bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of soft condensed materials with living cells: Phenotype/transcriptome correlations for the hydrophobic effect
Authors: Allen Lorcan T. , Fox Edward J. P. , Blute Irena , Kelly Zoe D. , Rochev Yuri , Keenan Alan K. , Dawson Kenneth A. , Gallagher William M.
Published: May 2003

The assessment of biomaterial compatibility relies heavily on the analysis of macroscopic cellular responses to material interaction. However, new technologies have become available that permit a more profound understanding of the molecular basis of cell–biomaterial interaction. Here, both conventional phenotypic and contemporary transcriptomic (DNA microarray-based) analysis techniques were combined to examine the interaction of cells with a homologous series of copolymer films that subtly vary in terms of surface hydrophobicity. More specifically, we used differing combinations of
N-
isopropylacrylamide, which is presently used as an adaptive cell culture substrate, and the more hydrophobic, yet structurally similar, monomer
N-tert-
butylacrylamide. We show here that even discrete modifications with respect to the physiochemistry of soft amorphous materials can lead to significant impacts on the phenotype of interacting cells. Furthermore, we have elucidated putative links between phenotypic responses to cell–biomaterial interaction and global gene expression profile alterations. This case study indicates that high-throughput analysis of gene expression not only can greatly refine our knowledge of cell–biomaterial interaction, but also can yield novel biomarkers for potential use in biocompatibility assessment.
The phosphorylation of caveolin-2 on serines 23 and 36 modulates caveolin-1-dependent caveolae formation
Authors: Sowa Grzegorz , Pypaert Marc , Fulton David , Sessa William C.
Published: May 2003

Caveolin-1 and -2 are the two major coat proteins found in plasma membrane caveolae of most of cell types. Here, by using adenoviral transduction of either caveolin-1 or caveolin-2 or both isoforms into cells lacking both caveolins, we demonstrate that caveolin-2 positively regulates caveolin-1-dependent caveolae formation. More importantly, we show that caveolin-2 is phosphorylated
in vivo
at two serine residues and that the phosphorylation of caveolin-2 is necessary for its actions as a positive regulator of caveolin-1 during organelle biogenesis in prostate cancer cells. Mutation of the primary phosphorylation sites on caveolin-2, serine 23 and 36, reduces the number of plasmalemma-attached caveolae and increases the accumulation of noncoated vesicles, but does not affect trafficking of caveolin-2, interaction with caveolin-1 or its biophysical properties. Thus, the phosphorylation of caveolin-2 is a novel mechanism to regulate the dynamics of caveolae assembly.
Asialoerythropoietin is a nonerythropoietic cytokine with broad neuroprotective activity
in vivo
Authors: Erbayraktar Serhat , Grasso Giovanni , Sfacteria Alessandra , Xie Qiao-wen , Coleman Thomas , Kreilgaard Mads , Torup Lars , Sager Thomas , Erbayraktar Zubeyde , Gokmen Necati , Yilmaz Osman , Ghezzi Pietro , Villa Pia , Fratelli Maddalena , Casagrande Simona , Leist Marcel , Helboe Lone , Gerwein Jens , Christensen Søren , Geist Marie Aavang , Pedersen Lars Østergaard , Cerami-Hand Carla , Wuerth Jean-Paul , Cerami Anthony , Brines Michael
Published: May 2003

Erythropoietin (EPO) is a tissue-protective cytokine preventing vascular spasm, apoptosis, and inflammatory responses. Although best known for its role in hematopoietic lineages, EPO also affects other tissues, including those of the nervous system. Enthusiasm for recombinant human erythropoietin (rhEPO) as a potential neuroprotective therapeutic must be tempered, however, by the knowledge it also enlarges circulating red cell mass and increases platelet aggregability. Here we examined whether erythropoietic and tissue-protective activities of rhEPO might be dissociated by a variation of the molecule. We demonstrate that asialoerythropoietin (asialoEPO), generated by total enzymatic desialylation of rhEPO, possesses a very short plasma half-life and is fully neuroprotective. In marked contrast with rhEPO, this molecule at doses and frequencies at which rhEPO exhibited erythropoiesis, did not increase the hematocrit of mice or rats. AsialoEPO appeared promptly within the cerebrospinal fluid after i.v. administration; intravenously administered radioiodine-labeled asialoEPO bound to neurons within the hippocampus and cortex in a pattern corresponding to the distribution of the EPO receptor. Most importantly, asialoEPO exhibits a broad spectrum of neuroprotective activities, as demonstrated in models of cerebral ischemia, spinal cord compression, and sciatic nerve crush. These data suggest that nonerythropoietic variants of rhEPO can cross the blood–brain barrier and provide neuroprotection.
Adoptive transfer of apoptotic splenocytes worsens survival, whereas adoptive transfer of necrotic splenocytes improves survival in sepsis
Authors: Hotchkiss Richard S. , Chang Katherine C. , Grayson Mitchell H. , Tinsley Kevin W. , Dunne Benjamin S. , Davis Christopher G. , Osborne Dale F. , Karl Irene E.
Published: May 2003

