bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective reconstitution of gastrin-releasing peptide receptor with Gα
q
Authors: Hellmich Mark R. , Battey James F. , Northup John K.
Published: July 2002

Identification of the molecular mechanisms that determine specificity of coupling interactions between gastrin-releasing peptide receptors (GRPrs) and their cognate heterotrimeric GTP-binding proteins is a fundamental step in understanding the signal transduction cascade initiated by receptor–ligand interaction. To explore these mechanisms in greater detail, we have developed an
in situ
reconstitution assay in chaotrope-extracted membranes from mouse fibroblasts expressing the GRPr, and we have used it to measure GRPr-catalyzed binding of GTPγS to purified G protein α subunits. Binding studies with
125
I-labeled [
d
-Tyr
6
]bombesin(6–13) methyl ester (
125
I-Tyr-ME), a GRPr specific antagonist, show a single binding site with a
K
d
= 1.4 nM ± 0.4 (mean ± SD,
n
= 3) and capacity of 15–22 pmol of receptor per mg of protein in the extracted membrane preparations, representing a 2- to 3-fold enrichment of binding sites compared with the membranes before extraction. Quantitative ligand displacement analysis using various unlabeled GRPr agonists shows a rank order of potency characteristic of the GRPr: bombesin ≥ GRP ≫ neuromedin B. Reconstitution of urea extracted membranes with a purified Gα
q
showed that receptor-catalyzed binding of GTPγS was dependent on agonist (GRP) and Gβγ subunits. The EC
50
for GRP was 3.5 nM, which correlates well with the reported
K
d
of 3.1 nM for GRP binding to GRPr expressed in mouse fibroblasts [Benya, R. V.,
et al.
(1994)
Mol. Pharmacol.
46, 235–245]. The apparent
K
d
for bovine brain Gβγ in this assay was 60 nM, and the
K
m
for squid retinal Gα
q
was 90 nM. The GRPr-catalyzed binding of GTPγS is selective for Gα
q
, since we did not detect receptor-catalyzed exchange using either Gα
i/o
or Gα
t
. These data demonstrate that GRPr can functionally couple to Gα
q
but not to the pertussis toxin-sensitive Gα
i/o
or retinal specific Gα
t
. This
in situ
receptor reconstitution method will allow molecular characterization of G protein coupling to other heptahelical receptors.
Phylogenetic assessment of length variation at a microsatellite locus
Authors: Ortí Guillermo , Pearse Devon E. , Avise John C.
Published: July 2002

Sixty-six haplotypes at a locus containing a simple dinucleotide (CA)nmicrosatellite repeat were isolated by PCR–single-strand conformational polymorphism from populations of the horseshoe crabLimulus polyphemus. These haplotypes were sequenced to assess nucleotide variation directly. Thirty-four distinct sequences (alleles) were identified in a region 570 bp long that included the microsatellite motif. In the repeat region itself, CA-number varied in integer values from 5 to 11 across alleles, except that a (CA)8class was not observed. Differences among alleles were due also to polymorphisms at 22 sites in regions immediately flanking the microsatellite repeats. Nucleotide substitutions in these regions were used to estimate phylogenetic relationships among alleles, and the gene phylogeny was used to trace the evolution of length variation and CA repeat numbers. A low correlation between size variation and genealogical relationships among alleles suggests that absolute fragment size (as normally scored in microsatellite assays) is an unreliable indicator of historical affinities among alleles. This finding on the molecular fine structure of microsatellite variation suggests the need for caution in the use of repeat counts at microsatellite loci as secure indicators of allelic relationships.
Molding a peptide into an RNA site byin vivopeptide evolution
Authors: Harada Kazuo , Martin Shelley S. , Tan Ruoying , Frankel Alan D.
Published: July 2002

Short peptides corresponding to the arginine-rich domains of several RNA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities. In the case of the HIV-1 Rev-Rev response element (RRE) complex, the peptide forms a single α-helix that binds deeply in a widened, distorted RNA major groove and makes a substantial set of base-specific and backbone contacts. Using a reporter system based on antitermination by the bacteriophage λ N protein, it has been possible to identify novel arginine-rich peptides from combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, RSG-1.2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, andin vitrocompetition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial α-helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an “RNA world.”
A gene spans the pseudoautosomal boundary in mice
Authors: Palmer Steve , Perry Jo , Kipling David , Ashworth Alan
Published: July 2002

The X and Y chromosomes of the mouse, like those of other mammals, are heteromorphic over most of their length, but at the distal ends of the chromosomes is a region of sequence identity, the pseudoautosomal region (PAR), where the chromosomes pair and recombine during male meiosis. The point at which the PAR diverges into X- and Y-specific sequences is called the pseudoautosomal boundary. We have completed a genomic walk from the X-specific
Amelogenin
gene to the PAR. Analysis of this region revealed that the pseudoautosomal boundary of mice is located within an intron of a transcribed gene that encodes a novel RING finger protein. The first three of the exons of the gene are located on the X chromosome whereas the 3′ exons of the gene are located on both X and Y chromosomes. This unusual arrangement may indicate that the gene is in a state of transition from pseudoautosomal to X-unique and provides evidence for a process of attrition of the pseudoautosomal region on the Y chromosome.
Transglutaminase-catalyzed inactivation of glyceraldehyde 3-phosphate dehydrogenase and α-ketoglutarate dehydrogenase complex by polyglutamine domains of pathological length
Authors: Cooper Arthur J. L. , Sheu K.-F. Rex , Burke James R. , Onodera Osamu , Strittmatter Warren J. , Roses Allen D. , Blass John P.
Published: July 2002

