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Phosphatidylethanol (PEth) is formed only in the presence of ethanol, via the action of phospholipase D. We studied PEth in blood as a possible marker of alcohol abuse in 15 male alcoholics admitted for detoxification. Blood was drawn on the first day after admission and up to 28 days thereafter. PEth in whole blood was 13.2 +/- 2.2 mumol liter-1 (mean +/- SE) at first sampling and remained detectable up to 14 days after admission. Blood ethanol was 0 on the morning after admission. The time courses of PEth disappearance varied among individuals. No PEth could be found in blood of control persons who had abstained from ethanol for 4 days. Levels of PEth and carbohydrate-deficient transferrin or gamma-glutamyltranspeptidase did not correlate. Its high specificity and prolonged detectability suggest PEth in blood as a marker of recent alcohol abuse.
Leprechaunism is a rare autosomal recessive disorder characterized by marked intrauterine and postnatal growth retardation, severe insulin resistance, and altered glucose homeostasis. This syndrome is related to mutations in the insulin receptor (IR) gene that impair the transmission of the insulin signal by several mechanisms. There is no effective therapy and patients usually die within the first months of life. Here we report the prenatal diagnosis of leprechaunism in two unrelated families in which affected children were compound heterozygotes with two different deficient IR alleles. In family Par-1, the disease IR alleles carried a missense mutation located in exon 18 (Arg1092-->Trp) and exon 20 (Glu1179-->Lys). In family Als, a 3-basepair deletion causing the loss of Asn281 in exon 3 and a major deletion of exons 10-13 were present in the maternal and paternal mutant IR alleles, respectively. Prenatal diagnosis was made in each family by a specific approach combining denaturing gradient gel electrophoresis (DGGE) and Southern blotting. This methodology allowed us to correctly predict the genotype of the two fetuses at the IR locus.
Vertebrate soluble beta-galactoside-binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross-reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), even when followed by immunoblotting. However, their different isoelectric points allow their identification using isoelectric focusing and two-dimensional (2-D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2-D immunoblotting using anti-galectin 1 (Gal-1) and anti carbohydrate-binding protein 15 (CPB15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG-Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside-binding proteins even at basic pIs.
We studied the biological properties of insulin receptors (IRs) and insulin-like growth factor-I (IGF-I) receptors in cultured fibroblasts from a patient with leprechaunism (leprechaun Par-1). Patient cells displayed normal insulin binding capacity and affinity. Basal in vivo autophosphorylation and in vitro exogenous kinase activity of patient IRs were elevated twofold to threefold compared with control receptors, and insulin had no further effect on these processes. Moreover, patient IRs were unable to promote the stimulation of metabolic and mitogenic pathways. IR substrate-1 (IRS-1) and mitogen-activated protein (MAP) kinase tyrosine phosphorylation and glycogen and DNA synthesis were not increased in the basal state in patient fibroblasts and were also insensitive to the stimulatory effect of insulin. As for IGF-I, although binding and receptor kinase activity were normal, the ability to stimulate glycogen and DNA synthesis was altered in patient cells. Two mutant alleles of the IR gene were detected by denaturing gradient gel electrophoresis (DGGE) and direct sequencing. The maternal allele contained a point mutation in exon 18 encoding the tryptophan-for-arginine substitution at position 1092, and the paternal allele had a point mutation in exon 20 substituting lysine for glutamic acid at codon 1179. Thereby, leprechaun Par-1 was a compound heterozygote for two missense mutations located in the IR beta-subunit. The present investigation provides the first evidence that leprechaunism can be causally related to structural alterations in the tyrosine kinase domain of the IR. These alterations result in severe impairment of insulin and IGF-I action.
We studied the structure and function of the insulin receptor (IR) in two sisters with leprechaunism. The patients had inherited alterations in the IR gene and were compound heterozygotes. Their paternal IR allele carried a major deletion, including exons 10-13, which shifted the reading frame and introduced a premature chain termination codon in the IR sequence. This allele was expressed at a very low level in cultured fibroblasts (< 10% of total IR messenger ribonucleic acid content) and encoded a truncated protein lacking transmembrane and tyrosine kinase domains. The maternal IR allele was deleted of 3 bp in exon 3, causing the loss of Asn281 in the alpha-subunit. This allele generated levels of IR messenger ribonucleic acid and cell surface receptors similar to those seen in control fibroblasts. However, IRs from patients' cells had impaired insulin binding and exhibited in vivo and in vitro constitutive activation of autophosphorylation and tyrosine kinase activity. As a result of this IR-preactivated state, the cells were desensitized to insulin stimulation of glycogen and DNA syntheses. These findings strongly suggest that Asn281 of the IR alpha-subunit plays a critical role in the inhibitory constraint exerted by the extracellular alpha-subunit over the intracellular kinase activity.
