bluuebunny/crossref_metadata_2025 huggingface dataset:
Interaction of “readthrough” acetylcholinesterase with RACK1 and PKCβII correlates with intensified fear-induced conflict behavior
Authors: Birikh Klara R. , Sklan Ella H. , Shoham Shai , Soreq Hermona
Published: January 2003

Behavioral reactions to stress are altered in numerous psychiatric and neurodegenerative syndromes, but the corresponding molecular processes and signal transduction pathways are yet unknown. Here, we report that, in mice, the stress-induced splice variant of acetylcholinesterase, AChE-R, interacts intraneuronally with the scaffold protein RACK1 and through it, with its target, protein kinase CβII (PKCβII), which is known to be involved in fear conditioning. In stress-responsive brain regions of normal FVB/N mice, the mild stress of i.p. injection increased AChE and PKCβII levels in a manner suppressible by antisense prevention of AChE-R accumulation. Injection stress also prolonged conflict between escape and hiding in the emergence into an open field test. Moreover, transgenic FVB/N mice overexpressing AChE-R displayed prolonged delay to emerge into another field (fear-induced behavioral inhibition), associated with chronically intensified neuronal colabeling of RACK1 and PKCβII in stress-responsive brain regions. These findings are consistent with the hypothesis that stress-associated changes in cholinergic gene expression regulate neuronal PKCβII functioning, promoting fear-induced conflict behavior after stress.
Asymmetry of the central apparatus defines the location of active microtubule sliding in
Chlamydomonas
flagella
Authors: Wargo Matthew J. , Smith Elizabeth F.
Published: January 2003

Regulation of ciliary and flagellar motility requires spatial control of dynein-driven microtubule sliding. However, the mechanism for regulating the location and symmetry of dynein activity is not understood. One hypothesis is that the asymmetrically organized central apparatus, through interactions with the radial spokes, transmits a signal to regulate dynein-driven microtubule sliding between subsets of doublet microtubules. Based on this model, we hypothesized that the orientation of the central apparatus defines positions of active microtubule sliding required to control bending in the axoneme. To test this, we induced microtubule sliding in axonemes isolated from wild-type and mutant
Chlamydomonas
cells, and then used electron microscopy to determine the orientation of the central apparatus. Transverse sections of wild-type axonemes revealed that the C1 microtubule is predominantly oriented toward the position of active microtubule sliding. In contrast, the central apparatus is randomly oriented in axonemes isolated from radial spoke deficient mutants. For outer arm dynein mutants, the C1 microtubule is oriented toward the position of active microtubule sliding in low calcium buffer, but is randomly oriented in high calcium buffer. These results provide evidence that the central apparatus defines the position of active microtubule sliding, and may regulate the size and shape of axonemal bends through interactions with the radial spokes. In addition, our results indicate that in high calcium conditions required to generate symmetric waveforms, the outer dynein arms are potential targets of the central pair-radial spoke control system.
IL-7 induces bone loss
in vivo
by induction of receptor activator of nuclear factor κB ligand and tumor necrosis factor α from T cells
Authors: Toraldo Gianluca , Roggia Cristiana , Qian Wei-Ping , Pacifici Roberto , Weitzmann M. Neale
Published: January 2003

IL-7, a powerful lymphopoietic cytokine, is elevated in rheumatoid arthritis (RA) and known to induce bone loss when administered
in vivo
. IL-7 has been suggested to induce bone loss, in part, by stimulating the proliferation of B220
+
cells, a population capable of acting as early osteoclast (OC) precursors. However, the mechanism by which IL-7 leads to differentiation of precursors into mature OCs remains unknown. We previously reported that,
in vitro
, IL-7 up-regulated T cell cytokines including receptor activator of nuclear factor κB ligand (RANKL). To demonstrate the importance of T cells to the bone-wasting effect of IL-7
in vivo
, we have now examined IL-7-induced bone loss in T cell-deficient nude mice. We show that T cell-replete mice undergo significant osteoclastic bone loss after IL-7 administration, concurrent with induction of RANKL and tumor necrosis factor α (TNF-α) secretion by splenic T cells. In contrast, nude mice were resistant to IL-7-induced bone loss and showed no detectable increase in either RANKL or TNF-α, despite an up-regulation of B220
+
cells. Importantly, T cell adoptive transfer into nude mice restored IL-7-induced bone loss, and RANKL and TNF-α secretion, demonstrating that T cells are essential mediators of IL-7-induced bone loss
in vivo
.
Direct interaction with a nuclear protein and regulation of gene silencing by a variant of the Ca
2+
-channel β
4
subunit
Authors: Hibino H. , Pironkova R. , Onwumere O. , Rousset M. , Charnet P. , Hudspeth A. J. , Lesage F.
Published: January 2003

