bluuebunny/crossref_metadata_2025 huggingface dataset:
Recognition of pneumolysin by Toll-like receptor 4 confers resistance to pneumococcal infection
Authors: Malley Richard , Henneke Philipp , Morse Sarah C. , Cieslewicz Michael J. , Lipsitch Marc , Thompson Claudette M. , Kurt-Jones Evelyn , Paton James C. , Wessels Michael R. , Golenbock Douglas T.
Published: February 2003

Streptococcus pneumoniaeis one of the leading causes of invasive bacterial disease worldwide. Fragments of the cell wall and the cytolytic toxin pneumolysin have been shown to contribute substantially to inflammatory damage, although the interactions between pneumococcal components and host-cell structures have not been elucidated completely. Results of a previous study indicated that cell-wall components of pneumococci are recognized by Toll-like receptor (TLR)2 but suggested that pneumolysin induces inflammatory events independently of this receptor. In this study we tested the hypothesis that pneumolysin interacts with surface proteins of the TLR family other than TLR2. We found that pneumolysin stimulates tumor necrosis factor-α and IL-6 release in wild-type macrophages but not in macrophages from mice with a targeted deletion of the cytoplasmic TLR-adapter molecule myeloid differentiation factor 88, suggesting the involvement of the TLRs in pneumolysin recognition. Purified pneumolysin synergistically activated macrophage responses together with preparations of pneumococcal cell walls or staphylococcal peptidoglycan, which are known to activate TLR2. Furthermore, when compared with wild-type macrophages, macrophages from mice that carry a spontaneous mutation in TLR4 (P712H) were hyporesponsive to both pneumolysin alone and the combination of pneumolysin with pneumococcal cell walls. Finally, these TLR4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with pneumolysin-positive pneumococci than were control mice. We conclude that the interaction of pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus.
Base pairing among three cis-acting sequences contributes to template switching during hepadnavirus reverse transcription
Authors: Liu Ning , Tian Ru , Loeb Daniel D.
Published: February 2003

Synthesis of the relaxed-circular (RC) DNA genome of hepadnaviruses requires two template switches during plus-strand DNA synthesis: primer translocation and circularization. Although primer translocation and circularization use different donor and acceptor sequences, and are distinct temporally, they share the common theme of switching from one end of the minus-strand template to the other end. Studies of duck hepatitis B virus have indicated that, in addition to the donor and acceptor sequences, three other cis-acting sequences, named 3E, M, and 5E, are required for the synthesis of RC DNA by contributing to primer translocation and circularization. The mechanism by which 3E, M, and 5E act was not known. We present evidence that these sequences function by base pairing with each other within the minus-strand template. 3E base-pairs with one portion of M (M3) and 5E base-pairs with an adjacent portion of M (M5). We found that disrupting base pairing between 3E and M3 and between 5E and M5 inhibited primer translocation and circularization. More importantly, restoring base pairing with mutant sequences restored the production of RC DNA. These results are consistent with the model that, within duck hepatitis B virus capsids, the ends of the minus-strand template are juxtaposed via base pairing to facilitate the two template switches during plus-strand DNA synthesis.
Epitope and isotype specificities of antibodies to β-amyloid peptide for protection against Alzheimer's disease-like neuropathology
Authors: Bard Frédérique , Barbour Robin , Cannon Catherine , Carretto Robert , Fox Michael , Games Dora , Guido Teresa , Hoenow Kathleen , Hu Kang , Johnson-Wood Kelly , Khan Karen , Kholodenko Dora , Lee Celeste , Lee Mike , Motter Ruth , Nguyen Minh , Reed Amanda , Schenk Dale , Tang Pearl , Vasquez Nicki , Seubert Peter , Yednock Ted
Published: February 2003

Transgenic PDAPP mice, which express a disease-linked isoform of the human amyloid precursor protein, exhibit CNS pathology that is similar to Alzheimer's disease. In an age-dependent fashion, the mice develop plaques containing β-amyloid peptide (Aβ) and exhibit neuronal dystrophy and synaptic loss. It has been shown in previous studies that pathology can be prevented and even reversed by immunization of the mice with the Aβ peptide. Similar protection could be achieved by passive administration of some but not all monoclonal antibodies against Aβ. In the current studies we sought to define the optimal antibody response for reducing neuropathology. Immune sera with reactivity against different Aβ epitopes and monoclonal antibodies with different isotypes were examined for efficacy both
ex vivo
and
in vivo
. The studies showed that: (
i
) of the purified or elicited antibodies tested, only antibodies against the N-terminal regions of Aβ were able to invoke plaque clearance; (
ii
) plaque binding correlated with a clearance response and neuronal protection, whereas the ability of antibodies to capture soluble Aβ was not necessarily correlated with efficacy; (
iii
) the isotype of the antibody dramatically influenced the degree of plaque clearance and neuronal protection; (
iv
) high affinity of the antibody for Fc receptors on microglial cells seemed more important than high affinity for Aβ itself; and (
v
) complement activation was not required for plaque clearance. These results indicate that antibody Fc-mediated plaque clearance is a highly efficient and effective process for protection against neuropathology in an animal model of Alzheimer's disease.
Long-term regional suppression of pink bollworm byBacillus thuringiensiscotton
Authors: Carrière Yves , Ellers-Kirk Christa , Sisterson Mark , Antilla Larry , Whitlow Mike , Dennehy Timothy J. , Tabashnik Bruce E.
Published: February 2003

