bluuebunny/crossref_metadata_2025 huggingface dataset:
Senescent fibroblasts promote epithelial cell growth and tumorigenesis: A link between cancer and aging
Authors: Krtolica Ana , Parrinello Simona , Lockett Stephen , Desprez Pierre-Yves , Campisi Judith
Published: July 2002

Mammalian cells can respond to damage or stress by entering a state of arrested growth and altered function termed cellular senescence. Several lines of evidence suggest that the senescence response suppresses tumorigenesis. Cellular senescence is also thought to contribute to aging, but the mechanism is not well understood. We show that senescent human fibroblasts stimulate premalignant and malignant, but not normal, epithelial cells to proliferate in culture and form tumors in mice. In culture, the growth stimulation was evident when senescent cells comprised only 10% of the fibroblast population and was equally robust whether senescence was induced by replicative exhaustion, oncogenic RAS, p14

ARF

, or hydrogen peroxide. Moreover, it was due at least in part to soluble and insoluble factors secreted by senescent cells. In mice, senescent, much more than presenescent, fibroblasts caused premalignant and malignant epithelial cells to form tumors. Our findings suggest that, although cellular senescence suppresses tumorigenesis early in life, it may promote cancer in aged organisms, suggesting it is an example of evolutionary antagonistic pleiotropy.
Temporal and spatial overlap between monarch larvae and corn pollen
Authors: Oberhauser Karen S. , Prysby Michelle D. , Mattila Heather R. , Stanley-Horn Diane E. , Sears Mark K. , Dively Galen , Olson Eric , Pleasants John M. , Lam Wai-Ki F. , Hellmich Richard L.
Published: July 2002

To assess the likelihood that monarch larvae will be exposed to
Bacillus thuringiensis
(Bt) pollen, we studied milkweed and monarch densities in habitats which comprise much of the land available to breeding monarchs, e.g., cornfields, cornfield edges, other agricultural fields, and nonagricultural areas, in four regions of the monarch breeding range. We found that monarchs use milkweed in cornfields throughout their breeding season, and that per plant densities are as high or higher in agricultural habitats as in nonagricultural habitats. As a result of the prevalence of agricultural land, most of the monarchs produced in the upper Midwest are likely to originate in cornfields or other agricultural habitats. There was a greater temporal overlap between susceptible monarchs and corn anthesis in the northern than the southern part of the summer breeding range, because of earlier pollen shed in the south. The importance of agricultural habitats to monarch production suggests that, regardless of the impact of genetically modified crops, agricultural practices such as weed control and foliar insecticide use could have large impacts on monarch populations.
C/EBPα is required for differentiation of white, but not brown, adipose tissue
Authors: Linhart Heinz G. , Ishimura-Oka Kazumi , DeMayo Franco , Kibe Tetsuya , Repka David , Poindexter Brian , Bick Roger J. , Darlington Gretchen J.
Published: July 2002

The transcription factor CCAAT enhancer binding protein α (C/EBPα) is expressed at high levels in liver and adipose tissue. Cell culture studies show that C/EBPα is sufficient to trigger differentiation of preadipocytes into mature adipocytes, suggesting a central role for C/EBPα in the development of adipose tissue. C/EBPα knockout mice die within 7–12 h after birth. Defective gluconeogenesis of the liver and subsequent hypoglycemia contribute to the early death of these animals. This short life span impairs investigation of the development of adipose tissue in these mice. To improve the survival of C/EBPα−/− animals, we generated a transgenic line that expresses C/EBPα under the control of the albumin enhancer/promoter. This line was bred into the knockout strain to generate animals that express C/EBPα in the liver but in no other tissue. The presence of the transgene improved survival of C/EBPα−/− animals almost 3-fold. Transgenic C/EBPα−/− animals at 7 days of age show an absence of s.c., perirenal, and epididymal white fat despite excess lipid substrate in the serum, whereas brown adipose tissue is somewhat hypertrophied and shows minimal biochemical alterations. Interestingly, mammary gland fat tissue is present and exhibits normal morphology. The absence of white adipose tissue in many depots in the presence of high serum lipid levels shows that C/EBPα is required for the
in vivo
development of this tissue. In contrast, brown adipose tissue differentiation is independent of C/EBPα expression. The presence of lipid in brown adipose tissue serves as an internal nutritional control, indicating that neither nutritional intake nor lipoprotein composition is likely responsible for the absence of white fat.
FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse
Authors: Hentges Kathryn E. , Sirry Baheya , Gingeras Anne-Claude , Sarbassov Dos , Sonenberg Nahum , Sabatini David , Peterson Andrew S.
Published: July 2002

