bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated matrix metalloprotease and angiostatin levels in integrin α1 knockout mice cause reduced tumor vascularization
Authors: Pozzi Ambra , Moberg Philip E. , Miles Lindsey A. , Wagner Simone , Soloway Paul , Gardner Humphrey A.
Published: July 2002

Integrin α1β1 is a collagen receptor abundantly expressed on microvascular endothelial cells. As well as being the only collagen receptor able to activate the Ras/Shc/mitogen-activated protein kinase pathway promoting fibroblast cell proliferation, it also acts to inhibit collagen and metalloproteinase (MMP) synthesis. We have observed that in integrin α1-null mice synthesis of MMP7 and MMP9 was markedly increased compared with that of their wild-type counterparts. As MMP7 and MMP9 have been shown to generate angiostatin from circulating plasminogen, and angiostatin acts as a potent inhibitor of endothelial cell proliferation, we determined whether tumor vascularization was altered in the α1-null mice. Tumors implanted into α1-null mice showed markedly decreased vascularization, with a reduction in capillary number and size, which was accompanied by an increase in plasma levels of angiostatin due to the action of MMP7 and MMP9 on circulating plasminogen.In vitroanalysis of α1-null endothelial cells revealed a marked reduction of their proliferation on both integrin α1-dependent (collagenous) and independent (noncollagenous) substrata. This reduction was prevented by culturing α1-null cells with plasma derived from plasminogen-null animals, thus omitting the source from which to generate angiostatin. Plasma from tumor-bearing α1-null animals uniquely inhibited endothelial cell growth, and this inhibition was relieved by the coaddition of either MMP inhibitors, or antibody to angiostatin. Integrin α1-deficient mice thus provide a genetically characterized model for enhanced angiostatin production and serve to reveal an unwanted potential side effect of MMP inhibition, increased tumor angiogenesis.
Human single-chain Fv intrabodies counteract
in situ
huntingtin aggregation in cellular models of Huntington's disease
Authors: Lecerf Jean-Michel , Shirley Thomas L. , Zhu Quan , Kazantsev Aleksey , Amersdorfer Peter , Housman David E. , Messer Anne , Huston James S.
Published: July 2002

This investigation was pursued to test the use of intracellular
antibodies (intrabodies) as a means of blocking the pathogenesis of
Huntington's disease (HD). HD is characterized by abnormally elongated
polyglutamine near the N terminus of the huntingtin protein, which
induces pathological protein–protein interactions and aggregate
formation by huntingtin or its exon 1-containing fragments. Selection
from a large human phage display library yielded a single-chain Fv
(sFv) antibody specific for the 17 N-terminal residues of huntingtin,
adjacent to the polyglutamine in HD exon 1. This anti-huntingtin sFv
intrabody was tested in a cellular model of the disease in which
huntingtin exon 1 had been fused to green fluorescent protein (GFP).
Expression of expanded repeat HD-polyQ-GFP in transfected cells shows
perinuclear aggregation similar to human HD pathology, which worsens
with increasing polyglutamine length; the number of aggregates in these
transfected cells provided a quantifiable model of HD for this study.
Coexpression of anti-huntingtin sFv intrabodies with the abnormal
huntingtin-GFP fusion protein dramatically reduced the number of
aggregates, compared with controls lacking the intrabody.
Anti-huntingtin sFv fused with a nuclear localization signal retargeted
huntingtin analogues to cell nuclei, providing further evidence of the
anti-huntingtin sFv specificity and of its capacity to redirect the
subcellular localization of exon 1. This study suggests that
intrabody-mediated modulation of abnormal neuronal proteins may
contribute to the treatment of neurodegenerative diseases such as HD,
Alzheimer's, Parkinson's, prion disease, and the spinocerebellar
ataxias.
Rhesus monkeys know when they remember
Authors: Hampton Robert R.
Published: July 2002

Humans are consciously aware of some memories and can make verbal
reports about these memories. Other memories cannot be brought to
consciousness, even though they influence behavior. This conspicuous
difference in access to memories is central in taxonomies of human
memory systems but has been difficult to document in animal studies,
suggesting that some forms of memory may be unique to humans. Here I
show that rhesus macaque monkeys can report the presence or absence of
memory. Although it is probably impossible to document subjective,
conscious properties of memory in nonverbal animals, this result
objectively demonstrates an important functional parallel with human
conscious memory. Animals able to discern the presence and absence of
memory should improve accuracy if allowed to decline memory tests when
they have forgotten, and should decline tests most frequently when
memory is attenuated experimentally. One of two monkeys examined
unequivocally met these criteria under all test conditions, whereas the
second monkey met them in all but one case. Probe tests were used to
rule out “cueing” by a wide variety of environmental and
behavioral stimuli, leaving detection of the absence of memory
per se
as the most likely mechanism underlying the
monkeys' abilities to selectively decline memory tests when they had
forgotten.
Plastic- and liquid-crystalline architectures from dendritic receptor molecules
Authors: Elemans Johannes A. A. W. , Boerakker Mark J. , Holder Simon J. , Rowan Alan E. , Cho Wook-Dong , Percec Virgil , Nolte Roeland J. M.
Published: July 2002

