bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of toxicity of
Bacillus thuringiensis
pollen to black swallowtails under field conditions
Authors: Wraight C. L. , Zangerl A. R. , Carroll M. J. , Berenbaum M. R.
Published: July 2002

A single laboratory study on monarch butterflies has prompted widespread concern that corn pollen, engineered to express
Bacillus thuringiensis
(
Bt
) endotoxin, might travel beyond corn fields and cause mortality in nontarget lepidopterans. Among the lepidopterans at high potential risk from this technology is the black swallowtail butterfly,
Papilio polyxenes
, whose host plants in the midwestern U.S. are located principally in narrow strips between roads and crop fields. A field study was performed to assess whether mortality of early instar black swallowtails was associated either with proximity to a field of
Bt
corn or by levels of
Bt
pollen deposition on host plants. Potted host plants were infested with first instar black swallowtails and placed at intervals from the edge of a field of
Bt
corn (Pioneer 34R07 containing Monsanto event 810) at the beginning of anthesis. We confirmed by ELISA that pollen from these plants contained Cry1Ab endotoxin (2.125 ± 0.289 ng/g). Although many of the larvae died during the 7 days that the experiments were run, there was no relationship between mortality and proximity to the field or pollen deposition on host plants. Moreover, pollen from these same plants failed to cause mortality in the laboratory at the highest pollen dose tested (10,000 grains/cm
2
), a level that far exceeded the highest pollen density observed in the field (200 grains/cm
2
). We conclude that
Bt
pollen of the variety tested is unlikely to affect wild populations of black swallowtails. Thus, our results suggest that at least some potential nontarget effects of the use of transgenic plants may be manageable.
Intraspecific violation of genetic colinearity and its implications in maize
Authors: Fu Huihua , Dooner Hugo K.
Published: July 2002

Although allelic sequences can vary extensively, it is generally assumed that each gene in one individual will have an allelic counterpart in another individual of the same species. We report here that this assumption does not hold true in maize. We have sequenced over 100 kb from the
bz
genomic region of two different maize lines and have found dramatic differences between them. First, the retrotransposon clusters, which comprise most of the repetitive DNA in maize, differ markedly in make-up and location relative to the genes in the
bz
region. Second, and more importantly, the genes themselves differ between the two lines, demonstrating that genetic microcolinearity can be violated within the same species. Our finding has bearing on the underlying genetic basis of hybrid vigor in maize, and possibly other organisms, and on the measurement of genetic distances.
Specific binding sites for alcohols and anesthetics on ligand-gated ion channels
Authors: Mascia Maria Paola , Trudell James R. , Harris R. Adron
Published: July 2002

Ligand-gated ion channels are a target for inhaled anesthetics and alcohols in the central nervous system. The inhibitory strychnine-sensitive glycine and γ-aminobutyric acid type A receptors are positively modulated by anesthetics and alcohols, and site-directed mutagenesis techniques have identified amino acid residues important for the action of volatile anesthetics and alcohols in these receptors. A key question is whether these amino acids are part of an alcohol/anesthetic-binding site. In the present study, we used an alkanethiol anesthetic to covalently label its binding site by mutating selected amino acids to cysteine. We demonstrated that the anesthetic propanethiol, or alternatively, propyl methanethiosulfonate, covalently binds to cysteine residues introduced into a specific second transmembrane site in glycine receptor and γ-aminobutyric acid type A receptor subunits and irreversibly enhances receptor function. Moreover, upon permanent occupation of the site by propyl disulfide, the usual ability of octanol, enflurane, and isoflurane to potentiate the function of the ion channels was lost. This approach provides strong evidence that the actions of anesthetics in these receptors are due to binding at a single site.
Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems
Authors: Caplen Natasha J. , Parrish Susan , Imani Farhad , Fire Andrew , Morgan Richard A.
Published: July 2002

Short interfering RNAs (siRNAs) are double-stranded RNAs of
≈21–25 nucleotides that have been shown to function as key
intermediaries in triggering sequence-specific RNA degradation during
posttranscriptional gene silencing in plants and RNA interference in
invertebrates. siRNAs have a characteristic structure, with
5′-phosphate/3′-hydroxyl ends and a 2-base 3′ overhang on each strand
of the duplex. In this study, we present data that synthetic siRNAs can
induce gene-specific inhibition of expression in
Caenorhabditis elegans
and in cell lines
from humans and mice. In each case, the interference by siRNAs was
superior to the inhibition of gene expression mediated by
single-stranded antisense oligonucleotides. The siRNAs seem to avoid
the well documented nonspecific effects triggered by longer
double-stranded RNAs in mammalian cells. These observations may open a
path toward the use of siRNAs as a reverse genetic and therapeutic tool
in mammalian cells.
An Eulerian path approach to DNA fragment assembly
Authors: Pevzner Pavel A. , Tang Haixu , Waterman Michael S.
Published: July 2002

