bluuebunny/crossref_metadata_2025 huggingface dataset:
Ascorbic acid stimulates gonadotropin release by autocrine action by means of NO
Authors: Karanth Sharada , Yu Wen H. , Walczewska Anna , Mastronardi Claudio A. , McCann Samuel M.
Published: July 2002

Because high concentrations of ascorbic acid (AA) are found
in the adenohypophysis, we hypothesized that it might have an acute
effect on the secretion of follicle-stimulating hormone (FSH) and
luteinizing hormone (LH) from the gland, particularly because we have
reported that AA rapidly inhibits stimulated LH-releasing hormone
(LHRH) release from medial basal hypothalamic explants. Incubation of
anterior pituitary halves from adult male rats with graded
concentrations of AA for 1 h induced highly significant release of
both FSH and LH with a minimal effective concentration of
10
−5
M. Release remained on a plateau from
10
−5
to 10
−2
M. When both AA and an effective
concentration of LHRH were incubated together, there was no additive
response to LHRH and the response was the same as to either compound
alone. The FSH and LH release in response to AA was blocked by
incubation with
N
G
-monomethyl-
l
-arginine (NMMA)
(300 μM), a competitive inhibitor of NO synthase. NMMA also inhibited
LHRH-induced LH and FSH release and gonadotropin release in the
presence of both LHRH and AA, whereas sodium nitroprusside, a releaser
of NO, stimulated LH and FSH release. Membrane depolarization caused by
incubation in high potassium (K
+
= 28 or 56 mM) medium
stimulated release of FSH, LH, and AA that was blocked by NMMA. We
hypothesize that AA is released with FSH and LH from secretory
granules. AA is transported back into gonadotropes by the AA
transporter and increases intracellular [Ca
2+
]-activating
NO synthase that evokes exocytosis of gonadotropins and AA by cGMP .
A biophysical model of bidirectional synaptic plasticity: Dependence on AMPA and NMDA receptors
Authors: Castellani Gastone C. , Quinlan Elizabeth M. , Cooper Leon N , Shouval Harel Z.
Published: July 2002

In many regions of the brain, including the mammalian cortex, the magnitude and direction of activity-dependent changes in synaptic strength depend on the frequency of presynaptic stimulation (synaptic plasticity), as well as the history of activity at those synapses (metaplasticity). We present a model of a molecular mechanism of bidirectional synaptic plasticity based on the observation that long-term synaptic potentiation (LTP) and long-term synaptic depression (LTD) correlate with the phosphorylation/dephosphorylation of sites on the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor subunit protein GluR1. The primary assumption of the model, for which there is wide experimental support, is that postsynaptic calcium concentration and consequent activation of calcium-dependent protein kinases and phosphatases are the triggers for the induction of LTP/LTD. As calcium influx through the
n
-methyl-
d
-aspartate (NMDA) receptor plays a fundamental role in the induction of LTP/LTD, changes in the properties of NMDA receptor-mediated calcium influx will dramatically affect activity-dependent synaptic plasticity (metaplasticity). We demonstrate that experimentally observed metaplasticity can be accounted for by activity-dependent regulation of NMDA receptor subunit composition and function. Our model produces a frequency-dependent LTP/LTD curve with a sliding synaptic modification threshold similar to what has been proposed theoretically by Bienenstock, Cooper, and Munro and observed experimentally.
Genetic control of manno(fructo)kinase activity in
Escherichia coli
Authors: Sproul Andrew A. , Lambourne Linda T. M. , Jean-Jacques D Jims , Kornberg Hans L.
Published: July 2002

Mutants of
Escherichia coli
unable to use fructose
by means of the phosphoenolpyruvate/glycose phosphotransferase system
mutate further to permit growth on that ketose by derepression of a
manno(fructo)kinase (Mak
+
phenotype) present in only trace
amounts in the parent organisms (Mak-o phenotype). The
mak
gene was located at min 8.8 on the
E.
coli
linkage map as an ORF designated
yajF
, of
hitherto unknown function; it specifies a deduced polypeptide of 344
aa. The derepression of Mak activity was associated with a single base
change at position 71 (codon 24) of the gene, where GCC (alanine) in
Mak-o has been changed to GAC (aspartate) in Mak
+
. By
cloning selected portions of the total 1,032-bp
mak
gene
into a plasmid that also carried a temperature-sensitive promoter, we
showed that the mutation resided in a 117-bp region that does not
specify sequences necessary for Mak activity but was located 46 bp
upstream of a 915-bp portion that does. Mak
+
and Mak-o
strains differ greatly in the heat stability of the enzyme: at 61°C,
mak-o
cloned into a
mak-o
recipient loses
50% of its activity in approximately 6 min, whereas it takes over 30
min to achieve a similar reduction in the activity of
mak
+
cloned into a
mak-o
strain. However, the Mak activity of the cloned fragment specifying the
enzyme without the regulatory region lost activity with a half-life of
29 min irrespective of whether it was derived from a
mak
+
or a
mak-o
donor, which
indicates that the A24D mutation contributes to the high enzyme
activity of Mak
+
mutants by serving to protect Mak from
denaturation.
Mutagenesis and gene identification in Dictyostelium by shotgun antisense.
Authors: Spann T P , Brock D A , Lindsey D F , Wood S A , Gomer R H
Published: July 2002

