Cloning,Characterization, and Expression Levels of the <Emphasis Type="Italic">Nectin</Emphasis> Gene from the Tube Feet of the Sea Urchin <Emphasis Type="Italic">Paracentrotus Lividus</Emphasis>

Marine bioadhesives perform in ways that manmade products simply cannot match, especially in wet environments. Despite their technological potential, bioadhesive molecular mechanisms are still largely understudied, and sea urchin adhesion is no exception. These animals inhabit wave-swept shores, relying on specialized adhesive organs, tube feet, composed by an adhesive disc and a motile stem. The disc encloses a duo-gland adhesive system, producing adhesive and deadhesive secretions for strong reversible substratum attachment. The disclosure of sea urchin Paracentrotus lividus tube foot disc proteome led to the identification of a secreted adhesion protein, Nectin, never before reported in adult adhesive organs but, that given its adhesive function in eggs/embryos, was pointed out as a putative substratum adhesive protein in adults. To further understand Nectin involvement in sea urchin adhesion, Nectin cDNA was amplified for the first time from P. lividus adhesive organs, showing that not only the known Nectin mRNA, called Nectin-1 (GenBank AJ578435), is expressed in the adults tube feet but also a new mRNA sequence, called Nectin-2 (GenBank KT351732), differing in 15 missense nucleotide substitutions. Nectin genomic DNA was also obtained for the first time, indicating that both Nectin-1 and Nectin-2 derive from a single gene. In addition, expression analysis showed that both Nectins are overexpressed in tube feet discs, its expression being significantly higher in tube feet discs from sea urchins just after collection from the field relative to sea urchin from aquarium. These data further advocate for Nectin involvement in sea urchin reversible adhesion, suggesting that its expression might be regulated according to the hydrodynamic conditions.     

Functional characterization of thioredoxin h type 5 with antimicrobial activity from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A plant protein with effective antifungal activity was isolated from Arabidopsis thaliana leaves. The results of N-terminal sequencing analysis revealed that the isolated protein matches with a partial fragment of A. thaliana oxidoreductase thioredoxin h type 5; AtTrx-h5 (GenBank accession number AT1G45145). The AtTrx-h5 gene isolated from an Arabidopsis leaf cDNA library was transformed into Escherichia coli to investigate the functional characterizations of AtTrx-h5 protein. Recombinant AtTrx-h5 protein inhibited the conidia germination in 7 filamentous fungal cells and the proliferation in 3 pathogenic yeast cells. In addition, the cellular distribution of recombinant AtTrx-h5 protein in Fusarium solani showed pH-dependent cytosolic accumulation. Interestingly, AtTrx-h5 acted as an inhibitor of fungal growth via cellular reactive oxygen species (ROS) generation. We propose the novel functions of AtTrx-h5 as a potential candidate for natural antifungal material in the study.     

Identification of calmodulin binding proteins in the entomopathogenic fungus <Emphasis Type="Italic">Beauveria bassiana</Emphasis>

Calmodulin (CaM) is a primary Ca²⁺ receptor and plays a pivotal role in a variety of cellular responses in eukaryotes. Even though a large number of CaM-binding proteins are well known in yeast, plants, and animals, little is known regarding CaM-targeted proteins in filamentous fungi. To identify CaM-binding proteins in filamentous fungi, we used a proteomics method coupled with co-immunoprecipitation (CoIP) and MALDI-TOF/TOF mass spectrometry (MS) in Beauveria bassiana. Through this method, we identified ten CaM-binding proteins in B. bassiana. One of the CaM-targeted proteins was the heat shock protein 70 (BbHSP70) in B. bassiana. Our biochemical study showed that ATP inhibits the molecular interaction between BbHSP70 and CaM, suggesting a regulatory mechanism between CaM and ATP for regulating BbHSP70.     

