bluuebunny/crossref_metadata_2025 huggingface dataset:
IL-7 differentially regulates cell cycle progression and HIV-1-based vector infection in neonatal and adult CD4+T cells
Authors: Dardalhon Valérie , Jaleco Sara , Kinet Sandrina , Herpers Bjorn , Steinberg Marcos , Ferrand Christophe , Froger Delphine , Leveau Christelle , Tiberghien Pierre , Charneau Pierre , Noraz Nelly , Taylor Naomi
Published: July 2002

Differences in the immunological reactivity of umbilical cord (UC) and adult peripheral blood (APB) T cells are poorly understood. Here, we show that IL-7, a cytokine involved in lymphoid homeostasis, has distinct regulatory effects on APB and UC lymphocytes. Neither naive nor memory APB CD4+cells proliferated in response to IL-7, whereas naive UC CD4+lymphocytes underwent multiple divisions. Nevertheless, both naive and memory IL-7-treated APB T cells progressed into the G1bphase of the cell cycle, albeit at higher levels in the latter subset. The IL-7-treated memory CD4+lymphocyte population was significantly more susceptible to infection with an HIV-1-derived vector than dividing CD4+UC lymphocytes. However, activation through the T cell receptor rendered UC lymphocytes fully susceptible to HIV-1-based vector infection. These data unveil differences between UC and APB CD4+T cells with regard to IL-7-mediated cell cycle progression and HIV-1-based vector infectivity. This evidence indicates that IL-7 differentially regulates lymphoid homeostasis in adults and neonates.
Activation and autoregulation of DNA-PK from structured single-stranded DNA and coding end hairpins
Authors: Soubeyrand Sébastien , Torrance Heather , Giffin Ward , Gong Wenrong , Schild-Poulter Caroline , Haché Robert J. G.
Published: July 2002

DNA-dependent protein kinase (DNA-PK) acts through an essential
relationship with DNA to participate in the regulation of multiple
cellular processes. Yet the role of DNA as a cofactor in kinase
activity remains to be completely elucidated. For example, although
DNA-PK activity appears to be required for the resolution of hairpin
coding ends in variable diversity joining recombination, kinase
activity remains to be demonstrated from hairpin ends or other DNA
structures. In the present study we report that DNA-PK is strongly
activated from hairpin ends and structured single-stranded DNA, but
that the phosphorylation of many heterologous substrates is blocked
efficiently by inactivation of the kinase through autophosphorylation.
However, substrates that bound efficiently to single-stranded DNA such
as p53 and replication protein A were efficiently phosphorylated by
DNA-PK from structured DNA. DNA-PK also was found to be active toward
heterologous substrates from hairpin ends on double-stranded DNA under
conditions where autophosphorylation was minimized. These results
suggest that the role of DNA-PK in resolving coding end hairpins is
likely to be enzymatic rather than structural, expand understanding of
how DNA-PK binding to structured DNA relates to enzyme activity, and
suggest a mechanism for autoregulatory control of its kinase activity
in the cell.
Nitroxyl anion exerts redox-sensitive positive cardiac inotropy
in vivo
by calcitonin gene-related peptide signaling
Authors: Paolocci Nazareno , Saavedra Walter F. , Miranda Katrina M. , Martignani Cristian , Isoda Takayoshi , Hare Joshua M. , Espey Michael G. , Fukuto Jon M. , Feelisch Martin , Wink David A. , Kass David A.
Published: July 2002

Nitroxyl anion (NO
−
) is the one-electron reduction product of nitric oxide (NO
⋅
) and is enzymatically generated by NO synthase
in vitro
. The physiologic activity and mechanism of action of NO
−
in vivo
remains unknown. The NO
−
generator Angeli's salt (AS, Na
2
N
2
O
3
) was administered to conscious chronically instrumented dogs, and pressure–dimension analysis was used to discriminate contractile from peripheral vascular responses. AS rapidly enhanced left ventricular contractility and concomitantly lowered cardiac preload volume and diastolic pressure (venodilation) without a change in arterial resistance. There were no associated changes in arterial or venous plasma cGMP. The inotropic response was similar despite reflex blockade with hexamethonium or volume reexpansion, indicating its independence from baroreflex stimulation. However, reflex activation did play a major role in the selective venodilation observed under basal conditions. These data contrasted with the pure NO donor diethylamine/NO, which induced a negligible inotropic response and a more balanced veno/arterial dilation. AS-induced positive inotropy, but not systemic vasodilatation, was highly redox-sensitive, being virtually inhibited by coinfusion of
N
-acetyl-
l
-cysteine. Cardiac inotropic signaling by NO
−
was mediated by calcitonin gene-related peptide (CGRP), as treatment with the selective CGRP-receptor antagonist CGRP(8–37) prevented this effect but not systemic vasodilation. Thus, NO
−
is a redox-sensitive positive inotrope with selective venodilator action, whose cardiac effects are mediated by CGRP-receptor stimulation. This fact is evidence linking NO
−
to redox-sensitive cardiac contractile modulation by nonadrenergic/noncholinergic peptide signaling. Given its cardiac and vascular properties, NO
−
may prove useful for the treatment of cardiovascular diseases characterized by cardiac depression and elevated venous filling pressures.
Nanoscale imaging of chemical interactions: Fluorine on graphite
Authors: Kelly Kevin F. , Mickelson Edward T. , Hauge Robert H. , Margrave John L. , Halas Naomi J.
Published: July 2002

