D1 and D4 dopaminergic receptor interplay mediates coincident G protein-independent and dependent regulation of glutamate NMDA receptors in the lateral amygdala

Dopamine (DA) receptor and NMDA receptor (NMDAR) activation in the lateral (LA) nucleus of the amygdala plays a critical role in emotional processing. Several distinct mechanisms regulate the molecular cross-talk between DA receptors and NMDARs in different brain regions; however, the cellular mechanism through which DA modulates NMDARs in LA projection neurons has not been studied. Here, we investigated the effect of DA receptor activation on NMDAR currents in LA projection neurons recorded in amygdala slices obtained from young rats. We found that DA reduces NMDAR current amplitudes in an additive manner through the activation of both D1-like and D2-like receptors. The reduction of NMDAR current amplitudes by D1-like receptor activation is mediated by a protein-protein interaction between the D1R and the NMDAR, while the regulation of NMDAR activity by D2-like receptors is elicited through a G protein-dependent pathway controlled by D4R. The results of our investigation show for the first time a functional interplay between D1R and D4R that mediates coincident G protein-independent and dependent regulation of NMDARs.     

Phospholipid synthesis and degradation during the life-cycle of P815Y mast cells synchronized with excess of thymidine

1. P815Y cells synchronized with excess of thymidine incorporate choline, proline and uridine throughout the cell cycle; the rate increases two- to four-fold during the S phase, when thymidine incorporation increases more than 15-fold. 2. Choline incorporated at any stage of the cell cycle turns over in a biphasic manner; stable and unstable components are each labelled maximally during the S phase. Total phospholipid also doubles predominantly during the S phase. 3. It is concluded that, despite turnover, choline incorporation is a useful measure of net phospholipid formation during the cell cycle.     

European guidelines for quality assurance in cervical cancer screening: recommendations for cytology laboratories.

The quality of a cervical cytology laboratory depends on adequate handling and staining of the samples, screening and interpretation of the slides and reporting of the results. These guidelines give an overview of procedures recommended in Europe to manage the balance between best patient care possible, laboratory quality assurance and cost effectiveness and will be published as a chapter 4 in the European Guidelines for Quality Assurance in Cervical Cancer Screening. The laboratory guidelines include protocols for personnel and organisation, material requirements, handling and analysing cervical samples, recording of results, quality management and communication. The section on quality management is comprehensive and includes protocols for all aspects of internal and external quality assurance. The guidelines are extensively referenced and as far as possible the recommendations are evidence-based.     

Identification of a critical chaperoning region on an archaeal recombinant thermosome

Chaperone function in water-miscible organic co-solvents is useful for biocatalytic applications requiring enzyme stability in semi-aqueous media and for understanding chaperone behavior in hydrophobic environments. Previously, we have shown that a recombinant single subunit thermosome (rTHS) from Methanocaldococcus jannaschii functions in multiple co-solvents to hydrolyze ATP, prevent protein aggregation, and refold enzymes following solvent denaturation. For the present study, a truncated analog to the thermosome in which 70 N-terminal amino acids are removed is used to identify important regions within the thermosome for its chaperoning functions in organic co-solvents. Data presented herein indicate that the N-terminal region of rTHS is essential for the chaperone to restore the native state of the enzyme citrate synthase, but it is not a critical region for either binding of unfolded proteins or ATP hydrolysis. This is the first demonstration that direct refolding by a Group II chaperonin requires the N-terminal region of the protein.     

Effect of increased Populus cover on Abies regeneration in the Picea–feathermoss boreal forest

Question: Does the increase in Populus tremuloides cover within the Picea mariana–feathermoss domain enhance establishment and growth conditions for Abies balsamea regeneration? Location: Boreal forest of northwest Quebec, Canada. Method: To document the effect of Populus tremuloides on A. balsamea regeneration, mixed stands with a heterogeneous presence of P. tremuloides adjacent to Picea mariana‐dominated stands were selected. Abies balsamea regeneration, understorey environment and canopy composition were characterized from 531 sampling units distributed along transects covering the mixed–coniferous gradient. Abundance of understorey A. balsamea regeneration was described using three height groups: seedling (<30 cm), small sapling (30 to <100 cm) and tall sapling (100 to 300 cm). Growth characteristics were measured from 251 selected individuals of A. balsamea (<3 m). Results: Results showed that A. balsamea regeneration was generally more abundant when P. tremuloides was present in the canopy. Differences between seedling and sapling abundance along the mixed–coniferous gradient suggest that while establishment probably occurs over a wide range of substrates, the better growth conditions found under mixed stands ensure a higher survival rate for A. balsamea seedlings. Conclusions: The abundant A. balsamea regeneration observed within mixed stands of the Picea mariana–feathermoss domain suggests that the increase in P. tremuloides cover, favoured by intensive management practices and climatic change, could contribute to acceleration of the northward expansion of the A. balsamea–Betula papyrifera domain into the northern boreal forest dominated by Picea mariana.     