The β subunits of voltage-gated Ca
2+
channels are known to be regulators of the channels' gating properties. Here we report a striking additional function of a β subunit. Screening of chicken cochlear and brain cDNA libraries identified β
4c
, a short splice variant of the β
4
subunit. Although β
4c
occurs together with the longer isoforms β
4a
or β
4b
in the brain, eye, heart, and lung, the cochlea expresses exclusively β
4c
. The association of β
4c
with the Ca
2+
-channel α
1
subunit has slight but significant effects on the kinetics of channel activation and inactivation. Yeast two-hybrid and biochemical assays revealed that β
4c
interacts directly with the chromo shadow domain of chromobox protein 2/heterochromatin protein 1γ (CHCB2/HP1γ), a nuclear protein involved in gene silencing and transcriptional regulation. Coexpression of this protein specifically recruits β
4c
to the nuclei of mammalian cells. Furthermore, β
4c
but not β
4a
dramatically attenuates the gene-silencing activity of chromobox protein 2/heterochromatin protein 1γ. The β
4c
subunit is therefore a multifunctional protein that not only constitutes a portion of the Ca
2+
channel but also regulates gene transcription.
Quantum-mechanical calculations of the stabilities of fluxional isomers of C
4
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Authors: Casanova Joseph , Kent David R. , Goddard William A. , Roberts John D.
Published: January 2003

Although numerous quantum calculations have been made over the years of the stabilities of the fluxional isomers of C
4
H

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, none have been reported for other than the gas phase (which is unrealistic for these ionic species) that exhibit exceptional fluxional properties in solution. To be sure, quantum-mechanical calculations for solutions are subject to substantial uncertainties, but nonetheless it is important to see whether the trends seen for the gas-phase C
4
H

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species are also found in calculations for polar solutions. Of the C
4
H

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species, commonly designated bisected-cyclopropylcarbinyl 1,
unsym
-bicyclobutonium 2,
sym
-bicyclobutonium 3, allylcarbinyl 4, and pyramidal structure 6, the most advanced gas-phase calculations available thus far suggest that the order of stability is 1 ≥ 2 ≥ 3 ≫ 4 ≫ 6 with barriers of only ≈1 kcal/mol for interconversions among 1, 2, and 3. We report here that, when account is taken of solvation, 2 turns out to be slightly more stable than 1 or 3 in polar solvents. The pattern of the overall results is unexpected, in that despite substantial differences in structures and charge distributions between the primary players in the C
4
H

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equilibria and the large differences in solvation energies calculated for the solvents considered, the differential solvent effects from species to species are rather small.
Contribution of GIRK2-mediated postsynaptic signaling to opiate and α
2
-adrenergic analgesia and analgesic sex differences
Authors: Mitrovic Igor , Margeta-Mitrovic Marta , Bader Semon , Stoffel Markus , Jan Lily Y. , Basbaum Allan I.
Published: January 2003

The analgesia produced by inhibitory G protein-coupled receptor agonists involves coordinated postsynaptic inhibition via G protein-coupled inwardly rectifying potassium channels (GIRKs) and presynaptic inhibition of neurotransmitter release through regulation of voltage-gated Ca
2+
channels. Here, we used mice lacking the GIRK2 channel subunit to assess the relative contribution of these two effector systems to nociceptive processing in male and female mice. Compared with female WT mice, male WT mice exhibited higher pain thresholds and enhanced opioid (morphine) and α
2
-adrenergic (clonidine) receptor-induced antinociception in a spinal reflex test. The GIRK2-null mutation reduced the “pain” threshold in male but not in female mice, effectively eliminating the sex differences in pain threshold. In addition, deletion of GIRK2 channels in mutant mice largely eliminated clonidine antinociception and significantly decreased morphine antinociception. Furthermore, the more pronounced morphine and clonidine-induced antinociception in male mice disappeared in the GIRK2 mutants. Based on the almost complete loss of clonidine-induced antinociception in the mutant mice, we conclude that it is primarily mediated by postsynaptic α
2
-adrenergic receptors. In contrast, the significant residual morphine effect in the mutant mice points to the presynaptic μ opioid receptor as a major contributor to its analgesic action. Finally, our results suggest that the reduced pain responsiveness of male compared with female mice results in part from GIRK2-coupled postsynaptic receptors that are activated by endogenous antinociceptive systems.
Expression of Th1-mediated immunity in mouse lungs induces aMycobacterium tuberculosistranscription pattern characteristic of nonreplicating persistence
Authors: Shi Lanbo , Jung Yu-Jin , Tyagi Sanjay , Gennaro Maria Laura , North Robert J.
Published: January 2003

