bluuebunny/crossref_metadata_2025 huggingface dataset:
Siamois is required for formation of Spemann’s organizer
Authors: Kessler Daniel S.
Published: July 2002

Spemann’s organizer develops in response to dorsal determinants that act via maternal components of the wnt pathway. The function of siamois, a wnt-inducible homeobox gene, in Spemann’s organizer development was examined by fusion of defined transcriptional regulatory domains to the siamois homeodomain. Similar to native siamois, a VP16 activator fusion induced axis formation, indicating that siamois functions as a transcriptional activator in axis induction. Fusion of the engrailed repressor generated a dominant inhibitor that blocked axis induction by Xwnt8, β-catenin, and siamois, and repressed wnt activation of the goosecoid promoter. Dorsal injection of the engrailed-siamois fusion resulted in complete inhibition of dorsal development and organizer gene expression, an effect rescued by siamois, but not by Xwnt8 or β-catenin. Thus, as a zygotic mediator of maternal dorsal signals, siamois function is required for development of Spemann’s organizer.
αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization
Authors: Garcia K. Christopher , Tallquist Michelle D. , Pease Larry R. , Brunmark Anders , Scott Christopher A. , Degano Massimo , Stura Enrico A. , Peterson Per A. , Wilson Ian A. , Teyton Luc
Published: July 2002

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor inDrosophila melanogastercells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities fromKd= 10−4to 10−6M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.
Gene induction in response to unfolded protein in the endoplasmic reticulum is mediated through Ire1p kinase interaction with a transcriptional coactivator complex containing Ada5p
Authors: Welihinda Ajith A. , Tirasophon Witoon , Green Sarah R. , Kaufman Randal J.
Published: July 2002

In eukaryotic cells, accumulation of unfolded protein in the endoplasmic reticulum induces transcription of a family of genes encoding endoplasmic reticulum protein chaperones through a conserved unfolded protein response element. In
Saccharomyces cerevisiae,
activation of a transmembrane receptor kinase, Ire1p (Ern1p), initiates signaling, although the mediators immediately downstream of Ire1 kinase are unknown. Here we demonstrate interaction of Ire1p with the transcriptional coactivator, Gcn5p (for general control nonrepressed; also known as Ada4p). Gcn5p associates with other Ada (for alteration/deficiency in activation) gene products in a heteromeric complex and has histone acetyltransferase activity. We show that the Gcn5/Ada complex is selectively required for the unfolded protein response but not for the heat shock response. A novel mechanism is proposed in which activation of a receptor kinase recruits a transcription coactivator complex to a specific chromosomal locus to mediate localized histone acetylation, thus making specific gene sequences accessible for transcription.
Conduction-band-edge ionization thresholds of DNA components in aqueous solution
Authors: Fernando Harshica , Papadantonakis George A. , Kim Nancy S. , LeBreton Pierre R.
Published: July 2002

Numerous investigations have focused on DNA damage induced by ionizing radiation; however, photoionization threshold energies of nucleic acid components in aqueous solution are not known. Herein, data from gas-phase photoelectron experiments have been combined with results from self-consistent field and post-self-consistent field molecular orbital calculations and with theoretical Gibbs free energies of hydration to describe aqueous ionization energies of 2′-deoxythymidine 5′-phosphate (5′-dTMP
−
) and 2′-deoxycytidine 5′-phosphate (5′-dCMP
−
). For the test molecules, indole and tryptophan, this approach yields aqueous ionization energies (4.46 and 4.58 eV, respectively) in agreement with experimental values (4.35 and 4.45 eV). When uridine and 2′-deoxythymidine ionization energies are evaluated, the results agree with recent data from 193-nm laser measurements indicating that uridine ionization occurs via a one-photon event. For 5′-dCMP
−
and 5′-dTMP
−
, a comparison of aqueous ionization energies with gas-phase ionization potentials (IPs) indicates that hydration alters the relative energies of ionization events. In the gas phase, phosphate vertical IPs are ∼1.3 eV smaller than base IPs. In aqueous solution, the base and phosphate ionization energies are more similar, and only differ by ∼0.5 eV. For 5′-dCMP
−
and 5′-dTMP
−
, the increased favorableness of base ionization, which accompanies hydration, is consistent with experimental data indicating that, at 77 K in aqueous perchlorate glasses, the primary photoionization pathway involves base ionization followed by deprotonation.
Molecular cloning and characterization of an invertebrate cellular retinoic acid binding protein
Authors: Mansfield S. Gary , Cammer Steven , Alexander Steven C. , Muehleisen David P. , Gray Rosemary S. , Tropsha Alexander , Bollenbacher Walter E.
Published: July 2002