For the last 20 years, fragment assembly in DNA sequencing
followed the “overlap–layout–consensus” paradigm that is used
in all currently available assembly tools. Although this approach
proved useful in assembling clones, it faces difficulties in genomic
shotgun assembly. We abandon the classical
“overlap–layout–consensus” approach in favor of a new
euler
algorithm that, for the first time, resolves the
20-year-old “repeat problem” in fragment assembly. Our main
result is the reduction of the fragment assembly to a variation of the
classical Eulerian path problem that allows one to generate accurate
solutions of large-scale sequencing problems.
euler
, in
contrast to the
celera
assembler, does not mask such
repeats but uses them instead as a powerful fragment assembly tool.
Proliferation is necessary for both repair and mutation in transgenic mouse cells
Authors: Bielas Jason H. , Heddle John A.
Published: July 2002

Proliferating cells are often presumed to be more mutable than
quiescent cells because they have less time to repair DNA damage before
DNA replication. Direct tests of this hypothesis have been confounded
by the need for cell division before a mutation can be detected. We
have avoided this problem by showing that the Big Blue mouse cell line
permits the dynamic quantification of both lesions and mutations in the
complete absence of cell division. These cells carry the bacterial
lacI
gene in a λ shuttle vector. Mutant plaques
recovered by
in vitro
packaging of the mouse DNA can
arise from mutations sustained either in mouse cells or in the
bacteria. The proportion of mutant phage contained within a mutant
plaque can distinguish these two types of mutation. Mutations formed in
mouse cells yield >90% mutant phage because both DNA strands are
mutant. On the other hand, mutations formed in the bacteria from
adducted DNA yield ≤50% mutant phage, because one of the DNA strands
is wild type. Immediately after exposure to a test mutagen,
ethylnitrosourea, all induced mutations were formed in the bacteria,
but after approximately one cell division, the reverse was true and all
mutations arose in the mouse cells. Only one-fifth as many mutations
were recovered from quiescent cells and all arose in the bacteria,
showing that the mouse cells made no mutations in the absence of
proliferation. Surprisingly, the mouse cells did not repair any of the
premutagenic damage during 4 days of quiescence. When these quiescent
cells were induced to proliferate, however, both repair and mutation
fixation ensued.
Presynaptic
N
-methyl-
d
-aspartate receptors at the parallel fiber–Purkinje cell synapse
Authors: Casado M. , Dieudonné S. , Ascher P.
Published: July 2002

At the cerebellar synapse between the parallel fibers (PFs) and the
Purkinje cells in the cerebellum, we have found that application of
N
-methyl-
d
-aspartate (NMDA) reversibly
depresses the postsynaptic current. We present evidence that this
depression involves NMDA receptors located on the presynaptic axons and
requires that the NMDA application be combined with action potentials
in the PFs. Unexpectedly, unlike other modulations mediated by
presynaptic receptors, the NMDA-induced inhibition does not involve a
depression of transmitter release. Because it is blocked by both nitric
oxide synthase and soluble guanylate cyclase inhibitors, we propose
that it involves a trans-synaptic mechanism in which NO released by the
PFs decreases the glutamate sensitivity of the Purkinje cell.
Assessing the impact of Cry1Ab-expressing corn pollen on monarch butterfly larvae in field studies
Authors: Stanley-Horn Diane E. , Dively Galen P. , Hellmich Richard L. , Mattila Heather R. , Sears Mark K. , Rose Robyn , Jesse Laura C. H. , Losey John E. , Obrycki John J. , Lewis Les
Published: July 2002

Survival and growth of monarch larvae,
Danaus plexippus
(L.), after exposure to either Cry1Ab-expressing pollen from three
Bacillus thuringiensis
(
Bt
) corn (
Zea mays
L.) events differing in toxin expression or to the insecticide, λ-cyhalothrin, were examined in field studies. First instars exposed to low doses (≈22 grains per cm
2
) of event-176 pollen gained 18% less weight than those exposed to Bt11 or Mon810 pollen after a 5-day exposure period. Larvae exposed to 67 pollen grains per cm
2
on milkweed leaves from within an event-176 field exhibited 60% lower survivorship and 42% less weight gain compared with those exposed to leaves from outside the field. In contrast, Bt11 pollen had no effect on growth to adulthood or survival of first or third instars exposed for 5 days to ≈55 and 97 pollen grains per cm
2
, respectively. Similarly, no differences in larval survivorship were observed after a 4-day exposure period to leaves with 504–586 (within fields) or 18–22 (outside the field) pollen grains per cm
2
collected from Bt11 and non-
Bt
sweet-corn fields. However, survivorship and weight gain were drastically reduced in non-
Bt
fields treated with λ-cyhalothrin. The effects of Bt11 and Mon810 pollen on the survivorship of larvae feeding 14 to 22 days on milkweeds in fields were negligible. Further studies should examine the lifetime and reproductive impact of Bt11 and Mon810 pollen on monarchs after long-term exposure to naturally deposited pollen.
Impact of
Bt
corn pollen on monarch butterfly populations: A risk assessment
Authors: Sears Mark K. , Hellmich Richard L. , Stanley-Horn Diane E. , Oberhauser Karen S. , Pleasants John M. , Mattila Heather R. , Siegfried Blair D. , Dively Galen P.
Published: July 2002

