3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

A carbon-13 nuclear magnetic resonance investigation of the metabolic fluxes associated withy glucose metabolism in human erythrocytes

We have used [2-¹³C]d-glucose and carbon-13 nuclear magnetic resonance (NMR) spectroscopy to investigate metabolic fluxes through the major pathways of glucose metabolism in intact human erythrocytes and to determine the interactions among these pathways under conditions that perturb metabolism. Using the method described, we have been able to measure fluxes through the pentose phosphate pathway, phosphofructokinase, the 2,3-diphosphoglycerate bypass, and phosphoglycerate kinase, as well as glucose uptake, concurrently and in a single experiment. We have measured these fluxes in normal human erythrocytes under the following conditions: (1) fully oxygenated; (2) treated with methylene blue; and (3) deoxygenated. This method makes it possible to monitor various metabolic effects of stresses in normal and pathological states. Not only has ¹³C-NMR spectroscopy proved to be a useful method for measuring in vivo flux through the pentose phosphate pathway, but it has also provided additional information about the cycling of metabolites through the non-oxidative portion of the pentose phosphate pathway. Our evidence from experiments with [1-¹³C]-, [2-¹³C]-, and [3-¹³C]d-glucoses indicates that there is an observable reverse flux of fructose 6-phosphate through the reactions catalyzed by transketolase and transaldolase, even in the presence of a net flux through the pentose phosphate pathway.     

血清GDF-15、hs-cTnT对冠状动脉旁路移植术后新发房颤及近期主要心血管事件的预测效能研究

摘要 目的：分析血清生长分化因子15（GDF-15）、高敏心肌肌钙蛋白T（hs-cTnT）对冠状动脉旁路移植术后新发心房颤动（房颤）及近期主要心血管事件的预测效能。方法：选择自2020年1月至2022年1月在我院行冠状动脉旁路移植术的140例冠心病患者作为研究对象,根据术后是否新发房颤,分为房颤组（46例）和非房颤组（94例）。检测两组术前血清GDF-15、hs-cTnT水平,使用多因素Logistic回归分析血清GDF-15、hs-cTnT与术后新发房颤的关系;随访6个月,观察主要心血管事件发生情况,通过受试者工作特征曲线下面积（AUC）评价血清GDF-15、hs-cTnT对术后新发房颤及近期主要心血管事件的预测效能。结果：两组患者年龄、性别、体重指数等一般资料比较无差异（P＞0.05）;房颤组SYNTAX积分高于非房颤组,差异有统计学意义(P＜0.05);房颤组血清GDF-15、hs-cTnT水平均高于非房颤组（P＜0.05）;经多因素Logistic回归分析,SYNTAX积分、血清GDF-15、hs-cTnT均是冠状动脉旁路移植术后新发房颤的独立预测因素（P＜0.05）;经ROC曲线分析,血清GDF-15联合hs-cTnT预测冠状动脉旁路移植术后新发房颤的AUC为0.933,大于SYNTAX积分的0.790,预测近期主要心血管事件的AUC为0.925,大于SYNTAX积分的0.750（P＜0.05）。结论：血清GDF-15联合hs-cTnT对冠状动脉旁路移植术后新发房颤及近期主要心血管事件均具有良好的预测效能,值得临床予以重视。     

