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At least 379 records · Page 21Linked to original sources
PubMed2000

Observation of anti-stokes fluorescence cooling in thulium-doped glass

We report the first observation of anti-Stokes fluorescence cooling in a thulium-doped solid with pump excitation at 1.82 &mgr;m<lambda<1.97 &mgr;m. At a pump wavelength of 1.9 &mgr;m and incident average power of approximately 3 W, a Tm3+:ZBLANP ( ZrF4-BaF2-LaF3-AlF3-NaF-PbF2) sample cooled to -1.2 degrees C from room temperature for a single pass of the pump beam. This corresponds to an absorbed pump power of approximately 40 mW and a peak temperature change per absorbed power of approximately -30 degrees C/W from room temperature.

Journal Article↗
PubMed2005

Molecular mechanisms of constitutive activity: mutations at position 111 of the angiotensin AT1 receptor.

A possible molecular mechanism for the constitutive activity of mutants of the angiotensin type 1 receptor (AT1) at position 111 was suggested by molecular modeling. This involves a cascade of conformational changes in spatial positions of side chains along transmembrane helix (TM3) from L112 to Y113 to F117, which in turn, results in conformational changes in TM4 (residues I152 and M155) leading to the movement of TM4 as a whole. The mechanism is consistent with the available data of site-directed mutagenesis, as well as with correct predictions of constitutive activity of mutants L112F and L112C. It was also predicted that the double mutant N111G/L112A might possess basal constitutive activity comparable with that of the N111G mutant, whereas the double mutants N111G/Y113A, N111G/F117A, and N111G/I152A would have lower levels of basal activity. Experimental studies of the above double mutants showed significant constitutive activity of N111G/L112A and N111G/F117A. The basal activity of N111G/I152A was higher than expected, and that of N111G/Y113A was not determined due to poor expression of the mutant. The proposed mechanism of constitutive activity of the AT(1) receptor reveals a novel nonsimplistic view on the general problem of constitutive activity, and clearly demonstrates the inherent complexity of the process of G protein-coupled receptor (GPCR) activation.

Amino Acid Sequence↗
PubMed2006

Effects of anesthetics on mutant N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Alcohols, inhaled anesthetics, and some injectable anesthetics inhibit the function of N-methyl-d-aspartate (NMDA) receptors, but the mechanisms responsible for this inhibition are not fully understood. Recently, it was shown that ethanol inhibition of NMDA receptors was reduced by mutation of residues in the transmembrane (TM) segment 3 of the NR1 subunit (F639A) or in TM4 of the NR2A subunit (A825W), suggesting putative ethanol binding sites. We hypothesized that the actions of other anesthetics might also require these amino acids and evaluated the effects of anesthetics on the NMDA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Effects of hexanol, octanol, isoflurane, halothane, chloroform, cyclopropane, 1-chloro-1,2,2-trifluorocyclobutane, and xenon were reduced or eliminated in the mutant NMDA receptors, whereas the inhibitory effects of nitrous oxide, ketamine, and benzene were not affected by these mutations. Rapid applications of glutamate and glycine by a T-tube device provided activation time constants, which suggested different properties of ketamine and isoflurane inhibition. Thus, amino acids in TM3 and TM4 are important for the actions of many anesthetics, but nitrous oxide, benzene, and ketamine seem to have distinct mechanisms for inhibition of the NMDA receptors.

Amino Acid Substitution↗
PubMed2004

A three-dimensional model for the substrate binding domain of the multidrug ATP binding cassette transporter LmrA.

Multidrug resistance presents a major obstacle to the treatment of infectious diseases and cancer. LmrA, a bacterial ATP-dependent multidrug transporter, mediates efflux of hydrophobic cationic substrates, including antibiotics. The substrate-binding domain of LmrA was identified by using photo-affinity ligands, proteolytic degradation of LmrA, and identification of ligand-modified peptide fragments with matrix-assisted laser desorption ionization/time of flight mass spectrometry. In the nonenergized state, labeling occurred in the alpha-helical transmembrane segments (TM) 3, 5 and 6 of the membrane-spanning domain. Upon nucleotide binding, the accessibility of TM5 for substrates increased, whereas that of TM6 decreased. Inverse changes were observed upon ATP-hydrolysis. An atomic-detail model of dimeric LmrA was generated based on the template structure of the homologous transporter MsbA from Vibrio cholerae, allowing a three-dimensional visualization of the substrate-binding domain. Labeling of TM3 of one monomer occurred in a predicted area of contact with TM5 or TM6 of the opposite monomer, indicating substrate-binding at the monomer/monomer interface. Inverse changes in the reactivity of TM segments 5 and 6 suggest that substrate binding and release involves a repositioning of these helices during the catalytic cycle.

