Production of human antithrombin-III in a serum-free culture of CHO cells.

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10-20 micrograms/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.     

Production of Human Antithrombin-III in a Serum-free Culture of CHO Cells

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10–20 µg/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.     

Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding

Two distinct forms of antithrombin III were isolated by chromatography of normal human plasma on heparin-Sepharose. The predominant antithrombin species present (AT-III alpha), which eluted from the affinity column in 1 M NaCl, was identified as the antithrombin III form which has been previously characterized. Ionic strength of the buffer was increased to elute a variant form of antithrombin III, designated as AT-III beta. The molecular weight of AT-III beta is less than that of AT-III alpha, but physicochemical studies do not indicate measureable differences in the polypeptide portion of the proteins. Carbohydrate determination revealed the sole detectable structural difference in the two antithrombins: levels of hexosamine, neutral sugars, and sialic acid in AT-III beta were all 25-30% less than in AT-III alpha. Kinetic studies of thrombin inactivation by both antithrombins, in the presence of nonsaturating amounts of heparin, indicated that AT-III beta inhibited thrombin more rapidly. AT-III beta is also distinguishable from AT-III alpha on the basis of heparin-binding affinity estimated from titration of protein fluorescence with heparin. Thus, antithrombin III exists as two molecular entities in human plasma which differ both structurally, in carbohydrate content, and functionally, in their heparin-binding behavior.     

Effect of antithrombin III on glycoprotein Ib/IX/V activation in human platelets: Suppression of thromboxane A(2) generation

We have previously shown that ristocetin, an activator of glycoprotein Ib/IX/V, induces release of soluble CD40 (sCD40) ligand via thromboxane (TX) A(2) production from human platelets. In the present study, we investigated the effect of antithrombin-III (AT-III), an anticoagulant, on the ristocetin-induced glycoprotein Ib/IX/V activation in human platelets. AT-III inhibited ristocetin-stimulated platelet aggregation. The ristocetin-induced production of 11-dehydro-TXB(2), a stable metabolite of TXA(2), and the release of sCD40 ligand were suppressed by AT-III. AT-III also reduced the ristocetin-stimulated secretion of platelet-derived growth factor (PDGF)-AB. AT-III failed to affect U46619-, a TXA(2) receptor agonist, induced levels of p38 mitogen-activated protein kinase phosphorylation or sCD40 ligand release. AT-III reduced the binding of SZ2, a monoclonal antibody to the sulfated sequence in the α-chain of glycoprotein Ib, to the ristocetin-stimulated platelets. These results strongly suggest that AT-III reduced ristocetin-stimulated release of sCD40 ligand due to inhibiting TXA(2) production in human platelets.     

Non-enzymatic glycation of antithrombin III in vitro

Non-enzymatic glycation of antithrombin III (AT-III) has been proposed as a significant contributor to the increased incidence of thrombo-occlusive events in diabetics. AT-III, isolated from normal human plasma by means of heparin affinity and ion-exchange chromatography, was incubated with 0-0.5 M glucose in neutral phosphate buffer at 37 degrees C. The extent of non-enzymatic glycation could be monitored by uptake of radioactivity as well as by binding to a phenylboronate affinity resin, which effectively retards AT-III containing ketoamine-linked glucose. Non-enzymatically glycated AT-III (approx. 1 mol glucose/mol protein) bound heparin nearly as efficiently as non-glycated AT-III. The two AT-III preparations were equally active in inhibiting thrombin cleavage of chromogenic substrate. Following incubation with [14C]glucose, structural analyses of cyanogen-bromide-cleaved peptides of enzymatically glycated AT-III showed that the [14C]glucose adducts were distributed over many sites on the molecule. This lack of specificity contrasts with the restricted sites of modification on hemoglobin, albumin and ribonuclease A, and explains why non-enzymatic glycation of AT-III has little if any effect on its function.     

Site-directed mutagenesis of alanine-382 of human antithrombin III

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.     

Physiological variant of antithrombin-III lacks carbohydrate sidechain at Asn 135

Both normal antithrombin-III (AT-III alpha) and the high heparin affinity form (AT-III beta) were isolated from pooled human plasma. AT-III beta had a lower negative charge and lower molecular mass than AT-III alpha. Sialidase and endo-F digestion indicated that the inherent difference resided in the oligosaccharide component of the molecule. CNBr fragmentation showed there was an oligosaccharide sidechain missing between residues 104 and 251, subdigestion with trypsin indicated that Asn 135 was not glycosylated in AT-III beta. Chromatography of total tryptic digests on concanavalin A-Sepharose confirmed that the high heparin affinity form of antithrombin lacked an oligosaccharide moiety at Asn 135.     