The lung is the primary target of infection withMycobacterium tuberculosis. It is well established that, in mouse lung, expression of adaptive, Th1-mediated host immunity inhibits further multiplication ofM. tuberculosis. Here, real-time RT-PCR was used to define the pattern of expression against time of lung infection of key genes involved in Th1-mediated immunity and of selected genes ofM. tuberculosis. Inhibition of bacterial multiplication was preceded by increased mRNA synthesis for IFN-γ and inducible NO synthase (NOS2) and by NOS2 protein synthesis in infected macrophages. Concurrently, the pattern of transcription of bacterial genes underwent dramatic changes. mRNA synthesis increased for α-crystallin (acr),rv2626c, andrv2623and decreased for superoxide dismutase C (sodC),sodA, and fibronectin-binding protein B (fbpB). This pattern ofM. tuberculosistranscription is characteristic of the nonreplicating persistence [Wayne, L. G. & Sohaskey, C. D. (2001)Annu. Rev. Microbiol.55, 139–163] associated with adaptation of tubercle bacilli to hypoxiain vitro. Based on this similarity, we infer that host immunity induces bacterial growth arrest. In IFN-γ gene-deleted mice, bacterial growth was not controlled; NOS2 protein was not detected in macrophages;sodC,sodA, andfbpBtranscription showed no decrease; andacr,rv2626c, andrv2623transcription increased only at the terminal stages of lung pathology. These findings define the transcription signature ofM. tuberculosisas it transitions from growth to persistence in the mouse lung. The bacterial transcription changes measured at onset of Th1-mediated immunity are likely induced, directly or indirectly, by nitric oxide generated by infected macrophages.
Insufficiently dehydrated hydrogen bonds as determinants of protein interactions
Authors: Fernández Ariel , Scheraga Harold A.
Published: January 2003

The prediction of binding sites and the understanding of interfaces associated with protein complexation remains an open problem in molecular biophysics. This work shows that a crucial factor in predicting and rationalizing protein–protein interfaces can be inferred by assessing the extent of intramolecular desolvation of backbone hydrogen bonds in monomeric structures. Our statistical analysis of native structures shows that, in the majority of soluble proteins, most backbone hydrogen bonds are thoroughly wrapped intramolecularly by nonpolar groups except for a few ones. These latter underwrapped hydrogen bonds may be dramatically stabilized by removal of water. This fact implies that packing defects are “sticky” in a way that decisively contributes to determining the binding sites for proteins, as an examination of numerous complexes demonstrates.
Genome-based peptide fingerprint scanning
Authors: Giddings Michael C. , Shah Atul A. , Gesteland Ray , Moore Barry
Published: January 2003

We have implemented a method that identifies the genomic origins of sample proteins by scanning their peptide-mass fingerprint against the theoretical translation and proteolytic digest of an entire genome. Unlike previously reported techniques, this method requires no predefined ORF or protein annotations. Fixed-size windows along the genome sequence are scored by an equation accounting for the number of matching peptides, the number of missed enzymatic cleavages in each peptide, the number of in-frame stop codons within a window, the adjacency between peptides, and duplicate peptide matches. Statistical significance of matching regions is assessed by comparing their scores to scores from windows matching randomly generated mass data. Tests with samples from
Saccharomyces cerevisiae
mitochondria and
Escherichia coli
have demonstrated the ability to produce statistically significant identifications, agreeing with two commonly used programs,
peptident
and
mascot
, in 86% of samples analyzed. This genome fingerprint scanning method has the potential to aid in genome annotation, identify proteins for which annotation is incorrect or missing, and handle cases where sequencing errors have caused framing mistakes in the databases. It might also aid in the identification of proteins in which recoding events such as frameshifting or stop-codon read-through have occurred, elucidating alternative translation mechanisms. The prototype is implemented as a client/server pair, allowing the distribution, among a set of cluster nodes, of a single or multiple genomes for concurrent analysis.
Disrupted-in-Schizophrenia-1 (DISC-1): Mutant truncation prevents binding to NudE-like (NUDEL) and inhibits neurite outgrowth
Authors: Ozeki Yuji , Tomoda Toshifumi , Kleiderlein John , Kamiya Atsushi , Bord Lyuda , Fujii Kumiko , Okawa Masako , Yamada Naoto , Hatten Mary E. , Snyder Solomon H. , Ross Christopher A. , Sawa Akira
Published: January 2003

Disrupted-in-Schizophrenia-1 (
DISC-1
) is a gene whose mutant truncation is associated with major psychiatric illness with a predominance of schizophrenic symptomatology. We have cloned and characterized rodent DISC-1. DISC-1 expression displays pronounced developmental regulation with the highest levels in late embryonic life when the cerebral cortex develops. In yeast two-hybrid analyses, DISC-1 interacts with a variety of cytoskeletal proteins. One of these, NudE-like (NUDEL), is associated with cortical development and is linked to LIS-1, the disease gene for a form of lissencephaly, a disorder of cortical development. The disease mutant form of DISC-1 fails to bind NUDEL. Expression of mutant, but not wild-type, DISC-1 in PC12 cells reduces neurite extension. As schizophrenia is thought to reflect defects in cortical development that are determined by cytoskeletal protein activities, the cellular disturbances we observe with mutant DISC-1 may be relevant to psychopathologic mechanisms.
Invariant representations of visual patterns in a temporal population code
Authors: Wyss Reto , König Peter , Verschure Paul F. M. J.
Published: January 2003

Mammalian visual systems are characterized by their ability to recognize stimuli invariant to various transformations. Here, we investigate the hypothesis that this ability is achieved by the temporal encoding of visual stimuli. By using a model of a cortical network, we show that this encoding is invariant to several transformations and robust with respect to stimulus variability. Furthermore, we show that the proposed model provides a rapid encoding, in accordance with recent physiological results. Taking into account properties of primary visual cortex, the application of the encoding scheme to an enhanced network demonstrates favorable scaling and high performance in a task humans excel at.
Optimal alignment for enzymatic proton transfer: Structure of the Michaelis complex of triosephosphate isomerase at 1.2-Å resolution
Authors: Jogl Gerwald , Rozovsky Sharon , McDermott Ann E. , Tong Liang
Published: January 2003

