bluuebunny/crossref_metadata_2025 huggingface dataset:
Solid phase epitaxial regrowth of boron-doped polycrystalline silicon deposited by low-pressure chemical vapor deposition
Authors: Ghannam M. Y. , Dutton R. W.
Published: July 2002

Solid phase epitaxial regrowth of polycrystalline silicon deposited on ‘‘oxide-free’’ (100) oriented single-crystal substrates and implanted with boron is investigated by means of secondary ion mass spectrometry, Rutherford backscattering channeling, and transmission electron microscopy. The effects of annealing ambients and heavy doping on the regrowth rate are also studied.
Atomic force microscopy of liquid-covered surfaces: Atomic resolution images
Authors: Marti O. , Drake B. , Hansma P. K.
Published: July 2002

Images of graphite surfaces that are covered with oil reveal the hexagonal rings of carbon atoms. Images of a sodium chloride surface, protected from moisture by oil, exhibit a monoatomic step. Together, these images demonstrate the potential of atomic force microscopy (AFM) for studying both conducting and nonconducting surfaces, even surfaces covered with liquids. Our AFM uses a cross of double wires with an attached diamond stylus as a force sensor. The force constant is ≊40 N/m. The resonant frequency is ≊3 kHz. The lateral and vertical resolutions are 0.15 nm and 5 pm.
Electrical properties of shallow p+-n junctions formed by BF2 ion implantation in germanium preamorphized silicon
Authors: Ozturk M. C. , Wortman J. J.
Published: July 2002

Shallow p+-n junctions were formed by BF2 ion implantation into both crystalline and Ge preamorphized substrates. Rapid thermal annealing (RTA) was used for dopant activation and damage removal. Secondary ion mass spectroscopy (SIMS) was used to measure the boron and fluorine distribution profiles. Based on SIMS analysis, junction depths as shallow as 0.12 μm can be formed using 25 keV/1×1015 cm−2 BF2 ion implantation into Ge preamorphized Si. The influence of Ge implantation parameters on the leakage current of junctions was investigated. The results show that if the Ge implantation conditions are optimized, high quality p+-n junctions can be formed in preamorphized substrates using RTA temperatures as low as 950 °C.
Evolution of sociality in a primitively eusocial lineage of bees
Authors: Danforth Bryan N.
Published: July 2002

Eusociality is a major evolutionary innovation involving alterations in life history, morphology, and behavior. Advanced eusocial insects, such as ants, termites, and corbiculate bees, cannot provide insights into the earliest stages of eusocial evolution because eusociality in these taxa evolved long ago (in the Cretaceous) and close solitary relatives are no longer extant. In contrast, primitively eusocial insects, such as halictid bees, provide insights into the early stages of eusocial evolution because eusociality has arisen recently and repeatedly. By mapping social behavior onto well-corroborated phylogenies, I show that eusociality has arisen only three times within halictid bees (contrary to earlier estimates of six or more origins). Reversals from eusocial to solitary behavior have occurred as many as 12 times, indicating that social reversals are common in the earliest stages of eusocial evolution. Important attributes of social complexity (e.g., colony size, queen/worker dimorphism) show no obvious association with phylogeny, and some reversals to solitary nesting are related to host-plant switches (from polylecty to oligolecty). These results provide a glimpse of social evolution in its earliest stages and provide insights into the early evolution of advanced eusocial organisms.
A genome-scale analysis for identification of genes required for growth or survival of
Haemophilus
influenzae
Authors: Akerley Brian J. , Rubin Eric J. , Novick Veronica L. , Amaya Kensey , Judson Nicholas , Mekalanos John J.
Published: July 2002

A high-density transposon mutagenesis strategy was applied to the
Haemophilus influenzae
genome to identify genes required for growth or viability. This analysis detected putative essential roles for the products of 259 ORFs of unknown function. Comparisons between complete genomes defined a subset of these proteins in
H. influenzae
having homologs in
Mycobacterium tuberculosis
that are absent in
Saccharomyces cerevisiae
, a distribution pattern that favors their use in development of antimicrobial therapeutics. Three genes within this set are essential for viability in other bacteria. Interfacing the set of essential gene products in
H. influenzae
with the distribution of homologs in other microorganisms can detect components of unrecognized cellular pathways essential in diverse bacteria. This genome-scale phenotypic analysis identifies potential roles for a large set of genes of unknown function.
Molecular basis of a progressive juvenile-onset hereditary cataract
Authors: Pande Ajay , Pande Jayanti , Asherie Neer , Lomakin Aleksey , Ogun Olutayo , King Jonathan A. , Lubsen Nicolette H. , Walton David , Benedek George B.
Published: July 2002

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human γD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J.,
et al
. (1999)
Proc. Natl. Acad. Sci. USA
96, 1008–1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human γD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in
Escherichia coli
and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.
The EntF and EntE adenylation domains of
Escherichia coli
enterobactin synthetase: Sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates
Authors: Ehmann David E. , Shaw-Reid Cathryn A. , Losey Heather C. , Walsh Christopher T.
Published: July 2002

