Changes of von Willebrand factor and antithrombin III levels in acute stroke: difference between thrombotic and haemorrhagic stroke.
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Acute Disease↗
Biomedical subjects
L Liu
Publications and source records attributed to L Liu.
Complex geometry, quantum tunneling, and time machines.
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Journal Article↗
Characterization of the interactions between procoagulant albumin and human endothelial cells.
Normal human plasma contains procoagulant albumin (PC-Al), an anionic form of albumin that induces tissue factor (TF) activity in human umbilical vein endothelial cells (HUVEC) and monocytes. In this study, we investigated both the interactions between HUVEC and PC-Al and the mechanism by which PC-Al induces TF activity. Binding of PC-Al to HUVEC was specific and reversible. Further studies indicated that membrane-bound PC-Al was not internalized by HUVEC. A potential receptor on HUVEC was suggested by studies in which the capacity of a variety of reagents to inhibit the activity of PC-Al was quantitated. Induction of TF activity by PC-Al was antagonized by dextran sulfate, heparin, fucoidan, and concanavalin A but not by ovalbumin, polyglutamic acid, or polyvinyl sulfate. This competition profile bears similarities to those reported for scavenger receptors that have been identified on both HUVEC and monocytes. Involvement of protein kinase C (PKC) in the PC-Al-induced enhancement of TF activity was suggested by experiments in which staurosporine, an inhibitor of PKC, suppressed the activity of PC-Al. The induction of TF activity by PC-Al was further characterized by using a quantitative polymerase chain reaction assay. Increased TF mRNA was first seen after 1 hour of incubation with PC-Al. Maximal observed expression occurred at 2 hours, but at 5 hours, expression had significantly decreased. Monocytes could also be induced to express TF mRNA after a 2-hour incubation with PC-Al. These results suggest that the functionally relevant binding of PC-Al to HUVEC may be mediated through interactions with a membrane constituent that has some of the properties of a scavenger receptor and that this interaction augments TF activity by enhancing transcription of TF mRNA, at least in part, by a mechanism that is dependent on activation of PKC.
Base Sequence↗
Nitric oxide synthase immunoreactive neurons in the rat kidney.
The presence of neurons with nitric oxide synthase (NOS) immunoreactivity was investigated in the rat kidney. Whole kidneys were examined by means of serial sections. The indirect immunocytochemical technique using polyclonal antibody raised against rat brain type Ia NOS and the histochemical technique for nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase are used in this study. NOS-immunoreactive (NOS-IR) neurons varied in size and were observed: (1) associated with nerve bundles at the hilus of the kidney, (2) in the proximity of the lower or middle portion of the interlobar arteries, and (3) on the wall of the renal pelvis. We are presenting anatomic evidence for the presence of neurons in the rat kidney. Their location is consistent with the existence of a parasympathetic innervation of the rat kidney.
Amino Acid Oxidoreductases↗
Multiple cytokines induce the tyrosine phosphorylation of Shc and its association with Grb2 in hemopoietic cells.
The identification and characterization of proteins that become tyrosine-phosphorylated in response to growth factor stimulation is critical to furthering our understanding of the signal transduction pathways involved in regulating cell proliferation and differentiation. In this report we demonstrate that interleukin-3, erythropoietin, and steel factor all induce the tyrosine phosphorylation of the SH2 containing protein, p52shc. These studies were carried out with various human and murine cell lines to document that this is a common event in hemopoietic cells. We also show that upon tyrosine phosphorylation, p52shc becomes associated with the adaptor protein, Grb2. The formation of this complex may directly link tyrosine phosphorylation events to Ras activation in hemopoietic progenitors and may be a critical step in stimulating these cells to transit through G1 into S phase.
Adaptor Proteins, Signal Transducing↗
Erythropoietin stimulates the tyrosine phosphorylation of Shc and its association with Grb2 and a 145-Kd tyrosine phosphorylated protein.
