bluuebunny/crossref_metadata_2025 huggingface dataset:
Magnetic and collisional effects on presheaths
Authors: Kim G.-H. , Hershkowitz N. , Diebold D. A. , Cho M.-H.
Published: July 2002

The effects of both magnetic field and collisions on presheath properties are experimentally investigated in plasmas with electron temperatures much greater than ion temperatures. Measurements of plasma potential in collisionless plasmas show presheath thicknesses at boundaries oblique to magnetic field to be approximately (Cs/ωci)sin ψ, where Cs is the ion sound speed, ωci is the ion gyrofrequency, and Ψ is the angle between the magnetic field and the normal to the wall boundary. Measurements of plasma potential in collisional plasmas find presheaths consisting of two distinctive regions. With ion–neutral collision mean-free path λn<(Cs/ωci)sin ψ, the presheath region next to the sheath has collisional characteristics and a thickness of approximately (0.5–0.6)λn. The corresponding presheath region adjacent to the bulk plasma has magnetic characteristics and a thickness of approximately (0.5–0.9)(Cs/ωci)sin ψ. Equipotential contours in the collisional region of this presheath are found to be parallel to the boundary, while those in the magnetic region are not.
Effects of radiation damage in ion-implanted thin films of metal-oxide superconductors
Authors: Clark G. J. , Marwick A. D. , Koch R. H. , Laibowitz R. B.
Published: July 2002

The effects of ion implantation into thin films of the superconductor YBa2Cu3Ox have been studied. Using oxygen and arsenic ions, the superconducting transition temperature Tc, the change in room-temperature electrical properties from conducting to insulating, and the crystalline to amorphous structural transition in the films were studied as a function of ion dose. The deposited energy required to change Tc was found to be 0.2 eV/atom, while 1–2 eV/atom was required to affect the room-temperature conductivity, and 4 eV/atom to render the film amorphous. This hierarchy of effects is discussed in terms of the damage mechanisms involved.
Isolation and characterization of powdery mildew-resistant
Arabidopsis
mutants
Authors: Vogel John , Somerville Shauna
Published: July 2002

A compatible interaction between a plant and a pathogen is the result of a complex interplay between many factors of both plant and pathogen origin. Our objective was to identify host factors involved in this interaction. These factors may include susceptibility factors required for pathogen growth, factors manipulated by the pathogen to inactivate or avoid host defenses, or negative regulators of defense responses. To this end, we identified 20 recessive
Arabidopsis
mutants that do not support normal growth of the powdery mildew pathogen,
Erysiphe cichoracearum
. Complementation analyses indicated that four loci, designated powdery mildew resistant 1–4 (
pmr1–4
), are defined by this collection. These mutants do not constitutively accumulate elevated levels of
PR1
or
PDF1.2
mRNA, indicating that resistance is not simply due to constitutive activation of the salicylic acid- or ethylene- and jasmonic acid-dependent defense pathways. Further Northern blot analyses revealed that some mutants accumulate higher levels of
PR1
mRNA than wild type in response to infection by powdery mildew. To test the specificity of the resistance, the
pmr
mutants were challenged with other pathogens including
Pseudomonas syringae
,
Peronospora parasitica
, and
Erysiphe orontii
. Surprisingly, one mutant,
pmr1,
was susceptible to
E. orontii
, a very closely related powdery mildew, suggesting that a very specific resistance mechanism is operating in this case. Another mutant,
pmr4
, was resistant to
P. parasitica
, indicating that this resistance is more generalized. Thus, we have identified a novel collection of mutants affecting genes required for a compatible interaction between a plant and a biotrophic pathogen.
Loss of circadian clock function decreases reproductive fitness in males of
Drosophila
melanogaster
Authors: Beaver L. M. , Gvakharia B. O. , Vollintine T. S. , Hege D. M. , Stanewsky R. , Giebultowicz J. M.
Published: July 2002

Circadian coordination of life functions is believed to contribute to an organism's fitness; however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. Here we examined the consequences of loss of clock function on reproductive fitness in
Drosophila melanogaster
with mutated
period
(
per

0

),
timeless
(
tim

0

),
cycle
(
cyc

0

), and
Clock
(
Clk

Jrk

) genes. Single mating among couples with clock-deficient phenotypes resulted in ≈40% fewer progeny compared with wild-type flies, because of a decreased number of eggs laid and a greater rate of unfertilized eggs. Male contribution to this phenotype was demonstrated by a decrease in reproductive capacity among
per

0

and
tim

0

males mated with wild-type females. The important role of clock genes for reproductive fitness was confirmed by reversal of the low-fertility phenotype in flies with rescued
per
or
tim
function. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles, and these tissues displayed rhythmic and autonomous expression of clock genes. By combining molecular and physiological approaches, we identified a circadian clock in the reproductive system and defined its role in the sperm release that promotes reproductive fitness in
D. melanogaster
.
Selective up-regulation of dopamine D1 receptors in dendritic spines by NMDA receptor activation
Authors: Scott Lena , Kruse Maria Sol , Forssberg Hans , Brismar Hjalmar , Greengard Paul , Aperia Anita
Published: July 2002

