Both phenolic and acyl glucuronidation pathways of diflunisal are impaired in liver cirrhosis

Summary The pharmacokinetics of diflunisal, a salicylate derivative that undergoes phenolic and acyl glucuronidation as well as sulphate conjugation, has been studied after a single oral dose (250 mg) in patients with cirrhosis (n=5) and in healthy controls (n=5).The plasma clearance of total (bound + unbound) diflunisal was 10.2 ml · min^–1 in the control subjects and it was not affected by cirrhosis (10.9 ml · min^–1). The plasma protein binding of diflunisal was significantly reduced in cirrhosis; the percentage of unbound diflunisal in plasma was 0.089 in the controls and 0.147 in the patients with cirrhosis. Plasma clearance of unbound diflunisal was significantly impaired in cirrhosis: 11.51 · min^–1 in control subjects vs 7.41 · min^–1 in cirrhotics.In cirrhotic patients, the unbound partial clearances to the phenolic and acyl glucuronides were both significantly reduced, by approximately 38%. The unbound partial clearance to the sulphate conjugate was not significantly affected by cirrhosis.The results show that both the phenolic and acyl glucuronidation pathways of diflunisal are equally susceptible to the effects of liver cirrhosis.     

正交试验优选白芨多糖硫酸酯化工艺的研究

目的优选白芨多糖硫酸酯化的最佳工艺。方法采用氯磺酸-吡啶法合成白芨多糖硫酸酯。以取代度和产物量为指标,采用L9(34)正交设计对试剂比例、反应温度和反应时间进行优选。结果吡啶和氯磺酸比例6∶1,反应温度85℃,反应时间2h为比较适宜的工艺条件。结论白芨多糖硫酸酯化优化的工艺条件方法可行,为进一步扩大试验提供了有价值的参数。     

星点设计-效应面法优化白鲜皮多糖硫酸酯化工艺及抗氧化活性

目的 利用星点设计-效应面法优化白鲜皮多糖（Dictamnus dasycarpus polysaccharide,DDP）硫酸酯化工艺,考察DDP修饰前后结构特征及抗氧化活性。方法 采用氯磺酸-吡啶法,以酯化试剂比例、酯化试剂与多糖溶液比例、反应温度、反应时间为自变量,硫酸根取代度（degree of substitution,DS）为因变量,对自变量各水平进行多元回归拟合,利用效应面法筛选最佳工艺并进行预测分析;采用红外光谱及扫描电镜观察其结构特点。测定硫酸酯化白鲜皮多糖（sulfated polysaccharides from Dictamnus dasycarpus,SDDP）对DPPH、OH·、O₂^-·清除能力,考察其抗氧化活性。结果 最佳工艺条件为酯化试剂比例1：4,酯化试剂与多糖溶液比例1：1,反应温度73℃,反应时间5 h。红外光谱显示SDDP在820 cm^-1和1 254 cm^-1附近出现C-O-S和S=O硫酸基特征吸收峰,表明DDP修饰成功;扫描电镜显示SDDP表面粗糙,排列紧密,且块状体积明显小于DDP;DDP和SDDP都有清除DPPH、OH·和O₂^-·能力,且SDDP对DPPH、OH·和O₂^-·清除率强于DDP。结论 星点设计-效应面法优化DDP硫酸酯化工艺方法简便且预测性良好,DDP经硫酸酯化后,抗氧化活性有所提高。     

Fate of goblet cells in experimental colitis

We sought to correlate the characteristic changes in goblet cell morphology in the chronically inflamed large intestine of IL10 ^–/– mice to specific changes in goblet cell gene expression. In healthy as well as IL10 ^–/– mice, marked differences were found among the large intestinal regions in goblet cell morphology and gene expression. The mucin Muc2, which is a major determinant of goblet cell morphology, was expressed in most goblet cells, yet only in cells staining positive for both Alcian blue and high iron diamine. TFF3 was expressed in only a small subset of goblet cells. Inflamed colon of IL10 ^–/– mice still contained high numbers of small, hypotrophic goblet cells with similar histochemical staining and Muc2 and TFF3 expression patterns, contradicting the often reported goblet cell depletion in colitis. Quantitatively, the Muc2 and TFF3 levels remained relatively stabile in IL10 ^–/– mice. Muc2 in distal IL10 ^–/– colon contained significantly less sulfate residues than in controls, which may compromise its protective properties.     

A high frequency missense SULT1B1 allelic variant (L145V) selectively expressed in African descendants exhibits altered kinetic properties

1.?Human cytosolic sulfotransferase 1B1 (SULT1B1) sulfates small phenolic compounds and bioactivates polycyclic aromatic hydrocarbons. To date, no SULT1B1 allelic variants have been well-characterized.

2.?While cloning SULT1B1 from human endometrial specimens, an allelic variant resulting in valine instead of leucine at the 145th amino acid position (L145V) was detected. NCBI reported this alteration as the highest frequency SULT1B1 allelic variant.