Host molecules with U-shaped receptor cavities have been derivatized at their convex side with two n-hydrocarbon tails (1), two first-generation (2), and two second-generation (3) monodendritic hydrocarbon tails. Although hosts 1 and 2 display plastic-crystalline behavior, evidence suggests that host 3 forms a cubic liquid-crystalline phase. In this phase, molecules of 3 are arranged in spherical supramacromolecular assemblies, in which the receptor cavities are situated in the core and the hydrocarbon tails at the periphery. The 1:1 host-guest complex of 3 with methyl 3,5dihydroxybenzoate forms a similar liquid-crystalline phase, with the guest included in the core of the assemblies.
Mitf and Tfe3, two members of the Mitf-Tfe family of bHLH-Zip transcription factors, have important but functionally redundant roles in osteoclast development
Authors: Steingrímsson Eiríkur , Tessarollo Lino , Pathak Bhavani , Hou Ling , Arnheiter Heinz , Copeland Neal G. , Jenkins Nancy A.
Published: July 2002

The Mitf-Tfe family of basic helix–loop–helix-leucine zipper (bHLH-Zip) transcription factors encodes four family members: Mitf, Tfe3, Tfeb, and Tfec.
In vitro
, each protein in the family can bind DNA as a homo- or heterodimer with other family members. Mutational studies in mice have shown that
Mitf
is essential for melanocyte and eye development, whereas
Tfeb
is required for placental vascularization. Here, we uncover a role for
Tfe3
in osteoclast development, a role that is functionally redundant with
Mitf
. Although osteoclasts seem normal in
Mitf
or
Tfe3
null mice, the combined loss of the two genes results in severe osteopetrosis. We also show that
Tfec
mutant mice are phenotypically normal, and that the
Tfec
mutation does not alter the phenotype of
Mitf
,
Tfeb
, or
Tfe3
mutant mice. Surprisingly, our studies failed to identify any phenotypic overlap between the different
Mitf
–
Tfe
mutations. These results suggest that heterodimeric interactions are not essential for Mitf-Tfe function in contrast to other bHLH-Zip families like Myc/Max/Mad, where heterodimeric interactions seem to be essential.
Early Pleistocene
40
Ar/
39
Ar ages for Bapang Formation hominins, Central Jawa, Indonesia
Authors: Larick Roy , Ciochon Russell L. , Zaim Yahdi , Sudijono  , Suminto  , Rizal Yan , Aziz Fachroel , Reagan Mark , Heizler Matthew
Published: July 2002

The Sangiran dome is the primary stratigraphic window for the
Plio-Pleistocene deposits of the Solo basin of Central Jawa. The dome
has yielded nearly 80
Homo erectus
fossils, around 50 of
which have known findspots. With a hornblende
40
Ar/
39
Ar plateau age of 1.66 ± 0.04
mega-annum (Ma) reportedly associated with two fossils [Swisher, C.C.,
III, Curtis, G. H., Jacob, T., Getty, A. G., Suprijo, A. &
Widiasmoro (1994)
Science
263, 1118–1121), the dome
offers evidence that early
Homo
dispersed to East Asia
during the earliest Pleistocene. Unfortunately, the hornblende pumice
was sampled at Jokotingkir Hill, a central locality with complex
lithostratigraphic deformation and dubious specimen provenance. To
address the antiquity of Sangiran
H. erectus
more
systematically, we investigate the sedimentary framework and hornblende
40
Ar/
39
Ar age for volcanic deposits in the
southeast quadrant of the dome. In this sector, Bapang (Kabuh)
sediments have their largest exposure, least deformation, and most
complete tephrostratigraphy. At five locations, we identify a sequence
of sedimentary cycles in which
H. erectus
fossils are
associated with epiclastic pumice. From sampled pumice, eight
hornblende separates produced
40
Ar/
39
Ar
plateau ages ranging from 1.51 ± 0.08 Ma at the Bapang/Sangiran
Formation contact, to 1.02 ± 0.06 Ma, at a point above the
hominin-bearing sequence. The chronological sequence of
40
Ar/
39
Ar ages follows stratigraphic order
across the southeast quadrant. An intermediate level yielding four
nearly complete crania has an age of about 1.25 Ma.
Jasmonic acid carboxyl methyltransferase: A key enzyme for jasmonate-regulated plant responses
Authors: Seo Hak Soo , Song Jong Tae , Cheong Jong-Joo , Lee Yong-Hwan , Lee Yin-Won , Hwang Ingyu , Lee Jong Seob , Choi Yang Do
Published: July 2002

Methyl jasmonate is a plant volatile that acts as an important
cellular regulator mediating diverse developmental processes and
defense responses. We have cloned the novel gene
JMT
encoding an
S
-adenosyl-
l
-methionine:jasmonic
acid carboxyl methyltransferase (JMT) from
Arabidopsis
thaliana
. Recombinant JMT protein expressed in
Escherichia coli
catalyzed the formation of methyl
jasmonate from jasmonic acid with
K
m
value
of 38.5 μM.
JMT
RNA was not detected in young
seedlings but was detected in rosettes, cauline leaves, and developing
flowers. In addition, expression of the gene was induced both locally
and systemically by wounding or methyl jasmonate treatment. This result
suggests that
JMT
can perceive and respond to local and
systemic signals generated by external stimuli, and that the signals
may include methyl jasmonate itself. Transgenic
Arabidopsis
overexpressing
JMT
had a
3-fold elevated level of endogenous methyl jasmonate
without altering jasmonic acid content. The transgenic plants exhibited
constitutive expression of jasmonate-responsive genes, including
VSP
and
PDF1.2
. Furthermore, the
transgenic plants showed enhanced level of resistance against the
virulent fungus
Botrytis cinerea
. Thus, our data suggest
that the jasmonic acid carboxyl methyltransferase is a key enzyme for
jasmonate-regulated plant responses. Activation of
JMT
expression leads to production of methyl jasmonate that could act as an
intracellular regulator, a diffusible intercellular signal transducer,
and an airborne signal mediating intra- and interplant communications.
The future of coral reefs
Authors: Knowlton Nancy
Published: July 2002

