On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

The roles of malate and aspartate in C4 photosynthetic metabolism of Flaveria bidentis (L.)

In C₄ grasses belonging to the NADP-malic enzyme-type subgroup, malate is considered to be the predominant C₄ acid metabolized during C₄ photosynthesis, and the bundle sheath cell chloroplasts contain very little photosystem-II (PSII) activity. The present studies showed that Flaveria bidentis (L.), an NADP-malic enzyme-type C₄ dicotyledon, had substantial PSII activity in bundle sheath cells and that malate and aspartate apparently contributed about equally to the transfer of CO₂ to bundle sheath cells. Preparations of bundle sheath cells and chloroplasts isolated from these cells evolved O₂ at rates between 1.5 and 2 mol · min^–1 · mg^–1 chlorophyll (Chl) in the light in response to adding either 3-phosphoglycerate plus HCO ₃ ^– or aspartate plus 2-oxoglutarate. Rates of more than 2 mol O₂ · min^–1 · mg^–1 Chl were recorded for cells provided with both sets of these substrates. With bundle sheath cell preparations the maximum rates of light-dependent CO₂ fixation and malate decarboxylation to pyruvate recorded were about 1.7 mol · min^–1 · mg^–1 Chl. Compared with NADP-malic enzyme-type grass species, F. bidentis bundle sheath cells contained much higher activities of NADP-malate dehydrogenase and of aspartate and alanine aminotransferases. Time-course and pulse-chase studies following the kinetics of radiolabelling of the C-4 carboxyl of C₄ acids from ¹⁴CO₂ indicated that the photosynthetically active pool of malate was about twice the size of the aspartate pool. However, there was strong evidence for a rapid flux of carbon through both these pools. Possible routes of aspartate metabolism and the relationship between this metabolism and PSII activity in bundle sheath cells are considered.Abbreviations DHAP dihydroxyacetone phosphate - NADP-ME(-type) NADP-malic enzyme (type) - NADP-MDH NADP-malate dehydrogenase - OAA oxaloacetic acid - 2-OG 2-oxoglutarate - PEP phosphoenolpyruvate - PGA 3-phosphoglycerate - Pi orthophosphate - Ru5P ribulose 5-phosphate     

Biologie und Verhalten zentralasiatischer Schneefinken (Montifringilla) und Erdsperlinge (Pyrgilauda)

Snow Finches and Mountain-steppe Sparrows differ in habitat selection, feeding, social and vocal behaviour. For these reasons, separation of the genusMontifringilla intoMontifringilla andPyrgilauda is recommended.     

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.     

Complete subunit sequences, structure and evolution of the 6 x 6-mer hemocyanin from the common house centipede, Scutigera coleoptrata.

Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure-function relationship and evolution. S. coleoptrata hemocyanin is a 6 x 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5-55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 x 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata.     

15N-ammonium and 15N-nitrate uptake of a 15-year-old Picea abies plantation

Throughfall nitrogen of a 15-year-old Picea abies (L.) Karst. (Norway spruce) stand in the Fichtelgebirge, Germany, was labeled with either ¹⁵N-ammonium or ¹⁵N-nitrate and uptake of these two tracers was followed during two successive growing seasons (1991 and 1992). ¹⁵N-labeling (62 mg ¹⁵N m^-2 under conditions of 1.5 g N m^-2 atmospheric nitrogen deposition) did not increase N concentrations in plant tissues. The ¹⁵N recovery within the entire stand (including soils) was 94%±6% of the applied ¹⁵N-ammonium tracer and 100%±6% of the applied ¹⁵N-nitrate tracer during the 1st year of investigation. This decreased to 80%±24% and 83%±20%, respectively, during the 2nd year. After 11 days, the ¹⁵N tracer was detectable in 1-year-old spruce needles and leaves of understory species. After 1 month, tracer was detectable in needle litter fall. At the end of the first growing season, more than 50% of the ¹⁵N taken up by spruce was assimilated in needles, and more than 20% in twigs. The relative distribution of recovered tracer of both ¹⁵N-ammonium and ¹⁵N-nitrate was similar within the different foliage age classes (recent to 11-year-old) and other compartments of the trees. ¹⁵N enrichment generally decreased with increasing tissue age. Roots accounted for up to 20% of the recovered ¹⁵N in spruce; no enrichment could be detected in stem wood. Although ¹⁵N-ammonium and ¹⁵N-nitrate were applied in the same molar quantities (¹⁵NH ₄ ⁺ : ¹⁵NO ₃ ^- =1:1), the tracers were diluted differently in the inorganic soil N pools (¹⁵NH ₄ ⁺ /NH ₄ ⁺ : ¹⁵NO ₃ ^- /NO ₃ ^- =1:9). Therefore the measured ¹⁵N amounts retained by the vegetation do not represent the actual fluxes of ammonium and nitrate in the soil solution. Use of the molar ammonium-to-nitrate ratio of 9:1 in the soil water extract to estimate ¹⁵N uptake from inorganic N pools resulted in a 2–4 times higher ammonium than nitrate uptake by P. abies.     

Intellectual disability-associated disruption of O-GlcNAc cycling impairs habituation learning in Drosophila

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of the O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognition-relevant habituation via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction, and suggests that blocking O-GlcNAc hydrolysis is a potential strategy to treat OGT-CDG.