Using C
60
-functionalized scanning tunneling microscope tips, we have investigated the adsorption of fluorine on graphite. Based on characteristics of the accompanying electron standing waves, we are able to distinguish the fluorine adatoms that have bonded ionically to the graphite surface from those that have formed covalent bonds with the surface. This result permits determination of the ratio of ionic to covalent C–F bonds on graphite obtained by gas phase fluorination, which seems to be temperatureindependent between 200 and 300°C under the reaction conditions used.
Prostatic intraepithelial neoplasia in mice expressing an androgen receptor transgene in prostate epithelium
Authors: Stanbrough Michael , Leav Irwin , Kwan Paul W. L. , Bubley Glenn J. , Balk Steven P.
Published: July 2002

Prostate cancer (PCa) is an androgen dependent disease that can be treated by androgen ablation therapy, and clinical trials are under way to prevent PCa through the reduction of androgen receptor (AR) activity. However, there are no animal models of AR-mediated prostatic neoplasia, and it remains unclear whether the AR is a positive or negative regulator of cell growth in normal prostate secretory epithelium. To assess the direct effects of the AR in prostate epithelium, a murine AR transgene regulated by the rat probasin promoter (Pb) was used to generate transgenic mice expressing increased levels of AR protein in prostate secretory epithelium. The prostates in younger (<1 year) Pb-mAR transgenic mice were histologically normal, but Ki-67 immunostaining revealed marked increases in epithelial proliferation in ventral prostate and dorsolateral prostate. Older (>1 year) transgenic mice developed focal areas of intraepithelial neoplasia strongly resembling human high-grade prostatic intraepithelial neoplasia (PIN), a precursor to PCa. These results demonstrate that the AR is a positive regulator of cell growth in normal prostate epithelium and provide a model system of AR-stimulated PIN that can be used for assessing preventative hormonal therapies and for identifying secondary transforming events relevant to human PCa.
Adaptive regulation of intestinal lysine metabolism
Authors: van Goudoever J. B. , Stoll B. , Henry J. F. , Burrin D. G. , Reeds P. J.
Published: July 2002

The metabolism of dietary essential amino acids by the gut has a
direct effect on their systemic availability and potentially limits
growth. We demonstrate that, in neonatal pigs bearing portal and
arterial catheters and fed a diet containing 23% protein [high
protein (HP) diet], more than half the intake of essential amino acids
is metabolized by the portal-drained viscera (PDV). Intraduodenal or
i.v. infusions of [U-
13
C]-lysine were used to measure the
appearance across and the use of the tracer by the PDV. In HP-fed pigs,
lysine use by the PDV was derived almost entirely from the arterial
input. In these animals, the small amount of dietary lysine used in
first pass was oxidized almost entirely. Even so, intestinal lysine
oxidation (24 μmol/kg per h) accounted for one-third of whole-body
lysine oxidation (77 μmol/kg per h). Total lysine use by the PDV
was not affected by low protein (LP) feeding (HP, 213 μmol/kg per
h; LP,186 μmol/kg per h). In LP-fed pigs, the use of lysine by the
PDV accounted for more than 75% of its intake. In contrast to HP
feeding, both dietary and arterial lysines were used by the PDV of
LP-fed pigs in nearly equal amounts. Intestinal lysine oxidation was
suppressed completely. We conclude that the PDV are key organs with
respect to amino acid metabolism and that the intestines use a
disproportionately large amount of the dietary supply of amino acids
during protein restriction.
An analysis of neural receptive field plasticity by point process adaptive filtering
Authors: Brown Emery N. , Nguyen David P. , Frank Loren M. , Wilson Matthew A. , Solo Victor
Published: July 2002

Neural receptive fields are plastic: with experience, neurons in many brain regions change their spiking responses to relevant stimuli. Analysis of receptive field plasticity from experimental measurements is crucial for understanding how neural systems adapt their representations of relevant biological information. Current analysis methods using histogram estimates of spike rate functions in nonoverlapping temporal windows do not track the evolution of receptive field plasticity on a fine time scale. Adaptive signal processing is an established engineering paradigm for estimating time-varying system parameters from experimental measurements. We present an adaptive filter algorithm for tracking neural receptive field plasticity based on point process models of spike train activity. We derive an instantaneous steepest descent algorithm by using as the criterion function the instantaneous log likelihood of a point process spike train model. We apply the point process adaptive filter algorithm in a study of spatial (place) receptive field properties of simulated and actual spike train data from rat CA1 hippocampal neurons. A stability analysis of the algorithm is sketched in the
Appendix
. The adaptive algorithm can update the place field parameter estimates on a millisecond time scale. It reliably tracked the migration, changes in scale, and changes in maximum firing rate characteristic of hippocampal place fields in a rat running on a linear track. Point process adaptive filtering offers an analytic method for studying the dynamics of neural receptive fields.
Fringe modulation of Jagged1-induced Notch signaling requires the action of β4galactosyltransferase-1
Authors: Chen Jihua , Moloney Daniel J. , Stanley Pamela
Published: July 2002

