bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in the polar clustering of the high- and low-abundance chemoreceptors of
Escherichia coli
Authors: Lybarger S. R. , Maddock J. R.
Published: July 2002

The chemosensory complexes in
Escherichia coli
are localized predominantly in large aggregates at one or both of the cell poles, however, neither the role of the polar localization nor the role of the clustering is understood. In
E. coli
, the two classes of chemoreceptors or transducers, high- and low-abundance, differ in their ability to support chemotaxis when expressed as the sole chemoreceptor type in the cell. In this study, we examined both the contribution of individual chemoreceptors to polar clustering and the ability of each chemoreceptor type to cluster in the absence of all others. We found that polar clustering of methyl-accepting chemotaxis proteins (MCPs) is not dependent on any one chemoreceptor type. Remarkably, when expressed individually at similar levels, the chemoreceptors display differential clustering abilities. The high-abundance transducers cluster at the cell pole almost as well as do the MCPs in cells expressing all four species, whereas the low-abundance transducers, although polar, are not particularly clustered. CheA and CheW distributions in strains expressing only one chemoreceptor type coincide with MCP localization, indicating that the low-abundance chemoreceptors are competent for ternary complex formation but are defective in aggregation. These studies reveal that, in contrast to our previous model, polarity of the chemoreceptors is independent of clustering, suggesting that the polar localization of the chemoreceptors is not simply caused by diffusion limitations on large protein aggregates.
A peptide derived from the non-receptor-binding region of urokinase plasminogen activator inhibits glioblastoma growth and angiogenesis
in vivo
in combination with cisplatin
Authors: Mishima Kazuhiko , Mazar Andrew P. , Gown Allen , Skelly Marilyn , Ji Xiang-Dong , Wang Xu-Dong , Jones Terence R. , Cavenee Webster K. , Huang H.-J. Su
Published: July 2002

The urokinase plasminogen activator system is involved in angiogenesis and tumor growth of malignant gliomas, which are highly neovascularized and so may be amenable to antiangiogenic therapy. In this paper, we describe the activity of Å6, an octamer capped peptide derived from the non-receptor-binding region of urokinase plasminogen activator. Å6 inhibited human microvascular endothelial cell migration but had no effect on the proliferation of human microvascular endothelial cells or U87MG glioma cells
in vitro
. In contrast, Å6 or cisplatin (CDDP) alone suppressed subcutaneous tumor growth
in vivo
by 48% and 53%, respectively, and, more strikingly, the combination of Å6 plus CDDP inhibited tumor growth by 92%. Such combination treatment also greatly reduced the volume of intracranial tumor xenografts and increased survival of tumor-bearing animals when compared with CDDP or Å6 alone. Tumors from the combination treatment group had significantly reduced neovascularization, suggesting a mechanism involving Å6-mediated inhibition of endothelial cell motility, thereby eliciting vascular sensitivity to CDDP-mediated toxicity. These data suggest that the combination of an angiogenesis inhibitor that targets endothelial cells with a cytotoxic agent may be a useful therapeutic approach.
Gel catalysts that switch on and off
Authors: Wang Guoqiang , Kuroda Kenichi , Enoki Takashi , Grosberg Alexander , Masamune Satoru , Oya Taro , Takeoka Yukikazu , Tanaka Toyoichi
Published: July 2002

We report development of a polymer gel with a catalytic activity that can be switched on and off when the solvent composition is changed. The gel consists of two species of monomers. The major component,
N
-isopropylacrylamide, makes the gel swell and shrink in response to a change in composition of ethanol/water mixtures. The minor component, vinylimidazole, which is capable of catalysis, is copolymerized into the gel network. The reaction rate for catalytic hydrolysis of
p
-nitrophenyl caprylate was small when the gel was swollen. In contrast, when the gel was shrunken, the reaction rate increased 5 times. The activity changes discontinuously as a function of solvent composition, thus the catalysis can be switched on and off by an infinitesimal change in solvent composition. The kinetics of catalysis by the gel in the shrunken state is well described by the Michaelis–Menten formula, indicating that the absorption of the substrate by the hydrophobic environment created by the
N
-isopropylacrylamide polymer in the shrunken gel is responsible for enhancement of catalytic activity. In the swollen state, the rate vs. active site concentration is linear, indicating that the substrate absorption is not a primary factor determining the kinetics. Catalytic activity of the gel is studied for substrates with various alkyl chain lengths; of those studied the switching effect is most pronounced for
p
-nitrophenyl caprylate.
Flavohemoglobin denitrosylase catalyzes the reaction of a nitroxyl equivalent with molecular oxygen
Authors: Hausladen Alfred , Gow Andrew , Stamler Jonathan S.
Published: July 2002

We have previously reported that bacterial flavohemoglobin (HMP) catalyzes both a rapid reaction of heme-bound O
2
with nitric oxide (NO) to form nitrate [HMP-Fe(II)O
2
+ NO → HMP-Fe(III) + NO

