bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterosynaptic metaplasticity in the hippocampus
in vivo
: A BCM-like modifiable threshold for LTP
Authors: Abraham Wickliffe C. , Mason-Parker Sara E. , Bear Mark F. , Webb Sarah , Tate Warren P.
Published: July 2002

The homeostatic maintenance of the “modification threshold” for inducing long-term potentiation (LTP) is a fundamental feature of the Bienenstock, Cooper, and Munro (BCM) model of synaptic plasticity. In the present study, two key features of the modification threshold, its heterosynaptic expression and its regulation by postsynaptic neural activity, were tested experimentally in the dentate gyrus of awake, freely moving rats. Conditioning stimulation ranging from 10 to 1,440 brief 400-Hz trains, when applied to medial perforant path afferents, raised the threshold for LTP induction heterosynaptically in the neighboring lateral perforant path synapses. This effect recovered slowly over a 7- to 35-day period. The same conditioning paradigms, however, did not affect the reversal of long-term depression. The inhibition of LTP by medial-path conditioning stimulation was
N
-methyl-
D
-aspartate (NMDA) receptor-dependent, but antidromic stimulation of the granule cells could also inhibit lateral path LTP induction, independently of NMDA receptor activation. Increased calcium buffering is a potential mechanism underlying the altered LTP threshold, but the levels of two important calcium-binding proteins did not increase after conditioning stimulation, nor was
de novo
protein synthesis required for generating the threshold shift. These data confirm, in an
in vivo
model, two key postulates of the BCM model regarding the LTP threshold. They also provide further evidence for the broad sensitivity of synaptic plasticity mechanisms to the history of prior activity, i.e., metaplasticity.
Social enhancement of fitness in yellow-bellied marmots
Authors: Armitage Kenneth B. , Schwartz Orlando A.
Published: July 2002

The yellow-bellied marmot (
Marmota flaviventris
) is
a social, ground-dwelling squirrel that lives either individually or in
kin groups of from two to five adult females. Philopatry and daughter
recruitment lead to the formation and persistence of matrilines at
habitat sites. By using 37 years of demographic data for 12 habitat
sites, we could determine long-term trends in the effects of group size
on two measures of fitness, survivorship and net reproductive rate,
which otherwise are obscured by annual fluctuations in these measures.
Both size and number of matrilines varied among sites and survivorship
and net reproductive rate varied among sites and among matriline sizes.
The role of social organization was explored further by examining the
effect of matriline size, averaged over all years and sites, on
fitness. For both survivorship and net reproductive rate the
relationship with matriline size was curvilinear. Fitness increased
with the increase in matriline size and then decreased in the largest
groups. Decreased fitness in matrilines of four or five was associated
with agonistic behavior, a large number of 2-year-old females in the
social group, and reproductive suppression. There is no evidence that
females acted to increase their fitness by increasing indirect fitness;
i.e., by assisting relatives, but attempted to increase direct fitness.
Direct fitness increased when mortality and fission of large matrilines
reduced group size and the surviving females increased reproduction.
Host–parasite relatedness shown by protein fingerprinting in a brood parasitic bird
Authors: Andersson Malte , Åhlund Matti
Published: July 2002

Brood parasitism as an alternative female breeding tactic is
particularly common in ducks, where hosts often receive eggs laid by
parasitic females of the same species and raise their offspring.
Herein, we test several aspects of a kin selection explanation for this
phenomenon in goldeneye ducks (
Bucephala clangula
) by
using techniques of egg albumen sampling and statistical bandsharing
analysis based on resampling. We find that host and primary parasite
are indeed often related, with mean
r
= 0.13, about
as high as between first cousins. Relatedness to the host is higher in
nests where a parasite lays several eggs than in those where she lays
only one. Returning young females parasitize their birth nestmates
(social mothers or sisters, which are usually also their genetic
mothers and sisters) more often than expected by chance. Such adult
relatives are also observed together in the field more often than
expected and for longer periods than other females. Relatedness and kin
discrimination, which can be achieved by recognition of birth
nestmates, therefore play a role in these tactics and probably
influence their success.
Phosphorylation and inactivation of glycogen synthase kinase 3 by protein kinase A
Authors: Fang Xianjun , Yu Shuang Xing , Lu Yiling , Bast Robert C. , Woodgett James R. , Mills Gordon B.
Published: July 2002

