bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite.
Authors: Szabó C , Zingarelli B , O'Connor M , Salzman A L
Published: July 2002

The free radicals nitric oxide and superoxide anion react to form peroxynitrite (ONOO-), a highly toxic oxidant species. In vivo formation of ONOO- has been demonstrated in shock and inflammation. Herein we provide evidence that cytotoxicity in cells exposed to ONOO- is mediated by DNA strand breakage and the subsequent activation of the DNA repair enzyme poly(ADP ribose) synthetase (PARS). Exposure to ONOO- (100 microM to 1 mM) inhibited mitochondrial respiration in cultured J774 macrophages and in rat aortic smooth muscle cells. The loss of cellular respiration was rapid, peaking 1-3 h after ONOO- exposure, and reversible, with recovery after a period of 6-24 h. The inhibition of mitochondrial respiration was paralleled by a dose-dependent increase in DNA strand breakage, reaching its maximum at 20-30 min after exposure to ONOO-. We observed a dose-dependent increase in the activity of PARS in cells exposed to ONOO-. Inhibitors of PARS such as 3-aminobenzamide (1 mM) prevented the inhibition of cellular respiration in cells exposed to ONOO-. Activation of PARS by ONOO--mediated DNA strand breakage resulted in a significant decrease in intracellular energy stores, as reflected by a decline of intracellular NAD+ and ATP content. 3-Aminobenzamide prevented the loss of NAD+ and ATP in cells exposed to ONOO-. In contrast, impairment of cellular respiration by the addition of the nitric oxide donors S-nitroso-N-acetyl-DL-penicillamine or diethyltriamine nitric oxide complex, was not associated with the development of DNA strand breaks, in concentrations up to 1 mM, and was largely refractory to PARS inhibition. Our results suggest that DNA damage and activation of PARS, an energy-consuming futile repair cycle, play a central role in ONOO--mediated cellular injury.
Agrobacterium VirE2 protein mediates nuclear uptake of single-stranded DNA in plant cells.
Authors: Zupan J R , Citovsky V , Zambryski P
Published: July 2002

Agrobacterium genetically transforms plant cells by transferring a single-stranded DNA (ssDNA) copy of the transferred DNA (T-DNA) element, the T-strand, in a complex with Agrobacterium proteins VirD2, bound to the 5' end, and VirE2. VirE2 binds single-stranded nucleic acid cooperatively, fully coating the T-strand, and the protein localizes to the plant cell nucleus when transiently expressed. The coupling of ssDNA binding and nuclear localizing activities suggests that VirE2 alone could mediate nuclear localization of ssDNA. In this study, fluorescently labeled ssDNA accumulated in the plant cell nucleus specifically when microinjected as a complex with VirE2. Microinjected ssDNA alone remained cytoplasmic. Import of VirE2-ssDNA complex into the nucleus via a protein import pathway was supported by (i) the inhibition of VirE2-ssDNA complex import in the presence of wheat germ agglutinin or a nonhydrolyzable GTP analog, both known inhibitors of protein nuclear import, and (ii) the retardation of import when complexes were prepared from a VirE2 mutant impaired in ssDNA binding and nuclear import.
The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.
Authors: Kato Y , Muto T , Tomura T , Tsumura H , Watarai H , Mikayama T , Ishizaka K , Kuroki R
Published: July 2002

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.
Amyloid-β peptide–Receptor for Advanced Glycation Endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: A proinflammatory pathway in Alzheimer disease
Authors: Du Yan Shi , Zhu Huaijie , Fu Jin , Yan Shi Fang , Roher Alex , Tourtellotte Wallace W. , Rajavashisth Tripathi , Chen Xi , Godman Gabriel C. , Stern David , Schmidt Ann Marie
Published: July 2002

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Aβ) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Aβ, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor κB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Aβ deposits, and in cerebrospinal fluid from AD patients there was ≈5-fold increased M-CSF antigen (
P
< 0.01), compared with age-matched controls. M-CSF released by Aβ-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Aβ, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Aβ on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.
Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence
Authors: Zucker-Franklin Dorothea , Pancake Bette A. , Marmor Michael , Legler Patricia M.
Published: July 2002

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities.
Spatial and temporal coherence in perceptual binding
Authors: Blake Randolph , Yang Yuede
Published: July 2002

Component visual features of objects are registered by distributed patterns of activity among neurons comprising multiple pathways and visual areas. How these distributed patterns of activity give rise to unified representations of objects remains unresolved, although one recent, controversial view posits temporal coherence of neural activity as a binding agent. Motivated by the possible role of temporal coherence in feature binding, we devised a novel psychophysical task that requires the detection of temporal coherence among features comprising complex visual images. Results show that human observers can more easily detect synchronized patterns of temporal contrast modulation within hybrid visual images composed of two components when those components are drawn from the same original picture. Evidently, time-varying changes within spatially coherent features produce more salient neural signals.
Movements of truncated kinesin fragments with a short or an artificial flexible neck
Authors: Inoue Yuichi , Toyoshima Yoko Yano , Iwane Atsuko Hikikoshi , Morimoto Sayuri , Higuchi Hideo , Yanagida Toshio
Published: July 2002