In sepsis, both necrotic and apoptotic cell death can occur. Apoptotic cells induce anergy that could impair the host response, whereas necrotic cells cause immune activation that might result in enhanced antimicrobial defenses. We determined whether adoptive transfer of apoptotic or necrotic cells impacted survival in a clinically relevant sepsis model. We also evaluated the effects of adoptive transfer of apoptotic or necrotic cells on the prototypical TH1 and TH2 cytokines IFN-γ and IL-4, respectively. C57BL6/J mice had adoptive transfer of apoptotic (irradiated) or necrotic (freeze thaw) splenocytes. Controls received saline. Apoptotic cells greatly increased mortality, whereas necrotic splenocytes markedly improved survival,P≤ 0.05. The contrasting effects that apoptotic or necrotic cells exerted on survival were mirrored by opposite effects on splenocyte IFN-γ production with greatly decreased and increased production, respectively. Importantly, either administration of anti-IFN-γ antibodies or use of IFN-γ knockout mice prevented the survival benefit occurring with necrotic cells. This study demonstrates that the type of cell death impacts survival in a clinically relevant model and identifies a mechanism for the immune suppression that is a hallmark of sepsis. Necrotic cells (and likely apoptotic cells) exert their effects via modulation of IFN-γ
Control of seed germination in transgenic plants based on the segregation of a two-component genetic system
Authors: Schernthaner Johann P. , Fabijanski Steven F. , Arnison Paul G. , Racicot Martine , Robert Laurian S.
Published: May 2003

We have developed a repressible seed-lethal (SL) system aimed at reducing the probability of transgene introgression into a population of sexually compatible plants. To evaluate the potential of this method, tobacco plants were transformed with an SL construct comprisinggene 1andgene 2fromAgrobacterium tumefacienswherebygene 1was controlled by the seed-specific phaseolin promoter modified to contain a binding site for theEscherichia coliTET repressor (R). The expression of this construct allows normal plant and seed development but inhibits seed germination. Plants containing the SL construct were crossed with plants containing thetetR gene to derive plant lines where the expression of the SL construct is repressed. Plant lines that contained both constructs allowed normal seed formation and germination, whereas seeds in which the SL construct was separated from the R gene through segregation did not germinate. The requirements of such a method to efficiently control the flow of novel traits among sexually compatible plants are discussed.
Biocomplexity and fisheries sustainability
Authors: Hilborn Ray , Quinn Thomas P. , Schindler Daniel E. , Rogers Donald E.
Published: May 2003

A classic example of a sustainable fishery is that targeting sockeye salmon in Bristol Bay, Alaska, where record catches have occurred during the last 20 years. The
stock complex
is an amalgamation of several hundred discrete spawning populations. Structured within lake systems, individual populations display diverse life history characteristics and local adaptations to the variation in spawning and rearing habitats. This biocomplexity has enabled the aggregate of populations to sustain its productivity despite major changes in climatic conditions affecting the freshwater and marine environments during the last century. Different geographic and life history components that were minor producers during one climatic regime have dominated during others, emphasizing that the biocomplexity of fish stocks is critical for maintaining their resilience to environmental change.
An EP2 receptor-selective prostaglandin E2agonist induces bone healing
Authors: Paralkar V. M. , Borovecki F. , Ke H. Z. , Cameron K. O. , Lefker B. , Grasser W. A. , Owen T. A. , Li M. , DaSilva-Jardine P. , Zhou M. , Dunn R. L. , Dumont F. , Korsmeyer R. , Krasney P. , Brown T. A. , Plowchalk D. , Vukicevic S. , Thompson D. D.
Published: May 2003

The morbidity and mortality associated with impaired/delayed fracture healing remain high. Our objective was to identify a small nonpeptidyl molecule with the ability to promote fracture healing and prevent malunions. Prostaglandin E2(PGE2) causes significant increases in bone mass and bone strength when administered systemically or locally to the skeleton. However, due to side effects, PGE2is an unacceptable therapeutic option for fracture healing. PGE2mediates its tissue-specific pharmacological activity via four different G protein-coupled receptor subtypes, EP1, -2, -3, and -4. The anabolic action of PGE2in bone has been linked to an elevated level of cAMP, thereby implicating the EP2 and/or EP4 receptor subtypes in bone formation. We identified an EP2 selective agonist, CP-533,536, which has the ability to heal canine long bone segmental and fracture model defects without the objectionable side effects of PGE2, suggesting that the EP2 receptor subtype is a major contributor to PGE2's local bone anabolic activity. The potent bone anabolic activity of CP-533,536 offers a therapeutic alternative for the treatment of fractures and bone defects in patients.
Mitochondrial succinic-semialdehyde dehydrogenase of the γ-aminobutyrate shunt is required to restrict levels of reactive oxygen intermediates in plants
Authors: Bouché Nicolas , Fait Aaron , Bouchez David , Møller Simon G. , Fromm Hillel
Published: May 2003

