bluuebunny/crossref_metadata_2025 huggingface dataset:
Proteomic survey of metabolic pathways in rice
Authors: Koller Antonius , Washburn Michael P. , Lange B. Markus , Andon Nancy L. , Deciu Cosmin , Haynes Paul A. , Hays Lara , Schieltz David , Ulaszek Ryan , Wei Jing , Wolters Dirk , Yates John R.
Published: September 2002

A systematic proteomic analysis of rice (
Oryza sativa
) leaf, root, and seed tissue using two independent technologies, two-dimensional gel electrophoresis followed by tandem mass spectrometry and multidimensional protein identification technology, allowed the detection and identification of 2,528 unique proteins, which represents the most comprehensive proteome exploration to date. A comparative display of the expression patterns indicated that enzymes involved in central metabolic pathways are present in all tissues, whereas metabolic specialization is reflected in the occurrence of a tissue-specific enzyme complement. For example, tissue-specific and subcellular compartment-specific isoforms of ADP-glucose pyrophosphorylase were detected, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms involved in the biosynthesis and breakdown of separate starch pools in different tissues. In addition, several previously characterized allergenic proteins were identified in the seed sample, indicating the potential of proteomic approaches to survey food samples with regard to the occurrence of allergens.
Formation of geometrically complex lipid nanotube-vesicle networks of higher-order topologies
Authors: Karlsson Mattias , Sott Kristin , Davidson Maximillian , Cans Ann-Sofie , Linderholm Pontus , Chiu Daniel , Orwar Owe
Published: September 2002

We present a microelectrofusion method for construction of fluid-state lipid bilayer networks of high geometrical complexity up to fully connected networks with genus = 3 topology. Within networks, self-organizing branching nanotube architectures could be produced where intersections spontaneously arrange themselves into three-way junctions with an angle of 120° between each nanotube. Formation of branching nanotube networks appears to follow a minimum-bending energy algorithm that solves for pathway minimization. It is also demonstrated that materials can be injected into specific containers within a network by nanotube-mediated transport of satellite vesicles having defined contents. Using a combination of microelectrofusion, spontaneous nanotube pattern formation, and satellite-vesicle injection, complex networks of containers and nanotubes can be produced for a range of applications in, for example, nanofluidics and artificial cell design. In addition, this electrofusion method allows integration of biological cells into lipid nanotube-vesicle networks.
A single origin of the peridinin- and fucoxanthin-containing plastids in dinoflagellates through tertiary endosymbiosis
Authors: Yoon Hwan Su , Hackett Jeremiah D. , Bhattacharya Debashish
Published: September 2002

The most widely distributed dinoflagellate plastid contains chlorophyll
c

2

and peridinin as the major carotenoid. A second plastid type, found in taxa such as
Karlodinium micrum
and
Karenia
spp., contains chlorophylls
c

1

+
c

2

and 19′-hexanoyloxy-fucoxanthin and/or 19′-butanoyloxy-fucoxanthin but lacks peridinin. Because the presence of chlorophylls
c

1

+
c

2

and fucoxanthin is typical of haptophyte algae, the second plastid type is believed to have originated from a haptophyte tertiary endosymbiosis in an ancestral peridinin-containing dinoflagellate. This hypothesis has, however, never been thoroughly tested in plastid trees that contain genes from both peridinin- and fucoxanthin-containing dinoflagellates. To address this issue, we sequenced the plastid-encoded
psa
A (photosystem I P700 chlorophyll
a
apoprotein A1),
psb
A (photosystem II reaction center protein D1), and “Form I”
rbc
L (ribulose-1,5-bisphosphate carboxylase/oxygenase) genes from various red and dinoflagellate algae. The combined
psa
A +
psb
A tree shows significant support for the monophyly of peridinin- and fucoxanthin-containing dinoflagellates as sister to the haptophytes. The monophyly with haptophytes is robustly recovered in the
psb
A phylogeny in which we increased the sampling of dinoflagellates to 14 species. As expected from previous analyses, the fucoxanthin-containing dinoflagellates formed a well-supported sister group with haptophytes in the
rbc
L tree. Based on these analyses, we postulate that the plastid of peridinin- and fucoxanthin-containing dinoflagellates originated from a haptophyte tertiary endosymbiosis that occurred before the split of these lineages. Our findings imply that the presence of chlorophylls
c

1

+
c

2

and fucoxanthin, and the Form I
rbc
L gene are in fact the primitive (not derived, as widely believed) condition in dinoflagellates.
Loss of microsatellite diversity and low effective population size in an overexploited population of New Zealand snapper (
Pagrus auratus
)
Authors: Hauser Lorenz , Adcock Greg J. , Smith Peter J. , Bernal Ramírez Julio H. , Carvalho Gary R.
Published: September 2002

