bluuebunny/crossref_metadata_2025 huggingface dataset:
Population genetics of Ice Age brown bears
Authors: Leonard Jennifer A. , Wayne Robert K. , Cooper Alan
Published: July 2002

The Pleistocene was a dynamic period for Holarctic mammal species, complicated by episodes of glaciation, local extinctions, and intercontinental migration. The genetic consequences of these events are difficult to resolve from the study of present-day populations. To provide a direct view of population genetics in the late Pleistocene, we measured mitochondrial DNA sequence variation in seven permafrost-preserved brown bear (
Ursus arctos
) specimens, dated from 14,000 to 42,000 years ago. Approximately 36,000 years ago, the Beringian brown bear population had a higher genetic diversity than any extant North American population, but by 15,000 years ago genetic diversity appears similar to the modern day. The older, genetically diverse, Beringian population contained sequences from three clades now restricted to local regions within North America, indicating that current phylogeographic patterns may provide misleading data for evolutionary studies and conservation management. The late Pleistocene phylogeographic data also indicate possible colonization routes to areas south of the Cordilleran ice sheet.
In vivonatural killer cell activities revealed by natural killer cell-deficient mice
Authors: Kim Sungjin , Iizuka Koho , Aguila Hector L. , Weissman Irving L. , Yokoyama Wayne M.
Published: July 2002

Studies of natural killer (NK) cell functionin vivohave been challenging primarily due to the lack of animal models in which NK cells are genetically and selectively deficient. Here, we describe a transgenic mouse with defective natural killing and selective deficiency in NK1.1+CD3−cells. Despite functionally normal B, T, and NK/T cells, transgenic mice displayed impaired acutein vivorejection of tumor cells. Adoptive transfer experiments confirmed that NK1.1+CD3−cells were responsible for acute tumor rejection, establishing the relationship of NK1.1+CD3−cells to NK cells. Additional studies provided evidence that (i) NK cells play an important role in suppressing tumor metastasis and outgrowth; (ii) NK cells are major producers of IFNγ in response to bacterial endotoxin but not to interleukin-12, and; (iii) NK cells are not essential for humoral responses to T cell-independent type 2 antigen or the generalized Shwartzman reaction, both of which were previously proposed to involve NK cells.
The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation
Authors: Forcet Christelle , Ye Xin , Granger Laure , Corset Véronique , Shin Hwain , Bredesen Dale E. , Mehlen Patrick
Published: July 2002

The expression of DCC (deleted in colorectal cancer) is often
markedly reduced in colorectal and other cancers. However, the rarity
of point mutations identified in DCC coding sequences and the lack of a
tumor predisposition phenotype in DCC hemizygous mice have raised
questions about its role as a tumor suppressor. DCC also mediates axon
guidance and functions as a dependence receptor; such receptors create
cellular states of dependence on their respective ligands by inducing
apoptosis when unoccupied by ligand. We now show that DCC
drives cell death independently of both the mitochondria-dependent
pathway and the death receptor/caspase-8 pathway. Moreover, we
demonstrate that DCC interacts with both caspase-3 and caspase-9 and
drives the activation of caspase-3 through caspase-9 without a
requirement for cytochrome
c
or Apaf-1. Hence, DCC
defines an additional pathway for the apoptosome-independent caspase
activation.
Altering dimerization specificity by changes in surface electrostatics
Authors: Nohaile M. J. , Hendsch Z. S. , Tidor B. , Sauer R. T.
Published: July 2002

Arc repressor forms a homodimer in which the subunits intertwine to
create a single globular domain. To obtain Arc sequences that fold
preferentially as heterodimers, variants with surface patches of excess
positive or negative charge were designed. Several but not all
oppositely charged sequence pairs showed preferential heterodimer
formation. In the most successful design pair, α helix B of one
subunit contained glutamic acids at positions 43, 46, 47, 48, and 50,
whereas the other subunit contained lysines or arginines at these
positions. A continuum electrostatic model captures many features of
the experimental results and suggests that the most successful designs
include elements of both positive and negative design.
Effect of rabies virus infection on gene expression in mouse brain
Authors: Prosniak Mikhail , Hooper D. Craig , Dietzschold Bernhard , Koprowski Hilary
Published: July 2002

A variety of molecular genetic approaches were used to study the
effect of rabies virus (RV) infection on host gene expression in mouse
brain. The down-regulation of gene expression was found to be a major
effect of RV infection by using subtraction hybridization. However, a
combination of techniques identified approximately 39 genes activated
by infection. These included genes involved in regulation of cell
metabolism, protein synthesis, synaptic activity, and cell growth and
differentiation. Northern blot analysis to monitor temporal activation
of several of these genes following infection revealed essentially two
patterns of activation: (
i
) an early response with
up-regulation beginning within 3 days after infection and correlating
with transcription of RV nuclear protein; and (
ii
) a
late response with enhanced expression occurring at days 6–7 after
infection and associated with peak RV replication. The gene activation
patterns and the known functions of their products suggest that a
number of host genes may be involved in the replication and spread of
RV in the brain.
Activity-dependent isolation of the presenilin– γ-secretase complex reveals nicastrin and a γ substrate
Authors: Esler William P. , Kimberly W. Taylor , Ostaszewski Beth L. , Ye Wenjuan , Diehl Thekla S. , Selkoe Dennis J. , Wolfe Michael S.
Published: July 2002