We have cloned a cDNA and gene from the tobacco hornworm,Manduca sexta, which is related to the vertebrate cellular retinoic acid binding proteins (CRABPs). CRABPs are members of the superfamily of lipid binding proteins (LBPs) and are thought to mediate the effects of retinoic acid (RA) on morphogenesis, differentiation, and homeostasis. This discovery of aManduca sextaCRABP (msCRABP) demonstrates the presence of a CRABP in invertebrates. Compared with bovine/murine CRABP I, the deduced amino acid sequence of msCRABP is 71% homologous overall and 88% homologous for the ligand binding pocket. The genomic organization of msCRABP is conserved with other CRABP family members and the larger LBP superfamily. Importantly, the promoter region contains a motif that resembles an RA response element characteristic of the promoter region of most CRABPs analyzed. Three-dimensional molecular modeling based on postulated structural homology with bovine/murine CRABP I shows msCRABP has a ligand binding pocket that can accommodate RA. The existence of an invertebrate CRABP has significant evolutionary implications, suggesting CRABPs appeared during the evolution of the LBP superfamily well before vertebrate/invertebrate divergence, instead of much later in evolution in selected vertebrates.
Role for G protein-coupled receptor kinase in agonist-specific regulation of μ-opioid receptor responsiveness
Authors: Zhang Jie , Ferguson Stephen S. G. , Barak Larry S. , Bodduluri Sobha R. , Laporte Stéphane A. , Law Ping-Yee , Caron Marc G.
Published: July 2002

The G protein-coupled μ-opioid receptor (μOR) mediates the physiological effects of endogenous opioid peptides as well as the structurally distinct opioid alkaloids morphine and etorphine. An intriguing feature of μOR signaling is the differential receptor trafficking and desensitization properties following activation by distinct agonists, which have been proposed as possible mechanisms related to opioid tolerance. Here we report that the ability of distinct opioid agonists to differentially regulate μOR internalization and desensitization is related to their ability to promote G protein-coupled receptor kinase (GRK)-dependent phosphorylation of the μOR. Although both etorphine and morphine effectively activate the μOR, only etorphine elicits robust μOR phosphorylation followed by plasma membrane translocation of β-arrestin and dynamin-dependent receptor internalization. In contrast, corresponding to its inability to cause μOR internalization, morphine is unable to either elicit μOR phosphorylation or stimulate β-arrestin translocation. However, upon the overexpression of GRK2, morphine gains the capacity to induce μOR phosphorylation, accompanied by the rescue of β-arrestin translocation and receptor sequestration. Moreover, overexpression of GRK2 also leads to an attenuation of morphine-mediated inhibition of adenylyl cyclase. These findings point to the existence of marked differences in the ability of different opioid agonists to promote μOR phosphorylation by GRK. These differences may provide the molecular basis underlying the different analgesic properties of opioid agonists and contribute to the distinct ability of various opioids to induce drug tolerance.
Lower intracellular hydrogen peroxide levels in cells overexpressing CuZn-superoxide dismutase
Authors: Teixeira Henrique D. , Schumacher Robert I. , Meneghini Rogerio
Published: July 2002

Transfection of V79 Chinese hamster cells produced clones in which CuZn-superoxide dismutase (CuZn-SOD) activities were 2.2- to 3.5-fold higher than in the parental cells. An overall reduction of antioxidant enzyme activities and both total and oxidized glutathione levels had been found in these clones. Aconitase activities in these cells were determined to indirectly measure the O2−steady-state levels. As expected, in cells overexpressing CuZn-SOD, both total and cytosolic aconitase activities have increased. Because these clones showed reduced oxidized glutathione contents, it is unlikely that they present higher H2O2steady-state levels as a consequence of the higher SOD levels. This was confirmed by measuring H2O2steady-state levels in cells by flow cytometric analysis of 2′,7′-dichlorofluorescein diacetate-treated cells. Despite the decreased antioxidant defenses, three of the clones overexpressing CuZn-SOD showed reduced H2O2steady-state levels. These reduced H2O2steady-state levels were found even when the cells were treated with the O2−generator 2,3-dimethoxy-1,4-naphthoquinone. These data providein vivosupport for the hypothesis proposed by Liochev and Fridovich [Liochev, S. I. & Fridovich, I. (1994)Free Radical Biol. Med.16, 29–33] that O2−dismutation prevents the formation of higher H2O2levels by other reactions.
Glutaredoxin function for the carboxyl-terminal domain of the plant-type 5′-adenylylsulfate reductase
Authors: Bick Julie-Ann , Åslund Fredrik , Chen Yichang , Leustek Thomas
Published: July 2002