Fringe modulates Notch signaling resulting in the establishment of compartmental boundaries in developing organisms. Fringe is a β3
N
-acetylglucosaminyltransferase (β3GlcNAcT) that transfers GlcNAc to
O
-fucose in epidermal growth factor-like repeats of Notch. Here we use five different Chinese hamster ovary cell glycosylation mutants to identify a key aspect of the mechanism of fringe action. Although the β3GlcNAcT activity of manic or lunatic fringe is shown to be necessary for inhibition of Jagged1-induced Notch signaling in a coculture assay, it is not sufficient. Fringe fails to inhibit Notch signaling if the disaccharide generated by fringe action, GlcNAcβ3Fuc, is not elongated. The trisaccharide, Galβ4GlcNAcβ3Fuc, is the minimal
O-
fucose glycan to support fringe modulation of Notch signaling. Of six β4galactosyltransferases (β4GalT) in Chinese hamster ovary cells, only β4GalT-1 is required to add Gal to GlcNAcβ3Fuc, identifying β4GalT-1 as a new modulator of Notch signaling.
Side-chain repacking calculations for predicting structures and stabilities of heterodimeric coiled coils
Authors: Keating Amy E. , Malashkevich Vladimir N. , Tidor Bruce , Kim Peter S.
Published: July 2002

An important goal in biology is to predict from sequence
data the high-resolution structures of proteins and the interactions
that occur between them. In this paper, we describe a computational
approach that can make these types of predictions for a series of
coiled-coil dimers. Our method comprises a dual strategy that augments
extensive conformational sampling with molecular mechanics
minimization. To test the performance of the method, we designed six
heterodimeric coiled coils with a range of stabilities and solved x-ray
crystal structures for three of them. The stabilities and structures
predicted by the calculations agree very well with experimental data:
the average error in unfolding free energies is <1 kcal/mol, and
nonhydrogen atoms in the predicted structures superimpose onto the
experimental structures with rms deviations <0.7 Å. We have also
tested the method on a series of homodimers derived from
vitellogenin-binding protein. The predicted relative stabilities of the
homodimers show excellent agreement with previously published
experimental measurements. A critical step in our procedure is to use
energy minimization to relax side-chain geometries initially selected
from a rotamer library. Our results show that computational methods can
predict interaction specificities that are in good agreement with
experimental data.
Novel unconventional binding site in the variable region of immunoglobulins.
Authors: Rajagopalan K , Pavlinkova G , Levy S , Pokkuluri P R , Schiffer M , Haley B E , Kohler H
Published: July 2002

The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies.
Induction of cytotoxic T lymphocytes by intramuscular immunization with plasmid DNA is facilitated by bone marrow-derived cells.
Authors: Doe B , Selby M , Barnett S , Baenziger J , Walker C M
Published: July 2002

Striated muscle is the predominant site of gene expression after i.m. immunization of plasmid DNA, but it is not clear if myocytes or professional antigen-presenting cells (APCs) of hematopoietic origin present the encoded antigens to class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL). To address this issue, CTL responses were assessed in mice engrafted with immune systems that were partially MHC matched with antigen-producing muscle cells. Spleen cells (sc) from immunocompetent F1 H-2bxd mice were infused into H-2b or H-2d mice carrying the severe combined immunodeficiency (scid) mutation, creating F1sc-->H-2b and F1sc-->H-2d chimeras, respectively. Immunization with DNA plasmids encoding the herpes simplex virus gB or the human immunodeficiency virus gp120 glycoproteins elicited antiviral CTL activity. F1sc-->H-2d chimeras responded to an H-2d-restricted gp120 epitope but not an H-2b restricted gB epitope, whereas F1sc-->H-2b chimeras responded to the H-2b but not the H-2d restricted epitope. This pattern of epitope recognition by the sc chimeras indicated that APCs of recipient (scid) origin were involved in initiation of CTL responses. Significantly, CTL responses against epitopes presented by the mismatched donor class I molecules were elicited if F1 bone marrow cells and sc were transferred into scid recipients before or several days to weeks after DNA immunization. Thus, bone marrow-derived APCs are sufficient for class I MHC presentation of viral antigens after i.m. immunization with plasmid DNA. Expression of plasmid DNA by these APCs is probably not a requirement for CTL priming. Instead, they appear to present proteins synthesized by other host cells.
Evolutionary conservation of sequence elements controlling cytoplasmic polyadenylylation.
Authors: Verrotti A C , Thompson S R , Wreden C , Strickland S , Wickens M
Published: July 2002

Cytoplasmic polyadenylylation is an evolutionarily conserved mechanism involved in the translational activation of a set of maternal messenger RNAs (mRNAs) during early development. In this report, we show by interspecies injections that Xenopus and mouse use the same regulatory sequences to control cytoplasmic poly(A) addition during meiotic maturation. Similarly, Xenopus and Drosophila embryos exploit functionally conserved signals to regulate polyadenylylation during early post-fertilization development. These experiments demonstrate that the sequence elements that govern cytoplasmic polyadenylylation, and hence one form of translational activation, function across species. We infer that the requisite regulatory sequence elements, and likely the trans-acting components with which they interact, have been conserved since the divergence of vertebrates and arthropods.
Peroxisome proliferators induce mouse liver stearoyl-CoA desaturase 1 gene expression.
Authors: Miller C W , Ntambi J M
Published: July 2002

