bluuebunny/crossref_metadata_2025 huggingface dataset:
Escherichia coli
RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates
Authors: Uptain Susan M. , Chamberlin Michael J.
Published: July 2002

Several models have been proposed for the mechanism of transcript termination by
Escherichia coli
RNA polymerase at rho-independent terminators. Yager and von Hippel (Yager, T. D. & von Hippel, P. H. (1991)
Biochemistry
30, 1097–118) postulated that the transcription complex is stabilized by enzyme–nucleic acid interactions and the favorable free energy of a 12-bp RNA–DNA hybrid but is destabilized by the free energy required to maintain an extended transcription bubble. Termination, by their model, is viewed simply as displacement of the RNA transcript from the hybrid helix by reformation of the DNA helix. We have proposed an alternative model where the RNA transcript is stably bound to RNA polymerase primarily through interactions with two single-strand specific RNA-binding sites; termination is triggered by formation of an RNA hairpin that reduces binding of the RNA to one RNA-binding site and, ultimately, leads to its ejection from the complex. To distinguish between these models, we have tested whether
E. coli
RNA polymerase can terminate transcription at rho-independent terminators on single-stranded DNA. RNA polymerase cannot form a transcription bubble on these templates; thus, the Yager–von Hippel model predicts that intrinsic termination will not occur. We find that transcript elongation on single-stranded DNA templates is hindered somewhat by DNA secondary structure. However,
E. coli
RNA polymerase efficiently terminates and releases transcripts at several rho-independent terminators on such templates at the same positions as termination occurs on duplex DNAs. Therefore, neither the nontranscribed DNA strand nor the transcription bubble is essential for rho-independent termination by
E. coli
RNA polymerase.
Hypersensitivity ofKu80-deficient cell lines and mice to DNA damage: The effects of ionizing radiation on growth, survival, and development
Authors: Nussenzweig André , Sokol Karen , Burgman Paul , Li Ligeng , Li Gloria C.
Published: July 2002

We recently have shown that mice deficient for the 86-kDa component (Ku80) of the DNA-dependent protein kinase exhibit growth retardation and a profound deficiency in V(D)J (variable, diversity, and joining) recombination. These defects may be related to abnormalities in DNA metabolism that arise from the inability ofKu80mutant cells to process DNA double-strand breaks. To further characterize the role of Ku80 in DNA double-strand break repair, we have generated embryonic stem cells and pre-B cells and examined their response to ionizing radiation.Ku80−/−embryonic stem cells are more sensitive than controls to γ-irradiation, and pre-B cells derived fromKu80mutant mice display enhanced spontaneous and γ-ray-induced apoptosis. We then determined the effects of ionizing radiation on the survival, growth, and lymphocyte development inKu80-deficient mice.Ku80−/−mice display a hypersensitivity to γ-irradiation, characterized by loss of hair pigmentation, severe injury to the gastrointestinal tract, and enhanced mortality. Exposure of newbornKu80−/−mice to sublethal doses of ionizing radiation enhances their growth retardation and results in the induction of T cell-specific differentiation. However, unlike severe combined immunodeficient mice, radiation-induced T cell development inKu80−/−mice is not accompanied by extensive thymocyte proliferation. The response ofKu80-deficient cell lines and mice to DNA-damaging agents provides important insights into the role of Ku80 in growth regulation, lymphocyte development, and DNA repair.
Molecular determinants of tissue selectivity in estrogen receptor modulators
Authors: Grese Timothy A. , Sluka James P. , Bryant Henry U. , Cullinan George J. , Glasebrook Andrew L. , Jones Charles D. , Matsumoto Ken , Palkowitz Alan D. , Sato Masahiko , Termine John D. , Winter Mark A. , Yang Na N. , Dodge Jeffrey A.
Published: July 2002

Interaction of the estrogen receptor/ligand complex with a DNA estrogen response element is known to regulate gene transcription. In turn, specific conformations of the receptor-ligand complex have been postulated to influence unique subsets of estrogen-responsive genes resulting in differential modulation and, ultimately, tissue-selective outcomes. The estrogen receptor ligands raloxifene and tamoxifen have demonstrated such tissue-specific estrogen agonist/antagonist effects. Both agents antagonize the effects of estrogen on mammary tissue while mimicking the actions of estrogen on bone. However, tamoxifen induces significant stimulation of uterine tissue whereas raloxifene does not. We postulate that structural differences between raloxifene and tamoxifen may influence the conformations of their respective receptor/ligand complexes, thereby affecting which estrogen-responsive genes are modulated in various tissues. These structural differences are 4-fold: (
A
) the presence of phenolic hydroxyls, (
B
) different substituents on the basic amine, (
C
) incorporation of the stilbene moiety into a cyclic benzothiophene framework, and (
D
) the imposition of a carbonyl “hinge” between the basic amine-containing side chain and the olefin. A series of raloxifene analogs that separately exemplify each of these differences have been prepared and evaluated in a series of
in vitro
and
in vivo
assays. This strategy has resulted in the development of a pharmacophore model that attributes the differences in effects on the uterus between raloxifene and tamoxifen to a low-energy conformational preference imparting an orthogonal orientation of the basic side chain with respect to the stilbene plane. This three-dimensional array is dictated by a single carbon atom in the hinge region of raloxifene. These data indicate that differences in tissue selective actions among benzothiophene and triarylethylene estrogen receptor modulators can be ascribed to discrete ligand conformations.
The effect of macromolecular crowding on chaperonin-mediated protein folding
Authors: Martin Jörg , Hartl F.-Ulrich
Published: July 2002