Despite the potentially profound impact of genetically modified crops on agriculture and the environment, we know little about their long-term effects. Transgenic crops that produce toxins fromBacillus thuringiensis(Bt) to control insects are grown widely, but rapid evolution of resistance by pests could nullify their benefits. Here, we present theoretical analyses showing that long-term suppression of pest populations is governed by interactions among reproductive rate, dispersal propensity, and regional abundance of a Bt crop. Supporting this theory, a 10-year study in 15 regions across Arizona shows that Bt cotton suppressed a major pest, pink bollworm (Pectinophora gossypiella), independent of demographic effects of weather and variation among regions. Pink bollworm population density declined only in regions where Bt cotton was abundant. Such long-term suppression has not been observed with insecticide sprays, showing that transgenic crops open new avenues for pest control. The debate about putative benefits of Bt crops has focused primarily on short-term decreases in insecticide use. The present findings suggest that long-term regional pest suppression after deployment of Bt crops may also contribute to reducing the need for insecticide sprays.
Dynamic recruitment of dynamin for final mitochondrial severance in a primitive red alga
Authors: Nishida Keiji , Takahara Manabu , Miyagishima Shin-ya , Kuroiwa Haruko , Matsuzaki Motomichi , Kuroiwa Tsuneyoshi
Published: February 2003

Dynamins are a eukaryote-specific family of GTPases. Some family members are involved in diverse and varied cellular activities. Here, we report that the primitive red alga
Cyanidioschyzon merolae
retains only one dynamin homolog, CmDnm1, belonging to the mitochondrial division subfamily. Previously, the bacterial cell division protein, FtsZ, was shown to localize at the mitochondrial division site in the alga. We showed that FtsZ and dynamin coexist as mitochondrial division-associated proteins that act during different phases of division. CmDnm1 was recruited from 10–20 cytoplasmic patches (dynamin patches) to the midpoint of the constricted mitochondrion-dividing ring (MD ring), which was observed as an electron-dense structure on the cytoplasmic side. CmDnm1 is probably not required for early constriction; it forms a ring or spiral when the outer mitochondrial membrane is finally severed, whereas the FtsZ and MD rings are formed before constriction. It is thought that the FtsZ, MD, and dynamin rings are involved in scaffolding, constriction, and final separation, respectively. In eukaryotes, mitochondrial severance is probably the most conserved role for the dynamin family.
Gene expression and viral prodution in latently infected, resting CD4
+
T cells in viremic versus aviremic HIV-infected individuals
Authors: Chun Tae-Wook , Justement J. Shawn , Lempicki Richard A. , Yang Jun , Dennis Glynn , Hallahan Claire W. , Sanford Christina , Pandya Punita , Liu Shuying , McLaughlin Mary , Ehler Linda A. , Moir Susan , Fauci Anthony S.
Published: February 2003

The presence of HIV-1 in latently infected, resting CD4
+
T cells has been clearly demonstrated in infected individuals; however, the extent of viral expression and the underlying mechanisms of the persistence of HIV-1 in this viral reservoir have not been fully delineated. Here, we show that resting CD4
+
T cells from the majority of viremic patients are capable of producing cell-free HIV-1 spontaneously
ex vivo
. The levels of HIV-1 released by resting CD4
+
T cells were not significantly reduced in the presence of inhibitors of cellular proliferation and viral replication. However, resting CD4
+
T cells from the majority of aviremic patients failed to produce virions, despite levels of HIV-1 proviral DNA and cell-associated HIV-1 RNA comparable to viremic patients. The DNA microarray analysis demonstrated that a number of genes involving transcription regulation, RNA processing and modification, and protein trafficking and vesicle transport were significantly upregulated in resting CD4
+
T cells of viremic patients compared to those of aviremic patients. These results suggest that active viral replication has a significant impact on the physiologic state of resting CD4
+
T cells in infected viremic patients and, in turn, allows release of HIV-1 without exogenous activation stimuli. In addition, given that no quantifiable virions were produced by the latent viral reservoir in the majority of aviremic patients despite the presence of cell-associated HIV-1 RNA, evidence for transcription of HIV-1 RNA in resting CD4
+
T cells of aviremic patients should not necessarily be taken as direct evidence for ongoing viral replication during effective therapy.
Single-cell nonphotochemical hole burning of ovarian surface epithelial carcinoma and normal cells
Authors: Walsh R. J. , Matsuzaki S. , Reinot T. , Hayes J. M. , Kalli K. R. , Hartmann L. C. , Small G. J.
Published: February 2003

