Intrathecal gene therapy in mouse models expressing CMT1X mutations.

Gap junction beta-1 (GJB1) gene mutations affecting the gap junction protein connexin32 (Cx32) cause the X-linked Charcot-Marie-Tooth disease (CMT1X), a common inherited neuropathy. Targeted expression of virally delivered Cx32 in Schwann cells following intrathecal injection of lentiviral vectors in the Cx32 knockout (KO) mouse model of the disease has led to morphological and functional improvement. To examine whether this approach could be effective in CMT1X patients expressing different Cx32 mutants, we treated transgenic Cx32 KO mice expressing the T55I, R75W or N175D CMT1X mutations. All three mutants were localized in the perinuclear compartment of myelinating Schwann cells consistent with retention in the ER (T55I) or Golgi (R75W, N175D) and loss of physiological expression in the non-compact myelin. Following intrathecal delivery of the GJB1 gene we detected the virally delivered wild-type (WT) Cx32 in non-compact myelin of T55I KO mice, but only rarely in N175D KO or R75W KO mice, suggesting dominant-negative effects of the R75W and N175D mutants but not of the T55I mutant on co-expressed WT Cx32. GJB1 treated T55I KO mice showed improved motor performance, lower ratios of abnormally myelinated fibers and reduction of inflammatory cells in spinal roots and peripheral nerves compared with mock-treated littermates. Either partial (N175D KO) or no (R75W KO) improvement was observed in the other two mutant lines. Thus, certain CMT1X mutants may interfere with gene addition therapy for CMT1X. Whereas gene addition can be used for non-interfering CMT1X mutations, further studies will be needed to develop treatments for patients harboring interfering mutations.

Structure of yeast plasma membrane H(+)-ATPase: comparison of activated and basal-level enzyme forms.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolising cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Structure of the two enzyme forms and the effects of beta, gamma-imidoadenosine 5'-triphosphate (AMP-PNP) and of diethylstilbestrol (DES) thereon were analysed by FT-IR spectroscopy. IR spectra revealed the presence of two populations of alpha-helices with different exposure to the solvent in both the A-ATPase and B-ATPase. AMP-PNP did not affect the secondary structure of A-ATPase while DES affected the ratio of the two alpha-helix populations. Thermal denaturation experiments suggested a more stable structure in the B-form than in the A-form. AMP-PNP stabilised the A-ATPase structure while DES destabilised both enzyme forms. IR spectra showed that 60% of the amide hydrogens were exchanged for deuterium in both forms at 20 degrees C. The remaining 40% were exchanged at higher temperatures. The maximum amount of H/D exchange was observed at 50-55 degrees C for both enzyme forms, while in the presence of DES it was observed at lower temperatures. The data do not contradict the possibility that the activation of H(+)-ATPase is due to the C-terminus of the enzyme dissociating from the ATP-binding site which is covered by it in the less active form.

Two-dimensional gel electrophoresis and FTIR spectroscopy reveal both forms of yeast plasma membrane H(+)-ATPase in activated and basal-level enzyme preparations.

Plasma membrane H(+)-ATPase of the yeast Saccharomyces cerevisiae was isolated and purified in its two forms, the activated A-ATPase from glucose-metabolizing cells, and the basal-level B-ATPase from cells with endogenous metabolism only. Using two-dimensional gel electrophoretic analysis, we showed that both enzyme preparations are actually mixtures of the non-active, i.e. non-phosphorylated, and the active, i.e. phosphorylated, forms of the enzyme. Previous deliberations suggesting that the B-ATPase displays some activity which is lower than that of A-ATPase were apparently wrong. It seems that, molecularly speaking, the B-form is actually not active at all, and what activity we measure in our preparation is due to an admixture of the true active form (A-form). Fourier transform infrared spectroscopic study of the secondary structure and particularly thermal denaturation data suggest the possibility that the two enzyme forms interact to form complexes less stable than the single forms. On the whole then, there apparently is a different ratio of the active and inactive forms and/or complexes between the two forms present in all enzyme preparations.

Accumulation of the proteolytic marker peptide ubiquitin in the trophoblast of mammalian blastocysts.

Ubiquitination is a universal protein degradation pathway in which the molecules of 8.5-kDa proteolytic peptide ubiquitin are covalently attached to the epsilon-amino group of the substrate's lysine residues. Little is known about the importance of this highly conserved mechanism for protein recycling in mammalian gametogenesis and fertilization. The data obtained by the students and faculty of the international training course Window to the Zygote 2000 demonstrate the accumulation of ubiquitin-cross-reactive structures in the trophoblast, but not in the inner cell mass of the expanding bovine and mouse blastocysts. This observation suggests that a major burst of ubiquitin-dependent proteolysis occurs in the trophoblast of mammalian peri-implantation embryos. This event may be important for the success of blastocyst hatching, differentiation of embryonic stem cells into soma and germ line, and/or implantation in both naturally conceived and reconstructed mammalian embryos.

Two forms of yeast plasma membrane H(+)-ATPase: comparison of yield and effects of inhibitors.

