bluuebunny/crossref_metadata_2025 huggingface dataset:
Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development
Authors: Lee Jaekwon , Prohaska Joseph R. , Thiele Dennis J.
Published: July 2002

The trace metal copper (Cu) plays an essential role in biology as a
cofactor for many enzymes that include Cu, Zn superoxide dismutase,
cytochrome oxidase, ceruloplasmin, lysyl oxidase, and dopamine
β-hydroxylase. Consequently, Cu transport at the cell surface and the
delivery of Cu to intracellular compartments are critical events for a
wide variety of biological processes. The components that orchestrate
intracellular Cu trafficking and their roles in Cu homeostasis have
been elucidated by the studies of model microorganisms and by the
characterizations of molecular basis of Cu-related genetic diseases,
including Menkes disease and Wilson disease. However, little is known
about the mechanisms for Cu uptake at the plasma membrane and the
consequences of defects in this process in mammals. Here, we show that
the mouse
Ctr1
gene encodes a component of the Cu
transport machinery and that mice heterozygous for
Ctr1
exhibit tissue-specific defects in copper accumulation and in the
activities of copper-dependent enzymes. Mice completely deficient for
Ctr1
exhibit profound growth and developmental defects
and die
in utero
in mid-gestation. These results
demonstrate a crucial role for Cu acquisition through the Ctr1
transporter for mammalian Cu homeostasis and embryonic development.
Insect herbivory, plant defense, and early Cenozoic climate change
Authors: Wilf Peter , Labandeira Conrad C. , Johnson Kirk R. , Coley Phyllis D. , Cutter Asher D.
Published: July 2002

Insect damage on fossil leaves from the Central Rocky Mountains,
United States, documents the response of herbivores to changing
regional climates and vegetation during the late Paleocene (humid, warm
temperate to subtropical, predominantly deciduous), early Eocene (humid
subtropical, mixed deciduous and evergreen), and middle Eocene
(seasonally dry, subtropical, mixed deciduous and thick-leaved
evergreen). During all three time periods, greater herbivory occurred
on taxa considered to have short rather than long leaf life spans,
consistent with studies in living forests that demonstrate the insect
resistance of long-lived, thick leaves. Variance in herbivory frequency
and diversity was highest during the middle Eocene, indicating the
increased representation of two distinct herbivory syndromes: one for
taxa with deciduous, palatable foliage, and the other for hosts with
evergreen, thick-textured, small leaves characterized by elevated
insect resistance. Leaf galling, which is negatively correlated with
moisture today, apparently increased during the middle Eocene, whereas
leaf mining decreased.
An isoleucine/leucine residue in the carboxyltransferase domain of acetyl-CoA carboxylase is critical for interaction with aryloxyphenoxypropionate and cyclohexanedione inhibitors
Authors: Zagnitko O. , Jelenska J. , Tevzadze G. , Haselkorn R. , Gornicki P.
Published: July 2002

cDNA fragments encoding the carboxyltransferase domain of the
multidomain plastid acetyl-CoA carboxylase (ACCase) from
herbicide-resistant maize and from herbicide-sensitive and
herbicide-resistant
Lolium rigidum
were cloned and
sequenced. A Leu residue was found in ACCases from herbicide-resistant
plants at a position occupied by Ile in all ACCases from sensitive
grasses studied so far. Leu is present at the equivalent position in
herbicide-resistant ACCases from other eukaryotes. Chimeric ACCases
containing a 1000-aa fragment of two ACCase isozymes found in a
herbicide-resistant maize were expressed in a yeast
ACC1
null mutant to test herbicide sensitivity of the enzyme
in
vivo
and
in vitr
o. One of the enzymes was
resistant/tolerant, and one was sensitive to haloxyfop and
sethoxydim, rendering the gene-replacement yeast strains resistant and
sensitive to these compounds, respectively. The sensitive enzyme has an
Ile residue, and the resistant one has a Leu residue at the putative
herbicide-binding site. Additionally, a single Ile to Leu replacement
at an equivalent position changes the wheat plastid ACCase from
sensitive to resistant. The effect of the opposite substitution, Leu to
Ile, makes
Toxoplasma gondii
apicoplast ACCase resistant
to haloxyfop and clodinafop. In this case, inhibition of the
carboxyltransferase activity of ACCase (second half-reaction) of a
large fragment of the Toxoplasma enzyme expressed in
Escherichia
coli
was tested. The critical amino acid residue is located
close to a highly conserved motif of the carboxyltransferase
domain, which is probably a part of the enzyme active site, providing
the basis for the activity of fop and dim herbicides.
A role for transcriptional repression of p21
CIP1
by c-Myc in overcoming transforming growth factor β-induced cell-cycle arrest
Authors: Claassen Gisela F. , Hann Stephen R.
Published: July 2002

