bluuebunny/crossref_metadata_2025 huggingface dataset:
Macrophage migration inhibitory factor (MIF) sustains macrophage proinflammatory function by inhibiting p53: Regulatory role in the innate immune response
Authors: Mitchell Robert A. , Liao Hong , Chesney Jason , Fingerle-Rowson Gunter , Baugh John , David John , Bucala Richard
Published: July 2002

The importance of the macrophage in innate immunity is underscored by its secretion of an array of powerful immunoregulatory and effector molecules. We report herein that macrophage migration inhibitory factor (MIF), a product of activated macrophages, sustains macrophage survival and function by suppressing activation-induced, p53-dependent apoptosis. Endotoxin administration to MIF−/−mice results in decreased macrophage viability, decreased proinflammatory function, and increased apoptosis when compared with wild-type controls. Moreover, inhibition of p53 in endotoxin-treated, MIF-deficient macrophages suppresses enhanced apoptosis and restores proinflammatory function. MIF inhibits p53 activity in macrophages via an autocrine regulatory pathway, resulting in a decrease in cellular p53 accumulation and subsequent function. Inhibition of p53 by MIF coincides with the induction of arachidonic acid metabolism and cyclooxygenase-2 (Cox-2) expression, which is required for MIF regulation of p53. MIF's effect on macrophage viability and survival provides a previously unrecognized mechanism to explain its critical proinflammatory action in conditions such as sepsis, and suggests new approaches for the modulation of innate immune responses.
Rescue of hereditary form of dilated cardiomyopathy by rAAV-mediated somatic gene therapy: Amelioration of morphological findings, sarcolemmal permeability, cardiac performances, and the prognosis of TO-2 hamsters
Authors: Kawada Tomie , Nakazawa Mikio , Nakauchi Sakura , Yamazaki Ken , Shimamoto Ryoichi , Urabe Masashi , Nakata Jumi , Hemmi Chieko , Masui Fujiko , Nakajima Toshiaki , Suzuki Jun-Ichi , Monahan John , Sato Hiroshi , Masaki Tomoh , Ozawa Keiya , Toyo-oka Teruhiko
Published: July 2002

The hereditary form comprises ≈1/5 of patients with dilated cardiomyopathy (DCM) and is a major cause of advanced heart failure. Medical and socioeconomic settings require novel treatments other than cardiac transplantation. TO-2 strain hamsters with congenital DCM show similar clinical and genetic backgrounds to human cases that have defects in the δ-sarcoglycan (δ-SG) gene. To examine the long-term
in vivo
supplement of normal δ-SG gene driven by cytomegalovirus promoter, we analyzed the pathophysiologic effects of the transgene expression in TO-2 hearts by using recombinant adeno-associated virus vector. The transgene preserved sarcolemmal permeability detected
in situ
by mutual exclusivity between cardiomyocytes taking up intravenously administered Evans blue dye and expressing the δ-SG transgene throughout life. The persistent amelioration of sarcolemmal integrity improved wall thickness and the calcification score postmortem. Furthermore,
in vivo
myocardial contractility and hemodynamics, measured by echocardiography and cardiac catheterization, respectively, were normalized, especially in the diastolic performance. Most importantly, the survival period of the TO-2 hamsters was prolonged after the δ-SG gene transduction, and the animals remained active, exceeding the life expectancy of animals without transduction of the responsible gene. These results provide the first evidence that somatic gene therapy is promising for human DCM treatment, if the rAAV vector can be justified for clinical use.
Metapopulation extinction caused by mutation accumulation
Authors: Higgins Kevin , Lynch Michael
Published: July 2002

Theory suggests that the risk of extinction by mutation
accumulation can be comparable to that by environmental stochasticity
for an isolated population smaller than a few thousand individuals.
Here we show that metapopulation structure, habitat loss or
fragmentation, and environmental stochasticity can be expected to
greatly accelerate the accumulation of mildly deleterious mutations,
lowering the genetic effective size to such a degree that even large
metapopulations may be at risk of extinction. Because of mutation
accumulation, viable metapopulations may need to be far larger and
better connected than would be required under just stochastic
demography.
A common pharmacophore for epothilone and taxanes: Molecular basis for drug resistance conferred by tubulin mutations in human cancer cells
Authors: Giannakakou Paraskevi , Gussio Rick , Nogales Eva , Downing Kenneth H. , Zaharevitz Daniel , Bollbuck Birgit , Poy George , Sackett Dan , Nicolaou K. C. , Fojo Tito
Published: July 2002

