栀子黄色素制备藏红花酸研究

以栀子黄色素为原料,对其水解反应制备藏红花酸及其纯化条件进行了研究,结果表明,栀子黄色素经碱水解法制备藏红花酸的最佳条件为:KOH溶液10%、温度为60℃、反应时间120 min,水解所得藏红花酸通过甲醇除杂,重结晶后,所得结晶其mp.、uv与文献报道一致,其质谱的诱导碰撞解离技术获得碎片裂解信息均表明所得到的结晶为藏红花酸,经HPLC检测纯度达到98.43%,得率为8.42%。     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

Volatile metabolic profiles of cell suspension cultures of Lavandula vera,Nicotiana tabacum and Helianthus annuus,cultivated under different regimes

Cell suspension cultures of Lavandula vera (Lamiaceae), Nicotiana tabacum (Solanaceae), and Helianthus annuus (Asteraceae) were cultivated in three different ways: in shake flasks both as free suspensions and in two‐phase systems (in the presence of Amberlite XAD‐4 resin as a second phase), as well as in 3‐L stirred tank reactor, and their volatile metabolic profiles were studied using GC‐MS. A number of compounds, some of them having allelochemical and biological activities, were identified in all the three cell suspension cultures under study. Also the presence of some compounds, unusual for the intact plants, was observed. It was found that the cultivation mode strongly influences the production and the transport (secretion into the culture medium) of the low‐molecular‐mass volatile metabolites. Principal component analyses of 12 common hydrocarbons showed discrimination between the different cultivation modes (shake flasks and two‐phase systems cultivation) by first principal component (PC1) and second principal component (PC2).     

Proteome and radioimmunoassay analyses of pituitary hormones and proteins in response to feed restriction of dairy cows

The hypothalamic-pituitary system controls homeostasis during feed energy reduction. In order to examine which pituitary proteins and hormone variants are potentially associated with metabolic adaptation, pituitary glands from ad libitum and energy restrictively fed dairy cows were characterized using RIA and 2-DE followed by MALDI-TOF-MS. We found 64 different spots of regulatory hormones: growth hormone (44), preprolactin (16), luteinizing hormone (LH) (1), thyrotropin (1), proopiomelanocortin (1) and its cleavage product lipotropin (1), but none of these did significantly differ between feeding groups. Quantification of total pituitary LH and prolactin concentrations by RIA confirmed the results obtained by proteome analysis. Also, feed energy restriction provoked increasing non-esterified fatty acid, decreasing prolactin, but unaltered glucose, LH and growth hormone plasma concentrations. Energy restriction decreased the expression of glial fibrillary acidic protein, triosephosphate isomerase, purine-rich element-binding protein A and elongation factor Tu, whereas it increased expression of proline synthetase co-transcribed homolog, peroxiredoxin III, β-tubulin and annexin A5 which is involved in the hormone secretion process. Our results indicate that in response to feed energy restriction the pituitary reservoir of all posttranslationally modified hormone forms remains constant. Changing plasma hormone concentrations are likely attributed to a regulated releasing process from the gland into the blood.     

Probing Interactions between CLIP-170, EB1, and Microtubules

Cytoplasmic linker protein 170 (CLIP-170) is a microtubule (MT) plus-end tracking protein (+ TIP) that dynamically localizes to the MT plus end and regulates MT dynamics. The mechanisms of these activities remain unclear because the CLIP-170-MT interaction is poorly understood, and even less is known about how CLIP-170 and other + TIPs act together as a network. CLIP-170 binds to the acidic C-terminal tail of α-tubulin. However, the observation that CLIP-170 has two CAP-Gly (cytoskeleton-associated protein glycine-rich) motifs and multiple serine-rich regions suggests that a single CLIP-170 molecule has multiple tubulin binding sites, and that these sites might bind to multiple parts of the tubulin dimer. Using a combination of chemical cross-linking and mass spectrometry, we find that CLIP-170 binds to both α-tubulin and β-tubulin, and that binding is not limited to the acidic C-terminal tails. We provide evidence that these additional binding sites include the H12 helices of both α-tubulin and β-tubulin and are significant for CLIP-170 activity. Previous work has shown that CLIP-170 binds to end-binding protein 1 (EB1) via the EB1 C-terminus, which mimics the acidic C-terminal tail of tubulin. We find that CLIP-170 can utilize its multiple tubulin binding sites to bind to EB1 and MT simultaneously. These observations help to explain how CLIP-170 can nucleate MTs and alter MT dynamics, and they contribute to understanding the significance and properties of the + TIP network.     

