bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of batrachotoxin with the local anesthetic receptor site in transmembrane segment IVS6 of the voltage-gated sodium channel
Authors: Linford Nancy J. , Cantrell Angela R. , Qu Yusheng , Scheuer Todd , Catterall William A.
Published: July 2002

The voltage-gated sodium channel is the site of action of more than six classes of neurotoxins and drugs that alter its function by interaction with distinct, allosterically coupled receptor sites. Batrachotoxin (BTX) is a steroidal alkaloid that binds to neurotoxin receptor site 2 and causes persistent activation. BTX binding is inhibited allosterically by local anesthetics. We have investigated the interaction of BTX with amino acid residues I1760, F1764, and Y1771, which form part of local anesthetic receptor site in transmembrane segment IVS6 of type IIA sodium channels. Alanine substitution for F1764 (mutant F1764A) reduces tritiated BTX-A-20-α-benzoate binding affinity, causing a 60-fold increase in
K
d
. Alanine substitution for I1760, which is adjacent to F1764 in the predicted IVS6 transmembrane alpha helix, causes only a 4-fold increase in
K
d
. In contrast, mutant Y1771A shows no change in BTX binding affinity. For wild-type and mutant Y1771A, BTX shifted the voltage for half-maximal activation ≈40 mV in the hyperpolarizing direction and increased the percentage of noninactivating sodium current to ≈60%. In contrast, these BTX effects were eliminated completely for the F1764A mutant and were reduced substantially for mutant I1760A. Our data suggest that the BTX receptor site shares overlapping but nonidentical molecular determinants with the local anesthetic receptor site in transmembrane segment IVS6 as well as having unique molecular determinants in transmembrane segment IS6, as demonstrated in previous work. Evidently, BTX conforms to a domain–interface allosteric model of ligand binding and action, as previously proposed for calcium agonist and antagonist drugs acting on
l
-type calcium channels.
A PII-like protein in
Arabidopsis
: Putative role in nitrogen sensing
Authors: Hsieh Ming-Hsiun , Lam Hon-Ming , van de Loo Frank J. , Coruzzi Gloria
Published: July 2002

PII is a protein allosteric effector in
Escherichia coli
and other bacteria that indirectly regulates glutamine synthetase at the transcriptional and post-translational levels in response to nitrogen availability. Data supporting the notion that plants have a nitrogen regulatory system(s) includes previous studies showing that the levels of mRNA for plant nitrogen assimilatory genes such as glutamine synthetase (
GLN
) and asparagine synthetase (
ASN
) are modulated by carbon and organic nitrogen metabolites. Here, we have characterized a PII homolog (
GLB1
) in two higher plants,
Arabidopsis thaliana
and
Ricinus communis
(Castor bean). Each plant PII-like protein has high overall identity to
E. coli
PII (50%). Western blot analyses reveal that the plant PII-like protein is a nuclear-encoded chloroplast protein. The PII-like protein of plants appears to be regulated at the transcriptional level in that levels of
GLB1
mRNA are affected by light and metabolites. To initiate studies of the
in vivo
function of the
Arabidopsis
PII-like protein, we have constructed transgenic lines in which PII expression is uncoupled from its native regulation. Analyses of these transgenic plants support the notion that the plant PII-like protein may serve as part of a complex signal transduction network involved in perceiving the status of carbon and organic nitrogen. Thus, the PII protein found in archaea, bacteria, and now in higher eukaryotes (plants) is one of the most widespread regulatory proteins known, providing evidence for an ancestral metabolic regulatory mechanism that may have existed before the divergence of these three domains of life.
Persistence of neutral polymorphisms in Lake Victoria cichlid fish
Authors: Nagl Sandra , Tichy Herbert , Mayer Werner E. , Takahata Naoyuki , Klein Jan
Published: July 2002

Phylogenetic trees for groups of closely related species often have different topologies, depending on the genes used. One explanation for the discordant topologies is the persistence of polymorphisms through the speciation phase, followed by differential fixation of alleles in the resulting species. The existence of transspecies polymorphisms has been documented for alleles maintained by balancing selection but not for neutral alleles. In the present study, transspecific persistence of neutral polymorphisms was tested in the endemic haplochromine species flock of Lake Victoria cichlid fish. Putative noncoding region polymorphisms were identified at four randomly selected nuclear loci and tested on a collection of 12 Lake Victoria species and their putative riverine ancestors. At all loci, the same polymorphism was found to be present in nearly all the tested species, both lacustrine and riverine. Different polymorphisms at these loci were found in cichlids of other East African lakes (Malawi and Tanganyika). The Lake Victoria polymorphisms must have therefore arisen after the flocks now inhabiting the three great lakes diverged from one another, but before the riverine ancestors of the Lake Victoria flock colonized the Lake. Calculations based on the mtDNA clock suggest that the polymorphisms have persisted for about 1.4 million years. To maintain neutral polymorphisms for such a long time, the population size must have remained large throughout the entire period.
Interferon-β gene therapy inhibits tumor formation and causes regression of established tumors in immune-deficient mice
Authors: Qin Xiao-Qiang , Tao Nianjun , Dergay Amie , Moy Pamela , Fawell Stephen , Davis Alan , Wilson James M. , Barsoum James
Published: July 2002