Glycogen synthase kinase 3 (GSK-3) is implicated in multiple
biological processes including metabolism, gene expression, cell fate
determination, proliferation, and survival. GSK-3 activity is inhibited
through phosphorylation of serine 21 in GSK-3α and serine 9 in
GSK-3β. These serine residues of GSK-3 have been previously
identified as targets of protein kinase B (PKB/Akt), a
serine/threonine kinase located downstream of phosphatidylinositol
3-kinase. Here, we show that serine 21 in GSK-3α and serine 9 in
GSK-3β are also physiological substrates of cAMP-dependent protein
kinase A. Protein kinase A physically associates with, phosphorylates,
and inactivates both isoforms of GSK-3. The results indicate that
depending on the stimulatory context, the activity of GSK-3 can be
modulated either by growth factors that work through the
phosphatidylinositol 3-kinase–protein kinase B cascade or by hormonal
stimulation of G protein-coupled receptors that link to changes in
intracellular cAMP levels.
Detection of mutations in transgenic fish carrying a bacteriophage λ
cII
transgene target
Authors: Winn Richard N. , Norris Michelle B. , Brayer Kathryn J. , Torres Cecilia , Muller Stacy L.
Published: July 2002

To address the dual needs for improved methods to assess potential
health risks associated with chemical exposure in aquatic environments
and for new models for
in vivo
mutagenesis studies, we
developed transgenic fish that carry multiple copies of a bacteriophage
λ vector that harbors the
cII
gene as a mutational
target. We adapted a forward mutation assay, originally developed for
λ transgenic rodents, to recover
cII
mutants
efficiently from fish genomic DNA by λ
in vitro
packaging. After infecting and plating phage on a
hfl
−
bacterial host,
cII
mutants were detected under
selective conditions. We demonstrated that many fundamental features of
mutation analyses based on λ transgenic rodents are shared by
transgenic fish. Spontaneous mutant frequencies, ranging from 4.3
× 10
−5
in liver, 2.9 × 10
−5
in whole
fish, to 1.8 × 10
−5
in testes, were comparable to
ranges in λ transgenic rodents. Treatment with ethylnitrosourea
resulted in concentration-dependent, tissue-specific, and
time-dependent mutation inductions consistent with known mechanisms of
action. Frequencies of mutants in liver increased insignificantly 5
days after ethylnitrosourea exposure, but increased 3.5-, 5.7- and
6.7-fold above background at 15, 20, and 30 days, respectively. Mutants
were induced 5-fold in testes at 5 days, attaining a peak 10-fold
induction 15 days after treatment. Spontaneous and induced mutational
spectra in the fish were also consistent with those of λ transgenic
rodent models. Our results demonstrate the feasibility of
in
vivo
mutation analyses using transgenic fish and illustrate the
potential value of fish as important comparative animal models.
Gene correction in hematopoietic progenitor cells by homologous recombination
Authors: Hatada Seigo , Nikkuni Koji , Bentley Stuart A. , Kirby Suzanne , Smithies Oliver
Published: July 2002

Homologous recombination (gene targeting) has many desirable features for gene therapy, because it can precisely correct mutant genes and restore their normal expression, and random nonhomologous integration of DNA is infrequent in cells in which homologous recombination has occurred. There are, however, no reports of attempts to use homologous recombination to correct mutant genes in normal hematopoietic stem cells (HSCs), which are prime cells for therapy of a variety of hematological and other conditions, presumably because of their low abundance and uncertainty that homologous recombination can occur at a usable frequency in these cells. The experiments reported here encourage optimism in this respect by demonstrating targeted correction of a defective hypoxanthine phosphoribosyltransferase gene in hematopoietic progenitor cells that can form colonies in methylcellulose culture. These clonogenic cells are in the same lineage as HSCs but are more abundant and more mature and so less pluripotent. Corrected colonies were identified by their survival in selective medium after electroporation of correcting DNA into unfractionated mouse bone marrow cells and were confirmed by reverse transcription–PCR and sequencing. The observed frequency (4.4 ± 3.3 × 10−5per treated clonogenic cell) is the same as in embryonic stem cells (2.3 ± 0.4 × 10−5) with the same DNA and mutation. These data suggest that gene targeting to correct mutant genes eventually will prove feasible in HSCs capable of long-term bone marrow reconstitution.
Redefinition of the mode Grüneisen parameter for polyatomic substances and thermodynamic implications
Authors: Hofmeister Anne M. , Mao Ho-kwang
Published: July 2002