Peroxisome proliferators induce stearoyl-CoA desaturase activity (EC 1.14.99.5) in liver [Kawashima, Y., Hanioka, N., Matsumura, M. & Kozuka, H. (1983) Biochim. Biophys. Acta 752, 259-264]. We analyzed the changes in stearoyl-CoA desaturase 1 (SCD1) mRNA to further define the molecular mechanism for the induction of stearoyl-CoA desaturase by peroxisome proliferators. SCD1 mRNA was analyzed from the livers of BALB/c mice that had been fed diets supplemented with clofibrate or gemfibrozil. Clofibrate was found to induce liver SCD1 mRNA levels 3-fold within 6 hr to a maximum of 22-fold in 30 hr. Gemfibrozil administration resulted in a similar induction pattern. This induction is primarily due to an increase in transcription of the SCD1 gene, as shown by nuclear run-on transcription assays and DNA deletion analysis of transfected SCD1-chloramphenicol acetyltransferase fusion genes. The cis-linked response element for peroxisome proliferator-activated receptor (PPAR) was localized to an AGGTCA consensus sequence between base pairs -664 to -642 of the SCD1 promoter. Clofibrate-mediated induction of SCD1 mRNA was shown to be independent of polyunsaturated fatty acids, with peroxisome proliferators and arachidonic acid having opposite effects on SCD1 mRNA levels. Additionally, the activation of SCD1 mRNA by clofibrate was inhibited 77% by cycloheximide administration. Levels of liver beta-actin and albumin mRNAs were unchanged by these dietary manipulations. Our data show that hepatic SCD1 gene expression is regulated by PPARs and suggest that peroxisome proliferators and poly-unsaturated fatty acids act through distinct mechanisms.
Elevation of intracellular calcium by muscarinic receptor activation induces a block of voltage-activated rat ether-à-go-go channels in a stably transfected cell line.
Authors: Stansfeld C E , Röper J , Ludwig J , Weseloh R M , Marsh S J , Brown D A , Pongs O
Published: July 2002

We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.
Pattern and inhibition-dependent invasion of pyramidal cell dendrites by fast spikes in the hippocampus in vivo.
Authors: Buzsáki G , Penttonen M , Nádasdy Z , Bragin A
Published: July 2002

The invasion of sodium spikes from the soma into dendrites was studied in hippocampal pyramidal cells by simultaneous extracellular and intracellular recordings in anesthetized rats and by simultaneous extracellular recordings of the somatic and dendritic potentials in freely behaving animals. During complex-spike patterns, recorded in the immobile or sleeping animal, dendritic invasion of successive spikes was substantially attenuated. Complex-spike bursts occurred in association with population discharge of CA3-CA1 pyramidal cells (sharp wave field events). Synaptic inhibition reduced the amplitude of sodium spikes in the dendrites and prevented the occurrence of calcium spikes. These findings indicate that (i) the voltage-dependent calcium influx into the dendrites is under the control of inhibitory neurons and (ii) the temporal coincidence of synaptic depolarization and activation of voltage-dependent calcium conductances by the backpropagating spikes during sharp wave bursts may be critical for synaptic plasticity in the intact hippocampus.
SNAP-25 and synaptotagmin involvement in the final Ca(2+)-dependent triggering of neurotransmitter exocytosis.
Authors: Mehta P P , Battenberg E , Wilson M C
Published: July 2002

In neurons, depolarization induces Ca2+ influx leading to fusion of synaptic vesicles docked at the active zone for neurotransmitter release. While a number of proteins have now been identified and postulated to participate in the assembly and subsequent disengagement of a vesicle docking complex for fusion, the mechanism that ultimately triggers neuroexocytosis remains elusive. Using a cell-free, lysed synaptosomal membrane preparation, we show that Ca2+ alone is sufficient to trigger secretion of glutamate and furthermore that Ca(2+)-signaled exocytosis is effectively blocked by antibodies and peptides to SNAP-25, a key constituent of the vesicle docking complex. In addition, Ca2+ inhibits the ability of synaptotagmin, a synaptic vesicle protein proposed as a calcium sensor and triggering device, to associate with this docking complex. These results support a model in which Ca(2+)-dependent triggering of neurotransmission at central synapses acts after ATP-dependent potentiation of the docking-fusion complex for membrane fusion.
Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis.
Authors: Vega Q C , Worby C A , Lechner M S , Dixon J E , Dressler G R
Published: July 2002

The receptor tyrosine kinase RET functions during the development of the kidney and the enteric nervous system, yet no ligand has been identified to date. This report demonstrates that the glial cell line-derived neurotrophic factor (GDNF) activates RET, as measured by tyrosine phosphorylation of the intracellular catalytic domain. GDNF also binds RET with a dissociation constant of 8 nM, and 125I-labeled GDNF can be coimmunoprecipitated with anti-RET antibodies. In addition, exogenous GDNF stimulates both branching and proliferation of embryonic kidneys in organ culture, whereas neutralizing antibodies against GDNF inhibit branching morphogenesis. These data indicate that RET and GDNF are components of a common signaling pathway and point to a role for GDNF in kidney development.
A simple light-driven transmembrane proton pump.
Authors: Sun K , Mauzerall D
Published: July 2002

