bluuebunny/crossref_metadata_2025 huggingface dataset:
Probing the assembly of transcription initiation complexes through changes in σ
N
protease sensitivity
Authors: Casaz Paul , Buck Martin
Published: July 2002

The alternative bacterial σ
N
RNA polymerase holoenzyme binds promoters as a transcriptionally inactive complex that is activated by enhancer-binding proteins. Little is known about how sigma factors respond to their ligands or how the responses lead to transcription. To examine the liganded state of σ
N
, the assembly of end-labeled
Klebsiella pneumoniae
σ
N
into holoenzyme, closed promoter complexes, and initiated transcription complexes was analyzed by enzymatic protein footprinting. V8 protease-sensitive sites in free σ
N
were identified in the acidic region II and bordering or within the minimal DNA binding domain. Interaction with core RNA polymerase prevented cleavage at noncontiguous sites in region II and at some DNA binding domain sites, probably resulting from conformational changes. Formation of closed complexes resulted in further protections within the DNA binding domain, suggesting close contact to promoter DNA. Interestingly, residue E36 becomes sensitive to proteolysis in initiated transcription complexes, indicating a conformational change in holoenzyme during initiation. Residue E36 is located adjacent to an element involved in nucleating strand separation and in inhibiting polymerase activity in the absence of activation. The sensitivity of E36 may reflect one or both of these functions. Changing patterns of protease sensitivity strongly indicate that σ
N
can adjust conformation upon interaction with ligands, a property likely important in the dynamics of the protein during transcription initiation.
Brain-derived neurotrophic factor regulates expression of androgen receptors in perineal motoneurons
Authors: Al-Shamma Hussien A. , Arnold Arthur P.
Published: July 2002

Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) express androgen receptors and innervate striated muscles attached to the penis. Previous studies indicated that androgen receptor immunoreactivity in the SNB motoneurons decreases after axotomy and returns to normal only in motoneurons allowed to reinnervate their muscle targets, suggesting that neuron–target interactions play a role in regulating steroid receptor expression in the central nervous system. This study demonstrates that (
i
) silencing the SNB neuromuscular system with tetrodotoxin did not affect androgen receptor expression in these motoneurons, suggesting that the regulation of androgen receptor is activity-independent; (
ii
) disruption of axonal transport with vinblastine caused a down-regulation of androgen receptor expression in the SNB motoneurons; and (
iii
) treatment with brain-derived neurotrophic factor, but not ciliary neurotrophic factor, neurotrophin-4, or glial cell line-derived neurotrophic factor, reversed the axotomy-induced down-regulation of androgen receptor expression. These findings demonstrate neurotrophin regulation of steroid receptor expression in the central nervous system
in vivo
.
Enhancer blocking activity located near the 3′ end of the sea urchin early H2A histone gene
Authors: Palla Franco , Melfi Raffaella , Anello Letizia , Di Bernardo Maria , Spinelli Giovanni
Published: July 2002

The sea urchin early histone repeating unit contains one copy of each of the five histone genes whose coordinate expression during development is regulated by gene-specific elements. To learn how within the histone repeating unit a gene-specific activator can be prevented to communicate with the heterologous promoters, we searched for domain boundaries by using the enhancer blocking assay. We focused on the region near the 3′ end of the H2A gene where stage-specific nuclease cleavage sites appear upon silencing of the early histone genes. We demonstrated that a DNA fragment of 265 bp in length, defined as
sns
(for silencing nucleoprotein structure), blocked the enhancer activity of the H2A modulator in microinjected sea urchin embryos only when placed between the enhancer elements and the promoter. We also found that
sns
silenced the modulator elements even when placed at 2.7 kb from the promoter. By contrast, the enhancer activity of the modulator sequences, located downstream to the coding region, was not affected when
sns
was positioned in close proximity to the promoter. Finally, the H2A
sns
fragment placed between the simian virus 40 regulative region and the
tk
promoter repressed chloramphenicol acetyltransferase expression in transfected human cell lines. We conclude that 3′ end of the H2A gene contains sequence elements that behave as functional barriers of enhancer function in the enhancer blocking assay. Furthermore, our results also indicate that the enhancer blocking function of
sns
lacks enhancer and species specificity and that it can act in transient assays.
Characterization of soluble forms of nonchimeric type V adenylyl cyclases
Authors: Scholich Klaus , Barbier Ann J. , Mullenix Jason B. , Patel Tarun B.
Published: July 2002