5′-Adenylylsulfate (APS) reductase (EC 1.8.99.-) catalyzes the reduction of activated sulfate to sulfite in plants. The evidence presented here shows that a domain of the enzyme is a glutathione (GSH)-dependent reductase that functions similarly to the redox cofactor glutaredoxin. The
APR1
cDNA encoding APS reductase from
Arabidopsis thaliana
is able to complement the cysteine auxotrophy of an
Escherichia coli cysH
[3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase] mutant, only if the
E. coli
strain produces glutathione. The purified recombinant enzyme (APR1p) can use GSH efficiently as a hydrogen donor
in vitro
, showing a K
m[GSH]
of ≈0.6 mM. Gene dissection was used to express separately the regions of APR1p from amino acids 73–327 (the R domain), homologous with microbial PAPS reductase, and from amino acids 328–465 (the C domain), homologous with thioredoxin. The R and C domains alone are inactive in APS reduction, but the activity is partially restored by mixing the two domains. The C domain shows a number of activities that are typical of
E. coli
glutaredoxin rather than thioredoxin. Both the C domain and APR1p are highly active in GSH-dependent reduction of hydroxyethyldisulfide, cystine, and dehydroascorbate, showing a K
m[GSH]
in these assays of ≈1 mM. The R domain does not show these activities. The C domain is active in GSH-dependent reduction of insulin disulfides and ribonucleotide reductase, whereas APR1p and R domain are inactive. The C domain can substitute for glutaredoxin
in vivo
as demonstrated by complementation of an
E. coli
mutant, underscoring the functional similarity between the two enzymes.
High-frequency, spin-label EPR of nonaxial lipid ordering and motion in cholesterol-containing membranes
Authors: Gaffney Betty J. , Marsh Derek
Published: July 2002

The EPR spectra of spin-labeled lipid chains in fully hydrated bilayer membranes of dimyristoyl phosphatidylcholine containing 40 mol % of cholesterol have been studied in the liquid-ordered phase at a microwave radiation frequency of 94 GHz. At such high field strengths, the spectra should be optimally sensitive to lateral chain ordering that is expected in the formation of in-plane domains. The high-field EPR spectra from random dispersions of the cholesterol-containing membranes display very little axial averaging of the nitroxide g-tensor anisotropy for lipids spin labeled toward the carboxyl end of the
sn
-2 chain (down to the 8-C atom). For these positions of labeling, anisotropic
14
N-hyperfine splittings are resolved in the
g
zz
and

g
yy

regions of the nonaxial EPR spectra. For positions of labeling further down the lipid chain, toward the terminal methyl group, the axial averaging of the spectral features systematically increases and is complete at the 14-C atom position. Concomitantly, the time-averaged 〈

A
zz

〉 element of the
14
N-hyperfine tensor decreases, indicating that the axial rotation at the terminal methyl end of the chains arises from correlated torsional motions about the bonds of the chain backbone, the dynamics of which also give rise to a differential line broadening of the
14
N-hyperfine manifolds in the

g
zz

region of the spectrum. These results provide an indication of the way in which lateral ordering of lipid chains in membranes is induced by cholesterol.
Interleukin 12 deficiency associated with recurrent infections
Authors: Haraguchi Soichi , Day Noorbibi K. , Nelson Robert P. , Emmanuel Patricia , Duplantier John E. , Christodoulou Christodoulos S. , Good Robert A.
Published: July 2002

A 3-yr-old female patient exhibited interleukin 12 (IL-12) deficiency that was associated with recurrent episodes of pneumococcal pneumonia with sepsis and other infections in the absence of fevers. The patient’s peripheral blood mononuclear cells (PBMCs) exhibited normal proliferative responses to antigens. Immune responses, includingin vivoproduction of antibodies to diphtheria, tetanus, or pneumococcal antigens, were normal. Ig levels and B cell and T cell phenotypes were also normal. In contrast, IL-12 p70 heterodimer production was undetectable by using supernatants of the patient’s stimulated PBMCs when compared with control cells treated similarly. Although present, interferon γ (IFN-γ) was reduced. The addition of recombinant IFN-γ to control cells enhanced the production of IL-12 by up to sixfold. By contrast, IL-12 was undetectable in supernatants of the patient’s cells in the presence of recombinant IFN-γ. IL-12 p40 subunit mRNA by using the patient’s PBMCs after stimulation withStaphylococcus aureusCowan strain 1 or lipopolysaccharide was also undetectable by reverse transcription–PCR when compared with control cells. Production of IL-2, IL-6, tumor necrosis factor α, or IFN-γ of the patient’s PBMCs after appropriate stimulation was observed. This patient has either a defect inStaphylococcus aureusCowan strain 1-lipopolysaccharide- or staphylococcal enterotoxin A-induced signaling pathways for the activation of IL-12 p40 gene expression, or an abnormality in the IL-12 p40 gene itself.
Complete mitochondrial genome suggests diapsid affinities of turtles
Authors: Zardoya Rafael , Meyer Axel
Published: July 2002