ATP-Binding Cassette Transporters↗
PubMed1998

Propofol and other intravenous anesthetics have sites of action on the gamma-aminobutyric acid type A receptor distinct from that for isoflurane.

Both volatile and intravenous general anesthetics allosterically enhance gamma-aminobutyric acid (GABA)-evoked chloride currents at the GABA type A (GABAA) receptor. Recent work has revealed that two specific amino acid residues within transmembrane domain (TM)2 and TM3 are necessary for positive modulation of GABAA and glycine receptors by the volatile anesthetic enflurane. We now report that mutation of these residues within either GABAA alpha2 (S270 or A291) or beta1 (S265 or M286) subunits resulted in receptors that retain normal or near-normal gating by GABA but are insensitive to clinically relevant concentrations of another inhaled anesthetic, isoflurane. To determine whether receptor modulation by intravenous general anesthetics also was affected by these point mutations, we examined the effects of propofol, etomidate, the barbiturate methohexital, and the steroid alphaxalone on wild-type and mutant GABAA receptors expressed in human embryonic kidney 293 cells. In most cases, these mutations had little or no effect on the actions of these intravenous anesthetics. However, a point mutation in the beta1 subunit (M286W) abolished potentiation of GABA by propofol but did not alter direct activation of the receptor by high concentrations of propofol. These data indicate that the receptor structural requirements for positive modulation by volatile and intravenous general anesthetics may be quite distinct.

Anesthetics, Inhalation↗
PubMed1998

Chimeric melanocortin MC1 and MC3 receptors: identification of domains participating in binding of melanocyte-stimulating hormone peptides.

The melanocortin receptors MC1 and MC3 are G protein-coupled receptors that have substantial structural similarities and bind melanocyte peptides but with different affinity profiles. We constructed a series of chimeric MC1/MC3 receptors to identify the epitopes that determine their selectivities for natural melanocyte peptides and synthetic analogues. The chimeric constructs were made by a polymerase chain reaction that used identical regions in or just outside transmembranes (TM) 1, 4, and 6 and divided the receptors into four segments. Saturation and competition studies on the expressed chimeric proteins indicate that TM1, TM2, TM3, and TM7 are involved in the subtype-specific binding of melanocyte peptides to these receptors. The results support the hypothesis that TM4 and TM5 may not contribute to the ligand-binding specificity of the MC receptors. This is the first report to describe the subtype-specific hormone-binding domains of the melanocortin receptor family.

Amino Acid Sequence↗
PubMed2001

Molecular mechanism for agonist-promoted alpha(2A)-adrenoceptor activation by norepinephrine and epinephrine.

We present a mechanism for agonist-promoted alpha(2A)-adrenergic receptor (alpha(2A)-AR) activation based on structural, pharmacological, and theoretical evidence of the interactions between phenethylamine ligands and alpha(2A)-AR. In this study, we have: 1) isolated enantiomerically pure phenethylamines that differ both in their chirality about the beta-carbon, and in the presence/absence of one or more hydroxyl groups: the beta-OH and the catecholic meta- and para-OH groups; 2) used [(3)H]UK-14,304 [5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; agonist] and [(3)H]RX821002 [2-(2-methoxy-1,4-benzodioxan-2-yl)-2-imidazoline; antagonist] competition binding assays to determine binding affinities of these ligands to the high- and low-affinity forms of alpha(2A)-AR; 3) tested the ability of the ligands to promote receptor activation by measuring agonist-induced stimulation of [(35)S]GTPgammaS binding in isolated cell membranes; and 4) used automated docking methods and our alpha(2A)-AR model to predict the binding modes of the ligands inside the alpha(2A)-AR binding site. The ligand molecules are sequentially missing different functional groups, and we have correlated the structural features of the ligands and ligand-receptor interactions with experimental ligand binding and receptor activation data. Based on the analysis, we show that structural rearrangements in transmembrane helix (TM) 5 could take place upon binding and subsequent activation of alpha(2A)-AR by phenethylamine agonists. We suggest that the following residues are important in phenethylamine interactions with alpha(2A)-AR: Asp113 (D(3.32)), Val114 (V(3.33)), and Thr118 (T(3.37)) in TM3; Ser200 (S(5.42)), Cys201 (C(5.43)), and Ser204 (S(5.46)) in TM5; Phe391 (F(6.52)) and Tyr394 (Y(6.55)) in TM6; and Phe411 (F(7.38)) and Phe412 (F(7.39)) in TM7.