Although the value of the thermal Grüneisen parameter (γth) should be obtained by averaging spectroscopic measurements of mode Grüneisen parameters [γi≡ (KT/νi)∂νi/∂P, whereKTis isothermal bulk modulus, ν is frequency, andPis pressure], in practice, the average 〈γi〉 is up to 25% lower than γth. This discrepancy limits the accuracy of inferring physical properties from spectroscopic data and their application to geophysics. The problem arises because the above formula is physically meaningful only for monatomic or diatomic solids. We redefine γito allow for the presence of functional groups in polyatomic crystal structures, and test the formula against spinel- and olivine-group minerals that have well-constrained spectra at pressure, band assignments, thermodynamic properties, and elastic moduli, and represent two types of functional groups. Our revised formula [γi≡ (KX/νi)∂νi/∂P] uses polyhedral bulk moduli (KX) appropriate to the particular atomic motion associated with each vibrational mode, which results in equal values for 〈γi〉, γth, and γLA(the Grüneisen parameter of the longitudinal acoustic mode). Similar revisions lead to the pressure derivatives of these parameters being equal. Accounting for differential compression intrinsic to structures with functional groups improves the accuracy with which spectroscopic models predict thermodynamic properties and link to elastic properties.
IL-15 plays a major role in the persistence of Tax-specific CD8 cells in HAM/TSP patients
Authors: Azimi Nazli , Nagai Masahiro , Jacobson Steven , Waldmann Thomas A.
Published: July 2002

IL-15 is a critical cytokine for the maintenance of memory-phenotype CD8 cells in mice. Here, we investigated the role of IL-15 in the neurological disease termed human T cell lymphotropic virus I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The high number of viral-specific CD8 cells in these patients is associated with inflammatory responses in the central nervous system. Because IL-15 is overexpressed in these patients, we asked whether IL-15 contributes to the persistence of human T cell lymphotropic virus I viral-specific CD8 cells. Usingex vivocultures of HAM/TSP peripheral blood mononuclear cells, we demonstrated that in the majority of patients examined here blocking IL-15 action resulted in a decrease in the number of viral-specific CD8 cells. This decrease was caused by both inhibition of proliferation and induction of apoptosis in these cells. The data indicate that IL-15 plays a major role in the maintenance of viral-specific CD8 cells in HAM/TSP.
Vpr protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers.
Authors: Piller S C , Ewart G D , Premkumar A , Cox G B , Gage P W
Published: July 2002

A small (96-aa) protein, virus protein R (Vpr), of human immunodeficiency virus type 1 contains one hydrophobic segment that could form a membrane-spanning helix. Recombinant Vpr, expressed in Escherichia coli and purified by affinity chromatography, formed ion channels in planar lipid bilayers when it was added to the cis chamber and when the trans chamber was held at a negative potential. The channels were more permeable to Na+ than to Cl- ions and were inhibited when the trans potential was made positive. Similar channel activity was caused by Vpr that had a truncated C terminus, but the potential dependence of channel activity was no longer seen. Antibody raised to a peptide mimicking part of the C terminus of Vpr (AbC) inhibited channel activity when added to the trans chamber but had no effect when added to the cis chamber. Antibody to the N terminus of Vpr (AbN) increased channel activity when added to the cis chamber but had no effect when added to the trans chamber. The effects of potential and antibodies on channel activity are consistent with a model in which the positive C-terminal end of dipolar Vpr is induced to traverse the bilayer membrane when the opposite (trans) side of the membrane is at a negative potential. The C terminus of Vpr would then be available for interaction with AbC in the trans chamber, and the N terminus would be available for interaction with AbN in the cis chamber. The ability of Vpr to form ion channels in vitro suggests that channel formation by Vpr in vivo is possible and may be important in the life cycle of human immunodeficiency virus type 1 and/or may cause changes in cells that contribute to AIDS-related pathologies.
Polynitrosylated proteins: characterization, bioactivity, and functional consequences.
Authors: Simon D I , Mullins M E , Jia L , Gaston B , Singel D J , Stamler J S
Published: July 2002

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.
The heart communicates with the kidney exclusively through the guanylyl cyclase-A receptor: acute handling of sodium and water in response to volume expansion.
Authors: Kishimoto I , Dubois S K , Garbers D L
Published: July 2002

Disruption of guanylyl cyclase-A (GC-A) results in mice displaying an elevated blood pressure, which is not altered by high or low dietary salt. However, atrial natriuretic peptide (ANP), a proposed ligand for GC-A, has been suggested as critical for the maintenance of normal blood pressure during high salt intake. In this report, we show that infusion of ANP results in substantial natriuresis and diuresis in wild-type mice but fails to cause significant changes in sodium excretion or urine output in GC-A-deficient mice. ANP, therefore, appears to signal through GC-A in the kidney. Other natriuretic/diuretic factors could be released from the heart. Therefore, acute volume expansion was used as a means to cause release of granules from the atrium of the heart. That granule release occurred was confirmed by measurements of plasma ANP concentrations, which were markedly elevated in both wild-type and GC-A-null mice. After volume expansion, urine output as well as urinary sodium and cyclic GMP excretion increased rapidly and markedly in wild-type mice, but the rapid increases were abolished in GC-A-deficient animals. These results strongly suggest that natriuretic/diuretic factors released from the heart function exclusively through GC-A.
Patterns of kinship in groups of free-living sperm whales (Physeter macrocephalus) revealed by multiple molecular genetic analyses.
Authors: Richard K R , Dillon M C , Whitehead H , Wright J M
Published: July 2002