3.?L145V frequency comprised 9% of 37 mixed-population human patients and was specific to African Americans with an allelic frequency of 25%. Structurally, replacement of leucine with valine potentially destabilizes a conserved helix (α8) that forms the “floor” of both the substrate and PAPS binding domains. This destabilization results in altered kinetic properties including a four-fold decrease in affinity for PAP (3′, 5′-diphosphoadenosine). K_ms for 3′-phosphoadenosine- 5′-phosphosulfate (PAPS) are similar; however, maximal turnover rate of the variant isoform (0.86?pmol/(min*μg)) is slower than wild-type (WT) SULT1B1 (1.26?pmol/(min*μg)). The L145V variant also displays altered kinetics toward small phenolic substrates, including a diminished p-nitrophenol K_m and increased susceptibility to 1-naphthol substrate inhibition.

4.?No significant correlation between genotype and prostate or colorectal cancer was observed in patients; however, the variant isoform could underlie specific pathologies in sub-Saharan African carriers.     

Regulation of MCF-7 breast cancer cell growth by beta-estradiol sulfation

Estrogen stimulation is an important factor in human breast cancer cell growth and development. Metabolism of -estradiol (E₂), the major endogenous human estrogen, is important in regulating both the level and activity of the hormone in breast tissues. Conjugation of E₂ with a sulfonate moiety is an inactivation process since the sulfate ester formed by this reaction can not bind and activate the estrogen receptor. In human tissues including the breast, estrogen sulfotransferase (EST, SULT1E1) is responsible for high affinity E₂ sulfation activity. EST is expressed in human mammary epithelial (HME) cells but not in most cultured breast cancer cell lines, including estrogen responsive MCF-7 cells. Stable expression of EST in MCF-7 cells at levels similar to those detected in HME cells significantly inhibits cell growth at physiologically relevant E₂ concentrations. The mechanism of cell growth inhibition involves the abrogation of responses observed in growth factor expression in MCF-7 cells following E₂ stimulation. MCF-7 cells expressing EST activity did not show a decrease in estrogen receptor- levels, nor a characteristic increase in progesterone receptor or decrease in transforming growth factor- expression upon exposure to 100 pM or 1 nM E₂. The lack of response in these MCF-7 cells is apparently due to the rapid sulfation and inactivation of free E₂ by EST. These results suggest that loss of EST expression in the transformation of normal breast tissues to breast cancer may be an important factor in increasing the growth responsiveness of preneoplastic or tumor cells to estrogen stimulation.     

Reevaluation of the determinants of tyrosine sulfation

The posttranslational sulfation of tyrosine has been though to be initiated by the recognition of specific consensus features by the sulfating enzyme tyrosylprotein sulfotransferase (TPST). However, using these recognition features to identify new tyrosine sulfation sites misses recently characterized sites that lack these features. Rigorous analysis of the amino acids surrounding the target tyrosin using the position-specific scoring matrix (PSSM) demonstrates that a consensus sequence does not contain all the information necessary to predict tyrosine sulfation. Instead, accurate prediction requires consideration of all residues within five amino acids on either side of the target tyrosine. These results support the notion that secondary structure is the major determinant of sulfation and that other residues within the sulfation site can compensate for deviations from commonly observed features. This view implies that specific consensus features are not critical for TPST substrate recognition but that TPST may instead broadly recognize any sufficiently exposed tyrosine residue.     

Lack of protein-tyrosine sulfation disrupts photoreceptor outer segment morphogenesis, retinal function and retinal anatomy

To investigate the role(s) of protein‐tyrosine sulfation in the retina, we examined retinal function and structure in mice lacking tyrosylprotein sulfotransferases (TPST) 1 and 2. Tpst double knockout (DKO; Tpst1^?/?/Tpst2 ^?/?) retinas had drastically reduced electroretinographic responses, although their photoreceptors exhibited normal responses in single cell recordings. These retinas appeared normal histologically; however, the rod photoreceptors had ultrastructurally abnormal outer segments, with membrane evulsions into the extracellular space, irregular disc membrane spacing and expanded intradiscal space. Photoreceptor synaptic terminals were disorganized in Tpst DKO retinas, but established ultrastructurally normal synapses, as did bipolar and amacrine cells; however, the morphology and organization of neuronal processes in the inner retina were abnormal. These results indicate that protein‐tyrosine sulfation is essential for proper outer segment morphogenesis and synaptic function, but is not critical for overall retinal structure or synapse formation, and may serve broader functions in neuronal development and maintenance.     

马尾松花粉硫酸酯化多糖调控脾细胞[Ca~(2+)]_i的研究

目的探讨马尾松花粉多糖硫酸酯化物(SPPM60-A)对小鼠脾细胞内游离钙离子浓度([Ca2+]i)调控的机制。方法热水浸提醇沉淀法提取马尾松花粉粗多糖,SephacrylS-400HR分离纯化,氯磺酸-吡啶法进行硫酸酯化,荧光分光光度计测定脾淋巴细胞[Ca2+]i。结果 LPS与SPPM60-A都能促进脾淋巴细胞[Ca2+]i升高,与对照相比升高率分别为11.7%和15.4%(P<0.01)。TAK-242、LY294002、U73122、低分子肝素与维拉帕米均可以抑制[Ca2+]i的升高。结论推测硫酸酯化马尾松花粉多糖可通过TLR4-PI3K-PLC-IP3R信号通路促进脾淋巴细胞内[Ca2+]i升高。     

PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor sulfolipid-1

Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL(1278), has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL(1278) and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL(1278) and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2'-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL(1278). Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the Delta papA2 and Delta papA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL(1278)) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.