The epothilones are naturally occurring antimitotic drugs
that share with the taxanes a similar mechanism of action without
apparent structural similarity. Although photoaffinity labeling and
electron crystallographic studies have identified the taxane-binding
site on β-tubulin, similar data are not available for epothilones. To
identify tubulin residues important for epothilone binding, we have
isolated two epothilone-resistant human ovarian carcinoma sublines
derived in a single-step selection with epothilone A or B. These
epothilone-resistant sublines exhibit impaired epothilone- and
taxane-driven tubulin polymerization caused by acquired β-tubulin
mutations (β274
Thr→Ile
and β282
Arg→Gln
)
located in the atomic model of αβ-tubulin near the taxane-binding
site. Using molecular modeling, we investigated the conformational
behavior of epothilone, which led to the identification of a common
pharmacophore shared by taxanes and epothilones. Although two binding
modes for the epothilones were predicted, one mode was identified as
the preferred epothilone conformation as indicated by the activity of a
potent pyridine-epothilone analogue. In addition, the
structure–activity relationships of multiple taxanes and epothilones
in the tubulin mutant cells can be fully explained by the model
presented here, verifying its predictive value. Finally, these
pharmacophore and activity data from mutant cells were used to model
the tubulin binding of sarcodictyins, a distinct class of microtubule
stabilizers, which in contrast to taxanes and the epothilones interact
preferentially with the mutant tubulins. The unification of taxane,
epothilone, and sarcodictyin chemistries in a single pharmacophore
provides a framework to study drug–tubulin interactions that should
assist in the rational design of agents targeting tubulin.
APC
mutations are sufficient for the growth of early colorectal adenomas
Authors: Lamlum Hanan , Papadopoulou Angeliki , Ilyas Mohammad , Rowan Andrew , Gillet Cheryl , Hanby Andrew , Talbot Ian , Bodmer Walter , Tomlinson Ian
Published: July 2002

It is not clear whether
APC
mutations are sufficient for early colorectal adenomas to grow or whether additional mutations at other loci are required. We previously have screened 210 early colorectal adenomas from familial adenomatous polyposis patients for mutations and allelic loss at
APC
. Here, we determined whether allelic loss at
APC
had any effect on the nearby α-catenin gene. However, loss on 5q in familial adenomatous polyposis adenomas rarely extended as far as α-catenin, and no differences in α-catenin protein expression were found in tumors that showed loss encompassing both
APC
and α-catenin. We then screened all 210 tumors for mutations at candidate loci other than
APC
(K-ras, β-catenin, and allelic loss at 1p33-p35 and 1p36) and for microsatellite instability (MSI). Each of these loci has been implicated previously in early colorectal tumorigenesis. One tumor harbored a β-catenin mutation and another MSI, but none showed K-ras mutation or allelic loss at 1p33-p35 or 1p36. These data support the following hypotheses derived from sporadic colorectal tumors: β-catenin mutations are generally an alternative to mutations at
APC
, MSI is not usually an early phenomenon in colorectal tumorigenesis, and K-ras mutations are more typical of large- and moderate-sized adenomas. Contrary to some previous reports, chromosome 1p allelic loss is infrequent in very early adenomas.
APC
mutations are generally sufficient for colorectal tumors to grow to about 1-cm diameter, although chance mutations at other loci can provide these early colorectal adenomas with a selective advantage, and some colorectal tumors may develop along a pathway not involving
APC
.
Whole-gene
APC
deletions cause classical familial adenomatous polyposis, but not attenuated polyposis or “multiple” colorectal adenomas
Authors: Sieber O. M. , Lamlum H. , Crabtree M. D. , Rowan A. J. , Barclay E. , Lipton L. , Hodgson S. , Thomas H. J. W. , Neale K. , Phillips R. K. S. , Farrington S. M. , Dunlop M. G. , Mueller H. J. , Bisgaard M. L. , Bulow S. , Fidalgo P. , Albuquerque C. , Scarano M. I. , Bodmer W. , Tomlinson I. P. M. , Heinimann K.
Published: July 2002

Familial adenomatous polyposis (FAP) is a dominantly inherited colorectal tumor predisposition that results from germ-line mutations in the
APC
gene (chromosome 5q21). FAP shows substantial phenotypic variability: classical polyposis patients develop more than 100 colorectal adenomas, whereas those with attenuated polyposis (AAPC) have fewer than 100 adenomas. A further group of individuals, so-called “multiple” adenoma patients, have a phenotype like AAPC, with 3–99 polyps throughout the colorectum, but mostly have no demonstrable germ-line
APC
mutation. Routine mutation detection techniques fail to detect a pathogenic
APC
germ-line mutation in approximately 30% of patients with classical polyposis and 90% of those with AAPC/multiple adenomas. We have developed a real-time quantitative multiplex PCR assay to detect
APC
exon 14 deletions. When this technique was applied to a set of 60 classical polyposis and 143 AAPC/multiple adenoma patients with no apparent
APC
germ-line mutation, deletions were found exclusively in individuals with classical polyposis (7 of 60, 12%). Fine-mapping of the region suggested that the majority (6 of 7) of these deletions encompassed the entire
APC
locus, confirming that haploinsufficiency can result in a classical polyposis phenotype. Screening for germ-line deletions in
APC
mutation-negative individuals with classical polyposis seems warranted.
A transcriptional corepressor of Stat1 with an essential LXXLL signature motif
Authors: Liu Bin , Gross Mitchell , ten Hoeve Johanna , Shuai Ke
Published: July 2002

Interferon (IFN) treatment induces tyrosine phosphorylation and
nuclear translocation of Stat1 (signal transducer and activator of
transcription) to activate or repress transcription. We report here
that a member of the protein inhibitor of activated STAT family, PIASy,
is a transcriptional corepressor of Stat1. IFN treatment triggers the
in vivo
interaction of Stat1 with PIASy, which represses
Stat1-mediated gene activation without blocking the DNA binding
activity of Stat1. An LXXLL coregulator signature motif located near
the NH
2
terminus of PIASy, although not involved in the
PIASy–Stat1 interaction, is required for the transrepression activity
of PIASy. Our studies identify PIASy as a transcriptional corepressor
of Stat1 and suggest that different PIAS proteins may repress
STAT-mediated gene activation through distinct mechanisms.
Arm-site binding by λ-integrase: Solution structure and functional characterization of its amino-terminal domain
Authors: Wojciak Jonathan M. , Sarkar Dibyendu , Landy Arthur , Clubb Robert T.
Published: July 2002

