改良法分离培养SD新生乳鼠原代心肌细胞*

目的:建立一种稳定、快速的SD新生乳鼠原代心肌细胞分离培养改良方法。 方法:取SD新生乳鼠心室,0.12%Ⅱ型胶原酶消化,Percoll密度梯度离心结合5-溴脱氧尿嘧啶(5-BrdU)化学抑制法纯化心肌细胞,体外培养于含5%马血清的改良DMEM/F12中,次日更换为普通含10%胎牛血清的高糖DMEM继续培养,并比较此改良方法与传统差速贴壁法的差异。 结果:改良法获得的心肌细胞生长良好,接种24 h 后几乎全部贴壁生长,细胞呈三角形、梭形或不规则形,个别细胞出现自主搏动,频率为10～30 beats/min不等。48 h后心肌细胞变长伸出伪足,部分细胞呈现同步搏动, 频率接近50～80 beats/min。72 h后心肌细胞成菊花样交织成网,自发搏动趋于同步,频率加快至80～100 beats/min;96 h后细胞聚集成簇,呈岛屿样,同步搏动频率在100～120 beats/min左右,一周内细胞状态良好。改良法纯化原代心肌细胞的得率((1.17±0.15)×10⁶ vs (1.21±0.22)×10⁶,P＞0.05)和存活率与传统差速贴壁法相当(93.3%±1.4% vs 92.2%±0.7%, P＞0.05 ),但是改良方法获得的原代心肌细胞纯度更高 (94.7%±2.1% vs 89.5%±1.3%, P＜0.05),且用时较短((3.1±0.4)h vs (4.3±0.3)h, P＜0.01)。 结论:改良法获得心肌细胞耗时短、纯度高、结构功能保存完整,且实验重复和稳定性好,是一种理想且简单易行的的原代心肌细胞分离培养方法。     

An improved simple technique for the collection of blood samples from rats and mice

The technique of blood collection from the lateral caudal vein has been improved. The method requires only moderate skill and no anaesthesia is necessary. Collection of blood samples causes little trauma and can be repeated at frequent (8 h) intervals thus making the method particularly suitable for pharmacokinetic and hormonal studies. Blood samples are uncontaminated by tissue fluids. Large volumes can be obtained, in mice up to 1.5 ml and in Wistar rats over 4 ml.     

An improved method for chemical alpha-oxidation

A nonisotopic assay for acetylserotonin methyltransferase (ASMT) was devised. Melatonin, the product of the enzyme reaction, is measured fluorometrically after its reaction with o-phthaldialdehyde (OPT). The reaction of melatonin with OPT is carried out in 1 n HCl to suppress the reaction of N-acetylserotonin, the substrate of ASMT, with OPT. The mixture is gassed with nitrogen just before incubation at 60°C for 60 min in order to secure the linear relationship between the concentration of melatonin and the fluorescence intensity. This method is much simpler than the isotopic assay and also has as much high sensitivity. Moreover, in this assay the enzyme can be well saturated with S-adenosylmethionine, whereas in the isotopic assay it cannot.     

An improved micropropagation protocol for teak

An improved micropropagation protocol has been developed for teak (Tectona grandis). Nodal explants placed on MS medium supplemented with 22.2 M benzylaminopurine and then serially transferred to fresh medium after 12, 24, 48 and 72 h gave maximum culture establishment (76.8%). Establishment was reduced when explants were retained in the initial culture medium longer than 12 h. Explants collected in May showed maximum (76.8%) response. Placement of the explants on MS medium supplemented with 22.2 M benzylaminopurine and 0.57 M indole-3-acetic acid resulted in the maximum average number of shoots. In vitro raised micro shoots were rooted ex vitro by dipping in indole-3-butyric acid (9.8 mM) for 2 min followed by planting in polyethylene pots containing a soil:vermiculite (1:1 v/v) mixture. This treatment resulted in 77.9% survival of the plantlets. They were weaned in a glasshouse and finally moved to an agro-net shade house.     

An improved heuristic for haplotype inference

We cloned a gene, kexD, that provides a multidrug-resistant phenotype from multidrug-resistant Klebsiella pneumoniae MGH78578. The deduced amino acid sequence of KexD is similar to that of the inner membrane protein, RND-type multidrug efflux pump. Introduction of the kexD gene into Escherichia coli KAM32 resulted in a MIC that was higher for erythromycin, novobiocin, rhodamine 6G, tetraphenylphosphonium chloride, and ethidium bromide than that of the control. Intracellular ethidium bromide levels in E. coli cells carrying the kexD gene were lower than that in the control cells under energized conditions, suggesting that KexD is a component of an energy-dependent efflux pump. RND-type pumps typically consist of three components: an inner membrane protein, a periplasmic protein, and an outer membrane protein. We discovered that KexD functions with a periplasmic protein, AcrA, from E. coli and K. pneumoniae, but not with the periplasmic proteins KexA and KexG from K. pneumoniae. KexD was able to utilize either TolC of E. coli or KocC of K. pneumoniae as an outer membrane component. kexD mRNA was not detected in K. pneumoniae MGH78578 or ATCC10031. We isolated erythromycin-resistant mutants from K. pneumoniae ATCC10031, and some showed a multidrug-resistant phenotype similar to the drug resistance pattern of KexD. Two strains of multidrug-resistant mutants were investigated for kexD expression; kexD mRNA levels were increased in these strains. We conclude that changing kexD expression can contribute to the occurrence of multidrug-resistant K. pneumoniae.     