The γ-aminobutyrate (GABA) shunt is a metabolic pathway that bypasses two steps of the tricarboxylic acid cycle, and it is present in both prokaryotes and eukaryotes. In plants the pathway is composed of the calcium/calmodulin-regulated cytosolic enzyme glutamate decarboxylase and the mitochondrial enzymes GABA transaminase and succinic-semialdehyde dehydrogenase (SSADH). The activity of the GABA shunt in plants is rapidly enhanced in response to various biotic and abiotic stresses. However the physiological role of this pathway remains obscure. To elucidate its role in plants, we analyzedArabidopsisT-DNA knockout mutants of SSADH, the ultimate enzyme of the pathway. Four alleles of thessadhmutation were isolated, and these exhibited a similar phenotype. When exposed to white light (100 μmol of photons per m2per s), they appear dwarfed with necrotic lesions. Detailed spectrum analysis revealed that UV-B has the most adverse effect on the mutant phenotype, whereas photosynthetic active range light has a very little effect. Thessadhmutants are also sensitive to heat, as they develop necrosis when submitted to such stress. Moreover, both UV and heat cause a rapid increase in the levels of hydrogen peroxide in thessadhmutants, which is associated with enhanced cell death. Surprisingly, our study also shows that trichomes are hypersensitive to stresses inssadhmutants. Our work establishes a role for the GABA shunt in preventing the accumulation of reactive oxygen intermediates and cell death, which appears to be essential for plant defense against environmental stress.
Genomewide view of gene silencing by small interfering RNAs
Authors: Chi Jen-Tsan , Chang Howard Y. , Wang Nancy N. , Chang Dustin S. , Dunphy Nina , Brown Patrick O.
Published: May 2003

RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5′ of the target region that has been observed in
Caenorhabditis elegans
, was unable to detect this phenomenon in human cells.
Exponential growth by cross-catalytic cleavage of deoxyribozymogens
Authors: Levy Matthew , Ellington Andrew D.
Published: May 2003

We have designed an autocatalytic cycle based on the highly efficient 10–23 RNA-cleaving deoxyribozyme that is capable of exponential amplification of catalysis. In this system, complementary 10–23 variants were inactivated by circularization, creating deoxyribozymogens. Upon linearization, the enzymes can act on their complements, creating a cascade in which linearized species accumulate exponentially. Seeding the system with a pool of linear catalysts resulted not only in amplification of function but in sequence selection and represents an
in vitro
selection experiment conducted in the absence of any protein enzymes.
Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein
Authors: Gibbs Daniel , Kitamoto Junko , Williams David S.
Published: May 2003

Mutations in the myosin VIIa gene (
MYO7A
) cause Usher syndrome type 1B (USH1B), a major type of the deaf-blind disorder, Usher syndrome. We have studied mutant phenotypes in the retinas of
Myo7a
mutant mice (shaker1), with the aim of elucidating the role(s) of myosin VIIa in the retina and what might underlie photoreceptor degeneration in USH1B patients. A photoreceptor defect has been described. Here, we report that the phagocytosis of photoreceptor outer segment disks by the retinal pigment epithelium (RPE) is abnormal in
Myo7a
null mice. Both
in vivo
and in primary cultures of RPE cells, the transport of ingested disks out of the apical region is inhibited in the absence of Myo7a. The results with the cultured RPE cells were the same, irrespective of whether the disks came from wild-type or mutant mice, thus demonstrating that the RPE is the source of this defect. The inhibited transport seems to delay phagosome–lysosomal fusion, as the degradation of ingested disks was slower in mutant RPE. Moreover, fewer packets of disk membranes were ingested
in vivo
, possibly because retarded removal of phagosomes from the apical processes inhibited the ingestion of additional disk membranes. We conclude that Myo7a is required for the normal processing of ingested disk membranes in the RPE, primarily in the basal transport of phagosomes into the cell body where they then fuse with lysosomes. Because the phagocytosis of photoreceptor disks by the RPE has been shown to be critical for photoreceptor cell viability, this defect likely contributes to the progressive blindness in USH1B.
ATP hydrolysis on actin-related protein 2/3 complex causes debranching of dendritic actin arrays
Authors: Le Clainche Christophe , Pantaloni Dominique , Carlier Marie-France
Published: May 2003

Extension of lamellipodia, an important dissipative process in cell motility, is driven by the turnover of a polarized dendritic array of actin filaments. Motility is driven by catalytic cycles of filament attachment to Wiskott–Aldrich syndrome protein (WASP)-activated actin-related protein (Arp)2/3 complex at the leading edge, branch formation, and detachment, allowing subsequent growth of branched filaments. The morphology, mechanical strength, and lifetime of the array are determined by the processes of filament branching, debranching, and treadmilling. All three processes are controlled by ATP hydrolysis. ATP hydrolysis on F-actin is known to be at the origin of treadmilling. Here, by using radiolabeled ATP covalently bound to Arp2/3, we show that ATP is hydrolyzed on Arp2, not on Arp3, after a delay following filament branching. Hydrolysis of ATP on Arp2 promotes debranching of filaments and acts as a clock that controls the stability of dendritic actin arrays in lamellipodia. Finally, we propose that hydrolysis of ATP on G-actin in the ternary G-actin–WASP–Arp2/3 complex on branch formation destabilizes the WASP–actin interface and energetically facilitates the detachment step in the branching reaction.
How the
Bfi
I restriction enzyme uses one active site to cut two DNA strands
Authors: Sasnauskas Giedrius , Halford Stephen E. , Siksnys Virginijus
Published: May 2003