Despite more than a century of debate, the evolutionary position of turtles (Testudines) relative to other amniotes (reptiles, birds, and mammals) remains uncertain. One of the major impediments to resolving this important evolutionary problem is the highly distinctive and enigmatic morphology of turtles that led to their traditional placement apart from diapsid reptiles as sole descendants of presumably primitive anapsid reptiles. To address this question, the complete (16,787-bp) mitochondrial genome sequence of the African side-necked turtle (
Pelomedusa subrufa
) was determined. This molecule contains several unusual features: a (TA)

n

microsatellite in the control region, the absence of an origin of replication for the light strand in the WANCY region of five tRNA genes, an unusually long noncoding region separating the
ND5
and
ND6
genes, an overlap between
ATPase 6
and
COIII
genes, and the existence of extra nucleotides in
ND3
and
ND4L
putative ORFs. Phylogenetic analyses of the complete mitochondrial genome sequences supported the placement of turtles as the sister group of an alligator and chicken (Archosauria) clade. This result clearly rejects the Haematothermia hypothesis (a sister-group relationship between mammals and birds), as well as rejecting the placement of turtles as the most basal living amniotes. Moreover, evidence from both complete mitochondrial rRNA genes supports a sister-group relationship of turtles to Archosauria to the exclusion of Lepidosauria (tuatara, snakes, and lizards). These results challenge the classic view of turtles as the only survivors of primary anapsid reptiles and imply that turtles might have secondarily lost their skull fenestration.
Identification of protein–protein interfaces by decreased amide proton solvent accessibility
Authors: Mandell Jeffrey G. , Falick Arnold M. , Komives Elizabeth A.
Published: July 2002

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity.
Termination of Ca
2+
release by a local inactivation of ryanodine receptors in cardiac myocytes
Authors: Sham James S. K. , Song Long-Sheng , Chen Ye , Deng Li-Hua , Stern Michael D. , Lakatta Edward G. , Cheng Heping
Published: July 2002

In heart, a robust regulatory mechanism is required to counteract the regenerative Ca
2+
-induced Ca
2+
release from the sarcoplasmic reticulum. Several mechanisms, including inactivation, adaptation, and stochastic closing of ryanodine receptors (RyRs) have been proposed, but no conclusive evidence has yet been provided. We probed the termination process of Ca
2+
release by using a technique of imaging local Ca
2+
release, or “Ca
2+
spikes”, at subcellular sites; and we tracked the kinetics of Ca
2+
release triggered by L-type Ca
2+
channels. At 0 mV, Ca
2+
release occurred and terminated within 40 ms after the onset of clamp pulses (0 mV). Increasing the open-duration and promoting the reopenings of Ca
2+
channels with the Ca
2+
channel agonist, FPL64176, did not prolong or trigger secondary Ca
2+
spikes, even though two-thirds of the sarcoplasmic reticulum Ca
2+
remained available for release. Latency of Ca
2+
spikes coincided with the first openings but not with the reopenings of L-type Ca
2+
channels. After an initial maximal release, even a multi-fold increase in unitary Ca
2+
current induced by a hyperpolarization to −120 mV failed to trigger additional release, indicating absolute refractoriness of RyRs. When the release was submaximal (e.g., at +30 mV), tail currents did activate additional Ca
2+
spikes; confocal images revealed that they originated from RyRs unfired during depolarization. These results indicate that Ca
2+
release is terminated primarily by a highly localized, use-dependent inactivation of RyRs but not by the stochastic closing or adaptation of RyRs in intact ventricular myocytes.
Minimal and optimal mechanisms for GroE-mediated protein folding
Authors: Ben-Zvi Anat Peres , Chatellier Jean , Fersht Alan R. , Goloubinoff Pierre
Published: July 2002

We have analyzed the effects of different components of the GroE chaperonin system on protein folding by using a nonpermissive substrate (i.e., one that has very low spontaneous refolding yield) for which rate data can be acquired. In the absence of GroES and nucleotides, the rate of GroEL-mediated refolding of heat- and DTT-denatured mitochondrial malate dehydrogenase was extremely low, but some three times higher than the spontaneous rate. This GroEL-mediated rate was increased 17-fold by saturating concentrations of ATP, 11-fold by ADP and GroES, and 465-fold by ATP and GroES. Optimal refolding activity was observed when the dissociation of GroES from the chaperonin complex was dramatically reduced. Although GroEL minichaperones were able to bind denatured mitochondrial malate dehydrogenase, they were ineffective in enhancing the refolding rate. The spectrum of mechanisms for GroE-mediated protein folding depends on the nature of the substrate. The minimal mechanism for permissive substrates (i.e., having significant yields of spontaneous refolding), requires only binding to the apical domain of GroEL. Slow folding rates of nonpermissive substrates are limited by the transitions between high- and low-affinity states of GroEL alone. The optimal mechanism, which requires holoGroEL, physiological amounts of GroES, and ATP hydrolysis, is necessary for the chaperonin-mediated folding of nonpermissive substrates at physiologically relevant rates under conditions in which retention of bound GroES prevents the premature release of aggregation-prone folding intermediates from the chaperonin complex. The different mechanisms are described in terms of the structural features of mini- and holo-chaperones.
Monomolecular collapse of plasmid DNA into stable virus-like particles
Authors: Blessing Thomas , Remy Jean-Serge , Behr Jean-Paul
Published: July 2002