c-Myc plays a vital role in cell-cycle progression. Deregulated expression of c-Myc can overcome cell-cycle arrest in order to promote cellular proliferation. Transforming growth factor β (TGFβ) treatment of immortalized human keratinocyte cells inhibits cell-cycle progression and is characterized by down-regulation of c-Myc followed by up-regulation of p21
CIP1
. A direct role of c-Myc in this pathway was demonstrated by the observation that ectopic expression of c-Myc overcame the cell-cycle block induced by TGFβ treatment. The induction of p21
CIP1
transcription by TGFβ was blocked in human keratinocyte cells stably expressing c-Myc. Furthermore, overexpression of c-Myc in NIH 3T3 cells repressed the basal levels of p21
CIP1
mRNA. Repression of p21
CIP1
transcription by c-Myc occurred at the promoter level in a region near the start site of transcriptional initiation and was independent of histone deacetylase activity. These data suggest that the down-regulation of c-Myc after TGFβ signaling is important for subsequent regulation of p21
CIP1
and cell-cycle inhibition. Thus, repression of the cell-cycle inhibitory gene p21
CIP1
plays a role in c-Myc-dependent cell-cycle progression.
A role of PDGFRα in basal cell carcinoma proliferation
Authors: Xie Jingwu , Aszterbaum Michelle , Zhang Xiaoli , Bonifas Jeannette M. , Zachary Christopher , Epstein Ervin , McCormick Frank
Published: July 2002

Activation of the hedgehog pathway, through the loss of patched (PTC) or the activation of smoothened (SMO), occurs frequently in basal cell carcinoma (BCC), the most common human cancer. However, the molecular basis of this neoplastic effect is not understood. The downstream molecule Gli1 is known to mediate the biological effect of the pathway and is itself up-regulated in all BCCs. Gli1 can drive the production of BCCs in the mouse when overexpressed in the epidermis. Here we show that Gli1 can activate platelet-derived growth factor receptor α (PDGFRα) in C3H10T½ cells. Functional up-regulation of PDGFRα by Gli1 is accompanied by activation of the ras-ERK pathway, a pathway associated with cell proliferation. The relevance of this mechanismin vivois supported by a high level expression of PDGFRα in BCCs of mice and humans. In the murine BCC cell line ASZ001, in which both copies of the PTC gene are inactivated, DNA synthesis and cell proliferation can be slowed by re-expression of PTC, which down-regulates PDGFRα expression, or by downstream inhibition of PDGFRα with neutralizing antibodies. Therefore, we conclude that increased expression of PDGFRα may be an important mechanism by which mutations in the hedgehog pathway cause BCCs.
A conserved RNA structure (
thi
box) is involved in regulation of thiamin biosynthetic gene expression in bacteria
Authors: Miranda-Ríos Juan , Navarro Margarito , Soberón Mario
Published: July 2002

The
thiCOGE
genes of
Rhizobium etli
code for enzymes involved in thiamin biosynthesis. These genes are
transcribed with a 211-base untranslated leader that contains the
thi
box, a 38-base sequence highly conserved in the 5′
regions of thiamin biosynthetic and transport genes of Gram-positive
and Gram-negative organisms. A deletion analysis of
thiC-lacZ
fusions revealed an unexpected relationship
between the degree of repression shown by the deleted derivatives and
the length of the
thiC
sequences present in the
transcript. Three regions were found to be important for regulation:
(
i
) the
thi
box sequence, which is
absolutely necessary for high-level expression of
thiC
;
(
ii
) the region immediately upstream to the translation
start codon of
thiC
, which can be folded into a
stem-loop structure that would mask the Shine-Dalgarno sequence; and
(
iii
) the proximal part of the coding region of
thiC
, which was shown to contain a putative
Rho-independent terminator. A comparative phylogenetic analysis
revealed a possible folding of the
thi
box sequence into
a hairpin structure composed of a hairpin loop, two helixes, and an
interior loop. Our results show that thiamin regulation of gene
expression involves a complex posttranscriptional mechanism and that
the
thi
box RNA structure is indispensable for
thiCOGE
expression.
Silent synapses in the developing hippocampus: Lack of functional AMPA receptors or low probability of glutamate release?
Authors: Gasparini Sonia , Saviane Chiara , Voronin Leon L. , Cherubini Enrico
Published: July 2002

At early developmental stages, silent synapses have been commonly found in different brain areas. These synapses are called silent because they do not respond at rest but are functional at positive membrane potentials. A widely accepted interpretation is that
N-
methyl-
d
-aspartate (NMDA) but not α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are functionally expressed on the subsynaptic membrane. Here we show that, in both CA3 and CA1 hippocampal regions, AMPA-mediated synaptic responses can be detected already at early stages of postnatal development. However, some synapses appear silent because of a very low probability of glutamate release. They can be converted into functional ones by factors that enhance release probability such as paired-pulse stimulation, increasing the temperature or cyclothiazide (CTZ), a drug that blocks AMPA receptor desensitization and increases transmitter release. Conversely, conducting synapses can be switched off by increasing the frequency of stimulation. Although we cannot exclude that “latent AMPA receptors” can become functional after activity-dependent processes, our results clearly indicate that, in the neonatal hippocampus, a proportion of glutamatergic synaptic connections are presynaptically rather than postsynaptically silent.
A cell-based system that recapitulates the dynamic light-dependent regulation of the vertebrate clock
Authors: Pando Matthew P. , Pinchak Anna B. , Cermakian Nicolas , Sassone-Corsi Paolo
Published: July 2002