Despite the potential of type 1 interferons (IFNs) for the treatment of cancer, clinical experience with IFN protein therapy of solid tumors has been disappointing. IFN-β has potent antiproliferative activity against most human tumor cellsin vitroin addition to its known immunomodulatory activities. The antiproliferative effect, however, relies on IFN-β concentrations that cannot be achieved by parenteral protein administration because of rapid protein clearance and systemic toxicities. We demonstrate here thatex vivoIFN-β gene transduction by a replication-defective adenovirus in as few as 1% of implanted cells blocked tumor formation. Directin vivoIFN-β gene delivery into established tumors generated high local concentrations of IFN-β, inhibited tumor growth, and in many cases caused complete tumor regression. Because the mice were immune-deficient, it is likely that the anti-tumor effect was primarily through direct inhibition of tumor cell proliferation and survival. Based on these studies, we argue that local IFN-β gene therapy with replication-defective adenoviral vectors might be an effective treatment for some solid tumors.
Perturbed dentate gyrus function in serotonin 5-HT
2C
receptor mutant mice
Authors: Tecott Laurence H. , Logue Sheree F. , Wehner Jeanne M. , Kauer Julie A.
Published: July 2002

Serotonin systems have been implicated in the regulation of hippocampal function. Serotonin 5-HT
2C
receptors are widely expressed throughout the hippocampal formation, and these receptors have been proposed to modulate synaptic plasticity in the visual cortex. To assess the contribution of 5-HT
2C
receptors to the serotonergic regulation of hippocampal function, mice with a targeted 5-HT
2C
-receptor gene mutation were examined. An examination of long-term potentiation at each of four principal regions of the hippocampal formation revealed a selective impairment restricted to medial perforant path–dentate gyrus synapses of mutant mice. This deficit was accompanied by abnormal performance in behavioral assays associated with dentate gyrus function. 5-HT
2C
receptor mutants exhibited abnormal performance in the Morris water maze assay of spatial learning and reduced aversion to a novel environment. These deficits were selective and were not associated with a generalized learning deficit or with an impairment in the discrimination of spatial context. These results indicate that a genetic perturbation of serotonin receptor function can modulate dentate gyrus plasticity and that plasticity in this structure may contribute to neural mechanisms underlying hippocampus-dependent behaviors.
Differential regulation of transcription: Repression by unactivated mitogen-activated protein kinase Kss1 requires the Dig1 and Dig2 proteins
Authors: Bardwell Lee , Cook Jeanette G. , Zhu-Shimoni Judith X. , Voora Deepak , Thorner Jeremy
Published: July 2002

Kss1, a yeast mitogen-activated protein kinase (MAPK), in its unphosphorylated (unactivated) state binds directly to and represses Ste12, a transcription factor necessary for expression of genes whose promoters contain filamentous response elements (FREs) and genes whose promoters contain pheromone response elements (PREs). Herein we show that two nuclear proteins, Dig1 and Dig2, are required cofactors in Kss1-imposed repression. Dig1 and Dig2 cooperate with Kss1 to repress Ste12 action at FREs and regulate invasive growth in a naturally invasive strain. Kss1-imposed Dig-dependent repression of Ste12 also occurs at PREs. However, maintenance of repression at PREs is more dependent on Dig1 and/or Dig2 and less dependent on Kss1 than repression at FREs. In addition, derepression at PREs is more dependent on MAPK-mediated phosphorylation than is derepression at FREs. Differential utilization of two types of MAPK-mediated regulation (binding-imposed repression and phosphorylation-dependent activation), in combination with distinct Ste12-containing complexes, contributes to the mechanisms by which separate extracellular stimuli that use the same MAPK cascade can elicit two different transcriptional responses.
mtDNA recombination in a natural population
Authors: Saville Barry J. , Kohli Yatika , Anderson James B.
Published: July 2002

Variation in mtDNA has been used extensively to draw inferences in
phylogenetics and population biology. In the majority of eukaryotes
investigated, transmission of mtDNA is uniparental and clonal, with
genotypic diversity arising from mutation alone. In other eukaryotes,
the transmission of mtDNA is biparental or primarily uniparental with
the possibility of “leakage” from the minority parent. In these
cases, heteroplasmy carries the potential for recombination between
mtDNAs of different descent. In fungi, such mtDNA recombination has
long been documented but only in laboratory experiments and only under
conditions in which heteroplasmy is ensured. Despite this experimental
evidence, mtDNA recombination has not been to our knowledge documented
in a natural population. Because evidence from natural populations is
prerequisite to understanding the evolutionary impact of mtDNA
recombination, we investigated the possibility of mtDNA recombination
in an organism with the demonstrated potential for heteroplasmy in
laboratory matings. Using nucleotide sequence data, we report here that
the genotypic structure of mtDNA in a natural population of the
basidiomycete fungus
Armillaria gallica
is inconsistent
with purely clonal mtDNA evolution and is fully consistent with mtDNA
recombination.
The neural development and organization of letter recognition: Evidence from functional neuroimaging, computational modeling, and behavioral studies
Authors: Polk Thad A. , Farah Martha J.
Published: July 2002