The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in
Escherichia coli
. Recent studies
in vitro
demonstrated that GroES binding to GroEL causes the displacement of unfolded polypeptide into the central volume of the GroEL cavity for folding in a sequestrated environment. Resulting native protein leaves GroEL upon GroES release, whereas incompletely folded polypeptide can be recaptured for structural rearrangement followed by another folding trial. Additionally, each cycle of GroES binding and dissociation is associated with the release of nonnative polypeptide into the bulk solution. Here we show that this loss of substrate from GroEL is prevented when the folding reaction is carried out in the presence of macromolecular crowding agents, such as Ficoll and dextran, or in a dense cytosolic solution. Thus, the release of nonnative polypeptide is not an essential feature of the productive chaperonin mechanism. Our results argue that conditions of excluded volume, thought to prevail in the bacterial cytosol, increase the capacity of the chaperonin to retain nonnative polypeptide throughout successive reaction cycles. We propose that the leakiness of the chaperonin system under physiological conditions is adjusted such that
E. coli
proteins are likely to complete folding without partitioning between different GroEL complexes. Polypeptides that are unable to fold on GroEL eventually will be transferred to other chaperones or the degradation machinery.
The adenovirus E4orf6 protein can promote E1A/E1B-induced focus formation by interfering with p53 tumor suppressor function
Authors: Nevels Michael , Rubenwolf Susanne , Spruss Thilo , Wolf Hans , Dobner Thomas
Published: July 2002

We have recently shown that the adenovirus type 5 E4orf6 protein interacts with the cellular tumor suppressor protein p53 and blocks p53 transcriptional functions. Here we report that the E4orf6 protein can promote focus formation of primary rodent epithelial cells in cooperation with adenovirus E1A and E1A plus E1B proteins. The E4orf6 protein can also inhibit p53-mediated suppression of E1A plus E1B-19kDa-induced focus formation. Mutant analysis of the E4orf6 protein demonstrates that these activities correlate with the ability of the adenovirus protein to relieve transcriptional repression mediated by the carboxyl-terminal region of p53 in transient transfection assays. We further demonstrate that expression of wild-type E4orf6 correlates with a dramatic reduction of p53 steady-state levels in transformed rat cells. Our data demonstrate that adenovirus type 5 encodes two different proteins, E1B-55kDa and E4orf6, that bind to p53 and contribute to transformation by modulating p53 transcriptional functions.
The
MUR1
gene of
Arabidopsis thaliana
encodes an isoform of GDP-
d
-mannose-4,6-dehydratase, catalyzing the first step in the
de novo
synthesis of GDP-
l
-fucose
Authors: Bonin Christopher P. , Potter Ian , Vanzin Gary F. , Reiter Wolf-Dieter
Published: July 2002

GDP-
l
-fucose is the activated nucleotide sugar form of
l
-fucose, which is a constituent of many structural polysaccharides and glycoproteins in various organisms. The
de novo
synthesis of GDP-
l
-fucose from GDP-
d
-mannose encompasses three catalytic steps, a 4,6-dehydration, a 3,5-epimerization, and a 4-reduction. The
mur1
mutant of
Arabidopsis
is deficient in
l
-fucose in the shoot and is rescued by growth in the presence of exogenously supplied
l
-fucose. Biochemical assays of the
de novo
pathway for the synthesis of GDP-
l
-fucose indicated that
mur1
was blocked in the first nucleotide sugar interconversion step, a GDP-
d
-mannose-4,6-dehydratase. An expressed sequence tag was identified that showed significant sequence similarity to proposed bacterial GDP-
d
-mannose-4,6-dehydratases and was tightly linked to the
mur1
locus. A full-length clone was isolated from a cDNA library, and its coding region was expressed in
Escherichia coli
. The recombinant protein exhibited GDP-
d
-mannose-4,6-dehydratase activity
in vitro
and was able to complement
mur1
extracts
in vitro
to complete the pathway for the synthesis of GDP-
l
-fucose. All seven
mur1
alleles investigated showed single point mutations in the coding region for the 4,6-dehydratase, confirming that it represents the
MUR1
gene.
Lymphotoxin-β receptor signaling complex: Role of tumor necrosis factor receptor-associated factor 3 recruitment in cell death and activation of nuclear factor κB
Authors: VanArsdale Todd L. , VanArsdale Sammee L. , Force Walker R. , Walter Barbara N. , Mosialos George , Kieff Elliott , Reed John C. , Ware Carl F.
Published: July 2002

