Mass spectrometric quantification of neutral and sialylated N-glycans from a recombinant therapeutic glycoprotein produced in the two Chinese hamster ovary cell lines

Quality control and assurance of glycan profiles of a recombinant glycoprotein from lot to lot is a critical issue in the pharmaceutical industry. To develop an easy and simple quantitative and qualitative glycan profile method based on matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), the modification with Girard’s reagent T (GT) was exploited. Because GT-derivatized quantification of oligosaccharides using MALDI-TOF MS is possible only with neutral glycans, sialylated glycans are not subjected to quantitative analysis with MALDI-TOF MS. To solve this problem, mild methyl esterification and subsequent GT derivatization were employed, enabling us to perform rapid qualitative and quantitative analysis of sialylated and neutral N-linked oligosaccharides using MALDI-TOF MS. This modified method was used in the comparative quantification of N-glycans from the recombinant therapeutic glycoprotein expressed in two different Chinese hamster ovary (CHO) cell lines. The percentages of sialylated N-glycans to total were 22.5 and 5.2% in CHO-I and CHO-II cells, respectively, resulting in a significant difference in the biological activity of the recombinant glycoprotein.     

Heterologous expression of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">NPR1</Emphasis> (<Emphasis Type="Italic">AtNPR1</Emphasis>) enhances oxidative stress tolerance in transgenic tobacco plants

In Arabidopsis, NPR1 (non-expressor of pathogenesis related genes 1, AtNPR1) functions downstream of salicylic acid (SA) and modulates the SA mediated systemic acquired resistance. It is also involved in a cross talk with the jasmonate pathway that is essential for resistance against herbivores and necrotrophic pathogens. Overexpression of AtNPR1 in transgenic plants resulted in enhanced disease resistance. Recently, tobacco transgenic plants expressing AtNPR1 were shown to be tolerant to the early instars of Spodoptera litura (Meur et al., Physiol Plant 133:765–775, 2008). In this communication, we show that the heterologous expression of AtNPR1 in tobacco has also enhanced the oxidative stress tolerance. The transgenic plants exhibited enhanced tolerance to the treatment with methyl viologen. This tolerance was associated with the constitutive upregulation of PR1, PR2 (glucanase), PR5 (thaumatin like protein), ascorbate peroxidase (APX) and Cu²⁺/Zn²⁺ superoxide dismutase (SOD). This is the first demonstration of the novel function of heterologous expression of AtNPR1 in oxidative stress tolerance in transgenic tobacco.     

Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor

Hairy root cultures of Salvia sclarea were grown in shake flasks and 10 L nutrient sprinkle bioreactor, running for 30 days and the effects of methyl jasmonate (MJ) on their growth and capacity to accumulate diterpenoids were measured. We found that MJ concentration and exposure time to the elicitor were factors that strongly affected the diterpenoid production. The highest diterpenoid accumulation (67.5 ± 7.1 mg g⁻¹ dry weight, calculated as a sum of ferruginol, salvipisone, aethiopinone and 1-oxoaethiopinone) without reduction of biomass, was achieved when the 23-day-old hairy roots in bioreactor culture were exposed to 125 μM MJ for 7 days. The roots produced 9 and 3.8 times as much aethiopinone (40 ± 5.9 mg g⁻¹ dry weight) and salvipisone (12.6 ± 0.4 mg g⁻¹ dry weight), respectively, as roots cultured in shake flasks. Our results imply that cultivation of S. sclarea hairy roots in sprinkle bioreactor after elicitation with MJ may be valuable to enhance production of the bioactive diterpenoids.     

Elicitor-induced accumulation of stilbenes in cell suspension cultures of Cayratia trifolia (L.) Domin

Cell cultures of Cayratia trifolia (Vitaceae), a tropical lianas, were maintained in Murashige and Skoog’s medium containing 0.25 mg l⁻¹ NAA, 0.2 mg l⁻¹ kinetin and casein hydrolysate 250 mg l⁻¹. Cell suspension cultures of C. trifolia accumulate stilbenes (piceid, resveratrol, viniferin, ampelopsin), which on elicitation by any of 500 μM salicylic acid, 100 μM methyl jasmonate, 500 μM ethrel and 500 mg l⁻¹ yeast extract, added on the 7th day, were enhanced by 3- to 6-fold (5–11 mg l⁻¹) by the 15th day.     

