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21.
The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function 总被引:6,自引:0,他引:6
Dr. Nancy L. Parenteau Patrick Bilbo Cynthia J. M. Nolte Valerie S. Mason Mireille Rosenberg 《Cytotechnology》1992,9(1-3):163-171
We describe an organotypic model of human skin comprised of a stratified layer of human epidermal keratinocytes and dermal
fibroblasts within a contracted collagen lattice. Feasible and reproducible production of the skin construct has required
the use of traditional as well as specialized culture techniques. The configuration of the construct has been engineered to
maintain polarity and permit extended culture at the air-liquid interface. Morphological, biochemical and kinetic parameters
were assessed and functional assays were performed to determine the degree of similarity to human skin. Light and ultrastructural
morphology of the epidermis closely resembled human skin. The immunocytochemical localization of a number of differentiation
markers and extracellular matrix proteins was also similar to human skin. Kinetic data showed a transition of the epidermal
layer to a morein vivo-like growth rate during the development of the construct at the air-liquid interface. The barrier properties of the construct
also increased with time reaching a permeability to water of less than 2%·h after approximately 2 weeks at the air-liquid
interface which is still on average 30-fold more water-permeable than normal human skin. The construct is currently used forin vitro research and testing and is also being tested in clinical applications. 相似文献
22.
A. Cortes D. C. Emery D. J. Halsall R. M. Jackson A. R. Clarke J. J. Holbrook 《Protein science : a publication of the Protein Society》1992,1(7):892-901
The proposal that the active site vacuole of NAD(+)-S-lactate dehydrogenase is unable to accommodate any imbalance in electrostatic charge was tested by genetically manipulating the cDNA coding for human muscle lactate dehydrogenase to make a protein with an aspartic acid introduced at position 140 instead of the wild-type asparagine. The Asn 140-Asp mutant enzyme has the same kcat as the wild type (Asn 140) at low pH (4.5), and at higher pH the Km for pyruvate increases 10-fold for each unit increase in pH up to pH 9. We conclude that the anion of Asp 140 is completely inactive and that it binds pyruvate with a Km that is over 1,000 times that of the Km of the neutral, protonated aspartic-140. Experimental results and molecular modeling studies indicate the pKa of the active site histidine-195 in the enzyme-NADH complex is raised to greater than 10 by the presence of the anion at position 140. Energy minimization and molecular dynamics studies over 36 ps suggest that the anion at position 140 promotes the opening of and the entry of mobile solvent beneath the polypeptide loop (98-110), which normally seals off the internal active site vacuole from external bulk solvent. 相似文献
23.
The optical spectra of the reaction center (RC) of Rhodopseudomonas viridis including absorption (A), linear dichrosism (LD), circular dichroism (CD), absorption-detected magnetic resonance (ADMR) and its linear dichroism (LD-ADMR) are simulated by an exciton model. It involves the Qy transitions of six prosthetic groups: four (bacteriochlorophyll-b molecules, two bacteriopheophytin-b molecules and the Soret bands By of the special-pair pigments, which couple most strongly with the corresponding Qy transitions. For the ADMR-spectra additional excitations of the special-pair triplet are introduced. While interactions between the Qy transitions and the By transitions are in principle determined from the structural arrangement of the pigments, the interactions to the other states are adjusted to fit the spectral features for the various transitions. The interaction of the newly introduced states are interpreted in terms of a simple model. 相似文献
24.
An oxygen-evolving complex has been highly purified from the thermophilic cyanobacterium Synechococcus sp. The complex, which reproducibly showed 5 major polypeptide bands of 47, 40, 35, 30 and 9 kDa on SDS-polyacrylamide gel electrophoresis and contained 3.2 Mn per QA, had an oxygen-evolving activity of 300–400 μmol/mg chl per h in the presence of 5 mM MnCl2; or CaCl2. The complex most likely represents a minimum functional unit of the photosynthetic oxygen evolution. 相似文献
25.
Spectrally pure reaction center preparations from Chloroflexus aurantiacus have been obtained in a stable form; however, the product contained several contaminating polypeptides. The reaction center pigment molecules (probably three bacteriochlorophyll a and three bacteriopheophytin a molecules) are associated with two polypeptides (Mr = 30000 and 28000) in a reaction center complex of Mr = 52000. No carotenoid is present in the complex. These data together with previous spectral data suggest that the Chloroflexus reaction center represents a more primitive evolutionary form of the purple bacterial reaction center, and that it has little if any relationship to the green bacterial component. A reaction center preparation from Rhodopseudomonas sphaeroides R26 was fully denatured at 50°C while the Chloroflexus reaction center required higher temperatures (70–75°C) for complete denaturation. Thus, an intrinsic membrane protein of a photosynthetic thermophile has been demonstrated to have greater thermal stability than the equivalent component of a mesophile. 相似文献
26.