Type V adenylyl cyclase (ACV) belongs to the family of Ca
2+
- inhibited cyclases. We have generated two soluble forms of the enzyme containing the C1 or C1a region (which lacks the C-terminal 112 amino acids) linked to the C2 domain and compared their regulation with the full-length ACV. All three forms of ACV were stimulated by the α subunit of the stimulatory G protein G
s
(G
sα
) and forskolin. However, the synergistic stimulation by both these activators was markedly enhanced in the soluble enzymes. Moreover, the α subunit of the inhibitory G protein G
i
(G
iα
) inhibited all forms of the enzyme, indicating that the regions for G
sα
and G
iα
interaction are preserved in the soluble forms. Ca
2+
inhibited forskolin-stimulated adenylyl cyclase (AC) activity of the full-length and C1-C2 forms of ACV but did not alter the activity of the C1a-C2 form. Maximal stimulation of AC activity by combination of G
sα
and forskolin obliterated the Ca
2+
-mediated inhibition of the full-length and C1-C2 forms of ACV. In
45
Ca
2+
overlay experiments, the C1-C2 but not the C1a-C2 soluble ACV bound Ca
2+
. Moreover, proteins corresponding to the C1a and C2 domains did not bind calcium. On the other hand, the proteins corresponding to C1 and its C-terminal 112 amino acids (C1b) bound
45
Ca
2+
. To our knowledge, this is the first report of nonchimeric soluble forms of AC in which regulation by G
sα
and G
iα
is preserved. Moreover, we demonstrate that the 112 amino acid C1b region of ACV is responsible for the binding of Ca
2+
and inhibition of enzyme activity.
Introduction of an RRHR motif into chicken urokinase-type plasminogen activator (ch-uPA) confers sensitivity to plasminogen activator inhibitor (PAI)-1 and PAI-2 and allows ch-uPA-mediated extracellular matrix degradation to be controlled by PAI-1
Authors: Sipley John D. , Alexander Daniela S. , Testa Jacqueline E. , Quigley James P.
Published: July 2002

Comparison of the amino acid sequence of the chicken and human urokinase-type plasminogen activators (uPAs) revealed that the putative PAI-binding site found in the variable region 1 (VR1) loop of mammalian PAs is absent in the homologous region of ch-uPA. ch-uPA, unlike mammalian PAs, also appears to be refractory to inhibition by human PAIs and as a naturally occurring PAI-resistant variant, constitutes a unique model system for assessing the functional relevance of the PAI-binding site. Therefore, we molecularly constructed a ch-uPA, ch-uPA
RRHR
, which contains the putative PAI-binding motif RRHR (residues 192–195) in its VR1 loop. As a result of this substitution, the second-order rate constant of inhibition of PAI-1 increased ≈700-fold from 4.50 × 10
4
M
−1
·s
−1
for wild-type ch-uPA to 3.02 × 10
7
M
−1
·s
−1
for ch-uPA
RRHR
, and the ability to form SDS-stable, uPA–PAI-1 complexes increased ≈1000-fold. Furthermore, the interaction of ch-uPA
RRHR
with PAI-2 was also substantially enhanced, while the interaction with other members of the serine proteinase inhibitor superfamily, protein nexin 1, α
1
-PI, and C
1
-inhibitor, was unaffected indicating that the RRHR motif is not a general serine proteinase inhibitor binding site. Finally, we show that extracellular matrix degradation by cells expressing ch-uPA
RRHR
is inhibited by PAI-1 in a dose-dependent manner, while matrix breakdown by cells expressing wild-type ch-uPA is unaffected by PAI-1. Thus acquisition of sensitivity to PAI-1 through a structural motif that enhances the specificity of the protease-inhibitor interaction confers to ch-uPA an added level of regulation in the context of the degradative cellular phenotype.
Synergy of Taxol and radioimmunotherapy with yttrium-90-labeled chimeric L6 antibody: Efficacy and toxicity in breast cancer xenografts
Authors: DeNardo Sally J. , Kukis David L. , Kroger Linda A. , O’Donnell Robert T. , Lamborn Kathleen R. , Miers Laird A. , DeNardo David G. , Meares Claude F. , DeNardo Gerald L.
Published: July 2002

Synergistic multimodality therapy is needed for breast cancer. Breast cancer frequently has p53 mutations that result in cells less likely to undergo apoptosis when exposed to DNA damaging therapies. Taxol (paclitaxel) is more effective in the presence of mutant p53.
90
Y-labeled DOTA-peptide-ChL6 (
90
Y-ChL6, where ChL6 is chimeric L6 antibody and DOTA is 1,4,7,10-tetraazacyclododecane-
N,N
′,
N
",
N
‴-tetraacetic acid) is a novel radioimmunoconjugate for targeting radiation to cancer. It has a stable metal chelator and a peptide linker that can be catabolized by hepatic lysozymes. This study was designed to assess potential synergism between Taxol and
90
Y-ChL6 in a highly anaplastic breast cancer model, HBT 3477. There was no tumor response in mice receiving ChL6 or Taxol alone. In mice receiving
90
Y-ChL6 alone, 79% (15 of 19) of tumors responded although none were cured. If Taxol was administered 24–72 hours before
90
Y-ChL6, again, 79% (23 of 29) of tumors responded but 21% were cured. When Taxol was administered 6 or 24 hours after
90
Y-ChL6, 100% (46 of 46) of tumors responded and 48% were cured. Taxol given with
90
Y-ChL6 did not substantially increase toxicity. Enhancement of the therapeutic effect when Taxol was added to
90
Y-ChL6 therapy for HBT 3477 xenografts was striking. The synergistic therapeutic effect of Taxol with
90
Y-ChL6 may relate to the p53 mutant status and
BCL2
expression in HBT 3477 cells, observations that increase the likelihood that the results of this study are relevant to therapy for breast cancer in patients. In conclusion, Taxol seemed to be synergistic with
90
Y-ChL6 in this human breast cancer model. Up to 50% of these anaplastic breast cancer xenografts were cured by combined modality therapy.
Direct searches for dark matter: Recent results
Authors: Rosenberg Leslie J.
Published: July 2002