The effect of long-term ethanol exposure on muscarinic receptors was investigated in human neuroblastoma SH-SY5Y cells. Exposure to 100 mM ethanol for 4 days enhanced both peak and steady-state levels of carbachol-stimulated inositol 1,4,5-bisphosphate increase. An ethanol concentration of 50 mM was sufficient for an enhancement of this event. The carbachol-stimulated decrease in [3H]inositol-labeled [3H]phosphatidylnositol 4,5-bisphosphate and increase [3H]inositol trisphosphate and [3H]inositol bisphosphate were also potentiated in ethanol-treated cells, which demonstrated that the receptor-stimulated activation of phospholipase C is augmented. Experiments with pirenzepine showed that carbachol-stimulated inositol 1,4,5-trisphosphate increase is mediated via M1 receptors both in ethanol-treated and control cells. Ethanol exposure for 2 or 4 days also caused an increase in [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate binding sites and elevation of [3H]pirenzepine binding, which indicated that the number of muscarinic M1 receptors is increased in ethanol-treated SH-SY5Y cells. These results demonstrate that long-term exposure to ethanol potentiates muscarinic M1 receptor-stimulated activation of phospholipase C in SH-SY5Y cells. This is likely to be explained by an increased number of muscarinic M1 receptors.
The coupling of glycolysis to serine and glycine metabolism was studied in fast-growing Zajdela hepatoma cultured cells. During the exponential phase of growth, occurring between 12 and 72 h, cells exhibited a decreased glycogen content together with a high glycolytic activity. Glycogen labelling, evaluated by 1 h-pulse experiments with [U-14C]glucose (5.5 mM), was minimal during the first 48 h and increased 2.5-fold at 72 h and 8-fold at 96 h, at which times it was also stimulated 2-fold by 10 nM insulin. [U-14C]Glucose carbons were incorporated into nucleic acid bases, with maximal incorporation at 72 h, the rate of nucleotide base labelling exceeding that of glycogen during the first 2 days of culture. Incubation of the cells with [U-14C]glucose resulted in the release into the medium of 14C-labelled glycine, the first intermediate formed on the route from serine to DNA. The rate of release per cell decreased as a function of cell growth, concomitantly with an increased rate of glucose carbon incorporation into nucleotide bases. The latter implied the intermediary formation of amino acids since the transaminase inhibitor cycloserine (10 mM), which totally inhibited [14C]glycine release, decreased by 65% nucleotide labelling from [U-14C]glucose. A dose-dependent inhibition by serine of the rate of [U-14C]glucose carbon incorporation into nucleotide bases was observed, which was maximal at 5 mM serine. These metabolic flux measurements indicate that glucose can be used as a precursor of nucleic acid synthesis. These results strongly suggest that this process is to a large extent mediated by a serine/glycine-biosynthesis-mediated pathway, and reinforce the hypothesis that glycolysis contributes to enhancing the provision of precursors required for cell proliferation.
A method for the purification and biotinylation of Fab fragments, using thiophilic adsorption (T-gel), is described. The T-gel was used to purify an IgG fraction directly in the buffer suitable for biotinylation, and to adsorb intact IgGs and papain after enzymatic digestion. For the final step, Fc fragments were removed with a protein A column.
