bluuebunny/crossref_metadata_2025 huggingface dataset:
Phase formation in mechanically alloyed Nb-Al powders
Authors: Hellstern E. , Schultz L. , Bormann R. , Lee D.
Published: July 2002

Nb-Al alloys have been prepared by mechanical alloying of elemental crystalline powders and investigated with x-ray diffraction and transmission electron microscopy. Crystalline equilibrium phases form for Al-rich alloys. Amorphous phase formation is observed for the Nb50Al50 composition. Mechanically alloyed powders with more than 70 at. % Nb exhibit the metastable bcc (A2) solid solution which transforms after thermal annealing into the A15 phase. Formation of the different phases can be understood by considering the free-energy curves of the amorphous and bcc phases as calculated from the phase diagram and additional thermodynamic data.
Methionine-independent initiation of translation in the capsid protein of an insect RNA virus
Authors: Sasaki Jun , Nakashima Nobuhiko
Published: July 2002

Protein synthesis is believed to be initiated with the amino
acid methionine because the AUG translation initiation codon of mRNAs
is recognized by the anticodon of initiator methionine transfer RNA. A
group of positive-stranded RNA viruses of insects, however, lacks an
AUG translation initiation codon for their capsid protein gene, which
is located at the downstream part of the genome. The capsid protein of
one of these viruses,
Plautia stali
intestine virus, is
synthesized by internal ribosome entry site-mediated translation. Here
we report that methionine is not the initiating amino acid in the
translation of the capsid protein in this virus. Its translation is
initiated with glutamine encoded by a CAA codon that is the first codon
of the capsid-coding region. The nucleotide sequence immediately
upstream of the capsid-coding region interacts with a loop segment in
the stem–loop structure located 15–43 nt upstream of the 5′ end of
the capsid-coding region. The pseudoknot structure formed by this base
pair interaction is essential for translation of the capsid protein.
This mechanism for translation initiation differs from the conventional
one in that the initiation step controlled by the initiator methionine
transfer RNA is not necessary.
spr-2
, a suppressor of the egg-laying defect caused by loss of
sel-12
presenilin in
Caenorhabditis
elegans
, is a member of the SET protein subfamily
Authors: Wen Chenhui , Levitan Diane , Li Xiajun , Greenwald Iva
Published: July 2002

Presenilin plays critical roles in the genesis of
Alzheimer's disease and in LIN-12/Notch signaling during
development. Here, we describe a screen for genes that influence
presenilin level or activity in
Caenorhabditis
elegans
. We identified four
spr
(suppressor of
presenilin) genes by reverting the egg-laying defective phenotype
caused by a null allele of the
sel-12
presenilin gene.
We analyzed the
spr-2
gene in some detail. We show that
loss of
spr-2
activity suppresses the egg-laying
defective phenotype of different
sel-12
alleles and
requires activity of the
hop-1
presenilin gene,
suggesting that suppression is accomplished by elevating presenilin
activity rather than by bypassing the need for presenilin activity. We
also show that SPR-2 is a nuclear protein and is a member of a protein
subfamily that includes human SET, which has been identified in
numerous different biochemical assays and at translocation breakpoints
associated with a subtype of acute myeloid leukemia.
The large GTPase dynamin regulates actin comet formation and movement in living cells
Authors: Orth James D. , Krueger E. W. , Cao H. , McNiven Mark A.
Published: July 2002

The large GTPase dynamin (Dyn2) has been demonstrated
by us and others to interact with several different actin-binding
proteins. To define how Dyn2 might participate in actin dynamics in
livings cells we have expressed green fluorescent protein (GFP)-tagged
Dyn2 in cultured cells and observed labeling of comet-like vesicles and
macropinosomes. The comet structures progressed with a constant
velocity and were reminiscent of actin comets associated with motile
vesicles in cells expressing type I phosphatidylinositol phosphate
5-kinases. Based on these observations we sought to determine whether
Dyn2 is an integral component of actin comets. Cells expressing type I
phosphatidylinositol phosphate 5-kinase and Dyn2-GFP revealed a
prominent colocalization of Dyn2 and actin in comet structures.
Interestingly, comet formation and motility were normal in cells
expressing wild-type Dyn2-GFP but altered markedly in Dyn2
mutant-expressing cells. Dyn2K44A-GFP mutant cells displayed a
significant reduction in comet number, length, velocity, and efficiency
of movement. In contrast, comets in cells expressing Dyn2ΔPRD-GFP
appeared dark and did not incorporate the mutant Dyn2 protein,
indicating that the proline-rich domain (PRD) is required for Dyn2
recruitment. Further, these comets were significantly longer and slower
than those in control cells. These findings demonstrate a role
for Dyn2 in actin-based vesicle motility.
Iron uptake in ferritin is blocked by binding of [Cr(TREN)(H
2
O)(OH)]
2+
, a slow dissociating model for [Fe(H
2
O)
6
]
2+
Authors: Barnés Carmen M. , Theil Elizabeth C. , Raymond Kenneth N.
Published: July 2002