There is abundant evidence for large amounts of unseen matter in
the universe. This dark matter, by its very nature, couples feebly to
ordinary matter and is correspondingly difficult to detect.
Nonetheless, several experiments are now underway with the sensitivity
required to detect directly galactic halo dark matter through their
interactions with matter and radiation. These experiments divide into
two broad classes: searches for weakly interacting massive particles
(WIMPs) and searches for axions. There exists a very strong theoretical
bias for supposing that supersymmetry (SUSY) is a correct description
of nature. WIMPs are predicted by this SUSY theory and have the
required properties to be dark matter. These WIMPs are detected from
the byproducts of their occasional recoil against nucleons. There are
efforts around the world to detect these rare recoils. The WIMP part of
this overview focuses on the cryogenic dark matter search (CDMS)
underway in California. Axions, another favored dark matter candidate,
are predicted to arise from a minimal extension of the standard model
that explains the absence of the expected large CP violating effects in
strong interactions. Axions can, in the presence of a large magnetic
field, turn into microwave photons. It is the slight excess of photons
above noise that signals the axion. Axion searches are underway in
California and Japan. The axion part of this overview focuses on the
California effort. Brevity does not allow me to discuss other WIMP and
axion searches, likewise for accelerator and satellite based searches;
I apologize for their omission.
Architecture and mechanism of the light-harvesting apparatus of purple bacteria
Authors: Hu Xiche , Damjanović Ana , Ritz Thorsten , Schulten Klaus
Published: July 2002

Photosynthetic organisms fuel their metabolism with light energy
and have developed for this purpose an efficient apparatus for
harvesting sunlight. The atomic structure of the apparatus, as it
evolved in purple bacteria, has been constructed through a combination
of x-ray crystallography, electron microscopy, and modeling. The
detailed structure and overall architecture reveals a hierarchical
aggregate of pigments that utilizes, as shown through femtosecond
spectroscopy and quantum physics, elegant and efficient mechanisms for
primary light absorption and transfer of electronic excitation toward
the photosynthetic reaction center.
Testing migration patterns and estimating founding population size in Polynesia by using human mtDNA sequences
Authors: Murray-McIntosh Rosalind P. , Scrimshaw Brian J. , Hatfield Peter J. , Penny David
Published: July 2002

The hypervariable 1 region of human mtDNA shows markedly reduced variability in Polynesians, and this variability decreases from western to eastern Polynesia. Fifty-four sequences from New Zealand Maori show that the mitochondrial variability with just four haplotypes is the lowest of any sizeable human group studied and that the frequency of haplotypes is markedly skewed. The Maori sequences, combined with 268 published sequences from the Pacific, are consistent with a series of founder effects from small populations settling new island groups. The distributions of haplotypes were used to estimate the number of females in founding population of New Zealand Maori. The three-step simulation used a randomly selected founding population from eastern Polynesia, an expansionary phase in New Zealand, and finally the random selection of 54 haplotypes. The results are consistent with a founding population that includes ≈70 women (between 50 and 100), and sensitivity analysis shows that this conclusion is robust to small changes in haplotype frequencies. This size is too large for models postulating a very small founding population of “castaways,” but it is consistent with a general understanding of Maori oral history as well as the results of recent canoe voyages recreating early trans-oceanic voyages.
Antiproliferative mechanism of action of cryptophycin-52: Kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends
Authors: Panda Dulal , DeLuca Keith , Williams Daniel , Jordan Mary Ann , Wilson Leslie
Published: July 2002

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (≥10 times the IC
50
), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC
50
= 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules
in vitro
(IC
50
= 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [
3
H]cryptophycin-52, we found that the compound bound to microtubule ends
in vitro
with high affinity (
K
d
, 47 nM, maximum of ≈19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that ≈5–6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics
in vitro
. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.
Polarity of DNA strand exchange promoted by recombination proteins of the RecA family
Authors: Gupta Ravindra C. , Golub Efim I. , Wold Marc S. , Radding Charles M.
Published: July 2002

Homologs of
Escherichia coli
RecA recombination protein, which have been found throughout the living kingdom, promote homologous pairing and strand exchange. The nucleoprotein filament, within which strand exchange occurs, has been conserved through evolution, but conservation of the polarity of exchange and the significance of that directionality has not been settled. Using oligonucleotides as substrates, and assays based on fluorescence resonance energy transfer (FRET), we distinguished the biased formation of homologous joints at either end of duplex DNA from the subsequent directionality of strand exchange. As with
E. coli
RecA protein, the homologous Rad51 proteins from both
Homo sapiens
(HsRad51) and
Saccharomyces cerevisiae
(ScRad51) propagated DNA strand exchange preferentially in the 5′ to 3′ direction. The data suggest that 5′ to 3′ polarity is a conserved intrinsic property of recombination filaments.
β-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein δ
Authors: Colangelo Anna M. , Johnson Peter F. , Mocchetti Italo
Published: July 2002

