Enzymes used for the determination of HbA1C

To develop an enzymatic measurement of HbA(1C), two key enzymes, i.e., fructosyl peptide oxidase and Aspergillus protease were characterized. Fructosyl peptide oxidase from Eupenicillium terrenum was a flavoenzyme that could catalyze the oxidation of N-(1-deoxyfructosyl)-Val-His. The enzyme showed high specificity toward alpha-glycated molecules, therefore it seemed suitable for the HbA(1C) assay. Since high levels of FPOX expression seemed toxic to host cells, we applied a gene expression system using a bacteriophage vector and achieved high levels of expression in Escherichia coli. Next, we found that Aspergillus protease was able to digest N-(1-deoxyfructosyl)-hexapeptide, a glycated peptide that was released from the beta-chain of HbA(1C) by Glu-C endoproteinase. We showed that the N-(1-deoxyfructosyl)-Val-His released from N-(1-deoxyfructosyl)-hexapeptide by Aspergillus protease could be assayed enzymatically using fructosyl peptide oxidase, therefore these enzymes could be applied to the enzymatic measurement of HbA(1C).     

Motif‐based search for a novel fructosyl peptide oxidase from genome databases

The measurement of glycated hemoglobin A1c (HbA1c) has important implications for diagnosis of diabetes and assessment of treatment effectiveness. We proposed specific sequence motifs to identify enzymes that oxidize glycated compounds from genome database searches. The gene encoding a putative fructosyl amino acid oxidase was found in the Phaeosphaeria nodorum SN15 genome and successfully expressed in Escherichia coli. The recombinant protein (XP_001798711) was confirmed to be a novel fructosyl peptide oxidase (FPOX) with high specificity for α‐glycated compounds, such as HbA1c model compounds fructosyl‐^αN‐valine (f‐^αVal) and fructosyl‐^αN‐valyl‐histidine (f‐^αVal‐His). Unlike previously reported FPOXs, the P. nodorum FPOX has a K_m value for f‐^αVal‐His (0.185 mM) that is considerably lower than that for f‐^αVal (0.458 mM). Based on amino acid sequence alignment, three dimensional structural modeling, and site‐directed mutagenesis, Gly60 was found to be a determining residue for the activity towards f‐^αVal‐His. A flexible surface loop region was also found to likely play an important role in accepting f‐^αVal‐His. Biotechnol. Bioeng. 2010; 106: 358–366. © 2010 Wiley Periodicals, Inc.     

Distribution and properties of novel deglycating enzymes for fructosyl peptide in fungi

Our fungal culture collection was screened for fructosyl peptide oxidase, an enzyme that could be used for the determination of glycated hemoglobin in diabetic subjects with hyperglycemia. Fructosyl peptide oxidases were found in strains of eight genera: Achaetomiella, Achaetomium, Chaetomium, Coniochaeta, Eupenicillium, Gelasinospora, Microascus and Thielavia. By their substrate specificity toward N-fructosyl valyl-histidine (-keto-amine) and N-fructosyl lysine (-keto-amine), fructosyl peptide oxidases could be categorized into two groups: (1) enzymes that oxidize both -keto-amine and -keto-amine, and (2) enzymes that preferably oxidize -keto-amine. A fructosyl peptide oxidase from Achaetomiella virescens ATCC 32393, active toward both N-fructosyl valyl-histidine and N-fructosyl lysine, was purified to homogeneity and characterized. The enzyme was monomeric (M_r=50,000), was most active at 40 °C and pH 8.0, and had a covalently bound flavin as a prosthetic group. Apparent K_m values for N-fructosyl valyl-histidine and N-fructosyl lysine were 2.30 and 1.69 mM, respectively. N-fructosyl valyl-histidine was consumed and the same molar amount of valyl-histidine was produced by the fructosyl peptide oxidase reaction. This enzyme could be useful for the measurement of hemoglobin A_1C, the N-terminal valine residue of the -subunit of which is glycated.Abbreviations HbA_1C Hemoglobin A_1C - FPOX Fructosyl peptide oxidase - FAOX Fructosyl amino acid oxidase - Fru-ValHis N-fructosyl valyl-histidine - Fru-Val N-fructosyl valine - Fru-Lys N-fructosyl lysine - Fru-Gly Fructosyl glycine - TOOS N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3-methylaniline, sodium salt     