Enterobactin, the
tris-(
N
-(2,3-dihydroxybenzoyl)serine) trilactone
siderophore of
Escherichia coli
, is synthesized by a
three-protein (EntE, B, F) six-module nonribosomal peptide synthetase
(NRPS). In this work, the 142-kDa four-domain protein EntF was bisected
into two double-domain fragments: a 108-kDa condensation and
adenylation construct, EntF C-A, and a 37-kDa peptidyl carrier protein
(PCP) and thioesterase protein, EntF PCP-TE. The adenylation domain
activity of EntF C-A formed seryl-AMP but lost the ability to transfer
the seryl moiety to the cognate EntF PCP-TE in
trans
.
Seryl transfer to heterologous PCP protein fragments, the SrfB1 PCP
from surfactin synthetase and Ybt PCP1 from yersiniabactin synthetase,
was observed at rates of 0.5 min
−1
and 0.01
min
−1
, respectively. The possibility that these slow
acylation rates reflected dissociation of acyl/aminoacyl-AMP followed
by adventitious thiolation by the heterologous PCPs in solution was
addressed by measuring catalytic turnover of pyrophosphate
(PP
i
) released from the adenylation domain. The holo SrfB1
PCP protein as well as Ybt PCP1 did not stimulate an increase in
PP
i
release from EntF C-A or EntE. In this light,
aminoacylations in
trans
between A and PCP domain
fragments of NRPS assembly lines must be subjected to kinetic scrutiny
to determine whether transfer is truly between protein domains or
results from slow aminoacyl-AMP release and subsequent nonenzymatic
thiol capture.
Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice
Authors: Hurwitz Arthur A. , Sullivan Timothy J. , Sobel Raymond A. , Allison James P.
Published: July 2002

We and others previously reported that cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the severity of peptide-induced experimental autoimmune encephalomyelitis (EAE) in mouse strains that are inherently susceptible to the disease. In this report, we show that CTLA-4 engagement also controls disease susceptibility in BALB/c mice, a strain considered to be resistant to EAE induction. Although immunization of BALB/c mice with syngeneic spinal cord homogenate or an I-Ad-binding myelin peptide antigen failed to result in EAE, immunization with either antigen preparation in conjunction with anti-CTLA-4 resulted in both clinical and histological EAE. CTLA-4 blockade also resulted in a preferential increase in the frequency of antigen-specific T cells secreting IFN-γ. We conclude that CTLA-4 controls susceptibility in BALB/c mice by limiting the expansion of autoreactive T cells present in the periphery, suggesting a mechanism whereby CTLA-4 contributes to the maintenance of peripheral T cell tolerance to self antigens.
Dynamics of bacterial phenotype selection in a colonized host
Authors: Webb G. F. , Blaser M. J.
Published: July 2002

The population dynamics of
Helicobacter pylori
during colonization in an infected animal host provide a quantifiable experimental model of
in vivo
microbial phenotype evolution. Phenotype variability in
H. pylori
populations can be typed as polymorphic expression of Lewis antigens on their cell surfaces. The high mutational frequency of
H. pylori
for Lewis expression provides substrate for differential selection by the host. Experimental challenge and successful colonization of mice and gerbils allows tracking of
H. pylori
phenotype variability from the initial inoculation to the ultimate establishment of a quasispecies. Colonization data provide a quantitative experimental model of phenotype evolution in a relatively large population (>10
4
individuals) over a relatively long evolutionary time scale (>10
3
generations). A mathematical model is developed to interpret the data in terms of the dynamic processes occuring during colonization. The mathematical model distinguishes the roles of selection and mutation; quantifies the effects of initial phenotype diversity, mutational frequency, and selective advantage; and applies generally to phenotype evolution in biological populations.
Electrostatic stress in catalysis: Structure and mechanism of the enzyme orotidine monophosphate decarboxylase
Authors: Wu Ning , Mo Yirong , Gao Jiali , Pai Emil F.
Published: July 2002

Orotidine 5′-monophosphate decarboxylase catalyzes the conversion of orotidine 5′-monophosphate to uridine 5′-monophosphate, the last step in biosynthesis of pyrimidine nucleotides. As part of a Structural Genomics Initiative, the crystal structures of the ligand-free and the6-azauridine 5′-monophosphate-complexed forms have been determined at 1.8 and 1.5 Å, respectively. The protein assumes a TIM-barrel fold with one side of the barrel closed off and the other side binding the inhibitor. A unique array of alternating charges (Lys-Asp-Lys-Asp) in the active site prompted us to apply quantum mechanical and molecular dynamics calculations to analyze the relative contributions of ground state destabilization and transition state stabilization to catalysis. The remarkable catalytic power of orotidine 5′-monophosphate decarboxylase is almost exclusively achieved via destabilization of the reactive part of the substrate, which is compensated for by strong binding of the phosphate and ribose groups. The computational results are consistent with a catalytic mechanism that is characterized by Jencks's Circe effect.
A protein-based therapeutic for human cytomegalovirus infection
Authors: Jean François , Thomas Laurel , Molloy Sean S. , Liu Gseping , Jarvis Michael A. , Nelson Jay A. , Thomas Gary
Published: July 2002

