Kinetic Basis for the Conjugation of Auxin by a GH3 Family Indole-acetic Acid-Amido Synthetase

The GH3 family of acyl-acid-amido synthetases catalyze the ATP-dependent formation of amino acid conjugates to modulate levels of active plant hormones, including auxins and jasmonates. Initial biochemical studies of various GH3s show that these enzymes group into three families based on sequence relationships and acyl-acid substrate preference (I, jasmonate-conjugating; II, auxin- and salicylic acid-conjugating; III, benzoate-conjugating); however, little is known about the kinetic and chemical mechanisms of these enzymes. Here we use GH3-8 from Oryza sativa (rice; OsGH3-8), which functions as an indole-acetic acid (IAA)-amido synthetase, for detailed mechanistic studies. Steady-state kinetic analysis shows that the OsGH3-8 requires either Mg²⁺ or Mn²⁺ for maximal activity and is specific for aspartate but accepts asparagine as a substrate with a 45-fold decrease in catalytic efficiency and accepts other auxin analogs, including phenyl-acetic acid, indole butyric acid, and naphthalene-acetic acid, as acyl-acid substrates with 1.4–9-fold reductions in k_cat/K_m relative to IAA. Initial velocity and product inhibition studies indicate that the enzyme uses a Bi Uni Uni Bi Ping Pong reaction sequence. In the first half-reaction, ATP binds first followed by IAA. Next, formation of an adenylated IAA intermediate results in release of pyrophosphate. The second half-reaction begins with binding of aspartate, which reacts with the adenylated intermediate to release IAA-Asp and AMP. Formation of a catalytically competent adenylated-IAA reaction intermediate was confirmed by mass spectrometry. These mechanistic studies provide insight on the reaction catalyzed by the GH3 family of enzymes to modulate plant hormone action.     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

微小根毛霉感染机理研究

本文在首次报告二例肺结核、糖尿病患者合并感染肺微小根毛霉的基础上,进一步探讨免疫功能受损与微小根毛霉感染关系。本文采用不同剂量~(60)Co-γ全身辐照小鼠,然后以不同途径注射同剂量的微小根毛霉的孢囊孢子,观察动物的感染情况和感染后的真菌检出率,结果发现各种辐射剂量均在辐射后7-14日感染菌的检出率最高;各种脏器感染菌的检出率以脾脏最高(66.7—81.8％);淋巴结最低(0.0—25.0％);其他脏器的感染菌检出率也有不同程度的差异。     

中国蚋属一新种（双翅目：蚋科）

本文报道中国蚋属绳蚋亚属一新种－重庆绳蚋,新种Ｓ．（Ｇ．）ｃｈｏｎｇｑｉｎｇｅｎｓｉｓｓｐ．ｎｏｖ．模式标本采自重庆四面山,保存于重庆市卫生防疫站。     

细胞自噬中关键蛋白乙酰化修饰与相关疾病诊治的研究进展

自噬是一种在进化上保守的溶酶体依赖的降解途径。近十多年来,自噬过程的分子机制研究得到了长足的发展。自噬过程中关键蛋白复合物的乙酰化修饰发挥了十分重要的作用。为此,该文阐述了细胞自噬过程中主要蛋白复合物的乙酰化修饰作用进展,并对蛋白质乙酰化修饰与肿瘤、神经退行性疾病等的关系作一总结。总之,自噬过程中蛋白乙酰化修饰已经成为自噬研究的热点之一,随着相关研究的不断深入,其必将为相关疾病的治疗提供重要的理论基础。     

Three isoforms of the Atg16L1 protein contribute different autophagic properties

The mammalian Atg16L1 protein consists of a coiled-coil domain and a tryptophan-aspartic acid (WD) repeat domain and is involved in the process of autophagy. However, the mechanisms underlying the effect of the Atg16L1 isoforms on autophagy remain to be elucidated in humans. In the present study, we successfully cloned three isoforms: Atg16L1-1, which contains the complete sequence; Atg16L1-2, which lacks all of exon 8; and Atg16L1-3, which lacks the coiled-coil domain. Subsequent experiments showed that the three isoforms of Atg16L1 were colocalised with MDC within the cells. Quantitative analysis of fluorescence showed that the average number of dots of Atg16L1-1 that colocalised with MDC was higher than those of Atg16L1-2 and Atg16L1-3. The three isoforms of Atg16L1 also colocalised with the lysosome within the cells. The average number of dots of Atg16L1-1 that colocalised with the lysosome was higher than those of Atg16L1-2 and Atg16L1-3. However, although Atg16L1-1 and Atg16L1-3 colocalised with the mitochondria, Atg16L1-2 did not. Functional analysis showed that overexpression of the three isoforms of Atg16L1 had a stimulative effect on autophagy. Significant increase in the number of positive LC3-II dots per cell was observed in Atg16L1-1 (70.2 ± 2.39 dots); this number was greater than those of the other two isoforms. Atg16L1-2 appeared to have an average of 59.25 ± 2.22 LC3-II dots per cell. Atg16L1-3 appeared to have the least number of LC3-II dots per cell (48.25 ± 2.22 dots) (P < 0.001). Our results indicated that the degree of autophagy varied with different Atg16L1 isoforms. The different domains of Atg16L1 played different roles in the process of autophagy. The coiled-coil domain of Atg16L1 was involved in the process of autophagy.     

Loss of microRNA-132 predicts poor prognosis in patients with primary osteosarcoma

??₂-glycoprotein I (??₂-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of ??₂-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of ??₂-GPI to endothelial cells, we investigated the effect of ??₂-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of ??₂-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that ??₂-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, ??₂-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, ??₂-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that ??₂-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.     

Microbial community responses reduce soil carbon loss in Tibetan alpine grasslands under short‐term warming

Changes in labile carbon (LC) pools and microbial communities are the primary factors controlling soil heterotrophic respiration (R_h) in warming experiments. Warming is expected to initially increase R_h but studies show this increase may not be continuous or sustained. Specifically, LC and soil microbiome have been shown to contribute to the effect of extended warming on R_h. However, their relative contribution is unclear and this gap in knowledge causes considerable uncertainty in the prediction of carbon cycle feedbacks to climate change. In this study, we used a two‐step incubation approach to reveal the relative contribution of LC limitation and soil microbial community responses in attenuating the effect that extended warming has on R_h. Soil samples from three Tibetan ecosystems—an alpine meadow (AM), alpine steppe (AS), and desert steppe (DS)—were exposed to a temperature gradient of 5–25°C. After an initial incubation period, soils were processed in one of two methods: (a) soils were sterilized then inoculated with parent soil microbes to assess the LC limitation effects, while controlling for microbial community responses; or (b) soil microbes from the incubations were used to inoculate sterilized parent soils to assess the microbial community effects, while controlling for LC limitation. We found both LC limitation and microbial community responses led to significant declines in R_h by 37% and 30%, respectively, but their relative contributions were ecosystem specific. LC limitation alone caused a greater R_h decrease for DS soils than AMs or ASs. Our study demonstrates that soil carbon loss due to R_h in Tibetan alpine soils—especially in copiotrophic soils—will be weakened by microbial community responses under short‐term warming.