Mature female sperm whales (Physeter macrocephalus) live in socially cohesive groups of 10-30, which include immature animals of both sexes, and within which there is communal care of the young. We examined kinship in such groups using analyses of microsatellite DNA, mitochondrial DNA sequence, and sex-linked markers on samples of sloughed skin collected noninvasively from animals in three groups off the coast of Ecuador. Social groups were defined through photographic identification of individuals. Each group contained about 26 members, mostly female (79%). Relatedness was greater within groups, as compared to between groups. Particular mitochondrial haplotypes were characteristic of groups, but all groups contained more than one haplotype. The data are generally consistent with each group being comprised of several matrillines from which males disperse at about the age of 6 years. There are indications of paternal relatedness among grouped individuals with different mitochondrial haplotypes, suggesting long-term associations between different matrilines.
Phosphorylation by protein kinase C of serine-23 of the alpha-1 subunit of rat Na+,K(+)-ATPase affects its conformational equilibrium.
Authors: Logvinenko N S , Dulubova I , Fedosova N , Larsson S H , Nairn A C , Esmann M , Greengard P , Aperia A
Published: July 2002

Phosphorylation of the alpha-1 subunit of rat Na+,K(+)-ATPase by protein kinase C has been shown previously to decrease the activity of the enzyme in vitro. We have now undertaken an investigation of the mechanism by which this inhibition occurs. Analysis of the phosphorylation of recombinant glutathione S-transferase fusion proteins containing putative cytoplasmic domains of the protein, site-directed mutagenesis, and two-dimensional peptide mapping indicated that protein kinase C phosphorylated the alpha-1 subunit of the rat Na+,K(+)-ATPase within the extreme NH2-terminal domain, on serine-23. The phosphorylation of this residue resulted in a shift in the equilibrium toward the E1 form, as measured by eosin fluorescence studies, and this was associated with a decrease in the apparent K+ affinity of the enzyme, as measured by ATPase activity assays. The rate of transition from E2 to E1 was apparently unaffected by phosphorylation by protein kinase C. These results, together with previous studies that examined the effects of tryptic digestion of Na+,K(+)-ATPase, suggest that the NH2-terminal domain of the alpha-1 subunit, including serine-23, is involved in regulating the activity of the enzyme.
From local to global in quasiconformal structures.
Authors: Heinonen J , Koskela P
Published: July 2002

We exhibit a large class of metric spaces whose infinitesimal quasiconformal structure is strong enough to capture the global quasiconformal structure. A sufficient condition for this to happen is described in terms of a Poincaré-type inequality.
The nuclear hormone receptor coactivator SRC-1 is a specific target of p300.
Authors: Yao T P , Ku G , Zhou N , Scully R , Livingston D M
Published: July 2002

p300 and its family member, CREB-binding protein (CBP), function as key transcriptional coactivators by virtue of their interaction with the activated forms of certain transcription factors. In a search for additional cellular targets of p300/CBP, a protein-protein cloning strategy, surprisingly identified SRC-1, a coactivator involved in nuclear hormone receptor transcriptional activity, as a p300/CBP interactive protein. p300 and SRC-1 interact, specifically, in vitro and they also form complexes in vivo. Moreover, we show that SRC-1 encodes a new member of the basic helix-loop-helix-PAS domain family and that it physically interacts with the retinoic acid receptor in response to hormone binding. Together, these results implicate p300 as a component of the retinoic acid signaling pathway, operating, in part, through specific interaction with a nuclear hormone receptor coactivator, SRC-1.
Identification of peptides specific for cerebrospinal fluid antibodies in multiple sclerosis by using phage libraries.
Authors: Cortese I , Tafi R , Grimaldi L M , Martino G , Nicosia A , Cortese R
Published: July 2002

The study of the origin and pathogenetic relevance of the oligoclonal antibodies present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients has been hampered by a lack of specific ligands. We recently reported a general strategy, based on phage-displayed random peptide libraries, to identify ligands for disease-specific antibodies even in the absence of any information on the nature of the pathologic antigen. With this procedure, we identified several peptides specifically recognized by antibodies present in the CSF of MS patients. Using these peptides as reagents, we demonstrated that they mimic different natural epitopes and react with antibodies enriched in the CSF of MS patients. Antibodies recognizing the selected peptides are commonly found with equal frequency in the sera of MS patients and of normal individuals. In contrast, the repertoire of CSF antibodies appears to be individual-specific and is probably the result of a nonspecific immunodysregulation rather than a stereotyped response to a single antigen/agent.
Transbilayer inhibition of protein kinase C by the lipophosphoglycan from Leishmania donovani.
Authors: Giorgione J R , Turco S J , Epand R M
Published: July 2002