To investigate the role of the neck domain of kinesin, we used optical trapping nanometry to perform high-resolution measurements of the movements and forces produced by recombinant kinesin fragments in which the neck domains were shortened or replaced by an artificial random coil. Truncated kinesin fragments (K351) that contain a motor domain consisting of ≈340 aa and a short neck domain consisting of ≈11 aa showed fast movement (800 nm/s) and 8-nm steps. Such behavior was similar to that of recombinant fragments containing the full-length neck domain (K411) and to that of native kinesin. Kinesin fragments lacking the short neck domain (K340), however, showed very slow movement (<50 nm/s), as previously reported. Joining an artificial 11-aa sequence that was expected to form a flexible random chain to the motor domain (K340–chain) produced normal fast (≈700 nm/s) and stepwise movement. The results suggest that the neck domain does not act as a rigid lever arm to magnify the structural change at the catalytic domain as has been believed for myosin, but it does act as a flexible joint to guarantee the mobility of the motor domain.
Cleavage patterns and the topology of the metazoan tree of life
Authors: Valentine James W.
Published: July 2002

Several major alliances of metazoan phyla have been identified by small subunit rRNA sequence comparisons. It is possible to arrange the phyla to produce a parsimonious distribution of cleavage types, requiring only one change from a radial ancestral condition to spiral cleavage and one other to “idiosyncratic” cleavage; this arrangement is consistent with most of the recent molecular phylogenies. The cleavage shifts are correlated with changes in many of the features that once were used to distinguish Protostomia and Deuterostomia. It is hypothesized that changes in cleavage direction are causally associated with changes in blastomere fates and thus that cleavage type correlates with such features as the identity of mesoderm founder cells, which in turn can constrain the mode of origination of the eucelom. Cleavage changes may also affect the timing of cell fate specification. In a tree that emphasizes cleavage parsimony, radial cleavage, regulative development, and enterocely are ancestral within the Bilateria, and spiral or idiosyncratic cleavages, mosaic development, and schizocely are associated with a change in cleavage direction. Deuterostomy is presumably ancestral and is correlated with radial cleavage for this reason, rather than mechanistically.
Loss of virulence inLeishmania donovanideficient in an amastigote-specific protein, A2
Authors: Zhang Wen-Wei , Matlashewski Greg
Published: July 2002

Leishmania donovaniis the etiologic agent of fatal visceral leishmaniasis in man. During their life cycle,Leishmaniaexist as flagellated promastigotes within the sandfly vector and as nonflagellated amastigotes in the macrophage phagolysosomal compartment of the mammalian host. The transformation from promastigotes to amastigotes is a critical step for the establishment of infection, and the molecular basis for this transformation is poorly understood. To define the molecular basis for amastigote survival in the mammalian host, we previously identified an amastigote stage-specific gene family termed “A2.” In the present study, we have inhibited the expression of A2 mRNA and A2 protein in amastigotes using antisense RNA and show that the resulting A2-deficient amastigotes are severely compromised with respect to virulence in mice. Amastigotes that did survive in the mice had restored A2 protein expression. These data demonstrate that A2 protein is required forL. donovanisurvival in a mammalian host, and this represents the first identified amastigote-specific virulence factor identified inLeishmania. This study also reveals that it is possible to study gene function inLeishmaniathrough the expression of antisense RNA.
A hypothalamic follicle-stimulating hormone-releasing decapeptide in the rat
Authors: Yu Wen H. , Karanth Sharada , Walczewska Anna , Sower Stacia A. , McCann Samuel M.
Published: July 2002

Previous studies indicated that there is a separate hypothalamic control of follicle-stimulating hormone (FSH) release distinct from that of luteinizing hormone (LH). An FSH-releasing factor (FSHRF) was purified from rat and sheep hypothalami, but has not been isolated. We hypothesized that FSHRF might be an analogue of mammalian luteinizing hormone-releasing hormone (m-LHRH) and evaluated the activity of many analogues of m-LHRH and of the known LHRHs found in lower forms. Here we demonstrate that lamprey (l) LHRH-III has a potent, dose-related FSH- but not LH-releasing action on incubated hemipituitaries of male rats. l-LHRH-I on the other hand, had little activity to release either FSH or LH. m-LHRH was equipotent to l-LHRH-III to release FSH, but also had a high potency to release LH in contrast to l-LHRH-III that selectively released FSH. Chicken LHRH-II had considerable potency to release both LH and FSH, but no selectivity in its action. Salmon LHRH had much less potency than the others tested, except for l-LHRH-I, and no selectivity in its action. Because ovariectomized, estrogen, progesterone-treated rats are a sensitive
in vivo
assay for FSH- and LH-releasing activity, we evaluated l-LHRH-III in this assay and found that it had a completely selective stimulatory effect on FSH release at the two doses tested (10 and 100 pmols). Therefore, l-LHRH-III is a highly potent and specific FSH-releasing peptide that may enhance fertility in animals and humans. It may be the long sought after m-FSHRF.
Functional association between promoter structure and transcript alternative splicing
Authors: Cramer Paula , Pesce C. Gustavo , Baralle Francisco E. , Kornblihtt Alberto R.
Published: July 2002