Classical isolation procedure for plasma membrane H(+)-ATPase of Saccharomyces cerevisiae based on fractional centrifugation yielded always a roughly two-fold greater amount of membranes when starting from glucitol-preincubated than from glucose-preincubated yeast. This difference persisted all the way to the purified plasma membranes and to the purified H(+)-ATPase. The ATP-hydrolyzing activity by plasma membranes was roughly twice greater in glucose-preincubated cells than in the D-glucitol-preincubated ones while the purified enzyme was 7 times more active after glucose than after glucitol. Effects of diethylstilbestrol, suloctidil, erythrosin B, vanadate and dicarbanonaboranuide were very similar on plasma membrane-localized and purified ATPases of both forms, suggesting that both preparations contain the two ATPase forms, the glucose-preincubated one being richer in the activated form while the glucitol-preincubated one contains less of it.

Extracellular acidification by Saccharomyces cerevisiae in normal and in heavy water.

Titratable acidity of the extracellular medium was compared with that calculated from pH changes in a suspension of Saccharomyces cerevisiae. After addition of cells to normal water the ratio of titratable acidity to the computed one was about 25, after addition of 50 mmol/L D-glucose it was about 13, after subsequent addition of K+ ions it was only 2. In heavy water the respective values were 30, 9, and 1. Apparently, the principal buffer-generating processes have to do with glucose metabolism but little with the K+/H+ exchange observed after addition of K+. D2O appears to block processes producing the buffering capacity of the medium, among them possibly extrusion of organic acids.

Effects of a ferrate-containing preparation on diverse metabolic processes in yeast.

A plant-sap-derived preparation containing bi- and tervalent ferrate anions was tested on growth, respiration on glucose, and membrane transport of 6-deoxy-D-glucose (6-dGlc) and 2-aminoisobutyric acid (Aib) in several yeast species, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Lodderomyces elongisporus, Rhodotorula gracilis, and Dipodascus magnusii. Growth was enhanced by as much as 65%, respiration was not affected significantly except for a decrease in R. gracilis, transport of 6-dGlc was not affected while that of Aib was increased by up to 45% in R. gracilis and up to 27% in L. elongisporus.

Glucose- and K(+)-induced acidification in different yeast species.

The process of acidification of the external medium after addition of glucose and subsequently of KCl to a suspension of yeast cells varies substantially from species to species. After glucose it is most pronounced in Saccharomyces cerevisiae and Schizosaccharomyces pombe but is very much lower in Lodderomyces elongisporus, Dipodascus magnusii and Rhodotorula gracilis. Both the buffering capacity and the varied effects of vanadate, suloctidil and erythrosin B indicate that the acidification is by about one-half due to the activity of plasma membrane H(+)-ATPase and by about one-half to the extrusion of acidic metabolites from cells. This is supported by the finding that a respiratory quotient greater than one (in various strains of S. cerevisiae and in S. pombe) is indicative of a greater buffering capacity and overall acidification of the medium. Taking into account the virtually negligible buffering capacity of the medium in the pH range where the effect of K+ is observed, the effect of K+ is generally of a similar magnitude as that of adding glucose. It is clearly dependent on (anaerobic) production of metabolic energy, quite distinct from the dependence of the H(+)-ATPase-caused acidification.

Subcellular shifts of trimeric G-proteins following activation of baker's yeast by glucose.

Addition of glucose to a resting cell suspension of the yeast Saccharomyces cerevisiae was accompanied by marked shifts of the G alpha-protein subunits from the plasma membrane to the cell interior. This process was rapid with half-times between < 10 and 20 s. The decrease of the plasma membrane pool of the Gi alpha/Go alpha- and Gq alpha/Gl 1 alpha-protein subunits correlated with an increase in acid-sensitive forms of these proteins which was recovered in the mitochondrial and/or lysosomal membrane fraction. In contrast to cells from higher organisms glucose-stimulated yeast exhibits an extremely rapid type of the redistribution (internalization). The question remains open as to the functional significance of the internalized forms of the G-proteins as these remain sequestered from the plasma membrane well after glucose has been consumed.

Dicarbanonaborates in yeast respiration and membrane transport.

Two derivatives of carborates, sodium 5,6-dichloro-7,8-dicarbanonaborate (CB-Cl) and sodium 5-mercapto-7,8-dicarbanonaborate (CB-SH) were found to inhibit endogenous as well as glucose-induced respiration of the yeast Saccharomyces cerevisiae. Both substances slightly increased endogenous acid production, were neutral toward H(+)-ATPase-associated acidification but pronouncedly inhibited the K(+)-stimulated acidification. The same effects were observed also with an ATPase-deficient mutant of the yeast. The ATP-hydrolyzing activity of yeast plasma membranes in vitro was severely reduced. The membrane potential was substantially increased toward more negative values. The H(+)-symporting uptake of glutamic acid was considerably decreased, that of adenine was diminished much less. The effects of the dicarbanonaborates are obviously pleiotropic but their inhibition of ATP hydrolysis and of uptake of H(+)-symported substances, on the one hand, and absolute lack of effect on ATPase-catalyzed acidification, on the other, pose an unresolved problem.

