Biomedical subjects
Publications and source records attributed to L J Li.
A new ceramide, named tuber-ceramide (2), along with a known cerebroside (1) were isolated from the seeds of Allium tuberosum. The structure of tuber-ceramide was determined on the basis of spectral data as N-(2',3'-dihydroxy-tetracosenoyl)-2-amino-1,3,4-trihydroxy octadecane (2). This is the first report of sphingosine derivatives isolated from the genus Allium.
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In the presence of montmorillonite K 10, 5(6)-unsaturated sterols (1) were heated under ultrasound at 45 degrees C in dichloromethane for 2-5.5 h to provide 3 beta,3'beta-5(6)/5'(6')-unsaturated disteryl ethers (2) in 35-79% yield. Meanwhile, 3 alpha,3'alpha-diandrost-5-en-17-one-3-yl ether (3a) and 3 alpha,3'alpha-dipregn-5-en-20-one-3-yl ether (3b) were also obtained as by-products from 3 beta-hydroxyl-androst-5-en-17-one (1a) and 3 beta-hydroxyl-pregnan-5-en-20-one (1b) respectively.
The nature of the antagonism by anticholinergic compounds of nicotine-induced convulsion in mice has not been defined clearly. Although, because they do not compete effectively for agonist binding to brain tissue in-vitro, these compounds are thought to be non-competitive antagonists in the brain, pharmacological evidence is lacking. This study describes the anti-nicotinic properties of the clinically used anticholinergic antiparkinson drugs, benztropine, biperiden, caramiphen, ethopropazine, procyclidine and trihexyphenidyl. Nicotine-induced convulsion and arecoline-induced tremor in mice were effectively prevented by these drugs. The concentrations of benztropine, biperiden, caramiphen, ethopropazine, procyclidine and trihexyphenidyl affording 50% prevention of nicotine-induced convulsion (ED50 values) were 7.4, 4.6, 7.8, 4.9, 3.1 and 3.3 mg kg(-1), respectively. The classical muscarinic receptor antagonist atropine had potent anti-muscarinic effects but very weak anti-nicotinic activity. The classical nicotinic receptor antagonist mecamylamine had potent anti-nicotinic activity but no anti-muscarinic effects. The pattern of shift of the dose-response curve for nicotine-induced convulsion in mice was determined in the presence of increasing concentrations of the anticholinergic antiparkinson drugs. These drugs were found to increase the ED50 (0.49 mg kg(-1)) of nicotine-induced convulsion in a dose-related manner. The maximum effect of nicotine and the slope of nicotine dose-response curve were not significantly influenced by either low or high doses of benztropine, procyclidine or trihexylphenidyl, which suggests competitive action. Biperiden, caramiphen and ethopropazine, at low doses which significantly increased the ED50 of nicotine, did not affect the maximum effect of nicotine or the slope of the nicotine dose-response curve; at higher doses, however, they reduced the maximum effect and the slope, which suggests that these drugs have both competitive and non-competitive properties in antagonizing nicotine-induced convulsion in mice. The experiments demonstrate that the anticholinergic antiparkinson drugs and mecamylamine effectively antagonize nicotine-induced convulsion, but atropine does not; some of these drugs have competitive properties whereas others seem to have both competitive and non-competitive properties in antagonizing nicotine-induced convulsion in mice.
The regulated expression of intercellular adhesion molecule-1 (ICAM-1) by cytokines such as tumor necrosis factor alpha (TNF-alpha) plays an important role in inflammation and immune responses. Induction of ICAM-1 gene transcription by TNF-alpha has previously been shown to be dependent upon a region of the ICAM-1 5'-flanking sequences that contains a modified kappaB site. We demonstrate here that this modified kappaB site alone is insufficient for induction of transcription by TNF-alpha. Site-directed mutagenesis of both the kappaB site and specific flanking nucleotides demonstrates that both the specific 5'- and 3'-flanking sequences and the modified kappaB site are necessary for TNF-alpha induction. Further, site-directed mutagenesis of this modified kappaB site to a consensus kappaB site allows it to mediate transcriptional activation in response to TNF-alpha, even in the absence of specific flanking sequences. Transcription through this minimal ICAM-1 TNF-alpha-responsive region can be driven by co-expression of p65, and the minimal response element interacts with p65 and p50 in supershift mobility shift assays. However, when in vitro transcription/translation products for the Rel proteins are used in an electrophoretic mobility shift assay, only p65 is capable of binding the minimal response element while both p50 and p65 bind a consensus kappaB oligonucleotide. Additionally, in the absence of the specific flanking nucleotides, the ICAM-1 kappaB site is incapable of DNA-protein complex formation in both electrophoretic mobility shift assay and UV cross-linking/SDS-polyacrylamide gel electrophoresis analysis. These results demonstrate the requirement for specific flanking sequences surrounding a kappaB binding site for functional transcription factor binding and transactivation and TNF-alpha-mediated induction of ICAM-1.