Expression of Human FGF18 by Fusion with Oleosin in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Seeds

Expression of recombinant human fibroblast growth factor 18 (hFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, but they are difficult, expensive. The expression of recombinant proteins fused to oleosin protein have distinct advantages, such as safety, ease, low cost. So we have expressed hFGF18 fused to oleosin protein in the oil bodies of Arabidopsis thaliana (A. thaliana) and screen the proliferation effect of NIH3T3 cells. The vector of oleosinhFGF18 fusion gene was constructed and transformed into wild A. thaliana. Transformed A. thaliana lines were obtained by the floral dip method and confirmed using polymerase chain reaction (PCR). The PCR results indicated that the oleosin-hFGF18 fusion gene was integrated into the A. thaliana genome. The oil bodies expression of oleosin-hFGF18 was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting. The biological activity showed that oil bodies expressing oleosin-hFGF18 could stimulate the proliferation of NIH3T3 cells.     

Centrin scaffold in Chlamydomonas reinhardtii revealed by immunoelectron microscopy

In the flagellate green alga Chlamydomonas reinhardtii the Ca(2+)-binding EF-hand protein centrin is encoded by a single-copy gene. Previous studies have localized the protein to four distinct structures in the flagellar apparatus: the nucleus-basal body connector, the distal connecting fiber, the flagellar transitional region, and the axoneme. To explain the disjunctive distribution of centrin, the interaction of centrin with as yet unknown specific centrin-binding proteins has been implied. Here, we demonstrate using serial section postembedding immunoelectron microscopy of isolated cytoskeletons that centrin is located in additional structures (transitional fibers and basal body lumen) and that the centrin-containing structures of the basal apparatus are likely part of a continuous filamentous scaffold that extends from the nucleus to the flagellar bases. In addition, we show that centrin is located in the distal lumen of the basal body in a rotationally asymmetric structure, the V-shaped filament system. This novel centrin-containing structure has also been detected near the distal end of the probasal bodies. Taken together, these results suggest a role for a rotationally asymmetric centrin "seed" in the growth and development of the centrin scaffold following replication of the basal apparatus.     

Cytoskeletal dynamics ofApedinella radians (Pedinellophyceae) II. Cell division and maintenance of cell polarity and symmetry

Summary The dynamics of the cytoskeletal proteins centrin, actin, and tubulin were followed during cell division in the unicellular phytoflagellateApedinella radians (Pedinellophyceae). Three centrin, or centrin-like, components appear to coordinate independent developmental events during cell division. The first component, basal body centrin, maintains a physical link between basal bodies and the anterior nuclear membrane. Basal body centrin divides in two at metaphase, and each portion segregates with two basal bodies at anaphase. As the positioning of basal bodies defines the anterior region of the cell, basal body centrin appears to play a role in maintaining cell polarity throughout the cell cycle. The second centrin component consists of an array of filamentous bundles arranged as a six-pointed star. During cell division, the star undergoes a conformational change resulting in two distinct centrin triangles, one distributed to each daughter cell, suggesting that centrin filamentous bundles are involved in maintaining cell (radial) symmetry. The third centrin component is transient and associates with the spindle poles, emerging prior to mitosis and remaining until late anaphase/early telophase. Spindle pole centrin establishes temporary horizontal bipolarity, thereby establishing the spindle axis. Unlike centrin filamentous bundles, actin filamentous bundles depolymerize prior to mitosis, indicating they do not influence cell symmetry during cell division. Mitosis is described for the first time in a pedinellid and features a closed spindle, the absence of rhizoplasts and a persistent spindle.     

Isolation and functional analysis of apple MdHMGR1 and MdHMGR4 gene promoters in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Cereal grains offer great potential as a storage system for production of highly valuable proteins using biotechnological approaches, but such applications require tight temporal and spatial control of transgene expression. Towards this aim, we have undertaken a detailed analysis of α-kafirin (α-kaf) promoter and α-kaf signal peptide (sp) in transgenic sorghum plants, using green fluorescent protein gene (gfp) as a reporter. Constructs containing either the α-kaf promoter or the constitutive maize ubiquitin-1 (ubi) promoter driving either gfp or sp-gfp translational fusion were introduced into Sorghum bicolor inbred line Tx430 by particle bombardment. We show for the first time that the α-kaf promoter directs endosperm-specific transgene expression, with activity first detected at 10 days post-anthesis (dpa), peaking at 20 dpa, and remaining active through to physiological maturity. Furthermore, we demonstrate for the first time that the α-kafirin sp is sufficient to direct foreign protein to protein bodies in the endosperm. The evidence is also provided for possible mis-targeting by α-kaf sp in vegetative tissues of transgenic lines with ubi-sp-gfp, resulting in loss of reporter gene translational activity that no GFP signal was observed. These results demonstrate that α-kaf promoter and α-kaf sp are well suited for seed bioengineering to produce recombinant proteins in sorghum endosperm or deposit foreign proteins into sorghum protein bodies.     

