Approximating the Non-Null Distribution of the Likelihood Ratio Test Statistic

This paper is concerned with investigation into the behavior of the likelihood ratio test statistic G² when the alternative hypothesis M (QQ) is the true model. Exact moments of G² are computed empirically and three approximations are considered for approximating the non-null distribution of G². Our results show that the two parameter gamma distribution provides a closer approximation to the exact powers of G². A randomized procedure was employed to obtain critical values based on 1000 simulated samples.     

TheRT1.G locus in the rat encodes a Qa/TL-like antigen

A new antigenic system in the rat homologous to theQa/TL antigen system in the mouse has been characterized. It was detected by antibodies raised in donor-recipient combinations that were matched for theRT1. A, B, D, E loci in the major histocompatibility complex (MHC): (R11×BN)F₁ anti-BN.1L(LEW), (R18×BN)F₁ anti-BN.1L, and BN.1LV1(F344) anti-BN.1L. Absorption analyses using these antisera and a variety of inbred, congenic and recombinant strains identified three alleles,RT1.G ^a ,G ^b ,G ^c , of whichG ^c is a null allele. The strain distribution of these alleles was determined, using 37 strains of rats representative of all of the prototypic haplotypes and a number of congenic and recombinant strains. The use of the congenic and recombinant strains showed that theRT1.G locus was linked to the MHC and that the most probable gene order wasA-E-G. Testcross analysis showed that the map distance betweenA andG was 1.4 cM(4/285 recombinants). The RT1.G antigen has a heavy chain ofM _r 46 000 and is present on both T and B cells.     

Cyanide Inhibition of Chloride Conductance Across Toad Skin

The effect of cyanide (CN⁻) on voltage-activated or cAMP-induced passive chloride conductance (G ^Cl ) was analyzed in isolated toad skin. Comparatively low concentrations of CN⁻ inhibited G ^Cl almost completely and fully reversibly, regardless of whether it was applied from the mucosal or serosal side. The IC₅₀ was 180 ± 12 μm for voltage-activated G ^Cl and 305 ± 30 μm for the cAMP-inducted conductance. At [CN] <100 μm, the initial inhibition frequently declined partly in the continuous presence of CN⁻. Inhibition was independent of the presence of Ca²⁺. Inhibition was stronger at more alkaline pH, which suggests that dissociated CN⁻ is the effective inhibitor. The onset of the inhibition of voltage-activated or cAMP-induced G ^Cl by CN⁻ occurred with half-times of 34 ± 10 sec, whereas reversibility upon washout was twice as fast (18 ± 7 sec). If [CN⁻] <200 μm was applied under inactivating conditions (serosa −30 mV), the reduction of G ^Cl was stronger upon subsequent voltage-activation than under steady-state activated conditions. This effect was essentially complete less than 30 sec after apical addition of CN⁻, but G _t recovered thereafter partially in the continuous presence of CN⁻. Dinitrophenol inhibited G ^Cl similarly, while omission of oxygen did not affect it. These observations, as well as the time course of inhibition and the full reversibility, suggest that interference of CN⁻ with oxidative phosphorylation and subsequent metabolic depletion is not the reason for the inhibition of G ^Cl . We propose that the inhibition is directly on G ^Cl , presumably by competition with Cl⁻ at a rate-limiting site in the pathway. Location and molecular nature of this site remain to be identified. Received: 8 February 1999/Revised: 22 September 1999     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Hybrid Oligomer of Cyclonucleotides and Deoxynucleotides. A High Anti Left-handed Double Helical DNA Structure

Abstract

It has been shown by us that oligonucleotides containing cyclonucleosides with a high anti glycosidic conformation take left-handed, single and double helical structures (S. Uesugi, J. Yano, E. Yano and M. Ikehara, J. Am. Chem. Soc. 99, 2313 (1977) and references therein). In order to see whether DNA can adopt the high anti left-handed double helical structure or not, a self-complementary hexanucleotide containing 6,2′-O-cyclocytidine (C^○), 8,2′-O-cyclo- guanosine (G^○), deoxycytidine and deoxyguanosine, C^○G^{○dCdGC ○}G^○, was synthesized. Corresponding hexanucleotide containing only cyclonucleosides, C^○G^○C^○G^○C^○G^○, was also synthesized. Their conformation was examined by UV, CD and ¹H NMR spectroscopy. C^○G^○C^○G^○C^○G^○ forms an unusually stable, left-handed duplex. Imino proton NMR spectra and the results of nuclear Overhauser effect experiments strongly suggest that C^○G^○dCdGC^○G^○ take a left-handed double helical structure where the deoxynucleoside residues are involved in hydrogen bonding and take a high anti glycosidic conformation. Thus it is revealed that DNA could form a high anti, left-handed double helix which is different from that of Z-DNA under some constrained conditions.     

