Biosynthesis of pseudoisoeugenols in tissue cultures ofPimpinella anisum

The genusPimpinella contains pseudoisoeugenols, phenylpropanoids with a rare 2,5-dioxy substitution pattern on the phenyl ring. To study the biosynthesis of these compounds, we set up a leaf-differentiating tissue culture ofPimpinella anisum. These cultures mainly produce epoxy-pseudoisoeugenol-(2-methylbutyrate). To corroborate the biosynthetic pathway of epoxy-pseudoisoeugenol-(2-methylbutyrate) as proposed on the basis of investigations with¹³C/¹⁴C-labelled precursors, the key steps of the pathway were investigated at an enzyme level. Experiments with cell-free homogenates clearly revealed that L-phenylalanine is converted to (E)-cinnamic acid by phenylalanine ammonia lyase and that (E)-cinnamic acid is converted top-coumaric acid by cinnamic acid 4-hydroxylase. L-2-aminooxy-3-phenylpropionic acid, an analogue of L-phenylalanine, inhibited the incorporation of L-[3-¹³C]phenylalanine into epoxy-pseudoisoeugenol-(2-methylbutyrate). Up to 2% of the precursor DL-[3-¹³C]phenyllactate was incorporated into epoxy-pseudoisoeugenol-(2-methylbutyrate). Inhibition experiments with oxalacetic acid clearly showed that cinnamic acid is not formed by dehydration of phenyllactic acid in this leaf-differentiating tissue culture ofP. anisum.     

柱形藻酸钙载体复合细胞修复异体兔膝关节软骨缺损

目的：应用柱形藻酸钙载体复合骨细胞俱复兔膝关节软骨缺损,为骨关节病的组织工程学修复提供一定的实验依据,方法,应用自行制作的柱形藻酸钙载体复合消化分离的兔关节软骨细胞,共同植入异体兔膝关节软骨缺损处,分别于术后4,8和12wk观察大体标本及组织学修复结果：结果：术后8wk时缺损处可见一定程度的修复,到12wk时大体标本修复较好,修复组织和周围关节面界限不清,组织学见缺损处表现为软骨组织修复,结论：通过短期观察说明藻酸钙可以作为软骨的细胞的革体材料,用于关节软骨缺损修复的组织工程学研究。     

Ex-vivo evaluation of alginate microparticles for Polymyxin B oral administration

A crosslinked alginate microparticle system for the targeting to the lymphatic system by Peyer's patches (PP) uptake was designed in order to improve the oral absorption of Polymyxin B (PMB). To verify mucoadhesion and PP uptake, microparticles labelled with fluorescein isothiocyanate (FITC) were prepared by spray-drying technique and crosslinking reactions with calcium ions and chitosan (CS), in vitro characterized and assayed by an ex vivo method. Microparticles showed a size less then 3 μm, an antibiotic loading level of 11.86 ± 0.70%, w/w, a sustained drug release behaviour in simulated gastro-intestinal (GI) fluids and a preserved biological activity throughout the manufacture. The ex vivo study was performed by a perfusion method on intestinal tracts of just sacrificed adult rats. The recovered samples were analysed by epifluorescence microscope for mucoadhesion and PP uptake and by microbiological analysis for antibiotic activity preservation, providing evidence of mucoadhesion at the level of both PP and non-PP epithelium, uptake by PP and PMB microbiological activity in PP tissue. Furthermore, the study revealed the involvement of transport pathways across villous enterocytes.     

Application of whey protein micro-bead coatings for enhanced strength and probiotic protection during fruit juice storage and gastric incubation

Context: Coated whey protein micro-beads may improve probiotic protection and provide delayed cell-release mechanisms.

Objective: Lactobacillus rhamnosus GG was encapsulated in whey protein micro-beads by droplet extrusion with coating via electrostatic deposition: primary-polysaccharide and secondary-whey protein.

