bluuebunny/crossref_metadata_2025 huggingface dataset:
Monitoring of implanted stem cell migration
in vivo
: A highly resolved
in vivo
magnetic resonance imaging investigation of experimental stroke in rat
Authors: Hoehn Mathias , Küstermann Ekkehard , Blunk James , Wiedermann Dirk , Trapp Thorsten , Wecker Stefan , Föcking Melanie , Arnold Heinz , Hescheler Jürgen , Fleischmann Bernd K. , Schwindt Wolfram , Bührle Christian
Published: December 2002

In vivo
monitoring of stem cells after grafting is essential for a better understanding of their migrational dynamics and differentiation processes and of their regeneration potential. Migration of endogenous or grafted stem cells and neurons has been described in vertebrate brain, both under normal conditions from the subventricular zone along the rostral migratory stream and under pathophysiological conditions, such as degeneration or focal cerebral ischemia. Those studies, however, relied on invasive analysis of brain sections in combination with appropriate staining techniques. Here, we demonstrate the observation of cell migration under
in vivo
conditions, allowing the monitoring of the cell dynamics within individual animals, and for a prolonged time. Embryonic stem (ES) cells, constitutively expressing the GFP, were labeled by a lipofection procedure with a MRI contrast agent and implanted into rat brains. Focal cerebral ischemia had been induced 2 weeks before implantation of ES cells into the healthy, contralateral hemisphere. MRI at 78-μm isotropic spatial resolution permitted the observation of the implanted cells with high contrast against the host tissue, and was confirmed by GFP registration. During 3 weeks, cells migrated along the corpus callosum to the ventricular walls, and massively populated the borderzone of the damaged brain tissue on the hemisphere opposite to the implantation sites. Our results indicate that ES cells have high migrational dynamics, targeted to the cerebral lesion area. The imaging approach is ideally suited for the noninvasive observation of cell migration, engraftment, and morphological differentiation at high spatial and temporal resolution.
The 2B domain of the
Escherichia coli
Rep protein is not required for DNA helicase activity
Authors: Cheng Wei , Brendza Katherine M. , Gauss George H. , Korolev Sergey , Waksman Gabriel , Lohman Timothy M.
Published: December 2002

The
Escherichia coli
Rep protein is a 3′ to 5′ SF1 DNA helicase required for replication of bacteriophage φX174 in
E. coli
, and is structurally homologous to the
E. coli
UvrD helicase and the
Bacillus stearothermophilus
PcrA helicase. Previous crystallographic studies of Rep protein bound to single-stranded DNA revealed that it can undergo a large conformational change consisting of an ≈130° rotation of its 2B subdomain about a hinge region connected to the 2A subdomain. Based on crystallographic studies of PcrA, its 2B subdomain has been proposed to form part of its duplex DNA binding site and to play a role in duplex destabilization. To test the role of the 2B subdomain in Rep-catalyzed duplex DNA unwinding, we have deleted its 2B subdomain, replacing it with three glycines, to form the RepΔ2B protein. This RepΔ2B protein can support φX174 replication in a
rep
−
E. coli
strain, although the growth rate of
E. coli
containing the
rep
Δ
2B
gene is ≈1.5-fold slower than with the wild-type
rep
gene. Pre-steady-state, single-turnover DNA unwinding kinetics experiments show that purified RepΔ2B protein has DNA helicase activity
in vitro
and unwinds an 18-bp DNA duplex with rates at least as fast as wild-type Rep, and with higher extents of unwinding and higher affinity for the DNA substrate. These studies show that the 2B domain of Rep is not required for DNA helicase activity
in vivo
or
in vitro
, and that it does not facilitate DNA unwinding
in vitro
.
Conflict between nuclear and mitochondrial DNA phylogenies of a recent species radiation: What mtDNA reveals and conceals about modes of speciation in Hawaiian crickets
Authors: Shaw Kerry L.
Published: December 2002

It has been asserted that recent mtDNA phylogenies support the plausibility of sympatric speciation, long considered a controversial mechanism of the origin of species. If such inferences are reliable, mtDNA phylogenies should be congruent with phylogenies based on other data. In previous work, a mtDNA phylogeny suggested that diversification of the Hawaiian cricket genus
Laupala
was initiated by single invasions into each of several Hawaiian islands, followed by multiple sympatric divergences within each island. In contrast, a systematic hypothesis based on morphology argues that speciation in
Laupala
has occurred primarily in allopatry, with two independent species radiations diversifying across the archipelago. In this study, I analyze nuclear DNA (nDNA) sequences from
Laupala
to compare with sequences from the mtDNA. The nDNA phylogeny corroborates the hypothesis of allopatric divergence and multiple invasions, and when compared with mtDNA patterns, suggests that interspecific hybridization is a persistent feature of the history of
Laupala
. The discrepancy between mtDNA and nDNA phylogenies reveals that speciation histories based on mtDNA alone can be extensively misleading.
Large-scale comparison of intron positions among animal, plant, and fungal genes
Authors: Fedorov Alexei , Merican Amir Feisal , Gilbert Walter
Published: December 2002

