The phosphofructokinase-B (MJ0406) from <Emphasis Type="Italic">Methanocaldococcus jannaschii</Emphasis> represents a nucleoside kinase with a broad substrate specificity

Recently, unusual non-regulated ATP-dependent 6-phosphofructokinases (PFK) that belong to the PFK-B family have been described for the hyperthermophilic archaea Desulfurococcus amylolyticus and Aeropyrum pernix. Putative homologues were found in genomes of several archaea including the hyperthermophilic archaeon Methanocaldococcus jannaschii. In this organism, open reading frame MJ0406 had been annotated as a PFK-B sugar kinase. The gene encoding MJ0406 was cloned and functionally expressed in Escherichia coli. The purified recombinant enzyme is a homodimer with an apparent molecular mass of 68 kDa composed of 34 kDa subunits. With a temperature optimum of 85°C and a melting temperature of 90°C, the M. jannaschii nucleotide kinase represents one of the most thermoactive and thermostable members of the PFK-B family described so far. The recombinant enzyme was characterized as a functional nucleoside kinase rather than a 6-PFK. Inosine, guanosine, and cytidine were the most effective phosphoryl acceptors. Besides, adenosine, thymidine, uridin and xanthosine were less efficient. Extremely low activity was found with fructose-6-phosphate. Further, the substrate specificity of closely related PFK-Bs from D. amylolyticus and A. pernix were reanalysed.     

Characterization of terminal deoxynucleotidyl transferase and polymerase μ in zebrafish

Terminal deoxynucleotidyl transferase (TdT) contributes to the junctional diversity of immunoglobulin and T-cell receptors by incorporating nucleotides in a template-independent manner. A closely related enzyme, polymerase μ (polμ), a template-directed polymerase, plays a role in general end-joining double-strand break repair. We cloned zebrafish TdT and polμ and found them to be 43% identical in amino acid sequence. Comparisons with sequences of other species revealed conserved residues typical for TdT in the zebrafish sequence that support the template independence of this enzyme. Some but not all of these features were identified in zebrafish polμ. In adult fish, TdT expression was most prominent in thymus, pro- and mesonephros, the primary lymphoid organs in teleost fish and in spleen, intestine, and the tissue around the intestine. Polμ expression was detected not only in pro- and mesonephros, the major sites for B-lymphocyte development, but also in ovary and testis and in all tissue preparations to a low extent. TdT expression starts at 4 dpf and increases thereafter. Polμ is expressed at all times to a similar extent. In situ studies showed a strong expression of TdT and polμ in the thymic cortex of 8-week-old fish. The characterization of zebrafish TdT and polμ provide new insights in fish lymphopoiesis and addresses the importance and evolution of TdT and polμ themselves.     

A new approach to the production of the recombinant SOD protein by methylotrophic <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The gene for the copper, zinc–superoxide dismutase (SOD) from the yeast Saccharomyces cerevisiae was cloned, characterized, and overexpressed in the methylotrophic Pichia pastoris. The sod gene sequence obtained is 465 bp and encodes 154 amino acid residues. The sod gene sequence was cloned into the pPIC9K vector, yielding pAB22. The linearized pAB22 DNA, digested with restriction enzyme SacI, was transformed into the genome of the GS115 strain of yeast P. pastoris. The overexpressed SOD protein was shown to have immunologically biological activity and to be enzymatically active. The SOD protein was purified from the cultured yeast by ammonium sulfate precipitation and diethylaminoethyl–cellulose column chromatography. This relatively simple purification method produced a single band on analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which indicated that the SOD protein obtained attained to higher purity and specific activity.     

Stability of <Emphasis Type="Italic">Potato virus X</Emphasis> expression vectors is related to insert size: implications for replication models and risk assessment

We investigated the stability of expression constructs based on Potato virus X (PVX) as a function of insert length. Five different inserts ranging in length from 261 to 1,758 bp (human proinsulin, murine interleukin-10, HIV-1 nef, petunia expansin-1 and human gad65) were expressed using a PVX vector in Nicotiana benthamiana plants for three sequential passages. Using a competitive RT-PCR approach we demonstrated that insert–deletion could occur in the first infection cycle for all inserts, but that this was much more likely to be the case for longer ones. This suggested a negative correlation between insert length and vector stability. Sequence analysis of the deleted constructs suggested that recombination usually occurred at sites close to the duplicated sub-genomic promoter, but in a smaller number of cases the foreign gene itself was probably involved, resulting in partially deleted constructs containing transgene fragments. The implications of these results in the context of recombinant protein expression and its risks are discussed.     

Hybridoma Ped-2E9 cells cultured under modified conditions can sensitively detect <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Bacillus cereus</Emphasis>

Lymphocyte origin hybridoma Ped-2E9 cell-based cytotoxicity assay can detect virulent Listeria or Bacillus species, and its application in a cell-based biosensor for onsite use would be very attractive. However, maintaining enough viable cells on a sensor platform for a prolonged duration is a challenging task. In this study, key factors affecting the survival and growth of Ped-2E9 cells under modified conditions were investigated. When the Ped-2E9 cells were grown in media containing 5% fetal bovine serum in sealed tubes without any replenishment of nutrients or exogenous CO₂ supply, a large portion of the cells remained viable for 6 to 7 days and cells entered into G0/G1 resting phase. The media pH change was negligible and no cell death was observed in the first 4 days, then cells sequentially underwent apoptotic (fourth day onward) phase until day 7 after which a majority was dead. Subsequent cytotoxicity testing of 3- to 7-day stored Ped-2E9 cells sensitively detected virulent Listeria and Bacillus species. These data strongly suggest that Ped-2E9 cells can be maintained in viable state for 6 days in a sealed tube mimicking the environment in a potential sensor device for onsite use without the need for expensive cell culture facilities.     