In response to interferon gamma (IFNgamma), intercellular adhesion molecule-1 (ICAM-1) is expressed on human keratinocytes, a cell type that is critically involved in cutaneous inflammation. An ICAM-1 5' regulatory region palindromic response element, pIgammaRE, has been shown to confer IFNgamma-dependent transcription enhancement. By electrophoretic mobility shift assays (EMSA), pIgammaRE forms a distinct complex with proteins from IFNgamma-treated human keratinocytes, termed gamma response factor (GRF). Binding of GRF is tyrosine phosphorylation-dependent, and mutations of pIgammaRE that disrupt the palindromic sequence or alter its spatial relationship abrogate GRF binding. Supershift EMSAs using antibodies to characterized STAT proteins suggest that GRF contains a Stat1alpha-like protein; however, non-ICAM-1 IFNgamma-responsive elements (REs) known to bind Stat1alpha homodimers fail to compete for GRF binding in EMSA, and pIgammaRE does not cross-compete with these REs that complex with homodimeric stat1alpha. The pIgammaRE x GRF complex also displays a distinctly different electrophoretic mobility compared to that of IFNgammaREs complexed to homodimeric Stat1alpha. These findings indicate that a distinct complex containing a Stat1alpha-like protein mediates IFNgamma-induced ICAM-1 gene transcription and identifies a subset of IFNgamma-responsive genes that appear to be regulated by this complex.
We conducted a randomized, open-label, controlled, multicenter study to compare sulbactam/cefoperazone with cefotaxime in terms of efficacy and safety for the treatment of hospitalized patients with moderate-to-severe bacterial infections. More than two-thirds of the pathogens recovered from these patients produced beta-lactamase. Two hundred-seven (88.1%) of the 235 patients enrolled completed the study and were included in the efficacy and safety evaluations. One hundred-three patients received sulbactam/cefoperazone (2-4 g/d) administered in evenly divided doses every 12 hours by a 30-minute intravenous drip; 104 patients received cefotaxime (6-12 g/d) administered in evenly divided doses every 6 or 8 hours by a 30-minute intravenous drip. The overall efficacy rates (i.e., cure or markedly improved) were 95% for the sulbactam/cefoperazone group and 90% for the cefotaxime group (P = .186), whereas the bacterial eradication rates were 85% for the sulbactam/cefoperazone group and 81% for the cefotaxime group (P = .467). Both drug regimens were well tolerated. Sulbactam/cefoperazone is effective and safe for the treatment of moderate-to-severe bacterial infections caused mainly by beta-lactamase-producing organisms.
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Promoter elements that are important for the G1-S induction of the human thymidine kinase (htk) promoter reside within the core of the cell cycle regulatory unit, positioned between -110 and -84 upstream of the TATA element. Within this 27-bp region are three GC-rich motifs, which resemble the E2F binding site. By site-directed mutagenesis, we identified a 14-bp region, between -97 and -84, critical for the htk promoter transcriptional activity. Methylation interference studies indicate that the sequences between -97 and -84 are major protein contact points, correlating with the functional significance of this sequence in vivo. Although the core of the cell cycle regulatory unit contains three E2F-like sites and can form minor S-phase-specific complexes containing p107, cyclin A, and cdk2, the major complex that binds to this region is not competed by E2F binding sites. Through DNA affinity chromatography, we identified a set of protein species of approximately 40 kDa that copurified with the htk DNA binding activity. From gel shift assays and Western blot analysis, this protein species is antigenically distinct from E2F-1, E2F-2, E2F-3, and E2F-4. Our studies raise the possibility that other members of the E2F protein family or a novel protein(s) with preferred binding affinity for the htk promoter exert(s) control on the G1 to S regulation of the htk promoter through their interactions with cyclins and kinases.