Lipophosphoglycan (LPG), the predominant molecule on the surface of the parasite Leishmania donovani, has previously been shown to be a potent inhibitor of protein kinase C (PKC) isolated from rat brain. The mechanism by which LPG inhibits PKC was further investigated in this study. LPG was found to inhibit the PKC alpha-catalyzed phosphorylation of histone in assays using large unilamellar vesicles composed of 1-palmitoyl, 2-oleoyl phosphatidylserine and 1-palmitoyl, 2-oleoyl phosphatidylcholine either with or without 1% 1,2 diolein added. The results also indicated that while PKC binding to sucrose-loaded vesicles was not substantially reduced in the presence of LPG at concentrations of 1-2%, the activity of membrane-bound PKC was inhibited by 70%. This inhibition of the membrane-bound form of PKC is not a consequence of reduced substrate availability to the membrane. However, Km shifted from approximately 31 +/- 4 microM to 105 +/- 26 microM in the presence of 5% LPG. LPG caused PKC to bind to membranes without inducing a conformational change as revealed by the lack of an increased susceptibility to trypsin. An LPG fragment containing only one repeating disaccharide unit was not as effective as the entire LPG molecule or of larger fragments in inhibiting the membrane-bound form of the enzyme. The shorter fragments were also less potent in raising the bilayer to hexagonal phase transition temperature of a model membrane. LPG is also able to inhibit the membrane-bound form of PKC alpha from the inner monolayer of large unilamellar vesicles, the opposite monolayer to which the enzyme binds in our assay. Inhibition is likely a result of alterations in the physical properties of the membrane. To our knowledge, this is the first example of a membrane additive that can inhibit the membrane-bound form of PKC in the presence of other lipid cofactors.
Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.
Authors: Zini S , Fournie-Zaluski M C , Chauvel E , Roques B P , Corvol P , Llorens-Cortes C
Published: July 2002

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.
Expansion of a recurrent V beta 5.3+ T-cell population in newly diagnosed and untreated HLA-DR2 multiple sclerosis patients.
Authors: Musette P , Bequet D , Delarbre C , Gachelin G , Kourilsky P , Dormont D
Published: July 2002

We have used a PCR-based technology to study the V beta 5 and V beta 17 repertoire of T-cell populations in HLA-DR2 multiple sclerosis (MS) patients. We have found that the five MS DR2 patients studied present, at the moment of diagnosis and prior to any treatment, a marked expansion of a CD4+ T-cell population bearing V beta 5-J beta 1.4 beta chains. The sequences of the complementarity-determining region 3 of the expanded T cells are highly homologous. One shares structural features with that of the T cells infiltrating the central nervous system and of myelin basic protein-reactive T cells found in HLA-DR2 MS patients. An homologous sequence was not detectable in MS patients expressing DR alleles other than DR2. However, it is detectable but not expanded in healthy DR2 individuals. The possible mechanisms leading to its in vivo proliferation at the onset of MS are discussed.
NRF2, a member of the NFE2 family of transcription factors, is not essential for murine erythropoiesis, growth, and development
Authors: Chan Kaimin , Lu Ronghua , Chang Judy C. , Kan Yuet Wai
Published: July 2002

The locus control region of the β-globin gene is composed of four
erythroid-specific hypersensitive sites. Hypersensitive site 2 has been
shown to be a powerful enhancer and contains a tandem repeat sequence
for the transcription factors AP1 and NFE2 (activating protein 1 and
nuclear factor erythroid 2, respectively). The human NRF2 (NFE2 related
factor 2) has been isolated by bacterial expression screening using
this core sequence as a probe. p45-NFE2, NRF1, and NRF2 belong to the
CNC (“cap ‘n’ collar”) subfamily of the basic region–leucine
zipper transcription factors, which exhibits strong homology at
specific regions such as the “CNC” and the DNA binding and
leucine zipper domains. Although the erythroid-specific p45-NFE2 has
been implicated in globin gene regulation, p45-NFE2 null mice succumb
to bleedings due to lack of platelets and those that survive exhibit
only a mild anemia. To determine the function of NRF2, which we found
to be widely expressed
in vivo
, we have characterized
the genomic structure of the mouse NRF2 gene, disrupted the Nrf2 gene
by homologous recombination in mouse embryonic stem cells (ES cells),
and generated NRF2
−/−
mice. Homozygous mutant mice
developed normally, were not anemic, reached adulthood, and reproduced.
Our studies indicate that NRF2 is dispensable for mouse development.
Potential brain neuronal targets for amphetamine-, methylphenidate-, and modafinil-induced wakefulness, evidenced by c-
fos
immunocytochemistry in the cat
Authors: Lin Jiang-Sheng , Hou Yiping , Jouvet Michel
Published: July 2002