Light-induced lipophilic porphyrin/aqueous acceptor charge separation across a single lipid-water interface can pump protons across the lipid bilayer when the hydrophobic weak acids, carbonylcyanide m-chlorophenylhydrazone and its p-trifluoromethoxyphenyl analogue, are present. These compounds act as proton carriers across lipid bilayers. In their symmetric presence across the bilayer, the positive currents and voltages produced by the photogeneration of porphyrin cations are replaced by larger negative currents and voltages. The maximum negative current and voltage occur at the pH of maximum dark conductance. The reversed larger current and voltage show a positive ionic charge transport in the same direction as the electron transfer. This transport can form an ion concentration gradient. The movement of protons is verified by an unusual D2O isotope effect that increases the negative ionic current by 2- to 3-fold. These effects suggest that an interfacial pK shift of the weak acid caused by the local electric field of photoformed porphyrin cations/acceptor anions functions as the driving force. The estimated pumping efficiency is 10-30%. Time-resolved results show that proton pumping across the bilayer occurs on the millisecond time scale, similar to that of biological pumps. This light-driven proteinless pump offers a simple model for a prebiological energy transducer.
Anthrax toxin-mediated delivery of a cytotoxic T-cell epitope in vivo.
Authors: Ballard J D , Collier R J , Starnbach M N
Published: July 2002

The protective antigen (PA) component of anthrax toxin mediates entry of the toxin's lethal factor (LF) and edema factor into the cytosolic compartment of mammalian cells. The amino-terminal domain of LF (LFn; 255 amino acids) binds LF to PA, and when fused to heterologous proteins, the LFn domain delivers such proteins to the cytoplasm in the presence of PA. In the current study, we fused a 9-amino acid cytotoxic T-lymphocyte (CTL) epitope (LLO91-99) from an intracellular pathogen, Listeria monocytogenes, to LFn and measured the ability of the resulting LFn-LLO91-99 fusion protein to stimulate a CTL response against the epitope in BALB/c mice. As little as 300 fmol of fusion could stimulate a response. The stimulation was PA-dependent and occurred with the peptide fused to either the amino terminus or the carboxyl terminus of LFn. Upon challenge with L. monocytogenes, mice previously injected with LFn-LLO91-99 and PA showed a reduction of colony-forming units in spleen and liver, relative to nonimmunized control mice. These results indicate that anthrax toxin may be useful as a CTL-peptide delivery system for research and medical applications.
Ca
2+
-induced rebound potentiation of γ-aminobutyric acid-mediated currents requires activation of Ca
2+
/calmodulin-dependent kinase II
Authors: Kano Masanobu , Kano Misao , Fukunaga Kohji , Konnerth Arthur
Published: July 2002

In cerebellar Purkinje neurons, γ-aminobutyric acid
(GABA)-mediated inhibitory synaptic transmission undergoes a
long-lasting “rebound potentiation” after the activation of
excitatory climbing fiber inputs. Rebound potentiation is triggered by
the climbing-fiber-induced transient elevation of intracellular
Ca
2+
concentration and is expressed as a long-lasting
increase of postsynaptic GABA
A
receptor sensitivity. Herein
we show that inhibitors of the
Ca
2+
/calmodulin-dependent protein kinase II
(CaM-KII) signal transduction pathway effectively block the induction
of rebound potentiation. These inhibitors have no effect on the once
established rebound potentiation, on voltage-gated Ca
2+
channel currents, or on the basal inhibitory transmission itself.
Futhermore, a protein phosphatase inhibitor and the intracellularly
applied CaM-KII markedly enhanced GABA-mediated currents in Purkinje
neurons. Our results demonstrate that CaM-KII activation and the
following phosphorylation are key steps for rebound potentiation.
Symmetry, stability, and dynamics of multidomain and multicomponent protein systems
Authors: Blundell Tom L. , Srinivasan N.
Published: July 2002

Symmetry is commonly observed in many biological systems. Here we
discuss representative examples of the role of symmetry in structural
molecular biology. Point group symmetries are observed in many protein
oligomers whose three-dimensional atomic structures have been
elucidated by x-ray crystallography. Approximate symmetry also occurs
in multidomain proteins. Symmetry often confers stability on the
molecular system and results in economical usage of basic components to
build the macromolecular structure. Symmetry is also associated with
cooperativity. Mild perturbation from perfect symmetry may be essential
in some systems for dynamic functions.
Equilibrium constants and free energies in unfolding of proteins in urea solutions
Authors: Klotz Irving M.
Published: July 2002

A novel thermodynamic approach to the reversible unfolding of
proteins in aqueous urea solutions has been developed based on the
premise that urea ligands are bound cooperatively to the macromolecule.
When successive stoichiometric binding constants have values larger
than expected from statistical effects, an equation for moles of bound
urea can be derived that contains imaginary terms. For a very steep
unfolding curve, one can then show that the fraction of
protein unfolded,
B̄
, depends on the square of the
urea concentration,
U
, and is given by

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\begin{equation*}\bar {B}=\frac{{\mathit{A}}^{{\mathit{2}}}_{{\mathit{1}}}{\mathit{e}}^{{\mathrm{{\lambda}}}n\bar {B}}{\mathit{U}}^{{\mathit{2}}}}{{\mathrm{1\hspace{.167em}+\hspace{.167em}}}{\mathit{A}}^{{\mathrm{2}}}_{{\mathrm{1}}}{\mathit{e}}^{{\mathrm{{\lambda}}}\bar {B}}{\mathit{U}}^{{\mathrm{2}}}}{\mathrm{.}}\end{equation*}\end{document}

