Total synthesis of L-2,2-difluoro-2-deoxy-MYO-inositol 1,4,5-trisphosphate, a potent inhibitor of the enzymes of d-MYO-inositol 1,4,5-trisphosphate metabolism

The synthesis of the enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate is reported. L-2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate is a potent inhibitor of 3-kinase and 5-phosphatase.     

Myo-inositol 1,4,6-trisphosphate: A new synthetic Ca-mobilising inositol phosphate

The synthesis of myo-inositol 1,4,6-trisphosphate from myo-inositol is described; this novel trisphosphate is a potent Ca²⁺-mobilising agonist at the Ins(1,4,5)P₃ receptor and is derived from structure-activity considerations of myo-inositol 1,3,4,6-tetrakisphosphate.     

Sensitive fluorescent quantitation of myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate by high-performance thin-layer chromatography

A non-radioactive micro-assay for the cyclic phosphodiesterase reaction catalyzed by Bacillus cereus phosphatidylinositol-specific phospholipase C is described. The assay involves high-performance thin-layer chromatography on silica gel to resolve the substrate (myo-inositol 1,2-cyclic phosphate) and the product (myo-inositol 1-phosphate), followed by detection with a lead tetraacetate–fluorescein stain. The quantitation of these inositol phosphates in sample spots relative to a series of standards is accomplished by analysis of the fluorescent plate image with a commercial phosphoimager and associated software. The experimental considerations for reliable quantitation of inositol monophosphates in the range of 0.1 to 50 nmol are presented.     

EXAFS studies and gaussian view energy calculations of the complexation of europium with 1,3,5-trideoxy-1,3,5-tris((2-hydroxybenzyl)amino)-cis-inositol

Complexation of 1,3,5-trideoxy-1,3,5-tris((2-hydroxybenzyl)amino)-cis-inositol (thci) in I = 1.00 M (NaClO₄) with Eu(III) and La(III) was studied by EXAFS measurements and gaussian view energy calculations.EXAFS studies indicated that two complexes Eu(thci)²⁺ and are formed with thci and Eu(III). At lower pH, Eu(III) is bonded to thci via two hydroxyl groups (cyclohexanetriol or 2-hydroxybenzylamino moieties) and 7 H₂O molecules, for a total of 9 Eu–O bonds. However, at higher pH there are two hydroxyl groups (cyclohexanetriol or 2-hydroxybenzylamino moieties), two amino groups, and five water molecules for a total Eu(III) coordination number of 9. gaussian view energy calculations indicate that in the Eu(thci)²⁺, thci bonds to Eu(III) in a similar manner and is in good correlation with the EXAFS data.     

A fluorescent substrate for the assay of phosphatidylinositol-specific phospholipase C: 4-(1-pyreno)butylphosphoryl-1-myo-inositol

Racemic 4-(1-pyreno)butylphosphoryl-1-myo-inositol was synthesized from a pentaprotected inositol-1-dimethylphosphite by phosphite coupling with 4-(1-pyreno)butanol. It is a good substrate for a very sensitive assay of phosphatidylinositol-specific phospholipase C.     

High concentrations of d-pinitol or d-chiro-inositol inhibit the biosynthesis of raffinose family oligosaccharides in maturing smooth tare (Vicia tetrasperma [L.] Schreb.) seeds

In the present study we have investigated the effect of exogenous cyclitols on accumulation of their galactosides and raffinose family oligosaccharides (RFOs) in maturing smooth tare (Vicia tetrasperma [L.] Schreb) seeds. Feeding d-pinitol to pods of smooth tare increased the amount of free d-pinitol and its galactosides: galactopinitol A, galactopinitol B, di- and trigalactopinitol A in seeds. Similarly, feeding d-chiro-inositol, which does not occur naturally in Vicia seeds, resulted in the transport of this cyclitol in the seed, and caused accumulation of high levels of d-chiro-inositol galactosides (fagopyritol B1, B2 and B3). Accumulation of both cyclitols and their galactosides drastically reduced accumulation of verbascose and, to a lesser extent, stachyose and di-galactosyl- myo-inositol. Feeding d-chiro-inositol also decreased accumulation of di- and tri-galactosyl pinitols, naturally occurring in seeds. Inhibition of RFOs accumulation by elevated levels of free cyclitols indicates competition between biosynthesis of both types galactosides, and similarity of both biosynthetic pathways in smooth tare seeds.     