The primary hallmark of circadian clocks is their ability to entrain to environmental stimuli. The dominant, and therefore most physiologically important, entraining stimulus comes from environmental light cycles. Here we describe the establishment and characterization of a new cell line, designated Z3, which derives from zebrafish embryos and contains an independent, light-entrainable circadian oscillator. Using this system, we show distinct and differential light-dependent gene activation for several central clock components. In particular, activation of
Per2
expression is shown to be strictly regulated and dependent on light. Furthermore, we demonstrate that
Per1
,
Per2
, and
Per3
all have distinct responses to light–dark (LD) cycles and light-pulse treatments. We also show that
Clock
,
Bmal1
, and
Bmal2
all oscillate under LD and dark–dark conditions with similar kinetics, but only
Clock
is significantly induced while initiating a light-induced circadian oscillation in Z3 cells that have never been exposed to a LD cycle. Finally, our results suggest that
Per2
is responsible for establishing the phase of a circadian rhythm entraining to an alternate LD cycle. These findings not only underscore the complexity by which central clock genes are regulated, but also establishes the Z3 cells as an invaluable system for investigating the links between light-dependent gene activation and the signaling pathways responsible for vertebrate circadian rhythms.
A genome-wide scan for linkage to human exceptional longevity identifies a locus on chromosome 4
Authors: Puca Annibale A. , Daly Mark J. , Brewster Stephanie J. , Matise Tara C. , Barrett Jeffrey , Shea-Drinkwater Maureen , Kang Sammy , Joyce Erin , Nicoli Julie , Benson Erica , Kunkel Louis M. , Perls Thomas
Published: July 2002

Substantial evidence supports the familial aggregation of exceptional longevity. The existence of rare families demonstrating clustering for this phenotype suggests that a genetic etiology may be an important component. Previous attempts at localizing loci predisposing for exceptional longevity have been limited to association studies of candidate gene polymorphisms. In this study, a genome-wide scan for such predisposing loci was conducted by using 308 individuals belonging to 137 sibships demonstrating exceptional longevity. By using nonparametric analysis, significant evidence for linkage was noted for chromosome 4 at D4S1564 with a MLS of 3.65 (
P
= 0.044). The analysis was corroborated by a parametric analysis (
P
= 0.052). These linkage results indicate the likelihood that there exists a gene, or genes, that exerts a substantial influence on the ability to achieve exceptional old age. Identification of the genes in humans that allow certain individuals to live to extreme old age should lead to insights on cellular pathways that are important to the aging process.
Telomerase reverse transcriptase promotes cardiac muscle cell proliferation, hypertrophy, and survival
Authors: Oh Hidemasa , Taffet George E. , Youker Keith A. , Entman Mark L. , Overbeek Paul A. , Michael Lloyd H. , Schneider Michael D.
Published: July 2002

Cardiac muscle regeneration after injury is limited by “irreversible” cell cycle exit. Telomere shortening is one postulated basis for replicative senescence, via down-regulation of telomerase reverse transcriptase (TERT); telomere dysfunction also is associated with greater sensitivity to apoptosis. Forced expression of TERT in cardiac muscle in mice was sufficient to rescue telomerase activity and telomere length. Initially, the ventricle was hypercellular, with increased myocyte density and DNA synthesis. By 12 wk, cell cycling subsided; instead, cell enlargement (hypertrophy) was seen, without fibrosis or impaired function. Likewise, viral delivery of TERT was sufficient for hypertrophy in cultured cardiac myocytes. The TERT virus and transgene also conferred protection from apoptosis,
in vitro
and
in vivo
. Hyperplasia, hypertrophy, and survival all required active TERT and were not seen with a catalytically inactive mutation. Thus, TERT can delay cell cycle exit in cardiac muscle, induce hypertrophy in postmitotic cells, and promote cardiac myocyte survival.
Multidimensional epistasis and the disadvantage of sex
Authors: Kondrashov Fyodor A. , Kondrashov Alexey S.
Published: July 2002

Sex is thought to facilitate accumulation of initially rare beneficial mutations by allowing simultaneous allele replacements at many loci. However, this advantage of sex depends on a restrictive assumption that the fitness of a genotype is determined by fitness potential, a single intermediate variable to which all loci contribute additively, so that new alleles can accumulate in any order. Individual-based simulations of sexual and asexual populations reveal that under generic selection, sex often retards adaptive evolution. When new alleles are beneficial only if they accumulate in a prescribed order, a sexual population may evolve two or more times slower than an asexual population because only asexual reproduction allows some overlap of successive allele replacements. Many other fitness surfaces lead to an even greater disadvantage of sex. Thus, either sex exists in spite of its impact on the rate of adaptive allele replacements, or natural fitness surfaces have rather specific properties, at least at the scale of intrapopulation genetic variability.
Minisatellite marker analysis of
Trypanosoma brucei
: Reconciliation of clonal, panmictic, and epidemic population genetic structures
Authors: MacLeod Annette , Tweedie Alison , Welburn Susan C. , Maudlin Ian , Turner C. Michael R. , Tait Andy
Published: July 2002