In enzyme catalysis, where exquisitely positioned functionality is the
sine qua non
, atomic coordinates for a Michaelis complex can provide powerful insights into activation of the substrate. We focus here on the initial proton transfer of the isomerization reaction catalyzed by triosephosphate isomerase and present the crystal structure of its Michaelis complex with the substrate dihydroxyacetone phosphate at near-atomic resolution. The active site is highly compact, with unusually short and bifurcated hydrogen bonds for both catalytic Glu-165 and His-95 residues. The carboxylate oxygen of the catalytic base Glu-165 is positioned in an unprecedented close interaction with the ketone and the α-hydroxy carbons of the substrate (C… O ≈ 3.0 Å), which is optimal for the proton transfer involving these centers. The electrophile that polarizes the substrate, His-95, has close contacts to the substrate's O1 and O2 (N… O ≤ 3.0 and 2.6 Å, respectively). The substrate is conformationally relaxed in the Michaelis complex: the phosphate group is out of the plane of the ketone group, and the hydroxy and ketone oxygen atoms are not in the
cisoid
configuration. The ɛ ammonium group of the electrophilic Lys-12 is within hydrogen-bonding distance of the substrate's ketone oxygen, the bridging oxygen, and a terminal phosphate's oxygen, suggesting a role for this residue in both catalysis and in controlling the flexibility of active-site loop.
Clearance of replicating hepatitis C virus replicon RNAs in cell culture by small interfering RNAs
Authors: Randall Glenn , Grakoui Arash , Rice Charles M.
Published: January 2003

RNA interference is a cellular process of gene silencing in which small duplexes of RNA specifically target a homologous sequence for cleavage by cellular ribonucleases. The introduction of ≈22-nt small interfering RNAs (siRNAs) into mammalian cells can specifically silence cellular mRNAs without induction of the nonspecific IFN responses that are activated by longer RNA duplexes. We investigate in this article whether siRNAs can also silence the expression of the cytoplasmically replicating hepatitis C virus (HCV) RNAs by using a replicon system that supports robust HCV replication, but not the production of infectious virions. We report the efficient silencing of both cellular lamin A/C and HCV RNAs in Huh-7 hepatoma cell lines supporting HCV replication. Silencing of HCV RNAs was dose dependent and specific, inasmuch as two HCV variants that differ by 3 nt within the target sequence were only silenced by the exact homologous sequence for each. siRNAs designed to target HCV RNA triggered an exponential decrease in HCV RNA, resulting in an 80-fold decrease in HCV RNA after 4 days. The introduction of siRNAs into cells with established HCV replication cured >98% of these cells of detectable HCV antigen and replication-competent HCV RNAs. These data support the principle of siRNA-based HCV antiviral therapy.
β-Arrestin 1 down-regulation after insulin treatment is associated with supersensitization of β2 adrenergic receptor Gαs signaling in 3T3-L1 adipocytes
Authors: Hupfeld Christopher J. , Dalle Stephane , Olefsky Jerrold M.
Published: January 2003

β-Arrestin 1 is required for internalization and mitogen-activated protein (MAP) kinase activation by the β2 adrenergic receptor (β2AR). Our previous studies have shown that chronic insulin treatment down-regulates cellular β-arrestin 1 levels, leading to a marked impairment in G protein-coupled receptor and insulin-like growth factor-1 receptor-mediated MAP kinase and mitogenic signaling. In this study, we show that chronic insulin-treated, β-arrestin 1depleted 3T3-L1 adipocytes display (
i
) increased isoproterenol-induced cAMP generation (53 ± 38% at 1.5 min, 25 ± 19% at 5 min, 63 ± 14% at 30 min, and 59 ± 2% at 60 min), a Gα
s
-associated pathway; (
ii
) impaired isoproterenol-induced β2AR internalization (reduced by 98 ± 4%), which is required for MAP kinase signaling, a Gα
i
-associated pathway; and (
iii
) increased β-arrestin 1 phosphorylation at Ser-412. Taken together, these findings represent a hitherto unknown mechanism (degradation and phosphorylation of β-arrestin, whereby the activation of the insulin receptor, belonging to the family of receptor tyrosine kinases, causes supersensitization of Gα
s
-associated signaling and inhibition of Gα
i
-associated signaling by the β2AR, a prototypical G protein-coupled receptor.
Block of
Shaker
potassium channels by external calcium ions
Authors: Gomez-Lagunas Froylan , Melishchuk Alexey , Armstrong Clay M.
Published: January 2003

