Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

A new approach for estimating the crosslink density of covalently crosslinked ionic polysaccharide gels

For an ideal polysaccharide gel with a known total polymer chain contour length, crosslinks all of the same functionality and elastic chains all with the same contour length and stiffness, the gel crosslink density can readily be determined from measurements of the maximum volume of the swollen gel (Moe et al., (1991) Food Hydrocolloids, 5, (1/2), 119–123. In the case of randomly crosslinked polysaccharide gels, where the chain contour length between two adjacent crosslinks may vary greatly, it is often much more difficult to determine the crosslink density. This paper reports on an attempt to extend the use of maximum gel volume measurements to estimate crosslink density for the latter type of gel. This is done by calculating the maximum swelling volume for polymer networks with four-functional crosslinks, known elastic chain mean contour length and standard deviation. The numerical analysis involves the calculation of the equilibrium force at each crosslink as the network expands. This allows a detailed study of how the distribution of individual polymer chain contour lengths affects the maximum swelling volume. The computer simulation results are compared with the results from experimental measurements of the maximum volume of swollen covalently crosslinked sodium alginate gels.     

The influence of inorganic silicon (Si) on pituitary-thyroid axis

The influence of silicon-treatment on the levels of TSH and thyroid hormones was studied in rats. Concentrations of thyrotropin (TSH), triiodothyronine (T₃), and thyroxine (T₄) were estimated in sera of rats receiving per os a soluble silicon compound—sodium metasilicate nonahydrate (Na₂SiO₃·9H₂O), dissolved in the animals' drinking water. An increase in the TSH level in the tested group was observed, without statistically significant differences in T₃ and T₄ concentrations between the two groups of animals. The results provide evidence for the influence of silicon on the endocrine balance. They could also prove that this chemical element is capable of modifying the rate of some hormones' synthesis.     

Flash-induced reduction of cytochrome b-559 by Q infB sup− in chloroplasts in the presence of protonophores

Flash-induced, fast (t _1/2 1 ms), reversible reduction of the high potential cytochrome b-559 (cyt b-559HP) was observed in chloroplasts in the presence of 2 M protonophore, FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), CCCP (carbonylcyanide 3-chlorophenylhydrazone) or SF 6847 (2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)phenol). These protonophores promote autooxidation of cyt b-559HP in the dark (Arnon and Tang 1988, Proc Natl Acad Sci USA 85: 9524). No fast photoreduction could, however, be observed if the molecules were oxidized with ferricyanide in the absence of protonophores. This suggests that the molecules must be deprotonated to be capable for fast photoreduction.Photoreduction of cyt b-559HP was largely insensitive to DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), but was inhibited by DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea). With a train of flashes, no oscillation could be observed in the amplitudes of photoreduction. These data strongly suggest that cyt b-559HP is reduced by the semireduced secondary quinone acceptor (Q_B ^–) of Photosystem 2.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis - Ant 2p- 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene - cyt- cyto-chrome - CCCP- carbonylcyanide 3-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl-6-iso-propyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimehtylurea - FCCP- carbonylcyanide p-trifluoromethoxyphenylhydrazone - FeCy- ferricyanide - HP- high potential form - HQ- hydroquinone - PQ- plastoquinone - PS 2- Photosystem 2 - SF 6847- 2,6-di-(t-butyl)-4-(2,2-dicyanovinyl)-phenol     

A strong protein unfolding activity is associated with the binding of precursor chloroplast proteins to chloroplast envelopes

Protein conformational changes related to transport into chloroplasts have been studied. Two chimaeric proteins carrying the transit peptide of either ferredoxin or plastocyanin linked to the mouse cytosolic enzyme dihydrofolate reductase (EC 1.5.1.3.) were employed. In contrast to observations in mitochondria, we found in chloroplasts that transport of a purified ferredoxin-dihydrofolate reductase fusion protein is not blocked by the presence of methotrexate, a folate analogue that stabilizes the structural conformation of dihydrofolate reductase. It is shown that transport competence of this protein in the presence of methotrexate is not a consequence of alteration of the folding characteristics or methotrexate binding properties of dihydrofolate reductase by fusion to the ferredoxin transit peptide. Binding of dihydrofolate reductase fusion proteins to chloroplast envelopes is not inhibited by low temperature and it is only partially diminished by methotrexate. It is demonstrated that the dihydrofolate reductase fusion proteins unfold, despite the presence of methotrexate, on binding to the chloroplast envelopes. We propose the existence of a strong protein unfolding activity associated to the chloroplast envelopes.     

IPG (inositolphosphate glycan) as a cellular signal for TGF-β1 modulation of chondrocyte cell cycle