The integrase protein (Int) from bacteriophage λ catalyzes the insertion and excision of the viral genome into and out of
Escherichia coli
. It is a member of the λ-Int family of site-specific recombinases that catalyze a diverse array of DNA rearrangements in archaebacteria, eubacteria, and yeast and belongs to the subset of this family that possesses two autonomous DNA-binding domains. The heterobivalent properties of Int can be decomposed into a carboxyl-terminal domain that executes the DNA cleavage and ligation reactions and a smaller amino-terminal domain that binds to an array of conserved DNA sites within the phage arms, thereby arranging Int protomers within the higher-order recombinogenic complex. We have determined that residues Met-1 to Leu-64 of Int constitute the minimal arm-type DNA-binding domain (INT-DBD
1–64
) and solved the solution structure by using NMR. We show that the INT-DBD
1–64
is a novel member of the growing family of three-stranded β-sheet DNA-binding proteins, because it supplements this motif with a disordered amino-terminal basic tail that is important for arm-site binding. A model of the arm-DNA-binding domain recognizing its cognate DNA site is proposed on the basis of similarities with the analogous domain of Tn916 Int and is discussed in relation to other features of the protein.
Structure-based design of mutant
Methanococcus jannaschii
tyrosyl-tRNA synthetase for incorporation of
O
-methyl-
l
-tyrosine
Authors: Zhang Deqiang , Vaidehi Nagarajan , Goddard William A. , Danzer Joseph F. , Debe Derek
Published: July 2002

Although incorporation of amino acid analogs provides a powerful means of producing new protein structures with interesting functions, many amino acid analogs cannot be incorporated easily by using the wild-type aminoacyl-tRNA synthetase (aaRS). To be able to incorporate specific amino acid analogs site-specifically, it is useful to build a mutant aaRS that preferentially activates the analog compared with the natural amino acids. Experimental combinatorial studies to find such mutant aaRSs have been successful but can easily become costly and time-consuming. In this article, we describe the clash opportunity progressive (COP) computational method for designing a mutant aaRS to preferentially take up the analog compared with the natural amino acids. To illustrate this COP procedure, we apply it to the design of mutant
Methanococcus jannaschii
tyrosyl-tRNA synthetase (
M.jann
-TyrRS). Because the three-dimensional structure for
M.jann
-TyrRS was not available, we used the STRUCTFAST homology modeling procedure plus molecular dynamics with continuum solvent forces to predict the structure of wild-type
M.jann
-TyrRS. We validate this structure by predicting the binding site for tyrosine and calculating the binding energies of the 20 natural amino acids, which shows that tyrosine binds the strongest. With the COP design algorithm we then designed a mutant tyrosyl tRNA synthetase to activate
O
-methyl-
l
-tyrosine preferentially compared with
l
-tyrosine. This mutant [Y32Q, D158A] is similar to the mutant designed with combinatorial experiments, [Y32Q, D158A, E107T, L162P], by Wang
et al.
[Wang, L., Brock, A., Herberich, B. & Schultz, P. G. (2001)
Scienc
e 292, 498–500]. We predict that the new one will have much greater activity while retaining significant discrimination between
O
-methyl-
l
-tyrosine and tyrosine.
Impaired cardiac hypertrophic response in
Calcineurin Aβ
-deficient mice
Authors: Bueno Orlando F. , Wilkins Benjamin J. , Tymitz Kevin M. , Glascock Betty J. , Kimball Thomas F. , Lorenz John N. , Molkentin Jeffery D.
Published: July 2002

Calcineurin is a calcium–calmodulin-regulated, serine–threonine phosphatase that functions as a key inducer of stress responsive gene expression in multiple cell types through a direct activation of nuclear factor of activated T cells and myocyte enhancer factor 2 transcription factors. In cardiomyocytes, calcineurin signaling has been implicated in the regulation of the hypertrophic response caused by pressure overload or neuroendocrine stimulation. Three separate genes encode the catalytic subunit of calcineurin in mammalian cells,
CnAα, CnAβ,
and
CnAγ
. To evaluate the necessary function of calcineurin as a hypertrophic regulatory factor, the
CnAβ
gene was disrupted in the mouse.
CnAβ
-deficient mice were viable, fertile, and overtly normal well into adulthood, but displayed a 80% decrease in calcineurin enzymatic activity in the heart that was associated with a 12% reduction in basal heart size.
CnAβ
-deficient mice were dramatically impaired in their ability to mount a productive hypertrophic response induced by pressure overload, angiotensin II infusion, or isoproterenol infusion. Analysis of marker genes associated with the hypertrophic response revealed a partial defect in the molecular program of hypertrophy. Collectively, these data solidify the hypothesis that calcineurin functions as a central regulator of the cardiac hypertrophic growth response
in vivo
.
Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase
Authors: Barradeau Sébastien , Imaizumi-Scherrer Tereza , Weiss Mary C. , Faust Daniela M.
Published: July 2002

