bluuebunny/crossref_metadata_2025 huggingface dataset:
Higher temporal variability of forest breeding bird communities in fragmented landscapes
Authors: Boulinier Thierry , Nichols James D. , Hines James E. , Sauer John R. , Flather Curtis H. , Pollock Kenneth H.
Published: July 2002

Understanding the relationship between animal community dynamics and landscape structure has become a priority for biodiversity conservation. In particular, predicting the effects of habitat destruction that confine species to networks of small patches is an important prerequisite to conservation plan development. Theoretical models that predict the occurrence of species in fragmented landscapes, and relationships between stability and diversity do exist. However, reliable empirical investigations of the dynamics of biodiversity have been prevented by differences in species detection probabilities among landscapes. Using long-term data sampled at a large spatial scale in conjunction with a capture-recapture approach, we developed estimates of parameters of community changes over a 22-year period for forest breeding birds in selected areas of the eastern United States. We show that forest fragmentation was associated not only with a reduced number of forest bird species, but also with increased temporal variability in the number of species. This higher temporal variability was associated with higher local extinction and turnover rates. These results have major conservation implications. Moreover, the approach used provides a practical tool for the study of the dynamics of biodiversity.
Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens
Authors: Lorito Matteo , Woo Sheridan L. , Fernandez Irene Garcia , Colucci Gabriella , Harman Gary E. , Pintor-Toro José A. , Filippone Edgardo , Muccifora Simona , Lawrence Christopher B. , Zoina Astolfo , Tuzun Sadik , Scala Felice
Published: July 2002

Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus
Trichoderma harzianum
was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens
Alternaria alternata
,
A. solani
,
Botrytis cinerea
, and the soilborne pathogen
Rhizoctonia solani
. The high level and the broad spectrum of resistance obtained with a single chitinase gene from
Trichoderma
overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.
A 1-deoxy-d-xylulose 5-phosphate reductoisomerase catalyzing the formation of 2-C-methyl-d-erythritol 4-phosphate in an alternative nonmevalonate pathway for terpenoid biosynthesis
Authors: Takahashi Shunji , Kuzuyama Tomohisa , Watanabe Hiroyuki , Seto Haruo
Published: July 2002

Several eubacteria includingEsherichia coliuse an alternative nonmevalonate pathway for the biosynthesis of isopentenyl diphosphate instead of the ubiquitous mevalonate pathway. In the alternative pathway, 2-C-methyl-d-erythritol or its 4-phosphate, which is proposed to be formed from 1-deoxy-d-xylulose 5-phosphate via intramolecular rearrangement followed by reduction process, is one of the biosynthetic precursors of isopentenyl diphosphate. To clone the gene(s) responsible for synthesis of 2-C-methyl-d-erythritol 4-phosphate, we prepared and selectedE. colimutants with an obligatory requirement for 2-C-methylerythritol for growth and survival. All the DNA fragments that complemented the defect in synthesizing 2-C-methyl-d-erythritol 4-phosphate of these mutants contained theyaeMgene, which is located at 4.2 min on the chromosomal map ofE. coli. The gene product showed significant homologies to hypothetical proteins with unknown functions present inHaemophilus influenzae,Synechocystissp. PCC6803,Mycobacterium tuberculosis,Helicobacter pyroli, andBacillus subtilis. The purified recombinantyaeMgene product was overexpressed inE. coliand found to catalyze the formation of 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate in the presence of NADPH. Replacement of NADPH with NADH decreased the reaction rate to about 1% of the original rate. The enzyme required Mn2+, Co2+, or Mg2+as well. These data clearly show that theyaeMgene encodes an enzyme, designated 1-deoxy-d-xylulose 5-phosphate reductoisomerase, that synthesizes 2-C-methyl-d-erythritol 4-phosphate from 1-deoxy-d-xylulose 5-phosphate, in a single step by intramolecular rearrangement and reduction and that this gene is responsible for terpenoid biosynthesis inE. coli.
DNA sequence-dependent deformability deduced from protein–DNA crystal complexes
Authors: Olson Wilma K. , Gorin Andrey A. , Lu Xiang-Jun , Hock Lynette M. , Zhurkin Victor B.
Published: July 2002

The deformability of double helical DNA is critical for its packaging in the cell, recognition by other molecules, and transient opening during biochemically important processes. Here, a complete set of sequence-dependent empirical energy functions suitable for describing such behavior is extracted from the fluctuations and correlations of structural parameters in DNA–protein crystal complexes. These elastic functions provide useful stereochemical measures of the local base step movements operative in sequence-specific recognition and protein-induced deformations. In particular, the pyrimidine-purine dimers stand out as the most variable steps in the DNA–protein complexes, apparently acting as flexible “hinges” fitting the duplex to the protein surface. In addition to the angular parameters widely used to describe DNA deformations (i.e., the bend and twist angles), the translational parameters describing the displacements of base pairs along and across the helical axis are analyzed. The observed correlations of base pair bending and shearing motions are important for nonplanar folding of DNA in nucleosomes and other nucleoprotein complexes. The knowledge-based energies also offer realistic three-dimensional models for the study of long DNA polymers at the global level, incorporating structural features beyond the scope of conventional elastic rod treatments and adding a new dimension to literal analyses of genomic sequences.
Fc receptors are required in passive and active immunity to melanoma
Authors: Clynes Raphael , Takechi Yoshizumi , Moroi Yoichi , Houghton Alan , Ravetch Jeffrey V.
Published: July 2002

