Functional implications of the human T-lymphotropic virus type 1 transmembrane glycoprotein helical hairpin structure

Retrovirus entry into cells follows receptor binding by the surface exposed envelope glycoprotein (Env) subunit (SU), which triggers the membrane fusion activity of the transmembrane (TM) protein. TM protein fragments expressed in the absence of SU adopt helical hairpin structures comprising a central coiled coil, a region of chain reversal containing a disulfide-bonded loop, and a C-terminal segment that packs onto the exterior of the coiled coil in an antiparallel manner. Here we used in vitro mutagenesis to test the functional role of structural elements observed in a model helical hairpin, gp21 of human T-lymphotropic virus type 1. Membrane fusion activity requires the stabilization of the N and C termini of the central coiled coil by a hydrophobic N cap and a small hydrophobic core, respectively. A conserved Gly-Gly hinge motif preceding the disulfide-bonded loop, a salt bridge that stabilizes the chain reversal region, and interactions between the C-terminal segment and the coiled coil are also critical for fusion activity. Our data support a model whereby the chain reversal region transmits a conformational signal from receptor-bound SU to induce the fusion-activated helical hairpin conformation of the TM protein.

Characterization of an ATP-dependent pathway of activation for the heterocyclic amine carcinogen N-hydroxy-2-amino-3-methylimidazo[4,5-f]quinoline

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is one of several mutagenic and carcinogenic heterocyclic amines formed during the cooking process of protein-rich foods, These compounds are highly mutagenic and have been shown to produce tumours in various tissues in rodents and non-human primates. Metabolic activation of IQ is a two-step process involving N-hydroxylation by CYP1A2 followed by esterification to a more reactive species capable of forming adducts with DNA, To date, acetylation and sulphation have been proposed as important pathways in the formation of N-hydroxy esters, In this study we have demonstrated the presence of an ATP-dependent activation pathway for N-hydroxy-IQ (N-OH-IQ) leading to DNA adduct formation measured by covalent binding of [H-3]N-OH-IQ to DNA, ATP-dependent DNA binding of N-OH-IQ was greatest in the cytosolic fraction of rat liver, although significant activity was also seen in colon, pancreas and lung. ATP was able to activate N-OH-IQ almost 10 times faster than N-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (7.7 +/- 0.3 and 0.9 +/- 0.1 pmol/mg protein/min, respectively). Using reported intracellular concentrations of cofactor, the ability of ATP to support DNA binding was similar to that seen with 3'-phosphoadenosine 5'-phosphosulphate and similar to 50% of that seen with acetyl coenzyme A (AcCoA), In addition to DNA binding, HPLC analysis of the reaction mixtures using ATP as co-factor showed the presence of two stable, polar metabolites, With AcCoA, only one metabolite was seen. The kinase inhibitors genistein, tyrphostin A25 and rottlerin significantly inhibited both DNA binding and metabolite formation with ATP. However, inhibition was unlikely to be due to effects on enzyme activity since the broad spectrum kinase inhibitor staurosporine had no effect and the inactive analogue of genistein, daidzein, was as potent as genistein, The effects of genistein and daidzein, which are naturally occurring isoflavones from soy and other food products, on DNA adduct formation may potentially be useful in the prevention of heterocyclic amine-induced carcinogenesis.

Ni-II and Zn-II complexes of the hexadentate macrocyclic ligand cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetra- decane-6,13-diamine

The title pendent-arm macrocyclic hexaamine ligand binds stereospecifically in a hexadentate manner, and we report here its isomorphous Ni-II and Zn-II complexes (both as perchlorate salts), namely (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)nickel(II) diperchlorate, [Ni(C12H30N6)](ClO4)(2), and (cis-6,13-dimethyl-1,4,8,11-tetraazacyclotetradecane-6,13-diamine-kappa(6)N)zinc(II) diperchlorate, [Zn(C-12 H30N6)](ClO4)(2). Distortion of the N-M-N valence angles from their ideal octahedral values becomes more pronounced with increasing metal-ion size and the present results are compared with other structures of this ligand.

The line structure of balanced ternary designs with block size three, index three and rho(2) = 1, 2

In this paper necessary and sufficient conditions for a vector to be the fine structure of a balanced ternary design with block size 3, index 3 and rho(2) = 1 and 2 are determined with one unresolved case.

