Outer dense fibres of human spermatozoa: partial characterization and possible physiological functions

The outer dense fibres are accessory fibres in the spermatozoon. They represent up to 30% of the protein portion in human spermatozoa and are involved in sperm progressive motility. If outer dense fibres are missing or developed poorly, spermatozoa are only locally motile. For isolation of the outer dense fibres, human spermatozoa were sonicated at 25 kHz and the flagella were separated by density gradient centrifugation in Percoll. Thereafter, membranes and fibrous sheath were dissolved under reducing conditions in the cationic detergent cetyltrimethylammonium bromide for 30, 60 and 90 min, respectively. The isolation steps were monitored by phase contrast microscopy and electron microscopy. After SDS-polyacrylamide gel electrophoresis and silver staining of isolated outer dense fibres, two protein bands at 55 and 67 kDa could be detected. By means of rhodamine B staining, no phosphorus could be detected in the outer dense fibre proteins.     

方格星虫多糖分离纯化及性质鉴定

以方格星虫（Sipunculus nudus）体壁和血液为试验材料，经胰蛋白酶水解，Sevage法去除蛋白质，乙醇沉淀得到粗多糖，透析并冷冻干燥后分别得到方格星虫体壁多糖（SWP）和血液多糖（SBP）。经凝胶渗透色谱分析，SWP和SBP均为单一组分，相对分子质量分别为284528和198212。红外光谱分析表明，两种多耱结构相似，不含硫酸基团，均含有葡萄耱、半乳糖、木糖和阿拉伯糖。SBP含有乙酰氨基，而SWP不含乙酰氨基，它们是以α-糖苷键或β-糖苷键连接的呋喃多糖。     

Rapid and simple isolation of DNA from agarose gels

Summary We present a simple method for the isolation of DNA from agarose gels that is economic, fast, and independent of electrical equipment. DNA fragments of up to 6 kb can be easily extracted within 5 min using a disposable plastic syringe and filter paper. Total extraction of DNA fragments between 10 and 20 kb in size is achieved by concentrating the DNA flushed from the gel in a DNA-binding column.     

鸡蛋清中卵转铁蛋白的分离提取

采用固定化金属螯合亲和色谱法(IMAC),从蛋清中分离和提取卵转铁蛋白(OVT).结果表明,采用均质处理蛋清液并结合硅藻土过滤澄清的预处理方法,去除了蛋清中的粘蛋白等一些影响柱分离的杂质蛋白,减轻了色谱柱的污染,延长了柱的使用寿命,从而降低了生产成本.SDS-PAGE电泳分析显示,分离提取的OVT纯度达到96%,远高于商品OVT;电喷雾质谱分析显示,OVT的相对分子质量为76 500.     

人血浆前白蛋白的分离和纯化

建立一种新的分离纯化人血浆前白蛋白（ＰＡ）的方法。由酚试剂沉淀后的血浆，通过离子交换树脂分离所得ＰＡ，用高效液相色谱仪（ＨＰＬＣ）进行纯化，经ＳＤＳ－聚丙烯酰胺凝胶电泳测定，其分子量在５５０００左右，纯度达到电泳纯和免疫纯。此法步骤简单，用血量少，适用于实验室常规操作和临床人体研究。     

Isolation and differentiation of embryonic stem cells from BALB/c mouse

Objective To invest the efficient method which can culture and induce embryonic stem cells to neurocyte in vitro. Methods Isolate the blastula of 3.5 d from BALB/c species mouse. Culture the cells from inner cell mass (inner cell mass, ICM) which were isolated by mechanical method on the mouse embryonic fibroblaste cell (MEF) feeder layer or 0.1% gelatin coated dishes. The stem cells were identified by characterized morphology, alkaline phosphatase stain, differential potency in vivo and immunochemistry stain. The isolated cells were differentiated by serial induction method that mimicking the intrinsic developmental process of the neural system. Results The isolated cells were positive for alkaline phosphatatse and SSEA-1 (stage specific embryonic antigen 1). Moreover they were identified pluripotent by differentiation in vivo. Therefore the isolated cells presented the characters of ESCs. Then the isolated cells were able to differentiate into neurocytes in vitro. Conclusion Mouse embryonic stem cells isolation, culture and differentiation system has been established.     