Although much of the brain’s functional organization is
genetically predetermined, it appears that some noninnate functions can
come to depend on dedicated and segregated neural tissue. In this
paper, we describe a series of experiments that have investigated the
neural development and organization of one such noninnate function:
letter recognition. Functional neuroimaging demonstrates that letter
and digit recognition depend on different neural substrates in some
literate adults. How could the processing of two stimulus categories
that are distinguished solely by cultural conventions become segregated
in the brain? One possibility is that correlation-based learning in the
brain leads to a spatial organization in cortex that reflects the
temporal and spatial clustering of letters with letters in the
environment. Simulations confirm that environmental co-occurrence does
indeed lead to spatial localization in a neural network that uses
correlation-based learning. Furthermore, behavioral studies confirm one
critical prediction of this co-occurrence hypothesis, namely, that
subjects exposed to a visual environment in which letters and digits
occur together rather than separately (postal workers who process
letters and digits together in Canadian postal codes) do indeed show
less behavioral evidence for segregated letter and digit processing.
A role for B cells in the development of T cell helper function in a malaria infection in mice
Authors: Langhorne Jean , Cross Caroline , Seixas Elsa , Li Ching , von der Weid Thierry
Published: July 2002

B cell knockout mice are unable to clear a primary erythrocytic infection ofPlasmodium chabaudi chabaudi. However, the early acute infection is controlled to some extent, giving rise to a chronic relapsing parasitemia that can be reduced either by drug treatment or by adoptive transfer of B cells. Similar to mice rendered B-cell deficient by lifelong treatment with anti-μ antibodies, B cell knockout mice (μMT) retain a predominant CD4+Th1-like response to malarial antigens throughout a primary infection. This contrasts with the response seen in control C57BL/6 mice in which the CD4+T-cell response has switched to that characteristic of Th2 cells at the later stages of infection, manifesting efficient help for specific antibodiesin vitroand interleukin 4 production. Both chloroquine and adoptive transfer of immune B cells reduced parasite load. However, the adoptive transfer of B cells resulted in a Th2 response in recipient μMT mice, as indicated by a relative increase in the precursor frequency of helper cells for antibody production. These data support the idea that B cells play a role in the regulation of CD4+T subset responses.
Ectopic induction of Clb2 in early G
1
phase is sufficient to block prereplicative complex formation in
Saccharomyces cerevisiae
Authors: Detweiler Corrella S. , Li Joachim J.
Published: July 2002

Eukaryotic cells ensure the stable propagation of their genome by coupling each round of DNA replication (S phase) to passage through mitosis (M phase). This control is exerted at the initiation of replication, which occurs at multiple origins throughout the genome. Once an origin has initiated, reinitiation is blocked until the completion of mitosis, ensuring that DNA is replicated at most once per cell cycle. Recent studies in several organisms have suggested a model in which S- and M-phase promoting cyclin-dependent kinases prevent reinitiation by blocking the repetition of an early step in the initiation reaction. In budding yeast, this regulation is thought to involve inhibition of prereplicative complex (pre-RC) formation at origins by S and M phase-promoting Clb kinases. To date, however, there has been no direct demonstration that these kinases can perform such an important function. In this report we provide such a confirmation by showing that ectopic induction in G
1
phase of a mitotic Clb, Clb2, is sufficient to inhibit DNA replication and does so by preventing pre-RC formation. This inhibition requires that Clb2 be induced before Cdc6, an initiation protein required for pre-RC formation; once pre-RCs have formed, Clb2 can no longer inhibit initiation. These results support the notion that during the normal cell cycle reassembly of the pre-RC, and hence reinitiation at an origin, is directly inhibited by S and M phase-promoting cyclin-dependent kinases.
How Cro and λ-repressor distinguish between operators: The structural basis underlying a genetic switch
Authors: Albright Ronald A. , Matthews Brian W.
Published: July 2002