It has been assumed that constitutive and regulated splicing of RNA polymerase II transcripts depends exclusively on signals present in the RNA molecule. Here we show that changes in promoter structure strongly affect splice site selection. We investigated the splicing of the ED I exon, which encodes a facultative type III repeat of fibronectin, whose inclusion is regulated during development and in proliferative processes. We used an alternative splicing assay combined with promoter swapping to demonstrate that the extent of ED I splicing is dependent on the promoter structure from which the transcript originated and that this regulation is independent of the promoter strength. Thus, these results provide the first evidence for coupling between alternative splicing and promoter-specific transcription, which agrees with recent cytological and biochemical evidence of coordination between splicing and transcription.
Recurrent excitatory postsynaptic potentials induced by synchronized fast cortical oscillations
Authors: Whittington Miles A. , Traub Roger D. , Faulkner Howard J. , Stanford Ian M. , Jefferys John G. R.
Published: July 2002

Gamma frequency (about 20–70 Hz) oscillations occur during novel sensory stimulation, with tight synchrony over distances of at least 7 mm. Synchronization in the visual system has been proposed to reflect coactivation of different parts of the visual field by a single spatially extended object. We have shown that intracortical mechanisms, including spike doublet firing by interneurons, can account for tight long-range synchrony. Here we show that synchronous gamma oscillations in two sites also can cause long-lasting (>1 hr) potentiation of recurrent excitatory synapses. Synchronous oscillations lasting >400 ms in hippocampal area CA1 are associated with an increase in both excitatory postsynaptic potential (EPSP) amplitude and action potential afterhyperpolarization size. The resulting EPSPs stabilize and synchronize a prolonged beta frequency (about 10–25 Hz) oscillation. The changes in EPSP size are not expressed during non-oscillatory behavior but reappear during subsequent gamma-oscillatory events. We propose that oscillation-induced EPSPs serve as a substrate for memory, whose expression either enhances or blocks synchronization of spatially separated sites. This phenomenon thus provides a dynamical mechanism for storage and retrieval of stimulus-specific neuronal assemblies.
Advances in apoptosis research
Authors: Peter Marcus E. , Heufelder Armin E. , Hengartner Michael O.
Published: July 2002

Apoptosis, also called programmed cell death, has attracted
great attention in recent years. After its discovery by Carl Vogt in
1842, apoptosis research was dormant for more than a century.
Its rediscovery in the second half of this century, and the coining of
the term
apoptosis
in 1972 by Kerr, Wyllie, and
Currie, ignited an unparalleled interest in this field of science. The
number of publications related to apoptosis has been growing
exponentially every year ever since. This is mainly due to three major
advances, two of which have been made recently and one that is
currently seen. First, studies with the small nematode
Caenorhabditis elegans
have identified a
number of apoptosis regulating genes—the first evidence that
cell death is an active process under genetic control. Many of these
genes have mammalian homologs that, like their worm counterparts, seem
to regulate mammalian apoptosis. Second, elucidation of the
signal transduction pathways of apoptosis has lead especially
to the identification of specific death signaling molecules such as a
new family of cysteine proteases, the caspases. Third, it has now
become clear that many diseases are characterized by dysregulation of
apoptotic programs. Many of these programs involve a family of
receptors and their ligands, the death receptor/ligand family.
The hope now is to interfere with apoptosis regulation in these
systems and to develop new therapeutic concepts.
A new mechanism of sound generation in songbirds
Authors: Goller Franz , Larsen Ole N.
Published: July 2002

Our current understanding of the sound-generating mechanism in the songbird vocal organ, the syrinx, is based on indirect evidence and theoretical treatments. The classical avian model of sound production postulates that the medial tympaniform membranes (MTM) are the principal sound generators. We tested the role of the MTM in sound generation and studied the songbird syrinx more directly by filming it endoscopically. After we surgically incapacitated the MTM as a vibratory source, zebra finches and cardinals were not only able to vocalize, but sang nearly normal song. This result shows clearly that the MTM are not the principal sound source. The endoscopic images of the intact songbird syrinx during spontaneous and brain stimulation-induced vocalizations illustrate the dynamics of syringeal reconfiguration before phonation and suggest a different model for sound production. Phonation is initiated by rostrad movement and stretching of the syrinx. At the same time, the syrinx is closed through movement of two soft tissue masses, the medial and lateral labia, into the bronchial lumen. Sound production always is accompanied by vibratory motions of both labia, indicating that these vibrations may be the sound source. However, because of the low temporal resolution of the imaging system, the frequency and phase of labial vibrations could not be assessed in relation to that of the generated sound. Nevertheless, in contrast to the previous model, these observations show that both labia contribute to aperture control and strongly suggest that they play an important role as principal sound generators.
Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein
Authors: Von Ohlen Tonia , Lessing Derek , Nusse Roel , Hooper Joan E.
Published: July 2002