Several adult-onset neurodegenerative diseases are caused by genes with expanded CAG triplet repeats within their coding regions and extended polyglutamine (Q

n

) domains within the expressed proteins. Generally, in clinically affected individuals
n
≥ 40. Glyceraldehyde 3-phosphate dehydrogenase binds tightly to four Q

n

disease proteins, but the significance of this interaction is unknown. We now report that purified glyceraldehyde 3-phosphate dehydrogenase is inactivated by tissue transglutaminase in the presence of glutathione
S
-transferase constructs containing a Q

n

domain of pathological length (
n
= 62 or 81). The dehydrogenase is less strongly inhibited by tissue transglutaminase in the presence of constructs containing shorter Q

n

domains (
n
= 0 or 10). Purified α-ketoglutarate dehydrogenase complex also is inactivated by tissue transglutaminase plus glutathione
S
-transferase constructs containing pathological-length Q

n

domains (
n
= 62 or 81). The results suggest that tissue transglutaminase-catalyzed covalent linkages involving the larger poly-Q domains may disrupt cerebral energy metabolism in CAG/Q

n

expansion diseases.
The extended environment of mononuclear metal centers in protein structures
Authors: Karlin Samuel , Zhu Zhan-Yang , Karlin Kenneth D.
Published: July 2002

The objectives of this and the following paper are to identify commonalities and disparities of the extended environment of mononuclear metal sites centering on Cu, Fe, Mn, and Zn. The extended environment of a metal site within a protein embodies at least three layers: the metal core, the ligand group, and the second shell, which is defined here to consist of all residues distant less than 3.5 Å from some ligand of the metal core. The ligands and second-shell residues can be characterized in terms of polarity, hydrophobicity, secondary structures, solvent accessibility, hydrogen-bonding interactions, and membership in statistically significant residue clusters of different kinds. Findings include the following: (
i
) Both histidine ligands of type I copper ions
exclusively
attach the N
δ1
nitrogen of the histidine imidazole ring to the metal, whereas histidine ligands for
all
mononuclear iron ions and nearly all type II copper ions are ligated via the N
ɛ2
nitrogen. By contrast, multinuclear copper centers are coordinated predominantly by histidine N
ɛ2
, whereas diiron histidine contacts are predominantly N
δ1
. Explanations in terms of steric differences between N
δ1
and N
ɛ2
are considered. (
ii
) Except for blue copper (type I), the second-shell composition favors polar residues. (
iii
) For blue copper, the second shell generally contains multiple methionine residues, which are elements of a statistically significant histidine–cysteine–methionine cluster. Almost half of the second shell of blue copper consists of solvent-accessible residues, putatively facilitating electron transfer. (
iv
) Mononuclear copper atoms are never found with acidic carboxylate ligands, whereas single Mn
2+
ion ligands are predominantly acidic and the second shell tends to be mostly buried. (
v
) The extended environment of mononuclear Fe sites often is associated with histidine–tyrosine or histidine–acidic clusters.
Cloning and functional characterization of mouse IκBɛ
Authors: Simeonidis Simos , Liang Stanley , Chen Guoying , Thanos Dimitris
Published: July 2002

The biological activity of the transcription factor NF-κB is mainly controlled by the IκB proteins IκBα and IκBβ, which restrict NF-κB in the cytoplasm and enter the nucleus where they terminate NF-κB-dependent transcription. In this paper we describe the cloning and functional characterization of mouse IκBɛ. Mouse IκBɛ contains 6 ankyrin repeats required for its interaction with the Rel proteins and is expressed in different cell types where we found that it is up-regulated by NF-κB inducers, as is the case for IκBα and human IκBɛ. IκBɛ functions as a bona fide IκB protein by restricting Rel proteins in the cytoplasm and inhibiting their
in vitro
DNA binding activity. Surprisingly, IκBɛ did not inhibit transcription of genes regulated by the p50/p65 heterodimer efficiently, such as the human interferon-β gene. However, IκBɛ was a strong inhibitor of interleukin-8 expression, a gene known to be regulated by p65 homodimers. In addition, IκBɛ appears to function predominantly in the cytoplasm to sequester p65 homodimers, in contrast with the other two members of the family, IκBα and IκBβ, which also function in the nucleus to terminate NF-κB-dependent transcriptional activation.
d
-
myo
-Inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to
Salmonella
invasion inhibits phosphoinositide 3-kinase signaling pathways
Authors: Eckmann Lars , Rudolf Marco T. , Ptasznik Andrzej , Schultz Carsten , Jiang Tao , Wolfson Nora , Tsien Roger , Fierer Joshua , Shears Stephen B. , Kagnoff Martin F. , Traynor-Kaplan Alexis E.
Published: July 2002