Much experimental and clinical data suggest that the
pharmacological profile of modafinil, a newly discovered waking
substance, differs from those of amphetamine and methylphenidate, two
classical psychostimulants. The brain targets on which modafinil acts
to induce wakefulness, however, remain unknown. A double-blind study
using the protooncogene c-
fos
as experimental marker in
the cat was, therefore, carried out to identify the potential target
neurons of modafinil and compare them with those for amphetamine and
methylphenidate. Cats were sacrificed after a single oral
administration of amphetamine, methylphenidate, or modafinil at
equivalent doses for wake induction (1, 2.5, or 5 mg/kg,
respectively) and brain sections examined for Fos by
immunocytochemistry. Administration of either amphetamine or
methylphenidate evoked Fos-like immunoreactivity in a large number of
neurons in the striatum and whole cortex, especially in the caudate
nucleus and mediofrontal cortex, which are known to be dopaminergic
targets. In contrast, administration of modafinil resulted in the
labeling of few cells in these structures, but did induce marked Fos
labeling in neurons of the anterior hypothalamic nucleus and adjacent
areas. These results provide evidence for the potential brain targets
of modafinil, which differ from those of amphetamine or
methylphenidate, and suggest that modafinil induces wakefulness by
mechanisms distinct from those of the two stimulants.
Ordered water molecules as key allosteric mediators in a cooperative dimeric hemoglobin
Authors: Royer William E. , Pardanani Animesh , Gibson Quentin H. , Peterson Eric S. , Friedman Joel M.
Published: July 2002

One of the most remarkable structural aspects of
Scapharca
dimeric hemoglobin is the disruption of a very
well-ordered water cluster at the subunit interface upon ligand
binding. We have explored the role of these crystallographically
observed water molecules by site-directed mutagenesis and osmotic
stress techniques. The isosteric mutation of Thr-72 → Val in the
interface increases oxygen affinity more than 40-fold with a surprising
enhancement of cooperativity. The only significant structural effect of
this mutation is to destabilize two ordered water molecules in the
deoxy interface. Wild-type
Scapharca
hemoglobin is
strongly sensitive to osmotic conditions. Upon addition of glycerol,
striking changes in Raman spectrum of the deoxy form are observed that
indicate a transition toward the liganded form. Increased osmotic
pressure, which lowers the oxygen affinity in human hemoglobin, raises
the oxygen affinity of
Scapharca
hemoglobin regardless
of whether the solute is glycerol, glucose, or sucrose. Analysis of
these results provides an estimate of six water molecules lost upon
oxygen binding to the dimer, in good agreement with eight predicted
from crystal structures. These experiments suggest that the observed
cluster of interfacial water molecules plays a crucial role in
communication between subunits.
Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.
Authors: de Vera M E , Shapiro R A , Nussler A K , Mudgett J S , Simmons R L , Morris S M , Billiar T R , Geller D A
Published: July 2002

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.
Persistent elevations of cerebrospinal fluid concentrations of corticotropin-releasing factor in adult nonhuman primates exposed to early-life stressors: implications for the pathophysiology of mood and anxiety disorders.
Authors: Coplan J D , Andrews M W , Rosenblum L A , Owens M J , Friedman S , Gorman J M , Nemeroff C B
Published: July 2002

There is increasing evidence for an important role of adverse early experience on the development of major psychiatric disorders in adulthood. Corticotropin-releasing factor (CRF), an endogenous neuropeptide, is the primary physiological regulator of the mammalian stress response. Grown nonhuman primates who were exposed as infants to adverse early rearing conditions were studied to determine if long-term alterations of CRF neuronal systems had occurred following the early stressor. In comparison to monkeys reared by mothers foraging under predictable conditions, infant monkeys raised by mothers foraging under unpredictable conditions exhibited persistently elevated cerebrospinal fluid (CSF) concentrations of CRF. Because hyperactivity of CRF-releasing neurons has been implicated in the pathophysiology of certain human affective and anxiety disorders, the present finding provides a potential neurobiological mechanism by which early-life stressors may contribute to adult psychopathology.
Expression of the human acute myeloid leukemia gene AML1 is regulated by two promoter regions.
Authors: Ghozi M C , Bernstein Y , Negreanu V , Levanon D , Groner Y
Published: July 2002

