Identification and characterization of an ATP-dependent hexokinase with broad substrate specificity from the hyperthermophilic archaeon Sulfolobus tokodaii

As a new member of the glucose-phosphorylating enzymes, the ATP-dependent hexokinase from the hyperthermophilic crenarchaeon Sulfolobus tokodaii was purified, identified, and characterized. Our results revealed that the enzyme differs from other known enzymes in primary structure and its broad substrate specificity for both phosphoryl donors and acceptors.     

Root aerobic respiration and growth characteristics of three Typha species in response to hypoxia

The responses of root aerobic respiration to hypoxia in three common Typha species were examined. Typha latifolia L., T. orientalis Presl, and T. angustifolia L. were hydroponically cultivated under both aerobic and hypoxic growth conditions to measure root oxygen consumption rates. Hypoxia significantly enhanced the root aerobic respiration capacity of the two deep-water species, T. orientalis and T. angustifolia, while it did not affect that of the shallow-water species, T. latifolia. T. angustifolia increased its root porosity and root mass ratio, while T. latifolia increased its root diameter under the hypoxic growth conditions. The relative growth rates in biomass of T. orientalis and T. angustifolia were 59 and 39% higher, respectively, under the hypoxic growth conditions than under the aerobic growth conditions. In contrast, that of T. latifolia did not differ between the two conditions. In T. orientalis and T. angustifolia, enhanced root aerobic respiration rates under the hypoxic growth conditions would have increased the nutrient uptake, and thus higher relative growth rates were obtained. For the deep-water species, T. orientalis and T. angustifolia, the root aerobic respiration capacity was enhanced, probably in order to maintain the generation of respiratory energy under hypoxia.     

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.     

Adaptive significance of death feigning posture as a specialized inducible defence against gape-limited predators

Death feigning is fairly common in a number of taxa, but the adaptive significance of this behaviour is still unclear and has seldom been tested. To date, all proposed hypotheses have assumed that prey manage to escape predation by sending a death-mimicking signal, although death-feigning postures are markedly different from those of dead animals. Moreover, the efficacy of this technique may largely depend on the foraging mode of the predator; death feigning seldom works with sit-and-wait predators that make the decision to attack and consume prey within a very brief time. We examined whether death feigning in the pygmy grasshopper Criotettix japonicus Haan was an inducible defence behaviour against the frog Rana nigromaculata, a sit-and-wait, gape-limited predator. The characteristic posture assumed by the grasshopper during death feigning enlarges its functional body size by stretching each of three body parts (pronotum, hind legs and lateral spines) in three different directions, thereby making it difficult for the predator to swallow the prey. Our result is the first consistent explanation for why death-mimicking animals do not always mimic the posture of dead animals.     

Synthesis and activity of N-oxalylglycine and its derivatives as Jumonji C-domain-containing histone lysine demethylase inhibitors

N-Oxalylglycine (NOG) derivatives were synthesized, and their inhibitory effect on histone lysine demethylase activity was evaluated. NOG and compound 1 inhibited histone lysine demethylases JMJD2A, 2C and 2D in enzyme assays, and their dimethyl ester prodrugs DMOG and 21 exerted histone lysine methylating activity in cellular assays.     

Growth characteristics of Sakhalin spruce (Picea glehnii) planted on the northern Japanese hillsides exposed to strong winds

Sakhalin spruce (Picea glehnii), a native species typically found in northern Japan, has been used in reforestation on hillsides exposed to strong winds. In the reforestation areas, there are south-facing (S-slope) and northwest-facing slopes (NW-slope). Climatic conditions are contrasting between the two slopes, with shallower snow cover on the S-slopes. We compared growth responses of the spruce to micro-environment between the S- and NW-slopes through soil nutrients, needle longevity, water status, photosynthesis, and nutrients in the needles. These parameters were measured in needles exposed above the snow in winter and in lower needles protected by snow cover. High-position needles suffered from drought stress, especially in winter, and needles were shed early in the year on both slopes. Low-position needles did not suffer from drought stress, and maintained a high photosynthetic rate on both slopes. However, needle longevity was reduced on the S-slope, and concentrations of nitrogen, phosphorus, and potassium in the needles decreased with needle age. Soil nutrient concentrations were low on the S-slope, which suggests that the needles on the S-slope may remobilize nutrients from aged needles to younger needles prior to shedding. This characteristic is probably an adaptation in Sakhalin spruce to poor soil conditions.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

We investigated proinflammatory cytokine TNFα production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the β-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFα production in human whole blood, IC₅₀ = 1.86 μM) and in vivo (inhibition of LPS induced TNFα production in mice, ID₅₀ = 5.98 mg/kg).     

Essential role of STIM1 in the development of cardiomyocyte hypertrophy

Store-operated Ca²⁺ entry (SOCE) through transient receptor potential (TRP) channels is important in the development of cardiac hypertrophy. Recently, stromal interaction molecule 1 (STIM1) was identified as a key regulator of SOCE. In this study, we examined whether STIM1 is involved in the development of cardiomyocyte hypertrophy. RT-PCR showed that cultured rat cardiomyocytes constitutively expressed STIM1. Endothelin-1 (ET-1) treatment for 48 h enhanced TRPC1 expression, SOCE, and nuclear factor of activated T cells activation without upregulating STIM1. However, the knockdown of STIM1 suppressed these effects, thereby preventing a hypertrophic response. These results suggest that STIM1 plays an essential role in the development of cardiomyocyte hypertrophy.     