燕麦-绿豆间作效应及氮素转移特性

为探究燕麦(Avena sativa)-绿豆(Phaseolus radiatus)间作效应及氮素转移特性, 在不施氮肥的大田试验条件下, 设置3种种植模式(燕麦单作、绿豆单作和燕麦-绿豆间作), 采用传统挖根法和¹⁵N同位素标记法进行研究。结果表明, 间作系统中燕麦侵袭力强于绿豆, 绿豆生长受到抑制。整个生育期, 间作燕麦地上部干物质积累量比单作增加14.9%-33.1%, 2年成熟期间作燕麦的氮素积累量比单作分别提高53.1%和44.8%; 间作减少了开花结荚期绿豆氮素积累量和根瘤重量, 降低了绿豆的固氮效率, 绿豆的固氮效率2年平均降低23.7%, 生物固氮量平均减少11.66%。间作绿豆向燕麦的氮素转移率2年平均值达31.7%, 氮素转移量为212.16 kg∙hm^-2。燕麦-绿豆间作降低了开花结荚期绿豆的根瘤固氮酶活性和固氮效率, 但绿豆体内氮素转移增加了燕麦对氮素的吸收利用, 实现了地上部与地下部生长的相互调节和促进, 优化了农田生态系统的氮素管理。     

Soil 13C–15N dynamics in an N2-fixing clover system under long-term exposure to elevated atmospheric CO2

Reduced soil N availability under elevated CO₂ may limit the plant's capacity to increase photosynthesis and thus the potential for increased soil C input. Plant productivity and soil C input should be less constrained by available soil N in an N₂‐fixing system. We studied the effects of Trifolium repens (an N₂‐fixing legume) and Lolium perenne on soil N and C sequestration in response to 9 years of elevated CO₂ under FACE conditions. ¹⁵N‐labeled fertilizer was applied at a rate of 140 and 560 kg N ha^?1 yr^?1 and the CO₂ concentration was increased to 60 Pa pCO₂ using ¹³C‐depleted CO₂. The total soil C content was unaffected by elevated CO₂, species and rate of ¹⁵N fertilization. However, under elevated CO₂, the total amount of newly sequestered soil C was significantly higher under T. repens than under L. perenne. The fraction of fertilizer‐N (f_N) of the total soil N pool was significantly lower under T. repens than under L. perenne. The rate of N fertilization, but not elevated CO₂, had a significant effect on f_N values of the total soil N pool. The fractions of newly sequestered C (f_C) differed strongly among intra‐aggregate soil organic matter fractions, but were unaffected by plant species and the rate of N fertilization. Under elevated CO₂, the ratio of fertilizer‐N per unit of new C decreased under T. repens compared with L. perenne. The L. perenne system sequestered more ¹⁵N fertilizer than T. repens: 179 vs. 101 kg N ha^?1 for the low rate of N fertilization and 393 vs. 319 kg N ha^?1 for the high N‐fertilization rate. As the loss of fertilizer‐¹⁵N contributed to the ¹⁵N‐isotope dilution under T. repens, the input of fixed N into the soil could not be estimated. Although N₂ fixation was an important source of N in the T. repens system, there was no significant increase in total soil C compared with a non‐N₂‐fixing L. perenne system. This suggests that N₂ fixation and the availability of N are not the main factors controlling soil C sequestration in a T. repens system.     

Gas chromatographic-mass spectrometric assay for N-2-chloroethylaziridine, a volatile cytotoxic metabolite of cyclophosphamide, in rat plasma

A sensitive and specific method for the quantitative analysis of N-2-chloroethylaziridine (CEA), a volatile cytotoxic metabolite of cyclophosphamide, has been developed using gas chromatography-mass spectrometry and stable isotope dilution techniques. The high volatility problem of CEA during isolation procedure was overcome by the combined use of a deuterium-labeled analog as the internal standard and a Snyder column-concentrator assembly. The assay was found to be linear from 16.7 to 2667 ng/ml in rat plasma with a routine detection limit of 5 ng/ml. The within-run precision at 33, 333 and 1333 ng/ml (n=6) was found to be 4.8, 4.9, and 6.1%, respectively. The between-run precision was 6.4% (n=6). The dichloromethane extraction recoveries at 33, 333, and 1333 ng/ml were found to be 101, 98, and 91%, respectively (all at n=6). However, the overall recovery through extraction and evaporation was only 18.3, 15.2, and 27.7% at 33, 333, and 1333 ng/ml levels, respectively. The analytical method was used to evaluate the generation of CEA from its precursors in sodium phosphate buffer, in cell culture media, and the degradation of CEA in these media. In pH 7.4, 0.067 M sodium phosphate buffer at 37°C, both phosphoramide mustard (PM) and nornitrogen mustard (NNM) were degraded in an apparent first-order fashion with half-lives of 24.8 and 14.5 min, respectively. The generated CEA was rather stable in this buffer and degraded with a half-life of 20 h. It was found that 32% PM and 91% NNM were converted to CEA in pH 7.4, 0.067 M sodium phosphate buffer at 37°C, respectively, and 41% PM was transformed into CEA in RPMI 1640 tissue culture media containing 10% FBS at 37°C. The generated CEA was very stabble in the culture media with a degradation half-life of 265 h.     

