Lifespan in male ants linked to mating syndrome

Male ants have long been thought to leave the nest, swarm, mate and die in quick succession (male aggregation syndrome). However, the ancestors of the ants likely used female calling, where females advertise with pheromones for longer lived and presumably free living males. In this study, male lifespan was compared in four species from a Panama rain forest. Males of two species with aggregation syndrome (Atta colombica and Azteca sp.) lived only days after collection at a light trap, and their lifespan failed to increase when supplied sugar water ad libitum. In contrast, two species with female calling syndrome (Ectatomma ruidum and Paraponera clavata) lived up to 116 days when fed. These results link male lifespan to mating systems, and provide a framework to examine variation in how ant colonies invest in males.     

Liquid chromatography-mass spectrometry to study chondroitin lyase action pattern

Liquid chromatography-mass spectrometry was applied to determine the action pattern of different chondroitin lyases. Two commercial enzymes, chondroitinase ABC (Proteus vulgaris) and chondroitinase ACII (Arthrobacter aurescens), having action patterns previously determined by viscosimetry and gel electrophoresis were first examined. Next, the action patterns of recombinant lyases, chondroitinase ABC from Bacteroides thetaiotaomicron (expressed in Escherichia coli) and chondroitinase AC from Flavobacterium heparinum (expressed in its original host), were examined. Chondroitin sulfate A (CS-A, also known as chondroitin-4-sulfate) was used as the substrate for these four lyases. Aliquots taken at various time points were analyzed. The products of chondroitinase ABC (P. vulgaris) and chondroitinase AC (F. heparinum) contained unsaturated oligosaccharides of sizes ranging from disaccharide to decasaccharide, demonstrating that both are endolytic enzymes. The products afforded by chondroitinase ABC (B. thetaiotaomicron) and chondroitinase ACII (A. aurescens) contained primarily unsaturated disaccharide. These two exolytic enzymes showed different minor products, suggesting some subtle specificity differences between the actions of these two exolytic lyases on chondroitin sulfate A.     

Molecular Cloning of Cyanobacterial Pteridine Glycosyltransferases That Catalyze the Transfer of either Glucose or Xylose to Tetrahydrobiopterin