The FKBP-12-rapamycin associated protein (FRAP, also known as mTOR and RAFT-1) is a member of the phosphoinositide kinase related kinase family. FRAP has serine/threonine kinase activity and mediates the cellular response to mitogens through signaling to p70s6 kinase (p70
s6k
) and 4E-BP1, resulting in an increase in translation of subsets of cellular mRNAs. Translational up-regulation is blocked by inactivation of FRAP signaling by rapamycin, resulting in G
1
cell cycle arrest. Rapamycin is used as an immunosuppressant for kidney transplants and is currently under investigation as an antiproliferative agent in tumors because of its ability to block FRAP activity. Although the role of FRAP has been extensively studied
in vitro
, characterization of mammalian FRAP function
in vivo
has been limited to the immune system and tumor models. Here we report the identification of a loss-of-function mutation in the mouse FRAP gene, which illustrates a requirement for FRAP activity in embryonic development. Our studies also determined that rapamycin treatment of the early embryo results in a phenotype indistinguishable from the FRAP mutant, demonstrating that rapamycin has teratogenic activity.
Functional muscle ischemia in neuronal nitric oxide synthase-deficient skeletal muscle of children with Duchenne muscular dystrophy
Authors: Sander Mikael , Chavoshan Bahman , Harris Shannon A. , Iannaccone Susan T. , Stull James T. , Thomas Gail D. , Victor Ronald G.
Published: July 2002

Duchenne muscular dystrophy (DMD) is a fatal disease caused by
mutation of the gene encoding the cytoskeletal protein dystrophin.
Despite a wealth of recent information about the molecular basis of
DMD, effective treatment for this disease does not exist because the
mechanism by which dystrophin deficiency produces the clinical
phenotype is unknown. In both mouse and human skeletal muscle,
dystrophin deficiency results in loss of neuronal nitric oxide
synthase, which normally is localized to the sarcolemma as part of the
dystrophin–glycoprotein complex. Recent studies in mice suggest that
skeletal muscle-derived nitric oxide may play a key role in the
regulation of blood flow within exercising skeletal muscle by blunting
the vasoconstrictor response to α-adrenergic receptor activation.
Here we report that this protective mechanism is defective in children
with DMD, because the vasoconstrictor response (measured as a decrease
in muscle oxygenation) to reflex sympathetic activation was not blunted
during exercise of the dystrophic muscles. In contrast, this protective
mechanism is intact in healthy children and those with polymyositis or
limb-girdle muscular dystrophy, muscle diseases that do not result in
loss of neuronal nitric oxide synthase. This clinical investigation
suggests that unopposed sympathetic vasoconstriction in exercising
human skeletal muscle may constitute a heretofore unappreciated
vascular mechanism contributing to the pathogenesis of DMD.
Genetic evidence for the
in planta
role of phloem-specific plasma membrane sucrose transporters
Authors: Gottwald Jennifer R. , Krysan Patrick J. , Young Jeffery C. , Evert Ray F. , Sussman Michael R.
Published: July 2002

A major question in plant physiology is how the large amount of
sucrose made in leaves is transported to the rest of the plant.
Although physiological, biochemical, and anatomical investigations have
been performed in this field, to date there have been very few genetic
studies. Using a reverse genetic screen, we have identified mutant
Arabidopsis
plants containing transferred DNA insertions
in the gene encoding a phloem-specific sucrose transporter,
SUC2
. SUC2 is thought to function in loading sugar from
the apoplast into the conducting sieve tubes. In the homozygous state,
these mutations resulted in stunted growth, retarded development, and
sterility. The source leaves of mutant plants contained a great excess
of starch, and radiolabeled sugar failed to be transported efficiently
to roots and inflorescences. These data provide genetic proof that
apoplastic phloem loading is critical for growth, development, and
reproduction in
Arabidopsis
and that SUC2 is at least
partially responsible for this step.
Import of amber and ochre suppressor tRNAs into mammalian cells: A general approach to site-specific insertion of amino acid analogues into proteins
Authors: Köhrer Caroline , Xie Liang , Kellerer Susanne , Varshney Umesh , RajBhandary Uttam L.
Published: July 2002

A general approach to site-specific insertion of amino acid
analogues into proteins
in vivo
would be the import into
cells of a suppressor tRNA aminoacylated with the analogue of choice.
The analogue would be inserted at any site in the protein specified by
a stop codon in the mRNA. The only requirement is that the suppressor
tRNA must not be a substrate for any of the cellular aminoacyl–tRNA
synthetases. Here, we describe conditions for the import of amber and
ochre suppressor tRNAs derived from
Escherichia coli
initiator tRNA into mammalian COS1 cells, and we present evidence for
their activity in the specific suppression of amber (UAG) and ochre
(UAA) codons, respectively. We show that an aminoacylated amber
suppressor tRNA (
supF
) derived from the
E.
coli
tyrosine tRNA can be imported into COS1 cells and acts as
a suppressor of amber codons, whereas the same suppressor tRNA imported
without prior aminoacylation does not, suggesting that the
supF
tRNA is not a substrate for any mammalian
aminoacyl–tRNA synthetase. These results open the possibility of using
the
supF
tRNA aminoacylated with an amino acid analogue
as a general approach for the site-specific insertion of amino acid
analogues into proteins in mammalian cells. We discuss the possibility
further of importing a mixture of amber and ochre suppressor tRNAs for
the insertion of two different amino acid analogues into a protein and
the potential use of suppressor tRNA import for treatment of some of
the human genetic diseases caused by nonsense mutations.
Up-regulation of TNF-producing T cells in the bone marrow: A key mechanism by which estrogen deficiency induces bone loss
in vivo
Authors: Roggia Cristiana , Gao Yuhao , Cenci Simone , Weitzmann M. Neale , Toraldo Gianluca , Isaia Giancarlo , Pacifici Roberto
Published: July 2002