Glutamate, by activating
N
-methyl-
d
-aspartate (NMDA) receptors, alters the balance between dopamine D1 and D2 receptor signaling, but the mechanism responsible for this effect has not been known. We report here, using immunocytochemistry of primary cultures of rat neostriatal neurons, that activation of NMDA receptors recruits D1 receptors from the interior of the cell to the plasma membrane while having no effect on the distribution of D2 receptors. The D1 receptors were concentrated in spines as shown by colocalization with phalloidin-labeled actin filaments. The effect of NMDA on D1 receptors was abolished by incubation of cells in calcium-free medium and was mimicked by the calcium ionophore ionomycin. Recruitment of D1 receptors from the interior of the cell to the membrane was confirmed by subcellular fractionation. The recruited D1 receptors were functional as demonstrated by an increase in dopamine-sensitive adenylyl cyclase activity in membranes derived from cells that had been pretreated with NMDA. These results provide evidence for regulated recruitment of a G protein-coupled receptor in neurons, provide a cell biological basis for the effect of NMDA on dopamine signaling, and reconcile the conflicting hyperdopaminergic and hypoglutamatergic hypotheses of schizophrenia.
The analysis of 100 genes supports the grouping of three highly divergent amoebae:
Dictyostelium
,
Entamoeba
, and
Mastigamoeba
Authors: Bapteste Eric , Brinkmann Henner , Lee Jennifer A. , Moore Dorothy V. , Sensen Christoph W. , Gordon Paul , Duruflé Laure , Gaasterland Terry , Lopez Philippe , Müller Miklós , Philippe Hervé
Published: July 2002

The phylogenetic relationships of amoebae are poorly resolved. To address this difficult question, we have sequenced 1,280 expressed sequence tags from
Mastigamoeba balamuthi
and assembled a large data set containing 123 genes for representatives of three phenotypically highly divergent major amoeboid lineages: Pelobionta, Entamoebidae, and Mycetozoa. Phylogenetic reconstruction was performed on ≈25,000 aa positions for 30 species by using maximum-likelihood approaches. All well-established eukaryotic groups were recovered with high statistical support, validating our approach. Interestingly, the three amoeboid lineages strongly clustered together in agreement with the Conosa hypothesis [as defined by T. Cavalier-Smith (1998)
Biol. Rev. Cambridge Philos. Soc.
73, 203–266]. Two amitochondriate amoebae, the free-living
Mastigamoeba
and the human parasite
Entamoeba
, formed a significant sister group to the exclusion of the mycetozoan
Dictyostelium
. This result suggested that a part of the reductive process in the evolution of
Entamoeba
(e.g., loss of typical mitochondria) occurred in its free-living ancestors. Applying this inexpensive expressed sequence tag approach to many other lineages will surely improve our understanding of eukaryotic evolution.
Adeno-associated virus effectively mediates conditional gene modification in the brain
Authors: Kaspar Brian K. , Vissel Bryce , Bengoechea Tasha , Crone Steven , Randolph-Moore Lynne , Muller Rolf , Brandon Eugene P. , Schaffer David , Verma Inder M. , Lee Kuo-Fen , Heinemann Stephen F. , Gage Fred H.
Published: July 2002

The Cre/
lox
P system is increasingly showing promise for investigating genes involved in neural function. Here, we demonstrate that
in vivo
modification of genes in the mouse brain can be accomplished in a spatial- and temporal-specific manner by targeted delivery of an adeno-associated virus (AAV) encoding a green fluorescent protein/Cre recombinase (GFP/Cre) fusion protein. By using a reporter mouse, in which Cre recombinase activates β-galactosidase expression, we demonstrate long-term recombination of neurons in the hippocampus, striatum, and septum as early as 7 days after stereotaxic injection of virus. Recombined cells were observed for at least 6 months postinjection without evidence of cell loss or neural damage. AAV-mediated delivery of GFP/Cre provides a valuable approach to alter the mouse genome, as AAV delivers genes efficiently to neurons with low toxicity. This approach will greatly facilitate the study of genetic modifications in the mouse brain.
Differences in quantal amplitude reflect GluR4- subunit number at corticothalamic synapses on two populations of thalamic neurons
Authors: Golshani Peyman , Liu Xiao-Bo , Jones Edward G.
Published: July 2002

Low-frequency thalamocortical oscillations that underlie drowsiness
and slow-wave sleep depend on rhythmic inhibition of relay cells by
neurons in the reticular nucleus (RTN) under the influence of
corticothalamic fibers that branch to innervate RTN neurons and relay
neurons. To generate oscillations, input to RTN predictably should be
stronger so disynaptic inhibition of relay cells overcomes direct
corticothalamic excitation. Amplitudes of excitatory postsynaptic
conductances (EPSCs) evoked in RTN neurons by minimal stimulation of
corticothalamic fibers were 2.4 times larger than in relay neurons, and
quantal size of RTN EPSCs was 2.6 times greater. GluR4-receptor
subunits labeled at corticothalamic synapses on RTN neurons outnumbered
those on relay cells by 3.7 times, providing a basis for differences in
synaptic strength.
Embryonic stem cells and somatic cells differ in mutation frequency and type
Authors: Cervantes Rachel B. , Stringer James R. , Shao Changshun , Tischfield Jay A. , Stambrook Peter J.
Published: July 2002

