RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site

Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.     

Tartrate dehydrogenase reductive decarboxylation: stereochemical generation of diastereotopically deuterated hydroxymethylenes

Tartrate dehydrogenase catalyzes the reductive decarboxylation of meso-tartrate to glycerate. Concomitant with the ketonization of the intermediate enolate the C3 hydroxymethylene of glycerate necessarily acquires a proton from solvent. In D2O, the proton is shown to be added stereospecifically to form (2R,3R)-[3-2H]glycerate. The 1H-NMR assignments of the diastereotopic C3 protons of glycerate were confirmed by the enzymatic conversion of [1R-2H]fructose-6-phosphate to (2R,3R)-[3-2H]glycerate. The decarboxylation-protonation occurs with retention of configuration, implying that the general acid is positioned on the same face of the intermediate as the departing carboxylate. The stereochemically pure (2R,3R)-[3-2H]glycerate is readily synthesized and serves as a chiral hydroxymethylene synthon as demonstrated by the synthesis of (2S,3R)-[3-2H]serine.     

Phosphotyrosyl peptides and analogues as substrates and inhibitors of purple acid phosphatases

Purple acid phosphatases are metal-containing hydrolases. While their precise biological role(s) is unknown, the mammalian enzyme has been linked in a variety of biological circumstances (e.g., osteoporosis) with increased bone resorption. Inhibition of the human enzyme is a possible strategy for the treatment of bone-resorptive diseases such as osteoporosis. Previously, we determined the crystal structure of pig purple acid phosphatase to 1.55A and we showed that it is a good model for the human enzyme. Here, a study of the pH dependence of its kinetic parameters showed that the pig enzyme is most efficient at pH values similar to those encountered in the osteoclast resorptive space. Based on the observation that phosphotyrosine-containing peptides are good substrates for pig purple acid phosphatase, peptides containing a range of phosphotyrosine mimetics were synthesized. Kinetic analysis showed that they act as potent inhibitors of mammalian and plant purple acid phosphatases, with the best inhibitors exhibiting low micromolar inhibition constants at pH 3-5. These compounds are thus the most potent organic inhibitors yet reported for the purple acid phosphatases.     

Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo. However its molecular interaction with pre-adipocytes remains unknown. Therefore, this study aimed to investigate membrane interaction of TRAP and its endocytosis routes in pre-adipocytes. Confocal and/or electron microscopy were used to detect TRAP in untreated or TRAP 5a/b treated pre-adipocytes under conditions that allow or inhibit endocytosis in combination with co-staining of endocytotic vesicles. TRAP interaction with heparin/heparan sulfate was verified by gel filtration. It could be shown that TRAP 5a, but not 5b, binds to the membrane of pre-adipocytes where it co-localizes with heparin-sulfate proteoglycan glypican-4. Also in vitro, TRAP 5a exhibited affinity for both heparin and heparan sulfate with heparin inhibiting its enzyme activity. Upon caveolae-mediated endocytosis of saturating levels of TRAP 5a, TRAP 5a co-localized intracellularly with glypican-4 and late endosomal marker Rab-7 positive vesicles. The protein was also located in multivesicular bodies (MVBs) but did not co-localize with lysosomal marker LAMP-1. TRAP 5a endocytosis was also detectable in pre-osteoblasts, but not fibroblasts, embryonic MSCs or mature adipocytes. These results indicate that TRAP 5a exhibits binding to cell surface, endocytosis and affinity to glucosaminoglycans (GAGs) in pre-adipocyte and pre-osteoblast lineage cells in a manner similar to other heparin-binding growth factors.     

Anaerobic growth of Salmonella typhimurium on l(+)- and d(−)-tartrate involves an oxaloacetate decarboxylase Na+ pump

We show here that the Enterobacterium Salmonella typhimurium LT2 has the capacity to grow anaerobically on l(+)- or d(-)-tartrate as sole carbon and energy source. Growth on these substrates was Na⁺-dependent and involved the l(+)- or d(-)-tartrate-inducible expression of oxaloacetate decarboxylase. The induced decarboxylase was closely related to the oxaloacetate decarboxylase Na⁺ pump of Klebsiella pneumoniae as shown by the sensitivity towards avidin, the location in the cytoplasmic membrane, activation by Na⁺ ions, and Western blot analysis with antiserum raised against the K. pneumoniae oxaloacetate decarboxylase. Participation of an oxaloacetate decarboxylase Na⁺ pump in l(+)-tartrate degradation by S. typhimurium is in accord with results from DNA analyses. The deduced protein sequence of the open reading frame identified upstream of the recently sequenced oxaloacetate decarboxylase genes is clearly homologous with the -subunit of l-tartrate dehydratase from Escherichia coli. Southern blot analysis with S. typhimurium chromosomal DNA indicated the presence of probably more than one gene for oxaloacetate decarboxylase.     