Although the erythropoietin receptor (EpR) lacks a tyrosine kinase consensus sequence within its proline-rich intracellular domain, addition of its ligand to Ep-responsive cells stimulates the rapid and transient tyrosine phosphorylation of a number of cellular proteins. The characterization of these phosphorylatable substrates, which include 5 major phosphoproteins with molecular masses of approximately 145, 130, 97, 72, and 56 Kd is an essential step in understanding the signal transduction pathways used by Ep. Recently, we and others have shown that the major 72-Kd tyrosine phosphorylated protein is the EpR itself. We now report, using both murine DA-3 and human MO7E cell lines engineered to express high levels of biologically responsive EpRs (and designated DA-ER and MO7-ER, respectively), that the major 56-Kd tyrosine phosphorylated protein is the recently identified SH2-containing protein, p52shc. Interestingly, in Ep-stimulated cells, anti-Shc antibodies coprecipitate the major 145-Kd tyrosine phosphorylated protein in both DA-ER and MO7-ER cells. Tyrosine phosphorylation of both proteins is detectable within 30 seconds of incubation with Ep at 37 degrees C, reaches a maximum between 2 and 5 minutes, and declines by 30 minutes. In addition, tyrosine phosphorylated Shc appears capable of associating with the activated EpR, but this could only be shown in MO7-ER cells. Lastly, as has been shown previously with the tyrosine kinase containing receptors for epidermal growth factor, platelet derived growth factor, and insulin, activation of the EpR leads to the association of p52shc with the 25-Kd polypeptide, Grb2. Taken together, our data suggest that the previously reported increases in rasGTP observed with Ep result, in part, from the tyrosine phosphorylation of Shc and its association with Grb2 and/or a tyrosine phosphorylated 145-Kd protein.
Adaptor Proteins, Signal Transducing↗
Asparagine 229 in thymidylate synthase contributes to, but is not essential for, catalysis.
The conserved Asn-229 (N229) of thymidylate synthase (TS, EC 2.1.1.45) provides the only side chain that directly hydrogen bonds with the pyrimidine ring of the substrate dUMP. The carboxamide moiety forms a cyclic hydrogen bond network with the NH-3 and O-4 of the base and is a prime candidate for assisting proton-transfer reactions that occur at O-4 of the pyrimidine ring of dUMP. A complete replacement set of mutants at position 229 of Lactobacillus casei TS (N229 mutants) has been prepared, purified, and characterized. Fifteen of the 19 TS mutants were catalytically active. Steady-state kinetic parameters of N229 mutants varied 17- and 115-fold in the Km values for 5,10-methylene-5,6,7,8-tetrahydrofolate and dUMP, respectively, 1000-fold in kcat values, and 10,000-fold in kcat/Km values. Wild-type TS possesses lower Km and higher kcat and kcat/Km values than any of the TS N229 mutants. We conclude that N229 contributes to, but is not essential for, binding and catalysis. When the wild-type enzyme was not considered, there were excellent correlations between log kcat and the hydrophobicity of the side chains at position 229, in which the more hydrophobic side chains showed higher values. Our results suggest a unique interaction between N229 and the substrates that seems important in appropriately positioning the uracil heterocycle for catalysis. We propose that in the absence of N229, the electrophilic catalyst that transfers protons to the O-4 and stabilizes enol intermediates is a highly conserved molecule of water.
Amino Acid Sequence↗
Exclusion of 2'-deoxycytidine 5'-monophosphate by asparagine 229 of thymidylate synthase.