A collaborative research effort by scientists in several states and in Canada has produced information to develop a formal risk assessment of the impact of
Bt
corn on monarch butterfly (
Danaus plexippus
) populations. Information was sought on the acute toxic effects of
Bt
corn pollen and the degree to which monarch larvae would be exposed to toxic amounts of
Bt
pollen on its host plant, the common milkweed,
Asclepias syriaca
, found in and around cornfields. Expression of Cry proteins, the active toxicant found in
Bt
corn tissues, differed among hybrids, and especially so in the concentrations found in pollen of different events. In most commercial hybrids,
Bt
expression in pollen is low, and laboratory and field studies show no acute toxic effects at any pollen density that would be encountered in the field. Other factors mitigating exposure of larvae include the variable and limited overlap between pollen shed and larval activity periods, the fact that only a portion of the monarch population utilizes milkweed stands in and near cornfields, and the current adoption rate of
Bt
corn at 19% of North American corn-growing areas. This 2-year study suggests that the impact of
Bt
corn pollen from current commercial hybrids on monarch butterfly populations is negligible.
The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily
Authors: Interthal Heidrun , Pouliot Jeffrey J. , Champoux James J.
Published: July 2002

The phospholipase D (PLD) superfamily is a diverse group of proteins that includes enzymes involved in phospholipid metabolism, a bacterial toxin, poxvirus envelope proteins, and bacterial nucleases. Based on sequence comparisons, we show here that the tyrosyl-DNA phosphodiesterase (Tdp1) that has been implicated in the repair of topoisomerase I covalent complexes with DNA contains two unusual HKD signature motifs that place the enzyme in a distinct class within the PLD superfamily. Mutagenesis studies with the human enzyme in which the invariant histidines and lysines of the HKD motifs are changed confirm that these highly conserved residues are essential for Tdp1 activity. Furthermore, we show that, like other members of the family for which it has been examined, the reaction involves the formation of an intermediate in which the cleaved substrate is covalently linked to the enzyme. These results reveal that the hydrolytic reaction catalyzed by Tdp1 occurs by the phosphoryl transfer chemistry that is common to all members of the PLD superfamily.
Direct real-time observation of actin filament branching mediated by Arp2/3 complex using total internal reflection fluorescence microscopy
Authors: Amann Kurt J. , Pollard Thomas D.
Published: July 2002

Existing methods for studying actin filament dynamics have allowed
analysis only of bulk samples or individual filaments after treatment
with the drug phalloidin, which perturbs filament dynamics.
Total internal reflection fluorescence microscopy with
rhodamine-labeled actin allowed us to observe polymerization in real
time, without phalloidin. Direct measurements of filament growth
confirmed the rate constants measured by electron microscopy and
established that rhodamine actin is a kinetically inactive tracer for
imaging. In the presence of activated Arp2/3 complex, growing actin
filaments form branches at random sites along their sides, rather than
preferentially from their barbed ends.
Critical involvement of a carbamylated lysine in catalytic function of class D β-lactamases
Authors: Golemi Dasantila , Maveyraud Laurent , Vakulenko Sergei , Samama Jean-Pierre , Mobashery Shahriar
Published: July 2002

β-Lactamases are the resistance enzymes for β-lactam
antibiotics, of which four classes are known. β-lactamases hydrolyze
the β-lactam moieties of these antibiotics, rendering them inactive.
It is shown herein that the class D OXA-10 β-lactamase depends
critically on an unusual carbamylated lysine as the basic residue for
both the enzyme acylation and deacylation steps of catalysis. The
formation of carbamylated lysine is reversible. Evidence is presented
that this enzyme is dimeric and carbamylated in living bacteria.
High-resolution x-ray structures for the native enzyme were determined
at pH values of 6.0, 6.5, 7.5, and 8.5. Two dimers are present per
asymmetric unit. One monomer in each dimer was carbamylated at pH 6.0,
whereas all four monomers were fully carbamylated at pH 8.5. At the
intermediate pH values, one monomer of each dimer was carbamylated, and
the other showed a mixture of carbamylated and non-carbamylated
lysines. It would appear that, as the pH increased for the sample,
additional lysines were “titrated” by carbamylation. A handful of
carbamylated lysines are known from protein crystallographic data, all
of which have been attributed roles in structural stabilization (mostly
as metal ligands) of the proteins. This paper reports a previously
unrecognized role for a noncoordinated carbamylate lysine as a
basic residue involved in mechanistic reactions of an enzyme, which
indicates another means for expansion of the catalytic capabilities of
the amino acids in nature beyond the 20 common amino acids in
development of biological catalysts.
Rapid shoreward encroachment of salt marsh cordgrass in response to accelerated sea-level rise
Authors: Donnelly Jeffrey P. , Bertness Mark D.
Published: July 2002