Coral reefs, with their millions of species, have changed profoundly because of the effects of people, and will continue to do so for the foreseeable future. Reefs are subject to many of the same processes that affect other human-dominated ecosystems, but some special features merit emphasis: (i) Many dominant reef builders spawn eggs and sperm into the water column, where fertilization occurs. They are thus particularly vulnerable to Allee effects, including potential extinction associated with chronic reproductive failure. (ii) The corals likely to be most resistant to the effects of habitat degradation are small, short-lived “weedy” corals that have limited dispersal capabilities at the larval stage. Habitat degradation, together with habitat fragmentation, will therefore lead to the establishment of genetically isolated clusters of inbreeding corals. (iii) Increases in average sea temperatures by as little as 1°C, a likely result of global climate change, can cause coral “bleaching” (the breakdown of coral–algal symbiosis), changes in symbiont communities, and coral death. (iv) The activities of people near reefs increase both fishing pressure and nutrient inputs. In general, these processes favor more rapidly growing competitors, often fleshy seaweeds, and may also result in explosions of predator populations. (v) Combinations of stress appear to be associated with threshold responses and ecological surprises, including devastating pathogen outbreaks. (vi) The fossil record suggests that corals as a group are more likely to suffer extinctions than some of the groups that associate with them, whose habitat requirements may be less stringent.
Efficient pyrophosphorolysis by a hepatitis B virus polymerase may be a primer-unblocking mechanism
Authors: Urban Sinisa , Urban Severin , Fischer Karl P. , Tyrrell D. Lorne
Published: July 2002

Effective antiviral agents are thought to inhibit hepatitis B virus
(HBV) DNA synthesis irreversibly by chain termination because reverse
transcriptases (RT) lack an exonucleolytic activity that can remove
incorporated nucleotides. However, since the parameters governing this
inhibition are poorly defined, fully delineating the catalytic
mechanism of the HBV-RT promises to facilitate the development of
antiviral drugs for treating chronic HBV infection. To this end,
pyrophosphorolysis and pyrophosphate exchange, two nonhydrolytic RT
activities that result in the removal of newly incorporated
nucleotides, were characterized by using endogenous avian
HBV replication complexes assembled
in vivo
. Although
these activities are presumed to be physiologically irrelevant for
every polymerase examined, the efficiency with which they are catalyzed
by the avian HBV-RT strongly suggests that it is the first known
polymerase to catalyze these reactions under replicative conditions.
The ability to remove newly incorporated nucleotides during replication
has important biological and clinical implications: these activities
may serve a primer-unblocking function
in vivo
. Analysis
of pyrophosphorolysis on chain-terminated DNA revealed that the potent
anti-HBV drug β-
l
-(−)-2′,3′-dideoxy-3′-thiacytidine
(3TC) was difficult to remove by pyrophosphorolysis, in contrast to
ineffective chain terminators such as ddC. This disparity may account
for the strong antiviral efficacy of 3TC versus that of ddC. The HBV-RT
pyrophosphorolytic activity may therefore be a novel determinant of
antiviral drug efficacy, and could serve as a target for future
antiviral drug therapy. The strong inhibitory effect of cytoplasmic
pyrophosphate concentrations on viral DNA synthesis may also partly
account for the apparent slow rate of HBV genome replication.
Construction of neocentromere-based human minichromosomes by telomere-associated chromosomal truncation
Authors: Saffery Richard , Wong Lee H. , Irvine Danielle V. , Bateman Melissa A. , Griffiths Belinda , Cutts Suzanne M. , Cancilla Michael R. , Cendron Angela C. , Stafford Angela J. , Choo K. H. Andy
Published: July 2002

Neocentromeres (NCs) are fully functional centromeres that arise
ectopically in noncentromeric regions lacking α-satellite DNA. Using
telomere-associated chromosome truncation, we have produced a series of
minichromosomes (MiCs) from a mardel(10) marker chromosome containing a
previously characterized human NC. These MiCs range in size from ≈0.7
to 1.8 Mb and contain single-copy intact genomic DNA from the 10q25
region. Two of these NC-based Mi-Cs (NC-MiCs) appear circular whereas
one is linear. All demonstrate stability in both structure and mitotic
transmission in the absence of drug selection. Presence of a functional
NC is shown by binding a host of key centromere-associated proteins.
These NC-MiCs provide direct evidence for mitotic segregation function
of the NC DNA and represent examples of stable mammalian MiCs lacking
centromeric repeats.
MIP-1α, MIP-1β, RANTES, and ATAC/lymphotactin function together with IFN-γ as type 1 cytokines
Authors: Dorner Brigitte G. , Scheffold Alexander , Rolph Michael S. , Hüser Martin B. , Kaufmann Stefan H. E. , Radbruch Andreas , Flesch Inge E. A. , Kroczek Richard A.
Published: July 2002

