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1.
Human centromere protein B (CENP-B) has a sequence-specific DNA binding activity. We previously reported several CENP-B binding motifs by analysing synthetic oligonucleotides as well as alphoid DNA isolated from the human genomic library. Here, we examined the size requirement and nucleotide specificity of human CENP-B binding sequences in vitro. We synthesized three sets of mixed oligonucleotides containing diverged authentic binding sites (CTTCGTTGGAAACGGGA) in which certain pairs of nucleotides (underlined) were degenerated. Each oligonucleotide with a defined sequence was separately introduced into a plasmid and mixed with GST-fused recombinant CENP-B. The DNA--protein complex formed was affinity purified with glutathione Sepharose. Any nucleotide substitutions at the positions 1, 2 and 17 did not significantly influence the recovery, while the substitutions at positions 3, 4 and 16 did, suggesting that the internal 14-bp motif (TCGTTGGAAACGGG) constituted the minimum requirement. However, it showed a lower affinity to CENP-B, compared with the authentic motif. The inclusion of T at the 5 end greatly increased the affinity, and the further addition of A or T at the 3 end (TTCGTTGGAAACGGGA/T) offered affinity similar to the authentic motif. The first nucleotide of the 17-bp authentic binding motif may not be essential for CENP-B binding. 相似文献
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Yeonsil Yu Yoon Shin Park Han Su Kim Ha Yeong Kim Yoon Mi Jin Sung-Chul Jung Kyung-Ha Ryu Inho Jo 《Journal of anatomy》2014,225(5):510-518
Although mesenchymal stem cells (MSC) isolated from bone marrow and adipose tissues are known to be subjected to in vitro culture-related alterations in their stem cell properties, such data have not been reported in human tonsil-derived MSC (T-MSC). Here, we investigated the culture-related changes of phenotypes, the senescence, and the differentiation potential of T-MSC. T-MSC were serially passaged by a standard protocol, and their characteristics were assessed, including MSC-specific surface antigen profiles, the senescence, and the differentiation potentials into adipocytes, chondrocytes and osteocytes. Up to at least passage 15, we found no alterations in either MSC-specific surface marker, CD14, CD34, CD45, CD73 and CD90, or the mRNA expression of embryonic stem cell gene markers, Nanog, Oct4-A and Sox-2. However, the expression of CD146, recently identified another MSC marker, dramatically decreased with increasing passages from ∼ 23% at passage 3 to ∼ 1% at passage 15. The average doubling time increased significantly from ∼ 38 h at passage 10 to ∼ 46 h at passage 15. From passage 10, the cell size increased slightly and SA-β-gal staining was evident. Both Alizarin Red S staining and osteocalcin expression showed that the osteogenic differentiation potential increased up to passage 10 and decreased thereafter. However, the adipogenic and chondrogenic differentiation potential decreased passage-dependently from the start, as evidenced by staining of Oil Red O and Alcian Blue, respectively. Consistent with a passage-dependent osteogenic differentiation, the expression of CCN1, an angiogenic protein known to be related to both senescence and osteogenesis, also increased up to passage 10. Furthermore, ectopic expression of small interfering RNA against CCN1 at passage 10 significantly reversed Alizarin Red S staining and osteocalcin expression. Altogether, our study demonstrates the characterization of long-term in vitro cultured T-MSC and that CCN1 may be involved in mediating a passage-dependent increase in osteogenic potential of T-MSC. 相似文献
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Ipek Duman
mer Tanyeli Yuksel Dereli Pembe Oltulu Hatice Toy Ayse Saide Sahin 《Archives of Medical Science》2022,18(6):1607