Ferritin concentrates iron as a hydrous ferric oxide in a protein cavity (8 nm in diameter) by using eight pores along the threefold symmetry axes of the octahedral supramolecular structure. The role of ligand exchange in the entry of Fe(II) hexahydrate into ferritin protein has been studied with [Cr(TREN)(H
2
O)(OH)]
2+
[TREN = N(CH
2
CH
2
NH
2
)
3
], a model for Fe(H
2
O)
6
2+
with only two exchangeable ligands. The results show that five different ferritin proteins, varying in pore structure, oxidation sites, and nucleation sites, bind Cr(TREN) at functional protein sites, based on inhibition of iron mineralization and oxidation. Properties of Cr(TREN)–ferritin adducts include an increased isoelectric point, a shift in the Cr(TREN) UV/vis spectrum consistent with exchange of water for protein carboxylate or thiolate ligands, binding affinities of 50–250 μM, and a slow rate of dissociation (
k
= 4 × 10
−6
sec
−1
). The relationship of Cr(TREN) inhibition of iron oxidation and mineralization by Cr(TREN) to the known structures of the various ferritins tested suggests that Cr(TREN) plugs the ferritin pores, obstructing Fe(II) entry in folded and unfolded pores. Because only two exchangeable waters are sufficient for pore binding of Cr(TREN), the physiological Fe(II) donor must bind to the pore with few exchangeable ligands. These results show the advantage of using stable model complexes to explore properties of transient Fe–protein complexes during Fe mineralization in ferritin.
The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity
Authors: Staudinger Jeff L. , Goodwin Bryan , Jones Stacey A. , Hawkins-Brown Diane , MacKenzie Kathleen I. , LaTour Anne , Liu Yaping , Klaassen Curtis D. , Brown Kathleen K. , Reinhard John , Willson Timothy M. , Koller Beverly H. , Kliewer Steven A.
Published: July 2002

The pregnane X receptor (PXR) is the molecular target for catatoxic
steroids such as pregnenolone 16α-carbonitrile (PCN), which induce
cytochrome P450 3A (
CYP3A
) expression and protect the
body from harmful chemicals. In this study, we demonstrate that PXR is
activated by the toxic bile acid lithocholic acid (LCA) and its 3-keto
metabolite. Furthermore, we show that PXR regulates the expression of
genes involved in the biosynthesis, transport, and metabolism of bile
acids including cholesterol 7α-hydroxylase (
Cyp7a1
)
and the Na
+
-independent organic anion transporter 2
(
Oatp2
). Finally, we demonstrate that activation of PXR
protects against severe liver damage induced by LCA. Based on these
data, we propose that PXR serves as a physiological sensor of LCA, and
coordinately regulates gene expression to reduce the concentrations of
this toxic bile acid. These findings suggest that PXR agonists may
prove useful in the treatment of human cholestatic liver disease.
Solution structure of the A loop of 23S ribosomal RNA
Authors: Blanchard Scott C. , Puglisi Joseph D.
Published: July 2002

The A loop is an essential RNA component of the ribosome
peptidyltransferase center that directly interacts with aminoacyl
(A)-site tRNA. The A loop is highly conserved and contains a ubiquitous
2′-
O
-methyl ribose modification at position U2552. Here,
we present the solution structure of a modified and unmodified A-loop
RNA to define both the A-loop fold and the structural impact of the
U2552 modification. Solution data reveal that the A-loop RNA has a
compact structure that includes a noncanonical base pair between C2556
and U2552. NMR evidence is presented that the N3 position of C2556 has
a shifted pKa and that protonation at C2556-N3 changes the C-U pair
geometry. Our data indicate that U2552 methylation modifies the A-loop
fold, in particular the dynamics and position of residues C2556 and
U2555. We compare our structural data with the structure of the A loop
observed in a recent 50S crystal structure [Ban, N., Nissen, P.,
Hansen, J., Moore, P. B. & Steitz, T. A. (2000)
Science
289, 905–920; Nissen, P., Hansen, J.,
Ban, N., Moore, P. B. & Steitz, T. A. (2000)
Science
289, 920–930]. The solution and crystal
structures of the A loop are dramatically different, suggesting that a
structural rearrangement of the A loop must occur on docking into the
peptidyltransferase center. Possible roles of this docking event, the
shifted pKa of C2556 and the U2552 2′-
O
-methylation in the
mechanism of translation, are discussed.
Genes, pseudogenes, and Alu sequence organization across human chromosomes 21 and 22
Authors: Chen Chingfer , Gentles Andrew J. , Jurka Jerzy , Karlin Samuel
Published: July 2002

Human chromosomes 21 and 22 (mainly the q-arms) were the first complete parts of the human genome released. Our analysis of genes, pseudogenes (Ψg), and Alu repeats across these chromosomes include the following findings: The number of gene structures containing untranslated exons exceeds 25%; the terminal exon tends to be the largest among exons, whereas, the initial intron tends to be the largest among introns; single-exon gene length is approximately the mean gene exon number times the mean internal exon length; processed Ψg lengths are on average approximately the same as single-exon gene length; and the G+C content and length of genes are uncorrelated. The counts and distribution of genes, Ψg, and Alu sequences and G+C variation are evaluated with respect to clusters and overdispersions. Other assessments concern comparisons of intergenic lengths, properties of Ψg sequences, and correlations between Alu and Ψg sequences.
The gas phase reaction of singlet dioxygen with water: A water-catalyzed mechanism
Authors: Xu Xin , Muller Richard P. , Goddard William A.
Published: July 2002