Unlike other restriction enzymes,
Bfi
I functions without metal ions. It recognizes an asymmetric DNA sequence, 5′-ACTGGG-3′, and cuts top and bottom strands at fixed positions downstream of this sequence. Many restriction enzymes are dimers of identical subunits, with one active site for each DNA strand. Others, like
Fok
I, dimerize transiently during catalysis.
Bfi
I is also a dimer but it has only one active site, at the dimer interface. We show here that
Bfi
I remains a dimer as it makes double-strand breaks in DNA and that its single active site acts sequentially, first on the bottom and then the top strand. Hence, after cutting the bottom strand, a rearrangement of either the protein and/or the DNA in the
Bfi
I–DNA complex must switch the active site to the top strand. Low pH values selectively block top-strand cleavage, converting
Bfi
I into a nicking enzyme that cleaves only the bottom strand. The switch to the top strand may depend on the ionization of the cleaved 5′ phosphate in the bottom strand.
Bfi
I thus uses a mechanism for making double-strand breaks that is novel among restriction enzymes.
Human cathepsin S, but not cathepsin L, degrades efficiently MHC class II-associated invariant chain in nonprofessional APCs
Authors: Bania Jacek , Gatti Evelina , Lelouard Hugues , David Alexandre , Cappello Fanny , Weber Ekkehard , Camosseto Voahirana , Pierre Philippe
Published: May 2003

MHC class II-restricted antigen presentation plays a central role in the immune response against exogenous antigens. The association of invariant (Ii) chain with MHC class II dimers is required for proper antigen presentation to CD4
+
T cells by antigen-presenting cells. MHC class II complexes first traffic through the endocytic pathway to allow Ii chain degradation and antigenic peptide loading before their arrival at the cell surface. In recent years, a considerable effort has been directed toward the identification of proteases responsible for Ii chain degradation. Targeted gene deletion in mice has allowed a precise description of the cysteine proteases involved in the last step of Ii chain degradation. By using nonspecialized cellular models expressing MHC II molecules, we are now exploring the contribution of known cysteine proteases to human Ii chain processing. Surprisingly and contrary to the situation in mouse, cathepsin S was found to be the only human cysteine protease able to efficiently degrade the Ii-p10 fragment in epithelial cells. This selectivity has implications for thymic selection and indicates that differences between man and mice are probably more profound at this level than expected.
Short- and long-term enhancement of excitatory transmission in the spinal cord dorsal horn by nicotinic acetylcholine receptors
Authors: Genzen Jonathan R. , McGehee Daniel S.
Published: May 2003

Spinal administration of nicotinic agonists can produce both hyperalgesic and analgesic effects
in vivo
. The cellular mechanisms underlying these behavioral phenomena are not understood. As a possible explanation for nicotinic hyperalgesia, we tested whether nicotinic acetylcholine receptors (nAChRs) could enhance excitatory transmission onto spinal cord dorsal horn neurons. Whole-cell patch–clamp recordings were performed in neonatal rat spinal cord slices. Activation of nAChRs enhanced glutamatergic synaptic transmission in 59% of dorsal horn neurons tested, and this effect was blocked by methyllycaconitine (10 nM), suggesting a key role for α7 nAChRs. Inhibition of acetylcholinesterase with methamidophos also enhanced transmission, demonstrating a similar effect of endogenous acetylcholine. nAChR activation also enhanced transmission by dorsal root entry zone stimulation, suggesting that α7 nAChRs on the central terminals of DRG afferents mediate this effect. Paired pre- and postsynaptic stimulation induced long-term potentiation of excitatory inputs to some of the dorsal horn neurons. Long-term potentiation induction was much more prevalent when nicotine was applied during stimulation. This effect also depended on both α7 nAChRs and
N
-methyl-
d
-aspartate glutamate receptors. Our findings demonstrate that α7 nAChRs can contribute to both short- and long-term enhancement of glutamatergic synaptic transmission in the spinal cord dorsal horn and provide a possible mechanism for nicotinic hyperalgesia.
A dual altered peptide ligand down-regulates myasthenogenic T cell responses by up-regulating CD25- and CTLA-4-expressing CD4+T cells
Authors: Paas-Rozner Miri , Sela Michael , Mozes Edna
Published: May 2003

Immunization of mice with two myasthenogenic peptides, p195–212 and p259–271, which are sequences of the human acetylcholine receptor, resulted in myasthenia gravis (MG)-associated immune responses. A dual altered peptide ligand (APL) composed of the two APLs of the myasthenogenic peptides inhibited,in vitroandin vivo, those responses. The aims of this study were to further elucidate the mechanism/s by which the dual APL down-regulates MG-associated responsesin vivoand characterize the cell population/s involved in this immunomodulatory suppressive effect. We have shown here that s.c. administration of the dual APL activates CD4CD25-expressing cells in lymph nodes (LN) of SJL mice. Furthermore, depletion of these cells diminished significantly the inhibitory effect of the APL on p195–212-specific proliferative responses. Depletion of the CD4+CD25+cells was accompanied with a decrease in the secretion of the immunosuppressive cytokine, transforming growth factor (TGF)-β. Administration of the dual APL resulted also in the up-regulation of the expression of cytotoxic T lymphocyte antigen (CTLA)-4 and in a down-regulated expression of CD28 on LN cells. Blockade of the CTLA-4 function,in vitro, abrogated the inhibitory effect of the dual APL on the proliferative responses specific to p195–212. Thus, our results suggest that the active suppression exerted by the dual APL is mediated by the CD4+CD25+immunoregulatory cell population, either directly through the CTLA-4 molecule expressed on these cells, and/or indirectly by causing the differentiation of other regulatory T cell population/s that secrete immunosuppressive cytokines.
Immunization with hepatitis C virus-like particles protects mice from recombinant hepatitis C virus-vaccinia infection
Authors: Murata Kazumoto , Lechmann Martin , Qiao Ming , Gunji Toshiaki , Alter Harvey J. , Liang T. Jake
Published: May 2003