Knowledge of the three-dimensional structures of the λ-Cro and λ-repressor proteins in complex with DNA has made it possible to evaluate how these proteins discriminate between different operators in phage λ. As anticipated in previous studies, the helix–turn–helix units of the respective proteins bind in very different alignments. In Cro the recognition helices are 29 Å apart and are tilted by 55° with respect to each other, but bind parallel to the major groove of the DNA. In λ-repressor [Beamer, L. J. & Pabo, C. O. (1992)
J. Mol. Biol
. 227, 177–196] the helices are 34 Å apart and are essentially parallel to each other, but are inclined to the major grooves. The DNA is much more bent when bound by Cro than in the case with λ-repressor. The first two amino acids of the recognition helices of the two proteins, Gln-27 and Ser-28 in Cro, and Gln-44 and Ser-45 in λ-repressor, make very similar interactions with the invariant bps 2 and 4. There are also analogous contacts between the thymine of bp 5 and, respectively, the backbone of Ala-29 of Cro and the backbone of Gly-46 of λ-repressor. Otherwise, however, unrelated parts of the two proteins are used in sequence-specific recognition. It appears that similar contacts to the invariant or almost invariant bps (especially 2 and 4) are used by both Cro and λ-repressor to differentiate the operator sites as a group from other sites on the DNA. The discrimination of Cro and λ-repressor between their different operators is more subtle and seems to be achieved primarily through differences in van der Waals contacts at bp 3′, together with weaker, less direct effects at bps 5′ and 8′, all in the nonconsensus half of the operators. The results provide further support for the idea that there is no simple code for DNA-protein recognition.
Regulation of the
Escherichia coli
water channel gene
aqpZ
Authors: Calamita Giuseppe , Kempf Bettina , Bonhivers Mélanie , Bishai William R. , Bremer Erhard , Agre Peter
Published: July 2002

Osmotic movement of water across bacterial cell membranes is postulated to be a homeostatic mechanism for maintaining cell turgor. The molecular water transporter remained elusive until discovery of the
Escherichia coli
water channel, AqpZ, however the regulation of the
aqpZ
gene expression and physiological function of the AqpZ protein are unknown. Northern analysis revealed a transcript of 0.7 kb, confirming the monocistronic nature of
aqpZ.
Regulatory studies performed with an
aqpZ
∷
lacZ
low copy plasmid demonstrate enhanced expression during mid-logarithmic growth, and expression of the gene is dependent upon the extracellular osmolality, which increased in hypoosmotic environments but strongly reduced in hyperosmolar NaCl or KCl. While disruption of the chromosomal
aqpZ
is not lethal for
E. coli
, the colonies of the
aqpZ
knockout mutant are smaller than those of the parental wild-type strain. When cocultured with parental wild-type
E. coli,
the
aqpZ
knockout mutant exhibits markedly reduced colony formation when grown at 39°C. Similarly, the
aqpZ
knockout mutant also exhibits greatly reduced colony formation when grown at low osmolality, but this phenotype is reversed by overexpression of AqpZ protein. These results implicate AqpZ as a participant in the adaptive response of
E. coli
to hypoosmotic environments and indicate a requirement for AqpZ by rapidly growing cells.
Marine latitudinal diversity gradients: Tests of causal hypotheses
Authors: Roy Kaustuv , Jablonski David , Valentine James W. , Rosenberg Gary
Published: July 2002

Latitudinal diversity gradients are first-order expressions of diversity patterns both on land and in the oceans, although the current hypotheses that seek to explain them are based chiefly on terrestrial data. We have assembled a database of the geographic ranges of 3,916 species of marine prosobranch gastropods living on the shelves of the western Atlantic and eastern Pacific Oceans, from the tropics to the Arctic Ocean. Western Atlantic and eastern Pacific diversities are similar, and the diversity gradients are strikingly similar despite many important physical and historical differences between the oceans. This shared diversity pattern cannot be explained by: (i) latitudinal differences in species range-length (Rapoport’s rule); (ii) species-area effects; or (iii) recent geologic histories. One parameter that does correlate significantly with diversity in both oceans is solar energy input, as represented by average sea surface temperature. If this correlation is causal, sea surface temperature is probably linked to diversity through some aspect of productivity. In this case, diversity is an evolutionary outcome of trophodynamic processes inherent in ecosystems, and not just a byproduct of physical geographies.
Developmental rescue ofDrosophilacephalic defects by the humanOtxgenes
Authors: Nagao Tomoko , Leuzinger Sandra , Acampora Dario , Simeone Antonio , Finkelstein Robert , Reichert Heinrich , Furukubo-Tokunaga Katsuo
Published: July 2002

The molecular mechanisms of head development are a central question in vertebrate and invertebrate developmental biology. The anteriorly expressed homeobox geneotdinDrosophilaand its homologOtxin mouse are required for the early development of the most anterior part of the body, suggesting that a fundamental genetic program of cephalic development might be conserved between vertebrates and invertebrates. We have examined this hypothesis by introducing the humanOtxgenes into flies. By inducing expression of the humanOtxhomologs with a heat shock promoter, we found that bothOtx1andOtx2functionally complement the cephalic defects of a flyotdmutant through specific activation and inactivation of downstream genes. Combined with previous morphological studies, these results are consistent with the view that a common molecular ground plan of cephalization was invented before the diversification of the protostome and the deuterostome in the course of metazoan evolution.
Effect of intravenous infusions of 12-O-tetradecanoylphorbol-13-acetate (TPA) in patients with myelocytic leukemia: Preliminary studies on therapeutic efficacy and toxicity
Authors: Han Zheng Tao , Zhu Xiao Xiu , Yang Ru Yu , Sun Jun Zhong , Tian Guo Fang , Liu Xin Jian , Cao Guo Shun , Newmark Harold L. , Conney Allan H. , Chang Richard L.
Published: July 2002