Stimulated by the recent surprising results from Wentworth
et al.
[Wentworth, A. D., Jones, L. H., Wentworth, P., Janda, K. D. & Lerner, R. A. (2000)
Proc. Natl. Acad. Sci. USA
97, 10930–10935] that Abs efficiently catalyze the conversion of molecular singlet oxygen (
1
O
2
) plus water to hydrogen peroxide (HOOH), we used quantum chemical methods (B3LYP density functional theory) to delineate the most plausible mechanisms for the observed efficient conversion of water to HOOH. We find two reasonable pathways. In Pathway I, (
i
) H
2
O catalyzes the reaction of
1
O
2
with a second water to form HOOOH; (
ii
) two HOOOH form a dimer, which rearranges to form the HOO-HOOO + H
2
O complex; (
iii
) HOO-HOOO rearranges to HOOH-OOO, which subsequently reacts with H
2
O to form H
2
O
4
+ HOOH; and (
iv
) H
2
O
4
rearranges to the cyclic dimer (HO
2
)
2
, which in turn forms HOOH plus
1
O
2
or
3
O
2
. Pathway II differs in that step
ii
is replaced with the reaction between HOOOH and
1
O
2
, leading to the formation of HOO-HOOO. This then proceeds to similar products. For a system with
18
O H
2
O, Pathway I leads to a 2.2:1 ratio of
16
O:
18
O in the product HOOH, whereas Pathway II leads to 3:1. These ratios are in good agreement with the 2.2:1 ratio observed in isotope experiments by Wentworth
et al.
These mechanisms lead to two HOOH per initial
1
O
2
or one, depending on whether the product of step
iv
is
1
O
2
or
3
O
2
, in good agreement with the experimental result of 2.0. In addition to the Ab-induced reactions, the hydrogen polyoxides (H
2
O
3
and H
2
O
4
) formed in these mechanisms and their decomposition product polyoxide radicals (HO
2
, HO
3
) may play a role in combustion, explosions, atmospheric chemistry, and the radiation chemistry in aqueous systems.
Comparative evaluation of the antitumor activity of antiangiogenic proteins delivered by gene transfer
Authors: Kuo Calvin J. , Farnebo Filip , Yu Evan Y. , Christofferson Rolf , Swearingen Rebecca A. , Carter Robert , von Recum Horst A. , Yuan Jenny , Kamihara Junne , Flynn Evelyn , D'Amato Robert , Folkman Judah , Mulligan Richard C.
Published: July 2002

Although the systemic administration of a number of different gene
products has been shown to result in the inhibition of angiogenesis and
tumor growth in different animal tumor models, the relative potency of
those gene products has not been studied rigorously. To address this
issue, recombinant adenoviruses encoding angiostatin, endostatin, and
the ligand-binding ectodomains of the vascular endothelial growth
factor receptors Flk1, Flt1, and neuropilin were generated and used to
systemically deliver the different gene products in several different
preexisting murine tumor models. Single i.v. injections of viruses
encoding soluble forms of Flk1 or Flt1 resulted in ≈80% inhibition
of preexisting tumor growth in murine models involving both murine
(Lewis lung carcinoma, T241 fibrosarcoma) and human (BxPC3 pancreatic
carcinoma) tumors. In contrast, adenoviruses encoding angiostatin,
endostatin, or neuropilin were significantly less effective. A strong
correlation was observed between the effects of the different viruses
on tumor growth and the activity of the viruses in the inhibition of
corneal micropocket angiogenesis. These data underscore the need for
comparative analyses of different therapeutic approaches that target
tumor angiogenesis and provide a rationale for the selection of
specific antiangiogenic gene products as lead candidates for use in
gene therapy approaches aimed at the treatment of malignant and ocular
disorders.
Distinct SNARE complexes mediating membrane fusion in Golgi transport based on combinatorial specificity
Authors: Parlati Francesco , Varlamov Oleg , Paz Keren , McNew James A. , Hurtado David , Söllner Thomas H. , Rothman James E.
Published: July 2002

Syntaxin-5 (Sed5) is the only syntaxin needed for transport into and across the yeast Golgi, raising the question of how a single syntaxin species could mediate vesicle transport in both the anterograde and the retrograde direction within the stack. Sed5 is known to combine with two light chains (Bos1 and Sec22) to form the t-SNARE needed to receive vesicles from the endoplasmic reticulum. However, the yeast Golgi contains several other potential light chains with which Sed5 could potentially combine to form other t-SNAREs. To explore the degree of specificity in the choice of light chains by a t-SNARE, we undertook a comprehensive examination of the capacity of all 21 Sed5-based t-SNAREs that theoretically could assemble in the yeast Golgi to fuse with each of the 7 potential v-SNAREs also present in this organelle. Only one additional of these 147 combinations was fusogenic. This functional proteomic strategy thereby revealed a previously uncharacterized t-SNARE in which Sed5 is the heavy chain and Gos1 and Ykt6 are the light chains, and whose unique cognate v-SNARE is Sft1. Immunoprecipitation experiments confirmed the existence of this complex
in vivo
. Fusion mediated by this second Golgi SNAREpin is topologically restricted, and existing genetic and morphologic evidence implies that it is used for transport across the Golgi stack. From this study, together with the previous functional proteomic analyses which have tested 275 distinct quaternary SNARE combinations, it follows that the fusion potential and transport pathways of the yeast cell can be read out from its genome sequence according to the SNARE hypothesis with a predictive accuracy of about 99.6%.
Evidence for interpersonal violence in the St. Césaire Neanderthal
Authors: Zollikofer Christoph P. E. , Ponce de León Marcia S. , Vandermeersch Bernard , Lévêque François
Published: July 2002