We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstratein vitroand in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin are cosecreted to a high degree with IFN-γ by activated individual natural killer (NK), CD8+T, and CD4+T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-γ in the up-regulation of CD40, IL-12, and tumor necrosis factor-α, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1α, MIP-1β, RANTES, and ATAC are not only chemoattractants but also coactivators of macrophages, (ii) MIP-1α, MIP-1β, RANTES, and ATAC constitute together with IFN-γ a group of “type 1 cytokines,” and (iii) these cytokines act together as a functional unit that is used by NK cells in the innate phase and then “handed over” to CD8+T cells in the antigen-specific phase of the immune defense, thus bridging the two components of a Th1 immune reaction.
Separation of presenilin function in amyloid β-peptide generation and endoproteolysis of Notch
Authors: Kulic Luka , Walter Jochen , Multhaup Gerd , Teplow David B. , Baumeister Ralf , Romig Helmut , Capell Anja , Steiner Harald , Haass Christian
Published: July 2002

Most of the genetically inherited Alzheimer's disease cases are caused by mutations in the presenilin genes, PS1 and PS2. PS mutations result in the enhanced production of the highly amyloidogenic 42/43 amino acid variant of amyloid β-peptide (Aβ). We have introduced arbitrary mutations at position 286 of PS1, where a naturally occurring PS1 mutation has been described (L286V). Introduction of charged amino acids (L286E or L286R) resulted in an increase of Aβ42/43 production, which reached almost twice the level of the naturally occurring PS1 mutation. Although pathological Aβ production was increased, endoproteolysis of Notch and nuclear transport of its cytoplasmic domain was significantly inhibited. These results demonstrate that the biological function of PS proteins in the endoproteolysis of β-amyloid precursor protein and Notch can be separated.
Declines of biomes and biotas and the future of evolution
Authors: Woodruff David S.
Published: July 2002

Although panel discussants disagreed whether the biodiversity crisis constitutes a mass extinction event, all agreed that current extinction rates are 50–500 times background and are increasing and that the consequences for the future evolution of life are serious. In response to the on-going rapid decline of biomes and homogenization of biotas, the panelists predicted changes in species geographic ranges, genetic risks of extinction, genetic assimilation, natural selection, mutation rates, the shortening of food chains, the increase in nutrient-enriched niches permitting the ascendancy of microbes, and the differential survival of ecological generalists. Rates of evolutionary processes will change in different groups, and speciation in the larger vertebrates is essentially over. Action taken over the next few decades will determine how impoverished the biosphere will be in 1,000 years when many species will suffer reduced evolvability and require interventionist genetic and ecological management. Whether the biota will continue to provide the dependable ecological services humans take for granted is less clear. The discussants offered recommendations, including two of paramount importance (concerning human populations and education), seven identifying specific scientific activities to better equip us for stewardship of the processes of evolution, and one suggesting that such stewardship is now our responsibility. The ultimate test of evolutionary biology as a science is not whether it solves the riddles of the past but rather whether it enables us to manage the future of the biosphere. Our inability to make clearer predictions about the future of evolution has serious consequences for both biodiversity and humanity.
Selection bias in gene extraction on the basis of microarray gene-expression data
Authors: Ambroise Christophe , McLachlan Geoffrey J.
Published: July 2002

In the context of cancer diagnosis and treatment, we consider the problem of constructing an accurate prediction rule on the basis of a relatively small number of tumor tissue samples of known type containing the expression data on very many (possibly thousands) genes. Recently, results have been presented in the literature suggesting that it is possible to construct a prediction rule from only a few genes such that it has a negligible prediction error rate. However, in these results the test error or the leave-one-out cross-validated error is calculated without allowance for the selection bias. There is no allowance because the rule is either tested on tissue samples that were used in the first instance to select the genes being used in the rule or because the cross-validation of the rule is not external to the selection process; that is, gene selection is not performed in training the rule at each stage of the cross-validation process. We describe how in practice the selection bias can be assessed and corrected for by either performing a cross-validation or applying the bootstrap external to the selection process. We recommend using 10-fold rather than leave-one-out cross-validation, and concerning the bootstrap, we suggest using the so-called .632+ bootstrap error estimate designed to handle overfitted prediction rules. Using two published data sets, we demonstrate that when correction is made for the selection bias, the cross-validated error is no longer zero for a subset of only a few genes.
The aflatoxin B
1
formamidopyrimidine adduct plays a major role in causing the types of mutations observed in human hepatocellular carcinoma
Authors: Smela Maryann E. , Hamm Michelle L. , Henderson Paul T. , Harris Constance M. , Harris Thomas M. , Essigmann John M.
Published: July 2002

A G to T mutation has been observed at the third position of codon 249 of the p53 tumor-suppressor gene in over 50% of the hepatocellular carcinoma cases associated with high exposure to aflatoxin B
1
(AFB
1
). Hypotheses have been put forth that AFB
1
, in concert with hepatitis B virus (HBV), may play a role in the formation of, and/or the selection for, this mutation. The primary DNA adduct of AFB
1
is 8,9-dihydro-8-(
N
7
-guanyl)-9-hydroxyaflatoxin B
1
(AFB
1
-N7-Gua), which is converted naturally to two secondary lesions, an apurinic site and an AFB
1
-formamidopyrimidine (AFB
1
-FAPY) adduct. AFB
1
-FAPY is detected at near maximal levels in rat DNA days to weeks after AFB
1
exposure, underscoring its high persistence
in vivo
. The present study reveals two striking properties of this DNA adduct: (
i
) AFB
1
-FAPY was found to cause a G to T mutation frequency in
Escherichia coli
approximately 6 times higher than that of AFB
1
-N7-Gua, and (
ii
) one proposed rotamer of AFB
1
-FAPY is a block to replication, even when the efficient bypass polymerase MucAB is used by the cell. Taken together, these characteristics make the FAPY adduct the prime candidate for both the genotoxicity of aflatoxin, because mammalian cells also have similar bypass mechanisms for combating DNA damage, and the mutagenicity that ultimately may lead to liver cancer.
Identification of residues in TIP47 essential for Rab9 binding
Authors: Hanna John , Carroll Kate , Pfeffer Suzanne R.
Published: July 2002

