A comprehensive siRNA screen for kinases that suppress macroautophagy in optimal growth conditions

Macroautophagy is a catabolic process that maintains cellular homeostasis and protects cells against various external stresses including starvation. Except for the identification of the Akt-mTORC1 pathway as a major negative regulator, little is known about signaling networks that control macroautophagy under optimal growth conditions. Therefore, we screened a human kinome siRNA library for siRNAs that increase the number of autophagosomes in normally growing MCF-7 human breast carcinoma cells, and identified 10 kinases as regulators of constitutive macroautophagy. Further analysis of these kinases with respect to the autophagic flux, kinase signaling and endolysosomal function identified WNK2 as a positive regulator of autophagosome maturation and nine others as macroautophagy inhibitors. The depletion of MK2, PACSIN1, DAPK2, CDKL3 and SCYL1 functioned upstream of Akt-mTORC1 pathway, whereas CSNK1A1, BUB1, PKLR and NEK4 suppressed autophagosome formation downstream or independent of mTORC1. Importantly, all identified kinases except for BUB1 regulated macroautophagy also in immortalized MCF-10A breast epithelial cells. The kinases identified here shed light to the complex regulation of macroautophagy and open new possibilities for its pharmacological manipulation.     

A large set of estrogen receptor β-interacting proteins identified by tandem affinity purification in hormone-responsive human breast cancer cell nuclei

Estrogen receptors α (ER-α) and β (ER-β) play distinct biological roles in onset and progression of hormone-responsive breast cancer, with ER-β exerting a modulatory activity on ER-α-mediated estrogen signaling and stimulation of cell proliferation by mechanisms still not fully understood. We stably expressed human ER-β fused to a tandem affinity purification-tag in estrogen-responsive MCF-7 cells and applied tandem affinity purification and nanoLC-MS/MS to identify the ER-β interactome of this cell type. Functional annotation by bioinformatics analyses of the 303 proteins that co-purify with ER-β from nuclear extracts identify several new molecular partners of this receptor subtype that represents nodal points of a large protein network controlling multiple processes and functions in breast cancer cells.     

Effects of transmitters and amyloid-beta peptide on calcium signals in rat cortical astrocytes: Fura-2AM measurements and stochastic model simulations

Background

To better understand the complex molecular level interactions seen in the pathogenesis of Alzheimer''s disease, the results of the wet-lab and clinical studies can be complemented by mathematical models. Astrocytes are known to become reactive in Alzheimer''s disease and their ionic equilibrium can be disturbed by interaction of the released and accumulated transmitters, such as serotonin, and peptides, including amyloid- peptides (A). We have here studied the effects of small amounts of A25–35 fragments on the transmitter-induced calcium signals in astrocytes by Fura-2AM fluorescence measurements and running simulations of the detected calcium signals.

Methodology/Principal Findings

Intracellular calcium signals were measured in cultured rat cortical astrocytes following additions of serotonin and glutamate, or either of these transmitters together with A25–35. A25–35 increased the number of astrocytes responding to glutamate and exceedingly increased the magnitude of the serotonin-induced calcium signals. In addition to A25–35-induced effects, the contribution of intracellular calcium stores to calcium signaling was tested. When using higher stimulus frequency, the subsequent calcium peaks after the initial peak were of lower amplitude. This may indicate inadequate filling of the intracellular calcium stores between the stimuli. In order to reproduce the experimental findings, a stochastic computational model was introduced. The model takes into account the major mechanisms known to be involved in calcium signaling in astrocytes. Model simulations confirm the principal experimental findings and show the variability typical for experimental measurements.

Conclusions/Significance

Nanomolar A25–35 alone does not cause persistent change in the basal level of calcium in astrocytes. However, even small amounts of A25–35, together with transmitters, can have substantial synergistic effects on intracellular calcium signals. Computational modeling further helps in understanding the mechanisms associated with intracellular calcium oscillations. Modeling the mechanisms is important, as astrocytes have an essential role in regulating the neuronal microenvironment of the central nervous system.     

New insights into Alzheimer's disease progression: a combined TMS and structural MRI study

Background:

Combination of structural and functional data of the human brain can provide detailed information of neurodegenerative diseases and the influence of the disease on various local cortical areas.