Effective tumor immunity requires recognition of tumor cells coupled with the activation of host effector responses. Fc receptor (FcR) γ−/−mice, which lack the activating FcγR types I and III, did not demonstrate protective tumor immunity in models of passive and active immunization against a relevant tumor differentiation antigen, the brown locus protein gp75. In wild-type mice, passive immunization with mAb against gp75 or active immunization against gp75 prevented the development of lung metastases. This protective response was completely abolished in FcRγ-deficient mice. Immune responses were intact in γ−/−mice because IgG titers against gp75 develop normally in γ−/−mice immunized with gp75. However, uncoupling of the FcγR effector pathway from antibody recognition of tumor antigens resulted in a loss of protection against tumor challenge. These data demonstrate an unexpected and critical role for FcRs in mediating tumor cytotoxicityin vivoand suggest that enhancement of FcγR-mediated antibody-dependent cellular cytotoxicity by inflammatory cells is a key step in the development of effective tumor immunotherapeutics.
Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1
Authors: Starr Robyn , Metcalf Donald , Elefanty Andrew G. , Brysha Marta , Willson Tracy A. , Nicola Nicos A. , Hilton Douglas J. , Alexander Warren S.
Published: July 2002

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1
in vivo
, we have used gene targeting to generate mice lacking this protein. SOCS-1
−/−
mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1
−/−
mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for
in vivo
regulation of multiple cell types and is indispensable for normal postnatal growth and survival.
Accelerated apoptosis of lymphocytes by augmented induction of Bax in SSI-1 (STAT-induced STAT inhibitor-1) deficient mice
Authors: Naka Tetsuji , Matsumoto Tomoshige , Narazaki Masashi , Fujimoto Minoru , Morita Yoshiaki , Ohsawa Yoshiyuki , Saito Hiroshi , Nagasawa Takashi , Uchiyama Yasuo , Kishimoto Tadamitsu
Published: July 2002

Growth, differentiation, and programmed cell death (apoptosis) are mainly controlled by cytokines. The Janus kinase–signal transducers and activators of transcription (JAK-STAT) signal pathway is an important component of cytokine signaling. We have previously shown that STAT3 induces a molecule designated as SSI-1, which inhibits STAT3 functions. To clarify the physiological roles of SSI-1
in vivo
, we generated, here, mice lacking SSI-1. These SSI-1−/− mice displayed growth retardation and died within 3 weeks after birth. Lymphocytes in the thymus and spleen of the SSI-1−/− mice exhibited accelerated apoptosis with aging, and their number was 20–25% of that in SSI-1+/+ mice at 10 days of age. However, the differentiation of lymphocytes lacking SSI-1 appeared to be normal. Among various pro- and anti-apoptotic molecules examined, an up-regulation of Bax was found in lymphocytes of the spleen and thymus of SSI-1−/− mice. These findings suggest that SSI-1 prevents apoptosis by inhibiting the expression of Bax.
Basal expression of cyclooxygenase-2 and nuclear factor–interleukin 6 are dominant and coordinately regulated by interleukin 1 in the pancreatic islet
Authors: Sorli Christopher H. , Zhang Hui-Jian , Armstrong Michael B. , Rajotte Ray V. , Maclouf Jacques , Robertson R. Paul
Published: July 2002

The enzyme cyclooxygenase (COX)-1 is constitutive whereas COX-2 is regulated in virtually all tissues. To assess whether this dogma holds true in the pancreatic islet, we examined basal and interleukin (IL)-1-regulated expression of COX-2 in HIT-T15 cells, Syrian hamster and human islets, and other Syrian hamster tissues. We found that COX-2, and not COX-1, gene expression is dominant in pancreatic islet tissue under both basal and IL-1-stimulated conditions. Control tissues (liver, spleen, and kidney) showed the expected predominance of COX-1 gene expression. Basal and IL-1-stimulated prostaglandin E
2
synthesis were blocked by a specific COX-2 inhibitor. IL-1 stimulation had a biphasic effect on COX-2 mRNA levels with an initial mild increase at 2–4 hr followed by a more dramatic decrease below basal level by 24 hr. The IL-1-induced increase in COX-2 mRNA levels was accompanied by a parallel increase in NF-κB binding to COX-2 promoter elements. The subsequent decrease in COX-2 mRNA levels was accompanied by a parallel decrease in NF-IL-6 binding activity and COX-2 promoter activity. Specific mutation of the NF-IL-6 binding motif within the COX-2 promoter reduced basal promoter activity by 50% whereas mutation of the NF-κB motif had no effect. These studies provide documentation of NF-IL-6 in the pancreatic islet and that COX-2, rather than COX-1, is dominantly expressed. They suggest coordinate regulation by IL-1 of COX-2 mRNA, NF-κB, and NF-IL-6 and raise the issue of whether intrinsically high levels of COX-2 gene expression predisposes the normal islet for microenvironmentally induced overproduction of islet prostaglandin E
2
.
Assisting developing countries toward food self-reliance
Authors: Herdt Robert W.
Published: July 2002

