bluuebunny/crossref_metadata_2025 huggingface dataset:
Glucose transporter GLUT3: Ontogeny, targeting, and role in the mouse blastocyst
Authors: Pantaleon M. , Harvey M. B. , Pascoe W. S. , James D. E. , Kaye P. L.
Published: July 2002

The first differentiative event in mammalian development is segregation of the inner cell mass and trophectoderm (TE) lineages. The epithelial TE cells pump fluid into the spherical blastocyst to form the blastocyst cavity. This activity is fuelled by glucose supplied through facilitative glucose transporters. However, the reported kinetic characteristics of blastocyst glucose transport are inconsistent with those of the previously identified transporters and suggested the presence of a high-affinity glucose carrier. We identified and localized the primary transporter in TE cells. It is glucose transporter GLUT3, previously described in the mouse as neuron-specific. In the differentiating embryo, GLUT3 is targeted to the apical membranes of the polarized cells of the compacted morula and then to the apical membranes of TE cells where it has access to maternal glucose. In contrast, GLUT1 was restricted to basolateral membranes of the outer TE cells in both compacted morulae and blastocysts. Using antisense oligodeoxynucleotides to specifically block protein expression, we confirmed that GLUT3 and not GLUT1 is the functional transporter for maternal glucose on the apical TE. More importantly, however, GLUT3 expression is required for blastocyst formation and hence this primary differentiation in mammalian development. This requirement is independent of its function as a glucose transporter because blastocysts will form in the absence of glucose. Thus the vectorial expression of GLUT3 into the apical membrane domains of the outer cells of the morula, which in turn form the TE cells of the blastocyst, is required for blastocyst formation.
Transport of a novel complex in the cytoplasmic matrix of
Chlamydomonas
flagella
Authors: Piperno Gianni , Mead Kara
Published: July 2002

Proteins necessary for maintenance and function of eukaryotic flagella are synthesized in the cell body. Transport of the inner dynein arm subunit p28

IDA4

in
Chlamydomonas
flagella requires the activity of the kinesin KHP1

FLA10

, a protein inactive at restrictive temperature in
fla
10, a temperature-dependent mutant of flagellar assembly. To identify other molecules involved in active transport of inner dynein arms within flagella we searched for polypeptides of the cytoplasmic matrix of flagella that fulfill two conditions: they bind to p28 and require the activity of KHP1 to be present in flagella. We found that the cytoplasmic matrix of flagella contains a previously unidentified “17S” complex of at least 13 polypeptides that in part is associated with p28. The 17S complex is present at permissive but not at restrictive temperature in
fla
10 flagella. It also turns over in the cytoplasmic matrix more frequently than dynein arms within the axoneme. This evidence suggests that the 17S complex transports precursors of inner dynein arms within flagella.
ETR2
is an
ETR1
-like gene involved in ethylene signaling in
Arabidopsis
Authors: Sakai Hajime , Hua Jian , Chen Qianhong G. , Chang Caren , Medrano Leonard J. , Bleecker Anthony B. , Meyerowitz Elliot M.
Published: July 2002

The plant hormone ethylene regulates a variety of processes of growth and development. To identify components in the ethylene signal transduction pathway, we screened for ethylene-insensitive mutants in
Arabidopsis thaliana
and isolated a dominant
etr2-1
mutant. The
etr2-1
mutation confers ethylene insensitivity in several processes, including etiolated seedling elongation, leaf expansion, and leaf senescence. Double mutant analysis indicates that
ETR2
acts upstream of
CTR1
, which codes for a Raf-related protein kinase. We cloned the
ETR2
gene on the basis of its map position, and we found that it exhibits sequence homology to the ethylene receptor gene
ETR1
and the
ETR1
-like
ERS
gene.
ETR2
may thus encode a third ethylene receptor in
Arabidopsis
, transducing the hormonal signal through its “two-component” structure. Expression studies show that
ETR2
is ubiquitously expressed and has a higher expression in some tissues, including inflorescence and floral meristems, petals, and ovules.
An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene
Authors: Morsy Manal A. , Gu MingCheng , Motzel Sherri , Zhao Jing , Lin Jing , Su Qin , Allen Henry , Franlin Laura , Parks Robin J. , Graham Frank L. , Kochanek Stefan , Bett Andrew J. , Caskey C. Thomas
Published: July 2002

Adenoviral (Ad)-mediated
in vivo
gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and
ob/ob
(leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.
Bloom’s and Werner’s syndrome genes suppress hyperrecombination in yeast
sgs1
mutant: Implication for genomic instability in human diseases
Authors: Yamagata Kazutsune , Kato Jun-ichi , Shimamoto Akira , Goto Makoto , Furuichi Yasuhiro , Ikeda Hideo
Published: July 2002