Pluripotent embryonic stem (ES) cells have been used to produce genetically modified mice as experimental models of human genetic diseases. Increasingly, human ES cells are being considered for their potential in the treatment of injury and disease. Here we have shown that mutation in murine ES cells, heterozygous at the selectable
Aprt
locus, differs from that in embryonic somatic cells. The mutation frequency in ES cells is significantly lower than that in mouse embryonic fibroblasts, which is similar to that in adult cells
in vivo
. The distribution of spontaneous mutagenic events is remarkably different between the two cell types. Although loss of the functional allele is the predominant mutation type in both cases, representing about 80% of all events, mitotic recombination accounted for all loss of heterozygosity events detected in somatic cells. In contrast, mitotic recombination in ES cells appeared to be suppressed and chromosome loss/reduplication, leading to uniparental disomy (UPD), represented more than half of the loss of heterozygosity events. Extended culture of ES cells led to accumulation of cells with adenine phosphoribosyltransferase deficiency and UPD. Because UPD leads to reduction to homozygosity at multiple recessive disease loci, including tumor suppressor loci, in the affected chromosome, the increased risk of tumor formation after stem cell therapy should be viewed with concern.
Diversity and evolution of the green fluorescent protein family
Authors: Labas Y. A. , Gurskaya N. G. , Yanushevich Y. G. , Fradkov A. F. , Lukyanov K. A. , Lukyanov S. A. , Matz M. V.
Published: July 2002

The family of proteins homologous to the green fluorescent protein (GFP) from
Aequorea victoria
exhibits striking diversity of features, including several different types of autocatalytically synthesized chromophores. Here we report 11 new members of the family, among which there are 3 red-emitters possessing unusual features, and discuss the similarity relationships within the family in structural, spectroscopic, and evolutionary terms. Phylogenetic analysis has shown that GFP-like proteins from representatives of subclass Zoantharia fall into at least four distinct clades, each clade containing proteins of more than one emission color. This topology suggests multiple recent events of color conversion. Combining this result with previous mutagenesis and structural data, we propose that (
i
) different chromophore structures are alternative products synthesized within a similar autocatalytic environment, and (
ii
) the phylogenetic pattern and color diversity in reef Anthozoa is a result of a balance between selection for GFP-like proteins of particular colors and mutation pressure driving the color conversions.
Substance P antagonist (CP-96,345) inhibits HIV-1 replication in human mononuclear phagocytes
Authors: Lai Jian-Ping , Ho Wen-Zhe , Zhan Guan-Xia , Yi Yanjie , Collman Ronald G. , Douglas Steven D.
Published: July 2002

Substance P (SP) is a potent modulator of neuroimmunoregulation. We recently reported that human immune cells express SP and its receptor. We have now investigated the possible role that SP and its receptor plays in HIV infection of human mononuclear phagocytes. SP enhanced HIV replication in human blood-isolated mononuclear phagocytes, whereas the nonpeptide SP antagonist (CP-96,345) potently inhibited HIV infectivity of these cells in a concentration-dependent fashion. CP-96,345 prevented the formation of typical giant syncytia induced by HIV Bal strain replication in these cells. This inhibitory effect of CP-96,345 was because of the antagonism of neurokinin-1 receptor, a primary SP receptor. Both CP-96,345 and anti-SP antibody inhibited SP-enhanced HIV replication in monocyte-derived macrophages (MDM). Among HIV strains tested (both prototype and primary isolates), only the R5 strains (Bal, ADA, BL-6, and CSF-6) that use the CCR5 coreceptor for entry into MDM were significantly inhibited by CP-96,345; in contrast, the X4 strain (UG024), which uses CXCR4 as its coreceptor, was not inhibited. In addition, the M-tropic ADA (CCR5-dependent)-pseudotyped HIV infection of MDM was markedly inhibited by CP-96,345, whereas murine leukemia virus-pseudotyped HIV was not affected, indicating that the major effect of CP-96,345 is regulated by Env-determined early events in HIV infection of MDM. CP-96,345 significantly down-regulated CCR5 expression in MDM at both protein and mRNA levels. Thus, SP–neurokinin-1 receptor interaction may play an important role in the regulation of CCR5 expression in MDM, affecting the R5 HIV strain infection of MDM.
The human brm protein is cleaved during apoptosis: The role of cathepsin G
Authors: Biggs Joseph R. , Yang Jie , Gullberg Urban , Muchardt Christian , Yaniv Moshe , Kraft Andrew S.
Published: July 2002

The human brm (hbrm) protein (homologue of the
Drosophila
melanogaster
brahma and
Saccharomyces cervisiae
SNF-2 proteins) is part of a polypeptide complex believed to regulate
chromatin conformation. We have shown that the hbrm protein is cleaved
in NB4 leukemic cells after induction of apoptosis by
UV-irradiation, DNA damaging agents, or staurosporine. Because hbrm is
found only in the nucleus, we have investigated the nature of the
proteases that may regulate the degradation of this protein during
apoptosis. In an
in vitro
assay, the hbrm
protein could not be cleaved by caspase-3, -7, or -6, the
“effector” caspases generally believed to carry out the cleavage
of nuclear protein substrates. In contrast, we find that cathepsin G, a
granule enzyme found in NB4 cells, cleaves hbrm in a pattern similar to
that observed
in vivo
during apoptosis. In
addition, a peptide inhibitor of cathepsin G blocks hbrm cleavage
during apoptosis but does not block activation of caspases or
cleavage of the nuclear protein polyADP ribose polymerase (PARP).
Although localized in granules and in the Golgi complex in untreated
cells, cathepsin G becomes diffusely distributed during
apoptosis. Cleavage by cathepsin G removes a 20-kDa fragment
containing a bromodomain from the carboxyl terminus of hbrm. This
cleavage disrupts the association between hbrm and the nuclear matrix;
the 160-kDa hbrm cleavage fragment is less tightly associated with the
nuclear matrix than full-length hbrm.
Local osmotic gradients drive the water flux associated with Na
+
/glucose cotransport
Authors: Duquette Pierre-Pascal , Bissonnette Pierre , Lapointe Jean-Yves
Published: July 2002

