Localization of a New Type of X-Linked Liver Glycogenosis to the Chromosomal Region Xp22 Containing the Liver α-Subunit of Phosphorylase Kinase (PHKA2)

We describe here a new type of X-linked liver glycogen storage disease. The main symptoms include liver enlargement and growth retardation. The clinical and biochemical abnormalities of this glycogenosis are similar to those of classical X-linked liver glycogenosis due to phosphorylase kinase deficiency (XLG). However, in contrast to patients with XLG, the patients described here have no reduced phosphorylase kinase activity in erythrocytes and leukocytes, and no enzyme deficiency could be found. Linkage analysis of four families with this X-linked type of liver glycogenosis assigned the disease gene to Xp22. Lod scores obtained with the markers DXS987, DXS207, and DXS999 were 3.97, 2.71, and 2.40, respectively, all at 0% recombination. Multipoint linkage analysis localized the disease gene between DXS143 and DXS989 with a maximum lod score of 4.70 at θ = 0, relative to DXS987. As both the classical XLG gene and the liver α-subunit of PHK (PHKA2) are also located in Xp22, this variant type of XLG may be allelic to classical XLG, and both diseases may be caused by mutations in PHKA2. Therefore, we propose to classify XLG as XLG type I (the classical type of XLG) and XLG type II (the variant type of XLG).     

Kinetics of growth and sugar consumption in yeasts

An overview is presented of the steady- and transient state kinetics of growth and formation of metabolic byproducts in yeasts.Saccharomyces cerevisiae is strongly inclined to perform alcoholic fermentation. Even under fully aerobic conditions, ethanol is produced by this yeast when sugars are present in excess. This so-called Crabtree effect probably results from a multiplicity of factors, including the mode of sugar transport and the regulation of enzyme activities involved in respiration and alcoholic fermentation. The Crabtree effect inS. cerevisiae is not caused by an intrinsic inability to adjust its respiratory activity to high glycolytic fluxes. Under certain cultivation conditions, for example during growth in the presence of weak organic acids, very high respiration rates can be achieved by this yeast.S. cerevisiae is an exceptional yeast since, in contrast to most other species that are able to perform alcoholic fermentation, it can grow under strictly anaerobic conditions.Non-Saccharomyces yeasts require a growth-limiting supply of oxygen (i.e. oxygen-limited growth conditions) to trigger alcoholic fermentation. However, complete absence of oxygen results in cessation of growth and therefore, ultimately, of alcoholic fermentation. Since it is very difficult to reproducibly achieve the right oxygen dosage in large-scale fermentations, non-Saccharomyces yeasts are therefore not suitable for large-scale alcoholic fermentation of sugar-containing waste streams. In these yeasts, alcoholic fermentation is also dependent on the type of sugar. For example, the facultatively fermentative yeastCandida utilis does not ferment maltose, not even under oxygen-limited growth conditions, although this disaccharide supports rapid oxidative growth.     

Expression of insulin-like growth factor II (IGF-II) and histological changes in the thymus and spleen of transgenic mice overexpressing IGF-II

 Previously, transgenic mice were constructed overexpressing human insulin-like growth factor II (IGF-II) under control of the H₂k^b promoter. The IGF-II transgene was highly expressed in thymus and spleen, and these organs showed an increase in weight. In the current study we have analyzed the sites of IGF-II mRNA expression, the distribution of IGF-II, IGF-I, and both IGF receptors, and histomorphometrical changes in thymus and spleen. With in situ mRNA hybridization, expression of the IGF-II transgene is found with high intensity in the thymic medulla and in the white pulp/marginal zone of the spleen, whereas there were scattered positive cells in the thymic cortex and in the splenic red pulp. Hybridization was restricted to non-lymphocytic cells. Immunohistochemistry revealed intense IGF-II peptide staining with the same distribution as IGF-II mRNA. There was additional intense IGF-II staining of all elements in the splenic red pulp (including trabeculae) and diffuse, low level staining in the thymic cortex. These findings were not observed in control mice. In the thymic medulla, most IGF-II producing cells co-labelled with keratin, whereas a minor population also stained for the monocyte/macrophage marker MOMA-2. In the spleen, co-labelling of IGF-II producing cells was found with MOMA-1 (marginal zone), or with the dendritic cell marker NLDC-145 (red pulp). IGF-I and both IGF receptors were found in these organs in nearly all cell types, with a similar pattern in transgenic mice and in control animals. Histomorphometric analysis revealed a marked increase of thymus cortex size and an increased trabecular size in the spleen. This suggests that IGF-II overproduction induces local effects (auto/paracrine) in the thymic cortex, but not in the thymic medulla. Trabecular growth in the spleen most likely is a distant effect (paracrine or endocrine) of IGF-II overproduction. Accepted: 5 September 1996     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Discrimination of flash-induced fast and slow electric potential components

