A novel F8 -/- rat as a translational model of human hemophilia A.

BACKGROUND: In preclinical hemophilia research, an animal model that reflects both the phenotype and the pathology of the disease is needed. OBJECTIVES: Here, we describe the generation and characterization of a novel genetically engineered F8(-/-) rat model. METHODS: The rats were produced on a Sprague Dawley background with the zinc finger nuclease technique. A founder with a 13-bp deletion in exon 16 causing a premature translational stop in the C-terminal part of the A3 domain of factor VIII was selected, and a breeding colony was established. RESULTS: Seventy per cent of the homozygous rats had clinically manifest spontaneous hemorrhagic episodes that needed treatment. The F8(-/-) rats had no detectable FVIII activity, and had a significantly prolonged activated partial thromboplastin time (APTT) and clot formation time as compared with wild-type (WT)/WT rats. In vitro spiking of rat plasma with human recombinant FVIII resulted in dose-dependent normalization of the APTT. CONCLUSION: On the basis of the targeted deletion in F8, and the distinct physical and analytic characteristics of the rat, we conclude that an FVIII-deficient rat strain has been generated that has the potential to contribute greatly to translational research.

Enhanced satellite cell proliferation with resistance training in elderly men and women.

In addition to the well-documented loss of muscle mass and strength associated with aging, there is evidence for the attenuating effects of aging on the number of satellite cells in human skeletal muscle. The aim of this study was to investigate the response of satellite cells in elderly men and women to 12 weeks of resistance training. Biopsies were collected from the m. vastus lateralis of 13 healthy elderly men and 16 healthy elderly women (mean age 76+/-SD 3 years) before and after the training period. Satellite cells were visualized by immunohistochemical staining of muscle cross-sections with a monoclonal antibody against neural cell adhesion molecule (NCAM) and counterstaining with Mayer's hematoxylin. Compared with the pre-training values, there was a significant increase (P<0.05) in the number of NCAM-positively stained cells per fiber post-training in males (from 0.11+/-0.03 to 0.15+/-0.06; mean+/-SD) and females (from 0.11+/-0.04 to 0.13+/-0.05). These results suggest that 12 weeks of resistance training is effective in enhancing the satellite cell pool in skeletal muscle in the elderly.

Interpretation of the gamma interferon test for diagnosis of subclinical paratuberculosis in cattle.

A group of 252 cattle without clinical signs of paratuberculosis (paraTB) in 10 herds infected with paraTB and a group of 117 cattle in 5 herds without paraTB were selected. Whole-blood samples were stimulated with bovine, avian, and johnin purified protein derivative (PPD) and examined for gamma interferon (IFN-gamma) release. For diagnosis of paraTB, satisfactory estimated specificities (95 to 99%) could be obtained by johnin PPD stimulation irrespective of interpretation relative to bovine PPD or no-antigen stimulation alone, but numbers of test positives in the infected herds varied from 64 to 112 with different interpretation criteria. For a limited number of test-positive animals, no change in the test results could be observed with increasing antigen concentrations but IFN-gamma responses were significantly reduced (P < 0.0001) and four out of seven reactors tested negative when stimulation was performed on day-old samples. Denmark is free of bovine tuberculosis, but cross-reactivity with paraTB could be documented for cattle more than 14 months old in paraTB-infected herds compared with those in non-paraTB-infected herds. In both paraTB-free and paraTB-infected herds, false positives were observed when the test was applied to calves less than 15 months of age. Until novel antigen formulations more specific for these diseases are available, interpretation of the IFN-gamma test must be individually adjusted to fit specific needs and the context within which the test is applied and, for paraTB, the test seems most appropriate for use as a supportive tool for evaluation of disease-preventive measures in young stock.

Grp78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum.

The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL receptors are unknown. In the present study we show that the molecular chaperone Grp78/BiP co-immunoprecipitates with both the wild type and two different mutant (W556S and C646Y) LDL receptors in lysates obtained from human liver cells overexpressing wild type or mutant LDL receptors. A pulse-chase study shows that the interaction between the wild type LDL receptor and Grp78 is no longer detectable after 2(1/2) h, whereas it persists for more than 4 h with the mutant receptors. Furthermore, about five times more Grp78 is co-immunoprecipitated with the mutant receptors than with the wild type receptor suggesting that Grp78 is involved in retention of mutant LDL receptors in the ER. Overexpression of Grp78 causes no major alterations on the steady state level of active LDL receptors at the cell surface. However, overexpression of Grp78 decreases the processing rate of newly synthesized wild type LDL receptors. This indicates that the Grp78 interaction is a rate-limiting step in the maturation of the wild type LDL receptor and that Grp78 may be an important factor in the quality control of newly synthesized LDL receptors.