Adrenergic Agonists↗
PubMed2003

Mutational analysis and molecular modeling of the binding pocket of the metabotropic glutamate 5 receptor negative modulator 2-methyl-6-(phenylethynyl)-pyridine.

Metabotropic glutamate (mGlu) 5 is a G-protein-coupled metabotropic glutamate receptor that plays an important role as a modulator of synaptic plasticity, ion channel activity, and excitotoxicity. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) is a highly potent, noncompetitive, selective, and systemically active antagonist of mGlu5 receptors. It binds to a novel allosteric site that resides within the seven-transmembrane domain of mGlu5 receptors. Using site-directed mutagenesis, [3H]MPEP binding, a functional Ca2+ mobilization assay, and rhodopsin-based homology modeling, we identified eight residues (Pro-6543.36, Tyr-6583.40, Leu-7435.47, Thr-7806.44, Trp-7846.48, Phe-7876.51, Tyr-7916.55, and Ala-8097.47) that are crucial for MPEP-binding to rat mGlu5 receptors. Four mutations, Y6583.40V, W7846.48A, F7876.51A, and A8097.47V, caused complete loss of [3H]MPEP binding and also blocked the MPEP-mediated inhibition of quisqualate-induced intracellular Ca2+ mobilization. To visualize these experimental findings, we have constructed a homology model based on the X-ray crystal of bovine rhodopsin and have suggested a possible binding mode of MPEP. We propose that MPEP via its interactions with a network of the aromatic residues including Phe-6583.40 in transmembrane (TM) 3 helix and Trp-7986.48, Phe-7876.51, and Tyr-7916.55 in TM6 helix prevents the movement of TM6 helix relative to TM3 helix, a step that is required for receptor activation, and consequently stabilizes the inactive conformation of mGlu5 receptor. In the TM6 region, we observed a striking similarity between the critical residues involved in MPEP-binding site with those of previously identified as 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydropyrimidine-5-carbonitrile-binding pocket of mGlu1, pointing to a common mechanism of inhibition shared by both antagonists.

Allosteric Site↗
PubMed2002

Crystal structure of Escherichia coli MscS, a voltage-modulated and mechanosensitive channel.

The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.

Amino Acid Sequence↗
PubMed1993

B epitopes and selection pressures in feline immunodeficiency virus envelope glycoproteins.

In order to map linear B epitopes in feline immunodeficiency virus (FIV) envelope glycoproteins (Env), a random library of FIV Env polypeptides fused to beta-galactosidase and expressed in Escherichia coli was screened by using sera from experimentally FIV-infected cats. We mapped five antibody-binding domains in the surface envelope glycoprotein (SU1 to SU5) and four in the transmembrane envelope glycoprotein (TM1 to TM4). Immunological analysis with 48 serum samples from naturally or experimentally infected cats of diverse origins revealed a broad group reactivity for epitopes SU2, TM2, and TM3, whereas SU3 appeared as strictly type specific. To study selection pressures acting on the identified immunogenic domains, we analyzed structural constraints and distribution of synonymous and nonsynonymous mutations (amino acids unchanged or changed). Two linear B epitopes (SU3 and TM4) appeared to be submitted to positive selection for change, a pattern of evolution predicting their possible involvement in antiviral protection. These experiments provide a pertinent choice of oligopeptides for further analysis of the protective response against FIV envelope glycoproteins, as a model to understand the role of antibody escape in lentiviral persistence and to design feline AIDS vaccines.

Animals↗
PubMed2006

Building of the tetraspanin web: distinct structural domains of CD81 function in different cellular compartments.