It recently was proposed [Loo, D. D. F., Zeuthen, T.,
Chandy, G. & Wright, E. M. (1996)
Proc. Natl. Acad. Sci.
USA
93, 13367–13370] that SGLT1, the high affinity intestinal
and renal sodium/glucose cotransporter carries water molecules along
with the cosubstrates with a strict stoichiometry of two
Na
+
, one glucose, and ≈220 water molecules per transport
cycle. Using electrophysiology together with sensitive volumetric
measurements, we investigated the nature of the driving force behind
the cotransporter-mediated water flux. The osmotic water permeability
of oocytes expressing human SGLT1 (
L
p
± SE)
averaged 3.8 ± 0.3 × 10
−4
cm⋅s
−1
(
n
= 15) and addition of 100
μM phlorizin (a specific SGLT1 inhibitor) reduced the permeability to
2.2 ± 0.2 × 10
−4
cm⋅s
−1
(
n
= 15), confirming the presence of a significant
water permeability closely associated with the cotransporter. Addition
of 5 mM α-methyl-glucose (αMG) induced an average inward current of
800 ± 10 nA at −50 mV and a water influx reaching 120 ± 20 pL
cm
−2
⋅s
−1
within 5–8 min. After rapidly
inhibiting the Na
+
/glucose cotransport with phlorizin,
the water flux remained significantly elevated, clearly indicating the
presence of a local osmotic gradient (Δπ) estimated at 16 ± 2
mOsm. In short-term experiments, a rapid depolarization from −100 to 0
mV in the presence of αMG decreased the cotransport current by 94%
but failed to produce a comparable reduction in the swelling rate. A
mathematical model depicting the intracellular accumulation of
transported osmolytes can accurately account for these observations. It
is concluded that, in SGLT1-expressing oocytes, αMG-dependent water
influx is induced by a local osmotic gradient by using both
endogenous and SGLT1-dependent water permeability.
Lack of tissue glucocorticoid reactivation in 11β-hydroxysteroid dehydrogenase type 1 knockout mice ameliorates age-related learning impairments
Authors: Yau Joyce L. W. , Noble June , Kenyon Christopher J. , Hibberd Carina , Kotelevtsev Yuri , Mullins John J. , Seckl Jonathan R.
Published: July 2002

11β-hydroxysteroid dehydrogenase type 1 (11β-HSD-1)
intracellularly regenerates active corticosterone from circulating
inert 11-dehydrocorticosterone (11-DHC) in specific tissues. The
hippocampus is a brain structure particularly vulnerable to
glucocorticoid neurotoxicity with aging. In intact hippocampal cells in
culture, 11β-HSD-1 acts as a functional 11β-reductase reactivating
inert 11-DHC to corticosterone, thereby potentiating kainate
neurotoxicity. We examined the functional significance of 11β-HSD-1
in the central nervous system by using knockout mice. Aged wild-type
mice developed elevated plasma corticosterone levels that correlated
with learning deficits in the watermaze. In contrast, despite elevated
plasma corticosterone levels throughout life, this
glucocorticoid-associated learning deficit was ameliorated in aged
11β-HSD-1 knockout mice, implicating lower intraneuronal
corticosterone levels through lack of 11-DHC reactivation. Indeed, aged
knockout mice showed significantly lower hippocampal tissue
corticosterone levels than wild-type controls. These findings
demonstrate that tissue corticosterone levels do not merely reflect
plasma levels and appear to play a more important role in hippocampal
functions than circulating blood levels. The data emphasize the crucial
importance of local enzymes in determining intracellular glucocorticoid
activity. Selective 11β-HSD-1 inhibitors may protect against
hippocampal function decline with age.
Candidate tumor suppressor HYAL2 is a glycosylphosphatidylinositol (GPI)-anchored cell-surface receptor for jaagsiekte sheep retrovirus, the envelope protein of which mediates oncogenic transformation
Authors: Rai Sharath K. , Duh Fuh-Mei , Vigdorovich Vladimir , Danilkovitch-Miagkova Alla , Lerman Michael I. , Miller A. Dusty
Published: July 2002

Jaagsiekte sheep retrovirus (JSRV) can induce rapid,
multifocal lung cancer, but JSRV is a simple retrovirus having no known
oncogenes. Here we show that the envelope (
env
) gene of
JSRV has the unusual property that it can induce transformation in rat
fibroblasts, and thus is likely to be responsible for oncogenesis in
animals. Retrovirus entry into cells is mediated by Env interaction
with particular cell-surface receptors, and we have used phenotypic
screening of radiation hybrid cell lines to identify the candidate lung
cancer tumor suppressor HYAL2/LUCA2 as the receptor for JSRV. HYAL2
was previously described as a lysosomal hyaluronidase, but we show that
HYAL2 is actually a glycosylphosphatidylinositol (GPI)-anchored
cell-surface protein. Furthermore, we could not detect hyaluronidase
activity associated with or secreted by cells expressing HYAL2, whereas
we could easily detect such activity from cells expressing the related
serum hyaluronidase HYAL1. Although the function of HYAL2 is currently
unknown, other GPI-anchored proteins are involved in signal
transduction, and some mediate mitogenic responses, suggesting a
potential role of HYAL2 in JSRV Env-mediated oncogenesis. Lung cancer
induced by JSRV closely resembles human bronchiolo-alveolar carcinoma,
a disease that is increasing in frequency and now accounts for ≈25%
of all lung cancer. The finding that JSRV
env
is
oncogenic and the identification of HYAL2 as the JSRV receptor provide
tools for further investigation of the mechanism of JSRV oncogenesis
and its relationship to human bronchiolo-alveolar carcinoma.
A peptide template as an allosteric supramolecular catalyst for the cleavage of phosphate esters
Authors: Scarso Alessandro , Scheffer Ute , Göbel Michael , Broxterman Quirinus B. , Kaptein Bernard , Formaggio Fernando , Toniolo Claudio , Scrimin Paolo
Published: July 2002