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] and, under anaerobic conditions, a slower reduction of heme-bound NO to an NO
−
equivalent (followed by the formation of N
2
O), thereby protecting against nitrosative stress under both aerobic and anaerobic conditions, and rationalizing our finding that NO is rapidly consumed across a wide range of O
2
concentrations. It has been alternatively suggested that HMP activity is inhibited at low pO
2
because the enzyme is then in the relatively inactive nitrosyl form [
k
off
/
k
on
for NO (0.000008 μM) ≪
k
off
/
k
on
for O
2
(0.012 μM) and
K
M
for O
2
= 30–100 μM]. To resolve this discrepancy, we have directly measured heme-ligand turnover and NADH consumption under various O
2
/NO concentrations. We find that, at biologically relevant O
2
concentrations, HMP preferentially binds NO (not O
2
), which it then reacts with oxygen to form nitrate (in essence NO
−
+ O
2
→ NO

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). During steady-state turnover, the enzyme can be found in the ferric (FeIII) state. The formation of a heme-bound nitroxyl equivalent and its subsequent oxidation is a novel enzymatic function, and one that dominates the oxygenase activity under biologically relevant conditions. These data unify the mechanism of HMP/NO interaction with those recently described for the nematode Ascaris and mammalian hemoglobins, and more generally suggest that the peroxidase (FeIII)-like properties of globins have evolved for handling of NO.
Avicins, a family of triterpenoid saponins from
Acacia victoriae
(Bentham), suppress H-
ras
mutations and aneuploidy in a murine skin carcinogenesis model
Authors: Hanausek Malgorzata , Ganesh Pattabhiraman , Walaszek Zbigniew , Arntzen Charles J. , Slaga Thomas J. , Gutterman Jordan U.
Published: July 2002

We tested the ability of avicins, a family of triterpenoid saponins
obtained from
Acacia victoriae
(Bentham) (Leguminosae:
Mimosoideae), to inhibit chemically induced mouse skin carcinogenesis.
Varying doses of avicins were applied to shaved dorsal skin of SENCAR
mice 15 min before application of 100 nmol of
7,12-dimethylbenz[
a
]anthracene (DMBA) twice a week for
4 weeks (complete carcinogenesis model). The dorsal skin of a second
group of mice was treated with one dose of 10 nmol of DMBA. Avicins
were then applied 15 min before repetitive doses of 2 μg of phorbol
12-tetradecanoate 13-acetate (TPA) twice a week for 8 weeks
(initiation/promotion model). At 12 weeks, avicins produced a 70%
decrease in the number of mice with papillomas and a greater than 90%
reduction in the number of papillomas per mouse in both protocols. We
also observed a 62% and 74% reduction by avicins in
H-
ras
mutations at codon 61 in the DMBA and DMBA/TPA
models, respectively, as well as a significant inhibition of the
modified DNA base formation (8-OH-dG) in both protocols. Marked
suppression of aneuploidy occurred with treatment at 16 weeks in the
initiation/promotion experiment. These findings, when combined with
the proapoptotic property of these compounds and their ability
to inhibit hydrogen peroxide (H
2
O
2
) generation,
nuclear factor-κB (NF-κB) activation, and inducible nitric oxide
synthase (iNOS) induction reported elsewhere, suggest that avicins
could prove exciting in reducing oxidative and nitrosative stress and
thereby suppressing the development of human skin cancer and other
epithelial malignancies.
Caterpillar talk: Acoustically mediated territoriality in larval Lepidoptera
Authors: Yack J. E. , Smith M. L. , Weatherhead P. J.
Published: July 2002

We provide evidence for conspecific acoustic communication in
caterpillars. Larvae of the common hook-tip moth,
Drepana
arcuata
(Drepanoidea), defend silk nest sites from conspecifics
by using ritualized acoustic displays. Sounds are produced by drumming
the mandibles and scraping the mandibles and specialized anal
“oars” against the leaf surface. Staged interactions between a
resident and intruder resulted in escalated acoustic “duels” that
were typically resolved within minutes, but sometimes extended for
several hours. Resident caterpillars generally won territorial
disputes, regardless of whether they had built the nest, but relatively
large intruders occasionally displaced residents from their nests. All
evidence is consistent with acoustic signaling serving a territorial
function. As with many vertebrates, ritualized signaling appears to
allow contestants to resolve contests without physical harm.
Comparative evidence indicates that larval acoustic signaling may be
widespread throughout the Lepidoptera, meriting consideration as a
principal mode of communication for this important group of insects.
Remarkable species diversity in Malagasy mouse lemurs (primates,
Microcebus
)
Authors: Yoder Anne D. , Rasoloarison Rodin M. , Goodman Steven M. , Irwin Jodi A. , Atsalis Sylvia , Ravosa Matthew J. , Ganzhorn Jörg U.
Published: July 2002

Phylogenetic analysis of mtDNA sequence data confirms the
observation that species diversity in the world's smallest living
primate (genus
Microcebus
) has been greatly
underestimated. The description of three species new to science, and
the resurrection of two others from synonymy, has been justified on
morphological grounds and is supported by evidence of reproductive
isolation in sympatry. This taxonomic revision doubles the number of
recognized mouse lemur species. The molecular data and phylogenetic
analyses presented here verify the revision and add a historical
framework for understanding mouse lemur species diversity. Phylogenetic
analysis revises established hypotheses of ecogeographic constraint for
the maintenance of species boundaries in these endemic Malagasy
primates. Mouse lemur clades also show conspicuous patterns of regional
endemism, thereby emphasizing the threat of local deforestation to
Madagascar's unique biodiversity.
The state diagram for cell adhesion under flow: Leukocyte rolling and firm adhesion
Authors: Chang Kai-Chien , Tees David F. J. , Hammer Daniel A.
Published: July 2002