Several inositol-containing compounds play key roles in receptor-mediated cell signaling events. Here, we describe a function for a specific inositol polyphosphate,
d
-
myo
-inositol 1,4,5,6-tetrakisphosphate [Ins(1,4,5,6)P
4
], that is produced acutely in response to a receptor-independent process. Thus, infection of intestinal epithelial cells with the enteric pathogen
Salmonella
, but not with other invasive bacteria, induced a multifold increase in Ins(1,4,5,6)P
4
levels. To define a specific function of Ins(1,4,5,6)P
4
, a membrane-permeant, hydrolyzable ester was used to deliver it to the intracellular compartment, where it antagonized epidermal growth factor (EGF)-induced inhibition of calcium-mediated chloride (Cl
−
) secretion (CaMCS) in intestinal epithelia. This EGF function is likely mediated through a phosphoinositide 3-kinase (PtdIns3K)-dependent mechanism because the EGF effects are abolished by wortmannin, and three different membrane-permeant esters of the PtdIns3K product phosphatidylinositol 3,4,5-trisphosphate mimicked the EGF effect on CaMCS. We further demonstrate that Ins(1,4,5,6)P
4
antagonized EGF signaling downstream of PtdIns3K because Ins(1,4,5,6)P
4
interfered with the PtdInsP
3
effect on CaMCS without affecting PtdIns3K activity. Thus, elevation of Ins(1,4,5,6)P
4
in
Salmonella
-infected epithelia may promote Cl
−
flux by antagonizing EGF inhibition mediated through PtdIns3K and PtdInsP
3
.
Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations
Authors: Wallace Douglas C. , Stugard Carol , Murdock Deborah , Schurr Theodore , Brown Michael D.
Published: July 2002

Nuclear-localized mtDNA pseudogenes might explain a recent report
describing a heteroplasmic mtDNA molecule containing five linked
missense mutations dispersed over the contiguous mtDNA CO1 and CO2
genes in Alzheimer’s disease (AD) patients. To test this hypothesis,
we have used the PCR primers utilized in the original report to amplify
CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell
lines. CO1 and CO2 sequences amplified from both of the ρ° cells,
demonstrating that these sequences are also present in the human
nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then
tested for each of the five “AD” missense mutations by
restriction endonuclease site variant assays. All five mutations were
found in the nuclear CO1 and CO2 PCR products from ρ° cells, but
none were found in the PCR products obtained from cells with normal
mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products
were cloned and sequenced, all five missense mutations were found, as
well as a linked synonymous mutation. Unlike the findings in the
original report, an additional 32 base substitutions were found,
including two in adjacent tRNAs and a two base pair deletion in the CO2
gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences
revealed that they diverged from modern human mtDNAs early in hominid
evolution about 770,000 years before present. These data would be
consistent with the interpretation that the missense mutations proposed
to cause AD may be the product of ancient mtDNA variants preserved as
nuclear pseudogenes.
Transgenic mice expressing rabbit C-reactive protein are resistant to endotoxemia
Authors: Xia Dongyuan , Samols David
Published: July 2002

C-reactive protein (CRP), the prototypic acute-phase reactant in humans, is synthesized in liver in response to a wide variety of inflammatory stimuli. We have generated a line of transgenic mice that express rabbit CRP from the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter in response to gluconeogenic signals. Here we show that transgenic mice expressing high levels of CRP were partially protected from a lethal challenge of bacterial lipopolysaccharide compared with littermates in which CRP expression had been suppressed. Similar protection was observed with challenges from platelet-activating factor (PAF) and the combination of tumor necrosis factor α (TNF-α) plus interleukin 1β, but not with TNF-α alone. We further demonstrate that although PAF was able to bind CRP, the mechanism by which CRP provides protection probably does not involve sequestration of PAF. The biologically inactive precursor of PAF, lyso-PAF, also bound CRP but did not render the transgenic mice sensitive to PAF when CRP-expressing animals were simultaneously challenged with PAF and an excess of lyso-PAF. These results suggest that CRP functionsin vivoby modulating host defense systems.
Early mortality surge in protein-deprived females causes reversal of sex differential of life expectancy in Mediterranean fruit flies
Authors: Müller Hans-Georg , Wang Jane-Ling , Capra William B. , Liedo Pablo , Carey James R.
Published: July 2002

Experiments based on over 400,000 medflies revealed that females
maintained on a normal diet (sucrose plus protein) have a higher life
expectancy than males maintained on a normal diet, with a difference of
1.30 ± 0.27 days in favor of females. However, this sex
differential reverses under protein deprivation, with a difference of
2.24 ± 0.18 days in favor of males. The reversal of the
male–female life expectancy differential is caused by a sustained
surge in early female mortality under protein deprivation that is tied
to egg-laying and physiological processes. In contrast, male mortality
and life expectancy are only mildly affected by protein deprivation.
The surge in early mortality for female medfly cohorts is an instance
of a vulnerable period. These vulnerable periods are linked with
patterns in hazard rates.
Peptide-specific killing of antigen-presenting cells by a recombinant antibody–toxin fusion protein targeted to major histocompatibility complex/peptide class I complexes with T cell receptor-like specificity
Authors: Reiter Yoram , Di Carlo Angelina , Fugger Lars , Engberg Jan , Pastan Ira
Published: July 2002