Antithrombin III,but not C1 esterase inhibitor reduces inflammatory response in lipopolysaccharide-stimulated human monocytes in an ex-vivo whole blood setting

In order to examine the immunomodulatory effects of antithrombin III (AT-III) and C1 esterase inhibitor (C1-INH) in human monocytes, we investigated the intracellular expression of interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF)-α in an ex-vivo laboratory study in a whole blood setting.Heparinized whole blood samples from 23 healthy male and female volunteers (mean age: 27 ± 7 years) were pre-incubated with clinically relevant concentrations of AT-III (n = 11) and C1-INH (n = 12), then stimulated with 0.2 ng/mL lipopolysaccharide (LPS) for 3 h. After phenotyping CD14⁺ monocytes, intracellular expression of IL-6, IL-8, and TNF-α was assessed using flow cytometry. In addition, 12 whole blood samples (AT-III and C1-INH, n = 6 each) were examined using hirudin for anticoagulation; all samples were processed in the same way. To exclude cytotoxicity effects, 7-amino-actinomycin D and Nonidet P40 staining were used to investigate probes.This study is the first to demonstrate the influence of C1-INH and AT-III on the monocytic inflammatory response in a whole blood setting, which mimics the optimal physiological setting. Cells treated with AT-III exhibited significant downregulation of the proportion of gated CD14⁺ monocytes for IL-6 and IL-8, in a dose-dependent manner; downregulation for TNF-α did not reach statistical significance. There were no significant effects on mean fluorescence intensity (MFI). In contrast, C1-INH did not significantly reduce the proportion of gated CD14⁺ monocytes or the MFI regarding IL-6, TNF-α, and IL-8. When using hirudin for anticoagulation, no difference in the anti-inflammatory properties of AT-III and C1-INH in monocytes occurs.Taken together, in contrast to TNF-α, IL-6 and IL-8 were significantly downregulated in monocytes in an ex-vivo setting of human whole blood when treated with AT-III. This finding implicates monocytes as an important point of action regarding the anti-inflammatory properties of AT-III in sepsis. C1-INH was unable to attenuate the monocytic response, which supports the hypothesis that other cellular components in whole blood (e.g., neutrophils) might be responsible for the known effects of C1-INH in inflammation.     

Neonatal manipulation of oxytocin alters oxytocin levels in the pituitary of adult rats.

The neuropeptide oxytocin (OT) and its OT antagonists (OTA) in infant rats affect their behavior as adults. In this study we attempted to determine whether treating rats on the day of birth (postnatal day 1) with OT or OTA would affect brain OT levels of these rats as adults. Rat pups were injected with OT (3 microg), OTA (0.3 microg) or saline vehicle ip on postnatal day 1. As 60-day-old adults, treated rats were killed, and the OT content in their medial preoptic areas (MPOAs), medial hypothalami (MH) and pituitaries were assayed. In females, treatment with OTA on postnatal day 1 significantly decreased pituitary OT levels as adults. In males, by contrast, treatment with OTA on postnatal day 1 resulted in increased pituitary OT levels when they become adults compared to male rats treated with OT on postnatal day 1. There were no significant effects of neonatal treatment on OT levels in either the MH or MPOA. Day 1 postnatal treatment with OT or OTA had a long-term sexually dimorphic effect on OT levels in the pituitary.     

Application of immobilized heparins for isolation of human antithrombin III

Immobilized heparins were prepared by six different methods, and these were utilized for affinity purification of human antithrombin III (AT-III). Affinity support capacities (mg AT-III/g support) were strongly influenced by immobilized active heparin concentrations. In the temperature range 5-37 degrees C, colder temperatures favored affinity adsorption of AT-III as well as nonspecific interactions of all proteins. For representative human-plasma-derived feed solutions the selectivity for AT-III on the affinity support was dependent on relative concentrations of non-AT-III proteins as well as the specific mode of adsorption and elution (batch/continuous).     