The African trypanosome,
Trypanosoma brucei
, has
been shown to undergo genetic exchange in the laboratory, but
controversy exists as to the role of genetic exchange in natural
populations. Much of the analysis to date has been derived from
isoenzyme or randomly amplified polymorphic DNA data with parasite
material from a range of hosts and geographical locations. These
markers fail to distinguish between the human infective (
T. b.
rhodesiense
) and nonhuman infective (
T. b.
brucei
) “subspecies” so that parasites derived from hosts
other than humans potentially contain both subspecies. To overcome some
of the inherent problems with the use of such markers and diverse
populations, we have analyzed a well-defined population from a discrete
geographical location (Busoga, Uganda) using three recently described
minisatellite markers. The parasites were primarily isolated from
humans and cattle with the latter isolates further characterized by
their ability to resist lysis by human serum (equivalent to human
infectivity). The minisatellite markers show high levels of
polymorphism, and from the data obtained we conclude that
T. b.
rhodesiense
is genetically isolated from
T. b.
brucei
and can be unambiguously identified by its multilocus
genotype. Analysis of the genotype frequencies in the separated
T. b. brucei
and
T. b. rhodesiense
populations shows the former has an epidemic population structure
whereas the latter is clonal. This finding suggests that the strong
linkage disequilibrium observed in previous analyses, where human and
nonhuman infective trypanosomes were not distinguished, results from
the treatment of two genetically isolated populations as a single
population.
Expression of cyclins E1 and E2 during mouse development and in neoplasia
Authors: Geng Yan , Yu Qunyan , Whoriskey Wendy , Dick Fred , Tsai Kenneth Y. , Ford Heide L. , Biswas Debajit K. , Pardee Arthur B. , Amati Bruno , Jacks Tyler , Richardson Andrea , Dyson Nicholas , Sicinski Piotr
Published: July 2002

Cyclin E1 (formerly called cyclin E) and the recently described cyclin E2 belong to the family of E-type cyclins that operate during the G
1
/S phase progression in mammalian cells. The two E-cyclins share a catalytic partner, cyclin-dependent kinase 2 (CDK2), and activate their associated kinase activities at similar times during cell cycle progression. Despite these similarities, it is unknown whether the two proteins perform distinct functions, or, alternatively, they control S-phase entry of different cell types in a tissue-specific fashion. To start addressing
in vivo
functions of E-cyclins, we determined the expression pattern of cyclins E1 and E2 during normal mouse development. We found that the two E-cyclins showed very similar patterns of expression; both were expressed within the proliferating compartment during embryo development. Analyses of cells and tissues lacking members of the retinoblastoma (pRB) family of proteins revealed that the expression of both cyclins is controlled in a pRB-dependent, but p107- and p130-independent fashion, likely through the pRB-dependent E2F transcription factors. We also found that cyclins E1 and E2 are expressed at high levels in mouse breast tumors driven by the
Myc
oncogene. Last, we found that cyclin E2 is overexpressed in ≈24% of analyzed human mammary carcinomas. Collectively these findings suggest that the expression of cyclins E1 and E2 is governed by similar molecular circuitry.
Chaotic flow: The physics of species coexistence
Authors: Károlyi György , Péntek Áron , Scheuring István , Tél Tamás , Toroczkai Zoltán
Published: July 2002

Hydrodynamical phenomena play a keystone role in the population
dynamics of passively advected species such as phytoplankton and
replicating macromolecules. Recent developments in the field of chaotic
advection in hydrodynamical flows encourage us to revisit the
population dynamics of species competing for the same resource in an
open aquatic system. If this aquatic environment is homogeneous and
well-mixed then classical studies predict competitive exclusion of all
but the most perfectly adapted species. In fact, this homogeneity is
very rare, and the species of the community (at least on an ecological
observation time scale) are in nonequilibrium coexistence. We argue
that a peculiar small-scale, spatial heterogeneity generated by chaotic
advection can lead to coexistence. In open flows this imperfect mixing
lets the populations accumulate along fractal filaments, where
competition is governed by an “advantage of rarity” principle.
The possibility of this generic coexistence sheds light on the
enrichment of phytoplankton and the information integration in early
macromolecule evolution.
Silent information regulator 2 family of NAD- dependent histone/protein deacetylases generates a unique product, 1-
O-
acetyl-ADP-ribose
Authors: Tanner Kirk G. , Landry Joseph , Sternglanz Rolf , Denu John M.
Published: July 2002

