Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Analysis of the role of RecQ helicases in RNAi in mammals

The identity of mammalian genes involved in RNA interference (RNAi), the targeted sequence-specific mRNA degradation by double-stranded RNA (dsRNA), is poorly defined. Here we report the analysis of mice with null mutations of Wrn, Blm, and RecQ1 genes that are related to Mut-7 and Qde3, two genes essential for RNAi in Caenorhabditis elegans and quelling in Neurospora, respectively. Our results suggest that Wrn, Blm, and RecQ1 are not involved in sequence-specific mRNA degradation in mammals in response to dsRNA, suggesting potential differences in the mammalian RNAi pathway.     

Mechanistic insights into Sky1p,a yeast homologue of the mammalian SR protein kinases

The SRPK family is distinguished from typical eukaryotic protein kinases by several unique structural features recently elucidated by X-ray diffraction methods [Nolen et al. (2001) Nat. Struct. Biol. 8, 176-183]. To determine whether these features impart unique catalytic function, the phosphorylation of the physiological Sky1p substrate, Npl3p, was monitored using steady-state and pre-steady-state kinetic techniques. While Sky1p has a low apparent affinity for ATP compared to other protein kinases, it binds Npl3p with very high affinity. The latter is achieved through a combination of local and distal factors in the protein substrate. The phosphoryl donor ATP has access to the nucleotide pocket in the absence or presence of Npl3p, indicating that a large protein substrate does not enforce an ordered addition of ligands. Sky1p binds two Mg(2+)-the first is essential whereas the second further enhances catalysis. While the turnover number is low (0.5 s(-1)), Npl3p is rapidly phosphorylated in the active site (40 s(-1)) based on single turnover experiments. These results indicate that Sky1p employs a catalytic pathway involving fast phosphoryl transfer followed by slow net release of products. These studies represent the first kinetic investigation of a member of the SRPK family and the first pre-steady-state kinetic study of a protein kinase using a natural protein substrate.     

N-linked glycans direct the cotranslational folding pathway of influenza hemagglutinin

For proteins that traverse the secretory pathway, folding commences cotranslationally upon translocation into the endoplasmic reticulum. In this study, we have comprehensively analyzed the earliest maturation steps of the model glycoprotein influenza hemagglutinin (HA). These steps include cleavage of the signal sequence, glycosylation, binding by the chaperones calnexin and calreticulin, and the oxidoreductase ERp57, and oxidation. Our results show that the molecular choreography of the nascent HA chain is largely directed by multiple glycans that are strategically placed to elicit the binding of lectin chaperones. These chaperones are recruited to specific nascent chain locations to regulate and facilitate glycoprotein folding, thereby suggesting that the positioning of N-linked glycans in critical regions has evolved to optimize the folding process in the cell.     

Nitrogen Export from Forested Watersheds in the Oregon Coast Range: The Role of N<Subscript>2</Subscript>-fixing Red Alder

Variations in plant community composition across the landscape can influence nutrient retention and loss at the watershed scale. A striking example of plant species importance is the influence of N₂-fixing red alder (Alnus rubra) on nutrient cycling in the forests of the Pacific Northwest. To understand the influence of red alder on watershed nutrient export, we studied the chemistry of 26 small watershed streams within the Salmon River basin of the Oregon Coast Range. Nitrate and dissolved organic nitrogen (DON) concentrations were positively related to broadleaf cover (dominated by red alder: 94% of basal area), particularly when near-coastal sites were excluded (r ² = 0.65 and 0.68 for nitrate-N and DON, respectively). Nitrate and DON concentrations were more strongly related to broadleaf cover within entire watersheds than broadleaf cover within the riparian area alone, which indicates that leaching from upland alder stands plays an important role in watershed nitrogen (N) export. Nitrate dominated over DON in hydrologic export (92% of total dissolved N), and nitrate and DON concentrations were strongly correlated. Annual N export was highly variable among watersheds (2.4–30.8 kg N ha^–1 y^–1), described by a multiple linear regression combining broadleaf and mixed broadleaf–conifer cover (r² = 0.74). Base cation concentrations were positively related to nitrate concentrations, which suggests that nitrate leaching increases cation losses. Our findings provide evidence for strong control of ecosystem function by a single plant species, where leaching from N saturated red alder stands is a major control on N export from these coastal watersheds.     

Apoptosis of CD4+ and CD8+ T cells during experimental infection with Mycobacterium avium is controlled by Fas/FasL and Bcl-2-sensitive pathways, respectively

Both CD4+ and CD8+ T cells from mice infected with Mycobacterium avium suffered a high rate of apoptosis, beginning with the onset of the immune response and culminating in the loss of T cells from the tissues and loss of IFN-gamma production. Fas expression increased over the course of infection on both T cell populations, as did their susceptibility to the induction of apoptosis in vitro by anti-Fas mAb. Nevertheless, although the rate of apoptosis among CD4+ T cells from infected mice was reduced to normal levels in lpr mice with a defective Fas, CD8+ T cells were unaffected, implying that Fas/FasL interaction was not important in these cells in vivo. Conversely, over-expression of B-cell lymphoma-2 (Bcl-2), which is known to protect T cells from apoptosis signalled through the TNF receptor or due to the withdrawal of cytokines, totally protected CD8+ T cells from infected mice but had no effect on CD4+. It is of interest that these two contrasting pathways of T-cell apoptosis operate at the same time during a single infection.     

Domains of estrogen receptor alpha (ERalpha) required for ERalpha/Sp1-mediated activation of GC-rich promoters by estrogens and antiestrogens in breast cancer cells

Estrogen receptor alpha (ERalpha)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on activation function 1 (AF1) of ERalpha, and this study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ERalpha on activation of ERalpha/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ERalpha (hERalpha or MOR), but not ERbeta and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hERalpha/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ERalpha. Antiestrogen-induced activation of hERalpha/Sp1 was lost using hERalpha mutants deleted in zinc finger 1 [amino acids (aa) 185-205], zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hERalpha/Sp1 required the C-terminal F domain (aa 579-595), which contains a beta-strand structural motif. Moreover, in peptide competition experiments overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hERalpha/Sp1, suggesting that F domain interactions with nuclear cofactors are required for ERalpha/Sp1 action.     

Quantification of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma using liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An accurate and reliable liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been developed and validated for the determination of 6-deoxy-6-demethyl-4-dedimethylaminotetracycline (COL-3) in human plasma. The assay used chrysin as an internal standard (I.S.). The analyte and the I.S. were extracted from acidified plasma by methyl-t-butyl ether. Separation was achieved on a YMCbasic column using acetonitrile-water-formic acid mobile phase. The MS-MS detection was by monitoring fragmentation 372.1-->326.2 (m/z) for COL-3 and 255.1-->153.1 (m/z) for the I.S. on a Sciex API 365 using a Turbo Ionspray in positive ion mode. The retention times were approximately 1.7 min for COL-3 and 1.8 min for the I.S. The validated dynamic range was 0.03-10.0 microg/ml using 0.25-ml plasma with correlation coefficients of >or=0.9985. The precision and accuracy for the calibration standards (n=3) were RSD相似文献