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.     

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.     

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.     

<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.     

<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.     

Constitutive expression of <Emphasis Type=Italic>SlTrxF</Emphasis> increases starch content in transgenic <Emphasis Type=Italic>Arabidopsis</Emphasis>

The plastidic thioredoxin F-type (TrxF) protein plays an important role in plant saccharide metabolism. In this study, a gene encoding the TrxF protein, named SlTrxF, was isolated from tomato. The coding region of SlTrxF was cloned into a binary vector under the control of 35S promoter and then transformed into Arabidopsis thaliana. The transgenic Arabidopsis plants exhibited increased starch accumulation compared to the wild-type (WT). Real-time quantitative PCR analysis showed that constitutive expression of SlTrxF up-regulated the expression of ADP-glucose pyrophosphorylase (AGPase) small subunit (AtAGPase-S1 and AtAGPase-S2), AGPase large subunit (AtAGPase-L1 and AtAGPase-L2) and soluble starch synthase (AtSSS I, AtSSS II, AtSSS III and AtSSS IV) genes involved in starch biosynthesis in the transgenic Arabidopsis plants. Meanwhile, enzymatic analyses showed that the major enzymes (AGPase and SSS) involved in the starch biosynthesis exhibited higher activities in the transgenic plants compared to WT. These results suggest that SlTrxF may improve starch content of Arabidopsis by regulating the expression of the related genes and increasing the activities of the major enzymes involved in starch biosynthesis.     

The expression of analgesic-antitumor peptide (<Emphasis Type=Italic>AGAP</Emphasis>) from Chinese <Emphasis Type=Italic>Buthus</Emphasis><Emphasis Type=Italic>martensii</Emphasis> Karsch in transgenic tobacco and tomato

The present study aimed to obtain analgesic-antitumor peptide (AGAP) gene expression in plants. The analgesic-antitumor peptide (AGAP) gene was from the venom of Buthus martensii Karsch. Previous studies showed that AGAP has both analgesic and antitumor activities, suggesting that AGAP would be useful in clinical situations as an antitumor drug. Given that using a plant as an expression vector has more advantages than prokaryotic expression, we tried to obtain transgenic plants containing AGAP. In the present study, the AGAP gene was cloned into the plasmid pBI121 to obtain the plant expression vector pBI-AGAP. By tri-parental mating and freeze–thaw transformation, pBI-AGAP was transformed into Agrobacterium tumefaciens LBA4404. Tobacco (Nicotiana tabacum) and tomato (Lycopersicom esculentum) were transformed by the method of Agrobacterium-mediated leaf disc transformation. The transformants were then screened to grow and root on media containing kanamycin. Finally, transformations were confirmed by analysis of PCR, RT-PCR and western blotting. The results showed that the AGAP gene was integrated into the genomic DNA of tobacco and tomato and was successfully expressed. Therefore, the present study suggests a potential industrial application of AGAP expressed in plants.     

Constitutive expression of <Emphasis Type=Italic>ZmsHSP</Emphasis> in <Emphasis Type=Italic>Arabidopsis</Emphasis> enhances their cytokinin sensitivity

A small HSP gene, ZmsHSP, was isolated from Zea mays. Sequence analysis revealed that the open reading frame of ZmsHSP was 477 bp and that it encodes a protein composed of 159 amino acid residues with a calculated molecular mass of 18.17 kD and a predicated isoelectric point (pI) of 5.63. ZmsHSP contains a CS domain (p23-like domain) and shares similarity with the HSP90 co-chaperone p23. The expression level of ZmsHSP was different among various tissues with the highest expression in leaves and the lowest in silks. Results also showed that the expression of ZmsHSP in maize was significantly up-regulated by dehydration. Transgenic Arabidopsis plants overexpressing ZmsHSP under the control of the CaMV 35S promoter had lower endogenous cytokinin content and showed more sensitivity to cytokinin during the germination and early seedling stage than wild-type plants, suggesting that ZmsHSP might has a function in cytokinin response in Zea mays.     

Cloning and expression of <Emphasis Type=Italic>Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type=Italic>Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

Maternal care and infant development in <Emphasis Type=Italic>Callimico goeldii</Emphasis> and <Emphasis Type=Italic>Callithrix jacchus</Emphasis>

Callimico goeldii gives birth to single offspring, whereas other callitrichids, including Callithrix jacchus, twin. This study compares maternal effort and infant development in C. goeldii and C. jacchus; it is the first study to look at nursing frequency. Infants were observed from birth for 7 weeks in two captive groups each of C. goeldii and C. jacchus. C. goeldii mothers physiologically invested the same or less than C. jacchus mothers. C. goeldii mothers gained the same amount of weight during pregnancy in absolute terms as did the smaller C. jacchus. This results in a smaller gain in proportion to maternal weight but an equivalent proportional gain on a per fetus basis. C. goeldii mothers nursed their infants less based on duration of nursing bouts compared with C. jacchus mothers. C. goeldii mothers transported their infants exclusively through the first 2 weeks of life, which is longer than C. jacchus mothers, who exclusively transported infants only during the first week of life. As maternal infant carriage declined, other group members transported offspring in both species. C. goeldii infants engaged in independent locomotive sequences later in development and tasted solid foods less frequently than C. jacchus infants when compared at equivalent ages. A single, opportunistic milk sample obtained from a C. goeldii mother when her infant was 48 days old indicates that C. goeldii milk contains gross energy from crude protein within the range of variation observed in Callithrix milk. Despite the similarities in milk quality and prenatal effort in individual fetuses, C. goeldii infants gain weight faster from 0 to 18 months than do C. jacchus infants. A reduction in litter size allows C. goeldii mothers to spend more time carrying their infant and to delay weaning, thereby allowing accelerated infant and juvenile growth rates compared with C. jacchus.