Although the effects of overfishing on species diversity and abundance are well documented, threats to the genetic diversity of marine fish populations have so far been largely neglected. Indeed, there seems to be little cause for concern, as even “collapsed” stocks usually consist of several million individuals, whereas population genetics theory suggests that only very small populations suffer significant loss of genetic diversity. On the other hand, in many marine species the genetically effective population size (
N
e
), which determines the genetic properties of a population, may be orders of magnitude smaller than the census population size (
N
). Here, microsatellite analyses of a time series of archived scales demonstrated a significant decline in genetic diversity in a New Zealand snapper population during its exploitation history. Effective population sizes estimated both from the decline in heterozygosity and from temporal fluctuations in allele frequency were five orders of magnitude smaller than census population sizes from fishery data. If such low
N
e
/
N
ratios are commonplace in marine species, many exploited marine fish stocks may be in danger of losing genetic variability, potentially resulting in reduced adaptability, population persistence, and productivity.
Mutations and common polymorphisms in
ADAMTS13
gene responsible for von Willebrand factor-cleaving protease activity
Authors: Kokame Koichi , Matsumoto Masanori , Soejima Kenji , Yagi Hideo , Ishizashi Hiromichi , Funato Masahisa , Tamai Hiroshi , Konno Mutsuko , Kamide Kei , Kawano Yuhei , Miyata Toshiyuki , Fujimura Yoshihiro
Published: September 2002

von Willebrand factor (VWF) is synthesized primarily in vascular endothelial cells and secreted into the plasma as unusually large VWF multimers. Normally, these multimers are quickly degraded into smaller forms by a plasma metalloproteinase, VWF-cleaving protease (VWF-CP). Decreases in the activity of this enzyme result in congenital and acquired thrombotic thrombocytopenic purpura (TTP). The human VWF-CP has recently been purified. Cloning of the corresponding cDNA revealed that the 1,427-aa polypeptide is a member of the ADAMTS gene family, termed
ADAMTS13
. Twelve rare mutations in this gene have been identified in patients with congenital TTP. Here, we report missense and nonsense mutations in two Japanese families with Upshaw–Schulman syndrome, congenital TTP with neonatal onset and frequent relapses. The comparison of individual
ADAMTS13
genotypes and plasma VWF-CP activities indicated that the R268P, Q449stop, and C508Y mutations abrogated activity of the enzyme, whereas the P475S mutant retained low but significant activity. The effects of these mutations were further confirmed by expression analysis in HeLa cells. Recombinant VWF-CP containing either the R268P or C508Y mutations was not secreted from cells. In contrast, Q449stop and P475S mutants were normally secreted but demonstrated minimal activity. Genotype analysis of 364 Japanese subjects revealed that P475S is heterozygous in 9.6% of individuals, suggesting that approximately 10% of the Japanese population possesses reduced VWF-CP activity. We report on a single-nucleotide polymorphism associated with alterations in VWF-CP activity; it will be important to assess this single-nucleotide polymorphism as a risk factor for thrombotic disorders.
Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure
Authors: MacRobbie Enid A. C.
Published: September 2002

Protein tyrosine phosphatases (PTPases) exist in plants, but their role in plant signaling processes is unknown. One of the most important signaling networks in plants concerns the regulation of stomatal aperture, by which closure of stomatal pores restricts water loss in dry conditions, a process essential for plant survival. Closure is achieved by reduction in guard cell volume as a consequence of net efflux of potassium salt at both plasmalemma and tonoplast. To test whether protein tyrosine phosphorylation has any role in guard cell signaling processes, the effects on stomatal aperture and on guard cell K(Rb) fluxes of a number of specific inhibitors of PTPases have been investigated. Stomatal closure induced by abscisic acid, high external Ca
2+
, hydrogen peroxide, and dark were all prevented by one such inhibitor, phenylarsine oxide, which added to closed stomata promoted reopening. Flux measurements with
86
Rb
+
identified the efflux across the tonoplast as the sensitive process, implying that protein tyrosine dephosphorylation must occur at or downstream of the Ca
2+
signal responsible for triggering ion efflux from the vacuole. There was no inhibition of efflux at the plasmalemma. A second inhibitor of PTPases, 3,4 dephosphatin, gave very similar effects, inhibiting closure induced by abscisic acid, high external Ca
2+
, and dark, and promoting reopening if added to closed stomata. Again, the efflux of K(Rb) at the tonoplast was the sensitive process. These results provide clear evidence for the involvement of PTPases in a major signaling network in plants.
The clothing effect: Tactile neurons in the precentral gyrus do not respond to the touch of the familiar primate chair
Authors: Graziano Michael S. A. , Alisharan Shalani E. , Hu Xintian , Gross Charles G.
Published: September 2002