Persistent spectral nonphotochemical hole-burning (NPHB) spectroscopy has recently been applied to dye molecules in cells. The sensitivity of NPHB to the nanoenvironment of the probe is well established. It has been shown that NPHB applied to bulk suspensions of cultured human cells can distinguish between normal and cancer cells. Thus, NPHB has potential as a diagnostic cancer tool. For this reason, the methodology is referred to as hole-burning imaging, by analogy with MRI. The optical dephasing time (
T
2
) of the dye in hole-burning image replaces the proton
T
1
relaxation time in MRI. In addition to the
T
2
mode of operation, there are four other modes including measurement of the spectral hole growth kinetics (HGK). Reported here is that the selectivity and sensitivity of NPHB operating in the HGK mode allow for distinction between normal and carcinoma cells at the single-cell level. The ovarian cell lines are ovarian surface epithelial cells with temperature-sensitive large T antigens (analogously normal) and ovarian surface epithelial carcinoma (OV167) cells. The mitochondrial specific dye used was rhodamine 800 (Molecular Probes). This carbocationic dye is highly specific for the outer and inner membranes of mitochondria. In line with the results for bulk suspensions of the two cell lines, the hole-burning efficiency for OV167 cells was found to be significantly higher than that for normal cells. Theoretical analysis of the HGK data leads to the conclusion that the degree of structural heterogeneity for the probe–host configurations in OV167 cells is lower than in the normal cells. Possible reasons for this are given.
The soluble epoxide hydrolase encoded by EPXH2 is a bifunctional enzyme with novel lipid phosphate phosphatase activity
Authors: Newman John W. , Morisseau Christophe , Harris Todd R. , Hammock Bruce D.
Published: February 2003

The gene
EPXH2
encodes for the soluble epoxide hydrolase (sEH), an enzyme involved in the regulation of cardiovascular and renal physiology containing two distinct domains connected via a proline-rich linker. The C-terminal domain containing the EH catalytic activity has been well studied. In contrast, a function for the N-terminal domain, which has high homology to the haloacid dehalogenase family of phosphatases, has not been definitively reported. In this study we describe the N-terminal domain as a functional phosphatase unaffected by a number of classic phosphatase inhibitors. Assuming a functional association between these catalytic activities, dihydroxy lipid phosphates were rationalized as potential endogenous substrates. A series of phosphorylated hydroxy lipids were therefore synthesized and found to be excellent substrates for the human sEH. The best substrate tested was the monophosphate of dihydroxy stearic acid (
threo
-9/10-phosphonoxy-hydroxy-octadecanoic acid) with
K
m
= 21 ± 0.3 μM,
V
Max
= 338 ± 12 nmol⋅min
−1
⋅mg
−1
, and
k
cat
= 0.35 ± 0.01 s
−1
. Therefore dihydroxy lipid phosphates are possible candidates for the endogenous substrates of the sEH N-terminal domain, which would represent a novel branch of fatty acid metabolism with potential signaling functions.
Regulatory gene networks and the properties of the developmental process
Authors: Davidson Eric H. , McClay David R. , Hood Leroy
Published: February 2003

Genomic instructions for development are encoded in arrays of regulatory DNA. These specify large networks of interactions among genes producing transcription factors and signaling components. The architecture of such networks both explains and predicts developmental phenomenology. Although network analysis is yet in its early stages, some fundamental commonalities are already emerging. Two such are the use of multigenic feedback loops to ensure the progressivity of developmental regulatory states and the prevalence of repressive regulatory interactions in spatial control processes. Gene regulatory networks make it possible to explain the process of development in causal terms and eventually will enable the redesign of developmental regulatory circuitry to achieve different outcomes.
Pretarget radiotherapy with an anti-CD25 antibody-streptavidin fusion protein was effective in therapy of leukemia/lymphoma xenografts
Authors: Zhang Meili , Zhang Zhuo , Garmestani Kayhan , Schultz Jody , Axworthy Donald B. , Goldman Carolyn K. , Brechbiel Martin W. , Carrasquillo Jorge A. , Waldmann Thomas A.
Published: February 2003

Although radioimmunotherapy with radiolabeled intact monoclonal antibodies has demonstrated efficacy in the treatment of lymphoma, it provides low tumor-to-normal-tissue radionuclide target ratios and unwanted prolonged radiation exposure to the bone marrow. To overcome these obstacles, the administration of the radionuclide was separated from that of the antibody by using an anti-IL-2 receptor α antibody single chain Fv-streptavidin fusion protein, followed by radiolabeled biotin to treat lymphoma or leukemia xenografted mice. This Pretarget approach provided extremely rapid and effective tumor targeting, permitting the use of short-lived α-emitting radionuclides. With the β-emitter
90
Y, all of the 10 lymphoma-xenografted mice were cured. With the α-emitter
213
Bi, significant efficacy was obtained in treating leukemic mice, and, furthermore, when combined with immunotherapy, 7 of 10 leukemic mice were cured. Thus, Pretarget radioimmunotherapy is very promising and could represent the next generation in the treatment of lymphoma and leukemia.
Regulation of photoperiodic flowering byArabidopsisphotoreceptors
Authors: Mockler Todd , Yang Hongyun , Yu XuHong , Parikh Dhavan , Cheng Ying-chia , Dolan Sarah , Lin Chentao
Published: February 2003

