HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Identification of N-terminal helix capping boxes by means of 13C chemical shifts

Summary We have examined the ¹³C and ¹³C chemical shifts of a number of proteins and found that their values at the N-terminal end of a helix provide a good predictor for the presence of a capping box. A capping box consists of a hydrogen-bonded cycle of four amino acids in which the side chain of the N-cap residue forms a hydrogen bond with the backbone amide of the N3 residue, whose side chain in turn may accept a hydrogen bond from the amide of the N-cap residue. The N-cap residue exhibits characteristic values for its backbone torsion angles, with and clustering around 94±15° and 167±5°, respectively. This is manifested by a 1–2 ppm upfield shift of the ¹³C resonance and a 1–4 ppm downfield shift of the ¹³C resonance, relative to their random coil values, and is mainly associated with the unusually large value of . The residues following the N-cap residue exhibit downfield shifts of 1–3 ppm for the ¹³C resonances and small upfield shifts for the ¹³C ones, typical of an -helix.     

Carbon distribution in grass (Festuca pratensis L.) during regrowth after cutting—utilization of stored and newly assimilated carbon

Distribution of net assimilated C in meadow fescue (Fectuca pratensi L.) was followed before and after cutting of the shoots. Plants were continuously labelled in a growth chamber with ¹⁴C-labelled CO₂ in the atmosphere from seedling to cutting and with ¹³C-labelled CO₂ in the atmosphere during regrowth after the cutting. Labelled C, both ¹⁴C and ¹³C, was determined at the end of the two growth periods in shoots, crowns, roots, soil and rhizosphere respiration. Distribution of net assimilated C followed almost the same pattern at the end of the two growth periods, i.e. at the end of the ¹⁴C- and the ¹³C-labelling periods. Shoots retained 71–73% of net assimilated C while 9% was detected in the roots and 11–14% was released from the roots, determined as labelled C in soil and as rhizosphere respiration. At the end of the 2nd growth period, after cutting and regrowth, 21% of the residual plant ¹⁴C at cutting (¹⁴C in crowns and roots) was found in the new shoot biomass. A minor part of the residual plant ¹⁴C, 12%, was lost from the plants. The decreases in ¹⁴C in crowns and roots during the regrowth period suggest that ¹⁴C in both crowns and roots was translocated to new shoot tissue. Approximately half of the total root C at the end of the regrowth period after cutting was ¹³C-labelled C and thus represents new root growth. Root death after cutting could not be determined in this experiment, since the decline in root ¹⁴C during the regrowth period may also be assigned to root respiration, root exudation and translocation to the shoots. ei]{gnH}{fnLambers} ei]{gnA C}{fnBorstlap}     

Cerebral Metabolic Compartmentation as Revealed by Nuclear Magnetic Resonance Analysis of D-[1-13C]Glucose Metabolism

Abstract: Nuclear magnetic resonance (NMR) was used to study the metabolic pathways involved in the conversion of glucose to glutamate, γ-aminobutyrate (GABA), glutamine, and aspartate. d -[1^-13C]Glucose was administered to rats intraperitoneally, and 6, 15, 30, or 45 min later the rats were killed and extracts from the forebrain were prepared for ¹³C-NMR analysis and amino acid analysis. The absolute amount of ¹³C present within each carbon-atom pool was determined for C-2, C-3, and C-4 of glutamate, glutamine, and GABA, for C-2 and C-3 of aspartate, and for C-3 of lactate. The natural abundance ¹³C present in extracts from control rats was also determined for each of these compounds and for N-acetylaspartate and taurine. The pattern of labeling within glutamate and GABA indicates that these amino acids were synthesized primarily within compartments in which glucose was metabolized to pyruvate, followed by decarboxylation to acetyl-CoA for entry into the tricarboxylic acid cycle. In contrast, the labeling pattern for glutamine and aspartate indicates that appreciable amounts of these amino acids were synthesized within a compartment in which glucose was metabolized to pyruvate, followed by carboxylation to oxaloacetate. These results are consistent with the concept that pyruvate carboxylase and glutamine synthetase are glia-specific enzymes, and that this partially accounts for the unusual metabolic compartmentation in CNS tissues. The results of our study also support the concept that there are several pools of glutamate, with different metabolic turnover rates. Our results also are consistent with the concept that glutamine and/or a tricarboxylic acid cycle intermediate is supplied by astrocytes to neurons for replenishing the neurotransmitter pool of GABA. However, a similar role for astrocytes in replenishing the transmitter pool of glutamate was not substantiated, possibly due to difficulties in quantitating satellite peaks arising from ¹³C-¹³C coupling.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Biosynthesis of 12α-and 13-hydroxylated gibberellins in a cell-free system from Cucurbita maxima endosperm and the identification of new endogenous gibberellins