Genetic tools for selective labeling of proteins with α-15N-amino acids

Summary A collection of genetic tools that can be used to manipulate amino acid metabolism in Escherichia coli is described. The set comprises 21 strains of bacteria, each containing a different genetic defect that is closely linked to a selectable transposon marker. These tools can be used to construct strains of E. coli with ideal genotypes for residue-specific, selective labeling of proteins with nearly any ¹⁵N-amino acid. By using strains which have been modified to contain the appropriate genetic lesions to control amino acid biosynthesis, dilution of the isotope by endogenous amino acid biosynthesis and scrambling of the label to other types of residues can be avoided.Abbreviations ¹⁵N-amino acid -¹⁵N-amino acid - Cam^R chloramphenicol-resistant - DPA diaminopimelic acid - Hfr high-frequency recombinant - LB Luria broth - Kan^R kanamycin resistant - P1 bacteriophage P1 - pfu plaque-forming units - Str^R streptomycin-resistant - Tet^R tetracycline-resistant     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

1H and 15N NMR resonance assignments and solution secondary structure of oxidized Desulfovibrio desulfuricans flavodoxin

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for 137 of the 146 nonprolyl residues in oxidized Desulfovibrio desulfuricans [Essex 6] flavodoxin. Assignments were obtained by a concerted analysis of the heteronuclear three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets, recorded on uniformly ¹⁵N-enriched protein at 300 K. Numerous side-chain resonances have been partially or fully assigned. Residues with overlapping ¹H^N chemical shifts were resolved by a three-dimensional ¹H-¹⁵N HMQC-NOESY-HMQC spectrum. Medium-and long-range NOEs, ³J_NH coupling constants, and ¹H^N exchange data indicate a secondary structure consisting of five parallel -strands and four -helices with a topology similar to that of Desulfovibrio vulgaris [Hidenborough] flavodoxin. Prolines at positions 106 and 134, which are not conserved in D. vulgaris flavodoxin, contort the two C-terminal -helices.Abbreviations CSI chemical shift index - DQF-COSY double-quantum-filtered correlation spectroscopy - DIPSI decoupling in the presence of scalar interactions - FMN flavin mononucleotide - GARP globally optimized alternating phase rectangular pulse - HMQC heteronuclear multiple-quantum coherence - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser effect - NOESY nuclear Overhauser enhancement spectroscopy - TOCSY total correlation spectroscopy - TPPI time-proportional phase increments - TSP 3-(trimethylsilyl)propionic-2,2,3,3-d ₄ acid, sodium salt     

N-(2-吡啶基)-N'-苯基脲对几种作物种子根、芽生长影响的回归分析

N-（2-吡啶基）-N'-苯基脲（2PU）,属苯基脲衍生物,具有细胞分裂素活性,有关其合成和应用的研究未见报道。作者合成了2PU并进行了生物效应试验研究。结果表明,2PU对作物种子萌发时胚根、胚芽的生长具有明显的促进作用,其根、芽生长与2PU浓度之间的关系符合Y=a＋bX＋eX2＋的数学模型。2PU对小麦根芽和赤豆根芽生长最佳浓度分别是0.001～0.0lppm、0.005～0.05ppm,对黄豆根生长以0.001～0.06ppm最佳。