Photoperiodism is a day-length-dependent seasonal change of physiological or developmental activities that is widely found in plants and animals. Photoperiodic flowering in plants is regulated by photosensory receptors including the red/far-red light-receptor phytochromes and the blue/UV-A light-receptor cryptochromes. However, the molecular mechanisms underlying the specific roles of individual photoreceptors have remained poorly understood. Here, we report a study of the day-length-dependent response of cryptochrome 2 (cry2) and phytochrome A (phyA) and their role as day-length sensors inArabidopsis. The protein abundance of cry2 and phyA showed a diurnal rhythm in plants grown in short-day but not in plants grown in long-day. The short-day-specific diurnal rhythm of cry2 is determined primarily by blue light-dependent cry2 turnover. Consistent with a proposition that cry2 and phyA are the major day-length sensors inArabidopsis, we show that phyA mediates far-red light promotion of flowering with modes of action similar to that of cry2. Based on these results and a finding that the photoperiodic responsiveness of plants depends on light quality, a model is proposed to explain how individual phytochromes and cryptochromes work together to confer photoperiodic responsiveness inArabidopsis.
Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens
Authors: Wentworth Paul , Wentworth Anita D. , Zhu Xueyong , Wilson Ian A. , Janda Kim D. , Eschenmoser Albert , Lerner Richard A.
Published: February 2003

Recent work in our laboratory showed that products formed by the antibody-catalyzed water-oxidation pathway can kill bacteria. Dihydrogen peroxide, the end product of this pathway, was found to be necessary, but not sufficient, for the observed efficiency of bacterial killing. The search for further bactericidal agents that might be formed along the pathway led to the recognition of an oxidant that, in its interaction with chemical probes, showed the chemical signature of ozone. Here we report that the antibody-catalyzed water-oxidation process is capable of regioselectively converting antibody-bound benzoic acid into
para
-hydroxy benzoic acid as well as regioselectively hydroxylating the 4-position of the phenyl ring of a single tryptophan residue located in the antibody molecule. We view the occurrence of these highly selective chemical reactions as evidence for the formation of a short-lived hydroxylating radical species within the antibody molecule. In line with our previously presented hypothesis according to which the singlet-oxygen (
1
O
*
2
) induced antibody-catalyzed water-oxidation pathways proceeds via the formation of dihydrogen trioxide (H
2
O
3
), we now consider the possibility that the hydroxylating species might be the hydrotrioxy radical HO

\documentclass[12pt]{minimal}
\usepackage{amsmath}
\usepackage{wasysym}
\usepackage{amsfonts}
\usepackage{amssymb}
\usepackage{amsbsy}
\usepackage{mathrsfs}
\setlength{\oddsidemargin}{-69pt}
\begin{document}
\begin{equation*}{\mathrm{_{_{3}}^{{\bullet}}}}\end{equation*}\end{document}

, and we point to the remarkable potential of this either H
2
O
3
- or O
3
-derivable species to act as a masked hydroxyl radical (HO
•
) in a biological environment.
MyoD is functionally linked to the silencing of a muscle-specific regulatory gene prior to skeletal myogenesis
Authors: Mal Asoke , Harter Marian L.
Published: February 2003

Most of the genes that are central to the process of skeletal muscle differentiation remain in a transcriptionally silent or “off” state until muscle cells (myoblasts) are induced to differentiate. Although the mechanisms that contribute to this phenomenon are still unclear, it is likely that histone deacetylases (HDACs), which play an important role in the repression of genes, are principally involved. Recent studies indicate that the initiator of the myogenic program, namely MyoD, can associate with the deacetylase HDAC1
in vivo
, and because HDACs are usually recruited to promoters by specific proteins, we considered the possibility that these two proteins might be acting together at the promoters of muscle-specific genes to repress their transcription in myoblasts. In this work, we show by chromatin immunoprecipitation (ChIP) assays that MyoD and HDAC1 are both occupying the promoter of myogenin and that this gene is in a region of repressed chromatin, as revealed by enrichment in histone H3 lysine 9 (Lys-9) methylation and the underacetylation of histones. Surprisingly, after the myoblasts are induced to differentiate, the promoter becomes absent of HDAC1, and eventually the acetyltransferase P/CAF takes it place alongside MyoD. In addition, enrichment of histone H3 acetylation (Lys-9/14) and phosphorylation of Ser-10 can now be observed at the myogenin promoter. These data strongly suggest that in addition to its widely accepted role as an activator of differentiation-specific genes, MyoD also can perform as a transcriptional repressor in proliferating myoblasts while in partnership with a HDAC.
Lysine-79 of histone H3 is hypomethylated at silenced loci in yeast and mammalian cells: A potential mechanism for position-effect variegation
Authors: Ng Huck Hui , Ciccone David N. , Morshead Katrina B. , Oettinger Marjorie A. , Struhl Kevin
Published: February 2003