Plasma protease inhibitors in premature infants: influence of gestational age, postnatal age, and health status

In newborn infants, the influence of gestational age (GA), postnatal age (PA), and health status on the plasma protease inhibitors alpha 2-macroglobulin (alpha 2-M), alpha 1-antitrypsin (alpha 1-AT), C1 esterase inhibitor (C1E-INH), alpha 2-antiplasmin (alpha 2-AP), and antithrombin III (AT-III) was investigated. Inhibitor levels were measured by radial-immunodiffusion and expressed as a percentage of pooled plasma from adults (mean +/- SEM). In total, 54 premature infants (28-36 weeks gestation) were classified at birth as healthy (N = 22) (IV fluids, antibiotics only) or sick (N = 32) (all other support, but excluding infants with disseminated intravascular coagulation (DIC] and studied on Days 1 and/or 7 of life. Healthy term infants (N = 18) and infants with DIC (N = 10) were studied on Day 1 only. All inhibitors except C1E-INH increased with increasing gestational age (P less than 0.01). In healthy premature infants all inhibitor levels reached the normal adult range by 1 week of age. In contrast, at 1 week of age, sick infants had lower levels of alpha 2-M and alpha 2-AP, and higher levels of alpha 1-AT compared to healthy infants (P less than 0.01). The presence of DIC depressed all of the inhibitors on Day 1 except alpha 1-AT when compared to healthy controls (P less than 0.01). Thus, gestational age, postnatal age, and health status all significantly influenced the levels of these plasma protease inhibitors.     

A comparison of internal and external lipids of nondiapausing and diapause initiation phase adult Colorado potato beetles, Leptinotarsa decemlineata

The Colorado potato beetle, Leptinotarsa decemlineata, reared under diapause-inducing conditions will emerge from the soil as an adult and enter the diapause initiation phase, a period where metabolic reserves are stockpiled before the beetles enter the nonfeeding diapause maintenance phase. Internal and external lipids were characterized during the diapause initiation phase (IP) and compared to the lipid profiles of nondiapausing adults. The primary internal lipids of both diapause IP and nondiapausing adults are triacylglycerols. Only trace amounts of internal lipids were detected in day 1 diapause IP adults. A dramatic increase in internal lipids was observed between day 7 and day 15 post-emergence in the diapause IP adults. The majority of the triacylglycerol isomers were identified as C50, C52 and C54 chain lengths by GC-MS. There were no observed differences in the isomeric distribution of the major internal lipids between diapause IP and nondiapausing adults. External lipids were mainly methyl-branched alkanes containing a 25 to 53 carbon backbone. The quantity of external lipids increased from day 1 to day 7 post-emergence in both the diapause IP and nondiapausing adults, with the bulk of the increase occurring in the longer chain-length methylalkanes.     

Strongyloides ratti: transplantation of adults recovered from the small intestine at different days after infection into the colon of naive and infection-primed Wistar rats, and the effect of antioxidant treatment on large intestinal parasitism

Strongyloides ratti (Nagoya strain) is unique in that a portion of adults parasitizing the small intestine withstands 'worm expulsion', which starts at around day 8 post-infection (p.i.) by host immunity, and establishes in the large intestine after day 19 p.i. To investigate the mechanism, adults obtained from the small intestine at day 7 or 19 p.i. were transplanted into the colon of infection-primed immune rats. Adults obtained at day 7 p.i. were rejected quickly, whereas those obtained at day 19 p.i. could establish infection. Moreover, the body length and the number of intrauterine eggs increased in the large intestine. In a separate experiment, large intestinal parasitism was abolished by the treatment of host rats with an anti-oxidant, butylated hydroxyanisole. These results indicate that small intestinal adults between days 7 and 19 p.i. acquired the ability to parasitize the large intestine of immune rats, and that free radicals produced by the host may have played a significant role in the process.     

