Inhibition of subsets of G protein-coupled receptors by empty mutants of G protein alpha subunits in g(o), G(11), and G(16)

We previously reported that the xanthine nucleotide binding G(o)alpha mutant, G(o)alphaX, inhibited the activation of G(i)-coupled receptors. We constructed similar mutations in G(11)alpha and G(16)alpha and characterized their nucleotide binding and receptor interaction. First, we found that G(11)alphaX and G(16)alphaX expressed in COS-7 cells bound xanthine 5'-O-(thiotriphosphate) instead of guanosine 5'-O-(thiotriphosphate). Second, we found that G(11)alphaX and G(16)alphaX interacted with betagamma subunits in the presence of xanthine diphosphate. These experiments demonstrated that G(11)alphaX and G(16)alphaX were xanthine nucleotide-binding proteins, similar to G(o)alphaX. Third, in COS-7 cells, both G(11)alphaX and G(16)alphaX inhibited the activation of G(q)-coupled receptors, whereas only G(16)alphaX inhibited the activation of G(i)-coupled receptors. Therefore, when in the nucleotide-free state, empty G(11)alphaX and G(16)alphaX appeared to retain the same receptor binding specificity as their wild-type counterparts. Finally, we found that G(o)alphaX, G(11)alphaX, and G(16)alphaX all inhibited the endogenous thrombin receptors and lysophosphatidic acid receptors in NIH3T3 cells, whereas G(11)alphaX and G(16)alphaX, but not G(o)alphaX, inhibited the activation of transfected m1 muscarinic receptor in these cells. We conclude that these empty G protein mutants of G(o)alpha, G(11)alpha, and G(16)alpha can act as dominant negative inhibitors against specific subsets of G protein-coupled receptors.     

10-Year risk of colorectal cancer: Development and validation of a prediction model in middle-aged Japanese men

Background: To estimate an individual's probability of developing colorectal cancer (CRC) may aid health professionals and individuals in improving lifestyle behaviors or deciding the screening regimens. As fewer studies on cancer risk prediction were seen so far, we initially developed an assessment tool with synthesizing key information from a variety of CRC risk factors through a large population-based cohort study. Method: The prediction model was derived from 28,115 men in the Japan Public Health Center-based (JPHC) Prospective Study Cohort II (follow-up: 1993–2005), with risk factors selected by Cox proportion hazard regression. 18,256 men in the JPHC Study Cohort I (follow-up: 1995–2005) were used to evaluate the model's performance. Results: 543 and 398 CRCs were diagnosed during the follow-up period in Cohorts II and I, respectively. The prediction model, including age, BMI, alcohol consumption, smoking status, and the daily physical activity level, showed modest discrimination ability for CRC (C = 0.70; 95% confidential interval, 0.68–0.72) in Cohort II and well calibrated in Cohort I (Hosmer–Lemeshow χ² = 14.2, P = 0.08). Conclusion: The 10-year CRC risk prediction model may be used to estimate CRC risk in Japanese men. It may also play a role in the promotion of CRC prevention strategies.     

Dual signaling of human Mel1a melatonin receptors via G(i2), G(i3), and G(q/11) proteins

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.     

Polyamine prevention of inhibition of rat liver isoleucyl-tRNA formation by poly(G), poly(I) or ribosomes.

It is shown that rat liver isoleucyl-tRNA formation in the presence of Mg²⁺ is inhibited by poly(G), poly(I) or ribosomes and that this inhibition is prevented by polyamines. The inhibition is found to be noncompetitive with respect to tRNA.     

Development and crystallization of a minimal thermostabilised G protein-coupled receptor

Structure determination of G protein-coupled receptors is still in its infancy and many factors affect whether crystals are obtained and whether the diffraction is of sufficient quality for structure determination. We recently solved the structure of a thermostabilised turkey β₁-adrenergic receptor by crystallization in the presence of the detergent octylthioglucoside. Three factors were essential for this success. Firstly, truncations were required at the N-terminus to give optimal expression. Secondly, 6 thermostabilising point mutations were incorporated to make the receptor sufficiently stable in short-chain detergents to allow crystallization. Thirdly, truncations at the C-terminus and within cytoplasmic loop 3, in combination with the removal of the palmitoylation site, were required to obtain well-diffracting crystals in octylthioglucoside. Here, we describe the strategy employed and the utility of thermostability assays in assessing how point mutations, truncations, detergents and ligands combine to develop a construct that forms diffraction-grade crystals.     

GTP-binding proteins G(salpha), G(ialpha), and Ran identified in mitochondria of human placenta.