Studies by several investigators have shown that 12–0-tetradecanoylphorbol-13-acetate (TPA) is an extraordinarily potent stimulator of differentiation of cultured human promyelocytic leukemia cells
in vitro
. In the present study, TPA was administered to humans by i.v. infusion without irreversible toxicity, and it was shown to have pharmacological activity for the treatment of myelocytic leukemia in patients refractory to cytosine arabinoside (Ara C), retinoic acid, and other antileukemic drugs. Marked decreases in bone marrow myeloblasts as well as temporary remission of disease symptoms were observed when TPA was administered alone or in combination with vitamin D
3
and Ara C. Additional studies with TPA after the determination of optimum dosing regimens are needed to determine whether long-lasting or permanent remissions of myelocytic leukemia can be achieved. Transient and reversible side effects were observed after a 1-mg i.v. dose of TPA, but these adverse effects became less intense or disappeared when a lower dose of TPA was used. The results of this study indicate a therapeutic effect of TPA in patients with myelocytic leukemia.
12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced increase in depressed white blood cell counts in patients treated with cytotoxic cancer chemotherapeutic drugs
Authors: Han Zheng Tao , Tong Yun Ke , He Lin Min , Zhang Yang , Sun Jun Zhong , Wang Tian Yi , Zhang Hong , Cui Ya Ling , Newmark Harold L. , Conney Allan H. , Chang Richard L.
Published: July 2002

Fifty-two patients with solid tumors had depressed white blood cell and neutrophil counts because of prior treatment with cytotoxic cancer chemotherapeutic drugs. These patients were given one or more i.v. infusions of 0.125–0.25 mg of 12-O-tetradecanoylphorbol-13-acetate (TPA), and this treatment increased the low white blood cell and neutrophil counts toward the normal range. The average white blood cell and neutrophil counts were 2.55 × 10
9
/liter and 1.76 × 10
9
/liter, respectively, before treatment with TPA. After one or more i.v. infusions of TPA, the white blood cell and neutrophil counts increased to peak values of 5.92 × 10
9
/liter and 4.76 × 10
9
/liter, respectively, within a few days. Most patients had increased levels of white blood cells and neutrophils by 24 hr after a single i.v. infusion of 0.25 mg TPA. Elevated levels were observed for at least 3 days. This study demonstrates that treatment with parenteral TPA is feasible with useful biological activity. Only mild and reversible side effects were observed.
The SRm160/300 splicing coactivator is required for exon-enhancer function
Authors: Eldridge Adam G. , Li Yu , Sharp Phillip A. , Blencowe Benjamin J.
Published: July 2002

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing “coactivator,” SRm160/300, which contains SRm160 (the
SR
nuclear
m
atrix protein of
160
kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the
Drosophila doublesex
gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the
Drosophila
alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound “activators” and the snRNP machinery of the spliceosome.
The
Arabidopsis thaliana HY1
locus, required for phytochrome-chromophore biosynthesis, encodes a protein related to heme oxygenases
Authors: Davis Seth J. , Kurepa Jasmina , Vierstra Richard D.
Published: July 2002

The
hy1
mutants of
Arabidopsis thaliana
fail to make the phytochrome-chromophore phytochromobilin and therefore are deficient in a wide range of phytochrome-mediated responses. Because this defect can be rescued by feeding seedlings biliverdin IXα, it is likely that the mutations affect an enzyme that converts heme to this phytochromobilin intermediate. By a combination of positional cloning and candidate-gene isolation, we have identified the
HY1
gene and found it to be related to cyanobacterial, algal, and animal heme oxygenases. Three independent alleles of
hy1
contain DNA lesions within the
HY1
coding region, and a genomic sequence spanning the
HY1
locus complements the
hy1–1
mutation.
HY1
is a member of a gene family and is expressed in a variety of
A. thaliana
tissues. Based on its homology, we propose that
HY1
encodes a higher-plant heme oxygenase, designated
At
HO1, responsible for catalyzing the reaction that opens the tetrapyrrole ring of heme to generate biliverdin IXα.
Vortices in rotating superfluid
3
He
Authors: Lounasmaa Olli V. , Thuneberg Erkki
Published: July 2002

In this review we first present an introduction to
3
He and to the ROTA collaboration under which most of the knowledge on vortices in superfluid
3
He has been obtained. In the physics part, we start from the exceptional properties of helium at millikelvin temperatures. The dilemma of rotating superfluids is presented. In
4
He and in
3
He-B the problem is solved by nucleating an array of singular vortex lines. Their experimental detection in
3
He by NMR is described next. The vortex cores in
3
He-B have two different structures, both of which have spontaneously broken symmetry. A spin-mass vortex has been identified as well. This object is characterized by a flow of spins around the vortex line, in addition to the usual mass current. A great variety of vortices exist in the A phase of
3
He; they are either singular or continuous, and their structure can be a line or a sheet or fill the whole liquid. Altogether seven different types of vortices have been detected in
3
He by NMR. We also describe briefly other experimental methods that have been used by ROTA scientists in studying vortices in
3
He and some important results thus obtained. Finally, we discuss the possible applications of experiments and theory of
3
He to particle physics and cosmology. In particular, we report on experiments where superfluid
3
He-B was heated locally by absorption of single neutrons. The resulting events can be used to test theoretical models of the Big Bang at the beginning of our universe.
Binding of buried structural water increases the flexibility of proteins
Authors: Fischer Stefan , Verma Chandra S.
Published: July 2002