Specificity in the immune system is dictated and regulated by specific recognition of peptide/major histocompatibility complex (MHC) complexes by the T cell receptor. Such peptide/MHC complexes are a desirable target for novel approaches in immunotherapy because of their highly restricted fine specificity. Recently, phage display was used to isolate an antibody that has T cell receptor-like specificity. It recognizes mouse MHC class I H-2K
k
molecules complexed with a H-2K
k
-restricted influenza virus-derived hemagglutinin peptide (Ha
255–262
) but does not bind to class I H-2K
k
alone, peptide alone, or H-2K
k
complexed with other peptides. We have used this antibody to make a recombinant antibody–toxin fusion protein (immunotoxin) and show herein that it specifically kills antigen-presenting cells in a peptide-dependent manner and with T cell receptor-like specificity. We find a striking correlation between the fine specificity of binding of the antibody and the cytotoxic activity of the recombinant immunotoxin. We also show specific killing of influenza virus-infected target cells. The results suggest that it should be possible to develop novel immunotherapeutic strategies against human cancer by making recombinant antibodies that will recognize cancer-related peptides complexed with MHC class I molecules on the surface of cancer cells and using these to deliver toxins, radioisotopes, or cytotoxic drugs to the cancer cells.
Negative control of bacterial DNA replication by a cell cycle regulatory protein that binds at the chromosome origin
Authors: Quon Kim C. , Yang Bing , Domian Ibrahim J. , Shapiro Lucy , Marczynski Gregory T.
Published: July 2002

Caulobacter crescentus
divides asymmetrically generating two distinct cell types at each cell division: a stalked cell competent for DNA replication, and a swarmer cell that is unable to initiate DNA replication until it differentiates into a stalked cell later in the cell cycle. The CtrA protein, a member of the response regulator family of the two-component signal transduction system, controls multiple cell cycle processes in
Caulobacter
and is present in swarmer cells but absent from stalked cells. We report that CtrA binds five sites within the chromosome replication origin
in vitro
. These sites overlap an essential DnaA box and a promoter in the origin that is essential for replication initiation. Analysis of mutant alleles of
ctrA
and point mutations in one of the CtrA binding sites in the origin demonstrate that CtrA represses replication
in vivo
. CtrA-mediated repression at the origin thus restricts replication to the stalked cell type. Thus, the direct coupling of chromosome replication with the cell cycle is mediated by the ubiquitous two-component signaling proteins.
A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes
Authors: Gunn Michael D. , Tangemann Kirsten , Tam Carmen , Cyster Jason G. , Rosen Steven D. , Williams Lewis T.
Published: July 2002

Preferential homing of naive lymphocytes to secondary lymphoid organs is thought to involve the action of chemokines, yet no chemokine has been shown to have either the expression pattern or the activities required to mediate this process. Here we show that a chemokine represented in the EST database, secondary lymphoid-tissue chemokine (SLC), is expressed in the high endothelial venules of lymph nodes and Peyer’s patches, in the T cell areas of spleen, lymph nodes, and Peyer’s patches, and in the lymphatic endothelium of multiple organs. SLC is a highly efficacious chemoattractant for lymphocytes with preferential activity toward naive T cells. Moreover, SLC induces firm adhesion of naive T lymphocytes via β2 integrin binding to the counter receptor, intercellular adhesion molecule-1, a necessary step for lymphocyte recruitment. SLC is the first chemokine demonstrated to have the characteristics required to mediate homing of lymphocytes to secondary lymphoid organs. In addition, the expression of SLC in lymphatic endothelium suggests that the migration of lymphocytes from tissues into efferent lymphatics may be an active process mediated by this molecule.
Histidine kinase activity of the ETR1 ethylene receptor from
Arabidopsis
Authors: Gamble Rebekah L. , Coonfield Michael L. , Schaller G. Eric
Published: July 2002

ETR1 represents a prototypical ethylene receptor. Homologues of ETR1 have been identified in
Arabidopsis
as well as in other plant species, indicating that ethylene perception involves a family of receptors and that the mechanism of ethylene perception is conserved in plants. The amino-terminal half of ETR1 contains a hydrophobic domain responsible for ethylene binding and membrane localization. The carboxyl-terminal half of the polypeptide contains domains with homology to histidine kinases and response regulators, signaling motifs originally identified in bacteria. The putative histidine kinase domain of ETR1 was expressed in yeast as a fusion protein with glutathione
S
-transferase and affinity purified. Autophosphorylation of the purified fusion protein was observed on incubation with radiolabeled ATP. The incorporated phosphate was resistant to treatment with 3 M NaOH, but was sensitive to 1 M HCl, consistent with phosphorylation of histidine. Autophosphorylation was abolished by mutations that eliminated either the presumptive site of phosphorylation (His-353) or putative catalytic residues within the kinase domain. Truncations were used to delineate the region required for histidine kinase activity. An examination of cation requirements indicated that ETR1 requires Mn
2+
for autophosphorylation. These results demonstrate that higher plants contain proteins with histidine kinase activity. Furthermore, these results indicate that aspects of ethylene signaling may be regulated by changes in histidine kinase activity of the receptor.
Dioxygen activation and bond cleavage by mixed-valence cytochrome
c
oxidase
Authors: Proshlyakov Denis A. , Pressler Michelle A. , Babcock Gerald T.
Published: July 2002

