Different Structural Requirements for Melanin‐Concentrating Hormone (MCH) Interacting with Rat MCH‐R1 (SLC‐1) and Mouse B16 Cell MCH‐R

Abstract

Melanin‐concentrating hormone (MCH) is a neuropeptide occurring in all vertebrates and some invertebrates and is now known to stimulate pigment aggregation in teleost melanophores and food‐intake in mammals. Whereas the two MCH receptor subtypes hitherto cloned, MCH‐R₁ and MCH‐R₂, are thought to mediate mainly the central effects of MCH, the MCH‐R on pigment cells has not yet been identified, although in some studies MCH‐R₁ was reported to be expressed by human melanocytes and melanoma cells. Here we present data of a structure‐activity study in which 12 MCH peptides were tested on rat MCH‐R₁ and mouse B16 melanoma cell MCH‐R, by comparing receptor binding affinities and biological activities. For receptor binding analysis with HEK‐293 cells expressing rat MCH‐R₁ (SLC‐1), the radioligand was [¹²⁵I]–[Tyr¹³]‐MCH with the natural sequence. For B16 cells (F1 and G4F sublines) expressing B16 MCH‐R, the analog [¹²⁵I]–[D‐Phe¹³, Tyr¹⁹]‐MCH served as radioligand. The bioassay used for MCH‐R₁ was intracellular Ca²⁺ mobilization quantified with the FLIPR instrument, whereas for B16 MCH‐R the signal determined was MAP kinase activation. Our data show that some of the peptides displayed a similar relative increase or decrase of potency in both cell types tested. For example, linear MCH with Ser residues at positions 7 and 16 was almost inactive whereas a slight increase in side‐chain hydrophilicity at residues 4 and 8, or truncation of MCH at the N‐terminus by two residues hardly changed binding affinity or bioactivity. On the other hand, salmonic MCH which also lacks the first two residues of the mammalian sequence but in addition has different residues at positions 4, 5, 9, and 18 exhibited a 5‐ to 10‐fold lower binding activity than MCH in both cell systems. A striking difference in ligand recognition between MCH‐R₁ and B16 MCH‐R was however observed with modifications at position 13 of MCH: whereas L‐Phe¹³ in [Phe¹³, Tyr¹⁹]‐MCH was well tolerated by both MCH‐R₁ and B16 MCH‐R, change of configuration to D‐Phe¹³ in [D‐Phe¹³, Tyr¹⁹]‐MCH or [D‐Phe¹³]‐MCH led to a complete loss of biological activity and to a 5‐ to 10‐fold lower binding activity with MCH‐R₁. By contrast, the D‐Phe¹³ residue increased the affinity of [D‐Phe¹³, Tyr¹⁹]‐MCH to B16 MCH‐R about 10‐fold and elicited MAP kinase activation as observed with [Phe¹³, Tyr¹⁹]‐MCH or MCH. These data demonstrate that ligand recognition by B16 MCH‐R differs from that of MCH‐R₁ in several respects, indicating that the B16 MCH‐R represents an MCH‐R subtype different from MCH‐R₁.     

Regulation by cyclic adenosine monophosphate of functional activity of the adenylyl cyclase system in the infusorian <Emphasis Type="Italic">Dileptus anser</Emphasis>

In some unicellular eukaryotes, cAMP performs functions not only of the secondary messenger, but also of hormone, the primary messenger. We have found that cAMP is bound to surface receptors of the free-living infusorian Dileptus anser and stimulates activity of the adenylyl cyclase signaling system (AC-system) including heterotrimeric G-proteins and the enzyme, adenylyl cyclase (AC). The binding of cAMP to receptor is performed with a high affinity (K _D = 27 nM) and is highly specific, as cGMP and adenosine do not produce a marked effect on it. The infusorian cAMP-receptors have been shown to be coupled to G-proteins, which is indicated by a decrease of their affinity to the ligand in the presence of GTP, stimulation of the GTP-binding of G-proteins with the cyclic nucleotide, and block of the cAMP regulatory effects with suramin, an inhibitor of heterotrimeric G-proteins. cAMP stimulates dose-dependently the AC activity, its effect remaining virtually unchanged in the presence of cGMP, AMP, GMP, and adenosine. N⁶,O^2′-dibutyryl-cAMP, a non-hydrolyzed cAMP analogue, only at comparatively high concentrations competes with cAMP for binding sites and decreases the cAMP stimulating effects on the AC activity and GTP binding. Thus, we have shown for the first time that the AC system of the infusorians D. anser is stimulated by the extracellular cAMP that in this case functions as the external signal regulates activity of extracellular cAMP-dependent effector systems.     