Current antiviral strategies target viral gene products. Although
initially successful, their severe toxicity and susceptibility to
circumvention by the generation of drug-resistant variants limit their
usefulness. By contrast, the central role of the host cell serine
endoprotease furin in the proteolytic activation of numerous pathogens
points to the endoprotease as a strategic target for therapeutics.
Herein, we show that the production of infectious human cytomegalovirus
is dramatically reduced by exogenous addition of a bioengineered
serpin, α
1
-PDX. This protein is a potent and selective
furin inhibitor (
K
i
= 0.6 nM) and is
10-fold more effective than currently used antiherpetic agents in
cell-culture models. The requirement of furin for the processing of
envelope glycoproteins from many pathogenic viruses and for the
activation of several bacterial toxins suggests that selective
inhibitors of furin have potential as broad-based anti-pathogens.
Dynamic localization of a cytoplasmic signal transduction response regulator controls morphogenesis during the
Caulobacter
cell cycle
Authors: Jacobs Christine , Hung Dean , Shapiro Lucy
Published: July 2002

We present evidence that a bacterial signal transduction cascade
that couples morphogenesis with cell cycle progression is regulated by
dynamic localization of its components. Previous studies have
implicated two histidine kinases, DivJ and PleC, and the response
regulator, DivK, in the regulation of morphogenesis in the dimorphic
bacterium
Caulobacter crescentus
. Here, we show that the
cytoplasmic response regulator, DivK, exhibits a dynamic, cyclical
localization that culminates in asymmetric distribution of DivK within
the two cell types that are characteristic of the
Caulobacter
cell cycle; DivK is dispersed throughout the
cytoplasm of the progeny swarmer cell and is localized to the pole of
the stalked cell. The membrane-bound DivJ and PleC histidine kinases,
which are asymmetrically localized at the opposite poles of the
predivisional cell, control the temporal and spatial localization of
DivK. DivJ mediates DivK targeting to the poles whereas PleC controls
its release from one of the poles at times and places that are
consistent with the activities and location of DivJ and PleC in the
late predivisional cell. Thus, dynamic changes in subcellular location
of multiple components of a signal transduction cascade may constitute
a novel mode of prokaryotic regulation to generate and maintain
cellular asymmetry.
Identification and dissection of an enhancer controlling epithelial gene expression in skin
Authors: Sinha Satrajit , Fuchs Elaine
Published: July 2002

Keratins 14 and 5 are the structural hallmarks of the basal
keratinocytes of the epidermis and outer root sheath (ORS) of the hair
follicle. Their genes are controlled in a tissue-specific manner and
thus serve as useful tools to elucidate the regulatory mechanisms
involved in keratinocyte-specific transcription. Previously we
identified several keratinocyte-specific DNase I hypersensitive sites
(HSs) in the 5′ regulatory sequences of the K14 gene and showed that a
700-bp regulatory domain encompassing HSs II and III can confer
epidermal and ORS-specific gene expression in transgenic mice
in
vivo
. Although HS II harbored much of the transactivation
activity
in vitro
, it was not sufficient to restrict
expression to keratinocytes
in vivo
. We now explore the
HS III regulatory element. Surprisingly, this element on its own
confers gene expression to the keratinocytes of the inner root sheath
(IRS) of the hair follicle, whereas a 275-bp DNA fragment containing
both HSs II and III shifts the expression from the IRS to the basal
keratinocytes and ORS
in vivo
. Electrophoretic
mobility-shift assays and mutational studies of HSs III reveal a role
for CACCC-box binding proteins, Sp1 family members, and other factors
adding to the list of previously described factors that are involved in
keratinocyte-specific gene expression. These studies highlight a
cooperative interaction of the two HSs domains and strengthen the
importance of combinatorial play of transcription factors that govern
keratinocyte-specific gene regulation.
Crystal structure of the yeast cytochrome
bc
1
complex with its bound substrate cytochrome
c
Authors: Lange Christian , Hunte Carola
Published: July 2002

Small diffusible redox proteins facilitate electron transfer in respiration and photosynthesis by alternately binding to integral membrane proteins. Specific and transient complexes need to be formed between the redox partners to ensure fast turnover. In respiration, the mobile electron carrier cytochrome
c
shuttles electrons from the cytochrome
bc
1
complex to cytochrome
c
oxidase. Despite extensive studies of this fundamental step of energy metabolism, the structures of the respective electron transfer complexes were not known. Here we present the crystal structure of the complex between cytochrome
c
and the cytochrome
bc
1
complex from
Saccharomyces cerevisiae.
The complex was crystallized with the help of an antibody fragment, and its structure was determined at 2.97-Å resolution. Cytochrome
c
is bound to subunit cytochrome
c
1
of the enzyme. The tight and specific interactions critical for electron transfer are mediated mainly by nonpolar forces. The close spatial arrangement of the c-type hemes unexpectedly suggests a direct and rapid heme-to-heme electron transfer at a calculated rate of up to 8.3 × 10
6
s
−1
. Remarkably, cytochrome
c
binds to only one recognition site of the homodimeric multisubunit complex. Interestingly, the occupancy of quinone in the Qi site is higher in the monomer with bound cytochrome
c
, suggesting a coordinated binding and reduction of both electron-accepting substrates. Obviously, cytochrome
c
reduction by the cytochrome
bc
1
complex can be regulated in response to respiratory conditions.
Allosteric crosstalk between peptide-binding, transport, and ATP hydrolysis of the ABC transporter TAP
Authors: Gorbulev Stanislav , Abele Rupert , Tampé Robert
Published: July 2002