Structural and biochemical bases of photorespiration in C<Subscript>4</Subscript> plants: quantification of organelles and glycine decarboxylase

In C₄ plants, photorespiration is decreased relative to C₃ plants. However, it remains unclear how much photorespiratory capacity C₄ leaf tissues actually have. We thoroughly investigated the quantitative distribution of photorespiratory organelles and the immunogold localization of the P protein of glycine decarboxylase (GDC) in mesophyll (M) and bundle sheath (BS) cells of various C₄ grass species. Specific differences occurred in the proportions of mitochondria and peroxisomes in the BS cells (relative to the M cells) in photosynthetic tissues surrounding a vein: lower in the NADP-malic enzyme (NADP-ME) species having poorly formed grana in the BS chloroplasts, and higher in the NAD-malic enzyme (NAD-ME) and phosphoenolpyruvate carboxykinase (PCK) species having well developed grana. In all C₄ species, GDC was localized mainly in the BS mitochondria. When the total amounts of GDC in the BS mitochondria per unit leaf width were estimated from the immunogold labeling density and the quantity of mitochondria, the BSs of NADP-ME species contained less GDC than those of NAD-ME or PCK species. This trend was also verified by immunoblot analysis of leaf soluble protein. There was a high positive correlation between the degree of granal development (granal index) in the BS chloroplasts and the total amount of GDC in the BS mitochondria. The variations in the structural and biochemical features involved in photorespiration found among C₄ species might reflect differences in the O₂/CO₂ partial pressure and in the potential photorespiratory capacity of the BS cells.Abbreviations BS Bundle sheath - GDC Glycine decarboxylase - M Mesophyll - NAD-ME NAD-malic enzyme - NADP-ME NADP-malic enzyme - PCK Phosphoenolpyruvate carboxykinase     

Differential inhibition of photosynthesis under drought stress in <Emphasis Type="Italic">Flaveria</Emphasis> species with different degrees of development of the C<Subscript>4</Subscript> syndrome

The effect of drought stress (DS) on photosynthesis and photosynthesis-related enzyme activities was investigated in F. pringlei (C₃), F. floridana (C₃–C₄), F. brownii (C₄-like), and F. trinervia (C₄) species. Stomatal closure was observed in all species, probably being the main cause for the decline in photosynthesis in the C₃ species under ambient conditions. In vitro ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) and stromal fructose 1,6-bisphosphatase (sFBP) activities were sufficient to interpret the net photosynthetic rates (P _N), but, from the decreases in P _N values under high CO₂ (C _a = 700 μmol mol^{− 1}) it is concluded that a decrease in the in vivo rate of the RuBPCO reaction may be an additional limiting factor under DS in the C₃ species. The observed decline in the photosynthesis capacity of the C₃–C₄ species is suggested to be associated both to in vivo decreases of RuBPCO activity and of the RuBP regeneration rate. The decline of the maximum P _N observed in the C₄-like species under DS was probably attributed to a decrease in maximum RuBPCO activity and/or to decrease of enzyme substrate (RuBP or PEP) regeneration rates. In the C₄ species, the decline of both in vivo photosynthesis and photosynthetic capacity could be due to in vivo inhibition of the phosphoenolpyruvate carboxylase (PEPC) by a twofold increase of the malate concentration observed in mesophyll cell extracts from DS plants.     