Utilization of metabolic scope in relation to feeding and activity by individual and grouped zebrafish, Brachydanio rerio (Hamilton-Buchanan)

Metabolic scope and its utilization in relation to feeding and activity were measured in individual and grouped zebrafish (weight range, 430–551 mg) at 24° C by respirometry. Mean maximum metabolic rate, induced by swimming to exhaustion, R_max(i), was 1223 (s.d. , 157) mg O₂, kg^?1 h^?1 for individuals. Standard metabolic rate, R_s. was 364 mg O₂ kg^?1 h^?1, as estimated by extrapolating to zero activity from measurements of unfed, spontaneously active individuals. Mean routine metabolic rate, R_rout, of individuals was 421 (s.d. , 58) mg O₂, kg_-1 h_-1. The mean voluntary maximum metabolic rate, R_max(v), following transfer of minimally exercised fish to the respirometer, was 1110 (s.d. , 83) mg O₂ kg ^?1 h^?1 for groups of six fish, and was not significantly different from the value measured for individuals, 1066 (s.d. , 122) mg O₂, kg^?1 h^?1. Grouped fish acclimated to the respirometer more slowly than individual fish and exhibited significantly higher R_rout, apparently a result of greater social interaction and activity in groups. Mean R_rout for groups was 560 (s.d. , 78) mg O₂, kg^?1 h^?1. While groups of zebrafish fed a ration of 5% wet body weight day^?1 exhibited consistently higher metabolic rates than fish fed rations of 2.5% wet body weight day^?1 the high ration group still used only a maximum of 77% of the metabolic scope. Zebrafish of the size studied do not appear to demonstrate a high degree of conflict in utilization of metabolic scope by different respiratory components. The metabolic rates measured for zebrafish are among the highest yet measured for fish of similar size and at similar temperatures.     

Comparison of the structure of ribosomal 5S RNA from E. coli and from rat liver using X-ray scattering and dynamic light scattering

The structures of eukaryotic ribosomal 5S RNA from rat liver and of prokaryotic 5S RNA from E. coli (A-conformer) have been investigated by scattering methods. For both molecules, a molar mass of 44,500±4,000 was determined from small angle X-ray scattering as well as from dynamic light scattering. The shape parameters of the two rRNAs, volume V _c, surface O _c, radius of gyration R _s, maximum dimension of the molecule L, thickness D, and cross section radius of gyration R _sq, agree within the experimental error limits. The mean values are V _c=57±3 nm³, O _c=165±10 nm², R _s=3.37±0.05 nm, L=10.8±0.7 nm, D=1.57±0.07 nm, R _sa=0.92±0.01 nm.Identical structures for the E. coli 5S rRNA and the rat liver 5S rRNA at a resolution of 1 nm can be deduced from this agreement and from the comparison of experimental X-ray scattering curves and of experimental electron distance distribution function. The flat shape model derived for prokaryotic and eukaryotic 5S rRNA shows a compact region and two protruding arms. Double helical stems are eleven-fold helices with a mean base pair distance of 0.28 nm. Combining the shape information obtained from X-ray scattering with the information about the frictional behaviour of the molecules, deduced from the diffusion coefficients D _20,w ⁰ =(5.9±0.2)·10^-7 cm²s^-1 and (6.2±0.2)·10^-7 cm²s^-1 for rat liver 5S rRNA and E. coli 5S rRNA, respectively, a solvation shell of about 0.3 nm thickness around both molecules is determined. This structural similarity and the consensus secondary structure pattern derived from comparative sequence analyses suggest that all 5S rRNAs may indeed have conserved essentially the same type of folding of their polynucleotide strands during evolution, despite having very different sequences.     

Effect of Intracluster Correlation on the R-Square Statistic

R²-statistic is a popular and very widely used statistic in regression analysis to estimate the square multiple correlation (SMC), ρ², between a response variable Y and p predictor variables, _X1, …, _Xp. Numerous articles are available in the statistical literature on the properties of R² as an estimator of ρ² when the observations are uncorrelated. However, relatively little is known about the behavior of R² when the available observations are correlated such as the data that result from complex sampling schemes. In this paper, we study the behavior R² in the presence of two-stage sampling data. An approximate expressions for the variance and the bias of R² in the presence of two-stage cluster sampling data with positive intracluster correlation (ρ*) are obtained. It is evident from these formulas and from a simulation study that R² is a poor estimator of ρ² except when ρ* is small. As such, we consider several alternative estimators of ρ² and evaluate their theoretical properties and finite sample performance using a simulation study.     