We have recently demonstrated that immunization with hepatitis C virus-like particles (HCV-LPs) generated in insect cells can elicit both humoral and cellular immune responses in BALB/c mice. Here, we evaluate the immunogenicity of HCV-LPs in HLA2.1 transgenic (AAD) mice in comparison to DNA immunization. HCV-LP immunization elicited a significantly stronger humoral immune response than DNA immunization. HCV-LP-immunized mice also developed stronger HCV-specific cellular immune responses than DNA-immunized mice as determined by using quantitative enzyme-linked immunospot (ELISpot) assay and intracellular cytokine staining. In BALB/c mice, immunization with HCV-LPs resulted in a >5 log10reduction in vaccinia titer when challenged with a recombinant vaccinia expressing the HCV structural proteins (vvHCV.S), as compared to 1 log10decrease in DNA immunization. In HLA2.1 transgenic mice, a 1–2 log10reduction resulted from HCV-LP immunization, whereas no reduction was seen from DNA immunization. Adoptive transfer of lymphocytes from HCV-LP-immunized mice to naive mice provided protection against vvHCV.S challenge, and this transferred immunity can be abrogated by either CD4 or CD8 depletion. Our results suggest that HCV-LPs can induce humoral and cellular immune responses that are protective in a surrogate HCV challenge model and that a strong cellular immunity provided by both CD4 and CD8 effector lymphocytes may be important for protection from HCV infection.
Subcellular localization of yeast ribonucleotide reductase regulated by the DNA replication and damage checkpoint pathways
Authors: Yao Ruojin , Zhang Zhen , An Xiuxiang , Bucci Brigid , Perlstein Deborah L. , Stubbe JoAnne , Huang Mingxia
Published: May 2003

The fidelity of DNA replication and repair processes is critical for maintenance of genomic stability. Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in dNTP production and thus plays an essential role in DNA synthesis. The level and activity of RNR are highly regulated by the cell cycle and DNA damage checkpoints, which maintain optimal dNTP pools required for genetic fidelity. RNRs are composed of a large subunit that binds the nucleoside diphosphate substrates and allosteric effectors and a small subunit that houses the di-iron tyrosyl radical cofactor essential for the reduction process. In
Saccharomyces cerevisiae
, there are two large subunits (Rnr1 and Rnr3) and two small subunits (Rnr2 and Rnr4). Here we report the subcellular localization of Rnr1–4 during normal cell growth and the redistribution of Rnr2 and Rnr4 in response to DNA damage and replicational stress. During the normal cell cycle, Rnr1 and Rnr3 are predominantly localized to the cytoplasm and Rnr2 and Rnr4 are predominantly present in the nucleus. Under genotoxic stress, Rnr2 and Rnr4 become redistributed to the cytoplasm in a checkpoint-dependent manner. Subcellular redistribution of Rnr2 and Rnr4 can occur in the absence of the transcriptional induction of the
RNR
genes after DNA damage and likely represents a posttranslational event. These results suggest a mechanism by which DNA damage checkpoint modulates RNR activity through the temporal and spatial regulation of its subunits.
Understanding tubulin–Taxol interactions: Mutations that impart Taxol binding to yeast tubulin
Authors: Gupta Mohan L. , Bode Claudia J. , Georg Gunda I. , Himes Richard H.
Published: May 2003

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in
Saccharomyces cerevisiae
tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from
S. cerevisiae
, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.
TwoArabidopsiscircadian oscillators can be distinguished by differential temperature sensitivity
Authors: Michael Todd P. , Salomé Patrice A. , McClung C. Robertson
Published: May 2003

Circadian rhythms are widespread in nature and reflect the activity of an endogenous biological clock. In metazoans, the circadian system includes a central circadian clock in the brain as well as distinct clocks in peripheral tissues such as the retina or liver. Similarly, plants have distinct clocks in different cell layers and tissues. Here, we show that two different circadian clocks, distinguishable by their sensitivity to environmental temperature signals, regulate the transcription of genes that are expressed in theArabidopsis thalianacotyledon. One oscillator, which regulatesCAB2expression, responds preferentially to light–dark versus temperature cycles and fails to respond to the temperature step associated with release from stratification. The second oscillator, which regulatesCAT3expression, responds preferentially to temperature versus light–dark cycles and entrains to the release from stratification. Finally, the phase response curves of these two oscillators to cold pulses are distinct. The phase response curve of the oscillator componentTOC1to cold pulses is similar to that ofCAB2, indicating thatCAB2is regulated by a TOC1-containing clock. The existence of two clocks, distinguishable on the basis of their sensitivity to temperature, provides an additional means by which plants may integrate both photoperiodic and temperature signals to respond to the changing seasons.
Trehalose 6-phosphate is indispensable for carbohydrate utilization and growth in
Arabidopsis thaliana
Authors: Schluepmann Henriette , Pellny Till , van Dijken Anja , Smeekens Sjef , Paul Matthew
Published: May 2003

