bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and isolation of candidate human keratinocyte stem cells based on cell surface phenotype
Authors: Li Amy , Simmons Paul J. , Kaur Pritinder
Published: July 2002

Despite the central role of human epidermal stem cells in tissue homeostasis, wound repair, and neoplasia, remarkably little is known about these cells, largely due to the absence of molecular markers that distinguish them from other proliferative cells within the germinative/basal layer. Epidermal stem cells can be distinguished from other cells in the basal layer by their quiescent naturein vivoand their greater overall proliferative capacity. In this study, we demonstrate enrichment and isolation of a subpopulation of basal epidermal cells from neonatal human foreskin based on cell surface phenotype, which satisfy these criteria. These putative stem cells are distinguished from other basal cells by their characteristic expression of high levels of the adhesion molecule α6, a member of the integrin family (α6bri), and low levels of a proliferation-associated cell surface marker recognized by recently described mAb 10G7 (10G7dim). We conclude that cells with the phenotype α6bri10G7dimrepresent the epidermal stem cell population based on the demonstration that these cells (i) exhibit the greatest regenerative capacity of any basal cells, (ii) represent a minor subpopulation (≈10%) of immature epidermal cells, which (iii) are quiescent at the time of isolation from the epidermis, as determined by cell cycle analysis.
Epithelial attachment alters the outcome of
Helicobacter pylori
infection
Authors: Guruge Janaki L. , Falk Per G. , Lorenz Robin G. , Dans Maria , Wirth Hans-Peter , Blaser Martin J. , Berg Douglas E. , Gordon Jeffrey I.
Published: July 2002

Genetically defined
in vivo
models are needed to assess the importance of target cell attachment in bacterial pathogenesis. Gastric colonization by
Helicobacter pylori
in human populations is common and persistent, and has various outcomes including peptic ulcers and cancer. The impact of attachment on the course of infection was examined in transgenic mice expressing a human receptor for
H. pylori
in their gastric epithelium. Persistent infection by a clinical isolate occurred at comparable microbial densities in transgenic and nontransgenic littermates. However, microbial attachment in transgenic mice resulted in production of autoantibodies to Lewis
x
carbohydrate epitopes shared by bacteria and acid-secreting parietal cells, chronic gastritis, and parietal cell loss. This model should help identify bacterial and host genes that produce attachment-related pathology.
Plant nuclear gene knockout reveals a role in plastid division for the homolog of the bacterial cell division protein FtsZ, an ancestral tubulin
Authors: Strepp René , Scholz Sirkka , Kruse Sven , Speth Volker , Reski Ralf
Published: July 2002

Little is known about the division of eukaryotic cell organelles and up to now neither in animals nor in plants has a gene product been shown to mediate this process. A cDNA encoding a homolog of the bacterial cell division protein FtsZ, an ancestral tubulin, was isolated from the eukaryote
Physcomitrella patens
and used to disrupt efficiently the genomic locus in this terrestrial seedless plant. Seven out of 51 transgenics obtained were knockout plants generated by homologous recombination; they were specifically impeded in plastid division with no detectable effect on mitochondrial division or plant morphology. Implications on the theory of endosymbiosis and on the use of reverse genetics in plants are discussed.
Mediator protein mutations that selectively abolish activated transcription
Authors: Myers Lawrence C. , Gustafsson Claes M. , Hayashibara Kathleen C. , Brown Patrick O. , Kornberg Roger D.
Published: July 2002

Deletion of any one of three subunits of the yeast Mediator of transcriptional regulation, Med2, Pgd1 (Hrs1), and Sin4, abolished activation by Gal4–VP16
in vitro
. By contrast, other Mediator functions, stimulation of basal transcription and of TFIIH kinase activity, were unaffected. A different but overlapping Mediator subunit dependence was found for activation by Gcn4. The genetic requirements for activation
in vivo
were closely coincident with those
in vitro
. A whole genome expression profile of a
Δmed2
strain showed diminished transcription of a subset of inducible genes but only minor effects on “basal” transcription. These findings make an important connection between transcriptional activation
in vitro
and
in vivo
, and identify Mediator as a “global” transcriptional coactivator.
Long-term trend toward earlier breeding in an American bird: A response to global warming?
Authors: Brown Jerram L. , Li Shou-Hsien , Bhagabati Nirmal
Published: July 2002

In regions with severe winters, global warming may be expected to cause earlier onset of breeding in most animals, yet no documentation of such a trend exists in North America. In a study of marked individuals of the Mexican jay (
Aphelocoma ultramarina
) in southeastern Arizona, from 1971 to 1998, the mean Julian date of first clutch in the population declined significantly by 10.1 days. The date of the first nest in the population also became earlier, by 10.8 days. These changes were associated with significant trends toward increased monthly minimum temperatures on the study area, traits that are associated with the onset of breeding in this population. Significant trends from 1971 to 1997 toward warmer minimum temperatures in the months before and during the initiation of breeding were observed. These trends parallel changes in minimum temperatures and community composition in a recent study of grassland ecology in the western United States. Together, they suggest that more attention should be given to the possible ecological importance of global change in minimum temperatures.
World food and agriculture: Outlook for the medium and longer term
Authors: Alexandratos Nikos
Published: July 2002