Conflicting reports have suggested that the silent information
regulator 2 (SIR2) protein family employs NAD
+
to
ADP-ribosylate histones [Tanny, J. C., Dowd, G. J., Huang,
J., Hilz, H. & Moazed, D. (1999)
Cell
99, 735–745;
Frye, R. A. (1999)
Biochem. Biophys. Res. Commun.
260, 273–279], deacetylate histones [Landry, J., Sutton, A., Tafrov,
S. T., Heller, R. C., Stebbins, J., Pillus, L. & Sternglanz,
R. (2000)
Proc. Natl. Acad. Sci. USA
97, 5807–5811;
Smith, J. S., Brachmann, C. B., Celic, I., Kenna, M. A.,
Muhammad, S., Starai, V. J., Avalos, J. L.,
Escalante-Semerena, J. C., Grubmeyer, C., Wolberger, C. & Boeke,
J. D. (2000)
Proc. Natl. Acad. Sci. USA
97,
6658–6663], or both [Imai, S., Armstrong, C. M., Kaeberlein, M.
& Guarente, L. (2000)
Nature (London)
403, 795–800].
Uncovering the true enzymatic function of SIR2 is critical to the basic
understanding of its cellular function. Therefore, we set out to
authenticate the reaction products and to determine the intrinsic
catalytic mechanism. We provide direct evidence that the efficient
histone/protein deacetylase reaction is tightly coupled to the
formation of a previously unidentified acetyl-ADP-ribose product
(1-
O
-acetyl-ADP ribose). One molecule of
NAD
+
and one molecule of acetyl-lysine are readily
catalyzed to one molecule of deacetylated lysine, nicotinamide, and
1-
O
-acetyl-ADP-ribose. A unique reaction mechanism
involving the attack of enzyme-bound acetate or the direct attack of
acetyl-lysine on an oxocarbenium ADP-ribose intermediate is proposed.
We suggest that the reported histone/protein ADP-ribosyltransferase
activity is a low-efficiency side reaction that can be explained
through the partial uncoupling of the intrinsic deacetylation and
acetate transfer to ADP-ribose.
The temperature-signaling cascade in sponges involves a heat-gated cation channel, abscisic acid, and cyclic ADP-ribose
Authors: Zocchi Elena , Carpaneto Armando , Cerrano Carlo , Bavestrello Giorgio , Giovine Marco , Bruzzone Santina , Guida Lucrezia , Franco Luisa , Usai Cesare
Published: July 2002

Sponges (phylum Porifera) are the phylogenetically oldest
metazoan animals, their evolution dating back to 600 million years ago.
Here we demonstrate that sponges express ADP-ribosyl cyclase activity,
which converts NAD
+
into cyclic ADP-ribose, a potent and
universal intracellular Ca
2+
mobilizer. In
Axinella polypoides
(Demospongiae, Axinellidae),
ADP-ribosyl cyclase was activated by temperature increases by means of
an abscisic acid-induced, protein kinase A-dependent mechanism. The
thermosensor triggering this signaling cascade was a heat-activated
cation channel. Elucidation of the complete thermosensing pathway in
sponges highlights a number of features conserved in higher organisms:
(
i
) the cation channel thermoreceptor, sensitive to
heat, mechanical stress, phosphorylation, and anesthetics, shares all
of the functional characteristics of the mammalian heat-activated
background K
+
channel responsible for central and
peripheral thermosensing; (
ii
) involvement of the
phytohormone abscisic acid and cyclic ADP-ribose as its second
messenger is reminiscent of the drought stress signaling pathway in
plants. These results suggest an ancient evolutionary origin of this
stress-signaling cascade in a common precursor of modern Metazoa and
Metaphyta.
The contrasting roles of ICE family proteases and interleukin-1beta in apoptosis induced by trophic factor withdrawal and by copper/zinc superoxide dismutase down-regulation.
Authors: Troy C M , Stefanis L , Prochiantz A , Greene L A , Shelanski M L
Published: July 2002

We compare here the mechanisms of apoptotic death of PC12 cells induced by down-regulation of Cu2+,Zn2+ superoxide dismutase (SOD1) and withdrawal of trophic support (serum/nerve growth factor). Our previous results indicated that the initiating causes of death are different in each paradigm. However, bcl-2 rescues cells in either paradigm, suggesting common downstream elements to the cell death pathway. To determine whether the ICE [interleukin 1beta converting enzyme] family of proteases, which is required for apoptosis on trophic factor withdrawal, is also required for apoptosis induced by oxidative stress, we have developed a novel peptide inhibitor that mimics the common catalytic site of these enzymes and thereby blocks their access to substrates. This differs from the more usual pseudosubstrate approach to enzyme inhibition. Blockade of ICE family proteases by either this inhibitor or by a permeant competitive ICE family antagonist rescues PC12 cells from apoptotic death following apoptosis induced by down-regulation of SOD1, as well as from trophic factor/nerve growth factor deprivation. SOD1 down-regulation results in an increase in interleukin 1beta (IL- 1beta) production by the cells, and cell death under these conditions can be prevented by either blocking antibodies against IL-1beta or the IL-1 receptor antagonist (IL-1Ralpha). In contrast, trophic factor withdrawal does not increase IL-1beta secretion, and the blocking antibody failed to protect PC12 cells from trophic factor withdrawal, whereas the receptor antagonist was only partially protective at very high concentrations. There were substantial differences in the concentrations of pseudosubstrate inhibitors which rescued cells from SOD1 down-regulation and trophic factor deprivation. These results suggest the involvement of different members of the ICE family, different substrates, or both in the two different initiating causes of cell death.
A point mutation in the HIV-1 Tat responsive element is associated with postintegration latency.
Authors: Emiliani S , Van Lint C , Fischle W , Paras P , Ott M , Brady J , Verdin E
Published: July 2002