Elucidating the structures of intermediates in the reduction of O
2
to water by cytochrome
c
oxidase is crucial to understanding both oxygen activation and proton pumping by the enzyme. In the work here, the reaction of O
2
with the mixed-valence enzyme, in which only heme
a
3
and Cu
B
in the binuclear center are reduced, has been followed by time-resolved resonance Raman spectroscopy. The results show that O=O bond cleavage occurs within the first 200 μs after reaction initiation; the presence of a uniquely stable Fe—O—O(H) peroxy species is not detected. The product of this rapid reaction is a heme
a
3
oxoferryl (Fe
IV
=O) species, which requires that an electron donor in addition to heme
a
3
and Cu
B
must be involved. The available evidence suggests that the additional donor is an amino acid side chain. Recent crystallographic data [Yoshikawa, S., Shinzawa-Itoh, K., Nakashima, R., Yaono, R., Yamashita, E., Inoue, N., Yao, M., Fei, M. J., Libeu, C. P., Mizushima, T.,
et al.
Science
, in press; Ostermeier, C., Harrenga, A., Ermler, U. & Michel, H. (1997)
Proc. Natl. Acad. Sci. USA
94, 10547–10553] show that one of the Cu
B
ligands, His240, is cross-linked to Tyr244 and that this cross-linked tyrosyl is ideally positioned to participate in dioxygen activation. We propose a mechanism for O—O bond cleavage that proceeds by concerted hydrogen atom transfer from the cross-linked His—Tyr species to produce the product oxoferryl species, Cu
B
2+
—OH
−
, and the tyrosyl radical. This mechanism provides molecular structures for two key intermediates that drive the proton pump in oxidase; moreover, it has clear analogies to the proposed O—O bond forming chemistry that occurs during O
2
evolution in photosynthesis.
ClC-5, the chloride channel mutated in Dent’s disease, colocalizes with the proton pump in endocytotically active kidney cells
Authors: Günther Willy , Lüchow Anke , Cluzeaud Françoise , Vandewalle Alain , Jentsch Thomas J.
Published: July 2002

Loss-of-function mutations of the ClC-5 chloride channel lead to Dent’s disease, a syndrome characterized by low molecular weight proteinuria, hypercalciuria, and kidney stones. We show that ClC-5 is expressed in renal proximal tubule cells, which normally endocytose proteins passing the glomerular filter. Expression is highest below the brush border in a region densely packed with endocytotic vesicles, where ClC-5 colocalizes with the H
+
-ATPase and with internalized proteins early after uptake. In intercalated cells of the collecting duct it again localizes to apical intracellular vesicles and colocalizes with the proton pump in α-intercalated cells. In transfected cells, ClC-5 colocalizes with endocytosed α
2
-macroglobulin. Cotransfection with a GTPase-deficient rab5 mutant leads to enlarged early endosomes that stain for ClC-5. We suggest that ClC-5 may be essential for proximal tubular endocytosis by providing an electrical shunt necessary for the efficient acidification of vesicles in the endocytotic pathway, explaining the proteinuria observed in Dent’s disease.
ZK200775: A phosphonate quinoxalinedione AMPA antagonist for neuroprotection in stroke and trauma
Authors: Turski Lechoslaw , Huth Andreas , Sheardown Malcolm , McDonald Fiona , Neuhaus Roland , Schneider Herbert H. , Dirnagl Ulrich , Wiegand Frank , Jacobsen Poul , Ottow Eckhard
Published: July 2002

Stroke and head trauma are worldwide public health problems and leading causes of death and disability in humans, yet, no adequate neuroprotective treatment is available for therapy. Glutamate antagonists are considered major drug candidates for neuroprotection in stroke and trauma. However,
N
-methyl-
d
-aspartate antagonists failed clinical trials because of unacceptable side effects and short therapeutic time window. α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) antagonists derived from the quinoxalinedione scaffold cannot be used in humans because of their insolubility and resulting renal toxicity. Therefore, achieving water solubility of quinoxalinediones without loss of selectivity and potency profiles becomes a major challenge for medicinal chemistry. One of the major tenets in the chemistry of glutamate antagonists is that the incorporation of phosphonate into the glutamate framework results in preferential
N
-methyl-
d
-aspartate antagonism. Therefore, synthesis of phosphonate derivatives of quinoxalinediones was not pursued because of a predicted loss of their selectivity toward AMPA. Here, we report that introduction of a methylphosphonate group into the quinoxalinedione skeleton leaves potency as AMPA antagonists and selectivity for the AMPA receptor unchanged and dramatically improves solubility. One such novel phosphonate quinoxalinedione derivative and competitive AMPA antagonist ZK200775 exhibited a surprisingly long therapeutic time window of >4 h after permanent occlusion of the middle cerebral artery in rats and was devoid of renal toxicity. Furthermore, delayed treatment with ZK200775 commencing 2 h after onset of reperfusion in transient middle cerebral artery occlusion resulted in a dramatic reduction of the infarct size. ZK200775 alleviated also both cortical and hippocampal damage induced by head trauma in the rat. These observations suggest that phosphonate quinoxalinedione-based AMPA antagonists may offer new prospects for treatment of stroke and trauma in humans.
Promoter specificity determinants of T7 RNA polymerase
Authors: Rong Minqing , He Biao , McAllister William T. , Durbin Russell K.
Published: July 2002