The human chromosome 21 AML1 gene is expressed predominantly in the hematopoietic system. In several leukemia-associated translocations AML1 is fused to other genes and transcription of the fused regions is mediated by upstream sequences that normally regulate the expression of AML1. The 5' genomic region of AML1 was cloned and sequenced. The two 5' untranslated regions (UTRs) previously identified in AML1 cDNAs were located in this region and the distance between them was established. The distal 5' UTR maps over 7 kb upstream of the proximal one. Using primer extension with mRNA, transcription start sites were identified at two distinct sites above these 5' uTRs. Sequence analysis revealed the absence of a TATA motif and the presence of Sp1, PU.1, Oct, CRE, Myb, Ets, and Ets-like binding sites in both upstream regions. Several initiator elements (Inr) that overlap the transcription start sites were also identified. These proximal and distal upstream regions and their deletion mutants were cloned in front of a luciferase reporter gene and used in transfection assays. We demonstrate that both upstream regions function as promoters in hematopoietic (Jurkat) and nonhematopoietic (HEK) cell lines. The activity of both promoters was orientation dependent and was enhanced, in a cell-type specific manner, by a heterologous enhancer sequence. These results indicate that additional control elements, either negative or positive, regulate the tissue-specific expression of AML1.
Logarithm of odds (lods) for linkage in complex inheritance.
Authors: Morton N E
Published: July 2002

Lod scores provide a method to unify linkage tests based on identity by descent and identity in marker state while permitting selection of the most informative individuals through their disease-related phenotypes and markers in relatives. After parametric lods are reviewed, a nonparametric approach that depends on a single logistic parameter beta is introduced. Lods for parents tested or unknown are derived, multiple pairwise mapping is presented, and power is shown to be good even for moderately small values of beta. Comparison of parametric and nonparametric approaches (yet to be made) will provide for polygenes the efficiency and reliability that lod scores gave to mapping of major loci 40 years ago.
On the controllability of distributed systems
Authors: Lions J. L.
Published: July 2002

To “control” a system is to make it behave (hopefully)
according to our “wishes,” in a way compatible with safety and
ethics, at the least possible cost. The systems considered here are
distributed—i.e., governed (modeled) by partial differential equations
(PDEs) of evolution. Our “wish” is to drive the system in a given
time, by an adequate choice of the controls, from a given initial state
to a final given state, which is the target. If this can be achieved
(respectively, if we can reach any “neighborhood” of the target)
the system, with the controls at our disposal, is exactly
(respectively, approximately) controllable. A very general (and fuzzy)
idea is that the more a system is “unstable” (chaotic, turbulent)
the “simplest,” or the “cheapest,” it is to achieve exact
or approximate controllability. When the PDEs are the Navier–Stokes
equations, it leads to conjectures, which are presented and explained.
Recent results, reported in this expository paper, essentially prove
the conjectures in two space dimensions. In three space dimensions, a
large number of new questions arise, some new results support (without
proving) the conjectures, such as generic controllability and cases of
decrease of cost of control when the instability increases. Short
comments are made on models arising in climatology, thermoelasticity,
non-Newtonian fluids, and molecular chemistry. The Introduction of the
paper and the first part of all sections are not technical. Many open
questions are mentioned in the text.
Activation of the orphan nuclear receptor steroidogenic factor 1 by oxysterols
Authors: Lala Deepak S. , Syka Peter M. , Lazarchik Steven B. , Mangelsdorf David J. , Parker Keith L. , Heyman Richard A.
Published: July 2002

Steroidogenic factor 1 (SF-1), an orphan member of the intracellular receptor superfamily, plays an essential role in the development and function of multiple endocrine organs. It is expressed in all steroidogenic tissues where it regulates the P450 steroidogenic genes to generate physiologically active steroids. Although many of the functions of SF-1
in vivo
have been defined, an unresolved question is whether a ligand modulates its transcriptional activity. Here, we show that 25-, 26-, or 27-hydroxycholesterol, known suppressors of cholesterol biosynthesis, enhance SF-1-dependent transcriptional activity. This activation is dependent upon the SF-1 activation function domain, and, is specific for SF-1 as several other receptors do not respond to these molecules. The oxysterols activate at concentrations comparable to those previously shown to inhibit cholesterol biosynthesis, and, can be derived from cholesterol by P450c27, an enzyme expressed within steroidogenic tissues. Recent studies have shown that the nuclear receptor LXR also is activated by oxysterols. We demonstrate that different oxysterols differ in their rank order potency for these two receptors, with 25-hydroxycholesterol preferentially activating SF-1 and 22(R)-hydroxycholesterol preferentially activating LXR. These results suggest that specific oxysterols may mediate transcriptional activation via different intracellular receptors. Finally, ligand-dependent transactivation of SF-1 by oxysterols may play an important role in enhancing steroidogenesis
in vivo
.
Differential ability of T cell subsets to undergo activation-induced cell death
Authors: Varadhachary Arun S. , Perdow Somia N. , Hu Chenggang , Ramanarayanan Malini , Salgame Padmini
Published: July 2002