Study of the mechanism of HIV-1 postintegration latency in the ACH2 cell line demonstrates that these cells failed to increase HIV-1 production following treatment with exogenous Tat. Reasoning that the defect in ACH2 cells involves the Tat response, we analyzed the sequence of tat cDNA and Tat responsive element (TAR) from the virus integrated in ACH2. Tat cDNA sequence is closely related to that of HIV LAI, and the encoded protein is fully functional in terms of long terminal repeat (LTR) transactivation. Cloning of a region corresponding to the 5'-LTR from ACH2, however, identified a point mutation (C37 -> T) in TAR. This mutation impaired Tat responsiveness of the LTR in transient transfection assays, and the measured defect was complemented in cells that had been treated with tetradecanoyl phorbol acetate or tumor necrosis factor type alpha (TNF-alpha). A compensatory mutation in TAR (G28 -> A), designed to reestablish base pairing in the TAR hairpin, restored wild-type Tat responsiveness. When the (C37 -> T) mutation was introduced in an infectious clone of HIV-1, no viral production was measured in the absence of TNF-alpha, whereas full complementation was observed when the infection was conducted in the presence of TNF-alpha or when a compensatory mutation (G28 -> A) was introduced into TAR. These experiments identify a novel mutation associated with HIV-1 latency and suggest that alterations in the Tat-TAR axis can be a crucial determinant of the latent phenotype in infected individuals.
In vitro motility from recombinant dynein heavy chain.
Authors: Mazumdar M , Mikami A , Gee M A , Vallee R B
Published: July 2002

The dyneins are a class of motor protein involved in ciliary and flagellar motility, organelle transport, and chromosome segregation. Because of their large size and subunit complexity, relatively little is known about their mechanisms of force production and regulation. We report here on the expression and analysis of the entire rat cytoplasmic dynein heavy chain (Mr 532,000). Full-length cDNAs were constructed from a series of partial clones and tagged at the C terminus with either a FLAG-epitope tag or a His6-tag. The recombinant polypeptides were expressed either in insect cells by baculovirus infection or in COS-7 cells by transient transfection. The recombinant protein was mostly soluble and showed good microtubule binding. It exhibited a broad sedimentation profile, indicative of the formation of dimers as well as higher order multimers. Good microtubule gliding motility activity was observed in assays of heavy chain expressed in either insect or COS-7 cells. Average microtubule gliding velocities of 1.2-1.8 microm/sec were observed, comparable with the rates determined for calf brain cytoplasmic dynein. These results represent the first indication that recombinant heavy chain alone is capable of force production, and should lead to rapid progress in defining the dynein motor domain.
Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.
Authors: Xia Y , Dawson V L , Dawson T M , Snyder S H , Zweier J L
Published: July 2002

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.
Structure of the ripple phase in lecithin bilayers.
Authors: Sun W J , Tristram-Nagle S , Suter R M , Nagle J F
Published: July 2002

The phases of the x-ray form factors are derived for the ripple (Pbeta') thermodynamic phase in the lecithin bilayer system. By combining these phases with experimental intensity data, the electron density map of the ripple phase of dimyristoyl-phosphatidylcholine is constructed. The phases are derived by fitting the intensity data to two-dimensional electron density models, which are created by convolving an asymmetric triangular ripple profile with a transbilayer electron density profile. The robustness of the model method is indicated by the result that many different models of the transbilayer profile yield essentially the same phases, except for the weaker, purely ripple (0,k) peaks. Even with this residual ambiguity, the ripple profile is well determined, resulting in 19 angstroms for the ripple amplitude and 10 degrees and 26 degrees for the slopes of the major and the minor sides, respectively. Estimates for the bilayer head-head spacings show that the major side of the ripple is consistent with gel-like structure, and the minor side appears to be thinner with lower electron density.
Agrobacterium tumefaciens VirB7 and VirB9 form a disulfide-linked protein complex.
Authors: Anderson L B , Hertzel A V , Das A
Published: July 2002

Agrobacterium tumefaciens VirB proteins are essential for gene transfer from bacteria to plants. These proteins are postulated to form a transport pore to allow transfer of the T-strand DNA intermediate. To study the function of the VirB proteins in DNA transfer, we developed an expression system in A. tumefaciens. Analysis of one VirB protein, VirB9, by Western blot assays showed that under nonreducing conditions VirB9, when expressed alone, migrates as a approximately 31-kDa band but that it migrates as a approximately 36-kDa band when expressed with all other VirB proteins. The 36-kDa band is converted to the 31-kDa band by the reducing agent 2-mercaptoethanol. Using strains that contain a deletion in a defined virB gene and strains that express specific VirB proteins, we demonstrate that the 36-kDa band is composed of VirB9 and VirB7 that are linked to each other by a disulfide bond. Mutational studies demonstrate that cysteine residues at positions 24 of VirB7 and 262 of VirB9 participate in the formation of this complex.
Epstein-Barr viral latency is disrupted by the immediate-early BRLF1 protein through a cell-specific mechanism.
Authors: Zalani S , Holley-Guthrie E , Kenney S
Published: July 2002