Effects of NH+4-N and NO+3-N on growth and metabolism of Sphagnum magellanicum

Photosynthetic CO₂-fixation, chlorophyll content, growth rate and nitrate reductase activity were used to examine the influence of NH⁺₄-N and NO⁻₃-N on Sphagnum magellanicum cultivated under defined conditions in phytotrons. NO⁻₃-concentrations up to 322 μ M were found to be favourable. Increased NH⁺₄ concentrations, however, resulted in growth inhibition and decreased chlorophyll content at concentrations ≧ 255 μ M ; e.g. 600 μ M NH⁺₄ caused a 20% reduction of nitrate reductase activity and net photosynthesis. For raised bog Sphagna an improved standard nutrient solution is proposed with the following ion concentrations (μ M ): 55 Na⁺; 17 K⁺; 95 NH⁺₄; 22 Ca²⁺; 22 Mg²⁺; 2 Fe³⁺; 20 Cl⁻; 100 NO⁻₃; 57 SO^2-₄; 7.4 H₂PO⁻₄; trace elements: A-Z solution (Hoagland) 50 μl 1000 ml⁻¹; pH 5.8.     

Effect of H2 evolution on 15N2 fixation, C2H2 reduction and relative efficiency of leguminous symbionts

The (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratios of 15 clover- Rhizobium symbionts. soybean, and black medick symbionts were measured. Relative efficiency based on the C2H4 production and on ¹⁵N₂ incorporation were compared, and in most symbionts there was little difference between the two measures of relative efficiency. Total measurable electron flux through nitrogenase during acetylene reduction and ¹⁵N₂ incorporation were nearly equal for most symbionts studied. The relative efficiency and the (C₂H₄+ H_2(C2H₂))/¹⁵N₂ ratio showed an inverse correlation. Use of this ratio appears preferable to use of the ratio of C2H2 reduction/N₂ reduction. Some evolution of H₂ was observed in the presence of C₂H₂.     

Regulation of nitrate uptake into Chara corallina cells via NH+4 stimulation of NO−3 efflux

Abstract. Net NO₃ uptake by NO⁻₃ deficient Chara cells was used to calculate [NO⁻₃]_c assuming that the cytoplasm occupies 10% total volume and that nitrate reduction and storage are negligible (i.e. maximum [NO⁻₃]_c was calculated). A linear relationship was found between NO⁻₃ efflux and [NO⁻₃]_c. There was an initial burst of NO⁻₃ efflux when NH⁺₄ was added, followed by a slower efflux rate which matched influx rate such that net NO⁻₃ uptake was zero. Over 50% of NO⁻₃ that had been taken up in 2 h was lost within the first 5 min of NH⁺₄ addition. The Nernst equation was used to predict the direction of the electrochemical driving force for NO⁻₃ entry. Under the experimental conditions used NO⁻₃ efflux is actively transported. The differential involvement of both NO⁻₃ influx and NO⁻₃ efflux in the regulation of NO⁻₃ uptake is discussed and a model is proposed to account for these results which envisages discrete NO⁻₃ influx and NO⁻₃ efflux carriers.     

A comparison of NH4+ and NO3– net fluxes along roots of rice and maize

Net fluxes of NH₄⁺ and NO₃^– along adventitious roots of rice ( Oryza sativa L.) and the primary seminal root of maize ( Zea mays L.) were investigated under nonperturbing conditions using ion-selective microelectrodes. The roots of rice contained a layer of sclerenchymatous fibres on the external side of the cortex, whereas this structure was absent in maize. Net uptake of NH₄⁺ was faster than that of NO₃^– at 1 mm behind the apex of both rice and maize roots when these ions were supplied together, each at 0·1 mol m^–3. In rice, NH₄⁺ net uptake declined in the more basal regions, whereas NO₃^– net uptake increased to a maximum at 21 mm behind the apex and then it also declined. Similar patterns of net uptake were observed when NH₄⁺ or NO₃^– was the sole nitrogen source, although the rates of NO₃^– net uptake were faster in the absence of NH₄⁺. In contrast to rice, rates of NH₄⁺ and NO₃^– net uptake in the more basal regions of maize roots were similar to those near the root apex. Hence, the layer of sclerenchymatous fibres may have limited ion absorption in the older regions of rice roots.     