GTP-binding proteins (GTPases) have been detected in the mitochondria of human placenta. It has been proposed that porin interacts with GTPases in the mitochondrion to modulate contact site function, however, their identity and location is not known. In this study, we investigated the location of GTPases in mitochondria from term placentae as well as the expression of mitochondrial GTPases in mid-term placentae. Mitochondria obtained from human term and mid-term placentae were purified by sedimentation. Sub-mitochondrial vesicles prepared from ruptured and sonicated mitochondria were separated by ultracentrifugation in sucrose density gradients. The location of membrane vesicles was determined using marker enzymes. Mitochondrial proteins were separated by SDS-PAGE. Western blots were incubated in [alpha-(32)P]-GTP and detected using autoradiography or antibodies against known GTPases and porin followed by enhanced chemiluminescence. [alpha-(32)P]-GTP bound 24 and 28 kDa proteins located in the outer membrane. The G(salpha)antibody detected 42.5, 53 and 67 kDa proteins. The G(ialpha)antibody identified a 40.5 kDa band in contact sites and the outer membrane, as well as 55 and 105 kDa proteins in contact site vesicles. The Ran antibody detected a 28 kDa protein, mainly in the outer membrane. Porin migrated at 30 kDa. G(ialpha)and Ran were detected in mitochondria from both term and mid-term placentae. The location of porin and GTPases leave open the possibility that these proteins interact in contact sites and may also be responding to extra-mitochondrial signals. Ran and G(ialpha)are expressed by mid-term in human placentae and may be necessary for placental functions at this stage of development. It will be important in future experiments to characterise the physiological functions of these GTP-binding proteins in the mitochondria of human placenta.     

A post-translational modification of nuclear proteins, N(G),N(G)-dimethyl-Arg, found in a natural HLA class I peptide ligand

Presentation of peptides derived from endogenous proteins by class I major histocompatibility complex molecules is essential both for immunological self-tolerance and induction of cytotoxic T-cell responses against intracellular parasites. Despite frequent and diverse post-translational modification of eukaryotic cell proteins, very few class I-bound peptides with post-translationally modified residues are known. Here we describe a natural dodecamer ligand of HLA-B39 (B*3910) derived from an RNA-binding nucleoprotein that carried N(G),N(G)-dimethyl-Arg. Although common among RNA-binding proteins, this modification was not previously known among natural class I ligands. The sequence of this peptide was determined by Edman degradation and electrospray ion trap mass spectrometry. The fragmentation pattern of the dimethyl-Arg side chain observed with this latter technique allowed us to unambiguously assign the isomeric form of the modified residue. The post-translationally modified ligand was a prominent component (1-2%) of the B*3910-bound peptide repertoire. The dimethyl-Arg residue was located in a central position of the peptide, amenable to interacting with T-cell receptors, and most other residues in the middle region of the peptide were Gly. These structural features strongly suggest that the post-translationally modified residue may have a major influence on the antigenic properties of this natural ligand.     

Thermodynamics and kinetics of phase separation of protein-RNA mixtures by a minimal model

Intracellular liquid-liquid phase separation enables the formation of biomolecular condensates, such as ribonucleoprotein granules, which play a crucial role in the spatiotemporal organization of biomolecules (e.g., proteins and RNAs). Here, we introduce a patchy-particle polymer model to investigate liquid-liquid phase separation of protein-RNA mixtures. We demonstrate that at low to moderate concentrations, RNA enhances the stability of RNA-binding protein condensates because it increases the molecular connectivity of the condensed-liquid phase. Importantly, we find that RNA can also accelerate the nucleation stage of phase separation. Additionally, we assess how the capacity of RNA to increase the stability of condensates is modulated by the relative protein-protein/protein-RNA binding strengths. We find that phase separation and multiphase organization of multicomponent condensates is favored when the RNA binds with higher affinity to the lower-valency proteins in the mixture than to the cognate higher-valency proteins. Collectively, our results shed light on the roles of RNA in ribonucleoprotein granule formation and the internal structuring of stress granules.     

Phosphorylation of neurofibromin by cAMP-dependent protein kinase is regulated via a cellular association of N(G),N(G)-dimethylarginine dimethylaminohydrolase

The neurofibromatosis type 1 (NF1) tumor suppressor (neurofibromin) is thought to play crucial roles in cellular Ras- and cAMP-dependent kinase (PKA)-associated signals. In this study, we identified a cellular neurofibromin-associating protein, N(G),N(G)-dimethylarginine dimethylaminohydrolase (DDAH) that is known as a cellular NO/NOS regulator. The interaction of DDAH was mainly directed to the C-terminal domain (CTD) and to the cysteine/serine-rich domain (CSRD) of neurofibromin, coinciding with the regions containing specific PKA phosphorylation sites. DDAH increased PKA phosphorylation of native neurofibromin in a dose-dependent manner, especially affecting the phosphorylation of CSRD. These findings suggest that the PKA accessibility of neurofibromin was regulated via DDAH interaction, and this regulation may modulate the cellular function of neurofibromin that is implicated in NF1-related pathogenesis.     