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, is a human herpesvirus associated with epithelial cell malignancies (nasopharyngeal carcinoma) as well as B-cell malignancies. Understanding how viral latency is disrupted is a central issue in herpesvirus biology. Epithelial cells are the major site of lytic EBV replication within the human host, and viral reactivation occurs in EBV-associated nasopharyngeal carcinomas. It is known that expression of a single viral immediate-early protein, BZLF1, is sufficient to initiate the switch from latent to lytic infection in B cells. Cellular regulation of BZLF1 transcription is therefore thought to play a key role in regulating the stringency of viral latency. Here we show that, unexpectedly, expression of another viral immediate-early protein, BRLF1, can disrupt viral latency in an epithelial cell-specific fashion. Therefore, the mechanisms leading to disruption of EBV latency appear to be cell-type specific.
The conserved role of Krox-20 in directing Hox gene expression during vertebrate hindbrain segmentation.
Authors: Nonchev S , Maconochie M , Vesque C , Aparicio S , Ariza-McNaughton L , Manzanares M , Maruthainar K , Kuroiwa A , Brenner S , Charnay P , Krumlauf R
Published: July 2002

Transient segmentation in the hindbrain is a fundamental morphogenetic phenomenon in the vertebrate embryo, and the restricted expression of subsets of Hox genes in the developing rhombomeric units and their derivatives is linked with regional specification. Here we show that patterning of the vertebrate hindbrain involves the direct upregulation of the chicken and pufferfish group 2 paralogous genes, Hoxb-2 and Hoxa-2, in rhombomeres 3 and 5 (r3 and r5) by the zinc finger gene Krox-20. We identified evolutionarily conserved r3/r5 enhancers that contain high affinity Krox-20. binding sites capable of mediating transactivation by Krox-20. In addition to conservation of binding sites critical for Krox-20 activity in the chicken Hoxa-2 and pufferfish Hoxb-2 genes, the r3/r5 enhancers are also characterized by the presence of a number of identical motifs likely to be involved in cooperative interactions with Krox-20 during the process of hindbrain patterning in vertebrates.
Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.
Authors: Schmidtmayerova H , Nottet H S , Nuovo G , Raabe T , Flanagan C R , Dubrovsky L , Gendelman H E , Cerami A , Bukrinsky M , Sherry B
Published: July 2002

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.
Cell cycling and patterned cell proliferation in the Drosophila wing during metamorphosis.
Authors: Milán M , Campuzano S , García-Bellido A
Published: July 2002

In metamorphosing wing discs, progression through the cell cycle takes place, as in larval discs, in nonclonally derived clusters of cells synchronized in the same cell cycle stage. Contrary to early discs, there are temporal and spatial heterogeneities in cell proliferation associated with wing margin, vein, intervein, and middle intervein territories. Within these territories, there are no indications of a wave progression of the cell cycle. Mitotic orientations are, as in early discs, at random but there is a preferential allocation of postmitotic cells along the proximodistal axis, thus explaining the elongated shape of the resulting clones along this axis. Shapes of clones in mature discs and in evaginated wings are similar, thus excluding major morphogenetic movements during evagination. After the proliferative period, all the cells are arrested in G1 phase. The final number of cells of the wing is fixed independently of experimental perturbations that alter the cell division schedule. These results are discussed in the context of a model of wing morphogenesis.
A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.
Authors: Shani N , Valle D
Published: July 2002

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.
A three-hybrid system for detecting small ligand–protein receptor interactions
Authors: Licitra Edward J. , Liu Jun O.
Published: July 2002

Small ligand–receptor interactions underlie many fundamental
processes in biology and form the basis for pharmacological
intervention of human diseases in medicine. We report herein a genetic
system, named the yeast three-hybrid system, for detecting
ligand–receptor interactions
in vivo
. This system is
adapted from the yeast two-hybrid system with which a third synthetic
hybrid ligand is combined. The feasibility of this system was
demonstrated using as the hybrid ligand a heterodimer of covalently
linked dexamethasone and FK506. Yeast expressing fusion proteins of the
hormone binding domain of the rat glucocorticoid receptor fused to the
LexA DNA-binding domain and of FKBP12 fused to a transcriptional
activation domain activated reporter genes when plated on medium
containing the dexamethasone–FK506 heterodimer. The reporter gene
activation is completely abrogated in a competitive manner by the
presence of excess FK506. Using this system, we screened a Jurkat cDNA
library fused to the transcriptional activation domain in yeast
expressing the hormone binding domain of rat glucocorticoid
receptor–LexA DNA binding domain fusion protein in the presence of
dexamethasone–FK506 heterodimer. We isolated overlapping clones of
human FKBP12. These results demonstrate that the three-hybrid system
can be used to discover receptors for small ligands and to screen for
new ligands to known receptors.
Melanotropic peptide-conjugated beads for microscopic visualization and characterization of melanoma melanotropin receptors
Authors: Sharma Shubh D. , Jiang Jinwen , Hadley Mac E. , Bentley David L. , Hruby Victor J.
Published: July 2002