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). Using a computational method called “Adhesive Dynamics,” we have simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor–ligand functional properties and the dynamics of adhesion. We express this relationship in a state diagram, a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors. Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. We varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate,kro, and the bond interaction length, γ, are the most important molecular properties controlling the dynamics of adhesion. Experimentalkroand γ values from the literature for molecules that are known to mediate rolling adhesion fall within the rolling region of the state diagram. We explain why L-selectin-mediated rolling, which has fasterkrothan other selectins, is accompanied by a smaller value for γ. We also show how changes in association rate, shear rate, and bond elasticity alter the dynamics of adhesion. The state diagram (which must be mapped for each receptor–ligand system) presents a concise and comprehensive means of understanding the relationship between bond functional properties and the dynamics of adhesion mediated by receptor–ligand bonds.
Pinpointing when T cell costimulatory receptor CTLA-4 must be engaged to dampen diabetogenic T cells
Authors: Lühder Fred , Chambers Cynthia , Allison James P. , Benoist Christophe , Mathis Diane
Published: July 2002

Engagement of the T cell costimulatory receptor CTLA-4 can potently down-regulate an immune response. For example, in a T cell receptor transgenic mouse model of autoimmune diabetes, CTLA-4 interactions keep pancreatic islet-reactive T cells in check, evidenced by the finding that mAb blockade of CTLA-4 rapidly provokes diabetes in animals that would not normally succumb until many months later. Interestingly, this effect is only observed early in the course of disease, before insulitis is stably entrenched. Here, we have exploited a highly synchronous and easily manipulable transfer system to determine precisely when CTLA-4 must be engaged to check the diabetogenicity of islet-reactive T cells. Our results indicate that CTLA-4 interactions during initial priming of the T cells in the pancreatic lymph nodes are not determinant. Rather, the critical interactions occur when the T cells secondarily reencounter their antigen in the target organ, the pancreatic islets. In addition, we made use of CTLA-4-deficient mice to bolster our interpretation that CTLA-4 engagement has a dampening rather than an enhancing influence on diabetes progression.
Modulation of nitric oxide bioavailability by erythrocytes
Authors: Huang Kuang-Tse , Han Tae H. , Hyduke Daniel R. , Vaughn Mark W. , Van Herle Helga , Hein Travis W. , Zhang Cuihua , Kuo Lih , Liao James C.
Published: July 2002

Nitric oxide (NO) activates soluble guanylyl cyclase in smooth
muscle cells to induce vasodilation in the vasculature. However, as
hemoglobin (Hb) is an effective scavenger of NO and is present in high
concentrations inside the red blood cell (RBC), the bioavailability of
NO would be too low to elicit soluble guanylyl cyclase activation in
the presence of blood. Therefore, NO bioactivity must be preserved.
Here we present evidence suggesting that the RBC participates in the
preservation of NO bioactivity by reducing NO influx. The NO uptake by
RBCs was increased and decreased by altering the degree of band 3
binding to the cytoskeleton. Methemoglobin and denatured hemoglobin
binding to the RBC membrane or cytoskeleton also were shown to
contribute to reducing the NO uptake rate of the RBC. These alterations
in NO uptake by the RBC, hence the NO bioavailability, were determined
to correlate with the vasodilation of isolated blood vessels. Our
observations suggest that RBC membrane and cytoskeleton associated
NO-inert proteins provide a barrier for NO diffusion and thus account
for the reduction in the NO uptake rate of RBCs.
Molecular determinants of the functional interaction between syntaxin and N-type Ca
2+
channel gating
Authors: Bezprozvanny Ilya , Zhong Pingyu , Scheller Richard H. , Tsien Richard W.
Published: July 2002

Syntaxin is a key presynaptic protein that binds to N- and
P/Q-type Ca
2+
channels in biochemical studies and affects
gating of these Ca
2+
channels in expression systems and in
synaptosomes. The present study was aimed at understanding the
molecular basis of syntaxin modulation of N-type channel gating.
Mutagenesis of either syntaxin 1A or the pore-forming α
1B
subunit of N-type Ca
2+
channels was combined with
functional assays of N-type channel gating in a
Xenopus
oocyte coexpression system and in biochemical binding experiments
in vitro
. Our analysis showed that the transmembrane
region of syntaxin and a short region within the H3 helical cytoplasmic
domain of syntaxin, containing residues Ala-240 and Val-244, appeared
critical for the channel modulation but not for biochemical association
with the “synprint site” in the II/III loop of
α
1B
. These results suggest that syntaxin and the
α
1B
subunit engage in two kinds of interactions: an
anchoring interaction via the II/III loop synprint site and a
modulatory interaction via another site located elsewhere in the
channel sequence. The segment of syntaxin H3 found to be involved in
the modulatory interaction would lie hidden within the four-helix
structure of the SNARE complex, supporting the hypothesis that
syntaxin's ability to regulate N-type Ca
2+
channels would
be enabled after SNARE complex disassembly after synaptic vesicle
exocytosis.
Pattern formation and traveling waves in myxobacteria: Theory and modeling
Authors: Igoshin Oleg A. , Mogilner Alex , Welch Roy D. , Kaiser Dale , Oster George
Published: July 2002