Oxidative DNA damage background estimated by a system model of base excision repair

Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level, based on measuring 8-oxoguanine lesions as a biomarker, have led to estimates that vary over three to four orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our findings show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.     

Biodistribution of p-Iodobenzoyl (PIP) labeled antibodies in a murine lymphoma model

A monoclonal antibody against the murine T-cell antigen Thy 1.1 was radioiodinated using N-succin-imidyl p-iodobenzoate (PIP) in an attempt to decrease deiodination of the labeled antibody. The biodistribution of the PIP labeled antibody was compared to Iodogen labeled antibody in Thy 1.1⁺ lymphoma bearing AKR/Cum mice, where the antibody was tumor specific, and AKR/J mice where the antibody reacted with both tumor and normal T-cells. PIP labeling resulted in decreased iodine concentrations in stomach and salivary gland as compared to Iodogen labeling. There was little difference in radioiodine concentrations between the two preparations in tumor, lymphoid tissues or other organs. These results suggest deiodination of intact antibody plays little role in the clearance of radioiodinated anti-Thy 1.1 antibody from tissues.     

Ribosomal binding and dipeptide formation by misacylated tRNA(Phe),S

Eight structurally modified peptidyl-tRNA(Phe),s were employed to study P-site binding and peptide bond formation in a cell-free system involving Escherichia coli ribosomes programmed with poly(uridylic acid). It was found that the two analogues (N-acetyl-D-phenylalanyl-tRNA(Phe) and N-acetyl-D-tyrosyl-tRNA(Phe] containing D-amino acids functioned poorly as donors in the peptidyltransferase reaction and that two N-acetyl-L-phenylalanyl-tRNA(Phe)'s differing from the prototype substrate in that they contained 2'- or 3'-deoxyadenosine at the 3'-terminus failed to form dipeptide at all when L-phenylalanyl-tRNA(Phe) was the acceptor tRNA. Interestingly, all four of these peptidyl-tRNA's bound to ribosomes to about the same extent as tRNA's that functioned normally as donors in the peptidyltransferase reaction, at least in the absence of competing peptidyl-tRNA species. Two peptidyl-tRNA's lacking an amino group were also tested. In comparison with N-acetyl-L-phenylalanyl-tRNA(Phe) it was found that trans-cinnamyl-tRNA(Phe) and 3-phenylpropionyl-tRNA(Phe)'s formed dipeptides to the extent of 53 and 80%, respectively, when L-phenylalanyl-tRNA(Phe)was used as the acceptor tRNA. N-Acetyl-beta-phenylalanyl-tRNA(Phe) was found to be the most efficient donor substrate studied. Both isomers transferred N-acetyl-beta-phenylalanine to L-phenylalanyl-tRNA(Phe); the nature of the dipeptides formed in each case was verified by HPLC in comparison with authentic synthetic samples. Further, the rate and extent of peptide bond formation in each case exceeded that observed with the control tRNA, N-acetyl-L-phenylalanyl-tRNA(Phe).     

A two-compartment model for the dependence of a postsynaptic potential on a postsynaptic current, measured by the patch-clamp method

As known, the dependence of a postsynaptic potential (PSP)1 on a postsynaptic current (PSC) is not satisfactorily approximated by simple Ohm's law due to a significant role of electrotonic propagation of currents along dendrites. The present work shows that a two-compartment model of a neuron, conjointly solving the two problems of voltage and current clamping, gives quite precisely the PSP-on-PSC dependence, in spite of inaccurate reconstruction of currents on dendritic terminals. The two-compartment model is compared with the neuron model consisting of a distributed cylindrical dendrite and a concentrated soma.     

