bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils
Authors: Cahoon Edgar B. , Shanklin John
Published: July 2002

We demonstrate that naturally occurring C
14
and
C
16
-specific acyl-acyl carrier protein (ACP) desaturases
from plants can complement the unsaturated fatty acid (UFA) auxotrophy
of an
Escherichia coli
fabA
/
fadR
mutant. Under the same growth
conditions, C
18
-specific Δ
9
-stearoyl
(18:0)-ACP desaturases are unable to complement the UFA auxotrophy.
This difference most likely results from the presence of sufficient
substrate pools of C
14
and C
16
acyl-ACPs but a
relative lack of C
18
acyl-ACP pools in
E.
coli
to support the activities of the plant fatty acid
desaturase. Based on this, a substrate-dependent selection system was
devised with the use of the
E. coli
UFA auxotroph to
isolate mutants of the castor Δ
9
-18:0-ACP desaturase that
display enhanced specificity for C
14
and C
16
acyl-ACPs. Using this selection system, a number of desaturase variants
with altered substrate specificities were isolated from pools of
randomized mutants. These included several G188L mutant isolates, which
displayed a 15-fold increase in specific activity with 16:0-ACP
relative to the wild-type castor Δ
9
-18:0-ACP desaturase.
Expression of this mutant in
Arabidopsis thaliana
resulted in the accumulation of unusual monounsaturated fatty acids to
amounts of >25% of the seed oil. The bacterial selection system
described here thus provides a rapid means of isolating variant fatty
acid desaturase activities for modification of seed oil
composition.
Different patterns of human discrimination learning for two interaural cues to sound-source location
Authors: Wright Beverly A. , Fitzgerald Matthew B.
Published: July 2002

Two of the primary cues used to localize the sources of sounds are interaural level differences (ILDs) and interaural time differences (ITDs). We conducted two experiments to explore how practice affects the human discrimination of values of ILDs and ongoing ITDs presented over headphones. We measured discrimination thresholds of 13 to 32 naive listeners in a variety of conditions during a pretest and again, 2 weeks later, during a posttest. Between those two tests, we trained a subset of listeners 1 h per day for 9 days on a single ILD or ITD condition. Listeners improved on both ILD and ITD discrimination. Improvement was initially rapid for both cue types and appeared to generalize broadly across conditions, indicating conceptual or procedural learning. A subsequent slower-improvement stage, which occurred solely for the ILD cue, only affected conditions with the trained stimulus frequency, suggesting that stimulus processing had fundamentally changed. These different learning patterns indicate that practice affects the attention to, or low-level encoding of, ILDs and ITDs at sites at which the two cue types are processed separately. Thus, these data reveal differences in the effect of practice on ILD and ITD discrimination, and provide insight into the encoding of these two cues to sound-source location in humans.
The
Arabidopsis
aldehyde oxidase 3 (
AAO3
) gene product catalyzes the final step in abscisic acid biosynthesis in leaves
Authors: Seo Mitsunori , Peeters Anton J. M. , Koiwai Hanae , Oritani Takayuki , Marion-Poll Annie , Zeevaart Jan A. D. , Koornneef Maarten , Kamiya Yuji , Koshiba Tomokazu
Published: July 2002

Abscisic acid (ABA) is a plant hormone involved in seed development
and germination and in responses to various environmental stresses. The
last step of ABA biosynthesis involves oxidation of abscisic aldehyde,
and aldehyde oxidase (EC
1.2.3.1
) is thought to catalyze this reaction.
An aldehyde oxidase isoform, AOδ, encoded by
AAO3
, one
of four
Arabidopsis
aldehyde oxidase genes
(
AAO1
,
AAO2
,
AAO3
, and
AAO4
), is the most likely candidate for the enzyme,
because it can efficiently catalyze the oxidation of abscisic aldehyde
to ABA. Here, we report the isolation and characterization of an
ABA-deficient
Arabidopsis
mutant that maps at the
AAO3
locus. The mutant exhibits a wilty phenotype in
rosette leaves, but seed dormancy is not affected. ABA levels were
significantly reduced in the mutant leaves, explaining the wilty
phenotype in rosettes, whereas the level in the mutant seeds was less
reduced. No AOδ activity could be detected in the rosette leaves of
the mutant. Sequence data showed that the mutant contains a G to A
substitution in the
AAO3
gene. The mutation causes
incorrect splicing of the ninth intron of
AAO3
mRNA. We
thus conclude that the ABA-deficient mutant is impaired in the
AAO3
gene and that the gene product, AOδ, is an
aldehyde oxidase that catalyzes the last step of ABA biosynthesis in
Arabidopsis
, specifically in rosette leaves. Other
aldehyde oxidases may be involved in ABA biosynthesis in other organs.
Identification of
neuropeptide-like protein
gene families in
Caenorhabditis
elegans
and other species
Authors: Nathoo Arif N. , Moeller Rachael A. , Westlund Beth A. , Hart Anne C.
Published: July 2002