The St. Césaire 1 Neanderthal skeleton of a young adult individual is unique in its association with Châtelperronian artifacts from a level dated to
ca.
36,000 years ago. Computer-tomographic imaging and computer-assisted reconstruction of the skull revealed a healed fracture in the cranial vault. When paleopathological and forensic diagnostic standards are applied, the bony scar bears direct evidence for the impact of a sharp implement, which was presumably directed toward the individual during an act of interpersonal violence. These findings add to the evidence that Neanderthals used implements not only for hunting and food processing, but also in other behavioral contexts. It is hypothesized that the high intra-group damage potential inherent to weapons might have represented a major factor during the evolution of hominid social behavior.
Commitment to natural killer cells requires the helix–loop–helix inhibitor Id2
Authors: Ikawa Tomokatsu , Fujimoto Shinji , Kawamoto Hiroshi , Katsura Yoshimoto , Yokota Yoshifumi
Published: July 2002

We have previously described how T and natural killer (NK) lineage commitment proceeds from common T/NK progenitors (p-T/NK) in the murine fetal thymus (FT), with the use of a clonal assay system capable of discriminating p-T/NK from unipotent T or NK lineage-committed progenitors (p-T and p-NK, respectively). The molecular mechanisms controlling the commitment processes, however, are yet to be defined. In this study, we investigated the progenitor activity of FT cells from Id2−/−mice that exhibit defective NK cell development. In the Id2−/−FT, NK cells were greatly reduced, and a cell population that exclusively contains p-NK in the wild-type thymus was completely missing. Id2−/−FT progenitors were unable to differentiate into NK cells in IL-2-supplemented-FT organ culture. Single progenitor analysis demonstrated that all Id2−/−fetal thymic progenitors are destined for the T cell lineage, whereas progenitors for T/NK, T, and NK cell lineages were found in the control. Interestingly, the total progenitor number was similar between Id2−/−and Id2+/+embryos analyzed. Expression of Id2 was correlated with p-NK activity. Our results suggest that Id2 is indispensable in thymic NK cell development, where it most probably restricts bipotent T/NK progenitors to the NK cell lineage.
Asymmetrical flagellar rotation in
Borrelia burgdorferi
nonchemotactic mutants
Authors: Li Chunhao , Bakker Richard G. , Motaleb Md. Abdul , Sartakova Marina L. , Cabello Felipe C. , Charon Nyles W.
Published: July 2002

The Lyme disease spirochete
Borrelia burgdorferi
has bundles of periplasmic flagella subpolarly located at each cell end. These bundles rotate in opposite directions during translational motility. When not translating, they rotate in the same direction, and the cells flex. Here, we present evidence that asymmetrical rotation of the bundles during translation does not depend upon the chemotaxis signal transduction system. The histidine kinase CheA is known to be an essential component in the signaling pathway for bacterial chemotaxis. Mutants of
cheA
in flagellated bacteria continually rotate their flagella in one direction.
B. burgdorferi
has two copies of
cheA
designated
cheA1
and
cheA2
. Both genes were found to be expressed in growing cells. We reasoned that if chemotaxis were essential for asymmetrical rotation of the flagellar bundles, and if the flagellar motors at both cell ends were identical, inactivation of the two
cheA
genes should result in cells that constantly flex. To test this hypothesis, the signaling pathway was completely blocked by constructing the double mutant
cheA1∷kan cheA2∷ermC
. This double mutant was deficient in chemotaxis. Rather than flexing, it failed to reverse, and it continually translated only in one direction. Video microscopy of mutant cells indicated that both bundles actively rotated. The results indicate that asymmetrical rotation of the flagellar bundles of spirochetes does not depend upon the chemotaxis system but rather upon differences between the two flagellar bundles. We propose that certain factors within the spirochete localize at the flagellar motors at one end of the cell to effect this asymmetry.
Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function
Authors: Kopke Richard D. , Jackson Ronald L. , Li Geming , Rasmussen Mark D. , Hoffer Michael E. , Frenz Dorothy A. , Costello Michael , Schultheiss Peter , Van de Water Thomas R.
Published: July 2002

The vestibules of adult guinea pigs were lesioned with gentamicin
and then treated with perilymphatic infusion of either of two growth
factor mixtures (i.e., GF I or GF II). GF I contained transforming
growth factor α (TGFα), insulin-like growth factor type one
(IGF-1), and retinoic acid (RA), whereas GF II contained those three
factors and brain-derived neurotrophic factor. Treatment with GF I
significantly enhanced vestibular hair cell renewal in ototoxin-damaged
utricles and the maturation of stereociliary bundle morphology. The
addition of brain-derived neurotrophic factor to the GF II infusion
mixture resulted in the return of type 1 vestibular hair cells in
ototoxin-damaged cristae, and improved vestibular function. These
results suggest that growth factor therapy may be an effective
treatment for balance disorders that are the result of hair cell
dysfunction and/or loss.
Identification and characterization of a second melanin-concentrating hormone receptor, MCH-2R
Authors: Sailer Andreas W. , Sano Hideki , Zeng Zhizhen , McDonald Terrence P. , Pan Jie , Pong Sheng-Shung , Feighner Scott D. , Tan Carina P. , Fukami Takehiro , Iwaasa Hisashi , Hreniuk Donna L. , Morin Nancy R. , Sadowski Sharon J. , Ito Makoto , Ito Masahiko , Bansal Alka , Ky Betty , Figueroa David J. , Jiang Qingping , Austin Christopher P. , MacNeil Douglas J. , Ishihara Akane , Ihara Masaki , Kanatani Akio , Van der Ploeg Lex H. T. , Howard Andrew D. , Liu Qingyun
Published: July 2002