We have developed a mutagenesis technique that uses antisense cDNA to identify genes required for development in Dictyostelium discoideum. We transformed Dictyostelium cells with a cDNA library made from the mRNA of vegetative and developing cells. The cDNA was cloned in an antisense orientation immediately downstream of a vegetative promoter, so that in transformed cells the promoter will drive the synthesis of an antisense RNA transcript. We find that individual transformants typically contain one or occasionally two antisense cDNAs. Using this mutagenesis technique, we have generated mutants that fail to aggregate, aggregate but fail to form fruiting bodies, or aggregate but form abnormal fruiting bodies. The individual cDNA molecules from the mutants were identified and cloned using PCR. Initial sequence analysis of the PCR products from 35 mutants has identified six novel Dictyostelium genes, each from a transformant with one antisense cDNA. When the PCR-isolated antisense cDNAs were ligated into the antisense vector and the resulting constructs transformed into cells, the phenotypes of the transformed cells matched those of the original mutants from which each cDNA was obtained. We made homologous recombinant gene disruption transformants for three of the novel genes, in each case generating mutants with phenotypes indistinguishable from those of the original antisense transformants. Shotgun antisense thus is a rapid way to identify genes in Dictyostelium and possibly other organisms.
Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).
Authors: McFarlane M B , Gilly W F
Published: July 2002

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.
Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.
Authors: Raz E , Tighe H , Sato Y , Corr M , Dudler J A , Roman M , Swain S L , Spiegelberg H L , Carson D A
Published: July 2002

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.
Differential effects of staphylococcal toxic shock syndrome toxin-1 on B cell apoptosis.
Authors: Hofer M F , Newell K , Duke R C , Schlievert P M , Freed J H , Leung D Y
Published: July 2002

Superantigens, such as toxic shock syndrome toxin 1 (TSST-1), have been implicated in the pathogenesis of several autoimmune and allergic diseases associated with polyclonal B cell activation. In this report, we studied the in vitro effects of TSST-1 on B cell activation. We show herein that TSST-1 produced antagonistic effects on Ig synthesis by peripheral blood mononuclear cells (PBMC) from normal subjects, depending on the concentration used; Ig production was inhibited at 1000 pg/ml (P < 0.01) and enhanced at 1 and 0.01 pg/ml (P < 0.01) of toxin. Cultures of PBMC were then examined for morphologic features and DNA fragmentation characteristic for apoptosis. B cells exhibited a significantly higher (P < 0.01) incidence of apoptosis after stimulation with 1000 pg/ml of TSST-1 compared with 1 or 0.01 pg/ml of toxin or medium alone. Abundant expression of Fas, a cell surface protein that mediates apoptosis, was detected on B cells after stimulation with 1000 pg/ml of TSST-1 and was significantly higher on B cells undergoing apoptosis than on live cells (P = 0.01). Additionally, increased Fas expression and B cell death occurred at concentrations of TSST-1 inducing the production of high amounts of gamma interferon (IFN-gamma), and both events could be blocked by neutralizing anti-IFN-gamma antibody. These findings suggest that high concentrations of TSST-1 can induce IFN-gamma-dependent B cell apoptosis, whereas at low concentrations it stimulates Ig synthesis by PBMC from normal subjects. These findings support the concept that staphylococcal toxins have a role in B cell hyperactivity in autoimmunity and allergy.
DNA vaccine for hepatitis B: evidence for immunogenicity in chimpanzees and comparison with other vaccines.
Authors: Davis H L , McCluskie M J , Gerin J L , Purcell R H
Published: July 2002