The binding of heterotrimeric lymphotoxin, LTα1β2, to the LTβ receptor (LTβR), a member of the tumor necrosis factor receptor (TNFR) superfamily, induces nuclear factor κB (NF-κB) activation and cell death in HT29 adenocarcinoma cells. We now show that treatment with LTα1β2or agonistic LTβR antibodies causes rapid recruitment of TNFR-associated factor 3 (TRAF3) to the LTβR cytoplasmic domain. Further, stable overexpression of a TRAF3 mutant that lacks the RING and zinc finger domains inhibits LTβR-mediated cell death. The inhibition is specific for LTβR cell death signaling, since NF-κB activation by LTα1β2and Fas-mediated apoptosis are not inhibited in the same cells. The mutant and endogenous TRAF3s are both recruited at equimolar amounts to the LTβR, suggesting that the mutant disrupts the function of the signaling complex. These results implicate TRAF3 as a critical component of the LTβR death signaling complex and indicate that at least two independent signaling pathways are initiated by LTβR ligation.
Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells
Authors: Politz Joan C. , Browne Elizabeth S. , Wolf David E. , Pederson Thoru
Published: July 2002

Fluorescein-labeled oligodeoxynucleotides (oligos) were introduced into cultured rat myoblasts, and their molecular movements inside the nucleus were studied by fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP). FCS revealed that a large fraction of both intranuclear oligo(dT) (43%) and oligo(dA) (77%) moves rapidly with a diffusion coefficient of 4 × 10
−7
cm
2
/s. Interestingly, this rate of intranuclear oligo movement is similar to their diffusion rates measured in aqueous solution. In addition, we detected a large fraction (45%) of the intranuclear oligo(dT), but not oligo(dA), diffusing at slower rates (≤1 × 10
−7
cm
2
/s). The amount of this slower-moving oligo(dT) was greatly reduced if the oligo(dT) was prehybridized in solution with (unlabeled) oligo(dA) prior to introduction to cells, presumably because the oligo(dT) was then unavailable for subsequent hybridization to endogenous poly(A) RNA. The FCS-measured diffusion rate for much of the slower oligo(dT) population approximated the diffusion rate in aqueous solution of oligo(dT) hybridized to a large polyadenylated RNA (1.0 × 10
−7
cm
2
/s). Moreover, this intranuclear movement rate falls within the range of calculated diffusion rates for an average-sized heterogeneous nuclear ribonucleoprotein particle in aqueous solution. A subfraction of oligo(dT) (15%) moved over 10-fold more slowly, suggesting it was bound to very large macromolecular complexes. Average diffusion coefficients obtained from FRAP experiments were in agreement with the FCS data. These results demonstrate that oligos can move about within the nucleus at rates comparable to those in aqueous solution and further suggest that this is true for large ribonucleoprotein complexes as well.
Fourier transform infrared spectroscopy reveals a rigid α-helical assembly for the tetrameric
Streptomyces lividans
K
+
channel
Authors: le Coutre Johannes , Kaback H. Ronald , Patel C. Kumar N. , Heginbotham Lise , Miller Christopher
Published: July 2002

The structure of the tetrameric K
+
channel from
Streptomyces lividans
in a lipid bilayer environment was
studied by polarized attenuated total reflection Fourier transform
infrared spectroscopy. The channel displays approximately 43%
α-helical and 25% β-sheet content. In addition, H/D exchange
experiments show that only 43% of the backbone amide protons are
exchangeable with solvent. On average, the α-helices are tilted 33°
normal to the membrane surface. The results are discussed in
relationship to the lactose permease of
Escherichia
coli
, a membrane transport protein.
Male fitness increases when females are eliminated from gene pool: Implications for the Y chromosome
Authors: Rice William R.
Published: July 2002

Because the two sexes share a common gene pool while performing many different biological functions, mutations benefiting one sex may not accumulate due to counter selection in the other sex. In these experiments 99% of a haploid genome of
Drosophila melanogaster
was constrained to segregate like a male-limited Y chromosome for 41 generations, thereby eliminating potential counter selection in females. The synthetic Y chromosomes rapidly accumulated genetic variation that increased male fitness and decreased female fitness. The survival and fertility of females declined when they were mated to males expressing the synthetic Y chromosomes. These results suggests that opposing selection between the sexes may substantially interfere with sex-specific adaptation. They also demonstrate how intersexual evolutionary conflict can lead to perpetual degeneration of the Y via genetic hitchhiking of deleterious mutations.
Analysis of human cytochrome P450 3A4 cooperativity: Construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics
Authors: Harlow Greg R. , Halpert James R.
Published: July 2002

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure–activity study from another laboratory using various effector–substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6β-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by α-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6β-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.
Static and dynamic lengths of neutrophil microvilli
Authors: Shao Jin-Yu , Ting-Beall Hie Ping , Hochmuth Robert M.
Published: July 2002