Melanin-concentrating hormone (MCH) is a 19-aa cyclic neuropeptide originally isolated from chum salmon pituitaries. Besides its effects on the aggregation of melanophores in fish several lines of evidence suggest that in mammals MCH functions as a regulator of energy homeostasis. Recently, several groups reported the identification of an orphan G protein-coupled receptor as a receptor for MCH (MCH-1R). We hereby report the identification of a second human MCH receptor termed MCH-2R, which shares about 38% amino acid identity with MCH-1R. MCH-2R displayed high-affinity MCH binding, resulting in inositol phosphate turnover and release of intracellular calcium in mammalian cells. In contrast to MCH-1R, MCH-2R signaling is not sensitive to pertussis toxin and MCH-2R cannot reduce forskolin-stimulated cAMP production, suggesting an exclusive G
α
q
coupling of the MCH-2R in cell-based systems. Northern blot and
in situ
hybridization analysis of human and monkey tissue shows that expression of MCH-2R mRNA is restricted to several regions of the brain, including the arcuate nucleus and the ventral medial hypothalamus, areas implicated in regulation of body weight. In addition, the human MCH-2R gene was mapped to the long arm of chromosome 6 at band 6q16.2–16.3, a region reported to be associated with cytogenetic abnormalities of obese patients. The characterization of a second mammalian G protein-coupled receptor for MCH potentially indicates that the control of energy homeostasis in mammals by the MCH neuropeptide system may be more complex than initially anticipated.
Analysis of telomerase catalytic subunit mutants
in vivo
and
in vitro
in
Schizosaccharomyces pombe
Authors: Haering Christian H. , Nakamura Toru M. , Baumann Peter , Cech Thomas R.
Published: July 2002

The chromosome end-replicating enzyme telomerase is composed of a
template-containing RNA subunit, a reverse transcriptase (TERT), and
additional proteins. The importance of conserved amino acid residues in
Trt1p, the TERT of
Schizosaccharomyces pombe
, was
tested. Mutation to alanine of the proposed catalytic aspartates in
reverse transcriptase motifs A and C and of conserved amino acids in
motifs 1 and B′ resulted in defective growth, progressive loss of
telomeric DNA, and loss of detectable telomerase enzymatic activity
in vitro
. Mutation of the phenylalanine (F) in the
conserved FYxTE of telomerase-specific motif T had no phenotype
in vivo
or
in vitro
whereas mutation of a
conserved amino acid in RT motif 2 had an intermediate effect. In
addition to identifying single amino acids of TERT required for
telomere maintenance in the fission yeast, this work provides useful
tools for
S. pombe
telomerase research: a functional
epitope-tagged version of Trt1p that allows detection of the protein
even in crude cellular extracts, and a convenient and robust
in
vitro
enzymatic activity assay based on immunopurification of
telomerase.
Glutamate translocation of the neuronal glutamate transporter EAAC1 occurs within milliseconds
Authors: Grewer Christof , Watzke Natalie , Wiessner Michael , Rauen Thomas
Published: July 2002

The activity of glutamate transporters is essential for the temporal and spatial regulation of the neurotransmitter concentration in the synaptic cleft, and thus, is crucial for proper excitatory signaling. Initial steps in the process of glutamate transport take place within a time scale of microseconds to milliseconds. Here we compare the steady-state and pre-steady-state kinetics of the neuronal heterologously expressed glutamate transporter EAAC1, cloned from the mammalian retina. Rapid transporter dynamics, as measured by using whole-cell current recordings, were resolved by applying the laser-pulse photolysis technique of caged glutamate with a time resolution of 100 μs. EAAC1-mediated pre-steady-state currents are composed of two components: A transport current generated by substrate-coupled charge translocation across the membrane and an anion current that is not stoichiometrically coupled to glutamate transport. The two currents were temporally resolved and studied independently. Our results indicate a rapid glutamate-binding step occurring on a submillisecond time scale that precedes subsequent slower electrogenic glutamate translocation across the membrane within a few milliseconds. The voltage-dependent steady-state turnover time constant of the transporter is about 1/10 as fast, indicating that glutamate translocation is not rate limiting. A third process, the transition to an anion-conducting state, is delayed with respect to the onset of glutamate transport. These rapid transporter reaction steps are summarized in a sequential shuttle model that quantitatively accounts for the results obtained here and are discussed regarding their functional importance for glutamatergic neurotransmission in the central nervous system.
Paradoxical intrathymic positive selection in mice with only a covalently presented agonist peptide
Authors: Viret Christophe , He Xin , Janeway Charles A.
Published: July 2002

The Y-AemAb and the 1H3.1 αβ T cell antigen receptor (TCR) are both specific for the I-Eα52–68 peptide bound to the I-Abmajor histocompatibility complex (MHC) class II molecule. Antigen-presenting cells (APCs) from I-Ab+mice with a natural or transgenic (Tg) I-Eα chain activate mature 1H3.1 T cells and cause the deletion of 1H3.1 TCR Tg thymocytes. However, 1H3.1 T cells were neither activated nor inactivated by confrontation with APCs from I-Ab-Ep mice in which I-Abmolecules are occupied only by the covalently associated Eα52–68 peptide. Instead, immature 1H3.1 TCR Tg thymocytes were efficiently positively selected into the CD4 lineage in the I-Ab-Ep thymus. This selection relied on specific recognition of the Eα52–68/I-Abcomplex because it was blocked by Y-Ae. 1H3.1 TCR Tg T cells maturing in the I-Ab-Ep thymus efficiently populated the periphery, displayed a naive phenotype, and were specifically reactive to the Eα52–68 peptide or to I-Ab+I-Eα+APCs, indicating that 1H3.1 T cells were not antagonized in I-Ab-Ep mice. The data identify major histocompatibility complex class II molecules with only a covalently attached self-peptide as a ligand forin vivopositive selection of T cells specific for the same peptide.
Relative role of heme nitrosylation and β-cysteine 93 nitrosation in the transport and metabolism of nitric oxide by hemoglobin in the human circulation
Authors: Gladwin Mark T. , Ognibene Frederick P. , Pannell Lewis K. , Nichols James S. , Pease-Fye Margaret E. , Shelhamer James H. , Schechter Alan N.
Published: July 2002