We describe an interaction between external Ca
2+
ions and
Shaker
K channels that is important in the gating of the channels. The interaction was first detected as a partial block of inward K
+
current in elevated Ca
2+
, beginning near −40 mV and becoming stronger at more negative voltage. Surprisingly, the time course of the block can be resolved as a rapid decay of inward current magnitude following a repolarizing step. The rapid decay of current is shown to be the result of channel block by using a two-pulse procedure that monitors the time course of gate closing. As a result of block, the decay of the tail current after repolarization is two to three times faster than gate closing. With physiological values for voltage and calcium concentration, block is readily detectable from tail time course, implying that it occurs as a normal concomitant of gate closing in
Shaker
. The slight voltage dependence of block from −60 to −100 mV suggests that Ca
2+
is bound (with low affinity) near the outer mouth of the channel. Elevated calcium quickens the inward gating current recorded as
Shaker
channels are closing; this current approximately doubles in amplitude and has a faster time course and quicker rising phase. When combined, the results suggest that calcium accelerates the first step in closing of the channel gate, perhaps by changing the channel's ion-occupancy state.
Substructure synthesis method for simulating large molecular complexes
Authors: Ming Dengming , Kong Yifei , Wu Yinghao , Ma Jianpeng
Published: January 2003

This paper reports a computational method for describing the conformational flexibility of very large biomolecular complexes using a reduced number of degrees of freedom. It is called the substructure synthesis method, and the basic concept is to treat the motions of a given structure as a collection of those of an assemblage of substructures. The choice of substructures is arbitrary and sometimes quite natural, such as domains, subunits, or even large segments of biomolecular complexes. To start, a group of low-frequency substructure modes is determined, for instance by normal mode analysis, to represent the motions of the substructure. Next, a desired number of substructures are joined together by a set of constraints to enforce geometric compatibility at the interface of adjacent substructures, and the modes for the assembled structure can then be synthesized from the substructure modes by applying the Rayleigh–Ritz principle. Such a procedure is computationally much more desirable than solving the full eigenvalue problem for the whole assembled structure. Furthermore, to show the applicability to biomolecular complexes, the method is used to study F-actin, a large filamentous molecular complex involved in many cellular functions. The results demonstrate that the method is capable of studying the motions of very large molecular complexes that are otherwise completely beyond the reach of any conventional methods.
Identification of pathogen-specific and conserved genes expressed
in vivo
by an avian pathogenic
Escherichia coli
strain
Authors: Dozois Charles M. , Daigle France , Curtiss Roy
Published: January 2003

Escherichia coli
is a diverse bacterial species that comprises commensal nonpathogenic strains such as
E. coli
K-12 and pathogenic strains that cause a variety of diseases in different host species. Avian pathogenic
E. coli
strain χ7122 (O78:K80:H9) was used in a chicken infection model to identify bacterial genes that are expressed in infected tissues. By using the cDNA selection method of selective capture of transcribed sequences and enrichment for the isolation of pathogen-specific (non-
E. coli
K-12) transcripts, pathogen-specific cDNAs were identified. Pathogen-specific transcripts corresponded to putative adhesins, lipopolysaccharide core synthesis, iron-responsive, plasmid- and phage-encoded genes, and genes of unknown function. Specific deletion of the aerobactin siderophore system and
E. coli iro
locus, which were identified by selective capture of transcribed sequences, demonstrated that these pathogen-specific systems contribute to the virulence of strain χ7122. Consecutive blocking to enrich for selection of pathogen-specific genes did not completely eliminate the presence of transcripts that corresponded to sequences also present in
E. coli
K-12. These
E. coli
conserved genes are likely to be highly expressed
in vivo
and contribute to growth or virulence. Overall, the approach we have used simultaneously provided a means to identify novel pathogen-specific genes expressed
in vivo
and insight regarding the global gene expression and physiology of a pathogenic
E. coli
strain in a natural animal host during the infectious process.
RNA interference machinery regulates chromosome dynamics during mitosis and meiosis in fission yeast
Authors: Hall Ira M. , Noma Ken-ichi , Grewal Shiv I. S.
Published: January 2003

The regulation of higher-order chromosome structure is central to cell division and sexual reproduction. Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis. In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region. In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis. We find that the RNAi machinery is required for the accurate segregation of chromosomes. Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres. Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells. Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase. This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis.
Imaging whole
Escherichia coli
bacteria by using single-particle x-ray diffraction
Authors: Miao Jianwei , Hodgson Keith O. , Ishikawa Tetsuya , Larabell Carolyn A. , LeGros Mark A. , Nishino Yoshinori
Published: January 2003

We report the first experimental recording, to our knowledge, of the diffraction pattern from intact
Escherichia coli
bacteria using coherent x-rays with a wavelength of 2 Å. By using the oversampling phasing method, a real space image at a resolution of 30 nm was directly reconstructed from the diffraction pattern. An
R
factor used for characterizing the quality of the reconstruction was in the range of 5%, which demonstrated the reliability of the reconstruction process. The distribution of proteins inside the bacteria labeled with manganese oxide has been identified and this distribution confirmed by fluorescence microscopy images. Compared with lens-based microscopy, this diffraction-based imaging approach can examine thicker samples, such as whole cultured cells, in three dimensions with resolution limited only by radiation damage. Looking forward, the successful recording and reconstruction of diffraction patterns from biological samples reported here represent an important step toward the potential of imaging single biomolecules at near-atomic resolution by combining single-particle diffraction with x-ray free electron lasers.
Warmer paleotemperatures for terrestrial ecosystems
Authors: Kowalski Elizabeth A. , Dilcher David L.
Published: January 2003