Recent experiments have provided new quantitative measurements of
the rippling phenomenon in fields of developing myxobacteria cells.
These measurements have enabled us to develop a mathematical model for
the ripple phenomenon on the basis of the biochemistry of the
C-signaling system, whereby individuals signal by direct cell contact.
The model quantitatively reproduces all of the experimental
observations and illustrates how intracellular dynamics,
contact-mediated intercellular communication, and cell motility can
coordinate to produce collective behavior. This pattern of waves is
qualitatively different from that observed in other social organisms,
especially
Dictyostelium discoideum
, which
depend on diffusible morphogens.
Paternity and relatedness in wild chimpanzee communities
Authors: Vigilant Linda , Hofreiter Michael , Siedel Heike , Boesch Christophe
Published: July 2002

The genetic structure of three contiguous wild chimpanzee communities in West Africa was examined to determine the extent to which the community, the mixed-sex social unit of chimpanzees, represents a closed reproductive unit. An analysis of paternity for 41 offspring resulted in 34 cases of paternity assignment to an adult male belonging to the same community. Among the 14 offspring for which all potential within-community fathers have been tested, one likely case of extra-group paternity (EGP) has been identified, suggesting an incidence of EGP of 7%. This more extensive analysis contradicts a previous genetic study of the Taï chimpanzees that inferred 50% extra-group fathers. We suggest, based on direct comparison of results for 33 individuals at 1 microsatellite locus and direct comparison of paternity assignments for 11 offspring, that the error rate in the previous study was too high to produce accurate genotypes and assignments of paternity and hence caused the false inference of a high rate of EGP. Thus, the community is the primary but not exclusive unit for reproduction in wild chimpanzees, and females do not typically reproduce with outside males. Despite the inferred low level of gene flow from extra-community males, relatedness levels among the community males are not significantly higher than among community females, and the distribution of genetic relationships within the group suggests that, rather than a primarily male-bonded social structure, the group is bonded through relationships between males and females. Kinship may explain cooperative behaviors directed against other communities, but is unlikely to explain the high levels of affiliation and cooperation seen for male within-community interactions.
Apical sorting of a voltage- and Ca
2+
-activated K
+
channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation
Authors: Bravo-Zehnder Marcela , Orio Patricio , Norambuena Andrés , Wallner Martin , Meera Pratap , Toro Ligia , Latorre Ramón , González Alfonso
Published: July 2002

The voltage- and Ca
2+
-activated K
+
(K
V,Ca
) channel is expressed in a variety of polarized
epithelial cells seemingly displaying a tissue-dependent
apical-to-basolateral regionalization, as revealed by
electrophysiology. Using domain-specific biotinylation and
immunofluorescence we show that the human channel K
V,Ca
α-subunit (human
Slowpoke
channel,
h
Slo
) is predominantly found in the apical plasma
membrane domain of permanently transfected Madin-Darby canine
kidney cells. Both the wild-type and a mutant h
Slo
protein lacking its only potential N-glycosylation site were
efficiently transported to the cell surface and concentrated in the
apical domain even when they were overexpressed to levels 200- to
300-fold higher than the density of intrinsic
Slo
channels. Furthermore, tunicamycin treatment did not prevent apical
segregation of h
Slo
, indicating that
endogenous glycosylated proteins (e.g., K
V,Ca
β-subunits) were not required. h
Slo
seems to display
properties for lipid-raft targeting, as judged by its buoyant
distribution in sucrose gradients after extraction with either
detergent or sodium carbonate. The evidence indicates that the
h
Slo
protein possesses intrinsic information for
transport to the apical cell surface through a mechanism that may
involve association with lipid rafts and that is independent of
glycosylation of the channel itself or an associated protein. Thus,
this particular polytopic model protein shows that
glycosylation-independent apical pathways exist for
endogenous membrane proteins in Madin-Darby canine kidney
cells.
Arp2/3 complex requires hydrolyzable ATP for nucleation of new actin filaments
Authors: Dayel Mark J. , Holleran Elizabeth A. , Mullins R. Dyche
Published: July 2002

The Arp2/3 complex, a seven-subunit protein complex
containing two actin-related proteins, Arp2 and Arp3, initiates
formation of actin filament networks in response to intracellular
signals. The molecular mechanism of filament nucleation, however, is
not well understood. Arp2 and Arp3 are predicted to bind ATP via
a highly conserved nucleotide-binding domain found in all members of
the actin superfamily and to form a heterodimer than mimics a
conventional actin dimer. We show here that adenosine nucleotides bind
with micromolar affinity to both Arp2 and Arp3 and that hydrolyzable
ATP is required for actin nucleation activity. Binding of N-WASP WA
increases the affinity of both Arp2 and Arp3 for ATP but does not alter
the stoichiometry of nucleotides bound in the presence of saturating
concentrations of ATP. The Arp2/3 complex bound to ADP or the
nonhydrolyzable ATP analogue AMP-PNP cannot nucleate actin filaments,
but addition of the phosphate analogue BeF
3
partially
restores activity to the ADP–Arp2/3 complex. Bound nucleotide also
regulates the affinity of the Arp2/3 complex for its upstream
activators N-WASP and ActA. We propose that the active nucleating form
of the Arp2/3 complex is the ADP-P
i
intermediate in the
ATPase cycle and that the ATPase activity of the Arp2/3 complex
controls both nucleation of new filaments and release of the Arp2/3
complex from membrane-associated activators.
Synaptonemal complex (SC) component Zip1 plays a role in meiotic recombination independent of SC polymerization along the chromosomes.
Authors: Storlazzi A , Xu L , Schwacha A , Kleckner N
Published: July 2002