We purge large databases of animal, plant, and fungal intron-containing genes to a 20% similarity level and then identify the most similar animal–plant, animal–fungal, and plant–fungal protein pairs. We identify the introns in each BLAST 2.0 alignment and score matched intron positions and slid (near-matched, within six nucleotides) intron positions automatically. Overall we find that 10% of the animal introns match plant positions, and a further 7% are “slides.” Fifteen percent of fungal introns match animal positions, and 13% match plant positions. Furthermore, the number of alignments with high numbers of matches deviates greatly from the Poisson expectation. The 30 animal–plant alignments with the highest matches (for which 44% of animal introns match plant positions) when aligned with fungal genes are also highly enriched for triple matches: 39% of the fungal introns match both animal and plant positions. This is strong evidence for ancestral introns predating the animal–plant–fungal divergence, and in complete opposition to any expectations based on random insertion. In examining the slid introns, we show that at least half are caused by imperfections in the alignments, and are most likely to be actual matches at common positions. Thus, our final estimates are that ≈14% of animal introns match plant positions, and that ≈17–18% of fungal introns match animal or plant positions, all of these being likely to be ancestral in the eukaryotes.
RETRACTED: Functional complementation of yeast cytosolic pyrophosphatase by bacterial and plant H
+
-translocating pyrophosphatases
Authors: Pérez-Castiñeira José R. , López-Marqués Rosa L. , Villalba José M. , Losada Manuel , Serrano Aurelio
Published: December 2002

Two types of proteins that hydrolyze inorganic pyrophosphate (PP
i
), very different in both amino acid sequence and structure, have been characterized to date: soluble and membrane-bound proton-pumping pyrophosphatases (sPPases and H
+
-PPases, respectively). sPPases are ubiquitous proteins that hydrolyze PP
i
releasing heat, whereas H
+
-PPases, so far unidentified in animal and fungal cells, couple the energy of PP
i
hydrolysis to proton movement across biological membranes. The budding yeast
Saccharomyces cerevisiae
has two sPPases that are located in the cytosol and in the mitochondria. Previous attempts to knock out the gene coding for a cytosolic sPPase (
IPP1
) have been unsuccessful, thus suggesting that this protein is essential for growth. Here, we describe the generation of a conditional
S. cerevisiae
mutant (named YPC-1) whose functional
IPP1
gene is under the control of a galactose-dependent promoter. Thus, YPC-1 cells become growth arrested in glucose but they regain the ability to grow on this carbon source when transformed with autonomous plasmids bearing diverse foreign H
+
-PPase genes under the control of a yeast constitutive promoter. The heterologously expressed H
+
-PPases are distributed among different yeast membranes, including the plasma membrane, functional complementation by these integral membrane proteins being consistently sensitive to external pH. These results demonstrate that hydrolysis of cytosolic PP
i
is essential for yeast growth and that this function is not substantially affected by the intrinsic characteristics of the PPase protein that accomplishes it. Moreover, this is, to our knowledge, the first direct evidence that H
+
-PPases can mediate net hydrolysis of PP
i
in vivo
. YPC-1 mutant strain constitutes a convenient expression system to perform studies aimed at the elucidation of the structure–function relationships of this type of proton pumps.
The spatial receptive field of thalamic inputs to single cortical simple cells revealed by the interaction of visual and electrical stimulation
Authors: Kara Prakash , Pezaris John S. , Yurgenson Sergey , Reid R. Clay
Published: December 2002

Electrical stimulation of the thalamus has been widely used to test for the existence of monosynaptic input to cortical neurons, typically with stimulation currents that evoke cortical spikes with high probability. We stimulated the lateral geniculate nucleus (LGN) of the thalamus and recorded monosynaptically evoked spikes from layer 4 neurons in visual cortex. We found that with moderate currents, cortical spikes were evoked with low to moderate probability and their occurrence was modulated by ongoing sensory (visual) input. Furthermore, when repeated at 8–12 Hz, electrical stimulation of the thalamic afferents caused such profound inhibition that cortical spiking activity was suppressed, aside from electrically evoked monosynaptic spikes. Visual input to layer 4 cortical cells between electrical stimuli must therefore have derived exclusively from LGN afferents. We used white-noise visual stimuli to make a 2D map of the receptive field of each cortical simple cell during repetitive electrical stimulation in the LGN. The receptive field of electrically evoked monosynaptic spikes (and thus of the thalamic input alone) was significantly elongated. Its primary subfield was comparable to that of the control receptive field, but secondary (flanking) subfields were weaker. These findings extend previous results from intracellular recordings, but also demonstrate the effectiveness of an extracellular method of measuring subthreshold afferent input to cortex.
An N-terminal domain of Dbf4p mediates interaction with both origin recognition complex (ORC) and Rad53p and can deregulate late origin firing
Authors: Duncker Bernard P. , Shimada Kenji , Tsai-Pflugfelder Monika , Pasero Philippe , Gasser Susan M.
Published: December 2002