The distribution of New England salt marsh communities is
intrinsically linked to the magnitude, frequency, and duration of tidal
inundation. Cordgrass (
Spartina alterniflora
)
exclusively inhabits the frequently flooded lower elevations, whereas a
mosaic of marsh hay (
Spartina patens
), spike grass
(
Distichlis spicata
), and black rush (
Juncus
gerardi
) typically dominate higher elevations. Monitoring plant
zonal boundaries in two New England salt marshes revealed that
low-marsh cordgrass rapidly moved landward at the expense of
higher-marsh species between 1995 and 1998. Plant macrofossils
from sediment cores across modern plant community boundaries provided a
2,500-year record of marsh community composition and documented the
migration of cordgrass into the high marsh. Isotopic dating revealed
that the initiation of cordgrass migration occurred in the
late 19th century and continued through the 20th century. The timing of
the initiation of cordgrass migration is coincident with an
acceleration in the rate of sea-level rise recorded by the New
York tide gauge. These results suggest that increased flooding
associated with accelerating rates of sea-level rise has stressed
high-marsh communities and promoted landward migration of cordgrass. If
current rates of sea-level rise continue or increase slightly over the
next century, New England salt marshes will be dominated by cordgrass.
If climate warming causes sea-level rise rates to increase
significantly over the next century, these cordgrass-dominated marshes
will likely drown, resulting in extensive losses of coastal wetlands.
Low ethanol concentrations enhance GABAergic inhibitory postsynaptic potentials in hippocampal pyramidal neurons only after block of GABAB receptors.
Authors: Wan F J , Berton F , Madamba S G , Francesconi W , Siggins G R
Published: July 2002

Despite considerable evidence that ethanol can enhance chloride flux through the gamma-aminobutyric acid type A (GABA/A/) receptor-channel complex in several central neuron types, the effect of ethanol on hippocampal GABAergic systems is still controversial. Therefore, we have reevaluated this interaction in hippocampal pyramidal neurons subjected to local monosynaptic activation combined with pharmacological isolation of the various components of excitatory and inhibitory synaptic potentials, using intracellular current- and voltage-clamp recording methods in the hippocampal slice. In accord with our previous findings, we found that ethanol had little effect on compound inhibitory postsynaptic potentials/currents (IPSP/Cs) containing both GABA/A/ and GABA/B/ components. However, after selective pharmacological blockade of the GABA/B/ component of the IPSP (GABA/B/-IPSP/C) by CGP-35348, low concentrations of ethanol (22-66 mM) markedly enhanced the peak amplitude, and especially the area, of the GABA/A/ component (GABA/A/-IPSP/C) in most CA1 pyramidal neurons. Ethanol had no significant effect on the peak amplitude or area of the pharmacologically isolated GABA/B/-inhibitory postsynaptic current (IPSC). These results provide new data showing that activation of GABAB receptors can obscure ethanol enhancement of GABA/A/ receptor function in hippocampus and suggest that similar methods of pharmacological isolation might be applied to other brain regions showing negative or mixed ethanol-GABA interactions.
A psychrophilic crenarchaeon inhabits a marine sponge: Cenarchaeum symbiosum gen. nov., sp. nov.
Authors: Preston C M , Wu K Y , Molinski T F , DeLong E F
Published: July 2002

Archaea, one of the three major domains of extant life, was thought to comprise predominantly microorganisms that inhabit extreme environments, inhospitable to most Eucarya and Bacteria. However, molecular phylogenetic surveys of native microbial assemblages are beginning to indicate that the evolutionary and physiological diversity of Archaea is far greater than previously supposed. We report here the discovery and preliminary characterization of a marine archaeon that inhabits the tissues of a temperate water sponge. The association was specific, with a single crenarchaeal phylotype inhabiting a single sponge host species. To our knowledge, this partnership represents the first described symbiosis involving Crenarchaeota. The symbiotic archaeon grows well at temperatures of 10 degrees C, over 60 degrees C below the growth temperature optimum of any cultivated species of Crenarchaeota. Archaea have been generally characterized as microorganisms that inhabit relatively circumscribed niches, largely high-temperature anaerobic environments. In contrast, data from molecular phylogenetic surveys, including this report, suggest that some crenarchaeotes have diversified considerably and are found in a wide variety of lifestyles and habitats. We present here the identification and initial description of Cenarchaeum symbiosum gen. nov., sp. nov., a symbiotic archaeon closely related to other nonthermophilic crenarchaeotes that inhabit diverse marine and terrestrial environments.
Nucleator-dependent intercellular assembly of adhesive curli organelles in Escherichia coli.
Authors: Hammar M , Bian Z , Normark S
Published: July 2002

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.
Phytosulfokine, sulfated peptides that induce the proliferation of single mesophyll cells of Asparagus officinalis L.
Authors: Matsubayashi Y , Sakagami Y
Published: July 2002