The heptapeptide H-Iva-Api-Iva-ATANP-Iva-Api-Iva-NHCH
3
(P1a), where Iva is (S)-isovaline, Api is 4-amino-4-carboxypiperidine, and ATANP is (S)-2-amino-3-[1-(1,4,7-triazacyclononane)]propanoic acid, has been synthesized. Its conformation in aqueous solution is essentially that of a 3
10
-helix. By connecting three copies of P1a to a functionalized Tris(2-aminoethyl)amine (Tren) platform a new peptide template, [T(P1)
3
], was obtained. This molecule is able to bind up to four metal ions (Cu
II
or Zn
II
): one in the Tren subsite and three in the azacyclononane subunits. The binding of the metals to the Tren platform induces a change from an open to a closed conformation in which the three short, helical peptides are aligned in a parallel manner with the azacyclonane units pointing inward within the pseudocavity they define. T(P1)
3
shows a peculiar behavior in the transphosphorylation of phosphate esters; the tetrazinc complex is a catalyst of the cleavage of 2-hydroxypropyl-
p
-nitrophenyl phosphate (HPNP), whereas the free ligand is a catalyst of the cleavage of an oligomeric RNA sequence with selectivity for pyrimidine bases. In the case of HPNP, Zn
II
acts as a positive allosteric effector by enhancing the catalytic efficiency of the system. In the case of the polyanionic RNA substrate, Zn
II
switches off the activity, thus behaving as a negative allosteric regulator. It is suggested that the opposite behavior of the catalyst induced by Zn
II
is associated with the change of conformation of the Tren platform, and consequently of the relative spatial disposition of the three linked peptides, that occurs after binding of the metal ion.
Neurogenesis in dentate subgranular zone and rostral subventricular zone after focal cerebral ischemia in the rat
Authors: Jin Kunlin , Minami Manabu , Lan Jing Q. , Mao Xiao Ou , Batteur Sophie , Simon Roger P. , Greenberg David A.
Published: July 2002

Because neurogenesis persists in the adult mammalian brain and can
be regulated by physiological and pathological events, we investigated
its possible involvement in the brain's response to focal cerebral
ischemia. Ischemia was induced by occlusion of the middle cerebral
artery in the rat for 90 min, and proliferating cells were labeled with
5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUrd) over 2-day periods
before sacrificing animals 1, 2 or 3 weeks after ischemia. Ischemia
increased the incorporation of BrdUrd into cells in two
neuroproliferative regions—the subgranular zone of the dentate gyrus
and the rostral subventricular zone. Both effects were bilateral, but
that in the subgranular zone was more prominent on the ischemic side.
Cells labeled with BrdUrd coexpressed the immature neuronal markers
doublecortin and proliferating cell nuclear antigen but did not express
the more mature cell markers NeuN and Hu, suggesting that they were
nascent neurons. These results support a role for ischemia-induced
neurogenesis in what may be adaptive processes that contribute to
recovery after stroke.
On the role of the K-proton transfer pathway in cytochrome
c
oxidase
Authors: Brändén Magnus , Sigurdson Håkan , Namslauer Andreas , Gennis Robert B. , Ädelroth Pia , Brzezinski Peter
Published: July 2002

Cytochrome
c
oxidase is a membrane-bound enzyme that
catalyzes the four-electron reduction of oxygen to water. This highly
exergonic reaction drives proton pumping across the membrane. One of
the key questions associated with the function of cytochrome
c
oxidase is how the transfer of electrons and protons
is coupled and how proton transfer is controlled by the enzyme. In this
study we focus on the function of one of the proton transfer pathways
of the
R. sphaeroides
enzyme, the so-called K-proton
transfer pathway (containing a highly conserved Lys(I-362) residue),
leading from the protein surface to the catalytic site. We have
investigated the kinetics of the reaction of the reduced enzyme with
oxygen in mutants of the enzyme in which a residue [Ser(I-299)] near
the entry point of the pathway was modified with the use of
site-directed mutagenesis. The results show that during the initial
steps of oxygen reduction, electron transfer to the catalytic site (to
form the “peroxy” state, P
r
) requires charge
compensation through the proton pathway, but no proton uptake from the
bulk solution. The charge compensation is proposed to involve a
movement of the K(I-362) side chain toward the binuclear center. Thus,
in contrast to what has been assumed previously, the results indicate
that the K-pathway is used during oxygen reduction and that K(I-362) is
charged at pH ≈ 7.5. The movement of the Lys is proposed to regulate
proton transfer by “shutting off” the protonic connectivity
through the K-pathway after initiation of the O
2
reduction
chemistry. This “shutoff” prevents a short-circuit of the
proton-pumping machinery of the enzyme during the subsequent reaction
steps.
Erythropoietin induces tumor regression and antitumor immune responses in murine myeloma models
Authors: Mittelman Moshe , Neumann Drorit , Peled Alpha , Kanter Pazit , Haran-Ghera Nechama
Published: July 2002

