A low trophic position of Japanese eel larvae indicates feeding on marine snow

What eel larvae feed on in the surface layer of the ocean has remained mysterious. Gut contents and bulk nitrogen stable isotope studies suggested that these unusual larvae, called leptocephali, feed at a low level in the oceanic food web, whereas other types of evidence have suggested that small zooplankton are eaten. In this study, we determined the nitrogen isotopic composition of amino acids of both natural larvae and laboratory-reared larvae of the Japanese eel to estimate the trophic position (TP) of leptocephali. We observed a mean TP of 2.4 for natural leptocephali, which is consistent with feeding on particulate organic matter (POM) such as marine snow and discarded appendicularian houses containing bacteria, protozoans and other biological materials. The nitrogen isotope enrichment values of the reared larvae confirm that the primary food source of natural larvae is consistent only with POM. This shows that leptocephali feed on readily available particulate material originating from various sources closely linked to ocean primary production and that leptocephali are a previously unrecognized part of oceanic POM cycling.     

The human apoptosis inhibitor NAIP induces pyroptosis in macrophages infected with Legionella pneumophila

Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals.     

Modeling the Expansion of an Introduced Tree Disease

Pine wilt disease is caused by the introduced pinewood nematode, Bursaphelenchus xylophilus, for which the vector is the pine sawyer beetle, Monochamus alternatus. Native Japanese pines, black pine (Pinus thunbergii) and red pine (P. densiflora), are extremely sensitive to the nematode's infection, and the parasite has been expanding nationwide in the last few decades, despite intensive control efforts. To understand the parasite's range expansion in Japan, we modeled the dynamics of the pines and the beetle that disperses the nematode, using an integro-difference equation in a one-dimensional space. Based on field data collected in Japan, we investigated the dependence of the parasite's rate of range expansion on the eradication rate of the beetle, the initial pine density, and the beetle dispersal ability. Our model predicts several results. (1) The Allee Effect operates on beetle reproduction, and consequently the parasite cannot invade a pine stand, once the beetle density decreases below a threshold. (2) The distribution of the dispersal distance of the beetles critically affects the expansion rate of the disease. As the fraction of the beetles that travel over long distance increases from zero, the range expansion accelerates sharply. (3) However, too frequent long-range dispersal results in a failure of the parasite invasion due to the Allee Effect, suggesting the importance of correctly assessing the beetle's mobility to predict the speed of range expansion of the parasite. (4) As the eradication rate is increased, the range expansion speed decreases gradually at first and suddenly drops to zero at a specific value of the eradication rate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Benzo[a]pyrene diol epoxide induces viral reactivation at concentrations that block DNA elongation in mammalian cells

The survival of UV-irradiated Simian virus 40 (SV40) in CV-1P African green monkey kidney cells treated with (+/-)7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-diol epoxide I) was studied. Enhanced survival of UV damaged SV40 was detected when CV-1P cells were treated with dose levels of BP-diol epoxide I corresponding to the exponential portion (0.33-1.11 microM) of a CV-1P cell survival curve. Dose levels of BP-diol epoxide I corresponding to the shoulder region (less than or equal to 0.16 microM) of a CV-1P survival curve did not induce viral reactivation. The shoulder region concentrations of BP-diol epoxide I selectively inhibited DNA initiation while the concentrations on the exponential portion of the curve preferentially inhibited DNA elongation. It was shown in a time course of enhanced viral survival at 0.66 microM BP-diol epoxide I that the reactivation response was fully induced by 24 h. In conclusion, the viral reactivation response was associated with concentrations of BP-diol epoxide I which induced lethal damage and preferentially inhibited DNA elongation.     

Dual Biosynthesis Pathway for Longer-Chain Polyamines in the Hyperthermophilic Archaeon Thermococcus kodakarensis