In this paper a number of experiments with the purple bacteria Rhodospirillum rubrum and Rhodopseudomonas capsulata is described in which the total fluorescence yield and/or the total fraction of reaction centers closed after a picosecond laser pulse were measured as a function of the pulse intensity. The conditions were such that the reaction centers were either all in the open or all in the closed state before the pulse arrived. These experiments are analysed using the theoretical formalism discussed in the preceding paper (Den Hollander, W.T.F., Bakker J.G.C., and Van Grondelle, R., Biochim. Biophys. Acta 725, 492–507). From the experimental results the number of connected photosynthetic units, λ, the rate of energy transfer between neighboring antenna molecules, kh, and the rate of trapping by an open reaction center, kot, can be estimated. For R. rubrum it is found that λ = 14−17, kh = (1−2)·1012 s−1 and kot = (4−6)·1011 s−1, for Rps. capsulata λ ≈ 30, kh ≈ 4·1011 s−1 and kot ≈ 3·1011 s−1. The findings are discussed in terms of current models for the structure of the antenna and the kinetic properties of the decay processes occurring in these purple bacteria. 相似文献
27.
We explored O2 flash yield in two cyanophytes, Anacystis nidulans and Agmenellum quadruplicatum. On a rate-measuring electrode, a single flash gave a contour of O2 evolution with a peak at about 10 ms which was maximum (100) for 680 nm background light. On 625 nm illumination the peak was smaller (62) but was followed by an increased tail of O2 attributed to enhancement of the background. After a period of darkness, repetitive flashes (5 Hz) gave a highly damped initial oscillation in individual flash yields which finally reached steady state at 94% of the yield for 680 nm illumination. When O2 of repetitive flashes was measured as an integrated flash yield the results was distinctive and similar to that for a continuous light 1 (680 nm). An apparent inhibition of respiration which persisted into the following dark period was taken as evidence for the Kok effect. With a concentration-measuring electrode, integrated flash yield vs. flash rate showed the same nonlinear behavior as O2 rate vs. intensity of light 1. We draw three conclusions about the two cyanophytes. (a) The plastoquinone pool is substantially reduced in darkness. (b) Because of a high ratio of reaction centers, reaction center 1 / reaction center 2, for the two photoreactions, saturating flashes behave as light 1. (c) Because repetitive flashes are light 1, they also give a Kok effect which must be guarded against in measurements designed to count reaction centers. 相似文献
28.
从堆肥和污泥中分离到一批抗药性高温细菌,经电泳检查,发现6株高温细菌细胞中有质粒存在。其中,嗜热脂肪芽孢杆菌T653的细胞DNA提取液电泳图谱上,有三条非染色体DNA条带,用电镜直接观察,证明它们是T653细胞中的三个质粒。测得两个较小质粒的分子量分别为3.6×10~6和45×10~6道尔顿。研究了嗜热脂肪芽孢杆的T653的温度生长条件与其细胞中质粒的关系。T653细胞中三个质粒的明确功能有待进一步探讨。 相似文献
29.
The reaction to C-banding was investigated throughout the mitotic cycle ofCrepis capillaris (2n=6): (1) 18–22 C-bodies or C-bands were found during mid telophase and interphase to prophase and metaphase, and also
12–14 at late anaphase to early telophase in the mitotic cycle. Fewer C-bands in late anaphase to early telophase were due
to the absence of minute bands; (2) large and medium sized C-bands were strongly stained by Giemsa, while small and minute
bands stained palely. It is suggested that inCrepis capillaris the difference of color in C-banded segments following Giemsa staining is referable to the amount of constitutive heterochromatin
rather than to the difference in the condensation and decondensation; (3) the size of C-bodies changed during telophase to
interphase and prophase. It is inferred that the extent of C-bodies is regulated by both the length of DNA sequences of constitutive
heterochromatin and the amount of proteins combined with C-banded DNA. It was shown that the reaction to C-banding is neither
due to the differential condensation of chromatin nor to a higher concentration of DNA in the C-banded regions, in the C-banding
mechanism as has been suggested so far at least. 相似文献
30.
Transformed plants with elevated levels of chloroplastic SOD are not more resistant to superoxide toxicity 总被引:29,自引:0,他引:29
The petunia nuclear gene which encodes the chloroplast isozyme of superoxide dismutase, SOD-1, has been fused with an efficient rbcS promoter fragment and 3 flanking region and introduced into tobacco and tomato cells. Transformed plants carrying this chimeric gene have up to 50-fold the levels of SOD-1 which occur in wild-type plants. However, tobacco plants with 30-to 50-fold the normal SOD-1 activity do not exhibit resistance to the light-activated herbicide paraquat. Similarly, tomato plants with 2-to 4-fold increases in SOD-1 do not exhibit tolerance to photoinhibitory conditions known to increase superoxide levels (high light, low temperatures and low CO2 concentrations). Our data indicate that increasing the chloroplastic SOD level in a plant cell is not sufficient to reduce the toxicity of superoxide. 相似文献