Per capita food availability in the developing world has increased by 20% since the early 1960s, according to the Food and Agriculture Organization, and today the world has twice as many people but 150 million fewer hungry people than in 1960. The world agricultural system has not done too bad a job over the past 35 years. It is likely that global agricultural production will continue to at least match growth in food demand over the next decade, assuming no major weather anomalies. Continued support of the Consultative Group for International Agricultural Research and programs involving U.S. universities is important to sharing knowledge about agriculture with colleagues in the developing world. This paper explores the reasons for providing agricultural development assistance, the benefits to the United States that come from doing so, and the special challenges facing the world over the next few decades.
Protein structure prediction by global optimization of a potential energy function
Authors: Liwo Adam , Lee Jooyoung , Ripoll Daniel R. , Pillardy Jaroslaw , Scheraga Harold A.
Published: July 2002

An approach based
exclusively
on finding the global minimum of an appropriate potential energy function has been used to predict the unknown structures of five globular proteins with sizes ranging from 89 to 140 amino acid residues. Comparison of the computed lowest-energy structures of two of them (HDEA and MarA) with the crystal structures, released by the Protein Data Bank
after
the predictions were made, shows that large fragments (61 residues) of both proteins were predicted with rms deviations of 4.2 and 6.0 Å for the C
α
atoms, for HDEA and MarA, respectively. This represents 80% and 53% of the observed structures of HDEA and MarA, respectively. Similar rms deviations were obtained for ∼60-residue fragments of the other three proteins. These results constitute an important step toward the prediction of protein structure based solely on
global optimization of a potential energy function
for a given amino acid sequence.
Chemistry for the analysis of protein–protein interactions: Rapid and efficient cross-linking triggered by long wavelength light
Authors: Fancy David A. , Kodadek Thomas
Published: July 2002

Chemical cross-linking is a potentially useful technique for probing the architecture of multiprotein complexes. However, analyses using typical bifunctional cross-linkers often suffer from poor yields, and large-scale modification of nucleophilic side chains can result in artifactual results attributable to structural destabilization. We report here the
de novo
design and development of a type of protein cross-linking reaction that uses a photogenerated oxidant to mediate rapid and efficient cross-linking of associated proteins. The process involves brief photolysis of tris-bipyridylruthenium(II) dication with visible light in the presence of the electron acceptor ammonium persulfate and the proteins of interest. Very high yields of cross-linked products can be obtained with irradiation times of <1 second. This chemistry obviates many of the problems associated with standard cross-linking reagents.
The α-helix folds on the millisecond time scale
Authors: Clarke David T. , Doig Andrew J. , Stapley Benjamin J. , Jones Gareth R.
Published: July 2002

It has long been believed that nucleation of the α-helix is a very fast reaction, occurring in around 10
−7
s. We show here that helix nucleation, in fact, takes place on the millisecond time scale. The rate of α-helix nucleation in two polyalanine-based peptides and in lysine and glutamic acid homopolymers was measured directly by stopped-flow deep UV CD with synchrotron radiation as the light source. Synchrotron radiation CD gives far superior signal to noise than a conventional instrument. The 16-aa AK peptide folds with first-order kinetics and a rate constant of 15 s
−1
at 0°C. The rate-determining step is presumably the initiation of a new helix, which occurs at least 10
5
times slower than expected. Helix folding occurs in at least two steps on the millisecond time scale for the longer peptides, with a transient overshoot of helix content significantly greater than at equilibrium, similar to that seen in the folding of several proteins. We suggest that the overshoot is caused by the formation of a single long helix followed by its breakage into the two or more helices present at equilibrium.
Neurosecretory control of aging in
Caenorhabditis
elegans
Authors: Ailion Michael , Inoue Takao , Weaver Carole I. , Holdcraft Robert W. , Thomas James H.
Published: July 2002

In the nematode
Caenorhabditis elegans
, an insulin receptor signaling pathway regulates adult life span and developmental arrest at the dauer larval stage. Here we show that the
unc-64
and
unc-31
genes also function in this pathway. These two genes are involved in mediating Ca
2+
-regulated secretion. Mutations in
unc-64
and
unc-31
increase adult life span and cause constitutive dauer formation. Both phenotypes are suppressed by mutations in
daf-16
, which also suppresses other mutations in this pathway. We present evidence that the site of action of
unc-64
is neuronal, suggesting that a neurosecretory signal regulates life span and dauer formation.
Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1
Authors: Biggs William H. , Meisenhelder Jill , Hunter Tony , Cavenee Webster K. , Arden Karen C.
Published: July 2002

Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. Here we show that the activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylated recombinant FKHR1
in vitro
at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) were resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus.
Folkecology and commons management in the Maya Lowlands
Authors: Atran Scott , Medin Douglas , Ross Norbert , Lynch Elizabeth , Coley John , Ek’ Edilberto Ucan , Vapnarsky Valentina
Published: July 2002

Three groups living off the same rainforest habitat manifest strikingly distinct behaviors, cognitions, and social relationships relative to the forest. Only the area’s last native Maya reveal systematic awareness of ecological complexity involving animals, plants, and people and practices clearly favoring forest regeneration. Spanish-speaking immigrants prove closer to native Maya in thought, action, and social networking than do immigrant Maya. There is no overriding “local,” “Indian,” or “immigrant” relationship to the environment. Results indicate that exclusive concern with rational self-interest and institutional constraints do not sufficiently account for commons behavior and that cultural patterning of cognition and access to relevant information are significant predictors. Unlike traditional accounts of relations between culture, cognition, and behavior, the models offered are not synthetic interpretations of people’s thoughts and behaviors but are emergent cultural patterns derived statistically from measurements of individual cognitions and behaviors.
CD4+T cells eliminate MHC class II-negative cancer cellsin vivoby indirect effects of IFN-γ
Authors: Mumberg Dominik , Monach Paul A. , Wanderling Sherry , Philip Mary , Toledano Alicia Y. , Schreiber Robert D. , Schreiber Hans
Published: July 2002

CD4+T cells can eliminate tumor cellsin vivoin the absence of CD8+T cells. We have CD4+T cells specific for a MHC class II-restricted, tumor-specific peptide derived from a mutant ribosomal protein expressed by the UV light-induced tumor 6132A-PRO. By using neutralizing mAb specific for murine IFN-γ and adoptive transfer of CD4+T cells into severe combined immunodeficient mice, we show that anti-IFN-γ treatment abolishes the CD4+T cell-mediated rejection of the tumor cellsin vivo. The tumor cells were MHC class II negative, and IFN-γ did not induce MHC class II expressionin vitro. Therefore, the tumor-specific antigenic peptide must be presented by host cells and not the tumor cells. Tumor cells transduced to secrete IFN-γ had a markedly reduced growth rate in severe combined immunodeficient mice, but IFN-γ did not inhibit the growth of the tumor cellsin vitro. Furthermore, tumor cells stably expressing a dominant-negative truncated form of the murine IFN-γ receptor α chain, and therefore insensitive to IFN-γ, nevertheless were rejected by the adoptively transferred CD4+T cells. Thus, host cells, and not tumor cells, seem to be the target of IFN-γ. Together, these results show that CD4+T cells can eliminate IFN-γ-insensitive, MHC class II-negative cancer cells by an indirect mechanism that depends on IFN-γ.
The role of MHC class II molecules in susceptibility to type I diabetes: Identification of peptide epitopes and characterization of the T cell repertoire
Authors: Chao Cheng-Chi , Sytwu Huey-Kang , Chen Emily Lichuan , Toma Jon , McDevitt Hugh O.
Published: July 2002

Susceptibility to type I diabetes is linked to class II MHC alleles in both mouse and man. However, the molecular mechanisms by which MHC molecules mediate disease susceptibility are unknown. To analyze how I-A alleles predispose to, or prevent, the development of type I diabetes, we have chosen, as the first step, to investigate the immune response to an important islet cell protein in diabetes-susceptible and diabetes-resistant mice. MHC class II alleles conferring susceptibility and resistance to diabetes select completely different sets of immunogenic epitopes from the β islet cell autoantigen glutamic acid decarboxylase 65. Peptide-binding studies, analysis of MHC restriction, and immunization with these peptide epitopes indicate that the two amino acid substitutions within the I-Aβchain that distinguish a diabetes-susceptibility from a diabetes-resistance allele are sufficient to alter peptide binding and MHC restriction and may also influence antigen presentation and the selection of the T cell repertoire. The data indicate that the molecular mechanisms for class II-mediated selection of immunodominant epitopes are complex and differ for each individual peptide epitope. Further study of the functional characteristics of the response to these epitopes should provide insight into mechanisms of MHC-mediated diabetes susceptibility.
Generation of influenza A viruses entirely from cloned cDNAs
Authors: Neumann Gabriele , Watanabe Tokiko , Ito Hiroshi , Watanabe Shinji , Goto Hideo , Gao Peng , Hughes Mark , Perez Daniel R. , Donis Ruben , Hoffmann Erich , Hobom Gerd , Kawaoka Yoshihiro
Published: July 2002