The transporter associated with antigen processing (TAP) is essential for intracellular transport of protein fragments into the endoplasmic reticulum for loading of major histocompatibility complex (MHC) class I molecules. On the cell surface, these peptide–MHC complexes are monitored by cytotoxic T lymphocytes. To study the ATP hydrolysis of TAP, we developed an enrichment and reconstitution procedure, by which we fully restored TAP function in proteoliposomes. A TAP-specific ATPase activity was identified that could be stimulated by peptides and blocked by the herpes simplex virus protein ICP47. Strikingly, the peptide-binding motif of TAP directly correlates with the stimulation of the ATPase activity, demonstrating that the initial peptide-binding step is responsible for TAP selectivity. ATP hydrolysis follows Michaelis–Menten kinetics with a maximal velocityVmaxof 2 μmol/min per mg TAP, corresponding to a turnover number of approximately 5 ATP per second. This turnover rate is sufficient to account for the role of TAP in peptide loading of MHC molecules and the overall process of antigen presentation. Interestingly, sterically restricted peptides that bind but are not transported by TAP do not stimulate ATPase activity. These results point to coordinated dialogue between the peptide-binding site, the nucleotide-binding domain, and the translocation site via conformational changes within the TAP complex.
Regulation of PKR and IRF-1 during hepatitis C virus RNA replication
Authors: Pflugheber Jill , Fredericksen Brenda , Sumpter Rhea , Wang Chunfu , Ware Felecia , Sodora Donald L. , Gale Michael
Published: July 2002

The virus–host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (
i
) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (
ii
) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.
A peptide-based fluorescence resonance energy transfer assay for
Bacillus anthracis
lethal factor protease
Authors: Cummings Richard T. , Salowe Scott P. , Cunningham Barry R. , Wiltsie Judyann , Park Young Whan , Sonatore Lisa M. , Wisniewski Douglas , Douglas Cameron M. , Hermes Jeffrey D. , Scolnick Edward M.
Published: July 2002

A fluorescence resonance energy transfer assay has been developed for monitoring
Bacillus anthracis
lethal factor (LF) protease activity. A fluorogenic 16-mer peptide based on the known LF protease substrate MEK1 was synthesized and found to be cleaved by the enzyme at the anticipated site. Extension of this work to a fluorogenic 19-mer peptide, derived, in part, from a consensus sequence of known LF protease targets, produced a much better substrate, cleaving approximately 100 times more efficiently. This peptide sequence was modified further on resin to incorporate donor/quencher pairs to generate substrates for use in fluorescence resonance energy transfer-based appearance assays. All peptides cleaved at similar rates with signal/background ranging from 9–16 at 100% turnover. One of these substrates, denoted (Cou)Consensus(K(QSY-35)GG)-NH
2
, was selected for additional assay optimization. A plate-based assay requiring only low nanomolar levels of enzyme was developed for screening and inhibitor characterization.
RAC, a stable ribosome-associated complex in yeast formed by the DnaK-DnaJ homologs Ssz1p and zuotin
Authors: Gautschi Matthias , Lilie Hauke , Fünfschilling Ursula , Mun Andrej , Ross Suzanne , Lithgow Trevor , Rücknagel Peter , Rospert Sabine
Published: July 2002

The yeast cytosol contains multiple homologs of the DnaK and DnaJ
chaperone family. Our current understanding of which homologs
functionally interact is incomplete. Zuotin is a DnaJ homolog bound to
the yeast ribosome. We have now identified the DnaK homolog
Ssz1p/Pdr13p as zuotin's partner chaperone. Zuotin and Ssz1p form a
ribosome-associated complex (RAC) that is bound to the ribosome via the
zuotin subunit. RAC is unique among the eukaryotic DnaK-DnaJ systems,
as the 1:1 complex is stable, even in the presence of ATP or ADP.
In vitro
, RAC stimulates the translocation of a
ribosome-bound mitochondrial precursor protein into mitochondria,
providing evidence for its chaperone-like effect on nascent chains. In
agreement with the existence of a functional complex, deletion of each
RAC subunit resulted in a similar phenotype
in vivo
.
However, overexpression of zuotin partly rescued the growth defect of
the Δ
ssz1
strain, whereas overexpression of Ssz1p did
not affect the Δ
zuo1
strain, suggesting a pivotal
function for the DnaJ homolog.
Inhibition of the expression of penicillin resistance in
Streptococcus pneumoniae
by inactivation of cell wall muropeptide branching genes
Authors: Filipe Sergio R. , Tomasz Alexander
Published: July 2002

