bluuebunny/crossref_metadata_2025 huggingface dataset:
Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1
Authors: Narayanan Karthikeyan , Srinivas Rampalli , Ramachandran Amsaveni , Hao Jianjun , Quinn Bruce , George Anne
Published: July 2002

Cells of the craniofacial skeleton are derived from a common
mesenchymal progenitor. The regulatory factors that control their
differentiation into various cell lineages are unknown. To investigate
the biological function of dentin matrix protein 1 (DMP1), an
extracellular matrix gene involved in calcified tissue formation,
stable transgenic cell lines and adenovirally infected cells
overexpressing DMP1 were generated. The findings in this paper
demonstrate that overexpression of DMP1 in pluripotent and
mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can
induce these cells to differentiate and form functional
odontoblast-like cells. Functional differentiation of odontoblasts
requires unique sets of genes being turned on and off in a growth- and
differentiation-specific manner. The genes studied include
transcription factors like core binding factor 1 (Cbfa1), bone
morphogenetic protein 2 (BMP2), and BMP4; early markers for
extracellular matrix deposition like alkaline phosphatase (ALP),
osteopontin, osteonectin, and osteocalcin; and late markers like DMP2
and dentin sialoprotein (DSP) that are expressed by terminally
differentiated odontoblasts and are responsible for the formation of
tissue-specific dentin matrix. However, this differentiation pathway
was limited to mesenchyme-derived cells only. Other cell lines tested
by the adenoviral expression system failed to express
odontoblast-phenotypic specific genes. An
in vitro
mineralized nodule formation assay demonstrated that overexpressed
cells could differentiate and form a mineralized matrix. Furthermore,
we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific
transcription factor) was not required for the expression of
odontoblast-specific genes, indicating the involvement of other
unidentified odontoblast-specific transcription factors or
coactivators. Cell lines that differentiate into odontoblast-like cells
are useful tools for studying the mechanism involved in the terminal
differentiation process of these postmitotic cells.
Self-assembly of a metal-ion-bound monolayer of trigonal connectors on mercury: An electrochemical Langmuir trough
Authors: Varaksa Natalia , Pospíšil Lubomír , Magnera Thomas F. , Michl Josef
Published: July 2002

The adsorption of the trigonal connector, 1,3,5-tris[10-(3-ethylthiopropyl)dimethylsilyl-1,10-dicarba-
closo
-decaboran-1-yl]benzene (1), from acetonitrile/0.1 M LiClO
4
on the surface of mercury at potentials ranging from +0.3 to −1.4 V (vs. aqueous Ag|AgCl|1 M LiCl) was examined by voltammetry, Langmuir isotherms at controlled potentials, and impedance measurements. No adsorption is observed at potentials more negative than ∼ −0.85 V. Physisorption is seen between ∼ −0.85 and 0 V. At positive potentials, adsorbate-assisted anodic dissolution of mercury occurs and an organized surface layer is formed. Although the mercury cations are reduced at −0.10 V, the surface layer remains metastable to potentials as negative as −0.85 V. Its surface areas per molecule and per redox center are compatible with a regular structure with the connectors 1 woven into a hexagonal network by RR′S→Hg

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←SRR′ or RR′S→Hg
2+
←SRR′ bridges. The structure is simulated closely by geometry optimization in the semiempirical AM1 approximation.
Protection from nitrosative stress by yeast flavohemoglobin
Authors: Liu Limin , Zeng Ming , Hausladen Alfred , Heitman Joseph , Stamler Jonathan S.
Published: July 2002

Yeast hemoglobin was discovered close to half a century ago, but its function has remained unknown. Herein, we report that this flavohemoglobin protects
Saccharomyces cerevisiae
from nitrosative stress. Deletion of the flavohemoglobin gene (
YHB1
) abolished the nitric oxide (NO)-consuming activity of yeast cells. Levels of protein nitrosylation were more than 10-fold higher in
yhb1
mutant yeast than in isogenic wild-type cells after incubation with NO donors. Growth of mutant cells was inhibited by a nitrosative challenge that had little effect on wild-type cells, whereas the resistance of mutant cells to oxidative stress was unimpaired. Protection conferred by yeast flavohemoglobin against NO and
S
-nitrosothiols was seen under both anaerobic and aerobic conditions, consistent with a primary function in NO detoxification. A phylogenetic analysis indicated that protection from nitrosative stress is likely to be a conserved function among microorganismal flavohemoglobins. Flavohemoglobin is therefore a potential target for antimicrobial therapy.
Anti-mosquito midgut antibodies block development of
Plasmodium falciparum
and
Plasmodium vivax
in multiple species of
Anopheles
mosquitoes and reduce vector fecundity and survivorship
Authors: Lal Altaf A. , Patterson Pamela S. , Sacci John B. , Vaughan Jefferson A. , Paul Chris , Collins William E. , Wirtz Robert A. , Azad Abdu F.
Published: July 2002