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-dependent and cation-independent mannose 6-phosphate receptors (MPRs) and is required for their transport from endosomes to the trans-Golgi network
in vitro
and in living cells. TIP47 recognizes distinct determinants in the cytoplasmic domains of these two receptors, and its ability to bind to the cation-independent MPR is enhanced by the concomitant binding of the Rab9 GTPase. We show here that TIP47 residues 161–169 are essential, but likely not sufficient, for Rab9 binding. Mutation of these residues led to a significant decrease in Rab9 binding, but did not alter the global folding of the protein. The most impaired mutant was indistinguishable from wild-type TIP47 in its circular dichroism spectrum, and mutant proteins that showed decreased Rab9 binding retained full capacity to bind to MPR cytoplasmic domains. Closely related sequences in a related protein, adipophilin, did not confer Rab9 binding capacity to that protein. Partial proteolysis of TIP47 and TIP47 mutant proteins revealed subtle conformational differences, suggesting that residues 161–169 reside in a portion of TIP47 that is important for its conformation. These experiments reveal distinct binding domains for the Rab9 GTPase and MPR cytoplasmic domains in the cargo selection protein TIP47.
Phospholipid:diacylglycerol acyltransferase: An enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants
Authors: Dahlqvist Anders , Ståhl Ulf , Lenman Marit , Banas Antoni , Lee Michael , Sandager Line , Ronne Hans , Stymne Sten
Published: July 2002

Triacylglycerol (TAG) is known to be synthesized in a reaction that uses acyl-CoA as acyl donor and diacylglycerol (DAG) as acceptor, and which is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase. We have found that some plants and yeast also have an acyl-CoA-independent mechanism for TAG synthesis, which uses phospholipids as acyl donors and DAG as acceptor. This reaction is catalyzed by an enzyme that we call phospholipid:diacylglycerol acyltransferase, or PDAT. PDAT was characterized in microsomal preparations from three different oil seeds: sunflower, castor bean, and
Crepis palaestina
. We found that the specificity of the enzyme for the acyl group in the phospholipid varies between these species. Thus,
C. palaestina
PDAT preferentially incorporates vernoloyl groups into TAG, whereas PDAT from castor bean incorporates both ricinoleoyl and vernoloyl groups. We further found that PDAT activity also is present in yeast microsomes. The substrate specificity of this PDAT depends on the head group of the acyl donor, the acyl group transferred, and the acyl chains of the acceptor DAG. The gene encoding the enzyme was identified. The encoded PDAT protein is related to lecithin:cholesterol acyltransferase, which catalyzes the acyl-CoA-independent synthesis of cholesterol esters. However, budding yeast PDAT and its relatives in fission yeast and
Arabidopsis
form a distinct branch within this protein superfamily, indicating that a separate PDAT enzyme arose at an early point in evolution.
High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides
Authors: Ellis Hilary M. , Yu Daiguan , DiTizio Tina , Court Donald L.
Published: July 2002

Homologous DNA recombination is a fundamental, regenerative process
within living organisms. However, in most organisms, homologous
recombination is a rare event, requiring a complex set of reactions and
extensive homology. We demonstrate in this paper that Beta protein of
phage λ generates recombinants in chromosomal DNA by using synthetic
single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA
recombination can be used to mutagenize or repair the chromosome with
efficiencies that generate up to 6% recombinants among treated cells.
Mechanistically, it appears that Beta protein, a Rad52-like protein,
binds and anneals the ssDNA donor to a complementary single-strand near
the DNA replication fork to generate the recombinant. This type of
homologous recombination with ssDNA provides new avenues for studying
and modifying genomes ranging from bacterial pathogens to eukaryotes.
Beta protein and ssDNA may prove generally applicable for repairing DNA
in many organisms.
Inhibition of glycolipid shedding rescues recognition of a CD1+T cell lymphoma by natural killer T (NKT) cells
Authors: Sriram Venkataraman , Cho Sungyoo , Li Ping , O'Donnell Patrick W. , Dunn Claire , Hayakawa Kyoko , Blum Janice S. , Brutkiewicz Randy R.
Published: July 2002

