Purification and antibacterial mechanism of fish-borne bacteriocin and its application in shrimp (<Emphasis Type="Italic">Penaeus vannamei</Emphasis>) for inhibiting <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus: is recognized as the main cause of gastroenteritis associated with consumption of seafood. Bacteriocin-producing Lactobacillus plantarum FGC-12 isolated from golden carp intestine had strong antibacterial activity toward V. parahaemolyticus. The fish-borne bacteriocin was purified by a three-step procedure consisting of ethyl acetate extraction, gel filtration chromatography and high performance liquid chromatography. Its molecular weight was estimated at 4.1 kDa using SDS-PAGE. The fish-borne bacteriocin reached the maximum production at stationary phase after 20 h. It was heat-stable (30 min at 121?°C) and remained active at pH range from 3.0 to 5.5, but was sensitive to nutrasin, papain and pepsin. Its minimum inhibitory concentration for V. parahaemolyticus was 6.0 mg/ml. Scanning electron microscopy analysis showed that the fish-borne bacteriocin disrupted cell wall of V. parahaemolyticus. The antibacterial mechanism of the fish-borne bacteriocin against V. parahaemolyticus might be described as action on membrane integrity in terms of the leakage of electrolytes, the losses of Na⁺K⁺-ATPase, AKP and proteins. The addition of the fish-borne bacteriocin to shrimps leaded V. parahaemolyticus to reduce 1.3 log units at 4?°C storage for 6 day. Moreover, a marked decline in total volatile base nitrogen and total viable counts was observed in bacteriocin treated samples than the control. It is clear that this fish-borne bacteriocin has promising potential as biopreservation for the control of V. parahaemolyticus in aquatic products.     

Detection and Quantification of <Emphasis Type="Italic">Vibrio cholerae,Vibrio parahaemolyticus</Emphasis>, <Emphasis Type="Italic">and Vibrio vulnificus</Emphasis> in Coastal Waters of Guinea-Bissau (West Africa)

V. cholerae, V. parahaemolyticus, and V. vulnificus are recognized human pathogens. Although several studies are available worldwide, both on environmental and clinical contexts, little is known about the ecology of these vibrios in African coastal waters. In this study, their co-occurrence and relationships to key environmental constraints in the coastal waters of Guinea-Bissau were examined using the most probable number-polymerase chain reaction (MPN-PCR) approach. All Vibrio species were universally detected showing higher concentrations by the end of the wet season. The abundance of V. cholerae (ISR 16S-23S rRNA) ranged 0–1.2 × 10⁴ MPN/L, whereas V. parahaemolyticus (toxR) varied from 47.9 to 1.2 × 10⁵ MPN/L. Although the presence of genotypes associated with virulence was found in environmental V. cholerae isolates, ctxA+ V. cholerae was detected, by MPN-PCR, only on two occasions. Enteropathogenic (tdh+ and trh+) V. parahaemolyticus were detected at concentrations up to 1.2 × 10³ MPN/L. V. vulnificus (vvhA) was detected simultaneously in all surveyed sites only at the end of the wet season, with maximum concentrations of 1.2 × 10⁵ MPN/L. Our results suggest that sea surface water temperature and salinity were the major environmental controls to all Vibrio species. This study represents the first detection and quantification of co-occurring Vibrio species in West African coastal waters, highlighting the potential health risk associated with the persistence of human pathogenic Vibrio species.     