Genes for trehalose metabolism are widespread in higher plants. Insight into the physiological role of the trehalose pathway outside of resurrection plant species is lacking. To address this lack of insight, we express
Escherichia coli
genes for trehalose metabolism in
Arabidopsis thaliana
, which manipulates trehalose 6-phosphate (T6P) contents in the transgenic plants. Plants expressing
otsA
[encoding trehalose phosphate synthase (TPS)] accumulate T6P whereas those expressing either
otsB
[encoding trehalose phosphate phosphatase (TPP)] or
treC
[encoding trehalose phosphate hydrolase (TPH)] contain low levels of T6P. Expression of
treF
(encoding trehalase) yields plants with unaltered T6P content and a phenotype not distinguishable from wild type when grown on soil. The marked phenotype obtained of plants accumulating T6P is opposite to that of plants with low T6P levels obtained by expressing either TPP or TPH and consistent with a critical role for T6P in growth and development. Supplied sugar strongly inhibits growth of plants with reduced T6P content and leads to accumulation of respiratory intermediates. Remarkably, sugar improves growth of TPS expressors over wild type, a feat not previously accomplished by manipulation of metabolism. The data indicate that the T6P intermediate of the trehalose pathway controls carbohydrate utilization and thence growth via control of glycolysis in a manner analogous to that in yeast. Furthermore, embryolethal
A. thaliana tps1
mutants are rescued by expression of
E. coli
TPS, but not by supply of trehalose, suggesting that T6P control over primary metabolism is indispensable for development.
Purine synthesis and increasedAgrobacterium tumefacienstransformation of yeast and plants
Authors: Roberts Radclyffe L. , Metz Matthew , Monks Dave E. , Mullaney Matthew Lockwood , Hall Tyler , Nester Eugene W.
Published: May 2003

The bacteriumAgrobacterium tumefacienstransforms eukaryotic hosts by transferring DNA to the recipient cell where it is integrated and expressed. Bacterial factors involved in this interkingdom gene transfer have been described, but less is known about host-cell factors. Using the yeastSaccharomyces cerevisiaeas a model host, we devised a genetic screen to identify yeast mutants with altered transformation sensitivities. Twenty-four adenine auxotrophs were identified that exhibited supersensitivity toA. tumefaciens-mediated transformation when deprived of adenine. We extended these results to plants by showing that purine synthesis inhibitors cause supersensitivity toA. tumefacienstransformation in three plant species. The magnitude of this effect is large and does not depend on prior genetic manipulations of host cells. These data indicate the utility of yeast as a model for the transformation process and identify purine biosynthesis as a key determinant of transformation efficiency. These findings should increase the utility ofA. tumefaciensin genetic engineering.
Conservation and molecular dissection of
ROUGH SHEATH2
and
ASYMMETRIC LEAVES1
function in leaf development
Authors: Theodoris George , Inada Noriko , Freeling Michael
Published: May 2003

Maize
ROUGH SHEATH2
(
RS2
) and
Arabidopsis ASYMMETRIC LEAVES1
(
AS1
) are orthologous Myb-related genes required for leaf development and act as negative regulators of class 1
KNOTTED1-like homeobox
(
KNOX
) genes in leaf primordia. Expression of
RS2
in
Arabidopsis
fully complements
as1
leaf phenotypes and represses the expression of the
KNOX
gene
KNAT1
in leaves. Whereas loss of AS1 function in
Arabidopsis
results in rounded, lobed leaves with shorter and wider petioles, overexpression of either
RS2
or
AS1
results in longer and narrower leaves with longer petioles than wild type. A conserved C-terminal domain (CTD) mediates homodimerization of both RS2 and AS1 and modulates leaf shape when expressed independently of the Myb domain in
Arabidopsis
. Homodimerization is not absolutely required for
KNAT1
repression. RS2:GFP fusion protein is biologically active, localized in discrete dynamic subnuclear foci and associates with DNA during cell division.
Programming peptidomimetic syntheses by translating genetic codes designed
de novo
Authors: Forster Anthony C. , Tan Zhongping , Nalam Madhavi N. L. , Lin Hening , Qu Hui , Cornish Virginia W. , Blacklow Stephen C.
Published: May 2003

Although the universal genetic code exhibits only minor variations in nature, Francis Crick proposed in 1955 that “the adaptor hypothesis allows one to construct, in theory, codes of bewildering variety.” The existing code has been expanded to enable incorporation of a variety of unnatural amino acids at one or two nonadjacent sites within a protein by using nonsense or frameshift suppressor aminoacyl-tRNAs (aa-tRNAs) as adaptors. However, the suppressor strategy is inherently limited by compatibility with only a small subset of codons, by the ways such codons can be combined, and by variation in the efficiency of incorporation. Here, by preventing competing reactions with aa-tRNA synthetases, aa-tRNAs, and release factors during translation and by using nonsuppressor aa-tRNA substrates, we realize a potentially generalizable approach for template-encoded polymer synthesis that unmasks the substantially broader versatility of the core translation apparatus as a catalyst. We show that several adjacent, arbitrarily chosen sense codons can be completely reassigned to various unnatural amino acids according to
de novo
genetic codes by translating mRNAs into specific peptide analog polymers (peptidomimetics). Unnatural aa-tRNA substrates do not uniformly function as well as natural substrates, revealing important recognition elements for the translation apparatus. Genetic programming of peptidomimetic synthesis should facilitate mechanistic studies of translation and may ultimately enable the directed evolution of small molecules with desirable catalytic or pharmacological properties.
Vam10p defines a Sec18p-independent step of priming that allows yeast vacuole tethering
Authors: Kato Masashi , Wickner William
Published: May 2003