The high specificity of T7 RNA polymerase (RNAP) for its promoter sequence is mediated, in part, by a specificity loop (residues 742–773) that projects into the DNA binding cleft (1). Previous work demonstrated a role for the amino acid residue at position 748 (N748) in this loop in discrimination of the base pairs (bp) at positions −10 and −11 (2). A comparison of the sequences of other phage RNAPs and their promoters suggested additional contacts that might be important in promoter recognition. We have found that changing the amino acid residue at position 758 in T7 RNAP results in an enzyme with altered specificity for the bp at position −8. The identification of two amino acid:base pair contacts (i.e., N748 with the bp at −10 and −11, and Q758 with the bp at −8) provides information concerning the disposition of the specificity loop relative to the upstream region of the promoter. The results suggest that substantial rearrangements of the loop (and/or the DNA) are likely to be required to allow these amino acids to interact with their cognate base pairs during promoter recognition.
Muscle protein synthesis by positron-emission tomography with
l
-[methyl-
11
C]methionine in adult humans
Authors: Fischman Alan J. , Yu Yong M. , Livni E. , Babich John W. , Young Vernon R. , Alpert Nathaniel M. , Tompkins Ronald G.
Published: July 2002

Existing methods for assessing protein synthetic rates (PSRs) in human skeletal muscle are invasive and do not readily provide information about individual muscle groups. Recent studies in canine skeletal muscle yielded PSRs similar to results of simultaneous stable isotope measurements using
l
-[1-
13
C, methyl-
2
H
3
]methionine, suggesting that positron-emission tomography (PET) with
l
-[methyl-
11
C]methionine could be used along with blood sampling and a kinetic model to provide a less invasive, regional assessment of PSR. We have extended and refined this method in an investigation with healthy volunteers studied in the postabsorptive state. They received ≈25 mCi of
l
-[methyl-
11
C]methionine with serial PET imaging of the thighs and arterial blood sampling for a period of 90 min. Tissue and metabolite-corrected arterial blood time activity curves were fitted to a three-compartment model. PSR (nmol methionine⋅min
−1
⋅g muscle tissue
−1
) was calculated from the fitted parameter values and the plasma methionine concentrations, assuming equal rates of protein synthesis and degradation. Pooled mean PSR for the anterior and posterior sites was 0.50 ± 0.040. When converted to a fractional synthesis rate for mixed proteins in muscle, assuming a protein-bound methionine content of muscle tissue, the value of 0.125 ± 0.01%⋅h
−1
compares well with estimates from direct tracer incorporation studies, which generally range from ≈0.05 to 0.09%⋅h
−1
. We conclude that PET can be used to estimate skeletal muscle PSR in healthy human subjects and that it holds promise for future
in vivo
, noninvasive studies of the influences of physiological factors, pharmacological manipulations, and disease states on this important component of muscle protein turnover and balance.
Chemical phenotype matching between a plant and its insect herbivore
Authors: Berenbaum May R. , Zangerl Arthur R.
Published: July 2002

Two potential outcomes of a coevolutionary interaction are an escalating arms race and stable cycling. The general expectation has been that arms races predominate in cases of polygenic inheritance of resistance traits and permanent cycling predominates in cases in which resistance is controlled by major genes. In the interaction between
Depressaria pastinacella
, the parsnip webworm, and
Pastinaca sativa
, the wild parsnip, traits for plant resistance to insect herbivory (production of defensive furanocoumarins) as well as traits for herbivore “virulence” (ability to metabolize furanocoumarins) are characterized by continuous heritable variation. Furanocoumarin production in plants and rates of metabolism in insects were compared among four midwestern populations; these traits then were classified into four clusters describing multitrait phenotypes occurring in all or most of the populations. When the frequency of plant phenotypes belonging to each of the clusters is compared with the frequency of the insect phenotypes in each of the clusters across populations, a remarkable degree of frequency matching is revealed in three of the populations. That frequencies of phenotypes vary among populations is consistent with the fact that spatial variation occurs in the temporal cycling of phenotypes; such processes contribute in generating a geographic mosaic in this coevolutionary interaction on the landscape scale. Comparisons of contemporary plant phenotype distributions with phenotypes of herbarium specimens collected 9–125 years ago from across a similar latitudinal gradient, however, suggest that for at least one resistance trait—sphondin concentration—interactions with webworms have led to escalatory change.
Insulin depletion leads to adipose-specific cell death in obese but not lean mice
Authors: Loftus Thomas M. , Kuhajda Francis P. , Lane M. Daniel
Published: July 2002

Mutation of the obese gene produces obesity, hyperinsulinemia, and compensatory “overexpression” of the defective gene. As insulin activates obese gene expression, it seemed possible that hyperinsulinemia might be responsible for overexpression of the gene. To address this question we rapidly neutralized circulating insulin by injection of an insulin antibody. Unexpectedly, insulin depletion in obese (
ob/ob
or
db/db
) mice caused massive adipose RNA degradation confirmed by histological analysis to result from adipocyte cell death by a largely necrotic mechanism. This effect was not observed in lean littermates and was completely corrected by coadministration of insulin. Comparison of multiple tissues demonstrated that the effect was restricted to adipose tissue. Insulin depletion in obese mice by administration of streptozotocin also led to cell death, but this death was less extensive and appeared to be apoptotic in mechanism. Thus insulin may promote the survival side of the physiological balance between adipocyte survival and death.
Attachment to the nuclear matrix mediates specific alterations in chromatin structure
Authors: Pemov Alexander , Bavykin Sergei , Hamlin Joyce L.
Published: July 2002