Water deeply buried in proteins is considered to be an integral part of the folded structure. Such structural water molecules make strong H bonds with polar groups of the surrounding protein and therefore are believed to tighten the protein matrix. Surprisingly, our computational analysis of the binding of a buried water molecule to bovine pancreatic trypsin inhibitor shows that the protein actually becomes more flexible, as revealed by an increase in the vibrational entropy. We find that this effect must be common in proteins, because the large entropic cost of immobilizing a single water molecule [−TΔS = 20
.
6 kcal/mol (1 kcal = 4.18 kJ) for the lost translational and rotational degrees of freedom] can only be partly compensated by water–protein interactions, even when they are nearly perfect, as in the case of bovine pancreatic trypsin inhibitor (ΔE = −19
.
8 kcal/mol), leaving no room for a further decrease in entropy from protein tightening. This study illustrates the importance of considering changes in protein flexibility (which in this case favor binding by 3
.
5 kcal/mol) for the prediction of ligand binding affinities.
The dynamics of structural deformations of immobilized single light-harvesting complexes
Authors: Bopp Martin A. , Sytnik Alexander , Howard Tina D. , Cogdell Richard J. , Hochstrasser Robin M.
Published: July 2002

Single assemblies of the intact light-harvesting complex LH2 from
Rhodopseudomonas acidophila
were bound to mica surfaces at 300 K and examined by observing their fluorescence after polarized light excitation. The complexes are generally not cylindrically symmetric. They act like elliptic absorbers, indicating that the high symmetry found in crystals of LH2 is not present when the molecules are immobilized on mica. The ellipticity and the principal axes of the ellipses fluctuate on the time scale of seconds, indicating that there is a mobile structural deformation. The B850 ring of cofactors shows significantly less asymmetry than B800. The photobleaching strongly depends on the presence of oxygen.
DNA specificity enhanced by sequential binding of protein monomers
Authors: Kohler Jennifer J. , Metallo Steven J. , Schneider Tanya L. , Schepartz Alanna
Published: July 2002

Transcriptional activation often requires the rapid assembly of complexes between dimeric transcription factors and specific DNA sites. Here we show that members of the basic region leucine zipper and basic region helix–loop–helix zipper transcription factor families follow an assembly pathway in which two protein monomers bind DNA sequentially and form their dimerization interface while bound to DNA. Nonspecific protein or DNA competitors have little effect on the rate of assembly along this pathway, but slow a competing pathway in which preformed dimers bind DNA. The sequential monomer-binding pathway allows the protein to search for and locate a specific DNA site more quickly, resulting in greater specificity prior to equilibrium.
Active-site structure of the soluble quinoprotein glucose dehydrogenase complexed with methylhydrazine: A covalent cofactor-inhibitor complex
Authors: Oubrie Arthur , Rozeboom Henriëtte J. , Dijkstra Bauke W.
Published: July 2002

Soluble glucose dehydrogenase (s-GDH) from the bacterium
Acinetobacter calcoaceticus
is a classical quinoprotein. It requires the cofactor pyrroloquinoline quinone (PQQ) to catalyze the oxidation of glucose to gluconolactone. The precise catalytic role of PQQ in s-GDH and several other PQQ-dependent enzymes has remained controversial because of the absence of comprehensive structural data. We have determined the crystal structure of a ternary complex of s-GDH with PQQ and methylhydrazine, a competitive inhibitor of the enzyme. This complex, refined at 1.5-Å resolution to an
R
factor of 16.7%, affords a detailed view of a cofactor-binding site of s-GDH. Moreover, it presents the first direct observation of covalent PQQ adduct in the active-site of a PQQ-dependent enzyme, thereby confirming previous evidence that the C5 carbonyl group of the cofactor is the most reactive moiety of PQQ.
Inhibitory role of the host apoptogenic gene
PKR
in the establishment of persistent infection by encephalomyocarditis virus in U937 cells
Authors: Yeung Michael C. , Chang Danny L. , Camantigue Randell E. , Lau Allan S.
Published: July 2002