In skeletal muscle, transcription of the gene encoding the mouse
type Iα (RIα) subunit of the cAMP-dependent protein kinase is
initiated from the alternative noncoding first exons 1a and 1b. Here,
we report that activity of the promoter upstream of exon 1a (Pa)
depends on two adjacent E boxes (E1 and E2) in NIH 3T3-transfected
fibroblasts as well as in intact muscle. Both basal activity and MyoD
transactivation of the Pa promoter require binding of the upstream
stimulating factors (USF) to E1. E2 binds either an unknown protein in
a USF/E1 complex-dependent manner or MyoD. Both E2-bound proteins
seem to function as repressors, but with different strengths, of the
USF transactivation potential. Previous work has shown localization of
the RIα protein at the neuromuscular junction. Using DNA injection
into muscle of plasmids encoding segments of RIα or RIIα fused to
green fluorescent protein, we demonstrate that anchoring at the
neuromuscular junction is specific to RIα subunits and requires the
amino-terminal residues 1–81. Mutagenesis of Phe-54 to Ala in the
full-length RIα–green fluorescent protein template abolishes
localization, indicating that dimerization of RIα is essential for
anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27,
are crucial for localization of RIα at the neuromuscular junction.
These amino acids are involved in the interaction of the
Caenorhabditis elegans
type Iα
homologue R
CE
with AKAP
CE
and for
in
vitro
binding of RIα to dual A-kinase anchoring protein 1. We
also show enrichment of dual A-kinase anchoring protein 1 at the
neuromuscular junction, suggesting that it could be responsible for
RIα tethering at this site.
The Huntington's disease protein interacts with p53 and CREB-binding protein and represses transcription
Authors: Steffan Joan S. , Kazantsev Aleksey , Spasic-Boskovic Olivera , Greenwald Marilee , Zhu Ya-Zhen , Gohler Heike , Wanker Erich E. , Bates Gillian P. , Housman David E. , Thompson Leslie M.
Published: July 2002

Huntington's Disease (HD) is caused by an expansion of a polyglutamine tract within the huntingtin (htt) protein. Pathogenesis in HD appears to include the cytoplasmic cleavage of htt and release of an amino-terminal fragment capable of nuclear localization. We have investigated potential consequences to nuclear function of a pathogenic amino-terminal region of htt (httex1p) including aggregation, protein–protein interactions, and transcription. httex1p was found to coaggregate with p53 in inclusions generated in cell culture and to interact with p53
in vitro
and in cell culture. Expanded httex1p represses transcription of the p53-regulated promoters,

p21
WAF1/CIP1

and
MDR-1
. httex1p was also found to interact
in vitro
with CREB-binding protein (CBP) and mSin3a, and CBP to localize to neuronal intranuclear inclusions in a transgenic mouse model of HD. These results raise the possibility that expanded repeat htt causes aberrant transcriptional regulation through its interaction with cellular transcription factors which may result in neuronal dysfunction and cell death in HD.
Lessons from the past: Evolutionary impacts of mass extinctions
Authors: Jablonski David
Published: July 2002

Mass extinctions have played many evolutionary roles, involving differential survivorship or selectivity of taxa and traits, the disruption or preservation of evolutionary trends and ecosystem organization, and the promotion of taxonomic and morphological diversifications—often along unexpected trajectories—after the destruction or marginalization of once-dominant clades. The fossil record suggests that survivorship during mass extinctions is not strictly random, but it often fails to coincide with factors promoting survival during times of low extinction intensity. Although of very serious concern, present-day extinctions have not yet achieved the intensities seen in the Big Five mass extinctions of the geologic past, which each removed ≥50% of the subset of relatively abundant marine invertebrate genera. The best comparisons for predictive purposes therefore will involve factors such as differential extinction intensities among regions, clades, and functional groups, rules governing postextinction biotic interchanges and evolutionary dynamics, and analyses of the factors that cause taxa and evolutionary trends to continue unabated, to suffer setbacks but resume along the same trajectory, to survive only to fall into a marginal role or disappear (“dead clade walking”), or to undergo a burst of diversification. These issues need to be addressed in a spatially explicit framework, because the fossil record suggests regional differences in postextinction diversification dynamics and biotic interchanges. Postextinction diversifications lag far behind the initial taxonomic and morphological impoverishment and homogenization; they do not simply reoccupy vacated adaptive peaks, but explore opportunities as opened and constrained by intrinsic biotic factors and the ecological and evolutionary context of the radiation.
Attenuated
APC
alleles produce functional protein from internal translation initiation
Authors: Heppner Goss Kathleen , Trzepacz Chris , Tuohy Thérèse M. F. , Groden Joanna
Published: July 2002

Some truncating mutations of the
APC
tumor suppressor gene are associated with an attenuated phenotype of familial adenomatous polyposis coli (AAPC). This work demonstrates that
APC
alleles with 5′ mutations produce APC protein that down-regulates β-catenin, inhibits β-catenin/T cell factor-mediated transactivation, and induces cell-cycle arrest. Transfection studies demonstrate that cap-independent translation is initiated internally at an AUG at codon 184 of
APC
. Furthermore,
APC
coding sequence between
AAPC
mutations and AUG 184 permits internal ribosome entry in a bicistronic vector. These data suggest that
AAPC
alleles
in vivo
may produce functional APC by internal initiation and establish a functional correlation between 5′
APC
mutations and their associated clinical phenotype.
Localization of GAR transformylase in
Escherichia coli
and mammalian cells
Authors: Gooljarsingh Lata T. , Ramcharan Joseph , Gilroy Simon , Benkovic Stephen J.
Published: July 2002