Methylation of lysine-79 (K79) within the globular domain of histone H3 by Dot1 methylase is important for transcriptional silencing and for association of the Sir silencing proteins in yeast. Here, we show that the level of H3-K79 methylation is low at all Sir-dependent silenced loci but not at other transcriptionally repressed regions. Hypomethylation of H3-K79 at the telomeric and silent mating-type loci, but not the ribosomal DNA, requires the Sir proteins. Overexpression of Sir3 concomitantly extends the domain of Sir protein association and H3-K79 hypomethylation at telomeres. In mammalian cells, H3-K79 methylation is found at loci that are active for V(D)J recombination, but not at recombinationally inactive loci that are heterochromatic. These results suggest that H3-K79 methylation is an evolutionarily conserved marker of active chromatin regions, and that silencing proteins block the ability of Dot1 to methylate histone H3. Further, they suggest that Sir proteins preferentially bind chromatin with hypomethylated H3-K79 and then block H3-K79 methylation. This positive feedback loop, and the reverse loop in which H3-K79 methylation weakens Sir protein association and leads to further methylation, suggests a model for position-effect variegation.
Suberoylanilide hydroxamic acid, a histone deacetylase inhibitor, ameliorates motor deficits in a mouse model of Huntington's disease
Authors: Hockly Emma , Richon Victoria M. , Woodman Benjamin , Smith Donna L. , Zhou Xianbo , Rosa Eddie , Sathasivam Kirupa , Ghazi-Noori Shabnam , Mahal Amarbirpal , Lowden Philip A. S. , Steffan Joan S. , Marsh J. Lawrence , Thompson Leslie M. , Lewis Cathryn M. , Marks Paul A. , Bates Gillian P.
Published: February 2003

Huntington's disease (HD) is an inherited, progressive neurological disorder that is caused by a CAG/polyglutamine repeat expansion and for which there is no effective therapy. Recent evidence indicates that transcriptional dysregulation may contribute to the molecular pathogenesis of this disease. Supporting this view, administration of histone deacetylase (HDAC) inhibitors has been shown to rescue lethality and photoreceptor neurodegeneration in a
Drosophila
model of polyglutamine disease. To further explore the therapeutic potential of HDAC inhibitors, we have conducted preclinical trials with suberoylanilide hydroxamic acid (SAHA), a potent HDAC inhibitor, in the R6/2 HD mouse model. We show that SAHA crosses the blood–brain barrier and increases histone acetylation in the brain. We found that SAHA could be administered orally in drinking water when complexed with cyclodextrins. SAHA dramatically improved the motor impairment in R6/2 mice, clearly validating the pursuit of this class of compounds as HD therapeutics.
Variation in gene expression patterns in follicular lymphoma and the response to rituximab
Authors: Bohen Sean P. , Troyanskaya Olga G. , Alter Orly , Warnke Roger , Botstein David , Brown Patrick O. , Levy Ronald
Published: February 2003

Analysis of the patterns of gene expression in follicular lymphomas from 24 patients suggested that two groups of tumors might be distinguished. All patients, whose biopsies were obtained before any treatment, were treated with rituximab, a monoclonal antibody directed against the B cell antigen, CD20. Gene expression patterns in the tumors that subsequently failed to respond to rituximab appeared more similar to those of normal lymphoid tissues than to gene expression patterns of tumors from rituximab responders. These findings suggest the possibility that the response of follicular lymphoma to rituximab treatment may be predicted from the gene expression pattern of tumors.
Hypersusceptibility of cystic fibrosis mice to chronic
Pseudomonas aeruginosa
oropharyngeal colonization and lung infection
Authors: Coleman Fadie T. , Mueschenborn Simone , Meluleni Gloria , Ray Christopher , Carey Vincent J. , Vargas Sara O. , Cannon Carolyn L. , Ausubel Frederick M. , Pier Gerald B.
Published: February 2003

No transgenic cystic fibrosis (CF) mouse model developed to date mimics the major clinical phenotype found in humans with CF, chronic
Pseudomonas aeruginosa
lung infection. In a transgenic CF transmembrane conductance regulator (
cftr
) mouse colony, we found WT, heterozygous, and homozygous CF mice housed in the same cage became chronically colonized in the oropharynx with environmental
P. aeruginosa
when the bacterium was present in drinking water. Elimination of
P. aeruginosa
from drinking water resulted in clearance in most WT and CF heterozygous, but not homozygous mice. For experimental evaluation, a combination of specific animal husbandry techniques and an oral infection route showed

cftr
−/−

mice but not WT mice can be chronically colonized by
P. aeruginosa
with subsequent lung translocation, yielding a pathologic picture indicative of chronic lung infection. In some instances, mucoid isolates of
P. aeruginosa
were recovered from lungs, indicating conditions were present for conversion to mucoidy. Overexpression of human CFTR in the lungs of WT mice markedly accelerated the clearance rate of
P. aeruginosa
, demonstrating that lung levels of CFTR play an important role in defense against infection.
P. aeruginosa
mutants unable to express the surface polysaccharide alginate or the global regulator GacA were deficient in their ability to colonize the mice. CF mice made potent immune responses to
P. aeruginosa
outer membrane antigens. Overall, we found that under the proper conditions, transgenic CF mice are hypersusceptible to
P. aeruginosa
colonization and infection and can be used for evaluations of lung pathophysiology, bacterial virulence, and development of therapies aimed at treating CF lung disease.
JNK-interacting protein-1 promotes transcription of Aβ protein precursor but not Aβ precursor-like proteins, mechanistically different than Fe65
Authors: Scheinfeld Meir H. , Matsuda Shuji , D'Adamio Luciano
Published: February 2003