The mosquito midgut plays a central role in the sporogonic
development of malaria parasites. We have found that polyclonal sera,
produced against mosquito midguts, blocked the passage of
Plasmodium falciparum
ookinetes across the midgut,
leading to a significant reduction of infections in mosquitoes.
Anti-midgut mAbs were produced that display broad-spectrum activity,
blocking parasite development of both
P. falciparum
and
Plasmodium vivax
parasites in five different species of
mosquitoes. In addition to their parasite transmission-blocking
activity, these mAbs also reduced mosquito survivorship and fecundity.
These results reveal that mosquito midgut-based antibodies have the
potential to reduce malaria transmission in a synergistic manner by
lowering both vector competence, through transmission-blocking effects
on parasite development, and vector abundance, by decreasing mosquito
survivorship and egg laying capacity. Because the intervention can
block transmission of different malaria parasite species in various
species of mosquitoes, vaccines against such midgut receptors may block
malaria transmission worldwide.
The crystal structure of exonuclease RecJ bound to Mn
2+
ion suggests how its characteristic motifs are involved in exonuclease activity
Authors: Yamagata Atsushi , Kakuta Yoshimitsu , Masui Ryoji , Fukuyama Keiichi
Published: July 2002

RecJ, a 5′ to 3′ exonuclease specific for single-stranded DNA, functions in DNA repair and recombination systems. We determined the crystal structure of RecJ bound to Mn
2+
ion essential for its activity. RecJ has a novel fold in which two domains are interconnected by a long helix, forming a central groove. Mn
2+
is located on the wall of the groove and is coordinated by conserved residues characteristic of a family of phosphoesterases that includes RecJ proteins. The groove is composed of residues conserved among RecJ proteins and is positively charged. These findings and the narrow width of the groove indicate that the groove binds single- instead of double-stranded DNA.
High frequency of hypermethylation at the 14-3-3 σ locus leads to gene silencing in breast cancer
Authors: Ferguson Anne T. , Evron Ella , Umbricht Christopher B. , Pandita Tej K. , Chan Timothy A. , Hermeking Heiko , Marks Jeffrey R. , Lambers Anouk R. , Futreal P. Andrew , Stampfer Martha R. , Sukumar Saraswati
Published: July 2002

Expression of 14-3-3 σ (σ) is induced in response to DNA damage, and causes cells to arrest in G
2
. By SAGE (serial analysis of gene expression) analysis, we identified σ as a gene whose expression is 7-fold lower in breast carcinoma cells than in normal breast epithelium. We verified this finding by Northern blot analysis. Remarkably, σ mRNA was undetectable in 45 of 48 primary breast carcinomas. Genetic alterations at σ such as loss of heterozygosity were rare (1/20 informative cases), and no mutations were detected (0/34). On the other hand, hypermethylation of CpG islands in the σ gene was detected in 91% (75/82) of breast tumors and was associated with lack of gene expression. Hypermethylation of σ is functionally important, because treatment of σ-non-expressing breast cancer cell lines with the drug 5-aza-2′-deoxycytidine resulted in demethylation of the gene and synthesis of σ mRNA. Breast cancer cells lacking σ expression showed increased number of chromosomal breaks and gaps when exposed to γ-irradiation. Therefore, it is possible that loss of σ expression contributes to malignant transformation by impairing the G
2
cell cycle checkpoint function, thus allowing an accumulation of genetic defects. Hypermethylation and loss of σ expression are the most consistent molecular alterations in breast cancer identified so far.
Repression of photosynthesis gene expression by formation of a disulfide bond in CrtJ
Authors: Masuda Shinji , Dong Chen , Swem Danielle , Setterdahl Aaron T. , Knaff David B. , Bauer Carl E.
Published: July 2002

Many species of purple photosynthetic bacteria repress synthesis of their photosystem in the presence of molecular oxygen. The bacterium
Rhodobacter capsulatus
mediates this process by repressing expression of bacteriochlorophyll, carotenoid, and light-harvesting genes via the aerobic repressor, CrtJ. In this study, we demonstrate that CrtJ forms an intramolecular disulfide bond
in vitro
and
in vivo
when exposed to oxygen. Mutational and sulfhydryl-specific chemical modification studies indicate that formation of a disulfide bond is critical for CrtJ binding to its target promoters. Analysis of the redox states of aerobically and anaerobically grown cells indicates that they have similar redox states of approximately −200 mV, thereby demonstrating that a change in midpoint potential is not responsible for disulfide bond formation.
In vivo
and
in vitro
analyses indicate that disulfide bond formation in CrtJ is insensitive to the addition of hydrogen peroxide but is sensitive to molecular oxygen. These results suggest that disulfide bond formation in CrtJ may differ from the mechanism of disulfide bond formation used by OxyR.
Base-specific fragmentation of amplified 16S rRNA genes analyzed by mass spectrometry: A tool for rapid bacterial identification
Authors: von Wintzingerode Friedrich , Böcker Sebastian , Schlötelburg Cord , Chiu Norman H. L. , Storm Niels , Jurinke Christian , Cantor Charles R. , Göbel Ulf B. , van den Boom Dirk
Published: July 2002