Materials and methods: Storage studies were performed in cranberry and pomegranate juice (pH 2.4; 28 days; 4 and 25°C) followed by simulated ex vivo porcine gastric (pH 1.6) and intestinal (pH 6.6) digestion.

Results and discussion: After storage and simulated gastro-intestinal digestion, free cells, cells suspended in protein and cells encapsulated in alginate micro-beads, illustrated complete probiotic mortality, while coated micro-beads enhanced probiotic viability after juice storage (8.6?±?0.1 log₁₀CFUmL^?1). Beads also showed significant binding of hydrophobic molecules. Coated micro-beads illustrated high gastric survival (9.5?±?0.1 log₁₀CFUmL^?1) with 30?min delayed intestinal release relative to non-coated micro-beads.

Conclusions: Micro-bead coatings could be applied in delayed cell-release for targeted intestinal probiotic delivery.     

Effect of cell density on the rate of glycosaminoglycan accumulation by disc and cartilage cells in vitro

Aggrecan concentration falls markedly during cartilage and disc degeneration with unfortunate biomechanical and physiological consequences. There is thus now an increasing interest in developing biological methods for its replacement. One approach is to stimulate aggrecan and hence glycosaminoglycan (GAG) production by resident cells through growth factor or genetic engineering. Another approach is to implant autologous cells into the cartilage or disc to enhance GAG production. In both instances GAG accumulation depends both on the number of active cells in the cartilage or disc and the rate of aggrecan synthesis per cell. Here we examine how cell density influences the rate of glycosaminoglycan accumulation in a three‐dimensional cell culture system. In the results, at cell densities found in vivo (standard condition) in the articular cartilage and the disc nucleus viz. 4 million cells/ml and at 21% oxygen the concentration of GAG in the bead reached 520.9±62.4 μg/ml and 649.0±40.1 μg/ml, respectively, in 5 days. These concentrations could be increased to 2‐4‐fold by raising cell density to 25 million cells/ml. However, rates of GAG production per cell decreased by 50‐60%. These results showed that rate of accumulation of glycosaminoglycan in this culture system in vitro is slow and is limited both by the rate of production of GAG per cell and by the cell density which can be maintained in a viable state. Although GAG production can be increased somewhat by use of higher cell densities, the consequent fall in concentration of oxygen and other nutrients in the center of three‐dimensional constructs slows metabolism and leads to apoptosis and cell death, which again limits the rate that the cells can produce matrix. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:493–503, 2008     

A novel method of comparing the healing properties of two hydrogels in chronic leg ulcers

Skin ulcers on the legs have a chronic, relapsing course and are often a significant management challenge. Novel methods of measuring and comparing the effects of different treatments can be of assistance in addressing this situation. A clinical pilot study using original methods was undertaken to compare the healing properties of the alginate gel Flaminal (test) and the hydrocolloid gel Intrasite (control) on chronic leg ulcers. The study was performed over a period of 28 days with two parallel groups of 10 patients. Both the surface (acetate tracing and planimetry) and the volume (Jeltrate mould impression and weighting) of each wound were measured at baseline and after 7, 14 and 28 days of treatment. On both parameters results were superior with the test product compared to the control, with volume reduction being the first parameter to change. Between groups, difference in wound volume reduction was detected as early as day 7 whereas difference in surface reduction was clearly apparent only at day 28. Correlation between wound surface and volume reductions was also better in the test group (r = 0.843 vs. 0.421) than in the control. In conclusion, this pilot study suggests that combining wound surface and volume evaluations allows a more precise analysis of the healing process in venous leg ulcers and that this method is able to detect very early differences in treatments even with limited sample size.     