Proliferation of dispersed plant cells in culture is strictly dependent on cell density, and cells in a low-density culture can only grow in the presence of conditioned medium (CM). No known plant hormones have been able to substitute for CM. To quantify the mitogenic activity of CM, we examined conditions for the assay system using mechanically dispersed mesophyll cells of Asparagus officinalis L. and established a highly sensitive bioassay method. By use of this method, the mitogenic activity of CM prepared from asparagus cells was characterized: it was heat-stable, susceptible to pronase digestion, and resistant to glycosidase treatment. On the basis of these results, the mitogenic activity in CM was purified 10(7)-fold by column chromatography, and two factors named phytosulfokine-alpha and -beta (PSK-alpha and PSK-beta) were obtained. By amino acid sequence analysis and mass spectrometry, the structures of these two factors were determined to be sulfated pentapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH) and sulfated tetrapeptide (H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH). PSK-alpha and PSK-beta were prepared by chemical synthesis and enzymatic sulfation. The synthetic peptides exhibited the same activity as the natural factors, confirming the structure for PSK-alpha and PSK-beta mentioned above. This is the first elucidation of the structure of a conditioned medium factor required for the growth of low-density plant cell cultures.
Differential expression of the ToxR regulon in classical and E1 Tor biotypes of Vibrio cholerae is due to biotype-specific control over toxT expression.
Authors: DiRita V J , Neely M , Taylor R K , Bruss P M
Published: July 2002

The two major disease-causing biotypes of Vibrio cholerae, classical and El Tor, exhibit differences in their epidemic nature. Their behavior in the laboratory also differs in that El Tor strains produce two major virulence factors, cholera toxin (CT) and the toxin coregulated pilus (TCP), only under very restricted growth conditions, whereas classical strains do so in standard laboratory medium. Expression of toxin and TCP is controlled by two activator proteins, ToxR and ToxT, that operate in cascade fashion with ToxR controlling the synthesis of ToxT. Both biotypes express equivalent levels of ToxR, but only classical strains appear to express ToxT when grown in standard medium. In this report we show that restrictive expression of CT and TCP can be overcome in El Tor strains by expressing ToxT independently of ToxR. An El Tor strain lacking functional ToxT does not express CT or TCP, ruling out existence of a cryptic pathway for virulence regulation in this biotype. These results may have implications for understanding the evolution of El Tor strains toward reduced virulence with respect to classical strains.
Cellular oxidative stress and the control of apoptosis by wild-type p53, cytotoxic compounds, and cytokines.
Authors: Lotem J , Peled-Kamar M , Groner Y , Sachs L
Published: July 2002

Apoptosis induced by wild-type p53 or cytotoxic compounds in myeloid leukemic cells can be inhibited by the cytokines interleukin 6, interleukin 3, granulocyte-macrophage colony-stimulating factor, and interferon gamma and by antioxidants. The antioxidants and cytokines showed a cooperative protective effect against induction of apoptosis. Cells with a higher intrinsic level of peroxide production showed a higher sensitivity to induction of apoptosis and required a higher cytokine concentration to inhibit apoptosis. Decreasing the intrinsic oxidative stress in cells by antioxidants thus inhibited apoptosis, whereas increasing this intrinsic stress by adding H2O2 enhanced apoptosis. Induction of apoptosis by wild-type p53 was not preceded by increased peroxide production or lipid peroxidation and the protective effect of cytokines was not associated with a decrease in these properties. The results indicate that the intrinsic degree of oxidative stress can regulate cell susceptibility to wild-type p53-dependent and p53-independent induction of apoptosis and the ability of cytokines to protect cells against apoptosis.
Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.
Authors: Proudfoot L , Nikolaev A V , Feng G J , Wei W Q , Ferguson M A , Brimacombe J S , Liew F Y
Published: July 2002

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.
Rapid preparation of giant unilamellar vesicles.
Authors: Moscho A , Orwar O , Chiu D T , Modi B P , Zare R N
Published: July 2002

We report here a rapid evaporation method that produces in high yield giant unilamellar vesicles up to 50 microns in diameter. The vesicles are obtained after only 2 min and can be prepared from different phospholipids, including L-alpha-phosphatidylcholine (lecithin), dipalmitoleoyl L-alpha-phosphatidylcholine, and beta-arachidonoyl gamma-palmitoyl L-alpha-phosphatidylcholine. Vesicles can be produced in distilled water and in Hepes, phosphate, and borate buffers in the pH range of 7.0 to 11.5 with ionic strengths up to 50 mM. The short preparation time allows encapsulation of labile molecular targets or enzymes with high catalytic activities. Cell-sized proteoliposomes have been prepared in which gamma-glutamyltransferase (EC 2.3.2.2) was functionally incorporated into the membrane wall.
The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner.
Authors: Xia K , Mukhopadhyay N K , Inhorn R C , Barber D L , Rose P E , Lee R S , Narsimhan R P , D'Andrea A D , Griffin J D , Roberts T M
Published: July 2002