Presenilin heterodimers apparently contain the active site of γ-secretase, a polytopic aspartyl protease involved in the transmembrane processing of both the Notch receptor and the amyloid-β precursor protein. Although critical to embryonic development and the pathogenesis of Alzheimer's disease, this protease is difficult to characterize, primarily because it is a multicomponent complex of integral membrane proteins. Here the functional γ-secretase complex was isolated by using an immobilized active site-directed inhibitor of the protease. Presenilin heterodimers and nicastrin bound specifically to this inhibitor under conditions tightly correlating with protease activity, whereas several other presenilin-interacting proteins (β-catenin, calsenilin, and presenilin-associated protein) did not bind. Moreover, anti-nicastrin antibodies immunoprecipitated γ-secretase activity from detergent-solubilized microsomes. Unexpectedly, C83, the major endogenous amyloid-β precursor protein substrate of γ-secretase, was also quantitatively associated with the complex. These results provide direct biochemical evidence that nicastrin is a member of the active γ-secretase complex, indicate that β-catenin, calsenilin, and presenilin-associated protein are not required for γ activity, and suggest an unprecedented mechanism of substrate–protease interaction.
Mechanism of topology simplification by type II DNA topoisomerases
Authors: Vologodskii Alexander V. , Zhang Wentao , Rybenkov Valentin V. , Podtelezhnikov Alexei A. , Subramanian Deepa , Griffith Jack D. , Cozzarelli Nicholas R.
Published: July 2002

Type II DNA topoisomerases actively reduce the fractions of knotted
and catenated circular DNA below thermodynamic equilibrium values. To
explain this surprising finding, we designed a model in which
topoisomerases introduce a sharp bend in DNA. Because the enzymes have
a specific orientation relative to the bend, they act like Maxwell's
demon, providing unidirectional strand passage. Quantitative analysis
of the model by computer simulations proved that it can explain much of
the experimental data. The required sharp DNA bend was demonstrated by
a greatly increased cyclization of short DNA fragments from
topoisomerase binding and by direct visualization with electron
microscopy.
Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease
Authors: Steiner Robert A. , Hohmann John G. , Holmes Andrew , Wrenn Craige C. , Cadd Gary , Juréus Anders , Clifton Donald K. , Luo Mulon , Gutshall Mitchell , Ma Shuang Y. , Mufson Elliott J. , Crawley Jacqueline N.
Published: July 2002

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimer's disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine β-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.
How amoeboids self-organize into a fruiting body: Multicellular coordination in
Dictyostelium discoideum
Authors: Marée Athanasius F. M. , Hogeweg Paulien
Published: July 2002

When individual amoebae of the cellular slime mold
Dictyostelium discoideum
are starving,
they aggregate to form a multicellular migrating slug, which moves
toward a region suitable for culmination. The culmination of the
morphogenesis involves complex cell movements that transform a mound of
cells into a globule of spores on a slender stalk. The movement has
been likened to a “reverse fountain,” whereby prestalk cells in
the upper part form a stalk that moves downwards and anchors to the
substratum, while prespore cells in the lower part move upwards to form
the spore head. So far, however, no satisfactory explanation has been
produced for this process. Using a computer simulation that we
developed, we now demonstrate that the processes that are essential
during the earlier stages of the morphogenesis are in fact sufficient
to produce the dynamics of the culmination stage. These processes are
cAMP signaling, differential adhesion, cell differentiation, and
production of extracellular matrix. Our model clarifies the processes
that generate the observed cell movements. More specifically, we show
that periodic upward movements, caused by chemotactic motion, are
essential for successful culmination, because the pressure waves they
induce squeeze the stalk downwards through the cell mass. The
mechanisms revealed by our model have a number of self-organizing and
self-correcting properties and can account for many previously
unconnected and unexplained experimental observations.
Brain-derived neurotrophic factor restores long-term potentiation in polysialic acid-neural cell adhesion molecule-deficient hippocampus
Authors: Muller D. , Djebbara-Hannas Z. , Jourdain P. , Vutskits L. , Durbec P. , Rougon G. , Kiss J. Z.
Published: July 2002

The neural cell adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM) contribute to long-term potentiation (LTP) in the CA1 hippocampus. Here we report that the deficient LTP found in slices prepared from NCAM knockout mice and in organotypic slice cultures treated with Endo-N, an enzyme that cleaves the PSA moiety of NCAM, can be rescued by brain-derived neurotrophic factor (BDNF). This effect is not reproduced by nerve growth factor, but can be obtained with high concentrations of NT4/5. The effect of BDNF cannot be accounted for by modifications of
N
-methyl-
d
-aspartate receptor-dependent responses or of high-frequency bursts. PSA-NCAM, however, could directly interact with BDNF. Exogenous application of PSA residues or recombinant PSA-NCAM also prevents LTP. Furthermore trkB phosphorylation, and thus BDNF signaling, is reduced in both NCAM knockout mice and Endo-N-treated slice cultures. These results suggest that one action of PSA-NCAM could be to sensitize pyramidal neurons to BDNF, thereby modulating activity-dependent synaptic plasticity.
Multiplication of antenna genes as a major adaptation to low light in a marine prokaryote
Authors: Garczarek Laurence , Hess Wolfgang R. , Holtzendorff Julia , van der Staay Georg W. M. , Partensky Frédéric
Published: July 2002