In vivo
studies have shown T cells to be central to the mechanism by which estrogen deficiency induces bone loss, but the mechanism involved remains, in part, undefined.
In vitro
, T cells from ovariectomized mice produce increased amounts of tumor necrosis factor (TNF), which augments receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis. However, both the mechanism and the relevance of this phenomenon
in vivo
remain to be established. In this study, we found that ovariectomy increased the number of bone marrow T cell-producing TNF without altering production of TNF per T cell. Attesting to the essential contribution of TNF, ovariectomy induced rapid bone loss in wild type (wt) mice but failed to do so in TNF-deficient (TNF
−/−
) mice. Furthermore, ovariectomy induced bone loss, which was absent in T cell-deficient nude mice, was restored by adoptive transfer of wt T cells, but not by reconstitution with T cells from TNF
−/−
mice. These findings demonstrate the key causal role of T cell-produced TNF in the bone loss after estrogen withdrawal. Finally, ovariectomy caused bone loss in wt mice and in mice lacking p75 TNF receptor but failed to do so in mice lacking the p55 TNF receptor. These findings demonstrate that enhanced T cell production of TNF resulting from increased bone marrow T cell number is a key mechanism by which estrogen deficiency induces bone loss
in vivo
. The data also demonstrate that the bone-wasting effect of TNF
in vivo
is mediated by the p55 TNF receptor.
An herbivore elicitor activates the gene for indole emission in maize
Authors: Frey Monika , Stettner Cornelia , Paré Paul W. , Schmelz Eric A. , Tumlinson James H. , Gierl Alfons
Published: July 2002

Maize and a variety of other plant species release volatile
compounds in response to herbivore attack that serve as chemical cues
to signal natural enemies of the feeding herbivore.
N-
(17-hydroxylinolenoyl)-
l
-glutamine is an
elicitor component that has been isolated and chemically characterized
from the regurgitant of the herbivore-pest beet armyworm. This fatty
acid derivative, referred to as volicitin, triggers the synthesis and
release of volatile components, including terpenoids and indole in
maize. Here we report on a previously unidentified enzyme,
indole-3-glycerol phosphate lyase (IGL), that catalyzes the formation
of free indole and is selectively activated by volicitin. IGL's
enzymatic properties are similar to BX1, a maize enzyme that serves as
the entry point to the secondary defense metabolites DIBOA and
DIMBOA. Gene-sequence analysis indicates that
Igl
and
Bx1
are evolutionarily related to the tryptophan
synthase alpha subunit.
Expression of cyclin D1 in epithelial tissues of transgenic mice results in epidermal hyperproliferation and severe thymic hyperplasia.
Authors: Robles A I , Larcher F , Whalin R B , Murillas R , Richie E , Gimenez-Conti I B , Jorcano J L , Conti C J
Published: July 2002

To study the involvement of cyclin D1 in epithelial growth and differentiation and its putative role as an oncogene in skin, transgenic mice were developed carrying the human cyclin D1 gene driven by a bovine keratin 5 promoter. As expected, all squamous epithelia including skin, oral mucosa, trachea, vaginal epithelium, and the epithelial compartment of the thymus expressed aberrant levels of cyclin D1. The rate of epidermal proliferation increased dramatically in transgenic mice, which also showed basal cell hyperplasia. However, epidermal differentiation was unaffected, as shown by normal growth arrest of newborn primary keratinocytes in response to high extracellular calcium. Moreover, an unexpected phenotype was observed in the thymus. Transgenic mice developed a severe thymic hyperplasia that caused premature death due to cardio-respiratory failure within 4 months of age. By 14 weeks, the thymi of transgenic mice increased in weight up to 40-fold, representing 10% of total body weight. The hyperplastic thymi had normal histology revealing a well-differentiated cortex and medulla, which supported an apparently normal T-cell developmental program based on the distribution of thymocyte subsets. These results suggest that proliferation and differentiation of epithelial cells are under independent genetic controls in these organs and that cyclin D1 can modulate epithelial proliferation without altering the initiation of differentiation programs. No spontaneous development of epithelial tumors or thymic lymphomas was perceived in transgenic mice during their first 8 months of life, although they continue under observation. This model provides in vivo evidence of the action of cyclin D1 as a pure mediator of proliferation in epithelial cells.
Differential distribution of two ATP-gated channels (P2X receptors) determined by immunocytochemistry.
Authors: Vulchanova L , Arvidsson U , Riedl M , Wang J , Buell G , Surprenant A , North R A , Elde R
Published: July 2002

