Phospholipases D1 and D2 coordinately regulate macrophage phagocytosis

Phagocytosis is a fundamental feature of the innate immune system, required for antimicrobial defense, resolution of inflammation, and tissue remodeling. Furthermore, phagocytosis is coupled to a diverse range of cytotoxic effector mechanisms, including the respiratory burst, secretion of inflammatory mediators and Ag presentation. Phospholipase D (PLD) has been linked to the regulation of phagocytosis and subsequent effector responses, but the identity of the PLD isoform(s) involved and the molecular mechanisms of activation are unknown. We used primary human macrophages and human THP-1 promonocytes to characterize the role of PLD in phagocytosis. Macrophages, THP-1 cells, and other human myelomonocytic cells expressed both PLD1 and PLD2 proteins. Phagocytosis of complement-opsonized zymosan was associated with stimulation of the activity of both PLD1 and PLD2, as demonstrated by a novel immunoprecipitation-in vitro PLD assay. Transfection of dominant-negative PLD1 or PLD2 each inhibited the extent of phagocytosis (by 55-65%), and their combined effects were additive (reduction of 91%). PLD1 and PLD2 exhibited distinct localizations in resting macrophages and those undergoing phagocytosis, and only PLD1 localized to the phagosome membrane. The COS-7 monkey fibroblast cell line, which has been used as a heterologous system for the analysis of receptor-mediated phagocytosis, expressed PLD2 but not PLD1. These data support a model in which macrophage phagocytosis is coordinately regulated by both PLD1 and PLD2, with isoform-specific localization. Human myelomonocytic cell lines accurately model PLD-dependent signal transduction events required for phagocytosis, but the heterologous COS cell system does not.     

1-Butanol interferes with phospholipase D1 and protein kinase Calpha association and inhibits phospholipase D1 basal activity

1-Butanol is commonly used as a substrate for phospholipase D (PLD) activity measurement. Surprisingly we found that, in the presence of 30 mM 1-butanol (standard PLD assay conditions), PLD1 activity in COS-7 cells was lost after incubation for 2 min. In contrast, in the presence of the protein kinase C (PKC) inhibitor staurosporine or dominant negative PKCalpha D481E, the activity was sustained for at least 30min. The binding between PLD1 and PKCalpha was also lost after 2 min incubation with 30 mM 1-butanol while staurosporine and D481E maintained the binding. 1-Butanol at 2 mM did not inhibit PLD1 basal activity or PLD1 binding to PKCalpha, and staurosporine and PKCalpha D481E produced a constant increase in PLD1 basal activity of 2-fold. These results indicate that 1-butanol is inhibitory to PLD1 activity by reducing its association with PKCalpha, and that the concentration of 1-butanol is an important consideration in assaying basal PLD1 activity.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

Delineation of the conserved functional properties of D1A, D1B and D1C dopamine receptor subtypes in vertebrates

The three main subtypes of dopamine D(1) receptor (D(1A), D(1B) and D(1C)) subtypes found in most vertebrate groups were generated by two major steps of gene duplications, early in evolution. To identify the functional characteristics contributing to conservation of these paralogous D(1) receptors in vertebrates, the pharmacological and functional properties of fish (Anguilla anguilla), amphibian (Xenopus laevis) and human receptors were systematically analysed in transfected cells. The ligand-binding parameters appeared essentially similar for orthologous receptors, but differed significantly among the subtypes. The D(1A) receptors from the three species displayed low intrinsic activity and a fast rate of agonist-induced desensitization. All the orthologous D(1B) receptors exhibited a similar desensitization time-course, but with smaller amplitude of decrease than D(1A) receptors, in agreement with their higher basal activity. In contrast, D(1C) receptors, which do not exist in mammals, have low intrinsic activity and exhibit only weak, but rapid, agonist-induced desensitization, without any changes upon longer treatment with agonist. Thus, each of the three D(1) receptor subtypes are characterized by activation and desensitization properties, in a sequence-specific manner, which has been probably acquired early after gene duplications, and constrained their conservation during vertebrate evolution. These properties have been instrumental to adapt dopamine system to the physiology of the numerous neuronal networks and functions they control in the large and complex brains of vertebrates.     