Two ecotypes of the prokaryote
Prochlorococcus
adapted to distinct light niches in the ocean have been described recently. These ecotypes are characterized by their different (divinyl-) chlorophyll (Chl)
a
to Chl
b
ratios and 16S rRNA gene signatures, as well as by their significantly distinct irradiance optima for growth and photosynthesis [Moore, L. R., Rocap, G. & Chisholm, S. W. (1998)
Nature (London)
393, 464–467]. However, the molecular basis of their physiological differences remained, so far, unexplained. In this paper, we show that the low-light-adapted
Prochlorococcus
strain SS120 possesses a gene family of seven transcribed genes encoding different Chl
a
/
b
-binding proteins (Pcbs). In contrast,
Prochlorococcus
sp. MED4, a high-light-adapted ecotype, possesses a single
pcb
gene. The presence of multiple antenna genes in another low-light ecotype (NATL2a), but not in another high-light ecotype (TAK9803–2), is demonstrated. Thus, the multiplication of
pcb
genes appears as a key factor in the capacity of deep
Prochlorococcus
populations to survive at extremely low photon fluxes.
The 1,800-year oceanic tidal cycle: A possible cause of rapid climate change
Authors: Keeling Charles D. , Whorf Timothy P.
Published: July 2002

Variations in solar irradiance are widely believed to explain
climatic change on 20,000- to 100,000-year time-scales in accordance
with the Milankovitch theory of the ice ages, but there is no
conclusive evidence that variable irradiance can be the cause of abrupt
fluctuations in climate on time-scales as short as 1,000 years. We
propose that such abrupt millennial changes, seen in ice and
sedimentary core records, were produced in part by well characterized,
almost periodic variations in the strength of the global oceanic
tide-raising forces caused by resonances in the periodic motions of the
earth and moon. A well defined 1,800-year tidal cycle is associated
with gradually shifting lunar declination from one episode of maximum
tidal forcing on the centennial time-scale to the next. An amplitude
modulation of this cycle occurs with an average period of about 5,000
years, associated with gradually shifting separation-intervals between
perihelion and syzygy at maxima of the 1,800-year cycle. We propose
that strong tidal forcing causes cooling at the sea surface by
increasing vertical mixing in the oceans. On the millennial time-scale,
this tidal hypothesis is supported by findings, from sedimentary
records of ice-rafting debris, that ocean waters cooled close to the
times predicted for strong tidal forcing.
Identification of a human recent thymic emigrant phenotype
Authors: McFarland Richard D. , Douek Daniel C. , Koup Richard A. , Picker Louis J.
Published: July 2002

The ability to measure human thymic output would be an invaluable tool for the study of the development of the naïve T cell repertoire, as well as naïve T cell regeneration after intensive cytotoxic chemotherapy or effective antiretroviral therapy of progressive HIV infection. We and others have demonstrated previously that quantification of T cell receptor rearrangement excision circles (TREC) within peripheral T cell populations provides insight into the frequency of recent thymic emigrants (RTE) and, therefore, into thymic function. However, measurement of RTE by this approach is complicated by the fact that TREC levels also are determined by turnover within the naïve T cell compartment. Here, we report a phenotypic approach to RTE measurement. We demonstrate that αE integrin (CD103) expression is up-regulated very late in thymic development on a subset of CD8+/CD4−thymocytes and also defines a distinct subset of naïve CD8+T cells in the periphery. The latter subset is differentiated from circulating CD103+mucosa-associated memory T cells by its naïve T cell phenotype (CD45RO−, CD62Lbright, CD27bright, CD11adim, CD95dim) and its high concentration of TREC. Indeed, sorted CD103+naïve CD8+cells display higher levels of TREC than their CD103−naïve counterparts, and these cells demonstrate an age-related decline in frequency that is enhanced significantly by thymectomy. The thymic dependence of this subset and the cells' relatively evanescent presence in the periphery suggest that these cells are a population of RTE and that quantification of their frequency in peripheral blood provides an estimate of the level of ongoing thymopoiesis.
Influenza A and B viruses expressing altered NS1 proteins: A vaccine approach
Authors: Talon Julie , Salvatore Mirella , O'Neill Robert E. , Nakaya Yurie , Zheng Hongyong , Muster Thomas , García-Sastre Adolfo , Palese Peter
Published: July 2002

We propose a rational approach to the generation of live viral vaccines: alteration of virally encoded type I IFN antagonists to attenuate virulence while retaining immunogenicity. We have explored this concept by using the influenza virus. Previously we have shown that the NS1 protein of influenza A virus possesses anti-IFN activity. We now present evidence that influenza A and B viruses encoding altered viral NS1 proteins are highly attenuated in the mouse host, yet provide protection from challenge with wild-type viruses.
Spatial and temporal emergence of high proliferative potential hematopoietic precursors during murine embryogenesis
Authors: Palis J. , Chan R. J. , Koniski A. , Patel R. , Starr M. , Yoder M. C.
Published: July 2002

During mouse embryogenesis, two waves of hematopoietic progenitors
originate in the yolk sac. The first wave consists of primitive
erythroid progenitors that arise at embryonic day 7.0 (E7.0), whereas
the second wave consists of definitive erythroid progenitors that arise
at E8.25. To determine whether these unilineage hematopoietic
progenitors arise from multipotential precursors, we investigated the
kinetics of high proliferative potential colony-forming cells
(HPP-CFC), multipotent precursors that give rise to macroscopic
colonies when cultured
in vitro
. No HPP-CFC were found
at presomite stages (E6.5–E7.5). Rather, HPP-CFC were detected first
at early somite stages (E8.25), exclusively in the yolk sac. HPP-CFC
were found subsequently in the bloodstream at higher levels than the
remainder of the embryo proper. However, the yolk sac remains the
predominant site of HPP-CFC expansion (>100-fold) until the liver
begins to serve as the major hematopoietic organ at E11.5. On secondary
replating, embryonic HPP-CFC give rise to definitive erythroid and
macrophage (but not primitive erythroid) progenitors. Our findings
support the hypothesis that definitive but not primitive hematopoietic
progenitors originate from yolk sac-derived HPP-CFC during late
gastrulation.
Fibronectin extension and unfolding within cell matrix fibrils controlled by cytoskeletal tension
Authors: Baneyx Gretchen , Baugh Loren , Vogel Viola
Published: July 2002