Penicillin-resistant strains of
Streptococcus pneumoniae
contain low affinity penicillin-binding proteins and often also produce abnormal indirectly crosslinked cell walls. However the relationship between cell wall abnormality and penicillin resistance has remained obscure. We now show that the genome of
S. pneumoniae
contains an operon composed of two genes (
murM
and
murN
) that encode enzymes involved with the biosynthesis of branched structured cell wall muropeptides. The sequences of
murMN
were compared in two strains: the penicillin-susceptible strain R36A producing the species-specific pneumococcal cell wall peptidoglycan in which branched stem peptides are rare, and the highly penicillin-resistant transformant strain Pen6, the cell wall of which is enriched for branched-structured stem peptides. The two strains carried different
murM
alleles:
murM
of the penicillin-resistant strain Pen6 had a “mosaic” structure encoding a protein that was only 86.5% identical to the product of
murM
identified in the isogenic penicillin-susceptible strain R36A. Mutants of R36A and Pen6 in which the
murMN
operon was interrupted by insertion-duplication mutagenesis produced peptidoglycan from which all branched muropeptide components were missing. The insertional mutant of Pen6 carried a
pbp2x
gene with the same “mosaic” sequence found in Pen6. On the other hand, inactivation of
murMN
in strain Pen6 and other resistant strains caused a virtually complete loss of penicillin resistance. Our observations indicate that the capacity to produce branched cell wall precursors plays a critical role in the expression of penicillin resistance in
S. pneumoniae
.
Increased IGF-II protein affectsp57kip2expressionin vivoandin vitro: Implications for Beckwith–Wiedemann syndrome
Authors: Grandjean Valérie , Smith Janet , Schofield Paul N. , Ferguson-Smith Anne C.
Published: July 2002

In both human and mouse, theIgf2gene, localized on chromosomes 11 and 7, respectively, is expressed from the paternally inherited chromosome in the majority of tissues. Insulin-like growth factor-II (IGF-II) plays an important role in embryonic growth, and aberrantIGF2expression has been documented in several human pathologies, such as Beckwith–Wiedemann syndrome (BWS), and a wide variety of tumors. Human and mouse genetic data strongly implicate another gene,CDKN1C(p57kip2), located in the same imprinted gene cluster on human chromosome II, in BWS. p57KIP2is a cyclin-dependent kinase inhibitor and is required for normal mouse embryonic development. Mutations inCDKN1C(p57kip2) have been identified in a small proportion of patients with BWS, and removal of the gene from mice by targeted mutagenesis produces a phenotype with elements in common with this overgrowth syndrome. Patients with BWS with biallelic expression ofIGF2or with aCDKN1C(p57kip2) mutation, as well as overlapping phenotypes observed in two types of mutant mice, thep57kip2knockout and IGF-II-overexpressing mice, strongly suggest that the genes may act in a common pathway of growth control in situations whereIgf2expression is abnormal. Herein, we show thatp57kip2expression is reduced on IGF-II treatment of primary embryo fibroblasts in a dose-dependent manner. In addition,p57kip2expression is down-regulated in mice with high serum levels of IGF-II. These data suggest that the effects of increased IGF-II in BWS may, in part, be mediated through a decrease inp57kip2gene expression.
Toward rules for 1:1 polyamide:DNA recognition
Authors: Urbach Adam R. , Dervan Peter B.
Published: July 2002

Polyamides composed of four amino acids, imidazole (Im), pyrrole
(Py), hydroxypyrrole (Hp), and β-alanine (β), are synthetic ligands
that form highly stable complexes in the minor groove of DNA. Although
specific pairing rules within the 2:1 motif can be used to distinguish
the four Watson⋅Crick base pairs, a comparable recognition code
for 1:1 polyamide:DNA complexes had not been described. To set a
quantitative baseline for the field, the sequence specificities of Im,
Py, Hp, and β for the four Watson⋅Crick base pairs were
determined for two polyamides, Im-β-ImPy-β-Im-β-ImPy-β-Dp (1,
for Im, Py, and β) and Im-β-ImHp-β-Im-β-ImPy-β-Dp (2, for
Hp), in a 1:1 complex within the DNA sequence context
5′-AAAGAGAAGAG-3′. Im residues do not distinguish G,C from A,T but bind
all four base pairs with high affinity. Py and β residues exhibit
≥10-fold preference for A,T over G,C base pairs. The Hp residue
displays a unique preference for a single A⋅T base pair with an
energetic penalty.
Stimulation of retinoic acid production and growth by ubiquitously expressed alcohol dehydrogenase
Adh3
Authors: Molotkov Andrei , Fan Xiaohong , Deltour Louise , Foglio Mario H. , Martras Sílvia , Farrés Jaume , Parés Xavier , Duester Gregg
Published: July 2002

Influence of vitamin A (retinol) on growth depends on its sequential oxidation to retinal and then to retinoic acid (RA), producing a ligand for RA receptors essential in development of specific tissues. Genetic studies have revealed that aldehyde dehydrogenases function as tissue-specific catalysts for oxidation of retinal to RA. However, enzymes catalyzing the first step of RA synthesis, oxidation of retinol to retinal, remain unclear because none of the present candidate enzymes have expression patterns that fully overlap with those of aldehyde dehydrogenases during development. Here, we provide genetic evidence that alcohol dehydrogenase (ADH) performs this function by demonstrating a role for
Adh3
, a ubiquitously expressed form.
Adh3
null mutant mice exhibit reduced RA generation
in vivo
, growth deficiency that can be rescued by retinol supplementation, and completely penetrant postnatal lethality during vitamin A deficiency. ADH3 was also shown to have
in vitro
retinol oxidation activity. Unlike the second step, the first step of RA synthesis is not tissue-restricted because it is catalyzed by ADH3, a ubiquitous enzyme having an ancient origin.
cdk-7
is required for mRNA transcription and cell cycle progression in
Caenorhabditis elegans
embryos
Authors: Wallenfang Matthew R. , Seydoux Geraldine
Published: July 2002