Recombinant human erythropoietin (rHuEpo) has been used successfully in the treatment of cancer-related anemia. Clinical observations with several patients with multiple-myeloma treated with rHuEpo has shown, in addition to the improved quality of life, a longer survival than expected, considering the poor prognostic features of these patients. Based on these observations, we evaluated the potential biological effects of rHuEpo on the course of tumor progression by using murine myeloma models (MOPC-315-IgAλ2and 5T33 MM-IgG2b). Here we report that daily treatment of MOPC-315 tumor-bearing mice with rHuEpo for several weeks induced complete tumor regression in 30–60% of mice. All regressors that were rechallenged with tumor cells rejected tumor growth, and this resistance was tumor specific. The Epo-triggered therapeutic effect was shown to be attributed to a T cell-mediated mechanism. Serum Ig analysis indicated a reduction in MOPC-315 λ light chain in regressor mice. Intradermal inoculation of 5T33 MM tumor cells followed by Epo treatment induced tumor regression in 60% of mice. The common clinical manifestation of myeloma bone disease in patients with multiple-myeloma was established in these myeloma models. Epo administration to these tumor-bearing mice markedly prolonged their survival and reduced mortality. Therefore, erythropoietin seems to act as an antitumor therapeutic agent in addition to its red blood cell-stimulating activity.
A DNA vector-based RNAi technology to suppress gene expression in mammalian cells
Authors: Sui Guangchao , Soohoo Christina , Affar El Bachir , Gay Frédérique , Shi Yujiang , Forrester William C. , Shi Yang
Published: July 2002

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants,
Caenorhabditis elegans
, and
Drosophila
. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates
in vivo
to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.
Computation, prediction, and experimental tests of fitness for bacteriophage T7 mutants with permuted genomes
Authors: Endy Drew , You Lingchong , Yin John , Molineux Ian J.
Published: July 2002

We created a simulation based on experimental data from bacteriophage T7 that computes the developmental cycle of the wild-type phage and also of mutants that have an altered genome order. We used the simulation to compute the fitness of more than 10
5
mutants. We tested these computations by constructing and experimentally characterizing T7 mutants in which we repositioned gene
1
, coding for T7 RNA polymerase. Computed protein synthesis rates for ectopic gene
1
strains were in moderate agreement with observed rates. Computed phage-doubling rates were close to observations for two of four strains, but significantly overestimated those of the other two. Computations indicate that the genome organization of wild-type T7 is nearly optimal for growth: only 2.8% of random genome permutations were computed to grow faster, the highest 31% faster, than wild type. Specific discrepancies between computations and observations suggest that a better understanding of the translation efficiency of individual mRNAs and the functions of qualitatively “nonessential” genes will be needed to improve the T7 simulation.
In silico
representations of biological systems can serve to assess and advance our understanding of the underlying biology. Iteration between computation, prediction, and observation should increase the rate at which biological hypotheses are formulated and tested.
Mirror self-recognition in the bottlenose dolphin: A case of cognitive convergence
Authors: Reiss Diana , Marino Lori
Published: July 2002

The ability to recognize oneself in a mirror is an exceedingly rare capacity in the animal kingdom. To date, only humans and great apes have shown convincing evidence of mirror self-recognition. Two dolphins were exposed to reflective surfaces, and both demonstrated responses consistent with the use of the mirror to investigate marked parts of the body. This ability to use a mirror to inspect parts of the body is a striking example of evolutionary convergence with great apes and humans.
Iris hypoplasia in mice that lack the alternatively spliced
Pax6(5a)
isoform
Authors: Singh Sanjaya , Mishra Rajnikant , Arango Nelson A. , Deng Jian Min , Behringer Richard R. , Saunders Grady F.
Published: July 2002

PAX6 is an evolutionarily conserved transcription factor that plays a critical role in vertebrate and invertebrate eye formation. Heterozygous null mutations in the
PAX6
gene result in aniridia in humans and a distinct small eye syndrome in rodents. Vertebrates primarily express two alternatively spliced isoforms of
Pax6
that differ by the presence or absence of exon 5a (e5A) that encodes an additional 14 aa residues within the paired domain. The e5a-containing isoform, PAX6(5a), is specific to and conserved in vertebrates. To determine the role of PAX6(5a), we have generated mice that lack
e5a
of the
Pax6
gene. Unlike
Pax6
null mice that exhibit anopthalmia with central nervous system defects and lethality, 5a isoform-null mice have iris hypoplasia and defects in the cornea, lens, and retina. Although invertebrates have structures that respond to light intensity and act to restrict light exposure of the eyes, a significant and distinct feature of the vertebrate eye is its ability to regulate the amount of incoming light through contractile pupils. This feature of the eye not only allows vertebrates to see in various light conditions but also enhances image resolution. The requirement of the 5a isoform in iris formation suggests that the evolution of this isoform contributed to advanced features of the vertebrate eye.
Use of double-stranded RNA interference in
Drosophila
cell lines to dissect signal transduction pathways
Authors: Clemens James C. , Worby Carolyn A. , Simonson-Leff Nancy , Muda Marco , Maehama Tomohiko , Hemmings Brian A. , Dixon Jack E.
Published: July 2002