[Symptomatic helium treatment of upper and lower airway obstruction]. / Symptomatisk heliumbehandling af øvre og nedre luftvejsobstruktion.

Since 1934 several clinical trials have been performed to investigate the effect of helium in the symptomatic treatment of upper and lower airway obstructions, especially in children. Controlled studies have only been produced during the last decade. Heliox, a mixture of helium and oxygen, has a significantly lower density than N2/O2-mixtures. This produces better flow and hence a decrease in respiratory work, improvement of distal gas exchange and theoretically less tendency to air-trapping and hyperinflation. When holding more than 40% O2 the clinical effect decreases. There are case reports of rapid subjective release, less stridor, lower respiratory rate and a normalization of hypercapnia and acidosis. Controlled studies confirm this and demonstrate a decrease in the need for intubation and mechanical ventilation. Time is bought until conventional therapy with steroids, epinephrine and beta 2-agonist inhalation works. Helium has its place in treatment of airway obstructions, but more clinical trials are needed to define the indication for symptomatic heliox treatment.

[Accidental cetirizine poisoning in a four-year-old boy]. / Accidentel cetirizinforgiftning hos en fireårig dreng.

Cetirizine is a commonly used non-sedating antihistamine for the symptomatic relief of allergic reactions. Few reports exist on the result of overdose in children. We would like to report the result of a 12 fold overdose of cetirizine in a four-year-old-boy (weight 20 kg) who accidentally ingested 60 mg. Vomiting was induced 1 1/2 hour after ingestion in the out-patient clinic at the local hospital because of severe drowsiness. Due to continued lethargy he was transferred to the referral paediatric department for further observation. He was fully recovered after five to six hours without any treatment. Electrocardiographic monitoring was normal. Five incidents of cetirizine overdose in children have been reported previously. Drowsiness and sedation were observed, but no other side effects. The risk of cardiac events related to an overdose of cetirizine is extremely small. A certain degree of sedation is to be expected.

GABAA agonists: resolution and pharmacology of (+)- and (-)-isoguvacine oxide.

(3SR,4RS)-3,4-Epoxypiperidine-4-carboxylic acid (isoguvacine oxide) is a potent and specific GABAA receptor agonist. Isoguvacine oxide, originally designed as a potentially alkylating agonist, turned out to interact with the GABAA receptor in a fully reversible manner. The protected form of isoguvacine oxide, benzyl (3SR,4RS)-1-(benzyl-oxycarbonyl)-3,4-epoxypiperidin e-4-carboxylate (1) (Scheme 1), has now been resolved by chiral chromatography using cellulose triacetate as a chiral stationary phase. The enantiomers of 1 (ee > or = 98.8%) were subsequently deprotected by hydrogenolysis. Whereas both enantiomers of isoguvacine oxide were inactive as inhibitors of the binding of [3H]GABA to GABAB receptor sites (IC50 > 100 microM), (+)-isoguvacine oxide (IC50 = 0.20 +/- 0.03 microM) and (-)-isoguvacine oxide (IC50 = 0.32 +/- 0.05 microM) showed comparable potencies as inhibitors of the binding of [3H]GABA to GABAA receptor sites. Furthermore, (+)-isoguvacine oxide (EC50 = 6 microM; 33% relative efficacy) and (-)-isoguvacine oxide (EC50 = 5 microM; 38% efficacy relative to 10 microM muscimol) were approximately equipotent and equiefficacious as stimulators of the binding of [3H]diazepam to the GABAA receptor-associated benzodiazepine site. This latter effect is an in vitro estimate of GABAA agonist efficacy. These pharmacological data for isoguvacine oxide and its enantiomers do not seem to support our earlier conception of the topography of the GABAA recognition site(s), derived from extensive structure-activity studies on GABAA agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Resolution, absolute stereochemistry, and pharmacology of the S-(+)- and R-(-)-isomers of the apparent partial AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid [(R,S)-APPA].