Bloom’s syndrome (BS) and Werner’s syndrome (WS) are genetic disorders in which an increased rate of chromosomal aberration is detected. The genes responsible for these diseases,
BLM
and
WRN
, have been found to be homologs of
Escherichia coli recQ
and
Saccharomyces cerevisiae SGS1
genes. Here we show that yeast Sgs1 helicase acts as a suppressor of illegitimate recombination through homologous recombination and that human BLM and WRN helicases can suppress the increased homologous and illegitimate recombinations in the
S. cerevisiae sgs1
mutant. The results imply a role of BLM and WRN helicases to control genomic stability in human cells. Similar to Sgs1 helicase, BLM helicase suppressed the cell growth in the
top3 sgs1
mutation background and restored the increased sensitivity of the
sgs1
mutant to hydroxyurea, but the WRN helicase did not. We discussed differential roles of BLM and WRN helicases in human cells. BLM- and WRN-bearing yeasts provide new useful models to investigate human BS and WS diseases.
Dual effect of interleukin 4 on HIV-1 expression: Implications for viral phenotypic switch and disease progression
Authors: Valentin Antonio , Lu Wenhong , Rosati Margherita , Schneider Ralf , Albert Jan , Karlsson Anders , Pavlakis George N.
Published: July 2002

We report that interleukin 4 (IL-4) inhibits the propagation of non-syncytia-inducing and increases the propagation of syncytia-inducing HIV-1 isolates by two mechanisms. It differentially regulates the two major HIV-1 coreceptors, CCR5 and CXCR4, in human peripheral blood mononuclear cells, increasing CXCR4 and decreasing CCR5 expression in primary CD4(+) T-lymphocytes. In addition, IL-4 stimulates the expression of all HIV-1 isolates via a transcriptional activation mechanism. The combination of these effects results in increased propagation of CXCR4-using and inhibition of CCR5-using HIV-1 strains. IL-4 also activates HIV-1 expression in primary monocytes/macrophages but does not affect CCR5 expression. These results identify IL-4 as an important regulator of HIV-1 and suggest a critical role for this cytokine in the control of viral evolution and in the phenotypic switch from non-syncytia-inducing to syncytia-inducing, which leads to accelerated disease progression.
Origin of the metazoan phyla: Molecular clocks confirm paleontological estimates
Authors: Ayala Francisco José , Rzhetsky Andrey , Ayala Francisco J.
Published: July 2002

The time of origin of the animal phyla is controversial. Abundant fossils from the major animal phyla are found in the Cambrian, starting 544 million years ago. Many paleontologists hold that these phyla originated in the late Neoproterozoic, during the 160 million years preceding the Cambrian fossil explosion. We have analyzed 18 protein-coding gene loci and estimated that protostomes (arthropods, annelids, and mollusks) diverged from deuterostomes (echinoderms and chordates) about 670 million years ago, and chordates from echinoderms about 600 million years ago. Both estimates are consistent with paleontological estimates. A published analysis of seven gene loci that concludes that the corresponding divergence times are 1,200 and 1,000 million years ago is shown to be flawed because it extrapolates from slow-evolving vertebrate rates to faster-evolving invertebrate rates, as well as in other ways.
Chlorophyll
a
oxygenase (
CAO
) is involved in chlorophyll
b
formation from chlorophyll
a
Authors: Tanaka Ayumi , Ito Hisashi , Tanaka Ryouichi , Tanaka Nobuaki K. , Yoshida Kazuichi , Okada Kiyotaka
Published: July 2002

Chlorophyll
b
is an ubiquitous accessory pigment in land plants, green algae, and prochlorophytes. Its biosynthesis plays a key role in the adaptation to various light environments. We isolated six chlorophyll
b
-less mutants by insertional mutagenesis by using the nitrate reductase or argininosuccinate lyase genes as tags and examined the rearrangement of mutant genomes. We found that an overlapping region of a nuclear genome was deleted in all mutants and that this encodes a protein whose sequence is similar to those of methyl monooxygenases. This coding sequence also contains putative binding domains for a [2Fe-2S] Rieske center and for a mononuclear iron. The results demonstrate that a chlorophyll
a
oxygenase is involved in chlorophyll
b
formation. The reaction mechanism of chlorophyll
b
formation is discussed.
Endothelin ET
A
receptor blockade restores NO-mediated endothelial function and inhibits atherosclerosis in apolipoprotein E-deficient mice
Authors: Barton Matthias , Haudenschild Christian C. , d’Uscio Livius V. , Shaw Sidney , Münter Klaus , Lüscher Thomas F.
Published: July 2002