Floras of predominantly wet-soil environments show a greater than expected proportion of toothed leaves, affecting the outcome of leaf physiognomically based temperature estimates. New analyses of foliar physiognomy of plants growing in predominantly wet soils in modern forests suggest that current methods of inferring paleotemperatures from fossil floras yield underestimates of 2.5–10°C. The changes we propose bring terrestrial paleotemperature estimates into agreement with temperatures inferred from other biological and geological proxies and strengthen the use of leaf physiognomy as a method for climate reconstruction.
Proteomics gives insight into the regulatory function of chloroplast thioredoxins
Authors: Balmer Yves , Koller Antonius , del Val Gregorio , Manieri Wanda , Schürmann Peter , Buchanan Bob B.
Published: January 2003

Thioredoxins are small multifunctional redox active proteins widely if not universally distributed among living organisms. In chloroplasts, two types of thioredoxins (
f
and
m
) coexist and play central roles in regulating enzyme activity. Reduction of thioredoxins in chloroplasts is catalyzed by an iron-sulfur disulfide enzyme, ferredoxin-thioredoxin reductase, that receives photosynthetic electrons from ferredoxin, thereby providing a link between light and enzyme activity. Chloroplast thioredoxins function in the regulation of the Calvin cycle and associated processes. However, the relatively small number of known thioredoxin-linked proteins (about 16) raised the possibility that others remain to be identified. To pursue this opportunity, we have mutated thioredoxins
f
and
m
, such that the buried cysteine of the active disulfide has been replaced by serine or alanine, and bound them to affinity columns to trap target proteins of chloroplast stroma. The covalently linked proteins were eluted with DTT, separated on gels, and identified by mass spectrometry. This approach led to the identification of 15 potential targets that function in 10 chloroplast processes not known to be thioredoxin linked. Included are proteins that seem to function in plastid-to-nucleus signaling and in a previously unrecognized type of oxidative regulation. Approximately two-thirds of these targets contained conserved cysteines. We also identified 11 previously unknown and 9 confirmed target proteins that are members of pathways known to be regulated by thioredoxin. In contrast to results with individual enzyme assays, specificity for thioredoxin
f
or
m
was not observed on affinity chromatography.
NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants
Authors: Moon Haejeong , Lee Boyoung , Choi Giltsu , Shin Dongjin , Prasad D. Theertha , Lee Oksun , Kwak Sang-Soo , Kim Doh Hoon , Nam Jaesung , Bahk Jeongdong , Hong Jong Chan , Lee Sang Yeol , Cho Moo Je , Lim Chae Oh , Yun Dae-Jin
Published: January 2003

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H
2
O
2
-mediated mitogen-activated protein kinase signaling in plants. H
2
O
2
stress strongly induces the expression of the NDPK2 gene in
Arabidopsis thaliana
(
AtNDPK2
). Proteins from transgenic plants overexpressing
AtNDPK2
showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking
AtNDPK2
had higher levels of ROS than wild type. H
2
O
2
treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H
2
O
2
-activated
A. thaliana
mitogen-activated protein kinases. In the absence of H
2
O
2
treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing
AtNDPK2
but markedly decreased in the
AtNDPK2
deletion mutant. Yeast two-hybrid and
in vitro
protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3
in vitro
. Finally, constitutive overexpression of AtNDPK2 in
Arabidopsis
plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation
in situ
. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H
2
O
2
-mediated MAPK signaling in plants.
RNA polymerase II-dependent transcription in trypanosomes is associated with a SNAP complex-like transcription factor
Authors: Das Anish , Bellofatto Vivian
Published: January 2003

Spliced leader RNA transcription is essential for cell viability in trypanosomes. The SL RNA genes are expressed from the only defined RNA polymerase II-dependent promoter identified to date in the trypanosome genome. The SL RNA gene promoter has been shown by
in vitro
and
in vivo
analyses to have a tripartite architecture. The upstream most cis-acting element, called PBP-1E, is located between 70 and 60 bp upstream from the transcription start site. This essential element functions along with two downstream elements to direct efficient and proper initiation of transcription. Electrophoretic mobility-shift studies detected a 122-kDa protein, called PBP-1, which interacts with PBP-1E. This protein is the first sequence-specific, double-stranded DNA-binding protein isolated in trypanosomes. Three polypeptides copurify with PBP-1 activity, suggesting that PBP-1 is composed of 57-, 46-, and 36-kDa subunits. We have cloned the genes that encode the 57- and 46-kDa subunits. The 46-kDa protein is a previously uncharacterized protein and may be unique to trypanosomes. Its predicted tertiary structure suggests it binds DNA as part of a complex. The 57-kDa subunit is orthologous to the human small nuclear RNA-activating protein (SNAP)50, which is an essential subunit of the SNAP complex (SNAPc). In human cells, SNAPc binds to the proximal sequence element in both RNA polymerase II- and III-dependent small nuclear RNA gene promoters. These findings identify a surprising link in the transcriptional machinery across a large evolutionary distance in the regulation of small nuclear RNA genes in eukaryotes.
Characterization of mouse clonogenic megakaryocyte progenitors
Authors: Nakorn Thanyaphong Na , Miyamoto Toshihiro , Weissman Irving L.
Published: January 2003