JAK2, a member of the Janus kinase superfamily was found to interact functionally with Raf-1, a central component of the ras/mitogen-activated protein kinase signal transduction pathway. Interferon-gamma and several other cytokines that are known to activate JAK2 kinase were also found to stimulate Raf-1 kinase activity toward MEK-1 in mammalian cells. In the baculovirus coexpression system, Raf-1 was activated by JAK2 in the presence of p21ras. Under these conditions, a ternary complex of p21ras, JAK2, and Raf-1 was observed. In contrast, in the absence of p21ras, coexpression of JAK2 and Raf-1 resulted in an overall decrease in the Raf-1 kinase activity. In addition, JAK2 phosphorylated Raf-1 at sites different from those phosphorylated by pp60v-src. In mammalian cells treated with either erythropoietin or interferon-gamma, a small fraction of Raf-1 coimmunoprecipitated with JAK2 in lysates of cells in which JAK2 was activated as judged by its state of tyrosine phosphorylation. Taken together, these data suggest that JAK2 and p21ras cooperate to activate Raf-1.
Zoidogamy in fossil gymnosperms
Authors: Poort Ruud J. , Visscher Henk , Dilcher David L.
Published: July 2002

This year is the centenary of the surprising discovery in 1896 of zoidogamy in extant cycadophytes and
Ginkgo
. But by coincidence, also in the same year, the concept of prepollen was introduced. The morphology of prepollen was considered justification for the probable production of motile antherozoids in extinct gymnosperms. In this paper, the history of the prepollen concept is briefly outlined. It is emphasized that, in addition to well-known examples in pteridosperms and cordaitaleans, a prepollen condition also occurred among late Paleozoic conifers.
Regional and cellular fractionation of working memory
Authors: Goldman-Rakic Patricia S.
Published: July 2002

This chapter recounts efforts to dissect the cellular and circuit
basis of a memory system in the primate cortex with the goal of
extending the insights gained from the study of normal brain
organization in animal models to an understanding of human cognition
and related memory disorders. Primates and humans have developed an
extraordinary capacity to process information “on line,” a
capacity that is widely considered to underlay comprehension, thinking,
and so-called executive functions. Understanding the interactions
between the major cellular constituents of cortical circuits—pyramidal
and nonpyramidal cells—is considered a necessary step in unraveling
the cellular mechanisms subserving working memory mechanisms and,
ultimately, cognitive processes. Evidence from a variety of sources is
accumulating to indicate that dopamine has a major role in regulating
the excitability of the cortical circuitry upon which the working
memory function of prefrontal cortex depends. Here, I describe several
direct and indirect intercellular mechanisms for modulating working
memory function in prefrontal cortex based on the localization of
dopamine receptors on the distal dendrites and spines of pyramidal
cells and on interneurons in the prefrontal cortex. Interactions
between monoamines and a compromised cortical circuitry may hold the
key to understanding the variety of memory disorders associated with
aging and disease.
Structure and function of declarative and nondeclarative memory systems
Authors: Squire Larry R. , Zola Stuart M.
Published: July 2002

This article reviews recent studies of memory systems in humans and
nonhuman primates. Three major conclusions from recent work are that
(
i
) the capacity for nondeclarative (nonconscious)
learning can now be studied in a broad array of tasks that assess
classification learning, perceptuomotor skill learning, artificial
grammar learning, and prototype abstraction; (
ii
)
cortical areas adjacent to the hippocampal formation, including
entorhinal, perirhinal, and parahippocampal cortices, are an essential
part of the medial temporal lobe memory system that supports
declarative (conscious) memory; and (
iii
) in humans,
bilateral damage limited to the hippocampal formation is nevertheless
sufficient to produce severe anterograde amnesia and temporally graded
retrograde amnesia covering as much as 25 years.
Involvement of the cyclin-dependent kinase inhibitor p16 (INK4a) in replicative senescence of normal human fibroblasts
Authors: Alcorta David A. , Xiong Yue , Phelps Dawn , Hannon Greg , Beach David , Barrett J. Carl
Published: July 2002

Human diploid fibroblasts (HDFs) can be grown in culture for a
finite number of population doublings before they cease proliferation
and enter a growth-arrest state termed replicative senescence. The
retinoblastoma gene product, Rb, expressed in these cells is
hypophosphorylated. To determine a possible mechanism by which
senescent human fibroblasts maintain a hypophosphorylated Rb, we
examined the expression levels and interaction of the Rb kinases, CDK4
and CDK6, and the cyclin-dependent kinase inhibitors p21 and p16 in
senescent HDFs. Cellular p21 protein expression increased dramatically
during the final two to three passages when the majority of cells lost
their growth potential and neared senescence but p21 levels declined in
senescent HDFs. During this period, p16 mRNA and cellular protein
levels gradually rose with the protein levels in senescent HDFs
reaching nearly 40-fold higher than early passage cells. In senescent
HDFs, p16 was shown to be complexed to both CDK4 and CDK6.
Immunodepletion analysis of p21 and p16 from the senescent cell
extracts revealed that p16 is the major CDK inhibitor for both CDK4 and
CDK6 kinases. Immunoprecipitation of CDK4 and CDK6 and their associated
proteins from radiolabeled extracts from senescent HDFs showed no other
CDK inhibitors. Based upon these results, we propose that senescence is
a multistep process requiring the expression of both p21 and p16. p16
up-regulation is a key event in the terminal stages of growth arrest in
senescence, which may explain why p16 but not p21 is commonly mutated
in immortal cells and human tumors.
Methionine residues as endogenous antioxidants in proteins
Authors: Levine Rodney L. , Mosoni Laurent , Berlett Barbara S. , Stadtman Earl R.
Published: July 2002