YOR
068c, termed
VAM
10 (altered vacuole morphology), lies within the
VPS
5 gene on the opposite DNA strand.
VAM
10 deletion causes vacuole fragmentation
in vivo
. The
in vitro
fusion of purified yeast vacuoles is stimulated by recombinant Vam10p and blocked by antibody to Vam10p. Vam10p acts early in the priming stage of fusion, independent of Sec18p. After priming, recombinant Vam10p will not stimulate fusion and anti-Vam10p antibodies will not inhibit; Vam10p provides a functional marker for this Sec18p-independent priming step. Pure Vam10p restores normal, Ypt7p-dependent tethering to vacuoles from a
vam
10Δ strain.
Calcium-permeable AMPA/kainate receptors mediate toxicity and preconditioning by oxygen-glucose deprivation in oligodendrocyte precursors
Authors: Deng Wenbin , Rosenberg Paul A. , Volpe Joseph J. , Jensen Frances E.
Published: May 2003

Hypoxic–ischemic brain injury in premature infants results in cerebral white matter lesions with prominent oligodendroglial injury and loss, a disorder termed periventricular leukomalacia (PVL). We have previously shown that glutamate receptors mediate hypoxic–ischemic injury to oligodendroglial precursor cells (OPCs) in a model of PVL in the developing rodent brain. We used primary OPC cultures to examine the mechanism of cellular toxicity induced by oxygen–glucose deprivation (OGD) to simulate brain ischemia. OPCs were more sensitive to OGD-induced toxicity than mature oligodendrocytes, and OPC toxicity was attenuated by nonselective [2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX), 6-cyano-7-nitroquinoxaline-2,3-dione], α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring (GYKI 52466), kainate-preferring (γ-d-glutamylaminomethanesulfonic acid), or Ca2+-permeable AMPA/kainate receptor antagonists (joro spider toxin, JSTx) administered either during or after OGD. Furthermore, NBQX or JSTx blocked OGD-induced Ca2+influx. Relevant to recurrent hypoxic–ischemic insults in developing white matter, we examined the effects of sublethal OGD preconditioning. A prior exposure of OPCs to sublethal OGD resulted in enhanced vulnerability to subsequent excitotoxic or OGD-induced injury associated with an increased Ca2+influx. AMPA/kainate receptor blockade with NBQX or JSTx either during or after sublethal OGD prevented its priming effect. Furthermore, OGD preconditioning resulted in a down-regulation of the AMPA receptor subunit GluR2 on cell surface that increased Ca2+permeability of the receptors. Overall, these data suggest that aberrantly enhanced activation of Ca2+-permeable AMPA/kainate receptors may be a major mechanism in acute and repeated hypoxic–ischemic injury to OPCs in disorders of developing cerebral white matter, such as PVL.
Development of teeth in chick embryos after mouse neural crest transplantations
Authors: Mitsiadis Thimios A. , Chéraud Yvonnick , Sharpe Paul , Fontaine-Pérus Josiane
Published: May 2003

Teeth were lost in birds 70–80 million years ago. Current thinking holds that it is the avian cranial neural crest-derived mesenchyme that has lost odontogenic capacity, whereas the oral epithelium retains the signaling properties required to induce odontogenesis. To investigate the odontogenic capacity of ectomesenchyme, we have used neural tube transplantations from mice to chick embryos to replace the chick neural crest cell populations with mouse neural crest cells. The mouse/chick chimeras obtained show evidence of tooth formation showing that avian oral epithelium is able to induce a nonavian developmental program in mouse neural crest-derived mesenchymal cells.
Alcohol potently inhibits the kainate receptor-dependent excitatory drive of hippocampal interneurons
Authors: Carta Mario , Ariwodola Olusegun J. , Weiner Jeff L. , Valenzuela C. Fernando
Published: May 2003