Containing most of the L-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) on their tips, microvilli are believed to promote the initial arrest of neutrophils on endothelium. At the rolling stage following arrest, the lifetimes of the involved molecular bonds depend on the pulling force imposed by the shear stress of blood flow. With two different methods, electron microscopy and micropipette manipulation, we have obtained two comparable neutrophil microvillus lengths, both ≈0.3 μm in average. We have found also that, under a pulling force, a microvillus can be extended (microvillus extension) or a long thin membrane cylinder (a tether) can be formed from it (tether formation). If the force is ≤34 pN (± 3 pN), the length of the microvillus will be extended; if the force is >61 pN (± 5 pN), a tether will be formed from the microvillus at a constant velocity, which depends linearly on the force. When the force is between 34 pN and 61 pN (transition zone), the degree of association between membrane and cytoskeleton in individual microvilli will dictate whether microvillus extension or tether formation occurs. When a microvillus is extended, it acts like a spring with a spring constant of ≈43 pN/μm. In contrast to a rigid or nonextendible microvillus, both microvillus extension and tether formation can decrease the pulling force imposed on the adhesive bonds, and thus prolonging the persistence of the bonds at high physiological shear stresses.
Supramolecular organization of immature and mature murine leukemia virus revealed by electron cryo-microscopy: Implications for retroviral assembly mechanisms
Authors: Yeager Mark , Wilson-Kubalek Elizabeth M. , Weiner Scott G. , Brown Patrick O. , Rein Alan
Published: July 2002

We have used electron cryo-microscopy and image analysis to examine the native structure of immature, protease-deficient (PR
−
) and mature, wild-type (WT) Moloney murine leukemia virus (MuLV). Maturational cleavage of the Gag polyprotein by the viral protease is associated with striking morphological changes. The PR
−
MuLV particles exhibit a rounded central core, which has a characteristic track-like shell on its surface, whereas the WT MuLV cores display a polygonal surface with loss of the track-like feature. The pleomorphic shape and inability to refine unique orientation angles suggest that neither the PR
−
nor the WT MuLV adheres to strict icosahedral symmetry. Nevertheless, the PR
−
MuLV particles do exhibit paracrystalline order with a spacing between Gag molecules of ≈45 Å and a length of ≈200 Å. Because of the pleomorphic shape and paracrystalline packing of the Gag–RNA complexes, we raise the possibility that assembly of MuLV is driven by protein–RNA, as well as protein–protein, interactions. The maturation process involves a dramatic reorganization of the packing arrangements within the ribonucleoprotein core with disordering and loosening of the individual protein components.
The allosteric mechanism of the chaperonin GroEL: A dynamic analysis
Authors: Ma Jianpeng , Karplus Martin
Published: July 2002

Normal mode calculations on individual subunits and a multisubunit construct are used to analyze the structural transitions that occur during the GroEL cycle. The normal modes demonstrate that the specific displacements of the domains (hinge bending, twisting) observed in the structural studies arise from the intrinsic flexibility of the subunits. The allosteric mechanism (positive cooperativity within a ring, negative cooperativity between rings) is shown to be based on coupled tertiary structural changes, rather than the quaternary transition found in classic allosteric proteins. The results unify static structural data from x-ray crystallography and cryoelectron microscopy with functional measurements of binding and cooperativity.
Experimental study of the interaction range and association rate of surface-attached cadherin 11
Authors: Pierres Anne , Feracci Hélène , Delmas Véronique , Benoliel Anne-Marie , Thiery Jean-Paul , Bongrand Pierre
Published: July 2002

We describe a method allowing quantitative determination of the interaction range and association rate of individual surface-attached molecules. Spherical beads (1.4 μm radius) were coated with recombinant outer domains of the newly described classical type II cadherin 11, a cell adhesion molecule. Beads were driven along cadherin-coated surfaces with a hydrodynamic force of ≈1 pN, i.e., much less than the mechanical strength of many ligand-receptor bonds. Spheres displayed periods of slow motion interspersed with arrests of various duration. Particle position was monitored with 50 Hz frequency and 0.025 μm accuracy. Nearly 1 million positions were recorded and processed. Comparison between experimental and computer-simulated trajectories suggested that velocity fluctuations might be related quantitatively to Brownian motion perpendicular to the surface. The expected amplitude of this motion was of order of 100 nm. Theoretical analysis of the relationship between sphere acceleration and velocity allowed simultaneous determination of the wall shear rate and van der Waals attraction between spheres and surface. The Hamaker constant was estimated at 2.9 × 10
−23
J. The frequency of bond formation was then determined as a function of sphere velocity. Experimental data were consistent with the view that the rate of association between a pair of adhesion molecules was ≈1.2 × 10
−3
s
−1
and the interaction range was ≈10 nm. It is concluded that the presented methodology allows sensitive measurement of sphere-to-surface interactions (with ≈10 fN sensitivity) as well as the effective range and rate of bond formation between individual adhesion molecules.
Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase
Authors: Couillard-Després Sébastien , Zhu Qinzhang , Wong Philip C. , Price Donald L. , Cleveland Don W. , Julien Jean-Pierre
Published: July 2002

To investigate the role of neurofilaments in motor neuron disease caused by superoxide dismutase (SOD1) mutations, transgenic mice expressing a amyotrophic lateral sclerosis-linked SOD1 mutant (SOD1
G37R
) were mated with transgenic mice expressing human neurofilament heavy (NF-H) subunits. Unexpectedly, expression of human NF-H transgenes increased by up to 65%, the mean lifespan of SOD1
G37R
mice. Microscopic examination corroborated the protective effect of NF-H protein against SOD1 toxicity. Although massive neurodegeneration occurred in 1-yr-old mice expressing SOD1
G37R
alone, spinal root axons and motor neurons were remarkably spared in doubly SOD1
G37R
;NF-H-transgenic littermates.
Episodic gonadotropin-releasing hormone gene expression revealed by dynamic monitoring of luciferase reporter activity in single, living neurons
Authors: Nuñez Lucia , Faught William J. , Frawley L. Stephen
Published: July 2002