Cationic lipids are being widely used for cell transfection
in vitro
. The lipid/DNA complexes, however, tend to aggregate into large and polydisperse particle mixtures; this hampers their use
in vivo
. Cationic detergents, on the contrary, do not mediate cell transfection
per se
, yet are capable of condensing individual DNA molecules into discrete entities. We have taken (only) the interesting features of both types of amphiphiles for the two-step formation of stable core particles reminiscent of viruses. Individual anionic plasmid molecules were cooperatively collapsed with a carefully tailored cationic cysteine-based detergent. The resulting 23-nm particles were then simply “frozen” by spontaneous aerobic dimerization of the cysteine-detergent into a cystine-lipid on the template DNA. The population of spherical particles is monodisperse and stable over days, in physiological conditions. Together with a negative surface potential, these properties should ensure good tissue dissemination and escape from the blood stream after i.v. injection.
Seminal fluid regulation of female sexual attractiveness in
Drosophila melanogaster
Authors: Tram Uyen , Wolfner Mariana F.
Published: July 2002

Finding a willing and suitable mate is critical for sexual reproduction. Visual, auditory, and chemical cues aid in locating and/or attracting partners. After mating, females from many insect species become less attractive. This is caused by changes in the quantity and/or quality of pheromones synthesized by the female and to changes in the female’s behavior. For example, female insects may stop releasing pheromones, assume a mate refusal posture, or move less in response to males. Many postmating changes in female insects are triggered by seminal fluid proteins from the male’s accessory gland proteins (Acps) and by sperm. To determine the role of seminal fluid components in mediating changes in attractiveness, we measured the attractiveness of
Drosophila melanogaster
females that had been mated to genetically altered males that lack sperm and/or Acps. We found that the drop in female attractiveness occurs in two phases. A short-term drop in attractiveness is triggered independent of the receipt of sperm and Acps. Maintenance of lowered attractiveness is dependent upon sperm.
The identification of a 9-
cis
retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-
cis
retinoic acid
Authors: Romert Anna , Tuvendal Paulina , Simon Andràs , Dencker Lennart , Eriksson Ulf
Published: July 2002

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-
trans
and 9-
cis
RA, are generated. Here we report the identification of a stereo-specific 9-
cis
retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-
cis
retinol into 9-
cis
retinaldehyde, an intermediate in 9-
cis
RA biosynthesis. Analysis by nonradioactive
in situ
hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-
cis
retinol is generated for subsequent oxidation to 9-
cis
RA.
Transient and sustained increases in inositol 1,4,5-trisphosphate precede the differential growth response in gravistimulated maize pulvini
Authors: Perera Imara Y. , Heilmann Ingo , Boss Wendy F.
Published: July 2002

The internodal maize pulvinus responds to gravistimulation with differential cell elongation on the lower side. As the site of both graviperception and response, the pulvinus is an ideal system to study how organisms sense changes in orientation. We observed a transient 5-fold increase in inositol 1,4,5-trisphosphate (IP
3
) within 10 s of gravistimulation in the lower half of the pulvinus, indicating that the positional change was sensed immediately. Over the first 30 min, rapid IP
3
fluctuations were observed between the upper and lower halves. Maize plants require a presentation time of between 2 and 4 h before the cells on the lower side of the pulvinus are committed to elongation. After 2 h of gravistimulation, the lower half consistently had higher IP
3,
and IP
3
levels on the lower side continued to increase up to ≈5-fold over basal levels before visible growth. As bending became visible after 8–10 h, IP
3
levels returned to basal values. Additionally, phosphatidylinositol 4-phosphate 5-kinase activity in the lower pulvinus half increased transiently within 10 min of gravistimulation, suggesting that the increased IP
3
production was accompanied by an up-regulation of phosphatidylinositol 4,5-bisphosphate biosynthesis. Neither IP
3
levels nor phosphatidylinositol 4-phosphate 5-kinase activity changed in pulvini halves from vertical control plants. Our data indicate the involvement of IP
3
and inositol phospholipids in both short- and long-term responses to gravistimulation. As a diffusible second messenger, IP
3
provides a mechanism to transmit and amplify the signal from the perceiving to the responding cells in the pulvinus, coordinating a synchronized growth response.
Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites
Authors: Peleg Gadi , Lewis Aaron , Linial Michal , Loew Leslie M.
Published: July 2002