Here, we report cloning of cyanobacterial genes encoding pteridine glycosyltransferases that catalyze glucosyl or xylosyl transfer from UDP-sugars to tetrahydrobiopterin. The genes were cloned by PCR amplification from genomic DNA which was isolated from culture and environmental samples and overexpressed in Escherichia coli for an in vitro activity assay.Tetrahydrobiopterin (BH4) is well known among pteridine compounds as a cofactor for aromatic amino acid hydroxylases and nitric oxide synthases in animals (19). Pteridine glycosides such as biopterin and 6-hydroxymethylpterin glycosides have been found in cyanobacteria and anaerobic photosynthetic bacteria (2, 4, 5, 8, 11, 13, 15, 17, 21). Although the function of these glycosides remains unknown, they are abundant and ubiquitous in cyanobacteria, implying some essential role (3, 6, 16-18, 21). There is a group of enzymes, named pteridine glycosyltransferases (PGTs), known to catalyze a variety of glycosyl transfers to pteridines. The first PGT isolated from the cyanobacterium Synechococcus sp. PCC 7942 was shown to catalyze a glucosyl transfer from UDP-glucose to BH4 and was therefore named UDP-glucose:BH4 glucosyltransferase (BGluT) (7). After cloning of the gene encoding BGluT (6), a PGT that catalyzes the transfer of glucuronic acid for cyanopterin synthesis was identified (12). In addition, there are many putative PGT homologs encoded in bacterial genomes, although their exact catalytic functions have not been determined. We recently found that BGluT is useful for the simultaneous detection of oxidized and reduced forms of BH4 in animal samples (14). Glycosyltransferases are also being studied intensively for applications in the design of novel pharmaceutical derivatives (1, 10). We were thus encouraged to find PGTs with new substrate specificities or enzymatic properties not only for study of protein structure and function but also for application in BH4 research. In this study, we succeeded in cloning four cyanobacterial genes encoding PGTs with either glucosyl- or xylosyltransferase activity, and here we report the results.PGT genes were cloned from Arthrospira platensis CY-007 (obtained through Hawaii Oceanic Institute sampling) and Arthrospira maxima CY-049 (UTEX 2342), which were cultured in the Korea Marine Microalgae Culture Center, and from environmental DNA sequences (designated UCNR-001 and UCNR-002) isolated from wild algal mats in the Nakdong River, South Korea. In order to amplify conserved internal sequences of the unknown PGT genes, degenerate PCR primers were designed from the nucleotide sequences of cyanobacterial PGT homologs using GeneFisher2 (9). A protein homology search with BGluT against the bacterial genome database in NCBI revealed more than a hundred PGT homologs. When a phylogenetic tree was constructed from the putative sequences, there was a separate group comprising cyanobacterial PGTs. Figure ​Figure11 shows the cyanobacterial cluster, in which members shared sequence identities of more than 34%. Because the degenerate primers designed from all of the cyanobacterial PGTs were too highly degenerate, the cluster was divided into four subgroups, as shown in Fig. ​Fig.1:1: this division allowed primers to be designed for each of the four subgroups. The PGTs in subgroup I were clearly distinguishable from the others, because they all originated from marine picocyanobacteria, which are abundant in the pelagic realm. Subgroup I could be divided further into two groups comprising PGTs from either Prochlorococcus species (CIA) or marine Synechococcus species (CIB). Subgroup II was also divided into two groups, CIIA, consisting mostly of PGTs from Synechococcus species, and CIIB, containing the other PGTs. Among the primers designed for each subgroup, those for the CIIA and CIIB subgroups successfully amplified DNA sequences of the expected sizes. The primer sequences were 5′-GTTCAGGAWTAGGAGGTGGAGT-3′ (CIIA-forward)/5′-CGCYTCAATWGCTACATTTCCA-3′ (CIIA-reverse) and 5′-ACGACTGGCTMYCGYTTTAYCTGA-3′ (CIIB-forward)/5′-GCYTCCACCCAYTTRGGGGTCA-3′ (CIIB-reverse). Based on the determined partial gene sequences, additional sets of primer pairs were designed for the inverse PCR method (20). The sequences were 5′-GATGAACTACAACAGGGTCTGCGTC-3′ (CY-007 forward)/5′-CGGCTTTTTAAGGCTTTTGCCATATTC-3′ (CY-007 reverse), 5′-GTCTGCGTGAATGTCGAGG-3′ (CY-047 forward)/5′-ATGACCTCGGCTGTGTAAG-3′ (CY-047 reverse), and 5′-CCTACAAAAAGAGCTAGGCGACTGTTTTG-3′ (UCNR forward)/5′-CCAAAGAAACGGAAGCCATGCTG-3′ (UCNR reverse). Total genomic DNA samples were partially digested with RsaI and then self-ligated to be used as templates for PCR amplification with the primer pairs. The amplified DNA sequences revealed the missing 5′- and 3′-end sequences of the genes.Open in a separate windowFIG. 