The Dbf4/Cdc7 kinase acts at the level of individual origins to promote the initiation of DNA replication. We demonstrate through both immunoprecipitation and two-hybrid assays that a domain comprising the first 296 aa of Dbf4p interacts with Orc2p and Orc3p subunits of the origin recognition complex (ORC). Given that the activation of Rad53 kinase in response to the DNA replication checkpoint leads to the release of Dbf4p from an ORC-containing chromatin fraction, we also examined interaction between Dbf4p and Rad53p. This same domain of Dbf4p binds specifically to the forkhead homology-associated (FHA) domains of Rad53p. Cell cycle arrest in G
2
/M, provoked by the overexpression of the Dbf4 domain, is suppressed in a
rad53
mutant. Moreover, its overexpression perturbs the regulation of late, but not early, origin firing in wild-type cells after treatment with hydroxyurea.
Magnetic resonance microimaging of intraaxonal water diffusion in live excised lamprey spinal cord
Authors: Takahashi Masaya , Hackney David B. , Zhang Guixin , Wehrli Suzanne L. , Wright Alex C. , O'Brien William T. , Uematsu Hidemasa , Wehrli Felix W. , Selzer Michael E.
Published: December 2002

Anisotropy of water diffusion in axon tracts, as determined by diffusion-weighted MRI, has been assumed to reflect the restriction of water diffusion across axon membranes. Reduction in this anisotropy has been interpreted as degeneration of axons. These interpretations are based primarily on
a priori
reasoning that has had little empirical validation. We used the experimental advantages of the sea lamprey spinal cord, which contains several very large axons, to determine whether intraaxonal diffusion is isotropic and whether anisotropy is attributable to restriction of water mobility by axon surface membranes. Through the application of magnetic resonance microimaging, we were able to measure the purely intraaxonal diffusion characteristics of the giant reticulospinal axons (20–40 μm in diameter). The intraaxonal apparent diffusion coefficients of water parallel (longitudinal ADC, l-ADC) and perpendicular (transverse ADC, t-ADC) to the long axis were 0.98 ± 0.06 (10
−3
mm
2
/sec) and 0.97 ± 0.11 (10
−3
mm
2
/sec), respectively. In white matter regions that included multiple axons, l-ADCs were almost identical regardless of axon density in the sampled axon tract. By comparison, t-ADCs were reduced and varied inversely with the number of axons (and thus axolemmas) in a fixed cross-sectional area. Thus, diffusion was found to be isotropic when measured entirely within a single axon and anisotropic when measured in regions that included multiple axons. These findings support the hypothesis that the cell membrane is the primary source of diffusion anisotropy in fiber tracts of the central nervous system.
De novo
designed peptide-based amyloid fibrils
Authors: López de la Paz Manuela , Goldie Kenneth , Zurdo Jesús , Lacroix Emmanuel , Dobson Christopher M. , Hoenger Andreas , Serrano Luis
Published: December 2002

Identification of therapeutic strategies to prevent or cure diseases associated with amyloid fibril deposition in tissue (Alzheimer's disease, spongiform encephalopathies, etc.) requires a rational understanding of the driving forces involved in the formation of these organized assemblies rich in β-sheet structure. To this end, we used a computer-designed algorithm to search for hexapeptide sequences with a high propensity to form homopolymeric β-sheets. Sequences predicted to be highly favorable on this basis were found experimentally to self-associate efficiently into β-sheets, whereas point mutations predicted to be unfavorable for this structure inhibited polymerization. However, the property to form polymeric β-sheets is not a sufficient requirement for fibril formation because, under the conditions used here, preformed β-sheets from these peptides with charged residues form well defined fibrils only if the total net charge of the molecule is ±1. This finding illustrates the delicate balance of interactions involved in the formation of fibrils relative to more disordered aggregates. The present results, in conjunction with x-ray fiber diffraction, electron microscopy, and Fourier transform infrared measurements, have allowed us to propose a detailed structural model of the fibrils.
RNA interference-mediated silencing of
Sod2
in
Drosophila
leads to early adult-onset mortality and elevated endogenous oxidative stress
Authors: Kirby Kim , Hu Jianguo , Hilliker Arthur J. , Phillips John P.
Published: December 2002

Oxidative stress has been widely implicated as an important factor in the aging process. Because mitochondrial respiration is the principal source of reactive oxygen within cells, the mitochondrially localized superoxide dismutase (SOD) 2 is thought to play an important front-line defensive role against aging-related oxidative stress. Although genetic studies with mutants deficient in SOD1, the predominantly cytosolic isoform of SOD, have been instrumental in elucidating the role of reactive oxygen metabolism in aging in
Drosophila
, the lack of available mutations in the
Sod2
gene has hampered an equivalent analysis of the participation of this important antioxidant enzyme in the
Drosophila
aging model. Here we report that ablation of mitochondrial SOD2 through expression of a GAL4-regulated, inverted-repeat
Sod2
RNA-interference transgene in an otherwise normal animal causes increased endogenous oxidative stress, resulting in loss of essential enzymatic components of the mitochondrial respiratory chain and the tricarboxylic acid cycle, enhances sensitivity to applied oxidative stress, and causes early-onset mortality in young adults. In sharp contrast, ablation of SOD2 has no overt effect on the development of larvae and pupae, which may reflect a fundamental transition in oxygen utilization and/or reactive oxygen metabolism that occurs during metamorphosis from larval to adult life.
Artificial human telomeres from DNA nanocircle templates
Authors: Lindström Ulf M. , Chandrasekaran Ravi A. , Orbai Lucian , Helquist Sandra A. , Miller Gregory P. , Oroudjev Emin , Hansma Helen G. , Kool Eric T.
Published: December 2002