Several P2X receptor subunits were recently cloned; of these, one was cloned from the rat vas deferens (P2X1) and another from pheochromocytoma (PC12) cells differentiated with nerve growth factor (P2X2). Peptides corresponding to the C-terminal portions of the predicted receptor proteins (P2X1 391-399 and P2X2 460-472) were used to generate antisera in rabbits. The specificities of antisera were determined by staining human embryonic kidney cells stably transfected with either P2X1 or P2X2 receptors and by absorption controls with the cognate peptides. In the vas deferens and the ileal submucosa, P2X1 immunoreactivity (ir) was restricted to smooth muscle, whereas P2X2-ir was restricted to neurons and their processes. Chromaffin cells of the adrenal medulla and PC12 cells contained both P2X1- and P2X2-ir. P2X1-ir was also found in smooth muscle cells of the bladder, cardiac myocytes, and nerve fibers and terminals in the superficial dorsal horn of the spinal cord. In contrast, P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function. The location of the P2X1 subunit in smooth muscle is consistent with its role as a postjunctional receptor in autonomic transmission, while in neurons, these receptors appear in both postsynaptic and presynaptic locations.
Glucocorticoids have state-dependent stimulant effects on the mesencephalic dopaminergic transmission.
Authors: Piazza P V , Rougé-Pont F , Deroche V , Maccari S , Simon H , Le Moal M
Published: July 2002

An increase in the activity of mesencephalic dopaminergic neurons has been implicated in the appearance of pathological behaviors such as psychosis and drug abuse. Several observations suggest that glucocorticoids might contribute to such an increase in dopaminergic activity. The present experiments therefore analyzed the effects of corticosterone, the major glucocorticoid in the rat, both on dopamine release in the nucleus accumbens of freely moving animals by means of microdialysis, and on locomotor activity, a behavior dependent on accumbens dopamine. Given that glucocorticoids have certain state-dependent neuronal effects, their action on dopamine was studied in situations differing in dopaminergic tonus, including during the light and dark phases of the circadian cycle, during eating, and in groups of animals differing in their locomotor reactivity to novelty. Dopaminergic activity is increased in the dark period, further increased during food-intake, and is higher in rats defined as high responders to novelty than in low responders. Corticosterone, peripherally administered in a dose that approximates stress-induced plasma concentrations, increased extracellular concentrations of dopamine, and this increase was augmented in the dark phase, during eating, and in high responder rats. Corticosterone had little or no effects in the light phase and in low responder rats. Corticosterone also stimulated locomotor activity, an effect that paralleled the release of dopamine and was abolished by neurochemical (6-hydroxydopamine) depletion of accumbens dopamine. In conclusion, glucocorticoids have state-dependent stimulant effects on mesencephalic dopaminergic transmission, and an interaction between these two factors might be involved in the appearance of behavioral disturbances.
Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation.
Authors: Zakhary R , Gaine S P , Dinerman J L , Ruat M , Flavahan N A , Snyder S H
Published: July 2002

Heme oxygenase 2 (HO-2), which synthesizes carbon monoxide (CO), has been localized by immunohistochemistry to endothelial cells and adventitial nerves of blood vessels. HO-2 is also localized to neurons in autonomic ganglia, including the petrosal, superior cervical, and nodose ganglia, as well as ganglia in the myenteric plexus of the intestine. Enzyme studies demonstrated that tin protoporphyrin-9 is a selective inhibitor of HO with approximately 10-fold selectivity for HO over endothelial nitric oxide synthase (NOS) and soluble guanylyl cyclase. Inhibition of HO activity by tin protoporphyrin 9 reverses the component of endothelial-derived relaxation of porcine distal pulmonary arteries not reversed by an inhibitor of NOS. Thus, CO, like NO, may have endothelial-derived relaxing activity. The similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiologic roles for these two mediators.
Role of cortical N-methyl-D-aspartate receptors in auditory sensory memory and mismatch negativity generation: implications for schizophrenia.
Authors: Javitt D C , Steinschneider M , Schroeder C E , Arezzo J C
Published: July 2002

Working memory refers to the ability of the brain to store and manipulate information over brief time periods, ranging from seconds to minutes. As opposed to long-term memory, which is critically dependent upon hippocampal processing, critical substrates for working memory are distributed in a modality-specific fashion throughout cortex. N-methyl-D-aspartate (NMDA) receptors play a crucial role in the initiation of long-term memory. Neurochemical mechanisms underlying the transient memory storage required for working memory, however, remain obscure. Auditory sensory memory, which refers to the ability of the brain to retain transient representations of the physical features (e.g., pitch) of simple auditory stimuli for periods of up to approximately 30 sec, represents one of the simplest components of the brain working memory system. Functioning of the auditory sensory memory system is indexed by the generation of a well-defined event-related potential, termed mismatch negativity (MMN). MMN can thus be used as an objective index of auditory sensory memory functioning and a probe for investigating underlying neurochemical mechanisms. Monkeys generate cortical activity in response to deviant stimuli that closely resembles human MMN. This study uses a combination of intracortical recording and pharmacological micromanipulations in awake monkeys to demonstrate that both competitive and noncompetitive NMDA antagonists block the generation of MMN without affecting prior obligatory activity in primary auditory cortex. These findings suggest that, on a neurophysiological level, MMN represents selective current flow through open, unblocked NMDA channels. Furthermore, they suggest a crucial role of cortical NMDA receptors in the assessment of stimulus familiarity/unfamiliarity, which is a key process underlying working memory performance.
Apicidin: A novel antiprotozoal agent that inhibits parasite histone deacetylase
Authors: Darkin-Rattray Sandra J. , Gurnett Anne M. , Myers Robert W. , Dulski Paula M. , Crumley Tami M. , Allocco John J. , Cannova Christine , Meinke Peter T. , Colletti Steven L. , Bednarek Maria A. , Singh Sheo B. , Goetz Michael A. , Dombrowski Anne W. , Polishook Jon D. , Schmatz Dennis M.
Published: July 2002