Zip1 is a yeast synaptonemal complex (SC) central region component and is required for normal meiotic recombination and crossover interference. Physical analysis of meiotic recombination in a zip1 mutant reveals the following: Crossovers appear later than normal and at a reduced level. Noncrossover recombinants, in contrast, seem to appear in two phases: (i) a normal number appear with normal timing and (ii) then additional products appear late, at the same time as crossovers. Also, Holliday junctions are present at unusually late times, presumably as precursors to late-appearing products. Red1 is an axial structure component required for formation of cytologically discernible axial elements and SC and maximal levels of recombination. In a red1 mutant, crossovers and noncrossovers occur at coordinately reduced levels but with normal timing. If Zip1 affected recombination exclusively via SC polymerization, a zip1 mutation should confer no recombination defect in a red1 strain background. But a red1 zip1 double mutant exhibits the sum of the two single mutant phenotypes, including the specific deficit of crossovers seen in a zip1 strain. We infer that Zip1 plays at least one role in recombination that does not involve SC polymerization along the chromosomes. Perhaps some Zip1 molecules act first in or around the sites of recombinational interactions to influence the recombination process and thence nucleate SC formation. We propose that a Zip1-dependent, pre-SC transition early in the recombination reaction is an essential component of meiotic crossover control. A molecular basis for crossover/noncrossover differentiation is also suggested.
Induction of apoptosis in rhabdomyosarcoma cells through down-regulation of PAX proteins
Authors: Bernasconi Michele , Remppis Andrew , Fredericks William J. , Rauscher Frank J. , Schäfer Beat W.
Published: July 2002

The expression of a number of human paired box-containing
(
PAX
) genes has been correlated with various types of
tumors. Novel fusion genes encoding chimeric fusion proteins have been
found in the pediatric malignant tumor alveolar rhabdomyosarcoma (RMS).
They are generated by two chromosomal translocations t(2;13) and
t(1;13) juxtaposing
PAX3
or
PAX7
,
respectively, with a forkhead domain gene
FKHR
. Here we
describe that specific down-regulation of the t(2;13) translocation
product in alveolar RMS cells by antisense oligonucleotides results in
reduced cellular viability. Cells of embryonal RMS, the other major
histiotype of this tumor, were found to express either wild type
PAX3
or
PAX7
at elevated levels when
compared with primary human myoblasts. Treatment of corresponding
embryonal RMS cells with antisense olignucleotides directed against the
mRNA translational start site of either one of these two transcription
factors similarly triggers cell death, which is most likely due to
induction of apoptosis. Retroviral mediated ectopic expression
of mouse
Pax3
in a
PAX7
expressing
embryonal RMS cell line could partially rescue antisense induced
apoptosis. These data suggest that the
PAX3
/
FKHR
fusion gene and wild-type
PAX
genes play a causative role in the formation of RMS
and presumably other tumor types, possibly by suppressing the
apoptotic program that would normally eliminate these cells.
Calcium-dependent switching of the specificity of phosphoinositide binding to synaptotagmin
Authors: Schiavo Giampietro , Gu Qu-Ming , Prestwich Glenn D. , Söllner Thomas H. , Rothman James E.
Published: July 2002

The synaptic vesicle membrane protein synaptotagmin (tagmin) is
essential for fast, calcium-dependent, neurotransmitter release and is
likely to be the calcium sensor for exocytosis, because of its many
calcium-dependent properties. Polyphosphoinositides are needed for
exocytosis, but it has not been known why. We now provide a possible
connection between these observations with the finding that the C2B
domain of tagmin I binds phosphatidylinositol-4,5-bisphosphate
(PIns-4,5-P
2
), its isomer
phosphatidylinositol-3,4-bisphosphate and
phosphatidylinositol-3,4,5-trisphosphate (PIns-3,4,5-P
3
).
Calcium ions switch the specificity of this binding from
PIns-3,4,5-P
3
(at calcium concentrations found in resting
nerve terminals) to PIns-4,5-P
2
(at concentration of
calcium required for transmitter release). Inositol polyphosphates,
known blockers of neurotransmitter release, inhibit the binding of both
PIns-4,5-P
2
and PIns-3,4,5-P
3
to tagmin. Our
findings imply that tagmin may operate as a bimodal calcium sensor,
switching bound lipids during exocytosis. This connection to
polyphosphoinositides, compounds whose levels are physiologically
regulated, could be important for long-term memory and learning.
Molecular cloning and analysis of two subunits of the human TFIID complex: hTAF
II
130 and hTAF
II
100
Authors: Tanese Naoko , Saluja Daman , Vassallo Milo F. , Chen Jin-Long , Admon Arie
Published: July 2002