The tetraspanin web is composed of a network of tetraspanins and their partner proteins that facilitate cellular interactions and fusion events by an unknown mechanism. Our aim was to unravel the web partnership between the tetraspanin CD81 and CD19, a cell surface signaling molecule in B lymphocytes. We found that CD81 plays multiple roles in the processing, intracellular trafficking, and membrane functions of CD19. Surprisingly, these different roles are embodied in distinct CD81 domains, which function in the different cellular compartments: the N-terminal tail of CD81 has an effect on the glycosylation of CD19; the first transmembrane domain of CD81 is sufficient to support the exit of CD19 from the endoplasmic reticulum, although the large extracellular loop (LEL) of CD81 associates physically with CD19 early during biosynthesis; and finally, the TM2 and TM3 domains of CD81 play a role in the transmission of signals initiated upon engagement of the LEL. The participation of distinct CD81 domains in varied functions may explain the pleiotropic effects of CD81 within the tetraspanin web.

Animals↗
PubMed1998

Solid-state NMR studies of magnetically aligned phospholipid membranes: taming lanthanides for membrane protein studies.

The addition of lanthanides (Tm3+, Yb3+, Er3+, or Eu3+) to a solution of long-chain phospholipids such as dimyristoylphosphatidylcholine (DMPC) and short-chain phospholipids such as dihexanoylphosphatidylcholine (DHPC) is known to result in a bilayer phase in which the average bilayer normal aligns parallel to an applied magnetic field. Lanthanide-doped bilayers have enormous potential for the study of membrane proteins by solid-state NMR, low-angle diffraction, and a variety of optical spectroscopic techniques. However, the addition of lanthanides poses certain challenges to the NMR spectroscopist: coexistence of an isotropic phase and hysteresis effects, direct binding of the paramagnetic ion to the peptide or protein of interest, and severe paramagnetic shifts and line broadening. Lower water concentrations and larger DMPC/DHPC ratios than those typically used in bicelles consistently yield a single oriented bilayer phase that is stable over a wide range of temperature (approximately 35-90 degrees C). Among the above choice of lanthanides, Yb3+ is found to give minimal paramagnetic shifts and line broadening at acceptably low concentrations necessary for alignment (i.e., Yb3+/DMPC mole ratios equal to or greater than 0.01). Finally, the addition of a phospholipid chelate, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine--diethylenetriaminepent aacetic acid, is observed to significantly reduce paramagnetic broadening and presumably prevent direct association of the peptide with the lanthanide ions.

Chelating Agents↗
PubMed2002

Preliminary mutational analysis of the human kinin B2 receptor for nonpeptide antagonist ligands recognition.

FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2 receptor. The kinin B2 receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2 receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr-) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MENI 1270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S1 17A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2 receptor.

Animals↗
PubMed1985

Urea and Na+ permeabilities in toad urinary bladder: one or two solute pathways?

It has been reported that toad urinary bladder amiloride not only reversibly blocks Na+ transport, but it also reversibly inhibits urea permeability (Pu). This finding prompted some queries: 1) Does Na+ transport rates and/or the cytosolic Na+ pool regulate Pu? 2) Does amiloride inhibit two different solute pathways or one pathway through which both Na+ and urea permeate? The following results were obtained in toad bladder. 1) The absence of mucosal Na+ does not interfere with basal or ADH-induced Pu. 2) Amiloride inhibits Pu in the absence of apical Na+. 3) The concentration of amiloride that produces half-maximal inhibition (K1/2) of the ADH-induced Na+ transport is 7 X 10(-7)M with 110 mM mucosal Na+, 7 X 10(-8)M with 1 mM apical Na+, and 2 X 10(-6)M in the absence of mucosal Ca2+; K1/2 of ADH-stimulated Pu is 9 X 10(-5)M and is not altered by lowering the mucosal Na+ concentration to 1 mM or deleting apical Ca2+. 4) Mucosal La3+ (10(-3)M): i) reversibly increases Na+ transport by 75%, potentiating the natriferic effect of ADH by 51%; ii) does not affect basal Pu and irreversibly blunts the ADH-mediated increase in Pu by 72%; and iii) has no effect on basal or ADH-induced osmotic flow. Identical results were obtained with Tm3+. These results suggest that the inhibitory effect of amiloride on amide permeation is not mediated by its effect on Na+ transport. There are three separate operational pathways for water, Na+, and urea; amiloride reversibly blocks those through which Na+ and urea permeate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amiloride↗
PubMedDate not supplied

A novel connexin 26 mutation associated with autosomal recessive sensorineural deafness.