A novel fungal metabolite, apicidin
[cyclo(
N
-
O
-methyl-
l
-tryptophanyl-
l
-isoleucinyl-
d
-pipecolinyl-
l
-2-amino-8-oxodecanoyl)],
that exhibits potent, broad spectrum antiprotozoal activity
in
vitro
against Apicomplexan parasites has been identified. It is
also orally and parenterally active
in vivo
against
Plasmodium berghei
malaria in mice. Many Apicomplexan
parasites cause serious, life-threatening human and animal diseases,
such as malaria, cryptosporidiosis, toxoplasmosis, and coccidiosis, and
new therapeutic agents are urgently needed. Apicidin’s antiparasitic
activity appears to be due to low nanomolar inhibition of Apicomplexan
histone deacetylase (HDA), which induces hyperacetylation of histones
in treated parasites. The acetylation–deacetylation of histones is a
thought to play a central role in transcriptional control in eukaryotic
cells. Other known HDA inhibitors were also evaluated and found to
possess antiparasitic activity, suggesting that HDA is an attractive
target for the development of novel antiparasitic agents.
Characterization of individual polynucleotide molecules using a membrane channel
Authors: Kasianowicz John J. , Brandin Eric , Branton Daniel , Deamer David W.
Published: July 2002

We show that an electric field can drive single-stranded RNA and
DNA molecules through a 2.6-nm diameter ion channel in a lipid bilayer
membrane. Because the channel diameter can accommodate only a single
strand of RNA or DNA, each polymer traverses the membrane as an
extended chain that partially blocks the channel. The passage of each
molecule is detected as a transient decrease of ionic current whose
duration is proportional to polymer length. Channel blockades can
therefore be used to measure polynucleotide length. With further
improvements, the method could in principle provide direct, high-speed
detection of the sequence of bases in single molecules of DNA or RNA.
A mutant cytochrome b5 with a lengthened membrane anchor escapes from the endoplasmic reticulum and reaches the plasma membrane.
Authors: Pedrazzini E , Villa A , Borgese N
Published: July 2002

Many resident membrane proteins of the endoplasmic reticulum (ER) do not have known retrieval sequences. Among these are the so-called tail-anchored proteins, which are bound to membranes by a hydrophobic tail close to the C terminus and have most of their sequence as a cytosolically exposed N-terminal domain. Because ER tail-anchored proteins generally have short (< or = 17 residues) hydrophobic domains, we tested whether this feature is important for localization, using cytochrome b5 as a model. The hydrophobic domain of cytochrome b5 was lengthened by insertion of five amino acids (ILAAV), and the localization of the mutant was analyzed by immunofluorescence in transiently transfected mammalian cells. While the wild-type cytochrome was localized to the ER, the mutant was relocated to the surface. This relocation was not due to the specific sequence introduced, as demonstrated by the ER localization of a second mutant, in which the original length of the membrane anchor was restored, while maintaining the inserted ILAAV sequence. Experiments with brefeldin A and with cycloheximide demonstrated that the extended anchor mutant reached the plasma membrane by transport along the secretory pathway. We conclude that the short membrane anchor of cytochrome b5 is important for its ER residency, and we discuss the relevance of this finding for other ER tail-anchored proteins.
A human protein related to yeast Cdc6p
Authors: Williams R. Sanders , Shohet Ralph V. , Stillman Bruce
Published: July 2002

The unstable proteins Cdc6p and cdc18
+
are essential and rate limiting for the initiation of DNA replication in
Saccharomyces cerevisiae
and
Schizosaccharomyces pombe
, respectively, and also participate in checkpoint controls that ensure DNA replication is completed before mitosis is initiated. We have identified Xenopus and human proteins closely related to Cdc6p/cdc18. The human protein, p62
cdc6
, is encoded on chromosome 17q21.3 and includes putative cyclin-dependent kinase phosphorylation sites, destruction boxes, a nucleotide binding/ATPase domain, and a potential leucine zipper. Expression of p62
cdc6
mRNA and protein is suppressed in human diploid fibroblasts made quiescent by serum starvation, and peaks as cells reenter the cell cycle and replicate DNA following serum stimulation. Conservation of structure among proteins involved in initiation suggests that fundamental features of replication complexes are maintained in all eukaryotes.
The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2
Authors: Sin Ny , Meng Lihao , Wang Margaret Q. W. , Wen James J. , Bornmann William G. , Crews Craig M.
Published: July 2002