A
1
2
is the binding constant as
B̄
→ 0, and λ is a parameter that reflects the
augmentation in affinities of protein for urea as the moles bound
increases to the saturation number,
n
. This equation
provides an analytic expression that reproduces the unfolding curve
with good precision, suggests a simple linear graphical procedure for
evaluating
A
1
2
and λ, and leads to the
appropriate standard free energy changes. The calculated
Δ
G
° values reflect the coupling of urea binding with
unfolding of the protein. Some possible implications of this analysis
to protein folding
in vivo
are
described.
Spontaneous human squamous cell carcinomas are killed by a human cytotoxic T lymphocyte clone recognizing a wild-type p53-derived peptide
Authors: Röpke Mads , Hald Jesper , Guldberg Per , Zeuthen Jesper , Nørgaard Lars , Fugger Lars , Svejgaard Arne , Burg Sjoerd Van Der , Nijman Hans W. , Melief Cornelis J. M. , Claesson Mogens H.
Published: July 2002

A cytotoxic T lymphocyte (CTL) clone generatedin vitrofrom the peripheral blood of a healthy HLA-A2-positive individual against a synthetic p53 protein-derived wild-type peptide (L9V) was shown to kill squamous carcinoma cell lines derived from two head and neck carcinomas, which expressed mutant p53 genes, in a L9V/HLA-A2 specific and restricted fashion. Thus, the normal tolerance against endogenously processed p53 protein-derived self-epitopes can be broken by peptide-specificin vitropriming. p53 protein-derived wild-type peptides might thus represent tumor associated target molecules for immunotherapeutical approaches.
Architectural limits on split genes
Authors: Sterner Deborah A. , Carlo Troy , Berget Susan M.
Published: July 2002

Exon/intron architecture varies across the eukaryotic kingdom
with large introns and small exons the rule in vertebrates and the
opposite in lower eukaryotes. To investigate the relationship between
exon and intron size in pre-mRNA processing, internally expanded exons
were placed in vertebrate genes with small and large introns. Both exon
and intron size influenced splicing phenotype. Intron size dictated if
large exons were efficiently recognized. When introns were large, large
exons were skipped; when introns were small, the same large exons were
included. Thus, large exons were incompatible for splicing if and only
if they were flanked by large introns. Both intron and exon size became
problematic at ≈500 nt, although both exon and intron sequence
influenced the size at which exons and introns failed to be recognized.
These results indicate that present-day gene architecture reflects at
least in part limitations on exon recognition. Furthermore, these
results strengthen models that invoke pairing of splice sites during
recognition of pre-mRNAs, and suggest that vertebrate consensus
sequences support pairing across either introns or exons.
Non-iron porphyrins cause tumbling to blue light by an Escherichia coli mutant defective in hemG.
Authors: Yang H , Sasarman A , Inokuchi H , Adler J
Published: July 2002

Previously we showed that an Escherichia coli hemH mutant, defective in the ultimate step of heme synthesis, ferrochelatase, is somewhat better than 100-fold more sensitive than its wild-type parent in tumbling to blue light. Here we explore the effect of a hemG mutant, defective in the penultimate step, protoporphyrinogen oxidase. We found that a hemG mutant also is somewhat better than 100-fold more sensitive in tumbling to blue light compared to its wild-type parent. The amount of non-iron porphyrins accumulated in hemG or hemH mutants was more than 100-fold greater than in wild type. The nature of these accumulated porphyrins is described. When heme was present, as in the wild type, the non-iron (non-heme) porphyrins were maintained at a relatively low concentration and tumbling to blue light at an intensity effective for hemG or hemH did not occur. The function of tumbling to light is most likely to allow escape from the lethality of intense light.
The translational function of nucleotide C1054 in the small subunit rRNA is conserved throughout evolution: genetic evidence in yeast.
Authors: Chernoff Y O , Newnam G P , Liebman S W
Published: July 2002

Mutations at position C1054 of 16S rRNA have previously been shown to cause translational suppression in Escherichia coli. To examine the effects of similar mutations in a eukaryote, all three possible base substitutions and a base deletion were generated at the position of Saccharomyces cerevisiae 18S rRNA corresponding to E. coli C1054. In yeast, as in E. coli, both C1054A (rdn-1A) and C1054G (rdn-1G) caused dominant nonsense suppression. Yeast C1054U (rdn-1T) was a recessive antisuppressor, while yeast C1054-delta (rdn-1delta) led to recessive lethality. Both C1054U and two previously described yeast 18S rRNA antisuppressor mutations, G517A (rdn-2) and U912C (rdn-4), inhibited codon-nonspecific suppression caused by mutations in eukaryotic release factors, sup45 and sup35. However, among these only C1054U inhibited UAA-specific suppressions caused by a UAA-decoding mutant tRNA-Gln (SLT3). Our data implicate eukaryotic C1054 in translational termination, thus suggesting that its function is conserved throughout evolution despite the divergence of nearby nucleotide sequences.
Hypothesis: "Rogue cell"-type chromosomal damage in lymphocytes is associated with infection with the JC human polyoma virus and has implications for oncopenesis.
Authors: Neel J V , Major E O , Awa A A , Glover T , Burgess A , Traub R , Curfman B , Satoh C
Published: July 2002