H+/PPi stoichiometry of a membrane‐bound pyrophosphatase of plant mitochondria

The H⁺/PP_i stoichiometry of the mitochondrial H⁺‐PP_iase from pea ( Pisum sativum L.) stem was determined by two kinetic approaches, and compared with the H⁺/substrate stoichiometries of the mitochondrial H⁺‐ATPase, and the vacuolar H⁺‐PP_iase and H⁺‐ATPase. Using sub‐mitochondrial particles or preparations enriched in vacuolar membranes, the rates of substrate‐dependent H⁺‐transport were evaluated: by a mathematical model, describing the time‐course of H⁺‐gradient (ΔpH) formation; or by determining the rate of H⁺‐leakage following H⁺‐pumping inhibition by EDTA at the steady‐state ΔpH. When the H⁺‐transport rates were divided by those of PP_i or ATP hydrolysis, measured under identical conditions, apparent stoichiometries of ca 2 were determined for the mitochondrial H⁺‐PP_iase and H⁺‐ATPase, and for the vacuolar H⁺‐ATPase. The stoichiometry of the vacuolar H⁺‐PP_iase was found to be ca 1. From these results, it is suggested that the mitochondrial H⁺‐PP_iase may, in theory, function as a primary H⁺‐pump poised towards synthesis of PP_i and, therefore, acting in parallel with the main H⁺‐ATPase.     

The sequence of RNA segment 1 of influenza virus A/NT/60/68 and its comparison with the corresponding segment of strains A/PR/8/34 and A/WSN/33.

The complete nucleotide sequence of RNA segment 1 of influenza virus A/NT/60/68, corresponding to the PB2 protein, has been determined. It is 2341 nucleotides long, encoding a predicted product of 759 amino acids with a net charge of +27 1/2 at neutral pH. The predicted amino acid sequence has been compared to the equivalent sequences in influenza viruses A/PR/8/34 and A/WSN/33. Evolutionary divergence, assuming a direct lineage from A/PR/8/34 and allowing for "laboratory drift", is 0.08% per year. The alignment of RNA segment 10 of A/NT/60/68 with segments 1 and 3 is completed, confirming that it is a mosaic of regions from these two segments.     

Ribonucieic Acid and Protein Metabolism of t12/t12 Embryos and T/t12 Spermatozoa

RNA precursor uptake and incorporation, amino acid uptake and incorporation, and the characterization of newly synthesized RNA and protein in pools of normal morulae and pools containing one-third t ¹²/ t ¹² morulae were compared. Maturing spermatoza of +/+ and T / t ¹² animals were analyzed for RNA and protein content, and the RNA characterized. No differences in these parameters could be ascribed to the t ¹² gene in homozygous embryos or haploid sperm.     

A/California/7/2009与A/California/4/2009病毒感染力比较

目的甲型H1N1流感病毒A/California/7/2009与A/California/4/2009病毒序列比较同源性在99%以上,本实验旨在比较两株病毒感染BALB/c小鼠研究感染力强弱。方法分别将A/California/7/2009（CA7）与A/California/4/2009（CA4）两株病毒分别连续10倍稀释后,对4~6周龄雌性BALB/c小鼠经乙醚麻醉后进行滴鼻攻毒,每个稀释度接种10只实验小鼠,测定CA7 MLD50为101.24/0.05 mL,检测小鼠感染、致病的多项指标,观察期为14 d。结果相同TCID50的CA7和CA4病毒感染小鼠,CA4感染小鼠后14 d内死亡率为20%,而CA7感染小鼠后8 d内死亡率为100%。CA7 106TCID50感染的小鼠病理表现为重度弥漫性间质性肺炎,CA4 106TCID50感染的小鼠病理表现为中度-重度间质性肺炎。结论在相同条件下,CA7感染力明显强于CA4。     

Induction of nitrate reductase in needles of Scots pine seedlings by NOX and NO3−

The possibility to induce nitrate reductase (NR; EC 1.6.6.2) in needles of Scots pine ( Pinus sylvestris L.) seedlings was studied. The NR activity was measured by an in vivo assay. Although increased NR activities were found in the roots after application of NO₃⁻, no such increase could be detected in the needles. Detached seedlings placed in NO₃⁻ solution showed increasing NR activities with increasing NO₃⁻ concentrations. Exposure of seedlings to NO_x (70–80 ppb NO₂ and 8–12ppb NO) resulted in an increase of the NR activity from 10–20 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹ to about 400 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. This level was reached after 2–4 days of exposure, thereafter the NR activity decreased to about 200 nmol NO₂⁻ (g fresh weight)⁻¹ h⁻¹. Analyses of free amino acids showed low concentrations of arginine and glutamine in NO_x-fumigated seedlings compared to corresponding controls.     