The world has been making progress in improving food security, as
measured by the per person availability of food for direct human
consumption. However, progress has been very uneven, and many
developing countries have failed to participate in such progress. In
some countries, the food security situation is today worse than 20
years ago. The persistence of food insecurity does not reflect so much
a lack of capacity of the world as a whole to increase food production
to whatever level would be required for everyone to have consumption
levels assuring satisfactory nutrition. The world already produces
sufficient food. The undernourished and the food-insecure persons are
in these conditions because they are poor in terms of income with which
to purchase food or in terms of access to agricultural resources,
education, technology, infrastructure, credit, etc., to produce their
own food. Economic development failures account for the persistence of
poverty and food insecurity. In the majority of countries with severe
food-security problems, the greatest part of the poor and food-insecure
population depend greatly on local agriculture for a living. In such
cases, development failures are often tantamount to failures of
agricultural development. Development of agriculture is seen as the
first crucial step toward broader development, reduction of poverty and
food insecurity, and eventually freedom from excessive economic
dependence on poor agricultural resources. Projections indicate that
progress would continue, but at a pace and pattern that would be
insufficient for the incidence of undernutrition to be reduced
significantly in the medium-term future. As in the past, world
agricultural production is likely to keep up with, and perhaps tend to
exceed, the growth of the effective demand for food. The problem will
continue to be one of persistence of poverty, leading to growth of the
effective demand for food on the part of the poor that would fall short
of that required for them to attain levels of consumption compatible
with freedom from undernutrition.
Stable exposure of the coreceptor-binding site in a CD4-independent HIV-1 envelope protein
Authors: Hoffman Trevor L. , LaBranche Celia C. , Zhang Wentao , Canziani Gabriella , Robinson James , Chaiken Irwin , Hoxie James A. , Doms Robert W.
Published: July 2002

We recently derived a CD4-independent virus from HIV-1/IIIB, termed IIIBx, which interacts directly with the chemokine receptor CXCR4 to infect cells. To address the underlying mechanism, a cloned Env from the IIIBx swarm (8x) was used to produce soluble gp120. 8x gp120 bound directly to cells expressing only CXCR4, whereas binding of IIIB gp120 required soluble CD4. Using an optical biosensor, we found that CD4-induced (CD4i) epitopes recognized by mAbs 17b and 48d were more exposed on 8x than on IIIB gp120. The ability of 8x gp120 to bind directly to CXCR4 and to react with mAbs 17b and 48d in the absence of CD4 indicated that this gp120 exists in a partially triggered but stable state in which the conserved coreceptor-binding site in gp120, which overlaps with the 17b epitope, is exposed. Substitution of the 8x V3 loop with that from the R5 virus strain BaL resulted in an Env (8x-V3BaL) that mediated CD4-independent CCR5-dependent virus infection and a gp120 that bound to CCR5 in the absence of CD4. Thus, in a partially triggered Env protein, the V3 loop can change the specificity of coreceptor use but does not alter CD4 independence, indicating that these properties are dissociable. Finally, IIIBx was more sensitive to neutralization by HIV-positive human sera, a variety of anti-IIIB gp120 rabbit sera, and CD4i mAbs than was IIIB. The sensitivity of this virus to neutralization and the stable exposure of a highly conserved region of gp120 suggest new strategies for the development of antibodies and small molecule inhibitors to this functionally important domain.
Blocking taste receptor activation of gustducin inhibits gustatory responses to bitter compounds
Authors: Ming Ding , Ninomiya Yuzo , Margolskee Robert F.
Published: July 2002

Gustducin, a transducin-like guanine nucleotide-binding regulatory protein (G protein), and transducin are expressed in taste receptor cells where they are thought to mediate taste transduction. Gustducin and transducin are activated in the presence of bovine taste membranes by several compounds that humans perceive to be bitter. We have monitored this activation with an
in vitro
assay to identify compounds that inhibited taste receptor activation of transducin by bitter tastants: AMP and chemically related compounds inhibited
in vitro
responses to several bitter compounds (e.g., denatonium, quinine, strychnine, and atropine). AMP also inhibited behavioral and electrophysiological responses of mice to bitter tastants, but not to NaCl, HCl, or sucrose. GMP, although chemically similar to AMP, inhibited neither the bitter-responsive taste receptor activation of transducin nor the gustatory responses of mice to bitter compounds. AMP and certain related compounds may bind to bitter-responsive taste receptors or interfere with receptor-G protein coupling to serve as naturally occurring taste modifiers.
Targeted disruption of the orphanin FQ/nociceptin gene increases stress susceptibility and impairs stress adaptation in mice
Authors: Köster Anja , Montkowski Alexandra , Schulz Stefan , Stübe Eva-Maria , Knaudt Kristina , Jenck Francois , Moreau Jean-Luc , Nothacker Hans-Peter , Civelli Olivier , Reinscheid Rainer K.
Published: July 2002