Neuropeptides play critical roles in synaptic signaling in all nervous systems. Unlike classical neurotransmitters, peptidergic neurotransmitters are encoded as preproproteins that are posttranslationally processed to yield bioactive neuropeptides. To identify novel peptidergic neurotransmitters, the
Caenorhabditis elegans
genome was searched for predicted proteins with the structural hallmarks of neuropeptide preproproteins. Thirty-two
C. elegans neuropeptide-like protein
(
nlp
) genes were identified. The
nlp
genes define at least 11 families of putative neuropeptides with unique motifs; similar expressed sequence tags were identified in other invertebrate species for all 11 families. Six of these families are defined by putative bioactive motifs (FAFA, GGxYamide, MRxamide, LQFamide, LxDxamide, and GGARAF); the remaining five families are related to allatostatin, myomodulin, buccalin/drosulfakinin, orcokinin, and APGWamide neuropeptides (MGL/Famide, FRPamide, MSFamide, GFxGF, and YGGWamide families, respectively). Most
C. elegans nlp
gene expression is in neurons. The
C. elegans nlp
genes and similar genes encoding putative neuropeptides in other species are likely to play diverse roles in nervous system function.
Insect herbivory accelerates nutrient cycling and increases plant production
Authors: Belovsky G. E. , Slade J. B.
Published: July 2002

Ecologists hold two views about the role of herbivory in ecosystem dynamics. First, from a food web perspective in population/community ecology, consumption by herbivores reduces plant abundance. Second, from a nutrient cycling perspective in ecosystem ecology, herbivory sometimes slows down cycling, which decreases plant abundance, but at other times speeds up cycling, which possibly increases plant abundance. The nutrient cycling perspective on herbivory has been experimentally addressed more thoroughly in aquatic systems than in terrestrial systems. We experimentally examined how grasshoppers influence nutrient cycling and, thereby, plant abundance and plant species composition over a period of 5 years. We examined how grasshoppers influence nutrient (nitrogen) cycling (i) by their excrement, (ii) by changing the abundance of and the decomposition rate of plant litter, and (iii) by both. Grasshoppers may speed up nitrogen cycling by changing the abundance and decomposition rate of plant litter, which increases total plant abundance (up to 32.9 g/m2or 18%), especially, the abundance of plants that are better competitors when nitrogen is more available. However, whether grasshoppers enhance plant abundance depends on how much they consume. Consequently, ecosystems and food web perspectives are not mutually exclusive. Finally, under some conditions, grasshoppers may decrease nutrient cycling and plant abundance.
Can race be erased? Coalitional computation and social categorization
Authors: Kurzban Robert , Tooby John , Cosmides Leda
Published: July 2002

Previous studies have established that people encode the race of each individual they encounter, and do so via computational processes that appear to be both automatic and mandatory. If true, this conclusion would be important, because categorizing others by their race is a precondition for treating them differently according to race. Here we report experiments, using unobtrusive measures, showing that categorizing individuals by race is not inevitable, and supporting an alternative hypothesis: that encoding by race is instead a reversible byproduct of cognitive machinery that evolved to detect coalitional alliances. The results show that subjects encode coalitional affiliations as a normal part of person representation. More importantly, when cues of coalitional affiliation no longer track or correspond to race, subjects markedly reduce the extent to which they categorize others by race, and indeed may cease doing so entirely. Despite a lifetime's experience of race as a predictor of social alliance, less than 4 min of exposure to an alternate social world was enough to deflate the tendency to categorize by race. These results suggest that racism may be a volatile and eradicable construct that persists only so long as it is actively maintained through being linked to parallel systems of social alliance.
Molecular cloning and expression of a cyclic AMP-activated chloride conductance regulator: a novel ATP-binding cassette transporter.
Authors: van Kuijck M A , van Aubel R A , Busch A E , Lang F , Russel F G , Bindels R J , van Os C H , Deen P M
Published: July 2002

Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-regulated, cAMP-activated chloride channel located in the apical membrane of many epithelial secretory cells. Here we report cloning of a cAMP-activated epithelial basolateral chloride conductance regulator (EBCR) that appears to be a basolateral CFTR counterpart. This novel chloride channel or regulator shows 49% identity with multidrug resistance-associated protein (MRP) and 29% identity with CFTR. On expression in Xenopus oocytes, EBCR confers a cAMP-activated chloride conductance that is inhibited by the chloride channel blockers niflumic acid, 5-nitro-2-(3-phenylpropylamine)benzoic acid, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid. Northern blot analysis reveals high expression in small intestine, kidney, and liver. In kidney, immunohistochemistry shows a conspicuous basolateral localization mainly in the thick ascending limb of Henle's loop, distal convoluted tubules and to a lesser extent connecting tubules. These data suggest that in the kidney EBCR is involved in hormone-regulated chloride reabsorption.
Bcl-2 potentiates the maximal calcium uptake capacity of neural cell mitochondria.
Authors: Murphy A N , Bredesen D E , Cortopassi G , Wang E , Fiskum G
Published: July 2002