This study investigated whether endothelin-1 (ET-1), a potent vasoconstrictor, which also stimulates cell proliferation, contributes to endothelial dysfunction and atherosclerosis. Apolipoprotein E (apoE)-deficient mice and C57BL/6 control mice were treated with a Western-type diet to accelerate atherosclerosis with or without ET
A
receptor antagonist LU135252 (50 mg/kg/d) for 30 wk. Systolic blood pressure, plasma lipid profile, and plasma nitrate levels were determined. In the aorta, NO-mediated endothelium-dependent relaxation, atheroma formation, ET receptor-binding capacity, and vascular ET-1 protein content were assessed. In apoE-deficient but not C57BL/6 mice, severe atherosclerosis developed within 30 wk. Aortic ET-1 protein content (
P
< 0.0001) and binding capacity for ET
A
receptors was increased as compared with C57BL/6 mice. In contrast, NO-mediated, endothelium-dependent relaxation to acetylcholine (56 ± 3 vs. 99 ± 2%,
P
< 0.0001) and plasma nitrate were reduced (57.9 ± 4 vs. 93 ± 10 μmol/liter,
P
< 0.01). Treatment with the ET
A
receptor antagonist LU135252 for 30 wk had no effect on the lipid profile or systolic blood pressure in apoE-deficient mice, but increased NO-mediated endothelium-dependent relaxation (from 56 ± 3 to 93 ± 2%,
P
< 0.0001 vs. untreated) as well as circulating nitrate levels (from 57.9 ± 4 to 80 ± 8.3 μmol/liter,
P
< 0.05). Chronic ET
A
receptor blockade reduced elevated tissue ET-1 levels comparable with those found in C57BL/6 mice and inhibited atherosclerosis in the aorta by 31% without affecting plaque morphology or ET receptor-binding capacity. Thus, chronic ET
A
receptor blockade normalizes NO-mediated endothelial dysfunction and reduces atheroma formation independent of plasma cholesterol and blood pressure in a mouse model of human atherosclerosis. ET
A
receptor blockade may have therapeutic potential in patients with atherosclerosis.
The
Arabidopsis thaliana AGRAVITROPIC 1
gene encodes a component of the polar-auxin-transport efflux carrier
Authors: Chen Rujin , Hilson Pierre , Sedbrook John , Rosen Elizabeth , Caspar Timothy , Masson Patrick H.
Published: July 2002

Auxins are plant hormones that mediate many aspects of plant growth and development. In higher plants, auxins are polarly transported from sites of synthesis in the shoot apex to their sites of action in the basal regions of shoots and in roots. Polar auxin transport is an important aspect of auxin functions and is mediated by cellular influx and efflux carriers. Little is known about the molecular identity of its regulatory component, the efflux carrier [Estelle, M. (1996)
Current Biol.
6, 1589–1591]. Here we show that mutations in the
Arabidopsis thaliana AGRAVITROPIC 1
(
AGR1
) gene involved in root gravitropism confer increased root-growth sensitivity to auxin and decreased sensitivity to ethylene and an auxin transport inhibitor, and cause retention of exogenously added auxin in root tip cells. We used positional cloning to show that
AGR1
encodes a putative transmembrane protein whose amino acid sequence shares homologies with bacterial transporters. When expressed in
Saccharomyces cerevisiae
,
AGR1
promotes an increased efflux of radiolabeled IAA from the cells and confers increased resistance to fluoro-IAA, a toxic IAA-derived compound.
AGR1
transcripts were localized to the root distal elongation zone, a region undergoing a curvature response upon gravistimulation. We have identified several
AGR1
-related genes in
Arabidopsis
, suggesting a global role of this gene family in the control of auxin-regulated growth and developmental processes.
Natal origins of migratory monarch butterflies at wintering colonies in Mexico: New isotopic evidence
Authors: Wassenaar Leonard I. , Hobson Keith A.
Published: July 2002

Each year, millions of monarch butterflies from eastern North America migrate to overwinter in 10–13 discrete colonies located in the Oyamel forests of central Mexico. For decades efforts to track monarch migration have relied on observations and tag-recapture methods, culminating with the discovery of the wintering colonies in 1975. Monarch tag returns from Mexico, however, are few and primarily from two accessible colonies, and therefore tag-recapture techniques have not quantified natal origins or distinctiveness among monarch populations at wintering sites. Such information would be invaluable in the conservation of the monarch and its migration phenomenon since the wintering sites currently are threatened by habitat alteration. Here we show that stable hydrogen (δD) and carbon (δ
13
C) isotope ratios of wintering monarchs can be used to evaluate natal origins on the summer breeding range. Stable-hydrogen and carbon isotopic values of 597 wintering monarchs from 13 wintering roost sites were compared with isotopic patterns measured in individuals at natal sites across their breeding range over a single migration cycle. We determined that all monarch wintering colonies were composed of individuals originating mainly from the Midwest, United States, thereby providing evidence for a panmictic model of wintering colony composition. However, two colonies showed more northerly origins, suggesting possible priority colonies for conservation efforts.
Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex
Authors: Kleinfeld David , Mitra Partha P. , Helmchen Fritjof , Denk Winfried
Published: July 2002