Hsc70 regulates accumulation of cyclin D1 and cyclin D1-dependent protein kinase

The cyclin D-dependent kinase is a critical mediator of mitogen-dependent G1 phase progression in mammalian cells. Given the high incidence of cyclin D1 overexpression in human neoplasias, the nature and complexity of cyclin D complexes in vivo have been subjects of intense interest. Besides its catalytic partner, the nature and complexity of cyclin D complexes in vivo remain ambiguous. To address this issue, we purified native cyclin D1 complexes from proliferating mouse fibroblasts by affinity chromatography and began to identify and functionally characterize the associated proteins. In this report, we describe the identification of Hsc70 and its functional importance for cyclin D1 and cyclin D1-dependent kinase maturation. We demonstrate that Hsc70 associates with newly synthesized cyclin D1 and is a component of a mature, catalytically active cyclin D1/CDK4 holoenzyme complex. Our data suggest that Hsc70 promotes stabilization of newly synthesized cyclin D1, thereby increasing its availability for assembly with CDK4. In addition, our data demonstrate that Hsc70 remains bound to cyclin D1 following its assembly with CDK4 and Cip/Kip proteins, where it ensures the formation of a catalytically active complex.     

Structure-affinity relationships of halogenated predicentrine and glaucine derivatives at D1 and D2 dopaminergic receptors: halogenation and D1 receptor selectivity

Halogenation of the aporphine alkaloid boldine at the 3-position leads to increased affinity for rat brain D(1)-like dopaminergic receptors with some selectivity over D(2)-like receptors. A series of 3-halogenated and 3,8-dihalogenated (halogen=Cl, Br or I) derivatives of predicentrine (9-O-methylboldine) and glaucine (2,9-di-O-methylboldine) were prepared and assayed for binding at D(1) and D(2) sites. Halogenation of predicentrine led to strong increases in affinity for D(1)-like receptors, while the affinities for D(2)-like receptors were either practically unchanged or reduced three- to fourfold. Halogenated glaucine derivatives did not show any clear trend towards enhanced selectivity, and the affinities were poor and similar to or worse than the values previously recorded for glaucine itself. Together with earlier work on boldine derivatives, these results suggest that the 2-hydroxy group on the aporphine skeleton may determine a binding mode favoring D(1)-like over D(2)-like receptors, with enhanced affinity when the C-3 position is halogenated.     

Bovine serum transferrin phenotypes AA,D1D1, D2D2, EE: Their carbohydrate compositions and electrophoretic multiplicity

Samples of homozygous bovine serum transferrins have been prepared and their purity has been ascertained by immunological techniques and electrophoretic analysis in SDS. Measurements of carbohydrate composition show that no significant differences exist among the phenotypic variants AA, D₁D₁, D₂D₂, and EE. Chromatography of transferrin AA on DEAE-cellulose separated four subfractions, each of which corresponded well with one band obtained by polyacrylamide gel electrophoresis. Carbohydrate analyses of the individual subfractions did not show significant differences in sialic acid, hexose, or hexosamine contents. After desialylation with neuraminidase, each subfraction was converted to a major band and a minor band on gel electrophoresis. From the relative band positions of the desialylated transferrins, it was concluded that possession of sialyl residues by bovine transferrin is not the primary cause of electrophoretic multiplicity. Rather, sialic acid masks an underlying heterogeneity which most likely resides within the polypeptide chain. Further characterization of this heterogeneity will best be undertaken with the isolated asialotransferrin subfractions.This research was supported by Grants MT-4074 and MA-5554 from the Medical Research Council of Canada and a Senior Fellowship (to M. W. C. H.) from the Ontario Heart Foundation.     

Molecular cloning and characterization of crustacean type-one dopamine receptors: D1alphaPan and D1betaPan

Dopamine (DA) differentially modulates identified neurons in the crustacean stomatogastric nervous system (STNS). While the electrophysiological actions of DA have been well characterized, little is known about the dopaminergic transduction cascades operating in this system. As a first step toward illuminating the molecular underpinnings of dopaminergic signal transduction in the crustacean STNS, we have cloned and characterized two type-one DA receptors (DARs) from the spiny lobster (Panulirus interruptus): D(1alphaPan) and D(1betaPan). We found that the structure and function of these arthropod DARs are well conserved across species. Using a heterologous expression system, we determined that DA, but not serotonin, octopamine, tyramine or histamine activates these receptors. When stably expressed in HEK cells, the D(1alphaPan) receptor couples with Gs, and DA elicits an increase in [cAMP]. The D(1betaPan) receptor responds to DA with a net increase in [cAMP] that is mediated by Gs and Gz.     