Evidence is emerging that mechanical stretching can alter the functional states of proteins. Fibronectin (Fn) is a large, extracellular matrix protein that is assembled by cells into elastic fibrils and subjected to contractile forces. Assembly into fibrils coincides with expression of biological recognition sites that are buried in Fn's soluble state. To investigate how supramolecular assembly of Fn into fibrillar matrix enables cells to mechanically regulate its structure, we used fluorescence resonance energy transfer (FRET) as an indicator of Fn conformation in the fibrillar matrix of NIH 3T3 fibroblasts. Fn was randomly labeled on amine residues with donor fluorophores and site-specifically labeled on cysteine residues in modules FnIII
7
and FnIII
15
with acceptor fluorophores. Intramolecular FRET was correlated with known structural changes of Fn in denaturing solution, then applied in cell culture as an indicator of Fn conformation within the matrix fibrils of NIH 3T3 fibroblasts. Based on the level of FRET, Fn in many fibrils was stretched by cells so that its dimer arms were extended and at least one FnIII module unfolded. When cytoskeletal tension was disrupted using cytochalasin D, FRET increased, indicating refolding of Fn within fibrils. These results suggest that cell-generated force is required to maintain Fn in partially unfolded conformations. The results support a model of Fn fibril elasticity based on unraveling and refolding of FnIII modules. We also observed variation of FRET between and along single fibrils, indicating variation in the degree of unfolding of Fn in fibrils. Molecular mechanisms by which mechanical force can alter the structure of Fn, converting tensile forces into biochemical cues, are discussed.
Collaboration of signal transducer and activator of transcription 1 (STAT1) and BRCA1 in differential regulation of IFN-γ target genes
Authors: Ouchi Toru , Lee Sam W. , Ouchi Mutsuko , Aaronson Stuart A. , Horvath Curt M.
Published: July 2002

Most of the activities of IFN-γ are the result of STAT1-mediated transcriptional responses. In this study, we show that the BRCA1 tumor suppressor acts in concert with STAT1 to differentially activate transcription of a subset of IFN-γ target genes and mediates growth inhibition by this cytokine. After IFN-γ treatment, induction of the cyclin-dependent kinase inhibitor,
p21WAF1,
was synergistically activated by BRCA1, whereas the
IRF-1
gene was unaffected. Importantly, the differential induction of
p21WAF1
was impaired in breast cancer cells homozygous for the mutant
BRCA1 5382C
allele. Biochemical analysis illustrated that the mechanism of this transcriptional synergy involves interaction between BRCA1 aa 502–802 and the C-terminal transcriptional activation domain of STAT1 including Ser-727 whose phosphorylation is crucial for transcriptional activation. Significantly, STAT1 proteins mutated at Ser-727 bind poorly to BRCA1, reinforcing the importance of Ser-727 in the recruitment of transcriptional coactivators by STAT proteins. These findings reveal a novel mechanism for BRCA1 function in the IFN-γ-dependent tumor surveillance system.
An important function of Nrf2 in combating oxidative stress: Detoxification of acetaminophen
Authors: Chan Kaimin , Han Xiao-Dong , Kan Yuet Wai
Published: July 2002

Nrf2, a member of the “cap ‘n collar” group of transcription
factors, is important for protecting cells against oxidative damage. We
investigated its role in the detoxification of acetaminophen
[
N
-acetyl-
p
-aminophenol (APAP)]-induced
hepatotoxicity. When Nrf2 knockout (Nrf2
−/−
) and
wild-type mice were given APAP by i.p. injection, the
Nrf2
−/−
mice were highly susceptible to APAP treatment.
With doses of APAP that were tolerated by wild-type mice, the
Nrf2
−/−
mice died of liver failure. When hepatic
glutathione was depleted after a dose of 400 mg/kg of APAP, the
wild-type mice were able to compensate and regain the normal
glutathione level. In contrast, the glutathione level in the
Nrf2
−/−
mice was not compensated and remained low. This
was because of the decrease in the gene expression of
gcs
H
and
gcs
L
as
well as
gss
in the livers of the Nrf2
−/−
mice. In addition, the expression of
ugt
1a6 and
gst
pi that detoxify APAP by conjugation was also
decreased. This increased susceptibility of the Nrf2
−/−
mice to APAP, because of an impaired capacity to replenish their
glutathione stores, compounded with a decreased detoxification
capability, highlights the importance of Nrf2 in the regulation of
glutathione synthesis and cellular detoxification processes.
Rapid transition in the structure of a coral reef community: The effects of coral bleaching and physical disturbance
Authors: Ostrander Gary K. , Armstrong Kelley Meyer , Knobbe Edward T. , Gerace Donald , Scully Erik P.
Published: July 2002