CDK7 is a cyclin-dependent kinase proposed to function in two essential cellular processes: transcription and cell cycle regulation. CDK7 is the kinase subunit of the general transcription factor TFIIH that phosphorylates the C-terminal domain (CTD) of RNA polymerase II, and has been shown to be broadly required for transcription in
Saccharomyces cerevisiae
. CDK7 can also phosphorylate CDKs that promote cell cycle progression, and has been shown to function as a CDK-activating kinase (CAK) in
Schizosaccharomyces pombe
and
Drosophila melanogaster
. That CDK7 performs both functions in metazoans has been difficult to prove because transcription is essential for cell cycle progression in most cells. We have isolated a temperature-sensitive mutation in
Caenorhabditis elegans cdk-7
and have used it to analyze the role of
cdk-7
in embryonic blastomeres, where cell cycle progression is independent of transcription. Partial loss of
cdk-7
activity leads to a general decrease in CTD phosphorylation and embryonic transcription, and severe loss of
cdk-7
activity blocks all cell divisions. Our results support a dual role for metazoan CDK7 as a broadly required CTD kinase, and as a CAK essential for cell cycle progression.
Telomerase recognizes its template by using an adjacent RNA motif
Authors: Miller Michael C. , Collins Kathleen
Published: July 2002

Telomerase adds telomeric repeats to chromosome 3′ ends, forestalling the cellular senescence, apoptosis, and genomic instability that result from telomere loss caused by incomplete DNA replication. The telomerase ribonucleoprotein is dedicated to synthesis of tandem, simple-sequence repeats by virtue of its specialization for copying only a specific template region within the integral RNA. Here, using circularly permuted variants of
Tetrahymena thermophila
telomerase RNA, we identify the features that allow recognition of the template region within the RNA. We engineered a template-less telomerase ribonucleoprotein that can position and reverse transcribe an exchangeable RNA oligonucleotide template accurately. Only a short “template-recognition” element sequence tag is required to direct efficient use of adjacent 5′ residues as a template for telomeric repeat synthesis. Our findings reveal molecular requirements for template selection by telomerase and physically resolve templating from other RNA functions in catalysis.
Biomechanics of the movable pretarsal adhesive organ in ants and bees
Authors: Federle Walter , Brainerd Elizabeth L. , McMahon Thomas A. , Hölldobler Bert
Published: July 2002

Hymenoptera attach to smooth surfaces with a flexible pad, the
arolium, between the claws. Here we investigate its movement in Asian
weaver ants (
Oecophylla smaragdina
) and honeybees
(
Apis mellifera
).  When ants run upside down on a
smooth surface, the arolium is unfolded and folded back with each step.
Its extension is strictly coupled with the retraction of the claws.
Experimental pull on the claw-flexor tendon revealed that the
claw-flexor muscle not only retracts the claws, but also moves the
arolium. The elicited arolium movement comprises (
i
)
about a 90° rotation (extension) mediated by the interaction of the
two rigid pretarsal sclerites arcus and manubrium and
(
ii
) a lateral expansion and increase in volume. In
severed legs of
O. smaragdina
ants, an increase in
hemolymph pressure of 15 kPa was sufficient to inflate the arolium to
its full size. Apart from being actively extended, an arolium in
contact also can unfold passively when the leg is subject to a pull
toward the body.  We propose a combined mechanical–hydraulic
model for arolium movement: (
i
) the arolium is engaged
by the action of the unguitractor, which mechanically extends the
arolium; (
ii
) compression of the arolium gland reservoir
pumps liquid into the arolium; (
iii
) arolia partly in
contact with the surface are unfolded passively when the legs are
pulled toward the body; and (
iv
) the arolium deflates
and moves back to its default position by elastic recoil of the
cuticle.
A larger hippocampus is associated with longer-lasting spatial memory
Authors: Biegler Robert , McGregor Anthony , Krebs John R. , Healy Susan D.
Published: July 2002

Volumetric studies in a range of animals (London taxi-drivers,
polygynous male voles, nest-parasitic female cowbirds, and a number of
food-storing birds) have shown that the size of the hippocampus, a
brain region essential to learning and memory, is correlated with tasks
involving an extra demand for spatial learning and memory. In this
paper, we report the quantitative advantage that food storers gain from
such an enlargement. Coal tits (
Parus ater
) a
food-storing species, performed better than great tits (
Parus
major
), a nonstoring species, on a task that assessed memory
persistence but not on a task that assessed memory resolution or on one
that tested memory capacity. These results show that the advantage to
the food-storing species associated with an enlarged hippocampus is one
of memory persistence.
Two recombination-dependent DNA replication pathways of bacteriophage T4, and their roles in mutagenesis and horizontal gene transfer
Authors: Mosig Gisela , Gewin John , Luder Andreas , Colowick Nancy , Vo Daniel
Published: July 2002