Human telomerase is a reverse-transcriptase enzyme that synthesizes the multikilobase repeating hexamer telomere sequence (TTAGGG)
n
at the ends of chromosomes. Here we describe a designed approach to mimicry of telomerase, in which synthetic DNA nanocircles act as essentially infinite catalytic templates for efficient synthesis of long telomeres by DNA polymerase enzymes. Results show that the combination of a nanocircle and a DNA polymerase gives a positive telomere-repeat amplification protocol assay result for telomerase activity, and similar to the natural enzyme, it is inhibited by a known telomerase inhibitor. We show that artificial telomeres can be engineered on human chromosomes by this approach. This strategy allows for the preparation of synthetic telomeres for biological and structural study of telomeres and proteins that interact with them, and it raises the possibility of telomere engineering in cells without expression of telomerase itself. Finally, the results provide direct physical support for a recently proposed rolling-circle mechanism for telomerase-independent telomere elongation.
A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata
Authors: Aoki Koh , Kragler Friedrich , Xoconostle-Cázares Beatriz , Lucas William J.
Published: December 2002

Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three
Cucurbita maxima
Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins.
STAT1 deficiency unexpectedly and markedly exacerbates the pathophysiological actions of IFN-α in the central nervous system
Authors: Wang Jianping , Schreiber Robert D. , Campbell Iain L.
Published: December 2002

Although signal transducer and activator of transcription 1 (STAT1) is an essential signaling molecule in many IFN-α-regulated processes, some biological responses to IFN-α can occur independently of STAT1. To establish the role of STAT1 in mediating the biological actions of IFN-α in the CNS, transgenic mice [termed glial fibrillary acidic protein (GFAP)-IFN-α] with astrocyte production of IFN-α were bred to be null for the STAT1 gene. Surprisingly, GFAP-IFN-α mice deficient for STAT1 developed earlier onset and more severe neurological disease with increased lethality compared with GFAP-IFN-α mice sufficient for STAT1. Whereas the brain of 2- to 3-month-old GFAP-IFN-α mice showed little, if any abnormality, the brain from GFAP-IFN-α mice deficient for STAT1 had severe neurodegeneration, inflammation, calcification with increased apoptosis, and glial activation. However, the cerebral expression of a number of IFN-regulated STAT1-dependent genes increased in GFAP-IFN-α mice but was reduced markedly in GFAP-IFN-α STAT1-null mice. Of many other signaling molecules examined, STAT3 alone was activated significantly in the brain of GFAP-IFN-α STAT1-null mice. Thus, in the absence of STAT1, alternative signaling pathways mediate pathophysiological actions of IFN-α in the living brain, giving rise to severe encephalopathy. Finally, STAT1 or a downstream component of the JAK/STAT pathway may protect against such IFN-α-mediated injury in the CNS.
Scanning surface confocal microscopy for simultaneous topographical and fluorescence imaging: Application to single virus-like particle entry into a cell
Authors: Gorelik J. , Shevchuk A. , Ramalho M. , Elliott M. , Lei C. , Higgins C. F. , Lab Max J. , Klenerman D. , Krauzewicz N. , Korchev Y.
Published: December 2002

We have developed a method for simultaneous recording of high-resolution topography and cell surface fluorescence in a single scan which we call scanning surface confocal microscopy. The resolution of the system allows imaging of individual fluorescent particles in the nanometer range on fixed or live cells. We used this technique to record the interaction of single virus-like particles with the cell surface and demonstrated that single particles sink into the membrane in invaginations reminiscent of caveolae or pinocytic vesicles. This method provides a technique for elucidating the interaction of individual viruses and other nanoparticles, such as gene therapy vectors, with target cells. Furthermore, this technique should find widespread application for studying the relationship of fluorescently tagged molecules with components of the cell plasma membrane.
A new role for an old enzyme: Nitrate reductase-mediated nitric oxide generation is required for abscisic acid-induced stomatal closure in
Arabidopsis
thaliana
Authors: Desikan Radhika , Griffiths Rachael , Hancock John , Neill Steven
Published: December 2002