Neurons in a restricted zone in the precentral gyrus of macaque monkeys respond to tactile, visual, and auditory stimuli. The tactile receptive fields of these multimodal cells are usually located on the face, arm, or upper torso. In the present study, in awake monkeys sitting in a primate chair, the neurons responded to a tactile probe touching the skin within the tactile receptive field. However, the same neurons did not respond when the tactile receptive field was touched by the primate chair, to which the monkey was habituated.
The C2A domain of JFC1 binds to 3′-phosphorylated phosphoinositides and directs plasma membrane association in living cells
Authors: Catz Sergio D. , Johnson Jennifer L. , Babior Bernard M.
Published: September 2002

Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3′-phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3′-phosphoinositidesin vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3′-phosphoinositidesin vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3′-phosphoinositidesin vitrowith similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Aktin vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking.
Prospective isolation of human clonogenic common myeloid progenitors
Authors: Manz Markus G. , Miyamoto Toshihiro , Akashi Koichi , Weissman Irving L.
Published: September 2002

The hierarchical development from hematopoietic stem cells to mature cells of the hematolymphoid system involves progressive loss of self-renewal capacity, proliferation ability, and lineage potentials. Here we show the prospective isolation of early developmental intermediates, the human clonogenic common myeloid progenitors and their downstream progeny, the granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. All three populations reside in the lineage-negative (lin−) CD34+CD38+fraction of adult bone marrow as well as in cord blood. They are distinguishable by the expression of the IL-3Rα chain, the receptor of an early-acting hematopoietic cytokine, and CD45RA, an isoform of a phosphotyrosine phosphatase involved in negative regulation of cytokine signaling. Multipotent progenitors, early lymphoid progenitors, and the here-defined myeloid progenitors express distinct profiles of hematopoiesis-affiliated genes. The isolation of highly purified hematopoietic intermediates provides tools to better understand developmental programs underlying normal and leukemic hematopoiesis.
Restriction of lentivirus in monkeys
Authors: Besnier Caroline , Takeuchi Yasuhiro , Towers Greg
Published: September 2002

Retroviruses are able to cross species barriers and have done so many times throughout evolution. Perhaps as a consequence, dominant mechanisms have arisen to block infection by murine retroviruses in mice (restriction factor Fv1) and humans (restriction factor Ref1), as well as in other mammals. Here we describe a block to HIV and simian immunodeficiency virus in monkeys. Like previously described restrictions the block is saturable and gives rise to multiple-hit infection kinetics. Furthermore, like restriction of murine leukemia virus in humans, the block is before reverse transcription. Intriguingly, African green monkey cells are able to block both HIV and simian immunodeficiency virus, and each virus is able to saturate and abrogate the restriction of the other, suggesting that a common factor is responsible.
Synergistic interaction between adenosine A2A and glutamate mGlu5 receptors: Implications for striatal neuronal function
Authors: Ferré Sergi , Karcz-Kubicha Marzena , Hope Bruce T. , Popoli Patrizia , Burgueño Javier , Gutiérrez M. Angeles , Casadó Vicent , Fuxe Kjell , Goldberg Steven R. , Lluis Carme , Franco Rafael , Ciruela Francisco
Published: September 2002

The physiological meaning of the coexpression of adenosine A2A receptors and group I metabotropic glutamate receptors in γ- aminobutyric acid (GABA)ergic striatal neurons is intriguing. Here we provide
in vitro
and
in vivo
evidence for a synergism between adenosine and glutamate based on subtype 5 metabotropic glutamate (mGluR5) and adenosine A2A (A2AR) receptor/receptor interactions. Colocalization of A2AR and mGluR5 at the membrane level was demonstrated in nonpermeabilized human embryonic kidney (HEK)-293 cells transiently cotransfected with both receptors by confocal laser microscopy. Complexes containing A2AR and mGluR5 were demonstrated by Western blotting of immunoprecipitates of either Flag-A2AR or hemagglutinin-mGluR5 in membrane preparations from cotransfected HEK-293 cells and of native A2AR and mGluR5 in rat striatal membrane preparations. In cotransfected HEK-293 cells a synergistic effect on extracellular signal-regulated kinase 1/2 phosphorylation and c-
fos
expression was demonstrated upon A2AR/mGluR5 costimulation. No synergistic effect was observed at the second messenger level (cAMP accumulation and intracellular calcium mobilization). Accordingly, a synergistic effect on c-
fos
expression in striatal sections and on counteracting phencyclidine-induced motor activation was also demonstrated after the central coadministration of A2AR and mGluR5 agonists to rats with intact dopaminergic innervation. The results suggest that a functional mGluR5/A2AR interaction is required to overcome the well-known strong tonic inhibitory effect of dopamine on striatal adenosine A2AR function.
Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants
Authors: Dinkova-Kostova Albena T. , Holtzclaw W. David , Cole Robert N. , Itoh Ken , Wakabayashi Nobunao , Katoh Yasutake , Yamamoto Masayuki , Talalay Paul
Published: September 2002