The inhibition of new blood vessel formation (angiogenesis) is an effective means of limiting both the size and metastasis of solid tumors. The leading anti-angiogenic compound, TNP-470, has proven to be effective in
in vitro
and in animal model studies, and is currently being tested in phase III antitumor clinical trials. Despite many detailed pharmacological studies, little is known of the molecular mode of action of TNP-470. Using a derivative of the TNP-470 parent compound, the fungal metabolite, fumagillin, we have purified a mammalian protein that is selectively and covalently bound by this natural product. This fumagillin binding protein was found to be a metalloprotease, methionine aminopeptidase (MetAP-2), that is highly conserved between human and
Saccharomyces cerevisiae
. In the absence of MetAP-1, a distantly related methionine aminopeptidase, MetAP-2 function is essential for vegetative growth in yeast. We demonstrate that fumagillin selectively inhibits the
S. cerevisiae
MetAP-2 protein
in vivo
. The binding is highly specific as judged by the failure of fumagillin to inhibit MetAP-1
in vivo
. Hence, these results identify MetAP-2 as an important target of study in the analysis of the potent biological activities of fumagillin.
Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium
Authors: Cooke P. S. , Buchanan D. L. , Young P. , Setiawan T. , Brody J. , Korach K. S. , Taylor J. , Lubahn D. B. , Cunha G. R.
Published: July 2002

Estradiol-17β (E2) acts through the estrogen receptor (ER) to regulate uterine growth and functional differentiation. To determine whether E2elicits epithelial mitogenesis through epithelial ER versus indirectly via ER-positive stromal cells, uteri from adult ER-deficient ER knockout (ko) mice and neonatal ER-positive wild-type (wt) BALB/c mice were used to produce the following tissue recombinants containing ER in epithelium (E) and/or stroma (S), or lacking ER altogether: wt-S + wt-E, wt-S + ko-E, ko-S + ko-E, and ko-S + wt-E. Tissue recombinants were grown for 4 weeks as subrenal capsule grafts in intact female nude mice, then the hosts were treated with either E2or oil a week after ovariectomy. Epithelial labeling index and ER expression were determined by [3H]thymidine autoradiography and immunohistochemistry, respectively. In tissue recombinants containing wt-S (wt-S + wt-E, wt-S + ko-E), E2induced a similar large increase in epithelial labeling index compared with oil-treated controls in both types of tissue recombinants despite the absence of epithelial ER in wt-S + ko-E tissue recombinants. This proliferative effect was blocked by an ER antagonist, indicating it was mediated through ER. In contrast, in tissue recombinants prepared with ko-S (ko-S + ko-E and ko-S + wt-E), epithelial labeling index was low and not stimulated by E2despite epithelial ER expression in ko-S + wt-E grafts. In conclusion, these data demonstrate that epithelial ER is neither necessary nor sufficient for E2-induced uterine epithelial proliferation. Instead, E2induction of epithelial proliferation appears to be a paracrine event mediated by ER-positive stroma. These data in the uterus and similar studies in the prostate suggest that epithelial mitogenesis in both estrogen and androgen target organs are stromally mediated events.
The hippocampus and memory for orderly stimulus relations
Authors: Dusek Jeffery A. , Eichenbaum Howard
Published: July 2002

Human declarative memory involves a systematic organization of information that supports generalizations and inferences from acquired knowledge. This kind of memory depends on the hippocampal region in humans, but the extent to which animals also have declarative memory, and whether inferential expression of memory depends on the hippocampus in animals, remains a major challenge in cognitive neuroscience. To examine these issues, we used a test of transitive inference pioneered by Piaget to assess capacities for systematic organization of knowledge and logical inference in children. In our adaptation of the test, rats were trained on a set of four overlapping odor discrimination problems that could be encoded either separately or as a single representation of orderly relations among the odor stimuli. Normal rats learned the problems and demonstrated the relational memory organization through appropriate transitive inferences about items not presented together during training. By contrast, after disconnection of the hippocampus from either its cortical or subcortical pathway, rats succeeded in acquiring the separate discrimination problems but did not demonstrate transitive inference, indicating that they had failed to develop or could not inferentially express the orderly organization of the stimulus elements. These findings strongly support the view that the hippocampus mediates a general declarative memory capacity in animals, as it does in humans.
A single amino acid substitution converts a carboxylesterase to an organophosphorus hydrolase and confers insecticide resistance on a blowfly
Authors: Newcomb R. D. , Campbell P. M. , Ollis D. L. , Cheah E. , Russell R. J. , Oakeshott J. G.
Published: July 2002

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly,
Lucilia cuprina
, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly
137
→ Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains.
In vitro
expression of two natural and two synthetic chimeric alleles shows that the Asp
137
substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp
137
in the homologous position in acetylcholinesterase suggests that Asp
137
may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.
Potential responses of soil organic carbon to global environmental change
Authors: Trumbore Susan E.
Published: July 2002