Expression of the human protooncogene bcl-2 protects neural cells from death induced by many forms of stress, including conditions that greatly elevate intracellular Ca2+. Considering that Bcl-2 is partially localized to mitochondrial membranes and that excessive mitochondrial Ca2+ uptake can impair electron transport and oxidative phosphorylation, the present study tested the hypothesis that mitochondria from Bcl-2-expressing cells have a higher capacity for energy-dependent Ca2+ uptake and a greater resistance to Ca(2+)-induced respiratory injury than mitochondria from cells that do not express this protein. The overexpression of bcl-2 enhanced the mitochondrial Ca2+ uptake capacity using either digitonin-permeabilized GT1-7 neural cells or isolated GT1-7 mitochondria by 1.7 and 3.9 fold, respectively, when glutamate and malate were used as respiratory substrates. This difference was less apparent when respiration was driven by the oxidation of succinate in the presence of the respiratory complex I inhibitor rotenone. Mitochondria from Bcl-2 expressors were also much more resistant to inhibition of NADH-dependent respiration caused by sequestration of large Ca2+ loads. The enhanced ability of mitochondria within Bcl-2-expressing cells to sequester large quantities of Ca2+ without undergoing profound respiratory impairment provides a plausible mechanism by which Bcl-2 inhibits certain forms of delayed cell death, including neuronal death associated with ischemia and excitotoxicity.
UV-induced ubiquitination of RNA polymerase II: a novel modification deficient in Cockayne syndrome cells.
Authors: Bregman D B , Halaban R , van Gool A J , Henning K A , Friedberg E C , Warren S L
Published: July 2002

Damage to actively transcribed DNA is preferentially repaired by the transcription-coupled repair (TCR) system. TCR requires RNA polymerase II (Pol II), but the mechanism by which repair enzymes preferentially recognize and repair DNA lesions on Pol II-transcribed genes is incompletely understood. Herein we demonstrate that a fraction of the large subunit of Pol II (Pol II LS) is ubiquitinated after exposing cells to UV-radiation or cisplatin but not several other DNA damaging agents. This novel covalent modification of Pol II LS occurs within 15 min of exposing cells to UV-radiation and persists for about 8-12 hr. Ubiquitinated Pol II LS is also phosphorylated on the C-terminal domain. UV-induced ubiquitination of Pol II LS is deficient in fibroblasts from individuals with two forms of Cockayne syndrome (CS-A and CS-B), a rare disorder in which TCR is disrupted. UV-induced ubiquitination of Pol II LS can be restored by introducing cDNA constructs encoding the CSA or CSB genes, respectively, into CS-A or CS-B fibroblasts. These results suggest that ubiquitination of Pol II LS plays a role in the recognition and/or repair of damage to actively transcribed genes. Alternatively, these findings may reflect a role played by the CSA and CSB gene products in transcription.
Intervention of carbohydrate recognition by proteins and nucleic acids.
Authors: Sears P , Wong C H
Published: July 2002

Carbohydrates in biological systems are often associated with specific recognition and signaling processes leading to important biological functions and diseases. Considerable efforts have been directed toward understanding and mimicking the recognition processes and developing effective agents to control the processes. The pace of discovery research in glycobiology and development of carbohydrate-based therapeutics, however, has been relatively slow due to the lack of appropriate strategies and methods available for carbohydrate-related research. This review summarizes some of the most recent developments in the field, with particular emphasis on work from our laboratories regarding the use of chemoenzymatic strategies to tackle the carbohydrate recognition problem. Highlights include the study of selectin-carbohydrate and aminoglycoside-RNA interactions and development of agents for the intervention of these recognition processes.
Adsorption from a one-dimensional lattice gas and the Brunauer–Emmett–Teller equation
Authors: Hill Terrell L.
Published: July 2002

An exact treatment of adsorption from a one-dimensional lattice gas is used to eliminate and correct a well-known inconsistency in the Brunauer–Emmett–Teller (B.E.T.) equation—namely, Gibbs excess adsorption is not taken into account and the Gibbs integral diverges at the transition point. However, neither model should be considered realistic for experimental adsorption systems.
On the molecular basis and regulation of cellular capacitative calcium entry: Roles for Trp proteins
Authors: Birnbaumer Lutz , Zhu Xi , Jiang Meisheng , Boulay Guylain , Peyton Michael , Vannier Brigitte , Brown Darren , Platano Daniela , Sadeghi Hamid , Stefani Enrico , Birnbaumer Mariel
Published: July 2002

During the last 2 years, our laboratory has worked on the
elucidation of the molecular basis of capacitative calcium entry (CCE)
into cells. Specifically, we tested the hypothesis that CCE channels
are formed of subunits encoded in genes related to the Drosophila
trp
gene. The first step in this pursuit was to search
for mammalian
trp
genes. We found not one but six
mammalian genes and cloned several of their cDNAs, some in their full
length. As assayed in mammalian cells, overexpression of some mammalian
Trps increases CCE, while expression of partial
trp
cDNAs in antisense orientation can interfere with
endogenous CCE. These findings provided a firm connection
between CCE and mammalian Trps. This article reviews the known forms of
CCE and highlights unanswered questions in our understanding of
intracellular Ca
2+
homeostasis and the physiological roles
of CCE.
Long-circulating bacteriophage as antibacterial agents.
Authors: Merril C R , Biswas B , Carlton R , Jensen N C , Creed G J , Zullo S , Adhya S
Published: July 2002