Purification and properties of glycolate oxidase from plants with different photosynthetic pathways: Distinctness of C4 enzyme from that of a C3 species and a C3–C4 intermediate

Glycolate oxidase (GO; EC 1.1.3.1) was purified from the leaves of three plant species:Amaranthus hypochondriacus L.(NAD-ME type C₄ dicot),Pisum sativum L. (C₃ species) andParthenium hysterophorus L. (C₃–C₄. intermediate). A flavin moiety was present in the enzyme from all the three species. The enzyme from the C₄ plant had a low specific activity, exhibited lower K_M for glycolate, and required a lower pH for maximal activity, compared to the C₃ enzyme. The enzyme from the C₄ species oxidized glyoxylate at <10% of the rate with glycolate, while the GO from the C₃ plant oxidized glyoxylate at a rate of about 35 to 40% of that with glycolate. The sensitivity of GO from C₄ plant to -hydroxypyridinemethane sulfonate, 2-hydroxy-3-butynoate and other inhibitors was less than that of the enzyme from C₃ source. The properties of GO fromParthenium hysterophorus, were similar to those of the enzyme fromPisum sativum. The characteristics of glycolate oxidase from leaves of a C₄ plant,Amaranthus hypochondriacus are different from those of the C₃ species or the C₃–C₄ intermediate.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.     

C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic pathways and life form types for native species from agro-forestry region,Northeastern China

Of the total 570 species, 194 species in 116 genera and 52 families were found with C₃ photosynthesis, 24 species in 17 genera and 6 families with C₄ photosynthesis, and 2 species in 1 genera and 1 family with CAM photosynthesis. 90 % of the total species can be found in Changbai Mountain flora, more a half (69 %) in North China flora, and about 1/3 in Mongolian flora and Xinan flora, respectively. The occurrence of C₄ species was not as common as that in adjacent grasslands and deserts, but relatively more than in the adjacent forests. Of the total 24 C₄ species, 63 % C₄ species (15 of 24) was found in Gramineae. Nine life form types can be found, reflecting the moist climate in the region, especially the occurrence of epiphyte and liana forms. Relatively more geophyte life form plants suggested the winter in the region was much colder than in grasslands. These indicated that both ecological studies and land management decisions must take into account plant photosynthetic pathway and life form patterns, for both of them are closely related to climatic changes and land use.     

Purification and characterization of an H<Subscript>2</Subscript>O-forming NADH oxidase from <Emphasis Type="Italic">Clostridium aminovalericum</Emphasis>: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria

Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O₂. When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O₂-free N₂ carrier-gas) to microoxic (sparged with 3% O₂/97% N₂ mixed carrier-gas) growth conditions in the mid exponential phase (OD₆₆₀=1.0). When the strain grew under 3% O₂/97% N₂, the medium remains anoxic. Thirty minutes after beginning aeration with 3% O₂, the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration. We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe. The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts. The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa. The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor. The final product of NADH oxidation was H₂O, and the estimated K_m for oxygen was 61.9 M. These data demonstrate that an O₂-response enzyme that is capable of detoxifying oxygen to water exists in C. aminovalericum.Abbreviations NRIC NODAI Research Institute-Culture Collection Center, Tokyo University of Agriculture, Tokyo, Japan - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonyl fluoride     

Performance of a generalist grasshopper on a C<Subscript>3</Subscript> and a C<Subscript>4</Subscript> grass: compensation for the effects of elevated CO<Subscript>2</Subscript> on plant nutritional quality

