Purification and characterization of Kurloff cell sialoglycoproteins with acid phosphatase activity

The majora2–6 sialoglycoproteins in detergent-extracts of Kurloff cells were purified by anion-exchange andSambucus nigra agglutinin-affinity chromatographies. The similar ultrastructural localisations of (1)S. nigra agglutinin-gold conjugates and (2) acid phosphatase activities on the Kurloff body and particularly on its myelin figures indicated that the majora2-6 sialoglycoproteins of the Kurloff cell had acid phosphatase activity. Two-dimensional electrophoresis showed that these tartrate-sensitive phosphatases corresponded to 2 acidic (pI 3.4–3.7) polypeptides of 36 and 34 kDa. Hydrolysis with peptide-N-glycosidases F gave a 33 kDa apoprotein rich in alanine, glutamic acid, tyrosine and lysin. A lectin-affinity study demonstrated that they contained hybrid type bisected and fucosylatedN-linked oligosaccharides. Cytotoxic properties were previously attributed to Kurloff cells and other studies suggested that not only acid phosphatases but alsoa2-6-linked sialic acid residues themselves may participate in natural killer activity.     

Interstitial noradrenaline concentration of rat hearts as influenced by cellular catecholamine uptake mechanisms

Marked concentration differences of noradrenaline (NA) between the vascular and the interstitial compartment were detected by sampling interstitial transudate from isolated perfused rat hearts. The ratios of vascular/interstitial concentration amounted to 7.4 to 1.3 depending on the concentration of NA administered (3 × 10^–9 to 10^–6 M). These concentration differences were abolished by inhibitors of uptake₁ desipramine (DMI) I and uptake, (O-methyl-isoprenaline (OMI)). Neuronal uptake, was characterized by a K_m of 0.22 mol/l and a V_max of 370 pmol × min^–1 × gWWT^–1, extraneuronal uptake₂ by a K_UPTAKE of = 0.313 min^–4.The apparent permeability surface area (P×S)-product calculated from uptake rate and transcapillary concentration difference was significantly decreased by administrating 100 mol/l (NA) in presence of DMI. A presumed endothelial uptake mechanism contributing to catecholamine translocation was investigated in endothelial cells in culture. These cells showed a specific noradrenaline uptake with a K_m of 4.35 mol/l and a V_max of about 75 pmol × min^–1 x gWWT^–1. Any inhibiton by inhibitors of both of the two noradrenaline uptakes was lacking. The uptake rate of this mechanism is insufficient to contribute to the diffusive conductivity of the capillary wall (P × S-product). We conclude from our investigations on interstitial concentrations of catecholamines and transcapillary concentration differences, that the capillary wall, owing to its metabolic and diffusional characteristics, influences the exchange of catecholamines to a substantial and physiologically relevant extent.     

Kurloff cell lysosomal arylsulphatases: Presence of both cationic and highly anionic isoforms of the sole B class

Following the previous ultrastructural demonstration of the presence of arylsulphatase (Asase) activities in Kurloff cells (KC) and of their quasi-exclusive localization in the Kurloff body (KB), this work investigates their biochemical and zymographic properties after extraction from purified KC suspensions. Using the discriminative inhibitory conditions of both the Baum or LeeVaupel and Conzelmann methods, nitrocatechol sulphate hydrolyzing enzymes of the KC were assumed to belong to the B class of the type II Asase alone. After electrophoretic separation under non-denaturing conditions in a 4–23% polyacrylamide gel, they were characterized by 55 kDa and 62 kDa zymographic bands. After isoelectric focusing, ‘classical’ cationic isoforms (pI 8.5) and two anionic isoforms (pI 4.4 and 4.6) were observed on zymograms. As expected for class B Asase, the different zymographic forms of KC Asase were only recovered in the unadsorbed fraction after anion-exchange chromatography on DEAE-cellulose column equilibrated with high ionic strength buffer. Their K_m (2.1 mM), their optimum pH (5.8) and their inhibitions by sulfite, phosphate, sulphate and ascorbic acid as well as their slight stimulation by AgNO₃ were also characteristic of this class of Asase. Finally, chondroitin4-sulphate was shown to potentially be a physiological substrate for these lysosomal enzymes.     

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.     

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