Kainate receptors (KA-Rs) are members of the glutamate-gated family of ionotropic receptors, which also includesN-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. KA-Rs are important modulators of interneuron excitability in the CA1 region of the hippocampus. Activation of these receptors enhances interneuron firing, which robustly increases spontaneous inhibitory postsynaptic currents in pyramidal neurons. We report here that ethanol (EtOH) potently inhibits this KA-R-mediated effect at concentrations as low as those that can be achieved in blood after the ingestion of just 1–2 drinks (5–10 mM). Pressure application of kainate, in the presence of AMPA and NMDA receptor antagonists, evoked depolarizing responses in interneurons that triggered repetitive action potential firing. EtOH potently inhibited these responses to a degree that was sufficient to abolish action potential firing. This effect appears to be specific for KA-Rs, as EtOH did not affect action potential firing triggered by AMPA receptor-mediated depolarizing responses. Importantly, EtOH inhibited interneuron action potential firing in response to KA-R activation by synaptically released glutamate, suggesting that our findings are physiologically relevant. KA-R-dependent modulation of glutamate release onto pyramidal neurons was not affected by EtOH. Thus, EtOH increases excitability of pyramidal neurons indirectly by inhibiting the KA-R-dependent drive of γ-aminobutyric acid (GABA)ergic interneurons. We postulate that this effect may explain, in part, some of the paradoxical excitatory actions of this widely abused substance. The excitatory actions of EtOH may be perceived as positive by some individuals, which could contribute to the development of alcoholism.
β3 integrin deficiency promotes atherosclerosis and pulmonary inflammation in high-fat-fed, hyperlipidemic mice
Authors: Weng Sherry , Zemany Laura , Standley Kara N. , Novack Deborah V. , La Regina Marie , Bernal-Mizrachi Carlos , Coleman Trey , Semenkovich Clay F.
Published: May 2003

Hyperlipidemia promotes the chronic inflammatory disease atherosclerosis through poorly understood mechanisms. Atherogenic lipoproteins activate platelets, but it is unknown whether platelets contribute to early inflammatory atherosclerotic lesions. To address the role of platelet aggregation in diet-induced vascular disease, we studied β3 integrin-deficient mice (lacking platelet integrin αIIbβ3 and the widely expressed nonplatelet integrin αvβ3) in two models of atherosclerosis, apolipoprotein E (apoE)-null and low-density lipoprotein receptor (LDLR)-null mice. Unexpectedly, a high-fat, Western-type (but not a low-fat) diet caused death in two-thirds of the β3
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/
-
apoE
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/
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and half of the β3
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/
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LDLR
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/
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mice due to noninfectious pneumonitis. In animals from both models surviving high-fat feeding, pneumonitis was absent, but aortic atherosclerosis was 2- to 6-fold greater in β3
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compared with β
+
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+
littermates. Expression of CD36, CD40L, and CD40 was increased in lungs of β3
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LDLR
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mice. Each was also increased in smooth muscle cells cultured from β3-deficient mice and suppressed by retroviral reconstitution of β3. These data show that the platelet defect caused by αIIbβ3 deficiency does not impair atherosclerotic lesion initiation. They also suggest that αvβ3 has a suppressive effect on inflammation, the loss of which induces atherogenic mediators that are amplified by diet-induced hyperlipidemia.
Evolution of the army ant syndrome: The origin and long-term evolutionary stasis of a complex of behavioral and reproductive adaptations
Authors: Brady Seán G.
Published: May 2003

The army ant syndrome of behavioral and reproductive traits (obligate collective foraging, nomadism, and highly specialized queens) has allowed these organisms to become the premiere social hunters of the tropics, yet we know little about how or why these strategies evolved. The currently accepted view holds that army ants evolved multiple times on separate continents. I generated data from three nuclear genes, a mitochondrial gene, and morphology to test this hypothesis. Results strongly indicate that the suite of behavioral and reproductive adaptations found in army ants throughout the world is inherited from a unique common ancestor, and did not evolve convergently in the New World and Old World as previously thought. New Bayesian methodology for dating the antiquity of lineages by using a combination of fossil and molecular information places the origin of army ants in the mid-Cretaceous, consistent with a Gondwanan origin. Because no known army ant species lacks any component of the army ant syndrome, this group represents an extraordinary case of long-term evolutionary stasis in these adaptations.
Quality control of ribosomal RNA mediated by polynucleotide phosphorylase and RNase R
Authors: Cheng Zhuan-Fen , Deutscher Murray P.
Published: May 2003

Despite their overall accuracy, errors in macromolecular processes, such as rRNA synthesis and ribosome assembly, inevitably occur. However, whether these errors are remediated and how this might be accomplished is not known. In previous work, we showed that a double mutant strain lacking both polynucleotide phosphorylase (PNPase) and RNase R activities is inviable. In the course of examining the molecular basis for this phenotype, we found that shifting a temperature-sensitive mutant strain to 42°C led to cessation of growth and loss of cell viability. Northern analysis of RNA isolated from such cells after the temperature shift revealed that fragments of 16S and 23S rRNA accumulated to a high level, and that the amount of ribosomes and ribosomal subunits decreased due to defects in ribosome assembly. rRNA fragments were not detected at 31°C or when single mutant strains were grown at 42°C. Pulse–chase analysis showed that the rRNA fragments appeared within 5 min at 42°C, and that they accumulated before the loss of cell viability. The data are consistent with a model in which PNPase and RNase R mediate a previously unknown quality control process that normally removes defective rRNAs as soon as they are generated. In the absence of these RNases, rRNA fragments accumulate, leading to interference with ribosome maturation and ultimately to cell death.
Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin, β-catenin, and the phosphatase DEP-1/CD148
Authors: Lampugnani Maria Grazia , Zanetti Adriana , Corada Monica , Takahashi Takamune , Balconi Giovanna , Breviario Ferruccio , Orsenigo Fabrizio , Cattelino Anna , Kemler Rolf , Daniel Thomas O. , Dejana Elisabetta
Published: May 2003

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in β-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. β-Catenin–null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density–enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin–β-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell–cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin–null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.