Vaccination of two chimpanzees against hepatitis B virus (HBV) by intramuscular injection of plasmid DNA encoding the major and middle HBV envelope proteins induced group-, subtype- and preS2-specific antibodies. These were initially of IgM isotype, and then they were of IgG (predominantly IgGl) isotype. The chimpanzee injected with 2 mg of DNA attained >100 milli-international units/ml of anti-HBs antibody after one injection and 14,000 milli-international units/ml after four injections. A smaller dose (400 microg) induced lower and transient titers, but a strong anamnestic response occurred 1 year later. Comparison with responses in 23 chimpanzees receiving various antigen-based HBV vaccines suggests that the DNA approach is promising for prophylactic immunization against HBV.
Targeted disruption of the mouse beta1-adrenergic receptor gene: developmental and cardiovascular effects.
Authors: Rohrer D K , Desai K H , Jasper J R , Stevens M E , Regula D P , Barsh G S , Bernstein D , Kobilka B K
Published: July 2002

At least three distinct beta-adrenergic receptor (beta-AR) subtypes exist in mammals. These receptors modulate a wide variety of processes, from development and behavior, to cardiac function, metabolism, and smooth muscle tone. To understand the roles that individual beta-AR subtypes play in these processes, we have used the technique of gene targeting to create homozygous beta 1-AR null mutants (beta 1-AR -/-) in mice. The majority of beta 1-AR -/- mice die prenatally, and the penetrance of lethality shows strain dependence. Beta l-AR -/- mice that do survive to adulthood appear normal, but lack the chronotropic and inotropic responses seen in wild-type mice when beta-AR agonists such as isoproterenol are administered. Moreover, this lack of responsiveness is accompanied by markedly reduced stimulation of adenylate cyclase in cardiac membranes from beta 1-AR -/- mice. These findings occur despite persistent cardiac beta 2-AR expression, demonstrating the importance of beta 1-ARs for proper mouse development and cardiac function, while highlighting functional differences between beta-AR subtypes.
The mode of action and the structure of a herbicide in complex with its target: binding of activated hydantocidin to the feedback regulation site of adenylosuccinate synthetase.
Authors: Fonné-Pfister R , Chemla P , Ward E , Girardet M , Kreuz K E , Honzatko R B , Fromm H J , Schär H P , Grütter M G , Cowan-Jacob S W
Published: July 2002

(+)-Hydantocidin, a recently discovered natural spironucleoside with potent herbicidal activity, is shown to be a proherbicide that, after phosphorylation at the 5' position, inhibits adenylosuccinate synthetase, an enzyme involved in de novo purine synthesis. The mode of binding of hydantocidin 5'-monophosphate to the target enzyme was analyzed by determining the crystal structure of the enzyme-inhibitor complex at 2.6-A resolution. It was found that adenylosuccinate synthetase binds the phosphorylated compound in the same fashion as it does adenosine 5'-monophosphate, the natural feedback regulator of this enzyme. This work provides the first crystal structure of a herbicide-target complex reported to date.
Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.
Authors: Xu C , Zipfel W , Shear J B , Williams R M , Webb W W
Published: July 2002

Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described.
A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation.
Authors: Mocarski E S , Kemble G W , Lyle J M , Greaves R F
Published: July 2002

Human cytomegalovirus (CMV) replication begins with the expression of two regulatory proteins, IE1(491aa) and IE2(579aa), produced from differentially spliced transcripts under control of the ie1/ie2 promoter-enhancer. A deletion mutation removing all 406 IE1(491aa)-specific amino acids was engineered into the viral genome and this mutant (RC303 delta Acc) was propagated on an IE1(491aa)-expressing human fibroblast cell line (ihfie1.3). RC303 delta Acc failed to replicate on normal human fibroblasts at low multiplicities of infection (mois). At mois > 3 plaque-forming units per cell, virus replication and production of progeny were comparable to wild type. However, at mois between 0.01 and 1, mutant virus replicated slowly on normal fibroblasts, a pattern that suggested initiation of productive infection required multiple hits. Replication of RC303 delta Acc correlated with the ability to express IE2(579aa), consistent with a role for IE1(491aa) in positive autoregulation of the ie1/ie2 promoter-enhancer and with data suggesting that virion transactivators compensate for the lack of IE1(491aa) under high moi conditions. ie1-deficient CMV should be completely avirulent, suggesting its utility as a gene therapy vector for hematopoietic progenitors that are normal sites of CMV latency.
Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography.
Authors: Uzunov D P , Cooper T B , Costa E , Guidotti A
Published: July 2002

