), the testing of resistance to viral pathogens such as pea seed-borne mosaic virus (PSbMV) is included in the breeding process. Resistance to the common strains of PSbMV is conferred by a single recessive gene (eIF4E), localized on LG VI (sbm-1 locus). We have analyzed for variation in the eIF4E genomic sequences from 43 pea varieties and breeding lines, reported as donors of resistance. This enabled a comprehensive investigation of the eIF4E gene structure and mutations responsible for PSbMV resistance were identified. Subsequently, PCR-based and gene-specific single nucleotide polymorphism and co-dominant amplicon length polymorphism markers were developed. All together 60 accessions were analyzed using sequence data and/or allele specific DNA markers. Developed allele specific markers were reproducibly amplified across a broad spectra of pea varieties and breeding lines. These were found to be 100% accurate in detecting the presence of the respective alleles when compared to symptomology and ELISA, testing (74% reliable). Hence, these molecular markers will substantially speed-up PSbMV diagnosis and resistance breeding processes in pea.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.     

Chemical composition and antimicrobial activity of <Emphasis Type=Italic>Thymus pannonicus</Emphasis> All. (<Emphasis Type=Italic>Lamiaceae</Emphasis>) essential oil

This paper presents the results of a study on chemical composition and antimicrobial activity of Thymus pannonicus All. (Lamiaceae) essential oil from Vojvodina province (north of Serbia). The investigated oil was hydrodistilled from a flowering plant and analysed by GC and GC-MS. Fifty-three constituents were identified (>97% of total oil), with geranial (41.42%, w/w) and neral (29.61%, w/w) as the most prominent. The antimicrobial activity of the oil was evaluated using agar disc diffusion and broth microdilution method against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Escherichia coli, two strains of Klebsiella pneumoniae and two strains of Candida albicans. The essential oil exhibited antimicrobial activity to varying degrees against all tested strains. The maximum activity of T. Pannonicus oil was observed against E. coli, S. aureus and both tested strains of C. Albicans (MIC = 50 μ/ml, each). Moderate activity was observed against P. aeruginosa and one of the tested strains of K. Pneumoniae (MIC = 200 μ/ml), while E. faecalis and the other strain of K. Pneumoniae expressed a higher degree of resistance (MIC > 200 μ/ml). This study confirms that essential oil of T. pannonicus possesses remarkable in vitro antimicrobial activity against several medicinally important pathogens. This is attributable to lemon-scented citral, a mixture of geranial and neral, which has well-documented antimicrobial activity against a range of bacteria and fungi.     

Molecular evidence of symbiotic activity between <Emphasis Type=Italic>Symbiodinium</Emphasis> and <Emphasis Type=Italic>Tridacna maxima</Emphasis> larvae

Dinoflagellates in the genus Symbiodinium (zooxanthellae) provide the photosynthesis that sustains the majority of primary production in coral reefs. They occur symbiotically with several phyla, including mollusks such as giant clams (Tridacna spp.). This mutualistic association is obligatory for the giant clams, but the exact point in which this symbiosis is established and the main translocated photosynthate are unknown. In this study, we tracked the expression of specific genes for symbiosis and glycerol synthesis during a time course experiment. Giant clam larvae were raised until 75 h post-fertilization and then infected with cultured isolates of Symbiodinium clade A3. Expression of symbiosis-specific and housekeeping genes was monitored at four time points. The expression of H⁺-ATPase, a symbiosis-specific gene in Symbiodinium, was observed at 24 h after symbiont acquisition by the clam larvae. The expression of an enzyme responsible for glycerol synthesis was also observed. Together, these results show that the symbiotic relationship was already in place 24 h after Symbiodinium acquisition, during veliger larval stage. This is the first report using a molecular symbiosis-specific marker that supports symbiotic activity between Symbiodinium and a metazoan larva of an organism that acquires symbionts horizontally. From the expression of the glycerol-synthesizing gene, it was qualitatively determined that Symbiodinium cells may produce glycerol regardless of whether they are free-living or in symbiosis.     

