The 3D-QSAR analysis of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains on thymidylate synthase

Thymidylate synthase (TS) is a critical enzyme for DNA biosynthesis and many nonclassical lipophilic antifolates targeting this enzyme are quite efficient and encouraging as antitumor drug. In this paper, the binding model of 14 antifolates of 4(3H)-quinazolinone derivatives with dithiocarbamate side chains was examined using molecular simulation methods--FlexiDock and SCORE2.0. The resulted conformation and orientation of these antifolates were directly applied to CoMFA study. A good correlation between the calculated binding energies of these antifolates complexed with TS and their inhibitory activities was derived. The robust QSAR model, its three-dimensional contour map, and binding score for these antifolates derived from SCORE2.0 provided guidelines for structural optimization of current antifolates.     

Granulocyte-macrophage colony-stimulating factor （GM-CSF） and T-cell responses： what we do and don＇t know

Granulocyte-macrophage colony-stimulating factor （GM-CSF） is an important hematopoietic growth factor and immune modulator. GM-CSF also has profound effects on the functional activities of various circulating leukocytes. It is produced by a variety of cell types including T cells, macrophages, endothelial cells and fibroblasts upon receiving immune stimuli. Although GM-CSF is produced locally, it can act in a paracrine fashion to recruit circulating neutrophils, monocytes and lymphocytes to enhance their functions in host defense. Recent intensive investigations are centered on the application of GM-CSF as an immune adjuvant for its ability to increase dendritic cell （DC） maturation and function as well as macrophage activity. It is used clinically to treat neutropenia in cancer patients undergoing chemotherapy, in AIDS patients during therapy, and in patients after bone marrow transplantation. Interestingly, the hematopoietic system of GM-CSF-deficient mice appears to be normal; the most significant changes are in some specific T cell responses. Although molecular cloning of GM-CSF was carried out using cDNA library oft cells and it is well known that the T cells produce GM-CSF after activation, there is a lack of systematic investigation of this cytokine in production by T cells and its effect on T cell function. In this article, we will focus mainly on the immunobiology of GM-CSF in T cells.     

Cell-surface protein disulfide isomerase is required for transnitrosation of metallothionein by S-nitroso-albumin in intact rat pulmonary vascular endothelial cells

S-nitrosation of the metal binding protein, metallothionein (MT) appears to be a critical link in affecting endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS)-derived nitric oxide (NO)-induced changes in cytoplasmic and nuclear labile zinc, respectively. Although low molecular weight S-nitrosothiols also appear to affect this signaling system, less is known about the ability of extracellular protein nitrosothiols to transnitrosate MT. Accordingly, we synthesized fluorescently labeled S-nitroso-albumin (SNO-albumin, a major protein S-nitrosothiol in plasma) and determined, via confocal microscopy in fixed tissue, that it is transported into cultured rat pulmonary vascular endothelial cells in a temperature sensitive fashion. The cells were transfected with an expression vector that encodes human MT-IIa cDNA sandwiched between enhanced cyan (donor) and yellow (acceptor) fluorescent proteins (FRET-MT) that can detect conformational changes in MT through fluorescence resonance energy transfer (FRET). SNO-albumin and the membrane-permeant low molecular weight S-nitroso-l-cysteine ethyl ester (l-SNCEE) caused a conformational change in FRET-MT as ascertained by full spectral laser scanning confocal microscopy in live rat pulmonary vascular endothelial cells, a result which is consistent with transnitrosation of the reporter molecule. Transnitrosation of FRET-MT by SNO-albumin, but not l-SNCEE, was sensitive to antisense oligonucleotide-mediated inhibition of the expression of cell surface protein disulfide isomerase (csPDI). These results extend the original observations of Ramachandran et al. (Ramachandran N, Root P, Jiang XM, Hogg PJ, Mutus B. Proc Natl Acad Sci U S A 98: 9539-9544, 2001) and suggest that csPDI-mediated denitrosation helps to regulate the ability of the major plasma NO carrier (SNO-albumin) to transnitrosate endothelial cell molecular targets (e.g. MT).     

Prediction of Signal Peptides Using Bio-Basis Function Neural Networks and Decision Trees

Signal peptide identification is of immense importance in drug design. Accurate identification of signal peptides is the first critical step to be able to change the direction of the targeting proteins and use the designed drug to target a specific organelle to correct a defect. Because experimental identification is the most accurate method, but is expensive and time-consuming, an efficient and affordable automated system is of great interest. In this article, we propose using an adapted neural network, called a bio-basis function neural network, and decision trees for predicting signal peptides. The bio-basis function neural network model and decision trees achieved 97.16% and 97.63% accuracy respectively, demonstrating that the methods work well for the prediction of signal peptides. Moreover, decision trees revealed that position P(1'), which is important in forming signal peptides, most commonly comprises either leucine or alanine. This concurs with the (P(3)-P(1)-P(1')) coupling model.     