Although it has been shown that unfractionated bone marrow, hematopoietic stem cells, common myeloid progenitors, and bipotent megakaryocyte/erythrocyte progenitors can give rise to megakaryocyte colonies in culture, monopotent megakaryocyte-committed progenitors (MKP) have never been prospectively isolated from the bone marrow of adult mice. Here, we use a monoclonal antibody to the megakaryocyte-associated surface protein, CD9, to purify MKPs from the c-kit
+
Sca-1
−
IL7Rα
−
Thy1.1
−
Lin
−
fraction of adult C57BL/Ka-Thy1.1 bone marrow. The CD9
+
fraction contained a subset of CD41
+
FcγR
lo
CD34
+
CD38
+
cells that represent ≈0.01% of the total nucleated bone marrow cells. They give rise mainly to colony-forming unit–megakaryocytes and occasionally burst-forming unit–megakaryocytes, with a plating efficiency >60% at the single-cell level.
In vivo
, MKPs do not have spleen colony-forming activity nor do they contribute to long-term multilineage hematopoiesis; they give rise only to platelets for ≈3 weeks. Common myeloid progenitors and megakaryocyte/erythrocyte progenitors can differentiate into MKPs after 72 h in stromal cultures, indicating that MKPs are downstream of these two progenitors. These isolatable MKPs will be very useful for further studies of megakaryopoiesis as well as the elucidation of their gene expression patterns.
Automatic classification of protein structure by using Gauss integrals
Authors: Røgen Peter , Fain Boris
Published: January 2003

We introduce a method of looking at, analyzing, and comparing protein structures. The topology of a protein is captured by 30 numbers inspired by Vassiliev knot invariants. To illustrate the simplicity and power of this topological approach, we construct a measure (scaled Gauss metric, SGM) of similarity of protein shapes. Under this metric, protein chains naturally separate into fold clusters. We use SGM to construct an automatic classification procedure for the CATH2.4 database. The method is very fast because it requires neither alignment of the chains nor any chain–chain comparison. It also has only one adjustable parameter. We assign 95.51% of the chains into the proper C (class), A (architecture), T (topology), and H (homologous superfamily) fold, find all new folds, and detect no false geometric positives. Using the SGM, we display a “map” of the space of folds projected onto two dimensions, show the relative locations of the major structural classes, and “zoom into” the space of proteins to show architecture, topology, and fold clusters. The existence of a simple measure of a protein fold computed from the chain path will have a major impact on automatic fold classification.
Protective Effect of Topical Iodine Preparations Upon Heat-Induced and Hydrofluoric Acid-Induced Skin Lesions
Authors: Wormser Uri , Sintov Amnon , Brodsky Berta , Amitai Yona , Nyska Abraham
Published: January 2003

In this study, the protectiveprophylacti c and post-exposure effects of novel topical iodine preparations were demonstrated uponheat- and hydrofluoric acid-induced skin lesions in the haired guinea pig. Prophylactic treatment of thermal burns with a liquid iodine preparation resulted in statistically significant reductions of 39% and 30%, respectively, in acute inflammation and hemorrhage-microscopi c dermal parameters indicative of acute tissue damage. A clear trend of iodine-induced reduction in dermal necrosis occurred, and the epidermal healing markers, acanthosis and hyperkeratosis, were increased. Postexposure treatment of thermal burns with an iodine ointment preparation immediately after occurrence also conferred significant therapeutic reduction in parameters of tissue damage such as epidermal ulceration (87%), acute inflammation (58%), and hemorrhage (30%). Gross pathological evaluation showed that prophylactic and postexposure treatments with the liquid iodine preparation significantly reduced the heat-induced ulceration area by 97% and 65%, respectively. In addition, immediate treatment with an ointment iodine formulation significantly decreased the ulceration area by 98%; its tetraglycol vehicle also had a beneficial effect. Postexposure treatment with the iodine ointment proved efficacious upon hydrofluoric acid-induced skin burns. We observed statistically significant reductions of 76% and 68% in ulceration areas at intervals of 5 and 10 minutes between exposure and treatment, whereas a weaker effect was observed at a longer time interval of 15 minutes. Our findings suggest the therapeutic usage of these newly developed iodine preparations for thermally induced and hydrofluoric acid-induced skin burns.
Evaluation of Carcinogenic Responses in the Eker Rat following Short-Term Exposure to Selected Nephrotoxins and Carcinogens
Authors: Morton Laura Dill , Youssef Ashraf F. , Lloyd Eric , Kiorpes Anthony L. , Goldsworthy Thomas L. , Fort Farrel L.
Published: January 2003