Stimulation of β-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (
i
) CLE increased C/EBPδ-binding activity, (
ii
) NGF mRNA levels were increased by overexpressing C/EBPδ, and (
iii
) C/EBPδ increased the activity of an NGF promoter–reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPδ site in the proximal region of the NGF promoter. C/EBPδ appears to be responsible for the BAR-mediated activation of the NGF gene
in vivo
, since CLE elicited a time-dependent increase in C/EBPδ-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPδ is a critical component determining the area-specific expression of NGF in response to BAR stimulation.
Identification of thyroid hormone response elements in the human fatty acid synthase promoter
Authors: Xiong Shunbin , Chirala Subrahmanyam S. , Hsu Matthew H. , Wakil Salih J.
Published: July 2002

To investigate the regulation of the human fatty acid synthase gene by the thyroid hormone triiodothyronine, various constructs of the human fatty acid synthase promoter and the luciferase reporter gene were transfected in combination with plasmids expressing the thyroid hormone and the retinoid X receptors in HepG2 cells. The reporter gene was activated 25-fold by the thyroid hormone in the presence of the thyroid hormone receptor. When both the thyroid hormone and the retinoid X receptors were expressed in HepG2 cells, there was about a 100-fold increase in reporter gene expression. 5′-Deletion analysis disclosed two thyroid hormone response elements, TRE1 (nucleotides −870 to −650) and TRE2 (nucleotides −272 to −40), in the human fatty acid synthase promoter. The presence of thyroid hormone response elements in these two regions of the promoter was confirmed by cloning various fragments of these two regions in the minimal thymidine kinase promoter−luciferase reporter gene plasmid construct and determining reporter gene expression. The results of this cloning procedure and those of electrophoretic mobility shift assays indicated that the sequence GGGTTAcgtcCGGTCA (nucleotides −716 to −731) represents TRE1 and that the sequence GGGTCC (nucleotides −117 to −112) represents TRE2. The sequence of TRE1 is very similar to the consensus sequence of the thyroid hormone response element, whereas the sequence of TRE2 contains only a half-site of the thyroid hormone response element consensus motif because it lacks the direct repeat. The sequences on either side of TRE2 seem to influence its response to the thyroid hormone and retinoid X receptors.
Noncytopathic Sindbis virus RNA vectors for heterologous gene expression
Authors: Agapov Eugene V. , Frolov Ilya , Lindenbach Brett D. , Prágai Béla M. , Schlesinger Sondra , Rice Charles M.
Published: July 2002

Infection of vertebrate cells with alphaviruses normally leads to prodigious expression of virus-encoded genes and a dramatic inhibition of host protein synthesis. Recombinant Sindbis viruses and replicons have been useful as vectors for high level foreign gene expression, but the cytopathic effects of viral replication have limited their use to transient studies. We recently selected Sindbis replicons capable of persistent, noncytopathic growth in BHK cells and describe here a new generation of Sindbis vectors useful for long-term foreign gene expression based on such replicons. Foreign genes of interest as well as the dominant selectable marker puromycinN-acteyltransferase, which confers resistance to the drug puromycin, were expressed as subgenomic transcripts of noncytopathic replicons or defective-interfering genomes complemented in trans by a replicon. Based on these strategies, we developed vectors that can be initiated via either RNA or DNA transfection and analyzed them for their level and stability of foreign gene expression. Noncytopathic Sindbis vectors express reasonably high levels of protein in nearly every cell. These vectors should prove to be flexible tools for the rapid expression of heterologous genes under conditions in which cellular metabolism is not perturbed, and we illustrate their utility with a number of foreign proteins.
TAF
II
mutations disrupt Dorsal activation in the
Drosophila
embryo
Authors: Zhou Jumin , Zwicker Jörk , Szymanski Paul , Levine Michael , Tjian Robert
Published: July 2002

In this study, we present evidence that the Dorsal activator interacts with limiting amounts of the TFIID complex in the
Drosophila
embryo.
In vitro
transcription reactions and protein binding assays implicate the TAF
II
110 and TAF
II
60 subunits of the TFIID complex in contributing to Dorsal-mediated activation. Mutations in TAF
II
110 and TAF
II
60 result in altered patterns of
snail
and
twist
transcription in embryos derived from
dl
/+ females. These results suggest that TAF
II
s contribute to the activation of transcription
in vivo
and support the hypothesis that subunits of TFIID may serve as targets of enhancer binding proteins.
Vaccinia locomotion in host cells: Evidence for the universal involvement of actin-based motility sequences ABM-1 and ABM-2
Authors: Zeile William L. , Condit Richard C. , Lewis Jackie I. , Purich Daniel L. , Southwick Frederick S.
Published: July 2002

