Three-dimensional constructs induce high cellular activity: Structural stability and the specific production of proteins and cytokines

Naofen has recently been identified from the rat brain/spinal cord cDNA library as a substance reactive against an anti-shigatoxin (Stx)-2 antibody. Naofen mRNA is composed of 4620 nucleotides and encodes 1170 amino acids. Naofen contains four WD-repeat domains in its N-terminus and is ubiquitously distributed in many tissues of the rat. Tumor necrosis factor (TNF)-α enhanced the expression of naofen mRNA in HEK293 cells in a dose-dependent manner. Furthermore, naofen siRNA, which predominantly knocked down the expression of naofen mRNA, significantly reduced both TNF-α-induced caspase-3 activation and apoptosis in HEK293 cells. Overexpression of naofen in HEK293 cells (FLAG-NF) spontaneously induced caspase -3 activation and apoptosis, and showed extremely high susceptibility to TNF-α-induced apoptosis. These results indicated that naofen may function as a novel modulator activating caspase-3, and promoting TNF-α-stimulated apoptosis.     

NudC is required for Plk1 targeting to the kinetochore and chromosome congression

The equal distribution of chromosomes during mitosis is critical for maintaining the integrity of the genome. Essential to this process are the capture of spindle microtubules by kinetochores and the congression of chromosomes to the metaphase plate . Polo-like kinase 1 (Plk1) is a mitotic kinase that has been implicated in microtubule-kinetochore attachment, tension generation at kinetochores, tension-responsive signal transduction, and chromosome congression . The tension-sensitive substrates of Plk1 at the kinetochore are unknown. Here, we demonstrate that human Nuclear distribution protein C (NudC), a 42 kDa protein initially identified in Aspergillus nidulans and shown to be phosphorylated by Plk1 , plays a significant role in regulating kinetochore function. Plk1-phosphorylated NudC colocalizes with Plk1 at the outer plate of the kinetochore. Depletion of NudC reduced end-on microtubule attachments at kinetochores and resulted in defects in chromosome congression at the metaphase plate. Importantly, NudC-deficient cells exhibited mislocalization of Plk1 and the Kinesin-7 motor CENP-E from prometaphase kinetochores. Ectopic expression of wild-type NudC, but not NudC containing mutations in the Plk1 phosphorylation sites, recovered Plk1 localization at the kinetochore and rescued chromosome congression. Thus, NudC functions as both a substrate and a spatial regulator of Plk1 at the kinetochore to promote chromosome congression.     

SSR-based linkage map with new markers using an intraspecific population of common wheat

Simple sequence repeats (SSRs) are valuable molecular markers in many plant species. In common wheat (Triticum aestivum L.), which is characteristic of its large genomes and alloploidy, SSRs are one of the most useful markers. To increase SSR marker sources and construct an SSR-based linkage map of appropriate density, we tried to develop new SSR markers from SSR-enriched genomic libraries and the public database. SSRs having (GA)n and (GT)n motifs were isolated from enriched libraries, and di- and tri-nucleotide repeats were mined from expressed sequence tags (ESTs) and DNA sequences of Triticum species in the public database. Of the 1,147 primer pairs designed, 842 primers gave accurate amplification products, and 478 primers showed polymorphism among the nine wheat lines examined. Using a doubled haploid (DH) population from an intraspecific cross between Kitamoe and Münstertaler (KM), we constructed an SSR-based linkage map that consisted of 464 loci: 185 loci from genomic libraries, 65 loci from the sequence database including ESTs, 213 loci from the SSR markers already reported, and 1 locus of morphological marker. Although newly developed SSR loci were distributed throughout all chromosomes, clustering of them around putative centromeric regions was found on several chromosomes. The total length of the KM map spanned 3,441 cM and corresponded to approximately 86% genome coverage. The KM map comprised of 23 linkage groups because two gaps of over 50 cM distance remained on chromosome 6A. This is a first report of SSR-based linkage map using single intraspecific population of common wheat. This mapping result suggests that it becomes possible to construct linkage maps with sufficient genome coverage using only SSR markers without RFLP markers, even in an intraspecific population of common wheat. Moreover, the new SSR markers will contribute to the enrichment of molecular marker resources in common wheat.     

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.     