Enzymes of the
de novo
purine biosynthetic pathway
may form a multienzyme complex to facilitate substrate flux through the
ten serial steps constituting the pathway. One likely strategy for
complex formation is the use of a structural scaffold such as the
cytoskeletal network or subcellular membrane of the cell to mediate
protein–protein interactions. To ascertain whether this strategy
pertains to the
de novo
purine enzymes, the localization
pattern of the third purine enzyme, glycinamide ribonucleotide
transformylase (GAR Tfase) was monitored in live
Escherichia
coli
and mammalian cells. Genes encoding human as well as
E. coli
GAR Tfase fused with green fluorescent protein
(GFP) were introduced into their respective cells with regulated
expression of proteins and localization patterns monitored by using
confocal fluorescence microscopy. In both instances images showed
proteins to be diffused throughout the cytoplasm. Thus, GAR Tfase is
not localized to an existing cellular architecture, so this device is
probably not used to concentrate the members of the pathway. However,
discrete clusters of the pathway may still exist throughout the
cytoplasm.
Evolution and divergence of sodium channel genes in vertebrates
Authors: Lopreato Gregory F. , Lu Ying , Southwell Amber , Atkinson Nigel S. , Hillis David M. , Wilcox Thomas P. , Zakon Harold H.
Published: July 2002

Invertebrate species possess one or two Na
+
channel genes, yet there are 10 in mammals. When did this explosive growth come about during vertebrate evolution? All mammalian Na
+
channel genes reside on four chromosomes. It has been suggested that this came about by multiple duplications of an ancestral chromosome with a single Na
+
channel gene followed by tandem duplications of Na
+
channel genes on some of these chromosomes. Because a large-scale expansion of the vertebrate genome likely occurred before the divergence of teleosts and tetrapods, we tested this hypothesis by cloning Na
+
channel genes in a teleost fish. Using an approach designed to clone all of the Na
+
channel genes in a genome, we found six Na
+
channel genes. Phylogenetic comparisons show that each teleost gene is orthologous to a Na
+
channel gene or gene cluster on a different mammalian chromosome, supporting the hypothesis that four Na
+
channel genes were present in the ancestors of teleosts and tetrapods. Further duplications occurred independently in the teleost and tetrapod lineages, with a greater number of duplications in tetrapods. This pattern has implications for the evolution of function and specialization of Na
+
channel genes in vertebrates. Sodium channel genes also are linked to homeobox (Hox) gene clusters in mammals. Using our phylogeny of Na
+
channel genes to independently test between two models of Hox gene evolution, we support the hypothesis that Hox gene clusters evolved as (AB) (CD) rather than {D[A(BC)]}.
In vitrogenerated antibodies specific for telomeric guanine-quadruplex DNA react withStylonychia lemnaemacronuclei
Authors: Schaffitzel Christiane , Berger Imre , Postberg Jan , Hanes Jozef , Lipps Hans J. , Plückthun Andreas
Published: July 2002

Most eukaryotic telomeres contain a repeating motif with stretches of guanine residues that form a 3′-terminal overhang extending beyond the telomeric duplex region. The telomeric repeat of hypotrichous ciliates, d(T4G4), forms a 16-nucleotide 3′-overhang. Such sequences can adopt parallel-stranded as well as antiparallel-stranded quadruplex conformationsin vitro. Although it has been proposed that guanine-quadruplex conformations may have important cellular roles including telomere function, recombination, and transcription, evidence for the existence of this DNA structurein vivohas been elusive to date. We have generated high-affinity single-chain antibody fragment (scFv) probes for the guanine-quadruplex formed by theStylonychiatelomeric repeat, by ribosome display from the Human Combinatorial Antibody Library. Of the scFvs selected, one (Sty3) had an affinity ofKd= 125 pM for the parallel-stranded guanine-quadruplex and could discriminate with at least 1,000-fold specificity between parallel or antiparallel quadruplex conformations formed by the same sequence motif. A second scFv (Sty49) bound both the parallel and antiparallel quadruplex with similar (Kd= 3–5 nM) affinity. Indirect immunofluorescence studies show that Sty49 reacts specifically with the macronucleus but not the micronucleus ofStylonychia lemnae. The replication band, the region where replication and telomere elongation take place, was also not stained, suggesting that the guanine-quadruplex is resolved during replication. Our results provide experimental evidence that the telomeres ofStylonychiamacronuclei adoptin vivoa guanine-quadruplex structure, indicating that this structure may have an important role for telomere functioning.
Crystal structure of the dimeric extracellular domain of human carbonic anhydrase XII, a bitopic membrane protein overexpressed in certain cancer tumor cells
Authors: Whittington Douglas A. , Waheed Abdul , Ulmasov Barbara , Shah Gul N. , Grubb Jeffrey H. , Sly William S. , Christianson David W.
Published: July 2002