Proton/hydroxide conductance through lipid bilayer membranes

Summary A simple method of measuring proton/hydroxide conductance (G _H/OH) through planar lipid bilayer membranes is described. First the total conductance (G _m ) is measured electrically. Then the H⁺/OH^– transference number (T _H/OH) is estimated from the diffusion potential (V _m ) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H⁺ and OH^–. Thus,V _m is due entirely to H⁺/OH^– diffusion andG _H/OH can be calculated from the relations,V _m =T _H/OH E _H/OH andG _H/OH=T _H/OH G _m , whereE _H/OH is the equilibrium potential for H⁺ and OH^–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G _H/OH is nearly independent of pH, ranging from about 10^–9 S cm^–2 at pH 1.6 to 10^–8 S cm^–2 at pH 10.5. BecauseG _H/OH is nearly independent of pH, the calculated permeability coefficients to H⁺ and/or OH^– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G _H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG _H/OH.G _H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H⁺/OH^– conductance is not known, the relation betweenG _H/OH and water activity suggests that several water molecules are involved in the H⁺/OH^– transport process.     

Adenosine A1 Agonists and the Ca2+ Channel Agonist Bay K 8644 Produce a Synergistic Stimulation of the GTPase Activity of Go in Rat Frontal Cortical Membranes

Abstract: The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10^?5M, the increase above basal GTPase in frontal cortex was 25 ± 4, 20 ± 3, and 8 ± 1%, respectively, and in the cerebellum 55 ± 2, 41 ± 4, and 22 ± 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A₁ antagonists (76–96% reduction), (2) pretreatment with pertussis toxin (90–100% reduction), and (3) antibodies raised against the α-subunit of G_i and G_o (55–57% reduction by each), suggesting that A₁ receptors interact equally with G_i and G_o. (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of G_i or G_o. Previously, (±)-Bay K 8644 enhanced GTP hydrolysis by G_o but not G_i. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (±)-Bay K 8644 (10^?7 to 10^?5M). (±)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G_o. It appears that a positive cooperative stimulation of G_o occurs when it is first activated by A₁ receptors and subsequently interacts with the L-type Ca²⁺ channel.     

Caprine blood groups. 2. The C,G, H,I, J,K, L,N, and Q systems

Nine blood group systems of goats were identified using 12 caprine reagents produced by absorption of alloimmune antisera. The caprine C blood group system, possibly homologous to the ovine C blood group system, was characterized by two reagents and shown to be controlled by three alleles,C ¹²,C ²⁵, andC ⁻. A more complex blood group system of goats, designated G, was identified using three reagents and shown to be controlled by six codominant alleles (G ^10.19.20,G ^10.19,G ^10.20,G ¹⁰,G ¹⁹,G ²⁰) and a recessive allele (G ⁻). A further seven one-factor two-allelic systems were identified by seven reagents. The nine genetic systems provided exclusion probabilities of 0.479, 0.492, 0.548, and 0.572 in Australian Angora, Dairy, Cashmere, and Texan Angora goat breeds, respectively. This work was supported by a grant from the Australian Stud Book, Alison Road, Randwick, New South Wales 2031, Australia.     