The increasing CO₂ concentration in Earths atmosphere is expected to cause a greater decline in the nutritional quality of C₃ than C₄ plants. As a compensatory response, herbivorous insects may increase their feeding disproportionately on C₃ plants. These hypotheses were tested by growing the grasses Lolium multiflorum C₃) and Bouteloua curtipendula C₄) at ambient (370 ppm) and elevated (740 ppm) CO₂ levels in open top chambers in the field, and comparing the growth and digestive efficiencies of the generalist grasshopper Melanoplus sanguinipes on each of the four plant × CO₂ treatment combinations. As expected, the nutritional quality of the C₃ grass declined to a greater extent than did that of the C₄ grass at elevated CO₂; protein levels declined in the C₃ grass, while levels of carbohydrates (sugar, fructan and starch) increased. However, M. sanguinipes did not significantly increase its consumption rate to compensate for the lower nutritional quality of the C₃ grass grown under elevated CO₂. Instead, these grasshoppers appear to use post-ingestive mechanisms to maintain their growth rates on the C₃ grass under elevated CO₂. Consumption rates of the C₃ and C₄ grasses were also similar, demonstrating a lack of compensatory feeding on the C₄ grass. We also examined the relative efficiencies of nutrient utilization from a C₃ and C₄ grass by M. sanguinipes to test the basis for the C₄ plant avoidance hypothesis. Contrary to this hypothesis, neither protein nor sugar was digested with a lower efficiency from the C₄ grass than from the C₃ grass. A novel finding of this study is that fructan, a potentially large carbohydrate source in C₃ grasses, is utilized by grasshoppers. Based on the higher nutrient levels in the C₃ grass and the better growth performance of M. sanguinipes on this grass at both CO₂ levels, we conclude that C₃ grasses are likely to remain better host plants than C₄ grasses in future CO₂ conditions.     

Characterization of the NADP malic enzyme gene family in the facultative,single-cell C<Subscript>4</Subscript> monocot <Emphasis Type="Italic">Hydrilla verticillata</Emphasis>

Hydrilla verticillata has a facultative single-cell system that changes from C₃ to C₄ photosynthesis. A NADP⁺-dependent malic enzyme (NADP-ME) provides a high [CO₂] for Rubisco fixation in the C₄ leaf chloroplasts. Of three NADP-ME genes identified, only hvme1 was up-regulated in the C₄ leaf, during the light period, and it possessed a putative transit peptide. Unlike obligate C₄ species, H. verticillata exhibited only one plastidic isoform that may perform housekeeping functions, but is up-regulated as the photosynthetic decarboxylase. Of the two cytosolic forms, hvme2 and hvme3, the latter exhibited the greatest expression, but was not light-regulated. The mature isoform of hvme1 had a pI of 6.0 and a molecular mass of 64 kD, as did the recombinant rHVME1m, and it formed a tetramer in the chloroplast. The recombinant photosynthetic isoform showed intermediate characteristics between isoforms in terrestrial C₃ and C₄ species. The catalytic efficiency of rHVME1m was four-fold higher than the cytosolic rHVME3 and two-fold higher than recombinant cytosolic isoforms of rice, but lower than plastidic forms of maize. The K _m (malate) of 0.6 mM for rHVME1 was higher than maize plastid isoforms, but four-fold lower than found with rice. A comprehensive phylogenetic analysis of 25 taxa suggested that chloroplastic NADP-ME isoforms arose from four duplication events, and hvme1 was derived from cytosolic hvme3. The chloroplastic eudicot sequences were a monophyletic group derived from a cytosolic clade after the eudicot and monocot lineages separated, while the monocots formed a polyphyletic group. The findings support the hypothesis that a NADP-ME isoform with specific and unusual regulatory properties facilitates the functioning of the single-cell C₄ system in H. verticillata. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Presence of ATP-sensitive and ATP-insensitive Ribonucleases in the Fruit Body of Flammulina velutipes

Neonatally streptozotocin-induced diabetic (n-STZ) rats were given food containing Lactobacillus GG cells (GG) or a control diet (control), from 9 to 18 weeks of age. The GG cells significantly lowered the blood hemoglobin A_1C (HbA_1C) level and improved glucose tolerance in n-STZ rats (p<0.05). In the GG group, the serum insulin level at 30 min after glucose loading was significantly higher than in the control group (p<0.05).     