Hedgehog (Hh) is a member of a family of secreted proteins that direct patterning at multiple stages in both
Drosophila
and vertebrate development. During
Drosophila
embryogenesis, Hh protein is secreted by the cells of the posterior compartment of each segment.
hh
activates transcription of
wingless
(
wg
),
gooseberry
(
gsb
), and
patched
(
ptc
) in the cells immediately adjacent to Hh-secreting cells. Hh signaling is thought to involve the segment polarity gene
cubitus interruptus
(
ci
).
ci
encodes a zinc finger protein of the Gli family of sequence-specific DNA binding proteins.
ci
mRNA is expressed in all non-Hh expressing cells. Here we demonstrate
ci
activity is both necessary and sufficient to drive expression of Hh-responsive genes in the
Drosophila
embryos. We show that Ci is a sequence-specific DNA binding protein that drives transcription from the
wg
promoter in transiently transfected cells. We demonstrate that Ci binding sites in the
wg
promoter are necessary for this transcriptional activation. These data taken together provide strong evidence that Ci is a transcriptional effector of Hh signaling.
Caveolin mRNA levels are up-regulated by free cholesterol and down-regulated by oxysterols in fibroblast monolayers
Authors: Fielding Christopher J. , Bist Anita , Fielding Phoebe E.
Published: July 2002

In confluent fibroblast monolayers, an increase in the selective uptake of free cholesterol (FC) from plasma low density lipoprotein (LDL) was accompanied by an increase in FC efflux. The rate of FC efflux was proportional to the FC content of the cell surface caveolae and to mRNA levels of caveolin, an FC-binding protein of caveolae. Inhibitors of LDL–FC internalization reduced the increase in caveolin mRNA levels and FC efflux. Oxysterols reduced caveolin mRNA levels, as well as transport of FC to the cell surface and FC efflux. DNA antisense to caveolin reduced caveolin mRNA and inhibited FC efflux. These data suggest that regulation of FC efflux can contribute to cellular FC homeostasis when LDL levels are modified over the physiological range, and they link this regulation to caveolin.
Convergent evolution of antifreeze glycoproteins in Antarctic notothenioid fish and Arctic cod
Authors: Chen Liangbiao , DeVries Arthur L. , Cheng Chi-Hing C.
Published: July 2002

Antarctic notothenioid fishes and several northern cods are phylogenetically distant (in different orders and superorders), yet produce near-identical antifreeze glycoproteins (AFGPs) to survive in their respective freezing environments. AFGPs in both fishes are made as a family of discretely sized polymers composed of a simple glycotripeptide monomeric repeat. Characterizations of the AFGP genes from notothenioids and the Arctic cod show that their AFGPs are both encoded by a family of polyprotein genes, with each gene encoding multiple AFGP molecules linked in tandem by small cleavable spacers. Despite these apparent similarities, detailed analyses of the AFGP gene sequences and substructures provide strong evidence that AFGPs in these two polar fishes in fact evolved independently. First, although Antarctic notothenioid AFGP genes have been shown to originate from a pancreatic trypsinogen, Arctic cod AFGP genes share no sequence identity with the trypsinogen gene, indicating trypsinogen is not the progenitor. Second, the AFGP genes of the two fish have different intron–exon organizations and different spacer sequences and, thus, different processing of the polyprotein precursors, consistent with separate genomic origins. Third, the repetitive AFGP tripeptide (Thr-Ala/Pro-Ala) coding sequences are drastically different in the two groups of genes, suggesting that they arose from duplications of two distinct, short ancestral sequences with a different permutation of three codons for the same tripeptide. The molecular evidence for separate ancestry is supported by morphological, paleontological, and paleoclimatic evidence, which collectively indicate that these two polar fishes evolved their respective AFGPs separately and thus arrived at the same AFGPs through convergent evolution.
Obese gene expression at
in vivo
levels by fat pads derived from s.c. implanted 3T3-F442A preadipocytes
Authors: Mandrup Susanne , Loftus Thomas M. , MacDougald Ormond A. , Kuhajda Francis P. , Lane M. Daniel
Published: July 2002