To quantify the reactions of nitric oxide (NO) with hemoglobin under physiological conditions and to test models of NO transport on hemoglobin, we have developed an assay to measure NO–hemoglobin reaction products in normal volunteers, under basal conditions and during NO inhalation. NO inhalation markedly raised total nitrosylated hemoglobin levels, with a significant arterial–venous gradient, supporting a role for hemoglobin in the transport and delivery of NO. The predominant species accounting for this arterial–venous gradient is nitrosyl(heme)hemoglobin. NO breathing increases
S
-nitrosation of hemoglobin β-chain cysteine 93, however only to a fraction of the level of nitrosyl(heme)hemoglobin and without a detectable arterial–venous gradient. A strong correlation between methemoglobin and plasma nitrate formation was observed, suggesting that NO metabolism is a primary physiological cause of hemoglobin oxidation. Our results demonstrate that NO–heme reaction pathways predominate
in vivo
, NO binding to heme groups is a rapidly reversible process, and
S
-nitrosohemoglobin formation is probably not a primary transport mechanism for NO but may facilitate NO release from heme.
Single-cell analysis of signal transduction in CD4 T cells stimulated by antigenin vivo
Authors: Zell Traci , Khoruts Alexander , Ingulli Elizabeth , Bonnevier Jody L. , Mueller Daniel L. , Jenkins Marc K.
Published: July 2002

Flow cytometry was used to study signaling events in individual CD4 T cells after antigen recognition in the body. Phosphorylation of c-jun and p38 mitogen-activated protein kinase was detected within minutes in all antigen-specific CD4 T cells in secondary lymphoid tissues after injection of peptide antigen into the bloodstream. The remarkable rapidity of this response correlated with the finding that most naive T cells are in constant contact with dendritic antigen-presenting cells. Contrary to predictions fromin vitroexperiments, antigen-induced c-jun and p38 mitogen-activated protein kinase phosphorylation did not depend on CD28 signals and was insensitive to inhibition by cyclosporin A. Our results highlight the efficiency of thein vivoimmune response and underscore the need to verify which signaling pathways identifiedin vitroactually operate under physiological conditions.
Spatially localized generation of nucleotide sequence-specific DNA damage
Authors: Oh Dennis H. , King Brett A. , Boxer Steven G. , Hanawalt Philip C.
Published: July 2002

Psoralens linked to triplex-forming oligonucleotides (psoTFOs) have
been used in conjunction with laser-induced two-photon excitation (TPE)
to damage a specific DNA target sequence. To demonstrate that TPE can
initiate photochemistry resulting in psoralen–DNA photoadducts, target
DNA sequences were incubated with psoTFOs to form triple-helical
complexes and then irradiated in liquid solution with pulsed 765-nm
laser light, which is half the quantum energy required for conventional
one-photon excitation, as used in psoralen + UV A radiation
(320–400 nm) therapy. Target DNA acquired strand-specific psoralen
monoadducts in a light dose-dependent fashion. To localize DNA damage
in a model tissue-like medium, a DNA–psoTFO mixture was prepared in a
polyacrylamide gel and then irradiated with a converging laser beam
targeting the rear of the gel. The highest number of photoadducts
formed at the rear while relatively sparing DNA at the front of the
gel, demonstrating spatial localization of sequence-specific DNA damage
by TPE. To assess whether TPE treatment could be extended to cells
without significant toxicity, cultured monolayers of normal human
dermal fibroblasts were incubated with tritium-labeled psoralen without
TFO to maximize detectable damage and irradiated by TPE. DNA from
irradiated cells treated with psoralen exhibited a 4- to 7-fold
increase in tritium activity relative to untreated controls. Functional
survival assays indicated that the psoralen–TPE treatment was not
toxic to cells. These results demonstrate that DNA damage can be
simultaneously manipulated at the nucleotide level and in three
dimensions. This approach for targeting photochemical DNA damage may
have photochemotherapeutic applications in skin and other optically
accessible tissues.
Ultrafast folding of WW domains without structured aromatic clusters in the denatured state
Authors: Ferguson Neil , Johnson Christopher M. , Macias Maria , Oschkinat Hartmut , Fersht Alan
Published: July 2002

Ultrafast-folding proteins are important for combining experiment and simulation to give complete descriptions of folding pathways. The WW domain family comprises small proteins with a three-stranded antiparallel β-sheet topology. Previous studies on the 57-residue YAP 65 WW domain indicate the presence of residual structure in the chemically denatured state. Here we analyze three minimal core WW domains of 38–44 residues. There was little spectroscopic or thermodynamic evidence for residual structure in either their chemically or thermally denatured states. Folding and unfolding kinetics, studied by using rapid temperature-jump and continuous-flow techniques, show that each domain folds and unfolds very rapidly in a two-state transition through a highly compact transition state. Folding half-times were as short as 17 μs at 25°C, within an order of magnitude of the predicted maximal rate of loop formation. The small size and topological simplicity of these domains, in conjunction with their very rapid two-state folding, may allow us to reduce the difference in time scale between experiment and theoretical simulation.
Functional significance of concomitant inactivation of
hMLH1
and
hMSH6
in tumor cells of the microsatellite mutator phenotype
Authors: Baranovskaya Svetlana , Soto Jose Luis , Perucho Manuel , Malkhosyan Sergei R.
Published: July 2002