Living inside a sponge skeleton: the association of a sponge,a macroalga and a diatom

The relationships between sponges and macroalgae have been poorly investigated, especially in temperate waters. This work describes the consortium between the dictyoceratid sponge Dysidea pallescens and the red alga Acrochaetium spongicola permeating spongin fibres in the Mediterranean Sea; moreover, this is the first report of a diatom, Amphora pediculus, living also inside the spongin skeleton. The annual trend of the total algal biomass did not vary over time in relation to the temperature, irradiance and incorporation of foreign bodies. Our analyses, conducted by light and electron microscopy, suggest that both the macroalga and the diatom invade the skeleton of the sponge from the external environment, and that the benthic diatom, epiphyte of the macroalga, is passively carried inside the fibres through the growth of Acrochaetium spongicola. All the examined samples of D. pallescens showed that the macroalga permeated at least some fibres, while the presence of the diatom was occasional. The superficial layer of the sponge, thin and reticulate, likely allows the passage of the light and ensures the algal survival inside the sponge tissue. The high occurrence of the association with A. spongicola, together with the morphological adaptations of the sponge favouring the algal life, suggest that the relationship is mutualistic. The possible benefits for the involved partners are hypothesized. The taxonomy and ecology of endozoic Acrochaetiales are controversial due to the reduced size of thalli, the absence of peculiar diagnostic characters and unknown reproductive structures. Therefore, detailed studies on the relationships between the algae and their hosts will provide helpful information for the algal identification.     

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon <Emphasis Type="Italic">Tyr-Cre</Emphasis>-Mediated Deletion of the Autophagy Gene <Emphasis Type="Italic">Atg7</Emphasis>

Defects in autophagy and the resulting deposition of protein aggregates have been implicated in aging and neurodegenerative diseases. While gene targeting in the mouse has facilitated the characterization of these processes in different types of neurons, potential roles of autophagy and accumulation of protein substrates in neuroepithelial cells have remained elusive. Here we report that Atg7^f/f Tyr-Cre mice, in which autophagy-related 7 (Atg7) is conditionally deleted under the control of the tyrosinase promoter, are a model for accumulations of the autophagy adapter and substrate sequestosome-1/p62 in both neuronal and neuroepithelial cells. In the brain of Atg7^f/f Tyr-Cre but not of fully autophagy competent control mice, p62 aggregates were present in sporadic neurons in the cortex and other brain regions as well in epithelial cells of the choroid plexus and the ependyma. Western blot analysis confirmed a dramatic increase of p62 abundance and formation of high-molecular weight species of p62 in the brain of Atg7^f/f Tyr-Cre mice relative to Atg7^f/f controls. Immuno-electron microscopy showed that p62 formed filamentous aggregates in neurons and ependymal cells. p62 aggregates were also highly abundant in the ciliary body in the eye. Atg7^f/f Tyr-Cre mice reached an age of more than 2 years although neurological defects manifesting in abnormal hindlimb clasping reflexes were evident in old mice. These results show that p62 filaments form in response to impaired autophagy in vivo and suggest that Atg7^f/f Tyr-Cre mice are a model useful to study the long-term effects of autophagy deficiency on the homeostasis of different neuroectoderm-derived cells.     