Fluoxetine administered intraperitoneally to sham-operated or adrenalectomized/castrated (ADX/CX) male rats dose-dependently (2.9-58 mumol/kg i.p.) increased the brain content of the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone, 3 alpha, 5 alpha-TH PROG). The increase of brain 3 alpha, 5 alpha-TH PROG content elicited by 58 mumol/kg fluoxetine lasted more than 2 hr and the range of its extent was comparable in sham-operated (approximately 3-10 pmol/g) and ADX/CX rats (2-9 pmol/g) and was associated with a decrease (from 2.8 to 1.1 pmol/g) in the 5 alpha-pregnan-3,20-dione (5 alpha-dihydroprogesterone, 5 alpha-DH PROG) content. The pregnenolone, progesterone, and dehydroepiandrosterone content failed to change in rats receiving fluoxetine. The extent of 3 alpha, 5 alpha-TH PROG accumulation elicited by fluoxetine treatment differed in various brain regions, with the highest increase occurring in the olfactory bulb. Importantly, fluoxetine failed to change the 3 alpha, 5 alpha-TH PROG levels in plasma, which in ADX/CX rats were at least two orders of magnitude lower than in the brain. Two other serotonin re-uptake inhibitors, paroxetine and imipramine, in doses equipotent to those of fluoxetine in inhibiting brain serotonin uptake, were either significantly less potent than fluoxetine (paroxetine) or failed to increase (imipramine) 3 alpha, 5 alpha-TH PROG brain content. The addition of 10 microM of 5 alpha-DH PROG to brain slices of ADX/CX rats preincubated with fluoxetine (10 microM, 15 min) elicited an accumulation of 3 alpha, 5 alpha-TH PROG greater than in slices preincubated with vehicle. A fluoxetine stimulation of brain 3 alpha, 5 alpha-TH PROG biosynthesis might be operative in the anxiolytic and antidysphoric actions of this drug.
Activation of the JC virus Tat-responsive transcriptional control element by association of the Tat protein of human immunodeficiency virus 1 with cellular protein Purα
Authors: Krachmarov Chavdar P. , Chepenik Lara G. , Barr-Vagell Sharon , Khalili Kamel , Johnson Edward M.
Published: July 2002

JC virus is activated to replicate in glial cells of many AIDS patients with neurological disorders. In human glial cells, the human immunodeficiency virus 1 (HIV-1) Tat protein activates the major late promoter of JC virus through a Tat-responsive DNA element, termed
upTAR
, which is a recognition site for cellular Purα, a sequence-specific single-stranded DNA binding protein implicated in cell cycle control of DNA replication and transcription. Tat interacts with two leucine-rich repeats in Purα to form a complex that can be immunoprecipitated from cell extracts. Tat enhances the ability of purified glutathione
S
-transferase-Purα (GST-Purα) to bind the
upTAR
element. Tat acts synergistically with Purα, in a cell-cycle-dependent manner, to activate transcription at an
upTAR
element placed upstream of a heterologous promoter. Since Purα is ubiquitously expressed in human cells and since
PUR
elements are located near many promoters and origins of replication, the Tat-Purα interaction may be implicated in effects of HIV-1 throughout the full range of HIV-1-infected cells.
Biosynthesis of vitamin B
12
: Concerning the identity of the two-carbon fragment eliminated during anaerobic formation of cobyrinic acid
Authors: Wang Jianji , Stolowich Neal J. , Santander Patricio J. , Park Jeong Ho , Scott A. Ian
Published: July 2002

It has been proved that, during anaerobic biosynthesis of the
corrin macrocycle, the two-carbon fragment excised from the precursor,
precorrin-3, is acetaldehyde, which originates from C-20 and its
attached methyl group. This apparently contradictory finding is
rationalized in terms of the subsequent enzymatic oxidation of
acetaldehyde to acetic acid, which was previously regarded as the
volatile fragment released by the action of the biosynthetic enzymes of
Propionibacterium shermanii
. The observation that
acetaldehyde (rather than acetic acid) is extruded during anaerobic
B
12
synthesis is in full accord with the structure of
factor IV, a new intermediate on the pathway.
Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination
Authors: Hegde Shrikanth P. , Qin Ming-hui , Li Xin-hui , Atkinson Mark A. L. , Clark Alvin J. , Rajagopalan Malini , Madiraju Murty V. V. S.
Published: July 2002

The products of the
recF, recO,
and
recR
genes are thought to interact and assist RecA in
the utilization of single-stranded DNA precomplexed with
single-stranded DNA binding protein (Ssb) during synapsis. Using
immunoprecipitation, size-exclusion chromatography, and Ssb protein
affinity chromatography in the absence of any nucleotide cofactors, we
have obtained the following results: (
i
) RecF interacts
with RecO, (
ii
) RecF interacts with RecR in the presence
of RecO to form a complex consisting of RecF, RecO, and RecR
(RecF–RecO–RecR); (
iii
) RecF interacts with Ssb
protein in the presence of RecO. These data suggested that RecO
mediates the interactions of RecF protein with RecR and with Ssb
proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in
the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded.
Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb
protein resulted in the formation of complexes consisting of RecF,
RecO, RecR, and Ssb proteins. These data suggest that one role of RecF
is to stabilize the interaction of RecR with RecO in the presence of
Ssb protein. Finally, we found that interactions of RecF with RecO are
lost in the presence of ATP. We discuss these results to explain how
the RecF–RecO–RecR complex functions as an anti-Ssb factor.
Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life.
Authors: Margulis L
Published: July 2002