Recent improvements in our understanding of the dynamics of soil
carbon have shown that 20–40% of the approximately 1,500 Pg of C
stored as organic matter in the upper meter of soils has turnover times
of centuries or less. This fast-cycling organic matter is largely
comprised of undecomposed plant material and hydrolyzable components
associated with mineral surfaces. Turnover times of fast-cycling carbon
vary with climate and vegetation, and range from <20 years at low
latitudes to >60 years at high latitudes. The amount and turnover
time of C in passive soil carbon pools (organic matter strongly
stabilized on mineral surfaces with turnover times of millennia and
longer) depend on factors like soil maturity and mineralogy, which, in
turn, reflect long-term climate conditions. Transient sources or sinks
in terrestrial carbon pools result from the time lag between
photosynthetic uptake of CO
2
by plants and the subsequent
return of C to the atmosphere through plant, heterotrophic, and
microbial respiration. Differential responses of primary production and
respiration to climate change or ecosystem fertilization have the
potential to cause significant interrannual to decadal imbalances in
terrestrial C storage and release. Rates of carbon storage and release
in recently disturbed ecosystems can be much larger than rates in more
mature ecosystems. Changes in disturbance frequency and regime
resulting from future climate change may be more important than
equilibrium responses in determining the carbon balance of terrestrial
ecosystems.
Gases in ice cores
Authors: Bender Michael , Sowers Todd , Brook Edward
Published: July 2002

Air trapped in glacial ice offers a means of reconstructing
variations in the concentrations of atmospheric gases over time scales
ranging from anthropogenic (last 200 yr) to glacial/interglacial
(hundreds of thousands of years). In this paper, we review the
glaciological processes by which air is trapped in the ice and discuss
processes that fractionate gases in ice cores relative to the
contemporaneous atmosphere. We then summarize concentration–time
records for CO
2
and CH
4
over the last 200 yr.
Finally, we summarize concentration–time records for CO
2
and CH
4
during the last two glacial–interglacial cycles,
and their relation to records of global climate change.
Buoyancy-driven, rapid exhumation of ultrahigh-pressure metamorphosed continental crust
Authors: Ernst W. G. , Maruyama S. , Wallis S.
Published: July 2002

Preservation of ultrahigh-pressure (UHP) minerals formed at depths of 90–125 km require unusual conditions. Our subduction model involves underflow of a salient (250 ± 150 km wide, 90–125 km long) of continental crust embedded in cold, largely oceanic crust-capped lithosphere; loss of leading portions of the high-density oceanic lithosphere by slab break-off, as increasing volumes of microcontinental material enter the subduction zone; buoyancy-driven return toward midcrustal levels of a thin (2–15 km thick), low-density slice; finally, uplift, backfolding, normal faulting, and exposure of the UHP terrane. Sustained over ≈20 million years, rapid (≈5 mm/year) exhumation of the thin-aspect ratio UHP sialic sheet caught between cooler hanging-wall plate and refrigerating, downgoing lithosphere allows withdrawal of heat along both its upper and lower surfaces. The intracratonal position of most UHP complexes reflects consumption of an intervening ocean basin and introduction of a sialic promontory into the subduction zone. UHP metamorphic terranes consist chiefly of transformed, yet relatively low-density continental crust compared with displaced mantle material—otherwise such complexes could not return to shallow depths. Relatively rare metabasaltic, metagabbroic, and metacherty lithologies retain traces of phases characteristic of UHP conditions because they are massive, virtually impervious to fluids, and nearly anhydrous. In contrast, H
2
O-rich quartzofeldspathic, gneissose/schistose, more permeable metasedimentary and metagranitic units have backreacted thoroughly, so coesite and other UHP silicates are exceedingly rare. Because of the initial presence of biogenic carbon, and its especially sluggish transformation rate, UHP paragneisses contain the most abundantly preserved crustal diamonds.
Structural mimicry of a native protein by a minimized binding domain
Authors: Starovasnik Melissa A. , Braisted Andrew C. , Wells James A.
Published: July 2002

The affinity between molecules depends both on the nature and presentation of the contacts. Here, we observe coupling of functional and structural elements when a protein binding domain is evolved to a smaller functional mimic. Previously, a 38-residue form of the 59-residue B-domain of protein A, termed Z38, was selected by phage display. Z38 contains 13 mutations and binds IgG only 10-fold weaker than the native B-domain. We present the solution structure of Z38 and show that it adopts a tertiary structure remarkably similar to that observed for the first two helices of B-domain in the B-domain/Fc complex [Deisenhofer, J. (1981)
Biochemistry
20, 2361–2370], although it is significantly less stable. Based on this structure, we have improved on Z38 by designing a 34-residue disulfide-bonded variant (Z34C) that has dramatically enhanced stability and binds IgG with 9-fold higher affinity. The improved stability of Z34C led to NMR spectra with much greater chemical shift dispersion, resulting in a more precisely determined structure. Z34C, like Z38, has a structure virtually identical to the equivalent region from native protein A domains. The well-defined hydrophobic core of Z34C reveals key structural features that have evolved in this small, functional domain. Thus, the stabilized two-helix peptide, about half the size and having one-third of the remaining residues altered, accurately mimics both the structure and function of the native domain.
DNA polymerase δ isolated from
Schizosaccharomyces pombe
contains five subunits
Authors: Zuo Shaojun , Gibbs Emma , Kelman Zvi , Wang Teresa S.-F. , O’Donnell Mike , MacNeill Stuart A. , Hurwitz Jerard
Published: July 2002

