Isolation of human epidermal stem cells by adherence and the reconstruction of skin equivalents

The isolation of human epidermal stem cells is critical for their clinical applications. In the present study, we isolated three populations of epidermal keratinocytes according to their ability to adhere to collagen type IV: i.e., rapidly adhering (RA), slowly adhering (SA), and non-adhering (NA) cells. The aim of this study was to characterize RA cells and to investigate the possibility of using these cells for epidermis reconstruction. To identify RA cells, flow cytometric analysis was performed using anti-₆ integrin and anti-CD71 antibodies. RA cells express high levels of ₆ integrin and low levels of CD71, which are considered as markers of an epidermal stem cell nature. Furthermore, electron microscopy showed that RA cells are small and have a high nuclear to cytoplasmic ratio, whereas SA and NA cells have well-developed cellular organelles and abundant tonofilaments. Western blot analysis showed that RA cells are slow cycling and express p63, a putative epidermal stem cell marker, whereas SA and NA cells express c-Myc, which is known to regulate stem cell fate. To compare epidermal regenerative abilities, skin equivalents (SEs) were made using RA, SA, and NA cells. The epidermis constructed from RA cells was well formed compared to those formed from SA or NA cells. In addition, only SEs with RA cells expressed ₆ integrin and ₁ integrin at the basal layer. These results indicate that RA cells represent epidermal stem cells and are predominately comprised of stem cells. Therefore, the isolation of RA cells using a simple technique offers a potential route to their clinical application, because they are easily isolated and provide a high yield of epidermal stem cells.Received 2 July 2004; received after revision 20 August 2004; accepted 10 September 2004     

Horizontal gene transfer from Eukarya to Bacteria and domain shuffling: the α-amylase model

-Amylases are present in all kingdoms of the living world. Despite strong conservation of the tertiary structure, only a few amino acids are conserved in interkingdom comparisons. Animal -amylases are characterized by several typical motifs and biochemical properties. A few cases of such -amylases have been previously reported in some eubacterial species. We screened the bacterial genomes available in the sequence databases for new occurrences of animal-like -amylases. Three novel cases were found, which belong to unrelated bacterial phyla: Chloroflexus aurantiacus, Microbulbifer degradans, and Thermobifida fusca. All the animal-like -amylases in Bacteria probably result from repeated horizontal gene transfer from animals. The M. degradans genome also contains bacterial-type and plant-type -amylases in addition to the animal-type one. Thus, this species exhibits -amylases of animal, plant, and bacterial origins. Moreover, the similarities in the extra C-terminal domains (different from both the -amylase domain C and the starch-binding domain), when present, also suggest interkingdom as well as intragenomic shuffling.Received 17 October 2003; accepted 6 November 2003     

Profiling of the secreted proteins during 3T3-L1 adipocyte differentiation leads to the identification of novel adipokines

Adipose tissue is an endocrine organ capable of secreting a number of adipokines with a role in the regulation of adipose tissue and whole-body metabolism. We used two-dimensional gel electrophoresis combined with mass spectrometry to profile the secreted proteins from (pre)adipocytes. The culture medium of 3T3-L1 cells during adipocyte differentiation was screened, and 41 proteins that responded to blocking of secretion by 20°C treatment and/or brefeldin A treatment were identified. Prohibitin, stress-70 protein, and adhesion-regulating molecule 1 are reported for the first time as secreted proteins. In addition, procollagen C-proteinase enhancer protein, galectin-1, cyclophilin A and C, and SF20/IL-25 are newly identified as adipocyte secreted factors. Secretion profiles indicated a dynamic environment including an actively remodeling extracellular matrix and several factors involved in growth regulation.Received 15 June 2004; received after revision 26 July 2004; accepted 2 August 2004     

Distribution of HIV-1 in the genomes of AIDS patients

The localization of HIV-1 proviruses in compositional DNA fractions from 27 AIDS patients during the chronic phase of the disease with depletion of CD4+ and different levels of viremia showed the following. (1) At low viremia, proviruses are predominantly localized in the GC-richest isochores, which are characterized by an open chromatin structure; this result mimics findings on HIV-1 integration in early infected cells in culture. (2) At higher viremia, an increased distribution of proviruses in GC-poor isochores (which match the GC poorness of HIV-1) was found; this suggests a selection of cells in which the isopycnic localization leads to a higher expression of proviruses and, in turn, to higher viremia. (3) At the highest viremia, integrations in GC-rich isochores are often predominant again, but generally not at the same level as in (1); this may be the consequence of new integrations from the extremely abundant RNA copies.Received 21 November 2003; received after revision 13 January 2004: accepted 15 January 2004     