The neuropeptide orphanin FQ (also known as nociceptin; OFQ/N) has been implicated in modulating stress-related behavior. OFQ/N was demonstrated to reverse stress-induced analgesia and possess anxiolytic-like activity after central administration. To further study physiological functions of OFQ/N, we have generated OFQ/N-deficient mice by targeted disruption of the OFQ/N gene. Homozygous mice display increased anxiety-like behavior when exposed to a novel and threatening environment. OFQ/N-null mice show elevated basal pain threshold but develop normal stress-induced analgesia. Interestingly, these mice show impaired adaptation to repeated stress when compared with wild-type mice, whereas their performance in spatial learning remained unaffected. Basal and poststress plasma corticosterone levels were found to be elevated in OFQ/N-deficient animals. Thus, OFQ/N appears to be crucially involved in the neurobiological regulation of stress-coping behavior and fear.
Disruption of myoglobin in mice induces multiple compensatory mechanisms
Authors: Gödecke Axel , Flögel Ulrich , Zanger Klaus , Ding Zhaoping , Hirchenhain Jens , Decking Ulrich K. M. , Schrader Jürgen
Published: July 2002

Myoglobin may serve a variety of functions in muscular oxygen supply, such as O
2
storage, facilitated O
2
diffusion, and myoglobin-mediated oxidative phosphorylation. We studied the functional consequences of a myoglobin deficiency on cardiac function by producing myoglobin-knockout (myo
−/−
) mice. To genetically inactivate the myoglobin gene, exon 2 encoding the heme binding site was deleted in embryonic stem cells via homologous recombination. Myo
−/−
mice are viable, fertile, and without any obvious signs of functional limitations. Hemoglobin concentrations were significantly elevated in myo
−/−
mice. Cardiac function and energetics were analyzed in isolated perfused hearts under resting conditions and during β-adrenergic stimulation with dobutamine. Myo
−/−
hearts showed no alteration in contractile parameters either under basal conditions or after maximal β-adrenergic stimulation (200 nM dobutamine). Tissue levels of ATP, phosphocreatine (
31
P-NMR), and myocardial O
2
consumption were not altered. However, coronary flow {6.4 ± 1.3 ml⋅min
−1
⋅g
−1
[wild-type (WT)] vs. 8.5 ± 2.4 ml⋅min
−1
⋅g
−1
[myo
−/−
]} and coronary reserve [17.1 ± 2.1 (WT) vs. 20.8 ± 1.1 (myo
−/−
) ml⋅min
−1
⋅g
−1
were significantly elevated in myo
−/−
hearts. Histological examination revealed that capillary density also was increased in myo
−/−
hearts [3,111 ± 400 mm
−2
(WT) vs. 4,140 ± 140 mm
−2
(Myo
−/−
)]. These data demonstrate that disruption of myoglobin results in the activation of multiple compensatory mechanisms that steepen the pO
2
gradient and reduce the diffusion path length for O
2
between capillary and the mitochondria; this suggests that myoglobin normally is important for the delivery of oxygen.
The maximal affinity of ligands
Authors: Kuntz I. D. , Chen K. , Sharp K. A. , Kollman P. A.
Published: July 2002

We explore the question of what are the best ligands for macromolecular targets. A survey of experimental data on a large number of the strongest-binding ligands indicates that the free energy of binding increases with the number of nonhydrogen atoms with an initial slope of ≈−1.5 kcal/mol (1 cal = 4.18 J) per atom. For ligands that contain more than 15 nonhydrogen atoms, the free energy of binding increases very little with relative molecular mass. This nonlinearity is largely ascribed to nonthermodynamic factors. An analysis of the dominant interactions suggests that van der Waals interactions and hydrophobic effects provide a reasonable basis for understanding binding affinities across the entire set of ligands. Interesting outliers that bind unusually strongly on a per atom basis include metal ions, covalently attached ligands, and a few well known complexes such as biotin–avidin.
Cellular mechanisms of β-amyloid production and secretion
Authors: Sinha Sukanto , Lieberburg Ivan
Published: July 2002

The major constituent of senile plaques in Alzheimer’s disease is
a 42-aa peptide, referred to as β-amyloid (Aβ). Aβ is generated
from a family of differentially spliced, type-1 transmembrane domain
(TM)-containing proteins, called APP, by endoproteolytic processing.
The major, relatively ubiquitous pathway of APP metabolism in cell
culture involves cleavage by α-secretase, which cleaves within the
Aβ sequence, thus precluding Aβ formation and deposition. An
alternate secretory pathway, enriched in neurons and brain, leads to
cleavage of APP at the N terminus of the Aβ peptide by β-secretase,
thus generating a cell-associated β-C-terminal fragment (β-CTF). A
pathogenic mutation at codons 670/671 in APP (APP “Swedish”)
leads to enhanced cleavage at the β-secretase scissile bond and
increased Aβ formation. An inhibitor of vacuolar ATPases,
bafilomycin, selectively inhibits the action of β-secretase in cell
culture, suggesting a requirement for an acidic intracellular
compartment for effective β-secretase cleavage of APP. β-CTF is
cleaved in the TM domain by γ-secretase(s), generating both Aβ
1–40 (90%) and Aβ 1–42 (10%). Pathogenic mutations in APP at
codon 717 (APP “London”) lead to an increased proportion of Aβ
1–42 being produced and secreted. Missense mutations in PS-1,
localized to chromosome 14, are pathogenic in the majority of familial
Alzheimer’s pedigrees. These mutations also lead to increased
production of Aβ 1–42 over Aβ 1–40. Knockout of PS-1 in
transgenic animals leads to significant inhibition of production of
both Aβ 1–40 and Aβ 1–42 in primary cultures, indicating that
PS-1 expression is important for γ-secretase cleavages. Peptide
aldehyde inhibitors that block Aβ production by inhibiting
γ-secretase cleavage of β-CTF have been discovered.
Association of HIV-1 Tat with the cellular protein, Purα, is mediated by RNA
Authors: Gallia Gary L. , Darbinian Nune , Tretiakova Anna , Ansari Sameer A. , Rappaport Jay , Brady John , Wortman Margaret J. , Johnson Edward M. , Khalili Kamel
Published: July 2002