In thymidylate synthase (TS, EC 2.1.1.45), the only side chain in direct hydrogen bonding with the pyrimidine ring of the substrate dUMP is asparagine 229 (N229). In binary and ternary complexes, the carboxamide moiety of the side chain of N229 forms a cyclic hydrogen bond network bridging N-3 and O-4 of the uracil heterocycle. Most of the N229 mutants of TS bind dUMP and catalyze dTMP formation as well as the wild-type enzyme; thus, N229 does not contribute to binding of dUMP. Wild-type TS binds dCMP weakly and does not accept dCMP as a substrate. Mutations at N229 of TS modify the interaction of TS with dCMP. TS N229D and TS N229E catalyze the methylation of dCMP [Liu, L., & Santi, D. V. (1992) Biochemistry 31, 5010-5014]. With the exception of the TS N229Q, most of the N229 mutants bind dCMP as well as or tighter than dUMP and bind dCMP 300-3000-fold tighter than wild-type TS. We conclude that TS discriminates binding of dUMP versus dCMP by a 3-4 kcal mol-1 difference in binding energy by exclusion of dCMP from the active site. We propose that this exclusion is a consequence of untoward interactions between dCMP and the side-chain carboxamide group of the Asn or Gln at position 229 of TS. We speculate that exclusion of cytosine versus uracil by Asn or Gln may account for specificity observed in other protein-pyrimidine interactions.
Asparagine↗
Identification of acetylcholine receptors in adult rat trigeminal ganglion neurons.
Nicotinic acetylcholine receptors (nAChRs) were identified in a subpopulation of cultured adult rat trigeminal ganglia (TG) neurons by whole-cell patch-clamp recordings. Dimethylphenylpiperazinium (DMPP), a nAChR agonist, induced inward currents in 21/68 of TG neurons having soma diameters greater than 28 microns. These currents were inhibited by hexamethonium, mecamylamine and atropine, indicating the presence of neuronal ganglionic-type nAChRs. This interpretation is consistent with the finding that the nicotine- or DMPP-induced currents were not inhibited by alpha-bungarotoxin (alpha-Bng) in 5 of the 9 cells tested with this compound. However, in 2 of the 9 cells tested, the DMPP-induced currents were completely inhibited by alpha-Bng, and in the remaining two cells tested, the currents were partially inhibited by alpha-Bng. About 22% of the cells having diameters > or = 28 microns were specifically labeled with FITC-labeled alpha-Bng, whereas only 2% of the cells with soma diameters < 28 microns were labeled. These data taken together suggest that more than one subtype of nAChR is present in TG.
Acetylcholine↗
Posttranslational processing and targeting of transgenic human defensin in murine granulocyte, macrophage, fibroblast, and pituitary adenoma cell lines.
Human defensins are 29 to 30 amino acid (aa) antimicrobial peptides that are among the principal constituents of the neutrophil's azurophil granules. To determine the tissue specificity of posttranslational processing and subcellular targeting of defensins, the cDNA for a 94 aa human preprodefensin was transduced into murine cell lines (NIH 3T3 embryonic fibroblasts, AtT-20 pituitary adenoma, J774.1 and RAW 264.7 macrophages, and 32D and 32D cl3 granulocytes) using retroviral vectors. All transduced cell types expressed and to a variable extent constitutively secreted a 75 aa prodefensin formed by the removal of the amino terminal signal sequence. In AtT-20 cells, the 75 aa form accumulated intracellularly in granules and was releasable by secretagogues. Proteolytic processing to mature defensins was seen only in myeloid cells (J774.1, RAW 264.7, 32D, and 32D cl3). Newly formed mature defensin was rapidly degraded in J774.1 and RAW 264.7 macrophages, but accumulated stably in multivesicular bodies in 32D cells and in cytoplasmic granules of 32D cl3 cells. Our data suggest that the enzymatic and transport machinery required to process preprodefensin to mature defensin and to store it in cytoplasmic granules is a specialized feature of cells of granulocytic lineage.
3T3 Cells↗
The structure of neutrophil defensin genes.
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Amino Acid Sequence↗
Axon growth is enhanced by NCAM lacking the VASE exon when expressed in either the growth substrate or the growing axon.