We describe a new reverse-genetics system that allows one to efficiently generate influenza A viruses entirely from cloned cDNAs. Human embryonic kidney cells (293T) were transfected with eight plasmids, each encoding a viral RNA of the A/WSN/33 (H1N1) or A/PR/8/34 (H1N1) virus, flanked by the human RNA polymerase I promoter and the mouse RNA polymerase I terminator—together with plasmids encoding viral nucleoprotein and the PB2, PB1, and PA viral polymerases. This strategy yielded >1 × 10
3
plaque-forming units (pfu) of virus per ml of supernatant at 48 hr posttransfection. The addition of plasmids expressing all of the remaining viral structural proteins led to a substantial increase in virus production, 3 × 10
4
–5 × 10
7
pfu/ml. We also used reverse genetics to generate a reassortant virus containing the PB1 gene of the A/PR/8/34 virus, with all other genes representing A/WSN/33. Additional viruses produced by this method had mutations in the PA gene or possessed a foreign epitope in the head of the neuraminidase protein. This efficient system, which does not require helper virus infection, should be useful in viral mutagenesis studies and in the production of vaccines and gene therapy vectors.
Expression of the
p48
xeroderma pigmentosum gene is p53-dependent and is involved in global genomic repair
Authors: Hwang Byung Joon , Ford James M. , Hanawalt Philip C. , Chu Gilbert
Published: July 2002

In human cells, efficient global genomic repair of DNA damage induced by ultraviolet radiation requires the p53 tumor suppressor, but the mechanism has been unclear. The
p48
gene is required for expression of an ultraviolet radiation-damaged DNA binding activity and is disrupted by mutations in the subset of xeroderma pigmentosum group E cells that lack this activity. Here, we show that
p48
mRNA levels strongly depend on basal p53 expression and increase further after DNA damage in a p53-dependent manner. Furthermore, like p53
−/−
cells, xeroderma pigmentosum group E cells are deficient in global genomic repair. These results identify p48 as the link between p53 and the nucleotide excision repair apparatus.
IL-18 binding and inhibition of interferon γ induction by human poxvirus-encoded proteins
Authors: Xiang Yan , Moss Bernard
Published: July 2002

Molluscum contagiosum virus (MCV) is a common, human poxvirus that causes small papular skin lesions that persist for long periods without signs of inflammation. Previous studies revealed that MCV encodes a family of proteins with homology to mammalian IL-18 binding proteins. IL-18 is a proinflammatory cytokine that induces synthesis of interferon γ, activates NK cells, and is required for a T-lymphocyte helper type 1 response. We expressed and purified the proteins encoded by the MC53L and MC54L genes of MCV, as well as their human and murine homologs. All four recombinant proteins were able to bind with high affinity to human and murine IL-18 molecules and inhibited IL-18 mediated interferon γ production in a dose-dependent manner. The pirating of IL-18 binding proteins by poxviruses and their use as decoy receptors is consistent with the critical role of IL-18 in defense against virus infections and provides a mechanism for evasion of the immune system by MCV.
Profound misregulation of muscle-specific gene expression in facioscapulohumeral muscular dystrophy
Authors: Tupler Rossella , Perini Giovanni , Pellegrino Maria Antonietta , Green Michael R.
Published: July 2002

Facioscapulohumeral muscular dystrophy (FSHD) is a neuromuscular disorder characterized by an insidious onset and progressive course. The disease has a frequency of about 1 in 20,000 and is transmitted in an autosomal dominant fashion with almost complete penetrance. Deletion of an integral number of tandemly arrayed 3.3-kb repeat units (D4Z4) on chromosome 4q35 is associated with FSHD but otherwise the molecular basis of the disease and its pathophysiology remain obscure. Comparison of mRNA populations between appropriate cell types can facilitate identification of genes relevant to a particular biological or pathological process. In this report, we have compared mRNA populations of FSHD and normal muscle. Unexpectedly, the dystrophic muscle displayed profound alterations in gene expression characterized by severe underexpression or overexpression of specific mRNAs. Intriguingly, many of the deregulated mRNAs are muscle specific. Our results suggest that a global misregulation of gene expression is the underlying basis for FSHD, distinguishing it from other forms of muscular dystrophy. The experimental approach used here is applicable to any genetic disorder whose pathogenic mechanism is incompletely understood.
Molecular evidence for multiple origins of woodiness and a New World biogeographic connection of the Macaronesian Island endemicPericallis(Asteraceae: Senecioneae)
Authors: Panero Jose L. , Francisco-Ortega Javier , Jansen Robert K. , Santos-Guerra Arnoldo
Published: July 2002