Coral reef communities are in a state of change throughout their geographical range. Factors contributing to this change include bleaching (the loss of algal symbionts), storm damage, disease, and increasing abundance of macroalgae. An additional factor for Caribbean reefs is the aftereffects of the epizootic that reduced the abundance of the herbivorous sea urchin,
Diadema antillarum
. Although coral reef communities have undergone phase shifts, there are few studies that document the details of such transitions. We report the results of a 40-month study that documents changes in a Caribbean reef community affected by bleaching, hurricane damage, and an increasing abundance of macroalgae. The study site was in a relatively pristine area of the reef surrounding the island of San Salvador in the Bahamas. Ten transects were sampled every 3–9 months from November 1994 to February 1998. During this period, the corals experienced a massive bleaching event resulting in a significant decline in coral abundance. Algae, especially macroalgae, increased in abundance until they effectively dominated the substrate. The direct impact of Hurricane Lili in October 1996 did not alter the developing community structure and may have facilitated increasing algal abundance. The results of this study document the rapid transition of this reef community from one in which corals and algae were codominant to a community dominated by macroalgae. The relatively brief time period required for this transition illustrates the dynamic nature of reef communities.
Regulation of the actin cycle
in vivo
by actin filament severing
Authors: McGrath James L. , Osborn Eric A. , Tardy Yanik S. , Dewey C. Forbes , Hartwig John H.
Published: July 2002

Cycling of actin subunits between monomeric and filamentous phases is essential for cell crawling behavior. We investigated actin filament turnover rates, length, number, barbed end exposure, and binding of cofilin in bovine arterial endothelial cells moving at different speeds depending on their position in a confluent monolayer. Fast-translocating cells near the wound edge have short filament lifetimes compared with turnover values that proportionately increase in slower moving cells situated at increasing distances from the wound border. Contrasted with slow cells exhibiting slow actin filament turnover speeds, fast cells have less polymerized actin, shorter actin filaments, more free barbed ends, and less actin-associated cofilin. Cultured primary fibroblasts manifest identical relationships between speed and actin turnover as the endothelial cells, and fast fibroblasts expressing gelsolin have higher actin turnover rates than slow fibroblasts that lack this actin-severing protein. These results implicate actin filament severing as an important control mechanism for actin cycling in cells.
The ADP ribosylation factor nucleotide exchange factor ARNO promotes β-arrestin release necessary for luteinizing hormone/choriogonadotropin receptor desensitization
Authors: Mukherjee Sutapa , Gurevich Vsevolod V. , Jones Jonathan C. R. , Casanova James E. , Frank Scott R. , Maizels Evelyn T. , Bader Marie-France , Kahn Richard A. , Palczewski Krzysztof , Aktories Klaus , Hunzicker-Dunn Mary
Published: July 2002

Desensitization of guanine nucleotide binding protein-coupled receptors is a ubiquitous phenomenon characterized by declining effector activity upon persistent agonist stimulation. The luteinizing hormone/choriogonadotropin receptor (LH/CGR) in ovarian follicles exhibits desensitization of effector adenylyl cyclase activity in response to the mid-cycle surge of LH. We have previously shown that uncoupling of the agonist-activated LH/CGR from the stimulatory G protein (G
s
) is dependent on GTP and attributable to binding of β-arrestin present in adenylyl cyclase-rich follicular membrane fraction to the third intracellular (3i) loop of the receptor. Here, we report that LH/CGR-dependent desensitization is mimicked by ADP ribosylation factor nucleotide-binding site opener, a guanine nucleotide exchange factor of the small G proteins ADP ribosylation factors (Arfs) 1 and 6, and blocked by synthetic N-terminal Arf6 peptide, suggesting that the GTP-dependent step of LH/CGR desensitization is receptor-dependent Arf6 activation. Arf activation by GTP and ADP ribosylation factor nucelotide-binding site opener promotes the release of docked β-arrestin from the membrane, making β-arrestin available for LH/CGR; Arf6 but not Arf1 peptides block β-arrestin release from the membrane. Thus, LH/CGR appears to activate two membrane delimited signaling cascades via two types of G proteins: heterotrimeric G
s
and small G protein Arf6. Arf6 activation releases docked β-arrestin necessary for receptor desensitization, providing a feedback mechanism for receptor self-regulation.
Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity
Authors: Tansey J. T. , Sztalryd C. , Gruia-Gray J. , Roush D. L. , Zee J. V. , Gavrilova O. , Reitman M. L. , Deng C.-X. , Li C. , Kimmel A. R. , Londos C.
Published: July 2002

Perilipin coats the lipid droplets of adipocytes and is thought to
have a role in regulating triacylglycerol hydrolysis. To study the role
of perilipin
in vivo
, we have created a
perilipin
knockout mouse.
Perilipin
null
(
peri
−/−
) and wild-type
(
peri
+/+
) mice consume equal amounts of
food, but the adipose tissue mass in the null animals is reduced to
≈30% of that in wild-type animals. Isolated adipocytes of
perilipin
null mice exhibit elevated basal lipolysis
because of the loss of the protective function of perilipin. They also
exhibit dramatically attenuated stimulated lipolytic activity,
indicating that perilipin is required for maximal lipolytic activity.
Plasma leptin concentrations in null animals were greater than expected
for the reduced adipose mass. The
peri
−/−
animals have a greater lean body mass and increased metabolic rate but
they also show an increased tendency to develop glucose intolerance and
peripheral insulin resistance. When fed a high-fat diet, the
perilipin
null animals are resistant to diet-induced
obesity but not to glucose intolerance. The data reveal a major role
for perilipin in adipose lipid metabolism and suggest perilipin as a
potential target for attacking problems associated with obesity.
Mammalian mitogenomic relationships and the root of the eutherian tree
Authors: Arnason Ulfur , Adegoke Joseph A. , Bodin Kristina , Born Erik W. , Esa Yuzine B. , Gullberg Anette , Nilsson Maria , Short Roger V. , Xu Xiufeng , Janke Axel
Published: July 2002