DNA polymerase δ (pol δ) plays an essential role in DNA replication, repair, and recombination. We have purified pol δ from
Schizosaccharomyces pombe
more than 10
3
-fold and demonstrated that the polymerase activity of purified
S. pombe
pol δ is completely dependent on proliferating cell nuclear antigen and replication factor C. SDS/PAGE analysis of the purified fraction indicated that the pol δ complex consists of five subunits that migrate with apparent molecular masses of 125, 55, 54, 42, and 22 kDa. Western blot analysis indicated that the 125, 55, and 54 kDa proteins are the large catalytic subunit (Pol3), Cdc1, and Cdc27, respectively. The identity of the other two subunits, p42 and p22, was determined following proteolytic digestion and sequence analysis of the resulting peptides. The peptide sequences derived from the p22 subunit indicated that this subunit is identical to Cdm1, previously identified as a multicopy suppressor of the temperature-sensitive
cdc1
-P13 mutant, whereas peptide sequences derived from the p42 subunit were identical to a previously uncharacterized ORF located on
S. pombe
chromosome 1.
Schizosaccharomyces pombe cdc
20
+
encodes DNA polymerase ɛ and is required for chromosomal replication but not for the S phase checkpoint
Authors: D’Urso Gennaro , Nurse Paul
Published: July 2002

In fission yeast both DNA polymerase alpha (pol α) and delta (pol δ) are required for DNA chromosomal replication. Here we demonstrate that
Schizosaccharomyces pombe cdc20
+
encodes the catalytic subunit of DNA polymerase epsilon (pol ɛ) and that this enzyme is also required for DNA replication. Following a shift to the restrictive temperature,
cdc20
temperature-sensitive mutant cells block at the onset of DNA replication, suggesting that
cdc20
+
is required early in S phase very near to the initiation step. In the budding yeast
Saccharomyces cerevisiae
, it has been reported that in addition to its proposed role in chromosomal replication, DNA pol ɛ (encoded by POL2) also functions directly as an S phase checkpoint sensor [Navas, T. A., Zhou, Z. & Elledge, S. J. (1995)
Cell
80, 29–39]. We have investigated whether
cdc20
+
is required for the checkpoint control operating in fission yeast, and our data indicate that pol ɛ does not have a role as a checkpoint sensor coordinating S phase with mitosis. In contrast, germinating spores disrupted for the gene encoding pol α rapidly enter mitosis in the absence of DNA synthesis, suggesting that in the absence of pol α, normal coordination between S phase and mitosis is lost. We propose that the checkpoint signal operating in S phase depends on assembly of the replication initiation complex, and that this signal is generated prior to the elongation stage of DNA synthesis.
Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation
Authors: Hering Steffen , Aczél Stefan , Kraus Richard L. , Berjukow Stanislav , Striessnig Jörg , Timin Eugen N.
Published: July 2002

The role of channel inactivation in the molecular mechanism of calcium (Ca
2+
) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca
2+
channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995)
J. Biol. Chem.
270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca
2+
channels in
Xenopus
oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I
Ba
) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I
Ba
recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I
Ba
recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca
2+
channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca
2+
channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.
From tropics to tundra: Global convergence in plant functioning
Authors: Reich Peter B. , Walters Michael B. , Ellsworth David S.
Published: July 2002

Despite striking differences in climate, soils, and evolutionary history among diverse biomes ranging from tropical and temperate forests to alpine tundra and desert, we found similar interspecific relationships among leaf structure and function and plant growth in all biomes. Our results thus demonstrate convergent evolution and global generality in plant functioning, despite the enormous diversity of plant species and biomes. For 280 plant species from two global data sets, we found that potential carbon gain (photosynthesis) and carbon loss (respiration) increase in similar proportion with decreasing leaf life-span, increasing leaf nitrogen concentration, and increasing leaf surface area-to-mass ratio. Productivity of individual plants and of leaves in vegetation canopies also changes in constant proportion to leaf life-span and surface area-to-mass ratio. These global plant functional relationships have significant implications for global scale modeling of vegetation–atmosphere CO
2
exchange.
Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: Implications for cancer and neuronal damage
Authors: Blount Benjamin C. , Mack Matthew M. , Wehr Carol M. , MacGregor James T. , Hiatt Robert A. , Wang Gene , Wickramasinghe Sunitha N. , Everson Richard B. , Ames Bruce N.
Published: July 2002

Folate deficiency causes massive incorporation of uracil into human
DNA (4 million per cell) and chromosome breaks. The likely mechanism is
the deficient methylation of dUMP to dTMP and subsequent incorporation
of uracil into DNA by DNA polymerase. During repair of uracil in DNA,
transient nicks are formed; two opposing nicks could lead to chromosome
breaks. Both high DNA uracil levels and elevated micronucleus frequency
(a measure of chromosome breaks) are reversed by folate administration.
A significant proportion of the U.S. population has low folate levels,
in the range associated with elevated uracil misincorporation and
chromosome breaks. Such breaks could contribute to the increased risk
of cancer and cognitive defects associated with folate deficiency in
humans.