Structural Factors that Distinguish Dopamine D1 and D2 Agonists

To determine the structural features responsible for their selectivity as dopamine D1 agonists, a conformational analysis has been performed on an analog of nomifensine, dihydrexidine, a benzergoline, and an isochroman using the MM2-87 program. The preferred three dimensional structure of the hydroxylated phenyl ring of the nomifensine analog was found to differ from the other compounds with a substantial energy barrier to achieving the planar conformation of the other compounds which may explain its weak potency for D1 receptors. The preferred three dimensional structures of dihydrexidine and the benzergoline were found to differ significantly despite their molecular similarity. These conformational differences were also evident in crystal structures of the compounds or their analogs. The hypothesis that an equatorial ammonium hydrogen (or amine lone pair) is required for D1 agonist selectivity was tested by performing calculations on N-methyl equatorial and N-methyl axial analogs of the compounds. Calculations were also performed on nonselective dopamine agonists (apomorphine and 5,6-diOH- and 6,7-diOH aminotetralin) and dopamine D2-selective agonists ((+)-PHNO and an analog of quinpirole). The energy difference for the N-methyl axial conformations (or their equivalent) were found to be relatively small for the nonselective agonists and more substantial for the D2-selective agonists. This suggests that D2-selectivity may be associated with the relative unfavorability of the N-methyl axial conformation and provides an explanation for the decreased potency of tertiary amine analogs of the D1-selective agonists. In the benzergoline, where the energy difference is computed to be smaller, the addition of the N-methyl group appears to have a smaller deleterious effect on D1 activity. An N-methyl axial conformation has also been observed for the benzergoline in the crystal state suggesting that this conformation is energetically accessible.     

Resolvin D series and protectin D1 mitigate acute kidney injury

Omega-3 fatty acid docosahexaenoic acid is converted to potent resolvins (Rv) and protectin D1 (PD1), two newly identified families of natural mediators of resolution of inflammation. We report that, in response to bilateral ischemia/reperfusion injury, mouse kidneys produce D series resolvins (RvDs) and PD1. Administration of RvDs or PD1 to mice before the ischemia resulted in a reduction in functional and morphological kidney injury. Initiation of RvDs and RvD1 administration 10 min after reperfusion also resulted in protection of the kidney as measured by serum creatinine 24 and 48 h later. Interstitial fibrosis after ischemia/reperfusion was reduced in mice treated with RvDs. Both RvDs and PD1 reduced the number of infiltrating leukocytes and blocked TLR-mediated activation of macrophages. Thus, the renal production of Rv and protectins, a previously unrecognized endogenous anti-inflammatory response, may play an important role in protection against and resolution of acute kidney injury. These data may also have therapeutic implications for potentiation of recovery from acute kidney injury.     

Estrogen-induced cyclin D1 and D3 gene expressions during mouse uterine cell proliferation in vivo: Differential induction mechanism of cyclin D1 and D3

D-type cyclins are involved in the regulation of the G₁/S transition of the cell cycle in various cell types cultured in vitro. Little is, however, known about the expression pattern and functional role of D-type cyclins in physiological processes in vivo. In this report, we studied whether the expression of murine D-type cyclins correlates with the states of mouse uterine cell proliferation in vivo. Time-course changes in cyclin D1 and D3 mRNA levels in the uterine tissues of immature mice primed with 17β-estradiol (E₂) were examined by Northern blot hybridization. c-fos and thymidine kinase (TK) mRNA levels were also examined as markers for the transition from G₀ to G₁ and the onset of S phase, respectively. Cyclin D1 and D3 mRNAs were induced 2.5-fold between c-fos and TK mRNA peaks. The E₂-induced cyclin D1 and D3 gene expressions were blocked by antiestrogens tamoxifen and ICI 182,780. We also investigated the effects of cycloheximide (CHX), a protein synthesis inhibitor, on cyclin D1 and D3 gene expressions. When CHX was treated alone, cyclin D3, but not cyclin D1, mRNA was immediately superinduced. The E₂-induced cyclin D3 gene expression was shifted by approximately 6 h when CHX was pretreated 1 hr before E₂ administration. Interestingly, the ³H-thymidine incorporation experiment showed that the mouse uterine cell cycle progression also shifted by 6 hr with pretreatment of CHX. The overall results suggest that both cyclin D1 and D3 mRNAs are constitutively expressed in uterine tissues and induced by E₂ at G₁ phase of the mouse uterine cell cycle. However, the superinducibility and temporal shift of cyclin D3 by CHX suggest that there is a different regulatory mechanism underlying cyclin D1 and D3 gene expressions in the mouse uterine cell cycle progression. Mol. Reprod. Dev. 46:450–458, 1997. © 1997 Wiley-Liss, Inc.     