The strict orthology of mitochondrial (mt) coding sequences has promoted their use in phylogenetic analyses at different levels. Here we present the results of a mitogenomic study (i.e., analysis based on the set of protein-coding genes from complete mt genomes) of 60 mammalian species. This number includes 11 new mt genomes. The sampling comprises all but one of the traditional eutherian orders. The previously unrepresented order Dermoptera (flying lemurs) fell within Primates as the sister group of Anthropoidea, making Primates paraphyletic. This relationship was strongly supported. Lipotyphla (“insectivores”) split into three distinct lineages: Erinaceomorpha, Tenrecomorpha, and Soricomorpha. Erinaceomorpha was the basal eutherian lineage. Sirenia (dugong) and Macroscelidea (elephant shrew) fell within the African clade. Pholidota (pangolin) joined the Cetferungulata as the sister group of Carnivora. The analyses identified monophyletic Pinnipedia with Otariidae (sea lions, fur seals) and Odobenidae (walruses) as sister groups to the exclusion of Phocidae (true seals).
Parathyroid hormone-related protein induces spontaneous osteoclast formation via a paracrine cascade
Authors: Nakchbandi Inaam A. , Weir Eleanor E. , Insogna Karl L. , Philbrick William M. , Broadus Arthur E.
Published: July 2002

Experiments
in vivo
have established that tooth eruption fails in the absence of parathyroid hormone (PTH)-related protein (PTHrP) action in the microenvironment of the tooth because of the failure of osteoclastic bone resorption on the coronal tooth surface to form an eruption pathway. To elucidate the effects of PTHrP on osteoclast regulation in this environment, we established primary cultures of epithelial stellate reticulum cells and mesenchymal dental follicle (DF) cells surrounding the teeth. When cocultured, these cells are fully capable of supporting the formation of functional osteoclasts in the absence of added splenic osteoclast precursors, osteoblasts, or vitamin D/PTH/PTHrP. Neutralizing the effects of PTHrP resulted in a decrease in the number of osteoclasts formed, suggesting that stellate reticulum-derived PTHrP drives osteoclast formation. DF cells were found to express functional PTH/PTHrP type I receptors, and conditioned media collected from PTHrP-treated DF cells were able to induce bone resorption in the fetal-rat long-bone assay. PTHrP treatment also induced an increase in osteoclast differentiation factor expression and a concomitant decrease in osteoclastogenesis inhibitory factor expression in DF cells. The addition of osteoclastogenesis inhibitory factor resulted in a decrease in the number of osteoclasts formed in the cocultures, suggesting that osteoclast formation is mediated by osteoclast differentiation factor. Thus, PTHrP seems to regulate osteoclast formation via mediation of the DF, in a manner analogous to the osteoblast-mediated process in the peripheral skeleton. The primary coculture system of dental crypt cells also offers a system for the study of osteoclast formation and regulation.
The
in vivo
neuromodulatory effects of the herbal medicine ginkgo biloba
Authors: Watanabe Coran M. H. , Wolffram Siegfried , Ader Peter , Rimbach Gerald , Packer Lester , Maguire John J. , Schultz Peter G. , Gohil Kishorchandra
Published: July 2002

Extracts of
Ginkgo biloba
leaves are consumed as
dietary supplements to counteract chronic, age-related neurological
disorders. We have applied high-density oligonucleotide microarrays to
define the transcriptional effects in the cortex and hippocampus of
mice whose diets were supplemented with the herbal extract. Gene
expression analysis focused on the mRNAs that showed a more than 3-fold
change in their expression. In the cortex, mRNAs for neuronal
tyrosine/threonine phosphatase 1, and microtubule-associated τ were
significantly enhanced. Hyperphosphorylated τ is the major
constituent of the neurofibrillary tangles in the brains of
Alzheimer's disease patients. The expression of
α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-2,
calcium and chloride channels, prolactin, and growth hormone (GH), all
of which are associated with brain function, were also up-regulated. In
the hippocampus, only transthyretin mRNA was upregulated.
Transthyretin plays a role in hormone transport in the brain and
possibly a neuroprotective role by amyloid-β sequestration. This
study reveals that diets supplemented with Ginkgo biloba extract have
notable neuromodulatory effects
in vivo
and illustrates
the utility of genome-wide expression monitoring to investigate the
biological actions of complex extracts.
Identification of gene expression profile of dorsal root ganglion in the rat peripheral axotomy model of neuropathic pain
Authors: Xiao Hua-Sheng , Huang Qiu-Hua , Zhang Fang-Xiong , Bao Lan , Lu Ying-Jin , Guo Chao , Yang Liang , Huang Wein-Jing , Fu Gang , Xu Shu-Hua , Cheng Xi-Ping , Yan Qing , Zhu Zhi-Dong , Zhang Xin , Chen Zhu , Han Ze-Guang , Zhang Xu
Published: July 2002