Coordinate induction of phase 2 proteins and elevation of glutathione protect cells against the toxic and carcinogenic effects of electrophiles and oxidants. All inducers react covalently with thiols at rates that are closely related to their potencies. Inducers disrupt the cytoplasmic complex between the actin-bound protein Keap1 and the transcription factor Nrf2, thereby releasing Nrf2 to migrate to the nucleus where it activates the antioxidant response element (ARE) of phase 2 genes and accelerates their transcription. We cloned, overexpressed, and purified murine Keap1 and demonstrated on native gels the formation of complexes of Keap1 with the Neh2 domain of Nrf2 and their concentration-dependent disruption by inducers such as sulforaphane and bis(2-hydroxybenzylidene)acetone. The kinetics, stoichiometry, and order of reactivities of the most reactive of the 25 cysteine thiol groups of Keap1 have been determined by tritium incorporation from [
3
H]dexamethasone mesylate (an inducer and irreversible modifier of thiols) and by UV spectroscopy with sulforaphane, 2,2′-dipyridyl disulfide and 4,4′-dipyridyl disulfide (titrants of thiol groups), and two closely related Michael reaction acceptors [bis(2- and 4-hydroxybenzylidene)acetones] that differ 100-fold in inducer potency and the UV spectra of which are bleached by thiol addition. With large excesses of these reagents nearly all thiols of Keap1 react, but sequential reaction with three successive single equivalents (per cysteine residue) of dipyridyl disulfides revealed excellent agreement with pseudo-first order kinetics, rapid successive declines in reaction velocity, and the stoichiometric formation of two equivalents of thiopyridone per reacted cysteine. This finding suggests that reaction of cysteine thiols is followed by rapid formation of protein disulfide linkages. The most reactive residues of Keap1 (C
257
, C
273
, C
288
, and C
297
) were identified by mapping the dexamethasone-modified cysteines by mass spectrometry of tryptic peptides. These residues are located in the intervening region between BTB and Kelch repeat domains of Keap1 and probably are the direct sensors of inducers of the phase 2 system.
Febrile temperatures induce cytoadherence of ring-stage
Plasmodium falciparum
-infected erythrocytes
Authors: Udomsangpetch Rachanee , Pipitaporn Busaba , Silamut Kamolrat , Pinches Robert , Kyes Sue , Looareesuwan Sornchai , Newbold Christopher , White Nicholas J.
Published: September 2002

In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of
Plasmodium falciparum
are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence
in vitro
of
P. falciparum
-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40°C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37°C did not. Heating to 40°C accelerated cytoadherence and doubled the maximum cytoadherence observed (
P
< 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4
var
also did not cytoadhere at 37°C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of
var
mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance.
Structural insights into peptide bond formation
Authors: Hansen Jeffrey L. , Schmeing T. Martin , Moore Peter B. , Steitz Thomas A.
Published: September 2002

The large ribosomal subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-Å resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the
Haloarcula marismortui
50S ribosomal subunit. A P site substrate, CCA-Phe-caproic acid–biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The α-NH
2
group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2′ OH of the A-76 ribose in the P site or with the 2′ OH of A2486 (2451). These interactions position the α amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.
SALSA, a variant of yeast SAGA, contains truncated Spt7, which correlates with activated transcription
Authors: Sterner David E. , Belotserkovskaya Rimma , Berger Shelley L.
Published: September 2002

Spt-Ada-Gcn5 acetyltransferase (SAGA) is a previously described histone acetyltransferase/transcriptional coactivator complex in yeast. At promoters of certain genes (
HIS3
and
TRP3
), SAGA has an inhibitory function involving a nonproductive TATA-binding protein interaction mediated by the Spt3 and Spt8 subunits. Related to this, Spt8-less SAGA is a major form of the complex under activating conditions for these genes. In the present study, we purify this activation-specific complex, called SALSA (SAGA altered, Spt8 absent). Besides lacking Spt8, SALSA contains Spt7 subunit that is truncated. Examining the role of this subunit, we find that C-terminally truncated
SPT7
resulted in derepressed
HIS3
transcription. Furthermore, when grown in rich media (repressing conditions), wild-type cells yielded predominantly SAGA, but Spt7 C-terminal truncations resulted primarily in a form of complex similar to SALSA. Thus, SALSA-like structure and activating function can be partially recapitulated in yeast by truncating the C terminus of Spt7. Overall, these results lead to a model that for a subset of promoters SAGA is inhibitory through Spt3, Spt8, and an Spt8-interacting subdomain of Spt7, whereas SALSA is a form of complex for positive transcriptional regulation. These data clarify a mechanism by which a transcriptional regulatory complex can switch between positive and negative modulation.
Isolation of high-affinity GTP aptamers from partially structured RNA libraries
Authors: Davis Jonathan H. , Szostak Jack W.
Published: September 2002