A symbiosis-based phylogeny leads to a consistent, useful classification system for all life. "Kingdoms" and "Domains" are replaced by biological names for the most inclusive taxa: Prokarya (bacteria) and Eukarya (symbiosis-derived nucleated organisms). The earliest Eukarya, anaerobic mastigotes, hypothetically originated from permanent whole-cell fusion between members of Archaea (e.g., Thermoplasma-like organisms) and of Eubacteria (e.g., Spirochaeta-like organisms). Molecular biology, life-history, and fossil record evidence support the reunification of bacteria as Prokarya while subdividing Eukarya into uniquely defined subtaxa: Protoctista, Animalia, Fungi, and Plantae.
Protein binding and signaling properties of RIN1 suggest a unique effector function
Authors: Han Limin , Wong Derrick , Dhaka Ajay , Afar Daniel , White Michael , Xie Wielin , Herschman Harvey , Witte Owen , Colicelli John
Published: July 2002

Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required for efficient RAS binding. RIN1 interacts with the “effector domain” of RAS and employs some RAS determinants that are common to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1 that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence shows preferential binding to the ABL–SH3 domain
in vitro
. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition, RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1 regulation.
SOS factors involved in translesion synthesis
Authors: Napolitano Rita L. , Lambert Iain B. , Fuchs Robert P. P.
Published: July 2002

Mutations are permanent DNA sequence changes that can be induced when replication occurs on a damaged DNA template. In
Escherichia coli
, the process of translesion synthesis past a lesion that hinders replication requires the induction of SOS-controlled gene products, among which are those of the
umuDC
operon. To study translesion synthesis
in vivo
, we have constructed single-stranded vectors containing single 2-acetylaminofluorene adducts located within −1 and −2 frameshift mutation hot spots formed by short repetitive sequences. These adducts strongly hinder DNA replication as only 2–5% of the molecules give rise to progeny under non-SOS-induced conditions. Induction of the SOS response lead to a 10-fold increase in survival. Adducts present within repetitive sequences trigger the formation of misaligned primer/template replication intermediates which, upon elongation, will result in the fixation of frameshift errors (mutagenic translesion synthesis). Surprisingly we find that elongation from the nonslipped intermediate depends upon functional
umuDC
+
gene products, whereas elongation from the slipped intermediate is
umuDC
+
independent but requires another, as yet biochemically uncharacterized, SOS function. These data are discussed in terms of the different steps involved during translesion synthesis through a replication-blocking lesion.
Exon/intron structure of aldehyde dehydrogenase genes supports the “
introns-late
” theory
Authors: Rzhetsky Andrey , Ayala Francisco José , Hsu Lily C. , Chang Cheng , Yoshida Akira
Published: July 2002

Whether or not nuclear introns predate the divergence of bacteria
and eukaryotes is the central argument between the proponents of the
“introns-early” and “introns-late” theories. In this study
we compared the goodness-of-fit of each theory with a probabilistic
model of exon/intron evolution and multiple nonallelic genes encoding
human aldehyde dehydrogenases (ALDHs). Using a reconstructed
phylogenetic tree of ALDH genes, we computed the likelihood of
obtaining the present-day ALDH sequences under the assumptions of each
competing theory. Although on the grounds of its own assumptions each
theory accounted for the ALDH data significantly better than its rival,
the introns-early model required frequent intron slippage, and the
estimated slippage rates were too high to be consistent with reported
correlations between the boundaries of ancient protein modules and the
ends of ancient exons. Because the molecular mechanisms proposed to
explain intron slippage are incapable of providing such high rates and
are incompatible with the observed distribution of introns in higher
eukaryotes, the ALDH data support the introns-late theory.
Selective binding of bacterial toxins to major histocompatibility complex class II-expressing cells is controlled by invariant chain and HLA-DM
Authors: Lavoie Pascal M. , Thibodeau Jacques , Cloutier Isabelle , Busch Robert , Sékaly Rafick-P.
Published: July 2002