A rapid approach to the 16S rRNA gene (16S rDNA)-based bacterial identification has been developed that combines uracil-DNA-glycosylase (UDG)-mediated base-specific fragmentation of PCR products with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). 16S rDNA signature sequences were PCR-amplified from both cultured and as-yet-uncultured bacteria in the presence of dUTP instead of dTTP. These PCR products then were immobilized onto a streptavidin-coated solid support to selectively generate either sense or antisense templates. Single-stranded amplicons were subsequently treated with uracil-DNA-glycosylase to generate T-specific abasic sites and fragmented by alkaline treatment. The resulting fragment patterns were analyzed by MALDI-TOF MS. Mass signals of 16S rDNA fragments were compared with patterns calculated from published 16S rDNA sequences. MS of base-specific fragments of amplified 16S rDNA allows reliable discrimination of sequences differing by only one nucleotide. This approach is fast and has the potential for high-throughput identification as required in clinical, pharmaceutical, or environmental microbiology. In contrast to identification by MS of intact whole bacterial cells, this technique allows for the characterization of both cultured and as-yet-uncultured bacteria.
The development of immunity in a social insect: Evidence for the group facilitation of disease resistance
Authors: Traniello James F. A. , Rosengaus Rebeca B. , Savoie Keely
Published: July 2002

The extraordinary diversity and ecological success of the social insects has been attributed to their ability to cope with the rich and often infectious microbial community inhabiting their nests and feeding sites. Mechanisms of disease control used by eusocial species include antibiotic glandular secretions, mutual grooming, removal of diseased individuals from the nest, and the innate and adaptive immune responses of colony members. Here we demonstrate that after a challenge exposure to the entomopathogenic fungus
Metarhizium anisopliae
, dampwood termites
Zootermopsis angusticollis
have higher survivorship when individuals develop immunity as group members. Furthermore, termites significantly improve their ability to resist infection when they are placed in contact with previously immunized nestmates. This “social transfer” of infection resistance, a previously unrecognized mechanism of disease control in the social insects, could explain how group living may improve the survivorship of colony members despite the increased risks of pathogen transmission that can accompany sociality.
Bidirectional cooperative motion of molecular motors
Authors: Badoual M. , Jülicher F. , Prost J.
Published: July 2002

Recently, in a beautiful set of experiments, it has been shown that a Ncd mutant, NK11, which lacks directionality in its individual motion, was able to exhibit a new kind of directed motion in motility assays (Endow, S. A. & Higuchi, H. (2000)
Nature (London)
406, 913–916): the filaments keep a given velocity for a while and then suddenly move in the opposite direction with similar velocity. We show that these observations nicely illustrate the concept of dynamic transitions in motor collections introduced earlier in the case of an infinite number of motors. We investigate the experimentally relevant case of a finite number of motors both when directionality is present (kinesins, myosins, Ncd) and absent (NK11). Using a symmetric two-state model, we demonstrate that bidirectional motion is the signature of a dynamic transition that results from the collective behavior of many motors acting on the same filament. For motors exhibiting directional bias individually, an asymmetric two-state model is appropriate. In that case, dynamic transitions exist for motor collections in the presence of an external force. We give predictions for the dependence of motion on ATP concentration, external forces, and the number of motors involved. In particular, we show that the reversal time grows exponentially with the number of motors per filament.
Creating a dynamic picture of the sliding clamp during T4 DNA polymerase holoenzyme assembly by using fluorescence resonance energy transfer
Authors: Trakselis Michael A. , Alley Stephen C. , Abel-Santos Ernesto , Benkovic Stephen J.
Published: July 2002

The coordinated assembly of the DNA polymerase (gp43), the sliding clamp (gp45), and the clamp loader (gp44/62) to form the bacteriophage T4 DNA polymerase holoenzyme is a multistep process. A partially opened toroid-shaped gp45 is loaded around DNA by gp44/62 in an ATP-dependent manner. Gp43 binds to this complex to generate the holoenzyme in which gp45 acts to topologically link gp43 to DNA, effectively increasing the processivity of DNA replication. Stopped-flow fluorescence resonance energy transfer was used to investigate the opening and closing of the gp45 ring during holoenzyme assembly. By using two site-specific mutants of gp45 along with a previously characterized gp45 mutant, we tracked changes in distances across the gp45 subunit interface through seven conformational changes associated with holoenzyme assembly. Initially, gp45 is partially open within the plane of the ring at one of the three subunit interfaces. On addition of gp44/62 and ATP, this interface of gp45 opens further in-plane through the hydrolysis of ATP. Addition of DNA and hydrolysis of ATP close gp45 in an out-of-plane conformation. The final holoenzyme is formed by the addition of gp43, which causes gp45 to close further in plane, leaving the subunit interface open slightly. This open interface of gp45 in the final holoenzyme state is proposed to interact with the C-terminal tail of gp43, providing a point of contact between gp45 and gp43. This study further defines the dynamic process of bacteriophage T4 polymerase holoenzyme assembly.
Protein misfolding and temperature up-shift cause G
1
arrest via a common mechanism dependent on heat shock factor in
Saccharomyces
cerevisiae
Authors: Trotter Eleanor W. , Berenfeld Lumilla , Krause Sue Ann , Petsko Gregory A. , Gray Joseph V.
Published: July 2002