The prevalence of woody species in oceanic islands has attracted the attention of evolutionary biologists for more than a century. We used a phylogeny based on sequences of the internal-transcribed spacer region of nuclear ribosomal DNA to trace the evolution of woodiness inPericallis(Asteraceae: Senecioneae), a genus endemic to the Macaronesian archipelagos of the Azores, Madeira, and Canaries. Our results show that woodiness inPericallisoriginated independently at least twice in these islands, further weakening some previous hypotheses concerning the value of this character for tracing the continental ancestry of island endemics. The same data suggest that the origin of woodiness is correlated with ecological shifts from open to species-rich habitats and that the ancestor ofPericalliswas an herbaceous species adapted to marginal habitats of the laurel forest. Our results also supportPericallisas closely related to New World genera of the tribe Senecioneae.
A membrane lipid imbalance plays a role in the phenotypic expression of cystic fibrosis in
cftr
−/−
mice
Authors: Freedman Steven D. , Katz Mark H. , Parker Eliza M. , Laposata Michael , Urman Mark Y. , Alvarez Juan G.
Published: July 2002

A deficiency in essential fatty acid metabolism has been reported
in plasma from patients with cystic fibrosis (CF). However, its
etiology and role in the expression of disease is unknown. The
objective of this study was to determine whether alterations in fatty
acid metabolism are specific to CF-regulated organs and whether they
play a role in the expression of disease. A membrane lipid imbalance
was found in ileum, pancreas, and lung from
cftr
−/−
mice characterized by an
increase in phospholipid-bound arachidonic acid and a decrease in
phospholipid-bound docosahexaenoic acid (DHA). This lipid imbalance was
observed in organs pathologically affected by CF including lung,
pancreas, and ileum and was not secondary to impaired intestinal
absorption or hepatic biosynthesis of DHA. As proof of concept, oral
administration of DHA to
cftr
−/−
mice
corrected this lipid imbalance and reversed the observed pathological
manifestations. These results strongly suggest that certain phenotypic
manifestations of CF may result from remediable alterations in
phospholipid-bound arachidonic acid and DHA levels.
Conserved characteristics of heterochromatin-forming DNA at the 15q11-q13 imprinting center
Authors: Greally John M. , Gray Todd A. , Gabriel James M. , Song Li qun , Zemel Sharon , Nicholls Robert D.
Published: July 2002

Nuclear matrix binding assays (NMBAs) define certain DNA sequences
as matrix attachment regions (MARs), which often have cis-acting
epigenetic regulatory functions. We used NMBAs to analyze the
functionally important 15q11-q13 imprinting center (IC). We find that
the IC is composed of an unusually high density of MARs, located in
close proximity to the germ line elements that are proposed to direct
imprint switching in this region. Moreover, we find that the
organization of MARs is the same at the homologous mouse locus,
despite extensive divergence of DNA sequence. MARs of this size are not
usually associated with genes but rather with heterochromatin-forming
areas of the genome. In contrast, the 15q11-q13 region contains
multiple transcribed genes and is unusual for being subject to genomic
imprinting, causing the maternal chromosome to be more
transcriptionally silent, methylated, and late replicating than the
paternal chromosome. We suggest that the extensive MAR sequences at the
IC are organized as heterochromatin during oogenesis, an organization
disrupted during spermatogenesis. Consistent with this model,
multicolor fluorescence
in situ
hybridization to halo
nuclei demonstrates a strong matrix association of the maternal IC,
whereas the paternal IC is more decondensed, extending into the nuclear
halo. This model also provides a mechanism for spreading of the
imprinting signal, because heterochromatin at the IC on the maternal
chromosome may exert a suppressive position effect in cis. We propose
that the germ line elements at the 15q11-q13 IC mediate their effects
through the candidate heterochromatin-forming DNA identified in this
study.
A human cell-surface receptor for xenotropic and polytropic murine leukemia viruses: Possible role in G protein-coupled signal transduction
Authors: Battini Jean-Luc , Rasko John E. J. , Miller A. Dusty
Published: July 2002

Although present in many copies in the mouse genome, xenotropic
murine leukemia viruses cannot infect cells from laboratory mice
because of the lack of a functional cell surface receptor required for
virus entry. In contrast, cells from many nonmurine species, including
human cells, are fully permissive. Using an expression library
approach, we isolated a cDNA from HeLa cell RNA that conferred
susceptibility to xenotropic envelope protein binding and virus
infection when expressed in nonpermissive cells. The deduced product is
a 696-aa multiple-membrane spanning molecule, is widely expressed in
human tissues, and shares homology with nematode, fly, and plant
proteins of unknown function as well as with the yeast SYG1 protein,
which has been shown to interact with a G protein. This molecule also
acts as a receptor for polytropic murine leukemia viruses, consistent
with observed interference between xenotropic and polytropic viruses in
some cell types. This xenotropic and polytropic retrovirus receptor
(XPR1) is the fourth identified molecule having multiple membrane
spanning domains among mammalian type C oncoretrovirus receptors and
may play a role in G protein-coupled signal transduction, as do the
chemokine receptors required for HIV entry.
Regulation of endogenous E2F1 stability by the retinoblastoma family proteins
Authors: Martelli Fabio , Livingston David M.
Published: July 2002