Neoplastic transformation of cells is accompanied by an aberration of cell surface glycolipid composition. These tumor-associated, altered glycosphingolipids are often shed into the tumor cell microenvironment and mediate immunosuppressive activity. The nature and form of glycolipids shed by a variety of tumor cell lines and the mechanism(s) of shedding have been well characterized. The murine T cell lymphoma line, L5178Y-R, is known to shed a tumor-associated glycolipid, gangliotriaosylceramide, into the culture medium. We analyzed the effect of glycolipids from L5178Y-R on antigen presentation by murine CD1d1 molecules. CD1d1 molecules present glycolipid antigens to a specialized class of T cells called natural killer T (NKT) cells that mainly express a T cell receptor α chain (Vα14Jα281) associated with Vβ chains of limited diversity. In the current report, we found that L5178Y-R cells express CD1 on their cell surface yet are unable to stimulate CD1d1-specific NKT cells. We hypothesized that the glycolipid(s) shed by L5178Y-R inhibited antigen presentation by CD1d1. Pretreatment of CD1d1+cells with conditioned medium from L5178Y-R inhibited CD1-specific stimulation of canonical (Vα14+) but not noncanonical (Vα5+) NKT cells. Exogenous addition of lipids extracted from L5178Y-R cells as well as purified gangliotriaosylceramide mimicked this effect. Inhibition of glycolipid shedding in L5178Y-R cells withd-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol resulted in the rescue of CD1d1 recognition by canonical (but not noncanonical) NKT cells. These results suggest that one means by which certain tumor cells can evade the host's innate antitumor immune response is by shedding glycolipids that inhibit CD1-mediated antigen presentation to NKT cells.
St. John's wort induces hepatic drug metabolism through activation of the pregnane X receptor
Authors: Moore Linda B. , Goodwin Bryan , Jones Stacey A. , Wisely G. Bruce , Serabjit-Singh Cosette J. , Willson Timothy M. , Collins Jon L. , Kliewer Steven A.
Published: July 2002

St. John's wort (
Hypericum perforatum
) is an herbal remedy used widely for the treatment of depression. Recent clinical studies demonstrate that hypericum extracts increase the metabolism of various drugs, including combined oral contraceptives, cyclosporin, and indinavir. In this report, we show that hyperforin, a constituent of St. John's wort with antidepressant activity, is a potent ligand (
K
i
= 27 nM) for the pregnane X receptor, an orphan nuclear receptor that regulates expression of the cytochrome P450 (CYP) 3A4 monooxygenase. Treatment of primary human hepatocytes with hypericum extracts or hyperforin results in a marked induction of
CYP3A4
expression. Because CYP3A4 is involved in the oxidative metabolism of >50% of all drugs, our findings provide a molecular mechanism for the interaction of St. John's wort with drugs and suggest that hypericum extracts are likely to interact with many more drugs than previously had been realized.
Two-dimensional infrared spectroscopy of peptides by phase-controlled femtosecond vibrational photon echoes
Authors: Asplund M. C. , Zanni M. T. , Hochstrasser R. M.
Published: July 2002

Two-dimensional infrared spectra of peptides are introduced that are the direct analogues of two- and three-pulse multiple quantum NMR. Phase matching and heterodyning are used to isolate the phase and amplitudes of the electric fields of vibrational photon echoes as a function of multiple pulse delays. Structural information is made available on the time scale of a few picoseconds. Line narrowed spectra of acyl-proline-NH
2
and cross peaks implying the coupling between its amide-I modes are obtained, as are the phases of the various contributions to the signals. Solvent-sensitive structural differences are seen for the dipeptide. The methods show great promise to measure structure changes in biology on a wide range of time scales.
The affective component of pain in rodents: Direct evidence for a contribution of the anterior cingulate cortex
Authors: Johansen Joshua P. , Fields Howard L. , Manning Barton H.
Published: July 2002

Numerous human and animal studies indirectly implicate neurons in the anterior cingulate cortex (ACC) in the encoding of the affective consequences of nociceptor stimulation. No causal evidence, however, has been put forth linking the ACC specifically to this function. Using a rodent pain assay that combines the hind-paw formalin model with the place-conditioning paradigm, we measured a learned behavior that directly reflects the affective component of pain in the rat (formalin-induced conditioned place avoidance) concomitantly with “acute” formalin-induced nociceptive behaviors (paw lifting, licking, and flinching) that reflect the intensity and localization of the nociceptive stimulus. Destruction of neurons originating from the rostral, but not caudal, ACC reduced formalin-induced conditioned place avoidance without reducing acute pain-related behaviors. These results provide evidence indicating that neurons in the ACC are necessary for the “aversiveness” of nociceptor stimulation.
A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of
Saccharomyces cerevisiae
to an antifungal plant defensin from dahlia (
Dahlia merckii
)
Authors: Thevissen Karin , Cammue Bruno P. A. , Lemaire Katleen , Winderickx Joris , Dickson Robert C. , Lester Robert L. , Ferket Kathelijne K. A. , Van Even Frederic , Parret Annabel H. A. , Broekaert Willem F.
Published: July 2002

We have previously identified a
Saccharomyces cerevisiae
mutant that is markedly more resistant than wild-type to
Dahlia merckii
antimicrobial peptide 1 (DmAMP1), an antifungal plant defensin isolated from seeds of dahlia (
Dahlia merckii
). A complementation approach was followed that consisted of the introduction of a genomic library of DmAMP1-sensitive wild-type yeast into the DmAMP1-resistant yeast mutant and screening for restored sensitivity to DmAMP1. The gene determining sensitivity of
S. cerevisiae
to DmAMP1 was identified as
IPT1
, a gene encoding an enzyme involved in the last step of the synthesis of the sphingolipid mannose-(inositol-phosphate)
2
-ceramide. Strains with a nonfunctional
IPT1
allele lacked mannose-(inositol-phosphate)
2
-ceramide in their plasma membranes, bound significantly less DmAMP1 compared with wild-type strains, and were highly resistant to DmAMP1-mediated membrane permeabilization. All of these phenotypic deviations could be restored by reintroduction of a functional
IPT1
gene. Our data support a model in which membrane patches containing sphingolipids act as binding sites for DmAMP1 or, alternatively, are required to anchor membrane or cell wall-associated proteins, which themselves interact with DmAMP1.
Altered expression of expansin modulates leaf growth and pedicel abscission inArabidopsis thaliana
Authors: Cho Hyung-Taeg , Cosgrove Daniel J.
Published: July 2002