Cortical blood flow at the level of individual capillaries and the coupling of neuronal activity to flow in capillaries are fundamental aspects of homeostasis in the normal and the diseased brain. To probe the dynamics of blood flow at this level, we used two-photon laser scanning microscopy to image the motion of red blood cells (RBCs) in individual capillaries that lie as far as 600 μm below the pia mater of primary somatosensory cortex in rat; this depth encompassed the cortical layers with the highest density of neurons and capillaries. We observed that the flow was quite variable and exhibited temporal fluctuations around 0.1 Hz, as well as prolonged stalls and occasional reversals of direction. On average, the speed and flux (cells per unit time) of RBCs covaried linearly at low values of flux, with a linear density of ≈70 cells per mm, followed by a tendency for the speed to plateau at high values of flux. Thus, both the average velocity and density of RBCs are greater at high values of flux than at low values. Time-locked changes in flow, localized to the appropriate anatomical region of somatosensory cortex, were observed in response to stimulation of either multiple vibrissae or the hindlimb. Although we were able to detect stimulus-induced changes in the flux and speed of RBCs in some single trials, the amplitude of the stimulus-evoked changes in flow were largely masked by basal fluctuations. On average, the flux and the speed of RBCs increased transiently on stimulation, although the linear density of RBCs decreased slightly. These findings are consistent with a stimulus-induced decrease in capillary resistance to flow.
Grandmothering, menopause, and the evolution of human life histories
Authors: Hawkes K. , O’Connell J. F. , Jones N. G. Blurton , Alvarez H. , Charnov E. L.
Published: July 2002

Long postmenopausal lifespans distinguish humans from all other primates. This pattern may have evolved with mother–child food sharing, a practice that allowed aging females to enhance their daughters’ fertility, thereby increasing selection against senescence. Combined with Charnov’s dimensionless assembly rules for mammalian life histories, this hypothesis also accounts for our late maturity, small size at weaning, and high fertility. It has implications for past human habitat choice and social organization and for ideas about the importance of extended learning and paternal provisioning in human evolution.
Bcl-2 expression causes redistribution of glutathione to the nucleus
Authors: Voehringer D. W. , McConkey D. J. , McDonnell T. J. , Brisbay S. , Meyn R. E.
Published: July 2002

In this study we used HeLa cells transfected with a conditional Bcl-2 expression construct to study the effects of Bcl-2 on reduced glutathione (GSH) metabolism. Our previous work demonstrated that depletion of GSH by culturing cells in tissue culture medium lacking the amino acids cysteine and methionine, essential for GSH biosynthesis, caused cells overexpressing Bcl-2 to become sensitized to apoptotic induction. Here we report that Bcl-2 also dramatically alters GSH compartmentalization. Cellular distribution of GSH, assayed by confocal microscopy, revealed that when Bcl-2 expression was suppressed GSH was uniformly distributed primarily in the cytosol, whereas overexpression of Bcl-2 led to a relocalization of GSH into the nucleus. Isolated nuclei readily accumulated radiolabeled GSH and maintained higher nuclear GSH concentration in direct relation to Bcl-2 nuclear protein levels. Moreover, exogenous GSH blocked apoptotic changes and caspase activity in isolated nuclei exposed to the pro-apoptotic protease granzyme B. Our results indicate that one of the functions of Bcl-2 is to promote sequestration of GSH into the nucleus, thereby altering nuclear redox and blocking caspase activity as well as other nuclear alterations characteristic of apoptosis. We speculate that this mechanism contributes to the suppression of apoptosis in cells with elevated Bcl-2 levels.
HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte
Authors: Claycomb William C. , Lanson Nicholas A. , Stallworth Beverly S. , Egeland Daniel B. , Delcarpio Joseph B. , Bahinski Anthony , Izzo Nicholas J.
Published: July 2002

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase–PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of α-cardiac myosin heavy chain, α-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (
I
Kr
) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at ≫+20 mV, and were highly sensitive to dofetilide (IC
50
of 46.9 nM). Specific binding of [
3
H]dofetilide was saturable and fit a one-site binding isotherm with a
K
d
of 140 +/− 60 nM and a
B
max
of 118 fmol per 10
5
cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
Intestinal trefoil factor controls the expression of the adenomatous polyposis coli–catenin and the E-cadherin–catenin complexes in human colon carcinoma cells
Authors: Efstathiou J. A. , Noda M. , Rowan A. , Dixon C. , Chinery R. , Jawhari A. , Hattori T. , Wright N. A. , Bodmer W. F. , Pignatelli M.
Published: July 2002