U-q[(sl(2)over-cap vertical bar 1)] vertex operators, screen currents, and correlation functions at an arbitrary level

Bosonized q-vertex operators related to the four-dimensional evaluation modules of the quantum affine superalgebra U-q[sl((2) over cap\1)] are constructed for arbitrary level k=alpha, where alpha not equal 0,-1 is a complex parameter appearing in the four-dimensional evaluation representations. They are intertwiners among the level-alpha highest weight Fock-Wakimoto modules. Screen currents which commute with the action of U-q[sl((2) over cap/1)] up to total differences are presented. Integral formulas for N-point functions of type I and type II q-vertex operators are proposed. (C) 2000 American Institute of Physics. [S0022-2488(00)00608-3].

Level-one highest weight representation of U-q[sl((N)over-cap vertical bar 1)] and Bosonization of the multicomponent Super t-J model

We study the level-one irreducible highest weight representations of the quantum affine superalgebra U-q[sl((N) over cap\1)], and calculate their characters and supercharacters. We obtain bosonized q-vertex operators acting on the irreducible U-q[sl((N) over cap\1)] modules and derive the exchange relations satisfied by the vertex operators. We give the bosonization of the multicomponent super t-J model by using the bosonized vertex operators. (C) 2000 American Institute of Physics. [S0022- 2488(00)00508-9].

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa - 1. Isolation, characterization and chemical synthesis

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transl-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMR solution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide A beta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native A beta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length A beta peptides A beta(1-40) and A beta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which A beta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of A beta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation. (C) 2000 Academic Press.

Chronic ethanol has region-selective effects on Egr-1 and Egr-3 DNA-binding activity and protein expression in the rat brain

This study focused on the DNA-binding activity and protein expression of the transcription factors Egr-1 and Egr-3 in the rat brain cortex and hippocampus after chronic or acute ethanol exposure. DNA-binding activity was reduced in both regions after chronic ethanol exposure and was restored to the level of the pair-fed group at 16 h of withdrawal. Cortical Egr-1 protein levels were not altered by chronic ethanol exposure but increased 16 h after withdrawal, thus mirroring DNA-binding activity. In contrast, Egr-3 protein levels did not undergo any change. There was no change in the level of either protein in the hippocampus. Immunohistochemistry revealed a region-selective change in immunopositive cells in the cortex and hippocampus. Finally, an acute bolus dose of ethanol did not affect Egr DNA-binding activity and ethanol treatment did not alter the DNA-binding activity or protein levels of the transcription factor Spl. These observations suggest that chronic exposure to ethanol has region-selective effects on the DNA-binding activity and protein expression of Egr-1 and Egr-3 transcription factors in the rat brain. These changes occur after prolonged ethanol exposure and may thus reflect neuroadaptive changes associated with physical dependency and withdrawal. These effects are also transcription factor-selective. Clearly, protein expression is not the sole mediator of the changes in DNA-binding activity and chronic ethanol exposure must have effects on modulatory agents of Egr DNA-binding activity. (C) 2000 Elsevier Science Ltd, All rights reserved.

Both beta(2)- and beta(1)-adrenergic receptors mediate hastened relaxation and phosphorylation of phospholamban and troponin I in ventricular myocardium of fallot infants, consistent with selective coupling of beta(2)-adrenergic receptors to G(s)-protein

Background-In adult human heart, both beta(1)- and beta(2)-adrenergic receptors mediate hastening of relaxation; however, it is unknown whether this also occurs in infant heart. We compared the effects of stimulation of beta(1)- and beta(2)-adrenergic receptors on relaxation and phosphorylation of phospholamban and troponin I in ventricle obtained from infants with tetralogy of Fallot. Methods and Results-Myocardium dissected from the right ventricular outflow tract of 27 infants (age range 2-1/2 to 35 months) with tetralogy of Fallot was set up to contract 60 times per minute. Selective stimulation of beta(1)-adrenergic receptors with (-)-norepinephrine (NE) and beta(2)-adrenergic receptors with (-)-epinephrine (EPI) evoked phosphorylation of phospholamban (at serine-16 and threonine-17) and troponin I and caused concentration-dependent increases in contractile force (-log EC50 [mol/L] NE 5.5+/-0.1, n=12; -EPI 5.6+/-0.1, n=13 patients), hastening of the time to reach peak force (-log EC50 [mol/L] NE 5.8+/--0.2; EPI 5.8+/-0.2) and 50% relaxation (-log EC50 [mol/L] NE 5.7+/-0.2: EPI 5.8+/-0.1), Ventricular membranes from Fallot infants, labeled with (-)-[I-125]-cyanopindolol, revealed a greater percentage of beta(1)- (71%) than beta(2)-adrenergic receptors (29%). Binding of (-)-epinephrine to beta(2)-receptors underwent greater GTP shifts than binding of (-)-norepinephrine to beta(1)-receptors. Conclusions-Despite their low density, beta(2)-adrenergic receptors are nearly as effective as beta(1)-adrenergic receptors of infant Fallot ventricle in enhancing contraction, relaxation, and phosphorylation of phospholamban and troponin I, consistent with selective coupling to G(s)-protein.