Vaccinia uses actin-based motility for virion movement in host cells, but the specific protein components have yet to be defined. A cardinal feature of
Listeria
and
Shigella
actin-based motility is the involvement of vasodilator-stimulated phosphoprotein (VASP). This essential adapter recognizes and binds to actin-based motility 1 (ABM-1) consensus sequences [(D/E)FPPPPX(D/E), X = P or T] contained in
Listeria
ActA and in the p90 host-cell vinculin fragment generated by
Shigella
infection. VASP, in turn, provides the ABM-2 sequences [XPPPPP, X = G, P, L, S, A] for binding profilin, an actin-regulatory protein that stimulates actin filament assembly. Immunolocalization using rabbit anti-VASP antibody revealed that VASP concentrates behind motile virions in HeLa cells. Profilin was also present in these actin-rich rocket tails, and microinjection of 10 μM (intracellular) ABM-2 peptide (GPPPPP)
3
blocked vaccinia actin-based motility. Vinculin did not colocalize with VASP on motile virions and remained in focal adhesion contacts; however, another ABM-1-containing host protein, zyxin, was concentrated at the rear of motile virions. We also examined time-dependent changes in the location of these cytoskeletal proteins during vaccinia infection. VASP and zyxin were redistributed dramatically several hours before the formation of actin rocket tails, concentrating in the viral factories of the perinuclear cytoplasm. Our findings underscore the universal involvement of ABM-1 and ABM-2 docking sites in actin-based motility of
Listeria
,
Shigella
, and now vaccinia.
Stomatal plugs ofDrimys winteri(Winteraceae) protect leaves from mist but not drought
Authors: Feild Taylor S. , Zwieniecki Maciej A. , Donoghue Michael J. , Holbrook N. Michele
Published: July 2002

Two outstanding features of the flowering plant family Winteraceae are the occlusion of their stomatal pores by cutin plugs and the absence of water-conducting xylem vessels. An adaptive relationship between these two unusual features has been suggested whereby stomatal plugs restrict gas exchange to compensate for the presumed poor conductivity of their vesselless wood. This hypothesized connection fueled evolutionary arguments that the vesselless condition is ancestral in angiosperms. Here we show that inDrimys winteri, a tree common to wet forests, these stomatal occlusions pose only a small fixed resistance to water loss. In addition, they modify the humidity response of guard cells such that under high evaporative demand, leaves with plugs lose water at a faster rate than leaves from which the plugs have been experimentally removed. Instead of being adaptations for drought, we present evidence that these cuticular structures function to maintain photosynthetic activity under conditions of excess water on the leaf surface. Stomatal plugs decrease leaf wettability by preventing the formation of a continuous water film that would impede diffusion of CO2into the leaf. Misting of leaves had no effect on photosynthetic rate of leaves with plugs, but resulted in a marked decrease (≈40%) in leaves from which the plugs had been removed. These findings do not support a functional association between stomatal plugs and hydraulic competence and provide a new perspective on debates surrounding the evolution of vessels in angiosperms.
Distinct functional stoichiometry of potassium channel β subunits
Authors: Xu Jia , Yu Weifeng , Wright Jerry M. , Raab Ronald W. , Li Min
Published: July 2002

Shaker-type potassium channels play important roles in determining the electrical excitability of cells. The native channel complex is thought to be formed by four pore-forming α subunits that provide four interaction sites for auxiliary modulatory Kvβ subunits. Because Kvβ subunits possess diverse modulatory activities including either up-regulation or down-regulation of potassium currents, differential assembly of the α–β complex could give rise to diverse current properties. However, the detailed physical and functional stoichiometry of the α–β complex remains unknown. Kvβ1 subunits reduce potassium currents through inactivation, whereas Kvβ2 subunits enhance potassium currents by inhibiting the Kvβ1-mediated inactivation and at the same time by promoting the surface expression of certain potassium channels. In this report we show that Kvβ1 and Kvβ2 of the Shaker-type potassium channels display distinct functional stoichiometry to interact with the Kv1 α subunits, a subfamily of Shaker-type potassium channels. The interaction of Kvβ1 subunits with α subunits is consistent with the α
4
β

n

model, where
n
equals 0, 1, 2, 3, or 4, depending upon the relative concentration of α and β subunits. The α
4
β

n

stoichiometry allows for gradual changes of the Kvβ1-mediated inactivation. In contrast, Kvβ2 subunits self-associate to form oligomers and interact with the α subunits via α
4
β
4
stoichiometry, which permits effective multivalent associations with α subunits. Such distinct functional stoichiometry of Kvβ1 and Kvβ2 provides a molecular mechanism that is well suited to their contrasting activities of up-regulation or down-regulation of potassium currents.
HIV gp120 inhibits the somatotropic axis: A possible GH-releasing hormone receptor mechanism for the pathogenesis of AIDS wasting
Authors: Mulroney Susan E. , McDonnell Kevin J. , Pert Candace B. , Ruff Michael R. , Resch Zachary , Samson Willis K. , Lumpkin Michael D.
Published: July 2002

