Coordinate regulation of sugar flux and translation by PAS kinase

PAS kinase is a serine/threonine kinase regulated in cis by a PAS domain. A genetic study of the two PAS kinase genes in budding yeast gave evidence of the involvement of these enzymes in the control of sugar metabolism and translation. Using a biochemical screen for PAS kinase substrates, three translation factors were identified as direct phosphorylation targets. PAS kinase was also found to phosphorylate UDP-glucose pyrophosphorylase and glycogen synthase, the enzymes catalyzing the two final steps in the glycogen biosynthetic pathway. Genetic, biochemical, and physiological data provide evidence that both of these enzymes are inhibited by PAS kinase-dependent phosphorylation, thereby downregulating carbohydrate storage. These studies provide evidence of a cell-autonomous signaling system that both controls and connects the balance of fuel consumption/storage to protein synthesis.     

The carboxyterminus of the ATP-binding cassette transporter A1 interacts with a beta2-syntrophin/utrophin complex

Recent work identified ABCA1 as the major regulator of plasma HDL-cholesterol responsible for the removal of excess choline-phospholipids and cholesterol from peripheral cells and tissues. ABCA1 function may depend on the association with heteromeric proteins and to identify these candidates a human liver yeast two-hybrid library was screened with the carboxyterminal 144 amino acids of ABCA1. Beta2-syntrophin was found to interact with ABCA1 and the C-terminal five amino acids of ABCA1 proned to represent a perfect tail for binding to syntrophin PDZ domains. Immunoprecipitation further confirmed the association of ABCA1 and beta2-syntrophin and in addition utrophin, known to couple beta2-syntrophin and its PDZ ligands to the F-actin cytoskeleton, was identified as a constituent of this complex. ABCA1 in the plasmamembrane of human macrophages was found to be partially associated with Lubrol rafts and effluxed choline-phospholipids involve these microdomains. Beta2-syntrophin does not colocalize in these rafts indicating that beta2-syntrophin may participate in the retaining of ABCA1 in cytoplasmic vesicles and for the targeting of ABCA1 to plasmamembrane microdomains when ABCA1 is released from beta2-syntrophin.     

Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells.

It was shown previously [Riedinger, H. J., van Betteraey-Nikoleit, M & Probst, H. (2002) Eur. J. Biochem.269, 2383-2393] that initiation of in vivo SV40 DNA replication is reversibly suppressed by hypoxia in a state where viral minichromosomes exhibit a nearly complete set of replication proteins. Reoxygenation triggers fast completion and post-translational modifications. Trying to reveal such fast changes of chromatin-bound replication proteins in the much more complex replication of the cellular genome itself, we developed a protocol to extend these studies using the human bladder carcinoma cell line T24, which was presynchronized in G1 by starvation. Concomitantly with stimulation of the cells by medium renewal, hypoxia was established. This treatment induced T24 cells to contain a large amount of replicons arrested in the 'hypoxic preinitiation state', ready to initiate replication as soon as normal pO2 was restored. Replicons in other stages of replicative activity were not detectable. Consequently the arrested replicons were rapidly released into synchronous initiation and succeeding elongation. Extraction of T24 nuclei with a Triton X-100 buffer yielded a fraction containing the cellular chromatin, including DNA-bound replication proteins, while unbound proteins were removed. The usefulness of this protocol was tested by the proliferation marker PCNA. We demonstrate here that this protein switches from the remainder cellular protein pool into the Triton-extracted nuclear fraction upon reoxygenation. Employing this protocol, analyses of chromatin-bound MCM2, MCM3, Cdc6 and cdk2 suggests that the 'classical' prereplication complex is already formed during hypoxia.     

Genetic mapping refines DFNB3 to 17p11.2, suggests multiple alleles of DFNB3, and supports homology to the mouse model shaker-2.

The nonsyndromic congenital recessive deafness gene, DFNB3, first identified in Bengkala, Bali, was mapped to a approximately 12-cM interval on chromosome 17. New short tandem repeats (STRs) and additional DNA samples were used to identify recombinants that constrain the DFNB3 interval to less, similar6 cM on 17p11.2. Affected individuals from Bengkala and affected members of a family with hereditary deafness who were from Bila, a village neighboring Bengkala, were homozygous for the same alleles for six adjacent STRs in the DFNB3 region and were heterozygous for other distal markers, thus limiting DFNB3 to an approximately 3-cM interval. Nonsyndromic deafness segregating in two unrelated consanguineous Indian families, M21 and I-1924, were also linked to the DFNB3 region. Haplotype analysis indicates that the DFNB3 mutations in the three pedigrees most likely arose independently and suggests that DFNB3 makes a significant contribution to hereditary deafness worldwide. On the basis of conserved synteny, mouse deafness mutations shaker-2 (sh2) and sh2J are proposed as models of DFNB3. Genetic mapping has refined sh2 to a 0.6-cM interval of chromosome 11. Three homologous genes map within the sh2 and DFNB3 intervals, suggesting that sh2 is the homologue of DFNB3.     