Certain E2F transcription factor species play a pivotal role in regulating cell-cycle progression. The activity of E2F1, a protein with neoplastic transforming activity when unregulated, is tightly controlled at the transcriptional level during G
0
exit. In addition, during this interval, the stability of endogenous E2F1 protein increased markedly. E2F1 stability also was dynamically regulated during myogenic differentiation and in response to gamma irradiation. One or more retinoblastoma family proteins likely participate in the stability process, because simian virus 40 T antigen disrupted E2F1 stability regulation during G
1
exit in a manner dependent on its ability to bind to pocket proteins. Thus, endogenous E2F1 function is regulated by both transcriptional and posttranscriptional control mechanisms.
Absence of tumor necrosis factor rescues RelA-deficient mice from embryonic lethality
Authors: Doi Takahiro S. , Marino Michael W. , Takahashi Toshitada , Yoshida Toshimichi , Sakakura Teruyo , Old Lloyd J. , Obata Yuichi
Published: July 2002

Mice lacking the RelA (p65) subunit of NF-κB die between days 14 and 15 of embryogenesis because of massive liver destruction. Fibroblasts and macrophages isolated fromrelA−/− embryos were found to be highly sensitive to tumor necrosis factor (TNF) cytotoxicity, raising the possibility that endogenous TNF is the cause of liver cell apoptosis. To test this idea, we generated mice lacking both TNF and RelA. Embryogenesis proceeds normally in such mice, and TNF/RelA double-deficient mice are viable and have normal livers. Thus, the RelA-mediated antiapoptotic signal that protects normal cells from TNF injuryin vitrocan be shown to be operativein vivo.
Insulin-like growth factor 1 is required for G
2
progression in the estradiol-induced mitotic cycle
Authors: Adesanya O. O. , Zhou J. , Samathanam C. , Powell-Braxton L. , Bondy C. A.
Published: July 2002

Insulin-like growth factor 1 (IGF1) has been proposed as a “G
1
-progression factor” and as a mediator of estradiol’s (E2) mitogenic effects on the uterus. To test these hypotheses, we compared E2’s mitogenic effects on the uteri of
Igf1
-targeted gene deletion (null) and wild-type littermate mice. The proportion of uterine cells involved in the cell cycle and G
1
- and S-phase kinetics were not significantly different in wild-type and
Igf1
-null mice. However, the appearance of E2-induced mitotic figures and cell number increases were profoundly retarded in
Igf1
-null uterine tissue. There was a significant increase in nuclear DNA concentration in
Igf1
-null cells, consistent with a G
2
arrest. Interestingly, apoptotic cells were also significantly reduced in abundance, and the normal massive apoptotic response to E2 withdrawal was absent in the
Igf1
-null uterus. These data show that Igf1 is an essential mediator of E2’s mitogenic effects, with a critical role not in G
1
progression but in G
2
progression.
Rings and filaments of β protein from bacteriophage λ suggest a superfamily of recombination proteins
Authors: Passy Sophia I. , Yu Xiong , Li Zhufang , Radding Charles M. , Egelman Edward H.
Published: July 2002

The β protein of bacteriophage λ acts in homologous genetic recombination by catalyzing the annealing of complementary single-stranded DNA produced by the λ exonuclease. It has been shown that the β protein binds to the products of the annealing reaction more tightly than to the initial substrates. We find that β protein exists in three structural states. In the absence of DNA, β protein forms inactive rings with ≈12 subunits. The active form of the β protein in the presence of oligonucleotides or single-stranded DNA is a ring, composed of ≈15–18 subunits. The double-stranded products of the annealing reaction catalyzed by the rings are bound by β protein in a left-handed helical structure, which protects the products from nucleolytic degradation. These observations suggest structural homology for a family of proteins, including the phage P22 erf, the bacterial RecT, and the eukaryotic Rad52 proteins, all of which are involved in homologous recombination.
NMR solution structure of the human prion protein
Authors: Zahn Ralph , Liu Aizhuo , Lührs Thorsten , Riek Roland , von Schroetter Christine , López García Francisco , Billeter Martin , Calzolai Luigi , Wider Gerhard , Wüthrich Kurt
Published: July 2002

The NMR structures of the recombinant human prion protein, hPrP(23–230), and two C-terminal fragments, hPrP(90–230) and hPrP(121–230), include a globular domain extending from residues 125–228, for which a detailed structure was obtained, and an N-terminal flexibly disordered “tail.” The globular domain contains three α-helices comprising the residues 144–154, 173–194, and 200–228 and a short anti-parallel β-sheet comprising the residues 128–131 and 161–164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167–171, the residues 187–194 at the end of helix 2, and the residues 219–228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187–193 in helix 2 and 219–226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23–230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167–171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.
Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA
Authors: Herrick John , Michalet Xavier , Conti Chiara , Schurra Catherine , Bensimon Aaron
Published: July 2002