The analysis of the carcinoembryonic antigen (CEA) promoter in the colon carcinoma lines HT-29 and SW403, using HeLa as a control, was performed using gel mobility shift assays and in vitro and in vivo footprinting before and after IFN-gamma treatment. Using a 332-bp probe extending from the start of translation (+1 to -331), we detected 4-5 specific complexes that increased with intensity with IFN-gamma treatment as measured by a gel mobility shift assay. In contrast, no complexes were observed for probes covering the regions -500 to -1000 and -1000 to -1500. DNase I in vitro footprinting with the 332-bp probe revealed three footprints, FP-I to FP-III, none of which changed during the course of IFN-gamma treatment. Using probes corresponding to each footprint, 6-7 specific complexes were observed by gel mobility shift assays. Although minor changes were observed on IFN-gamma treatment, no consistent pattern was observed for all cell lines tested. Several of the proteins involved in the promoter complexes were identified by antibody super shifts, UV cross-linking, and Southwestern blotting. FP-I bound an Sp1-like protein, binding to a GT box sequence, and USF (upstream regulatory factor). FP-II and FP-III bound Sp1, binding through the consensus sequence for a GC box. Lower molecular weight complexes of an unknown nature were observed with sequence specificity for both single- and double-stranded DNA. DNase I in vivo footprints confirmed the boundaries of FP-I to FP-III and revealed a fourth but weaker footprint, FP-IV. The strongest in vivo footprints were observed for SW403 cells, with weaker and no footprints observed for HeLa cells, thus correlating with the degree of CEA transcriptional activity (HeLa cells make no CEA mRNA). DNase I hypersensitive sites correlated well with the boundaries of the footprints and also revealed activity around the start of transcription (-110). The specific pattern for DNase I hypersensitivity for Sp1 in the CEA promoter was the same as observed for the SV40 early promoter. In vivo footprinting with dimethyl sulfate revealed protein binding at the Sp1 consensus sequences in FP-II and FP-III and at the USF consensus sequence in FP-I. We conclude that in vivo footprinting is the most accurate predictor of the state of transcriptional activity of the CEA gene. It is also likely that Sp1 and USF play a major role in CEA transcriptional activation and that the majority of IFN-gamma effects are at the posttranscriptional level.
The surface glycoprotein intercellular adhesion molecule-1 (ICAM-1) mediates important immunologic cell interactions during cutaneous inflammatory processes by binding to the leukocyte integrin lymphocyte function-associated antigen-1. The expression of ICAM-1 is induced in epidermal keratinocytes by certain pro-inflammatory stimuli, and this modulation is transcriptionally regulated. To identify the molecular mechanisms involved in the regulation of ICAM-1 gene expression, we have previously cloned the transcriptional regulatory region of the human ICAM-1-gene and have characterized a functional promoter. Here we have used the phorbol ester phorbol-12-myristate-13-acetate (PMA) to further evaluate the transcriptional mechanisms of ICAM-1 gene induction in A431 cells. Exposure to PMA induced ICAM-1 both at the mRNA and cell surface level. Promoter activity and PMA-enhanced effects were assessed by transiently transfecting A431 cells with chloramphenicol acetyl transferase reporter gene constructs containing a series of sequential ICAM-1 5' deletions. Constructs containing ICAM-1 5' fragments from -1162/+1 (relative to the transcription start site) to -277/+1 displayed a threefold increase in promoter activity when cells were stimulated with PMA. Inducibility dropped below 1.5-fold in chloramphenicol acetyl transferase construct -182/+1. Using electrophoretic mobility shift assays, a PMA-inducible binding site was identified for an NF kappa B-like complex within positions -186/-177. A -199/-170 fragment containing this NF kappa B-like element conferred PMA responsiveness when cloned into a thymidine kinase-driven chloramphenicol acetyl transferase vector, indicating that the region containing this NF kappa B-like element is not only necessary but also sufficient for PMA induction of ICAM-1.
Endothelial cells rest on a basement membrane that anchors them to the vessel wall. The alpha 6 beta 4 integrin complex has been described on epithelial cells, frequently localizes to basement-membrane structures, and appears to play a role in binding epithelial cells to laminin. We have determined that human microvascular endothelial cells express the beta 4 integrin chain in vivo and that it preferentially localizes to the endothelial basement membrane. Human microvascular endothelial cells and human umbilical vein endothelial cells also express cell-surface beta 4 in vitro. In addition, the expression of beta 4 appears to be polarized to the undersurface of endothelial cell monolayers in vitro, mimicking its in vivo localization. Stimulation of microvascular endothelial cells with basic fibroblast growth factor or phorbol 12-myristate 13-acetate, agents previously shown to induce endothelial cell migration in vitro, resulted in a marked decrease in cell-surface expression of the beta 4 integrin chain, associated with a decrease in beta 4 mRNA. These data demonstrate that human endothelial cells express the beta 4 integrin chain in vivo and in vitro, the expression of this integrin chain is polarized, and its expression is regulated on microvascular endothelial cells by factors important in wound healing and vascular regeneration.
Using antibody raised against putative Form I phosphatidylinositide-specific phospholipase C (PI-PLC) and direct amino acid sequencing of the protein recognized by this antibody, we have shown that the antibody reacts with luminal endoplasmic reticulum (ER) proteins, including ERp61. ERp61 possesses a COOH-terminal QEDL sequence that acts as an ER retention signal. Additional experiments have shown, however, that PI-PLC activity is separable from ERp61 and that rat or murine ERp61 expressed in COS cells failed to produce an increase in PI-PLC activity in the COS cells. Finally, we have identified ERp61 as GRP58, a 58-kDa protein inducible by glycosylation block and treatment with the Ca2+ ionophore, A23187.