Persistent infections by viruses such as HIV-1 and hepatitis B virus can pose long-term health hazards. Because establishment of persistent infections involves close interactions and adjustments in both host and virus, it would be informative to establish a paradigm with which a normally cytolytic viral infection can be easily converted to persistent infection, so that the different stages in developing persistent infection can be examined. Such a model system is described in this paper. Highly cytolytic encephalomyocarditis virus (EMCV) infection was shifted to persistent infection as a result of repressed expression of the double-stranded RNA-dependent protein kinase (PKR) in the promonocytic U937 cells. Because of the apoptogenic potential of PKR, a deficiency of PKR resulted in a delay in virus-induced apoptosis in EMCV-infected U937 cells, allowing the eventual establishment of persistent EMCV infection in these cells (U9K-AV2). That this was a bona fide persistent infection was demonstrated by the ability of infected cells to propagate as long-term virus-shedding cultures; electron microscopy studies showing presence of intracellular EMCV virions and chromatin condensation; detection of virus-induced chromosomal DNA fragmentation and sustained expression of apoptogenic p53 and IL-1β converting enzyme; and demonstration of active EMCV transcription by reverse transcription–PCR. In addition, a host-virus coevolution was observed in U9K-AV2 cultures over time: U9K-AV2 cells exhibited slower growth rates, resistance to viral super-infection, and cessation of IFN-α synthesis, whereas the infectivity of EMCV was drastically attenuated. Finally, data are presented on the suitability of this model to study establishment of persistent infection by other viruses such as Sendai virus and reovirus.
Visualizing the kinetics of tumor-cell clearance in living animals
Authors: Sweeney Thomas J. , Mailänder Volker , Tucker Amanda A. , Olomu Adesuwa B. , Zhang Weisheng , Cao Yu-an , Negrin Robert S. , Contag Christopher H.
Published: July 2002

Evaluation of potential antineoplastic therapies would be enhanced by noninvasive detection of tumor cells in living animals. Because light is transmitted through mammalian tissues, it was possible to use bioluminescence to monitor (both externally and quantitatively) growth and regression of labeled human cervical carcinoma (HeLa) cells engrafted into immunodeficient mice. The efficacy of both chemotherapy and immunotherapeutic treatment withex vivoexpanded human T cell-derived effector cells was evaluated. In the absence of therapy, animals showed progressive increases in signal intensity over time. Animals treated with cisplatin had marked reductions in tumor signal; 5′-fluorouracil was less effective, and cyclophosphamide was ineffective. Immunotherapy dramatically reduced signals at high effector-to-target cell ratios, and significant decreases were observed with lower ratios. This model system allowed sensitive, quantitative, real-time spatiotemporal analyses of the dynamics of neoplastic cell growth and facilitated rapid optimization of effective treatment regimens.
Energetic considerations of ciliary beating and the advantage of metachronal coordination
Authors: Gueron Shay , Levit-Gurevich Konstantin
Published: July 2002

The internal mechanism of cilia is among the most ancient biological motors on an evolutionary scale. It produces beat patterns that consist of two phases: during the effective stroke, the cilium moves approximately as a straight rod, and during the recovery stroke, it rolls close to the surface in a tangential motion. It is commonly agreed that these two phases are designed for efficient functioning: the effective stroke encounters strong viscous resistance and generates thrust, whereas the recovery stroke returns the cilium to starting position while avoiding viscous resistance. Metachronal coordination between cilia, which occurs when many of them beat close to each other, is believed to be an autonomous result of the hydrodynamical interactions in the system. Qualitatively, metachronism is perceived as a way for reducing the energy expenditure required for beating. This paper presents a quantitative study of the energy expenditure of beating cilia, and of the energetic significance of metachronism. We develop a method for computing the work done by model cilia that beat in a viscous fluid. We demonstrate that for a single cilium, beating in water, the mechanical work done during the effective stroke is approximately five times the amount of work done during the recovery stroke. Investigation of multicilia configurations shows that having neighboring cilia beat metachronally is energetically advantageous and perhaps even crucial for multiciliary functioning. Finally, the model is used to approximate the number of dynein arm attachments that are likely to occur during the effective and recovery strokes of a beat cycle, predicting that almost all of the available dynein arms should participate in generating the motion.
Kinesin’s processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains
Authors: Hancock William O. , Howard Jonathon
Published: July 2002

Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3–6 s
−1
and that detachment from the microtubule occurs at a similar rate (3 s
−1
). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP⋅P
i
state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP⋅P
i
state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.
Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases
Authors: Zindy Frederique , Cunningham Justine J. , Sherr Charles J. , Jogal Sachin , Smeyne Richard J. , Roussel Martine F.
Published: July 2002

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19
Ink4d
and p27
Kip1
, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of
Ink4d
- null with
Kip1
-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G
2
and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19
Ink4d
and p27
Kip1
cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.
Sequential and coordinated action of phytochromes A and B during
Arabidopsis
stem growth revealed by kinetic analysis
Authors: Parks Brian M. , Spalding Edgar P.
Published: July 2002