This study examined the response of the Eker rat to nephrotoxic compounds and to genotoxic nonrenal carcinogens. Groups of male Eker rats received either no treatment; a vehicle treatment; treatment with a noncarcinogeni c nephrotoxin (aluminum nitrilotriacetate, 2 mg/kg/day of aluminum, intraperitoneally, 3 days per week or cyclosporine A, 30 mg/kg/day, orally by gavage, 7 days/week); or treatment with a genotoxic nonrenal carcinogen (furan, 8 mg/kg/day, orally by gavage, 5 days/week or 2,4-diaminotoluene, 6.5 mg/kg/day, orally by gavage, 7 days/week or 2-nitropropane, 89 mg/kg/day, orally by gavage, 3 days/week). Duration of treatment was 4 and/or 6 months. Tissues from the Eker rats were evaluated microscopically and numbers of proliferative renal lesions were counted. Administration of nephrotoxic compounds (Al-NTA and cyclosporine) significantly increased the number of preneoplasti c and neoplasti c renal lesions in the Eker rat compared to concurrent vehicle controls. The genotoxi c nonrenal carcinogens had no consistent effect on numbers of preneoplastic or neoplastic renal lesions and did not produce neoplasms in the expected target organ (liver).
Invited Review: Cell Volume Control and Signal Transduction in Apoptosis
Authors: Gómez-Angelats Mireia , Cidlowski John A.
Published: January 2003

Apoptosis is a physiological form of death in which cells turn-on an intrinsic genetic program that eventually leads to their destruction in a highly regulated manner. This process renders elimination of “unwanted cells” in the body, and accounts for cellular turnover and homeostasi s of tissues in multicellular organisms. Consequently, an imbalance in the apoptotic rate in a particular tissue can lead to profound effects in the whole organism. Exposure of cells to apoptotic stimuli induces a rapid loss of cell volume (apoptotic volume decrease) that plays a pivotal role in the decision of a cell to undergo apoptosis. Interestingly, the apoptotic volume decrease is driven by changes in ionic fluxes across the plasma membrane that promote a decrease in the intracellular ions that ultimately also leads to a reduction in intracellular ionic strength. Despite an intensive research effort however, the cellular and molecular mechanisms that trigger changes in cell volume during apoptosis remain poorly understood. Nevertheless, this apoptotic volume decrease has been shown to be a necessary component of the apoptotic cascade and an important point of modulation for the entire cell death process. In this review, we will focus on the importance of the apoptotic volume decrease in the context of signaling and modulation of programmed cell death.
Propylene Glycol Monomethyl Ether (PGME): Inhalation Toxicity and Carcinogenicity in Fischer 344 Rats and B6C3F1 Mice
Authors: Spencer Pamela J. , Crissman James W. , Stott William T. , Corley Richard A. , Cieszlak Frank S. , Schumann Alan M. , Hardisty Jerry F.
Published: January 2003

A series of inhalation studies with propylene glycol monomethyl ether (PGME) vapor were undertaken to characterize its subchronic toxicity in mice and chronic toxicity/oncogenicity in rats and mice. Groups of male and female Fischer 344 rats and B6C3F1 mice were exposed to 0,300, 1,000, or 3,000 ppm vapor from 1 week to 2 years. Primary treatment-related effects included: initial sedation of animals exposed to 3,000 ppm and its subsequent resolution correlating with induction of hepatic mixed function oxidase activity and S-phase DNA synthesis; elevated mortality in high-exposure male rats and mice (chronic study); elevated deposition of alpha2U-globulin ( α2U-G) and associated nephropathy and S-phase DNA synthesis in male rat kidneys; accelerated atrophy of the adrenal gland X-zone infemale mice (subchronic study only); and increased occurrence and/or severity of eosinophilic foci of altered hepatocytes in male rats. No toxicologically relevant statistically significant increases in neoplasia occurred in either species. A numerical increase in the incidence of kidney adenomas occurred in intermediate-exposure male rats; however, the association with α2U-G nephropathy, a male rat specific effect, indicated a lack of relevance for human risk assessment.
4-Vinylphenol-Induced Pneumotoxicity and Hepatotoxicity in Mice
Authors: Carlson Gary P. , Ullman Mary , Mantick Nancy A. , Snyder Paul W.
Published: January 2003

4-Vinylphenol (4-hydroxystyrene , 4-ethenylphenol , 4-VP) occurs naturally in some foods and has been used as a flavoring agent in food products. It is used synthetically in the production of polymers and resins. It has also been reported to be a minor metabolite of styrene in rats and humans. Varying doses of 4-vinylpheno l were administered ip to mice. Hepatotoxicity was assessed by measuring serum sorbitol dehydrogenas e (SDH) and by light microscopy. Pneumotoxicity was assessed by measuring proteins, cells, and lactate dehydrogenas e activity in bronchoalveolar lavage fluid (BALF) and by light microscopy. 4-VP caused a dose-dependent increase in serum SDH and mild hepatocellular swelling. It caused an increase in cell number and lactate dehydrogenas e activity in BALF. Microscopically, there was widespread and severe necrosis of the bronchioles by 12 hours. Re-epithelialzation of the bronchioles was evident by 48 hours. These studies indicate that 4-vinylphenol is both hepatotoxi c and pneumotoxic.