Photoautotrophic growth of sugarcane plantlets <Emphasis Type="Italic">in vitro</Emphasis> as affected by photosynthetic photon flux and vessel air exchanges

Summary Explants of sugarcane, a C₄ plant, were cultured in vitro for 18d on Floridalite (a solid cube consisting of vermiculite and cellulose fibers) used as supporting material with sugar-free Murashige and Skoog liquid medium with double-strength KH₂PO₄, MgSO₄, FeSO₄, and Na₂-EDTA in the vessel with enhanced natural ventilation. CO₂ concentration in the culture room was kept at 1500 μmol mol⁻¹ (four times the atmospheric CO₂ concentration) during the photoperiod. A factorial experiment was designed with two levels of photosynthetic photon flux (PPF) and three levels of N (number of air exchanges of the vessel). The results were compared with those in the control treatment (photomixotrophic culture using sugar-containing agar medium under low PPF and low N). PPF and N showed significant positive effects on the growth of sugarcane plantlets in vitro. In the photoautotrophic (using sugar-free medium) treatments with relatively high PPF (200–400 μmol m⁻² s⁻¹) and high N (2–10 h⁻¹), the growth of plantlets was four to seven times greater than that in the control. Also, the culture period for multiplication and rooting was shortened from 30 d in the control to 18 d or less in the photoautotrophic, high PPF, and high N treatments. Use of porous supporting material in photoautotrophic treatments promoted rooting and plantlet growth significantly.     

Salicylhydroxamic acid (SHAM) inhibits O<Subscript>2</Subscript> photoreduction which protects nitrogenase activity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. RF-1

Synechococcus sp. RF-1, a unicellular N₂-fixing cyanobacterium, can grow photosynthetically and diazotrophically in continuous light. How the organism protects its nitrogenase from damage by oxygen is unclear. In cyanobacerial cells, electron transport carriers associated with photosynthesis and respiration are all on the thylakoid membranes and share some common components, including plastoquinone pool and cytochrome b ₆ f complex, and the pathways are interacting with each other. In this work, a pulse amplitude modulation (PAM) fluorometer (PAM-101) and an O₂ electrode are used simultaneously to study the chlorophyll a fluorescence and to monitor O₂ exchanges in Synechococcus sp. RF-1 cells. At the CO₂ compensation point, the photochemical quenching activity remained high unless the O₂ was exhausted by the glucose oxidase system (GOS). It indicates that in addition to CO₂, O₂ can also act as electron acceptor to receive electrons derived from Q_A. Studies with various inhibitors of the electron transport chain demonstrated that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) and salicylhydroxamic acid (SHAM) inhibited the photoreduction of O₂, while glycolaldehyde, disalicylidenepropanediamine (DSPD), methyl viologen (MV) and KCN did not. These results imply that a KCN-resistant and SHAM-sensitive oxidase transfers electrons generated from Photosystem II to O₂ between cytochrome b ₆ f complex and ferredoxin. When SHAM blocked this alternative electron transport pathway, the dinitrogen-fixing activity decreased significantly. The results indicate that a novel oxidase may function as an intracellular O₂-scavenger in Synechococcus sp. RF-1 cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Molecular properties of phosphoenolpyruvate carboxylase from C3, C3−C4 intermediate,and C4 Flaveria species

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C₄), F. floridana Johnston (C₃–C₄), and F. cronquistii Powell (C₃) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C₃ and C₃–C₄ plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C₃–C₄ intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C₃ and C₃–C₄ intermediate PEPCases. The PEPCase from the C₄ plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C₃–C₄ intermediate species as C₃-like plants with some C₄-like photosynthetic characteristics, but there are differences from the C₃ plant in the quantity and properties of the PEPCase from the C₃–C₄ intermediate plant.Abbreviations IEF isoelectric focusing - kDa kilodalton - PEPCase phosphoenolpyruvate carboxylase - Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase     

Light and growth temperature alter carbon isotope discrimination and estimated bundle sheath leakiness in C<Subscript>4</Subscript> grasses and dicots