Genetic or epigenetic inactivation of one of the DNA mismatch
repair (MMR) genes in tumor precursor cells causes a profound
mutator phenotype, known as the microsatellite mutator phenotype (MMP).
This mutator phenotype induces mutations not only in cancer genes that
drive tumorigenesis but also in other DNA repair genes. The functional
significance of these successive DNA repair gene mutations, however,
has not been substantiated. Here we show that the concomitant
inactivation of two DNA MMR genes (
hMLH1
and
hMSH6
) increases the mutator phenotype. We isolated cell
clones of the SW48 MMP-positive cell line with either active or
inactive
hMSH6
. All of these clones lacked expression of
hMLH1
because of promoter hypermethylation. Compared
with inactivation of
hMLH1
alone, the additional
inactivation of
hMSH6
produced a higher mutation rate
and a different spectrum of mutations in the endogenous
hprt
gene. These results confirm our model that the
mutator phenotype can increase during tumorigenesis by the consecutive
inactivation of different members of the DNA MMR system. Thus, a
stronger mutator phenotype accelerates the accumulation of mutations in
target cancer genes, which, in turn, speeds up tumor progression. The
results of this study also have significant impact on our understanding
of the mechanism of DNA MMR.
Neurotrophins are key mediators of the myelination program in the peripheral nervous system
Authors: Chan Jonah R. , Cosgaya José Miguel , Wu Yong Jian , Shooter Eric M.
Published: July 2002

Although knowledge of the functions of neurotrophins has advanced
rapidly in recent years, studies concerning the involvement of
neurotrophins in glial–neuronal interactions rarely extend further
than their roles in supporting the survival and differentiation of
neuronal cells. In this study endogenous
brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) were
identified in Schwann cell/dorsal root ganglia neuronal cocultures
and shown to modulate the myelination program of the peripheral nervous
system. The differential expression of BDNF and NT3 were examined and
compared with the expression profiles of myelin proteins in the
cocultures throughout the myelination process. BDNF levels correlated
with active myelin formation, whereas NT3 expression was initially high
and then down regulated throughout the proliferation and premyelination
periods. Addition of exogenous BDNF enhanced myelination, whereas the
removal of the endogenous BDNF by using the BDNF receptor
TrkB-Fc fusion protein inhibited the formation of mature myelin
internodes. Interestingly, exogenous NT3 significantly inhibited
myelination, whereas the removal of the endogenous NT3 by
using the NT3 receptor TrkC-Fc fusion protein resulted in an
enhancement similar to that obtained with the addition of BDNF. In
addition,
in vivo
studies were performed during the
development of the mouse sciatic nerve. Subcutaneous injections of BDNF
resulted in an enhancement of myelin formation in the sciatic nerve,
whereas the removal of the endogenous BDNF dramatically
inhibited myelination. Injections of NT3 inhibited myelin formation,
and the removal of the endogenous NT3 enhanced myelination.
These results demonstrate that BDNF and NT3 possess different
modulatory roles in the myelination program of the peripheral nervous
system and that their mechanisms of action are specific and highly
regulated.
The regulatory role for the ERCC3 helicase of general transcription factor TFIIH during promoter escape in transcriptional activation
Authors: Fukuda Aya , Nogi Yasuhisa , Hisatake Koji
Published: July 2002

Eukaryotic transcriptional activators have been proposed to function, for the most part, by promoting the assembly of preinitiation complex through the recruitment of the RNA polymerase II transcriptional machinery to the promoter. Previous studies have shown that transcriptional activation is critically dependent on transcription factor IIH (TFIIH), which functions during promoter opening and promoter escape, the steps following preinitiation complex assembly. Here we have analyzed the role of TFIIH in transcriptional activation and show that the excision repair cross-complementing (ERCC) 3 helicase activity of TFIIH plays a regulatory role to stimulate promoter escape in activated transcription. The stimulatory effect of the ERCC3 helicase is observed until ≈10-nt RNA is synthesized, and the helicase seems to act throughout the entire course of promoter escape. Analyses of the early phase of transcription show that a majority of the initiated complexes abort transcription and fail to escape the promoter; however, the proportion of productive complexes that escape the promoter apparently increases in response to activation. Our results establish that promoter escape is an important regulatory step stimulated by the ERCC3 helicase activity in response to activation and reveal a possible mechanism of transcriptional synergy.
Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.
Authors: Yan J , Lawson J E , Reed L J
Published: July 2002

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.
The human multidrug resistance-associated protein functionally complements the yeast cadmium resistance factor 1.
Authors: Tommasini R , Evers R , Vogt E , Mornet C , Zaman G J , Schinkel A H , Borst P , Martinoia E
Published: July 2002