The neural cell adhesion molecule NCAM exists as several related peptides formed by alternative splicing of the single NCAM gene. Here the ability of NCAM containing and lacking the alternatively spliced VASE exon to act as a permissive growth substrate was tested by examining retinal axon outgrowth on normal L cell fibroblasts and L cells expressing stably transfected 140 kD NCAM +/- VASE. L cells expressing either NCAM form were a more permissive substrate than control L cells. At higher substrate cell densities, greater axon outgrowth occurred on substrate cells expressing NCAM - VASE than on those expressing NCAM + VASE. Similar experiments tested retinal axon growth on neuronal substrates by utilizing clonal B35 cells, C3 cells that are NCAM lacking variants of B35, and C3 cells into which 140 kD NCAM +/- VASE has been restored by transfection. Axon growth on C3 cells transfected with NCAM - VASE was greater than that on all other substrates including cells transfected with NCAM + VASE. In these experiments C3 cells and transfected C3 expressing NCAM + VASE cell promoted similar outgrowth. The influence on neurite growth of the NCAM isoform of the neurite itself was tested by examining neurite formation using combinations of C3 cells and C3 NCAM transfectants both in the growth monolayer and as responding cells. C3 cells were able to extend neurites, indicating NCAM is not required for neurite growth. However, C3 derivatives transfected with NCAM +/- VASE had greater neurite outgrowth. The most extensive neurite growth was found when NCAM - VASE was expressed by both substrate cells and the responding neurite growing cells. Thus NCAM enhances axon or neurite outgrowth when present either in the growth substrate or on the growing axon. NCAM - VASE has a significantly greater growth promoting capability than NCAM + VASE. The expression of NCAM + VASE by more mature neural cells could thus be a significant factor in the reduced axonation capabilities of mature neurons.
Animals↗
Amplification and overexpression of the MDM2 gene in a subset of human malignant gliomas without p53 mutations.
The MDM2 (murine double minute 2) gene has recently been shown to code for a cellular protein that can complex the p53 tumor suppressor gene product and inhibit its function. We studied a series of 157 primary brain tumors and report here that the MDM2 gene is amplified and overexpressed in 8-10% of glioblastomas and anaplastic astrocytomas. Thus, MDM2 represents the second most frequently amplified gene after the epidermal growth factor receptor gene in these tumor types. Sequencing of the p53 transcripts in the cases with MDM2 amplification revealed no mutations and restriction fragment length polymorphism analysis showed, with one exception, no losses of alleles on chromosome 17. Our results indicate that amplification and overexpression of MDM2 may be an alternative molecular mechanism by which a subset of human malignant gliomas escapes from p53-regulated growth control.
Astrocytoma↗
The structure of neutrophil defensin genes.
Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and -2) or Paneth cells (HD-5) are organized differently: HD-5 had only two exons, and MCP-1 and -2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression.
Amino Acid Sequence↗
Solid-phase separation of 14C-labelled urea and aminoisobutyric acid for membrane transport studies.
A sorbent extraction method has been developed for separating 14C-labelled urea and aminoisobutyric acid (AIB) in blood. The use of commercial solid-phase extraction cartridges containing aminopropyl-bonded silica provided a convenient and rapid separation of urea and AIB with better than 92% recovery of each and less than 5% cross-contamination. This allows these compounds, together with [3H]methylglucose, to be used as marker compounds for investigating three aspects of membrane transport. The facility to separate any two of the three compounds permits their simultaneous measurement, greatly increasing the amount of data obtainable from each in vivo preparation.
3-O-Methylglucose↗
Duplicated KOX zinc finger gene clusters flank the centromere of human chromosome 10: evidence for a pericentric inversion during primate evolution.