The existence of an intrinsic oscillator for pulsatile gonadotropin-releasing hormone (GnRH) secretion in normal and transformed GnRH neurons raises the question of whether the corresponding gene also is expressed in an episodic manner. To resolve this question, we used a modification of conventional luciferase technology, which enabled continuous monitoring of GnRH gene activity in single, living neurons. With this method, the relative rate of endogenous gene expression is estimated by quantification of photons emitted by individual neurons microinjected with a GnRH promoter-driven luciferase reporter construct. Immortalized GT1–1 neurons, which secrete the decapeptide GnRH in a pulsatile manner conceptually identical to that of their nontransformed counterparts
in vivo
, were chosen as the model for these studies. First, we injected individual cells with purified luciferase protein and established that the reporter half-life was sufficiently short (50 min) to enable detection of transient changes in gene expression. Next, we subjected transfected GT1–1 cells to continuous monitoring of reporter activity for 16 h and found that the majority of them exhibited spontaneous fluctuations of photonic activity over time. Finally, we established that photonic activity accurately reflected endogenous GnRH gene expression by treating transfected GT1–1 cells with phorbol 12-myristate 13 acetate (a consensus inhibitor of GnRH gene expression) and observing a dramatic suppression of photonic emissions from continuously monitored cells. Taken together, these results demonstrate the validity of our “real-time” strategy for dynamically monitoring GnRH gene activity in living neurons. Moreover, our findings indicate that GnRH gene expression as well as neuropeptide release can occur in an intermittent manner.
ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function
Authors: Franco Michel , Boretto Joëlle , Robineau Sylviane , Monier Solange , Goud Bruno , Chardin Pierre , Chavrier Philippe
Published: July 2002

Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF
in vitro
, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (
i
) fragmentation of the Golgi apparatus, (
ii
) redistribution of Golgi resident proteins as well as the coat component β-COP, and (
iii
) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells.
A recipe for randomness
Authors: Pincus Steve , Singer Burton H.
Published: July 2002

Despite many diverse theories that address closely related
themes—e.g., probability theory, algorithmic complexity,
cryptoanalysis, and pseudorandom number generation—a near-void remains
in constructive methods certified to yield the desired “random”
output. Herein, we provide explicit techniques to produce broad sets of
both highly irregular finite and normal infinite sequences, based on
constructions and properties derived from approximate entropy (ApEn), a
computable formulation of sequential irregularity. Furthermore, for
infinite sequences, we considerably refine normality, by providing
methods for constructing diverse classes of normal numbers, classified
by the extent to which initial segments deviate from maximal
irregularity.
Identification of the gene (
lgtG
) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from
Neisseria gonorrhoeae
Authors: Banerjee Asesh , Wang Rong , Uljon Sacha N. , Rice Peter A. , Gotschlich Emil C. , Stein Daniel C.
Published: July 2002

The lipooligosaccharide from
Neisseria gonorrhoeae
(GC), consists of lipid A, an oligosaccharide core and three branches,
α, β, and γ. We report the cloning of the gene
(
lgtG
, lipooligosaccharide glycosyl transferase G)
encoding the glucosyl transferase of GC that initiates the β chain
which consists of a lactosyl moiety. This gene contains a homopolymeric
tract of cytidine [poly(C)] and we demonstrate that changes in the
number of Cs in poly(C) account for the variation of β chain
expression in different GC strains. Biochemical analyses and mass
spectrometry clearly attribute the reactivity of mAb 2C7 to the
presence of the lactosyl β chain. In addition, we demonstrate that in
the absence of the lactosyl group, a phosphoethanolamine is added to
generate a new antigenic epitope as evidenced by the gain of reactivity
to mAb 2-L1–8. These results show that, like the α chain, the β
chain of lipooligosaccharide is subject to antigenic variation.
The 2.8-Å structure of rat liver F
1
-ATPase: Configuration of a critical intermediate in ATP synthesis/hydrolysis
Authors: Bianchet Mario A. , Hullihen Joanne , Pedersen Peter L. , Amzel L. Mario
Published: July 2002

During mitochondrial ATP synthesis, F
1
-ATPase—the portion of the ATP synthase that contains the catalytic and regulatory nucleotide binding sites—undergoes a series of concerted conformational changes that couple proton translocation to the synthesis of the high levels of ATP required for cellular function. In the structure of the rat liver F
1
-ATPase, determined to 2.8-Å resolution in the presence of physiological concentrations of nucleotides, all three β subunits contain bound nucleotide and adopt similar conformations. This structure provides the missing configuration of F
1
necessary to define all intermediates in the reaction pathway. Incorporation of this structure suggests a mechanism of ATP synthesis/hydrolysis in which configurations of the enzyme with three bound nucleotides play an essential role.
Essential role of mitochondrially encoded large rRNA for germ-line formation in
Drosophila
embryos
Authors: Iida Takako , Kobayashi Satoru
Published: July 2002