Membrane potential around single molecules has been measured by using the nonlinear optical phenomenon of second harmonic generation. This advance results from the interaction between a highly dipolar molecule with a selectively directed highly polarizable 1-nm gold particle. With this approach, a second harmonic signal, which is enhanced by the nanoparticle, is detected from a volume of nanometric dimensions. This present work clearly shows that functional cellular imaging around single molecules is possible by selectively directing an antibody with a 1-nm gold label to a specific membrane protein. The results of this work open the way for three-dimensional, high resolution functional imaging of membrane electrophysiology in cells and cellular networks.
AKT3, a phloem-localized K
+
channel, is blocked by protons
Authors: Marten I. , Hoth S. , Deeken R. , Ache P. , Ketchum K. A. , Hoshi T. , Hedrich R.
Published: July 2002

The potassium-channel gene,
AKT3,
has recently been isolated from an
Arabidopsis thaliana
cDNA library. By using the whole-mount and
in situ
hybridization techniques, we found AKT3 predominantly expressed in the phloem. To study the physiological role of this channel type, AKT3 was heterologously expressed in
Xenopus
oocytes, and the electrical properties were examined with voltage-clamp techniques. Unlike the plant inward-rectifying guard cell K
+
channels KAT1 and KST1, the AKT3 channels were only weakly regulated by the membrane potential. Furthermore, AKT3 was blocked by physiological concentrations of external Ca
2+
and showed an inverted pH regulation. Extracellular acidification decreased the macroscopic AKT3 currents by reducing the single-channel conductance. Because assimilate transport in the vascular tissue coincides with both H
+
and K
+
fluxes, AKT3 K
+
channels may be involved in K
+
transport accompanying phloem loading and unloading processes.
The
nop-1
gene of
Neurospora crassa
encodes a seven transmembrane helix retinal-binding protein homologous to archaeal rhodopsins
Authors: Bieszke Jennifer A. , Braun Edward L. , Bean Laura E. , Kang Seogchan , Natvig Donald O. , Borkovich Katherine A.
Published: July 2002

Opsins are a class of retinal-binding, seven transmembrane helix proteins that function as light-responsive ion pumps or sensory receptors. Previously, genes encoding opsins had been identified in animals and the Archaea but not in fungi or other eukaryotic microorganisms. Here, we report the identification and mutational analysis of an opsin gene,
nop-1
, from the eukaryotic filamentous fungus
Neurospora crassa
. The
nop-1
amino acid sequence predicts a protein that shares up to 81.8% amino acid identity with archaeal opsins in the 22 retinal binding pocket residues, including the conserved lysine residue that forms a Schiff base linkage with retinal. Evolutionary analysis revealed relatedness not only between NOP-1 and archaeal opsins but also between NOP-1 and several fungal opsin-related proteins that lack the Schiff base lysine residue. The results provide evidence for a eukaryotic opsin family homologous to the archaeal opsins, providing a plausible link between archaeal and visual opsins. Extensive analysis of Δ
nop-1
strains did not reveal obvious defects in light-regulated processes under normal laboratory conditions. However, results from Northern analysis support light and conidiation-based regulation of
nop-1
gene expression, and NOP-1 protein heterologously expressed in
Pichia pastoris
is labeled by using all-
trans
[
3
H]retinal, suggesting that NOP-1 functions as a rhodopsin in
N. crassa
photobiology.
Protein preservation and DNA retrieval from ancient tissues
Authors: Poinar Hendrik N. , Stankiewicz B. Artur
Published: July 2002

The retrieval of DNA from fossils remains controversial. To substantiate claims of DNA recovery, one needs additional information on the preservation of other molecules within the same sample. Flash pyrolysis with GC and MS was used to assess the quality of protein preservation in 11 archaeological and paleontological remains, some of which have yielded ancient DNA sequences authenticated via a number of criteria and some of which have consistently failed to yield any meaningful DNA. Several samples, including the Neanderthal-type specimen from which DNA sequences were recently reported, yielded abundant pyrolysis products assigned to 2,5-diketopiperazines of proline-containing dipeptides. The relative amounts of these products provide a good index of the amount of peptide hydrolysis and DNA preservation. Of these samples, four stem from arctic or subarctic regions, emphasizing the importance of cooler temperatures for the preservation of macromolecules. Flash pyrolysis with GC and MS offers a rapid and effective method for assessing fossils for the possibility of DNA preservation.
The poly(A)-limiting element is a conserved cis-acting sequence that regulates poly(A) tail length on nuclear pre-mRNAs
Authors: Gu Haidong , Das Gupta Jaydip , Schoenberg Daniel R.
Published: July 2002