Intestinal trefoil factor 3 (TFF3) is a member of the trefoil family of peptides, small molecules constitutively expressed in epithelial tissues, including the gastrointestinal tract. TFF3 has been shown to promote migration of intestinal epithelial cells
in vitro
and to enhance mucosal healing and epithelial restitution
in vivo
. In this study, we evaluated the effect of recombinant TFF3 (rTFF3) stimulation on the expression and cellular localization of the epithelial (E)-cadherin–catenin complex, a prime mediator of Ca
2+
dependent cell–cell adhesion, and the adenomatous polyposis coli (APC)–catenin complex in HT29, HCT116, and SW480 colorectal carcinoma cell lines. Stimulation by rTFF3 (10
−9
M and 10
−8
M) for 20–24 hr led to cell detachment and to a reduction in intercellular adhesion in HT29 and HCT116 cells. In both cell lines, E-cadherin expression was down-regulated. The expression of APC, α-catenin and β-catenin also was decreased in HT29 cells, with a translocation of APC into the nucleus. No change in either cell adhesion or in the expression of E-cadherin, the catenins, and APC was detected in SW480 cells. In addition, TFF3 induced DNA fragmentation and morphological changes characteristic of apoptosis in HT29. Tyrphostin, a competitive inhibitor of protein tyrosine kinases, inhibited the effects of TFF3. Our results indicate that by perturbing the complexes between E-cadherin, β-catenin, and associated proteins, TFF3 may modulate epithelial cell adhesion, migration, and survival.
Multilocus sequence typing: A portable approach to the identification of clones within populations of pathogenic microorganisms
Authors: Maiden Martin C. J. , Bygraves Jane A. , Feil Edward , Morelli Giovanna , Russell Joanne E. , Urwin Rachel , Zhang Qing , Zhou Jiaji , Zurth Kerstin , Caugant Dominique A. , Feavers Ian M. , Achtman Mark , Spratt Brian G.
Published: July 2002

Traditional and molecular typing schemes for the characterization of pathogenic microorganisms are poorly portable because they index variation that is difficult to compare among laboratories. To overcome these problems, we propose multilocus sequence typing (MLST), which exploits the unambiguous nature and electronic portability of nucleotide sequence data for the characterization of microorganisms. To evaluate MLST, we determined the sequences of ≈470-bp fragments from 11 housekeeping genes in a reference set of 107 isolates of
Neisseria meningitidis
from invasive disease and healthy carriers. For each locus, alleles were assigned arbitrary numbers and dendrograms were constructed from the pairwise differences in multilocus allelic profiles by cluster analysis. The strain associations obtained were consistent with clonal groupings previously determined by multilocus enzyme electrophoresis. A subset of six gene fragments was chosen that retained the resolution and congruence achieved by using all 11 loci. Most isolates from hyper-virulent lineages of serogroups A, B, and C meningococci were identical for all loci or differed from the majority type at only a single locus. MLST using six loci therefore reliably identified the major meningococcal lineages associated with invasive disease. MLST can be applied to almost all bacterial species and other haploid organisms, including those that are difficult to cultivate. The overwhelming advantage of MLST over other molecular typing methods is that sequence data are truly portable between laboratories, permitting one expanding global database per species to be placed on a World-Wide Web site, thus enabling exchange of molecular typing data for global epidemiology via the Internet.
Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL
Authors: Yasuda Hisataka , Shima Nobuyuki , Nakagawa Nobuaki , Yamaguchi Kyoji , Kinosaki Masahiko , Mochizuki Shin-ichi , Tomoyasu Akihiro , Yano Kazuki , Goto Masaaki , Murakami Akihiko , Tsuda Eisuke , Morinaga Tomonori , Higashio Kanji , Udagawa Nobuyuki , Takahashi Naoyuki , Suda Tatsuo
Published: July 2002

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.
Association of the
Arabidopsis
CTR1 Raf-like kinase with the ETR1 and ERS ethylene receptors
Authors: Clark Karen L. , Larsen Paul B. , Wang Xiaoxia , Chang Caren
Published: July 2002

In
Arabidopsis thaliana
, signal transduction of the hormone ethylene involves at least two receptors, ETR1 and ERS, both of which are members of the two-component histidine protein kinase family that is prevalent in prokaryotes. The pathway also contains a negative regulator of ethylene responses, CTR1, which closely resembles members of the Raf protein kinase family. CTR1 is thought to act at or downstream of ETR1 and ERS based on double mutant analysis; however, the signaling mechanisms leading from ethylene perception to the regulation of CTR1 are unknown. By using the yeast two-hybrid assay, we detected a specific interaction between the CTR1 amino-terminal domain and the predicted histidine kinase domain of ETR1 and ERS. We subsequently verified these interactions by using an
in vitro
protein association assay(s). In addition, we determined that the amino-terminal domain of CTR1 can associate with the predicted receiver domain of ETR1
in vitro
. Based on deletion analysis, the portion of CTR1 that interacts with ETR1 roughly aligns with the regulatory region of Raf kinases. These physical associations support the genetic evidence that CTR1 acts in the pathway of ETR1 and ERS and suggest that these interactions could be involved in the regulation of CTR1 activity.
Continuation of neurogenesis in the hippocampus of the adult macaque monkey
Authors: Kornack David R. , Rakic Pasko
Published: July 2002