Processing of the amyloid β protein precursor (AβPP) by the β and γ secretases leads to the production of two small peptides, amyloid β and the AβPP intracellular domain (AID, or called elsewhere AICD). Whereas the role of amyloid β in the pathogenesis of Alzheimer's disease has been studied extensively, only recently has information begun to accumulate as to the role of AID. Functions identified for AID include its ability to trigger apoptosis and a role in regulating gene transcription, particularly in combination with the AβPP binding protein Fe65. Here, we report that AID in combination with Janus kinase interacting protein-1 (JIP-1) can activate gene expression. We demonstrate that the mechanism is different from activation in combination with Fe65 by first showing that although Fe65 enters the nucleus in the absence of full-length AβPP, JIP-1 does not. Additionally, JIP-1-induced activation is Tip60 independent, whereas a complex with AID, Fe65, and Tip60 is formed for Fe65-induced activation. Finally, and probably most interestingly, we show that although the AβPP family members APLP1 and APLP2 (for amyloid β precursor-like protein) can cause activation in combination with Fe65, APLP1 and APLP2 show little or no activation in combination with JIP-1. This activity for the AID fragment may help explain the unique functions of AβPP relative to its other family members, and changes in gene expression found in Alzheimer's disease.
Arabidopsis
LEAFY COTYLEDON1 represents a functionally specialized subunit of the CCAAT binding transcription factor
Authors: Lee Hyeseung , Fischer Robert L. , Goldberg Robert B. , Harada John J.
Published: February 2003

LEAFY COTYLEDON1 (LEC1) is a central regulator that is required for many aspects of
Arabidopsis
embryogenesis and sufficient to induce embryo development in vegetative cells when expressed ectopically. We previously showed that LEC1 encodes an HAP3 subunit of the CCAAT binding factor and that the 10
Arabidopsis
HAP3 (AHAP3) subunits can be divided into two classes, the LEC1-type and the non-LEC1-type, based on sequence similarity within their B domains. By analyzing the ability of chimeric HAP3 subunits containing different combinations of domains from LEC1 and a non-LEC1-type AHAP3 subunit to suppress genetically the
lec1
mutation, we show that the B domain of LEC1 is necessary and sufficient within the context of the protein for its activity in embryogenesis. Moreover, we identify one amino acid residue, Asp-55, specific to the LEC1-type B domain that is required for LEC1 activity in embryogenesis and sufficient to confer partial LEC1 activity to a non-LEC1-type B domain. Based on structural similarities between the HAP3 B domain and histone fold motif, we discuss how the Asp-55 residue may functionally differentiate LEC1 from the non-LEC1-type AHAP3 subunits.
Electrophile and oxidant damage of mitochondrial DNA leading to rapid evolution of homoplasmic mutations
Authors: Mambo Elizabeth , Gao Xiangqun , Cohen Yoram , Guo Zhongmin , Talalay Paul , Sidransky David
Published: February 2003

mtDNA mutations occur in a wide variety of degenerative diseases and cancer. mtDNA seems to be more susceptible to DNA damage and consequently sustains higher rates of mutation than does nuclear DNA (nDNA). Many of the somatic mtDNA mutations in human cancers are located in the displacement loop (D-loop) and in particular in a polycytidine stretch (C-tract) termed D310. The D310 region exhibits polymorphic length variation among individuals and has been described as a “hot spot” for somatic mutations in many cancer types. We used real-time quantitative PCR to analyze mtDNA integrity, damage repair, and induced mutations after exposure of human adult retinal pigment epithelial (ARPE)-19 cells to 4-nitroquinoline 1-oxide, a UV-mimetic and adduct-forming carcinogen, and
tert
-butyl hydroperoxide, an oxidant. The mtDNA-damage profile depended on the region. Thus, the tRNA coding for glycine (tRNA
G
) was the least affected region, whereas the D-loop, and especially its D310 region, were most sensitive to damage. The time course of repair of mutations of the D-loop and especially the D310 region after exposure to DNA-damaging agents was delayed when compared with other regions and gave rise to common D310 C-tract frame-shift mutations. The induced mutations in the D310 region were predominantly homoplasmic only 7 days after exposure to damage. Our results establish that the D-loop (especially its D310 region) is highly susceptible to mutations because of its vulnerability to DNA damage and inefficient repair mechanisms. Our findings may explain the high frequency of homoplasmic D310 somatic mutations in many tumor types.
The structural basis of cysteine aminoacylation of tRNA
Pro
by prolyl-tRNA synthetases
Authors: Kamtekar Satwik , Kennedy W. Dexter , Wang Jimin , Stathopoulos Constantinos , Söll Dieter , Steitz Thomas A.
Published: February 2003

Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in
Methanocaldococcus jannaschii
,
Methanothermobacter thermautotrophicus
, or
Methanopyrus kandleri
. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 Å: apo
M. jannaschii
ProRS, and
M. thermautotrophicus
ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for
M. thermautotrophicus
ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3′-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that
M. jannaschii
ProRS misacylates tRNA
Pro
with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA
Pro
and tRNA
Cys
with their cognate amino acids.
A general method for gene knockdown in mice by using lentiviral vectors expressing small interfering RNA
Authors: Tiscornia Gustavo , Singer Oded , Ikawa Masahito , Verma Inder M.
Published: February 2003

We describe the use of lentiviral vectors expressing small interfering RNAs (siRNAs) to knock down the expression of specific genes
in vitro
and
in vivo
. A lentiviral vector capable of generating siRNA specific for GFP after transduction of 293T-GFP cell lines showed no GFP fluorescence. Furthermore, no GFP-specific RNA could be detected. When eggs from GFP-positive transgenic mice were transduced with lentivirus-expressing siGFP virus, reduced fluorescence could be seen in blastocysts. More interestingly, pups from F
1
progeny, which expressed siGFP, showed considerably diminished fluorescence and decreased GFP. We propose that an approach of combining transgenesis by lentiviral vectors expressing siRNAs can be used successfully to generate a large number of mice in which the expression of a specific gene(s) can be down-regulated substantially. We believe that this approach of generating “knockdown” mice will aid in functional genomics.
Designed
FHIT
alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding
Authors: Trapasso Francesco , Krakowiak Agnieszka , Cesari Rossano , Arkles Jeffrey , Yendamuri Sai , Ishii Hideshi , Vecchione Andrea , Kuroki Tamotsu , Bieganowski Pawel , Pace Helen C. , Huebner Kay , Croce Carlo M. , Brenner Charles
Published: February 2003

The
FHIT
gene is inactivated early in the development of many human tumors, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the active-site histidine of which is not required for tumor suppression. To provide a rigorous test of the hypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding and/or hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele. The allele series covered defects as great as 100,000-fold in
k
cat
and increases as large as 30-fold in
K
M
. Nonetheless, when mutant
FHIT
genes were expressed in two human cancer cell lines containing
FHIT
deletions, reductions in apoptotic activity correlated exclusively with
K
M
. Mutants with 2- and 7-fold increases in
K
M
significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in
K
M
retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.
Decreased embryonic retinoic acid synthesis results in a DiGeorge syndrome phenotype in newborn mice
Authors: Vermot Julien , Niederreither Karen , Garnier Jean-Marie , Chambon Pierre , Dollé Pascal
Published: February 2003

Retinoic acid (RA), the active derivative of vitamin A, is involved in various developmental and homeostatic processes. To define whether certain developmental events are particularly sensitive to a decrease in embryonic RA levels, we generated mice bearing a hypomorphic allele of the RA-synthesizing enzymeRaldh2. The resulting mutant mice, which die perinatally, exhibit the features of the human DiGeorge syndrome (DGS) with heart outflow tract septation defects and anomalies of the aortic arch-derived head and neck arteries, laryngeal-tracheal cartilage defects, and thymus/parathyroid aplasia or hypoplasia. Analysis ofRaldh2hypomorph embryos reveal selective defects of the posterior (third to sixth) branchial arches, including absence or hypoplasia of the corresponding aortic arches and pharyngeal pouches, and local down-regulation of RA-target genes. Thus, a decreased level of embryonic RA (through genetic and/or nutritional causes) could represent a major modifier of the expressivity of human 22q11del-associated DiGeorge/velocardiofacial syndromes and, if severe enough, could on its own lead to the clinical features of the DiGeorge syndrome.
The effect of anandamide on prolactin secretion is modulated by estrogen
Authors: Scorticati Camila , Mohn Claudia , De Laurentiis Andrea , Vissio Paula , Fernández Solari Javier , Seilicovich Adriana , McCann Samuel M. , Rettori Valeria
Published: February 2003