The increased prevalence of multidrug-resistant bacterial pathogens motivated us to attempt to enhance the therapeutic efficacy of bacteriophages. The therapeutic application of phages as antibacterial agents was impeded by several factors: (i) the failure to recognize the relatively narrow host range of phages; (ii) the presence of toxins in crude phage lysates; and (iii) a lack of appreciation for the capacity of mammalian host defense systems, particularly the organs of the reticuloendothelial system, to remove phage particles from the circulatory system. In our studies involving bacteremic mice, the problem of the narrow host range of phage was dealt with by using selected bacterial strains and virulent phage specific for them. Toxin levels were diminished by purifying phage preparations. To reduce phage elimination by the host defense system, we developed a serial-passage technique in mice to select for phage mutants able to remain in the circulatory system for longer periods of time. By this approach we isolated long-circulating mutants of Escherichia coli phage lambda and of Salmonella typhimurium phage P22. We demonstrated that the long-circulating lambda mutants also have greater capability as antibacterial agents than the corresponding parental strain in animals infected with lethal doses of bacteria. Comparison of the parental and mutant lambda capsid proteins revealed that the relevant mutation altered the major phage head protein E. The use of toxin-free, bacteria-specific phage strains, combined with the serial-passage technique, may provide insights for developing phage into therapeutically effective antibacterial agents.
The wagon wheel illusion in movies and reality.
Authors: Purves D , Paydarfar J A , Andrews T J
Published: July 2002

Wheels turning in the movies or in other forms of stroboscopic presentation often appear to be rotating backward. Remarkably, a similar illusion is also seen in continuous light. The occurrence of this perception in the absence of intermittent illumination suggests that we normally see motion, as in movies, by processing a series of visual episodes.
DNA transport by a type II topoisomerase: direct evidence for a two-gate mechanism.
Authors: Roca J , Berger J M , Harrison S C , Wang J C
Published: July 2002

Recent biochemical and crystallographic results suggest that a type II DNA topoisomerase acts as an ATP-modulated clamp with two sets of jaws at opposite ends: a DNA-bound enzyme can admit a second DNA through one set of jaws; upon binding ATP, this DNA is passed through an enzyme-mediated opening in the first DNA and expelled from the enzyme through the other set of jaws. Experiments based on the introduction of reversible disulfide links across one dimer interface of yeast DNA topoisomerase II have confirmed this mechanism. The second DNA is found to enter the enzyme through the gate formed by the N-terminal parts of the enzyme and leave it through the gate close to the C termini.
Macrophage killing is an essential virulence mechanism of Salmonella typhimurium.
Authors: Lindgren S W , Stojiljkovic I , Heffron F
Published: July 2002

Phagocytic cells are a critical line of defense against infection. The ability of a pathogen to survive and even replicate within phagocytic cells is a potent method of evading the defense mechanisms of the host. A number of pathogens survive within macrophages after phagocytosis and this contributes to their virulence. Salmonella is one of these pathogens. Here we report that 6-14 hr after Salmonella enters the macrophage and replicates, it resides in large vacuoles and causes the destruction of these cells. Furthermore, we identified four independently isolated MudJ-lacZ insertion mutants that no longer cause the formation of these vacuoles or kill the macrophages. All four insertions were located in the ompR/envZ regulon. These findings suggest that killing and escape from macrophages may be as important steps in Salmonella pathogenesis as are survival and replication in these host cells.
Microtubule release from the centrosome
Authors: Keating T. J. , Peloquin J. G. , Rodionov V. I. , Momcilovic D. , Borisy G. G.
Published: July 2002

Although microtubules (MTs) are generally thought to originate at the centrosome, a number of cell types have significant populations of MTs with no apparent centrosomal connection. The origin of these noncentrosomal MTs has been unclear. We applied kinetic analysis of MT formation
in vivo
to establish their mode of origin. Time-lapse fluorescence microscopy demonstrated that noncentrosomal MTs in cultured epithelial cells arise primarily by constitutive nucleation at, and release from, the centrosome. After release, MTs moved away from the centrosome and tended to depolymerize. Laser-marking experiments demonstrated that released MTs moved individually with their plus ends leading, suggesting that they were transported by minus end-directed motors. Released MTs were dynamic. The laser marking experiments demonstrated that plus ends of released MTs grew, paused, or shortened while the minus ends were stable or shortened. Microtubule release may serve two kinds of cellular function. Release and transport could generate the noncentrosomal MT arrays observed in epithelial cells, neurons, and other asymmetric, differentiated cells. Release would also contribute to polymer turnover by exposing MT minus ends, thereby providing additional sites for loss of subunits. The noncentrosomal population of MTs may reflect a steady-state of centrosomal nucleation, release, and dynamics.
Homo- and heterodimeric interactions between the gene products of
PKD1
and
PKD
2
Authors: Tsiokas Leonidas , Kim Emily , Arnould Thierry , Sukhatme Vikas P. , Walz Gerd
Published: July 2002

PKD1
and
PKD2
are two recently identified genes that are responsible for the vast majority of autosomal polycystic kidney disease, a common inherited disease that causes progressive renal failure.
PKD1
encodes polycystin, a large glycoprotein that contains several extracellular motifs indicative of a role in cell–cell or cell–matrix interactions, and the
PKD2
encodes a protein with homology to a voltage-activated calcium channel and to PKD1. It is currently unknown how mutations of either protein functionally cause autosomal polycystic kidney disease. We show that PKD1 and PKD2 interact through their C-terminal cytoplasmic tails. This interaction resulted in an up-regulation of PKD1 but not PKD2. Furthermore, the cytoplasmic tail of PKD2 but not PKD1 formed homodimers through a coiled–coil domain distinct from the region required for interaction with PKD1. These interactions suggest that PKD1 and PKD2 may function through a common signaling pathway that is necessary for normal tubulogenesis and that PKD1 may require the presence of PKD2 for stable expression.
Transforming growth factor β-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells
Authors: Liu Xuedong , Sun Yin , Constantinescu Stefan N. , Karam Edmund , Weinberg Robert A. , Lodish Harvey F.
Published: July 2002

