Toward a hypothesis-free understanding of how phosphorylation dynamically impacts protein turnover

The turnover measurement of proteins and proteoforms has been largely facilitated by workflows coupling metabolic labeling with mass spectrometry (MS), including dynamic stable isotope labeling by amino acids in cell culture (dynamic SILAC) or pulsed SILAC (pSILAC). Very recent studies including ours have integrated themeasurement of post-translational modifications (PTMs) at the proteome level (i.e., phosphoproteomics) with pSILAC experiments in steady state systems, exploring the link between PTMs and turnover at the proteome-scale. An open question in the field is how to exactly interpret these complex datasets in a biological perspective. Here, we present a novel pSILAC phosphoproteomic dataset which was obtained during a dynamic process of cell starvation using data-independent acquisition MS (DIA-MS). To provide an unbiased “hypothesis-free” analysis framework, we developed a strategy to interrogate how phosphorylation dynamically impacts protein turnover across the time series data. With this strategy, we discovered a complex relationship between phosphorylation and protein turnover that was previously underexplored. Our results further revealed a link between phosphorylation stoichiometry with the turnover of phosphorylated peptidoforms. Moreover, our results suggested that phosphoproteomic turnover diversity cannot directly explain the abundance regulation of phosphorylation during cell starvation, underscoring the importance of future studies addressing PTM site-resolved protein turnover.     

Effects of irrigation on the seasonal abundance of Empoasca vitis in north-Italian vineyards

The effect of irrigation on the abundance of Empoasca vitis (G?the) populations was investigated in four vineyards located in northeastern Italy. In two experiments, we compared leafhopper population densities in plots irrigated (micro-spray irrigation system) or nonirrigated. In another experiment, we studied the effect of various irrigation systems on E. vitis populations over two successive seasons. In particular, five treatments were compared: control (not irrigated), traditional drip system, three types of subirrigation varying in distance from the row (40, 135, and 95 cm). In this vineyard, stem water potential was monitored with a pressure chamber. E. vitis population densities were affected by irrigation, with higher densities of this pest recorded on irrigated vines. Highest E. vitis densities were detected in drip irrigation plots compared with nonirrigated plots where water stress was highest. Moderate water stress (subirrigation plots) was associated with intermediate leafhopper densities. Implications for integrated pest management are discussed.     

N-cadherin specifies first asymmetry in developing neurons

The precise polarization and orientation of developing neurons is essential for the correct wiring of the brain. In pyramidal excitatory neurons, polarization begins with the sprouting of opposite neurites, which later define directed migration and axo-dendritic domains. We here show that endogenous N-cadherin concentrates at one pole of the newborn neuron, from where the first neurite subsequently emerges. Ectopic N-cadherin is sufficient to favour the place of appearance of the first neurite. The Golgi and centrosome move towards this newly formed morphological pole in a second step, which is regulated by PI3K and the actin/microtubule cytoskeleton. Moreover, loss of function experiments in vivo showed that developing neurons with a non-functional N-cadherin misorient their cell axis. These results show that polarization of N-cadherin in the immediate post-mitotic stage is an early and crucial mechanism in neuronal polarity.     

Morphological and phytochemical features of secretory structures in Hypericum richeri (Clusiaceae)

Structural and complementary chemical studies were carried out on Hypericum richeri , a lesser known species amongst those reported for folk medical use. We found only one type of secretory glands consisting of black dots which are present even in early emerging leaves. In the fully expanded leaves the nodular structure appears to be composed by a cluster of cells. These become unfunctional and disassembled towards the end of their development, and are used only as reservoirs of secretion products. HPTLC analyses showed that flower buds and flowers are the plant parts richest in active compounds. However, the spectrum of active compounds accumulated by H. richeri was both quantitatively and qualitatively similar to those reported for the pharmaceutically utilized, H. perforatum , and thus could potentially represent a possible alternative to this species.     