1H-NMR study of GM2 ganglioside: evidence that an interresidue amide-carboxyl hydrogen bond contributes to stabilization of a preferred conformation

Several properties of the exchangeable amide protons of the ganglioside G_M2 were studied in detail by¹H-NMR spectroscopy in fully deuterated dimethylsulfoxide [²H₆]DMSO/2% H₂O, and compared with data obtained for the simpler constituent glycosphingolipids G_A2 and G_M3. In addition to chemical shifts,³ J _2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/²H chemical exchange were examined by following the disappearance of the amide resonances in [²H₆]DMSO/2%²H₂O. The results included observation of an increase in half-life of theN-acetylgalactosamine acetamido HN by more than an order of magnitude in G_M2 compared to G_A2, attributable to the presence of the additionalN-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of G_M2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the -acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesanet al. (1984,Can J Chem 62: 1034–45), and the models of G_M1 proposed more recently by Acquottiet al. (1990,J Am Chem Soc 112:7772–8) and Scarsdaleet al. (1990,Biochemistry 29:9843–55).     

Two Different Conductances Contribute to the Anion Currents in Coffea arabica Protoplasts

The anion conductance of the plasma membrane of Coffea arabica protoplasts was isolated and characterized using the whole-cell patch clamp technique. Voltage pulse protocols revealed two components: a voltage-gated conductance (G _s ) and a voltage-independent one (G _l ). G _s is activated upon depolarization (e-fold activation every +36 mV) with time constants of 1 sec and 5 sec at all potentials. G _l and G _s also differ by their kinetic and biophysical properties. In bi-ionic conditions the current associated with G _s shows strong outward rectification and its permeability sequence is F⁻ > NO₃ ⁻ > Cl⁻. In the same conditions the current associated with G _l does not rectify and its permeability sequence is F⁻≫ NO₃ ⁻= Cl⁻. Furthermore, at potentials over +50 mV G _s , but not G _l , increases with a time constant of several minutes. Finally the gating of G _s is affected by stretch of the membrane, which leads to an increased activation and a reduced voltage sensitivity. Anion conductances similar to the ones described here have been found in many plant preparations but G _l -type components have been generally interpreted as the background activation of the slow voltage-gated channels (corresponding to G _s ). We show that in coffee protoplasts G _l and G _s are kinetically and biophysically distinct, suggesting that they correspond to two different molecular entities. Received: 25 November 1996/Revised: 9 April 1997     

Dynamic light-scattering studies of internal motions in DNA. I. Applicability of the rouse-zimm model

The intermediate scattering function G(K,t) for any polymer model obeying a linear separable Langevin equation can be expressed in terms of the eigenvalues and eigenvectors of its normal coordinate transformation. An algorithm for the extract numerical evaluation of G(K,t) for linear Rouse-Zimm chains in the presence of hydrodynamic interaction has been developed. The computed G(K,t)² were fit to C(t) = A exp(?t/τA) + B, and apparent diffusion coefficients calculated according to D_app ≡ 1/(2τ_AK²). G(K,t)² was surprisingly well-fit by single-exponential decays, especially at both small and large values of Kb, where K is the scattering vector and b the root-mean-squared subunit extension. Plots of D_app vs K² in-variably showed a sigmoidal rise from D₀ at K² = O up to a constant plateau value at large K²b². Analytical expression for G(K,t), exact in the limit of short times, were obtained for circular Rouse-Zimm chains with and without hydrodynamic interaction, and also for free-draining linear chains, and in addition for the independent-segment-mean-force (ISMF) model. The predicted behaviors for G(K,t) at large Kb (or KR_G) was found in all cases to be single-exponential with 1/τ ∝ K² at large Kb, in agreement with the computational results. A simple procedure for estamating all parameter of the Rouse-Zimm model from a plot of D_app vs K² is proposed. Experimental data for both native and pH-denatured calf-thymus DNA in 1.0M Nacl with and without EDTA clearly plateau behavior of D_app at large values of K, in harmony with the present Rouse-Zimm and ISMF theories, and in sharp contrast to previous predictions based on the Rouse-Zimm model.     