Two major pathways of recombination-dependent DNA replication, “join-copy” and “join-cut-copy,” can be distinguished in phage T4: join-copy requires only early and middle genes, but two late proteins, endonuclease VII and terminase, are uniquely important in the join-cut-copy pathway. In wild-type T4, timing of these pathways is integrated with the developmental program and related to transcription and packaging of DNA. In primase mutants, which are defective in origin-dependent lagging-strand DNA synthesis, the late pathway can bypass the lack of primers for lagging-strand DNA synthesis. The exquisitely regulated synthesis of endo VII, and of two proteins from its gene, explains the delay of recombination-dependent DNA replication in primase (as well as topoisomerase) mutants, and the temperature-dependence of the delay. Other proteins (e.g., the single-stranded DNA binding protein and the products of genes
46
and
47
) are important in all recombination pathways, but they interact differently with other proteins in different pathways. These homologous recombination pathways contribute to evolution because they facilitate acquisition of any foreign DNA with limited sequence homology during horizontal gene transfer, without requiring transposition or site-specific recombination functions. Partial heteroduplex repair can generate what appears to be multiple mutations from a single recombinational intermediate. The resulting sequence divergence generates barriers to formation of viable recombinants. The multiple sequence changes can also lead to erroneous estimates in phylogenetic analyses.
whmD
is an essential mycobacterial gene required for proper septation and cell division
Authors: Gomez James E. , Bishai William R.
Published: July 2002

A study of potential mycobacterial regulatory genes led to the isolation of the
Mycobacterium smegmatis whmD
gene, which encodes a homologue of WhiB, a
Streptomyces coelicolor
protein required for sporulation. Unlike its
Streptomyces
homologue, WhmD is essential in
M. smegmatis
. The
whmD
gene could be disrupted only in the presence of a plasmid supplying
whmD
in trans. A plasmid that allowed chemically regulated expression of the WhmD protein was used to generate a conditional
whmD
mutant. On withdrawal of the inducer, the conditional
whmD
mutant exhibited irreversible, filamentous, branched growth with diminished septum formation and aberrant septal placement, whereas WhmD overexpression resulted in growth retardation and hyperseptation. Nucleic acid synthesis and levels of the essential cell division protein FtsZ were unaltered by WhmD deficiency. Together, these phenotypes indicate a role for WhmD in mycobacterial septum formation and cell division.
A role for muscarinic excitation: Control of specific singing behavior by activation of the adenylate cyclase pathway in the brain of grasshoppers
Authors: Heinrich Ralf , Wenzel Beate , Elsner Norbert
Published: July 2002

Muscarinic acetylcholine receptors exert slow and prolonged
synaptic effects in both vertebrate and invertebrate nervous systems.
Through activation of G proteins, they typically decrease intracellular
cAMP levels by inhibition of adenylate cyclase or stimulate
phospholipase C and the turnover of inositol phosphates. In insects,
muscarinic receptors have been credited with two main functions:
inhibition of transmitter release from sensory neuron terminals and
regulation of the excitability of motoneurons and interneurons. Our
pharmacological studies with intact and behaving grasshoppers revealed
a functional role for muscarinic acetylcholine receptors as being the
basis for specific arousal in defined areas of the brain, underlying
the selection and control of acoustic communication behavior. Periodic
injections of acetylcholine into distinct areas of the brain elicited
songs of progressively increasing duration. Coinjections of the
muscarinic receptor antagonist scopolamine and periodic stimulations
with muscarine identified muscarinic receptor activation as being the
basis for the underlying accumulation of excitation. In contrast to
reports from other studies on functional circuits, muscarinic
excitation was apparently mediated by activation of the adenylate
cyclase pathway. Stimulation of adenylate cyclase with forskolin and of
protein kinase A with 8-Br-cAMP mimicked the stimulatory effects of
muscarine whereas inhibition of adenylate cyclase with SQ22536 and of
protein kinase A with H-89 and Rp-cAMPs suppressed muscarine-stimulated
singing behavior. Activation of adenylate cyclase by muscarinic
receptors has previously been reported from studies on membrane
preparations and heterologous expression systems, but a physiological
significance of this pathway remained to be demonstrated in an
in vivo
preparation.
Rolling-circle transposons in eukaryotes
Authors: Kapitonov Vladimir V. , Jurka Jerzy
Published: July 2002

All eukaryotic DNA transposons reported so far belong to a single category of elements transposed by the so-called “cut-and-paste” mechanism. Here, we report a previously unknown category of eukaryotic DNA transposons,
Helitron
, which transpose by rolling-circle replication. Autonomous
Helitrons
encode a 5′-to-3′ DNA helicase and nuclease/ligase similar to those encoded by known rolling-circle replicons.
Helitron
-like transposons have conservative 5′-TC and CTRR-3′ termini and do not have terminal inverted repeats. They contain 16- to 20-bp hairpins separated by 10–12 nucleotides from the 3′-end and transpose precisely between the 5′-A and T-3′, with no modifications of the AT target sites. Together with their multiple diverged nonautonomous descendants,
Helitrons
constitute ≈2% of both the
Arabidopsis thaliana
and
Caenorhabditis elegans
genomes and also colonize the
Oriza sativa
genome. Sequence conservation suggests that
Helitrons
continue to be transposed.
The role of antigen and IL-12 in sustaining Th1 memory cellsin vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge
Authors: Stobie Laura , Gurunathan Sanjay , Prussin Calman , Sacks David L. , Glaichenhaus Nicolas , Wu Chang-You , Seder Robert A.
Published: July 2002