Aptamers, RNA sequences that bind to target ligands, are typically isolated by
in vitro
selection from RNA libraries containing completely random sequences. To see whether higher-affinity aptamers can be isolated from partially structured RNA libraries, we selected for aptamers that bind GTP, starting from a mixture of fully random and partially structured libraries. Because stem-loops are common motifs in previously characterized aptamers, we designed the partially structured library to contain a centrally located stable stem-loop. We used an off-rate selection protocol designed to maximize the enrichment of high-affinity aptamers. The selection produced a surprisingly large number of distinct sequence motifs and secondary structures, including seven different aptamers with
K
d
s ranging from 500 to 25 nanomolar. The engineered stem-loop was present in the three highest affinity aptamers, and in 12 of 13 independent isolates with a single consensus sequence, suggesting that its inclusion increased the abundance of high-affinity aptamers in the starting pool.
A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from
Entacmaea quadricolor
(Anthozoa, Actinaria)
Authors: Wiedenmann Jörg , Schenk Andreas , Röcker Carlheinz , Girod Andreas , Spindler Klaus-Dieter , Nienhaus G. Ulrich
Published: September 2002

We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone
Entacmaea quadricolor
cloned in
Escherichia coli
. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4–10. Red fluorophore maturation reaches a level of 90% after ≈12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS/PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for
in vivo
labeling in eukaryotic systems was shown by expression in a mammalian cell culture.
Evidence for a susceptibility gene (SLEH1) on chromosome 11q14 for systemic lupus erythematosus (SLE) families with hemolytic anemia
Authors: Kelly Jennifer A. , Thompson Kevin , Kilpatrick Jeff , Lam Tom , Nath Swapan K. , Gray-McGuire Courtney , Reid Jeff , Namjou Bahram , Aston Christopher E. , Bruner Gail R. , Scofield R. Hal , Harley John B.
Published: September 2002

Hemolytic anemia is aforme frusteof systemic lupus erythematosus (SLE), being observed months or even years before the onset of other clinical manifestations in some patients. We hypothesized that hemolytic anemia in those SLE-affected patients would identify a group of SLE pedigrees that share a high degree of genetic homogeneity. From 160 multiplex SLE pedigrees, we sought evidence for linkage in 35 (16 African-American, 17 European-American, and 2 Hispanic) who had at least one SLE-affected patient with hemolytic anemia. Significant linkage was present at 11q14 in the 16 African-American pedigrees, yielding a maximum two-point logarithm of odds (LOD) score of 4.5 at D11S2002. The segregation pattern of SLE in these African-American pedigrees suggested a dominant mode of inheritance and, when maximized across penetrance and disease allele frequencies, produced a multipoint LOD of 4.7. Multipoint analysis yielded a multipoint heterogeneity LOD score of 3.6 (α = 0.63), again with maximum LOD at D11S2002. Finally, markers typed 7 centimorgans to either side of D11S2002 achieved LOD scores of 3 or better by using the maximized model, supporting linkage to 11q14. Clearly, pedigree ascertainment based on select clinical manifestations is an important tool, capable of revealing otherwise cryptic genetic linkages in complex genetic diseases. Thus, we show strong evidence for an SLE susceptibility gene,SLEH1, near D11S2002 in African-American pedigrees multiplex for SLE that have at least one SLE-affected patient with hemolytic anemia.
Sleep forms memory for finger skills
Authors: Fischer Stefan , Hallschmid Manfred , Elsner Anna Lisa , Born Jan
Published: September 2002

Practicing a motor skill triggers a process of memory consolidation that continues for hours after practice has ended, and becomes manifest in an improved skill at later testing. We used a sequential motor task (finger-to-thumb opposition task) to show that, in humans, the formation of motor skill memories essentially benefits from sleep. Independent of whether placed during daytime or nighttime, sleep after practice enhanced speed of sequence performance on average by 33.5% and reduced error rate by 30.1% as compared with corresponding intervals of wakefulness. The effect of sleep after learning proved to be stable when retesting was postponed for another night, to exclude effects of sleep loss and to assure that all subjects had sufficient sleep before retrieval testing. Also, the consolidating effect of sleep was specific for the motor sequence learned. It did not generalize to a similar sequence containing identical movement segments in a different order. Retention periods of wakefulness improved performance only moderately and only if placed during daytime. The observations demonstrate a critical role of sleep for storing and optimizing motor skills.
Short-term antigen presentation and single clonal burst limit the magnitude of the CD8+ T cell responses to malaria liver stages
Authors: Hafalla Julius C. R. , Sano Gen-ichiro , Carvalho Luzia H. , Morrot Alexandre , Zavala Fidel
Published: September 2002