The plant hormone abscisic acid (ABA), synthesized in response to water-deficit stress, induces stomatal closure via activation of complex signaling cascades. Recent work has established that nitric oxide (NO) is a key signaling molecule mediating ABA-induced stomatal closure. However, the biosynthetic origin of NO in guard cells has not yet been resolved. Here, we provide pharmacological, physiological, and genetic evidence that NO synthesis in
Arabidopsis
guard cells is mediated by the enzyme nitrate reductase (NR). Guard cells of wild-type
Arabidopsis
generate NO in response to treatment with ABA and nitrite, a substrate for NR. Moreover, NR-mediated NO synthesis is required for ABA-induced stomatal closure. However, in the NR double mutant,
nia1
,
nia2
that has diminished NR activity, guard cells do not synthesize NO nor do the stomata close in response to ABA or nitrite, although stomatal opening is still inhibited by ABA. Furthermore, by using the ABA-insensitive (ABI)
abi1–1
and
abi2–1
mutants, we show that the ABI1 and ABI2 protein phosphatases are downstream of NO in the ABA signal-transduction cascade. These data demonstrate a previously uncharacterized signaling role for NR, that of mediating ABA-induced NO synthesis in
Arabidopsis
guard cells.
Air pollution induces heritable DNA mutations
Authors: Somers Christopher M. , Yauk Carole L. , White Paul A. , Parfett Craig L. J. , Quinn James S.
Published: December 2002

Hundreds of thousands of people worldwide live or work in close proximity to steel mills. Integrated steel production generates chemical pollution containing compounds that can induce genetic damage (1, 2). Previous investigations of herring gulls in the Great Lakes demonstrated elevated DNA mutation rates near steel mills (3, 4) but could not determine the importance of airborne or aquatic routes of contaminant exposure, or eliminate possible confounding factors such as nutritional status and disease burden. To address these issues experimentally, we exposed laboratory mice
in situ
to ambient air in a polluted industrial area near steel mills. Heritable mutation frequency at tandem-repeat DNA loci in mice exposed 1 km downwind from two integrated steel mills was 1.5- to 2.0-fold elevated compared with those at a reference site 30 km away. This statistically significant elevation was due primarily to an increase in mutations inherited through the paternal germline. Our results indicate that human and wildlife populations in proximity to integrated steel mills may be at risk of developing germline mutations more frequently because of the inhalation of airborne chemical mutagens.
In vitro
reconstitution of the activated zeaxanthin state associated with energy dissipation in plants
Authors: Aspinall-O'Dea Mark , Wentworth Mark , Pascal Andy , Robert Bruno , Ruban Alexander , Horton Peter
Published: December 2002

Dissipation of excess light energy in plant photosynthetic membranes plays an important role in the response of plants to the environment, providing short-term balancing between the intensity of sunlight and photosynthetic capacity. The carotenoid zeaxanthin and the photosystem II subunit PsbS play vital roles in this process, but the mechanism of their action is largely unexplained. Here we report that the isolated photosystem II subunit PsbS was able to bind exogenous zeaxanthin, the binding resulting in a strong red shift in the absorption spectrum, and the appearance of characteristic features in the resonance Raman spectrum and a distinct circular dichroism spectrum, indicating pigment–protein, as well as specific pigment–pigment, interaction. A strong shift in the absorption spectrum of PsbS phenylalanine residues after zeaxanthin binding was observed. It is concluded that zeaxanthin binding to PsbS is the origin of the well known energy dissipation-related 535-nm absorption change that we showed
in vivo
to arise from activation of 1–2 molecules of this pigment. The altered properties of zeaxanthin and PsbS that result from this interaction provide the first direct indication about how they regulate energy dissipation.
Murine apolipoprotein serum amyloid A in solution forms a hexamer containing a central channel
Authors: Wang Limin , Lashuel Hilal A. , Walz Thomas , Colón Wilfredo
Published: December 2002

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins in the serum. Although SAA seems to play a role in host defense and lipid transport and metabolism, its specific functions have not been defined. Despite the growing implications that SAA plays a role in the pathology of various diseases, a high-resolution structure of SAA is lacking because of limited solubility in the high-density lipoprotein-free form. In this study, complementary methods including glutaraldehyde cross-linking, size-exclusion chromatography, and sedimentation-velocity analytical ultracentrifugation were used to show that murine SAA2.2 in aqueous solution exists in a monomer–hexamer equilibrium. Electron microscopy of hexameric SAA2.2 revealed that the subunits are arranged in a ring forming a putative central channel. Limited trypsin proteolysis and mass spectrometry analysis identified a significantly protease-resistant SAA2.2 region comprising residues 39–86. The isolated 39–86 SAA2.2 fragment did not hexamerize, suggesting that part of the N terminus is involved in SAA2.2 hexamer formation. Circular-dichroism spectrum deconvolution and secondary-structure prediction suggest that SAA2.2 contains ≈50% of its residues in α-helical conformation and <10% in β-structure. These findings are consistent with the recent discovery that human SAA1.1 forms a membrane channel and have important implications for understanding the 3D structure, multiple functions, and pathological roles of this highly conserved protein.
Large-scale molecular dynamics simulations of general anesthetic effects on the ion channel in the fully hydrated membrane: The implication of molecular mechanisms of general anesthesia
Authors: Tang Pei , Xu Yan
Published: December 2002