Distribution and Developmental Regulation of Metabotropic Glutamate Receptor 7a in Rat Brain

Abstract: To determine the regional and cellular distribution of the metabotropic glutamate receptor mGluR7a, we used rabbit anti-peptide polyclonal-targeted antibodies against the C-terminal domain of mGluR7a. Here we report that immunocytochemistry at the light-microscopic level revealed that mGluR7a is widely distributed throughout the adult rat brain, with a high level of expression in sensory areas, such as piriform cortex, superior colliculus, and dorsal cochlear nucleus. In most brain structures, mGluR7a immunoreactivity is characterized by staining of puncta and fibers. However, in some regions, including the locus ceruleus, cerebellum, and thalamic nuclei, both cell bodies and fibers are immunopositive. The changes in levels of mGluR7a during development were investigated with immunoblotting and immunocytochemical analysis. Immunoblot analysis revealed that the levels of mGluR7a are differentially regulated across brain regions during postnatal development. In cortical regions (hippocampus, neocortex, and olfactory cortex), mGluR7a levels were highest at postnatal day 7 (P7) and P14, then declined in older rats. In contrast, mGluR7a levels were highest at P7 in pons/medulla and cerebellum and decreased markedly between P7 and P14. In these regions, mGluR7a immunoreactivity was at similar low levels at P14 and P21 and in adults. Immunocytochemical analysis revealed that staining for mGluR7a was exceptionally high in fiber tracts in P7 animals relative to adults. Furthermore, the pattern of mGluR7a immunoreactivity in certain brain structures, including cerebellum, piriform cortex, and hippocampus, was significantly different in P7 and adult animals. In summary, these data suggest that mGluR7a is widely distributed throughout the rat brain and that this receptor undergoes a dynamic, regionally specific regulation during postnatal development.     

Immunoelectrophoretic quantitation of antithrombin III. Electroimmunodiffusion in agarose gels containing heparin.

Antithrombin III (AT-III), being an alpha2-globulin, will have an electrophoretic mobility in the presence of heparin like prealbumin in agarose gels. This phenomenon was utilized to quantitate AT-III from serum and plasma by electroimmunodiffusion (EID) for 90 min agarose gels containing 75 USP units of heparin/ml gel. The method permits a rapid quantitation of AT-III from serum, citrated plasma and EDTA plasma, and a positive correlation was observed between these values and those obtained by single radial immunodiffusion (SRI). This is in contrast to quantitation of AT-III by EID in gels containing no heparin where the values for plasma showed poor correlation with those obtained by SRI.     

Ontogeny of endocrine responses to methadone in rats

The effects of methadone (METH) on serum levels of prolactin (PRL), growth hormone (GH), corticosterone (CS) and TSH were determined in developing rats. METH increased PRL, GH and CS and decreased TSH at all ages tested, but the time course and magnitude of these effects changed during ontogeny. METH effects on day 10 were lower in magnitude than those observed in adults. In 20 day old pups, METH effects on GH and CS were comparable to those of adults, but TSH effects were still blunted. METH effects on hormone secretion in both 10 and 20 day old pups lasted longer than those observed in adults. Naloxone blocked all hormonal responses in adults, but did not completely block METH effects on CS secretion in 10 day old pups.     

Evidence for a heparin-induced conformational change on antithrombin III

The effect of heparin on the conformation of antithrombin III (AT-III) was investigated. Solvent perturbation difference spectroscopy shows that the binding of heparin to AT-III results in exposure of two tyrosine residues and a partial burial of a tryptophan residue. The occurrence of a conformational change suggested by this study is also substantiated by circular dichroism (CD) findings in the aromatic and peptide regions. The data in the peptide region show that heparin produces a decrease in the β-structure of AT-III, with a compensatory increase in random coil.     

Comparison of plasma ACTH and corticosterone levels after embryo bursectomy

Plasma levels of both adrenocorticotropic hormone (ACTH) and corticosterone (B) were determined in embryos (day 15 of incubation), chicks (day 3 after hatch) and young chickens (8 weeks). Experimental animals were bursectomized at 80 hr of incubation, i.e., before any anlage of the bursa of Fabricius could develop. Bursectomized (BFX) animals were compared to sham-operated controls (T), in basal, resting condition and 7 (ACTH) or 14 min (B) after ether stress was delivered for 30 sec. Basal B and ACTH levels seemed not to be significantly modified in BFX embryos, chicks and chickens. Hypophysial and adrenocortical response to stress appeared more precociously in BFX embryos (day 15 of incubation) than in intact ones (day 19). The non stress-responsive period that was observed for one week after hatch of T birds did not appear in 3-day-old BFX chicks whose both B and ACTH stress-induced levels were as high as in intact adults. In contrast, adrenocortical and pituitary corticotropic responses to stress were markedly impaired (by 50%) in adult BFX chickens as compared to intact controls.