Drosophila
Mad proteins are intracellular signal transducers of decapentaplegic (dpp), the
Drosophila
transforming growth factor β (TGF-β)/bone morphogenic protein (BMP) homolog. Studies in which the mammalian Smad homologs were transiently overexpressed in cultured cells have implicated Smad2 in TGF-β signaling, but the physiological relevance of the Smad3 protein in signaling by TGF-β receptors has not been established. Here we stably expressed Smad proteins at controlled levels in epithelial cells using a novel approach that combines highly efficient retroviral gene transfer and quantitative cell sorting. We show that upon TGF-β treatment Smad3 becomes rapidly phosphorylated at the SSVS motif at its very C terminus. Either attachment of an epitope tag to the C terminus or replacement of these three serine residues with alanine abolishes TGF-β-induced Smad3 phosphorylation; these proteins act in a dominant-negative fashion to block the antiproliferative effect of TGF-β in mink lung epithelial cells. A Smad3 protein in which the three C-terminal serines have been replaced by aspartic acids is also a dominant inhibitor of TGF-β signaling, but can activate plasminogen activator inhibitor 1 (PAI-1) transcription in a ligand-independent fashion when its nuclear localization is forced by transient overexpression. Phosphorylation of the three C-terminal serine residues of Smad3 by an activated TGF-β receptor complex is an essential step in signal transduction by TGF-β for both inhibition of cell proliferation and activation of the PAI-1 promoter.
Developmental changes in DNA methylation of the two tobacco pollen nuclei during maturation
Authors: Oakeley Edward J. , Podestà Adriano , Jost Jean-Pierre
Published: July 2002

Changes in DNA methylation during tobacco pollen development have been studied by confocal fluorescence microscopy using a monoclonal anti-5-methylcytosine (anti-m5C) antibody and a polyclonal anti-histone H1 (anti-histone) antibody as an internal standard. The specificity of the anti-m5C antibody was demonstrated by a titration series against both single-stranded DNA and double-stranded DNA substrates in either the methylated or unmethylated forms. The antibody was found to show similar kinetics against both double- and single-stranded DNA, and the fluorescence was proportional to the amount of DNA used. No signal was observed with unmethylated substrates. The extent of methylation of the two pollen nuclei remained approximately constant after the mitotic division that gave rise to the vegetative and generative nuclei. However, during the subsequent development of the pollen, the staining of the generative nucleus decreased until it reached a normalized value of\documentclass[12pt]{minimal} \usepackage{wasysym} \usepackage{amsmath} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}\frac{1}{5}\end{equation*}\end{document}of that of the vegetative nucleus. The use of a confocal microscope makes these data independent of possible focusing artefacts. The anti-histone antibody was used as a control to show that, while the antibody staining directed against 5-methylcytosine changed dramatically during pollen maturation, the histone signal did not. We observed the existence of structural dimorphism amongst tobacco pollen grains, the majority having three pollen apertures and the rest with four. However, the methylation changes observed occurred to the same extent in both subclasses.
Nonmuscle myosin II-B is required for normal development of the mouse heart
Authors: Tullio Antonella N. , Accili Domenico , Ferrans Victor J. , Yu Zu-Xi , Takeda Kazuyo , Grinberg Alexander , Westphal Heiner , Preston Yvette A. , Adelstein Robert S.
Published: July 2002

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the
NMHC-B
−/−
embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in
NMHC-B
+/−
mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 ± 1.8 μm (right ventricle) and 7.8 ± 1.3 μm (left ventricle) in
NMHC-B
+/+
and
B
+/−
mice to 14.7 ± 1.1 μm and 13.8 ± 2.3 μm, respectively, in
NMHC-B
−/−
mice (in both cases,
P
< 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 ± 0.2 μm for
NMHC-B
+/+
and
B
+/−
vs. 7.2 ± 0.6 μm for
NMHC-B
−/−
mice (
P
< 0.01)). Six of seven
NMHC-B
−/−
newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of
NMHC-B
−/−
mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.
Overexpressed human CD44s promotes lung colonization during micrometastasis of murine fibrosarcoma cells: Facilitated retention in the lung vasculature
Authors: Kogerman Priit , Sy Man-Sun , Culp Lloyd A.
Published: July 2002

Normally nonmetastatic murine
sis
-transformed BALB/c 3T3 cells, transfected with human CD44s gene (hCD44s), acquire spontaneous metastatic capacity to the lung. The mechanism(s) of this facilitated micrometastasis was analyzed in an experimental metastasis model. Human CD44s overexpression promoted the earliest stages severalfold (initial implantation and subsequent stabilization of tumor cells) but was irrelevant for later stages (subsequent outgrowth) of lung experimental micrometastasis. By injecting mixed populations of parental (nonmetastatic) and CD44s-transfected cells, it was shown that cell–cell adhesion between tumor and parental cells was not promoted by hCD44s but that promotion of cell–cell adhesion to lung endothelium or specifically between transfected cells (via hyaluronan) are likely mechanisms. Results obtained with hCD44s-negative primary tumor cells and hCD44s-positive or -negative variants of lung micrometastatic cells (after s.c. injection of transfectants) confirmed the importance of CD44s overexpression for early but not late stages of experimental lung metastasis. Therefore, CD44s represents a metastasis-facilitating molecule that is irrelevant for primary tumor outgrowth but that promotes micrometastasis to the lungs at the very earliest stages.
Cooperative effects of photodynamic treatment of cells in microcolonies
Authors: Dahle Jostein , Kaalhus Olav , Moan Johan , Steen Harald B.
Published: July 2002