Human T cell clones were analyzed for their susceptibility to activation-induced cell death (AICD) in response to CD3/T cell receptor ligation. AICD was observed only in Th1 clones and was Fas-mediated, whereas Th2 clones resisted AICD. Analysis of a panel of Th0 clones, characterized by their ability to secrete both Th1 and Th2 cytokines, revealed that this subset included both AICD-sensitive (type A) and -resistant (type B) clones. Resistance to AICD by Th2 and Th0-type B clones was not due to lack of expression of either Fas receptor or its ligand. Paradoxically, the AICD-resistant clones were susceptible to apoptosis when Fas receptor was directly ligated by anti-Fas antibodies. However, prior activation of the resistant clones by monoclonal antibodies to CD3/TCR complex induced resistance against Fas-mediated apoptosis. Thus, the Fas–FasL pathway is critical for the induction of AICD in T cells, and moreover this pathway can be negatively regulated in the AICD-resistant clones by signals that are generated from ligation of the CD3/TCR complex.
Thymineless death in colon carcinoma cells is mediated via Fas signaling
Authors: Houghton Janet A. , Harwood Franklin G. , Tillman David M.
Published: July 2002

Fas is expressed constitutively in colonic epithelial cells and is also expressed in colon carcinomas and in cultured colon carcinoma cell lines. However, the potential role of Fas signaling in mediating apoptosis in cells of this type remains unknown. We have developed human colon carcinoma cell models deficient in thymidylate synthase that demonstrate acute (TS−cells) or delayed (Thy4 cells) apoptosis following DNA damage induced by thymineless stress. Complete protection of cells from acute apoptosis and prolongation of delayed apoptosis was obtained following exposure to the NOK-1 monoclonal antibody (inhibitory to Fas signaling) during the period of dThd deprivation. These results suggested that apoptosis induced by thymineless stress was regulated by autocrine signaling via Fas–FasL interactions. Fas expression was high in both TS−and Thy4 cells. However, FasL, undetectable in synchronous cultures, was up-regulated in TS−cells at 48 hr, when cells were undergoing acute apoptosis, and in Thy4 cells at 96 hr, correlating with the delayed onset of thymineless death. FasL expression also correlated with acute apoptosis induced in parental GC3/cl cells, commencing at 48 hr, following thymidylate synthase inhibition by 5-fluorouracil/leucovorin exposure. Fas-mediated apoptosis induced by the cytotoxic anti-Fas monoclonal antibody CH-11 was inhibited following adenoviral delivery of a Bcl-2 cDNA, and Bcl-2 also protected cells from acute apoptosis induced by dThd deprivation. Taken together, these data demonstrate a functional Fas system in these cultured colon carcinoma cell models, and they demonstrate that Fas–FasL interactions can link DNA damage induced by thymineless stress to the apoptotic machinery of colon carcinoma cells.
DNA typing in thirty seconds with a microfabricated device
Authors: Schmalzing Dieter , Koutny Lance , Adourian Aram , Belgrader Phillip , Matsudaira Paul , Ehrlich Daniel
Published: July 2002

We report the development of a practical ultrafast allelic profiling assay for the analysis of short tandem repeats (STRs) by using a highly optimized microfluidic electrophoresis device. We have achieved baseline-resolved electrophoretic separations of single-locus STR samples in 30 sec. Analyses of PCR samples containing the four loci
CSF1PO
,
TPOX
,
THO1
, and
vWA
(abbreviated as
CTTv
) were performed in less than 2 min. This constitutes a 10- to 100-fold improvement in speed relative to capillary or slab gel systems. The separation device consists of a microfabricated channel 45 μm × 100 μm in cross section and 26 mm in length, filled with a replaceable polyacrylamide matrix operated under denaturing conditions at 50°C. A fluorescently labeled STR ladder was used as an internal standard for allele identification. Samples were prepared by standard procedures and only 4 μl was required for each analysis. The device is capable of repetitive operation and is suitable for automated high-speed and high-throughput applications.
Visualization of supercoiled DNA with atomic force microscopy
in situ
Authors: Lyubchenko Yuri L. , Shlyakhtenko Luda S.
Published: July 2002

Tertiary structure of supercoiled DNA is a significant factor in a number of genetic functions and is apparently affected by environmental conditions. We applied atomic force microscopy (AFM) for imaging the supercoiled DNA deposited at different ionic conditions. We have employed a technique for the sample preparation that permits high-resolution AFM imaging of DNA bound to the surface in buffer solutions without drying the sample (AFM
in situ
). The AFM data show that at low ionic strength, DNA molecules are loosely interwound supercoils with an irregular shape. Plectonemic superhelices are formed in high-concentration, near-physiological salt solutions. At such ionic conditions, superhelical loops are typically separated by regions of close helix–helix contacts. The data obtained show directly and unambiguously that overall geometry of supercoiled DNA depends dramatically on ionic conditions. This fact and the formation of close contacts between DNA helices are important features of supercoiled DNA related to its biological functions.