1.Neighbor-joining phylogenetic tree of cyanobacterial PGT protein sequences, identified by NCBI accession numbers. Bootstrap values are presented at the nodes. The names of strains whose PGTs are characterized are in bold.The deduced protein sequences were multiply aligned with BGluT (Fig. ​(Fig.2).2). Amino acid identities for all sequences in pairwise comparisons are given as percentages in Fig. ​Fig.2.2. Recently, draft assemblies of the genome sequences of Arthrospira platensis strain Paraca and Arthrospira maxima CS-328 (UTEX 2342) were announced. The annotated PGT (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"EDZ91868","term_id":"209491491","term_text":"EDZ91868"}}EDZ91868) of Arthrospira maxima CS-328 was identical to the PGT of CY-049 at both the amino acid and nucleotide levels, proving that the two organisms originated from the same UTEX stock (UTEX 2342). On the other hand, the PGTs of Arthrospira platensis strains Paraca and CY-007 were different at nine individual nucleotides, resulting in seven amino acid differences. A phylogenetic analysis showed that CY-007 and CY-049 PGTs belonged to the CIIB subgroup and that UCNR-001 and UCNR-002 PGTs clustered in the CIIA subgroup (data not shown).Open in a separate windowFIG. 2.Alignment of multiple PGT sequences. Conserved sequences are shaded at four levels using GeneDoc software. At the end of the alignment, amino acid identities in percentages are given for all sequences in pairwise comparisons.In order to identify the catalytic function of the putative PGTs, the recombinant proteins were produced in Escherichia coli. The complete open reading frame (ORF) sequences were amplified by PCR from the genomic DNA samples, cloned into the pGEM-T vector, and subsequently cloned as NdeI/BamHI restriction fragments into pET-28b (for CY-007 and CY-049 sequences) or pET-15b (for UCNR-001 and UCNR-002 sequences). E. coli BL21(DE3)/pLysS transformants were induced with 0.05 to 0.2 mM isopropyl-β-d-thiogalactopyranoside and were cultured for 8 h at 22°C. The recombinant proteins were purified by chromatography on Ni-nitrilotriacetic acid gel according to the instructions of the manufacturer (Qiagen). The proteins were eluted with 250 mM imidazole, dialyzed against a mixture of 20 mM Tris-HCl (pH 7.5) and 30% (vol/vol) glycerol, and stored in aliquots at −70°C until use. Purification of the proteins was confirmed by electrophoresis on an SDS-polyacrylamide gel (Fig. ​(Fig.3A).3A). BGluT from a previous purification was used (6). Aliquots of PGT were assayed at 37°C for 10 min in a reaction mixture of 100 μl containing 50 mM sodium phosphate, pH 7.5, 10 mM MnCl₂, 0.2% ascorbic acid, 1 μM BH4 (Schircks Lab, Switzerland), and 100 μM UDP-glucose or UDP-xylose. The reaction mixture was combined with an equal volume of acidic iodine solution (2% KI and 1% I₂ in 1 N HCl) for 1 h in the dark. After centrifugation, the supernatant was mixed in a 10:1 volume ratio with 5% ascorbic acid and subjected to high-performance liquid chromatography (HPLC). HPLC was performed with a Gilson 321 pump equipped with an Inertsil ODS-3 column (150 by 2.3 mm; particle size, 5 μm [GL Science, Japan]) and a fluorescence detector (Shimadzu RF-10AXL). Pteridines were eluted with 10 mM potassium phosphate buffer (pH 6.0) at a flow rate of 1.2 ml/min and were monitored at excitation and emission wavelengths of 350 and 450 nm, respectively.Open in a separate windowFIG. 3.Analysis of purified recombinant PGTs on an SDS-12.5% polyacrylamide gel (A) and HPLC analysis of the enzymatic products (B).The enzymatic products of PGTs (Fig. ​(Fig.3)3) appeared only when enzymes were incubated with BH4 as a sugar acceptor and either UDP-glucose (for CY-007, UCNR-001, and UNCR-002 PGTs) or UDP-xylose (for CY-049 PGT) as a sugar donor. HPLC analysis of cultured CY-007 and CY-049 cells confirmed the presence of the corresponding biopterin glycosides (data not shown), supporting the conclusion that the PGTs exhibited genuine in vivo activities. This is the first report of a gene encoding a PGT that catalyzes xylosyl transfer to BH4. Although the data are not shown here, we found additional xylosyl transfer PGTs in Anabaena sp. PCC 7120, Gloeobacter violaceus PCC 7421, and Thermosynechococcus elongatus BP-1, whose genomic sequences were determined. The putative PGT genes (represented in Fig. ​Fig.1)1) were amplified by PCR from the genomic DNA, which was a kind gift from the Kazusa DNA Research Institute (http://genome.kazusa.or.jp/cyanobase/). The recombinant proteins for the in vitro activity assay were prepared by cloning the genes into pET-28b and overexpressing the proteins in E. coli according to the same procedures performed for the other PGTs. Interestingly, CY-007 and CY-049 PGTs exhibited different substrate specificities, although they share 93% protein sequence identity, and they also had higher specific activities than the other PGTs (Fig. ​(Fig.4).4). The three-dimensional structures of the proteins are currently being investigated to further understanding of the structural properties involved. Considering the cyanobacterial PGTs hitherto identified, there seems to be little correlation between their substrate preferences and phylogenetic classification. However, the CI group PGTs, which diverged early from the CII group PGTs, might have some distinctive features. Finally, the successful cloning of PGT genes from environmental DNA allows for potentially new PGTs to be isolated from cyanobacteria, which are abundant in nature.Open in a separate windowFIG. 4.Comparative analysis of PGT activities. The maximal activity (100%) corresponds to complete glycosylation of 1 μM BH4 in the reaction mixture, which contained 0.5 mM UDP-xylose for CY-049 PGT or 0.5 mM UDP-glucose for the other PGTs. The mixtures were incubated for 10 min with the indicated amounts of proteins.     