AIDS is often associated with growth retardation in children and wasting in adults. The dissociated envelope protein of the HIV (HIV-1), gp120, can be found in significant concentrations in the parenchyma and cerebrospinal fluid of brains in infected individuals, even in the earliest stages of HIV-1 disease. On the basis of this and the fact that we observed pentapeptide sequence homology between GH-releasing hormone (GHRH) and the V2 receptor-binding region of gp120, we initiated experiments to determine whether gp120 could affect GH secretion and growthin vivoand/or interact with anterior pituitary GHRH receptorsin vitro. Although acute IV administration of gp120 in conscious rats had no effect on plasma GH levels, acute administration of gp120 (400 ng) into the brain significantly suppressed pulsatile GH release over a 6-h period compared with saline-injected controls. Furthermore, the putative gp120 antagonist, Peptide T (DAPTA), prevented the suppression of GH by gp120. In support of thesein vivofindings, gp120 also significantly (P< 0.05) suppressed GHRH-stimulated GH release in static cultures of dispersed pituitary cells and from cells undergoing perifusion with the peptides. DAPTA prevented the GH suppression by gp120 in both of the pituitary cell paradigms. Furthermore, chronic administration of gp120 into the third ventricle significantly reduced body weight in juvenile rats, compared with saline-injected controls. Thus, gp120 appears to act both at the hypothalamus and pituitary to suppress GH release, and its action at these two locations is associated with a significant loss in body weight in chronically treated young animals. These findings may suggest a specific mechanism for the pathogenesis of wasting in HIV-1 patients that involves blockade of endogenous GHRH receptors by gp120.
Polymerase recognition of synthetic oligodeoxyribonucleotides incorporating degenerate pyrimidine and purine bases
Authors: Hill F. , Loakes D. , Brown D. M.
Published: July 2002

A universal base that is capable of substituting for any of the four natural bases in DNA would be of great utility in both mutagenesis and recombinant DNA experiments. This paper describes the properties of oligonucleotides incorporating two degenerate bases, the pyrimidine base 6
H
,8
H
-3,4-dihydropyrimido[4,5-
c
][1,2]oxazin-7-one and the purine base
N
6
-methoxy-2,6-diaminopurine, designated P and K, respectively. An equimolar mixture of the analogues P and K (called M) acts, in primers, as a universal base. The thermal stability of oligonucleotide duplexes were only slightly reduced when natural bases were replaced by P or K. Templates containing the modified bases were copied by
Taq
polymerase; P behaved as thymine in 60% of copying events and as cytosine in 40%, whereas K behaved as if it were guanine (13%) or adenine (87%). The dUTPase gene of
Caenorhabditis elegans
, which we have found to contain three nonidentical homologous repeats, was used as a model system to test the use of these bases in primers for DNA synthesis. A pair of oligodeoxyribonucleotides, each 20 residues long and containing an equimolar mixture of P and K at six positions, primed with high specificity both T7 DNA polymerase in sequencing reactions and
Taq
polymerase in PCRs; no nonspecific amplification was obtained on genomic DNA of
C. elegans
. Use of P and K can significantly reduce the complexity of degenerate oligonucleotide mixtures, and when used together, P and K can act as a universal base.
Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade
Authors: Purbhoo Marco A. , Sewell Andrew K. , Klenerman Paul , Goulder Philip J. R. , Hilyard Katherine L. , Bell John I. , Jakobsen Bent K. , Phillips Rodney E.
Published: July 2002

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.
Structure of the catalytic domain of avian sarcoma virus integrase with a bound HIV-1 integrase-targeted inhibitor
Authors: Lubkowski Jacek , Yang Fan , Alexandratos Jerry , Wlodawer Alexander , Zhao He , Burke Terrence R. , Neamati Nouri , Pommier Yves , Merkel George , Skalka Anna Marie
Published: July 2002

The x-ray structures of an inhibitor complex of the catalytic core domain of avian sarcoma virus integrase (ASV IN) were solved at 1.9- to 2.0-Å resolution at two pH values, with and without Mn
2+
cations. This inhibitor (Y-3), originally identified in a screen for inhibitors of the catalytic activity of HIV type 1 integrase (HIV-1 IN), was found in the present study to be active against ASV IN as well as HIV-1 IN. The Y-3 molecule is located in close proximity to the enzyme active site, interacts with the flexible loop, alters loop conformation, and affects the conformations of active site residues. As crystallized, a Y-3 molecule stacks against its symmetry-related mate. Preincubation of IN with metal cations does not prevent inhibition, and Y-3 binding does not prevent binding of divalent cations to IN. Three compounds chemically related to Y-3 also were investigated, but no binding was observed in the crystals. Our results identify the structural elements of the inhibitor that likely determine its binding properties.
Autologously up-regulated Fc receptor expression and action in airway smooth muscle mediates its altered responsiveness in the atopic asthmatic sensitized state
Authors: Hakonarson H. , Grunstein M. M.
Published: July 2002