Regulation of Transforming Growth Factor-��-dependent Cyclooxygenase-2 Expression in Fibroblasts

Abnormal transforming growth factor-β (TGF-β) signaling is a critical contributor to the pathogenesis of various human diseases ranging from tissue fibrosis to tumor formation. Excessive TGF-β signaling stimulates fibrotic responses. Recent research has focused in the main on the antiproliferative effects of TGF-β in fibroblasts, and it is presently understood that TGF-β-stimulated cyclooxygenase-2 (COX-2) induction in fibroblasts is essential for antifibroproliferative effects of TGF-β. Both TGF-β and COX-2 have been implicated in tumor growth, invasion, and metastasis, and therefore tumor-associated fibroblasts are a recent topic of interest. Here we report the identification of positive and negative regulatory factors of COX-2 expression induced by TGF-β as determined using proteomic approaches. We show that TGF-β coordinately up-regulates three factors, heterogeneous nuclear ribonucleoprotein A/B (HNRPAB), nucleotide diphosphate kinase A (NDPK A), and nucleotide diphosphate kinase A (NDPK B). Functional pathway analysis showed that HNRPAB augments mRNA and protein levels of COX-2 and subsequent prostaglandin E₂ (PGE₂) production by suppressing degradation of COX-2 mRNA. In contrast, NDPK A and NDPK B attenuated mRNA and protein levels of COX-2 by affecting TGF-β-Smad2/3/4 signaling at the receptor level. Collectively, we report on a new regulatory pathway of TGF-β in controlling expression of COX-2 in fibroblasts, which advances our understanding of pathophysiological mechanisms of TGF-β.     

Eukaryotic nirK Genes Encoding Copper-Containing Nitrite Reductase: Originating from the Protomitochondrion?

Although denitrification or nitrate respiration has been found among a few eukaryotes, its phylogenetic relationship with the bacterial system remains unclear because orthologous genes involved in the bacterial denitrification system were not identified in these eukaryotes. In this study, we isolated a gene from the denitrifying fungus Fusarium oxysporum that is homologous to the bacterial nirK gene responsible for encoding copper-containing nitrite reductase (NirK). Characterization of the gene and its recombinant protein showed that the fungal nirK gene is the first eukaryotic ortholog of the bacterial counterpart involved in denitrification. Additionally, recent genome analyses have revealed the occurrence of nirK homologs in many fungi and protozoa, although the denitrifying activity of these eukaryotes has never been examined. These eukaryotic homolog genes, together with the fungal nirK gene of F. oxysporum, are grouped in the same branch of the phylogenetic tree as the nirK genes of bacteria, archaea, and eukaryotes, implying that eukaryotic nirK and its homologs evolved from a single ancestor (possibly the protomitochondrion). These results show that the fungal denitrifying system has the same origin as its bacterial counterpart.Denitrification plays an important role in the global nitrogen cycle and reduces nitrate (NO₃⁻) and/or nitrite (NO₂⁻) to a gaseous form of nitrogen, generally to dinitrogen (N₂) or nitrous oxide (N₂O) (27). It typically follows four reduction stages, NO₃⁻ → NO₂⁻ → NO → N₂O → N₂, each of which is catalyzed by a specific reductase: dissimilatory NO₃⁻ reductase (dNaR), dissimilatory NO₂⁻ reductase (dNiR), nitric oxide (NO) reductase (NoR), and N₂O reductase, respectively. These enzymes receive electrons from a respiratory chain functioning as a “terminal reductase.” Thus, denitrification exhibits a physiological significance in its ability to anaerobically respire through the processes of nitrate respiration, nitrite respiration, and so forth. Denitrification was previously thought to be a characteristic of bacteria; however, similar reactions have been found to occur in a few eukaryotes and archaea (6, 27). Eukaryotic nitrate respiration was first found in protozoa that reside in an anaerobic freshwater habitat (8). The organism particularly reduces NO₃⁻ to NO₂⁻ in a single step, a process which recovers dNaR activity in the mitochondrial fraction but does not result in denitrification. Eukaryotic denitrification was first found to occur among fungi (19, 20), which generally form N₂O from NO₃⁻ or NO₂⁻. Recently, eukaryotic denitrification was also found in a benthic foraminifer that forms N₂ from NO₃⁻ (18). The fungal denitrification system localizes in the mitochondria and couples to the mitochondrial electron transport chain to produce ATP (12, 21), thus exhibiting properties similar to those of the bacterial systems in its ability to respire anaerobically. Moreover, the mechanism of anaerobic respiration in the “aerobic” organelle of eukaryotes (mitochondrion) evokes interest regarding the origin and evolution of the mitochondrion.The main components of the fungal denitrifying system, the dNaR, dNiR, and NoR proteins, were either completely or partially purified from Fusarium oxysporum. Fungal NoR of the cytochrome P450 (P450) type, referred to as P450nor (CYP55) (11, 16), is a distinct species of bacterial cytochrome cb-type NoR. By contrast, the previously isolated fungal dNiR protein is a copper-containing type (NirK) that closely resembles its bacterial counterpart (13). Furthermore, dNaR activity partially purified from the mitochondrial membrane fraction showed that fungal dNaR possibly resembles its bacterial counterpart, NarGHI (12, 23). Therefore, while a portion of the fungal system appears to resemble its bacterial counterpart, the phylogenetic relationship between the fungal and bacterial denitrification systems remained unclear because the genes of the fungal components (dNaR and dNiR) have not been sequenced.Recent genome analyses have revealed the presence of nirK homolog genes in many eukaryotes (fungi and protozoa), a finding consistent with our previous findings on the isolation of the fungal NirK protein (13). Therefore, whether these eukaryotes containing the nirK homolog gene exhibit denitrification activity and whether the denitrifying fungus F. oxysporum really contains a nirK gene deserve a great deal of attention. To address this issue, we used the suppression subtractive hybridization (SSH) technique (7) and succeeded in isolating the nirK gene from the denitrifying fungus F. oxysporum.