Transcription factor TFIID is a multiprotein complex composed of
the TATA box-binding protein (TBP) and multiple TBP-associated factors
(TAFs). TFIID plays an essential role in mediating transcriptional
activation by gene-specific activators. Numerous transcriptional
activators have been characterized from mammalian cells; however,
molecular analysis of the components of mammalian TFIID has been
incomplete. Here we describe isolation of cDNAs encoding two TAF
subunits of the human transcription factor TFIID. The first cDNA is
predicted to encode the C-terminal 947 residues of the 130-kDa human
TAF subunit, hTAF
II
130. The second cDNA encodes the
C-terminal 801 residues of the 100-kDa subunit, hTAF
II
100.
Recombinant TAFs expressed in human cells by transient transfections
are capable of associating with the endogenous TAFs and TBP
to form a TFIID complex
in vivo
. Protein binding
experiments demonstrate that hTAF
II
130, like its
Drosophila
homolog dTAF
II
110, interacts with
the glutamine-rich activation domains of the human transcription factor
Sp1. Furthermore, hTAF
II
130 shows reduced binding to the
Sp1 mutants with impaired ability to activate transcription, suggesting
a role for hTAF
II
130 as a direct coactivator target for
Sp1.
Identification of residues that control specific binding of the Shc phosphotyrosine-binding domain to phosphotyrosine sites.
Authors: van der Geer P , Wiley S , Gish G D , Lai V K , Stephens R , White M F , Kaplan D , Pawson T
Published: July 2002

The Shc adaptor protein contains two phosphotyrosine [Tyr(P)]binding modules--an N-terminal Tyr(P) binding (PTB) domain and a C-terminal Src homology 2 (SH2) domain. We have compared the ability of the Shc PTB domain to bind the receptors for nerve growth factor and insulin, both of which contain juxtamembrane Asn-Pro-Xaa-Tyr(P) motifs implicated in PTB binding. The Shc PTB domain binds with high affinity to a phosphopeptide corresponding to the nerve growth factor receptor Tyr-490 autophosphorylation site. Analysis of individual residues within this motif indicates that the Asn at position -3 [with respect to Tyr(P)], in addition to Tyr(P), is critical for PTB binding, while the Pro at position -2 plays a less significant role. A hydrophobic amino acid 5 residues N-terminal to the Tyr(P) is also essential for high-affinity binding. In contrast, the Shc PTB domain does not bind stably to the Asn-Pro-Xaa-Tyr(P) site at Tyr-960 in the activated insulin receptor, which has a polar residue (Ser) at position -5. Substitution of this Ser at position -5 with Ile markedly increased binding of the insulin receptor Tyr-960 phosphopeptide to the PTB domain. These results suggest that while the Shc PTB domain recognizes a core sequence of Asn-Pro-Xaa-Tyr(P), its binding affinity is modulated by more N-terminal residues in the ligand, which therefore contribute to the specificity of PTB-receptor interactions. An analysis of residues in the Shc PTB domain required for binding to Tyr(P) sites identified a specific and evolutionarily conserved Arg (Arg-175) that is uniquely important for ligand binding and is potentially involved in Tyr(P) recognition.
Oxidation states of the manganese cluster during the flash-induced S-state cycle of the photosynthetic oxygen-evolving complex.
Authors: Roelofs T A , Liang W , Latimer M J , Cinco R M , Rompel A , Andrews J C , Sauer K , Yachandra V K , Klein M P
Published: July 2002

The Mn K-edge x-ray absorption spectra for the pure S states of the tetranuclear Mn cluster of the oxygen-evolving complex of photosystem II during flash-induced S-state cycling have been determined. The relative S-state populations in samples given 0, 1, 2, 3, 4, or 5 flashes were determined from fitting the flash-induced electron paramagnetic resonance (EPR) multiline signal oscillation pattern to the Kok model. The edge spectra of samples given 0, 1, 2, or 3 flashes were combined with EPR information to calculate the pure S-state edge spectra. The edge positions (defined as the zero-crossing of the second derivatives) are 6550.1, 6551.7, 6553.5, and 6553.8 eV for S0, S1, S2, and S3, respectively. In addition to the shift in edge position, the S0--> S1 and S1--> S2 transitions are accompanied by characteristic changes in the shape of the edge, both indicative of Mn oxidation. The edge position shifts very little (0.3 eV) for the S2--> S3 transition, and the edge shape shows only subtle changes. We conclude that probably no direct Mn oxidation is involved in this transition. The proposed Mn oxidation state assignments are as follows: S0 (II, III, IV, IV) or (III, III, III, IV), S1 (III, III, IV, IV), S2 (III, IV, IV, IV), S3 (III, IV, IV, IV).
Nonexponential kinetics of a single tRNA
Phe
molecule under physiological conditions
Authors: Jia Yiwei , Sytnik Alexander , Li Liangquan , Vladimirov Serguei , Cooperman Barry S. , Hochstrasser Robin M.
Published: July 2002

The fluorescence decay functions of individual, specifically labeled tRNA
Phe
molecules exhibit nonexponential character as a result of conformational dynamics occurring during the measurement on a single molecule. tRNA
Phe
conformational states that interchange much more slowly are evidenced by the distribution of lifetimes observed for many individual molecules. A structural model for the nonexponential decay indicates that the tRNA
Phe
–probe adduct fluctuates between two states, one of which provides conditions that quench the probe fluorescence.
The hepatitis B virus X gene induces p53-mediated programmed cell death
Authors: Chirillo Paolo , Pagano Sabrina , Natoli Gioacchino , Puri Pier Lorenzo , Burgio Vito Lelio , Balsano Clara , Levrero Massimo
Published: July 2002

