In vitro localization of the protein synthesis defect associated with experimental phenylketonuria

We have used a cell-free system derived from hamster brain to investigate protein synthesis during experimental phenylketonuria. In such a system the elongation inhibitor emetine impeded translation in extracts derived from both treated and control animals. On the other hand the initiation inhibitor aurintricarboxylic acid showed no effects on protein synthesis activity of treated hamsters, although it was severely inhibiting in controls. This suggests that initiation is the altered step in brain protein synthesis failure consecutive to phenylketonuria.Abbreviations ATA aurintricarboxylic acid - HPA hyperphenylalaninaemia (hyperphenylalaninaemic) - PHE phenylalanine - PKU phenylketonuria (phenylketonuric) - PR polyribosome     

Neurofibromatosis type 2 appears to be a genetically homogeneous disease

Neurofibromatosis type 2 (NF2) is an autosomal dominant syndrome characterized by the development of vestibular schwannomas and other tumors of the nervous system, including cranial and spinal meningiomas, schwannomas, and ependymomas. The presence of bilateral vestibular schwannomas is sufficient for the diagnosis. Skin manifestations are less common than in neurofibromatosis type 1 (NF1; von Recklinghausen disease). The apparent clinical distinction between NF1 and NF2 has been confirmed at the level of the gene locus by linkage studies; the gene for NF1 maps to chromosome 17, whereas the gene for NF2 has been assigned (in a single family) to chromosome 22. To increase the precision of the genetic mapping of NF2 and to determine whether additional susceptibility loci exist, we have performed linkage analysis on 12 families with NF2 by using four polymorphic markers from chromosome 22 and a marker at the NF1 locus on chromosome 17. Our results confirm the assignment of the gene for NF2 to chromosome 22 and do not support the hypothesis of genetic heterogeneity. We believe that chromosome 22 markers can now be used for presymptomatic diagnosis in selected families. The NF2 gene is tightly linked to the D22S32 locus (maximum lod score 4.12; recombination fraction 0). A CA-repeat polymorphism at the CRYB2 locus was the most informative marker in our families (lod score 5.99), but because the observed recombination fraction between NF2 and CRYB2 was 10 cM, predictions using this marker will need to be interpreted with caution.     

Identification of α2 adrenergic receptors in human frontal cortex membranes by binding of [H]RX 821002, the 2-methoxy analog of [H]idazoxan

The antagonist [³H]idazoxan binds with comparable affinity to α₂ adrenergic receptors and to phentolamine-displaceable non-stereoselective sites in human frontal cortex membranes. In contrast, idazoxan analogs possessing alkyl and alkoxy substituents at the 2-position of the benzodioxan moiety (i.e. RX 821002: 2-methoxy-1,4-[6,7-³H]benzodioxan-2-yl-2-imidazolin HCl, 43.8 Ci/mmol) possess 300–1200 times lower affinity for the non-stereoselective sites. Their affinity for the α₂ receptors is increased as well, resulting in more than a 1000-fold selectivity towards the receptors as compared to the non-stereoselective sites. [³H]RX 821002, the 2-methoxy analog of idazoxan possesses an approx. 10-fold higher affinity for the α₂ receptors (K_D = 2.8 nM than [³H]idazoxan (K_D = 24 nM) and about equal affinity as [³H]rauwolscine (K_D = 3.6 nM).[³H]Rauwolscine binds with comparable affinity to α₂ receptors and to 5-HT_1A receptors, and competition studies indicate that the K_i value of unlabelled RX 821002 for the 5-HT_1A receptors (30 nM) is about one order in magnitude above its K_i value for the α₂ receptors (4.1 nM). Labelling of the 5-HT_1A receptors by [³H]RX 821002 and by [³H]rauwolscine can be prevented by selective masking with 8-OH-DPAT (30 nM) or 5-HT (0.3 μM). Under these conditions, specific binding of [³H]RX 821002 to the α₂ receptors represents 84% of total binding (at its K_D), as compared to 77% for [³H]rauwolscine and 20% for [³H]idazoxan.[³H]RX 821002 labels the α₂ receptors as a single class of non-cooperative sites. Association and dissociation kinetics are very fast at 37°C. Antagonist competition curves are steep with Hill coefficients close to one and the agonist curves can be analysed in terms of two affinity sites, confirming the antagonistic properties of [³H]RX821002. About 60% of the α₂ receptors possess high agonist affinity.     