Phenotypic modification of dorsal root ganglion (DRG) neurons represents an important mechanism underlying neuropathic pain. However, the nerve injury-induced molecular changes are not fully identified. To determine the molecular alterations in a broader way, we have carried out cDNA array on the genes mainly made from the cDNA libraries of lumbar DRGs of normal rats and of rats 14 days after peripheral axotomy. Of the 7,523 examined genes and expressed sequence tags (ESTs), the expression of 122 genes and 51 expressed sequence tags is strongly changed. These genes encompass a large number of members of distinct families, including neuropeptides, receptors, ion channels, signal transduction molecules, synaptic vesicle proteins, and others. Of particular interest is the up-regulation of γ-aminobutyric acid
A
receptor α5 subunit, peripheral benzodiazepine receptor, nicotinic acetylcholine receptor α7 subunit, P2Y1 purinoceptor, Na
+
channel β2 subunit, and L-type Ca
2+
channel α2δ-1 subunit. Our findings therefore reveal dynamic and complex changes in molecular diversity among DRG neurons after axotomy.
C75 increases peripheral energy utilization and fatty acid oxidation in diet-induced obesity
Authors: Thupari Jagan N. , Landree Leslie E. , Ronnett Gabriele V. , Kuhajda Francis P.
Published: July 2002

C75, a known inhibitor of fatty acid synthase is postulated to cause significant weight loss through decreased hypothalamic neuropeptide Y (NPY) production. Peripherally, C75, an α-methylene-γ-butyrolactone, reduces adipose tissue and fatty liver, despite high levels of malonyl-CoA. To investigate this paradox, we studied the effect of C75 on fatty acid oxidation and energy production in diet-induced obese (DIO) mice and cellular models. Whole-animal calorimetry showed that C75-treated DIO mice had a 50% greater weight loss, and a 32.9% increased production of energy because of fatty acid oxidation, compared with paired-fed controls. Etomoxir, an inhibitor of carnitine
O
-palmitoyltransferase-1 (CPT-1), reversed the increased energy expenditure in DIO mice by inhibiting fatty acid oxidation. C75 treatment of rodent adipocytes and hepatocytes and human breast cancer cells increased fatty acid oxidation and ATP levels by increasing CPT-1 activity, even in the presence of elevated concentrations of malonyl-CoA. Studies in human cancer cells showed that C75 competed with malonyl-CoA, as measured by CPT-1 activity assays. Thus, C75 acts both centrally to reduce food intake and peripherally to increase fatty acid oxidation, leading to rapid and profound weight loss, loss of adipose mass, and resolution of fatty liver. The pharmacological stimulation of CPT-1 activity is a novel finding. The dual action of the C75 class of compounds as fatty acid synthase inhibitors and CPT-1 agonists has therapeutic implications in the treatment of obesity and type II diabetes.
Crystal structure of the Holliday junction DNA in complex with a single RuvA tetramer
Authors: Ariyoshi Mariko , Nishino Tatsuya , Iwasaki Hiroshi , Shinagawa Hideo , Morikawa Kosuke
Published: July 2002

In the major pathway of homologous DNA recombination in prokaryotic cells, the Holliday junction intermediate is processed through its association with RuvA, RuvB, and RuvC proteins. Specific binding of the RuvA tetramer to the Holliday junction is required for the RuvB motor protein to be loaded onto the junction DNA, and the RuvAB complex drives the ATP-dependent branch migration. We solved the crystal structure of the Holliday junction bound to a single
Escherichia coli
RuvA tetramer at 3.1-Å resolution. In this complex, one side of DNA is accessible for cleavage by RuvC resolvase at the junction center. The refined junction DNA structure revealed an open concave architecture with a four-fold symmetry. Each arm, with B-form DNA, in the Holliday junction is predominantly recognized in the minor groove through hydrogen bonds with two repeated helix-hairpin-helix motifs of each RuvA subunit. The local conformation near the crossover point, where two base pairs are disrupted, suggests a possible scheme for successive base pair rearrangements, which may account for smooth Holliday junction movement without segmental unwinding.
Subnucleus-specific loss of neurons in medial thalamus of schizophrenics
Authors: Popken Gregory J. , Bunney William E. , Potkin Steven G. , Jones Edward G.
Published: July 2002

The hypoactivity of dorsolateral prefrontal cortex in schizophrenics is well known. One cause of this hypoactivity may be defective corticocortical or thalamocortical connections. Recent imaging studies of the thalamus suggest reductions in volume of the whole thalamus and reduced activity in the medial group of thalamic nuclei, which may indicate loss of functional input to the cortex. Using stereological techniques in six pairs of individually matched brains from schizophrenics and controls, we measured the volumes and obtained estimates of the number of neurons in the three subnuclei (parvocellular, pc; densocellular, dc; magnocellular, mc) of the mediodorsal nucleus (MD) and from the ventral posterior medial nucleus. There was a significant reduction in total neuron number in MD as a whole but this neuron loss was largely restricted to MDpc and MDdc [−30.9 and −24.5%, respectively (P≤ 0.01)]. MDmc and the control ventral posterior medial nucleus showed no significant changes in cell number. Because the subnuclei of MD have different connections and project to different areas of the frontal cortex, the specific loss of neurons in MDpc and MDdc has implications for the functional defects observed in schizophrenia.
Structural insights into the catalytic mechanism of a family 18 exo-chitinase
Authors: van Aalten D. M. F. , Komander D. , Synstad B. , Gåseidnes S. , Peter M. G. , Eijsink V. G. H.
Published: July 2002

