Digital roentgen analysis as a tool for kinematic studies of the shoulder--a study of radiation dosage

The radiation exposure during biomechanical investigation of the shoulder joint done with a digital radiography system (Polytron 1000 VR - Siemens) was measured experimentally. Total radiation dose and surface dose were determined by phantom measurements. The radiation exposure per image is about 1/6 to 1/7 of that of a conventional study. The digital radiography system used is thus suitable for kinesiologic studies of large joints in healthy volunteers.     

多头绒泡菌微原质团瞬时表达系统的构建

真核生物多头绒泡菌的原质团是研究细胞周期的好材料。但尚无合适的表达体系可供选择。本研究用多头绒泡菌ardC actin基因启动子和终止子分别替换哺乳动物细胞表达质粒pDsRed1-N1的CMVIE和SV40 polyA片段,构建了多头绒泡菌红色荧光蛋白(RFP)表达质粒pXM1;用PardC-MCS-DsRed1-TardC替换pTB38表达盒PardC-hph-TardC,构建了多头绒泡菌RFP表达质粒pXM2。将多头绒泡菌转录延伸因子类似蛋白(PELF1)基因与质粒pXM2重组,构建了PELF1红色荧光融合蛋白(PELF1-RFP)表达质粒pXM2-pelf1。通过荧光显微镜和激光扫描共聚焦显微镜观察RFP表达发现,电转参数为4kV/cm(电场)、1A(电流)、70μs(电击时间)时,质粒pXM1和pXM2电转多头绒泡菌微原质团(≤500μm)后24～48h内,RFP荧光最显著;而PELF1-RFP则主要聚集在多头绒泡菌细胞核,说明本试验建立的表达系统可以用于研究特定蛋白在多头绒泡菌内的瞬时表达。     

Properties of a new crystal form of the complex of concanavalin A with methyl alpha-D-glucopyranoside

The complex of concanavalin A with methyl alpha-D-glucopyranoside crystallizes as regular rhombic dodecahedra containing 35% protein by weight. The crystal is of space group I23 with a = 167.8 A (1 A = 0.1 nm) and contains one concanavalin A dimer per asymmetric unit. It diffracts to a resolution of 1.9 A and is suitable for crystallographic investigation of the structure of the saccharide-binding site.     

Crystallization of hen egg-white avidin in a tetragonal form

Hen egg-white avidin has been crystallized at pH 5.7 from ammonium sulfate solutions. The crystals belong to the tetragonal space group P4(2)2(1)2, with unit cell edges a = b = 79.6 A, c = 84.3 A. Assuming a molecular weight of 15,600 per avidin monomer, this crystal form is compatible with the presence of a dimer in the asymmetric unit, and is suitable for a crystallographic structural investigation at high resolution.     

Neonatal rabbit kidney cortex in culture as tool for the study of collecting duct formation and nephron differentiation

By stripping off the capsula fibrosa of neonatal rabbit kidneys a consistently thin tissue layer consisting of collecting duct anlagen, S-shaped bodies and nephrogenic blastema is obtained. This thin layer seems to be an excellent object for investigation of epithelium formation and nephron differentiation. Three different tissue culture protocols are described: 1. A polarly differentiated collecting duct epithelium with 'tight' characteristics consisting only of principal cells, grown on specific renal support 2. A morphologically dedifferentiated collecting duct principal cell monolayer grown on the unspecific bottom of a plastic culture dish 3. An embryonic tissue layer with numerous S-shaped bodies which might be a suitable model for investigation of the development of maturing nephron structures in serum-free culture medium.     

Next-generation spirobenzazepines: identification of RWJ-676070 as a balanced vasopressin V1a/V2 receptor antagonist for human clinical studies

We have continued to explore spirobenzazepines as vasopressin receptor antagonists to follow up on RWJ-339489 (2), which had advanced into preclinical development. Further structural modifications were pursued to find a suitable backup compound for human clinical studies. Thus, we identified carboxylic acid derivative 3 (RWJ-676070; JNJ-17158063) as a potent, balanced vasopressin V(1a)/V(2) receptor antagonist with favorable properties for clinical development. Compound 3 is currently undergoing human clinical investigation.     