This work aimed at the resolution of the multi-component electric potential changes induced by single-turnover flash illumination of Photosystem-I-enriched subchloroplast vesicles. If supplemented with ferredoxin and under carefully adjusted redox poising, these vesicles show a pronounced slow-rising and -decaying electric potential component, as monitored by endogenous and exogenous field-sensitive probes, carotenoids and oxonol VI, respectively. The fast and slow potential components can be easily discriminated without the need for computer-assisted deconvolution after selective presaturation of the slow component by preillumination or a transmembrane ΔpH, after selective suppression of the slow component by low valinomycin or uncoupler concentrations or in the absence of ferredoxin. The slow electric potential component, as compared to the fast one, is relatively sensitive to low concentrations of ionophores and uncouplers, detergent, ageing and lower temperatures (4–12°C), is associated with electrogenic proton displacements and is interpreted to respond to a field that is more located on the membrane-bulk interface. Temperature effects show transition temperatures around 20°C for both the rise and decay of the slow potential component. The results provide further evidence that the carotenoids and oxonol VI sense the same (slow) electric field, but may be differently located in the thylakoid membrane.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Development of mitochondrial energy metabolism in rat brain

1. The development of pyruvate dehydrogenase and citrate synthase activity in rat brain mitochondria was studied. Whereas the citrate synthase activity starts to increase at about 8 days after birth, that of pyruvate dehydrogenase starts to increase at about 15 days. Measurements of the active proportion of pyruvate dehydrogenase during development were also made. 2. The ability of rat brain mitochondria to oxidize pyruvate follows a similar developmental pattern to that of the pyruvate dehydrogenase. However, the ability to oxidize 3-hydroxybutyrate shows a different developmental pattern (maximal at 20 days and declining by half in the adult), which is compatible with the developmental pattern of the ketone-body-utilizing enzymes. 3. The developmental pattern of both the soluble and the mitochondrially bound hexokinase of rat brain was studied. The total brain hexokinase activity increases markedly at about 15 days, which is mainly due to an increase in activity of the mitochondrially bound form, and reaches the adult situation (approx. 70% being mitochondrial) at about 30 days after birth. 4. The release of the mitochondrially bound hexokinase under different conditions by glucose 6-phosphate was studied. There was insignificant release of the bound hexokinase in media containing high KCl concentrations by glucose 6-phosphate, but in sucrose media half-maximal release of hexokinase was achieved by 70μm-glucose 6-phosphate 5. The production of glucose 6-phosphate by brain mitochondria in the presence of Mg²⁺+glucose was demonstrated, together with the inhibition of this by atractyloside. 6. The results are discussed with respect to the possible biological significance of the similar developmental patterns of pyruvate dehydrogenase and the mitochondrially bound kinases, particularly hexokinase, in the brain. It is suggested that this association may be a mechanism for maintaining an efficient and active aerobic glycolysis which is necessary for full neural expression.     

Effect of road traffic on the breeding site-tenacity of male Willow Warblers (Phylloscopus trochilus)

Summary The breeding site-tenacity of colour-ringed male Willow Warblers (Phylloscopus trochilus) was studied in an homogenous area of 154 ha, which is crossed by a heavily travelled road (45 000 cars per day). Close to the road (<200 m) the dispersal of the males is much higher than for birds further away. There is also some evidence that most of the near-to-the-road males are yearlings. The differences indicate that adjacent to the road the habitat is less favourable for the Willow Warbler and can be considered as marginal.

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Zusammenfassung Untersucht wurde die Brutortstreue von individuell fabrberingter Männchen des Fitis (Phylloscopus trochilus) in einem 154 ha großen Gebiet, das von einer Autobahn mit regem Verkehr (45 000 Fahrzeuge pro Tag) durchschnitten wird. In der Nähe der Autobahn (<200 m) zeigten die Reviermännchen eine viel größere Dismigration als die Revierinhaber in größerem Abstand von der Verkehrstraße. Auch gab es Hinweise, daß in der Nähe der Autobahn überwiegend einjährige Männchen die Reviere besetzten. Für den Fitis kann man Habitate entlang der Autobahn als marginal betrachten.

     

Nucleotide sequence of the genome of the filamentous bacteriophage I2-2: Module evolution of the filamentous phage genome

Summary The nucleotide sequence of the circular single-stranded genome of the filamentous Escherichia coli phage I2-2 has been determined and compared with those of the filamentous E. coli phages Ff(M13, fl, or fd) and IKe. The I2-2 DNA sequence comprises 6744 nucleotides; 139 nucleotides less than that of the N- and I2-plasmid-specific phage IKe, and 337 (336) nucleotides more than that of the F-plasmid-specific phage Ff. Nucleotide sequence comparisons have indicated that I2-2, IKe, and Ff have a similar genetic organization, and that the genomes of I2-2 and IKe are evolutionarily more closely related than those of I2-2 and Ff. The studies have further demonstrated that the I2-2 genome is a composite replicon, composed of only two-thirds of the ancestral genome of IKe. Only a contiguous I2-2 DNA sequence of 4615 nucleotides encompassing not only the coat protein and phage assembly genes, but also the signal required for efficient phage morphogenesis, was found to be significantly homologous to sequences in the genomes of IKe and Ff. No homology was observed between the consecutive DNA sequence that contains the origins for viral and complementary strand replication and the replication genes. Although other explanations cannot be ruled out, our data strongly suggest that the ancestor filamentous phage genome of phages I2-2 and IKe has exchanged its replication module during evolution with that of another replicon, e.g., a plasmid that also replicates via the so-called rolling circle mechanism. Offprint requests to: R.N.H. Konings     

Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate.

Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.