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Purα and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Purα includes the region demonstrated to bind Tat’s viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Purα derived from human U-87MG cells plays an important role in the Purα:Tat interaction as examined by an
in vitro
reconstitution assay. Furthermore, expression of the Purα-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Purα.
Glucocorticoid receptor inhibits transforming growth factor-β signaling by directly targeting the transcriptional activation function of Smad3
Authors: Song Chao-Zhong , Tian Xin , Gelehrter Thomas D.
Published: July 2002

The transforming growth factor-β (TGF-β) family of cytokines and glucocorticoids regulate diverse biological processes through modulating the expression of target genes. Here we report that glucocorticoid receptor (GR) represses TGF-β transcriptional activation of the type-1 plasminogen activator inhibitor (PAI-1) gene in a ligand-dependent manner. Similarly, GR represses TGF-β activation of the TGF-β responsive sequence containing Smad3/4-binding sites. Using mammalian two-hybrid assays, we demonstrate that GR inhibits transcriptional activation by both Smad3 and Smad4 C-terminal activation domains. Finally, we show that GR interacts with Smad3 both
in vitro
and
in vivo
. These results suggest a molecular basis for the cross-regulation between glucocorticoid and TGF-β signaling pathways.
Exploring drug-induced alterations in gene expression in
Mycobacterium tuberculosis
by microarray hybridization
Authors: Wilson Michael , DeRisi Joseph , Kristensen Hans-Henrik , Imboden Paul , Rane Sangeeta , Brown Patrick O. , Schoolnik Gary K.
Published: July 2002

Tuberculosis is a chronic infectious disease that is transmitted by cough-propelled droplets that carry the etiologic bacterium,
Mycobacterium tuberculosis.
Although currently available drugs kill most isolates of
M. tuberculosis
, strains resistant to each of these have emerged, and multiply resistant strains are increasingly widespread. The growing problem of drug resistance combined with a global incidence of seven million new cases per year underscore the urgent need for new antituberculosis therapies. The recent publication of the complete sequence of the
M. tuberculosis
genome has made possible, for the first time, a comprehensive genomic approach to the biology of this organism and to the drug discovery process. We used a DNA microarray containing 97% of the ORFs predicted from this sequence to monitor changes in
M. tuberculosis
gene expression in response to the antituberculous drug isoniazid. Here we show that isoniazid induced several genes that encode proteins physiologically relevant to the drug’s mode of action, including an operonic cluster of five genes encoding type II fatty acid synthase enzymes and
fbpC
, which encodes trehalose dimycolyl transferase. Other genes, not apparently within directly affected biosynthetic pathways, also were induced. These genes,
efp
A,
fadE23, fadE24
, and
ahpC
, likely mediate processes that are linked to the toxic consequences of the drug. Insights gained from this approach may define new drug targets and suggest new methods for identifying compounds that inhibit those targets.
Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation
Authors: Lein E. S. , Finney E. M. , McQuillen P. S. , Shatz C. J.
Published: July 2002

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.
Forced unfolding of the fibronectin type III module reveals a tensile molecular recognition switch
Authors: Krammer André , Lu Hui , Isralewitz Barry , Schulten Klaus , Vogel Viola
Published: July 2002

The 10th type III module of fibronectin possesses a
β
-sandwich structure consisting of seven
β
-strands (A–G) that are arranged in two antiparallel
sheets. It mediates cell adhesion to surfaces via its integrin binding
motif, Arg
78
, Gly
79
, and Asp
80
(RGD), which is placed at the apex of the loop connecting
β
-strands F and G. Steered molecular dynamics
simulations in which tension is applied to the protein’s terminal ends
reveal that the
β
-strand G is the first to break away
from the module on forced unfolding whereas the remaining fold
maintains its structural integrity. The separation of strand G from the
remaining fold results in a gradual shortening of the distance between
the apex of the RGD-containing loop and the module surface, which
potentially reduces the loop’s accessibility to surface-bound
integrins. The shortening is followed by a straightening of the
RGD-loop from a tight
β
-turn into a linear
conformation, which suggests a further decrease of affinity and
selectivity to integrins. The RGD-loop therefore is located
strategically to undergo strong conformational changes in the early
stretching stages of the module and thus constitutes a mechanosensitive
control of ligand recognition.
Effective treatment of autoimmune disease and progressive renal disease by mixed bone-marrow transplantation that establishes a stable mixed chimerism in BXSB recipient mice
Authors: Wang Bingyan , Yamamoto Yoshihisa , El-Badri Nagwa S. , Good Robert A.
Published: July 2002