METHYLATED AMINO ACID RESIDUES OF PROTEINS OF BRAIN AND OTHER ORGANS

—Methods for the determination of methyl-lysine, methyllarginine and methylhistidine residues of tissue proteins are described. They consist of preliminary purification of basic amino acids, enzymic removal of lysine, arginine and histidine followed by amino acid analysis. Recovery rates and specificities of the method were satisfactory. The contents of methylamino acids in proteins of mammalian organs were determined. The distribution of proteins containing the methylamino acids in human brain showed that the concentrations of methyl-lysine and N^G,N′^G-dimethylarginine were highest in the gray matter of the cerebellar cortex and relatively high in regions rich in gray matter, while those of N^G-mono- and N^G,N′^G-dimethylarginine were highest in the white matter. The following findings suggest that most of the N^G-mono- and N^G,N′^G-dimethylarginine was associated with the myelin basic protein. The distribution of the methylarginine residues of acid-soluble proteins in bovine brains coincided with the cerebroside pattern. The concentrations of the amino acids in acid-soluble proteins of rat brain increased concomitantly with the increase of cerebroside. The methylamino acid content in proteins increased during the purification of the myelin basic protein from the white matter of human and bovine brains. Proteins containing N^G,N^G-dimethyiarginine and di- and trimethyl-lysine are concentrated in cell nuclei. The first amino acid was found mainly in nucleoplasmic proteins and the other two were found in histones. The concentration of 3-methylhistidine residue, highest in muscular proteins, is low in cerebral proteins and is probably derived from proteins of walls of blood vessels in the brain.     

The γ-irradiation of aqueous solutions of urea. Implications for chemical evolution

0.05 mole dm^–3, O₂-free aqueous solutions of urea were studies after receiving various doses of⁶⁰Co gamma rays (0.14–600 kGy). Urea was found to be relatively stable under radiation; its radiation chemical yield of decomposition was 0.47. Hydrogen (G=0.50), carbon dioxide (G=0.44), ammonia (G=0.22), oxalic acid (G=0.0054), malonic acid (G=0.000064) and three unidentified oligomers were found to be the main radiolytic products. The origin of these products is explained by free radical reactions initiated by the transients from water radiolysis (H·,·OH,e _aq ^– ).     

Proton conductance through phospholipid bilayers: Water wires or weak acids?

The proton/hydroxide (H⁺/OH^–) permeability of phospholipid bilayer membranes at neutral pH is at least five orders of magnitude higher than the alkali or halide ion permeability, but the mechanism(s) of H⁺/OH^– transport are unknown. This review describes the characteristics of H⁺/OH^– permeability and conductance through several types of planar phospholipid bilayer membranes. At pH7, the H⁺/OH^– conductances (G _H/OH) range from 2–6 nS cm^–2, corresponding to net H⁺/OH^– permeabilities of (0.4–1.7)×10^–5 cm sec^–1. Inhibitors ofG _H/OH include serum albumin, phloretin, glycerol, and low pH. Enhancers ofG _H/OH include chlorodecane, fatty acids, gramicidin, and voltages >80 mV. Water permeability andG _H/OH are not correlated. The characteristics ofG _H/OH in fatty acid (weak acid) containing membranes are qualitatively similar to the controls in at least eight different respects. The characteristics ofG _H/OH in gramicidin (water wire) containing membranes are qualitatively different from the controls in at least four different respects. Thus, the simplest explanation for the data is thatG _H/OH in unmodified bilayers is due primarily to weakly acidic contaminants which act as proton carriers at physiological pH. However, at low pH or in the presence of inhibitors, a residualG _H/OH remains which may be due to water wires, hydrated defects, or other mechanisms.     

Note on a combinatorial problem

In the first part of this paper we have assembled some properties of the quantitiesR _m ⁿ , whereR _m ⁿ denotes the number of distributions ofn different objects intom indifferent parcels, with no empty parcels allowed. We then discuss the following problem (N. Rashevsky, 1954, 1955 a,b, 1956): to find the total number,G _n , of graphs that can be obtained from the biotopological transformation (T ⁽¹⁾ X) for a given value of the parametern. This is related to the distribution ofn indifferent objects intom different boxes. A formula forG _n is given which, however, is not very convenient for practical computations because it involves a summation over certain “admissible partitions” of the numbermn (m is a second parameter of the transformation). Some theorems are derived; with their help we can simplify the calculation ofG _n to a small extent. The numbersG _n are calculated forn≤9 and estimated forn=10. It is found thatG ₇≈5.4×10⁴,G ₈≈8.3×10⁵,G ₉≈1.4×10⁷, andG ₁₀≈3×10⁸. These values ofn are those which might be used in connection with N. Rashevsky’s work (cf. Rashevsky, 1956).     