Gibberellin (GA) biosynthesis in cell-free systems from Cucurbita maxima L. endosperm was reinvestigated using incubation conditions different from those employed in previous work. The metabolism of GA₁₂ yielded GA₁₃, GA₄₃ and 12α-hydroxyGA₄₃ as major products, GA₄, GA₃₇, GA₃₉, GA₄₆ and four unidentified compounds as minor products. The intermediates GA₁₅, GA₂₄ and GA₂₅ accumulated at low protein concentrations. The structure of the previously uncharacterised 12α-hydroxyGA₄₃ was inferred from its mass spectrum and by its formation from both GA₃₉ and GA₄₃. Gibberellin A₃₉ and 12α-hydroxyGA₄₃ were formed by a soluble 12α-hydroxylase that had not been detected before. Gibberellin A₁₂-aldehyde was metabolised to essentially the same products as GA₁₂ but with less efficiency. A new 13-hydroxylation pathway was found. Gibberellin A₅₃, formed from GA₁₂ by a microsomal oxidase, was converted by soluble 2-oxoglutarate-dependent oxidases to GA₁ GA₂₃, GA₂₈, GA₄₄, and putative 2β-hydroxyGA₂₈. Minor products were GA₁₉, GA₂₀, GA₃₈ and three unidentified GAs. Microsomal 13-hydroxylation (the formation of GA₅₃) was suppressed by the cofactors for 2-oxoglutarate-dependent enzymes. Reinvestigation of the endogenous GAs confirmed the significance of the new metabolic products. In addition to the endogenous GAs reported by Blechschmidt et al. (1984, Phytochemistry 23, 553–558), GA₁, GA₈, GA₂₅, GA₂₈, GA₃₆, GA₄₈ and 12α-hydroxyGA₄₃ were identified by full-scan capillary gas chromatography-mass spectrometry and Kovats retention indices. Thus both the 12α-hydroxylation and the 13-hydroxylation pathways found in the cell-free system operate also in vivo, giving rise to 12α-hydroxyGA₄₃ and GA₁ (or GA₈), respectively, as their end products. Evidence for endogenous GA₂₀ and GA₂₄ was also obtained but it was less conclusive due to interference.     

Cell wall synthesis during growth and maturation of Nitella internodal cells

An improved ¹³C-density-labeling method was used to study cell wall synthesis in rapidly expanding, slowly expanding and recently mature internodes of Nitella translucens var axillaris (A.Br.) R.D.W. As cells matured, the rate of wall synthesis slowed and the deposition of cellulose microfibrils changed from a predominantly transverse direction in the primary wall of rapidly expanding internodes to a helicoidal array in the secondary wall of mature internodes. The secondary wall was characterized by relatively higher rates of cellulose synthesis and lower rates of pectin synthesis than the primary wall. The synthesis of xyloglucan also decreased markedly at the transition to secondary wall synthesis, while the synthesis of mannose-rich hemicellulose increased. Even though structural differences were striking between the primary and secondary walls of Nitella, compositional differences between the two types of wall were quantitative rather than qualitative. The authors appreciate the assistance of Martin Yousef with the electron microscopy.     

黄花棘豆的喹诺里西定生物碱

豆科棘豆属有毒植物含多种有毒生物碱,有关棘豆属中生物碱成分的研究,早在1929年Couch由兰伯氏棘豆(Oxytropis lambertii DC.)中分离到一种多羟基化的含氮有机化合物,这是最早报道由棘豆属植物中分离出的生物碱成分,由于当时条件限制未能确定其结构。1982年美国农业部西部研究中心的植物化学家Molyneux从绢毛棘豆(Oxytropis sericea)中分离到苦马豆碱(swainsonine)等吲哚里西定生物碱(indolizidinealkaloids);1989年沈阳药学院于荣敏等由小花棘豆(Oxytropis glabra DC.)中分离出多种喹诺里西定生物碱。喹诺里西定生物碱具有多种生理活性,对试验动物中枢神经系统产生抑制,呼吸抑制或兴奋,致幻、流产和致畸等作用。本文报道由黄花棘豆(Oxytropis ochrocephala Bunge)中测得4种喹诺里西定生物碱。     

Lipopolysaccharide Isolated from a New O-Antigenic Form (O13) of Vibrio parahaemolyticus

The chemical properties of a lipopolysaccharide (LPS) isolated from a new O-antigenic form (O13) of Vibrio parahaemolyticus were investigated. The LPS contained glucose, galactose, L -glycero-D -manno-heptose and glucosamine. 2-Keto-3-deoxy-octonate (KDO) was not detected in the LPS by the periodate-thiobarbituric acid test (Weissbach's reaction) under conventional hydrolysis conditions. Instead, phosphorylated KDO (X1 and X2) was found in its strong-acid hydrolysate. This sugar composition was identical to that of V. parahaemolyticus O3, O5 and O11 LPS, indicating that, based on the sugar composition, O13 LPS belongs to Chemotype III to which O3, O5 and O11 belong. In addition, structural study demonstrated the presence of KDO 4-phosphate in its inner-core region.     

Rice ubiquitin-conjugating enzyme OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a

Ubc13 is required for Lys63-linked polyubiquitination and innate immune responses in mammals, but its functions in plant immunity still remain largely unknown. Here, we used molecular biological, pathological, biochemical, and genetic approaches to evaluate the roles of rice OsUbc13 in response to pathogens. The OsUbc13-RNA interference (RNAi) lines with lesion mimic phenotypes displayed a significant increase in the accumulation of flg22- and chitin-induced reactive oxygen species, and in defence-related genes expression or hormones as well as resistance to Magnaporthe oryzae and Xanthomonas oryzae pv oryzae. Strikingly, OsUbc13 directly interacts with OsSnRK1a, which is the α catalytic subunit of SnRK1 (sucrose non-fermenting-1-related protein kinase-1) and acts as a positive regulator of broad-spectrum disease resistance in rice. In the OsUbc13-RNAi plants, although the protein level of OsSnRK1a did not change, its activity and ABA sensitivity were obviously enhanced, and the K63-linked polyubiquitination was weaker than that of wild-type Dongjin (DJ). Overexpression of the deubiquitinase-encoding gene OsOTUB1.1 produced similar effects with inhibition of OsUbc13 in affecting immunity responses, M. oryzae resistance, OsSnRK1a ubiquitination, and OsSnRK1a activity. Furthermore, re-interfering with OsSnRK1a in one OsUbc13-RNAi line (Ri-3) partially restored its M. oryzae resistance to a level between those of Ri-3 and DJ. Our data demonstrate OsUbc13 negatively regulates immunity against pathogens by enhancing the activity of OsSnRK1a.