Malaria sporozoites induce swift activation of antigen-specific CD8+ T cells that inhibit the intracellular development of liver-stage parasites. The length of time of functionalin vivoantigen presentation, estimated by monitoring the activation of antigen-specific CD8+ T cells, is of short duration, with maximum T cell activation occurring within the first 8 h after immunization and lasting approximately 48 h. Although the magnitude of the CD8+ T cell response closely correlates with the number of parasites used for immunization, increasing the time of antigen presentation by daily immunizations does not enhance the magnitude of this response. Thus, once a primary clonal burst is established, the CD8+ T cell response becomes refractory or unresponsive to further antigenic stimulation. These findings strongly suggest that the most efficient strategy for the induction of primary CD8+ T cell responses is the delivery of a maximal amount of antigen in a single dose, thereby ensuring a clonal burst that involves the largest number of precursors to become memory cells.
Identification of macrophage liver X receptors as inhibitors of atherosclerosis
Authors: Tangirala Rajendra K. , Bischoff Eric D. , Joseph Sean B. , Wagner Brandee L. , Walczak Robert , Laffitte Bryan A. , Daige Chris L. , Thomas Diane , Heyman Richard A. , Mangelsdorf David J. , Wang Xuping , Lusis Aldons J. , Tontonoz Peter , Schulman Ira G.
Published: September 2002

Recent studies have identified the liver X receptors (LXRα and LXRβ) as important regulators of cholesterol metabolism and transport. LXRs control transcription of genes critical to a range of biological functions including regulation of high density lipoprotein cholesterol metabolism, hepatic cholesterol catabolism, and intestinal sterol absorption. Although LXR activity has been proposed to be critical for physiologic lipid metabolism and transport, direct evidence linking LXR signaling pathways to the pathogenesis of cardiovascular disease has yet to be established. In this study bone marrow transplantations were used to selectively eliminate macrophage LXR expression in the context of murine models of atherosclerosis. Our results demonstrate that LXRs are endogenous inhibitors of atherogenesis. Additionally, elimination of LXR activity in bone marrow-derived cells mimics many aspects of Tangier disease, a human high density lipoprotein deficiency, including aberrant regulation of cholesterol transporter expression, lipid accumulation in macrophages, splenomegaly, and increased atherosclerosis. These results identify LXRs as targets for intervention in cardiovascular disease.
High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants
Authors: Klahre Ulrich , Crété Patrice , Leuenberger Sabrina A. , Iglesias Victor A. , Meins Frederick
Published: September 2002

Posttranscriptional gene silencing (PTGS) in transgenic plants is an epigenetic form of RNA degradation related to PTGS and RNA interference (RNAi) in fungi and animals. Evidence suggests that transgene loci and RNA viruses can generate double-stranded RNAs similar in sequence to the transcribed region of target genes, which then undergo endonucleolytic cleavage to generate small interfering RNAs (siRNA) that promote degradation of cognate RNAs. The silent state in transgenic plants and inCaenorhabditis eleganscan spread systemically, implying that mobile silencing signals exist. Neither the chemical nature of these signals nor their exact source in the PTGS pathway is known. Here, we use a positive marker system and real-time monitoring of green fluorescent protein expression to show that large sense, antisense, and double-stranded RNAs as well as double-stranded siRNAs delivered biolistically into plant cells trigger silencing capable of spreading locally and systemically. Systemically silenced leaves show greatly reduced levels of target RNA and accumulate siRNAs, confirming that RNA can induce systemic PTGS. The induced siRNAs represent parts of the target RNA that are outside of the region of homology with the triggering siRNA. Our results imply that siRNAs themselves or intermediates induced by siRNAs could comprise silencing signals and that these signals induce self-amplifying production of siRNAs.
Evidence against stabilization of the transition state oxyanion by a pKa-perturbed RNA base in the peptidyl transferase center
Authors: Parnell K. Mark , Seila Amy C. , Strobel Scott A.
Published: September 2002

The crystal structure of the ribosomal 50S subunit from
Haloarcula marismortui
in complex with the transition state analog CCdA-phosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an “oxyanion hole” to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000)
Science
289, 920–929]. In the model, close proximity (3 Å) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.
VEGF increases engraftment of bone marrow-derived endothelial progenitor cells (EPCs) into vasculature of newborn murine recipients
Authors: Young Pampee P. , Hofling A. Alex , Sands Mark S.
Published: September 2002