Most vertebrate mRNAs exit the nucleus with a 200+-residue poly(A) tail and are deadenylated to yield heterogeneous polymers of 50–200 adenosine residues on any given mRNA. We previously reported that
Xenopus
albumin mRNA and pre-mRNA have an unusually short, discrete 17-residue poly(A) tail and showed that regulation of poly(A) length is controlled independently by two cis-acting poly(A)-limiting elements (PLE A and PLE B) located in the terminal exon. The present study sought to determine the generality of this regulatory mechanism. Transferrin mRNA also has a discrete <20-nt poly(A) tail, and deletion mapping experiments identified an element homologous to the albumin gene PLE B within the terminal exon of the transferrin gene that conferred poly(A) length regulation on a globin reporter mRNA. Based on this similarity the PLE B sequence was used in a database search to identify candidate mRNA targets for regulated polyadenylation. Of the several hundred sequences identified in this manner we focused on
HIV-EP2/Schnurri-2
, a member of a family of genes encoding related zinc finger transcription factors. A striking feature of the PLE-like element in these genes is its location 10–33 bp upstream of the translation stop codon. We demonstrate that
HIV-EP2
mRNA has a <20-nt poly(A) tail, for which the identified PLE-like sequence is responsible. These results indicate that the presence of a PLE can predict mRNAs with <20-nt poly(A) tails, and that nuclear regulation of poly(A) tail length is a feature of many mRNAs.
A cooperative oxygen-binding hemoglobin from
Mycobacterium tuberculosis
Authors: Couture Manon , Yeh Syun-Ru , Wittenberg Beatrice A. , Wittenberg Jonathan B. , Ouellet Yannick , Rousseau Denis L. , Guertin Michel
Published: July 2002

Two putative hemoglobin genes,
glbN
and
glbO
, were recently discovered in the complete genome sequence of
Mycobacterium tuberculosis
H37Rv. Here, we show that the
glbN
gene encodes a dimeric hemoglobin (HbN) that binds oxygen cooperatively with very high affinity (P
50
= 0.013 mmHg at 20°C) because of a fast combination (25 μM
−1
⋅s
−1
) and a slow dissociation (0.2 s
−1
) rate. Resonance Raman spectroscopy and ligand association/dissociation kinetic measurements, along with mutagenesis studies, reveal that the stabilization of the bound oxygen is achieved through a tyrosine at the B10 position in the distal pocket of the heme with a conformation that is unique among the globins. Physiological studies performed with
Mycobacterium bovis
bacillus Calmette–Guérin demonstrate that the expression of HbN is greatly enhanced during the stationary phase in aerobic cultures but not under conditions of limited oxygen availability. The results suggest that, physiologically, the primary role of HbN may be to protect the bacilli against reactive nitrogen species produced by the host macrophage.
DNA packing in stable lipid complexes designed for gene transfer imitates DNA compaction in bacteriophage
Authors: Schmutz M. , Durand D. , Debin A. , Palvadeau Y. , Etienne A. , Thierry A. R.
Published: July 2002

The structure of complexes made from DNA and suitable lipids (lipoplex, Lx) was examined by cryo-electron microscopy (cryoEM). We observed a distinct concentric ring-like pattern with striated shells when using plasmid DNA. These spherical multilamellar particles have a mean diameter of 254 nm with repetitive spacing of 7.5 nm with striation of 5.3 nm width. Small angle x-ray scattering revealed repetitive ordering of 6.9 nm, suggesting a lamellar structure containing at least 12 layers. This concentric and lamellar structure with different packing regimes also was observed by cryoEM when using linear double-stranded DNA, single-stranded DNA, and oligodeoxynucleotides. DNA chains could be visualized in DNA/lipid complexes. Such specific supramolecular organization is the result of thermodynamic forces, which cause compaction to occur through concentric winding of DNA in a liquid crystalline phase. CryoEM examination of T4 phage DNA packed either in T4 capsides or in lipidic particles showed similar patterns. Small angle x-ray scattering suggested an hexagonal phase in Lx-T4 DNA. Our results indicate that both lamellar and hexagonal phases may coexist in the same Lx preparation or particle and that transition between both phases may depend on equilibrium influenced by type and length of the DNA used.
Macroevolution of insect–plant associations: The relevance of host biogeography to host affiliation
Authors: Becerra Judith X. , Venable D. Lawrence
Published: July 2002

Identifying the factors that have promoted host shifts by phytophagous insects at a macroevolutionary scale is critical to understanding the associations between plants and insects. We used molecular phylogenies of the beetle genus
Blepharida
and its host genus
Bursera
to test whether these insects have been using hosts with widely overlapping ranges over evolutionary time. We also quantified the importance of host range coincidence relative to host chemistry and host phylogenetic relatedness. Overall, the evolution of host use of these insects has not been among hosts that are geographically similar. Host chemistry is the factor that best explains their macroevolutionary patterns of host use. Interestingly, one exceptional polyphagous species has shifted among geographically close chemically dissimilar plants.
Signaling of need, sibling competition, and the cost of honesty
Authors: Johnstone Rufus A.
Published: July 2002

