Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.

RENATAL DIAGNOSIS IS USEFULfor managing a pregnancy with an identified fetal abnormality and may allow for planning and coordinating care during delivery and the neonatal period. 1 A variety of prenatal diagnostic tests are available but have limitations. Noninvasive tests such as maternal serum marker testing and ultrasound can be used to screen for the presence of chromosomal abnormalities but are not definitive. [2][3][4][5] On the other hand, invasive diagnostic tests (eg, amniocentesis, chorionic villus sampling, percutaneous umbilical blood sampling) for fetal chromosomal abnormalities are highly reliable, but the procedure used for each test carries a risk for loss of pregnancy. 6,7 Many patients who are candidates for these tests decline them because of the risk of pregnancy loss.An alternative to existing methods for prenatal diagnosis is to use fetal cells and fetal DNA that exist in the maternal circulation. [8][9][10][11][12][13][14][15] Circulating fetal DNA has been used to determine the sex of the fetus through detection of sequences present on the Y chromosome. 13 In addition, several studies have

Representational difference analysis of the glomerular endothelial cell response to transforming growth factor-beta1 (TGF-beta1) revealed a novel gene, TIMAP (TGF-beta-inhibited membrane-associated protein), which contains 10 exons and maps to human chromosome 20.q11.22. By Northern blot, TIMAP mRNA is highly expressed in all cultured endothelial and hematopoietic cells. The frequency of the TIMAP SAGE tag is much greater in endothelial cell SAGE databases than in nonendothelial cells. Immunofluorescence studies of rat tissues show that anti-TIMAP antibodies localize to vascular endothelium. TGF-beta1 represses TIMAP through a protein synthesis- and histone deacetylase-dependent process. The TIMAP protein contains five ankyrin repeats, a protein phosphatase-1 (PP1)-interacting domain, a COOH-terminal CAAX box, a domain arrangement similar to that of MYPT3, and a PP1 inhibitor. A green fluorescent protein-TIMAP fusion protein localized to the plasma membrane in a CAAX box-dependent fashion. Hence, TIMAP is a novel gene highly expressed in endothelial and hematopoietic cells and regulated by TGF-beta1. On the basis of its domain structure, TIMAP may serve a signaling function, potentially through interaction with PP1.

Objective-Intercellular adhesion molecule-1 (ICAM-1) is upregulated rapidly on endothelial cells during ischemiareperfusion (I-R) and mediates tissue leukocyte accumulation. The ICAM-1 proximal promoter contains a signal transducer and activator of transcription (Stat) binding motif (gamma-interferon activation site [GAS] sequence), which flanks a specificity protein 1 (Sp1) binding site. We examined the roles of Stat and Sp1 in the regulation of ICAM-1 after myocardial I-R. Methods and Results-Open-chest anesthetized rats underwent coronary artery occlusion for 35 minutes and reperfusion for 0 to 240 minutes. Stat became activated within 15 minutes after reperfusion, primarily in vascular endothelial cells; the activated Stat protein was identified as Stat3 (␣-isoform). After phosphorylation on serine 727 (p-S727), Stat3␣ was found in association with the transcriptional regulator Sp1, and the complex bound to an ICAM-1-GAS probe. ICAM-1 expression increased after I-R and lagged shortly behind Stat3␣ activation. In cultured human umbilical vein endothelial (HUVE) cells, activation of Stat3␣ after hypoxia-reoxygenation (H-R) was dependent on the small GTPase Rac1. Transfection of a dominant-negative Stat3 (Y705F) adenovirus or a GAS decoy oligonucleotide reduced ICAM-1 mRNA expression after H-R. Using a reporter gene transfected into HUVE cells, mutation of the GAS element in the ICAM-1 promoter resulted in reduced transcriptional activity after H-R. Sp1 coimmunoprecipitated with p-S727 Stat3 during H-R, and Sp1 or Stat3␣ interfering RNA markedly reduced ICAM-1 mRNA expression. Key Words: adhesion molecule Ⅲ signal transduction Ⅲ ischemia-reperfusion Ⅲ myocardium Ⅲ endothelial cell I ntercellular adhesion molecule-1 (ICAM-1) is a cell surface glycoprotein that is highly expressed in vascular endothelial cells, promotes leukocyte activity in a variety of inflammatory reactions, and plays an important role in mediating neutrophil adherence and tissue injury during reperfusion after ischemia. 1,2 The ICAM-1 proximal promoter contains a signal transducer and activator of transcription 1 (Stat1)/Stat3 binding motif (GAS sequence or palindromic interferon response element), which flanks a specificity protein 1 (Sp1) binding site and a promoter cis-acting sequence (TATAA) box. Reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ) and superoxide, and proinflammatory cytokines such as interleukin-6 (IL-6) and interferon-␥ (IFN-␥), are known to induce transcriptional complexes that bind to the ICAM-1 GAS sequence. [3][4][5] Mutation or deletion of this element decreases ICAM-1 promoter activity. 4,5 In the past 10 years, Stat proteins have been studied intensively as cellular transcriptional regulators. Various Stat proteins, including Stat1 and Stat3, have been reported to be activated in ischemia-reperfusion (I-R). 6,7 However, the role of Stats in I-R is unclear because Stats appear to mediate a broad range of seemingly conflicting processes, including apoptosis, survival pathways, proliferation, angiog...

