Maturation of human lactase-phlorizin hydrolase Proteolytic cleavage of precursor occurs after passage through the Golgi complex

AbstractMaturation of human intestinal lactase-phlorizin hydrolase (LPH) requires that a precursor (pro-LPH) be proteolytically processed to the mature microvillus membrane enzyme (m-LPH). The subcellular site of this processing is unknown. Using low-temperature experiments and brefeldin A (BFA), intracellular transport was blocked in intestinal epithelial cells. In Caco-2 cells incubated at 18°C pro-LPH was complex-glycosylated but not cleaved, while at 20°C small amounts of proteolytically processed LPH were observed. These data exclude a pre-Golgi proteolytic event. BFA completely blocked proteolytic maturation of LPH and lead to an aberrant form of pro-LPH in both Caco-2 cells and intestinal explants. Therefore, proteolytic processing of LPH is a post-Golgi event, occuring either in the trans-Golgi network, transport vesicles, or after insertion of pro-LPH into the microvillus membrane

Fission Yeast CSL Transcription Factors: Mapping Their Target Genes and Biological Roles

BACKGROUND: Cbf11 and Cbf12, the fission yeast CSL transcription factors, have been implicated in the regulation of cell-cycle progression, but no specific roles have been described and their target genes have been only partially mapped. METHODOLOGY/PRINCIPAL FINDINGS: Using a combination of transcriptome profiling under various conditions and genome-wide analysis of CSL-DNA interactions, we identify genes regulated directly and indirectly by CSL proteins in fission yeast. We show that the expression of stress-response genes and genes that are expressed periodically during the cell cycle is deregulated upon genetic manipulation of cbf11 and/or cbf12. Accordingly, the coordination of mitosis and cytokinesis is perturbed in cells with genetically manipulated CSL protein levels, together with other specific defects in cell-cycle progression. Cbf11 activity is nutrient-dependent and Δcbf11-associated defects are mitigated by inactivation of the protein kinase A (Pka1) and stress-activated MAP kinase (Sty1p38) pathways. Furthermore, Cbf11 directly regulates a set of lipid metabolism genes and Δcbf11 cells feature a stark decrease in the number of storage lipid droplets. CONCLUSIONS/SIGNIFICANCE: Our results provide a framework for a more detailed understanding of the role of CSL proteins in the regulation of cell-cycle progression in fission yeast

CSL protein regulates transcription of genes required to prevent catastrophic mitosis in fission yeast.

For every eukaryotic cell to grow and divide, intricately coordinated action of numerous proteins is required to ensure proper cell-cycle progression. The fission yeast Schizosaccharomyces pombe has been instrumental in elucidating the fundamental principles of cell-cycle control. Mutations in S. pombe 'cut' (cell untimely torn) genes cause failed coordination between cell and nuclear division, resulting in catastrophic mitosis. Deletion of cbf11, a fission yeast CSL transcription factor gene, triggers a 'cut' phenotype, but the precise role of Cbf11 in promoting mitotic fidelity is not known. We report that Cbf11 directly activates the transcription of the acetyl-coenzyme A carboxylase gene cut6, and the biotin uptake/biosynthesis genes vht1 and bio2, with the former two implicated in mitotic fidelity. Cbf11 binds to a canonical, metazoan-like CSL response element (GTGGGAA) in the cut6 promoter. Expression of Cbf11 target genes shows apparent oscillations during the cell cycle using temperature-sensitive cdc25-22 and cdc10-M17 block-release experiments, but not with other synchronization methods. The penetrance of catastrophic mitosis in cbf11 and cut6 mutants is nutrient-dependent. We also show that drastic decrease in biotin availability arrests cell proliferation but does not cause mitotic defects. Taken together, our results raise the possibility that CSL proteins play conserved roles in regulating cell-cycle progression, and they could guide experiments into mitotic CSL functions in mammals

1.000.000 Fußballfans in einer Stadt mit 120.000 Einwohnern - ein notfallmedizinischer Albtraum?: Die Euro 2008 und das "Oranje-Wunder von Bern"