Interactions of volatile anesthetics with the central nervous system are characterized by low yet specific binding affinities. Although neurotransmitter-gated ion channels are considered the primary anesthetic targets, the mechanism of action at the molecular level remains elusive. We consider here the theoretical implications of channel dynamics on anesthetic action in a simplified membrane-channel system. Large-scale 2.2-ns all-atom molecular dynamics simulations were performed to study the effects of halothane, a clinical anesthetic, on a gramicidin A (gA) channel in a fully hydrated dimyristoyl phosphatidylcholine membrane. In agreement with experimental results, anesthetics preferentially target the anchoring residues at the channel–lipid–water interface. Although the anesthetic effect on channel structure is minimal, the presence of halothane profoundly affects channel dynamics. For 2.2-ns simulation, the rms fluctuation of gA backbone in the lipid core increases from ≈1 Å in the absence of anesthetics to ≈1.5 Å in the presence of halothane. Autocorrelation analysis reveals that halothane (
i
) has no effect on the subpicosecond librational motion, (
ii
) prolongs the backbone autocorrelation time in the 10- to 100-ps time scale, and (
iii
) significantly decreases the asymptotic values of generalized order parameter and correlation time of nanosecond motions for the inner but not the outer residues. The simulation results discount the viewpoint of a structure–function paradigm that overrates the importance of structural fitting between general anesthetics and yet-unidentified hydrophobic protein pockets. Instead, the results underscore the global, as opposed to local, effects of anesthetics on protein dynamics as the underlying mechanisms for the action of general anesthetics and possibly of other low-affinity drugs.
A pectin glucuronyltransferase gene is essential for intercellular attachment in the plant meristem
Authors: Iwai Hiroaki , Masaoka Nobutaka , Ishii Tadashi , Satoh Shinobu
Published: December 2002

Intercellular attachment is an essential process in the morphogenesis of multicellular organisms. A unique mutant,nolac-H18(nonorganogenic callus with loosely attached cells), generated by T-DNA transformation using leaf-disk cultures of haploidNicotiana plumbaginifolia, lost the ability to form tight intercellular attachments and adventitious shoots. The gene tagged with T-DNA, namedNpGUT1(glucuronyltransferase 1), was similar to the gene for the catalytic domains of animal glucuronyltransferases and was expressed predominantly in shoot and root apical meristems. The transformation ofNpGUT1complemented thenolac-H18mutation, and the expression of antisenseNpGUT1RNA produced crumbled shoots. The mutation caused defects in the glucuronic acid of rhamnogalacturonan II of pectin, which drastically reduced the formation of borate cross-linking of rhamnogalacturonan II.NpGUT1, which encodes a unique glucuronyltransferase, is a glycosyltransferase gene identified in pectin biosynthesis and is essential for intercellular attachment in plant meristems and tissues.
Consensus-derived structural determinants of the ankyrin repeat motif
Authors: Mosavi Leila K. , Minor Daniel L. , Peng Zheng-yu
Published: December 2002

The ankyrin repeat is one of the most common, modular, protein–protein interaction motifs in nature. To understand the structural determinants of this family of proteins and extract the consensus information that defines the architecture of this motif, we have designed a series of idealized ankyrin repeat proteins containing one, two, three, or four repeats by using statistical analysis of ≈4,000 ankyrin repeat sequences from the PFAM database. Biophysical and x-ray crystallographic studies of the three and four repeat constructs (3ANK and 4ANK) to 1.26 and 1.5 Å resolution, respectively, demonstrate that these proteins are well-folded, monomeric, display high thermostability, and adopt a very regular, tightly packed ankyrin repeat fold. Mapping the degree of amino acid conservation at each position on the 4ANK structure shows that most nonconserved residues are clustered on the surface of the molecule that has been designated as the binding site in naturally occurring ankyrin repeat proteins. Thus, the consensus amino acid sequence contains all information required to define the ankyrin repeat fold. Our results suggest that statistical analysis and the consensus sequence approach can be used as an effective method to design proteins with complex topologies. These generic ankyrin repeat proteins can serve as prototypes for dissecting the rules of molecular recognition mediated by ankyrin repeats and for engineering proteins with novel biological functions.
Disruption of
Abcg5
and
Abcg8
in mice reveals their crucial role in biliary cholesterol secretion
Authors: Yu Liqing , Hammer Robert E. , Li-Hawkins Jia , von Bergmann Klaus , Lutjohann Dieter , Cohen Jonathan C. , Hobbs Helen H.
Published: December 2002

Cholesterol and other sterols exit the body primarily by secretion into bile. In patients with sitosterolemia, mutations in either of two ATP-binding cassette (ABC) half-transporters, ABCG5 or ABCG8, lead to reduced secretion of sterols into bile, implicating these transporters in this process. To elucidate the roles of ABCG5 and ABCG8 in the trafficking of sterols, we disrupted
Abcg5
and
Abcg8
in mice (
G5G8
−/−
). The
G5G8
−/−
mice had a 2- to 3-fold increase in the fractional absorption of dietary plant sterols, which was associated with an ≈30-fold increase in plasma sitosterol. Biliary cholesterol concentrations were extremely low in the
G5G8
−/−
mice when compared with wild-type animals (mean = 0.4 vs. 5.5 μmol/ml) and increased only modestly with cholesterol feeding. Plasma and liver cholesterol levels were reduced by 50% in the chow-fed
G5G8
−/−
mice and increased 2.4- and 18-fold, respectively, after cholesterol feeding. These data indicate that ABCG5 and ABCG8 are required for efficient secretion of cholesterol into bile and that disruption of these genes increases dramatically the responsiveness of plasma and hepatic cholesterol levels to changes in dietary cholesterol content.
African rice (Oryza glaberrima): History and future potential
Authors: Linares Olga F.
Published: December 2002