We demonstrate the efficacy of double-stranded RNA-mediated interference (RNAi) of gene expression in generating “knock-out” phenotypes for specific proteins in several
Drosophila
cell lines. We prove the applicability of this technique for studying signaling cascades by dissecting the well-characterized insulin signal transduction pathway. Specifically, we demonstrate that inhibiting the expression of the DSOR1 (mitogen-activated protein kinase kinase, MAPKK) prevents the activation of the downstream ERK-A (MAPK). In contrast, blocking ERK-A expression results in increased activation of DSOR1. We also show that
Drosophila
AKT (DAKT) activation depends on the insulin receptor substrate, CHICO (IRS1–4). Finally, we demonstrate that blocking the expression of
Drosophila
PTEN results in the activation of DAKT. In all cases, the interference of the biochemical cascade by RNAi is consistent with the known steps in the pathway. We extend this powerful technique to study two proteins, DSH3PX1 and
Drosophila
ACK (DACK). DSH3PX1 is an SH3, phox homology domain-containing protein, and DACK is homologous to the mammalian activated Cdc42 tyrosine kinase, ACK. Using RNAi, we demonstrate that DACK is upstream of DSH3PX1 phosphorylation, making DSH3PX1 an identified downstream target/substrate of ACK-like tyrosine kinases. These experiments highlight the usefulness of RNAi in dissecting complex biochemical signaling cascades and provide a highly effective method for determining the function of the identified genes arising from the
Drosophila
genome sequencing project.
Manipulating the mammalian genome by homologous recombination
Authors: Vasquez Karen M. , Marburger Kathleen , Intody Zsofia , Wilson John H.
Published: July 2002

Gene targeting in mammalian cells has proven invaluable in biotechnology, in studies of gene structure and function, and in understanding chromosome dynamics. It also offers a potential tool for gene-therapeutic applications. Two limitations constrain the current technology: the low rate of homologous recombination in mammalian cells and the high rate of random (nontargeted) integration of the vector DNA. Here we consider possible ways to overcome these limitations within the framework of our present understanding of recombination mechanisms and machinery. Several studies suggest that transient alteration of the levels of recombination proteins, by overexpression or interference with expression, may be able to increase homologous recombination or decrease random integration, and we present a list of candidate genes. We consider potentially beneficial modifications to the vector DNA and discuss the effects of methods of DNA delivery on targeting efficiency. Finally, we present work showing that gene-specific DNA damage can stimulate local homologous recombination, and we discuss recent results with two general methodologies—chimeric nucleases and triplex-forming oligonucleotides—for stimulating recombination in cells.
Effect of COMT Val
108/158
Met genotype on frontal lobe function and risk for schizophrenia
Authors: Egan Michael F. , Goldberg Terry E. , Kolachana Bhaskar S. , Callicott Joseph H. , Mazzanti Chiara M. , Straub Richard E. , Goldman David , Weinberger Daniel R.
Published: July 2002

Abnormalities of prefrontal cortical function are prominent
features of schizophrenia and have been associated with genetic risk,
suggesting that susceptibility genes for schizophrenia may impact on
the molecular mechanisms of prefrontal function. A potential
susceptibility mechanism involves regulation of prefrontal dopamine,
which modulates the response of prefrontal neurons during working
memory. We examined the relationship of a common functional
polymorphism (Val
108/158
Met) in the
catechol-
O
-methyltransferase (COMT) gene, which accounts
for a 4-fold variation in enzyme activity and dopamine catabolism, with
both prefrontally mediated cognition and prefrontal cortical
physiology. In 175 patients with schizophrenia, 219 unaffected
siblings, and 55 controls, COMT genotype was related in allele dosage
fashion to performance on the Wisconsin Card Sorting Test of executive
cognition and explained 4% of variance (
P
= 0.001)
in frequency of perseverative errors. Consistent with other evidence
that dopamine enhances prefrontal neuronal function, the load of the
low-activity Met allele predicted enhanced cognitive performance. We
then examined the effect of COMT genotype on prefrontal physiology
during a working memory task in three separate subgroups
(
n
= 11–16) assayed with functional MRI. Met
allele load consistently predicted a more efficient physiological
response in prefrontal cortex. Finally, in a family-based association
analysis of 104 trios, we found a significant increase in transmission
of the Val allele to the schizophrenic offspring. These data suggest
that the COMT Val allele, because it increases prefrontal dopamine
catabolism, impairs prefrontal cognition and physiology, and by this
mechanism slightly increases risk for schizophrenia.
A role for estrogen receptor β in the regulation of growth of the ventral prostate
Authors: Weihua Zhang , Mäkelä Sari , Andersson Leif C. , Salmi Saija , Saji Shigehira , Webster Jeanette I. , Jensen Elwood V. , Nilsson Stefan , Warner Margaret , Gustafsson Jan-Åke
Published: July 2002