An improved fluorometric assay for DNA

A modification of the fluorometric assay of Kissane and Robins is described. The modified procedure is very accurate, widely applicable, and reasonably easy to use. A standard cell type instead of a standard DNA solution makes the assay universally reproducible. DNA content is calculated by a simple method from the slope of a DNA concentration series. This can be used to detect technique erros.     

An improved heuristic for haplotype inference

Haplotypes include essential SNP information used for a variety of purposes such as investigating potential links between certain diseases and genetic variations. Given a set of genotypes, the haplotype inference problem based on pure parsimony is the problem of finding a minimum set of haplotypes that explains all the given genotypes. The problem is especially important because, while it is fairly inexpensive to obtain genotypes, other approaches to obtaining haplotypes are significantly expensive. There are two types of methods proposed for the problem, namely exact and inexact methods. Existing exact methods guarantee obtaining purely parsimonious solutions but have exponential time-complexities and are not practical for large number or length of genotypes. However, inexact methods are relatively fast but do not always obtain optimum solutions. In this paper, an improved heuristic is proposed, based on which new inexact and exact methods are provided. Experimental results indicate that the proposed methods replace the state-of-the-art inexact and exact methods for the problem.     

An improved micromethod for tyrosine estimation

A modified and improved micromethod for tyrosine determination has been developed. The method is sensitive, economic and applicable for estimation of tyrosine released in enzymatic reactions and in tissue. A range of Folin-Ciocalteu (FC) reagent was used to optimize the conditions for the development of blue color. Thus in 1.5 ml of the assay system, the suitably diluted FC reagent at the final concentration of 0.2 N gave a rapid optimum color development with an absorption maximum at 750 nm. Color development showed a linear relationship in the range of 2 to 16 microg tyrosine for a described assay system under optimized conditions. Thus, the method is 3-fold more sensitive in terms of its estimation range than a conventional method. The blue color formed was stable up to 24 h. The applicability of the method for tyrosine determination in the assay of lysosomal cathepsin D and in tissue was checked by comparison to the conventional procedure. Under both systems the results obtained by the micromethod were identical to those obtained by the conventional method. In general the method that produces quantitatively a blue color, not only is rapid and economical in terms of chemical usage but also has application for routine biochemical analysis.     

An improved method for butyrylcholinesterase phenotyping

An improved method for the identification of butyrylcholinesterase phenotypes is proposed. It is based on modifications of a method that uses -naphthyl acetate as substrate anddl-propranolol and Ro2-0683 as inhibitors. The proposed modifications make the method more rapid and increase the accuracy of the determinations of the phenotypes tested (BCHE U, BCHE UF, BCHE UA, BCHE AK, BCHE AF, and BCHE A). These modifications make the method even more adequate for population studies and clinical routine.We are grateful to the CNPq for research grants and scholarships.A preliminary report was presented at the Third International Meeting on Cholinesterases, France, May 12–16, 1990.     

一种改进的适用于膜片钳记录的成年大鼠海马神经元急性分离法

本文建立了一种快速、可靠的急性分离成年大鼠海马神经细胞的方法。此法可将实验大鼠的年龄提高到500d以上,体重300g以上;不损伤神经细胞膜的电学特性：形态上有差异的细胞易于分辨。用膜片钳技术的单通道和全细胞模式证实,在本实验条件下,约95％左右的健康细胞均能形成高阻抗封接,并成功地记录了电压依赖性钾、钠、钙通道,外向整流氯通道和大电导的钙激活钾通道电流。     

An improved glucoseoxidase--peroxidase-coupled assay for -fructofuranosidase activity

An improved glucoseoxidase-peroxidase-coupled assay for the determination of β-fructofuranosidase activity is described. The method makes use of the double effect of Tris (2-amino-2-hydroxymethylpropane-1,3-diol) as an inhibitor of both invertase and contaminating glucosidases. The method is very sensitive and is suitable for routine determinations. The total time needed for a single analysis is less than half an hour.     

An improved culture medium for mouse blastocysts

Summary Eagle's basal medium, modified to contain essential amino acids at the concentrations optimal for mouse blastocyst hatching, attachment, and outgrowth, supported in vitro development of the mouse blastocyst better than other tissue culture media tested. This medium was improved for growth and differentiation of the inner cell mass by doubling the concentration of amino acids and glucose and by adding uridine (10⁻⁵ M) and β-mercaptoethanol (10⁻⁵ M). In this improved medium nearly all blastocysts grown from the two-cell stage hatched and formed trophoblast outgrowths, and 62% developed into two-layer egg cylinders. This work was supported by the U.S. Department of Energy.