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Abstract-Fractalkine (FKN) is a membrane-bound chemokine that can be released by proteolysis to produce soluble FKN (s-FKN). FKN and its receptor, CX3CR1, are believed to be important factors in atherosclerosis and may play a role in acute inflammatory responses. Although FKN is expressed on endothelial cells (ECs), CX3CR1 is reported to reside mainly on certain leukocyte populations. RT-PCR and Western blotting demonstrated that both human coronary artery and umbilical vein ECs expressed CX3CR1 mRNA and protein. Confocal microscopy showed that CX3CR1 was located at the cell membrane and to a lesser extent in the cytoplasm. Following exposure of both types of ECs to hypoxia and reoxygenation, FKN expression increased rapidly and s-FKN was shed into the culture medium. The addition of s-FKN protein to cultured ECs resulted in a dose-dependent increase in intercellular adhesion molecule (ICAM)-1 mRNA. Perfusion of mouse hearts with s-FKN protein increased expression of ICAM-1 protein in vascular endothelium. Transfection of ECs with CX3CR1-interfering RNA to knockdown the receptor resulted in decreased ICAM-1 expression after s-FKN stimulation. In addition, when ECs were stimulated with s-FKN, greater adhesion of human neutrophils to the ECs was observed. This increase was ICAM-1 dependent and was blocked by CX3CR1 knockdown. Key Words: endothelial cell Ⅲ CX3CR1 Ⅲ fractalkine Ⅲ adhesion Ⅲ Stat5 F ractalkine (FKN) is a unique membrane-bound molecule expressed on endothelial cells (ECs), possessing a chemokine domain and an extended mucin-like stalk that allows it to function as both a chemoattractant and an adhesion molecule. 1 The chemokine domain contains 2 cysteines separated by 3 other amino acids (CXXXC) and is therefore designated as CX3C ligand-1. Its receptor was identified as a G protein-coupled receptor (GPCR) with high sequence similarity to the genes encoding human chemokine receptors for monocyte attractant protein-1 and macrophage inflammatory protein-1A, 2 and has been designated CX3C receptor1 (CX3CR1). CX3CR1 is believed to reside mainly on certain leukocyte populations, including macrophages, lymphocytes, and natural killer cells but has not been definitively described on ECs. 3,4 FKN has both membrane-bound and soluble forms. Membrane-bound FKN can act as an adhesion molecule to mediate firm adhesion by binding with its receptor on leukocytes. 1,3,4 This membrane-bound form can be cleaved by metalloproteinases 5,6 to create circulating soluble (s)-FKN, a potential chemoattractant. 5 Plasma s-FKN is increased in patients with certain inflammatory conditions, including coronary artery disease, allergic asthma and rhinitis, and brain inflammation. [7][8][9][10] Several studies have reported that FKN and CX3CR1 play an important role in vascular inflammation and injury 4,11-14 and contribute to atherosclerosis. 11,13,14 Although expressed by ECs, it is unknown whether FKN can participate in the activation of ECs or their conversion to a proinflammatory phenotype. FKN-induced endothelial activation co...

DNA-protein complexes (DPCs

No abstract