Bacterial superantigens (SAgs) bind to major histocompatibility complex (MHC) class II molecules and activate T cells in a Vβ-restricted fashion. We recently identified subsets of HLA-DR1 molecules that show selectivity for SAgs. Here, we extend these observations by showing that different cell lineages demonstrate distinct SAg-binding specificities although they all express HLA-DR1. Indeed, B cells bind staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin 1 (TSST-1) with high affinity while staphylococcal enterotoxin B (SEB) binding is barely detectable. In contrast, DR1-transfected HeLa cells show efficient binding of SEB, but not of SEA or TSST-1. We investigated the class II maturation events required for efficient interaction with SAgs and found that the ability of cells to bind and present the toxins can be drastically modulated by coexpression of the class II-associated invariant chain (Ii) and HLA-DM. SEA binding to DR1 molecules required coexpression of Ii, whereas TSST-1 binding was selectively enhanced by DM. Binding of SEB was affected by cell type-specific factors other than Ii or DM. The selectivity of SAgs for different MHC class II populations was minimally affected by HLA-DR intrinsic polymorphism and could not be explained by binding to alternative sites on DR molecules. Our results indicate that SAgs are sensitive to structural heterogeneity in class II molecules, which is consequent to the differential regulation of expression of antigen processing cofactors. Therefore, we speculate thatStaphylococcus aureushave retained the ability to express numerous SAgs in adaptation to the micro-heterogeneity displayed by MHC class II molecules and that this may relate to their ability to infect different tissues.
Recombinant parvovirus-like particles as an antigen carrier: A novel nonreplicative exogenous antigen to elicit protective antiviral cytotoxic T cells
Authors: Sedlik C. , Saron M.-F. , Sarraseca J. , Casal I. , Leclerc C.
Published: July 2002

To develop a strategy that promotes efficient antiviral immunity, hybrid virus-like particles (VLP) were prepared by self-assembly of the modified porcine parvovirus VP2 capsid protein carrying a CD8+T cell epitope from the lymphocytic choriomeningitis virus nucleoprotein. Immunization of mice with these hybrid pseudoparticles, without adjuvant, induced strong cytotoxic T lymphocyte (CTL) responses against both peptide-coated- or virus-infected-target cells. This CD8+class I-restricted cytotoxic activity persistedin vivofor at least 9 months. Furthermore, the hybrid parvovirus-like particles were able to induce a complete protection of mice against a lethal lymphocytic choriomeningitis virus infection. To our knowledge, this study represents the first demonstration that hybrid nonreplicative VLP carrying a single viral CTL epitope can induce protection against a viral lethal challenge, in the absence of any adjuvant. These recombinant particles containing a single type of protein are easily produced by the baculovirus expression system and, therefore, represent a promising and safe strategy to induce strong CTL responses for the elimination of virus-infected cells.
A DnaB intein in
Rhodothermus marinus
: Indication of recent intein homing across remotely related organisms
Authors: Liu Xiang-Qin , Hu Zhuma
Published: July 2002

A
dnaB
gene encoding a homologue of the
Escherichia coli
DNA helicase DnaB was cloned and sequenced in the thermophilic eubacterium
Rhodothermus marinus
, predicting a DnaB protein that harbors an intein. This DnaB intein is 428 amino acid residues long, has several putative intein sequence motifs (including two putative endonuclease motifs), and is capable of protein splicing when produced in
E. coli
cells. The
R. marinus
DnaB intein is a close homologue of a DnaB intein in the cyanobacterium
Synechocystis
sp. strain PCC6803. The two inteins are positioned identically in their respective DnaB proteins. They also share a 54% sequence identity (74% sequence similarity) that is markedly higher than the 37% sequence identity shared by the extein sequences of the two DnaB proteins. Horizontal intein transfer (homing) is therefore invoked to relate these two DnaB inteins. The codon usage of
R. marinus
DnaB intein coding sequence differs markedly from the codon usages of its flanking extein coding sequences and other genes in the same genome, suggesting more recent acquisition of the DnaB intein in this organism.
The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain
Authors: Guadaño-Ferraz Ana , Obregón Maria Jesus , Germain Donald L. St. , Bernal Juan
Published: July 2002

Thyroid hormone plays an essential role in mammalian brain maturation and function, in large part by regulating the expression of specific neuronal genes. In this tissue, the type 2 deiodinase (D2) appears to be essential for providing adequate levels of the active thyroid hormone 3,5,3′-triiodothyronine (T3) during the developmental period. We have studied the regional and cellular localization of D2 mRNA in the brain of 15-day-old neonatal rats. D2 is expressed in the cerebral cortex, olfactory bulb, hippocampus, caudate, thalamus, hypothalamus, and cerebellum and was absent from the white matter. At the cellular level, D2 is expressed predominantly, if not exclusively, in astrocytes and in the tanycytes lining the third ventricle and present in the median eminence. These results suggest a close metabolic coupling between subsets of glial cells and neurons, whereby thyroxine is taken up from the blood and/or cerebrospinal fluid by astrocytes and tanycytes, is deiodinated to T3, and then is released for utilization by neurons.
Oxidative damage during aging targets mitochondrial aconitase
Authors: Yan Liang-Jun , Levine Rodney L. , Sohal Rajindar S.
Published: July 2002