Crystallization and preliminary X-ray study of AMP nucleosidase

Adenosine-5'-monophosphate nucleosidase from Escherichia coli has been crystallized in the presence of its strong competitive inhibitor formycin 5'-monophosphate and its allosteric activator adenosine 5'-triphosphate. Crystals are tetragonal bipyramids which grow to 1.2 mm in the longest dimension, are resistant to radiation damage, and diffract to a resolution of 3.5 A. The space group is P4(1)2(1)2 or P4(3)2(1)2, and the unit cell dimensions are a = 120.1 A and c = 243.7 A. The asymmetric unit is estimated to contain four subunits of 52,000 daltons. The crystals appear suitable for single crystal x-ray structure investigation.     

微载体规模化培养细胞的研究

通过实验探索使用微载体进行动物细胞规模化培养,以期达到建立规模化生产病毒疫苗的目的。实验研究了Vero细胞的生长曲线,以及对细胞生长过程中影响细胞生长的葡萄糖、氨含量两个主要因素的变化规律以及微载体浓度与细胞密度的关系。通过实验发现微载体规模化培养细胞易于操作,比传统转瓶培养的细胞密度高,封闭式的培养方式不但减少了污染几率,而且可以充分保证疫苗的质量。最终找出适宜疫苗培养的微载体使用浓度为2.5g/L,适宜的细胞接种浓度为:1~5×105cell/m l。     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Synthesis of 3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles, displaying combined 5-HT uptake inhibiting and alpha2-adrenoceptor antagonistic activities. Part 2: Further exploration on the cinnamyl moiety

In our previous paper we have described the synthesis of a series of 3-piperazinylmethyl-3a,4-dihydro-3H-[1]benzopyrano[4,3-c]isoxazoles, as novel dual 5-HT reuptake inhibitors and alpha2-adrenoceptor antagonists. That investigation led to the identification of the cinnamyl fragment as the most suitable moiety for combined activity. This paper outlines a further optimisation programme, focused on the exploration of the aromatic ring present on the cinnamyl moiety of compounds 1, 2 and 3.     

1型CRISPR位点间区序列分析在嗜热链球菌菌株分型中的应用

为了建立适用于嗜热链球菌菌株资源多样性调查的菌株分型方法,尝试将1型CRISPR位点间区序列分析用于嗜热链球菌的菌株分型,并与常用ERIC-PCR指纹图谱方法进行了比较。结果表明,1型CRISPR位点间区序列分析可以把30株从三个不同样品中分离的嗜热链球菌分成三种差异明显的类型:不同类型菌株之间没有相同的间区序列;而同一类型菌株之间,间区序列的组成和排列则基本一致,并且上述分型的结果与用ERIC-PCR指纹图谱技术获得的结果完全一致。因此,1型CRISPR位点间区序列分析是嗜热链球菌分型鉴定的可靠方法,并适用于大量菌株的分型鉴定和多样性调查。     

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

The aim of the present investigation was to evaluate whether routinely frozen biopsies of human skeletal muscle may be suitable for morphological and immunocytochemical analyses at transmission electron microscopy. The fixation/embedding protocols we successfully used for decades to process fresh mammalian tissues have been applied to frozen muscle biopsies stored for one to four years in liquid nitrogen. After 2.5% glutaraldehyde -2% paraformaldehyde - 1% OsO₄ fixation and embedding in epoxy resin, the ultrastructural morphology of myofibres and satellite cells as well as of their organelles and inclusions proved to be well preserved. As expected, after 4% paraformaldehyde - 0.5% glutaraldehyde fixation and embedding in LR White resin, the morphology of membrane-bounded organelles was relatively poor, although myofibrillar and sarcomeric organization was still recognizable. On the contrary, the myonuclei were excellently preserved and, after conventional staining with uranyl acetate, showed an EDTA-like effect, i.e. the bleaching of condensed chromatin, which allows the visualization of RNP-containing structures. These samples proved to be suitable for immunocytochemical analyses of both cytoskeletal and nuclear components, whereas the poor mitochondrial preservation makes unreliable any in situ investigation on these organelles.Keeping in mind the limitations found, these results open promising perspectives in the study of frozen skeletal muscle samples stored in the tissue banks; this would be especially interesting for rare muscle diseases, where the limited number of biopsies suitable for ultrastructural investigation has so far represented a great restriction in elucidating the cellular mechanisms responsible for the pathological phenotype.Key words: frozen biopsy, electron microscopy, fixation, immunocytochemistry, skeletal muscle.     