IL-17A modulates osteoclast precursors' apoptosis through autophagy-TRAF3 signaling during osteoclastogenesis

Osteoclasts play an important role in bone remodeling. The inflammatory cytokine IL-17A could modulate the RANKL-induced osteoclastogenesis by regulating the autophagic activity. It is well accepted that protective autophagy has an anti-apoptotic effect. It is necessary to elucidate whether IL-17A can influence the apoptosis of osteoclast precursors (OCPs) through autophagy responses during osteoclastogenesis. The results showed that apoptosis of RAW264.7-derived OCPs was promoted by high levels of IL-17A, but the opposite anti-apoptotic function was shown by low levels of IL-17A. Furthermore, the enhanced apoptosis by high levels of IL-17A was reversed by overexpression of autophagy protein Beclin1; conversely, the inhibited apoptosis by low levels of IL-17A was restored by knockdown of Beclin1. It was also found that Beclin1 suppression with Beclin1 inhibitor (spautin1) could block the reduced apoptosis by low levels of IL-17A, which was recovered by TRAF3 knockdown. Moreover, the enhanced apoptosis by high levels of IL-17A decreased following the downregulation of TRAF3. Importantly, overexpression of caspase3 further attenuated osteoclastogenesis treated by high levels of IL-17A, without significantly affecting osteoclastogenesis stimulated by low levels of IL-17A. In conclusion, IL-17A modulates apoptosis of OCPs through Beclin1-autophagy-TRAF3 signaling pathway, thereby influencing osteoclastogenesis. Therefore, our study sheds lights on the improvement of clinical strategies of dental implantation or orthodontic treatment by revealing the novel targets in the bone remodeling.     

Metabolism of the phthalocyanine textile dye remazol turquoise blue by Phanerochaete chrysosporium

Three tartrate utilization regions from Agrobacterium vitis strains involved in host specificity have been compared, to clearly define the borders of these regions and eventually identify specific sequences that could provide a mechanism of duplication of this region. A 10.8-kb conserved DNA fragment called the TAR element, found in different genetic contexts, was defined. A comparison of the two tartrate dehydrogenase genes (ttuC and ttuC') in each of the three TAR elements suggests that these genes co-evolve.     

沙库巴曲缬沙坦联合小剂量美托洛尔治疗中青年高血压的临床观察

目的:观察沙库巴曲缬沙坦钠片联合小剂量美托洛尔治疗中青年高血压的临床疗效。方法:选择2018年12月-2019年12月间入院的92例中青年高血压患者,采用随机数表法将其分为试验组和对照组,每组46例。对照组口服沙库巴曲缬沙坦钠片,50 mg/次,2次/d;试验组在服用相同剂量沙库巴曲缬沙坦钠片的基础上增加服用小剂量美托洛尔12.5 mg/次,2次/d,两组用药时间均为8 w。治疗结束后,比较两组试验前后收缩压(systolic blood pressure,SBP)、舒张压(diastolic blood pressure,DBP)的变化情况,讨论两种治疗方法的有效性,并记录不良反应发生情况。结果:(1)试验组和对照组治疗后收缩压、舒张压较治疗前均有显著降低(P<0.05);(2)治疗后,试验组收缩压、舒张压的数值明显低于对照组(P<0.05);(3)试验组总有效率为97.83%,高于对照组的73.91%(P<0.05);(4)两组不良反应发生率为试验组8.68%,对照组6.51%,无统计学差异(P>0.05)。结论:采用沙库巴曲缬沙坦联合小剂量美托洛尔治疗中青年高血压较单独服用沙库巴曲缬沙坦有更明显的临床优势,建议推广。     

硝苯地平片联合酒石酸托特罗定片对TURP术后膀胱过度活动症的应用效果分析

目的:探讨硝苯地平片联合酒石酸托特罗定片用于经尿道前列腺电切术(TURP)术后膀胱过度活动症的临床效果及安全性。方法:选择2015年8月至2017年8月我院接诊的103例TURP术后出现膀胱过度活动症的患者作为本研究对象,通过随机数表法将其分为试验组52例和对照组51例。两组均给予常规处理,对照组在此基础上口服酒石酸托特罗定片,2 mg/次,2次/d;试验组在对照组基础上联合硝苯地平片口服,5 mg/次,3次/d;两组均连续用药7d。比较两组临床疗效,治疗前后膀胱过度活动症(OABSS)评分、国际前列腺症(IPSS)评分、膀胱痉挛次数、排尿情况的变化以及不良反应的发生情况。结果:治疗后,试验组和对照组临床疗效总有效率分别为92.31%(48/52)和76.47%(39/51),OABSS评分分别为(2.69±0.58)分和(4.76±0.62)分,IPSS评分分别为(5.02±0.80)分和(7.86±1.15)分,24h膀胱痉挛次数分别为(0.65±0.48)次和(1.10±0.61)次,24h尿急次数分别为(0.88±0.32)次和(1.59±0.54)次,24h排尿次数分别为(5.52±1.02)次和(7.24±0.97)次,夜间排尿次数分别为(0.73±0.45)次和(1.39±0.70)次,24 h平均尿量分别为(227.07±16.68)mL和(196.65±15.07)mL,试验组临床总有效率和24 h平均尿量均显著高于对照组(P0.05),OABSS评分、IPSS评分、24 h膀胱痉挛次数、24 h尿急次数及24h排尿次数均显著低于对照组(P0.05)。两组不良反应发生率比较差异无统计学意义(P0.05)。结论:硝苯地平片联合酒石酸托特罗定片治疗TURP术后膀胱过度活动症患者的临床疗效明显优于单用酒石酸托特罗定片,其可更有效促进膀胱功能恢复,且不增加不良反应。     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.