Different sources of acidity in glucose-elicited extracellular acidification in the yeast Saccharomyces cerevisiae.

Three wild-type strains of Saccharomyces cerevisiae, viz. K, Y55 and sigma 1278b, two mutants lacking one or both of the putative K+ transporters, trk1 delta and trk1 delta trk2 delta, and a mutant in the plasma membrane H(+)-ATPase, viz. pma1-105, were compared in their extracellular acidification following addition of glucose and subsequent addition of KCl; in ATPase activity in purified plasma membranes; and in respiration on glucose. The glucose-induced acidification was the greater the higher the respiratory quotient, i.e. the higher the anaerobic metabolism. A markedly lower acidification was found in the ATPase-deficient pma1-105 strain but also in the TRK-deficient double mutant. The acidification pattern after addition of KCl corresponds to expectations in the TRK mutants; however, a similarly decreased acid production was found in the ATPase-deficient mutant pma1-105. The highest rate of ATP hydrolysis in vitro was found with the trk1 delta trk2 delta mutant where glucose-, as well as KCl-induced acidification were lowest. Likewise, the pma1-105 mutant with extremely low acidification showed only a minutely lower ATP hydrolysis than did its parent Y55 strain. Apparently, several different sources of acidity are involved in the glucose-induced acidification (including extrusion of organic acids); in fact, contrary to the general belief, the H(+)-ATPase may play a minor role in this process in some strains.

Univalent cation fluxes in yeast.

Transport of H+, K+, Rb+ and Tl+ ions was studied in a wild-type strain of Saccharomyces cerevisiae and in its mutants defective in the high-affinity K+ transport system TRK1 and in the double mutant with an additional deletion in the TRK2 gene. In the absence of glucose K+, Rb+ and Tl+ elicited a more or less stoichiometric exchange outflow of H+, in the mutants K+ moved out of cells even in the presence of 10 mM KCl or KNO3. In the presence of glucose in the wild type, K+, Rb+ and Tl+ brought about a massive outflow of H+ while being transported inward against high concentration gradients. In the trk1 delta mutant the exchange fluxes were reduced by 65-85%, in the double mutant those of K+, Rb+ and Tl+ practically cease but outflow of H+ caused by Tl+ remained at the level of the trk1 delta mutant. It appears that, in addition to the H+ export by the PMA1-coded plasma membrane H(+)-ATPase, at least three different univalent-cation involving activities are present: the high-affinity transport system for K+ (TRK1), another system (possibly TRK2) with different responses to K+ and Rb+, vs. Tl+, and an active system for K+ export. The first two are apparently active exchange systems for K+, Rb+, and Tl+ against H+. The source of energy for these highly active transports (acting against gradients of 1000:1 and 5000:1, respectively) is unclear.

Analysis of the constitution of the beer yeast genome by PCR, sequencing and subtelomeric sequence hybridization.

The lager brewing yeasts, Saccharomyces pastorianus (synonym Saccharomyces carlsbergensis), are allopolyploid, containing parts of two divergent genomes. Saccharomyces cerevisiae contributed to the formation of these hybrids, although the identity of the other species is still unclear. The presence of alleles specific to S. cerevisiae and S. pastorianus was tested for by PCR/RFLP in brewing yeasts of various origins and in members of the Saccharomyces sensu stricto complex. S. cerevisiae-type alleles of two genes, HIS4 and YCL008c, were identified in another brewing yeast, S. pastorianus CBS 1503 (Saccharomyces monacensis), thought to be the source of the other contributor to the lager hybrid. This is consistent with the hybridization of S. cerevisiae subtelomeric sequences X and Y' to the electrophoretic karyotype of this strain. S. pastorianus CBS 1503 (S. monacensis) is therefore probably not an ancestor of S. pastorianus, but a related hybrid. Saccharomyces bayanus, also thought to be one of the contributors to the lager yeast hybrid, is a heterogeneous taxon containing at least two subgroups, one close to the type strain, CBS 380T, the other close to CBS 395 (Saccharomyces uvarum). The partial sequences of several genes (HIS4, MET10, URA3) were shown to be identical or very similar (over 99%) in S. pastorianus CBS 1513 (S. carlsbergensis), S. bayanus CBS 380T and its close derivatives, showing that S. pastorianus and S. bayanus have a common ancestor. A distinction between two subgroups within S. bayanus was made on the basis of sequence analysis: the subgroup represented by S. bayanus CBS 395 (S. uvarum) has 6-8% sequence divergence within the genes HIS4, MET10 and MET2 from S. bayanus CBS 380T, indicating that the two S. bayanus subgroups diverged recently. The detection of specific alleles by PCR/RFLP and hybridization with S. cerevisiae subtelomeric sequences X and Y' to electrophoretic karyotypes of brewing yeasts and related species confirmed our findings and revealed substantial heterogeneity in the genome constitution of Czech brewing yeasts used in production.