We have recently identified oligoclonal IgG antibodies that are related to a human brain lectin (HBL14) from serum and cerebrospinal fluid of patients with neurological disorders. They were termed lectin-like IgG (L-IgG) (Joubert-Caron et al., 1994a,b). In this paper, the occurrence of antibodies reactive both towards HBL14 and L-IgG was investigated. Binding of antibodies to HBL14 was demonstrated by solid-phase ELISA and chromatography on immobilized HBL14. Fab fragments of these antibodies were also shown to bind to HBL14. The specificity of the antibodies towards HBL14 was studied using a panel of different antigens. Our data show that individual sera from healthy people as well as a pool of immunoglobulins from 80 blood donors contain an IgG autoreactivity to HBL14, while no IgM autoreactivity was detected. Anti-HBL14 antibodies from sera were purified using affinity chromatography on immobilized HBL14. Affinity chromatography further allowed us to demonstrate that the binding of anti-HB14 antibodies was mediated through their Fab fragments. A higher amount of anti-HBL14 antibodies was purified using a L-IgG-depleted fraction of sera. The binding of anti-HBL14 antibodies to L-IgG was confirmed by ELISA. Finally, anti-HBL14 antibodies were found to be polyreactive. These results indicate the occurrence of a novel class of natural antibodies reactive towards a human brain lectin and suggest that these antibodies may participate in immunoregulatory mechanisms probably though idiotypic/anti-idiotypic interaction.
Histological brain sections were probed with human oligoclonal lectin-like IgGs (L-IgG) purified from normal serum. In intact brain, antigenic determinants for these IgG were restricted to some blood vessel endothelial cells. By contrast, during the inflammatory reaction following a surgical injury, these determinants were detected at the cell surface of different cell types, within and near the lesion site. The cells reacting with L-IgG consisted of endothelial cell, mature astrocytes, activated microglial and ependymal cells.
Experimental traumatic brain injury produces a series of cellular events contributing to a neurochemical and neurometabolic cascade. This cascade is defined by the release of neurotransmitters resulting in a massive ionic flux, which, consequently, produces an increase in glycolysis. This increase in glycolysis is followed by a metabolic diaschisis, which is related to the degree and extent of behavioral deficits. Clinical efforts have now determined that a similar cascade occurs in human head injury, validating the animal model as well as providing new assessment strategies for the management and treatment of brain injury.
"The links" takes on several meanings to avid sportsfan and networking guru Mark Caron, director of networking and personal computing for Healthsource, Inc., Hookset, N.H. Caron is attempting to lead the way among integrated healthcare delivery networks by setting up a client/server architecture, videoconferencing and Internet connections between Healthsource sites.
The binding of epidermal growth factor (EGF) or an EGF-like growth factor to the EGF receptor is the initial event which leads to receptor activation, and consequently the induction of cell growth. In order to study this binding interaction in detail, we produced the extracellular domain of the EGF receptor (EGFR) using the baculovirus expression system. Affinity-labeling and Western-blot analyses revealed that the baculovirus-infected insect cells secrete active EGFR extracellular domain relatively efficiently, however a significant amount of inactive EGFR extracellular domain is retained within the cells. The apparent dissociation constant (Kd) of the secreted EGFR extracellular domain for EGF and transforming growth factor alpha (TGF-alpha), as determined using an immobilized receptor binding assay, was approximately 200 nM. Interestingly, this Kd value is 30-40-fold lower than that of the full-length EGFR derived from detergent-solubilized A431 cell membranes. The stoichiometry of binding of the EGFR extracellular domain to EGF and TGF-alpha was examined by band-shift analysis on non-denaturing PAGE and was estimated to be 1:1. We have also shown, using sedimentation equilibrium analysis, that ligand binding induces significant dimerization of the EGFR extracellular domain. Finally, we carried out site-specific mutagenesis on the EGFR extracellular domain in order to define the ligand-binding region. We identified amino acid residues which are close to the binding site since they are common to the epitopes of several ligand-competitive monoclonal antibodies. However, these residues do not contribute directly to ligand binding since the affinity of the mutated EGFR extracellular domain for EGF and TGF-alpha was unaffected.
