类弹性蛋白多肽的从头设计、非色谱纯化及盐效应

近年来,因病毒侵害人类每年都要蒙受巨大的经济损失和社会损失。犬肾细胞MDCK以其具有的培养容易、增殖快、流感病毒感染效率高等特点,成为适用于流感病毒疫苗生产的重要细胞系之一。以MDCK细胞为研究对象,在自制无血清培养基中成功实现了MDCK细胞从有血清培养到无血清培养的驯化;并通过单细胞悬浮培养驯化过程实现了MDCK细胞的无血清单细胞悬浮培养,获得了适于无血清单细胞悬浮生长的ssf-MDCK细胞株,无血清单细胞悬浮批培养最大活细胞密度可达3.81×106 cells/mL,最大比生长速率可达0.056 h?     

Determination of malonyl-coenzyme A in rat heart,kidney, and liver: A comparison between acetyl-coenzyme A and butyryl-coenzyme A as fatty acid synthase primers in the assay procedure

The malonyl-CoA assay was nonlinear at low malonyl-CoA concentrations when labeled acetyl-CoA was used as fatty acid synthase primer. Linearity was obtained with low concentrations of both fatty acid synthase and labeled acetyl-CoA, but then the assay was disturbed by the diluting effect of endogenous acetyl-CoA. The problems of nonlinearity and dilution of radioactivity by endogenous compounds were absent when labeled butyryl-CoA was used as primer. The levels of malonyl-CoA in rat heart, kidney, and liver were determined. The use of butyryl-CoA gave higher values of malonyl-CoA.     

A Novel Isoindoline,Porritoxin Sulfonic Acid,from Alternaria porri and the Structure-phytotoxicity Correlation of Its Related Compounds

Novel zinniol-related compound 3, named porritoxin sulfonic acid, with an isoindoline skeleton was isolated from the culture liquid of Alternaria porri. The structure was determined to be 2-(2″-sulfoethyl)-4-methoxy-5-methyl-6-(3′-methyl-2′-butenyloxy)-2,3-dihydro-1H-isoindol-1-one. The phytotoxic activities of three isoindolines (1-3) were evaluated in a seedling-growth assay against stone leek and lettuce.     

下调脂肪酸合成酶表达对膀胱癌UMUC3细胞增殖、迁移及侵袭能力的影响*

目的: 研究脂肪酸合成酶(FASN)表达对膀胱癌UMUC3细胞增殖、迁移、侵袭的影响,探讨其内在可能机制。方法:免疫组化法检测30例膀胱癌和15例正常膀胱组织FASN蛋白的表达;用脂质体2000分别转染FASN siRNA和无义siRNA至UMUC3细胞,筛选、鉴定siFASN和siControl稳定的细胞,siFASN组细胞设为实验组,siControl组设为对照组;采用蛋白印迹法(Western blot)和实时荧光定量PCR(RT-PCR)法分别检测siFASN组和siControl组细胞FASN蛋白及mRNA的表达,MTT法检测siFASN组和siControl组细胞增殖情况,划痕试验、Transwell试验分别检测siFASN组和siControl组细胞迁移、侵袭能力。结果:FASN蛋白在膀胱癌组织中过表达,且与病理分期、分级密切相关(P＜0.05)。与siControl组相比,siFASN组细胞FASN mRNA及蛋白表达下调(P＜0.05),细胞增殖活力明显下降(P＜0.05),迁移能力明显下降(P＜0.05),穿膜细胞数量明显减少(P＜0.05)。结论:FASN过表达在膀胱癌发生、发展中发挥重要作用,下调FASN表达能抑制膀胱癌细胞的增殖、迁移、侵袭能力,抑制FASN表达有望成为一种新的膀胱癌治疗方法。     

Role of dietary oleic acid from two different sources on fatty acid composition of erythrocyte membrane and blood pressure in healthy subjects