Male BXSB mice spontaneously develop autoimmune disease with features similar to systemic lupus erythematosus. To determine whether this autoimmune disease can be treated as well as prevented by bone-marrow transplantation (BMT) and, at the same time, whether the immunity functions of lethally irradiated recipients can be reconstituted fully, male BXSB mice were engrafted with mixed T cell-depleted marrow (TCDM) both from fully allogeneic autoimmune-resistant BALB/c mice and from syngeneic autoimmune-prone BXSB mice, after the onset of autoimmune disease in the recipient mice. BMT with mixed TCDM from both resistant and susceptible strains of mice (mixed BMT) established stable mixed chimerism, prolonged the median life span, and arrested development of glomerulonephritis in BXSB mice. BMT with mixed TCDM also reduced the formation of anti-DNA antibodies that are observed typically in male mice of this strain. Furthermore, mixed BMT reconstituted the primary antibody production in BXSB recipients impressively. These findings indicate that transplantation of allogeneic autoimmune-resistant TCDM plus syngeneic autoimmune-prone TCDM into lethally irradiated BXSB mice can be used to treat autoimmune and renal disease in this strain of mice. In addition, this dual bone-marrow transplantation reconstitutes the immunity functions and avoids the immunodeficiencies that occur regularly in fully allogeneic chimeras after total body irradiation. This report describes an effective treatment of progressive renal disease and autoimmunity by establishing a stable mixed chimerism of TCDM transplantation from allogeneic autoimmune-resistant BALB/c mice plus syngeneic autoimmune-prone BXSB mice into BXSB mice.
The
NDUFA1
gene product (MWFE protein) is essential for activity of complex I in mammalian mitochondria
Authors: Au Harry C. , Seo Byoung Boo , Matsuno-Yagi Akemi , Yagi Takao , Scheffler Immo E.
Published: July 2002

The MWFE polypeptide of mammalian complex I (the proton-translocating NADH-quinone oxidoreductase) is 70 amino acids long, and it is predicted to be a membrane protein. The
NDUFA1
gene encoding the MWFE polypeptide is located on the X chromosome. This polypeptide is 1 of approximately 28 “accessory proteins” identified in complex I, which is composed of 42 unlike subunits. It was considered accessory, because it is not one of the 14 polypeptides making up the core complex I; a homologous set of 14 polypeptides can make a fully functional proton-translocating NADH-quinone oxidoreductase in prokaryotes. One MWFE mutant has been identified and isolated from a collection of respiration-deficient Chinese hamster cell mutants. The CCL16-B2 mutant has suffered a deletion that would produce a truncated and abnormal MWFE protein. In these mutant cells, complex I activity is reduced severely (<10%). Complementation with hamster
NDUFA1
cDNA restored the rotenone-sensitive complex I activity of these mutant cells to ≈100% of the parent cell activity. Thus, it is established that the MWFE polypeptide is absolutely essential for an active complex I in mammals.
Human RNA-specific adenosine deaminase
ADAR1
transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible
Authors: George Cyril X. , Samuel Charles E.
Published: July 2002

RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible ≈150-kDa protein and a constitutively expressed N-terminally truncated ≈110-kDa protein. We have now identified alternative exon 1 structures of human
ADAR1
transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5′-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of
ADAR1
and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B–exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A–exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated P
C
and P
I
. Exon 1B transcripts were initiated from the P
C
promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible P
I
promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the
ADAR1
gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed ≈110-kDa ADAR1 protein (exon 1B) or the interferon-induced ≈150-kDa ADAR1 protein (exon 1A).
Regulatory interaction of PRL1 WD protein with
Arabidopsis
SNF1-like protein kinases
Authors: Bhalerao Rishikesh P. , Salchert Klaus , Bakó László , Ökrész László , Szabados László , Muranaka Toshiya , Machida Yasunori , Schell Jeff , Koncz Csaba
Published: July 2002