Recent research has revealed that endogenous cannabinoid receptors (CB1 and CB2) react with the active ingredient of marijuana, Δ
9
-tetrahydrocannabinol. Two endogenous ligands activate these receptors. The principal one, anandamide (AEA), activates CB1. AEA and CB1 are localized to various neurons within the brain. Because Δ
9
-tetrahydrocannabinol inhibited prolactin (Prl) secretion following its intraventricular injection into male rats, we hypothesized that AEA would have a similar effect. Estrogen modifies many hormonal responses and is known to increase Prl secretion. Therefore, we hypothesized that responses to intraventricular AEA would change depending on the gonadal steroid environment. Consequently, we evaluated the effects of lateral cerebral ventricular microinjection of AEA (20 ng) into male, ovariectomized (OVX), and estrogen-primed (OVX-E) rats. AEA decreased plasma Prl in male rats, had little effect in OVX females, and increased Prl in OVX-E rats. The results were at least partially mediated by changes in dopaminergic turnover, altering the inhibitory dopaminergic control of Prl release by the anterior pituitary gland. Thus, dopamine turnover was increased in the male rats and decreased significantly in OVX and in OVX-E rats. The changes in Prl may be caused not only by altered dopamine input to the anterior pituitary gland but also by effects of AEA on other transmitters known to alter Prl release. Importantly, in OVX-E rats, the elevated Prl release and the response to AEA were blocked by the AEA antagonist, indicating that AEA is a synaptic transmitter released from neurons that decrease inhibitory control of Prl release.
An α-syntrophin-dependent pool of AQP4 in astroglial end-feet confers bidirectional water flow between blood and brain
Authors: Amiry-Moghaddam Mahmood , Otsuka Takashi , Hurn Patricia D. , Traystman Richard J. , Haug Finn-Mogens , Froehner Stanley C. , Adams Marvin E. , Neely John D. , Agre Peter , Ottersen Ole Petter , Bhardwaj Anish
Published: February 2003

The water channel AQP4 is concentrated in perivascular and subpial membrane domains of brain astrocytes. These membranes form the interface between the neuropil and extracerebral liquid spaces. AQP4 is anchored at these membranes by its carboxyl terminus to α-syntrophin, an adapter protein associated with dystrophin. To test functions of the perivascular AQP4 pool, we studied mice homozygous for targeted disruption of the gene encoding α-syntrophin (α-
Syn
−/−
). These animals show a marked loss of AQP4 from perivascular and subpial membranes but no decrease in other membrane domains, as judged by quantitative immunogold electron microscopy. In the basal state, perivascular and subpial astroglial end-feet were swollen in brains of α-
Syn
−/−
mice compared to WT mice, suggesting reduced clearance of water generated by brain metabolism. When stressed by transient cerebral ischemia, brain edema was attenuated in α-
Syn
−/−
mice, indicative of reduced water influx. Surprisingly, AQP4 was strongly reduced but α-syntrophin was retained in perivascular astroglial end-feet in WT mice examined 23 h after transient cerebral ischemia. Thus α-syntrophin-dependent anchoring of AQP4 is sensitive to ischemia, and loss of AQP4 from this site may retard the dissipation of postischemic brain edema. These studies identify a specific, syntrophin-dependent AQP4 pool that is expressed at distinct membrane domains and which mediates bidirectional transport of water across the brain–blood interface. The anchoring of AQP4 to α-syntrophin may be a target for treatment of brain edema, but therapeutic manipulations of AQP4 must consider the bidirectional water flux through this molecule.
Identification of frame-shift intermediate mutant cells
Authors: Gasche Christoph , Chang Christina L. , Natarajan Loki , Goel Ajay , Rhees Jennifer , Young Dennis J. , Arnold Christian N. , Boland C. Richard
Published: February 2003

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA)⋅poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the (CA)
13
microsatellite. Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both (CA)
13
and (CA)
12
microsatellites. It is likely that M1 cells represent intermediate mutants that carry (CA)
13
⋅(GT)
12
heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 × 10
−4
) was 30 times higher than in HCT116+chr3 (1.9 × 10
−5
), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 × 10
−3
) and HCT116+chr3 (4.13 × 10
−3
), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency.
Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats
Authors: Oakes Christopher C. , Smiraglia Dominic J. , Plass Christoph , Trasler Jacquetta M. , Robaire Bernard
Published: February 2003

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.
Nanodissection and high-resolution imaging of the
Rhodopseudomonas viridis
photosynthetic core complex in native membranes by AFM
Authors: Scheuring Simon , Seguin Jérôme , Marco Sergio , Lévy Daniel , Robert Bruno , Rigaud Jean-Louis
Published: February 2003

In photosynthesis, highly organized multiprotein assemblies convert sunlight into biochemical energy with high efficiency. A challenge in structural biology is to analyze such supramolecular complexes in native membranes. Atomic force microscopy (AFM) with high lateral resolution, high signal-to-noise ratio, and the possibility to nanodissect biological samples is a unique tool to investigate multiprotein complexes at molecular resolution
in situ
. Here we present high-resolution AFM of the photosynthetic core complex in native
Rhodopseudomonas viridis
membranes. Topographs at 10-Å lateral and ≈1-Å vertical resolution reveal a single reaction center (RC) surrounded by a closed ellipsoid of 16 light-harvesting (LH1) subunits. Nanodissection of the tetraheme cytochrome (4Hcyt) subunit from the RC allows demonstration that the L and M subunits exhibit an asymmetric topography intimately associated to the LH1 subunits located at the short ellipsis axis. This architecture implies a distance distribution between the antenna and the RC compared with a centered location of the RC within a circular LH1, which may influence the energy transfer within the core complex. The LH1 subunits rearrange into a circle after removal of the RC from the core complex.