3T3-F442A preadipocytes implanted s.c. into athymic mice develop into fat pads that are indistinguishable from normal adipose tissue. Implanted preadipocytes harboring a β-galactosidase transgene gave rise to fat pads in which almost all adipocytes expressed β-galactosidase. This finding proved that the implanted 3T3-F442A preadipocytes, rather than endogenous preadipose cells, gave rise to the newly developed “adipose tissue.” 3T3-F442A preadipocytes, when differentiated into adipocytes in cell culture, express the obese gene at an unexpectedly low level, i.e., ≤1% the level in adipose tissue. However, adipose tissue derived from s.c. implanted 3T3-F442A preadipocytes expressed leptin mRNA at a level comparable to that in epididymal adipose tissue. These findings indicate that a factor(s) or condition, present in the tissue context and necessary for maximal obese gene expression, is lacking in cell culture. Furthermore, adipocytes derived from the implanted cells were hormonally responsive in that leptin mRNA levels were up-regulated 3- to 8-fold by glucocorticoid injection into the host animal. Thus, these findings indicate that adipose-specific promoter–reporter constructs, transfected into 3T3-F442A preadipocytes, can be tested in an
in vivo
context during and after development of these cells into adipose tissue. Furthermore, the effect of transgenes on the adipogenic development of the implanted preadipocytes can be assessed. Thus, this approach offers a faster and less costly alternative to the transgenic mouse method for assessing adipose gene function.
A double-stranded RNA element from a hypovirulent strain of
Rhizoctonia solani
occurs in DNA form and is genetically related to the pentafunctional AROM protein of the shikimate pathway
Authors: Lakshman Dilip K. , Jian Jianhua , Tavantzis Stellos M.
Published: July 2002

M2 is a double-stranded RNA (dsRNA) element occurring in the hypovirulent isolate Rhs 1A1 of the plant pathogenic basidiomycete
Rhizoctonia solani
. Rhs 1A1 originated as a sector of the virulent field isolate Rhs 1AP, which contains no detectable amount of the M2 dsRNA. The complete sequence (3,570 bp) of the M2 dsRNA has been determined. A 6.9-kbp segment of total DNA from either Rhs 1A1 or Rhs 1AP hybridizes with an M2-specific cDNA probe. The sequences of M2 dsRNA and of PCR products generated from Rhs 1A1 total DNA were found to be identical. Thus this report describes a fungal host containing full-length DNA copies of a dsRNA element. A major portion of the M2 dsRNA is located in the cytoplasm, whereas a smaller amount is found in mitochondria. Based on either the universal or the mitochondrial genetic code of filamentous fungi, one strand of M2 encodes a putative protein of 754 amino acids. The resulting polypeptide has all four motifs of a dsRNA viral RNA-dependent RNA polymerase (RDRP) and is phylogenetically related to the RDRP of a mitochondrial dsRNA associated with hypovirulence in strain NB631 of
Cryphonectria parasitica
, incitant of chestnut blight. This polypeptide also has significant sequence similarity with two domains of a pentafunctional polypeptide, which catalyzes the five central steps of the shikimate pathway in yeast and filamentous fungi.
Gene-for-gene disease resistance without the hypersensitive response in
Arabidopsis dnd1
mutant
Authors: Yu I-ching , Parker Jane , Bent Andrew F.
Published: July 2002

The cell death response known as the hypersensitive response (HR) is a central feature of gene-for-gene plant disease resistance. A mutant line of
Arabidopsis thaliana
was identified in which effective gene-for-gene resistance occurs despite the virtual absence of HR cell death. Plants mutated at the
DND1
locus are defective in HR cell death but retain characteristic responses to avirulent
Pseudomonas syringae
such as induction of pathogenesis-related gene expression and strong restriction of pathogen growth. Mutant
dnd1
plants also exhibit enhanced resistance against a broad spectrum of virulent fungal, bacterial, and viral pathogens. The resistance against virulent pathogens in
dnd1
plants is quantitatively less strong and is differentiable from the gene-for-gene resistance mediated by resistance genes
RPS2
and
RPM1
. Levels of salicylic acid compounds and mRNAs for pathogenesis-related genes are elevated constitutively in
dnd1
plants. This constitutive induction of systemic acquired resistance may substitute for HR cell death in potentiating the stronger gene-for-gene defense response. Although cell death may contribute to defense signal transduction in wild-type plants, the
dnd1
mutant demonstrates that strong restriction of pathogen growth can occur in the absence of extensive HR cell death in the gene-for-gene resistance response of
Arabidopsis
against
P. syringae
.
Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection
Authors: Gradi Alessandra , Svitkin Yuri V. , Imataka Hiroaki , Sonenberg Nahum
Published: July 2002