To elucidate the role of IgE-dependent mechanisms in inducing altered airway responsiveness in the atopic asthmatic state, the expression and actions of Fc receptor activation were examined in isolated rabbit tracheal smooth muscle (TSM) tissue and cultured cells passively sensitized with sera from atopic asthmatic patients or nonatopic/nonasthmatic (control) subjects. Relative to control tissues, the atopic asthmatic-sensitized TSM exhibited significantly increased maximal isometric contractility to acetylcholine (P< 0.01) and attenuated maximal relaxation responses and sensitivity (i.e., −log ED50) to isoproterenol (P< 0.005). These changes in agonist responsiveness in atopic sensitized TSM were ablated by pretreating the tissues with a blocking mAb to the low affinity receptor for IgE, FcɛRII (i.e., CD23) or by depleting the sensitizing serum of its immune complexes. Moreover, in complimentary experiments, exogenous administration of IgE immune complexes to naive TSM produced changes in agonist responsiveness that were qualitatively similar to those obtained in the atopic asthmatic-sensitized state. Extended studies further demonstrated that, in contrast to their respective controls, atopic asthmatic serum-sensitized human and rabbit TSM tissue and cultured cells exhibited markedly induced mRNA and cell surface expression of FcɛRII, whereas constitutive expression of the IgG receptor subtype, FcγRIII, was unaltered. Finally, the up-regulated mRNA expression of FcɛRII observed following exposure of TSM to atopic asthmatic serum or to exogenously administered IgE immune complexes was significantly inhibited by pretreating the tissues or cells with anti-CD23 mAb. Collectively, these observations provide evidence demonstrating that the altered agonist responsiveness in atopic asthmatic sensitized airway smooth muscle is largely attributed to IgE-mediated induction of the autologous expression and activation of FcɛRII receptors in the airway smooth muscle itself.
Converting enzyme-independent release of tumor necrosis factor α and IL-1β from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3
Authors: Coeshott Claire , Ohnemus Christie , Pilyavskaya Anna , Ross Sherman , Wieczorek Maciej , Kroona Heather , Leimer Axel H. , Cheronis John
Published: July 2002

Two important cytokines mediating inflammation are tumor necrosis factor α (TNFα) and IL-1β, both of which require conversion to soluble forms by converting enzymes. The importance of TNFα-converting enzyme and IL-1β-converting enzyme in the production of circulating TNFα and IL-1β in response to systemic challenges has been demonstrated by the use of specific converting enzyme inhibitors. Many inflammatory responses, however, are not systemic but instead are localized. In these situations release and/or activation of cytokines may be different from that seen in response to a systemic stimulus, particularly because associations of various cell populations in these foci allows for the exposure of procytokines to the proteolytic enzymes produced by activated neutrophils, neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (Cat G). To investigate the possibility of alternative processing of TNFα and/or IL-1β by neutrophil-derived proteinases, immunoreactive TNFα and IL-1β release from lipopolysaccharide-stimulated THP-1 cells was measured in the presence of activated human neutrophils. Under these conditions, TNFα and IL-1β release was augmented 2- to 5-fold. In the presence of a specific inhibitor of NE and PR3, enhanced release of both cytokines was largely abolished; however, in the presence of a NE and Cat G selective inhibitor, secretory leucocyte proteinase inhibitor, reduction of the enhanced release was minimal. This finding suggested that the augmented release was attributable to PR3 but not NE nor Cat G. Use of purified enzymes confirmed this conclusion. These results indicate that there may be alternative pathways for the production of these two proinflammatory cytokines, particularly in the context of local inflammatory processes.
Cholecystokinin-8 protects central cholinergic neurons against fimbria–fornix lesion through the up-regulation of nerve growth factor synthesis
Authors: Tirassa Paola , Aloe Luigi , Stenfors Carina , Turrini Paolo , Lundeberg Thomas
Published: July 2002

In this study, we demonstrate that cholecystokinin-8 (CCK-8) induces an increase in both nerve growth factor (NGF) protein and NGF mRNA in mouse cortex and hippocampus when i.p. injected at physiological doses. By using fimbria–fornix-lesioned mice, we have also demonstrated that repeated CCK-8 i.p. injections result in recovery of lesion-induced NGF deficit in septum and restore the baseline NGF levels in hippocampus and cortex. Parallel to the effects on NGF, CCK-8 increases choline acetyltransferase (Chat) activity in forebrain when injected in unlesioned mice and counteract the septo-hippocampal Chat alterations in fimbria–fornix-lesioned mice. To assess the NGF involvement in the mechanism by which CCK-8 induces brain Chat, NGF antibody was administrated intracerebrally to saline- and CCK-8-injected mice. We observe that pretreatment with NGF antibody causes a marked reduction of NGF and Chat activity in septum and hippocampus of both saline- and CCK-8-injected mice. This evidence indicates that the CCK-8 effects on cholinergic cells are mediated through the synthesis and release of NGF. Taken together, our results suggest that peripheral administration of CCK-8 may represent a potential experimental model for investigating the effects of endogenous NGF up-regulation on diseases associated with altered brain cholinergic functions.
Functional interaction between human topoisomerase IIα and retinoblastoma protein
Authors: Bhat Uppoor G. , Raychaudhuri Pradip , Beck William T.
Published: July 2002