This study has been undertaken to determine the effect of a diet enriched with olive oil (OO) and high-oleic sunflower oil (HOSO) on fatty acid composition of erythrocyte membrane phospholipids and blood pressure in healthy women. OO and HOSO were used as natural sources of monounsaturated fatty acids (MUFAs) in a random-order sequence over two 4-week periods with a 4-week washout period between both MUFA diets. HOSO diet resulted in significant increases in oleic [(18:1n-9) 8.6%, P < 0.001], eicosenoic [(20:1n-9) 33.3%, P < 0.05], arachidonic [(20:4n-6) 6.2%, P < 0.05], and docosapentaenoic [(22:5n-6) 56.0%, P < 0.001] acids, whereas OO diet besides increased the content of stearic acid [(18:0) 13.6%, P < 0.01] and long-chain polyunsaturated fatty acids (PUFAs) of the n-3 family (22:5n-3 and 22:6n-3), when compared with the baseline [a diet high in saturated fatty acids (SFAs) and low in MUFAs]. In contrast, there was a significant decrease in linoleic acid [(18:2n-6) 21.8%, P < 0.001] for both MUFA diets. Consistent with these data, dietary intake of OO significantly raised total PUFAs (7.2%, P < 0.05), the n-3 fatty acids (22.2%, P < 0.01) and the PUFAs/SFAs ratio (9.3%, P < 0.01), as well as decreased the ratio of cholesterol to phospholipids (26,1%, P < 0.001) versus HOSO-based diet. Interestingly, dietary OO, but not HOSO, was able to significantly reduce the systolic (3%, P < 0.05) and diastolic (4%, P < 0.05) blood pressures. Although both vegetable oils provided a similar content of MUFAs (mainly oleic acid), our findings rather indicate that OO has important benefits to modulate the fatty acid composition of membranes and the mechanisms involved in the regulation of blood pressure in human.     

QTL detection on porcine chromosome 12 for fatty-acid composition and association analyses of the fatty acid synthase, gastric inhibitory polypeptide and acetyl-coenzyme A carboxylase alpha genes

Refinement of previous QTL on porcine chromosome 12 for fatty-acid composition and a candidate gene association analysis were conducted using an Iberian × Landrace cross. The concentrations of ten fatty acids were assayed in backfat tissue from which four metabolic ratios were calculated for 403 F₂ animals. Linkage analysis identified two significant QTL. The first QTL was associated with the average chain length ratio and the percentages of myristic, palmitic and gadoleic acids. The second QTL was associated with percentages of palmitoleic, stearic and vaccenic acids. Based upon its position on SSC12, fatty acid synthase was tested as a candidate gene for the first QTL and no significant effects were found. Similarly, gastric inhibitory polypeptide ( GIP ) and acetyl-coenzyme A carboxylase alpha ( ACACA ) were tested as candidate genes for the second QTL using three SNPs in GIP and 15 synonymous SNPs in ACACA cDNA sequences. Two missense SNPs in GIP showed significant effects with palmitoleic and stearic fatty-acid concentration. Highly significant associations were found for two SNPs in ACACA with stearic, palmitoleic and vaccenic fatty-acid concentrations. These associations could be due to linkage disequilibrium with the causal mutations.     

新型蛋白酶体抑制剂YSY-01A对肿瘤细胞促血管生成作用的抑制及其机制初探

YSY-01A是一种新型蛋白酶体抑制剂,前期研究已经证实其对肿瘤细胞的增殖有抑制作用．但是它对肿瘤血管生成是否有影响尚不明确．本研究旨在探明YSY-01A阻碍肿瘤细胞促进血管生成的作用及机制．我们首先将磺酰罗丹明B(sulforhodamine B,SRB)法与细胞共培养(Transwell)模型相结合,探讨YSY-01A抑制人结肠癌细胞(HT-29 cells)对人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的增殖促进作用;运用高内涵筛选(high content screening,HCS)法研究YSY-01A对HT-29细胞中NF-κB核转位的影响;利用Western blot法检测YSY-01A对HT-29细胞中缺氧诱导因子-1α(hypoxia- inducible factor-1α,HIF-1α)及血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达调控．为了观察YSY-01A对HUVEC增殖和运动有无直接抑制作用,我们采用SRB法观察YSY-01A对HUVEC的增殖抑制作用;运用HCS法分别考察YSY-01A对HUVEC的运动抑制和细胞毒作用．结果证实,YSY-01A可以阻碍HT-29细胞对HUVEC的增殖促进作用并具有浓度依赖性．YSY-01A还可抑制HT-29细胞中NF-κB的核转位,下调HIF-1α及VEGF的表达．进一步研究证实,YSY-01A能够浓度依赖地抑制HUVECs的增殖和运动,而不伴有明显的细胞毒作用．上述结果表明,YSY-01A可以通过抑制蛋白酶体活性下调肿瘤细胞中促血管生成因子的表达,进而在血管内皮细胞中发挥抗血管生成作用．     

Dinklagins A,B and C: three prenylated flavonoids and other constituents from the twigs of Dorstenia dinklagei

The dichloromethane extract of the aerial parts of Artabotrys hexapetalus afforded three beta-methoxy-gamma-methylene-alpha,beta-unsaturated-gamma-butyrolactones, which are proposed to be derived from a C(18) unsaturated fatty acid by a biosynthetic route similar to that proposed for the Annonaceous acetogenins. The structure of the unique beta-methoxy-gamma-methylene-substituted, alpha,beta-unsaturated-gamma-butyrolactone ring of artapetalins A-C (1-3) was determined by 2D-NMR spectroscopic analyses. Two unusual simple butyrolactones, (+)-tulipalin B and (2R,3R)- 3-hydroxy-2-methylbutyrolactone were also isolated from this species.     