Mutation of the
PRL1
gene, encoding a regulatory WD protein, results in glucose hypersensitivity and derepression of glucose-regulated genes in
Arabidopsis
. The yeast SNF1 protein kinase, a key regulator of glucose signaling, and
Arabidopsis
SNF1 homologs AKIN10 and AKIN11, which can complement the
Δsnf1
mutation, were found to interact with an N-terminal domain of the PRL1 protein in the two-hybrid system and
in vitro
. AKIN10 and AKIN11 suppress the yeast Δ
snf4
mutation and interact with the SNF4p-activating subunit of SNF1. PRL1 and SNF4 bind independently to adjacent C-terminal domains of AKIN10 and AKIN11, and these protein interactions are negatively regulated by glucose in yeast. AKIN10 and AKIN11, purified in fusion with glutathione
S
-transferase, undergo autophosphorylation and phosphorylate a peptide of sucrose phosphate synthase
in vitro
. The sucrose phosphate synthase-peptide kinase activity of AKIN complexes detected by immunoprecipitation is stimulated by sucrose in light-grown
Arabidopsis
plants. In comparison with wild type, the activation level of AKIN immunocomplexes is higher in the
prl1
mutant, suggesting that PRL1 is a negative regulator of
Arabidopsis
SNF1 homologs. This conclusion is supported by the observation that PRL1 is an inhibitor of AKIN10 and AKIN11
in vitro
.
Crystal structure of aStaphylococcus aureusprotein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity
Authors: Graille Marc , Stura Enrico A. , Corper Adam L. , Sutton Brian J. , Taussig Michael J. , Charbonnier Jean-Baptiste , Silverman Gregg J.
Published: July 2002

Staphylococcus aureusproduces a virulence factor, protein A (SpA), that contains five homologous Ig-binding domains. The interactions of SpA with the Fab region of membrane-anchored Igs can stimulate a large fraction of B cells, contributing to lymphocyte clonal selection. To understand the molecular basis for this activity, we have solved the crystal structure of the complex between domain D of SpA and the Fab fragment of a human IgM antibody to 2.7-Å resolution. In the complex, helices II and III of domain D interact with the variable region of the Fab heavy chain (VH) through framework residues, without the involvement of the hypervariable regions implicated in antigen recognition. The contact residues are highly conserved in human VH3 antibodies but not in other families. The contact residues from domain D also are conserved among all SpA Ig-binding domains, suggesting that each could bind in a similar manner. Features of this interaction parallel those reported for staphylococcal enterotoxins that are superantigens for many T cells. The structural homology between Ig VHregions and the T-cell receptor Vβregions facilitates their comparison, and both types of interactions involve lymphocyte receptor surface remote from the antigen binding site. However, T-cell superantigens reportedly interact through hydrogen bonds with T-cell receptor Vβbackbone atoms in a primary sequence-independent manner, whereas SpA relies on a sequence-restricted conformational binding with residue side chains, suggesting that this common bacterial pathogen has adopted distinct molecular recognition strategies for affecting large sets of B and T lymphocytes.
Normal cardiovascular development in mice deficient for 16 genes in 550 kb of the velocardiofacial/ DiGeorge syndrome region
Authors: Puech Anne , Saint-Jore Bruno , Merscher Sandra , Russell Robert G. , Cherif Dorra , Sirotkin Howard , Xu Hui , Factor Stephen , Kucherlapati Raju , Skoultchi Arthur I.
Published: July 2002

Hemizygous interstitial deletions in human chromosome 22q11 are associated with velocardiofacial syndrome and DiGeorge syndrome and lead to multiple congenital abnormalities, including cardiovascular defects. The gene(s) responsible for these disorders is thought to reside in a 1.5-Mb region of 22q11 in which 27 genes have been identified. We have used Cre-mediated recombination of LoxP sites in embryonic stem cells and mice to generate a 550-kb deletion encompassing 16 of these genes in the corresponding region on mouse chromosome 16. Mice heterozygous for this deletion are normal and do not exhibit cardiovascular abnormalities. Because mice with a larger deletion on mouse chromosome 16 do have heart defects, the results allow us to exclude these 16 genes as being solely, or in combination among themselves, responsible for the cardiovascular abnormalities in velocardiofacial/DiGeorge syndrome. We also generated mice with a duplication of the 16 genes that may help dissect the genetic basis of “cat eye” and derivative 22 syndromes that are characterized by extra copies of portions of 22q11, including these 16 genes. We also describe a strategy for selecting cell lines with defined chromosomal rearrangements. The method is based on reconstitution of a dominant selection marker after Cre-mediated recombination of LoxP sites. Therefore it should be widely applicable to many cell lines.
IL-18 regulates IL-1β-dependent hepatic melanoma metastasis via vascular cell adhesion molecule-1
Authors: Vidal-Vanaclocha Fernando , Fantuzzi Giamila , Mendoza Lorea , Fuentes Angela M. , Anasagasti Miren J. , Martín Javier , Carrascal Teresa , Walsh Patrick , Reznikov Leonid L. , Kim Soo-Hyun , Novick Daniela , Rubinstein Menachem , Dinarello Charles A.
Published: July 2002