Differentiation of Brucella species by random amplified polymorphic DNA analysis

Random amplification of polymorphic DNA (RAPD) was used for discrimination between 46 Brucella strains and 14 representatives of the alpha-2 and alpha-1 subgroups of Proteobacteria. To evaluate a relatively quick and exact method for Brucella identification, the authors specified the most suitable conditions for RAPD amplification of Brucella DNA with two 10-mer primers, containing lower and higher percentages of G and C. The software package PHYLIP 3.1 was used for cluster analysis of the RAPD fingerprints. The optimization of RAPD conditions resulted in PCR mixes suitable for reliable typing of Brucellae. The distance-based methods (Fitch-Margoliash, UPGMA and Neighbour-joining) gave clear discrimination between Brucella species. The constructed dendrograms put Br. canis and Br. suis bv. 1 in the same cluster and differentiated Brucella strains according to their host preferences. RAPD can be useful method to distinguish related bacterial species, and under strictly established conditions the reaction appears to be a simple, quick and sensitive technique for the epidemiological investigation of brucellosis.     

Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells

This article describes a quick basic method adapted for the purification of mammalian mitochondria from different sources. The organelles obtained using this protocol are suitable for the investigation of biogenetic activities such as enzyme activity, mtDNA, mtRNA, mitochondrial protein synthesis, and mitochondrial tRNA aminoacylation. In addition, these mitochondria are capable of efficient protein import and the investigation of mtDNA/protein interactions by DNA footprinting is also possible.     

流感病毒在Vero细胞系与MDCK细胞系增殖条件的比较

目的研究流感病毒H1N1及其他亚型在Vero细胞系和MDCK细胞系高效增殖的最适条件,比较两种细胞系对流感病毒的敏感性差异及影响敏感性差异的条件。方法在培养好的Vero细胞系与MDCK细胞系用不同的病毒感染复数(M.O.I)、胰酶浓度、病毒吸附时间、病毒维持液血清质量浓度等条件进行流感病毒在细胞上的增殖。结果在M.O.I为0.01接种流感病毒,吸附时间为1 h,胰酶质量浓度2μg/mL,血清质量浓度为8%时,流感病毒血凝素在MDCK细胞系可获得较高的滴度。结论 MDCK细胞系是适于流感病毒培养的细胞,它作为生产新型流感病毒疫苗的主要细胞基质需要进一步的研究。     

Characterization of MR (P) strains of Drosophila melanogaster: the number of intact P elements and their genetic effect.

To study the effect of mutagenic/carcinogenic agents on P-element transposition, the P strains used should be defined, especially with respect to the number of intact and functional P elements present. In this investigation, the relation between the number of complete P elements present in dysgenic males and P-insertion mutagenesis was studied in several MR (P) strains. The main conclusions from this investigation are: (1) Complete P elements can be present in the genome without genetic activity (even in a 'dysgenic' cross). As a consequence, the number of complete P elements present in particular dysgenic flies, is not necessarily an indication of their dysgenic genetic activity. (2) The MR-h12/Cy strain carries two complete P elements, one on the X chromosome without and one on the MR chromosome with genetic activity (making this strain most suitable for studies on P-transposition mechanisms).     

A library of novel hydroxamic acids targeting the metallo-protease family: design, parallel synthesis and screening

We report here the design and parallel synthesis of 217 compounds based on a malonic-hydroxamic acid template. These compounds are obtained via a two-step solution-phase procedure. The set of diverse building-blocks used makes this strategy suitable for the search of inhibitors of various metallo-proteases and for the investigation of the biological role of new metallo-proteases. As a proof of concept, we screened this library on Neutral Aminopeptidase (APN; EC 3.4.11.2), the prototypal enzyme of the M1 family. Several submicromolar inhibitors were identified.     

Crystallization and x-ray diffraction studies of a phosphate-binding protein involved in active transport in Escherichia coli

We have obtained single crystals of a phosphate-binding protein (Mr = 34,400) that serves as initial receptor in osmotic shock-sensitive active transport in Escherichia coli. The crystals, suitable for high resolution crystallographic analysis, belong to the space group P2(1)2(1)2(1). The unit cell has dimensions of a = 41.97, b = 64.66, and c = 124.6 A and contains four protein molecules. Including this phosphate-binding protein, there are now a total of six different binding protein structures currently under investigation in our laboratory, the others being those specific for L-arabinose, D-galactose, D-maltose, sulfate, or leucine/isoleucine/valine.