Long-chain and/or branched-chain polyamines are unique polycations found in thermophiles. Cytoplasmic polyamines were analyzed for cells cultivated at various growth temperatures in the hyperthermophilic archaeon Thermococcus kodakarensis. Spermidine [34] and N⁴-aminopropylspermine [3(3)43] were identified as major polyamines at 60°C, and the amounts of N⁴-aminopropylspermine [3(3)43] increased as the growth temperature rose. To identify genes involved in polyamine biosynthesis, a gene disruption study was performed. The open reading frames (ORFs) TK0240, TK0474, and TK0882, annotated as agmatine ureohydrolase genes, were disrupted. Only the TK0882 gene disruptant showed a growth defect at 85°C and 93°C, and the growth was partially retrieved by the addition of spermidine. In the TK0882 gene disruptant, agmatine and N¹-aminopropylagmatine accumulated in the cytoplasm. Recombinant TK0882 was purified to homogeneity, and its ureohydrolase characteristics were examined. It possessed a 43-fold-higher k_cat/K_m value for N¹-aminopropylagmatine than for agmatine, suggesting that TK0882 functions mainly as N¹-aminopropylagmatine ureohydrolase to produce spermidine. TK0147, annotated as spermidine/spermine synthase, was also studied. The TK0147 gene disruptant showed a remarkable growth defect at 85°C and 93°C. Moreover, large amounts of agmatine but smaller amounts of putrescine accumulated in the disruptant. Purified recombinant TK0147 possessed a 78-fold-higher k_cat/K_m value for agmatine than for putrescine, suggesting that TK0147 functions primarily as an aminopropyl transferase to produce N¹-aminopropylagmatine. In T. kodakarensis, spermidine is produced mainly from agmatine via N¹-aminopropylagmatine. Furthermore, spermine and N⁴-aminopropylspermine were detected in the TK0147 disruptant, indicating that TK0147 does not function to produce spermine and long-chain polyamines.Polyamines are positively charged aliphatic compounds. Putrescine [4], spermidine [34], and spermine [343] are common polyamines observed in various living organisms, from viruses to humans (16). Polyamines, which play important roles in cell proliferation and cell differentiation (19, 34), are thought to contribute to adaptation against various stresses (9, 26). In thermophilic microorganisms, polyamines contribute to growth under high-temperature conditions. Indeed, in the thermophilic bacterium Thermus thermophilus, a mutant strain lacking the enzyme related to polyamine biosynthesis shows defective growth at high temperatures (23). Furthermore, thermophilic archaea and bacteria possess long-chain and branched-chain polyamines such as N⁴-aminopropylspermidine [3(3)4], N⁴-aminopropylspermine [3(3)43], and N⁴-bis(aminopropyl)spermidine [3(3)(3)4], in addition to common polyamines (11, 13, 14). N⁴-aminopropylspermine was detected in the cells of thermophiles, such as Saccharococcus thermophilus, thermophilic Bacillus and Geobacillus spp. (Bacillus caldolyticus, B. caldotenax, B. smithii, Geobacillus stearothermophilus, and G. thermocatenulatus), Caldicellulosiruptor spp. (C. kristjanssonii and C. owensensis) and Calditerricola spp. (C. satsumensis and C. yamamurae) (10, 12, 22), but it was not detected in archaea. These unique polyamines are thought to support the growth of thermophilic microorganisms under high-temperature conditions. An in vitro study indicated that long-chain and branched-chain polyamines effectively stabilized DNA and RNA, respectively (32).Polyamines are synthesized from amino acids such as arginine, ornithine, and methionine (26). In most eukaryotes, putrescine is synthesized directly from ornithine by ornithine decarboxylase (34). Plants and some bacteria possess additional or alternative putrescine biosynthesis pathways in which putrescine is synthesized from arginine via agmatine (18, 31, 35). In this pathway, agmatine is synthesized by arginine decarboxylase, and agmatine is converted to putrescine by agmatine ureohydrolase or a combination of agmatine iminohydrolase and N-carbamoylputrescine amidohydrolase. Longer polyamines are then produced by the addition of the aminopropyl group from decarboxylated S-adenosylmethionine. This pathway is shown on the left in Fig. ​Fig.11 (pathway I). On the other hand, the thermophilic bacterium T. thermophilus possesses a unique polyamine-biosynthetic pathway (23) in which spermidine is synthesized from agmatine via N¹-aminopropylagmatine by aminopropyl transferase followed by ureohydrolase, as shown on the right in Fig. ​Fig.11 (pathway II).Open in a separate windowFIG. 1.Predicted biosynthetic pathway of polyamines in T. kodakarensis. (A) Predicted biosynthetic pathway. Pyruvoyl-dependent arginine decarboxylase proenzyme (TK0149), arginine/agmatine ureohydrolases (TK0240/TK0474/TK0882), aminopropyl transferase (TK0147), and pyruvoyl-dependent S-adenosylmethionine decarboxylase proenzyme (TK1592) are shown based on the genome analysis. (B) Structures of unique polyamines.A sulfur-reducing hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, was isolated from Kodakara Island, Kagoshima, Japan (1, 21). This archaeon grows at temperatures between 60°C and 100°C but optimally at 85°C. Under low- or high-temperature-stressed conditions, T. kodakarensis produces cold- or heat-inducible chaperones to adapt to unfavorable growth environments (4, 5, 30). The lipid composition of the membrane also changes depending on the growth shift (20). In addition to acting as such tolerance factors, polyamines have been suggested to play an important role in maintaining nucleosomes in high-temperature environments (15). A complete genome analysis of T. kodakarensis has been performed, and the pathway of polyamine biosynthesis has been predicted (Fig. ​(Fig.1)1) (6, 7). It has been speculated that putrescine is synthesized from arginine via agmatine by arginine decarboxylase (PdaD_Tk) and agmatine ureohydrolase. Long- and/or branched-chain polyamines are then produced by the addition of the aminopropyl group derived from decarboxylated S-adenosylmethionine. Previously, we revealed that PdaD_Tk catalyzed the first step of polyamine biosynthesis and was essential for cell growth (6). The strain DAD, which lacks the gene pdaD_Tk, does not grow in medium without agmatine. Archaeal cells are known to use agmatine to synthesize agmatidine, which is an agmatine-conjugated cytidine found at the anticodon wobble position of archaeal tRNA^Ile (17). Agmatine is important for agmatidine synthesis as well as long-chain polyamine. In the present study, we focused on the subsequent steps in polyamine biosynthesis, especially from agmatine to spermidine. T. kodakarensis possesses three agmatine ureohydrolase homologues (TK0240, TK0474, and TK0882); however, it is unclear which one is dominantly functional in T. kodakarensis cells. In a closely related genus, Pyrococcus, TK0474 and TK0882 orthologues have been identified, but the TK0240 orthologue is missing in Pyrococcus genomes. In Pyrococcus horikoshii, PH0083, which is an orthologue of TK0882, was shown to possess agmatine ureohydrolase activity (8). TK0882, hence, appears to possess agmatine ureohydrolase activity as well. It is unclear whether other agmatine ureohydrolase homologues (TK0240 and TK0474) are involved in polyamine synthesis and cell growth in T. kodakarensis. In addition to agmatine ureohydrolase, aminopropyl transferase plays a crucial role in the synthesis of polyamines. TK0147 was annotated first as spermidine synthase and shares sequence identity with aminopropyl transferase (PF0127) from Pyrococcus furiosus (3). It is therefore expected to harbor the function of aminopropyl transferase for long-chain-polyamine synthesis. Recombinant PF0127 showed broad amine acceptor specificity for agmatine, 1,3-diaminopropane (3), putrescine, cadaverine (5), sym-nor-spermidine (33), and spermidine. While maximal catalytic activity was observed with cadaverine, agmatine was most often preferred on the basis of the k_cat/K_m value (3), suggesting that pathway II is a dominant route for polyamine synthesis in P. furiosus. In the present study, various disruptants lacking genes for polyamine biosynthesis were constructed in order to understand the physiological roles of these enzymes in T. kodakarensis. The cell growth profiles and cytoplasmic polyamines of the wild type and the disruptants were analyzed and compared. Recombinant enzymes were also purified and characterized. The obtained results are expected to provide useful information regarding the specific roles of polyamines in thermophiles.     

Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.     

Globin genes in Micronesia: origins and affinities of Pacific Island peoples.

DNA polymorphisms and copy-number variants of alpha-, zeta-, and gamma-globin genes have been studied in seven Micronesian island populations and have been compared with those in populations from Southeast Asia, Melanesia, and Polynesia. Micronesians are not significantly different from Polynesians at these loci and appear to be intermediate between Southeast Asians and Melanesians. There is evidence of significant Melanesian input into the Micronesian gene pool and of substantial proto-Polynesian contact with Melanesia.     

A physical map of the 20q12-q13.1 region associated with type 2 diabetes

Several recent genetic studies have suggested linkage of Type 2 diabetes (non-insulin-dependent diabetes mellitus) susceptibility to a region of chromosome 20q12-q13.1. To facilitate the identification and cloning of a diabetes susceptibility gene(s) in this region, we have constructed correlated radiation hybrid and YAC/BAC contig physical maps of the region. A high-resolution radiation hybrid map encompassing 9.5 Mb between the PLC and the CEBPB genes was constructed using 68 markers: 25 polymorphic markers, 15 known genes, 21 ESTs, and 7 random genomic sequences. The physical order of the polymorphic markers within this radiation hybrid map is consistent with published genetic maps. A YAC/BAC contig that gives continuous coverage between PLC and CEBPB was also constructed. This contig was constructed from 24 YACs, 34 BACs, and 1 P1 phage clone onto which 71 markers were mapped: 23 polymorphic markers, 12 genes, 24 ESTs, and 12 random genomic sequences. The radiation hybrid map and YAC/BAC physical map enable precise mapping of newly identified transcribed sequences and polymorphic markers that will aid in linkage and linkage disequilibrium studies and facilitate identification and cloning of candidate Type 2 diabetes susceptibility genes residing in 20q12-q13.1.