The African species of rice (Oryza glaberrima) was cultivated long before Europeans arrived in the continent. At present,O. glaberrimais being replaced by the introduced Asian species of rice,Oryza sativa. Some West African farmers, including the Jola of southern Senegal, still grow African rice for use in ritual contexts. The two species of rice have recently been crossed, producing a promising hybrid.
Selenosugars are key and urinary metabolites for selenium excretion within the required to low-toxic range
Authors: Kobayashi Yayoi , Ogra Yasumitsu , Ishiwata Kazuya , Takayama Hiromitsu , Aimi Norio , Suzuki Kazuo T.
Published: December 2002

Essential micronutrient selenium is excreted into the urine and/or expired after being transformed to methylated metabolites. Monomethylated selenium is excreted into the urine in response to a supply within the required to low-toxic range, whereas tri- and dimethylated selenium increase with excessive supply at a toxic dose. Here we show that the major urinary selenium metabolite within the required to low-toxic range is a selenosugar. The structure of 1β-methylseleno-
N
-acetyl-
d
-galactosamine was deduced from the spectroscopic data and confirmed by chemical synthesis. This metabolite was also detected in the liver, and an additional metabolite increased with inhibition of methylation. The latter metabolite was again a selenosugar conjugated with glutathione instead of a methyl group and was assumed to be a precursor for methylation to the former metabolite. A metabolic pathway for the urinary excretion of selenium, i.e., from the glutathione-
S
-conjugated selenosugar to the methylated one, was proposed. Urinary monomethylated (selenosugar) and trimethylated selenium can be used as specific indices that increase within the required to low-toxic range and with a distinct toxic dose, respectively.
Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells
Authors: Hori Yuichi , Rulifson Ingrid C. , Tsai Bernette C. , Heit Jeremy J. , Cahoy John D. , Kim Seung K.
Published: December 2002

The use of embryonic stem cells for cell-replacement therapy in diseases like diabetes mellitus requires methods to control the development of multipotent cells. We report that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase, an essential intracellular signaling regulator, produced cells that resembled pancreatic β cells in several ways. These cells aggregated in structures similar, but not identical, to pancreatic islets of Langerhans, produced insulin at levels far greater than previously reported, and displayed glucose-dependent insulin release
in vitro
. Transplantation of these cell aggregates increased circulating insulin levels, reduced weight loss, improved glycemic control, and completely rescued survival in mice with diabetes mellitus. Graft removal resulted in rapid relapse and death. Graft analysis revealed that transplanted insulin-producing cells remained differentiated, enlarged, and did not form detectable tumors. These results provide evidence that embryonic stem cells can serve as the source of insulin-producing replacement tissue in an experimental model of diabetes mellitus. Strategies for producing cells that can replace islet functions described here can be adapted for similar uses with human cells.
Human cytomegalovirus gene expression during infection of primary hematopoietic progenitor cells: A model for latency
Authors: Goodrum Felicia D. , Jordan Craig T. , High Kevin , Shenk Thomas
Published: December 2002

Human cytomegalovirus (HCMV) resides latently in hematopoietic cells of the bone marrow. Although viral genomes can be found in CD14+ monocytes and CD34+ progenitor cells, the primary reservoir for latent cytomegalovirus is unknown. We analyzed human hematopoietic subpopulations infected
in vitro
with a recombinant virus that expresses a green fluorescent protein marker gene. Although many hematopoietic cell subsets were infected
in vitro
, CD14+ monocytes and various CD34+ subpopulations were infected with the greatest efficiency. We have developed an
in vitro
system in which to study HCMV infection and latency in CD34+ cells cultured with irradiated stromal cells. Marker gene expression was substantially reduced by 4 days postinfection, and infectious virus was not made during the culture period. However, viral DNA sequences were maintained in infected CD34+ cells for >20 days in culture, and, importantly, virus replication could be reactivated by coculture with human fibroblasts. Using an HCMV gene array, we examined HCMV gene expression in CD34+ cells. The pattern of viral gene expression was distinct from that observed during productive or nonproductive infections. Some of these expressed viral genes may function in latency and are targets for further analysis. Altered gene expression in hematopoietic progenitors may be indicative of the nature and outcome of HCMV infection.
Guiding neuronal growth with light
Authors: Ehrlicher A. , Betz T. , Stuhrmann B. , Koch D. , Milner V. , Raizen M. G. , Käs J.
Published: December 2002