Cell growth and P(3HB) accumulation from CO<Subscript>2</Subscript> of a carbon monoxide-tolerant hydrogen-oxidizing bacterium, <Emphasis Type="Italic">Ideonella</Emphasis> sp. O-1

Cell growth and accumulation of polyhydroxybutyric acid, P(3HB), from CO₂ in autotrophic condition of a newly isolated hydrogen-oxidizing bacterium, the strain O-1, was investigated. The bacterium, which was deposited in the Japan Collection of Microorganisms as JCM17105, autotrophically grows by assimilating H₂, O₂, and CO₂ as substrate. 16S rRNA gene sequence of the bacterium was the closest to Ideonella dechloratans (99%). Specific growth rate of the strain O-1 was faster than a hydrogen-oxidizing bacterium, Ralstonia eutropha, which is well-known P(3HB)-producing microorganism. The strain O-1 is tolerant to high O₂ concentration and it can grow above 30% (v/v) O₂, while the growth of R. eutropha and Alcaligenes latus was seriously inhibited. In culture medium containing 1 g/L (NH₄)₂SO₄, cell concentration of the strain O-1 and P(3HB) increased to 6.75 and 5.26 g/L, respectively. The content of P(3HB) in the cells was 77.9% (w/w). The strain O-1 was very tolerant to carbon monoxide (CO) and it grew even at 70% (v/v) CO, while the growth of R. eutropha and A. latus were seriously inhibited at 5% (v/v) CO. From these results, it is expected that the strain O-1 will be useful in the manufacture of P(3HB) because the industrial exhaust gas containing CO₂, H₂, and CO can be directly used as the substrate in the fermentation process.     

Stimulus-specific development of learning ability during habitat shift in pre to post-recruitment stage jack mackerel

Marine fishes often experience major habitat shifts during their life history, and previous studies have shown that the learning capability of fish change ontogenetically and in accordance with such habitat shifts. However, because all of these studies used a single type of conditioned stimuli (CS), they failed to detect qualitative changes in learning capability. Here we tested the hypothesis that preparedness for learning changes ontogenetically in jack mackerel Trachurus japonicus, which undergo a drastic change in habitat preference during their life history as they move from offshore pelagic waters to coastal and demersal rocky reefs. Groups of juveniles measuring 40 mm standard length (SL) (pelagic stage) and 60 mm SL (demersal stage) were conditioned to food rewards in response to three different CS; the presence of a surface structure, mid-water structure, and aeration. The results showed that small juveniles tended to become conditioned to the surface stimulus faster than they did to the mid-water stimulus. Conversely, large juveniles responded to the mid-water stimulus significantly more quickly than they did to the surface stimulus. These results suggest that stimulus-specific learning capability in T. japonicus changes ontogenetically, facilitating adaptation to their life-history strategy.     

Chromosome identification and comparative karyotypic analyses of four Pinus species

Chromosomal landmarks in four Pinus species: P. densiflora, P. thunbergii, P. sylvestris, and P. nigra were identified by fluorescence in situ hybridization (FISH) using hapten- or fluorochrome-labeled probes for the plant telomere repeat, centromeric repeat (PCSR), and rDNA. FISH landmarks were located at the interstitial and proximal regions of chromosomes and allowed us to identify nearly all of the homologous chromosomes in each species. A comparative analysis of the FISH karyotypes among the four species showed that the interstitial FISH signals obtained by hybridization with the telomere and rDNA sequences were stable and could be used to identify homologous chromosomes among species. The identification of homologous chromosomes among species facilitated a detailed comparative karyotype analysis. The results suggest that the degree of chromosomal differentiation among the four Pinus species is very low and that the proximal regions vary in their DNA sequences. The similarities and differences among FISH karyotypes are discussed in relation to phylogeny.     

Isolation and characterization of polyhydroxyalkanoates inclusions and their associated proteins in Pseudomonas sp. 61-3

Two types of polyester inclusions of poly(3-hydroxybutyrate) [P(3HB)] and poly(3HB-co-3-hydroxyalkanoates) [P(3HB-co-3HA)] were isolated from crude extract of Pseudomonas sp. 61-3. Proteins associated with each inclusion were separated by SDS-PAGE. PHA synthase 1 (PhaC1(Ps)), PhaF(Ps), and PhaI(Ps) were identified from P(3HB-co-3HA) inclusions by N-terminal amino acid sequences analyses, as well as PHB synthase (PhbC(Ps)) and 24-kDa unknown protein were identified from P(3HB) inclusions. The structural genes of PhaF(Ps) and PhaI(Ps) were located downstream of the pha locus. The relative PHA/PHB synthase activities of each inclusion were measured for various 3-hydroxyacyl-coenzyme As of 4-12 carbon atoms. Direct atomic force microscopy observation of P(3HB) and P(3HB-co-3HA) inclusions demonstrated that the two types of inclusions had different morphologies.