We present evidence for continuous generation of neurons, oligodendrocytes, and astrocytes in the hippocampal dentate gyrus of adult macaque monkeys, using immunohistochemical double labeling for bromodeoxyuridine and cell-type-specific markers. We estimate that the relative rate of neurogenesis is approximately 10 times less than that reported in the adult rodent dentate gyrus. Nevertheless, the generation of these three cell types in a discreet brain region suggests that a multipotent neural stem cell may be retained in the adult primate hippocampus. This demonstration of adult neurogenesis in nonhuman Old World primates—with their phylogenetic proximity to humans, long life spans, and elaborate cognitive abilities—establishes the macaque as an unexcelled animal model to experimentally investigate issues of neurogenesis in humans and offers new insights into its significance in the adult brain.
Broad patterns of gene expression revealed by clustering analysis of tumor and normal colon tissues probed by oligonucleotide arrays
Authors: Alon U. , Barkai N. , Notterman D. A. , Gish K. , Ybarra S. , Mack D. , Levine A. J.
Published: July 2002

Oligonucleotide arrays can provide a broad picture of the state of the cell, by monitoring the expression level of thousands of genes at the same time. It is of interest to develop techniques for extracting useful information from the resulting data sets. Here we report the application of a two-way clustering method for analyzing a data set consisting of the expression patterns of different cell types. Gene expression in 40 tumor and 22 normal colon tissue samples was analyzed with an Affymetrix oligonucleotide array complementary to more than 6,500 human genes. An efficient two-way clustering algorithm was applied to both the genes and the tissues, revealing broad coherent patterns that suggest a high degree of organization underlying gene expression in these tissues. Coregulated families of genes clustered together, as demonstrated for the ribosomal proteins. Clustering also separated cancerous from noncancerous tissue and cell lines from
in vivo
tissues on the basis of subtle distributed patterns of genes even when expression of individual genes varied only slightly between the tissues. Two-way clustering thus may be of use both in classifying genes into functional groups and in classifying tissues based on gene expression.
MyoR: A muscle-restricted basic helix–loop–helix transcription factor that antagonizes the actions of MyoD
Authors: Lu Jianrong , Webb Robert , Richardson James A. , Olson Eric N.
Published: July 2002

Skeletal muscle development is controlled by a family of muscle-specific basic helix–loop–helix (bHLH) transcription factors that activate muscle genes by binding E-boxes (CANNTG) as heterodimers with ubiquitous bHLH proteins, called E proteins. Myogenic bHLH factors are expressed in proliferating undifferentiated myoblasts, but they do not initiate myogenesis until myoblasts exit the cell cycle. We describe a bHLH protein, MyoR (for
myo
genic
r
epressor), that is expressed in undifferentiated myoblasts in culture and is down-regulated during differentiation. MyoR is also expressed specifically in the skeletal muscle lineage between days 10.5 and 16.5 of mouse embryogenesis and down-regulated thereafter during the period of secondary myogenesis. MyoR forms heterodimers with E proteins that bind the same DNA sequence as myogenic bHLH/E protein heterodimers, but MyoR acts as a potent transcriptional repressor that blocks myogenesis and activation of E-box-dependent muscle genes. These results suggest a role for MyoR as a lineage-restricted transcriptional repressor of the muscle differentiation program.
Circular permutation and receptor insertion within green fluorescent proteins
Authors: Baird Geoffrey S. , Zacharias David A. , Tsien Roger Y.
Published: July 2002

Many areas of biology and biotechnology have been revolutionized by the ability to label proteins genetically by fusion to the
Aequorea
green fluorescent protein (GFP). In previous fusions, the GFP has been treated as an indivisible entity, usually appended to the amino or carboxyl terminus of the host protein, occasionally inserted within the host sequence. The tightly interwoven, three-dimensional structure and intricate posttranslational self-modification required for chromophore formation would suggest that major rearrangements or insertions within GFP would prevent fluorescence. However, we now show that several rearrangements of GFPs, in which the amino and carboxyl portions are interchanged and rejoined with a short spacer connecting the original termini, still become fluorescent. These circular permutations have altered pKa values and orientations of the chromophore with respect to a fusion partner. Furthermore, certain locations within GFP tolerate insertion of entire proteins, and conformational changes in the insert can have profound effects on the fluorescence. For example, insertions of calmodulin or a zinc finger domain in place of Tyr-145 of a yellow mutant (enhanced yellow fluorescent protein) of GFP result in indicator proteins whose fluorescence can be enhanced severalfold upon metal binding. The calmodulin graft into enhanced yellow fluorescent protein can monitor cytosolic Ca
2+
in single mammalian cells. The tolerance of GFPs for circular permutations and insertions shows the folding process is surprisingly robust and offers a new strategy for creating genetically encodable, physiological indicators.
Case study of the effects of atmospheric aerosols and regional haze on agriculture: An opportunity to enhance crop yields in China through emission controls?
Authors: Chameides W. L. , Yu H. , Liu S. C. , Bergin M. , Zhou X. , Mearns L. , Wang G. , Kiang C. S. , Saylor R. D. , Luo C. , Huang Y. , Steiner A. , Giorgi F.
Published: July 2002