Recent evidence suggests that bone marrow-derived angioblasts or endothelial progenitor cells circulate in peripheral blood and can incorporate at sites of pathologic neovascularization or during the ovarian cycle. However, the incorporation of endothelial progenitor cells into vessels of nonischemic tissues in adult animals has not been observed. We hypothesized that the vascular microenvironment differs between newborn and adult animals, and that donor endothelial cell progenitors would engraft in rapidly growing normal tissues during the neonatal period. After nonablative administration of bone marrow cells either at birth or at 4 weeks of age, donor-derived endothelial cells were found only in the neovasculature of the newborn recipients. Both the incorporation of donor endothelial cells into the newborn neovasculature as well as tissue vascularity were significantly increased by coadministering vascular endothelial growth factor with bone marrow cells. These findings suggest that bone marrow-derived endothelial progenitor cells can contribute to neovascularization during the newborn period and are responsive to vascular endothelial growth factor.
Salvinorin A: A potent naturally occurring nonnitrogenous κ opioid selective agonist
Authors: Roth Bryan L. , Baner Karen , Westkaemper Richard , Siebert Daniel , Rice Kenner C. , Steinberg SeAnna , Ernsberger Paul , Rothman Richard B.
Published: September 2002

Salvia divinorum
, whose main active ingredient is the neoclerodane diterpene Salvinorin A, is a hallucinogenic plant in the mint family that has been used in traditional spiritual practices for its psychoactive properties by the Mazatecs of Oaxaca, Mexico. More recently,
S. divinorum
extracts and Salvinorin A have become more widely used in the U.S. as legal hallucinogens. We discovered that Salvinorin A potently and selectively inhibited
3
H-bremazocine binding to cloned κ opioid receptors. Salvinorin A had no significant activity against a battery of 50 receptors, transporters, and ion channels and showed a distinctive profile compared with the prototypic hallucinogen lysergic acid diethylamide. Functional studies demonstrated that Salvinorin A is a potent κ opioid agonist at cloned κ opioid receptors expressed in human embryonic kidney-293 cells and at native κ opioid receptors expressed in guinea pig brain. Importantly, Salvinorin A had no actions at the 5-HT
2A
serotonin receptor, the principal molecular target responsible for the actions of classical hallucinogens. Salvinorin A thus represents, to our knowledge, the first naturally occurring nonnitrogenous opioid-receptor subtype-selective agonist. Because Salvinorin A is a psychotomimetic selective for κ opioid receptors, κ opioid-selective antagonists may represent novel psychotherapeutic compounds for diseases manifested by perceptual distortions (e.g., schizophrenia, dementia, and bipolar disorders). Additionally, these results suggest that κ opioid receptors play a prominent role in the modulation of human perception.
Direct evidence for a G-quadruplex in a promoter region and its targeting with a small molecule to repress c-
MYC
transcription
Authors: Siddiqui-Jain Adam , Grand Cory L. , Bearss David J. , Hurley Laurence H.
Published: September 2002

The nuclease hypersensitivity element III
1
upstream of the P1 promoter of c-
MYC
controls 85–90% of the transcriptional activation of this gene. We have demonstrated that the purine-rich strand of the DNA in this region can form two different intramolecular G-quadruplex structures, only one of which seems to be biologically relevant. This biologically relevant structure is the kinetically favored chair-form G-quadruplex, which is destabilized when mutated with a single G → A transition, resulting in a 3-fold increase in basal transcriptional activity of the c-
MYC
promoter. The cationic porphyrin TMPyP4, which has been shown to stabilize this G-quadruplex structure, is able to suppress further c-
MYC
transcriptional activation. These results provide compelling evidence that a specific G-quadruplex structure formed in the c-
MYC
promoter region functions as a transcriptional repressor element. Furthermore, we establish the principle that c-
MYC
transcription can be controlled by ligand-mediated G-quadruplex stabilization.
Inactivation of CMP-
N
-acetylneuraminic acid hydroxylase occurred prior to brain expansion during human evolution
Authors: Chou Hsun-Hua , Hayakawa Toshiyuki , Diaz Sandra , Krings Matthias , Indriati Etty , Leakey Meave , Paabo Svante , Satta Yoko , Takahata Naoyuki , Varki Ajit
Published: September 2002