Microcolonies of 2-8 Madison–Darby canine kidney cells (MDCK II) and Chinese hamster lung fibroblasts (V79) cells were incubated with the photosensitizer Photofrin and exposed to light, and the resulting number of dead cells per colony was determined. The distribution of this number was found to be incompatible with the assumption that cells are inactivated independently. The experimental distributions were significantly different from the binomial distribution expected from this assumption, but in accordance with a model in which an inactivated cell can inactivate adjacent cells with a certain probability. These findings are contrary to the common view that damage caused by radiation is limited to the cell in which the primary damage takes place. Our findings clearly indicate some kind of cooperativity between cells treated with Photofrin and light.
Na
+
pump low and high ouabain affinity α subunit isoforms are differently distributed in cells
Authors: Juhaszova Magdalena , Blaustein Mordecai P.
Published: July 2002

Three isoforms (α1, α2, and α3) of the catalytic (α) subunit of the plasma membrane (PM) Na
+
pump have been identified in the tissues of birds and mammals. These isoforms differ in their affinities for ions and for the Na
+
pump inhibitor, ouabain. In the rat, α1 has an unusually low affinity for ouabain. The PM of most rat cells contains both low (α1) and high (α2 or α3) ouabain affinity isoforms, but precise localization of specific isoforms, and their functional significance, are unknown. We employed high resolution immunocytochemical techniques to localize α subunit isoforms in primary cultured rat astrocytes, neurons, and arterial myocytes. Isoform α1 was ubiquitously distributed over the surfaces of these cells. In contrast, high ouabain affinity isoforms (α2 in astrocytes, α3 in neurons and myocytes) were confined to a reticular distribution within the PM that paralleled underlying endoplasmic or sarcoplasmic reticulum. This distribution is identical to that of the PM Na/Ca exchanger. This raises the possibility that α1 may regulate bulk cytosolic Na
+
, whereas α2 and α3 may regulate Na
+
and, indirectly, Ca
2+
in a restricted cytosolic space between the PM and reticulum. The high ouabain affinity Na
+
pumps may thereby modulate reticulum Ca
2+
content and Ca
2+
signaling.
Plant diversity and ecosystem productivity: Theoretical considerations
Authors: Tilman David , Lehman Clarence L. , Thomson Kendall T.
Published: July 2002

Ecosystem processes are thought to depend on both the number and
identity of the species present in an ecosystem, but mathematical
theory predicting this has been lacking. Here we present three simple
models of interspecific competitive interactions in communities
containing various numbers of randomly chosen species. All three models
predict that, on average, productivity increases asymptotically with
the original biodiversity of a community. The two models that address
plant nutrient competition also predict that ecosystem nutrient
retention increases with biodiversity and that the effects of
biodiversity on productivity and nutrient retention increase with
interspecific differences in resource requirements. All three models
show that both species identity and biodiversity simultaneously
influence ecosystem functioning, but their relative importance varies
greatly among the models. This theory reinforces recent experimental
results and shows that effects of biodiversity on ecosystem functioning
are predicted by well-known ecological processes.
Targeting RNA decay with 2′,5′ oligoadenylate-antisense in respiratory syncytial virus-infected cells
Authors: Cirino Nick M. , Li Guiying , Xiao Wei , Torrence Paul F. , Silverman Robert H.
Published: July 2002

Treatment of human cells with 2′,5′ oligoadenylate covalently linked to antisense (2–5A-antisense) results in the selective cleavage of targeted RNA species by 2–5A-dependent RNase L. Here we show that 2–5A-antisense containing stabilizing modifications at both termini are effective in suppressing the replication of respiratory syncytial virus (RSV) in human tracheal epithelial cells. The affinity of 2–5A-antisense for different regions in the RSV M2 and L mRNAs was predicted from a computer-generated model of the RNA secondary structure. The most potent 2–5A-antisense molecule caused a highly effective, dose-dependent suppression of RSV yields when added to previously infected cells. In contrast, control oligonucleotides, including an inactive dimeric form of 2–5A linked to antisense, 2–5A linked to a randomized sequence of nucleotides, and antisense molecules lacking 2–5A, had minimal effects on virus replication. The specificity of this approach was shown by reverse transcriptase-coupled PCR analysis of RSV M2, P, and N mRNA and of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The RSV M2 mRNA amounts were depleted after treating RSV-infected cells with 2–5A-antisense targeted to this mRNA, whereas the amounts of the other RNA species were unchanged. These studies demonstrate that 2′,5′ oligoadenylate covalently linked to antisense (2–5A-antisense) can effectively suppress RSV replication by directing the cellular RNase L to selectively degrade an essential viral mRNA.
Greetings: 50 years of Atomic Bomb Casualty Commission–Radiation Effects Research Foundation studies
Authors: Shigematsu Itsuzo
Published: July 2002