Effect of darkness and CO2 starvation on NH4+ and NO3− assimilation in the unicellular alga Cyanidium caldarium

Cyanidium caldarium (Tilden) Geitler, a non-vacuolate unicellular alga, resuspended in medium flushed with air enriched with 5% CO₂, assimilated NH₄⁺ at high rates both in the light and in the dark. The assimilation of NO₃⁻, by contrast, was inhibited by 63% in the dark. In cell suspensions flushed with CO₂-free air, NH₄⁺ assimilation decreased with time both in the light and in the dark and ceased almost completely after 90 min. The addition of CO₂ completely restored the capacity of the alga to assimilate NH₄⁺. NO₃⁻ assimilation, by contrast, was 33% higher in the absence of CO₂ and was linear with time. It is suggested that NO₃⁻ and NH₄⁺ metabolism in C. caldarium are differently controlled in response to the light and carbon conditions of the cell.     

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.     

Promoter structure of mouse RANKL/TRANCE/OPGL/ODF gene

Receptor activator of NF-kappa B ligand (RANKL)/tumor necrosis factor-related activation induced cytokine (TRANCE)/osteoprotegerin ligand (OPGL)/osteoclast differentiation factor (ODF) is a membrane-bound signal transducer responsible for differentiation and maintenance of osteoclasts. To elucidate the mechanism regulating RANKL/TRANCE/OPGL/ODF gene expression, we cloned the 5'-flanking basic promoter region of the mouse RANKL/TRANCE/OPGL/ODF gene and characterized it by transient transfection studies and genomic Southern blot analysis. Inverted TATA- and CAAT-boxes and a putative Cbfa1/Osf2/AML3 binding domain constituted the basic promoter structure. The repeated half-sites for the vitamin D3 (VitD3) and glucocorticoid receptors were located at -935 and -640, respectively. Transient transfection studies revealed that short-term treatment with 1alpha,25(OH)2 VitD3 or dexamethasone increased luciferase activity up to 204% and 178%, respectively; on the other hand, treatment with dibutyryl cyclic AMP did not affect the promoter activity. Since the expression of Cbfa1/Osf2/AML3 is also regulated by VitD3, 1alpha,25(OH)2 VitD3 might affect RANKL/TRANCE/OPGL/ODF gene expression both directly and indirectly. CpG methylation was observed dominantly in mouse stromal cells, ST2, of a later passage which ceased to support in vitro osteoclastogenesis, suggesting that the methylation status of the CpG loci in the RANKL/TRANCE/OPGL/ODF gene promoter may be one of the influential cis-regulating factors.     

Relationship between C2H2 reduction, H2 evolution and 15N2 fixation in root nodules of pea (Pisum sativum)

The quantitative relationship between C₂H₂ reduction, H₂ evolution and ¹⁵N₂ fixation was investigated in excised root nodules from pea plants ( Pisum sativum L. cv. Bodil) grown under controlled conditions. The C₂H₂/N₂ conversion factor varied from 3.31 to 5.12 between the 32nd and the 67th day after planting. After correction for H₂ evolution in air, the factor (C₂H₂-H₂)/N₂ decreased to values near the theoretical value 3, or in one case to a value significantly ( P < 0.05) below 3. The proportion of the total electron flow through nitrogenase, which is not wasted in H₂ production but used for N₂ reduction, is often stated as the relative efficiency (1-H₂/C₂H₂). This factor varied significantly ( P < 0.05) during the growth period. The actual allocation of electrons to H₂ and N₂, expressed as the H₂/N₂ ratio, was independent of plant age, however. This discrepancy and the observation that the (C₂H₂-H₂)/N₂ conversion factor tended to be lower than 3, suggests that the C₂H₂reduction assay underestimates the total electron flow through nitrogenase.