Sequence variations in small-subunit ribosomal RNAs of Hartmannella vermiformis and their phylogenetic implications

Evidence of associations between free-living amoebas and human disease has been increasing in recent years. Knowledge about phylogenetic relationships that may be important for the understanding of pathogenicity in the genera involved is very limited at present. Consequently, we have begun to study these relationships and report here on the phylogeny of Hartmannella vermiformis, a free-living amoeba that can harbor the etiologic agent of Legionnaires' disease. Our analysis is based on studies of small-subunit ribosomal RNA genes (srDNA). Nucleotide sequences were determined for nuclear srDNA from three strains of H. vermiformis isolated from the United Kingdom, Germany, and the United States. These sequences then were compared with a sequence previously obtained for a North American isolate by J. H. Gunderson and M. L. Sogin. The four genes are 1,840 bp long, with an average GC content of 49.6%. Sequence differences among the strains range are 0.38%-0.76%. Variation occurs at 19 positions and includes 2 single-base indels plus 14 monotypic and 3 ditypic single-base substitutions. Variation is limited to eight helix/loop structures according to a current model for srRNA secondary structure. Parsimony, distance, and bootstrap analyses used to examine phylogenetic relationships between the srDNA sequences of H. vermiformis and other eukaryotes indicated that Hartmannella sequences were most closely related to those of Acanthamoeba and the alga Cryptomonas. All ditypic sites were consistent with a separation between European and North American strains of Hartmannella, but results of other tests of this relationship were statistically inconclusive.      

Short-term regulation of glycolysis by insulin and dexamethasone in cultured rat hepatocytes

Evidence for a direct metabolic effect of insulin in isolated liver preparations is scarce. The stimulation of glycolysis by insulin previously demonstrated in monolayer cultures of adult rat hepatocytes [(1982) Eur. J. Biochem. 126, 271-278] was further investigated. The degree of stimulation varied with the age of the culture and amounted to 250%, 200%, 500% and 200% of the control value using cells at the culture age of 2 h, 24 h, 48 h, and 72 h, respectively. Half-maximal dose of insulin was 0.1 nM. Maximal stimulation was reached within 5 min and lasted for at least 4 h. Dexamethasone acted both as a long-term and short-term modulator. Long-term pretreatment of the cells with dexamethasone proved necessary to permit insulin action. In addition to this permissive action, pretreatment with dexamethasone reduced the insulin-independent basal glycolytic rate. In short-term experiments dexamethasone decreased the basal glycolytic flux, however, it did not affect the absolute increase in glycolysis brought about by insulin. The half-maximal dose of dexamethasone was 10 nM. The stimulatory effects of insulin may in part be attributed to the activation of pyruvate kinase. Insulin produced a left-shift of the substrate saturation curve, decreasing the K0.5 value for phosphoenolpyruvate.     

Expression of a major surface protein of Trypanosoma brucei insect forms is controlled by the activity of mitochondrial enzymes

In cycling between the mammalian host and the tsetse fly vector, trypanosomes undergo major changes in energy metabolism and surface coat composition. Early procyclic (insect) forms in the tsetse fly midgut are coated by glycoproteins known as EP and GPEET procyclins. EP expression continues in late procyclic forms, whereas GPEET is down-regulated. In culture, expression of GPEET is modulated by glycerol or glucose. Here, we demonstrate that a glycerol-responsive element of 25 nucleotides within the 3' untranslated region of GPEET mRNA also controls expression by glucose and during development in the fly. In trypanosomes, mitochondrial ATP is produced mainly by the acetate: succinate-CoA transferase/succinyl-CoA synthetase (ASCT) cycle, the citric acid cycle, and the cytochromes. Silencing of the pyruvate dehydrogenase or succinyl-CoA synthetase from the ASCT cycle by RNA interference induces reexpression of GPEET in late procyclic forms, whereas inhibition of the citric acid cycle or the cytochromes has no effect. In contrast, inhibition of the alternative oxidase, the second branch of the electron transport chain, with salicylhydroxamic acid overrides the effect of glucose or glycerol and causes a reduction in the level of GPEET mRNA. Our results reveal a new mechanism by which expression of a surface glycoprotein is controlled by the activity of mitochondrial enzymes.     

Priming of CTLs by lymphocytic choriomeningitis virus depends on dendritic cells

Appropriate activation of naive CD8(+) T cells depends on the coordinated interaction of these cells with professional APC that present antigenic peptides in the context of MHC class I molecules. It is accepted that dendritic cells (DC) are efficient in activating naive T cells and are unique in their capacity to prime CD8(+) T cell responses against exogenous cell-associated Ags. Nevertheless, it is unclear whether epitopes, derived from endogenously synthesized proteins and presented by MHC class I molecules on the surface of other APC including B cells and macrophages, can activate naive CD8(+) T cells in vivo. By infecting transgenic CD11c-DTR/GFP mice that allow conditional depletion of DC with lymphocytic choriomeningitis virus (LCMV), which infects all types of APC and elicits a vigorous CTL response, we unambiguously show that priming of LCMV-specific CD8(+) T cells is crucially dependent on DC, despite ample presence of LCMV-infected macrophages and B cells in secondary lymphoid organs.     

Use of zonal centrifugation for preparing synchronous cultures from Ehrlich ascites cells grown in vivo

A method is described which permits the selection of Ehrlich ascites tumour cells belonging to different stages of the cell cycle. The cells are grown in vivo and separated by zonal centrifugation immediately after being withdrawn from the intraperitoneal cavity of mice. Further in vitro growth of cells derived from different fractions obtained by zonal centrifugation demonstrates that this method permits the preparation of synchronous cultures which begin to grow at different points of the cell cycle and which maintain synchrony for at least two further cycles. The selection procedure seems to cause the subsequent appearance of a shortened cell cycle which develops more gradually in asynchronous primary cultures. Advantages of this method are, the large capacity and easy availability of the starting material.