DNA topoisomerase II—an essential nuclear enzyme in DNA replication and transcription, chromatin segregation, and cell cycle progression—is also a target of clinically useful anticancer drugs. Preliminary observations of a positive correlation between the expression of topoisomerase (topo) IIα and the retinoblastoma protein (Rb) in a series of rhabdomyosarcoma cells prompted us to ask whether these two proteins interact
in vivo
. Using human rhabdomyosarcoma and leukemic cell lines, we found a physical association between topo IIα and Rb protein by reciprocal immunoprecipitation and immunoblotting, in which topo IIα appeared to interact primarily with the underphosphorylated form of Rb. Experiments with truncated glutathione
S
-transferase-Rb fusion proteins and nuclear extracts of Rh1 rhabdomyosarcoma cells indicated that topo IIα binds avidly to the A/B pocket domain of Rb, which contains the intact spacer amino acid sequence. To determine whether this interaction has functional consequences
in vivo
, we expressed wild-type and mutant Rb in human cervical carcinoma cells lacking functional Rb. Wild-type, but not mutant, Rb inhibited topo II activity in nuclear extracts of these transfected cells. Moreover, purified wild-type Rb inhibited the activity of purified human topo IIα, indicating a direct interaction between these two proteins. We conclude that topo IIα associates physically with Rb in interactions that appear to have functional significance.
Femtochemistry
Authors: Tanimura Yoshitaka , Yamashita Koichi , Anfinrud Philip A.
Published: July 2002

The topic of femtochemistry is surveyed from both theoretical and
experimental points of view. A time-dependent wave packet description
of the photodissociation of the O—C—S molecule reveals vibrational
motion in the transition-state region and suggests targets for direct
experimental observation. Theoretical approaches for treating
femtosecond chemical phenomena in condensed phases are featured along
with prospects for laser-controlled chemical reactions by using
tailored ultrashort chirped pulses. An experimental study of the
photoisomerization of retinal in the protein bacteriorhodopsin is
discussed with an aim to gain insight into the potential energy
surfaces on which this remarkably efficient and selective reactions
proceeds. Finally, a prospective view of new frontiers in
femtochemistry is given.
Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens
Authors: Smith Jeffrey S. , Keller Jonathan R. , Lohrey Nancy C. , McCauslin Christine S. , Ortiz Mariaestela , Cowan Kenneth , Spence Sally E.
Published: July 2002

The inability of adenovirus to infect primitive hematopoietic cells presents an obstacle to the use of adenovirus vectors for gene transfer to these cell types. Therefore, expanding the tropism of adenovirus vectors to unique cell surface antigens would be an important development for gene therapy protocols. In this study, we sought to redirect infection of adenovirus vectors to primitive human hematopoietic cells that universally express the c-Kit receptor on their cell surface. To accomplish this, a vector was constructed by covalently linking biotin molecules to recombinant adenovirus, followed by addition of the biotinylated ligand for the c-Kit receptor, stem cell factor (SCF), through an avidin bridge. Gene transfer was directed specifically to c-Kit-positive hematopoietic cell lines, resulting in up to a 2,440-fold increase in luciferase expression with frequencies equivalent to recombinant virus infection of permissive cells. Substitution of biotinylated antibodies directed against c-Kit, CD34 (binds
l
-selectin), and CD44 (hyaluronate receptor) receptors for biotinylated SCF resulted in 50-, 8-, and 260-fold increases in reporter gene expression, respectively, demonstrating that infection also could be redirected through antibody–antigen interactions and through antigens other than growth factor receptors. The versatility of this vector was demonstrated further by infection of primary T cells with vectors targeted with antibodies to CD44 (resting and activated T cells) and biotinylated IL-2 (activated T cells only). Taken together, directly biotinylated adenovirus vectors represent a versatile and efficient method for redirection of virus infection to specific cells.
p21
WAF1/Cip1
functions as a suppressor of malignant skin tumor formation and a determinant of keratinocyte stem-cell potential
Authors: Topley Gabrielle I. , Okuyama Ryuhei , Gonzales José G. , Conti Claudio , Dotto G. Paolo
Published: July 2002

p21
WAF1/Cip1
is one of the best characterized downstream targets of p53, and the growth suppressing function of this cyclin-dependent kinase inhibitor is well established. However, whether p21 exerts a tumor-suppressing function of its own remains to be established. We report here that, similarly to loss of p53, disruption of the p21
WAF1/Cip1
gene results in a markedly increased susceptibility to chemically induced skin carcinoma formation, whereas the number of papillomas is reduced. Previous evidence indicates that malignant versus benign keratinocyte tumor formation is likely to involve distinct target-cell populations with a different commitment to differentiation. In parallel with the increased susceptibility to carcinoma formation, loss of p21
WAF1/Cip1
was found to promote keratinocyte subpopulations with increased growth/differentiation potential, including clonal growth capability, reversible commitment to differentiation, and capability to generate all types of terminally differentiated keratinocytes present
in vivo
, not only in the interfollicular epidermis but also in hair follicles. Thus, these findings have revealed a function of p21 as a suppressor of malignant but not benign skin-tumor formation and a determinant of the growth/differentiation potential of keratinocyte subpopulations.