A synoptic review of the Eocene (Ypresian) cartilaginous fishes (Chondrichthyes: Holocephali,Elasmobranchii) of the Bolca Konservat-Lagerstätte,Italy

Here, we review and discuss the records and taxonomy of the Ypresian (Eocene) chondrichthyans from the famous Bolca Konservat-Lagerstätte in northeastern Italy. Despite the outstanding diversity and the numerous studies focusing on the actinopterygian faunas from Pesciara and Monte Postale, the current knowledge about the systematics, taxonomy and phylogenetic relationships of the cartilaginous fishes from these Eocene sites remains elusive and largely inadequate. The celebrated Eocene Bolca Lagerstätte has yielded several exquisitely preserved articulated remains of chondrichthyan fishes in which delicate structures and soft tissues are preserved, as well as isolated teeth. The cartilaginous fish assemblage of Bolca comprises at least 17 species-level taxa belonging to 10 families in 6 orders, including selachians (Carcharhiniformes, Lamniformes), batoids (Torpediniformes, Myliobatiformes, Rajiformes) and holocephalans (Chimaeriformes). The occurrence of holocephalans represented by an isolated fin-spine of the chimeroid Ischyodus in the Bolca assemblage is reported here for the first time and represents the first record of chimeroids in the Eocene of Italy and also southern Europe. The Bolca chondrichthyan assemblage is remarkably different from those of other contemporaneous Boreal or Tethyan deposits, suggesting that its taxonomic composition is largely influenced by the palaeoenvironmental context. However, this synoptic review also highlights the importance of detailed revisions of all chondrichthyan remains from the Bolca Konservat-Lagerstätten.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Inhibition of a nutrient-dependent pinocytosis in dictyostelium discoideum by the amino acid analogue hadacidin

In the present study we examine the effects of the drug hadacidin (N-formyl-N- hydroxyglycine) on pinocytosis in the eukaryotic microorganism dictyostelium discoideum. At concentrations of up to approximately 8 mg/ml, hadacidin inhibited the rate of pinocytosis of fluorescein isothiocyanate (FITC) dextran in cells in growth medium in a concentration-dependent manner but had no effect on cells in starvation medium. Because hadacidin also inhibits cellular proliferation at this concentration, the relationship between growth rate and pinocytosis was studied further using another drug, cerulenin, to produce growth-arrest. These experiments showed no changes in the rate pinocytosis even after complete cessation of cellular proliferation. Other studies showed that the transfer of cells from growth to starvation medium reduced the rate of pinocytosis by approximately 50 percent. A reduction of similar magnitude occurred if cells were transferred from growth to starvation medium containing hadacidin. Also, no additional reduction in pinocytosis occurred when cells that had been treated with hadacidin were transferred to starvation medium containing hadacidin. These cells were able to take up [(14)C]hadacidin in the starvation medium. In contrast to the results with hadacidin-treated cells, cells in a cerulenin-induced state of growth-arrest when transferred to starvation medium exhibited the same 50 percent reduction in pinocytosis observed in cells not previously exposed to either drug. Cells treated with azide, in either growth or starvation medium, exhibited an immediate inhibition of all pinocytotic activity. After the transfer of log-phase cells to starvation medium supplemented with glucose, the reduction in rate was only approximately 10-15 percent. In contrast, a 50 percent reduction was observed after supplementation of starvation medium with sucrose, KCl, or concanavalin A. Maintaining the cells in growth medium containing hadacidin for as long as 16 h had no effect on the rate at which cells aggregated. These results are consistent with the conclusion that D. discoideum exhibits two types of pinocytotic activity: one that is nutrient dependent and the other independent of nutrients. This latter activity persists in starvation medium and is unaffected by hadacidin, whereas the nutrient-dependent activity is present in growth medium and is inhibited by hadacidin.     

The binding of 9-aminoacridine to calf thymus DNA in aqueous solution. Electronic spectral studies

Absorption, circular dichroism and steady-state fluorescence spectra were determined of 9-aminoacridine solutions in the presence of DNA at an ionic strength of 0.001 mol dm-3. Up to a dye/DNA phosphorus ratio of about 0.2 the results are fully consistent with the requirements and predictions of a binding model already shown to apply to the binding of other aminoacridines to DNA. The apparently anomalous spectroscopic behaviour of the 9-aminoacridine/DNA system compared with proflavine/DNA, for example, can be satisfactorily explained from a consideration of the magnitudes of exciton interactions between dyes bound to DNA.