2-Phenylhydroxypropynyladenosine Derivatives as High Potent Agonists at A2B Adenosine Receptor Subtype

Abstract

Adenosine derivatives bearing in 2-position the (R,S)- phenylhydroxypropynyl chain were evaluated for their potency at human A_2B adenosine receptor, stably transfected on CHO cells, on the basis that (R,S)-2-phenylhydroxy-propynyl-5′-N-ethylcarboxyamidoadenosine [(R,S)-PHPNECA] was found to be a good agonist at the A_2B receptor subtype. Biological studies demonstrated that the presence of small alkyl groups in N ⁶-position of these molecules are well tolerated, whereas large groups abolished A_2B potency. On the other hand, the presence of an ethyl group in the 4′-carboxamido function seems to be optimal, the (S)-PHPNECA resulting the most potent agonist at A_2B receptor reported so far.     

Biohydrogen production from carbon monoxide and water byRhodopseudomonas palustris P4

A reactor-scale hydrogen (H₂) productionvia the water-gas shift reaction of carbon monoxide (CO) and water was studied using the purple nonsulfur bacterium,Rhodopseudomonas palustris P4. The experiment was conducted in a two-step process: an aerobic/chemoheterotrophic cell growth step and a subsequent anaerobic H₂ production step. Important parameters investigated included the agitation speed, inlet CO concentration and gas retention time. P4 showed a stable H₂ production capability with a maximum activity of 41 mmol H₂ g cell⁻¹h⁻¹ during the continuous reactor operation of 400 h. The maximal volumetric H₂ production rate was estimated to be 41 mmol H₂ L¹h⁻¹, which was about nine-fold and fifteen-fold higher than the rates reported for the photosynthetic bacteriaRhodospirillum rubrum andRubrivivax gelatinosus, respectively. This is mainly attributed to the ability of P4 to grow to a high cell density with a high specific H₂ production activity. This study indicates that P4 has an outstanding potential for a continuous H₂ productionvia the water-gas shift reaction once a proper bioreactor system that provides a high rate of gas-liquid mass transfer is developed.     

Analysis of theD-region products ofH-2444using monoclonal antibodies reveals the expression of a new class I-like molecule

To analyze how many D-region-encoded molecules could be detected inH-2 ^q , we produced a panel of nine monoclonal antibodies from AKR (K^kD^k) anti-AKR.M (K^kD^q) immunizations. All of the D_q region antibodies cross-reacted on D^d and/or L^d, and all except one cross-reacted on D^b, confirming the previously observed serologic and amino acid sequence homology between theD-region products ofH-2 ^d ,H-2 ^b , andH-2 ^q . All of these monoclonal antibodies precipitated 46 000 dalton molecules from both cell-surface-labeled and biosynthetically labeled BIO.AKM spleen cells, indicating that all were reactive with class I-like molecules. Sequential immunoprecipitation analysis with one of these antibodies, 66-3-5, reveals the presence of a previously unidentified class I-like molecule. Tryptic peptide map analysis reveals that this molecule may be the product of a newly describedH-2D ^q -region gene.     

In vitro amplification of scrapie and chronic wasting disease PrPres using baculovirus-expressed recombinant PrP as substrate