Polypeptides of the synaptic membrane antigens D1, D2, and D3

The rat brain synaptic membrane antigens D1, D2, and D3 were labelled by 125I and precipitated by antibodies in a crossed immunoelectrophoresis. The precipitates were stained, scraped off, reduced, and analysed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The D1 antigen was composed of two polypeptide chains, apparent molecular weights 50 300 and 116 000 D2 of only one polypeptide chain, apparent molecular weight 139 000, and D3 of three polypeptides, apparent molecular weights 14 100, 23 500, and 34 400. Higher apparent molecular weight polypeptides were present in variable amounts in the D3 precipitate, except when the synaptic membrane extracts had been pre-treated with phospholipase D.     

Cyclin D1与细胞周期调控

细胞周期是细胞生命活动中一个最重要的过程,其关键是G1 期的启动.细胞周期蛋白(Cyclin)、细胞周期蛋白依赖性激酶(CDKs)和CDK抑制因子(CKIs)是参与钿胞周期调控的主要因子.Cyclin D1是调控细胞周期G1期的关键蛋白,是一个比其他Cyclins更加敏感的指标,对细胞周期调控至关重要.综述Cyclin D1的结构和功能及其在肿瘤组织中的表达特征,初步分析Cyclin D在昆虫细胞周期调控的研究.     

Discriminating D1 and D2 agonists with a hydrophobic similarity index

Currently, methods for calculating molecular similarity indices have been developed for comparing steric, charge density, and molecular electrostatic potential (MEP) properties. Much of the existing technology may, however, be applied to the quantitative comparison of molecular hydrophobicities. In this article we present an empirical hydrophobic similarity index. We utilize atomic hydrophobic parameters derived from a quantum mechanical semiempirical wavefunction. Hydrophobicity at points on a grid is computed with a recently introduced “molecular lipophilicity potential”. The overlap of pairs of molecules is calculated with the metric introduced by Carbó. This approach is applied to a case in which steric and electrostatic criteria have already been shown to be inadequate in rationalizing selectivity, namely, requirements for recognition at the dopamine D1 and D2 receptors. We demonstrate that, for a set of dopamine agonists, D1 ligands show higher similarity in this property than D2 analogs. This indicator of similarity is more successful at accounting for D1 selectivity than previous methods.     

Differential expression of phospholipases D1 and D2 in mouse tissues

The differential expression of phospholipase D (PLD) isozymes, which include PLD1 and PLD2, was examined in various murine tissues, including the cerebrum, cerebellum, heart, lung, liver, spleen, stomach, pancreas, ileum, colon, adrenal gland, kidneys, testes, ovaries, and uterus. In Western blot analysis, only PLD1 was detected in the heart and ovary, while only PLD2 was detected in the pancreas and ileum. Both PLD1 and PLD2 were strongly expressed in the cerebrum, cerebellum, and lung, and both were also expressed in the liver, spleen, stomach, colon, kidney, testes, and uterus. Immunohistochemistry showed intense PLD immunostaining in the cerebrum, cerebellum, lungs, intestines, and testis, and weak PLD immunostaining in the liver, kidneys, spleen, and heart. These findings suggest that PLD1 and PLD2 are differentially expressed in the various organs of mice, and that each PLD isozyme plays a distinct role in each organ.     