A spin-label study of sciatic nerves from quaking,jimpy and trembler mice

The spin labels, 5-nitroxide stearic acid and 16-nitroxide stearic acid were incorporated into whole sciatic nerves dissected from normal, quaking, jimpy and trembler mice. With 5-nitroxide stearic acid, we have studied the thermal variation of the maximal apparent coupling constant ($\text{T}{}_6$) between 0°C and 50°C. Within this range of temperatures, we obtained identical values of 2 $\text{T}{}_6$ for nerves from normal and jimpy mice, whereas 2 $\text{T}{}_6$ was smaller for nerves from quaking and trembler mice. With 16-nitroxide stearic acid, composite spectra were recorded, particularly in the high-field range. A line characteristic of myelin was clearly observed in the spectra of nerves from normal and jimpy mice; its intensity was somewhat less in nerves from quaking mice and much less in spectra from trembler mice. A shoulder in the principal highfield line of the spectrum is modified only with nerves from jimpy mice.The results agree well with those obtained by electron microscopy, which reveal normal myelination in nerves from jimpy mice, a slight modification of the myelin from those of quaking mice and a practically complete demyelination in peripheral nerves from trembler mice. However, the structure of the nerves of jimpy mice also seems to be modified at an, as yet, undetermined level.     

Effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of neural stem cells derived from the cerebral cortex of embryonic mice

Salvianolic acid B is isolated from Salvia miltiorrhiza,the root of which is widely used as a traditional Chinese medicine to treat stroke.However,little is known about how salvianolic acid B influences growth characteristics of neural stem cells (NSCs).The purpose of the present study was to evaluate the effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of NSCs derived from the cerebral cortex of embryonic mice using MTT,flow cytometry,immunofluorescence and RT-PCR.It was found that 20 μg mL·1 and 40 μg mL·1 salvianolic acid B had similar effects on proliferation of NSCs,and a suitable concentration of salvianolic acid B increased the number of NSCs and their derivative neurospheres.The growth-promoting activity of salvianolic acid B was dependent on and associated with an accumulation in the G2/S-phase cell population.Salvianolic acid B also promoted the neurite outgrowth of NSCs and their differentiation into neurons.The mRNA for tau,GFAP and nestin were present in differentiating neurospheres induced by salvianolic acid B.However,high-level expression of tau mRNA and low-level expression of GFAP mRNA was detected in differentiated cells,in contrast to the control conditions.This collective evidence indicates that exogenous salvianolic acid B is capable of promoting proliferation of neurospheres and differentiation towards the neuronal lineage in vitro and may act in the proliferation of NSCs and may promote NSC differentiation into neuronal cells.     

Antifungal and Insecticidal Activities of Selected Plant Species from Cloud Forest of Veracruz,Mexico: A Contribution to the Search of Novel Control Agents against Ambrosia Pest Complexes

The antifungal and insecticidal activities of 34 extracts from 27 plant species were evaluated against fungal phytopathogens of the genus Fusarium and Xyleborus Scolytine ambrosia beetles involved in Fusarium dieback (FD) and laurel wilt (LW) diseases. Sixteen extracts caused mycelial growth inhibition (MGI) above 23 % at 2 mg mL⁻¹ against F. solani, those from S. nudum and M. argyrophylla exhibited the highest MGI (57 % and 49 %, respectively). Thirteen extracts displayed significant antifungal activity against F. kuroshium, those from C. nocturnum and M. argyrophylla exhibited the highest MGI (100 % and 54.9 %, respectively). Additionally, ten plants extracts caused mortality in at least one of the beetle species tested, mainly from Solanaceae species. In the most active species, 39 phenolics were identified that may have contributed to their biological effects. This study is one of the first to report the potential of plant-derived natural products against the causative agents of FD and LW.     

A Comparative Study on Hulled Adlay and Unhulled Adlay through Evaluation of Their LPS‐Induced Anti‐Inflammatory Effects,and Isolation of Pure Compounds

Coicis semen (=the hulled seed of Coix lacryma‐jobi L. var. ma‐yuen (Rom.Caill. ) Stapf ; Gramineae), commonly known as adlay and Job's tears, is widely used in traditional medicine and as a nutritious food. Bioassay‐guided fractionation of the AcOEt fraction of unhulled adlays, using measurement of nitric oxide (NO) production on lipopolysaccharide (LPS)‐stimulated RAW 264.7 macrophage cells, led to the isolation and identification of two new stereoisomers, (+)‐(7′S,8′R,7″S,8″R)‐guaiacylglycerol β‐O‐4′‐dihydrodisinapyl ether ( 1 ) and (+)‐(7′S,8′R,7″R,8″R)‐guaiacylglycerol β‐O‐4′‐dihydrodisinapyl ether ( 2 ), together with six known compounds, 3 – 8 . Compounds 3 and 4 exhibited inhibitory activities on LPS‐induced NO production with IC₅₀ values of 1.4 and 3.7 μM , respectively, and suppressed inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) protein expressions in RAW 264.7 macrophage cells. Simple high‐performance liquid chromatography with ultraviolet detection (HPLC/UV) was used to compare the AcOEt fraction of unhulled adlays responsible for the anti‐inflammatory activity in RAW 264.7 cells and the inactive AcOEt fraction of hulled adlays.     