The human hepatitis B virus (HBV) protein pX is a multifunctional regulatory protein that is known to affect both transcription and cell growth. Here we describe induction of apoptosis in NIH 3T3 polyclonal cell lines upon stimulation of pX expression from a dexamethasone inducible mouse mammary tumor virus (MMTV)-X expression vector. The effect of long-term pX expression on the cell survival of mouse fibroblasts was confirmed in colony generation assays. This effect is not shared either by the other HBV products and it is c-
myc
mediated, as shown by the use of a dominant negative deletion mutant of c-
myc
. pX also sensitize cells to programmed cell death after exposure to DNA damaging agents. Taking advantage of stable transfectants carrying the p53val135 temperature-sensitive allele, we directly demonstrate that induction of apoptosis by pX requires p53. In p53 null mouse embryo fibroblasts pX activates transcription and confers an evident growth advantage without loss of cell viability. Although pX protein was not detectable in the experimental conditions we used, our results indicate that its expression affects both cell growth and cell death control.
The high spontaneous mutation rate: Is it a health risk?
Authors: Crow James F.
Published: July 2002

The human mutation rate for base substitutions is much higher
in males than in females and increases with paternal age. This effect
is mainly, if not entirely, due to the large number of cell divisions
in the male germ line. The mutation-rate increase is considerably
greater than expected if the mutation rate were simply proportional to
the number of cell divisions. In contrast, those mutations that are
small deletions or rearrangements do not show the paternal age effect.
The observed increase with the age of the father in the incidence of
children with different dominant mutations is variable, presumably the
result of different mixtures of base substitutions and deletions. In
Drosophila
, the rate of mutations causing minor
deleterious effects is estimated to be about one new mutation per
zygote. Because of a larger number of genes and a much larger amount of
DNA, the human rate is presumably higher. Recently, the
Drosophila
data have been reanalyzed and the
mutation-rate estimate questioned, but I believe that the totality of
evidence supports the original conclusion. The most reasonable way in
which a species can cope with a high mutation rate is by
quasi-truncation selection, whereby a number of mutant genes are
eliminated by one “genetic death.”
Measurements of attractive forces between proteins and end-grafted poly(ethylene glycol) chains
Authors: Sheth S. R. , Leckband D.
Published: July 2002

The surface force apparatus was used to measure directly the molecular forces between streptavidin and lipid bilayers displaying grafted
M
r
2,000 poly(ethylene glycol) (PEG). These measurements provide direct evidence for the formation of relatively strong attractive forces between PEG and protein. At low compressive loads, the forces were repulsive, but they became attractive when the proteins were pressed into the polymer layer at higher loads. The adhesion was sufficiently robust that separation of the streptavidin and PEG uprooted anchored polymer from the supporting membrane. These interactions altered the properties of the grafted chains. After the onset of the attraction, the polymer continued to bind protein for several hours. The changes were not due to protein denaturation. These data demonstrate directly that the biological activity of PEG is not due solely to properties of simple polymers such as the excluded volume. It is also coupled to the competitive interactions between solvent and other materials such as proteins for the chain segments and to the ability of this material to adopt higher order intrachain structures.
The structure of the acto-myosin subfragment 1 complex: Results of searches using data from electron microscopy and x-ray crystallography
Authors: Mendelson Robert , Morris Edward P.
Published: July 2002

Surmises of how myosin subfragment 1 (S1) interacts with actin filaments in muscle contraction rest upon knowing the relative arrangement of the two proteins. Although there exist crystallographic structures for both S1 and actin, as well as electron microscopy data for the acto–S1 complex (AS1), modeling of this arrangement has so far only been done “by eye.” Here we report fitted AS1 structures obtained using a quantitative method that is both more objective and makes more complete use of the data. Using undistorted crystallographic results, the best-fit AS1 structure shows significant differences from that obtained by visual fitting. The best fit is produced using the F-actin model of Holmes
et al.
[Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990)
Nature (London)
347, 44–49]. S1 residues at the AS1 interface are now found at a higher radius as well as being translated axially and rotated azimuthally. Fits using S1 plus loops missing from the crystal structure were achieved using a homology search method to predict loop structures. These improved fits favor an arrangement in which the loop at the 50- to 20-kDa domain junction of S1 is located near the N terminus of actin. Rigid-body movements of the lower 50-kDa domain, which further improve the fit, produce closure of the large 50-kDa domain cleft and bring conserved residues in the lower 50-kDa domain into an apparently appropriate orientation for close interaction with actin. This finding supports the idea that binding of ATP to AS1 at the end of the ATPase cycle disrupts the actin binding site by changing the conformation of the 50-kDa cleft of S1.
Characterization of the α
1B
-adrenergic receptor gene promoter region and hypoxia regulatory elements in vascular smooth muscle
Authors: Eckhart Andrea D. , Yang Nengyu , Xin Xiaohua , Faber James E.
Published: July 2002