The extracellular proteases produced by <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis>

Vibrio parahaemolyticus, a Gram-negative bacterium, inhabits marine and estuarine environments and it is a major pathogen responsible globally for most cases of seafood-associated gastroenteritis in humans and acute hepatopancreatic necrosis syndrome in shrimps. There has been a dramatic worldwide increase in V. parahaemolyticus infections over the last two decades. The pathogenicity of V. parahaemolyticus has been linked to the expression of different kinds of virulence factors including extracellular proteases, such as metalloproteases and serine proteases. V. parahaemolyticus expresses the metalloproteases; PrtV, VppC, VPM and the serine proteases; VPP1/Protease A, VpSP37, PrtA. Extracellular proteases have been identified as potential virulence factors which directly digest many kinds of host proteins or indirectly are involved in the processing of other toxic protein factors. This review summarizes findings on the metalloproteases and serine proteases produced by V. parahaemolyticus and their roles in infections. Identifying the role of V. parahaemolyticus virulence-associated extracellular proteases deepens our understanding of diseases caused by this bacterium.     

Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

Electron paramagnetic resonance and Mössbauer spectroscopy of intact mitochondria from respiring <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Mitochondria from respiring cells were isolated under anaerobic conditions. Microscopic images were largely devoid of contaminants, and samples consumed O₂ in an NADH-dependent manner. Protein and metal concentrations of packed mitochondria were determined, as was the percentage of external void volume. Samples were similarly packed into electron paramagnetic resonance tubes, either in the as-isolated state or after exposure to various reagents. Analyses revealed two signals originating from species that could be removed by chelation, including rhombic Fe³⁺ (g = 4.3) and aqueous Mn²⁺ ions (g = 2.00 with Mn-based hyperfine). Three S = 5/2 signals from Fe³⁺ hemes were observed, probably arising from cytochrome c peroxidase and the a₃:Cu_b site of cytochrome c oxidase. Three Fe/S-based signals were observed, with averaged g values of 1.94, 1.90 and 2.01. These probably arise, respectively, from the [Fe₂S₂]⁺ cluster of succinate dehydrogenase, the [Fe₂S₂]⁺ cluster of the Rieske protein of cytochrome bc ₁, and the [Fe₃S₄]⁺ cluster of aconitase, homoaconitase or succinate dehydrogenase. Also observed was a low-intensity isotropic g = 2.00 signal arising from organic-based radicals, and a broad signal with g _ave = 2.02. Mössbauer spectra of intact mitochondria were dominated by signals from Fe₄S₄ clusters (60–85% of Fe). The major feature in as-isolated samples, and in samples treated with ethylenebis(oxyethylenenitrilo)tetraacetic acid, dithionite or O₂, was a quadrupole doublet with ΔE _Q = 1.15 mm/s and δ = 0.45 mm/s, assigned to [Fe₄S₄]²⁺ clusters. Substantial high-spin non-heme Fe²⁺ (up to 20%) and Fe³⁺ (up to 15%) species were observed. The distribution of Fe was qualitatively similar to that suggested by the mitochondrial proteome.     

Cloning and Functional Analysis of <Emphasis Type="Italic">SaCLCc1</Emphasis>, a Gene Belonging to the Chloride Channel Family (<Emphasis Type="Italic">CLC</Emphasis>), from the Halophyte <Emphasis Type="Italic">Suaeda altissima</Emphasis> (L.) Pall.

One of the genes of the CLC (Chloride Channel) family, SaCLCc1, from the halophyte Suaeda altissima (L.) Pall. was cloned. To investigate the function of SaCLCc1, it was expressed in the S. cerevisiae deletion mutant Δgef1::LEU2 for the only gene of the CLC family in this organism. The growth of the transformed SaCLCc1-expressing mutant Δgef1 was restored when cells were grown in Fe²⁺-deficient YPEG medium, in minimal synthetic media SD and SR (pH 7.0), and in rich YPD medium containing Mn²⁺. The complementation of the Δgef1 mutant phenotype with the SaClCc1 gene indicates the involvement of the SaClCc1 protein in the transport of Cl^– ions.     