Cysteine and methionine are the two sulfur-containing residues
normally found in proteins. Cysteine residues function in the catalytic
cycle of many enzymes, and they can form disulfide bonds that
contribute to protein structure. In contrast, the specific functions of
methionine residues are not known. We propose that methionine residues
constitute an important antioxidant defense mechanism. A variety of
oxidants react readily with methionine to form methionine sulfoxide,
and surface exposed methionine residues create an extremely high
concentration of reactant, available as an efficient oxidant scavenger.
Reduction back to methionine by methionine sulfoxide reductases would
allow the antioxidant system to function catalytically. The effect of
hydrogen peroxide exposure upon glutamine synthetase from
Escherichia coli
was studied as an
in
vitro
model system. Eight of the 16 methionine residues could
be oxidized with little effect on catalytic activity of the enzyme. The
oxidizable methionine residues were found to be relatively surface
exposed, whereas the intact residues were generally buried within the
core of the protein. Furthermore, the susceptible residues were
physically arranged in an array that guarded the entrance to the active
site.
Synthesis and biological activity of DNA damaging agents that form decoy binding sites for the estrogen receptor
Authors: Rink Stacia M. , Yarema Kevin J. , Solomon Marjorie S. , Paige Lisa A. , Tadayoni-Rebek B. Mitra , Essigmann John M. , Croy Robert G.
Published: July 2002

It is a goal of cancer chemotherapy to achieve the selective
killing of tumor cells while minimizing toxicity to normal tissues. We
describe the design of selective toxins forming DNA adducts that
attract the estrogen receptor (ER), a transcription factor that is
overexpressed in many human breast and ovarian tumors. The compounds
consist of
4-(3-aminopropyl)-
N
,
N
-(2-chloroethyl)-aniline
linked to 2-(4′-hydroxyphenyl)-3-methyl-5-hydroxy-indole. The former
moiety is a DNA damaging nitrogen mustard and the latter is a ligand
for the ER. The connection between these groups was refined to permit
DNA adducts formed by the mustard portion of the molecule to present
the ligand domain so that it was able to interact efficiently with the
ER. By using 16-mers containing specific DNA adducts, it was determined
that monoadducts and putative intrastrand crosslinks were preferred
targets for the ER over interstrand crosslinks. A series of
structurally related 2-phenylindole mustards was prepared, some of
which were selectively toxic to the ER-positive breast cancer cell line
MCF-7, as compared with the ER(−) negative line MDA-MB231. The ability
both to bind to DNA and to interact significantly with the ER were
essential to achieve selective lethality toward ER(+) cells. Compounds
forming DNA adducts without the ability to bind receptor showed similar
toxicities in the two cell lines. Several models could explain the
selective toxicity of the mustard–phenylindole compounds toward ER(+)
cells. The favored model suggests that a mustard–DNA adduct is
shielded by the ER from DNA repair enzymes and hence cells possessing
an abundance of the ER selectively retain the adduct and are killed.
Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage.
Authors: Playford R J , Marchbank T , Goodlad R A , Chinery R A , Poulsom R , Hanby A M
Published: July 2002

pS2 is a member of the trefoil peptide family, all of which are overexpressed at sites of gastrointestinal injury. We hypothesized that they are important in stimulating mucosal repair. To test this idea, we have produced a transgenic mice strain that expresses human pS2 (hpS2) specifically within the jejunum and examined the effect of this overexpression on proliferation and susceptibility to indomethacin-induced damage. A transgenic mouse was produced by microinjecting fertilized oocytes with a 1.7-kb construct consisting of rat intestinal fatty acid binding protein promoter (positions -1178 to +28) linked to full-length (490 bp) hpS2 cDNA. Screening for positive animals was by Southern blot analysis. Distribution of hpS2 expression was determined by using Northern and Western blot analyses and immunohistochemical staining. Proliferation of the intestinal mucosa was determined by assessing the crypt cell production rate. Differences in susceptibility to intestinal damage were analyzed in animals that had received indomethacin (85 mg/kg s.c.) 0-30 h previously. Expression of hpS2 was limited to the enterocytes of the villi within the jejunum. In the nondamaged intestine, villus height and crypt cell production rate were similar in transgenic and negative (control) litter mates. However, there was a marked difference in the amount of damage caused by indomethacin in control and transgenic animals in the jejunum (30% reduction in villus height in controls vs. 12% reduction in transgenic animals, P < 0.01) but the damage sustained in the non-hpS2-expressing ileal region was similar in control and transgenic animals. These studies support the hypothesis that trefoil peptides are important in stimulating gastrointestinal repair.
Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia.
Authors: Sanguinetti M C , Curran M E , Spector P S , Keating M T
Published: July 2002