Young birds and mammals frequently solicit food by means of extravagant and apparently costly begging displays. Much attention has been devoted to the idea that these displays are honest signals of need, and that their apparent cost serves to maintain their honesty. Recent analyses, however, have shown that the cost needed to maintain a fully informative, honest signal may often be so great that both offspring (signaler) and parent (receiver) would do better to refrain from communication. This apparently calls into question the relevance of the costly signaling hypothesis. Here, I show that this argument overlooks the impact of sibling competition. When multiple signalers must compete for the attention of a receiver (as is commonly the case in parent–offspring interactions), I show that (all other things being equal) individual equilibrium signal costs will typically be lower. The greater the number of competitors, the smaller the mean cost, though the maximum level of signal intensity employed by very needy signalers may actually increase with the number of competitors. At the same time, costs become increasingly sensitive to relatedness among signalers as opposed to relatedness between signalers and receivers. As a result of these trends, signaling proves profitable for signalers under a much wider range of conditions when there is competition (though it is still likely to be unprofitable for receivers).
Optical detection of cytochrome P450 by sensitizer-linked substrates
Authors: Dmochowski Ivan J. , Crane Brian R. , Wilker Jonathan J. , Winkler Jay R. , Gray Harry B.
Published: July 2002

The ability to detect, characterize, and manipulate specific biomolecules in complex media is critical for understanding metabolic processes. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. We have found that Ru photosensitizers linked to P450 substrates specifically recognize submicromolar cytochrome P450
cam
in the presence of other heme proteins. In the P450:Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. The crystal structure of a P450
cam
:Ru-adamantyl complex reveals access to the active center via a channel whose depth (Ru-Fe distance is 21 Å) is virtually the same as that extracted from an analysis of the energy-transfer kinetics. Suitably constructed libraries of sensitizer-linked substrates could be employed to probe the steric and electronic properties of buried active sites.
Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein
Authors: Fabian Heinz , Mantsch Henry H. , Schultz Christian P.
Published: July 2002

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.
The nuclear matrix prepared by amine modification
Authors: Wan Katherine M. , Nickerson Jeffrey A. , Krockmalnic Gabriela , Penman Sheldon
Published: July 2002

The nucleus is spatially ordered by attachments to a nonchromatin nuclear structure, the nuclear matrix. The nuclear matrix and chromatin are intimately connected and integrated structures, and so a major technical challenge in nuclear matrix research has been to remove chromatin while retaining a native nuclear matrix. Most methods for removing chromatin require first a nuclease digestion and then a salt extraction to remove cut chromatin. We have hypothesized that cut chromatin is held in place by charge interactions involving nucleosomal amino groups. We have tested this hypothesis by chemically modifying amino groups after nuclease digestion. By using this protocol, chromatin could be effectively removed at physiological ionic strength. We compared the ultrastructure and composition of this nuclear matrix preparation with the traditional high-salt nuclear matrix and with the third nuclear matrix preparation that we have developed from which chromatin is removed after extensive crosslinking. All three matrix preparations reveal internal nuclear matrix structures that are built on a network of branched filaments of about 10 nm diameter. That such different chromatin-removal protocols reveal similar principles of nuclear matrix construction increases our confidence that we are observing important architectural elements of the native structure in the living cell.
Confocal fluorescence coincidence analysis: An approach to ultra high-throughput screening
Authors: Winkler Thorsten , Kettling Ulrich , Koltermann Andre , Eigen Manfred
Published: July 2002

Fluorescence-based assay technologies play an increasing role
in high-throughput screening. They can be classified into different
categories: fluorescence polarization, time-resolved fluorescence,
fluorescence resonance energy transfer, and fluorescence correlation
spectroscopy. In this work we present an alternative analytical
technique for high-throughput screening, which we call confocal
fluorescence coincidence analysis. Confocal fluorescence coincidence
analysis extracts fluorescence fluctuations that occur coincidently in
two different spectral ranges from a tiny observation volume of below 1
fl. This procedure makes it possible to monitor whether an association
between molecular fragments that are labeled with different
fluorophores is established or broken. Therefore, it provides access to
the characterization of a variety of cleavage and ligation reactions in
biochemistry. Confocal fluorescence coincidence analysis is a very
sensitive and ultrafast technique with readout times of 100 ms and
below. This feature is demonstrated by means of a homogeneous assay for
restriction endonuclease
Eco
RI. The presented
achievements break ground for throughput rates as high as
10
6
samples per day with using only small amounts of
sample substance and therefore constitute a solid base for screening
applications in drug discovery and evolutionary biotechnology.