Biogas generation apple pulp

In view of the pressing problem that appears in our region (Asturias, north of Spain) with the residues from the cider production, it was decided to test this kind of material as a co-substrate joint with slaughterhouse waste in a laboratory unit.     

Distinct functions of maternal and somatic Pat1 protein paralogs

We previously identified Xenopus Pat1a (P100) as a member of the maternal CPEB RNP complex, whose components resemble those of P-(rocessing) bodies, and which is implicated in translational control in Xenopus oocytes. Database searches have identified Pat1a proteins in other vertebrates, as well as paralogous Pat1b proteins. Here we characterize Pat1 proteins, which have no readily discernable sequence features, in Xenopus oocytes, eggs, and early embryos and in human tissue culture cells. xPat1a and 1b have essentially mutually exclusive expression patterns in oogenesis and embryogenesis. xPat1a is degraded during meiotic maturation, via PEST-like regions, while xPat1b mRNA is translationally activated at GVBD by cytoplasmic polyadenylation. Pat1 proteins bind RNA in vitro, via a central domain, with a preference for G-rich sequences, including the NRAS 5′ UTR G-quadruplex-forming sequence. When tethered to reporter mRNA, both Pat proteins repress translation in oocytes. Indeed, both epitope-tagged proteins interact with the same components of the CPEB RNP complex, including CPEB, Xp54, eIF4E1b, Rap55B, and ePAB. However, examining endogenous protein interactions, we find that in oocytes only xPat1a is a bona fide component of the CPEB RNP, and that xPat1b resides in a separate large complex. In tissue culture cells, hPat1b localizes to P-bodies, while mPat1a-GFP is either found weakly in P-bodies or disperses P-bodies in a dominant-negative fashion. Altogether we conclude that Pat1a and Pat1b proteins have distinct functions, mediated in separate complexes. Pat1a is a translational repressor in oocytes in a CPEB-containing complex, and Pat1b is a component of P-bodies in somatic cells.     

水稻化感品种根分泌物中非酚酸类化感物质的鉴定与抑草活性

水稻化感品种能从根系分泌释放化感作用物质 ,长期以来 ,酚酸类物质被认为是水稻根分泌的主要化感物质 ,但这一结论常常被质疑。利用连续循环和直接树脂吸收两种方法采集典型的水稻化感品种 PI31 2 777幼苗的根分泌物 ,并用液相色谱 /质谱(L C/ MS)联用技术鉴定了根分泌物中的非酚酸类物质。结果显示 ,水稻 PI31 2 777幼苗根系能分泌释放 7-甲氧基羟基肟酸、羟基肟酸、3-异丙基 - 5 -乙酰氧基环己烯酮 - 1、5 ,7,4′-三羟基 - 3′,5′-二甲氧基黄酮、二萜内酯 A和二萜内酯 B6个非酚酸类化合物。经液相色谱 (HPL C)定量分析 ,这些化合物在水稻生长 1 0 d的根分泌物中的浓度为 5～ 1 9μmol/ L。进一步的生测结果显示 ,这些化合物在其释放的浓度范围能对稻田常见的稗草和异型莎草有抑制活性 ,尤其是这些化合物的等摩尔混合物的抑草活性增加 ,同时水稻根分泌物的抑草活性与土壤载体显著相关。表明羟基肟酸、环己烯酮、黄酮和二萜内酯四类非酚酸类物质是水稻的主要化感物质 ,这与近期愈来愈多的研究结果一致