Proteomic pattern-based analyses of light responses in Arabidopsis thaliana wild-type and photoreceptor mutants

Light critically affects the physiology of plants. Using two-dimensional gel electrophoresis, we used a proteomics approach to analyze the responses of Arabidopsis thaliana to red (660 nm), far-red (730 nm) and blue (450 nm) light, which are utilized by type II and type I phytochromes, and blue light receptors, respectively. Under specific light treatments, the proteomic profiles of 49 protein spots exhibited over 1.8-fold difference in protein abundance, significant at p <0.05. Most of these proteins were metabolic enzymes, indicating metabolic changes induced by light of specific wavelengths. The differentially-expressed proteins formed seven clusters, reflecting co-regulation. We used the 49 differentially-regulated proteins as molecular markers for plant responses to light, and by developing a procedure that calculates the Pearson correlation distance of cluster-to-cluster similarity in expression changes, we assessed the proteome-based relatedness of light responses for wild-type and phytochrome mutant plants. Overall, this assessment was consistent with the known physiological responses of plants to light. However, we also observed a number of novel responses at the proteomic level, which were not predicted from known physiological changes.     

Self-organized pullulan/deoxycholic acid nanogels: Physicochemical characterization and anti-cancer drug-releasing behavior

The objective of this study was to develop new self-organized nanogels as a means of drug delivery in patients with cancer. Pullulan (PUL) and deoxycholic acid (DOCA) were conjugated through an ester linkage between the hydroxyl group in PUL and the carboxyl group in DOCA. Three types of PUL/DOCA conjugates were obtained, differing in the number of DOCA substitutions (DS; 5, 8, or 11) per 100 PUL anhydroglucose units. The physicochemical properties of the resulting nanogels were characterized by dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The mean diameter of DS 11 was the smallest (approx. 100 nm), and the size distribution was unimodal. To determine the organizing behavior of these conjugates, we calculated their critical aggregation concentrations (CACs) in a 0.01-M phosphate buffered saline solution. They were 10.5×10⁻⁴ mg/mL, 7.2×10⁻⁴ mg/mL, and 5.6×10⁻⁴ mg/mL for DS 5, 8, and 11, respectively. This indicates that DOCA can serve as a hydrophobic moiety to create self-organized nanogels. To monitor the drug-releasing behavior of these nanogels, we loaded doxorubicin (DOX) onto the conjugates. The DOX-loading efficiency increased with the degree of DOCA substitution. The release rates of DOX from PUL/DOCA nanogels varied inversely with the DS. We concluded that the PUL/DOCA nanogel has some potential for use as an anticancer drug carrier because of its low CAC and satisfactory drug-loading capacity.     

Growth and tuberization of transgenic potato plants expressing sense and antisense sequences of Cu/Zn superoxide dismutase from lily chloroplasts

Overexpression of a chloroplast-localized Cu/Zn superoxide dismutase (chCu/ZnSOD) obtained from lily significantly affects the growth and shape of potato tubers from anin vitro culture system (Kim et al., 2007). Here, we further characterized the sense and antisense transgenic potatoes grown and pots and the greenhouse to investigate the potential for more practical field applications of such phenotypic manipulations. Underin vitro conditions, antisense transgenic plants showed increased shoot growth, delayed tuberization, and altered tuber shapes. When antisense plants were treated with paclobutrazol, an inhibitor of GA biosynthesis, tuberization efficiency and tuber shape were recovered to a status very similar to that ofin vitro- grown wild-type plants. Our results strongly support the idea that potato tuberization and shape is mediated by SOD-catalyzed reactive oxygen species, possibly via the GA biosynthesis pathway.     