Intestinal growth and pathology of Giardia duodenalis assemblage subtype A(I), A(II), B and E in the gerbil model

This study investigated the significance of the genetic differences between assemblages A, B and E on intestinal growth and virulence. Intestinal growth and virulence were studied in 2 laboratory (A(I): WB and B: GS/M-83-H7) and 6 field isolates of assemblage subtype A(I), A(II), B and E(III). Intestinal trophozoite burdens, body weight and faecal consistency were monitored until day 29 post-infection (p.i.), morphological (mucosal architecture and inflammation) and functional (disaccharidase and alkaline phosphatase enzyme activity) damage to the small intestine were evaluated on days 7 and 18 p.i. The assemblage subtypes A(I) and B were more infectious and produced higher trophozoite loads for a longer period compared to the subtypes A(II) and E(III). The body weight of infected gerbils was significantly reduced compared to uninfected controls, but did not differ between the assemblage subtypes. Consistent softening of the faeces was only observed with assemblage B. Assemblage B next to assemblage subtype A(I) elicited relatively higher pathogenicity, characterized by more extensive damage to mucosal architecture, decreased brush-border enzyme function and infiltration of inflammatory cells. Assemblage E(III) and A(II) isolates showed relatively low virulence. The Giardia assemblage subtypes exhibit different levels of growth and virulence in the gerbil model.     

Prediction of normal values for lactate threshold estimated by gas exchange in men and women

Lactate threshold (LT) is an index of exercise capacity and can be estimated from the gas exchange consequences of a metabolic acidosis (LT_GE). In recent years, it has emerged as a diagnostic tool in the evaluation of subjects with exercise limitation. The purpose of this study was to develop LT_GE prediction equations on a relatively large sample of adults and to cross-validate each equation. A total of 204 healthy, sedentary, nonsmoking subjects (103 men and 101 women), aged 20–70 years, underwent graded exercise testing on a cycle ergometer. The V-slope technique was used to detect LT_GE as the oxygen uptake (V˙O₂) at the breakpoint of the carbon dioxide output versus V˙O₂ relationship. Multiple linear regression was used to develop 12 equations with combinations of the following predictor variables: age, height, body mass, and fat-free mass. Eight of the equations are gender-specific and four are generalized with gender as a dummy variable. The equations were cross-validated using the predicted residual sum of squares (PRESS) method. The results demonstrate that the equations had relatively high multiple correlations (0.577–0.863) and low standard errors of the estimate (0.123–0.228 1 · min⁻¹). The PRESS method demonstrated that the equations are generalizable, i.e., can be used in future studies without a significant loss of accuracy. Since we tested only healthy, sedentary subjects, our equations can be used to predict the lower limit of normal for a given subject. Using individual data for healthy and diseased subjects from the literature, we found that our gender-specific equations rarely miscategorized subjects unless they were obese and mass was a predictor variable. We conclude that our equations provide accurate predictions of normal values for LT_GE and that they are generalizable to other subject populations. Accepted: 13 February 1997     

Spectral and thermodynamic properties of Ag(I), Au(III), Cd(II), Co(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(IV), and Zn(II) binding by methanobactin from Methylosinus trichosporium OB3b

Methanobactin (mb) is a novel chromopeptide that appears to function as the extracellular component of a copper acquisition system in methanotrophic bacteria. To examine this potential physiological role, and to distinguish it from iron binding siderophores, the spectral (UV–visible absorption, circular dichroism, fluorescence, and X-ray photoelectron) and thermodynamic properties of metal binding by mb were examined. In the absence of Cu(II) or Cu(I), mb will bind Ag(I), Au(III), Co(II), Cd(II), Fe(III), Hg(II), Mn(II), Ni(II), Pb(II), U(VI), or Zn(II), but not Ba(II), Ca(II), La(II), Mg(II), and Sr(II). The results suggest metals such as Ag(I), Au(III), Hg(II), Pb(II) and possibly U(VI) are bound by a mechanism similar to Cu, whereas the coordination of Co(II), Cd(II), Fe(III), Mn(II), Ni(II) and Zn(II) by mb differs from Cu(II). Consistent with its role as a copper-binding compound or chalkophore, the binding constants of all the metals examined were less than those observed with Cu(II) and copper displaced other metals except Ag(I) and Au(III) bound to mb. However, the binding of different metals by mb suggests that methanotrophic activity also may play a role in either the solubilization or immobilization of many metals in situ.     

Two approaches to modeling kinetics of biodegradation by growing cells and application of a two-compartment model for mineralization kinetics in sewage

The patterns of microbial mineralization of 0.3 to 30 ng of glucose, benzoate, and phenol per ml of sewage collected in late fall and winter were analyzed with the integrated Monod equation and a model in which growth of active organisms occurs at the expense of organic compounds other than the test substrate. Either model could be closely fit by nonlinear regression to the data from individual tests with one concentration of substrate added to one dilution of sewage. However, neither model accounted satisfactorily for differences in patterns of mineralization resulting from differences in substrate concentration and cell density between different tests. It is suggested that both the added substrates and other organics present in sewage contributed to the growth of the active organisms. The mineralization of glucose in sewage collected in summer was better described by a two-compartment model than by any other model tested.