Magnetic resonance imaging (MRI) and diseases of the liver and biliary tract. Part 1. Basic principles, MRI in the assessment of diffuse and focal hepatic disease

Magnetic resonance imaging (MRI) relies on the physical properties of unpaired protons in tissues to generate images. Unpaired protons behave like tiny bar magnets and will align themselves in a magnetic field. Radiofrequency pulses will excite these aligned protons to higher energy states. As they return to their original state, they will release this energy as radio waves. The frequency of the radio waves depends on the local magnetic field and by varying this over a subject, it is possible to build the images we are familiar with. In general, MRI has not been sufficiently sensitive or specific in the assessment of diffuse liver disease for clinical use. However, because of the specific characteristics of fat and iron, it may be useful in the assessment of hepatic steatosis and iron overload. Magnetic resonance imaging is useful in the assessment of focal liver disease, particularly in conjunction with contrast agents. Haemangiomas have a characteristic bright appearance on T-2 weighted images because of the slow flowing blood in dilated sinusoids. Focal nodular hyperplasia (FNH) has a homogenous appearance, and enhances early in the arterial phase after gadolinium injection, while the central scar typically enhances late. Hepatic adenomas have a more heterogenous appearance and also enhance in the arterial phase, but less briskly than FNH. Hepatocellular carcinoma is similar to an adenoma, but typically occurs in a cirrhotic liver and has earlier washout of contrast. The appearance of metastases depends on the underlying primary malignancy. Overall, MRI appears more sensitive and specific than computed tomography with contrast for the detection and evaluation of malignant lesions. (C) 2000 Blackwell Science Asia Pty Ltd.

Halogenated terpenoids. XXXII - Pentabromides from limonene two 1,2,8,9,9-pentabromo-p-menthanes

The problems of structure in diastereomers where one chiral centre is remote from another are further investigated in the 1,2,8,9,9-pentabromo-p-menthane series.

An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes

Arylamine N-acetyltransferase (NAT) was first identified as the inactivator of the anti-tubercular drug isoniazid, The enzyme was shown to catalyse the transfer of an acetyl group from acetyl-CoA to the terminal nitrogen of the hydrazine drug. The rate of inactivation of isoniazid was polymorphically distributed in the population and was one of the first examples of pharmacogenetic variation, NAT was identified recently in Mycobacterium tuberculosis and is a candidate for; modulating the response to isoniazid, Genome sequences have revealed many homologous members of this unique family of enzymes. The first three-dimensional structure of a member of the NAT family identifies a catalytic triad consisting of aspartate, histidine and cysteine proposed to form the activation mechanism. So far, all procaryotic NATs resemble the human enzyme which acetylates isoniazid (NAT2), Human NAT2 is characteristic of drug-metabolizing enzymes: it is found in liver and intestine, In humans and other mammals, there are up to three different isoenzymes. If only one isoenzyme is present, it is like human NAT1. Human NAT1 and its murine equivalent specifically acetylate the folate catabolite p-amino-benzoylglutamate. NAT1 and its murine homologue each have a ubiquitous tissue distribution and are expressed early in development at the blastocyst stage, During murine embryonic development, NAT is expressed in the developing neural tube. The proposed endogenous role of NAT in folate metabolism, and its multi-allelic nature, indicate that its role in development should be assessed further.

Three-dimensional structure of RK-1: A novel alpha-defensin peptide

NMR spectroscopy and simulated annealing calculations have been used to determine the three-dimensional structure of RK-1, an antimicrobial peptide from rabbit kidney recently discovered from homology screening based on the distinctive physicochemical properties of the corticostatins/defensins. RK-1 consists of 32 residues, including six cysteines arranged into three disulfide bonds. It exhibits antimicrobial activity against Escherichia coli and activates Ca2+ channels in vitro. Through its physicochemical similarity, identical cysteine spacing, and linkage to the corticostatins/defensins, it was presumed to be a member of this family. However, RK-1 lacks both a large number of arginines in the primary sequence and a high overall positive charge, which are characteristic of this family of peptides. The three-dimensional solution structure, determined by NMR, consists of a triple-stranded antiparallel beta -sheet and a series of turns and is similar to the known structures of other alpha -defensins. This has enabled the definitive classification of RK-1 as a member of this family of antimicrobial peptides. Ultracentrifuge measurements confirmed that like rabbit neutrophil defensins, RK-1 is monomeric in solution, in contrast to human neutrophil defensins, which are dimeric.