The regulation and post-translational modifications of the human dopamine D1 receptor were studied in the baculovirus-eukaryotic cell expression system. Baculovirus constructs containing either the DNA encoding the dopamine D1 receptor or a DNA encoding a c-myc epitope tagged dopamine D1 receptor (c-myc-dopamine D1 receptor) were used to infect Spodoptera frugiperda (Sf9) insect cells. Expressed dopamine D1 and c-myc-dopamine D1 receptors bound agonists and antagonists with affinities and a rank order of potency characteristic of a classical dopamine D1 receptor pharmacological profile. In membrane preparations from cells expressing c-myc-dopamine D1 receptor, the photoaffinity label [125I](3-methyl-2-[4'-azidophenyl]-2,3,5-tetrahydro-2H-3-benzazepine) ([125I]MAB) bound specifically upon photolysis. A major broad band of approximately 48 kDa was detected. This species was identified in immunoblots by the monoclonal antibody raised against the c-myc epitope of c-myc-dopamine D1 receptor was isolated by immunoprecipitation from whole cells and was shown to be post-translationally modified by phosphorylation and palmitoylation. Exposure of cells expressing c-myc-dopamine D1 receptor to dopamine for 15 min resulted in a reduction in the maximal dopamine stimulated adenylyl cyclase activity, which was accompanied by an increased phosphorylation of the receptor and a rapid redistribution of surface c-myc-dopamine D1 receptor as detected by in situ immunofluorescence. Dopamine exposure also resulted in an increased level of incorporation of [3H]palmitic acid into the receptor. Thus, we provide the first evidence that the human dopamine D1 receptor undergoes agonist-dependent desensitization, phosphorylation and palmitoylation.
Focal contusions following head injury may be associated with focal or diffuse cerebral edema. Early global hyperemia and perifocal hyperemia may play a role in cerebral edema, although causal relationships have yet to be clearly been defined. We studied 27 patients with head injury (admission GCS 3-12) resulting in focal contusions (without evidence of subarachnoid, intraventricular or intraparenchymal hemorrhage by CT). Patients were studied with ICP monitors, head CTs, and intravenous 133Xenon regional cerebral perfusion studies serially over several days post injury. Low cortical blood flow and a low mean CBF15 flow were evident on the day of the injury. Additionally, F1 analysis indicated significantly (p < 0.05) greater cortical blood flow in the surrounding brain (mean 60 cc/100 g/min) compared to the contusion area (mean 43 cc/100 g/min) on the day of trauma. Mean regional CBF remained below normal in the contused areas (CBF15 < 35 cc/100 g/min), however the cortical flow increased in the first few days post-injury (peak F1 = 95 cc/100 g/min on day 3) then decreased to sub-normal levels. The mean CBF in the surrounding brain was low on the day of injury (CBF15 = 29 cc/100 g/min), although higher than the contused area, and increased to a peak of 45 cc/1009/min on day 3 posttrauma. Cortical flow in the surrounding brain, however, exhibited a different trend. The mean F1 was low on the day of trauma and significantly higher one day after trauma (mean 105 cc/100 g/min). Only 15 of the 27 patients with focal contusions had evidence of cerebral edema. Eleven of these exhibited focal edema and 4 exhibited diffuse edema. Focal edema developed over the first few days posttrauma as seen in followup CT, whereas patients with diffuse edema exhibited edema on the admission CT. Initial oligemia in the contused areas was associated with a subsequent hyperemic rim about the contusion. Focal hyperemia was associated with focal edema in 41% of the patients, whereas diffuse edema appeared to be independent of the hyperemic response in contusions.
The distribution of a 14.4 kDa S-type lectin was examined in murine neuroblastoma cells, either undifferentiated or after differentiation induced by dibutyryl-cyclic adenosine monophosphate. In undifferentiated cells the immunoreactivity was detected extracellularly, associated with the plasma membrane and in bulges released into the extracellular milieu. Important modifications of the lectin localization were associated with the differentiation process that induced an increased cytosolic expression and a decreased externalization. Possible functions for the lectin expressed intracellularly in the differentiated cells are also considered.
1. An antiserum raised against a 14 kDa beta-galactoside specific lectin from human brain (HBL14) was used to probe blots from samples of serum and cerebrospinal fluid. The only HBL14-immunoreactive material detected was heavy and light chains of a beta-galactoside-binding IgG fraction (lectin-like IgG). 2. Lectin-like IgG, as well as IgG Fab fragments, compete with HBL14 for binding either to anti-HBL14 antibody or to a lactosyl polyacrylamide-based copolymer. 3. Purification of lectin-like IgG was obtained by affinity chromatography on immobilized rabbit anti-lectin immunoglobulins. The carbohydrate-binding specificity of the purified molecules was restricted to beta-Gal-containing structures and close to the HBL14 one.