Hepatocyte isolation from pig livers after warm ischaemic injury

Abstract Hepatocyte cultures have been used extensively for a wide variety of physiological, pharmacological and experimental studies. The warm ischaemic period before isolation is kept to a minimum to achieve a high yield of cells isolated and a good viability for culture. We have recently introduced a new concept of liver resuscitation after warm ischaemia that is based on a 3-h reperfusion period with an improved perfusate and simultaneous dialysis. In this study, we applied the new technique for hepatocyte isolation from livers subjected to 80 min of complete ischaemia at 37 °C. Cell yield was improved by a resuscitating perfusion from 58% to 73% and viability from 39% to 76%.     

Diagnosis of fetal rubella infection by nucleic acid hybridization

The efficacy of nucleic acid hybridization for the diagnosis of rubella infection in experimental and clinical materials was compared with immunoblot and virus isolation techniques. Our results showed that nucleic acid hybridization is specific and rapid but gives false-negative results when compared with conventional virus isolation in some experimental although not in clinical materials so far examined. For this reason, a failure to demonstrate rubella virus in fetal specimens by this method alone cannot yet be taken as a sole criterion for ruling out fetal rubella infection.     

Ion conductances of isolated cortical collecting duct cells

The study of ion conductances in the intact cortical collecting duct (CCD) with the patch-clamp method is rather difficult. An optimized method to isolate CCD cells from rat kidneys using an in vivo followed by an in vitro enzyme digestion is described. Individual CCD segments were collected after this digestion and incubated in EGTA-buffered medium. This procedure resulted in single cells or cell clusters. These freshly isolated CCD cells were studied with different modifications of the patch-clamp method. Membrane voltages measured in the cell-attached-nystatin configuration were –74 ±1mV (n=13) and –68±3 mV (n=22) in cells isolated from normal and mineralocorticoid-treated rats respectively. These values and those measured with the nystatin-perforated slow-whole-cell configuration (–79 ±1mV, n=23) are comparable to those measured in principal cells of isolated CCD segments. The cells hyperpolarized after the addition of amiloride and depolarized with the addition of adiuretin to the bath. The amiloride effect was enhanced when cells were isolated from deoxycorticosterone-acetate-treated rats. The cells were strongly depolarized upon elevation of the extracellular K⁺-concentration and did not demonstrate a measurable Cl^– conductance. A large-conductance K⁺ channel (174 pS, n=5, cell-attached, 145 mmol/l K⁺ in the pipette; 140 pS, n=12, cell-free, 3.6 mmol/l K⁺ in the bath) was seen. It had a very low activity on the cell, but a high open probability when excised into a solution with 1 mmol/l Ca²⁺ on the cytosolic side. More often a small-conductance K⁺ channel (36–52 pS, n=19, cell-attached; 30 pS, n=5, cell-free) with a high open probability was found on the cell. These freshly isolated cells seem to be a powerful preparation to study the properties and regulation of ion conductances of rat CCD with several electrophysiological methods. These freshly isolated CCD cells maintain the conductance properties known from principal cells of the intact CCD.     

The Community Links of a Sample of People with Intellectual Disabilities

Background In 1995, an unpublished study (S. Hewson & C. Waters) showed that the community links of residents in 11 local National Health Services (NHS) trust houses were meagre, despite the service's stated commitment to community presence and participation. The 1995 study was repeated for the same 11 houses in 2002 to examine whether any changes had occurred. Method A test–retest design, with repeated measures was used, involving, as closely as possible, the same participants at two time points. Community links referred to the time residents spent outside their house, and the time unpaid visitors spent inside the house in the presence of the residents. Results The community links of the people studied were no better in 2002 than in 1995. Conclusions These findings question whether current service provision can deliver policy objectives for social inclusion for people with intellectual disabilities.