Modulation of Thr phosphorylation of integrin beta1 during muscle differentiation

By using transient elevations of cytosolic free calcium levels triggered by integrin antibody or laminin (Kwon, M. S., Park, C. S., Choi, K., Park, C.-S., Ahnn, J., Kim, J. I., Eom, S. H., Kaufman, S. J., and Song, W. K. (2000) Mol. Biol. Cell 11, 1433-1443), we have demonstrated that protein phosphatase 2A (PP2A) is implicated in the regulation of reversible phosphorylation of integrin. In E63 skeletal myoblasts, the treatment of PP2A inhibitors such as okadaic acid and endothall induces an increase of phosphorylation of integrin beta1A and thereby inhibits integrin-induced elevation of cytosolic calcium level and formation of focal adhesions. None of these effects were in differentiated myotubes expressing the alternate beta1D isoform. In the presence of okadaic acid, PP2A in association with integrin beta1A was reduced on myoblasts, whereas beta1D on myotubes remained bound with PP2A. Both co-immunoprecipitation and in vitro phosphatase assays revealed that dephosphorylation of residues Thr788-Thr789 in the integrin beta1A cytoplasmic domain is dependent upon PP2A activity. Mutational analysis of the cytoplasmic domain and confocal microscopy experiments indicated that substitution of Thr788-Thr789 with Asn788-Asn789 is of critical importance for regulating the function of integrin beta1. These results suggest that PP2A may be a primary regulator of threonine phosphorylation of integrin beta1A and subsequent activation of downstream signaling molecules. Taken together, we propose that dephosphorylation of residues Thr788-Thr789 in the cytoplasmic domain of integrin beta1A may contribute to the linkage of integrins to focal adhesion sites and induce the association with cytoskeleton proteins. The switch of integrin beta1A to beta1D isoform in myotubes therefore may be a mechanism to escape from phospho-regulation by PP2A and promotes a more stable association of the cytoskeleton with the extracellular matrix.     

Genotoxic effects of volatile organic compounds in a chemical factory as evaluated by the Tradescantia micronucleus assay and by chemical analysis

The clastogenic effects of volatile organic compounds in the workplace air of a chemical factory were studied by means of the Tradescantia micronucleus (Trad-MCN) assay and chemical analysis. Sampling was performed at a chemical factory producing PVC film in Cheong-ju, South Korea. Inflorescences of Tradescantia BNL 4430 were placed for 2, 6, and 9 h at the height of 1.40 m at two locations in the workplace and one outdoor of the chemical industry. Air samplings were conducted in the same places and the collected tube samples were analyzed by automatic thermal desorption/gas chromatography/mass spectrometry (ATD/GC/MS). The frequencies of micronuclei in specimens exposed for 2 h in sites 1-3 were 6.13 +/- 0.47, 5.40 +/- 1.60, and 2.93 +/- 0.43 MCN per 100 tetrads, respectively. GC/MS analysis proved the presence of various volatile organic compounds such as trichloroethylene, toluene, ethyl benzene, (m, p, o)-xylene, styrene, 1,3,5-trimethyl benzene, and 1,2,4-trimethyl benzene. Mean values of toluene measured by 2 h sampling in sites 1-3 were 1946.6, 1368.3, and 340.1 microg/m3, respectively. The toluene concentrations in sites 1 and 2 were at least four to six times higher than that in site 3. The micronucleus frequencies increased with exposure time. In addition, there was a correlation between the micronucleus frequencies and toluene concentration in the air (R2 = 0.96). The results of this in situ monitoring proved the applicability of the Trad-MCN assay combined with chemical analysis for monitoring genotoxic chemicals in the work environment.     

Effect of (1-->3)- and (1-->4)-linkages of fully sulfated polysaccharides on their anticoagulant activity

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.