IntroductionAutologous saphenous vein (SV) and internal mammary artery (IMA) are used as bypass conduits during coronary artery bypass graft surgery. Vasospasm of the arterial and venous grafts may constitute a significant clinical problem. Pretreatment with a vasodilator drug of the graft ex vivo or intraluminal injection before implantation may be used for spasm prophylaxis. This in vitro study was designed to assess the vasoactive effects and time-dependent changes of botulinum toxin A (BTX-A) and papaverine pretreatment on vasospasm of human SV and IMA grafts. Also, histomorphology of the vessels was assessed.Material and methodsSV and IMA segments were suspended in organ baths, and isometric contraction responses to 2 different concentrations of 5-hydroxytryptamine (5-HT) and endothelin-1 (ET-1) were recorded after incubation with 2 different concentrations of BTX-A and papaverine at 2 time points (0 h and 2 h).ResultsThe results revealed the following: 1) incubation with BTX-A and papaverine relaxes both SV and IMA rings contracted with 5-HT and ET-1; 2) the duration of the relaxant effect of BTX-A lasts longer than papaverine; and 3) no apparent histomorphological changes were observed in the grafts under light microscopy.ConclusionsThis study demonstrates that in human SV and IMA grafts, pretreatment with both BTX-A and papaverine are safe and have a potent inhibitory effect depending on the vessel and vasoconstrictor agent. The long-lasting vasodilatory effect of BTX-A on vascular smooth muscle may provide promising results in the prevention of venous and arterial graft spasm. 相似文献
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M. A. Cremer Edward F. Rosloniec Andrew H. Kang 《Journal of molecular medicine (Berlin, Germany)》1998,76(3-4):275-288
This contribution reviews the structure and organization of collagen molecules found in cartilage and the roles that they
may play in rheumatic diseases. Cartilage is unique in its physical properties and molecular composition, and contains sufficient
amounts of types II, IX, X, and XI collagen to deem these molecules as ”cartilage-specific.” The vitreous body of the eye,
a ”cartilagelike” tissue is also rich in the same collagens but is type X deficient. Types VI and XII collagen are present
in cartilage as well as noncartilaginous tissues. Types II, IX, and XI collagen are organized into matrix fibrils, where type
II constitutes the bulk of the fibril, type XI regulates fibril size, and type IX facilitates fibril interaction with proteoglycan
macromolecules. Genetic defects in these collagens can produce mild to severe developmental abnormalities, including spondyloepiphyseal
dysplasia often accompanied by an accelerated form of osteoarthritis. Sensitization with collagen can produce experimental
rheumatic diseases. Type II collagen induces an erosive polyarthritis in certain strains of rats, mice, and higher primates
which can resemble rheumatoid arthritis and relapsing polychondritis. Type XI collagen is arthritogenic in rats but not mice;
type IX induces autoimmunity in both species but not arthritis. Arthritis is initiated by complement fixing antibodies that
bind to type II collagen in autologous cartilage, and the production of these antibodies is MHC restricted and T cell dependent.
It is unclear whether T cells alone can induce arthritis, although they probably help sustain it. Mapping and characterizing
the of T cell epitopes on type II collagen has resulted in the synthesis of small homolog and substituted peptides of type
II collagen which suppress arthritis in an antigen-specific manner by a variety of routes, including mucosal. Moreover, collagen-induced
arthritis has proven a valuable model to study the contribution of cytokines and other biological agents in the pathogenesis
of joint injury and how they might be used to develop new therapies. Collagen autoimmunity has been implicated in the pathogenesis
rheumatoid arthritis and polychondritis. Circulating antibodies to type II collagen are found in both diseases. Antibodies
to types IX and XI collagen are also present in rheumatoid sera but are less prevalent. Rheumatoid cartilage and synovium
contain antibodies to type II collagen at a prevalence far greater than serum, suggesting an intra-articular antigen-driven
immune process. Although effective in animal models, attempts to treat rheumatoid arthritis with orally administered type
II collagen have proven elusive. Different approaches using newer formulations and selected or modified oligopeptides remain
to be tested and could prove effective in the treatment of the human rheumatic diseases.