Divergence in spatial expression patterns and in response to stimuli of tandem-repeat paralogues encoding a novel class of proline-rich proteins in Oryza sativa

Gene duplication has been recognized as a major route to supply raw sequences for genome novelty in evolution, but the mechanism underlying the retention of duplicate genes is not fully understood yet. Divergence in spatial expression patterns was investigated here and in response to stimuli of the four members of a rice proline-rich protein gene family (OsPRP1), which encode a class of proline-rich proteins. The four paralogues are tandemly organized within a 20 kbp range of chromosome 10 without any interval of other open reading frames and with a median K(S) value of 0.474. These paralogues showed little similarity in their regulatory regions but high conservation in coding regions. Search of an intergenomic cis-element database predicted their promoter regions with divergent cis-element fingerprints. Further expression analyses involving different tissues/organs and nine types of stimuli by a promoter::GUS-fusion strategy revealed that the four paralogues were expressed mainly in vascular cylinders of different organs and showed diversity in tissue/organ specificity and in response to these stimuli, with some overlapping expression. Furthermore, these data show that OsPRP1.2 appeared to inherit most of the functions from their multifunctional progenitor, whereas the other three genes diverged after duplication events. Thus, the retention of paralogues in a multigene family seems to require a more complicated diversification process than originally thought. In addition, the promoter::GUS strategy is a powerful way to explore function divergence of a tandem-repeat gene family.     

Differential co-expression of AMPA receptor subunits in substance P receptor-containing neurons of basal forebrain regions of C57/BL mice

We are interested in cellular co-expression patterns of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptor subunits 1-4 (GluR1-4) in substance P receptor (SPR)-containing neurons of the basal forebrain, which may act as a morphological basis for interaction between neurokinins and glutamate-driven neuronal signaling and excitotoxicity. Immunohistochemistry and laser scanning confocal microscopy in adult C57/BL mice revealed that distribution of SPR-positive neurons overlapped with that of GluR1-4-containing ones in most basal forebrain regions, i.e. the medial septal nucleus, nucleus of diagonal band of Broca, magnocellular preoptic nucleus and substantia innominata. Neurons showing both SPR and GluR1-4-immunoreactivities were found in above cholinergic neurons-rich containing basal forebrain regions. Semi-quantification analysis indicated that about 57-95% of SPR-positive neurons displayed GluR1-4-immunoreactivity. The percentages of AMPA receptor subunits co-localizing in SPR-positive neurons were GluR4 (48%), GluR1 (47%), GluR2 (26%) and GluR3 (20%), respectively. However, the neurons co-expressing SPR and GluR1-4 were hardly detected in the basal nucleus of Meynert of the basal forebrain. The co-localization of SPR and AMPA receptors has provided a molecular basis for functional interaction between neurokinins and AMPA receptors-mediated signaling in basal forebrain neurons. This study has also implied that glutamate-driven neuronal transmission and excitotoxicity can be modulated by neurokinin peptides in most basal forebrain regions but not in the basal nucleus of Meynert, suggesting that neurokinins or SP may play certain roles in determining neuronal functional properties or excitotoxic susceptibility in the various basal forebrain regions of mammals.     

Identification and characterization of AGS4: a protein containing three G-protein regulatory motifs that regulate the activation state of Gialpha

Activators of G-protein signaling 1-3 (AGS1-3) were identified in a functional screen of mammalian cDNAs that activated G-protein signaling in the absence of a receptor. We report the isolation and characterization of an additional AGS protein (AGS4) from a human prostate leiomyosarcoma cDNA library. AGS4 is identical to G18.1b, which is encoded by a gene within the major histocompatibility class III region of chromosome 6. The activity of AGS4 in the yeast-based functional screen was selective for G(i2)/G(i3) and independent of guanine-nucleotide exchange by G(i)alpha. RNA blots indicated enrichment of AGS4/G18.1b mRNA in heart, placenta, lung, and liver. Immunocytochemistry with AGS4/G18.1b-specific antisera indicated a predominant nonhomogeneous, extranuclear distribution within the cell following expression in COS7 or Chinese hamster ovary cells. AGS4/G18.1b contains three G-protein regulatory motifs downstream of an amino terminus domain with multiple prolines. Glutathione S-transferase (GST)-AGS4/G18.1b fusion proteins interacted with purified G(i)alpha, and peptides derived from each of the G-protein regulatory motifs inhibited guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) binding to purified G(i)alpha(1). AGS4/G18.1b was also complexed with G(i)alpha(3) in COS7 cell lysates following cell transfection. However, AGS4/G18.1b did not alter the generation of inositol phosphates in COS7 cells cotransfected with the Gbetagamma-regulated effector phospholipase C-beta2. These data suggest either that an additional signal is required to position AGS4/G18.1b in the proper cellular location where it can access heterotrimer and promote subunit dissociation or that AGS4 serves as an alternative binding partner for G(i)alpha independent of Gbetagamma participating in G-protein signaling events that are independent of classical G-protein-coupled receptors at the cell surface.     

ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators

The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.