3-羟基-3-甲基戊二酸尿症患儿的临床诊治及3-羟基-3-甲基戊二酰裂解酶基因新突变分析

目的探讨3-羟基-3-甲基戊二酸尿症患儿的主要临床表现、生化特点及3-羟基-3-甲基戊二酰裂解酶(HMGCL)基因突变检测结果。 方法收集2014年12月7日于北京大学第一医院儿科就诊的1例3-羟基-3-甲基戊二酸尿症患儿的临床病历资料为研究对象。采用回顾性研究方法分析该患儿的入院查体及辅助检查结果、诊疗情况;对患儿及父母进行HMGCL基因检测,并于患儿出院后6个月,对其进行随访。本研究遵循的程序符合北京大学第一医院人体试验委员会制定的伦理学标准,得到该委员会批准,对患儿及其父母进行基因检测均获得受试对象及受试对象监护人的知情同意。 结果患儿年龄为1岁1个月,男性。因"不明原因腹泻、呕吐,反复呕血,发热2 d,昏迷3 h"收入本院。入院查体及辅助检查结果示：白细胞计数升高、肝功能损害、电解质紊乱、低血糖、凝血功能异常、低蛋白、血氨水平高、代谢性酸中毒;头颅MRI检查结果示：双侧侧脑室旁及双侧额颞顶皮层下异常信号。确诊为3-羟基-3-甲基戊二酸尿症。经限制蛋白质摄入,静脉滴注葡萄糖和左旋肉碱治疗后症状缓解。该患儿HMGCL基因c.509G >T(鸟嘌呤>胸腺嘧啶)发生点突变,c.348＋1G > C(鸟嘌呤>胞嘧啶)发生剪切位点突变,导致外显子拼接错误,其中c.509G >T是来自母亲的杂合突变,c.348＋1G > C是患儿新出现纯合突变,c.348＋1G > C剪切位点突变,导致第170位的半胱氨酸剪切突变为苯丙氨酸。出院后6个月后随访结果示：患儿年龄为1岁7个月,智力运动发育稍落后,但无明显倒退,无抽搐发作,全身情况良好,各项生化指标及尿3-羟基-3-甲基戊二酸水平降低。 结论3-羟基-3-甲基戊二酸尿症系少见的常染色体隐性遗传疾病,通过HMGCL基因检测及HMGCL基因分析不仅有助于诊断该病,对于指导家系遗传咨询及产前诊断亦至关重要。     

内源性硫化氢在糖尿病大鼠肾脏的变化及作用

新型内源性气体信号分子-硫化氢(内源性H₂S)具有类似氧化亚氮(nitric oxide,NO)的某些特征,如松弛血管、抑制血管平滑肌细胞增生、调节血管平滑肌细胞张力等作用.本系列研究观察了糖尿病肾病(diabetic nephropathy, DN)大鼠肾脏内源性H₂S的变化以及与H₂S合成酶-半胱氨酸胱硫醚2β2合成酶(cystathionine 2β2 synthase, CBS)、胱硫醚2γ2裂解酶(cystathionine 2γ2 lyase, CSE)的关系,对DN大鼠肾脏病理改变的影响,对成肌纤维细胞特征性标志物-a平滑肌肌动蛋白(a-smooth muscle actin, α-SMA)和细胞外基质成分-纤维粘连蛋白(fibronectin, FN)在大鼠肾组织分布和表达的影响,来探讨内源性H₂S在糖尿病肾病发病机制中的作用.结果显示:1DN大鼠内源性H₂S量明显减少,肾脏CSE的表达亦明显减少.2内源性H₂S的减少与DN大鼠临床表现和肾脏病理改变密切相关.3DN组大鼠肾组织中α-SMA 和肾小球基质中FN均增加,分布增多,补充外源性H₂S后,α-SMA和 FN明显下降.DN大鼠肾脏内源性H₂S降低.肾脏CSE的减少可能是导致DN大鼠肾脏内源性H₂S降低的直接因素.外源性补充H₂S可抑制糖尿病肾病大鼠肾脏中成肌纤维细胞的生成和细胞外基质的过度积聚.提示,内源性H₂S的减少与糖尿病肾病的发生有关.