In
Drosophila
, pole cells, the progenitors of the germ line, are induced by the factors localized in the posterior pole region of oocytes and cleavage embryos, or germ plasm. Polar granules in germ plasm are electron-dense structures and have been proposed to contain factors essential for pole cell formation. Mitochondrially encoded large ribosomal RNA (mtlrRNA) has been identified as a component of polar granules. We previously have shown that mtlrRNA is able to rescue embryos that fail to form pole cells as a result of UV irradiation. However, there is a possibility that the function of mtlrRNA is limited to UV-irradiated embryos, and the question of whether mtlrRNA is required for the normal pathway leading to pole cell formation remains unanswered. In this study, we report that the reduction of mtlrRNA in germ plasm by injecting anti-mtlrRNA ribozymes into cleavage embryos leads to their inability to form pole cells. Other components of germ plasm, namely
oskar
mRNA,
germ cell-less
mRNA, and Vasa and Tudor proteins appear to be unaffected in these ribozyme-injected embryos. These results support an essential role for mtlrRNA in pole cell formation. We propose that mitochondrially encoded molecules participate in a key event in early cell-type specification.
Both here and there: Simultaneous expression of autonomous spatial memories in rats
Authors: Fenton A. A. , Wesierska M. , Kaminsky Yu , Bures J.
Published: July 2002

Foraging rats learned to avoid footshock that was present in a part of a circular arena that was either stable or rotating slowly in a lighted room. The rotation dissociated spatial information in the separate reference frames of the room and arena. After learning to avoid the shocked region in either condition, in the absence of shock, memory for this place was expressed by simultaneous avoidance of an area defined in the reference frame of the room as well as of an area defined in the reference frame of the rotating arena. Spatial memories in these distinct reference frames were acquired, retrieved, and extinguished autonomously.
Subnuclear localization of the active variant surface glycoprotein gene expression site in
Trypanosoma brucei
Authors: Chaves Inês , Zomerdijk Joost , Dirks-Mulder Anita , Dirks Roeland W. , Raap Anton K. , Borst Piet
Published: July 2002

In
Trypanosoma brucei
, transcription by RNA polymerase II and 5′ capping of messenger RNA are uncoupled: a capped spliced leader is trans spliced to every RNA. This decoupling makes it possible to have protein-coding gene transcription driven by RNA polymerase I. Indeed, indirect evidence suggests that the genes for the major surface glycoproteins, variant surface glycoproteins (VSGs) in bloodstream-form trypanosomes, are transcribed by RNA polymerase I. In a single trypanosome, only one
VSG
expression site is maximally transcribed at any one time, and it has been speculated that transcription takes place at a unique site within the nucleus, perhaps in the nucleolus. We tested this by using fluorescence
in situ
hybridization. With probes that cover about 50 kb of the active
221
expression site, we detected nuclear transcripts of this site in a single fluorescent spot, which did not colocalize with the nucleolus. Analysis of marker gene-tagged active expression site DNA by fluorescent DNA
in situ
hybridization confirmed the absence of association with the nucleolus. Even an active expression site in which the promoter had been replaced by an rDNA promoter did not colocalize with the nulceolus. As expected, marker genes inserted in the rDNA array predominantly colocalize with the nucleolus, whereas the tubulin gene arrays do not. We conclude that transcription of the active
VSG
expression site does not take place in the nucleolus.
The helical domain of a G protein α subunit is a regulator of its effector
Authors: Liu Wei , Northup John K.
Published: July 2002

The α subunit (Gα) of heterotrimeric G proteins is a major determinant of signaling selectivity. The Gα structure essentially comprises a GTPase “Ras-like” domain (RasD) and a unique α-helical domain (HD). We used the vertebrate phototransduction model to test for potential functions of HD and found that the HD of the retinal transducin Gα (Gα
t
) and the closely related gustducin (Gα
g
), but not Gα
i1
, Gα
s
, or Gα
q
synergistically enhance guanosine 5′-γ[-thio]triphosphate bound Gα
t
(Gα
t
GTPγS) activation of bovine rod cGMP phosphodiesterase (PDE). In addition, both HD
t
and HD
g
, but not HD
i1
, HD
s
, or HD
q
attenuate the trypsin-activated PDE. Gα
t
GDP and HD
t
attenuation of trypsin-activated PDE saturate with similar affinities and to an identical 38% of initial activity. These data suggest that interaction of intact Gα
t
with the PDE catalytic core may be caused by the HD moiety, and they indicate an independent site(s) for the HD moiety of Gα
t
within the PDE catalytic core in addition to the sites for the inhibitory Pγ subunits. The HD moiety of Gα
t
GDP is an attenuator of the activated catalytic core, whereas in the presence of activated Gα
t
GTPγS the independently expressed HD
t
is a potent synergist. Rhodopsin catalysis of Gα
t
activation enhances the PDE activation produced by subsaturating levels of Gα
t
, suggesting a HD-moiety synergism from a transient conformation of Gα
t
. These results establish HD-selective regulations of vertebrate retinal PDE, and they provide evidence demonstrating that the HD is a modulatory domain. We suggest that the HD works in concert with the RasD, enhancing the efficiency of G protein signaling.
NF-κB activation provides the potential link between inflammation and hyperplasia in the arthritic joint
Authors: Miagkov Alexei V. , Kovalenko Dmitry V. , Brown Chadwick E. , Didsbury John R. , Cogswell John P. , Stimpson Stephen A. , Baldwin Albert S. , Makarov Sergei S.
Published: July 2002

