Design, synthesis and biological activity of novel peptidyl benzyl ketone FVIIa inhibitors

Herein is described the synthesis of a novel class of peptidyl FVIIa inhibitors having a C-terminal benzyl ketone group. This class is designed to be potentially suitable as stabilization agents of liquid formulations of rFVIIa, which is a serine protease used for the treatment of hemophilia A and B inhibitor patients. A library of compounds was synthesized with different tripeptide sequences, N-terminals and d-amino acids in the P3 position. Cbz-d-Phe-Phe-Arg-bk (33) was found to be the best candidate with a potency of K_i = 8 μM and no substantial inhibition of related blood coagulation factors (thrombin and FXa). Computational studies revealed that 33 has a very stable binding conformation due to intramolecular hydrogen bonds, which cannot be formed with l-Phe in the P3 position. Nonpolar amino acids were found to be superior, probably due to a minimization of the cost of desolvation upon binding to FVIIa.     

Academic and molecular matrices: a study of the transformations of connective tissue research at the University of Manchester (1947-1996)

This paper explores the different identities adopted by connective tissue research at the University of Manchester during the second half of the 20th century. By looking at the long-term redefinition of a research programme, it sheds new light on the interactions between different and conflicting levels in the study of biomedicine, such as the local and the global, or the medical and the biological. It also addresses the gap in the literature between the first biomedical complexes after World War II and the emergence of biotechnology. Connective tissue research in Manchester emerged as a field focused on new treatments for rheumatic diseases. During the 1950s and 60s, it absorbed a number of laboratory techniques from biology, namely cell culture and electron microscopy. The transformations in scientific policy during the late 70s and the migration of Manchester researchers to the US led them to adopt recombinant DNA methods, which were borrowed from human genetics. This resulted in the emergence of cell matrix biology, a new field which had one of its reference centres in Manchester. The Manchester story shows the potential of detailed and chronologically wide local studies of patterns of work to understand the mechanisms by which new biomedical tools and institutions interact with long-standing problems and existing affiliations.     

Organotypic collagen I assay: a malleable platform to assess cell behaviour in a 3-dimensional context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic. Furthermore, interaction with other cell types, including stromal fibroblasts and immune cells, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo. Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo. As such, they can serve an important role in reducing the need for experiments on living animals.     

bFGF/BMP/ 胶原膜加速颌骨骨折愈合的大体及X 线观察

目的:了解胶原膜作为生长因子缓释材料治疗颌骨骨折的应用前景。方法:将100μg的rhBMP-2用1ml的bFGF溶液完全溶解;用移液器移出40μl的该溶液,滴加到面积为0.5cm×1cm的胶原膜组织块中,冻干后制成生长因子缓释系统;在12只新西兰大白兔两侧制成人工下颌骨骨折模型,左侧置放bFGF/BMP/胶原膜;右侧均为空白对照;术后2、4、12周行临床大体观察及X线片观察。结果:实验组骨折愈合速度明显快于对照组。术后2周,X线结果显示bFGF/BMP/胶原膜组骨折断端边缘模糊。对照组骨折线明显。术后4周,X线结果显示bFGF/BMP/胶原膜骨折线基本不可见,骨折对位良好,断端边缘基本消失,骨折无错位。对照组骨折下缘可见纤维性骨痂形成,骨折线模糊。术后12周,各组X线结果无差异,骨折部位接近正常骨组织。结论:bFGF/BMP/胶原膜能加速骨折愈合,提高骨折愈合效果。     

Simultaneous observation of collagen and elastin in normal and pathological tissues: analysis of Sirius-red-stained sections by fluorescence microscopy

In order to observe collagen and elastic fibers simultaneously, sections of human aorta, skin, lung, liver, and bladder were stained by Sirius red and analyzed by fluorescence microscopy. In all cases, the fibers of collagen presented the characteristic fluorescent red-orange color that results from the interaction of this extracellular protein with the dye, whereas elastic fibers showed strong green fluorescence (intrinsic fluorescence). This method efficiently detects collagen and elastic fibers when these two structures are present and could have valuable applications in processes that involves both fibers.L.F.B. received a doctoral fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil.     

Complex between triple helix of collagen and double helix of DNA in aqueous solution

We demonstrate in this paper that one example of a biologically important and molecular self-assembling complex system is a collagen–DNA ordered aggregate which spontaneously forms in aqueous solutions. Interaction between the collagen and the DNA leads to destruction of the hydration shell of the triple helix and stabilization of the double helix structure. From a molecular biology point of view this nano-scale self-assembling superstructure could increase the stability of DNA against the nucleases during collagen diseases and the growth of collagen fibrills in the presence of DNA.     

Tissue remodeling in Guinea pig lateral prostate at different ages after estradiol treatment

Estrogen seems to have an essential role in the fibromuscular growth characteristic of benign prostatic hyperplasia (BPH). This paper describes the effects of chronic estradiol treatment on Guinea pig prostatic stroma at different ages. Tissues from experimental animals were studied by histological and histochemical procedures, morphometric-stereological analysis and transmission electron microscopy (TEM). Marked fibromuscular hypertrophy was observed after estradiol treatment in animals of pre-pubertal and adult ages. Increases in the density and thickness of the collagen and elastic fibers were observed by histochemistry. TEM revealed wide distributions of collagen fibrils and large elastic fibers adjacent to the epithelial basal lamina and between the stromal cells, establishing contacts between them. These results indicate that the Guinea pig prostate simulates the stromal modifications observed in BPH in some aged animals after estrogen treatment at different ages, making it a good model for this disease.     

Global gene expression profiling and cluster analysis in Xenopus laevis

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.     

Assembly of homotrimeric type XXI minicollagen by coexpression of prolyl 4-hydroxylase in stably transfected Drosophila melanogaster S2 cells

We established stably transfected insect cell lines containing cDNAs encoding the alpha and beta subunits of human prolyl 4-hydroxylase in both Trichoplusia ni and Drosophila melanogaster S2 cells. The expression level and enzymatic activity of recombinant prolyl 4-hydroxylase produced in the Drosophila expression system were significantly higher than those produced in the T. ni system. We further characterized the involvement of prolyl 4-hydroxylase in the assembly of the three alpha chains to form trimeric type XXI minicollagen, which comprises the intact C-terminal non-collagenous (NC1) and collagenous domain (COL1), in the Drosophila system. When minicollagen XXI was stably expressed in Drosophila S2 cells alone, negligible amounts of interchain disulfide-bonded trimers were detected in the culture media. However, minicollagen XXI was secreted as disulfide-bonded homotrimers by coexpression with prolyl 4-hydroxylase in the stably transfected Drosophila S2 cells. Minicollagen XXI coexpressed with prolyl 4-hydroxylase contained sufficient amounts of hydroxyproline to form thermal stable pepsin-resistant triple helices consisting of both interchain and non-interchain disulfide-bonded trimers. These results demonstrate that a sufficient amount of active prolyl 4-hydroxylase is required for the assembly of type XXI collagen triple helices in Drosophila cells and the trimeric assembly is governed by the C-terminal collagenous domain.     

Role of water content in dielectric properties and delayed luminescence of bovine Achilles' tendon

In order to investigate the role of water network in collagen structure, measurement of dielectric permittivity was performed on bovine Achilles' tendon as a function of water content. The data show a sudden decrease of the permittivity at each measured frequency value when the tendon humidity decreases. A similar behaviour is shown by the total number of photons emitted in delayed luminescence (DL) experiments. The comparison of the two results is in agreement with the hypothesis that DL is connected to the excitation and subsequent decay of collective electronic states, whose properties depend on the organized structure of the system.