Eukaryotic initiation factor (eIF) 4GI is a component of the cap-binding protein complex eIF4F, which is required for cap-dependent translation. Infection of cells by poliovirus results in a precipitous decline of host cell protein synthesis, which is preceded by the cleavage of eIF4GI. Cleavage of eIF4GI results in the inhibition of cap-dependent translation. Poliovirus translation is not affected by eIF4GI cleavage, however, because poliovirus mRNA is translated by a cap-independent mechanism. Cleavage of eIF4GI alone cannot explain the shutoff of host protein synthesis, because after infection in the presence of inhibitors of virus replication, eIF4GI is cleaved, yet host protein synthesis is only partially inhibited. Here we show that eIF4GII, a recently discovered functional homolog of eIF4GI, is more resistant to poliovirus-mediated cleavage than eIF4GI, and that its proteolysis is concomitant with the shutoff of host cell protein synthesis. Moreover, infection with poliovirus in the presence of inhibitors of virus replication resulted in efficient cleavage of eIF4GI, but only partial proteolysis of eIF4GII. Thus, cleavage of both eIF4GI and eIF4GII appears to be required for the shutoff of host protein synthesis after poliovirus infection. These results explain several earlier reports documenting the lack of correlation between eIF4GI cleavage and inhibition of cellular mRNA translation after poliovirus infection.
Receptors for anti-Müllerian hormone on Leydig cells are responsible for its effects on steroidogenesis and cell differentiation
Authors: Racine Chrystèle , Rey Rodolfo , Forest Maguelone G. , Louis Franck , Ferré Axelle , Huhtaniemi Ilpo , Josso Nathalie , di Clemente Nathalie
Published: July 2002

Strong overexpression of anti-Müllerian hormone (AMH) in transgenic mice leads to incomplete fetal virilization and decreased serum testosterone in the adult. Conversely, AMH-deficient mice exhibit Leydig cell hyperplasia. To probe the mechanism of action of AMH on Leydig cell steroidogenesis, we have studied the expression of mRNA for steroidogenic proteins
in vivo
and
in vitro
and performed a morphometric analysis of testicular tissue in mice overexpressing the hormone. We show that overexpression of AMH in male transgenic mice blocks the differentiation of Leydig cell precursors. Expression of steroidogenic protein mRNAs, mainly cytochrome P450 17α-hydroxylase/C17–20 lyase (P450c17), is decreased in transgenic mice overexpressing AMH and in AMH-treated purified Leydig cells. In contrast, transgenic mice in whom the AMH locus has been disrupted show increased expression of P450c17.
In vitro
, but not
in vivo
, AMH also decreases the expression of the luteinizing hormone receptor. The effect of AMH is explained by the presence of its receptor on Leydig cells. Our results provide insight into the action of AMH as a negative modulator of Leydig cell differentiation and function.
PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides
Authors: De Angelis Dino A. , Miesenböck Gero , Zemelman Boris V. , Rothman James E.
Published: July 2002

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins
in vivo
. The method, which we term
pr
oximity
im
aging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.
Cloning of inversion breakpoints in the
Anopheles gambiae
complex traces a transposable element at the inversion junction
Authors: Mathiopoulos Kostas D. , della Torre Alessandra , Predazzi Valentina , Petrarca Vincenzo , Coluzzi Mario
Published: July 2002

Anopheles arabiensis
, one of the two most potent malaria vectors of the
gambiae
complex, is characterized by the presence of chromosomal paracentric inversions. Elucidation of the nature and the dynamics of these inversions is of paramount importance for the understanding of the population genetics and evolutionary biology of this mosquito and of the impact on malaria epidemiology. We report here the cloning of the breakpoints of the naturally occurring polymorphic inversion
2Rd′
of
A. arabiensis
. A cDNA clone that cytologically mapped on the proximal breakpoint was the starting material for the isolation of a cosmid clone that spanned the breakpoint. Analysis of the surrounding sequences demonstrated that adjacent to the distal breakpoint lies a repetitive element that exhibits distinct distribution in different
A. arabiensis
strains. Sequencing analysis of that area revealed elements characteristic of transposable element terminal repeats. We called this presumed transposable element
Odysseus
. The presence of
Odysseus
at the junction of the naturally occuring inversion
2Rd′
suggests that the inversion may be the result of the transposable element’s activity. Characteristics of
Odysseus
’ terminal region as well as its cytological distribution in different strains may indicate a relatively recent activity of
Odysseus
.
The conducting form of gramicidin A is a right-handed double-stranded double helix
Authors: Burkhart B. M. , Li N. , Langs D. A. , Pangborn W. A. , Duax W. L.
Published: July 2002

The linear pentadecapeptide antibiotic, gramicidin D, is a naturally occurring product of
Bacillus brevis
known to form ion channels in synthetic and natural membranes. The x-ray crystal structures of the right-handed double-stranded double-helical dimers (DSDH
ℛ
) reported here agree with
15
N-NMR and CD data on the functional gramicidin D channel in lipid bilayers. These structures demonstrate single-file ion transfer through the channels. The results also indicate that previous crystal structure reports of a left-handed double-stranded double-helical dimer in complex with Cs
+
and K
+
salts may be in error and that our evidence points to the DSDH
ℛ
as the major conformer responsible for ion transport in membranes.
The role of left prefrontal cortex in language and memory
Authors: Gabrieli John D. E. , Poldrack Russell A. , Desmond John E.
Published: July 2002