(R,S)-2-Amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid ((R,S)-APPA) is the only partial agonist at the (R,S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) subtype of excitatory amino acid receptors so far described. In light of the pharmacological interest in partial agonists, we have now accomplished the resolution of (R,S)-APPA. (S)-(+)-APPA (5) and (R)-(-)-APPA (6) were obtained in high enantiomeric purity using (R)-(+)- and (S)-(-)-1-phenylethylamine, respectively, as resolving agents. The absolute stereochemistry of 6 was established by X-ray analysis of 6.HCl.0.25H2O. Compounds 5 and 6 were tested electropharmacologically using the rat cortical wedge preparation and in receptor-binding assays using [3H]-AMPA, [3H]kainic acid, and the N-methyl-D-aspartic acid (NMDA) receptor ligands [3H]CPP, [3H]MK-801, and [3H]glycine. Whereas 6 did not significantly affect the binding of any of these ligands (IC50 > 100 microM), compound 5 revealed affinity for only the [3H]AMPA-binding site (IC50 = 6 microM). In electropharmacological tests, 5 showed full AMPA receptor agonism (EC50 = 230 microM). This effect of 5 was insensitive to the NMDA antagonist CPP but was inhibited competitively by the non-NMDA antagonist NBQX (pKi = 6.30). Compound 6, on the other hand, turned out to be a non-NMDA receptor antagonist, inhibiting competitively depolarizations induced by AMPA (pKi = 3.54), kainic acid (pKi = 3.07), and 5 (pKi = 3.57).

GABAB antagonists: resolution, absolute stereochemistry, and pharmacology of (R)- and (S)-phaclofen.

Phaclofen, which is the phosphonic acid analogue of the GABAB agonist (RS)-3-(4-chlorophenyl)-4-aminobutyric acid (baclofen), is a GABAB antagonist. As part of our studies on the structural requirements for activation and blockade of GABAB receptors, we have resolved phaclofen using chiral chromatographic techniques. The absolute stereochemistry of (-)-(R)-phaclofen was established by X-ray crystallographic analysis. (-)-(R)-Phaclofen was shown to inhibit the binding of [3H]-(R)-baclofen to GABAB receptor sites on rat cerebellar membranes (IC50 = 76 +/- 13 microM), whereas (+)-(S)-phaclofen was inactive in this binding assay (IC50 > 1000 microM). (-)-(R)-Phaclofen (200 microM) was equipotent with (RS)-phaclofen (400 microM) in antagonizing the action of baclofen in rat cerebral cortical slices, while (+)-(S)-phaclofen (200 microM) was inactive. The structural similarity of the agonist (R)-baclofen and the antagonist (-)-(R)-phaclofen suggests that these ligands interact with the GABAB receptor sites in a similar manner. Thus, it may be concluded that the different pharmacological effects of these compounds essentially result from the different spatial and proteolytic properties of their acid groups.

The non-depolarizing D-form of bromohomoibotenic acid enhances depolarizations evoked by the L-form or quisqualate.

The D-enantiomer of bromohomoibotenic acid (Br-HIBO) was inactive in electrophysiological experiments when administered alone, but enhanced depolarizations evoked by L-Br-HIBO or quisqualate when co-administered with these agonists. In addition, quisqualate induced a long-lasting (> 120 min) sensitization of cortical wedge neurons to D-Br-HIBO. This latter effect of D-Br-HIBO was similar to, but significantly more potent and selective, than the earlier observed quisqualate-induced sensitization of cortical neurones to depolarization by (S)-2-amino-4-phosphonobutyric acid (L-AP4).

The inheritance of vegetative growth traits in strawberries (Fragaria x ananassa) grown at low temperatures and their relationship to field productivity.

The genetic relationship between vegetative growth at low temperatures and productivity was investigated for strawberries grown in controlled and field environments. Genotypes from 20 biparental crosses were grown in controlled environments with 11°, 14°, and 17 °C days, 11 °C nights, and 11-h daylength to simulate a range of winter growing conditions expected in mediterranean environments. Individual plants were scored for two initial runner traits and eight vegetative growth traits. Significant main effects of temperature and cross were detected for all growth chamber traits, and conservative estimates of the broad sense heritability (h(2)) for these traits were 0.10-0.28. None of the temperature x cross interaction effects were significant, suggesting that genetic potential for vegetative growth and vigor is expressed similarly at low and optimal growing temperatures. Highly significant genetic correlations were detected between many growth chamber trait pairs, indicating pleiotropic effects for the genes that condition these traits. Complementary field trials were established, and individual plants were scored for traits that describe yield, production pattern, and plant size. Significant negative genetic correlations were detected between traits that describe growth in the chambers and early production in the field trials, but genetic correlations between chamber growth traits and mid-season or total production were significantly positive and occasionally large. Several of the yield and field growth variables were genetically correlated to initial runner plant traits, suggesting that indirect selection using traits scored in the nursery can be used to improve yield and modify production pattern in the field.

Naturally-occurring excitatory amino acids as neurotoxins and leads in drug design.