In normal rats and mice, immunostaining with specific antibodies revealed that nuclei of most prostatic epithelial cells harbor estrogen receptor β (ERβ). In rat ventral prostate, 530- and 549-aa isoforms of the receptor were identified. These sediment in the 4S region of low-salt sucrose gradients, indicating that prostatic ERβ does not contain the same protein chaperones that are associated with ERα. Estradiol (E2) binding and ERβ immunoreactivity coincide on the gradient, with no indication of ERα. In prostates from mice in which the ERβ gene has been inactivated (BERKO), androgen receptor (AR) levels are elevated, and the tissue contains multiple hyperplastic foci. Most epithelial cells express the proliferation antigenKi-67. In contrast, prostatic epithelium from wild-type littermates is single layered with no hyperplasia, and very few cells expressKi-67. Rat ventral prostate contains an estrogenic component, which comigrates on HPLC with the testosterone metabolite 5α-androstane-3β,17β-diol (3βAdiol). This compound, which competes with E2for binding to ERβ and elicits an estrogenic response in the aorta but not in the pituitary, decreases the AR content in prostates of wild-type mice but does not affect the elevated levels seen in ERβ knockout (BERKO) mice. Thus ERβ, probably as a complex with 3βAdiol, is involved in regulating the AR content of the rodent prostate and in restraining epithelial growth. These findings suggest that ligands specific for ERβ may be useful in the prevention and/or clinical management of prostatic hyperplasia and neoplasia.
Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production
Authors: Flurkey Kevin , Papaconstantinou John , Miller Richard A. , Harrison David E.
Published: July 2002

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at thePit1(Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ × DW/J)F1background. MutantdwJ/dwanimals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for theGhrhrlitmutation, which like thePit1dwmutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposityper se. Further studies of thePit1dwmutant, and the closely related, long-livedProp-1df(Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.
Synthetic LXR ligand inhibits the development of atherosclerosis in mice
Authors: Joseph Sean B. , McKilligin Elaine , Pei Liming , Watson Michael A. , Collins Alan R. , Laffitte Bryan A. , Chen Mingyi , Noh Grace , Goodman Joanne , Hagger Graham N. , Tran Jonathan , Tippin Tim K. , Wang Xuping , Lusis Aldons J. , Hsueh Willa A. , Law Ronald E. , Collins Jon L. , Willson Timothy M. , Tontonoz Peter
Published: July 2002

The nuclear receptors LXRα and LXRβ have been implicated in the control of cholesterol and fatty acid metabolism in multiple cell types. Activation of these receptors stimulates cholesterol efflux in macrophages, promotes bile acid synthesis in liver, and inhibits intestinal cholesterol absorption, actions that would collectively be expected to reduce atherosclerotic risk. However, synthetic LXR ligands have also been shown to induce lipogenesis and hypertriglyceridemia in mice, raising questions as to the net effects of these compounds on the development of cardiovascular disease. We demonstrate here that the nonsteroidal LXR agonist GW3965 has potent antiatherogenic activity in two different murine models. In LDLR
−/−
mice, GW3965 reduced lesion area by 53% in males and 34% in females. A similar reduction of 47% was observed in male apoE
−/−
mice. Long-term (12-week) treatment with LXR agonist had differential effects on plasma lipid profiles in LDLR
−/−
and apoE
−/−
mice. GW3965 induced expression of ATP-binding cassettes A1 and G1 in modified low-density lipoprotein-loaded macrophages
in vitro
as well as in the aortas of hyperlipidemic mice, suggesting that direct actions of LXR ligands on vascular gene expression are likely to contribute to their antiatherogenic effects. These observations provide direct evidence for an atheroprotective effect of LXR agonists and support their further evaluation as potential modulators of human cardiovascular disease.
Noncoding RNA genes identified in AT-rich hyperthermophiles
Authors: Klein Robert J. , Misulovin Ziva , Eddy Sean R.
Published: July 2002

Noncoding RNA (ncRNA) genes that produce functional RNAs instead of encoding proteins seem to be somewhat more prevalent than previously thought. However, estimating their number and importance is difficult because systematic identification of ncRNA genes remains challenging. Here, we exploit a strong, surprising DNA composition bias in genomes of some hyperthermophilic organisms: simply screening for GC-rich regions in the AT-rich
Methanococcus jannaschii
and
Pyrococcus furiosus
genomes efficiently detects both known and new RNA genes with a high degree of secondary structure. A separate screen based on comparative analysis also successfully identifies noncoding RNA genes in
P. furiosus
. Nine of the 30 new candidate genes predicted by these screens have been verified to produce discrete, apparently noncoding transcripts with sizes ranging from 97 to 277 nucleotides.
Retroposon analysis of major cetacean lineages: The monophyly of toothed whales and the paraphyly of river dolphins
Authors: Nikaido Masato , Matsuno Fumio , Hamilton Healy , Brownell Robert L. , Cao Ying , Ding Wang , Zuoyan Zhu , Shedlock Andrew M. , Fordyce R. Ewan , Hasegawa Masami , Okada Norihiro
Published: July 2002

SINE (short interspersed element) insertion analysis elucidates contentious aspects in the phylogeny of toothed whales and dolphins (Odontoceti), especially river dolphins. Here, we characterize 25 informative SINEs inserted into unique genomic loci during evolution of odontocetes to construct a cladogram, and determine a total of 2.8 kb per taxon of the flanking sequences of these SINE loci to estimate divergence times among lineages. We demonstrate that: (
i
) Odontocetes are monophyletic; (
ii
) Ganges River dolphins, beaked whales, and ocean dolphins diverged (in this order) after sperm whales; (
iii
) three other river dolphin taxa, namely the Amazon, La Plata, and Yangtze river dolphins, form a monophyletic group with Yangtze River dolphins being the most basal; and (
iv
) the rapid radiation of extant cetacean lineages occurred some 28–33 million years B.P., in strong accord with the fossil record. The combination of SINE and flanking sequence analysis suggests a topology and set of divergence times for odontocete relationships, offering alternative explanations for several long-standing problems in cetacean evolution.