Chytrid infection reduces thoracic beat and heart rate of <Emphasis Type=Italic>Daphnia</Emphasis><Emphasis Type=Italic>pulicaria</Emphasis>

Zooplankters are hosts to numerous endo- and ectoparasites, some of which have dramatic impacts on their hosts. Epizootics on zooplankton are probably more widespread in lake systems than it is currently known, and few studies have explored the direct and indirect importance of parasitism in aquatic food webs. In addition, our understanding of the sublethal effects of parasitic infections on host organisms and populations is limited. We used a novel electro-chemical based technique to measure in the outflow of the feeding current changes in the beat rate of the thoracic appendages in female Daphnia pulicaria. We observed simultaneously the heart rates and compared chytrid infected animals with uninfected gravid and non-gravid ones. We found in uninfected animals a thoracic beat rate of 3.81 ± 018 Hz and a heart rate of 4.67 ± 0.42 Hz. Gravid daphnids had a 14% lower thoracic beat rate (3.27 ± 0.30 Hz) than non-gravid females while the heart rate did not significantly differ (4.48 ± 0.28 Hz). In contrast, infected animals showed a 22% lower thoracic beat rate (2.96 ± 0.47 Hz) and a 36% lower heart rate (2.98 ± 0.5 Hz) when compared with uninfected non-gravid females. We discuss the ways Daphnia are affected by Polycaryum leave infections on the individual and population level.     

Regulation of the <Emphasis Type=Italic>ALBINO3</Emphasis>-mediated transition to flowering in <Emphasis Type=Italic>Arabidopsis</Emphasis> depends on the expression of <Emphasis Type=Italic>CO</Emphasis> and <Emphasis Type=Italic>GA1</Emphasis>

ALBINO3, a homologue of PPF1 in Arabidopsis, encodes a chloroplast protein, and is essential for chloroplast differentiation. In the present study, ALBINO3(−) transgenic plants exhibited a significant decrease in both the number of rosette leaves at bolting and the days before bolting, suggesting the important roles of ALBINO3 in regulating flowering during non-inductive short-day photoperiods. ALBINO3 mRNA was apparently accumulated in shoot apical meristem and floral meristems around the shoot apical meristem in wild-type plants. ALBINO3 might be predominantly involved in inducing the floral repression pathway by activating the expression of TFL1, and by suppressing the expression of LFY, respectively, in the shoot apical meristem. Moreover, the function of ALBINO3 in regulating flowering transition depended on the expression of CO and GA1, because ALBINO3 might function in the downstream integration of the photoperiod-dependent and the photoperiod-independent pathways. These results suggest that ALBINO3 may have an important integrative function in the flowering process in Arabidopsis.     

<Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type=Italic>in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Transposon display supports transpositional activity of <Emphasis Type=Italic>P</Emphasis> elements in species of the <Emphasis Type=Italic>saltans</Emphasis> group of <Emphasis Type=Italic>Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

Presence of <Emphasis Type=Italic>C. albidus</Emphasis>, <Emphasis Type=Italic>C. laurentii</Emphasis> and <Emphasis Type=Italic>C. uniguttulatus</Emphasis> in Crop and Droppings of Pigeon Lofts (<Emphasis Type=Italic>Columba livia</Emphasis>)

Columba livia is an important reservoir and carrier of Cryptococcus neoformans, Cryptococcus uniguttulatus, Cryptococcus laurentii and Cryptococcus albidus. Upper digestive tract of this species is also known as a habitat for Cryptococcus neoformans. Given the increasing clinical interest of this microorganism, 331 swabs from crop and 174 dropping samples from pigeon lofts in Grand Canary Island have been studied. The obtained results show an extensive presence samples 81 positive (24.47%) of Cryptococcus spp. in analysed crops: 32 (9.66%) for C. neoformans, 24 (7.2%) for C. uniguttulatus, 23 (6.9%) for C. albidus and 2 (0.6%) for C. laurentii. In the same way, Cryptococcus spp was also isolated in 82 (47.13%), dropping samples: C. neoformans in 59 (33.9%), C. uniguttulatus, in 9 (5.17%), C. laurentii in 8 (4.59%) and C. albidus in 6 (3.44%) of the investigated samples, respectively. The cryptococcosis produced by species of cryptococci other than C. neoformans has become more important during the last decade, supporting the study on the role of pigeon in the epidemiology of this disease.