Methodology and Principal Findings:

To examine the relationship between structure and function of the brain the cortical thickness based on structural magnetic resonance images and motor cortex excitability assessed with transcranial magnetic stimulation were correlated in Alzheimer''s disease (AD) and mild cognitive impairment (MCI) patients as well as in age-matched healthy controls. Motor cortex excitability correlated negatively with cortical thickness on the sensorimotor cortex, the precuneus and the cuneus but the strength of the correlation varied between the study groups. On the sensorimotor cortex the correlation was significant only in MCI subjects. On the precuneus and cuneus the correlation was significant both in AD and MCI subjects. In healthy controls the motor cortex excitability did not correlate with the cortical thickness.

Conclusions:

In healthy subjects the motor cortex excitability is not dependent on the cortical thickness, whereas in neurodegenerative diseases the cortical thinning is related to weaker cortical excitability, especially on the precuneus and cuneus. However, in AD subjects there seems to be a protective mechanism of hyperexcitability on the sensorimotor cortex counteracting the prominent loss of cortical volume since the motor cortex excitability did not correlate with the cortical thickness. Such protective mechanism was not found on the precuneus or cuneus nor in the MCI subjects. Therefore, our results indicate that the progression of the disease proceeds with different dynamics in the structure and function of neuronal circuits from normal conditions via MCI to AD.     

Application of cation-exchange membranes for characterisation and imaging ammonia-oxidising bacteria in soils

A new approach, in which ammonia-oxidizing bacteria (AOB) are entrapped from soil onto cation-exchange membranes, was applied to identify terrestrial AOB by fluorescence in situ hybridization (FISH). An experimental hot spot of ammonia oxidation was developed by establishing a gradient of ammonium substrate (200 to <20 mg NH4+-N l(-1)) diffused through the cation-exchange membranes incubated in soil for 6 months. By this approach we were able to characterise and image indigenous AOB populations growing in heavily oil-polluted soil using FISH and sequence analysis of PCR-amplified 16S rRNA genes, respectively. The FISH results revealed that Nitrosospira-like AOB were dominant on the ammonium-enriched membranes incubated in the soil. Fourteen unique Nitrosospira-like 16S rRNA gene sequences belonging to clusters 2 and 3 were recovered from the soil-incubated membranes and from the soil, suggesting the importance of Nitrosospira-like AOB in the oil-polluted landfarming soil.     

Contagious mucocutaneous dermatitis of the mountain hare (Lepus timidus): pathology and cause

Contagious mucocutaneous dermatitis is a frequently encountered disease of mountain hares (Lepidus timidus) in Finland. We describe the histopathologic changes and propose an etiologic cause for this disorder. Fifty-three cases collected during 1982-2000 were examined histologically. Transmission electron microscopy was performed in one case. In fully developed lesions, keratinocytes in epidermis and follicular infundibula were swollen and contained large eosinophilic intracytoplasmic inclusion bodies with marked reticular and ballooning degeneration. In later stages, there was marked necrosis and ulceration with severe pyogranulomatous and suppurative inflammation. At this stage, no viral inclusions were detectable, but secondary Staphylococcus warnerii infection was present in most cases. In late lesions, there was dermal fibrosis with epidermal hyperplasia. No spiral-shaped bacteria suggesting treponematosis were detected at any stage. Ultrastructurally, swollen epidermal and follicle infundibular cells contained round intracytoplasmic inclusion bodies with a myriad of virions typical of poxvirus with a biconcave nucleocapsid core, two lateral bodies, and a clearly discernible outer lipoprotein capsule. The findings suggest that contagious mucocutaneous dermatitis in mountain hares is a viral disease caused by a poxvirus. The disease is often complicated by secondary bacterial infection, most commonly S. warneri.     

Eider females form non-kin brood-rearing coalitions

Kin selection is a powerful tool for understanding cooperation among individuals, yet its role as the sole explanation of cooperative societies has recently been challenged on empirical grounds. These studies suggest that direct benefits of cooperation are often overlooked, and that partner choice may be a widespread mechanism of cooperation. Female eider ducks (Somateria mollissima) may rear broods alone, or they may pool their broods and share brood-rearing. Females are philopatric, and it has been suggested that colonies may largely consist of related females, which could promote interactions among relatives. Alternatively, shared brood care could be random with respect to relatedness, either because brood amalgamations are accidental and nonadaptive, or through group augmentation, assuming that the fitness of all group members increases with group size. We tested these alternatives by measuring the relatedness of co-tending eider females in enduring coalitions with microsatellite markers. Females formed enduring brood-rearing coalitions with each other at random with respect to relatedness. However, based on previous data, partner choice is nonrandom and dependent on female body condition. We discuss potential mechanisms underlying eider communal brood-rearing decisions, which may be driven by the specific ecological conditions under which sociality has evolved in this species.     