Received: 16 April 1997 / Accepted: 15 January 1998 相似文献
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Tajima N Tezuka K Tanaka M Tanimoto M Miyai A Takeshima H Watanabe Y 《Human immunology》2008,69(7):399-408
Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator, ICOS) is the third homolog of the "professional" costimulatory molecule, CD28. To date, the characteristics and role of AILIM/ICOS, especially in effector function of T cells, have been determined through numerous studies in vitro and in vivo using mice. Considering potential differences among species, whether the AILIM/ICOS blockade acts as an efficacious immunomodulator for human diseases remains to be elucidated. In the present study, ability of AILIM/ICOS blockade to modulate immune responses of human and monkey cells was investigated using a fully human antibody (JTA-009), comparing the effect of CD28 blockade. JTA-009 blocked the response of human and monkey T cells co-stimulated with anti-CD3 and AILIM/ICOS ligand, B7h. AILIM/ICOS and CD28 blockade both inhibited human mixed lymphocyte reaction in different fashions, as well as cytokine production in T helper (Th) 1-/Th2-type recall responses. In monkeys however, CD28 blockade by CTLA4-Ig effectively prevented mixed lymphocyte reaction to a greater extent than AILIM/ICOS blockade. These results suggest that AILIM/ICOS blockade is valuable for suppressing both primary allogenic response and recall responses of T cell in human beings, and that there are differences between human and monkey use preferences for costimulatory molecules. 相似文献
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背景:EdU为新一代细胞核标记物,目前对此标记的研究较少。
目的:采取EdU标记人脐带间充质干细胞,筛选出EdU标记人脐带间充质干细胞的最适浓度。
方法:采用组织块法分离、纯化及传代培养人脐带间充质干细胞,倒置显微镜观察其形态及生长情况,流式细胞仪鉴定细胞表面标志物,并进行成脂诱导分化能力鉴定。分别采用5,10,20,50,100 µmol/L浓度的EdU标记脐带间充质干细胞24 h,选择标记率最高的优化浓度,绘制细胞增殖曲线。
结果与结论:倒置显微镜下观察细胞为贴壁生长,形态为长梭形,且EdU标记后的细胞形态与其一致,流式细胞仪检测CD44呈阳性表达,成脂诱导后具有向脂肪细胞分化的能力。5,10 µmol/L EdU的标记率最高,两种浓度标记的人脐带间充质干细胞生长曲线未见明显差异,故5,10 µmol/L是体外标记人脐带间充质干细胞的最适浓度。
中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程 相似文献
9.
Maria C. Bertomeu Sharon Gallo Davide Lauri Thomas A. Haas F. William Orr Eva Bastida Michael R. Buchanan 《Clinical & experimental metastasis》1993,11(3):243-250
Previously, we have demonstrated that stimulation of endothelial cells (ECs) with interleukin-la (IL-l) enhances the synthesis and expression of the vitronectin receptor (VnR), promotes VnR-dependent adhesion of human A549 adenocarcinoma cells to ECs, and is associated with decreased EC 13-hydroxyoctadecadienoic acid (13-HODE) synthesis in vitro. To determine whether these observations are relevant in vivo, we examined the acute retention and subsequent metastasis of intravenously-injected B16F10 melanoma cells in murine lungs, in relation to vessel wall 13-HODE. In C57BL/6 mice pretreated with IL-l, vessel wall 13-HODE was decreased and B16F10 lung entrapment and metastasis were increased. The latter two events were blocked by pretreating the animals with the GRGDS peptide. These data suggest a relationship between vessel wall 13-HODE synthesis, adhesion molecule expression, and adhesion of B16F10 cells to the endothelium. 相似文献
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MUC1 is a transmembrane glycoprotein abundantly expressed on the apical surface of human ductal epithelial cells and over
entire cell surface of tumors originating from those cells. It is 300 to 500 nm long and has a rigid, rod-like structure protruding
from the cell surface. MUC1 expressed by normal cells has heavily O-glycosylated tandem repeat domain while MUC1 on malignant
cells is aberrantly O-glycosylated. Substantially reduced (aberrant) glycosylation of the tandem repeat region of tumor MUC1
results in uncovering of the polypeptide core. This new structural feature may play an important role in the attachment of
metastasizing tumor cells to tissues at distant sites. We show that MDA-MB-231 cells attaching to the immobilized extracellular
matrix proteins (ECM) are higher MUC1 expressers than those non-attaching and that the attachment is inhibited by the addition