Expansins are cell-wall-loosening proteins that induce stress relaxation and extension of plant cell walls. To evaluate their hypothesized role in cell growth, we genetically manipulated expansin gene expression inArabidopsis thalianaand assessed the consequent changes in growth and cell-wall properties. Various combinations of promoters were used to drive antisense and sense sequences ofAtEXP10, which is maximally expressed in the growing leaf and at the base of the pedicel. Compared with controls, antisense lines had smaller rosettes because of shorter petioles and leaf blades and often acquired a twisted leaf morphology. Petiole cells from antisense plants were smaller than controls and their cell walls were significantly less extensiblein vitro. Sense plants had slightly longer petioles, larger leaf blades, and larger cells than controls. Abscission at the base of the pedicel, whereAtEXP10is endogenously expressed, was enhanced in sense plants but reduced in antisense lines. These results support the concept that expansins function endogenously as cell-wall-loosening agents and indicate that expansins have versatile developmental roles that include control of organ size, morphology, and abscission.
Altered expression of the ToxR-regulated porins OmpU and OmpT diminishes
Vibrio cholerae
bile resistance, virulence factor expression, and intestinal colonization
Authors: Provenzano Daniele , Klose Karl E.
Published: July 2002

The transmembrane transcriptional activators ToxR and TcpP modulate expression of
Vibrio cholerae
virulence factors by exerting control over
toxT
, which encodes the cytoplasmic transcriptional activator of the
ctx
,
tcp
, and
acf
virulence genes. However, ToxR, independently of TcpP and ToxT, activates and represses transcription of the genes encoding two outer-membrane porins, OmpU and OmpT. To determine the role of ToxR-dependent porin regulation in
V. cholerae
pathogenesis, the ToxR-activated
ompU
promoter was used to drive
ompT
transcription in a strain lacking OmpU. Likewise, the ToxR-repressed
ompT
promoter was used to drive
ompU
transcription in a strain lacking both ToxR and OmpT. This strategy allowed the generation of a
toxR
+
strain that expresses OmpT in place of OmpU, and a
toxR
−
strain that expresses OmpU in place of OmpT. Growth rates in the presence of bile salts and other anionic detergents were retarded for the
toxR
+
V. cholerae
expressing OmpT in place of OmpU, but increased in
toxR
−
V. cholerae
expressing OmpU in place of OmpT. Additionally, the
toxR
+
V. cholerae
expressing OmpT in place of OmpU expressed less cholera toxin and toxin-coregulated pilus, and this effect was shown to be caused by reduced
toxT
transcription in this strain. Finally, the
toxR
+
V. cholerae
expressing OmpT in place of OmpU was ≈100-fold reduced in its ability to colonize the infant-mouse intestine. Our results indicate that ToxR-dependent modulation of the outer membrane porins OmpU and OmpT is critical for
V. cholerae
bile resistance, virulence factor expression, and intestinal colonization.
Differential requirement of the cytoplasmic subregions of γc chain in T cell development and function
Authors: Tsujino Shiho , Di Santo James P. , Takaoka Akinori , McKernan Teresa L. , Noguchi Shigeru , Taya Choji , Yonekawa Hiromichi , Saito Takashi , Taniguchi Tadatsugu , Fujii Hodaka
Published: July 2002

The common cytokine receptor γ chain (γc), a shared component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15, is critical for the development and function of lymphocytes. The cytoplasmic domain of γc consists of 85 aa, in which the carboxyl-terminal 48 aa are essential for its interaction with and activation of the Janus kinase, Jak3. Evidence has been provided that Jak3-independent signals might be transmitted via the residual membrane-proximal region; however, its rolein vivoremains totally unknown. In the present study, we expressed mutant forms of γc, which lack either most of the cytoplasmic domain or only the membrane-distal Jak3-binding region, on aγcnull background. We demonstrate that, unlikeγcorJak3null mice, expression of the latter, but not the former mutant, restores T lymphopoiesisin vivo, accompanied by strong expression of Bcl-2. On the other hand, thein vitrofunctions of the restored T cells still remained impaired. These results not only reveal the hitherto unknown role of the γc membrane-proximal region, but also suggest the differential requirement of the cytoplasmic subregions of γc in T cell development and function.
Caspase cleavage of MST1 promotes nuclear translocation and chromatin condensation
Authors: Ura Seiji , Masuyama Norihisa , Graves Jonathan D. , Gotoh Yukiko
Published: July 2002

MST1, mammalian STE20-like kinase 1, is a serine/threonine kinase that is cleaved and activated by caspases during apoptosis. MST1 is capable of inducing apoptotic morphological changes such as chromatin condensation upon overexpression. In this study, we show that MST1 contains two functional nuclear export signals (NESs) in the C-terminal domain, which is released from the N-terminal kinase domain upon caspase-mediated cleavage. Full-length MST1 is excluded from the nucleus and localized to the cytoplasm. However, either truncation of the C-terminal domain, point mutation of the two putative NESs, or treatment with leptomycin B, an inhibitor of the NES receptor, results in nuclear localization of MST1. Staurosporine treatment induces chromatin condensation, MST1 cleavage, and nuclear translocation. Staurosporine-induced chromatin condensation is partially inhibited by expressing a kinase-negative mutant of MST1, suggesting an important role of MST1 in this process. Significantly, MST1 is more efficient at inducing chromatin condensation when it is constitutively localized to the nucleus by mutation of its NESs. Moreover, inhibition of MST1 nuclear translocation by mutation of its cleavage sites reduces its ability to induce chromatin condensation. Taken together, these results suggest that truncation of the C-terminal domain of MST1 by caspases may result in translocation of MST1 into the nucleus, where it promotes chromatin condensation.
Transportin-SR2 mediates nuclear import of phosphorylated SR proteins
Authors: Lai Ming-Chih , Lin Ru-Inn , Tarn Woan-Yuh
Published: July 2002