Long QT syndrome (LQT) is an autosomal dominant disorder that can cause sudden death from cardiac arrhythmias. We recently discovered that mutations in HERG, a K+-channel gene, cause chromosome 7-linked LQT. Heterologous expression of HERG in Xenopus oocytes revealed that HERG current was similar to a well-characterized cardiac delayed rectifier K+ current, IKr, and led to the hypothesis that mutations in HERG reduced IKr, causing prolonged myocellular action potentials. To define the mechanism of LQT, we injected oocytes with mutant HERG complementary RNAs, either singly or in combination with wild-type complementary RNA. Some mutations caused loss of function, whereas others caused dominant negative suppression of HERG function. These mutations are predicted to cause a spectrum of diminished IKr and delayed ventricular repolarization, consistent with the prolonged QT interval observed in individuals with LQT.
Imaging of single molecule diffusion.
Authors: Schmidt T , Schütz G J , Baumgartner W , Gruber H J , Schindler H
Published: July 2002

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.
A new pathway for vacuolar cadmium sequestration in
Saccharomyces cerevisiae
: YCF1-catalyzed transport of bis(glutathionato)cadmium
Authors: Li Ze-Sheng , Lu Yu-Ping , Zhen Rui-Guang , Szczypka Mark , Thiele Dennis J. , Rea Philip A.
Published: July 2002

The yeast cadmium factor (
YCF1
) gene encodes an MgATP-energized glutathione
S
-conjugate transporter responsible for the vacuolar sequestration of organic compounds after their
S
-conjugation with glutathione. However, while
YCF1
was originally isolated according to its ability to confer resistance to cadmium salts, neither its mode of interaction with Cd
2+
nor the relationship between this process and organic glutathione-conjugate transport are known. Here we show through direct comparisons between vacuolar membrane vesicles purified from
Saccharomyces cerevisiae
strain DTY167, harboring a deletion of the
YCF1
gene, and the isogenic wild-type strain DTY165 that YCF1 mediates the MgATP-energized vacuolar accumulation of Cd·glutathione complexes. The substrate requirements, kinetics and Cd
2+
/glutathione stoichiometry of cadmium uptake and the molecular weight of the transport-active complex demonstrate that YCF1 selectively catalyzes the transport of bis(glutathionato)cadmium (Cd·GS
2
). On the basis of these results—the Cd
2+
hypersensitivity of DTY167, versus DTY165, cells, the inducibility of YCF1-mediated transport, and the rapidity and spontaneity of Cd·GS
2
formation—this new pathway is concluded to contribute substantially to Cd
2+
detoxification.
Optical trapping and manipulation of neutral particles using lasers
Authors: Ashkin Arthur
Published: July 2002

The techniques of optical trapping and manipulation of neutral particles by lasers provide unique means to control the dynamics of small particles. These new experimental methods have played a revolutionary role in areas of the physical and biological sciences. This paper reviews the early developments in the field leading to the demonstration of cooling and trapping of neutral atoms in atomic physics and to the first use of optical tweezers traps in biology. Some further major achievements of these rapidly developing methods also are considered.
Association of arsenic-induced malignant transformation with DNA hypomethylation and aberrant gene expression
Authors: Zhao Christopher Q. , Young Matthew R. , Diwan Bhalchandra A. , Coogan Timothy P. , Waalkes Michael P.
Published: July 2002

Inorganic arsenic, a human carcinogen, is enzymatically methylated for detoxication, consuming
S
-adenosyl-methionine (SAM) in the process. The fact that DNA methyltransferases (MeTases) require this same methyl donor suggests a role for methylation in arsenic carcinogenesis. Here we test the hypothesis that arsenic-induced initiation results from DNA hypomethylation caused by continuous methyl depletion. The hypothesis was tested by first inducing transformation in a rat liver epithelial cell line by chronic exposure to low levels of arsenic, as confirmed by the development of highly aggressive, malignant tumors after inoculation of cells into Nude mice. Global DNA hypomethylation occurred concurrently with malignant transformation and in the presence of depressed levels of
S
-adenosyl-methionine. Arsenic-induced DNA hypomethylation was a function of dose and exposure duration, and remained constant even after withdrawal of arsenic. Hyperexpressibility of the
MT
gene, a gene for which expression is clearly controlled by DNA methylation, was also detected in transformed cells. Acute arsenic or arsenic at nontransforming levels did not induce global hypomethylation of DNA. Whereas transcription of DNA MeTase was elevated, the MeTase enzymatic activity was reduced with arsenic transformation. Taken together, these results indicate arsenic can act as a carcinogen by inducing DNA hypomethylation, which in turn facilitates aberrant gene expression, and they constitute a tenable theory of mechanism in arsenic carcinogenesis.