Photoreceptor proteins of the phytochrome family mediate light-induced inhibition of stem (hypocotyl) elongation during the development of photoautotrophy in seedlings. Analyses of overt mutant phenotypes have established the importance of phytochromes A and B (phyA and phyB) in this developmental process, but kinetic information that would augment emerging molecular models of phytochrome signal transduction is absent. We have addressed this deficiency by genetically dissecting phytochrome-response kinetics, after having solved the technical issues that previously limited growth studies of small
Arabidopsis
seedlings. We show here, with resolution on the order of minutes, that phyA initiated hypocotyl growth inhibition upon the onset of continuous red light. This primary contribution of phyA began to decrease after 3 hr of irradiation, the same time at which immunochemically detectable phyA disappeared and an exclusively phyB-dependent phase of inhibition began. The sequential and coordinated actions of phyA and phyB in red light were not observed in far-red light, which inhibited growth persistently through an exclusively phyA-mediated pathway.
Transgenic tobacco plants with reduced capability to detoxify reactive oxygen intermediates are hyperresponsive to pathogen infection
Authors: Mittler Ron , Herr Elza Hallak , Orvar Bjorn Larus , van Camp Wim , Willekens Hilde , Inzé Dirk , Ellis Brian E.
Published: July 2002

Reactive oxygen intermediates (ROI) play a critical role in the defense of plants against invading pathogens. Produced during the “oxidative burst,” they are thought to activate programmed cell death (PCD) and induce antimicrobial defenses such as pathogenesis-related proteins. It was shown recently that during the interaction of plants with pathogens, the expression of ROI-detoxifying enzymes such as ascorbate peroxidase (APX) and catalase (CAT) is suppressed. It was suggested that this suppression, occurring upon pathogen recognition and coinciding with an enhanced rate of ROI production, plays a key role in elevating cellular ROI levels, thereby potentiating the induction of PCD and other defenses. To examine the relationship between the suppression of antioxidative mechanisms and the induction of PCD and other defenses during pathogen attack, we studied the interaction between transgenic antisense tobacco plants with reduced APX or CAT and a bacterial pathogen that triggers the hypersensitive response. Transgenic plants with reduced capability to detoxify ROI (i.e., antisense APX or CAT) were found to be hyperresponsive to pathogen attack. They activated PCD in response to low amounts of pathogens that did not trigger the activation of PCD in control plants. Our findings support the hypothesis that suppression of ROI-scavenging enzymes during the hypersensitive response plays an important role in enhancing pathogen-induced PCD.
Niemann-Pick C1 protein: Obligatory roles for N-terminal domains and lysosomal targeting in cholesterol mobilization
Authors: Watari Hidemichi , Blanchette-Mackie E. Joan , Dwyer Nancy K. , Glick Jane M. , Patel Shutish , Neufeld Edward B. , Brady Roscoe O. , Pentchev Peter G. , Strauss Jerome F.
Published: July 2002

Niemann-Pick type C (NPC) disease is an inherited lipid storage disorder that affects the viscera and central nervous system. A characteristic feature of NPC cells is the lysosomal accumulation of low density lipoprotein-derived cholesterol. To elucidate important structural features of the recently identified
NPC1
gene product defective in NPC disease, we examined the ability of wild-type NPC1 and NPC1 mutants to correct the excessive lysosomal storage of low density lipoprotein-derived cholesterol in a model cell line displaying the NPC cholesterol-trafficking defect (CT60 Chinese hamster ovary cells). CT60 cells transfected with human wild-type NPC1 contained immunoreactive proteins of 170 and 190 kDa localized to the lysosomal/endosomal compartment. Wild-type NPC1 protein corrected the NPC cholesterol-trafficking defect in the CT60 cells. Mutation of conserved cysteine residues in the NPC1 N terminus to serine residues resulted in proteins targeted to lysosomal membranes encircling cholesterol-laden cores, whereas deletion of the C-terminal 4-aa residues containing the LLNF lysosome-targeting motif resulted in the expression of protein localized to the endoplasmic reticulum. None of these mutant NPC1 proteins corrected the NPC cholesterol-trafficking defect in CT60 cells. We conclude that transport of the NPC1 protein to the cholesterol-laden lysosomal compartment is essential for expression of its biological activity and that domains in the N terminus of the NPC1 protein are critical for mobilization of cholesterol from lysosomes.
Femtosecond dynamics of the DNA intercalator ethidium and electron transfer with mononucleotides in water
Authors: Fiebig Torsten , Wan Chaozhi , Kelley Shana O. , Barton Jacqueline K. , Zewail Ahmed H.
Published: July 2002

Ethidium (E) is a powerful probe of DNA dynamics and DNA-mediated
electron transfer (ET). Molecular dynamical processes, such as
solvation and orientation, are important on the time scale of ET. Here,
we report studies of the femtosecond and picosecond time-resolved
dynamics of E, E with 2′deoxyguanosine triphosphate (GTP) in water,
and E with 7-deaza-2′-deoxyguanosine triphosphate (ZTP) in water; E
undergoes ET with ZTP but not GTP. These studies elucidate the critical
role of relative orientational motions of the donor–acceptor complex
on ET processes in solution. For ET from ZTP to E, such motions are in
fact the rate-determining step. Our results indicate that these
complexes reorient before ET. The time scale for the solvation of E in
water is 1 ps, and the orientational relaxation time of E is 70 ps. The
impact of orientational and solvation effects on ET between E and
mononucleotides must be considered in the application of E as a probe
of DNA ET.