Humans are genetically deficient in the common mammalian sialic acid
N
-glycolylneuraminic acid (Neu5Gc) because of an
Alu
-mediated inactivating mutation of the gene encoding the enzyme CMP-
N
-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (CMAH). This mutation occurred after our last common ancestor with bonobos and chimpanzees, and before the origin of present-day humans. Here, we take multiple approaches to estimate the timing of this mutation in relationship to human evolutionary history. First, we have developed a method to extract and identify sialic acids from bones and bony fossils. Two Neandertal fossils studied had clearly detectable Neu5Ac but no Neu5Gc, indicating that the CMAH mutation predated the common ancestor of humans and Neandertals, ≈0.5–0.6 million years ago (mya). Second, we date the insertion event of the inactivating human-specific sah
Alu
Y element that replaced the ancestral
Alu
Sq element found adjacent to exon 6 of the CMAH gene in the chimpanzee genome. Assuming
Alu
source genes based on a phylogenetic tree of human-specific
Alu
elements, we estimate the sah
Alu
Y insertion time at ≈2.7 mya. Third, we apply molecular clock analysis to chimpanzee and other great ape CMAH genes and the corresponding human pseudogene to estimate an inactivation time of ≈2.8 mya. Taken together, these studies indicate that the CMAH gene was inactivated shortly before the time when brain expansion began in humankind's ancestry, ≈2.1–2.2 mya. In this regard, it is of interest that although Neu5Gc is the major sialic acid in most organs of the chimpanzee, its expression is selectively down-regulated in the brain, for as yet unknown reasons.
Ancient mitochondrial haplotypes and evidence for intragenic recombination in a gynodioecious plant
Authors: Städler Thomas , Delph Lynda F.
Published: September 2002

Because of their extremely low nucleotide mutation rates, plant mitochondrial genes are generally not expected to show variation within species. Remarkably, we found nine distinct cytochrome
b
sequence haplotypes in the gynodioecious alpine plant
Silene acaulis
, with two or more haplotypes coexisting locally in each of three sampled regions. Moreover, there is evidence for intragenic recombination in the history of the haplotype sample, implying at least transient heteroplasmy of mitochondrial DNA (mtDNA). Heteroplasmy might be achieved by one of two potential mechanisms, either continuous coexistence of subgenomic fragments in low stoichiometry, or occasional paternal leakage of mtDNA. On the basis of levels of synonymous nucleotide substitutions, the average divergence time between haplotypes is estimated to be at least 15 million years. Ancient coalescence of extant haplotypes is further indicated by the paucity of fixed differences in haplotypes obtained from related species, a pattern expected under trans-specific evolution. Our data are consistent with models of frequency-dependent selection on linked cytoplasmic male-sterility factors, the putative molecular basis of females in gynodioecious populations. However, associations between marker loci and the inferred male-sterility genes can be maintained only with very low rates of recombination. Heteroplasmy and recombination between divergent haplotypes imply unexplored consequences for the evolutionary dynamics of gynodioecy, a widespread plant breeding system.
RNAi functions in cultured mammalian neurons
Authors: Krichevsky Anna M. , Kosik Kenneth S.
Published: September 2002

In a wide range of organisms, double-stranded RNA triggers posttranscriptional gene silencing or RNA interference (RNAi). Small interfering RNAs, the 21-nt double-stranded RNA intermediates of this natural pathway, have became a powerful tool to knock down specific gene expression in mammalian cell lines and potentially will be useful for the analysis of loss-of-function phenotypes. In mammalian primary neuronal cultures, where genetic manipulations are especially difficult, RNAi might be developed into a highly efficacious tool to study the roles of specific genes in neuron development and functioning. Neurons, however, have been considered the most resistant to RNAi. We report here an application of RNAi to postmitotic primary neuronal cultures. Synthetic siRNA can be readily introduced into neurons and effectively inhibit the expression of endogenous and transfected genes.
Huntington's disease age-of-onset linked to polyglutamine aggregation nucleation
Authors: Chen Songming , Ferrone Frank A. , Wetzel Ronald
Published: September 2002

In Huntington's Disease and related expanded CAG repeat diseases, a polyglutamine [poly(Gln)] sequence containing 36 repeats in the corresponding disease protein is benign, whereas a sequence with only 2–3 additional glutamines is associated with disease risk. Above this threshold range, longer repeat lengths are associated with earlier ages-of-onset. To investigate the biophysical basis of these effects, we studied the
in vitro
aggregation kinetics of a series of poly(Gln) peptides. We find that poly(Gln) peptides in solution at 37°C undergo a random coil to β-sheet transition with kinetics superimposable on their aggregation kinetics, suggesting the absence of soluble, β-sheet-rich intermediates in the aggregation process. Details of the time course of aggregate growth confirm that poly(Gln) aggregation occurs by nucleated growth polymerization. Surprisingly, however, and in contrast to conventional models of nucleated growth polymerization of proteins, we find that the aggregation nucleus is a monomer. That is, nucleation of poly(Gln) aggregation corresponds to an unfavorable protein folding reaction. Using parameters derived from the kinetic analysis, we estimate the difference in the free energy of nucleus formation between benign and pathological length poly(Gln)s to be less than 1 kcal/mol. We also use the kinetic parameters to calculate predicted aggregation curves for very low concentrations of poly(Gln) that might obtain in the cell. The repeat-length-dependent differences in predicted aggregation lag times are in the same range as the length-dependent age-of-onset differences in Huntington's disease, suggesting that the biophysics of poly(Gln) aggregation nucleation may play a major role in determining disease onset.