Protein misfolding cyclic amplification (PMCA) is an in vitro simulation of prion replication, which relies on the use of normal brain homogenate derived from host species as substrate for the specific amplification of abnormal prion protein, PrP^Sc. Studies showed that recombinant cellular PrP, PrP^C, expressed in Escherichia coli lacks N-glycosylation and an glycophosphatidyl inositol anchor (GPI) and therefore may not be the most suitable substrate in seeded PMCA reactions to recapitulate prion conversion in vitro. In this study, we expressed 2 PRNP genotypes of sheep, V₁₃₆L₁₄₁R₁₅₄Q₁₇₁ and A₁₃₆F₁₄₁R₁₅₄Q₁₇₁, and one genotype of white-tailed deer (Q₉₅G₉₆, X₁₃₂,Y₂₁₆) using the baculovirus expression system and evaluated their suitability as substrates in seeded-PMCA. It has been reported that host-encoded mammalian RNA molecules and divalent cations play a role in the pathogenesis of prion diseases, and RNA molecules have also been shown to improve the sensitivity of PMCA assays. Therefore, we also assessed the effect of co-factors, such as prion-specific mRNA molecules and a divalent cation, manganese, on protein conversion. Here, we report that baculovirus-expressed recombinant PrP^C shows a glycoform and GPI-anchor profile similar to mammalian brain-derived PrP^C and supports amplification of PrP^Sc and PrP^CWD derived from prion-affected animals in a single round of seeded PMCA in the absence of exogenous co-factors. Addition of species-specific in vitro transcribed PrP mRNA molecules stimulated the conversion efficiency resulting in increased PrP^Sc or PrP^CWD production. Addition of 2 to 20 μM of manganese chloride (MnCl₂) to unseeded PMCA resulted in conversion of recombinant PrP^C to protease-resistant PrP. Collectively, we demonstrate, for the first time, that baculovirus expressed sheep and deer PrP can serve as a substrate in protein misfolding cyclic amplification for sheep and deer prions in the absence of additional exogenous co-factors.     

Electromyography of pongid shoulder muscles II. Deltoid, rhomboid and "rotator cuff"

Red cell antigen, serum protein and red cell enzyme groups were determined for a series of 1,821 individuals belonging to six language families in Western New Guinea. Three of the language families represent groupings of languages spoken by people in the swampy coastal plain of south central Western New Guinea, two belong to the Central Highlands and one to the Lake Plain area near the confluence of the Idenburg and Rouffaer Rivers. The distribution of genetic markers reveals similarities with other parts of New Guinea. The A₂ allele is absent in the ABO system, the frequency of Ns in the MNS system is very high as is the R₁ (CDe) allele in the Rh system. Hp¹ frequencies are high, and the transferrin allele Tf^D 1 is present as in other parts of New Guinea. In the red cell enzyme systems several alleles were detected which are characteristic of Papuan, and in some cases other Melanesian populations: these include MDH³, PGK⁴, PGK², PGM⁹₂, PGM¹⁰₂, as well as some very restricted alleles such as Peptidase B⁶ and Pep B². Three indices of genetic distance were computed. The most striking results are the genetic closeness of the Dani and Moni populations from the Central Highlands to the Asmat on the southern coastal plain, and the relative remoteness of the Awyu from the other south coastal populations. The results are discussed in terms of recent theories on the origin and dispersal of Papuan languages.     

Effect of soil water stress on soil respiration and its temperature sensitivity in an 18-year-old temperate Douglas-fir stand

We analyzed 17 months (August 2005 to December 2006) of continuous measurements of soil CO₂ efflux or soil respiration (R_S) in an 18‐year‐old west‐coast temperate Douglas‐fir stand that experienced somewhat greater than normal summertime water deficit. For soil water content at the 4 cm depth (θ) > 0.11 m³ m^?3 (corresponding to a soil water matric potential of ?2 MPa), R_S was positively correlated to soil temperature at the 2 cm depth (T_S). Below this value of θ, however, R_S was largely decoupled from T_S, and evapotranspiration, ecosystem respiration and gross primary productivity (GPP) began to decrease, dropping to about half of their maximum values when θ reached 0.07 m³ m^?3. Soil water deficit substantially reduced R_S sensitivity to temperature resulting in a Q₁₀ significantly < 2. The absolute temperature sensitivity of R_S (i.e. dR_S/dT_S) increased with θ up to 0.15 m³ m^?3, above which it slowly declined. The value of dR_S/dT_S was nearly 0 for θ < 0.08 m³ m^?3, thereby confirming that R_S was largely unaffected by temperature under soil water stress conditions. Despite the possible effects of seasonality of photosynthesis, root activity and litterfall on R_S, the observed decrease in its temperature sensitivity at low θ was consistent with the reduction in substrate availability due to a decrease in (a) microbial mobility, and diffusion of substrates and extracellular enzymes, and (b) the fraction of substrate that can react at high T_S, which is associated with low θ. We found that an exponential (van't Hoff type) model with Q₁₀ and R₁₀ dependent on only θ explained 92% of the variance in half‐hourly values of R_S, including the period with soil water stress conditions. We hypothesize that relating Q₁₀ and R₁₀ to θ not only accounted for the effects of T_S on R_S and its temperature sensitivity but also accounted for the seasonality of biotic (photosynthesis, root activity, and litterfall) and abiotic (soil moisture and temperature) controls and their interactions.