Overexpression of the zinc enzyme carbonic anhydrase (CA; EC
4.2.1.1
) XII is observed in certain human cancers. This bitopic
membrane protein contains an N-terminal extracellular catalytic domain,
a membrane-spanning α-helix, and a small intracellular C-terminal
domain. We have determined the three-dimensional structure of the
extracellular catalytic domain of human CA XII by x-ray
crystallographic methods at 1.55-Å resolution. The structure reveals a
prototypical CA fold; however, two CA XII domains associate to form an
isologous dimer, an observation that is confirmed by studies of the
enzyme in solution. The identification of signature GXXXG and GXXXS
motifs in the transmembrane sequence that facilitate helix–helix
association is additionally consistent with dimeric architecture. The
dimer interface is situated so that the active site clefts of each
monomer are clearly exposed on one face of the dimer, and the C termini
are located together on the opposite face of the dimer to facilitate
membrane interaction. The amino acid composition of the active-site
cleft closely resembles that of the other CA isozymes in the immediate
vicinity of the catalytic zinc ion, but differs in the region of the
nearby α-helical “130's segment.” The structure of the CA
XII–acetazolamide complex is also reported at 1.50-Å resolution, and
prospects for the design of CA XII-specific inhibitors of possible
chemotherapeutic value are discussed.
Attributes of γδ intraepithelial lymphocytes as suggested by their transcriptional profile
Authors: Fahrer Aude M. , Konigshofer Yves , Kerr Elizabeth M. , Ghandour Ghassan , Mack David H. , Davis Mark M. , Chien Yueh-hsiu
Published: July 2002

γδ T lymphocytes in the intestinal intraepithelial layer (γδ IELs) are thought to contribute to immune competence, but their actual function remains poorly understood. Here we used DNA microarrays to study the gene expression profile of γδ IELs in aYersiniainfection system to better define their roles. To validate this approach, mesenteric lymph node CD8+αβ T cells were similarly analyzed. The transcription profiles show that, whereas lymph node CD8+αβ T cells must be activated to become cytotoxic effectors, γδ IELs are constitutively activated and appear to use different signaling cascades. Our data suggest that γδ IELs may respond efficiently to a broad range of pathological situations irrespective of their diverse T cell antigen receptor repertoire. γδ IELs may modulate local immune responses and participate in intestinal lipid metabolism, cholesterol homeostasis, and physiology. This study provides a strong basis for further investigations of the roles of these cells as well as mucosal immune defense in general.
Nitric oxide-induced apoptosis in pancreatic β cells is mediated by the endoplasmic reticulum stress pathway
Authors: Oyadomari Seiichi , Takeda Kiyoshi , Takiguchi Masaki , Gotoh Tomomi , Matsumoto Makoto , Wada Ikuo , Akira Shizuo , Araki Eiichi , Mori Masataka
Published: July 2002

Excessive nitric oxide (NO) production in cytokine-activated β cells has been implicated in β cell disruption in type 1 diabetes. β cells are very vulnerable to NO-induced apoptosis. However, the mechanism underlying this phenomenon is unclear. Low concentrations of NO that lead to apoptosis apparently do not cause severe DNA damage in mouse MIN6 β cells. CHOP, a C/EBP homologous protein that is induced by endoplasmic reticulum (ER) stress and plays a role in growth arrest and cell death, was induced by a NO donor,
S
-nitroso-
N
-acetyl-
d,l
-penicillamine (SNAP). SNAP increased cytosolic Ca
2+
, and only agents depleting ER Ca
2+
induced CHOP expression and led to apoptosis, suggesting that NO depletes ER Ca
2+
. Overexpression of calreticulin increased the Ca
2+
content of ER and afforded protection to cells against NO-mediated apoptosis. Furthermore, pancreatic islets from CHOP knockout mice showed resistance to NO. We conclude that NO depletes ER Ca
2+
, causes ER stress, and leads to apoptosis. Thus, ER Ca
2+
stores are a new target of NO, and the ER stress pathway is a major mechanism of NO-mediated β cell apoptosis.
Functional cloning and characterization of a UDP- glucuronic acid decarboxylase: The pathogenic fungus
Cryptococcus neoformans
elucidates UDP-xylose synthesis
Authors: Bar-Peled Maor , Griffith Cara L. , Doering Tamara L.
Published: July 2002

UDP-xylose is a sugar donor required for the synthesis of diverse and important glycan structures in animals, plants, fungi, and bacteria. Xylose-containing glycans are particularly abundant in plants and in the polysaccharide capsule that is the major virulence factor of the pathogenic fungus
Cryptococcus neoformans
. Biosynthesis of UDP-xylose is mediated by UDP-glucuronic acid decarboxylase, which converts UDP-glucuronic acid to UDP-xylose. Although this enzymatic activity was described over 40 years ago it has never been fully purified, and the gene encoding it has not been identified. We used homology to a bacterial gene, hypothesized to encode a related function, to identify a cryptococcal sequence as putatively encoding a UDP-glucuronic acid decarboxylase. A soluble 47-kDa protein derived from bacteria expressing the
C. neoformans
gene catalyzed conversion of UDP-glucuronic acid to UDP-xylose, as confirmed by NMR analysis. NADH, UDP, and UDP-xylose inhibit the activity. Close homologs of the cryptococcal gene, which we termed
UXS1
, appear in genome sequence data from organisms ranging from bacteria to humans.
Allurin, a 21-kDa sperm chemoattractant from
Xenopus
egg jelly, is related to mammalian sperm-binding proteins
Authors: Olson John H. , Xiang Xueyu , Ziegert Tillmann , Kittelson Andrew , Rawls Alan , Bieber Allan L. , Chandler Douglas E.
Published: July 2002