We developed two solid-phase reagent systems for microscopic
visualization and characterization of melanocyte-stimulating hormone
(MSH) receptors of melanoma cells. Multiple copies of
[Nle-4,
d
-Phe-7]-α-MSH, a potent analog of α-MSH, were
conjugated to microspheres (latex beads) or macrospheres (polyamide
beads) through a thioether or disulfide bond. Binding between the beads
and mouse and human melanoma cells was examined by scanning electron
microscopy and by light microscopy. Each mouse and human melanoma cell
(of all cell lines) evinced binding to the beads. Binding of the
melanotropin conjugates was not restricted to any one phase of the cell
cycle. Specificity of binding was demonstrated by several studies.
Negative controls included cell types of nonmelanocyte origin (e.g.,
mammary cancer cells) and beads that lacked the melanotropic ligand or
had other attached ligands. Beads with a disulfide-linked melanotropin
analog served as a direct control. Treatment of these beads with DTT
during or before incubation of the beads with melanoma cells (resulting
in release of the MSH analog from the beads) eliminated binding of the
beads to melanoma cells. Binding interactions between melanoma cells
and melanotropin-bound beads also could be abolished by prior
incubation with unconjugated MSH analog. During these experiments,
certain membrane receptor-hormone associated phenomena, such as capping
(aggregation) of the receptor-ligand complex, also were observed. These
results provide visual evidence that MSH receptors are a property
common to melanoma cells. Normal human epidermal melanocytes and
keratinocytes were also shown to express melanotropin receptors by the
same criteria established for melanoma cells.
Steroidogenic acute regulatory protein (StAR) retains activity in the absence of its mitochondrial import sequence: Implications for the mechanism of StAR action
Authors: Arakane Futoshi , Sugawara Teruo , Nishino Hideaki , Liu Zhiming , Holt John A. , Pain Debkumar , Stocco Douglas M. , Miller Walter L. , Strauss Jerome F.
Published: July 2002

Steroidogenic acute regulatory protein (StAR) plays a critical role
in steroid hormone biosynthesis, presumably by facilitating the
delivery of cholesterol to P450scc in the inner mitochondrial
membranes. StAR is synthesized as a 37-kDa preprotein that is processed
to a 30-kDa mature form by cleavage of an N-terminal mitochondrial
import sequence. To identify structural features required for StAR
biological activity, we mutated the human StAR cDNA, including the
deletion of N- and C-terminal sequences, and examined the ability of
the mutants to promote steroidogenesis and enter the mitochondria of
transfected COS-1 cells. Deletion of up to 62 residues from the N
terminus (N-62) did not significantly affect steroidogenesis-enhancing
activity. The N-terminal deletion mutants were associated with
mitochondria-enriched fractions, but import and processing were
progressively impaired with increasing length of the deletion.
Immunogold electron microscopy and
in vitro
import
assays showed that the active N-62 mutant was not imported into the
mitochondria. Removal of the 28 C-terminal amino acids (C-28)
inactivated StAR. Deletion of the C-terminal 10 amino acids (C-10)
reduced steroidogenic activity by 53%, while truncation of the last 4
amino acids had no effect. The C-28 mutant StAR was not efficiently
imported into mitochondria or processed, whereas some of the C-10
mutant was processed, indicating that import had occurred. We conclude
that in the COS-1 cell system used, StAR does not need to enter into
mitochondria to stimulate steroidogenesis and that residues in the C
terminus are essential for steroidogenesis-enhancing activity. These
findings imply that StAR acts via C-terminal domains on the outside of
the mitochondria.
Identification and tissue-specific expression of PDE7 phosphodiesterase splice variants
Authors: Bloom Timothy J. , Beavo Joseph A.
Published: July 2002

Type 7 cyclic nucleotide phosphodiesterases (PDE7s) are a newly
described family of enzymes having high affinity and specificity for
cAMP. However, little is known about their structure, function, or
regulation. We have isolated a mouse skeletal muscle cDNA representing
a new alternative splice variant (PDE7A2) of the PDE7 gene. The ORF
encodes a 456-amino acid protein having a predicted molecular weight of
52.4 kDa. The 5′ end of the mouse PDE7A2 is divergent from the 5′ end
of the human PDE7A1 sequence and is more hydrophobic. A comparison of
the 5′ ends of the two cDNA clones with human genomic sequence
indicates that they represent alternate splice products rather than
species variation. RNase protection analysis of several mouse tissues
indicates that PDE7 is expressed widely with highest levels in skeletal
muscle. HPLC fractionation and Western blot analysis of two human
lymphocyte T-cell lines shows that an unknown PDE activity described by
Ichimura and Kase [Ichimura, M. & Kase, H. (1993)
Biochem.
Biophys. Res. Commun.
193, 985–990] is most likely to be
PDE7A1. A single immunoreactive band of ≈55 kDa, which comigrates
with PDE7A1, is seen in fractions of the HPLC profile containing this
activity suggesting that the original human PDE7A1 clone contains a
full-length ORF, and is not truncated at the 5′ end as was originally
postulated. In a human lymphocyte B-cell line and also in mouse
skeletal muscle, a large amount of PDE7 mRNA but little PDE7 protein or
activity is expressed suggesting that the translation or stability of
PDE7 protein may be highly regulated in these tissues.