This article reviews attempts to characterize the mental operations
mediated by left inferior prefrontal cortex, especially the anterior
and inferior portion of the gyrus, with the functional neuroimaging
techniques of positron emission tomography and functional magnetic
resonance imaging. Activations in this region occur during semantic,
relative to nonsemantic, tasks for the generation of words to semantic
cues or the classification of words or pictures into semantic
categories. This activation appears in the right prefrontal cortex of
people known to be atypically right-hemisphere dominant for language.
In this region, activations are associated with meaningful encoding
that leads to superior explicit memory for stimuli and deactivations
with implicit semantic memory (repetition priming) for words and
pictures. New findings are reported showing that patients with global
amnesia show deactivations in the same region associated with
repetition priming, that activation in this region reflects selection
of a response from among numerous relative to few alternatives, and
that activations in a portion of this region are associated
specifically with semantic relative to phonological processing. It is
hypothesized that activations in left inferior prefrontal cortex
reflect a domain-specific semantic working memory capacity that is
invoked more for semantic than nonsemantic analyses regardless of
stimulus modality, more for initial than for repeated semantic analysis
of a word or picture, more when a response must be selected from among
many than few legitimate alternatives, and that yields superior later
explicit memory for experiences.
Dynamical principles in biological processes
Authors: Schlag E. W. , Lin S. H. , Weinkauf R. , Rentzepis P. M.
Published: July 2002

The purpose of this paper is to propose certain dynamical principles in biological systems, which can be used to explain the effectiveness of charge transfer or excitation transfer in biological systems. Some of these systems are accessible experimentally.
Stabilization and activation of p53 are regulated independently by different phosphorylation events
Authors: Chernov Mikhail V. , Ramana Chilakamarti V. , Adler Victor V. , Stark George R.
Published: July 2002

Treatment of mouse or human cells with the protein kinase C (PKC) inhibitors H7 or bisindolylmaleimide I induced an increase in the lifetime of p53, leading to its accumulation. In inhibitor-treated cells, p53 translocated to the nuclei and bound to DNA but was not competent to induce transcription. However, transactivation could be induced by subsequent DNA damage. Phorbol ester, a potent activator of PKC, significantly inhibited the accumulation of p53 after DNA damage. Therefore, constitutive PKC-dependent phosphorylation of p53 itself, or of a protein that interacts with p53, is required for the rapid degradation of p53 in untreated cells. Furthermore, an increase in the lifetime of p53 is not accompanied necessarily by its activation. Treatment with the PKC inhibitors decreased the overall level of p53 phosphorylation but led to the appearance of a phosphopeptide not seen in tryptic digests of p53 from untreated cells. Therefore, the lifetime and activities of p53 are likely to be regulated by distinct alterations of the phosphorylation pattern of p53, probably caused by the actions of different kinases.
Two distinct pathways of positive selection for thymocytes
Authors: Akashi Koichi , Kondo Motonari , Weissman Irving L.
Published: July 2002

Most mouse thymocytes undergoing positive selection are found on one of two pathways; the c-Kit+and the c-Kit−pathways. Here, we show that c-Kit and interleukin-7 receptor (IL-7R)-mediated signals support positive selection during the transition from the subpopulation that first expresses cell surface T cell receptor (TCR)—the TCRα/βloCD4int/CD8int(DPint) c-Kit+cells to TCRα/βmedc-Kit+transitional intermediate cells (the c-Kit+pathway). Cells that fail positive selection on the c-Kit+pathway become TCRα/βloc-Kit−(DPhi) blasts that appear to undergo alternative TCRα rearrangements. The rare DPhic-Kit−blast cells that thus are salvaged for positive selection by expressing a self-major histocompatibility complex selectable TCRα/β up-regulate IL-7R, but not c-Kit, and are the principal progenitors on the c-Kit−pathway; this c-Kit−IL-7R+pathway is mainly CD4 lineage committed. Cell division is a feature of the TCRlo-medc-Kit+transition, but is not essential for CD4 lineage maturation from DPhic-Kit−blasts. In this view, positive selection on the c-Kit−path results from a salvage of cells that failed positive selection on the c-Kit+path.
The
rpoB
mutants destabilizing initiation complexes at stringently controlled promoters behave like “stringent” RNA polymerases in
Escherichia coli
Authors: Zhou Yan Ning , Jin Ding Jun
Published: July 2002