Involvement of the Dihydropyridine Receptor and Internal CaStores in Myoblast Fusion

The process of myoblast fusion during skeletal myogenesis is calcium regulated. Both dihydropyridine receptor and ryanodine receptor are already present on muscle precursors, at the prefusional stage, before they are required for excitation–contraction coupling. Previous pharmacological studies have shown the need for a special pool of Ca²⁺associated with the membrane for the fusion process to occur. We hypothesized that this pool of Ca²⁺is mobilized via a machinery similar to that involved in excitation–contraction coupling. The process of fusion in rat L6 muscle precursors was either totally or partially abolished in the presence of the L-type calcium channel inhibitors SR33557 and nifedipine (half inhibition towards 2 μM), respectively. The inhibition was reversible and dose-dependent. Drugs able to deplete internal calcium stores (caffeine, ryanodine, and thapsigargin) were also tested on the fusion. Both caffeine and thapsigargin drastically inhibited fusion whereas ryanodine had no effect. This suggests that fusion may be controlled by internal pools of Ca²⁺but that its regulation may be insensitive to ryanodine. We presumed that an early form of the ryanodine receptor may exist, with different pharmacological properties than the adult forms. Indeed, Western blot analysis of pre- and postfusional L6 cells demonstrated the presence, at the prefusional stage, of a transient form of the ryanodine receptor protein with an apparent molecular weight slightly different from those of the classical skeletal and cardiac forms. Taken together, these results support the hypothesis that the fusion process is driven by a mechanism involving both the dihydropyridine receptor (α1 subunit of the L-type Ca²⁺channel) and the internal stores of Ca²⁺. The machinery underlying this mechanism might consist of slightly different forms of the classic molecules that in adult muscle ensure excitation–contraction coupling. It remains to be seen, however, whether the mobilization of the internal pool of Ca²⁺is triggered by the type of mechanism already described in skeletal muscle.     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.     

Hydrodynamic characteristics and equilibrium rigidity of pullulan molecules

The hydrodynamic characteristics of the polysaccharide pullulan (polymaltotriose) in water have been investigated and its molecular characteristics have been determined. Experimental values varied over the following ranges: velocity sedimentation coefficient (S): 0.9 < S < 11.2, translational diffusion coefficient (10⁷ cm² s⁻¹): 1.1 < D < 14.7 and intrinsic viscosity (cm³ g⁻¹): 6.7 < [η] < 164, which corresponds to a change in molecular weight (× 10³) in the range 3.9 < M_SD < 644. On the basis of analysis of the literature and our experimental data, excluded volume effects have been shown to have a prevailing influence on the chain length of these polysaccharides. The equilibrium rigidity and hydrodynamic chain diameter of pullulan were evaluated on the basis of the theory of hydrodynamic properties of a wormlike necklace, taking into account excluded volume effects. At low M (< 30 × 10³) the translation friction data (in contrast to viscometric data) cannot be described in the framework of the theory of linear molecules.     

Effects of Ca(II) ions on Mn(II) dynamics in chick glia and rat astrocytes: Potential regulation of glutamine synthetase

Previous studies have demonstrated that in glia and astrocytes Mn(II) is distributed with ca. 30–40% in the cytoplasm, 60–70% in mitochondria. Ca(II) ions were observed to alter both the flux rates and distribution of Mn(II) ions in primary cultues of chick glia and rat astrocytes. External (influxing) Ca(II) ions had the greatest effect on Mn(II) uptake and efflux, compared to internal (effluxing) or internal-external equilibrated Ca(II) ions. External (influxing) Ca(II) ions inhibited the net rate and extent of Mn(II) uptake but enhanced Mn(II) efflux from mitochondria. These observations differ from Ca(II)–Mn(II) effects previously reported with brain (neuronal) mitochondria. Overall, increased cytoplasmic Ca(II) acts to block Mn(II) uptake and enhance Mn(II) release by mitochondria, which serve to increase the cytoplasmic concentration of free Mn(II). A hypothesis is presented involving external L-glutamate acting through membrane receptors to mobilize cell Ca(II), which in turn causes mitochondrial Mn(II) to be released. Because the concentration of free cytoplasmic Mn(II) is poised near the K_d for Mn(II) with glutamine synthetase, a slight increase in cytoplasmic Mn(II) will directly enhance the activity of glutamine synthetase, which catalyzes removal of neurotoxic glutamate and ammonia.     

Quantitative estimation of chimerism in mice using microsatellite markers

An embryonic stem cell line was established from SV129 mouse blastocysts and used to generate chimeric mice by injection into OF1 blastocysts; 18 out of the 30 resulting offspring appeared chimeric as judged from their coat color patterns, and 3 of the 13 males proved to be germ-line chimeras as they transmitted the SV129 agouti phenotype to all or part of their offspring. The degree of chimerism of these males was evaluated for different tissues using polymorphic microsatellite markers amplified by the polymerase chain reaction. It was shown that these new markers can be effectively used to quantitatively estimate levels of chimerism. The CKMM (creatine kinase, muscle) microsatellite system was used to distinguish the SV129 from the OF1 genotype. In all performed tests, the correlation between DNA ratio and signal ratio, expressed as a base 10 logarithm, was shown to exceed or equal 0.98 for known DNA ratios (SV129/OF1) ranging from 1/99 to 99/1. Linear calibration methods were used to predict the % SV129 DNA of a test sample based on the obtained signal ratio. The accuracy of the prediction was evaluated by performing repeated measurements. Differences among three repeated estimates ranged from 2 to 17% for a given sample. Microsatellite systems should be very useful to monitor chimerism involving strains that can not be discerned with coat color or biochemical markers. This will be particularly important when ES methodology becomes available in species other than mice. © 1993 Wiley-Liss, Inc.