The mechanisms that cause aging are not well understood. The oxidative stress hypothesis proposes that the changes associated with aging are a consequence of random oxidative damage to biomolecules. We hypothesized that oxidation of specific proteins is critical in controlling the rate of the aging process. Utilizing an immunochemical probe for oxidatively modified proteins, we show that mitochondrial aconitase, an enzyme in the citric acid cycle, is a specific target during aging of the housefly. The oxidative damage detected immunochemically was paralleled by a loss of catalytic activity of aconitase, an enzyme activity that is critical in energy metabolism. Experimental manipulations which decrease aconitase activity should therefore cause a decrease in life-span. This expected decrease was observed when flies were exposed to hyperoxia, which oxidizes aconitase, and when they were given fluoroacetate, an inhibitor of aconitase. The identification of a specific target of oxidative damage during aging allows for the assessment of the physiological age of a specific individual and provides a method for the evaluation of treatments designed to affect the aging process.
The amino-terminal region of Tyk2 sustains the level of interferon α receptor 1, a component of the interferon α/β receptor
Authors: Gauzzi M. Cristina , Barbieri Giovanna , Richter Marc F. , Uzé Gilles , Ling Leona , Fellous Marc , Pellegrini Sandra
Published: July 2002

Tyk2 belongs to the Janus kinase (JAK) family of receptor associated tyrosine kinases, characterized by a large N-terminal region, a kinase-like domain and a tyrosine kinase domain. It was previously shown that Tyk2 contributes to interferon-α (IFN-α) signaling not only catalytically, but also as an essential intracellular component of the receptor complex, being required for high affinity binding of IFN-α. For this function the tyrosine kinase domain was found to be dispensable. Here, it is shown that mutant cells lacking Tyk2 have significantly reduced IFN-α receptor 1 (IFNAR1) protein level, whereas the mRNA level is unaltered. Expression of the N-terminal region of Tyk2 in these cells reconstituted wild-type IFNAR1 level, but did not restore the binding activity of the receptor. Studies of mutant Tyk2 forms deleted at the N terminus indicated that the integrity of the N-terminal region is required to sustain IFNAR1. These studies also showed that the N-terminal region does not directly modulate the basal autophosphorylation activity of Tyk2, but it is required for efficientin vitroIFNAR1 phosphorylation and for rendering the enzyme activatable by IFN-α. Overall, these results indicate that distinct Tyk2 domains provide different functions to the receptor complex: the N-terminal region sustains IFNAR1 level, whereas the kinase-like domain provides a function toward high affinity ligand binding.
Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants
Authors: Shao Wei , Everitt Lorraine , Manchester Marianne , Loeb Daniel D. , Hutchison Clyde A. , Swanstrom Ronald
Published: July 2002

The retroviral proteases (PRs) have a structural feature called the flap, which consists of a short antiparallel β-sheet with a turn. The flap extends over the substrate binding cleft and must be flexible to allow entry and exit of the polypeptide substrates and products. We analyzed the sequence requirements of the amino acids within the flap region (positions 46–56) of the HIV-1 PR. The phenotypes of 131 substitution mutants were determined using a bacterial expression system. Four of the mutant PRs with mutations in different regions of the flap were selected for kinetic analysis. Our phenotypic analysis, considered in the context of published structures of the HIV-1 PR with a bound substrate analogs, shows that: (
i
) Met-46 and Phe-53 participate in hydrophobic interactions on the solvent-exposed face of the flap; (
ii
) Ile-47, Ile-54, and Val-56 participate in hydrophobic interactions on the inner face of the flap; (
iii
) Ile-50 has hydrophobic interactions at the distance of both the δ and γ carbons; (
iv
) the three glycine residues in the β-turn of the flap are virtually intolerant of substitutions. Among these mutant PRs, we have identified changes in both
k
cat
and
K
m
. These results establish the nature of the side chain requirements at each position in the flap and document a role for the flap in both substrate binding and catalysis.
Phosphorylation stimulates the cooperative DNA-binding properties of the transcription factor OmpR
Authors: Huang Ke-Jung , Lan Chung-Yu , Igo Michele M.
Published: July 2002

The two-component regulatory proteins OmpR and EnvZ of
Escherichia coli
K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of
ompF
transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the
ompF
promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in
ompF
regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the
ompF
regulatory region, thereby resulting in the activation or repression of
ompF
transcription under the appropriate environmental conditions.
Preferential localization of self-stimulation sites in striosomes/patches in the rat striatum
Authors: White Norman M. , Hiroi Noboru
Published: July 2002