Accumulation of misfolded proteins in the cell at high temperature may cause entry into a nonproliferating, heat-shocked state. The imino acid analog azetidine 2-carboxylic acid (AZC) is incorporated into cellular protein competitively with proline and can misfold proteins into which it is incorporated. AZC addition to budding yeast cells at concentrations sufficient to inhibit proliferation selectively activates heat shock factor (HSF). We find that AZC treatment fails to cause accumulation of glycogen and trehalose (Msn2/4-dependent processes) or to induce thermotolerance (a protein kinase C-dependent process). However, AZC-arrested cells can accumulate glycogen and trehalose and can acquire thermotolerance in response to a subsequent heat shock. We find that AZC treatment arrests cells in a viable state and that this arrest is reversible. We find that cells at high temperature or cells deficient in the ubiquitin-conjugating enzymes Ubc4 and Ubc5 are hypersensitive to AZC-induced proliferation arrest. We find that AZC treatment mimics temperature up-shift in arresting cells in G
1
and represses expression of
CLN1
and
CLN2
. Mutants with reduced G
1
cyclin-Cdc28 activity are hypersensitive to AZC-induced proliferation arrest. Expression of the hyperstable Cln3–2 protein prevents G
1
arrest upon AZC treatment and temperature up-shift. Finally, we find that the
EXA3–1
mutation, encoding a defective HSF, prevents efficient G
1
arrest in response to both temperature up-shift and AZC treatment. We conclude that nontoxic levels of misfolded proteins (induced by AZC treatment or by high temperature) selectively activate HSF, which is required for subsequent G
1
arrest.
Of the three tegument proteins that package mRNA in herpes simplex virions, one (VP22) transports the mRNA to uninfected cells for expression prior to viral infection
Authors: Sciortino Maria Teresa , Taddeo Brunella , Poon Alice P. W. , Mastino Antonio , Roizman Bernard
Published: July 2002

An earlier report has shown that herpes simplex virus 1 virions package RNA. Experiments designed to reveal the identity of the virion proteins capable of binding the RNA and to show whether the mRNA carried in the newly infected cells was expressed showed the following: (
i
)
32
P-labeled riboprobe generated by
in vitro
transcription of the U
S
8.5 ORF bound three proteins identified as the products of U
S
11, U
L
47, and U
L
49 (VP22) genes. (
ii
) Viral RNA was bound to U
L
47 or U
S
11 proteins immune precipitated from cells transduced with baculoviruses expressing U
L
47 or U
S
11 and then superinfected with HSV-1 under conditions that blocked DNA synthesis and assembly of virions. (
iii
) Virions were purified from cells transduced with a baculovirus encoding a U
S
8.5 protein fused to green fluorescent protein and superinfected with an HSV-1 mutant lacking the U
S
8–12 genes. HEp-2 cells infected with these virions expressed the chimeric protein in ≈1% of infected cells. (
iv
) In mixed cultures, untreated Vero cells acquired the mRNA encoding the green fluorescent–U
S
8.5 chimeric protein from HEp-2 cells doubly transduced with the genes encoding VP22 and the chimeric protein. The transfer was RNase sensitive and VP22 dependent, indicating that the RNA encoded by the chimeric gene was transferred to Vero cells as mRNA. We conclude that (
i
) three virion proteins are capable of binding RNA; (
ii
) the packaged RNA can be expressed in newly infected cells; and (
iii
) the U
L
47 protein was earlier reported to shuttle from nucleus to the cytoplasm and may transport RNA. VP22 thus appears to be a member of a new class of viral proteins whose major function is to bind and transport infected cell mRNA to uninfected cells to create the environment for effective initiation of infection.
Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia
Authors: Chen Yanwen , Molloy Sean S. , Thomas Laurel , Gambee Jay , Bächinger Hans Peter , Ferguson Betsy , Zonana Jonathan , Thomas Gary , Morris Nicholas P.
Published: July 2002

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the
ED-1
gene disrupting the morphogenesis of ectodermal structures. The
ED-1
gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence

-Arg-Val-Arg-

Arg
156

-Asn-Lys-Arg
159
-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg
↓
) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for ≈20% of all known XLHED cases, with mutations occurring most frequently at Arg
156
, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg
156
Asn-Lys-Arg
159↓
- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor α
1
-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.
Phosphate is a specific signal for induction of osteopontin gene expression
Authors: Beck George R. , Zerler Brad , Moran Elizabeth
Published: July 2002

Osteopontin is a phosphorylated glycoprotein secreted to the mineralizing extracellular matrix by osteoblasts during bone development. It is believed to facilitate the attachment of osteoblasts and osteoclasts to the extracellular matrix, allowing them to perform their respective functions during osteogenesis. Several other functions have been suggested for this protein, and its up-regulation is associated with various disease states related to calcification, including arterial plaque formation and the formation of kidney stones. Although expression of this gene has been demonstrated in multiple tissues, its regulation is not well understood. Our previous studies on the roles of the retinoblastoma protein (pRB) and p300/CBP in the regulation of osteoblast differentiation revealed a link between osteopontin induction and the synthesis of alkaline phosphatase. In this paper, we describe results specifically linking induction of osteopontin to the enzymatic activity of alkaline phosphatase in the medium, which results in the generation of free phosphate. This elevation of free phosphate in the medium is sufficient to signal induction of osteopontin RNA and protein. The strong and specific induction of osteopontin in direct response to increased phosphate levels provides a mechanism to explain how expression of this product is normally regulated in bone and suggests how it may become up-regulated in damaged tissue.
Transcription coupled repair of 8-oxoguanine in murine cells: The Ogg1 protein is required for repair in nontranscribed sequences but not in transcribed sequences
Authors: Le Page Florence , Klungland Arne , Barnes Deborah E. , Sarasin Alain , Boiteux Serge
Published: July 2002