E-selectin is an inducible endothelial cell adhesion protein that is a critical element in the binding of leukocytes to activated endothelial cells. It is induced by a variety of pro-inflammatory soluble substances including interleukin-1 (IL-1), tumor necrosis factor (TNF), or bacterial lipopolysaccharide (LPS). In vitro studies of a large vessel endothelial cells demonstrate that stimulation with TNF or IL-1 leads to a rapid, but transient, induction of E-selectin expression that disappears within 24 hours. However, in vivo studies have shown that microvascular endothelial cells persistently express E-selectin in chronic inflammatory states, particularly in the skin where it serves as a homing receptor for memory T cells. Stimulation of dermal-derived microvascular endothelial cells (HDMECs) with single doses of IL-1 alpha, TNF alpha, or LPS resulted in transient but slightly more persistent expression of E-selectin than seen after stimulation of large vessel derived umbilical vein endothelial cells (HUVECs). However, stimulation of either HDMECs or HUVECs with repetitive doses of IL-1 alpha, TNF alpha, or LPS in the presence of human serum or plasma resulted in persistent E-selectin expression in vitro. The persistent E-selectin cell surface expression was associated with persistent E-selectin mRNA expression and correlated with E-selectin-mediated HL-60 binding to endothelial cell monolayers. The effect of human plasma or serum was dose dependent, and fractionation of human plasma by gel filtration demonstrated that the E-selectin persistence activity resolved into high and low molecular peaks. These data demonstrate that human endothelial cells are capable of persistent E-selectin expression in vitro and that factors in human serum or plasma are critical in preventing cytokine refractoriness and loss of E-selectin expression. This study provides a basis to resolve the apparent discrepancies between previous in vivo and in vitro dynamics of E-selectin expression.
Although a portion of ultraviolet light (UV) penetrates into the dermis, histologic changes that occur within the dermal microvasculature have largely been attributed to the elaboration of biologic substances, such as interleukin 1 (IL-1), from other constitutive cells of the skin, as opposed to a direct effect of UV on the endothelial cell. As a potential model for understanding early molecular events occurring in UV-induced cutaneous inflammation, we have examined the direct effects of UVB, as well as cytokine-positive controls, upon human dermal microvascular endothelial cells (HDMEC) cell adhesion molecule (CAM) gene expression. Cultured HDMEC were exposed to varying dosages of UVB, and examined for cell surface and mRNA expression of the CAMs intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (formerly ELAM-1). Following UVB exposure, dose-dependent increases in baseline cell surface expression of ICAM-1 were demonstrated by fluorescence-activated cell sorter analysis with concomitant increases in ICAM-1 mRNA, as shown by Northern blot analysis; there was no induction of either E-selectin or VCAM-1. The UVB-induced ICAM-1 upregulation could not be blocked by antibodies to IL-1 or tumor necrosis factor alpha (TNF-alpha). In fact, ICAM-1 gene regulatory region based CAT reporter gene plasmids, including constructs containing IL-1- and TNF-alpha-responsive elements, did not display increased CAT expression after transfection into HDMEC followed by UVB exposure, though control cytokine-treated transfectants did. Thus, UVB selectively upregulates ICAM-1, but not E-selectin or VCAM-1, mRNA and cell surface expression in HDMEC, and this upregulation is not dependent upon the autologous secretion and activity of either IL-1 or TNF-alpha.
Modulation of the expression of the alpha v beta 3 complex on human dermal microvascular endothelial cells (HDMEC) may be crucial in wound healing and angiogenesis. Therefore, we examined the influence of basic fibroblast growth factor (bFGF), transforming growth factor beta, and interferon-gamma (IFN-gamma) on the expression of this complex. Stimulation of HDMEC with bFGF increased cell surface expression of both alpha v and beta 3 in a dose- and time-dependent manner associated with the development of a spindled, elongated cell morphology. Northern blot analysis of HDMEC stimulated with bFGF demonstrated a marked increase in beta 3 but not alpha v mRNA expression. Incubation of HDMEC with transforming growth factor-beta or interferon-gamma alone resulted in modest decreases in cell surface alpha v beta 3, and co-incubation of HDMEC with bFGF and transforming growth factor-beta or interferon-gamma inhibited bFGF-induced changes in cell morphology, increases in cell surface alpha v beta 3 expression, and increases in beta 3 mRNA. These data demonstrate that both growth factors and pro-inflammatory cytokines alter the expression of alpha v beta 3 on microvascular endothelial cells and that these alterations correlate with changes in cell morphology.
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