Development of an efficient method for screening microorganisms by using symbiotic association between <Emphasis Type="Italic">Nasutitermes takasagoensis</Emphasis> and intestinal microorganisms

Screening method of microorganisms that utilized the symbiotic association between insect (Nasutitermes takasagoensis: Nt) and intestinal microorganisms was developed. The existence of desired microorganisms that grew by degrading difficult-to-degrade materials in the gut was detected using survivability of Nt as an indicator. The desired microorganisms were isolated from the survived Nt. It was thought that guts of Nt behave as continuous culture systems whereby microorganisms that cannot degrade diet components are washed out, whereas those that can degrade it are retained and concentrated in the gut. About 60% of Nt fed with phenol artificial diet (PAD) died within 7 days, while 4% of termites survived for 9 days. The structure of intestinal microorganisms of the survived Nt fed with PAD differed from the bacterial communities obtained from enrichment culture (which contained phenol) of wood-feeding Nt. Relatively high colonies (650-times) were detected in the gut of Nt fed on phenol artificial diet compared with those obtained when Nt was fed on wood. Seven denaturing gradient gel electrophoresis (DGGE) bands were detected from gut of wood-feeding Nt, whereas 11 DGGE-bands were detected from that of phenol-feeding Nt. Out of 11 DGGE-bands, 5 of them were sequenced, and bacterial species including phenol-degrading bacteria were identified.     

Fatty acid efficiency profile in uncoupling of <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> mitochondria

A profile of free fatty acid (FFA) specificity in Acanthamoeba castellanii mitochondrial uncoupling is described. The FFA uncoupling specificity was observed as different abilities to stimulate resting respiration, to decrease resting membrane potential, and to decrease oxidative phosphorylation efficiency. Tested unsaturated FFA (C18–20) were more effective as uncouplers and protonophores when compared to tested saturated FFA (C8–18), with palmitic acid (C16:0) as the most active. As FFA efficiency in mitochondrial uncoupling is related to physiological changes of fatty acid composition (and thereby FFA availability) during growth of amoeba cells, it could be a way to regulate the activity of an uncoupling protein and thereby the efficiency of oxidative phosphorylation during a cell life of this unicellular organism. Aleksandra Swida and Małgorzata Czarna contributed equally to this work.     

Cloning and functional expression of the gene encoding an inhibitor against <Emphasis Type="Italic">Aspergillus flavus</Emphasis> α-amylase,a novel seed lectin from <Emphasis Type="Italic">Lablab purpureus</Emphasis> (<Emphasis Type="Italic">Dolichos lablab</Emphasis>)

Maize is one of the more important agricultural crops in the world and, under certain conditions, prone to attack from pathogenic fungi. One of these, Aspergillus flavus, produces toxic and carcinogenic metabolites, called aflatoxins, as byproducts of its infection of maize kernels. The alpha-amylase of A. flavus is known to promote aflatoxin production in the endosperm of these infected kernels, and a 36-kDa protein from the Lablab purpureus, denoted AILP, has been shown to inhibit alpha-amylase production and the growth of A. flavus. Here, we report the isolation of six full-length labAI genes encoding AILP and a detailed analysis of the activities of the encoded proteins. Each of the six labAI genes encoded sequences of 274 amino acids, with the deduced amino acid sequences showing approximately 95-99% identity. The sequences are similar to those of lectin members of a legume lectin-arcelin-alpha-amylase inhibitor family reported to function in plant resistance to insect pests. The labAI genes did not show any of the structures characteristic of conserved structures identified in alpha-amylase inhibitors to date. The recombinant proteins of labAI-1 and labAI-2 agglutinated human red blood cells and inhibited A. flavus alpha-amylase in a manner similar to that shown by AILP. These data indicate that labAI genes are a new class of lectin members in legume seeds and that their proteins have both lectin and alpha-amylase inhibitor activity. These results are a valuable contribution to our knowledge of plant-pathogen interactions and will be applicable for developing protocols aimed at controlling A. flavus infection.     

Impact of ploughing on soil seed bank dynamics in temporary pools

We examined the impact of ploughing on soil seed banks of plant communities living in temporary marshes located in agricultural fields. The quantity, quality and vertical distribution of seeds were quantified under ploughed or unploughed treatment at community level. We also focussed on a typical semi-aquatic species, Damasonium alisma, to investigate the impact of ploughing at population level. We used two complementary techniques to study seed banks: hand sorting and seedling emergence. We found that species richness of seeds, seed abundance and germination ability were strongly affected by ploughing at community level. Concerning D. alisma, most of the seeds (56%) were stored in the two deepest soil layers among the four considered in ploughed pools. Moreover, the germination rate was higher for buried seeds (84%) than for seeds collected at the surface (33.6%). These patterns were almost inverted in unploughed pools. Our results agree with the temporal storage effect generally suggested to describe the seed bank property of plant communities. But in addition, we showed that ploughing induces a spatial storage effect in accumulating species and individuals in the seed banks that favourably influence community dynamics. We conclude that, in contrast to what is usually thought, ploughing disturbance can be of benefit for such ephemeral wetland vegetation.