Polarization of immunity induced by direct injection of naked sequence-stabilized mRNA vaccines

In the context of developing a safe genetic vaccination strategy we tested and studied globin-stabilized mRNA-based vaccination in mice. This vaccination strategy has the advantages of genetic vaccination (easy production, adaptability to any disease and inexpensive storage when lyophilized), but not the drawbacks of DNA vaccination (long-term uncontrolled expression of a transgene, possibility of integration into the host genome and possible induction of anti-DNA antibodies). We report here that injection of naked -globin untranslated region (UTR)-stabilized mRNA coding for -galactosidase is followed by detectable translation in vivo. In addition, we show that such a vaccination strategy primes a T helper 2 (Th2) type of response which can be enhanced and shifted to a Th1-type immune response by application of recombinant granulocyte/macrophage colony-stimulating factor 1 day after mRNA injection. Our data demonstrate that the administration of globin UTR-stabilized mRNA is a versatile vaccination strategy that can be manipulated to fit the requirement of antiviral, antibacterial or antitumor immunity.Received 14 June 2004; received after revision 19 July 2004; accepted 9 August 2004     

Immunogenicity of recombinant fowl-pox virus co-expressing structural protein precursor P1-2A and proteinase 3C of FMDV

Two recombinant plasmids, pUTA2P1 and pUTAL3CP1, were constructed by inserting structural protein precursor P1-2A and proteinase 3C of foot-and-mouth disease virus (FMDV) into fowl-pox virus (FPV) recombinant vectors pUTA-2 and pUTA-16-LacZ respectively, and two recombinant FPVs (vUTA2P1 and vUTAL3CP1) screened by the RT-PCR, IFA assay and Western blotting assay were obtained successfully. Mice injected respectively with rFPVs were induced high level specific anti-FMDV antibodies, increasing of T subtypes, and higher cytotoxicities of splenocytes than those of control groups. These results indicated that a new method was used to construct a potential candidate vaccine of FMDV.     

“三环制“供热系统中热水一级网运行计算研究

针对"三环制"供热系统,建立了热水一级网("二环")运行调节曲线数学模型,从而实现"三环制"供热系统连锁调节和自动控制,达到最大限度的节能.     

Parlay的业务移动能力分析

基于Parlay业务规范的开放业务技术是下一代网络研究的重要内容。虽然Parlay业务规范目前已经被电信界普遍接受,但是,Parlay业务规范是否可以满足现在和未来电信业务发展的需求,这仍然是一个尚未解决的问题。论文通过构造两个典型的移动业务模型,并基于Parlay业务规范,设计了相应的实现模型,论证了Parlay业务规范对移动业务的支持能力。为了保证实现模型的正确性,论文采用通信顺序进程(CSP)理论描述了移动业务实现模型。最后得出结论:Parlay业务规范在跨业务管理域的业务调用、用户身份验证和用户业务逻辑传递方面仍然无法支持业务的移动性。     

含季节性虚拟变量的GM(1,1)模型及其应用

针对GM（1，1）模型难以准确预测季节性时间序列的问题，本文将季节性虚拟变量作为灰作用量引入传统GM（1，1）模型中，提出了含季节性虚拟变量的GM（1，1）模型（简记为GMSD（1，1））.在GMSD（1，1）模型定义型和白化型的基础上，推导了GMSD（1，1，x⁽¹⁾）模型、GMSD（1，1，x⁽⁰⁾）模型、GMSD（1，1，b）模型、GMSD（1，1，exp）模型、GMSD（1，1，C）模型等五种派生型GMSD（1，1）模型，构建了季节性虚拟变量GM（1，1）模型群.同时利用粒子群算法对GMSD（1，1）模型和GMSD（1，1，exp）模型中的解析式进行最优化求解.最后以中国水力发电量的季度数据为例，验证了含季节性虚拟变量的GM（1，1）模型及其派生模型的有效性和优越性.结果表明：相较于传统GM（1，1）模型，引入季节性虚拟变量的GMSD（1，1）模型及其派生模型更能准确地描述系统特征序列的季节性波动和周期性变化特征.