Previously, we demonstrated that a protein from
Xenopus
egg jelly exhibits sperm chemoattractant
activity when assayed by either video microscopy or by sperm passage
across a porous filter. Here we describe the isolation and purification
of allurin, the protein responsible for this activity. Freshly
oviposited jellied eggs were soaked in buffer, and the conditioned
medium was loaded onto an anion exchange column and eluted with an NaCl
gradient. The active fraction was purified further by RP-HPLC, the
chemoattractant protein appearing as a single sharp peak. The amino
acid sequence of the protein, determined by direct sequencing and
cloning of cDNAs coding for the protein, consisted of 184 amino acids
having a molecular mass of 21,073 Da. The protein shares homology with
the mammalian cysteine-rich secretory protein (CRISP) family that
includes testes-specific spermatocyte protein 1, a cell adhesion
protein which links spermatocytes to Seritoli cells, and acidic
epididymal glycoproteins that bind to sperm and have been implicated in
sperm–egg fusion. Phylogenetic analysis suggests that allurin evolved
from the ancestral protein that gave rise to the mammalian CRISP
family. Addition of allurin to this family portends that the CRISP
family represents a group of “sperm escort” proteins, which bind
to sperm at various steps in their life history, facilitating passage
from one functional stage to the next. Allurin stands out in this
regard, representing both the first vertebrate sperm chemoattractant to
be purified and sequenced and the first member of the CRISP family to
be found in the female reproductive tract.
Genome sequence of an industrial microorganism
Streptomyces avermitilis
: Deducing the ability of producing secondary metabolites
Authors: Ōmura Satoshi , Ikeda Haruo , Ishikawa Jun , Hanamoto Akiharu , Takahashi Chigusa , Shinose Mayumi , Takahashi Yoko , Horikawa Hiroshi , Nakazawa Hidekazu , Osonoe Tomomi , Kikuchi Hisashi , Shiba Tadayoshi , Sakaki Yoshiyuki , Hattori Masahira
Published: July 2002

Streptomyces avermitilis
is a soil bacterium that carries out not only a complex morphological differentiation but also the production of secondary metabolites, one of which, avermectin, is commercially important in human and veterinary medicine. The major interest in this genus
Streptomyces
is the diversity of its production of secondary metabolites as an industrial microorganism. A major factor in its prominence as a producer of the variety of secondary metabolites is its possession of several metabolic pathways for biosynthesis. Here we report sequence analysis of
S. avermitilis
, covering 99% of its genome. At least 8.7 million base pairs exist in the linear chromosome; this is the largest bacterial genome sequence, and it provides insights into the intrinsic diversity of the production of the secondary metabolites of
Streptomyces
. Twenty-five kinds of secondary metabolite gene clusters were found in the genome of
S. avermitilis
. Four of them are concerned with the biosyntheses of melanin pigments, in which two clusters encode tyrosinase and its cofactor, another two encode an ochronotic pigment derived from homogentiginic acid, and another polyketide-derived melanin. The gene clusters for carotenoid and siderophore biosyntheses are composed of seven and five genes, respectively. There are eight kinds of gene clusters for type-I polyketide compound biosyntheses, and two clusters are involved in the biosyntheses of type-II polyketide-derived compounds. Furthermore, a polyketide synthase that resembles phloroglucinol synthase was detected. Eight clusters are involved in the biosyntheses of peptide compounds that are synthesized by nonribosomal peptide synthetases. These secondary metabolite clusters are widely located in the genome but half of them are near both ends of the genome. The total length of these clusters occupies about 6.4% of the genome.
A newly discovered bacterium associated with parthenogenesis and a change in host selection behavior in parasitoid wasps
Authors: Zchori-Fein E. , Gottlieb Y. , Kelly S. E. , Brown J. K. , Wilson J. M. , Karr T. L. , Hunter M. S.
Published: July 2002

The symbiotic bacteriumWolbachia pipientishas been considered unique in its ability to cause multiple reproductive anomalies in its arthropod hosts. Here we report that an undescribed bacterium is vertically transmitted and associated with thelytokous parthenogenetic reproduction inEncarsia, a genus of parasitoid wasps. AlthoughWolbachiawas found in only one of seven parthenogeneticEncarsiapopulations examined, the “Encarsiabacterium” (EB) was found in the other six. Among seven sexually reproducing populations screened, EB was present in one, and none harboredWolbachia. Antibiotic treatment did not induce male production inEncarsia pergandiellabut changed the oviposition behavior of females. Cured females accepted one host type at the same rate as control females but parasitized significantly fewer of the other host type. Phylogenetic analysis based on the 16S rDNA gene sequence places the EB in a unique clade within the Cytophaga-Flexibacter-Bacteroid group and shows EB is unrelated to the Proteobacteria, whereWolbachiaand most other insect symbionts are found. These results imply evolution of the induction of parthenogenesis in a lineage other thanWolbachia. Importantly, these results also suggest that EB may modify the behavior of its wasp carrier in a way that enhances its transmission.
JAC
, a direct target of oncogenic transcription factor Jun, is involved in cell transformation and tumorigenesis
Authors: Hartl Markus , Reiter Fritz , Bader Andreas G. , Castellazzi Marc , Bister Klaus
Published: July 2002