Search for genes influencing the maintenance of the [<Emphasis Type="Italic">ISP</Emphasis><Superscript>+</Superscript>] prion-like antisuppressor determinant in yeast with the use of an insertion gene library

The prion-like determinant [ISP ⁺] manifests itself as an antisuppressor of certain sup35 mutations. To establish that [ISP ⁺] is indeed a new yeast prion, it is necessary to identify the gene that codes for the protein whose prion form is [ISP ⁺]. Analysis of the transformants obtained by transformation of an [ISP ⁺] strain with an insertion gene library revealed three genes controlling the [ISP ⁺] maintenance: UPF1, UPF2, and SFP1. SFP1 codes for a potentially prionogenic protein, which is enriched in Asn and Gln residues, and is thereby the most likely candidate for the [ISP ⁺] structural gene. UPF1 and UPF2 code for components of nonsense-mediated mRNA decay. The [ISP ⁺] elimination caused by UPF1 and UPF2 inactivation was reversible, and Upf1p and Upf2p were not functionally related to phosphatase Ppz1p, which influences the [ISP ⁺] manifestation. Possible mechanisms sustaining the influence of UPF1 and UPF2 on [ISP ⁺] maintenance are discussed.     

SacAcuA/SacSrtN system modulates the metabolism by controlling the special proteins in <Emphasis Type="Italic">Saccharopolyspora erythraea</Emphasis>

N^?-lysine acetylation is a dynamic, reversible, regulatory post-translational modification in prokaryotes and eukaryotes that modulates a variety of protein functions. As known, acetylation is introduced to lysine mainly through two ways in vivo: nonenzymatic acetylation and acetyltransferase/deacetylase system. Herein, we select the Gcn5-like protein acetyltransferase SacAcuA (encoded by SACE_5148) and the sirtuin-type NAD⁺-dependent deacetylase SacSrtN (encoded by SACE_3798) as the researching objects. By comparison of ΔSACE_3798 and ΔSACE_5148 to wild type of Saccharopolyspora erythraea, the growth and the synthesis of secondary metabolites were affected by SacAcuA/SacSrtN system. Moreover, 96 proteins were classified into three aspects of cellular components, molecular function, and biological processes. These findings suggest that the acetyltransferase/deacetyltransferase system could not only catalyze the acetylation/deacetylation of special proteins but also affect the protein level to modulate the primary and secondary metabolism in S. erythraea.     

The <Emphasis Type="Italic">alm1</Emphasis><Superscript><Emphasis Type="Italic">+</Emphasis></Superscript> gene from <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> encodes a coiled-coil protein that associates with the medial region during mitosis

We have isolated a cDNA that encodes a 142-kDa protein by immunoscreening of a Schizosaccharomyces pombe expression library with a new antibody, mAb8, that reveals spindle poles and equatorial ring-like structures in several organisms. This cDNA encodes a putative protein which we termed Alm (for abnormal long morphology). The protein is predicted to be a coiled-coil protein, containing a central α-helical domain flanked by non-helical terminal domains. Immunofluorescence analysis showed that Alm1 is localized in the medial region of the cell from anaphase to the end of cytokinesis. Cells carrying an alm1::ura4 ⁺ disruption are viable and exhibit an elongated morphology. Homozygous alm1::ura4 ⁺ diploids sporulated normally but the spores did not germinate. Spores that have inherited the disruption allele from a heterozygous alm1 ⁺ / alm1::ura4 ⁺ diploid germinated but generated smaller colonies. We propose that Alm1 participates in the structural organization of the medial region in S. pombe.     

Rnq1 protein protects [<Emphasis Type="Italic">PSI</Emphasis><Superscript>+</Superscript>] prion from effect of the <Emphasis Type="Italic">PNM</Emphasis> mutation

The interaction of [PSI ⁺] and [PIN ⁺] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [PIN ⁺] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [PSI ⁺] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [PSI ⁺] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [PSI ⁺].     