The Atomic Bomb Casualty Commission was established in Hiroshima in
1947 and in Nagasaki in 1948 under the auspices of the U.S. National
Academy of Sciences to initiate a long-term and comprehensive
epidemiological and genetic study of the atomic bomb survivors. It was
replaced in 1975 by the Radiation Effects Research Foundation which is
a nonprofit Japanese foundation binationally managed and supported with
equal funding by the governments of Japan and the United States. Thanks
to the cooperation of the survivors and the contributions of a
multitude of scientists, these studies flourish to this day in what
must be the most successful long-term research collaboration between
the two countries. Although these studies are necessarily limited to
the effects of acute, whole-body, mixed gamma-neutron radiation from
the atom bombs, their comprehensiveness and duration make them the most
definitive descriptions of the late effects of radiation in humans. For
this reason, the entire world relies heavily on these data to set
radiation standards. As vital as the study results are, they still
represent primarily the effects of radiation on older survivors.
Another decade or two should correct this deficiency and allow us to
measure definitively the human risk of heritable mutation from
radiation. We look to the worldwide radiation and risk community as
well as to the survivors who have contributed so much to what has been
done already to accomplish this goal.
Continuous amperometric monitoring of glucose in a brittle diabetic chimpanzee with a miniature subcutaneous electrode
Authors: Wagner J. G. , Schmidtke D. W. , Quinn C. P. , Fleming T. F. , Bernacky B. , Heller A.
Published: July 2002

The performance of an amperometric biosensor, consisting of a subcutaneously implanted miniature (0.29 mm diameter, 5 × 10−4cm2mass transporting area), 90 s 10–90% rise/decay time glucose electrode, and an on-the-skin electrocardiogram Ag/AgCl electrode was tested in an unconstrained, naturally diabetic, brittle, type I, insulin-dependent chimpanzee. The chimpanzee was trained to wear on her wrist a small electronic package and to present her heel for capillary blood samples. In five sets of measurements, averaging 5 h each, 82 capillary blood samples were assayed, their concentrations ranging from 35 to 400 mg/dl. The current readings were translated to blood glucose concentration by assaying, att= 1 h, one blood sample for each implanted sensor. The rms error in the correlation between the sensor-measured glucose concentration and that in capillary blood was 17.2%, 4.9% above the intrinsic 12.3% rms error of the Accu-Chek II reference, through which the illness of the chimpanzee was routinely managed. Linear regression analysis of the data points taken att>1 h yielded the relationship (Accu-Chek) = 0.98 × (implanted sensor) + 4.2 mg/dl,r2= 0.94. The capillary blood and the subcutaneous glucose concentrations were statistically indistinguishable when the rate of change was less than 1 mg/(dl⋅min). However, when the rate of decline exceeded 1.8 mg/(dl⋅min) after insulin injection, the subcutaneous glucose concentration was transiently higher.
Flavonoids: A class of modulators with bifunctional interactions at vicinal ATP- and steroid-binding sites on mouse P-glycoprotein
Authors: Conseil Gwenaelle , Baubichon-Cortay Helene , Dayan Guila , Jault Jean-Michel , Barron Denis , Di Pietro Attilio
Published: July 2002

A hexahistidine-tagged C-terminal nucleotide-binding domain (H
6
-NBD2) from mouse P-glycoprotein was designed, overexpressed, and purified as a highly soluble recombinant protein. Intrinsic fluorescence of its single tryptophan residue allowed monitoring of high-affinity binding of 2′(3′)-
N
-methylanthraniloyl-ATP (MANT-ATP), a fluorescent ATP derivative that induces a marked quenching correlated to fluorescence resonance-energy transfer. H
6
-NBD2 also bound all flavonoids known to modulate the multidrug resistance phenotype of P-glycoprotein-positive cancer cells, with similar affinities and relative efficiencies. Flavones (like quercetin or apigenin) bound more strongly than flavanones (naringenin), isoflavones (genistein), or glycosylated derivatives (rutin). Kaempferide, a 4′-methoxy 3,5,7-trihydroxy flavone, was even more reactive and induced a complete quenching of H
6
-NBD2 intrinsic fluorescence. Kaempferide binding was partly prevented by preincubation with ATP, or partly displaced upon ATP addition. Interestingly, kaempferide was also able to partly prevent the binding of the antiprogestin RU 486 to a hydrophobic region similar to that recently found, close to the ATP site, in the N-terminal cytosolic domain. Conversely, RU 486 partly prevented kaempferide binding, the effect being additive to the partial prevention by ATP. Furthermore, MANT-ATP binding, which occurred at the ATP site and extended to the vicinal steroid-interacting hydrophobic region, was completely prevented or displaced by kaempferide. All results indicate that flavonoids constitute a new class of modulators with bifunctional interactions at vicinal ATP-binding site and steroid-interacting region within a cytosolic domain of P-glycoprotein.
Adult fitness consequences of sexual selection inDrosophila melanogaster
Authors: Promislow Daniel E. L. , Smith Emily A. , Pearse Louise
Published: July 2002

Few experiments have demonstrated a genetic correlation between the process of sexual selection and fitness benefits in offspring, either through female choice or male competition. Those that have looked at the relationship between female choice and offspring fitness have focused on juvenile fitness components, rather than fitness at later stages in the life cycle. In addition, many of these studies have not controlled for possible maternal effects. To test for a relationship between sexual selection and adult fitness, we carried out an artificial selection experiment in the fruit fly,Drosophila melanogaster. We created two treatments that varied in the level of opportunity for sexual selection. Increased opportunity for female choice and male competition was genetically correlated with an increase in adult survivorship, as well as an increase in male and female body size. Contrary to previous, single-generation studies, we did not find an increase in larval competitive ability. This study demonstrates that mate choice and/or male–male competition are correlated with an increase in at least one adult fitness component of offspring.
U94 of human herpesvirus 6 is expressed in latently infected peripheral blood mononuclear cells and blocks viral gene expression in transformed lymphocytes in culture
Authors: Rotola Antonella , Ravaioli Tullia , Gonelli Arianna , Dewhurst Stephen , Cassai Enzo , Di Luca Dario
Published: July 2002