Mutations in the connexin 26 (Cx26) gene (GJB2) are a common cause of hereditary hearing impairment which affects approximately 1 in 2000 newborn children. We report the identification of a novel Cx26 point mutation (439 G-->A) linked to familial, autosomal recessive, sensorineural hearing loss. This missense mutation (E147K) is located in the highly conserved, putative K(+) channel lining sequence of the third transmembrane domain (TM3) of Cx26. Hearing impairment associated with this mutation was congenital, moderate to profound and showed no signs of progressive deterioration.

Adolescent↗
PubMed2003

Molecular characterization of three gonad cell lines.

To facilitate the study of the regulation and downstream interactions of genes involved in gonad development it is important to have a suitable cell culture model. We therefore aimed to characterize molecularly three different mouse gonad cell lines. TM3 and TM4 cells were originally isolated from prepubertal mouse gonads and were tentatively identified as being of Leydig cell and Sertoli cell origin, respectively, based upon their morphology and hormonal responses. The third line is a conditionally immortalized cell line, derived from 10.5-11.5 days post-coitum (dpc) male gonads of transgenic embryos carrying a temperature-sensitive SV40 large T-antigen. We studied by reverse transcription-polymerase chain reaction (RT-PCR) the expression profiles of a number of genes known to be important for early gonad development. Moreover, we assessed these cell lines for their capacity to induce SOX9 transcription upon expression of SRY, a key molecular event occurring during sex determination. We found that all three cell lines were unable to upregulate SOX9 expression upon transfection of SRY-expression constructs, even though these cells express many of the studied embryonic gonad genes. These observations point to a requirement for SRY cofactors for direct or indirect upregulation of SOX9 expression during testis determination.

Animals↗
PubMed2006

An extracellular loop of the human non-gastric H,K-ATPase alpha-subunit is involved in apical plasma membrane polarization.

The human non-gastric H,K-ATPase, ATP1AL1, belongs to the gene family of P-type ATPases. Consistent with their physiological roles in ion transport, members of this group, including the Na,KATPase and the gastric and non-gastric H,K-ATPases, are differentially polarized to either the basolateral or apical plasma membrane in epithelial cells. However, their polarized distribution is highly complex and depends on specific sorting signals or motifs which are recognized by the subcellular targeting machinery. For the gastric H,K-ATPase it has been suggested that the 4(th) transmembrane spanning domain (TM4) and its flanking regions induce conformational sorting motifs which direct the ion pump exclusively to the epithelial apical membrane. Here, we show in transfected Madin-Darby canine kidney (MDCK) cells that the related non-gastric H,KATPase, ATP1AL1, does contain similar sorting motifs in close proximity to TM4. A short extracellular loop between TM3 and TM4 is critical for this pump's apical delivery. A single point mutation in the corresponding region redirects ATP1AL1 to the basolateral membrane. In conclusion, our work provides further evidence that the cellular distribution of P-type ATPases is determined by conformational sorting motifs.

Amino Acid Sequence↗
PubMed1988

Experimental chemotherapy-induced skin necrosis in swine. Mechanistic studies of anthracycline antibiotic toxicity and protection with a radical dimer compound.

The reactivity of antitumor anthracycline and mitomycin C antibiotics with the oxomorpholinyl radical dimers, bi(3,5,5-trimethyl-2-oxomorpholin-3-yl) (TM3) and bi(3,5-dimethyl-5-hydroxymethyl-2-oxomorpholin-3-yl) (DHM3), was studied in vitro. The oxomorpholinyl radical reduced daunorubicin to a quinone methide intermediate that reacted with solvent to form 7-deoxydaunorubicinone. The solvolysis reaction followed first order kinetics, and the reactivity rate constants (k2) measured for seven anthracycline analogues ranged from 2 X 10(-2) s-1 to 8.0 X 10(-4) s-1. The chemical reactivity of each anthracycline quinone methide correlated with the total skin toxicity caused by the respective parent anthracycline following injection into swine skin. Microscopic examination of experimental lesions in swine skin resemble those observed in humans after inadvertant chemotherapy extravasation. Hydrocortisone sodium succinate was not effective for the treatment of doxorubicin-induced skin necrosis, whereas DHM3 was effective for the treatment of skin necrosis caused by all seven anthracyclines and by the quinone containing antibiotic, mitomycin C.

Animals↗