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such asMycobacterium tuberculosisandLeishmania majoris the requirements to sustain memory/effector Th1 cellsin vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity toL. majorafter vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generatedin vivoto mediate long-term protection to an intracellular pathogen.
The development of fatal myocarditis and polymyositis in mice heterozygous for IFN-γ and lacking the SOCS-1 gene
Authors: Metcalf Donald , Di Rago Ladina , Mifsud Sandra , Hartley Lynne , Alexander Warren S.
Published: July 2002

Mice lacking the gene encoding the suppressor of cytokine signaling-1 (SOCS-1 −/−) and heterozygous for the IFN–γ gene (IFN-γ +/−) avoided the IFN-γ-dependent preweaning death of SOCS-1 −/− IFN-γ +/+ mice but did not exhibit the good health of young adult SOCS-1 −/− IFN-γ −/− mice. SOCS-1 −/− IFN-γ +/− mice died within 160 days of birth with massive T lymphocyte, macrophage, and eosinophil infiltration of all skeletal muscles and a similar severe myocarditis. The cornea also developed inflammatory infiltration and often a corneal ulcer. The mice exhibited evidence of selective CD8 T lymphocyte activation in populations in the thymus, spleen, and lymph nodes and focal T- and B-lymphoid infiltrates developed in the lung and salivary gland without apparent tissue damage. Comparison of SOCS-1 −/− IFN-γ +/− mice with various control mice indicated that the development of tissue-damaging T lymphocyte, macrophage, and eosinophil infiltrates required loss of SOCS-1 and the presence of some IFN–γ, but that the lung lymphoid infiltrates required only loss of SOCS-1 to develop.
Structural rationale for the modulation of abscess formation byStaphylococcus aureuscapsular polysaccharides
Authors: Tzianabos Arthur O. , Wang Julia Y. , Lee Jean C.
Published: July 2002

Staphylococcus aureusis a medically important bacterial pathogen that is a common cause of superficial and deep-seated abscesses in humans. MostS. aureusisolates produce either a serotype 5 or 8 capsular polysaccharide (CP) that has been shown to enhance bacterial virulence. We investigated the role ofS. aureusCPs in modulating abscess formation in an experimental animal model of intraabdominal infection. Structural studies of CP8 revealed that it has a zwitterionic charge motif conferred by the negatively charged carboxyl group ofN-acetylmannosaminuronic acid and free amino groups available on partially N-acetylated fucosamine residues. We report that purified CP5 and CP8 facilitated intraabdominal abscess formation in animals when given i.p. with a sterile cecal contents adjuvant. Chemical modifications that neutralized the positively or negatively charged groups on CP8 abrogated its ability to provoke abscesses. Rats prophylactically treated with CP8 s.c. were protected against abscess formation induced by homologous or heterologous zwitterionic polysaccharides. Likewise, treatment with CP8 protected against challenge with viableS. aureusstrains PS80 (a capsule type 8 strain) or COL (a methicillin-resistant capsule type 5 strain). Purified CP8 was a potent activator of rat and human CD4+T cellsin vitro. When transferred to naïve rats, these activated T cells modulated the development of intraabdominal abscess formation. These results provide a structure/function rationale for abscess formation byS. aureusand expand the sphere of encapsulated organisms that interact directly with T cells to regulate this host response to bacterial infection.
Functional interaction between c-Jun and promoter factor Sp1 in epidermal growth factor-induced gene expression of human 12(
S
)-lipoxygenase
Authors: Chen Ben-Kuen , Chang Wen-Chang
Published: July 2002

The functional role of the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in epidermal growth factor (EGF)-induced expression of 12(
S
)-lipoxygenase gene in human epidermoid carcinoma A431 cells was studied. Coimmunoprecipitation experiments indicated that EGF stimulated interaction between c-Jun and Sp1 in a time-dependent manner. Overexpression of Ha-ras and c-Jun also enhanced the amount of c-Jun binding to Sp1. In addition, the c-Jun dominant negative mutant TAM-67 not only inhibited the coimmunoprecipitated c-Jun binding to Sp1 in a dose-dependent manner in cells overexpressing c-Jun but also reduced promoter activity of the 12(
S
)-lipoxygenase gene induced by c-Jun overexpression. Treatment of cells with EGF increased the interaction between the Sp1 oligonucleotide and nuclear c-Jun/Sp1 in a time-dependent manner. Furthermore, EGF activated the chimeric promoter consisting of 10 tandem GAL4-binding sites, which replaced the three Sp1-binding sites in the 12(
S
)lipoxygenase promoter only when coexpressed with GAL4-c-Jun () fusion proteins. These results indicate that the direct interaction between c-Jun and Sp1 induced by EGF cooperatively activated expression of the 12(
S
)-lipoxygenase gene, and that Sp1 may serve at least in part as a carrier bringing c-Jun to the promoter, thus transactivating the transcriptional activity of 12(
S
)-lipoxygenase gene.