To assess the role of the Ogg1 DNA glycosylase in the transcription-coupled repair (TCR) of the mutagenic lesion, 7,8-dihydro-8oxoguanine (8-OxoG), we have investigated the removal of this lesion in wild-type and
ogg1
−/−
null mouse embryo fibroblast (MEF) cell lines. We used nonreplicating plasmids containing a single 8-OxoG·C base pair in a different assay that allowed us to study the removal of 8-OxoG located in a transcribed sequence (TS) or in a nontranscribed sequence (NTS). The results show that the removal of 8-OxoG in a wild-type MEF cell line is faster in the TS than in the NTS, indicating TCR of 8-OxoG in murine cells. In the homozygous
ogg1
−/−
MEF cell line, 8-OxoG was not removed from the NTS whereas there was still efficient 8-OxoG repair in the TS. Expression of the mouse Ogg1 protein in the homozygous
ogg1
−/−
cell line restored the ability to remove 8-OxoG in the NTS. Therefore, we have demonstrated that Ogg1 is essential for the repair of 8-OxoG in the NTS but is not required in the TS. These results indicate the existence of an Ogg1-independent pathway for the TCR of 8-OxoG
in vivo
.
Multiple vesiculoviral matrix proteins inhibit both nuclear export and import
Authors: Petersen Jeannine M. , Her Lu-Shiun , Dahlberg James E.
Published: July 2002

The matrix (M) protein of vesicular stomatitis virus inhibits both nuclear import and export. Here, we demonstrate that this inhibitory property is conserved between the M proteins from two other vesiculoviruses, chandipura virus and spring viremia carp virus. All three M proteins completely block nuclear transport of spliced mRNA, small nuclear RNAs, and small nuclear ribonucleoproteins and slow the nuclear transport of many other cargoes. In all cases where transport was merely slowed by the M proteins, the chandipura virus M protein had the strongest inhibitory activity. When expressed in transfected HeLa cells, active M proteins displayed prominent association with the nuclear rim. Moreover, mutation of a conserved methionine abolished both the inhibitory activity and efficient targeting of the M proteins to the nuclear rim. We propose that all of the vesiculoviral M proteins associate with the same nuclear target, which is likely to be a component of the nuclear pore complex.
Transgenic overexpression of caveolin-3 in skeletal muscle fibers induces a Duchenne-like muscular dystrophy phenotype
Authors: Galbiati Ferruccio , Volonté Daniela , Chu Jeffrey B. , Li Maomi , Fine Samson W. , Fu Maofu , Bermudez Jorge , Pedemonte Marina , Weidenheim Karen M. , Pestell Richard G. , Minetti Carlo , Lisanti Michael P.
Published: July 2002

It recently was reported that Duchenne muscular dystrophy (DMD) patients and
mdx
mice have elevated levels of caveolin-3 expression in their skeletal muscle. However, it remains unknown whether increased caveolin-3 levels in DMD patients contribute to the pathogenesis of DMD. Here, using a genetic approach, we test this hypothesis directly by overexpressing wild-type caveolin-3 as a transgene in mice. Analysis of skeletal muscle tissue from caveolin-3- overexpressing transgenic mice reveals: (
i
) a dramatic increase in the number of sarcolemmal muscle cell caveolae; (
ii
) a preponderance of hypertrophic, necrotic, and immature/regenerating skeletal muscle fibers with characteristic central nuclei; and (
iii
) down-regulation of dystrophin and β-dystroglycan protein expression. In addition, these mice show elevated serum creatine kinase levels, consistent with the myo-necrosis observed morphologically. The Duchenne-like phenotype of caveolin-3 transgenic mice will provide an important mouse model for understanding the pathogenesis of DMD in humans.
Distinct mechanisms of cell cycle arrest control the decision between differentiation and senescence in human neuroblastoma cells
Authors: Wainwright Linda J. , Lasorella Anna , Iavarone Antonio
Published: July 2002

Retinoic acid (RA) induces cell cycle arrest and differentiation of human neuroblastoma (NB) cells. Typically, NB cells differentiate along the neuronal lineage, but quiescent, “flat” cell types frequently have been described after treatment with differentiating agents. Two indistinguishable subclones of the cell line SK-N-SH, SK-N-SH-N (SH-N) and SK-N-SH-F (SH-F), display dramatically different responses to RA. In SH-N, RA induces neuronal differentiation, but in SH-F it transforms the small neuroblastic cells into large, flattened, epithelium-like cells. Here we analyze the mechanistic basis for the different effects of RA in the two NB subclones. First, we show that the flattened RA-treated SH-F expresses markers of cells undergoing replicative senescence. Inhibition of DNA synthesis by RA is significantly more rapid in SH-F than in SH-N. SH-F, which expresses basal amounts of p16
INK4A
, responds to RA with elevation of p18
INK4C
, marked down-regulation of cyclin D1, and swift inhibition of cyclin D-dependent kinases (cdks). Conversely, after addition of RA, SH-N retains cell cycling due to high expression of cyclin D1, the absence of Ink4 inhibitors, and accumulation of p21
Cip1
. These changes result in sustained cdk activity. Accordingly, overexpression of p21
Cip1
but not p16
INK4A
induces neuronal differentiation of untreated NB cells. We propose that rapid inhibition of cdks by RA in NB leads to early cell cycle arrest, prevents neuronal differentiation, and results in a senescence-like state.
Nucleotide sequence and predicted functions of the entire
Sinorhizobium meliloti
pSymA megaplasmid
Authors: Barnett Melanie J. , Fisher Robert F. , Jones Ted , Komp Caridad , Abola A. Pia , Barloy-Hubler Frédérique , Bowser Leah , Capela Delphine , Galibert Francis , Gouzy Jérôme , Gurjal Mani , Hong Andrea , Huizar Lucas , Hyman Richard W. , Kahn Daniel , Kahn Michael L. , Kalman Sue , Keating David H. , Palm Curtis , Peck Melicent C. , Surzycki Raymond , Wells Derek H. , Yeh Kuo-Chen , Davis Ronald W. , Federspiel Nancy A. , Long Sharon R.
Published: July 2002