The DNA in eukaryotic chromosomes is organized into a series of loops that are permanently attached at their bases to the nuclear scaffold or matrix at sequences known as scaffold-attachment or matrix-attachment regions. At present, it is not clear what effect affixation to the nuclear matrix has on chromatin architecture in important regulatory regions such as origins of replication or the promoter regions of genes. In the present study, we have investigated cell-cycle-dependent changes in the chromatin structure of a well characterized replication initiation zone in the amplified dihydrofolate reductase domain of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Replication can initiate at any of multiple potential sites scattered throughout the 55-kilobase intergenic region in this domain, with two subregions (termed
ori-β
and
ori-γ
) being somewhat preferred. We show here that the chromatin in the
ori-β
and
ori-γ
regions undergoes dramatic alterations in micrococcal nuclease hypersensitivity as cells cross the G
1
/S boundary, but only in those copies of the amplicon that are affixed to the nuclear matrix. In contrast, the fine structure of chromatin in the promoter of the dihydrofolate reductase gene does not change detectably as a function of matrix attachment or cell-cycle position. We suggest that attachment of DNA to the nuclear matrix plays an important role in modulating chromatin architecture, and this could facilitate the activity of origins of replication.
Increased expression of adenylylcyclase type VI proportionately increases β-adrenergic receptor-stimulated production of cAMP in neonatal rat cardiac myocytes
Authors: Gao Meihua , Ping Peipei , Post Steven , Insel Paul A. , Tang Ruoying , Hammond H. Kirk
Published: July 2002

Cellular content of cAMP generated by activation of adenylylcyclase (AC; EC
4.6.1.1
) is a key determinant of functional responsiveness in the heart and other tissues. We have tested two hypotheses regarding the relationship between AC content and β-adrenergic receptor (βAR)-mediated signal transduction in cardiac myocytes. First, that AC content limits adrenergic signal transduction, and, second, that increased AC, independent of (βAR) number and G-protein content, yields a proportional increase in βAR-mediated transmembrane signaling. We used recombinant adenovirus to increase AC isoform VI (AC
VI
) expression in neonatal cardiac myocytes. Cells that overexpressed AC
VI
responded to agonist stimulation with marked increases in cAMP production in proportion to protein expressed. In parallel experiments performed on cells transfected with
lacZ
(control) or AC
VI
, [
3
H]forskolin binding, used to assess AC protein expression, was amplified 6-fold, while βAR-stimulated cAMP production from these cells was increased 7-fold. No changes in βAR number, or in the heterotrimeric GTP-binding proteins, Gαs or Gαi
2
, were observed. Previous studies indicate that increased cardiac expression of βAR or Gαs does not yield proportional increases in transmembrane adrenergic signaling. In contrast, the current data demonstrate that increased AC
VI
expression provides a proportional increase in β-adrenergic signal transduction. Our results show that the amount of AC sets a limit on transmembrane β-adrenergic signaling. We speculate that similar functional responses are possible in other cell types in which AC plays an important physiological role.
Escherichia coli
NusA is required for efficient RNA binding by phage HK022 Nun protein
Authors: Watnick Randolph S. , Gottesman Max E.
Published: July 2002

The Nun protein of phage HK022 is an RNA binding protein of the arginine-rich motif family. Nun binds the phage λ boxB RNA sequence (BOXB) on nascent λ transcripts and arrests transcription elongation. Binding to BOXB is inhibited by Zn
2+
and stimulated by the
Escherichia coli
NusA protein. Deletion of the Nun C-terminal region enhances BOXB binding and makes it independent of Zn
2+
and NusA. The C terminus of Nun thus appears to interfere with the N-terminal RNA binding motif. NusA relieves this interference by binding to the Nun C terminus and forming a complex with Nun and BOXB. However, NusA also inhibits transcription arrest
in vitro
, in the absence of the other Nus factors. Nun deleted for its C terminus fails to bind RNA polymerase (RNAP) (RNAP) or NusA
in vitro
or to arrest transcription
in vivo
or
in vitro
. Our findings are consistent with the idea that NusA inhibits transcription arrest by binding to the Nun C terminus, thus blocking the interaction between Nun and RNAP. NusG, NusB, and NusE factors restore transcription arrest, presumably by promoting transfer of Nun from NusA to RNAP.
Activation of Kv3.1 channels in neuronal spine-like structures may induce local potassium ion depletion
Authors: Wang Lu-Yang , Gan Li , Perney Teresa M. , Schwartz Ilsa , Kaczmarek Leonard K.
Published: July 2002

Spines are specialized neuronal membrane structures, often localized at sites where synaptic information is relayed from one cell to another in the central nervous system. By electron immunomicroscopy we have found that the mammalian
Shaw
family potassium channel Kv3.1 is localized on spine-like protrusions, adjacent to postsynaptic membranes of bushy cells in the cochlear nucleus. As direct characterization of the electrophysiological behavior of ion channels in such structures is difficult, we have used Kv3.1-transfected CHO cells to create artificial spine-like membrane compartments. Membrane patches were sucked into microelectrodes to form small, cell-attached vesicles with dimensions comparable to those of the neuronal structures. Currents mediated by the Kv3.1 channel in these vesicles undergo rapid and complete inactivation, in contrast to their noninactivating behavior in whole-cell recordings. This apparent inactivation is caused by the rapid depletion of K
+
from the vesicle and the slow refilling of K
+
into the vesicle compartment from the bulk cytoplasm. Our data provide evidence that compartmentalized ionic transients can be generated in spine-like membrane structures and support the view that the localization of ion channels in spine-like structures may influence responses to synaptic stimulation.
Retrotransposon R1Bm endonuclease cleaves the target sequence
Authors: Feng Qinghua , Schumann Gerald , Boeke Jef D.
Published: July 2002