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin β family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using
in vitro
import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles
in vitro
. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.
Genomic profiling of short- and long-term caloric restriction effects in the liver of aging mice
Authors: Cao Shelley X. , Dhahbi Joseph M. , Mote Patricia L. , Spindler Stephen R.
Published: July 2002

We present genome-wide microarray expression analysis of 11,000 genes in an aging potentially mitotic tissue, the liver. This organ has a major impact on health and homeostasis during aging. The effects of life- and health-span-extending caloric restriction (CR) on gene expression among young and old mice and between long-term CR (LT-CR) and short-term CR (ST-CR) were examined. This experimental design allowed us to accurately distinguish the effects of aging from those of CR on gene expression. Aging was accompanied by changes in gene expression associated with increased inflammation, cellular stress, and fibrosis, and reduced capacity for apoptosis, xenobiotic metabolism, normal cell-cycling, and DNA replication. LT-CR and just 4 weeks of ST-CR reversed the majority of these changes. LT-CR produced in young mice a pattern of gene expression that is a subset of the changes found in old LT-CR mice. It is possible that the early changes in gene expression, which extend into old age, are key to the life- and health-span-extending effects of CR. Further, ST-CR substantially shifted the “normo-aging” genomic profile of old control mice toward the “slow-aging” profile associated with LT-CR. Therefore, many of the genomic effects of CR are established rapidly. Thus, expression profiling should prove useful in quickly identifying CR- mimetic drugs and treatments.
Intracellular unesterified arachidonic acid signals apoptosis
Authors: Cao Yang , Pearman A. Terrece , Zimmerman Guy A. , McIntyre Thomas M. , Prescott Stephen M.
Published: July 2002

Cyclooxygenase-2 (COX-2) is up-regulated in many cancers and is a rate-limiting step in colon carcinogenesis. Nonsteroidal antiinflammatory drugs, which inhibit COX-2, prevent colon cancer and cause apoptosis. The mechanism for this response is not clear, but it might result from an accumulation of the substrate, arachidonic acid, an absence of a prostaglandin product, or diversion of the substrate into another pathway. We found that colon adenocarcinomas overexpress another arachidonic acid-utilizing enzyme, fatty acid-CoA ligase (FACL) 4, in addition to COX-2. Exogenous arachidonic acid caused apoptosis in colon cancer and other cell lines, as did triacsin C, a FACL inhibitor. In addition, indomethacin and sulindac significantly enhanced the apoptosis-inducing effect of triacsin C. These findings suggested that unesterified arachidonic acid in cells is a signal for induction of apoptosis. To test this hypothesis, we engineered cells with inducible overexpression of COX-2 and FACL4 as “sinks” for unesterified arachidonic acid. Activation of the enzymatic sinks blocked apoptosis, and the reduction of cell death was inversely correlated with the cellular level of arachidonic acid. Inhibition of the COX-2 component by nonsteroidal antiinflammatory drugs restored the apoptotic response. Cell death caused by exposure to tumor necrosis factor α or to calcium ionophore also was prevented by removal of unesterified arachidonic acid. We conclude that the cellular level of unesterified arachidonic acid is a general mechanism by which apoptosis is regulated and that COX-2 and FACL4 promote carcinogenesis by lowering this level.
Insulin-like growth factor 1 regulates the location, stability, and transcriptional activity of β-catenin
Authors: Playford Martin P. , Bicknell David , Bodmer Walter F. , Macaulay Valentine M.
Published: July 2002

The insulin-like growth factor (IGF) type 1 receptor is required
for growth, transformation, and protection from apoptosis. IGFs
can enhance cell migration, which is known to be influenced via
regulation of the E-cadherin/β-catenin complex. We sought
to investigate whether IGF-1 modulated the interaction between
E-cadherin and β-catenin in human colorectal cancer cells. We used
the C10 cell line, which we established and have previously shown to
lack adenomatous polyposis coli, E-cadherin, or β-catenin
mutations. We found that IGF-1 stimulation enhanced tyrosine
phosphorylation of two proteins, β-catenin and insulin-receptor
substrate 1, which formed a complex with E-cadherin. Tyrosine
phosphorylation of β-catenin was accompanied by rapid (<1 min)
dissociation from E-cadherin at the plasma membrane, followed by
relocation to the cellular cytoplasm. IGF-1 also enhanced the stability
of β-catenin protein. Despite this, we observed no enhancement of
transcriptional activity in complex with T-cell factor 4 (Tcf-4) in
human embryonic kidney 293 cells treated with IGF-1 or insulin alone.
IGF-1 did, however, enhance transcriptional activity in combination
with lithium chloride, an inhibitor of glycogen synthase kinase 3β,
which also stabilizes β-catenin. In conclusion, we have shown that
IGF-1 causes tyrosine phosphorylation and stabilization of β-catenin.
These effects may contribute to transformation, cell migration, and a
propensity for metastasis
in vivo
.