The symbiotic nitrogen-fixing soil bacterium
Sinorhizobium
meliloti
contains three replicons: pSymA, pSymB, and the
chromosome. We report here the complete 1,354,226-nt sequence of pSymA.
In addition to a large fraction of the genes known to be specifically
involved in symbiosis, pSymA contains genes likely to be involved in
nitrogen and carbon metabolism, transport, stress, and resistance
responses, and other functions that give
S. meliloti
an
advantage in its specialized niche.
A hemoglobin from plants homologous to truncated hemoglobins of microorganisms
Authors: Watts R. A. , Hunt P. W. , Hvitved A. N. , Hargrove M. S. , Peacock W. J. , Dennis E. S.
Published: July 2002

We have identified a nuclear-encoded Hb from plants (GLB3) that has a central domain similar to the “truncated” Hbs of bacteria, protozoa, and algae. The three-dimensional structure of these Hbs is a 2-on-2 arrangement of α-helices, distinct from the 3-on-3 arrangement of the standard globin fold [Pesce, A., Couture, M., Dewilde, S., Guertin, M., Yamauchi, K., Ascenzi, P., Moens, L. & Bolognesi, M. (2000)
EMBO J.
19, 2424–2434].
GLB3-
like genes are not found in animals or yeast, but our analysis reveals that they are present in a wide range of Angiosperms and a Bryophyte. Although cyanobacteria and
Chlamydomonas
have 2-on-2 Hbs (GLBN), GLB3 is more likely related to GLBO-type 2-on-2 Hbs from bacteria. Consequently,
GLB3
is unlikely to have arisen from a horizontal transfer between the chloroplast and nuclear genomes.
Arabidopsis thaliana
GLB3 protein exhibits unusual concentration-independent binding of O
2
and CO. The absorbance spectrum of deoxy-GLB3 is unique; the protein forms a transient six-coordinate structure after reduction and deoxygenation, which slowly converts to a five-coordinate structure. In
A. thaliana
,
GLB3
is expressed throughout the plant but responds to none of the treatments that induce plant 3-on-3 Hbs. Our analysis of the sequence, ligand interactions, and expression profile of GLB3 indicates that this protein has unique biochemical properties, evolutionary history, and, most likely, a function distinct from those of other plant Hbs.
Inhibition of transcription factor-DNA complexes and gene expression by a microgonotropen
Authors: White Christine M. , Satz Alexander L. , Bruice Thomas C. , Beerman Terry A.
Published: July 2002

Developing minor groove-binding drugs to selectively inhibit transcription factor (TF)/DNA interactions and accompanying gene expression is a current goal in drug development studies. Equipping minor groove-binding agents with positively charged, major groove-contacting side chains yields microgonotropens (MGTs). Previously, we demonstrated that MGTs were superior inhibitors of TF/DNA complexes in cell-free assays compared with “classical” groove binders, but MGTs showed limited ability to inhibit gene expression. To determine what chemical characteristics contribute to or improve activity, we evaluate five MGTs for their effectiveness in inhibiting TF complex formation and resultant transcription by using the c-fos serum response element (SRE) as a target. MGT L1 binds DNA via a bisbenzimidazole equipped with a tripyrrole moiety. It is compared with analog L2, which has been functionalized with propylamines on each of the three pyrroles. L2, which binds DNA at subpicomolar concentrations, was at least three orders of magnitude more potent than L1 at inhibiting TF binding to the c-fos SRE in cell-free assays. Unlike L1 and previous MGTs, L2 also inhibited endogenous c-fos expression in NIH 3T3 cells at micromolar levels. Structure/activity relationships suggest that, although the tripyrrole/polyamine functional group of L2 may be largely responsible for its inhibition of TF complexes in cell-free assays, its bisbenzimidazole moiety appears to impart improved cellular uptake and activity. These findings make L2 a promising lead candidate for future, rational MGT design.
Contribution of
Salmonella typhimurium
type III secretion components to needle complex formation
Authors: Kimbrough Tyler G. , Miller Samuel I.
Published: July 2002