Proinflammatory cytokines, including IL-1βand tumor necrosis factor-α(TNF-α), promote cancer cell adhesion and liver metastases by up-regulating the expression of vascular cell adhesion molecule-1 (VCAM-1) on hepatic sinusoidal endothelium (HSE). In this study, hepatic metastasis after intrasplenically injected mouse B16 melanoma (B16M) cells was reduced 84–95% in mice with null mutations for either IL-1βor the IL-1β-converting enzyme (ICE, caspase-1) compared with wild-type mice. On day 12, 47% of wild-type mice were dead compared with 19% of either IL-1βor ICE-deficient mice.In vitro, conditioned medium from B16M cells (B16M-CM) induced the release of TNF-αand IL-1βfrom cultures of primary murine HSE. The effect of B16M-CM on HSE resulted in increased numbers of B16M cells adhering to HSE, which was completely abrogated by a specific inhibitor of ICE, anti-IL-18 or IL-18-binding protein. Exogenous IL-18 added to HSE also increased the number of adhering melanoma cells; however, this was not affected by IL-1 receptor blockade or TNF neutralization but rather by anti-VCAM-1. These results demonstrate a role for IL-1βand IL-18 in the development of hepatic metastases of B16Min vivo.In vitro, soluble products from B16M cells stimulate HSE to sequentially release TNF-α, IL-1β, and IL-18. The IL-18 cytokine increases expression of VCAM-1 and the adherence of melanoma cells.
An
Arabidopsis
histone H2A mutant is deficient in
Agrobacterium
T-DNA integration
Authors: Mysore Kirankumar S. , Nam Jaesung , Gelvin Stanton B.
Published: July 2002

Agrobacterium tumefaciens
genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified. We characterized an
Arabidopsis
mutant generated by T-DNA insertional mutagenesis,
rat5
, that is resistant to
Agrobacterium
root transformation.
rat5
contains two copies of T-DNA integrated as a tandem direct repeat into the 3′ untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence. Transient and stable β-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and
rat5 Arabidopsis
plants indicated that the
rat5
mutant is deficient in T-DNA integration. We complemented the
rat5
mutation by expressing the
RAT5
histone H2A gene in the mutant plant. Overexpression of
RAT5
in wild-type plants increased
Agrobacterium
transformation efficiency. Furthermore, transient expression of a
RAT5
gene from the incoming T-DNA was sufficient to complement the
rat5
mutant and to increase the transformation efficiency of wild-type
Arabidopsis
plants.
Experimental diabetes in rats causes hippocampal dendritic and synaptic reorganization and increased glucocorticoid reactivity to stress
Authors: Magariños Ana María , McEwen Bruce S.
Published: July 2002

We report that 9 d of uncontrolled experimental diabetes
induced by streptozotocin (STZ) in rats is an endogenous
chronic stressor that produces retraction and simplification of apical
dendrites of hippocampal CA3 pyramidal neurons, an effect also observed
in nondiabetic rats after 21 d of repeated restraint stress or
chronic corticosterone (Cort) treatment. Diabetes also induces
morphological changes in the presynaptic mossy fiber terminals (MFT)
that form excitatory synaptic contacts with the proximal CA3 apical
dendrites. One effect, synaptic vesicle depletion, occurs in diabetes
as well as after repeated stress and Cort treatment. However, diabetes
produced other MFT structural changes that differ qualitatively and
quantitatively from other treatments. Furthermore, whereas 7 d of
repeated stress was insufficient to produce dendritic or synaptic
remodeling in nondiabetic rats, it potentiated both dendritic atrophy
and MFT synaptic vesicle depletion in STZ rats. These changes occurred
in concert with adrenal hypertrophy and elevated basal Cort release as
well as hypersensitivity and defective shutoff of Cort secretion after
stress. Thus, as an endogenous stressor, STZ diabetes not
only accelerates the effects of exogenous stress to alter hippocampal
morphology; it also produces structural changes that overlap only
partially with those produced by stress and Cort in the nondiabetic
state.
Detection of synchrony in the activity of auditory nerve fibers by octopus cells of the mammalian cochlear nucleus
Authors: Oertel Donata , Bal Ramazan , Gardner Stephanie M. , Smith Philip H. , Joris Philip X.
Published: July 2002

The anatomical and biophysical specializations of octopus cells allow them to detect the coincident firing of groups of auditory nerve fibers and to convey the precise timing of that coincidence to their targets. Octopus cells occupy a sharply defined region of the most caudal and dorsal part of the mammalian ventral cochlear nucleus. The dendrites of octopus cells cross the bundle of auditory nerve fibers just proximal to where the fibers leave the ventral and enter the dorsal cochlear nucleus, each octopus cell spanning about one-third of the tonotopic array. Octopus cells are excited by auditory nerve fibers through the activation of rapid, calcium-permeable, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate receptors. Synaptic responses are shaped by the unusual biophysical characteristics of octopus cells. Octopus cells have very low input resistances (about 7 MΩ), and short time constants (about 200 μsec) as a consequence of the activation at rest of a hyperpolarization-activated mixed-cation conductance and a low-threshold, depolarization-activated potassium conductance. The low input resistance causes rapid synaptic currents to generate rapid and small synaptic potentials. Summation of small synaptic potentials from many fibers is required to bring an octopus cell to threshold. Not only does the low input resistance make individual excitatory postsynaptic potentials brief so that they must be generated within 1 msec to sum but also the voltage-sensitive conductances of octopus cells prevent firing if the activation of auditory nerve inputs is not sufficiently synchronous and depolarization is not sufficiently rapid.In vivoin cats, octopus cells can fire rapidly and respond with exceptionally well-timed action potentials to periodic, broadband sounds such as clicks. Thus both the anatomical specializations and the biophysical specializations make octopus cells detectors of the coincident firing of their auditory nerve fiber inputs.
Bounds for cell entries in contingency tables given marginal totals and decomposable graphs
Authors: Dobra Adrian , Fienberg Stephen E.
Published: July 2002

