The intracellular location of adenylyl cyclase in the cellular slime molds Dictyostelium discoideum and Polysphondylium pallidum

Adenylyl cyclase activity was low or not detectable on intact cells and in isolated plasma membranes, phagocytic vacuoles and nuclei of the two slime mold species examined. The entire activity of homogenates was sedimentable and concentrated in a light membrane fraction. When this fraction was centrifugated through sucrose density gradients the adenylyl cyclase activity sedimented differently from all other enzymes measured. The gradient fractions with the highest specific activity of adenylyl cyclase consisted mainly of small vesicles. No changes in adenylyl cyclase distribution were associated with development. The possibility that cellular slime mold adenylyl cyclase activity is associated with vesicles in vivo, as already suggested by Maeda & Gerisch [10], is discussed.     

Nickel,cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum on H₂ and CO₂ as sole energy and carbon sources was found to be dependent on Ni, Co, and Mo. At low concentrations of Ni (<100 nM), Co (<10 nM) and Mo (<10 nM) the amount of cells formed was roughly proportional to the amount of transition metal added to the medium; for the formation of 1 g cells (dry weight) approximately 150 nmol NiCl₂, 20 nmol CoCl₂ and 20 nmol Na₂MoO₄ were required. A dependence of growth on Cu, Mn, Zn, Ca, Al, and B could not be demonstrated. Conditions are described under which the bacterium grew exponentially with a doubling time of 1.8 h up to a cell density of 2 g cells (dry weight)/1.     

Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.     

Protoprimulagenin a Als Aglykon des Hauptsaponins von Primula elatior

It was shown that the aglycone of the main saponin of Primula elatior (L.) Hill is not primulagenin A but protoprimulagenin A with an oxide ring between C-13 β and C-28 in the β amyrin ring system.     

Calf articular cartilage in organ culture in a chemically defined medium

In Vitro Cellular &; Developmental Biology - Plant - Articular cartilage from 6-month-old calves was maintained in organ culture in Eagle's minimum essential medium at different oxygen...     

Molecular Phylogeny Reveals the Past Transoceanic Voyages of Drywood Termites (Isoptera,Kalotermitidae)

Termites are major decomposers in terrestrial ecosystems and the second most diverse lineage of social insects. The Kalotermitidae form the second-largest termite family and are distributed across tropical and subtropical ecosystems, where they typically live in small colonies confined to single wood items inhabited by individuals with no foraging abilities. How the Kalotermitidae have acquired their global distribution patterns remains unresolved. Similarly, it is unclear whether foraging is ancestral to Kalotermitidae or was secondarily acquired in a few species. These questions can be addressed in a phylogenetic framework. We inferred time-calibrated phylogenetic trees of Kalotermitidae using mitochondrial genomes of ∼120 species, about 27% of kalotermitid diversity, including representatives of 21 of the 23 kalotermitid genera. Our mitochondrial genome phylogenetic trees were corroborated by phylogenies inferred from nuclear ultraconserved elements derived from a subset of 28 species. We found that extant kalotermitids shared a common ancestor 84 Ma (75–93 Ma 95% highest posterior density), indicating that a few disjunctions among early-diverging kalotermitid lineages may predate Gondwana breakup. However, most of the ∼40 disjunctions among biogeographic realms were dated at <50 Ma, indicating that transoceanic dispersals, and more recently human-mediated dispersals, have been the major drivers of the global distribution of Kalotermitidae. Our phylogeny also revealed that the capacity to forage is often found in early-diverging kalotermitid lineages, implying the ancestors of Kalotermitidae were able to forage among multiple wood pieces. Our phylogenetic estimates provide a platform for critical taxonomic revision and future comparative analyses of Kalotermitidae.     

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10–12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.Subject terms: Cancer metabolism, CNS cancer     

Monoacylglycerol Lipases Act as Evolutionarily Conserved Regulators of Non-oxidative Ethanol Metabolism

Fatty acid ethyl esters (FAEEs) are non-oxidative metabolites of ethanol that accumulate in human tissues upon ethanol intake. Although FAEEs are considered as toxic metabolites causing cellular dysfunction and tissue damage, the enzymology of FAEE metabolism remains poorly understood. In this study, we used a biochemical screen in Saccharomyces cerevisiae to identify and characterize putative hydrolases involved in FAEE catabolism. We found that Yju3p, the functional orthologue of mammalian monoacylglycerol lipase (MGL), contributes >90% of cellular FAEE hydrolase activity, and its loss leads to the accumulation of FAEE. Heterologous expression of mammalian MGL in yju3Δ mutants restored cellular FAEE hydrolase activity and FAEE catabolism. Moreover, overexpression or pharmacological inhibition of MGL in mouse AML-12 hepatocytes decreased or increased FAEE levels, respectively. FAEEs were transiently incorporated into lipid droplets (LDs) and both Yju3p and MGL co-localized with these organelles. We conclude that the storage of FAEE in inert LDs and their mobilization by LD-resident FAEE hydrolases facilitate a controlled metabolism of these potentially toxic lipid metabolites.