Potency of the mosquitocidal Cry46Ab toxin produced using a 4AaCter-tag,which facilitates formation of protein inclusion bodies in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A Cry46Ab toxin derived from Bacillus thuringiensis strain TK-E6 shows mosquitocidal activity against Culex pipiens pallens Coquillett (Diptera: Culicidae) larvae as well as preferential cytotoxicity against human cancer cells. In B. thuringiensis cells, Cry46Ab is produced and accumulates as a protein crystal that is processed into the active 29-kDa toxin upon solubilization in the alkaline environment of the insect midgut. The Cry46Ab protoxin is 30 kDa, and is therefore thought to require an accessory protein such as P20 and/or ORF2 for efficient crystal formation. In the present study, the potency of the 4AaCter-tag was investigated for the production of alkali-soluble inclusion bodies of recombinant Cry46Ab in Escherichia coli. The 4AaCter-tag is a polypeptide derived from the C-terminal region of the B. thuringiensis Cry4Aa toxin and facilitates the formation of alkali-soluble protein inclusion bodies in E. coli. Fusion with the 4AaCter-tag enhanced both Cry46Ab production and the formation of Cry46Ab inclusion bodies. In addition, upon optimization of protein expression procedures, the Cry46Ab–4AaCter inclusion bodies showed mosquitocidal activity and stability in aqueous environments comparable to Cry46Ab without the 4AaCter-tag. Our study suggests that use of the 4AaCter-tag is a straightforward approach for preparing formulations of smaller-sized Cry toxins such as Cry46Ab in E. coli.     

The Role of <Emphasis Type="Italic">sho1</Emphasis> in Polarized Growth of <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis>

Aspergillus fumigatus is an opportunistic pathogen that may cause severe invasive disease in immunocompromised patients. The filamentous fungi undergo polarized growth, searching for nutrients in the environment and causing invasive growth in tissue. Sho1 is a sensor of the high osmolarity glycerol pathway, and the sho1 mutant showed a decrease in growth rate. We found that sho1 is involved in the polarized growth of A. fumigatus. The sho1 mutation resulted in extended isotropic growth of germinating conidia followed by multiple germ tubes and wide hyphae with short intercalary cells by calcofluor white staining. The mechanism by which sho1 gene affected polarized growth is investigated. A reduced number of apical vesicles with greater dispersion were observed by transmission electron microscopy in the Spitzenkörper body of the sho1 mutant. Actin patches were distributed randomly at low density at early stages of mutant strain fungal development and reaggregated to the hyphal tip of later stages when long filamentous fungi formed. Actin patches located at the tip of polarized wild-type cells. RNA levels of polarized growth-related genes Rho GTPases were detected by real-time PCR. The sho1 gene did not affect the RNA expression when strains were cultured at 37°C for 6 h. At 17 h, the RNA expression of rho1, rho3 and CDC42 in the sho1 mutant were 0.18-, 0.18- and 0.33-fold of that in the wild type. The sho1 gene affected the polarized growth through affecting the expression of Rho GTPases, the distribution of actin cytoskeleton, vesicle quantity and distribution.     

On the multicomponent nature of <Emphasis Type="Italic">Halobacterium salinarum</Emphasis> flagella

Filaments of the flagellum of the halophilic archaeon Halobacterium salinarum consist of five flagellins: A1, A2, B1, B2, and B3, which are encoded by five genes localized in tandem in two flgA and flgB operons. While the role of flagellins A1 and A2 has been determined, the role of the proteins, B operon products, is still unclear. A mutant strain of H. salinarum with deleted A and B flagellin genes (ΔflgAΔflgB) has been obtained for the first time. This strain has been used to create and analyze the strains carrying only individual B1 or B3 flagellin genes. Cells of the ΔflgAΔflgB strain were shown to have short filamentous formations, 7–8 nm thick, which we have named as X-filaments. It has been shown that X-filaments consist of a protein immunologically related to flagellins A and B. Expression of the B1 and B3 genes is suppressed in the absence of A1, A2, and B2. It has been shown that flagellins B1 and B3 cannot be substituted for flagellin B2 upon the formation of a curved hook-like structure, which serves as a connecting element between the flagellar filament and the motor axis. The multicomponent nature of flagella is discussed in the light of their possible involvement in other cell processes besides providing motility.     

The promoter of filamentation (POF1) protein from <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> is an ATPase involved in the protein quality control process

Background

The gene YCL047C, which has been renamed promoter of filamentation gene (POF1), has recently been described as a cell component involved in yeast filamentous growth. The objective of this work is to understand the molecular and biological function of this gene.