We previously demonstrated that α
1B
-adrenergic receptor (AR) gene transcription, mRNA, and functionally coupled receptors increase during 3% O
2
exposure in aorta, but not in vena cava smooth muscle cells (SMC). We report here that α
1B
AR mRNA also increases during hypoxia in liver and lung, but not heart and kidney. A single 2.7-kb α
1B
AR mRNA was detected in aorta and vena cava during normoxia and hypoxia. The α
1B
AR 5′ flanking region was sequenced to −2,460 (relative to ATG +1). Transient transfection experiments identify the minimal promoter region between −270 and −143 and sequence between −270 and −248 that are required for transcription of the α
1B
AR gene in aorta and vena cava SMC during normoxia and hypoxia. An ATTAAA motif within this sequence specifically binds aorta, vena cava, and DDT
1
MF-2 nuclear proteins, and transcription primarily initiates downstream of this motif at approximately −160 in aorta SMC. Sequence between −837 and −273 conferred strong hypoxic induction of transcription in aorta, but not in vena cava SMC, whereas the
cis
-element for the transcription factor, hypoxia-inducible factor 1, conferred hypoxia-induced transcription in both aorta and vena cava SMC. These data identify sequence required for transcription of the α
1B
AR gene in vascular SMC and suggest the atypical TATA-box, ATTAAA, may mediate this transcription. Hypoxia-sensitive regions of the α
1B
AR gene also were identified that may confer the differential hypoxic increase in α
1B
AR gene transcription in aorta, but not in vena cava SMC.
Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin–ankyrin G119 skeleton in Madin Darby canine kidney cells
Authors: Devarajan Prasad , Stabach Paul R. , De Matteis Maria Antonietta , Morrow Jon S.
Published: July 2002

Spectrin (βIΣ∗) and ankyrin (Ank
G119
) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of βI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of α- and β-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of β-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as Ank
G119
, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular–tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin–ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.
Electrical stimulation of neonatal cardiomyocytes results in the sequential activation of nuclear genes governing mitochondrial proliferation and differentiation
Authors: Xia Yang , Buja L. Maximilian , Scarpulla Richard C. , McMillin Jeanie B.
Published: July 2002

Electrical stimulation of neonatal cardiac myocytes produces hypertrophy and cellular maturation with increased mitochondrial content and activity. To investigate the patterns of gene expression associated with these processes, cardiac myocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation [c-
fos
, c-
jun
,
JunB
, nuclear respiratory factor 1 (NRF-1)], mitochondrial proliferation [cytochrome
c
(Cyt
c
), cytochrome oxidase], and mitochondrial differentiation [carnitine palmitoyltransferase I (CPT-I) isoforms] were measured. The results establish a temporal pattern of mRNA induction beginning with c-
fos
(0.25–3 hr) and followed sequentially by c-
jun
(0.5–3 hr),
JunB
(0.5–6 hr), NRF-1 (1–12 hr), Cyt
c
(12–72 hr), and muscle-specific CPT-I (48–72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA, thus supporting the developmental fidelity of this pattern of gene regulation. Consistent with a transcriptional mechanism, electrical stimulation increased c-
fos
, β-myosin heavy chain, and Cyt
c
promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element, and Sp-1 site mutations within the Cyt
c
promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the NRF-1 and CRE sites inhibited the induction by electrical stimulation (5-fold and 2-fold, respectively) whereas mutation of the Sp-1 site maintained or increased the fold induction. This finding is consistent with the appearance of NRF-1 and
fos
/
jun
mRNAs prior to that of Cyt
c
and suggests that induction of these transcription factors is a prerequisite for the transcriptional activation of Cyt
c
expression. These results support a regulatory role for NRF-1 and possibly AP-1 in the initiation of mitochondrial proliferation.
Human fatty acid synthase: Assembling recombinant halves of the fatty acid synthase subunit protein reconstitutes enzyme activity
Authors: Jayakumar Arumugam , Chirala Subrahmanyam S. , Wakil Salih J.
Published: July 2002

Our model of the native fatty acid synthase (FAS) depicts it as a dimer of two identical multifunctional proteins (
M
r
≈ 272,000) arranged in an antiparallel configuration so that the active Cys-SH of the β-ketoacyl synthase of one subunit (where the acyl group is attached) is juxtaposed within 2 Å of the pantetheinyl-SH of the second subunit (where the malonyl group is bound). This arrangement generates two active centers for fatty acid synthesis and predicts that if we have two appropriate halves of the monomer, we should be able to reconstitute an active fatty acid-synthesizing site. We cloned, expressed, and purified catalytically active thioredoxin (TRX) fusion proteins of the NH
2
-terminal half of the human FAS subunit protein (TRX-hFAS-dI; residues 1–1,297;
M
r
≈ 166) and of the C-terminal half (TRX-hFAS-dII-III; residues 1,296–2,504;
M
r
≈ 155). Adding equivalent amounts of TRX-hFAS-dI and TRX-hFAS-dII-III to a reaction mixture containing acetyl-CoA, malonyl-CoA, and NADPH resulted in the synthesis of long-chain fatty acids. The rate of synthesis was dependent upon the presence of both recombinant proteins and reached a constant level when they were present in equivalent amounts, indicating that the reconstitution of an active fatty acid-synthesizing site required the presence of every partial activity associated with the subunit protein. Analyses of the product acids revealed myristate to be the most abundant with small amounts of palmitate and stearate, possibly because of the way the fused recombinant proteins interacted with each other so that the thioesterase hydrolyzed the acyl group in its myristoyl state. The successful reconstitution of the human FAS activity from its domain I and domains II and III fully supports our model for the structure–function relationship of FAS in animal tissues.