of non-glycosylated, MUC1 peptide. This 100 a.a. peptide composed of 5 tandem repeats from the tandem repeat domain mimics
the forms of MUC1 found in ascites fluid of cancer patients. We also show that this synthetic form of MUC1 inhibited attachment
of breast tumor cells to sections of normal human lung tissue and immobilized ECM. We did not find correlation between the
expression of Tn (GalNAc-Ser/Thr) epitope and the ability of tumor cells to adhere to the immobilized ECM. These results indicate
that the non-glycosylated form of MUC1 plays a role in the initial attachment of carcinoma cells to tissues at distant sites,
which may facilitate establishment of metastatic foci.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
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Franceschi C Olivieri F Marchegiani F Cardelli M Cavallone L Capri M Salvioli S Valensin S De Benedictis G Di Iorio A Caruso C Paolisso G Monti D 《Mechanisms of ageing and development》2005,126(2):351-361
In this paper, we review data of recent literature on the distribution in centenarians of candidate germ-line polymorphisms that likely affect the individual chance to reach the extreme limit of human life. On the basis of previous observations on the immunology, endocrinology and cellular biology of centenarians we focused on genes that regulate immune responses and inflammation (IL-6, IL-1 cluster, IL-10), genes involved in the insulin/IGF-I signalling pathway and genes that counteract oxidative stress (PON1). On the whole, data indicate that polymorphisms of these genes likely contribute to human longevity, in accord with observations emerging from a variety of animal models, and suggest that a common core of master genes and metabolic pathways are responsible for aging and longevity across animal species. Moreover, in the concern of our plan to discover new genetic factors related to longevity, we explored the possibility to by-pass the need of an a-priori choice of candidate genes, extending the search to genes and genomic regions of still unknown function. Alu sequences may be considered as good markers of highly variable and potentially unstable loci in functionally important genomic regions. We extensively screened Alu-rich genomic sites and found a new genomic region associated with longevity. 相似文献
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Cowey A Campana G Walsh V Vaina LM 《Experimental brain research. Experimentelle Hirnforschung. Expérimentation cérébrale》2006,171(4):558-562
Several published single case studies reveal a double dissociation between the effects of brain damage in separate extra-striate cortical visual areas on the perception of global visual motion defined by a difference in luminance (first-order motion) versus motion defined by a difference in contrast (second-order motion). In particular, the medial extrastriate cortical region V2/V3 seems to be crucial for the perception of first-order motion, but not for second-order, whereas a lateral and more anterior portion of the cortex close to the temporo–parieto–occipital junction (in the territory of the human motion area hV5/MT+) seems to be essential only for the perception of second-order motion. In order to test the hypothesis of a functional specialization of different visual areas for different types of motion, we applied repetitive transcranial magnetic stimulation (rTMS) unilaterally over areas V2/V3, V5/MT, or posterior parietal cortex (PPC) while subjects performed a 2AFC task with first- or second-order global motion displays in the contralateral visual field. Results showed a comparable disruption of the two types of motion, with both rTMS over V2/V3 or over MT/V5, and little or no effect with rTMS over PPC. The results suggest that either the previous psychophysical results with neurological patients are incorrect (highly unlikely) or that the lateral and medial regions are directly connected (as they are in macaque monkeys) such that stimulating one automatically affects the other, in this instance disruptively 相似文献