Upper and lower bounds on cell counts in cross-classifications of nonnegative counts play important roles in a number of practical problems, including statistical disclosure limitation, computer tomography, mass transportation, cell suppression, and data swapping. Some features of the Fréchet bounds are well known, intuitive, and regularly used by those working on disclosure limitation methods, especially those for two-dimensional tables. We previously have described a series of results relating these bounds to theory on loglinear models for cross-classified counts. This paper provides the actual theory and proofs for the special case of decomposable loglinear models and their related independence graphs. It also includes an extension linked to the structure of reducible graphs and a discussion of the relevance of other results linked to nongraphical loglinear models.
The structure of the chromophore within DsRed, a red fluorescent protein from coral
Authors: Gross Larry A. , Baird Geoffrey S. , Hoffman Ross C. , Baldridge Kim K. , Tsien Roger Y.
Published: July 2002

DsRed, a brilliantly red fluorescent protein, was recently cloned fromDiscosomacoral by homology to the green fluorescent protein (GFP) from the jellyfishAequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming —C⩵N—C⩵O at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the C⩵N bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The C⩵N bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS/PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging.
Widespread occurrence of structurally diverse tetraether membrane lipids: Evidence for the ubiquitous presence of low-temperature relatives of hyperthermophiles
Authors: Schouten Stefan , Hopmans Ellen C. , Pancost Richard D. , Damsté Jaap S. Sinninghe
Published: July 2002

Isoprenoid glycerol dialkyl glycerol tetraethers (GDGTs) and
branched glycerol dialkyl diethers are main membrane constituents of
cultured hyperthermophilic archaea and eubacteria, respectively, and
are found in environments with temperatures >60°C. Recently, we
developed a new technique for the analysis of intact core tetraether
lipids in cell material and sediments. The application of this
technique to recent sediments shows that known and newly identified
isoprenoid and branched GDGTs are widespread in low-temperature
environments (<20°C) and are structurally far more diverse than
previously thought. Their distribution indicates the ubiquitous
environmental presence of as yet uncultivated, nonthermophilic
organisms that may have independently evolved from hyperthermophilic
archaea and eubacteria. The structures of some of the new GDGTs
point to the hybridization of both typical archaeal and eubacterial
biosynthetic pathways in single organisms.
FIDDLEHEAD
, a gene required to suppress epidermal cell interactions in
Arabidopsis
, encodes a putative lipid biosynthetic enzyme
Authors: Pruitt Robert E. , Vielle-Calzada Jean-Philippe , Ploense Sara E. , Grossniklaus Ueli , Lolle Susan J.
Published: July 2002

In plants, the outer epidermal cell wall and cuticle presents a semipermeable barrier that maintains the external integrity of the plant and regulates the passage of various classes of molecules into and out of the organism. During vegetative development, the epidermal cells remain relatively inert, failing to respond to wounding or grafting. During reproductive development and fertilization, however, the epidermis is developmentally more labile and participates in two types of contact-mediated cell interactions: organ fusion and pollen hydration. Here we describe the isolation and characterization of one gene whose product normally functions in blocking both types of epidermal cell interactions during vegetative development: the
FIDDLEHEAD
gene. As suggested by previous biochemical analyses, the gene encodes a protein that is probably involved in the synthesis of long-chain lipids found in the cuticle and shows similarity to a large class of genes encoding proteins related to β-ketoacyl-CoA synthases and chalcone synthases.
In situ
hybridization reveals an epidermal pattern of expression consistent with a role for this protein in the synthesis of lipid components that are thought to localize extracellularly and probably modify the properties of the cuticle.
Detecting Holocene changes in thermohaline circulation
Authors: Keigwin L. D. , Boyle E. A.
Published: July 2002

Throughout the last glacial cycle, reorganizations of deep
ocean water masses were coincident with rapid millennial-scale changes
in climate. Climate changes have been less severe during the present
interglacial, but evidence for concurrent deep ocean circulation change
is ambiguous.
Parsing executive processes: Strategic vs. evaluative functions of the anterior cingulate cortex
Authors: Carter Cameron S. , Macdonald Angus M. , Botvinick Matthew , Ross Laura L. , Stenger V. Andrew , Noll Douglas , Cohen Jonathan D.
Published: July 2002

Event-related functional MRI and a version of the Stroop color naming task were used to test two conflicting theories of anterior cingulate cortex (ACC) function during executive processes of cognition. A response-related increase in ACC activity was present when strategic processes were less engaged, and conflict high, but not when strategic processes were engaged and conflict reduced. This is inconsistent with the widely held view that the ACC implements strategic processes to reduce cognitive conflicts, such as response competition. Instead, it suggests that the ACC serves an evaluative function, detecting cognitive states such as response competition, which may lead to poor performance, and representing the knowledge that strategic processes need to be engaged.