The knowledge of transforming growth factor (TGF)-β receptors has greatly progressed in the recent years. TGF-β receptors type I and II have been implicated in the modulation of cell proliferation, whereas type III (betaglycan) may act as a component presenting TGF-β to its signaling receptors. In addition, four other proteins that bind TGF-β1 or TGF-β2 have been recently identified in some cell lines, three being anchored to the membrane through a glycosylphosphatidylinositol (GPI). Despite this knowledge, the molecular mechanism of signal transduction through the TGF-β receptors remain an enigma. TGF-β family does not signal via any of the classical pathways. As GPI anchors of membrane proteins have been implicated in the transduction of some hormonal effects, we investigated the putative role of GPI in signaling the TGF-β effects on the proliferation of rabbit articular chondrocytes (RAC). We previously showed that TGF-β1 increased DNA replication rate of RAC, with a recruitment of cells in G2/M followed by a subsequent mitosis wave. Here, we find that the factor causes specific GPI hydrolysis, with correlated increase of inositolphosphate glycan (IPG). This effect was specifically inhibited by antibodies that bind TGF-β1. Using [³H]-inositol labeling and Triton X-114 extraction, we demonstrate that a hydrophobic material from the membrane is cleaved by treatment of cell cultures with phosphatidylinositol specific phospholipase C (PI-PLC) or by exposure to TGF-β, supporting that a PI-anchored molecule gives rise to IPG by TGF-β-induced hydrolysis. The biological relevance of this hydrolysis was demonstrated by the enhancing effect of purified IPG on the DNA synthesis rate, which mimicked the TGF-β action. These results demonstrate that IPG could be an early messenger in the cellular signaling that mediates the effect of TGF-β on RAC growth. © 1993 Wiley-Liss, Inc.     

Release of 6-KETO-PGF1α and thromboxane B2 in late appearing cardioprotection induced by the stable PGI analogue: 7-OXO-PGI

We have shown earlier that prostacylin (PGI₂) and its stable analogue: 7-oxo-prostacyclin(7-OXO) may induce a prolonged, late appearing (24–48 h after drug administration), dose dependent protection of the heart from harmful consequences of a subsequent severe ischaemic stress, such as myocardial ischaemia, life-threatening ventricular arrhythmias and early ischaemic morphological changes. In an other study we observed that a similar but shortlived (less than 1 h) cardioprotection, induced by preconditioning brief coronary artery occlusions, is greatly reduced by blockade of the cyclooxygenase pathway, suggesting that prostanoids might play a role in this shortlasting protection.Objective of our present study was to elucidate the importance of some arachidonic acid (AA) metabolites, such as PGI₂ and thromboxane A₂ (TXA₂) in the mechanism of the late appearing, prolonged cardioprotection. Estimation of the metabolites: 6-keto-PGF₁ (6-KETO) and thromboxane B₂ (TXB₂) was made from the perfusate of isolated Langendorff hearts of guinea-pigs pretreated with 50 g/kg 7-OXO, 24 and 48 h before preparation. Pretreatment alone produced a slight, but significant elevation of 6-KETO (from 206±11 to 284±19 pg/ml/min after 24 h, and to 261±18 pg/ml/min after 48 h). No change was seen in TXB₂ production. Global ischaemia for 25 min (followed by 25 min reperfusion) markedly increased the release of both AA metabolites; maximal values were observed in the third min of reperfusion (6-KETO from 206±11 to 1275±55 pg/ml/min and TXB₂ from 29±4 to 172±12 pg/ml/min). All values returned to the preischaemic level by the 25th min of reperfusion. Ischaemic increase in 6-KETO level was significantly higher in the perfusate of hearts from pretreated animals (1507±73 pg/ml/min after 24 h, and 1398±54 pg/ml/min after 48 h) that in those of untreated controls. There was no difference in TXB₂ values. Thus both basal and ischaemic release of PGI₂ increased 24 and 48 h after pretreatment with 7-OXO but not TXA₂ production. Results suggest that endogenous prostanoids might play a role in late appearing cardioprotection.     

Prosaposin deficiency: further characterization of the sphingolipid activator protein-deficient sibs

Sphingolipid activator protein (SAP) deficiency, previously described in two sibs and shown to be caused by the absence of the common saposin precursor (prosaposin), was further characterized by biochemical lipid and enzyme studies and by ultrastructural analysis. The 20 week old fetal sib had increased concentrations of neutral glycolipids, including mono-, di-, tri- and tetrahexosylceramide, in liver, kidney and cultured skin fibroblasts compared with the controls. Glucosylceramide and lactosylceramide were particularly elevated. The kidney of the affected fetus showed additional increases in the concentration of sulphatide, galactosylceramide and digalactosylceramide. Free ceramide was stored in the liver and kidney, and G_M3 and G_M2 gangliosides were elevated in the liver, but not the brain, of the fetus. Phospholipids, however, were normal in the affected fetus. In the liver biopsy of the propositus, who later died at 16 weeks of age, only a few lipids could be studied. Glucosylceramide, dihexosylceramide and ceramide were elevated in agreement with our previous study. Enzyme studies were undertaken using detergent free liposomal substrate preparations and fibroblast extracts. The sibs' -glucocerebrosidase and -galactocerebrosidase activities were clearly reduced, but their sphingomyelinase activities were normal. The normal activity of the latter enzyme and the almost normal tissue concentration of sphingomyelin in prosaposin deficiency suggest that the prosaposin derived SAPs are not required for sphingomyelinase activity in vivo. In keeping with the biochemical findings, skin biopsies from the sibs showed massive lysosomal storage with a vesicular and membranous ultrastructure. The function of SAPs in sphingolipid degradation and the role of SAPs for enzyme activity in vitro are discussed. In addition, the similarity in neutral glycolipid accumulations in Niemann Pick disease type C and in prosaposin deficiency are noted. The phenotype of the prosaposin deficient sibs resembled acute neuronopathic (type 2) Gaucher disease more than Farber disease in several aspects, but their genotype was unique.This paper is dedicated to Prof. Jürgen Pfeiffer on the occasion of his 70th birthday