Differential Secretion and Accumulation of Terpene Indole Alkaloids in Hairy Roots of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> Treated with Methyl Jasmonate

The induction of several secondary metabolites in plants is one of the most commonly observed effects after the external addition of methyl jasmonate (MeJA). After the elicitation of Catharanthus roseus hairy roots with different concentrations of MeJA, changes in the accumulation of alkaloids such as ajmalicine, serpentine, ajmaline and catharanthine were observed. In addition to the increased accumulation of alkaloids in the tissues, the root exudation of phytochemicals increased compared to that of the non-treated control hairy roots. Moreover, MeJA induced differential secretion of several C. roseus hairy root metabolites.     

DrosophilaCG10527 mutants are resistant to juvenile hormone and its analog methoprene

Juvenile hormone (JH) is critical for development, metamorphosis, and reproduction in insects. While the physiological importance of JH has been appreciated for decades, its biosynthetic pathway and molecular action remain poorly understood. DrosophilaCG10527 encodes a protein with high homology to crustacean farnesoic acid methyltransferase (FAMeT) that converts farnesoic acid to methyl farnesoate (MF), a precursor of JH, but its in vivo functions remain unclear. Here we report that CG10527 is expressed widely in secondary cells in the male accessory glands, in ovarian follicle cells, and in glial cells in the nervous system. Furthermore, CG10527 is expressed abundantly in the corpora allata where JH is synthesized. To understand the physiological functions of CG10527, we generated specific CG10527 deletions. Phenotypic analysis showed that CG10527 null mutants are fully viable and fertile in both sexes, indicating that CG10527 is not essential for survival and fertility. Surprisingly, CG10527 mutants showed no defects in the biosynthesis of MF and JH. However, CG10527 mutants were 3-5 times more resistant than wild-type flies to topically applied MF and JH as well as the JH analog methoprene at both sub-lethal and lethal doses. Taken together, our data indicate that DrosophilaCG10527 plays little, if any, role in JH biosynthesis but may participate in the JH signaling pathway.     

A combined nuclear magnetic resonance and molecular dynamics study of the two structural motifs for mixed-linkage β-glucans: methyl β-cellobioside and methyl β-laminarabioside

The conformational and hydration properties of the two disaccharides methyl β-cellobioside and methyl β-laminarabioside were investigated by NMR spectroscopy and explicit solvation molecular dynamics simulations using the carbohydrate solution force field (CSFF). Adiabatic maps produced with this force field displayed 4 minima A: (Φ = 300°, Ψ = 280°), B: (Φ = 280°, Ψ = 210°), C: (Φ = 260°, Ψ = 60°), and D: (Φ = 60°, Ψ = 260°) for methyl β-cellobioside and 3 minima A: (Φ = 290°, Ψ = 130°), B: (Φ = 270°, Ψ = 290°), and C: (Φ = 60°, Ψ = 120°) for methyl β-laminarabioside. Molecular dynamics simulations were initiated from all minima. For each disaccharide, the simulation started from the A minimum was conducted for 50 ns, while the other minima were explored for 10 ns. The simulations revealed two stable minima for both compounds. For methyl β-cellobioside, the simulation minima in aqueous solution were shifted from their adiabatic map counterparts, while the simulation minima for methyl β-laminarabioside coincided with the corresponding adiabatic map minima. To validate the simulation results, NMR-derived NOEs and coupling constants across the glycoside linkage, ³J_HC and ³J_CH, were compared with values calculated from the MD trajectories. For each disaccharide, the best agreement was obtained for the simulations started at the A minimum. For both compounds, inter-ring water bridges in combination with the direct hydrogen bonds between the same groups were found to be determining factors for the overall solution structure of the disaccharides which differed from solid-state structures. Comparison with helical parameters showed that the preferred glycosidic dihedral configurations in the methyl β-cellobioside simulation were not highly compatible with the structure of cellulose, but that curdlan helix structures agreed relatively well with the methyl β-laminarabioside simulation. Polymers generated using glycosidic dihedral angles from the simulations revealed secondary structure motifs that that may help to elucidate polymer associations and small-molecule binding.     