Synthesis, vibrational spectra and X-ray structures of copper(I) thiourea complexes

Complex formation of thiourea with copper takes place as an intermediate step in the preparation of copper sulfide thin films by spray pyrolysis starting from aqueous solutions of copper(II) chloride and thiourea. The stoichiometry of the complex and that of the resulting thin film primarily depends on the molecular ratio of the starting materials. For comparison, the structures of all copper(I) thiourea complexes found in the Cambridge Structural Database are classified in this paper. In addition, syntheses, structural (single crystal XRD also at low temperature 193 K) and spectroscopic studies (FTIR and Raman) of six copper-thiourea complexes are now reported. The copper to thiourea stoichiometric ratio is 1:3 in four of these complexes, but their structures are basically different as dimerization or polymer formation takes place depending on whether the water of crystallisation is present or absent. The structure of bis(μ-thiourea)tetrakis(thiourea)dicopper(I) dichloride dihydrate, [Cu₂(tu)₆]Cl₂ · 2H₂O (1) was determined at room and also at low temperature. Bis(μ-thiourea)tetrakis(thiourea)dicopper(I) dibromide dihydrate, [Cu₂(tu)₆]Br₂ · 2H₂O (2) is isomorphous with 1, like the anhydrous compounds chlorotris(thiourea)copper(I), [Cu(tu)₃]Cl (3) and bromotris(thiourea)copper(I), [Cu(tu)₃]Br (4) are isomorphous. In the third isomorphous pair of complexes the copper to thiourea stoichiometric ratio is 1:1, viz. chloro(thiourea)copper(I) hemihydrate, [Cu(tu)]Cl · 0.5H₂O (5) and bromo(thiourea)copper(I) hemihydrate, [Cu(tu)]Br · 0.5H₂O (6). During the preparation of chloro(thiourea)copper(I) hemihydrate (5) a reaction by product α,α^′-dithiobisformamidinium dichloride, [SC(NH₂)₂]₂Cl₂ (7) was identified and structurally characterized which made it possible to suggest a reaction path leading to complex formation.     

Identification of new Golgi complex specific proteins by direct organelle proteomic analysis

The Golgi complex is in the crossroad of the endocytic and secretory pathways. Its function is to post-translationally modify and sort proteins and lipids, and regulate the membrane balance in the cell. To understand the structure-function relationship of the Golgi complex the Golgi proteome has to be identified first. We have used a direct organelle proteomic analysis to identify new Golgi complex proteins. Enriched stacked Golgi membrane fractions from rat livers were isolated, and the proteins from these membranes were subsequently digested into peptides. The peptides were fractionated by cation-exchange chromatography followed by protein identification by automated capillary-LC/ESI-MS/MS analysis and database searches. Two different search programs, ProID and MASCOT were used. This resulted in a total of 1125 protein identifications in two experiments. In addition to the known Golgi resident proteins, a significant number of unknown proteins were identified. Some of these were further characterized in silico using different programs to provide insight into their structure, intracellular localization and biological functions. The Golgi localization of two of these newly identified proteins was also confirmed by indirect immunofluorescence.     

Systemic and mucosal antibody response in experimental Chlamydia pneumoniae infection of mice

Chlamydia pneumoniae is a common human respiratory pathogen, and sera from infected individuals recognize several proteins of C. pneumoniae. We produced C. pneumoniae-specific proteins in a Bacillus subtilis expression system. We then used these recombinant C. pneumoniae proteins and purified C. pneumoniae elementary bodies as antigens in enzyme immunoassays to assess the kinetics and protein specificity of the systemic and mucosal antibody responses induced by C. pneumoniae intranasal infection in BALB/c mice. The systemic antibodies in mice recognized strong 'key' immunogens of Chlamydia, Omp2 and Hsp60, but weakly targeted the MOMP protein, the major immunogen in chlamydial species other than C. pneumoniae. The IgA antibodies in bronchial secretions specifically recognized the putative surface protein of C. pneumoniae, Omp4. Our preliminary observations point to the necessity of further characterization of the mucosal antibody response during C. pneumoniae infection.