Two related zinc finger (ZNF) gene clusters from the pericentromeric region of human chromosome 10, defined by cDNAs of the KOX series, have been cloned in yeast artificial chromosomes (YACs). The two clusters evolved by duplication of an ancestral gene cluster before the divergence of the human and great ape lineages. Included in cluster A are the ZNF gene sequences ZNF11A, ZNF33A, and ZNF37A, while cluster B comprises the related sequences ZNF11B, ZNF33B and ZNF37B. Genes from both clusters are expressed: cDNAs KOX2, KOX31 and KOX21 derive from ZNF11B, ZNF33A and ZNF37A, respectively. Further YACs have been isolated which link ZNF11A and ZNF33A to another gene, ZNF25, defined by cDNA clone KOX19. Therefore ZNF25 also forms part of cluster A, but has no counterpart in cluster B. Surprisingly, the KOX ZNF gene clusters are located on opposite sides of the centromere: cluster A maps to 10p11.2, while cluster B is in 10q11.2. This suggests the occurrence during primate evolution of a previously undescribed pericentric inversion subsequent to the cluster duplication. The evolution of this subset of KOX ZNF genes has therefore involved three types of genetic event: local gene duplication, gene cluster duplication, and chromosome rearrangement.
Amino Acid Sequence↗
The L2/HNK-1 carbohydrate mediates adhesion of neural cells to laminin.
The L2/HNK-1 carbohydrate epitope shared by several neural adhesion molecules has been implicated in cell-to-cell and cell-to-laminin adhesion (Keilhauer et al., Nature, 316, 728-730, 1985; Künemund et al., J. Cell Biol., 106, 213-223, 1988). As demonstrated previously for chicken retinal ganglion cells (Cole et al., Neurosci. Lett., 93, 170-175, 1988), cerebral cortex astrocytes or cerebellar neurons could not be shown to adhere to the substrate-bound L2/HNK-1 carbohydrate. The cell-bound L2/HNK-1 carbohydrate, however, was a potent mediator of astrocytic and neuronal cell adhesion to laminin, which was strongly reduced in the presence of the L2/HNK-1 carbohydrate-carrying glycolipids or Fab fragments of a monoclonal antibody against it. Inhibition of adhesion could not be observed in the presence of the negatively charged gangliosides or sulphatide, but in the presence of heparin. To investigate whether the L2/HNK-1 carbohydrate and heparin use the same or different binding sites on laminin, adhesion of cells to laminin was determined in the presence of heparin and Fab fragments of a monoclonal L2 antibody, which gave an additive value of inhibition as compared to the inhibition caused by the single compounds. This result, as well as studies of the binding of the L2/HNK-1 glycolipids to laminin in the presence of heparin, indicates that the L2/HNK-1 carbohydrate and heparin are implicated in different aspects of neural cell adhesion to laminin.
Animals↗
Perinatal phenotype and hypothyroidism are associated with elevated levels of 21.5- to 22-kDa basic fibroblast growth factor in cardiac ventricles.
Control of cardiomyocyte growth and differentiation may be exercised, in part, at the level of expression of endogenous growth factors such as bFGF (basic fibroblast growth factor), believed to act locally, in an autocrine or paracrine fashion. Examination of bFGF accumulation by extract fractionation and immunoblotting indicated a 4-fold increase of an 18-kDa bFGF in adult compared to newborn rat heart ventricles. In contrast, a 22-kDa bFGF species, found to be the predominant form in newborn ventricles, displayed a 2.5-fold decrease in extracts from adult (compared to neonatal) ventricles. Since newborn rats are physiologically hypothyroid, the effect of thyroid status on the accumulation of 22-kDa bFGF in the heart was examined. A 21.5- to 22-kDa bFGF showed a 5-fold increase in extracts from hyperthyroid rat heart ventricles, compared to those from euthyroid controls. The 21.5- to 22-kDa bFGF was essentially unaffected by thyroid status in extracts from brain. Our data point to a correlation between immature or differentiated cardiac phenotype and the predominance of 21- to 22.5-kDa or 18-kDa bFGF species, respectively. Elevated levels of 21.5- to 22-kDa bFGF detected in cardiac ventricles in either physiological or experimentally induced hypothyroidism indicates that thyroid hormone may, directly or indirectly, down-regulate accumulation of the higher molecular mass forms of bFGF in the heart.
Animals↗