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.     

<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.     

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.     

<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.     

Remobilization of <Emphasis Type=Italic>Tol2</Emphasis> transposons in <Emphasis Type=Italic>Xenopus tropicalis</Emphasis>

Background  

The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency.     

Control of pyrimidine nucleotide formation in <Emphasis Type=Italic>Pseudomonas</Emphasis><Emphasis Type=Italic>fulva</Emphasis>

Control of pyrimidine formation was examined in Pseudomonas fulva ATCC 31418. Pyrimidine supplementation lowered pyrimidine biosynthetic pathway enzyme activities in cells grown on glucose or succinate as a carbon source indicating possible repression of enzyme synthesis. Pyrimidine limitation experiments were conducted using an orotidine 5′-monophosphate decarboxylase mutant strain isolated in this study. Compared to uracil-supplemented, glucose-grown mutant cells, pyrimidine limitation of this strain caused aspartate transcarbamoylase, dihydroorotase, dihydroorotate dehydrogenase and orotate phosphoribosyltransferase activities to increase about 6-, 13-, 3-, 15-fold, respectively, which confirmed regulation of enzyme synthesis by pyrimidines. At the level of enzyme activity, transcarbamoylase activity in Ps. fulva was strongly inhibited by pyrophosphate, CTP, GTP and GDP under saturating substrate concentrations.     

High-level expression of <Emphasis Type=Italic>Camelid</Emphasis> nanobodies in <Emphasis Type=Italic>Nicotiana benthamiana</Emphasis>

Nanobodies (or VHHs) are single-domain antigen-binding fragments derived from Camelid heavy chain-only antibodies. Their small size, monomeric behaviour, high stability and solubility, and ability to bind epitopes not accessible to conventional antibodies make them especially suitable for many therapeutic and biotechnological applications. Here we describe high-level expression, in Nicotiana benthamiana, of three versions of an anti-hen egg white lysozyme (HEWL) nanobody which include the original VHH from an immunized library (cAbLys3), a codon-optimized derivative, and a codon-optimized hybrid nanobody comprising the CDRs of cAbLys3 grafted onto an alternative ‘universal’ nanobody framework. His6- and StrepII-tagged derivatives of each nanobody were targeted for accumulation in the cytoplasm, chloroplast and apoplast using different pre-sequences. When targeted to the apoplast, intact functional nanobodies accumulated at an exceptionally high level (up to 30% total leaf protein), demonstrating the great potential of plants as a nanobody production system.     

<Emphasis Type=Italic>Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type=Italic>Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

<Emphasis Type=Italic>IXR1</Emphasis> and <Emphasis Type=Italic>HMO1</Emphasis> genes jointly control the level of spontaneous mutagenesis in yeast <Emphasis Type=Italic>Saccharomyces cerevisiae</Emphasis>

The yeast genes IXR1 and HMO1 encode proteins belonging to the family of chromatin nonhistone proteins, which are able to recognize and bind to irregular DNA structures. The full deletion of gene IXR1 leads to an increase in cell resistance to the lethal action of UV light, γ-rays, and MMS, increases spontaneous mutagenesis and significantlly decreases the level of UV-induced mutations. It was earlier demonstrated in our works that the hmo1 mutation renders cells sensitive to the lethal action of cisplatin and virtually does not affect the sensitivity to UV light. Characteristically, the rates of spontaneous and UV-induced mutagenesis in the mutant are increased. Epistatic analysis of the double mutation hmo1 ixr1 demonstrated that the interaction of these genes in relation to the lethal effect of cisplatin and UV light, as well as UV-induced mutagenesis, is additive. This suggests that the products of genes HMO1 and IXR1 participate in different repair pathways. The ixr1 mutation significantly increases the rate of spontaneous mutagenesis mediated by replication errors, whereas mutation hmo1 increases the rate of repair mutagenesis. In wild-type cells, the level of spontaneous mutagenesis was nearly one order of magnitude lower than that obtained in cells of the double mutant. Consequently, the combined activity of the Hmo1 and the Ixr1 proteins provides efficient correction of both repair and replication errors.