Salt oversensitivity derived from mutation breeding improves salinity tolerance in barley <Emphasis Type="Italic">via</Emphasis> ion homeostasis

Salt stress imposes a major environmental threat to agriculture, therefore, understanding the basic physiology and genetics of cell under salt stress is crucial for developing any breeding strategy. In the present study, the expression profile of genes involved in ion homeostasis including salt overly sensitive (HvSOS1, HvSOS2, HvSOS3), vacuolar Na⁺/H⁺ antiporter (HvNHX1), and H⁺-ATPase (HVA) along with ion content measurement were investigated in two genotypes of Hordeum vulgare under 300 mM NaCl. The gene expressions were measured in the roots and shoots of a salt-tolerant mutant genotype M4-73-30 and in its wild-type cv. Zarjou by real-time qPCR technique. The critical differences between the salt-tolerant mutant and its wild-type were observed in the expressions of HvSOS1 (105-fold), HvSOS2 (24-fold), HvSOS3 (31-fold), and HVA (202-fold) genes in roots after 6-h exposure to NaCl. The parallel early up-regulation of these genes in root samples of the salt-tolerant mutant genotype indicated induction of Na⁺/H⁺ antiporters activity and Na+ exclusion into apoplast and vacuole. The earlier up-regulation of HvSOS1, HVA, and HvNHX1 genes in shoot of the wild-type genotype corresponded to the relative accumulation of Na⁺ which was not observed in salt-tolerant mutant genotype because of efficient inhibitory role of the root in Na+ transport to the shoot. In conclusion, the lack of similarity in gene expression patterns between the two genotypes with similar genetic background may confirm the hypothesis that mutation breeding could change the ability of salt-tolerant mutant genotype for efficient ion homeostasis via salinity oversensitivity response.     

Banding and FISH in three species of <Emphasis Type="Italic">Vernonia</Emphasis>, subsection <Emphasis Type="Italic">Macrocephalae</Emphasis> (Asteraceae,Vernonieae)

To complement our knowledge about the karyotypes of the genus Vernonia Schreb., different techniques of chromosome banding, including AgNOR, triple staining with fluorochromes CMA/DA/DAPI (CDD), and fluorescence in situ hybridization (FISH) for the 45S rDNA probe, were applied to three species of subsection Macrocephalae. Vernonia bardanoides was collected from an area of cerrado (savanna) vegetation in Itirapina, São Paulo State, Brazil, and V. linearifolia and V. tomentella were collected from areas of rocky, open altitudinal vegetation in Joaquim Felicio and Diamantina, respectively, in Minas Gerais State. All species showed two terminal CMA⁺ and NOR bands. FISH indicated two terminal 45S rDNA sites in V. linearifolia and V. tomentella, and six in V. bardanoides.     

Structural organization characteristics of the <Emphasis Type="Italic">DIP1</Emphasis> gene in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> strains mutant for the <Emphasis Type="Italic">flamenco</Emphasis> gene

Molecular cloning of the DIP1 gene located in the 20A4-5 region has been performed from the following strains with the flamenco phenotype: flam ^SS (SS) and flam ^MS (MS) characterized by a high transposition rate of retrotransposon gypsy (mdg4), flam ^{py + (P)} carrying the insertion of a construction based on the P element into the region of the flamenco gene, and flamenco ⁺. The results of restriction analysis and sequencing cloned DNA fragments has shown that strains flam ^SS , flam ^MS considerably differ from flam ^{py + (P)}, and flamenco ⁺ in the structure of DIP1. Strains flam ^SS and flam ^MS have no DraI restriction site at position 1765 in the coding region of the gene, specifically, in the domain determining the signal of the nuclear localization of the DIP1 protein. This mutation has been found to consist in a nucleotide substitution in the recognition site of DraI restriction endonuclease, which is transformed from TTTAAA into TTTAAG and, hence, is not recognized by the enzyme. This substitution changes codon AAA into AAG and is translationally insignificant, because both triplets encode the same amino acid, lysine. The DIP1 gene of strains flam ^SS and flam ^MS has been found to contain a 182-bp insertion denoted IdSS (insertion in DIP1 strain SS); it is located in the second intron of the gene. The IdSS sequence is part of the open reading frame encoding the putative transposase of the mobile genetic element HB1 belonging to the Tc1/mariner family. This insertion is presumed to disturb the conformations of DNA and the chromosome, in particular, by forming loops, which alters the expression of DIP1 and, probably, neighboring genes. In strains flamenco ⁺ and flam ^{py + (P)}, the IdSS insertion within the HB1 sequence is deleted. The deletion encompasses five C-terminal amino acid residues of the conserved domain and the entire C-terminal region of the putative HB1 transposase. The obtained data suggest that DIP1 is involved in the control of gypsy transpositions either directly or through interaction with other elements of the genome.     