SABP2, a methyl salicylate esterase is required for the systemic acquired resistance induced by acibenzolar-S-methyl in plants

Tobacco SABP2, a 29 kDa protein catalyzes the conversion of methyl salicylic acid (MeSA) into salicylic acid (SA) to induce SAR. Pretreatment of plants with acibenzolar-S-methyl (ASM), a functional analog of salicylic acid induces systemic acquired resistance (SAR). Data presented in this paper suggest that SABP2 catalyzes the conversion of ASM into acibenzolar to induce SAR. Transgenic SABP2-silenced tobacco plants when treated with ASM, fail to express PR-1 proteins and do not induce robust SAR expression. When treated with acibenzolar, full SAR is induced in SABP2-silenced plants. These results show that functional SABP2 is required for ASM-mediated induction of resistance.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.     

甲基沿阶草酮甲对干眼症小鼠的治疗作用及机制

摘要 目的：探讨甲基沿阶草酮甲（Methyl ophiopogonanone A，MOA）对干眼症（Dry eye disease，DED）小鼠的治疗作用及机制。方法：将雄性BALB/c小鼠（7~8周龄，体重18-22 g）随机分为5组（n=12）：对照组、DED组、DED+10MOA组、DED+20MOA组和DED+40MOA组。对照组为正常小鼠，其他组小鼠双眼滴入质量浓度为0.2%的苯扎氯铵溶液诱导DED模型小鼠，每日1次，连续6周。建模后，对照组和DED组小鼠腹腔注射0.5 mL的1%二甲基亚砜溶液，DED+10MOA组、DED+20MOA组和DED+40MOA组小鼠依次腹腔注射0.5 mL剂量为10、20和40 mg/kg的MOA，每天1次，共给药28天。治疗结束后，检测各组小鼠的泪液分泌量和角膜荧光素钠染色分级，并进行角膜苏木素伊红（Hematoxylin and eosin，HE）染色、结膜过碘酸-雪夫（Periodic acid-Schiff，PAS）染色和角膜TUNEL染色。通过Western blot检测角膜K10蛋白表达水平。检测角膜氧化应激指标丙二醛（Malondialdehyde，MDA）和超氧化物歧化酶（Superoxide dismutase，SOD）水平。通过实时荧光定量逆转录聚合酶链式反应检测角膜中肿瘤坏死因子-α（Tumor necrosis factor-α，TNF-α）、白细胞介素-1β（Interleukin-1β，IL-1β）、白细胞介素-6（Interleukin-6，IL-6）、B淋巴细胞瘤-2相关X蛋白（Bcl2-associated X，Bax）和B淋巴细胞瘤-2（B-cell lymphoma-2，Bcl-2）的mRNA水平。结果：与对照组比较，DED组小鼠的泪液分泌量降低（P<0.05），角膜荧光素钠染色分级升高（P<0.05），角膜出现明显病变，结膜杯状细胞数量降低（P<0.05）。角膜Bcl-2 mRNA相对表达量、SOD水平降低（P<0.05），角膜TUNEL阳性率、Bax mRNA相对表达量、K10蛋白相对表达量、MDA、TNF-α、IL-1β和IL-6水平升高（P<0.05）。与DED组比较，DED+10MOA组、DED+20MOA组和DED+40MOA组的泪液分泌量升高（P<0.05），角膜荧光素钠染色分级降低（P<0.05），角膜病变减轻，结膜杯状细胞数量升高（P<0.05）。角膜TUNEL阳性率、Bax mRNA、K10蛋白相对表达量、MDA、TNF-α、IL-1β和IL-6水平降低（P<0.05），Bcl-2 mRNA相对表达量、SOD水平升高（P<0.05）。结论：甲基沿阶草酮甲有效减轻干眼症小鼠的症状及眼表病变，其机制可能与抑制杯状细胞凋亡和氧化应激及炎症反应有关。