The hemagglutination inhibition antibody titers against the JC and BK polyoma viruses (JCV and BKV, respectively) are significantly elevated in individuals exhibiting "rogue" cells among their cultured lymphocytes. However, the elevation is so much greater with respect to JCV that the BKV elevation could readily be explained by cross reactivity to the capsid protein of these two closely related viruses. The JCV exhibits high sequence homology with the simian papovavirus, simian virus 40 (SV40), and inoculation of human fetal brain cells with JCV produces polyploidy and chromosomal damage very similar to that produced by SV40. We suggest, by analogy with the effects of SV40, that these changes are due to the action of the viral large tumor antigen, a pluripotent DNA binding protein that acts in both transcription and replication. The implications of these findings for oncogenesis are briefly discussed.
Chemical egg defense in a green lacewing (Ceraeochrysa smithi)
Authors: Eisner T , Attygalle A B , Conner W E , Eisner M , MacLeod E , Meinwald J
Published: July 2002

The green lacewing Ceraeochrysa smithi (Neuroptera, Chrysopidae), like other members of its family, lays its eggs on stalks, but it is unusual in that it coats these stalks with droplets of an oily fluid. The liquid consists of a mixture of fatty acids, an ester, and a series of straight-chain aldehydes. Relative to the eggs of a congeneric chrysopid that lacks stalk fluid, the eggs of C. smithi proved well protected against ants. Components of the fluid, in an assay with a cockroach, proved potently irritant. Following emergence from the egg, C. smithi larvae imbibe the stalk fluid, thereby possibly deriving nutritive benefit, defensive advantage, or both.
Geometric incompatibility in a fault system.
Authors: Gabrielov A , Keilis-Borok V , Jackson D D
Published: July 2002

Interdependence between geometry of a fault system, its kinematics, and seismicity is investigated. Quantitative measure is introduced for inconsistency between a fixed configuration of faults and the slip rates on each fault. This measure, named geometric incompatibility (G), depicts summarily the instability near the fault junctions: their divergence or convergence ("unlocking" or "locking up") and accumulation of stress and deformations. Accordingly, the changes in G are connected with dynamics of seismicity. Apart from geometric incompatibility, we consider deviation K from well-known Saint Venant condition of kinematic compatibility. This deviation depicts summarily unaccounted stress and strain accumulation in the region and/or internal inconsistencies in a reconstruction of block- and fault system (its geometry and movements). The estimates of G and K provide a useful tool for bringing together the data on different types of movement in a fault system. An analog of Stokes formula is found that allows determination of the total values of G and K in a region from the data on its boundary. The phenomenon of geometric incompatibility implies that nucleation of strong earthquakes is to large extent controlled by processes near fault junctions. The junctions that have been locked up may act as transient asperities, and unlocked junctions may act as transient weakest links. Tentative estimates of K and G are made for each end of the Big Bend of the San Andreas fault system in Southern California. Recent strong earthquakes Landers (1992, M = 7.3) and Northridge (1994, M = 6.7) both reduced K but had opposite impact on G: Landers unlocked the area, whereas Northridge locked it up again.
Chromophore-bearing NH2-terminal domains of phytochromes A and B determine their photosensory specificity and differential light lability.
Authors: Wagner D , Fairchild C D , Kuhn R M , Quail P H
Published: July 2002

In early seedling development, far-red-light-induced deetiolation is mediated primarily by phytochrome A (phyA), whereas red-light-induced deetiolation is mediated primarily by phytochrome B (phyB). To map the molecular determinants responsible for this photosensory specificity, we tested the activities of two reciprocal phyA/phyB chimeras in diagnostic light regimes using overexpression in transgenic Arabidopsis. Although previous data have shown that the NH2-terminal halves of phyA and phyB each separately lack normal activity, fusion of the NH2-terminal half of phyA to the COOH-terminal half of phyB (phyAB) and the reciprocal fusion (phyBA) resulted in biologically active phytochromes. The behavior of these two chimeras in red and far-red light indicates: (i) that the NH2-terminal halves of phyA and phyB determine their respective photosensory specificities; (ii) that the COOH-terminal halves of the two photoreceptors are necessary for regulatory activity but are reciprocally inter-changeable and thus carry functionally equivalent determinants; and (iii) that the NH2-terminal halves of phyA and phyB carry determinants that direct the differential light lability of the two molecules. The present findings suggest that the contrasting photosensory information gathered by phyA and phyB through their NH2-terminal halves may be transduced to downstream signaling components through a common biochemical mechanism involving the regulatory activity of the COOH-terminal domains of the photoreceptors.
Molecular characterization of the sodium channel subunits expressed in mammalian cerebellar Purkinje cells
Authors: de Miera Eleazar Vega-Saenz , Rudy Bernardo , Sugimori Mutsuyuki , Llinás Rodolfo
Published: July 2002

Inactivating and noninactivating Na
+
conductances are known to generate, respectively, the rising phase and the prolonged plateau phase of cerebellar Purkinje cell (PC) action potentials. These conductances have different voltage activation levels, suggesting the possibility that two distinct types of ion channels are involved. Single Purkinje cell reverse transcription–PCR from guinea pig cerebellar slices identified two Na
+
channel α subunit transcripts, the orthologs of RBI (rat brain I) and Nach6/Scn8a. The latter we shall name CerIII.
In situ
hybridization histochemistry in rat brain demonstrated broad CerIII expression at high levels in many neuronal groups in the brain and spinal cord, with little if any expression in white matter, or nerve tracts. RBII (rat brain II), the most commonly studied recombinant Na
+
channel α subunit is not expressed in PCs. As the absence of Scn8a has been correlated with motor endplate disease (med), in which transient sodium currents are spared, RBI appears to be responsible for the transient sodium current in PC. Conversely, jolting mice with a mutated Scn8a message demonstrates PC abnormalities in rapid, simple spike generation, linking CerIII to the persistent sodium current.
Assortative fertilization in
Drosophila
Authors: Markow Therese Ann
Published: July 2002