Human herpesvirus 6 (HHV-6) like other herpesviruses, expresses sequentially immediate early (IE), early, and late genes during lytic infection. Evidence of ability to establish latent infection has not been available, but by analogy with other herpesviruses it could be expected that IE genes that regulate and transactivate late genes would not be expressed. We report that peripheral blood mononuclear cells of healthy individuals infected with HHV-6 express the U94 gene, transcribed under IE conditions. Transcription of other IE genes (U16/17, U39, U42, U81, U89/90, U91) was not detected. To verify that U94 may play a role in the maintenance of the latent state, we derived lymphoid cell lines that stably expressed U94. HHV-6 was able to infect these cells, but viral replication was restricted. No cytopathic effect developed. Furthermore, viral transcripts were present in the first days postinfection and declined thereafter. A similar decline in the level of intracellular viral DNA also was observed. These findings are consistent with the hypothesis that the U94 gene product of HHV-6 regulates viral gene expression and enables the establishment and/or maintenance of latent infection in lymphoid cells.
Cleaving DNA with DNA
Authors: Carmi Nir , Balkhi Shameelah R. , Breaker Ronald R.
Published: July 2002

A DNA structure is described that can cleave single-stranded DNA oligonucleotides in the presence of ionic copper. This “deoxyribozyme” can self-cleave or can operate as a bimolecular complex that simultaneously makes use of duplex and triplex interactions to bind and cleave separate DNA substrates. Bimolecular deoxyribozyme-mediated strand scission proceeds with a
k
obs
of 0.2 min
−1
, whereas the corresponding uncatalyzed reaction could not be detected. The duplex and triplex recognition domains can be altered, making possible the targeted cleavage of single-stranded DNAs with different nucleotide sequences. Several small synthetic DNAs were made to function as simple “restriction enzymes” for the site-specific cleavage of single-stranded DNA.
Glucocorticoid receptor and protein/RNA synthesis-dependent mechanisms underlie the control of synaptic plasticity by stress
Authors: Xu Lin , Holscher Christian , Anwyl Roger , Rowan Michael J.
Published: July 2002

Learning and memory are exquisitely sensitive to behavioral stress, but the underlying mechanisms are still poorly understood. Because activity-dependent persistent changes in synaptic strength are believed to mediate memory processes in brain areas such as the hippocampus we have examined the means by which stress affects synaptic plasticity in the CA1 region of the hippocampus of anesthetized rats. Inescapable behavioral stress (placement on an elevated platform for 30 min) switched the direction of plasticity, favoring low frequency stimulation-induced decreases in synaptic transmission (long-term depression, LTD), and opposing the induction of long-term potentiation by high frequency stimulation. We have discovered that glucocorticoid receptor activation mediates these effects of stress on LTD and long-term potentiation in a protein synthesis-dependent manner because they were prevented by the glucocorticoid receptor antagonist RU 38486 and the protein synthesis inhibitor emetine. Consistent with this, the ability of exogenously applied corticosterone in non-stressed rats to mimic the effects of stress on synaptic plasticity was also blocked by these agents. The enablement of low frequency stimulation-induced LTD by both stress and exogenous corticosterone was also blocked by the transcription inhibitor actinomycin D. Thus, naturally occurring synaptic plasticity is liable to be reversed in stressful situations via glucocorticoid receptor activation and mechanisms dependent on the synthesis of new protein and RNA. This indicates that the modulation of hippocampus-mediated learning by acute inescapable stress requires glucocorticoid receptor-dependent initiation of transcription and translation.
Light-activated rhodopsin induces structural binding motif in G protein α subunit
Authors: Kisselev Oleg G. , Kao Jeff , Ponder Jay W. , Fann Yang C. , Gautam N. , Marshall Garland R.
Published: July 2002

A large superfamily of transmembrane receptors control cellular responses to diverse extracellular signals by catalyzing activation of specific types of heterotrimeric GTP-binding proteins. How these receptors recognize and promote nucleotide exchange on G protein α subunits to initiate signal amplification is unknown. The three-dimensional structure of the transducin (Gt) α subunit C-terminal undecapeptide Gtα(340–350) IKENLKDCGLF was determined by transferred nuclear Overhauser effect spectroscopy while it was bound to photoexcited rhodopsin. Light activation of rhodopsin causes a dramatic shift from a disordered conformation of Gtα(340–350) to a binding motif with a helical turn followed by an open reverse turn centered at Gly-348, a helix-terminating C capping motif of an αLtype. Docking of the NMR structure to the GDP-bound x-ray structure of Gt reveals that photoexcited rhodopsin promotes the formation of a continuous helix over residues 325–346 terminated by the C-terminal helical cap with a unique cluster of crucial hydrophobic side chains. A molecular mechanism by which activated receptors can control G proteins through reversible conformational changes at the receptor–G protein interface is demonstrated.