The
prgHIJK
operon encodes components of the
Salmonella typhimurium
pathogenicity island 1 type III
secretion system (TTSS). Previously,
prgH
and
prgK
were shown to be required for formation of the
supramolecular type III secretion needle complex (NC) [Kubori, T.,
et al.
(1998)
Science
280, 602–605].
This work indicates that all
prg
operon genes are
required for NC formation. PrgH multimerizes into a distinct
tetrameric-shaped structure that may be an early intermediate of NC
assembly and may provide the structural foundation required for PrgK
oligomerization. PrgH and PrgK, in the absence of other TTSS
components, oligomerize into ring-shaped structures identical in
appearance and size to the base of the NC, indicating that they are
likely the major inner membrane structural components required for
secretion. PrgI and PrgJ cofractionate with the NC and are secreted
into the culture supernatant. NC from
prgI
and
prgJ
mutants have an identical morphology to the
envelope-spanning (basal body) NC components, but are missing the
external needle, indicating that PrgI and PrgJ are required for full NC
assembly and are likely components of the external needle. Therefore,
PrgI and PrgJ are secreted through the NC basal body, composed in part
of PrgH/K and InvG/H rings, to participate in assembly of the more
distal components of the NC.
Genetic conflict and conditional altruism in social aphid colonies
Authors: Abbot Patrick , Withgott James H. , Moran Nancy A.
Published: July 2002

Although kin selection is central to the modern study of social evolution, recent studies of social species have revealed that no simple relationship exists between levels of kinship and sociality. The soldier-producing aphids are unique among highly social animals because, barring movement by aphids between colonies, they occur in clonal groups of genetically identical individuals. Potentially, clonality simplifies efforts to understand social evolution in aphids by obviating issues of intragroup conflict. However, we report here high levels of clonal mixing and conflict in an aphid society. The gall-dwelling colonies of a social aphid species (Pemphigus obesinymphae) are not pure clones, but are invaded by large numbers of aphids from other clones. Intruders behave and develop selfishly once they have invaded a colony of nonkin. They refrain from risky defensive behaviors and accelerate their own development into reproductive rather than defensive stages. This conditionality in the social life ofP. obesinymphaereveals complex dynamics and a degree of behavioral plasticity not previously known in aphid societies.
Intracellular calcium dependence of large dense-core vesicle exocytosis in the absence of synaptotagmin I
Authors: Voets Thomas , Moser Tobias , Lund Per-Eric , Chow Robert H. , Geppert Martin , Südhof Thomas C. , Neher Erwin
Published: July 2002

Synaptotagmin I is a synaptic vesicle-associated protein essential
for synchronous neurotransmission. We investigated its impact on the
intracellular Ca
2+
-dependence of large dense-core vesicle
(LDCV) exocytosis by combining Ca
2+
-uncaging and membrane
capacitance measurements in adrenal slices from mouse synaptotagmin I
null mutants. Synaptotagmin I-deficient chromaffin cells displayed
prolonged exocytic delays and slow, yet Ca
2+
-dependent
fusion rates, resulting in strongly reduced LDCV release in response to
short depolarizations. Vesicle recruitment, the shape of individual
amperometric events, and endocytosis appeared unaffected. These
findings demonstrate that synaptotagmin I is required for rapid, highly
Ca
2+
-sensitive LDCV exocytosis and indicate that it
regulates the equilibrium between a slowly releasable and a readily
releasable state of the fusion machinery. Alternatively, synaptotagmin
I could function as calcium sensor for the readily releasable pool,
leading to the destabilization of the pool in its absence.
Mobile elements and chromosomal evolution in the
virilis
group of
Drosophila
Authors: Evgen'ev Michael B. , Zelentsova Helena , Poluectova Helena , Lyozin George T. , Veleikodvorskaja Vera , Pyatkov K. I. , Zhivotovsky Lev A. , Kidwell Margaret G.
Published: July 2002

Species of the
virilis
group of
Drosophila
differ by multiple inversions and chromosome
fusions that probably accompanied, or led to, speciation.
Drosophila virilis
has the primitive karyotype for the
group, and natural populations are exceptional in having no chromosomal
polymorphisms. We report that the genomic locations of
Penelope
and
Ulysses
transposons are
nonrandomly distributed in 12 strains of
D. virilis
.
Furthermore,
Penelope
and
Ulysses
insertion sites in
D. virilis
show a statistically
significant association with the breakpoints of inversions found in
other species of the
virilis
group. Sixteen newly
induced chromosomal rearrangements were isolated from the progeny of
D. virilis
hybrid dysgenic crosses, including 12
inversions, 2 translocations, and 2 deletions.
Penelope
and
Ulysses
were associated with the breakpoints of over
half of these new rearrangements. Many rearrangement breakpoints also
coincide with the chromosomal locations of
Penelope
and
Ulysses
insertions in the parental strains and with
breakpoints of inversions previously established for other species of
the group. Analysis of homologous sequences from
D.
virilis
and
Drosophila lummei
indicated that
Penelope
insertion sites were closely, but not
identically, located at the nucleotide sequence level. Overall, these
results indicate that
Penelope
and
Ulysses
insert in a limited number of genomic locations
and are consistent with the possibility that these elements play an
important role in the evolution of the
virilis
species
group.
Retrotransposition of a yeast group II intron occurs by reverse splicing directly into ectopic DNA sites
Authors: Dickson Lorna , Huang Hon-Ren , Liu Lu , Matsuura Manabu , Lambowitz Alan M. , Perlman Philip S.
Published: July 2002