Histological sections of the mammalian striatum reveal a “matrix” that is histochemically distinguishable from patches, or “striosomes”. The latter are cross sections of a compartment that consists primarily of tube-shaped structures radiating through the matrix. As a test of the hypothesis that the function of the striosome/patch compartment includes the mediation of behaviors related to reward, the present study examined electrical self-stimulation of the caudoputamen in rats with electrodes in either of the two compartments. Rats acquired and maintained bar-pressing responses that were contingent on stimulation through electrodes making contact with striosomes/patches more reliably than animals with electrodes terminating exclusively in the matrix. The results provide
in vivo
evidence that the striosome/patch compartment is functionally differentiated from the matrix compartment: Stimulation centered in or around the striosome/patch compartment but not in the matrix led to rapid acquisition of a new behavior.
Does the colonic H,K-ATPase also act as an Na,K-ATPase?
Authors: Cougnon Marc , Bouyer Patrice , Planelles Gabrielle , Jaisser Frédéric
Published: July 2002

We previously have demonstrated that the colonic P-ATPase α subunit cDNA encodes an H,K-ATPase when expressed in
Xenopus laevis
oocytes. Besides its high level of amino acid homology (75%) with the Na,K-ATPase, the colonic H,K-ATPase also shares a common pharmacological profile with Na,K-ATPase, because both are ouabain-sensitive and Sch 28080-insensitive. These features raise the possibility that an unrecognized property of the colonic H,K-ATPase would be Na
+
translocation. To test this hypothesis, ion-selective microelectrodes were used to measure the intracellular Na
+
activity of
X. laevis
oocytes expressing various combinations of P-ATPase subunits. The results show that expression in oocytes of the colonic H,K-ATPase affects intracellular Na
+
homeostasis in a way similar to the expression of the
Bufo marinus
Na,K-ATPase; intracellular Na
+
activity is lower in oocytes expressing the colonic H,K-ATPase or the
B. marinus
Na,K-ATPase than in oocytes expressing the gastric H,K-ATPase or a β subunit alone. In oocytes expressing the colonic H,K-ATPase, the decrease in intracellular Na
+
activity persists when diffusive Na
+
influx is enhanced by functional expression of the amiloride-sensitive epithelial Na
+
channel, suggesting that the decrease is related to increased active Na
+
efflux. The Na
+
decrease depends on the presence of K
+
in the external medium and is inhibited by 2 mM ouabain, a concentration that inhibits the colonic H,K-ATPase. These data are consistent with the hypothesis that the colonic H,K-ATPase may transport Na
+
, acting as an (Na,H),K-ATPase. Despite its molecular and functional characterization, the physiological role of the colonic (Na,H),K-ATPase in colonic and renal ion homeostasis remains to be elucidated.
The design, computer modeling, solution structure, and biological evaluation of synthetic analogs of bryostatin 1
Authors: Wender Paul A. , DeBrabander Jef , Harran Patrick G. , Jimenez Juan-Miguel , Koehler Michael F. T. , Lippa Blaise , Park Cheol-Min , Siedenbiedel Carsten , Pettit George R.
Published: July 2002

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity
in vitro
and
in vivo
. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with
K
i
= 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of
in vitro
growth inhibitory activity (1.8–170 ng/ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.
The RIIβ regulatory subunit of protein kinase A binds to cAMP response element: An alternative cAMP signaling pathway
Authors: Srivastava Rakesh K. , Lee Youl Nam , Noguchi Kohei , Park Yun Gyu , Ellis Matthew J. C. , Jeong Jin-Sook , Kim Se Nyun , Cho-Chung Yoon S.
Published: July 2002

cAMP, through the activation of cAMP-dependent protein kinase (PKA), is involved in transcriptional regulation. In eukaryotic cells, cAMP is not considered to alter the binding affinity of CREB/ATF to cAMP-responsive element (CRE) but to induce serine phosphorylation and consequent increase in transcriptional activity. In contrast, in prokaryotic cells, cAMP enhances the DNA binding of the catabolite repressor protein to regulate the transcription of several operons. The structural similarity of the cAMP binding sites in catabolite repressor protein and regulatory subunit of PKA type II (RII) suggested the possibility of a similar role for RII in eukaryotic gene regulation. Herein we report that RIIβ subunit of PKA is a transcription factor capable of interacting physically and functionally with a CRE. In contrast to CREB/ATF, the binding of RIIβ to a CRE was enhanced by cAMP, and in addition, RIIβ exhibited transcriptional activity as a Gal4-RIIβ fusion protein. These experiments identify RIIβ as a component of an alternative pathway for regulation of CRE-directed transcription in eukaryotic cells.