The transcription factor NF-κB is a pivotal regulator of inflammatory responses. While the activation of NF-κB in the arthritic joint has been associated with rheumatoid arthritis (RA), its significance is poorly understood. Here, we examine the role of NF-κB in animal models of RA. We demonstrate thatin vitro, NF-κB controlled expression of numerous inflammatory molecules in synoviocytes and protected cells against tumor necrosis factor α (TNFα) and Fas ligand (FasL) cytotoxicity. Similar to that observed in human RA, NF-κB was found to be activated in the synovium of rats with streptococcal cell wall (SCW)-induced arthritis.In vivosuppression of NF-κB by either proteasomal inhibitors or intraarticular adenoviral gene transfer of super-repressor IκBα profoundly enhanced apoptosis in the synovium of rats with SCW- and pristane-induced arthritis. This indicated that the activation of NF-κB protected the cells in the synovium against apoptosis and thus provided the potential link between inflammation and hyperplasia. Intraarticular administration of NF-kB decoys prevented the recurrence of SCW arthritis in treated joints. Unexpectedly, the severity of arthritis also was inhibited significantly in the contralateral, untreated joints, indicating beneficial systemic effects of local suppression of NF-κB. These results establish a mechanism regulating apoptosis in the arthritic joint and indicate the feasibility of therapeutic approaches to RA based on the specific suppression of NF-κB.
Real-time enzyme kinetics monitored by dual-color fluorescence cross-correlation spectroscopy
Authors: Kettling Ulrich , Koltermann Andre , Schwille Petra , Eigen Manfred
Published: July 2002

A method for sensitively monitoring enzyme kinetics and activities
by using dual-color fluorescence cross-correlation spectroscopy is
described. This universal method enables the development of highly
sensitive and precise assays for real-time kinetic analyses of any
catalyzed cleavage or addition reaction, where a chemical linkage is
formed or cleaved through an enzyme’s action between two fluorophores
that can be discriminated spectrally. In this work, a homogeneous assay
with restriction endonuclease
Eco
RI and a 66-bp
double-stranded DNA containing the GAATTC recognition site and
fluorophores at each 5′ end is described. The enzyme activity can be
quantified down to the low picomolar range (>1.6 pM) where the rate
constants are linearly dependent on the enzyme concentrations over two
orders of magnitude. Furthermore, the reactions were monitored on-line
at various initial substrate concentrations in the nanomolar range, and
the reaction rates were clearly represented by the Michaelis–Menten
equation with a
K
M
of 14 ± 1 nM and a
k
cat
of 4.6 ± 0.2 min
−1
.
In addition to kinetic studies and activity determinations, it is
proposed that enzyme assays based on the dual-color fluorescence
cross-correlation spectroscopy will be very useful for high-throughput
screening and evolutionary biotechnology.
Cdkn2a, the cyclin-dependent kinase inhibitor encoding p16INK4aand p19ARF, is a candidate for the plasmacytoma susceptibility locus,Pctr1
Authors: Zhang Shuling , Ramsay Edward S. , Mock Beverly A.
Published: July 2002

Plasma cell tumor induction in mice by pristane is under multigenic control. BALB/c mice are susceptible to tumor development; whereas DBA/2 mice are resistant. Restriction fragment length polymorphisms between BALB/c and DBA/2 forCdkn2a(p16) andCdkn2b(p15), and between BALB/c andMus spretusforCdkn2c(p18INK4c) were used to position these loci with respect to thePctr1locus. These cyclin-dependent kinase (CDK) inhibitors mapped to a 6 cM interval of chromosome 4 betweenIfnaandTal1. C.D2-Chr 4 congenic strains harboring DBA/2 alleles associated with thePctr1locus contained DBA/2 “resistant” alleles of the CDK4/CDK6 inhibitors p16 and p15. On sequencing p16 and p18 cDNAs, two different allelic variants within ankyrin repeat regions of p16 were found between BALB/c and DBA/2 mice. By using an assay involving PCR amplification and restriction enzyme digestion, allelic variants were typed among several inbred strains of mice. One of the variants, G232A, was specific to two inbred strains, BALB/cAn and ABP/Le, of mice and occurred in a highly conserved amino acid in both human and rat p16. When tested with wild-type (DBA/2) p16, both A134C and G232A BALB/c-specific variants of p16 were inefficient in their ability to inhibit the activity of cyclin D2/CDK4 in kinase assays with retinoblastoma protein, suggesting this defective, inherited allele plays an important role in the genetic susceptibility of BALB/c mice for plasmacytoma induction and that p16INK4ais a strong candidate for thePctr1locus.
Allosteric modulation of AMPA-type glutamate receptors increases activity of the promoter for the neural cell adhesion molecule, N-CAM
Authors: Holst Brent D. , Vanderklish Peter W. , Krushel Leslie A. , Zhou Wei , Langdon Ronald B. , McWhirter John R. , Edelman Gerald M. , Crossin Kathryn L.
Published: July 2002