The effect of atmospheric aerosols and regional haze from air pollution on the yields of rice and winter wheat grown in China is assessed. The assessment is based on estimates of aerosol optical depths over China, the effect of these optical depths on the solar irradiance reaching the earth’s surface, and the response of rice and winter wheat grown in Nanjing to the change in solar irradiance. Two sets of aerosol optical depths are presented: one based on a coupled, regional climate/air quality model simulation and the other inferred from solar radiation measurements made over a 12-year period at meteorological stations in China. The model-estimated optical depths are significantly smaller than those derived from observations, perhaps because of errors in one or both sets of optical depths or because the data from the meteorological stations has been affected by local pollution. Radiative transfer calculations using the smaller, model-estimated aerosol optical depths indicate that the so-called “direct effect” of regional haze results in an ≈5–30% reduction in the solar irradiance reaching some of China’s most productive agricultural regions. Crop-response model simulations suggest an ≈1:1 relationship between a percentage increase (decrease) in total surface solar irradiance and a percentage increase (decrease) in the yields of rice and wheat. Collectively, these calculations suggest that regional haze in China is currently depressing optimal yields of ≈70% of the crops grown in China by at least 5–30%. Reducing the severity of regional haze in China through air pollution control could potentially result in a significant increase in crop yields and help the nation meet its growing food demands in the coming decades.
The delayed resurgence of equatorial forests after the Permian–Triassic ecologic crisis
Authors: Looy C. V. , Brugman W. A. , Dilcher D. L. , Visscher H.
Published: July 2002

In conjunction with the Permian–Triassic ecologic crisis ≈250 million years ago, massive dieback of coniferous vegetation resulted in a degradation of terrestrial ecosystems in Europe. A 4- to 5-million-year period of lycopsid dominance followed, and renewed proliferation of conifers did not occur before the transition between Early and Middle Triassic. We document this delayed re-establishment of equatorial forests on the basis of palynological data. The reconstructed pattern of vegetational change suggests that habitat restoration, migration, and evolutionary processes acted synergistically, setting the stage for successional replacement of lycopsid dominants by conifers within a period of ≈0.5 million years.
Mutation rates among RNA viruses
Authors: Drake John W. , Holland John J.
Published: July 2002

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of μ
g
≈ 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.
Isometric contraction induces the Ca
2+
-independent activation of the endothelial nitric oxide synthase
Authors: Fleming Ingrid , Bauersachs Johann , Schäfer Andreas , Scholz Dimitri , Aldershvile Jan , Busse Rudi
Published: July 2002

Shear stress and tyrosine phosphatase inhibitors have been shown to activate the endothelial NO synthase (eNOS) in a Ca
2+
/calmodulin-independent manner. We report here that isometric contraction of rabbit aorta activates eNOS by a pharmacologically identical pathway. Endothelium-intact aortic rings were precontracted under isometric conditions up to 60% of the maximal phenylephrine-induced tone. The NO synthase inhibitor N
G
nitro-
l
-arginine (
l
-NA) and the soluble guanylyl cyclase inhibitor NS 2028 induced an additional contraction, the amplitude of which depended on the level of precontraction. The maximal production of NO by isometrically contracted aortic rings (as estimated by the increase in cGMP in detector smooth muscle cells in a superfusion bioassay) was observed during the initial phase of isometric contraction and was greater than that detected following the application of acetylcholine. The supplementary
l
-NA-induced increase in vascular tone was inhibited by the nonselective kinase inhibitor staurosporine and the tyrosine kinase inhibitors erbstatin A and herbimycin A. Another tyrosine kinase inhibitor, genistein, the calmodulin antagonist calmidazolium, and the selective protein kinase C inhibitor, Ro 31–8220, had no effect. Coincident with the enhanced NO formation during isometric contraction was an increase in the tyrosine phosphorylation of endothelial proteins, which also correlated with the level of precontraction. Thus, isometric contraction activates eNOS via a Ca
2+
-independent, tyrosine kinase inhibitor-sensitive pathway and, like shear stress, seems to be an independent determinant of mechanically induced NO formation.
Accelerated chemical synthesis of peptides and small proteins
Authors: Miranda Les P. , Alewood Paul F.
Published: July 2002