Control over neuronal growth is a fundamental objective in neuroscience, cell biology, developmental biology, biophysics, and biomedicine and is particularly important for the formation of neural circuits
in vitro
, as well as nerve regeneration
in vivo
[Zeck, G. & Fromherz, P. (2001)
Proc. Natl. Acad. Sci. USA
98, 10457–10462]. We have shown experimentally that we can use weak optical forces to guide the direction taken by the leading edge, or growth cone, of a nerve cell. In actively extending growth cones, a laser spot is placed in front of a specific area of the nerve's leading edge, enhancing growth into the beam focus and resulting in guided neuronal turns as well as enhanced growth. The power of our laser is chosen so that the resulting gradient forces are sufficiently powerful to bias the actin polymerization-driven lamellipodia extension, but too weak to hold and move the growth cone. We are therefore using light to control a natural biological process, in sharp contrast to the established technique of optical tweezers [Ashkin, A. (1970)
Phys. Rev. Lett.
24, 156–159; Ashkin, A. & Dziedzic, J. M. (1987)
Science
235, 1517–1520], which uses large optical forces to manipulate entire structures. Our results therefore open an avenue to controlling neuronal growth
in vitro
and
in vivo
with a simple, noncontact technique.
Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses
Authors: Garg Ajay K. , Kim Ju-Kon , Owens Thomas G. , Ranwala Anil P. , Choi Yang Do , Kochian Leon V. , Wu Ray J.
Published: December 2002

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant “resurrection plants,” trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of
Escherichia coli
trehalose biosynthetic genes (
otsA
and
otsB
) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3–10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mg/g fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.
A putative molecular-activation switch in the transmembrane domain of erbB2
Authors: Fleishman Sarel J. , Schlessinger Joseph , Ben-Tal Nir
Published: December 2002

Overexpression of the receptor tyrosine kinase (RTK) erbB2 (also designated
neu
or HER2) was implicated in causing a variety of human cancers, including mammary and ovarian carcinomas. Ligand-induced receptor dimerization is critical for stimulation of the intrinsic protein tyrosine kinase (PTK) of RTKs. It was therefore proposed that PTK activity is stimulated as a result of the reorientation of the cytoplasmic domains within receptor dimers, leading to transautophosphorylation and stimulation of enzymatic activity. Here, we propose a molecular mechanism for rotation-coupled activation of the erbB2 receptor. Using a computational exploration of conformation space of the transmembrane (TM) segments of an erbB2 homodimer, we found two stable conformations of the TM domain. We suggest that these conformations correspond to the active and inactive states of erbB2, and that the receptor molecules may switch from one conformation to the other without crossing exceedingly unfavorable states. This model provides an explanation for the biochemical and oncogenic properties of erbB2, such as the effects of erbB2 overexpression on kinase activity and cell transformation. Furthermore, the opposing effects of the
neu
* activating oncogenic point mutation and the Val-655→Ile single-nucleotide polymorphism shown to be linked to reduced risk of breast cancer are explained in terms of shifts in the equilibrium between the active and inactive states of erbB2
in vivo
.
The acquisition of multidimensional NMR spectra within a single scan
Authors: Frydman Lucio , Scherf Tali , Lupulescu Adonis
Published: December 2002

A scheme enabling the complete sampling of multidimensional NMR domains within a single continuous acquisition is introduced and exemplified. Provided that an analyte's signal is sufficiently strong, the acquisition time of multidimensional NMR experiments can thus be shortened by orders of magnitude. This could enable the characterization of transient events such as proteins folding, 2D NMR experiments on samples being chromatographed, bring the duration of higher dimensional experiments (e.g., 4D NMR) into the lifetime of most proteins under physiological conditions, and facilitate the incorporation of spectroscopic 2D sequences intoin vivoimaging investigations. The protocol is compatible with existing multidimensional pulse sequences and can be implemented by using conventional hardware; its performance is exemplified here with a variety of homonuclear 2D NMR acquisitions.
Attentional mechanisms of borderline personality disorder
Authors: Posner Michael I. , Rothbart Mary K. , Vizueta Nathalie , Levy Kenneth N. , Evans David E. , Thomas Kathleen M. , Clarkin John F.
Published: December 2002

We consider whether disruption of a specific neural circuit related to self-regulation is an underlying biological deficit in borderline personality disorder (BPD). Because patients with BPD exhibit a poor ability to regulate negative affect, we hypothesized that brain mechanisms thought to be involved in such self-regulation would function abnormally even in situations that seem remote from the symptoms exhibited by these patients. To test this idea, we compared the efficiency of attentional networks in BPD patients with controls who were matched to the patients in having very low self-reported effortful control and very high negative emotionality and controls who were average in these two temperamental dimensions. We found that the patients exhibited significantly greater difficulty in their ability to resolve conflict among stimulus dimensions in a purely cognitive task than did average controls but displayed no deficit in overall reaction time, errors, or other attentional networks. The temperamentally matched group did not differ significantly from either group. A significant correlation was found between measures of the ability to control conflict in the reaction-time task and self-reported effortful control.