Chitinase B (ChiB) from
Serratia marcescens
is a family 18 exo-chitinase whose catalytic domain has a TIM-barrel fold with a tunnel-shaped active site. We have solved structures of three ChiB complexes that reveal details of substrate binding, substrate-assisted catalysis, and product displacement. The structure of an inactive ChiB mutant (E144Q) complexed with a pentameric substrate (binding in subsites −2 to +3) shows closure of the “roof” of the active site tunnel. It also shows that the sugar in the −1 position is distorted to a boat conformation, thus providing structural evidence in support of a previously proposed catalytic mechanism. The structures of the active enzyme complexed to allosamidin (an analogue of a proposed reaction intermediate) and of the active enzyme soaked with pentameric substrate show events after cleavage of the glycosidic bond. The latter structure shows reopening of the roof of the active site tunnel and enzyme-assisted product displacement in the +1 and +2 sites, allowing a water molecule to approach the reaction center. Catalysis is accompanied by correlated structural changes in the core of the TIM barrel that involve conserved polar residues whose functions were hitherto unknown. These changes simultaneously contribute to stabilization of the reaction intermediate and alternation of the pKa of the catalytic acid during the catalytic cycle.
Modulation of Akt kinase activity by binding to Hsp90
Authors: Sato Saori , Fujita Naoya , Tsuruo Takashi
Published: July 2002

Serine/threonine kinase Akt/PKB is a downstream effector
molecule of phosphoinositide 3-kinase and is thought to mediate many
biological actions toward anti-apoptotic responses. We found
that Akt formed a complex with a 90-kDa heat-shock protein (Hsp90)
in vivo
. By constructing deletion mutants, we identified
that amino acid residues 229–309 of Akt were involved in the binding
to Hsp90 and amino acid residues 327–340 of Hsp90β were involved in
the binding to Akt. Inhibition of Akt-Hsp90 binding led to the
dephosphorylation and inactivation of Akt, which increased sensitivity
of the cells to apoptosis-inducing stimulus. The
dephosphorylation of Akt was caused by an increase in protein
phosphatase 2A (PP2A)-mediated dephosphorylation and not by a decrease
in 3-phosphoinositide-dependent protein kinase-1-mediated
phosphorylation. These results indicate that Hsp90 plays an important
role in maintaining Akt kinase activity by preventing PP2A-mediated
dephosphorylation.
Evaluating hypotheses of basal animal phylogeny using complete sequences of large and small subunit rRNA
Authors: Medina Mónica , Collins Allen G. , Silberman Jeffrey D. , Sogin Mitchell L.
Published: July 2002

We studied the evolutionary relationships among basal metazoan
lineages by using complete large subunit (LSU) and small subunit (SSU)
ribosomal RNA sequences for 23 taxa. After identifying competing
hypotheses, we performed maximum likelihood searches for trees
conforming to each hypothesis. Kishino–Hasegawa tests were used to
determine whether the data (LSU, SSU, and combined) reject any of the
competing hypotheses. We also conducted unconstrained tree searches,
compared the resulting topologies, and calculated bootstrap indices.
Shimodaira–Hasegawa tests were applied to determine whether the data
reject any of the topologies resulting from the constrained and
unconstrained tree searches. LSU, SSU, and the combined data strongly
contradict two assertions pertaining to sponge phylogeny. Hexactinellid
sponges are not likely to be the basal lineage of a monophyletic
Porifera or the sister group to all other animals. Instead,
Hexactinellida and Demospongia form a well-supported clade of siliceous
sponges, Silicea. It remains unclear, on the basis of these data alone,
whether the calcarean sponges are more closely related to Silicea or to
nonsponge animals. The SSU and combined data reject the hypothesis that
Bilateria is more closely related to Ctenophora than it is to Cnidaria,
whereas LSU data alone do not refute either hypothesis. LSU and SSU
data agree in supporting the monophyly of Bilateria, Cnidaria,
Ctenophora, and Metazoa. LSU sequence data reveal phylogenetic
structure in a data set with limited taxon sampling. Continued
accumulation of LSU sequences should increase our understanding of
animal phylogeny.
Accelerated regulatory gene evolution in an adaptive radiation
Authors: Barrier Marianne , Robichaux Robert H. , Purugganan Michael D.
Published: July 2002

The disparity between rates of morphological and molecular evolution remains a key paradox in evolutionary genetics. A proposed resolution to this paradox has been the conjecture that morphological evolution proceeds via diversification in regulatory loci, and that phenotypic evolution may correlate better with regulatory gene divergence. This conjecture can be tested by examining rates of regulatory gene evolution in species that display rapid morphological diversification within adaptive radiations. We have isolated homologues to theArabidopsis APETALA3(ASAP3/TM6) andAPETALA1(ASAP1) floral regulatory genes and theCHLOROPHYLL A/B BINDING PROTEIN9(ASCAB9) photosynthetic structural gene from species in the Hawaiian silversword alliance, a premier example of plant adaptive radiation. We have compared rates of regulatory and structural gene evolution in the Hawaiian species to those in related species of North American tarweeds. Molecular evolutionary analyses indicate significant increases in nonsynonymous relative to synonymous nucleotide substitution rates in theASAP3/TM6andASAP1regulatory genes in the rapidly evolving Hawaiian species. By contrast, no general increase is evident in neutral mutation rates for these loci in the Hawaiian species. An increase in nonsynonymous relative to synonymous nucleotide substitution rate is also evident in theASCAB9structural gene in the Hawaiian species, but not to the extent displayed in the regulatory loci. The significantly accelerated rates of regulatory gene evolution in the Hawaiian species may reflect the influence of allopolyploidy or of selection and adaptive divergence. The analyses suggest that accelerated rates of regulatory gene evolution may accompany rapid morphological diversification in adaptive radiations.