The R1Bm element, found in the silkworm
Bombyx mori
, is a member of a group of widely distributed retrotransposons that lack long terminal repeats. Some of these elements are highly sequence-specific and others, like the human L1 sequence, are less so. The majority of R1Bm elements are associated with ribosomal DNA (rDNA). R1Bm inserts into 28S rDNA at a specific sequence; after insertion it is flanked by a specific 14-bp target site duplication of the 28S rDNA. The basis for this sequence specificity is unknown. We show that R1Bm encodes an enzyme related to the endonuclease found in the human L1 retrotransposon and also to the apurinic/apyrimidinic endonucleases. We expressed and purified the enzyme from bacteria and showed that it cleaves
in vitro
precisely at the positions in rDNA corresponding to the boundaries of the 14-bp target site duplication. We conclude that the function of the retrotransposon endonucleases is to define and cleave target site DNA.
Evolutionary analyses of the 12-kDa acidic ribosomal P-proteins reveal a distinct protein of higher plant ribosomes
Authors: Szick Kathleen , Springer Mark , Bailey-Serres Julia
Published: July 2002

The P-protein complex of eukaryotic ribosomes forms a lateral stalk structure in the active site of the large ribosomal subunit and is thought to assist in the elongation phase of translation by stimulating GTPase activity of elongation factor-2 and removal of deacylated tRNA. The complex in animals, fungi, and protozoans is composed of the acidic phosphoproteins P0 (35 kDa), P1 (11–12 kDa), and P2 (11–12 kDa). Previously we demonstrated by protein purification and microsequencing that ribosomes of maize (Zea maysL.) contain P0, one type of P1, two types of P2, and a distinct P1/P2 type protein designated P3. Here we implemented distance matrices, maximum parsimony, and neighbor-joining analyses to assess the evolutionary relationships between the 12 kDa P-proteins of maize and representative eukaryotic species. The analyses identify P3, found to date only in mono- and dicotyledonous plants, as an evolutionarily distinct P-protein. Plants possess three distinct groups of 12 kDa P-proteins (P1, P2, and P3), whereas animals, fungi, and protozoans possess only two distinct groups (P1 and P2). These findings demonstrate that the P-protein complex has evolved into a highly divergent complex with respect to protein composition despite its critical position within the active site of the ribosome.
Linkage analysis and comparative mapping of canine progressive rod–cone degeneration (
prcd
) establishes potential locus homology with retinitis pigmentosa (RP17) in humans
Authors: Acland Gregory M. , Ray Kunal , Mellersh Cathryn S. , Gu Weikuan , Langston Amelia A. , Rine Jasper , Ostrander Elaine A. , Aguirre Gustavo D.
Published: July 2002

Progressive rod–cone degeneration (
prcd
) is the most widespread hereditary retinal disease leading to blindness in dogs and phenotypically is the canine counterpart of retinitis pigmentosa (RP) in humans. In previous efforts to identify the genetic locus for
prcd,
the canine homologs for many of the genes causally associated with RP in humans, such as
RHO
,
PDE6B
, and
RDS
/peripherin, have been excluded. In parallel with a recent undertaking to establish a framework map of the canine genome, multiple
prcd
-informative pedigrees have been typed with a panel of more than 100 anchor loci and microsatellite-based markers. Identification of a linkage group flanking
prcd
([
TK1
,
GALK1
,
prcd
]–[
MYL4
,
C09.173
,
C09.2263
]–
RARA–C09.250–C09.474–NF1
) localizes
prcd
close to the centromeric end of canine chromosome 9 (CFA9), and excludes
RARA
as a candidate gene. The conserved synteny of this region of CFA9 and distal human chromosome 17q establishes the potential locus homology of
prcd
in the dog with RP17, a human retinitis pigmentosa locus for which no gene has yet been identified. Assignment of the
prcd
disease locus to an identified canine autosome represents a powerful application of the developing canine linkage map in medical genetics. The usefulness of this approach is further demonstrated by identification of the correspondence of the
prcd
interval to homologous human and mouse chromosomal regions. The rapid progress that is now occurring in the field of canine genetics will expedite the identification of the genes underlying many of the inherited traits and diseases that make the dog a unique asset for the study of mammalian traits.
Codon reassignment and amino acid composition in hemichordate mitochondria
Authors: Castresana Jose , Feldmaier-Fuchs Gertraud , Pääbo Svante
Published: July 2002

In the mitochondrial genome of the hemichordateBalanoglossus carnosus, the codon AAA, which is assigned to lysine in most metazoans but to asparagine in echinoderms, is absent. Furthermore, the lysine tRNA gene carries an anticodon substitution that renders its gene product unable to decode AAA codons, whereas the asparagine tRNA gene has not changed to encode a tRNA with the ability to recognize AAA codons. Thus, the hemichordate mitochondrial genome can be regarded as an intermediate in the process of reassignment of mitochondrial AAA codons, where most metazoans represent the ancestral situation and the echinoderms the derived situation. This lends support to the codon capture hypothesis. We also show that the reassignment of the AAA codon is associated with a reduction in the relative abundance of lysine residues in mitochondrial proteins.