Interaction of gangliosides with plasma low density lipoproteins

The role of gangliosides in the reception of low density lipoproteins (LDL) was studied using as targets mouse ascites hepatoma 22a (MAH) cells which bind LDL through a specific high affinity receptor. Low density lipoprotein binding and uptake by MAH cells decreased after brief treatment of the cells with neuraminidase to partially remove surface sialic acid residues. The LDL uptake capability of the neuraminidasetreated MAH cells was fully restored after incorporation of exogeneous G_M1- and G_D1a-gangliosides into the cell surface. In contrast, free (extracellular) gangliosides inhibited LDL uptake by native MAH cells. This inhibitory effect was seen at ganglioside concentrations corresponding to the ganglioside content of serum and was most pronounced with gangliosides whose sialic acids were linked to a terminal galactose residue (G_M3, G_D1a, G_T1b) but was smaller or absent with gangliosides whose sialic acids were attached to an internal galactose (G_M1, G_M2). The binding of gangliosides to LDL was structure and concentration dependent, saturable and trypsin sensitive. The LDL-ganglioside interaction was further investigated by steady state fluorescence spectroscopy. Changes in the LDL fluorescence polarization were observed with as little as 0.01 M concentrations of the gangliosides. The magnitude and nature of the effect depended on the type of ganglioside. We conclude that the LDL surface possesses sites recognizing specific carbohydrate sequences of glycoconjugates and that changes in the cell surface concentrations of sialic acids significantly modulate the LDL uptake. It is postulated that shedding of gangliosides into the blood stream may be a factor involved in regulation of cholesterol homeostasis.Abbreviations MAH mouse ascites hepatoma 22a - LDL low density lipoprotein - ASM anthrylvinyl-labeled sphingomyelin [N-12-(9-anthryl-trans-dodecanoyl-sphingosine-1-phosphocholine] - RITC rhodamine isothiocyanate. The designation of gangliosides follows the IUPAC-IUB recommendation [1]: G_M3, II³NeuAc-LacCer, II³-N-acetylneuraminosyllactosylceramide - G_M2 II³-NeuAc-GgOse₃Cer, II³-N-acetylneuraminosylgangliotriaosylceramide - G_M1 II³-NeuAc-GgOse₄Cer, II³-N-acetylneuraminosylgangliotetraosylceramide - G_D1a, II³ IV³(NeuAc)₂-GgOse₄Cer, II³, IV³-di(N-acetylneuraminosyl)gangliotetraosylceramide - G_T1b II³(NeuAc)₂, IV³ NeuAc-GgOse₄Cer, II³-di-N-acetylneuraminosyl, IV³-N-acetylneuraminosylgangliotetraosylceramide     

The Xmn1 polymorphic site 5′ to the Gγ gene and its correlation to the Gγ:Aγ ratio, age at first blood transfusion and clinical features in β-Thalassemia patients from Western Iran

β-Thalassemia is the most common single gene disorder in Iran and more than 25,000 affected individuals have been reported. It has been reported that in patients with β-thalassemia in the presence of Xmn1 polymorphic site the level of Hb F and ^Gγ: ^Aγ ratio is increased. The prevalence of Xmn1 polymorphic site, ^Gγ: ^Aγ ratio and Hb F in 197 β-thalassemia major patients from the Kermanshah Province of Iran were studied. The Xmn1 polymorphic site was determined by PCR-RFLP procedure. The levels of ^Gγ and ^Aγ chains were detected by HPLC. The percent of Hb F was determined using electrophoresis method. In β-thalassemia major patients the frequency of presence Xmn1 was 0.39. The mean of ^Gγ: ^Aγ ratio was found to be 2.5. In the present study it was found that in the presence of Xmn1 polymorphic site ^Gγ percent and ^Gγ: ^Aγ ratio were significantly increased (P = 0.01) and the clinical features such as splenomegaly and bone marrow expansion were significantly improved (P = 0.01). We found that in the presence of Xmn1 polymorphic site on both chromosomes (+/+) the level of Hb F tended to be increased compared to the absence of Xmn1 (−/−). The present investigation has studied the frequency of Xmn1 polymorphic site in β-thalassemia major patients from Western Iran and has revealed that the presence of this polymorphic site caused a positive influence on Hb F production and the ^Gγ percent which could improve the clinical symptoms of β-thalassemia patients.