Zusammenfassung: Die Fußballeuropameisterschaft 2008 war die größte je in der Schweiz organisierte Sportveranstaltung. Eine Million Fußballfans besuchten in diesem Zeitraum Bern, und der lokale Flughafen Bern/Belp verzeichnete 261 zusätzliche Flüge. Pro Fußballspiel waren 33.000 Zuschauer im Stadion und 100.000 schauten in den Public-viewing-Zonen zu. Der Rettungsdienst und das Notfallzentrum des Inselspitals am Universitätsklinikum Bern waren für die medizinische Grundversorgung und die Notfallversorgung zuständig. Verletzungen und Krankheiten wurden mit einem standardisierten Score (NACA-Score, NACA National Advisory Committee of Aeronautics) analysiert. Details zu den Vorbereitungen, Kosten und Patientenzahlen werden im vorliegenden Artikel dargestellt. Insgesamt waren 30 zusätzlich Ambulanzfahrzeuge im Einsatz, 4723 zusätzliche Arbeitstage (1/3 davon durch medizinische Fachkräfte) wurden geleistet, 662 Ambulanzrufe gingen ein, 240 Personen benötigten eine medizinische Versorgung (62% Schweizer, 28% Niederländer, 10% anderer Nationalität). Von diesen wurden 51 Personen in einem der 4 städtischen Krankenhäuser behandelt. Es kamen keine Verletzungen der Grade NACAVI und VII vor (NACAI 4, NACAII 17, NACAIII 16, NACAIV 10, NACAV 4Patienten). Die Stadt Bern erstattete dem Inselspital 112.603EUR für die medizinische Versorgung. Die größten Anteile daran hatten die Sicherheitsmaßnahmen (50.300EUR) und die Kosten für medizinisches Personal (22.600EUR für Ärzte, 29.000EUR für Pflegefachkräfte). Aufgrund des schlechten Wetters und des vorbildlichen Verhaltens der Fans nahmen die Ereignisse einen eher friedlichen Verlau

Cdk9 and H2Bub1 signal to Clr6-CII/Rpd3S to suppress aberrant antisense transcription

Mono-ubiquitylation of histone H2B (H2Bub1) and phosphorylation of elongation factor Spt5 by cyclin-dependent kinase 9 (Cdk9) occur during transcription by RNA polymerase II (RNAPII), and are mutually dependent in fission yeast. It remained unclear whether Cdk9 and H2Bub1 cooperate to regulate the expression of individual genes. Here, we show that Cdk9 inhibition or H2Bub1 loss induces intragenic antisense transcription of ∼10% of fission yeast genes, with each perturbation affecting largely distinct subsets; ablation of both pathways de-represses antisense transcription of over half the genome. H2Bub1 and phospho-Spt5 have similar genome-wide distributions; both modifications are enriched, and directly proportional to each other, in coding regions, and decrease abruptly around the cleavage and polyadenylation signal (CPS). Cdk9-dependence of antisense suppression at specific genes correlates with high H2Bub1 occupancy, and with promoter-proximal RNAPII pausing. Genetic interactions link Cdk9, H2Bub1 and the histone deacetylase Clr6-CII, while combined Cdk9 inhibition and H2Bub1 loss impair Clr6-CII recruitment to chromatin and lead to decreased occupancy and increased acetylation of histones within gene coding regions. These results uncover novel interactions between co-transcriptional histone modification pathways, which link regulation of RNAPII transcription elongation to suppression of aberrant initiation

The fission yeast Rpb4 subunit of RNA polymerase II plays a specialized role in cell separation

RNA polymerase II is a complex of 12 subunits, Rpb1 to Rpb12, whose specific roles are only partly understood. Rpb4 is essential in mammals and fission yeast, but not in budding yeast. To learn more about the roles of Rpb4, we expressed the rpb4 gene under the control of regulatable promoters of different strength in fission yeast. We demonstrate that below a critical level of transcription, Rpb4 affects cellular growth proportional to its expression levels: cells expressing lower levels of rpb4 grew slower compared to cells expressing higher levels. Lowered rpb4 expression did not affect cell survival under several stress conditions, but it caused specific defects in cell separation similar to sep mutants. Microarray analysis revealed that lowered rpb4 expression causes a global reduction in gene expression, but the transcript levels of a distinct subset of genes were particularly responsive to changes in rpb4 expression. These genes show some overlap with those regulated by the Sep1-Ace2 transcriptional cascade required for cell separation. Most notably, the gene expression signature of cells with lowered rpb4 expression was highly similar to those of mcs6, pmh1, sep10 and sep15 mutants. Mcs6 and Pmh1 encode orthologs of metazoan TFIIH-associated cyclin-dependent kinase (CDK)-activating kinase (Cdk7-cyclin H-Mat1), while Sep10 and Sep15 encode mediator components. Our results suggest that Rpb4, along with some other general transcription factors, plays a specialized role in a transcriptional pathway that controls the cell cycle-regulated transcription of a specific subset of genes involved in cell division. ELECTRONIC SUPPLEMENTARY MATERIAL: Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00438-006-0161-5 and is accessible for authorized users

Slx8 removes Pli1-dependent protein-SUMO conjugates including SUMOylated Topoisomerase I to promote genome stability

Peer reviewedPublisher PD