Group II introns, the presumed ancestors of nuclear pre-mRNA introns, are site-specific retroelements. In addition to “homing” to unoccupied sites in intronless alleles, group II introns transpose at low frequency to ectopic sites that resemble the normal homing site. Two general mechanisms have been proposed for group II intron transposition, one involving reverse splicing of the intron RNA directly into an ectopic DNA site, and the other involving reverse splicing into a site in RNA followed by reverse transcription and integration of the resulting cDNA by homologous recombination. Here, by using an “inverted-site” strategy, we show that the yeast mtDNA group II intron aI1 retrotransposes by reverse splicing directly into an ectopic DNA site. This same mechanism could account for other previously described ectopic transposition events in fungi and bacteria and may have contributed to the dispersal of group II introns into different genes.
UV-induced replication arrest in the xeroderma pigmentosum variant leads to DNA double-strand breaks, γ-H2AX formation, and Mre11 relocalization
Authors: Limoli Charles L. , Giedzinski Erich , Bonner William M. , Cleaver James E.
Published: July 2002

UV-induced replication arrest in the xeroderma pigmentosum
variant (XPV) but not in normal cells leads to an accumulation of the
Mre11/Rad50/Nbs1 complex and phosphorylated histone H2AX
(γ-H2AX) in large nuclear foci at sites of stalled replication
forks. These complexes have been shown to signal the presence of DNA
damage, in particular, double-strand breaks (DSBs). This finding
suggests that UV damage leads to the formation of DSBs during the
course of replication arrest. After UV irradiation, XPV cells showed a
fluence-dependent increase in the yield of γ-H2AX foci that
paralleled the production of Mre11 foci. The percentage of
foci-positive cells increased rapidly (10–15%) up to fluences of 10
J⋅m
−2
before saturating at higher fluences.
Frequencies of γ-H2AX and Mre11 foci both reached maxima at 4 h
after UV irradiation. This pattern contrasts sharply to the
situation observed after x-irradiation, where peak levels of
γ-H2AX foci were found to precede the formation of Mre11 foci by
several hours. The nuclear distributions of γ-H2AX and Mre11 were
found to colocalize spatially after UV- but not x-irradiation.
UV-irradiated XPV cells showed a one-to-one correspondence between
Mre11 and γ-H2AX foci-positive cells. These results show that XPV
cells develop DNA DSBs during the course of UV-induced replication
arrest. These UV-induced foci occur in cells that are unable to carry
out efficient bypass replication of UV damage and may contribute to
further genetic variation.
Increased atherosclerosis in hyperlipidemic mice deficient in α-tocopherol transfer protein and vitamin E
Authors: Terasawa Yuko , Ladha Zuleika , Leonard Scott W. , Morrow Jason D. , Newland Dale , Sanan David , Packer Lester , Traber Maret G. , Farese Robert V.
Published: July 2002

Although lipid peroxidation in the subendothelial space has been
hypothesized to play a central role in atherogenesis, the role of
vitamin E in preventing lipid peroxidation and lesion development
remains uncertain. Here we show that in atherosclerosis-susceptible
apolipoprotein E knockout mice, vitamin E deficiency caused by
disruption of the α-tocopherol transfer protein gene
(
Ttpa
) increased the severity of atherosclerotic lesions
in the proximal aorta. The increase was associated with increased
levels of isoprostanes, a marker of lipid peroxidation, in aortic
tissue. These results show that vitamin E deficiency promotes
atherosclerosis in a susceptible setting and support the hypothesis
that lipid peroxidation contributes to lesion development.
Ttpa
−/−
mice are a genetic model of
vitamin E deficiency and should be valuable for studying other diseases
in which oxidative stress is thought to play a role.
Autosomal dominant myopathy: Missense mutation (Glu-706 → Lys) in the myosin heavy chain IIa gene
Authors: Martinsson Tommy , Oldfors Anders , Darin Niklas , Berg Kerstin , Tajsharghi Homa , Kyllerman Mårten , Wahlström Jan
Published: July 2002

We here report on a human myopathy associated with a mutation in a
fast myosin heavy chain (MyHC) gene, and also the genetic defect in a
hereditary inclusion body myopathy. The disorder has previously been
described in a family with an “autosomal dominant myopathy, with
joint contractures, ophthalmoplegia, and rimmed vacuoles.” Linkage
analysis and radiation hybrid mapping showed that the gene locus (Human
Genome Map locus name:
IBM3
) is situated in a 2-Mb
region of chromosome 17p13, where also a cluster of MyHC genes is
located. These include the genes encoding embryonic, IIa, IIx/d,
IIb, perinatal, and extraocular MyHCs. Morphological analysis of muscle
biopsies from patients from the family indicated to us that the type 2A
fibers frequently were abnormal, whereas other fiber types appeared
normal. This observation prompted us to investigate the
MyHC-IIa
gene, since MyHC-IIa is the major isoform in
type 2A fibers. The complete genomic sequence for this gene was deduced
by using an “in silico” strategy. The gene, found to consist of
38 exons, was subjected to a complete mutation scan in patients and
controls. We identified a missense mutation, Glu-706 → Lys, which is
located in a highly conserved region of the motor domain, the so-called
SH1 helix region. By conformational changes this region communicates
activity at the nucleotide-binding site to the neck region, resulting
in the lever arm swing. The mutation in this region is likely to result
in a dysfunctional myosin, compatible with the disorder in the family.
Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.
Authors: Li X J , Sharp A H , Li S H , Dawson T M , Snyder S H , Ross C A
Published: July 2002

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.