We combined measurements of short-term (during gas exchange) and long-term (from plant dry matter) carbon isotope discrimination to estimate CO₂ leakiness from bundle sheath cells in six C₄ species (three grasses and three dicots) as a function of leaf insertion level, growth temperature and short-term irradiance. The two methods for determining leakiness yielded similar results (P > 0.05) for all species except Setaria macrostachya, which may be explained by the leaf of this species not being accommodating to gas exchange. Leaf insertion level had no effect on leakiness. At the highest growth temperature (36°C) leakiness was lower than at the two lower growth temperatures (16°C and 26°C), between which no differences in leakiness were apparent. Higher irradiance decreased leakiness in three species, while it had no significant effect on the others (there was an opposite trend in two species). The inverse response to increasing irradiance was most marked in the two NAD-ME dicots (both Amaranthus species), which both showed almost 50% leakiness at low light (300 μmol quanta m⁻² s⁻¹) compared to about 30% at high light (1,600 μmol quanta m⁻² s⁻¹). NADP-ME subtype grasses had lower leakiness than NAD-ME dicots. Although there were exceptions, particularly in the effect of irradiance on leakiness in Sorghum and Boerhavia, we conclude that conditions favourable to C₄ photosynthesis (high temperature and high light) lead to a reduction in leakiness.     

Effects of elevated CO<Subscript>2</Subscript> and/or O<Subscript>3</Subscript> on hormone IAA in needles of Chinese pine

This study examined the effects of season-long exposure of Chinese pine (Pinus tabulaeformis) to elevated carbon dioxide (CO₂) and/or ozone (O₃) on indole-3-acetic acid (IAA) content, activities of IAA oxidase (IAAO) and peroxidase (POD) in needles. Trees grown in open-top chambers (OTC) were exposed to control (ambient O₃, 55 nmol mol⁻¹ + ambient CO₂, 350 μmol mol⁻¹, CK), elevated CO₂ (ambient O₃ + high CO₂, 700 μmol mol⁻¹, EC) and elevated O₃ (high O₃, 80 ± 8 nmol mol⁻¹ + ambient CO₂, EO) OTCs from 1 June to 30 September. Plants grown in elevated CO₂ OTC had a growth increase of axial shoot and needle length, compared to control, by 20% and 10% respectively, while the growth in elevated O₃ OTC was 43% and 7% less respectively, than control. An increase in IAA content and POD activity and decrease in IAAO activity were observed in trees exposed to elevated CO₂ concentration compared with control. Elevated O₃ decreased IAA content and had no significant effect on IAAO activity, but significantly increased POD activity. When trees pre-exposed to elevated CO₂ were transferred to elevated O₃ (EC–EO) or trees pre-exposed to elevated O₃ were transferred to elevated CO₂ (EO–EC), IAA content was lower while IAAO activity was higher than that transferred to CK (EC–CK or EO–CK), the change in IAA content was also related to IAAO activity. The results indicated that IAAO and POD activities in Chinese pine needles may be affected by the changes in the atmospheric environment, resulting in the change of IAA metabolism which in turn may cause changes in Chinese pine’s growth. An erratum to this article can be found at     

Relationship between allocation of absorbed light energy in PSII and photosynthetic rates of C3 and C4 plants

Two C₃ dicotyledonous crops and five C₄ monocotyledons treated with three levels of nitrogen were used to evaluate quantitatively the relationship between the allocation of absorbed light energy in PSII and photosynthetic rates (P _N) in a warm condition (25–26°C) at four to five levels [200, 400, 800, 1,200 (both C₃ and C₄) and 2,000 (C₄ only) μmol m⁻² s⁻¹] of photosynthetic photon flux density (PPFD). For plants of the same type (C₃ or C₄), there was a linear positive correlation between the fraction of absorbed light energy that was utilized in PSII photochemistry (P) and P _N, regardless of the broad range of their photosynthetic rates due to species-specific effect and/or nitrogen application; meanwhile, the fraction of absorbed light energy that was dissipated through non-photochemical quenching (D) showed a negative linear regression with P _N for each level of PPFD. The intercept of regression lines between P and P _N of C₃ and C₄ plants decreased, and that between D and P _N increased with increasing PPFD. With P and D as the main components of energy dissipation and complementary to each other, the fraction of excess absorbed light energy (E) was unchanged by P _N under the same level of PPFD. At the same level of P _N, C₄ plants had lower P and higher D than C₃ plants, due to the fact that C₄ plants with little or no photorespiration is considered a limited energy sink for electrons. Nevertheless there was a significant negative linear correlation between D and P when data from both C₃ and C₄ plants at varied PPFD levels was merged. The slope of regression lines between P and D was 0.85, indicating that in plants of both types, most of the unnecessary absorbed energy (ca. 85%) could dissipate through non-photochemical quenching, when P was inhibited by low P _N due to species-specific effect and nitrogen limitation at all levels of illumination used in the experiment.     