TPEN,a Specific Zn<Superscript>2+</Superscript> Chelator,Inhibits Sodium Dithionite and Glucose Deprivation (SDGD)-Induced Neuronal Death by Modulating Apoptosis,Glutamate Signaling,and Voltage-Gated K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> Channels

Hypoxia–ischemia-induced neuronal death is an important pathophysiological process that accompanies ischemic stroke and represents a major challenge in preventing ischemic stroke. To elucidate factors related to and a potential preventative mechanism of hypoxia–ischemia-induced neuronal death, primary neurons were exposed to sodium dithionite and glucose deprivation (SDGD) to mimic hypoxic–ischemic conditions. The effects of N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), a specific Zn²⁺-chelating agent, on SDGD-induced neuronal death, glutamate signaling (including the free glutamate concentration and expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor (GluR2) and N-methyl-d-aspartate (NMDA) receptor subunits (NR2B), and voltage-dependent K⁺ and Na⁺ channel currents were also investigated. Our results demonstrated that TPEN significantly suppressed increases in cell death, apoptosis, neuronal glutamate release into the culture medium, NR2B protein expression, and I _K as well as decreased GluR2 protein expression and Na⁺ channel activity in primary cultured neurons exposed to SDGD. These results suggest that TPEN could inhibit SDGD-induced neuronal death by modulating apoptosis, glutamate signaling (via ligand-gated channels such as AMPA and NMDA receptors), and voltage-gated K⁺ and Na⁺ channels in neurons. Hence, Zn²⁺ chelation might be a promising approach for counteracting the neuronal loss caused by transient global ischemia. Moreover, TPEN could represent a potential cell-targeted therapy.     

Cloning,purification and characterization of a cellulase-free xylanase from <Emphasis Type="Italic">Geobacillus thermodenitrificans</Emphasis> AK53

Geobacillus thermodenitrificans AK53 xyl gene encoding xylanase was isolated, cloned and expressed in Escherichia coli. After purifying recombinant xylanase from G. thermodenitrificans AK53 (GthAK53Xyl) to homogeneity by ammonium sulfate precipitation and ion exchange chromatography, biochemical properties of the enzyme were determined. The kinetic studies for GthAK53Xyl showed K_M value to be 4.34 mg/mL (for D-xylose) and V_max value to be 2028.9 μmoles mg^–1 min^–1. The optimal temperature and pH for enzyme activity were found out to be 70°C and 5.0, respectively. The expressed protein showed the highest sequence similarity with the xylanases of G. thermodenitrificans JK1 (JN209933) and G. thermodenitrificans T-2 (EU599644). Metal cations Mg²⁺ and Mn²⁺ were found to be required for the enzyme activity, however, Co²⁺, Hg²⁺, Fe²⁺ and Cu²⁺ ions caused inhibitor effect on it. GthAK53Xyl had no cellulolytic activity and degraded xylan in an endo-fashion. The action of the enzyme on xylan from oat spelt produced xylobiose and xylopentose. The reported results are suggestive of a xylanase exhibiting desirable kinetics, stability parameters and metal resistance required for the efficient production of xylobiose at industrial scale.