Comparison of the crystallin mRNA populations from rat,calf and duck lens. Evidence for a longer αA2-mRNA and two distinct αB2-mRNAs in the birds

Total cytoplasmic poly(A)-containing RNA from rat, calf and duck lens was fractionated by electrophoresis in methylmercury hydroxide-containing agarose gels. RNA electrophoresed in parallel lanes was either transferred onto nitrocellulose and hybridized with total cDNA synthesized on the initial mRNA or was recovered from individual gel fractions for in vitro translation in a reticulocyte cell-free system. This allowed the identification and size-characterization of individual mRNA species encoding α-, β-, γ- and δ-crystallin polypeptides. The 14 S mRNA fraction of rat lens comprises two αA₂-mRNAs of approximately 1250 and 1350 nucleotides and the αA^Ins-mRNA with a size similar to that of the largest αA₂-mRNA. The calf lens 14 S mRNA fraction harbors a heterogeneous population of αA₂-mRNA. In the same fraction another mRNA encoding a polypeptide, designated X, has been found sharing no homology with αA sequences. The duck lens αA₂-mRNA appears to be 400–450 bases longer than the rat and calf lens αA₂-mRNAs. Furthermore, in contrast to the single αB₂-mRNA in rat and calf lens, two αB₂-mRNAs have been identified in duck lens, one, the major species, similar in size to the αB₂-mRNA in rat and calf lens (800 bases), and the other species 700 nucleotides longer. The large size differences among the αA₂- and αB₂-mRNAs most likely reside in their 3′-untranslated sequences.     

Inhibition of mTOR down-regulates scavenger receptor,class B,type I (SR-BI) expression,reduces endothelial cell migration and impairs nitric oxide production

The mammalian target of rapamycin (mTOR) inhibiting drug rapamycin (Sirolimus) has severe side effects in patients including hyperlipidemia, an established risk factor for atherosclerosis. Recently, it was shown that rapamycin decreases hepatic LDL receptor (LDL-R) expression, which likely contributes to hypercholesterolemia. Scavenger receptor, class B, type I (SR-BI) is the major HDL receptor and consequently regulating HDL-cholesterol levels and the athero-protective effects of HDL. By using the mTOR inhibitor rapamycin, we show that SR-BI is down-regulated in human umbilical vein endothelial cells (HUVECs). This reduction of SR-BI protein as well as mRNA levels by about 50% did not alter HDL particle uptake or HDL-derived lipid transfer. However, rapamycin reduced HDL-induced activation of eNOS and stimulation of endothelial cell migration. The effects on cell migration could be counteracted by SR-BI overexpression, indicating that decreased SR-BI expression is in part responsible for the rapamycin-induced effects. We demonstrate that inhibition of mTOR leads to endothelial cell dysfunction and decreased SR-BI expression, which may contribute to atherogenesis during rapamycin treatment.     

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium.     

A current review of molecular mechanisms regarding osteoarthritis and pain

Osteoarthritis afflicts millions of individuals across the world resulting in impaired quality of life and increased health costs. To understand this disease, physicians have been studying risk factors, such as genetic predisposition, aging, obesity, and joint malalignment; however have been unable to conclusively determine the direct etiology. Current treatment options are short-term or ineffective and fail to address pathophysiological and biochemical mechanisms involved with cartilage degeneration and the induction of pain in arthritic joints. OA pain involves a complex integration of sensory, affective, and cognitive processes that integrate a variety of abnormal cellular mechanisms at both peripheral and central (spinal and supraspinal) levels of the nervous system Through studies examined by investigators, the role of growth factors and cytokines has increasingly become more relevant in examining their effects on articular cartilage homeostasis and the development of osteoarthritis and osteoarthritis-associated pain. Catabolic factors involved in both cartilage degradation in vitro and nociceptive stimulation include IL-1, IL-6, TNF-α, PGE2, FGF-2 and PKCδ, and pharmacologic inhibitors to these mediators, as well as compounds such as RSV and LfcinB, may potentially be used as biological treatments in the future. This review explores several biochemical mediators involved in OA and pain, and provides a framework for the understanding of potential biologic therapies in the treatment of degenerative joint disease in the future.