Discoidin domain receptor 1 expression in activated T cells is regulated by the ERK MAP kinase signaling pathway

The expression and function of discoidin domain receptor 1 (DDR1) in T cells are still poorly explored. We have recently shown that activation of primary human T cells via their T cell receptor leads to increased expression of DDR1, which promoted their migration in three-dimensional collagen. In the present study, we provide evidence that activated T cells bind collagen through DDR1. We found that the DDR1:Fc blocking molecule significantly reduced the ability of activated T cells to bind soluble biotinylated collagen. However, DDR1:Fc had no impact on the adhesion of activated T cells to collagen and overexpression of DDR1 in Jurkat T cells did not enhance their adhesion. Together, our results indicate that DDR1 can promote T cell migration without enhancing adhesion to collagen, suggesting that it can contribute to the previously described amoeboid movement of activated T cells in collagen matrices. Our results also show that CD28, in contrast to IL-2 expression, did not costimulate the expression of DDR1 in primary human T cells. Using specific inhibitors, we demonstrated that TCR-induced expression of DDR1 in T cells is regulated by the Ras/Raf/ERK MAP Kinase and PKC pathways but not by calcium/calcineurin signaling pathway or the JNK and P38 MAP Kinases. Thus, our study provides additional insights into the physiology of DDR1 in T cells and may therefore further our understanding of the regulatory mechanisms of T cell migration.     

Differential kinetics and sensitivity to chloroquine of receptor-mediated insulin and prolactin endocytosis in liver parenchymal cells

Systemically injected [125I]prolactin or [125I]insulin was accumulated and cleared from rat liver at different rates. Quantitative subcellular fractionation indicated a predominant accumulation of [125I]insulin in liver microsomes while [125I]prolactin was found in both the light-mitochondrial and microsomal fractions. The acidotropic agent chloroquine diminished the rate and extent of loss of each ligand from liver homogenates. In chloroquine treated rats, radiolabeled insulin accumulated in both the light-mitochondrial and the microsomal fractions. Subfraction of microsomes on discontinuous sucrose gradients revealed "early' endosomes in which ligand uptake was maximal at 2-5 min. In contrast, comparable subfraction of the of light mitochondrial fraction revealed "late' endosomes in which ligand uptake was maximal at 10-20 min. Chloroquine-treated rats showed a more marked enhancement of insulin compared to prolactin uptake in the "early' endosomes. It is suggested that "early' endosomes found in the Golgi-intermediate and -heavy fractions floated from parent microsomes may selectively degrade insulin but not prolactin. This could account for the apparently different kinetics of insulin and prolactin uptake into liver parenchyma.     

Proteomics characterization of abundant Golgi membrane proteins

A mass spectrometric analysis of proteins partitioning into Triton X-114 from purified hepatic Golgi apparatus (84% purity by morphometry, 122-fold enrichment over the homogenate for the Golgi marker galactosyl transferase) led to the unambiguous identification of 81 proteins including a novel Golgi-associated protein of 34 kDa (GPP34). The membrane protein complement was resolved by SDS-polyacrylamide gel electrophoresis and subjected to a hierarchical approach using delayed extraction matrix-assisted laser desorption ionization mass spectrometry characterization by peptide mass fingerprinting, tandem mass spectrometry to generate sequence tags, and Edman sequencing of proteins. Major membrane proteins corresponded to known Golgi residents, a Golgi lectin, anterograde cargo, and an abundance of trafficking proteins including KDEL receptors, p24 family members, SNAREs, Rabs, a single ARF-guanine nucleotide exchange factor, and two SCAMPs. Analytical fractionation and gold immunolabeling of proteins in the purified Golgi fraction were used to assess the intra-Golgi and total cellular distribution of GPP34, two SNAREs, SCAMPs, and the trafficking proteins GBF1, BAP31, and alpha(2)P24 identified by the proteomics approach as well as the endoplasmic reticulum contaminant calnexin. Although GPP34 has never previously been identified as a protein, the localization of GPP34 to the Golgi complex, the conservation of GPP34 from yeast to humans, and the cytosolically exposed location of GPP34 predict a role for a novel coat protein in Golgi trafficking.