A Saccharomyces cerevisiae strain with a disrupted yeast cadmium resistance factor (YCF1) gene (DTY168) is hypersensitive to cadmium. YCF1 resembles the human multidrug resistance-associated protein MRP (63% amino acid similarity), which confers resistance to various cytotoxic drugs by lowering the intracellular drug concentration. Whereas the mechanism of action of YCF1 is not known, MRP was recently found to transport glutathione S-conjugates across membranes. Here we show that expression of the human MRP cDNA in yeast mutant DTY168 cells restores cadmium resistance to the wild-type level. Transport of S-(2,4-dinitrobenzene)-glutathione into isolated yeast microsomal vesicles is strongly reduced in the DTY168 mutant and this transport is restored to wild-type level in mutant cells expressing MRP cDNA. We find in cell fractionation experiments that YCF1 is mainly localized in the vacuolar membrane in yeast, whereas MRP is associated both with the vacuolar membrane and with other internal membranes in the transformed yeast cells. Our results indicate that yeast YCF1 is a glutathione S-conjugate pump, like MRP, and they raise the possibility that the cadmium resistance in yeast involves cotransport of cadmium with glutathione derivatives.
kappa opioid receptors in human microglia downregulate human immunodeficiency virus 1 expression.
Authors: Chao C C , Gekker G , Hu S , Sheng W S , Shark K B , Bu D F , Archer S , Bidlack J M , Peterson P K
Published: July 2002

Microglial cells, the resident macrophages of the brain, play an important role in the neuropathogenesis of human immunodeficiency virus type 1 (HIV-1), and recent studies suggest that opioid peptides regulate the function of macrophages from somatic tissues. We report herein the presence of kappa opioid receptors (KORs) in human fetal microglia and inhibition of HIV-1 expression in acutely infected microglial cell cultures treated with KOR ligands. Using reverse transcriptase-polymerase chain reaction and sequencing analyses, we found that mRNA for the KOR was constitutively expressed in microglia and determined that the nucleotide sequence of the open reading frame was identical to that of the human brain KOR gene. The expression of KOR in microglial cells was confirmed by membrane binding of [3H]U69,593, a kappa-selective ligand, and by indirect immunofluorescence. Treatment of microglial cell cultures with U50,488 or U69,593 resulted in a dose-dependent inhibition of expression of the monocytotropic HIV-1 SF162 strain. This antiviral effect of the kappa ligands was blocked by the specific KOR antagonist, nor-binaltrophimine. These findings suggest that kappa opioid agonists have immunomodulatory activity in the brain, and that these compounds could have potential in the treatment of HIV-1-associated encephalopathy.
Ordered tandem arrangement of chromosomes in the sperm heads of monotreme mammals.
Authors: Watson J M , Meyne J , Graves J A
Published: July 2002

A very old unanswered question in classical cytology is whether chromosomes are arranged randomly in sperm or whether they occupy specific positions. Even with modern methods of chromosome painting, it is difficult to resolve this question for the very condensed and almost spherical sperm head of most mammals. We have taken advantage of the unusual fibrillar sperm head of monotreme mammals (echidna and platypus) to examine the position of chromosome landmarks in a two-dimensional array. We used fluorescence and radioactive in situ hybridization to telomeric, rDNA, and unique sequences to show that chromosomes are arranged tandemly and in a defined order in the sperm nucleus.
Expression cloning of Forssman glycolipid synthetase: a novel member of the histo-blood group ABO gene family.
Authors: Haslam D B , Baenziger J U
Published: July 2002

A phenotypic cloning approach was used to isolate a canine cDNA encoding Forssman glycolipid synthetase (FS; UDP-GalNAc:globoside alpha-1,3-N-acetylgalactosaminyltransferase; EC 2.4.1.88). The deduced amino acid sequence of FS demonstrates extensive identity to three previously cloned glycosyltransferases, including the enzymes responsible for synthesis of histo-blood group A and B antigens. These three enzymes, like FS, catalyze the addition of either N-acetylgalactosamine (GalNAc) or galactose (Gal) in alpha-1,3-linkage to their respective substrates. Despite the high degree of sequence similarity among the transferases, we demonstrate that the FS cDNA encodes an enzyme capable of synthesizing Forssman glycolipid, and demonstrates no GalNAc or Gal transferase activity when closely related substrates are examined. Thus, the FS cDNA is a novel member of the histo-blood group ABO gene family that encodes glycosyltransferases with related but distinct substrate specificity. Cloning of the FS cDNA will allow a detailed dissection of the roles Forssman glycolipid plays in cellular differentiation, development, and malignant transformation.
An essential role for the interferon-inducible, double-stranded RNA-activated protein kinase PKR in the tumor necrosis factor-induced apoptosis in U937 cells.
Authors: Yeung M C , Liu J , Lau A S
Published: July 2002

Tumor necrosis factor alpha (TNF-alpha) is well-characterized for its necrotic action against tumor cells; however, it has been increasingly associated with an apoptosis-inducing potential on target cells. While the signaling events and the actual cytolytic mechanism(s) for both TNF-alpha-induced necrosis and apoptosis remain to be fully elucidated, we report here on (i) the ability of TNF-alpha to induce apoptosis in the promonocytic U937 cells, (ii) the discovery of a cross-talk between the TNF-alpha and the interferon signaling pathways, and (iii) the pivotal role of interferon-inducible, double-stranded RNA-activated protein kinase (PKR) in the induction of apoptosis by TNF-alpha. Our data from microscopy studies, trypan blue exclusion staining, and apoptotic DNA ladder electrophoresis revealed that a subclone derived from U937 and carrying a PKR antisense expression vector was resistant to TNF-alpha-induced apoptosis. Further, TNF-alpha initiated a generalized RNA degradation process in which the participation of PKR was required. Finally, the PKR gene is a candidate "death gene" since overexpression of this gene could bring about apoptosis in U937 cells.