To study regulation
in vivo
of the promoter for the neural cell adhesion molecule, N-CAM, we have used homologous recombination to insert the bacterial
lacZ
gene between the transcription and translation initiation sites of the N-CAM gene. This insertion disrupts the gene and places the expression of β-galactosidase under the control of the N-CAM promoter. Animals homozygous for the disrupted allele did not express N-CAM mRNA or protein, but the pattern of β-galactosidase expression in heterozygous and homozygous embryos was similar to that of N-CAM mRNA in wild-type animals. The homozygotes exhibited many of the morphological abnormalities observed in previously reported N-CAM knockout mice, with the exception that hippocampal long-term potentiation in the Schaffer collaterals was identical in homozygous, heterozygous, and wild-type animals. Heterozygous mice were used to examine the regulation of the N-CAM promoter in response to enhanced synaptic transmission. Treatment of the mice with an ampakine, an allosteric modulator of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors that enhances normal glutamate-mediated synaptic transmission, increased the expression of β-galactosidase
in vivo
as well as in tissue slices
in vitro
. Similar treatments also increased the expression of N-CAM mRNA in the heterozygotes. The effects of ampakine in slices were strongly reduced in the presence of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an AMPA receptor antagonist. Taken together, these results indicate that facilitation of AMPA receptor-mediated transmission leads to activation of the N-CAM promoter and provide support for the hypothesis that N-CAM synthesis is regulated in part by synaptic activity.
Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent manner
Authors: An Woojin , Leuba Sanford H. , van Holde Kensal , Zlatanova Jordanka
Published: July 2002

The protection against micrococcal nuclease digestion afforded to chromatosomal DNA by the presence of a linker histone (H1
o
) has been quantitatively measured in two reconstituted systems. We have used chromatosomes reconstituted at two distinct positions on a DNA fragment containing the 5S rRNA gene from
Lytechinus variegatus
and at a specific position on a sequence containing Gal4- and USF-binding sites. In all cases, we find asymmetric protection, with ≈20 bp protected on one side of the core particle and no protection on the other. We demonstrated through crosslinking experiments that the result is not due to any sliding of the histone core caused by either linker histone addition or micrococcal nuclease cleavage. Because the core particle is itself a symmetric object, the preferred asymmetric location of a linker histone must be dictated by unknown elements in the DNA sequence.
HIV transcriptional activation by the accessory protein, VPR, is mediated by the p300 co-activator
Authors: Felzien Lisa K. , Woffendin Clive , Hottiger Michael O. , Subbramanian Ramu A. , Cohen Eric A. , Nabel Gary J.
Published: July 2002

The accessory protein, Vpr, is a virion-associated protein that is required for HIV-1 replication in macrophages and regulates viral gene expression in T cells. Vpr causes arrest of cell cycle progression at G
2
/M, presumably through its effect on cyclin B1⋅Cdc2 activity. Here, we show that the ability of Vpr to activate HIV transcription correlates with its ability to induce G
2
/M growth arrest, and this effect is mediated by the p300 transcriptional co-activator, which promotes cooperative interactions between the Rel A subunit of NF-κB and cyclin B1⋅Cdc2. Vpr cooperates with p300, which regulates NF-κB and the basal transcriptional machinery, to increase HIV gene expression. Similar effects are seen in the absence of Vpr with a kinase-deficient Cdc2, and overexpression of p300 increases levels of HIV Vpr
+
replication. Taken together, these data suggest that p300, through its interactions with NF-κB, basal transcriptional components, and Cdks, is modulated by Vpr and regulates HIV replication. The regulation of p300 by Vpr provides a mechanism to enhance viral replication in proliferating cells after growth arrest by increasing viral transcription.
Heparin-binding epidermal growth factor-like growth factor, a v-Jun target gene, induces oncogenic transformation
Authors: Fu Shu-ling , Bottoli Ivan , Goller Martin , Vogt Peter K.
Published: July 2002

Jun is a transcription factor belonging to the activator protein 1 family. A mutated version of Jun (v-Jun) transduced by the avian retrovirus ASV17 induces oncogenic transformation in avian cell cultures and sarcomas in young galliform birds. The oncogenicity of Jun probably results from transcriptional deregulation of v-Jun-responsive target genes. Here we describe the identification and characterization of a growth-related v-Jun target, a homolog of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF is strongly expressed in chicken embryo fibroblasts (CEF) transformed by v-Jun. HB-EGF expression is not detectable or is marginal in nontransformed CEF. Using a hormone-inducible Jun-estrogen receptor chimera, we found that HB-EGF expression is correlated with v-Jun activity. In this system, induction of v-Jun is followed within 1 hr by elevated levels of HB-EGF. In CEF infected with various Jun mutants, HB-EGF expression is correlated with the oncogenic potency of the mutant. Constitutive expression of HB-EGF conveys to CEF the ability to grow in soft agar and to form multilayered foci of transformed cells on a solid substrate. These observations suggest that HB-EGF is an effector of Jun-induced oncogenic transformation.