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.     

Phylogeny and substitution rates of angiosperm actin genes

Forty-four actin genes from five angiosperm species were PCR-amplified, cloned, and sequenced. Phylogenetic analysis of 34 of these actins, along with those previously published, indicates that angiosperm actin genes are monophyletic and underwent several duplications during evolution. Orthologues have been identified between Solanaceae species, as well as between Solanaceae species and soybean. These sequences were used to calculate nucleotide substitution rates. The synonymous rate (6.96 x 10(-9) substitutions/site/year) is similar to that of other nuclear protein-coding genes, but the nonsynonymous rate (0.19 x 10(-9) substitutions/site/year) is 6-19 times higher than that of mammalian actin genes. Relative rate tests indicate that actin genes are evolving at similar rates in monocots and in dicots. Evidence is also presented that some members of the maize actin multigene family have been involved in gene conversion events, that the potato genome contains 24 +/- 12 actin genes, and that potato and tomato diverged 11.6 +/- 3.6 MYA.      

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.     

Transfer of the broad-host-range IncQ plasmid RSF1010 and other plasmid vectors to the Gram-positive methylotroph Brevibacterium methylicum by electrotransformation

Gram-positive facultative methylotrophic coryneform bacterium Brevibacterium methylicum was efficiently transformed with various plasmids using electroporation of intact cells. In addition to the plasmid vectors pEC71 and pZ6-1 constructed on the basis of cryptic plasmids from coryneform bacteria, broad-host-range plasmids pLS5 (derivative of plasmid pMV158 from Streptococcus agalactiae) and RSF1010 belonging to the incompatibility group IncQ from Gram-negative bacteria were found to be present as autonomous structurally unchanged DNA molecules in B. methylicum transformants. With the exception of pZ6-1, all these plasmids were stably maintained in B. methylicum cells grown under non-selective conditions. When plasmid DNAs isolated from B. methylicum were used, the highest efficiency of transformation (10⁵ transformants/g DNA) was achieved. Correspondence to: J. Nevera     

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