Results

Here, we report that the protein encoded by the POF1 gene, Pof1p, is an ATPase that may be part of the Saccharomyces cerevisiae protein quality control pathway. According to the results, Δpof1 cells showed increased sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, heat shock and protein unfolding agents, such as dithiothreitol and tunicamycin. Besides, the overexpression of POF1 suppressed the sensitivity of Δpct1, a strain that lacks a gene that encodes a phosphocholine cytidylyltransferase, to heat shock. In vitro analysis showed, however, that the purified Pof1p enzyme had no cytidylyltransferase activity but does have ATPase activity, with catalytic efficiency comparable to other ATPases involved in endoplasmic reticulum-associated degradation of proteins (ERAD). Supporting these findings, co-immunoprecipitation experiments showed a physical interaction between Pof1p and Ubc7p (an ubiquitin conjugating enzyme) in vivo.

Conclusions

Taken together, the results strongly suggest that the biological function of Pof1p is related to the regulation of protein degradation.

     

Mutations in <Emphasis Type="Italic">CDC14</Emphasis> result in high sensitivity to cyclin gene dosage in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14.     

<Emphasis Type="Italic">Saccharothrix tharensis</Emphasis> sp. nov., an actinobacterium isolated from the Thar Desert,India

The taxonomic provenance of a filamentous actinobacterial strain isolated from a desert soil was established using a polyphasic approach. The strain has chemotaxonomic and morphological properties consistent with its classification in the genus Saccharothrix. It forms a distinct branch in the Saccharothrix 16S rRNA gene tree, related to the type strain of Saccharothrix saharensis (96.7%) but was distinguished readily from it using a combination of phenotypic properties. The genotypic and phenotypic data show that the strain represents a novel species in the genus Saccharothrix, for which the name Saccharothrix tharensis sp. nov. is proposed with the type strain TD-093^T (=?KCTC 39724^T?=?MCC 2832^T).     

Comprehensive analysis of fungal diversity and enzyme activity in <Emphasis Type="Italic">nuruk</Emphasis>, a Korean fermenting starter,for acquiring useful fungi

Nuruk is a fermenting starter that is involved in the production of alcoholic beverages, and has been used in South Korea for a very long time. To analyze the fungal diversity, we collected a total of 59 nuruk samples from several companies and persons in 2013 to 2014, and obtained 364 isolates. All of the single isolated fungi were identified, both morphologically and molecularly, based on the sequences of ribosomal RNA gene [18S, ITS1-5.8S-ITS2, and 26S (D1/D2 region)]. In 46 nuruk samples out of 59 (78%), Saccharomycopsis fibuligera, a dimorphic yeast, was most frequently isolated. Among the filamentous fungi, Aspergillus and Lichtheimia were found in more than 50% of the samples with lower colony forming unit (CFU/g of sample) than those of yeasts. The yeasts S. fibuligera and Wickerhamomyces anomalus were counted with maximum 1.3–1.8 × 10⁸ CFU/g. Among Mucorales fungi, Lichtheimia and Mucor were isolated in much higher numbers than Rhizopus and Rhizomucor. Overall, the home-made nuruks tend to contain more diverse filamentous fungi than the commercial nuruks. To acquire industrially useful filamentous fungi and yeasts, we analyzed the enzyme activities of α-amylase, glucoamylase and acid protease associated with brewing properties for 131 strains. Aspergillus oryzae and S. fibuligera had high α- and glucoamylase activities and most isolates of Lichtheimia ramosa had high acid protease activity. For further applications, 27 fungal strains were chosen based on isolation frequencies from nuruk, and the ability to produce useful enzyme.     

Phylogenetic relationships of sculpin <Emphasis Type="Italic">Cottus volki</Emphasis> Taranetz, 1933 (Scorpaeniformes,Cottidae) according to the results of analysis of control region in mitochondrial DNA

Sequencing of the fragment of control region in mitochondrial DNA in sculpin Cottus volki and the comparison of obtained data with homologous nucleotide sequences in the other species from genus Cottus demonstrated that C. volki occupies the basal position in the “poecilopus” group, which includes also typical sculpin C. poecilopus Heckel, 1837 from water bodies of Europe, sculpin C. szanaga Dybowski, 1869 from the Amur River basin, and sculpin Cottus cf. poecilopus from the Lena River basin. Early separation of C. volki line from common trunk of “poecilopus” group explains the presence in C. volki of several primitive (plesiomorphic for this group) morphological characters: long interior ray of ventral fin, practically complete lateral body line, and well-developed dentition of palatine bones.