The chemical synthesis of peptides and small proteins is a
powerful complementary strategy to recombinant protein overexpression
and is widely used in structural biology, immunology, protein
engineering, and biomedical research. Despite considerable improvements
in the fidelity of peptide chain assembly, side-chain protection, and
postsynthesis analysis, a limiting factor in accessing polypeptides
containing greater than 50 residues remains the time taken for chain
assembly. The ultimate goal of this work is to establish highly
efficient chemical procedures that achieve chain-assembly rates of
approximately 10–15 residues per hour, thus underpinning the rapid
chemical synthesis of long polypeptides and proteins, including
cytokines, growth factors, protein domains, and small enzymes. Here we
report Boc chemistry that employs
O
-(7-azabenzotriazol-1-yl)-
N,N,N
′
,N
′-tetramethyluronium
hexafluorophosphate (HATU)/dimethyl sulfoxide
in situ
neutralization as the coupling agent and
incorporates a protected amino acid residue every 5 min to produce
peptides of good quality. This rapid coupling chemistry was
successfully demonstrated by synthesizing several small to medium
peptides, including the “difficult” C-terminal sequence of HIV-1
proteinase (residues 81–99); fragment 65–74 of the acyl carrier
protein; conotoxin PnIA(A10L), a potent neuronal nicotinic receptor
antagonist; and the pro-inflammatory chemotactic protein CP10, an
88-residue protein, by means of native chemical ligation. The benefits
of this approach include enhanced ability to identify and characterize
“difficult couplings,” rapid access to peptides for biological
and structure–activity studies, and accelerated synthesis of tailored
large peptide segments (<50 residues) for use in chemoselective
ligation methods.
Quorum sensing in
Escherichia coli
,
Salmonella typhimurium
, and
Vibrio harveyi
: A new family of genes responsible for autoinducer production
Authors: Surette Michael G. , Miller Melissa B. , Bassler Bonnie L.
Published: July 2002

In bacteria, the regulation of gene expression in response to
changes in cell density is called quorum sensing. Quorum-sensing
bacteria produce, release, and respond to hormone-like molecules
(autoinducers) that accumulate in the external environment as the cell
population grows. In the marine bacterium
Vibrio harveyi
two parallel quorum-sensing systems exist, and each is composed of a
sensor–autoinducer pair.
V. harveyi
reporter strains
capable of detecting only autoinducer 1 (AI-1) or autoinducer 2 (AI-2)
have been constructed and used to show that many species of bacteria,
including
Escherichia coli
MG1655,
E.
coli
O157:H7,
Salmonella typhimurium
14028, and
S. typhimurium
LT2 produce autoinducers similar or
identical to the
V. harveyi
system 2 autoinducer AI-2.
However, the domesticated laboratory strain
E. coli
DH5α does not produce this signal molecule. Here we report the
identification and analysis of the gene responsible for AI-2 production
in
V. harveyi
,
S. typhimurium
, and
E. coli
. The genes, which we have named

luxS
V.h.

,

luxS
S.t.

, and

luxS
E.c.

respectively, are highly homologous
to one another but not to any other identified gene.
E.
coli
DH5α can be complemented to AI-2 production by the
introduction of the
luxS
gene from
V.
harveyi
or
E. coli
O157:H7. Analysis of the
E. coli
DH5α

luxS
E.c.

gene
shows that it contains a frameshift mutation resulting in premature
truncation of the LuxS

E.c.

protein. Our
results indicate that the
luxS
genes define a new family
of autoinducer-production genes.
The insulin-like growth factor (IGF)-dependent IGF binding protein-4 protease secreted by human fibroblasts is pregnancy-associated plasma protein-A
Authors: Lawrence James B. , Oxvig Claus , Overgaard Michael T. , Sottrup-Jensen Lars , Gleich Gerald J. , Hays Lara G. , Yates John R. , Conover Cheryl A.
Published: July 2002

Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action
in vitro,
and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.
Quantitative fine-structural analysis of olfactory cortical synapses
Authors: Schikorski T. , Stevens C. F.
Published: July 2002

To determine the extent to which hippocampal synapses are typical of those found in other cortical regions, we have carried out a quantitative analysis of olfactory cortical excitatory synapses, reconstructed from serial electron micrograph sections of mouse brain, and have compared these new observations with previously obtained data from hippocampus. Both superficial and deep layer I olfactory cortical synapses were studied. Although individual synapses in each of the areas—CA1 hippocampus, olfactory cortical layer Ia, olfactory cortical area Ib—might plausibly have been found in any of the other areas, the average characteristics of the three synapse populations are distinct. Olfactory cortical synapses in both layers are, on average, about 2.5 times larger than their hippocampal counterparts. The layer Ia olfactory cortical synapses have fewer synaptic vesicles than do the layer Ib synapses, but the absolute number of vesicles docked to the active zone in the layer Ia olfactory cortical synapses is about equal to the docked vesicle number in the smaller hippocampal synapses. As would be predicted from studies on hippocampus that relate paired-pulse facilitation to the number of docked vesicles, the synapses in layer 1a exhibit facilitation, whereas the ones in layer 1b do not. Although hippocampal synapses provide as a good model system for central synapses in general, we conclude that significant differences in the average structure of synapses from one cortical region to another exist, and this means that generalizations based on a single synapse type must be made with caution.