In
Escherichia coli
, stringently controlled genes are highly transcribed during rapid growth, but “turned off” under nutrient limiting conditions, a process called the stringent response. To understand how transcriptional initiation at these promoters is coordinately regulated, we analyzed the interactions between RNA polymerase (RNAP) (both wild type and mutants) and four stringently controlled promoters. Our results show that the interactions between RNAP and stringently controlled promoters are intrinsically unstable and can alternate between relatively stable and metastable states. The mutant RNAPs appear to specifically further weaken interactions with these promoters
in vitro
and behave like “stringent” RNAPs in the absence of the stringent response
in vivo
, constituting a novel class of mutant RNAPs. Consistently, these mutant RNAPs also activate the expression of other genes that normally require the response. We propose that the stability of initiation complexes is coupled to the transcription of stringently controlled promoters, and this unique feature coordinates the expression of genes positively and negatively regulated by the stringent response.
Ca
2+
-sensitive inactivation of L-type Ca
2+
channels depends on multiple cytoplasmic amino acid sequences of the α
1C
subunit
Authors: Zühlke Roger D. , Reuter Harald
Published: July 2002

Ca
2+
-dependent inactivation of Ca
2+
currents is a physiological phenomenon widely associated with L-type Ca
2+
channels. Although the pore-forming α
1C
subunit of the channel is the target for Ca
2+
binding, the amino acid sequences involved in the binding and/or in the coordination of Ca
2+
-dependent inactivation are still unclear. Based on previous experiments, we have prepared truncation mutants of a human α
1C
subunit by systematically deleting an EF-hand motif and sequences in a segment of 80 amino acids in the carboxyl-terminal tail. We found that the rate as well as the Ca
2+
dependence of inactivation of currents through these mutated channels were very different. We have identified three amino acid sequences, the presence of which is important for Ca
2+
-dependent inactivation: (
i
) a putative Ca
2+
-binding EF-hand motif, (
ii
) two hydrophilic residues (asparagine and glutamic acid) 77–78 amino acids downstream of the EF-hand motif, and (
iii
) a putative IQ calmodulin binding motif. We suggest that Ca
2+
-dependent inactivation is a cooperative process involving several amino acid sequences in cytoplasmic segments of the α
1C
subunit.
The glutathione redox couple modulates zinc transfer from metallothionein to zinc-depleted sorbitol dehydrogenase
Authors: Jiang Li-Juan , Maret Wolfgang , Vallee Bert L.
Published: July 2002

The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC
1.1.1.14
)
in vitro
has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in this process. Reduced glutathione (GSH) and glutathione disulfide (GSSG) are critical modulators of both the rate of zinc transfer and the ultimate number of zinc atoms transferred. GSSG increases the rate of zinc transfer 3-fold, and its concentration is the major determinant for efficient zinc transfer. GSH has a dual function. In the absence of GSSG, it inhibits zinc transfer from MT, indicating that MT is in a latent state under the relatively high cellular concentrations of GSH. In addition, it primes MT for the reaction with GSSG by enhancing the rate of zinc transfer 10-fold and by increasing the number of zinc atoms transferred to four.
65
Zn-labeling experiments confirm the release of one zinc from MT in the absence of glutathione and the more effective release of zinc in the presence of GSH and GSSG.
In vivo
, MT may keep the cellular concentrations of free zinc very low and, acting as a temporary cellular reservoir, release zinc in a process that is dynamically controlled by its interactions with both GSH and GSSG. These results suggest that a change of the redox state of the cell could serve as a driving force and signal for zinc distribution from MT.
The expression of matrix metalloproteinase 9 is enhanced by Epstein–Barr virus latent membrane protein 1
Authors: Yoshizaki Tomokazu , Sato Hiroshi , Furukawa Mitsuru , Pagano Joseph S.
Published: July 2002

Matrix metalloproteinases (MMPs) are frequently expressed in malignant tumor cells and are thought to play crucial roles in tumor invasion and metastasis. Here we report that expression of MMP9 is increased in Epstein-Barr virus (EBV)-infected type III latency lymphoma cell lines, but not in type I lines where latent viral gene expression is highly restricted. Type III cell lines express abundant EBV latent membrane protein 1 (LMP1), the principal EBV oncoprotein, as well as the other latency proteins including the transcriptional factor, EBV nuclear antigen 2, which is also required for cell immortalization. Transfection of an LMP1 expression plasmid in the C33A cell line increased MMP9 expression, whereas overexpression of EBV nuclear antigen 2 did not. Three motifs, homologous to the binding sites of NF-κB, SP-1, and AP-1 proteins, contribute to induction of the MMP9 promoter by 12-O-tetradecanoyl-phorbol-13-acetate and tumor necrosis factor α. Here we report that binding sites for NF-κB, SP-1, and AP-1 also contribute to induction of the MMP9 promoter by the viral protein, LMP1, mainly through the NF-κB and, to a lesser extent, the SP-1 and AP-1 sites. Moreover the AP-1 binding site is essential in that mutation of it abolished reporter gene induction by LMP1. The enhancement of MMP9 expression was blocked by cotransfection of an IκB expression plasmid. Thus in addition to its transforming properties, the oncoprotein LMP1 may contribute to invasiveness and metastasis of EBV-associated tumors such as nasopharyngeal carcinoma.