Comparison of 1D and 2D NMR spectroscopy for metabolic profiling

High-resolution, liquid state nuclear magnetic resonance (NMR) spectroscopy is a popular platform for metabolic profiling because the technique is nondestructive, quantitative, reproducible, and the spectra contain a wealth of biochemical information. Because of the large dynamic range of metabolite concentrations in biofluids, statistical analyses of one-dimensional (1D) proton NMR data tend to be biased toward selecting changes in more abundant metabolites. Although two-dimensional (2D) proton-proton experiments can alleviate spectral crowding, they have been mainly used for structural determination. In this study, 2D total correlation spectroscopy NMR was used to compare the global metabolic profiles of urine obtained from wild-type and Abcc6-knockout mice. The 2D data were compared to an improved 1D experiment in which signal contributions from macromolecules and the urea peak have been spectroscopically removed for more accurate quantitation of low-abundance metabolites. Although statistical models from both 1D and 2D data could differentiate samples acquired from the two groups of mice, only the 2D spectra allowed the characterization of statistically relevant changes in the low-abundance metabolites. While acquisition of the 2D data require more time, the data obtained resulted in a more meaningful and comprehensive metabolic profile, aided in metabolite identifications, and minimized ambiguities in peak assignments.     

Functional implications of post-translational modifications of phospholipases D1 and D2

Our previous studies showed that truncation of the N-terminal 168 amino acids of rat brain phospholipase D1 (rPLD1) abolishes its response to protein kinase C (PKC) and greatly diminishes its palmitoylation and Ser/Thr phosphorylation. In this study, we show that the response to PKC as well as the palmitoylation and Ser/Thr phosphorylation were restored when the truncated rPLD1 mutant (rPLD1(DeltaN168)) was coexpressed with a fragment containing the N-terminal 168 amino acids. Immunoprecipitation experiments showed that the N-terminal fragment associated with rPLD1(DeltaN168) when coexpressed in COS 7 cells and that palmitoylation of Cys(240) and Cys(241) was not necessary for the association. In addition, we found that rat PLD2 (rPLD2) was palmitoylated on Cys(223) and Cys(224) in COS 7 cells. Mutation of both these cysteines reduced the basal activity of rPLD2, however its response to PMA stimulation in vivo was retained. As in the case of rPLD1, loss of palmitoylation weakened membrane association of rPLD2. In summary, the N-terminal 168-amino-acid fragment of rPLD1 can associate with truncated rPLD1(DeltaN168) to restore its palmitoylation, Ser/Thr phosphorylation and PKC response. Although rPLD2 differs from rPLD1 in many properties, it is palmitoylated at the corresponding conserved cysteine residues in COS 7 cells.     

Photochemical activity of photosystem II and hydrogen photoproduction in sulfur-deprived Chlamydomonas reinhardtii mutants D1-R323D and D1-R323L

The role of photosystem II in hydrogen photoproduction by Chlamydomonas reinhardtii cells was studied in mutants with modified D1-protein. In D1-R323D and D1-R323L mutants, the replacement of arginine by aspartate or leucine, respectively, resulted in the disruption of electron transport at the donor side of photosystem II. The rate of oxygen evolution in D1-R323D decreased twice as compared to the pseudo-wild type (pWT), and in D1-R323L no oxygen evolution was detected. The latter mutant was not capable of photoautotrophical growth. The dynamics of changes in oxygen content, the reduction of photosystem II active reaction centers (deltaF/F(1)m), and hydrogen production rate in pWT were found to be similar to the wild type if cultivated under sulfur deprivation in a closed bioreactor. The observed gradual decrease in the deltaF/F(1)m value turned to a sharp drop almost to zero followed by a partial recovery during which the production of hydrogen set in. The transition to the anaerobic phase in D1-R323D cultured in a sulfur-deprived medium occurred earlier than it happened in pWt under the same conditions. However, the partial recovery of photosystem II activity and hydrogen production started at a later time, and the rate of hydrogen production was low. The D1-R323L mutant incapable of oxygen evolution entered the rapidly anaerobiosis but produced no hydrogen. The kinetics of photoinduced redox transitions in P700 was similar in all investigated strains and was not affected by diuron addition. This implies that the mutants had a pool of reducers, which could donate electrons through the quinone pool or cytochrome to photosystem I. However, in D1-R323L mutant lacking the active photosystem II, this condition was not sufficient to support hydrogenase activity.