Cellular expression of C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic enzymes in the amphibious sedge<Emphasis Type="Italic"> Eleocharis retroflexa</Emphasis> ssp.<Emphasis Type="Italic"> chaetaria</Emphasis>

The amphibious leafless sedge Eleocharis retroflexa ssp. chaetaria expresses C₄-like biochemical characteristics in both the terrestrial and submerged forms. Culms of the terrestrial form have Kranz anatomy, whereas those of the submerged form have Kranz-like anatomy combined with anatomical features of aquatic plant leaves. We examined the immunolocalization of C₃ and C₄ enzymes in culms of the two forms. In both forms, phosphoenolpyruvate carboxylase; pyruvate, Pi dikinase; and NAD-malic enzyme were compartmentalized between the mesophyll (M) and Kranz cells, but their levels were somewhat reduced in the submerged form. In the terrestrial form, ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) occurred mainly in the Kranz cells, and weakly in the M chloroplasts. In the submerged form, the rubisco occurred at higher levels in the M cells than in the terrestrial form. In both forms, the C₄ pattern of enzyme expression was clearer in the M cells adjacent to Kranz cells than in distant M cells. During the transition from terrestrial to submerged conditions, the enzyme expression pattern changed in submerged mature culms that had been formed in air before submergence, and matched that in culms newly developed underwater. It seems that effects of both environmental and developmental factors overlap in the C₄ pattern expression in this plant.     

Recent improvements in the development of A<Subscript>2B</Subscript> adenosine receptor agonists

Adenosine is known to exert most of its physiological functions by acting as local modulator at four receptor subtypes named A₁, A_2A, A_2B and A₃ (ARs). Principally as a result of the difficulty in identifying potent and selective agonists, the A_2B AR is the least extensively characterised of the adenosine receptors family. Despite these limitations, growing understanding of the physiological meaning of this target indicates promising therapeutic perspectives for specific ligands. As A_2B AR signalling seems to be associated with pre/postconditioning cardioprotective and anti-inflammatory mechanisms, selective agonists may represent a new therapeutic group for patients suffering from coronary artery disease. Herein we present an overview of the recent advancements in identifying potent and selective A_2B AR agonists reported in scientific and patent literature. These compounds can be classified into adenosine-like and nonadenosine ligands. Nucleoside-based agonists are the result of modifying adenosine by substitution at the N ⁶-, C²-positions of the purine heterocycle and/or at the 5′-position of the ribose moiety or combinations of these substitutions. Compounds 1-deoxy-1-{6-[N′-(furan-2-carbonyl)-hydrazino]-9H-purin-9-yl}-N-ethyl-β-D-ribofuranuronamide (19, hA₁ K _i = 1050 nM, hA_2A K _i = 1550 nM, hA_2B EC₅₀ = 82 nM, hA₃ K _i > 5 μM) and its 2-chloro analogue 23 (hA₁ K _i = 3500 nM, hA_2A K _i = 4950 nM, hA_2B EC₅₀ = 210 nM, hA₃ K _i > 5 μM) were confirmed to be potent and selective full agonists in a cyclic adenosine monophosphate (cAMP) functional assay in Chinese hamster ovary (CHO) cells expressing hA_2B AR. Nonribose ligands are represented by conveniently substituted dicarbonitrilepyridines, among which 2-[6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-ylsulfanyl]acetamide (BAY-60–6583, hA₁, hA_2A, hA₃ EC₅₀ > 10 μM; hA_2B EC₅₀ = 3 nM) is currently under preclinical-phase investigation for treating coronary artery disorders and atherosclerosis.