Using subtractive hybridization techniques, we have isolated a gene termed
JAC
that is strongly and specifically activated in avian fibroblasts transformed by the v-
jun
oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore,
JAC
is highly expressed in cell lines derived from
jun
-induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb
JAC
mRNA is induced rapidly upon activation of the oncogenic v-
jun
allele. Nucleotide sequence analysis and transcriptional mapping revealed that the
JAC
gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian high-sulfur keratin-associated proteins. The promoter region of
JAC
contains a consensus (5′-TGACTCA-3′) and a nonconsensus (5′-TGAGTAA-3′) AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. Mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of
JAC
in avian fibroblasts leads to anchorage-independent growth, strongly suggesting that deregulation of
JAC
is an essential event in
jun
-induced cell transformation and tumorigenesis.
An alpha-mercaptoacrylic acid derivative is a selective nonpeptide cell-permeable calpain inhibitor and is neuroprotective.
Authors: Wang K K , Nath R , Posner A , Raser K J , Buroker-Kilgore M , Hajimohammadreza I , Probert A W , Marcoux F W , Ye Q , Takano E , Hatanaka M , Maki M , Caner H , Collins J L , Fergus A , Lee K S , Lunney E A , Hays S J , Yuen P
Published: July 2002

Overactivation of calcium-activated neutral protease (calpain) has been implicated in the pathophysiology of several degenerative conditions, including stroke, myocardial ischemia, neuromuscular degeneration, and cataract formation. Alpha-mercaptoacrylate derivatives (exemplified by PD150606), with potent and selective inhibitory actions against calpain, have been identified. PD150606 exhibits the following characteristics: (i) Ki values for mu- and m-calpains of 0.21 microM and 0.37 microM, respectively, (ii) high specificity for calpains relative to other proteases, (iii) uncompetitive inhibition with respect to substrate, and (iv) it does not shield calpain against inactivation by the active-site inhibitor trans-(epoxysuccinyl)-L-leucyl-amido-3-methylbutane, suggesting a nonactive site action for PD150606. The recombinant calcium-binding domain from each of the large or small subunits of mu-calpain was found to interact with PD150606. In low micromolar range, PD15O6O6 inhibited calpain activity in two intact cell systems. The neuroprotective effects of this class of compound were also demonstrated by the ability of PD150606 to attenuate hypoxic/hypoglycemic injury to cerebrocortical neurons in culture and excitotoxic injury to Purkinje cells in cerebellar slices.
Dimorphism and haploid fruiting in Cryptococcus neoformans: association with the alpha-mating type.
Authors: Wickes B L , Mayorga M E , Edman U , Edman J C
Published: July 2002

Cryptococcus neoformans is a major opportunistic fungal pathogen in AIDS and other immunosuppressed patients. We have shown that wild-type haploid C. neoformans can develop an extensive hyphal phase under appropriate conditions. Hyphae produced under these conditions are monokaryotic, possess unfused clamp connections, and develop basidia with viable basidiospores. The ability to undergo this transition is determined by the presence of the alpha-mating type locus and is independent of serotype. The association of the hyphal phase with the alpha-mating type may explain the preponderance of this mating type in the environment and the nature of the infectious propagule of C. neoformans.
Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.
Authors: Chattoraj M , King B A , Bublitz G U , Boxer S G
Published: July 2002

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.
Negative-strand RNA viruses: genetic engineering and applications.
Authors: Palese P , Zheng H , Engelhardt O G , Pleschka S , García-Sastre A
Published: July 2002

The negative-strand RNA viruses are a broad group of animal viruses that comprise several important human pathogens, including influenza, measles, mumps, rabies, respiratory syncytial, Ebola, and hantaviruses. The development of new strategies to genetically manipulate the genomes of negative-strand RNA viruses has provided us with new tools to study the structure-function relationships of the viral components and their contributions to the pathogenicity of these viruses. It is also now possible to envision rational approaches--based on genetic engineering techniques--to design live attenuated vaccines against some of these viral agents. In addition, the use of different negative-strand RNA viruses as vectors to efficiently express foreign polypeptides has also become feasible, and these novel vectors have potential applications in disease prevention as well as in gene therapy.
hMSH2 forms specific mispair-binding complexes with hMSH3 and hMSH6
Authors: Acharya Samir , Wilson Teresa , Gradia Scott , Kane Michael F. , Guerrette Shawn , Marsischky Gerald T. , Kolodner Richard , Fishel Richard
Published: July 2002

The genetic and biochemical properties of three human MutS
homologues, hMSH2, hMSH3, and hMSH6, have been examined. The
full-length hMSH6 cDNA and genomic locus were isolated and
characterized, and it was demonstrated that the
hMSH6
gene consisted of 10 exons and mapped to chromosome 2p15-16. The hMSH3
cDNA was in some cases found to contain a 27-bp deletion resulting in a
loss of nine amino acids, depending on the individual from which the
cDNA was isolated. hMSH2, hMSH3, and hMSH6 all showed similar
tissue-specific expression patterns. hMSH2 protein formed a complex
with both hMSH3 and hMSH6 proteins, similar to protein complexes
demonstrated by studies of the
Saccharomyces cerevisiae
MSH2, MSH3, and MSH6. hMSH2 was also found to form a homomultimer
complex, but neither hMSH3 nor hMSH6 appear to interact with themselves
or each other. Analysis of the mismatched nucleotide-binding
specificity of the hMSH2–hMSH3 and hMSH2–hMSH6 protein complexes
showed that they have overlapping but not identical binding
specificity. These results help to explain the distribution of
mutations in different mismatch-repair genes seen in hereditary
nonpolyposis colon cancer.