The central excitatory neurotransmitter (S)-glutamic acid (Glu) activates at least three types of receptors the NMDA, AMPA, and kainic acid (KAIN) receptors. These receptors mediate the neurotoxicity of a number of naturally-occurring Glu analogues. Thus, domoic acid, a KAIN receptor agonist, has probably been the cause of severe neurologic illness in people who consumed domoic acid poisoned food. beta-N-oxalylaminoalanine (beta-ODAP), an AMPA receptor agonist, has been associated with lathyrism, a spastic paraparesis caused by dietary intake of Lathyrus sativus. The neurotoxic Amanita muscaria constituent ibotenic acid, a nonselective NMDA receptor agonist, has been used as a lead structure for the development of the specific NMDA receptor agonist AMAA, AMPA, and a number of therapeutically interesting AMPA and KAIN receptor agonists.

Stereoselective effects of AMOA on non-NMDA receptors expressed in Xenopus oocytes.

Pharmacological characterization of the action of the novel non-N-methyl-D-aspartate (non-NMDA) antagonist AMOA (2-amino-3-[3-(carboxymethoxy)-5-methylisoxazol-4-yl]propionate) on glutamate receptors was investigated in Xenopus oocytes injected with mouse brain mRNA. AMOA (150 microM) produced a nearly parallel shift to the right of the dose-response curve for kainate-induced currents. AMOA was found to have two different effects on AMPA receptors: 1) currents elicited by low concentrations of AMPA (6 microM) were inhibited by AMOA with an IC50 value of 160 +/- 19 microM and 2) currents elicited by high concentrations of AMPA (100 microM) were potentiated with an IC50 value of 88 +/- 22 microM. The maximal potentiating effect of AMOA on AMPA currents was around 170%. Furthermore, the two opposing effects of AMOA on AMPA responses are specific for the L-configuration of AMOA. This unusual antagonistic/agonistic property of AMOA may explain its unusual properties with regard to antagonism of non-NMDA receptor-mediated events previously described.

Hydroxylated analogues of 5-aminovaleric acid as 4-aminobutyric acidB receptor antagonists: stereostructure-activity relationships.

The (R) and (S) forms of 5-amino-2-hydroxyvaleric acid (2-OH-DAVA) and 5-amino-4-hydroxyvaleric acid (4-OH-DAVA) were designed as structural hybrids of the 4-aminobutyric acidB (GABAB) agonist (R)-(-)-4-amino-3-hydroxybutyric acid [(R)-(-)-3-OH-GABA] and the GABAB antagonist 5-aminovaleric acid (DAVA). (S)-(-)-2-OH-DAVA and (R)-(-)-4-OH-DAVA showed a moderately potent affinity for GABAB receptor sites in rat brain and showed GABAB antagonist effects in a guinea pig ileum preparation. The respective enantiomers, (R)-(+)-2-OH-DAVA and (S)-(+)-4-OH-DAVA, were markedly weaker in both test systems. All four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low-energy conformations of (R)-(-)-3-OH-GABA, (S)-(-)-2-OH-DAVA, and (R)-(-)-4-OH-DAVA can be superimposed. These conformations may reflect the shapes adopted by these conformationally flexible compounds during their interaction with GABAB receptors. The present studies emphasize the similar, but distinct, constraints imposed on agonists and antagonists for GABAB receptors.

Excitatory amino acid agonists. Enzymic resolution, X-ray structure, and enantioselective activities of (R)- and (S)-bromohomoibotenic acid.

The enantiomers of alpha-amino-4-bromo-3-hydroxy-5-isoxazolepropionic acid (4-bromohomoibotenic acid, Br-HIBO, 1) a selective and potent agonist at one class of the central (S)-glutamic acid receptors, were prepared with an enantiomeric excess higher than 98.8% via stereoselective enzymic hydrolysis of (RS)-alpha-(acetylamino)-4-bromo-3-methoxy-5-isoxazolepropionic acid (4) using immobilized aminoacylase. The absolute configuration of the enantiomers of Br-HIBO was established by X-ray crystallographic analysis, which confirmed the expected preference of the enzyme for the S form of the substrate 4. (S)- and (RS)-Br-HIBO were potent neuroexcitants on cat spinal neurones in vivo, while (R)-Br-HIBO was a very weak excitant. Correspondingly, the S enantiomer of Br-HIBO (IC50 = 0.34 microM) was considerably more potent than the R form (IC50 = 32 microM) as an inhibitor of [3H]-(RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([ 3H]AMPA) binding to rat brain synaptic membranes in vitro. In contrast, (S)- and (R)-Br-HIBO were approximately equipotent (IC50 values of 0.22 and 0.15 microM, respectively) as inhibitors of [3H]-(S)-glutamic acid binding in the presence of CaCl2. The enantiomers of Br-HIBO showed no significant affinity for those binding sites on rat brain membranes which are labeled by [3H]kainic acid or [3H]-(R)-aspartic acid.