The concept of gametic isolation has its origins in the 1937
edition of T. Dobzhansky’s
Genetics and the Origin of
Species
. Involving either positive assortative fertilization
(as opposed to self-incompatibility) or negative assortative
fertilization, it occurs after mating but prior to fertilization.
Gametic isolation is generally subsumed under either prezygotic or
postmating isolation and thus has not been the subject of extensive
investigation. Examples of assortative fertilization in
Drosophila
are reviewed and compared with those of other
organisms. Potential mechanisms leading to assortative fertilization
are discussed, as are their evolutionary implications.
Helical repeat of DNA in the region of homologous pairing
Authors: Kiianitsa Konstantin , Stasiak Andrzej
Published: July 2002

The process of DNA strand exchange during general genetic recombination is initiated within protein-stabilized synaptic filaments containing homologous regions of interacting DNA molecules. The RecA protein in bacteria and its analogs in eukaryotic organisms start this process by forming helical filamentous complexes on single-stranded or partially single-stranded DNA molecules. These complexes then progressively bind homologous double-stranded DNA molecules so that homologous regions of single- and double-stranded DNA molecules become aligned in register while presumably winding around common axis. The topological assay presented herein allows us to conclude that in synaptic complexes containing homologous single- and double-stranded DNA molecules, all three DNA strands have a helicity of approximately 19 nt per turn.
Allosteric intermediates indicate R2 is the liganded hemoglobin end state
Authors: Schumacher Maria A. , Zheleznova Ekaterina E. , Poundstone Kitty S. , Kluger Ronald , Jones Richard T. , Brennan Richard G.
Published: July 2002

Hemoglobin has been a long-standing paradigm for understanding protein allostery. Here, the x-ray structures of two chemically crosslinked, fully liganded hemoglobins, α
2
β
82
CA
82
β and α
2
β
82
ND
82
β, are described at 2.3 Å and 2.6 Å resolution, respectively. Strikingly, these crosslinked hemoglobins assume intermediate conformations that lie between those of R and the controversial liganded hemoglobin state R2 rather than between R and T. Thus, these structures support only a T ↔ R ↔ R2 allosteric pathway and underscore the physiological importance of the R2 conformation.
Inhibition of Reaper-induced apoptosis by interaction with inhibitor of apoptosis proteins (IAPs)
Authors: Vucic Domagoj , Kaiser William J. , Harvey Alex J. , Miller Lois K.
Published: July 2002

IAPs comprise a family of inhibitors of apoptosis found in viruses and animals.
In vivo
binding studies demonstrated that both baculovirus and
Drosophila
IAPs physically interact with an apoptosis-inducing protein of
Drosophila
, Reaper (RPR), through their baculovirus IAP repeat (BIR) region. Expression of IAPs blocked RPR-induced apoptosis and resulted in the accumulation of RPR in punctate perinuclear locations which coincided with IAP localization. When expressed alone, RPR rapidly disappeared from the cells undergoing RPR-induced apoptosis. Expression of P35, a caspase inhibitor, also blocked RPR-induced apoptosis and delayed RPR decline, but RPR remained cytoplasmic in its location. Mutational analysis of RPR demonstrated that caspases were not directly responsible for RPR disappearance. The physical interaction of IAPs with RPR provides a molecular mechanism for IAP inhibition of RPR’s apoptotic activity.
Tertiary hypothyroidism and hyperglycemia in mice with targeted disruption of the thyrotropin-releasing hormone gene
Authors: Yamada Masanobu , Saga Yumiko , Shibusawa Nobuyuki , Hirato Jyunko , Murakami Masami , Iwasaki Toshiharu , Hashimoto Koshi , Satoh Teturou , Wakabayashi Katsumi , Taketo Makoto M. , Mori Masatomo
Published: July 2002

Thyrotropin-releasing hormone (TRH) is a brain hypothalamic hormone that regulates thyrotropin (TSH) secretion from the anterior pituitary and is ubiquitously distributed throughout the brain and other tissues including pancreas. To facilitate studies into the role of endogenous TRH, we have used homologous recombination to generate mice that lack TRH. These TRH
−/−
mice are viable, fertile, and exhibit normal development. However, they showed obvious hypothyroidism with characteristic elevation of serum TSH level and diminished TSH biological activity. Their anterior pituitaries exhibited an apparent decrease in TSH immunopositive cells that was not due to hypothyroidism. Furthermore, this decrease could be reversed by TRH, but not thyroid hormone replacement, suggesting a direct involvement of TRH in the regulation of thyrotrophs. The TRH
−/−
mice also exhibited hyperglycemia, which was accompanied by impaired insulin secretion in response to glucose. These findings indicate that TRH
−/−
mice provide a model of exploiting tertiary hypothyroidism, and that TRH gene abnormalities cause disturbance of insulin secretion resulting in marked hyperglycemia.