An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.
A mammalian germ cell-specific RNA-binding protein interacts with ubiquitously expressed proteins involved in splice site selection
Authors: Elliott David J. , Bourgeois Cyril F. , Klink Albrecht , Stévenin James , Cooke Howard J.
Published: July 2002

RNA-binding motif
(
RBM
) genes are found on all mammalian Y chromosomes and are implicated in spermatogenesis. Within human germ cells, RBM protein shows a similar nuclear distribution to components of the pre-mRNA splicing machinery. To address the function of RBM, we have used protein–protein interaction assays to test for possible physical interactions between these proteins. We find that RBM protein directly interacts with members of the SR family of splicing factors and, in addition, strongly interacts with itself. We have mapped the protein domains responsible for mediating these interactions and expressed the mouse RBM interaction region as a bacterial fusion protein. This fusion protein can pull-down several functionally active SR protein species from cell extracts. Depletion and add-back experiments indicate that these SR proteins are the only splicing factors bound by RBM which are required for the splicing of a panel of pre-mRNAs. Our results suggest that RBM protein is an evolutionarily conserved mammalian splicing regulator which operates as a germ cell-specific cofactor for more ubiquitously expressed pre-mRNA splicing activators.
Thiol–disulfide exchange is involved in the catalytic mechanism of peptide methionine sulfoxide reductase
Authors: Lowther W. Todd , Brot Nathan , Weissbach Herbert , Honek John F. , Matthews Brian W.
Published: July 2002

Peptide methionine sulfoxide reductase (MsrA; EC
1.8.4.6
) reverses the inactivation of many proteins due to the oxidation of critical methionine residues by reducing methionine sulfoxide, Met(O), to methionine. MsrA activity is independent of bound metal and cofactors but does require reducing equivalents from either DTT or a thioredoxin-regenerating system. In an effort to understand these observations, the four cysteine residues of bovine MsrA were mutated to serine in a series of permutations. An analysis of the enzymatic activity of the variants and their free sulfhydryl states by mass spectrometry revealed that thiol–disulfide exchange occurs during catalysis. In particular, the strictly conserved Cys-72 was found to be essential for activity and could form disulfide bonds, only upon incubation with substrate, with either Cys-218 or Cys-227, located at the C terminus. The significantly decreased activity of the Cys-218 and Cys-227 variants in the presence of thioredoxin suggested that these residues shuttle reducing equivalents from thioredoxin to the active site. A reaction mechanism based on the known reactivities of thiols with sulfoxides and the available data for MsrA was formulated. In this scheme, Cys-72 acts as a nucleophile and attacks the sulfur atom of the sulfoxide moiety, leading to the formation of a covalent, tetracoordinate intermediate. Collapse of the intermediate is facilitated by proton transfer and the concomitant attack of Cys-218 on Cys-72, leading to the formation of a disulfide bond. The active site is returned to the reduced state for another round of catalysis by a series of thiol—disulfide exchange reactions via Cys-227, DTT, or thioredoxin.
Cyclooxygenase-2 mediates the cardioprotective effects of the late phase of ischemic preconditioning in conscious rabbits
Authors: Shinmura Ken , Tang Xian-Liang , Wang Yang , Xuan Yu-Ting , Liu Si-Qi , Takano Hitoshi , Bhatnagar Aruni , Bolli Roberto
Published: July 2002

We examined the role of cyclooxygenase-2 (COX-2) in the late phase of ischemic preconditioning (PC). A total of 176 conscious rabbits were used. Ischemic PC (six cycles of 4-min coronary occlusions/4-min reperfusions) resulted in a rapid increase in myocardial COX-2 mRNA levels (+231 ± 64% at 1 h; RNase protection assay) followed 24 h later by an increase in COX-2 protein expression (+216 ± 79%; Western blotting) and in the myocardial content of prostaglandin (PG)E
2
and 6-keto-PGF
1α
(+250 ± 85% and +259 ± 107%, respectively; enzyme immunoassay). Administration of two unrelated COX-2 selective inhibitors (NS-398 and celecoxib) 24 h after ischemic PC abolished the ischemic PC-induced increase in tissue levels of PGE
2
and 6-keto-PGF
1α
. The same doses of NS-398 and celecoxib, given 24 h after ischemic PC, completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that COX-2 activity is necessary for this phenomenon to occur. Neither NS-398 nor celecoxib lowered PGE
2
or 6-keto-PGF
1α
levels in the nonischemic region of preconditioned rabbits, indicating that constitutive COX-1 activity was unaffected. Taken together, these results demonstrate that, in conscious rabbits, up-regulation of COX-2 plays an essential role in the cardioprotection afforded by the late phase of ischemic PC. Therefore, this study identifies COX-2 as a cardioprotective protein. The analysis of arachidonic acid metabolites strongly points to PGE
2
and/or PGI
2
as the likely effectors of COX-2-dependent protection. The recognition that COX-2 mediates the antistunning and antiinfarct effects of late PC impels a reassessment of current views regarding this enzyme, which is generally regarded as detrimental.