bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes
Authors: Fruman David A. , Ferl Gregory Z. , An Sam S. , Donahue Amber C. , Satterthwaite Anne B. , Witte Owen N.
Published: July 2002

Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle.
Cytosine substituted calix[4]pyrroles: Neutral receptors for 5′-guanosine monophosphate
Authors: Sessler Jonathan L. , Král Vladimír , Shishkanova Tatiana V. , Gale Philip A.
Published: July 2002

The synthesis and characterization of two cytosine-substituted calix[4]pyrrole conjugates, bearing the appended cytosine attached at either a β- or
meso
-pyrrolic position, is described. These systems were tested as nucleotide-selective carriers and as active components of nucleotide-sensing ion-selective electrodes at pH 6.6. Studies of carrier selectivity were made using a Pressman-type model membrane system consisting of an initial pH 6.0 aqueous phase, an intervening dichloromethane barrier containing the calix[4]pyrrole conjugate, and a receiving basic aqueous phase. Good selectivity for the Watson–Crick complementary nucleotide, 5′-guanosine monophosphate (5′-GMP), was seen in the case of the
meso
-linked conjugate with the relative rates of through-membrane transport being 7.7:4.1:1 for 5′-GMP, 5′-AMP, and 5′-CMP, respectively. By contrast, the β-substituted conjugate, while showing a selectivity for 5′-GMP that was enhanced relative to unsubstituted calix[4]pyrrole, was found to transport 5′-CMP roughly 4.5 times more quickly than 5′-GMP. Higher selectivities were also found for 5′-CMP when both the β- and
meso
-substituted conjugates were incorporated into polyvinyl chloride membranes and tested as ion selective electrodes at pH 6.6, whereas near-equal selectivities were observed for 5′-CMP and 5′-GMP in the case of unsubstituted calix[4]pyrroles. These seemingly disparate results are consistent with a picture wherein the
meso
-substituted cytosine calix[4]pyrrole conjugate, but not its β-linked congener, is capable of acting as a ditopic receptor, binding concurrently both the phosphate anion and nucleobase portions of 5′-GMP to the calixpyrrole core and cytosine “tails” of the molecule, respectively, with the effect of this binding being most apparent under the conditions of the transport experiments.
The mutated human gene encoding hepatocyte nuclear factor 1β inhibits kidney formation in developing
Xenopus
embryos
Authors: Wild Wiltrud , Pogge von Strandmann Elke , Nastos Aristotelis , Senkel Sabine , Lingott-Frieg Anja , Bulman Michael , Bingham Coralie , Ellard Sian , Hattersley Andrew T. , Ryffel Gerhart U.
Published: July 2002

The transcription factor hepatocyte nuclear factor 1β (HNF1β) is a tissue-specific regulator that also plays an essential role in early development of vertebrates. In humans, four heterozygous mutations in the HNF1β gene have been identified that lead to early onset of diabetes and severe primary renal defects. The degree and type of renal defects seem to depend on the specific mutation. We show that the frameshift mutant P328L329fsdelCCTCT associated with nephron agenesis retains its DNA-binding properties and acts as a gain-of-function mutation with increased transactivation potential in transfection experiments. Expression of this mutated factor in the
Xenopus
embryo leads to defective development and agenesis of the pronephros, the first kidney form of amphibians. Very similar defects are generated by overexpressing in
Xenopus
the wild-type HNF1β, which is consistent with the gain-of-function property of the mutant. In contrast, introduction of the human HNF1β mutant R137-K161del, which is associated with a reduced number of nephrons with hypertrophy of the remaining ones and which has an impaired DNA binding, shows only a minor effect on pronephros development in
Xenopus
. Thus, the overexpression of both human mutants has a different effect on renal development in
Xenopus
, reflecting the variation in renal phenotype seen with these mutations. We conclude that mutations in human HNF1β can be functionally characterized in
Xenopus
. Our findings imply that HNF1β not only is an early marker of kidney development but also is functionally involved in morphogenetic events, and these processes can be investigated in lower vertebrates.
5-Methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex
Authors: Zhu Bing , Zheng Yong , Hess Daniel , Angliker Herbert , Schwarz Steffen , Siegmann Michel , Thiry Stéphane , Jost Jean-Pierre
Published: July 2002

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in
Escherichia coli
has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in
E. coli
and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.
Functional switch between motor tracts in the presence of the mAb IN-1 in the adult rat
Authors: Raineteau Olivier , Fouad Karim , Noth Pascal , Thallmair Michaela , Schwab Martin E.
Published: July 2002

Fine finger and hand movements in humans, monkeys, and rats are
under the direct control of the corticospinal tract (CST). CST lesions
lead to severe, long-term deficits of precision movements. We
transected completely both CSTs in adult rats and treated the animals
for 2 weeks with an antibody that neutralized the central nervous
system neurite growth inhibitory protein Nogo-A (mAb IN-1). Anatomical
studies of the rubrospinal tracts showed that the number of collaterals
innervating the cervical spinal cord doubled in the mAb IN-1- but not
in the control antibody-treated animals. Precision movements of the
forelimb and fingers were severely impaired in the controls, but almost
completely recovered in the mAb IN-1-treated rats. Low threshold
microstimulation of the motor cortex induced a rapid forelimb
electromyography response that was mediated by the red nucleus in the
mAb IN-1 animals but not in the controls. These findings demonstrate an
unexpectedly high capacity of the adult central nervous system motor
system to sprout and reorganize in a targeted and functionally
meaningful way.
The synthesis, discovery, and development of a highly promising class of microtubule stabilization agents: Curative effects of desoxyepothilones B and F against human tumor xenografts in nude mice
Authors: Chou Ting-Chao , O'Connor Owen A. , Tong William P. , Guan Yongbiao , Zhang Zui-Guo , Stachel Shawn J. , Lee Chulbom , Danishefsky Samuel J.
Published: July 2002

We have evaluated two synthetic epothilone analogues lacking the 12,13-epoxide functionality, 12,13-desoxyepothilone B (dEpoB), and 12,13-desoxyepothilone F (dEpoF). The concentrations required for 50% growth inhibition (IC
50
) for a variety of anticancer agents were measured in CCRF-CEM/VBL1000 cells (2,048-fold resistance to vinblastine). By using dEpoB, dEpoF, aza-EpoB, and paclitaxel, the IC
50
values were 0.029, 0.092, 2.99, and 5.17 μM, respectively. These values represent 4-, 33.5-, 1,423- and 3,133-fold resistance, respectively, when compared with the corresponding IC
50
in the parent [nonmultiple drug-resistant (MDR)] CCRF-CEM cells. We then produced MDR human lung carcinoma A549 cells by continuous exposure of the tumor cells to sublethal concentrations of dEpoB (1.8 yr), vinblastine (1.2 yr), and paclitaxel (1.8 yr). This continued exposure led to the development of 2.1-, 4,848-, and 2,553-fold resistance to each drug, respectively. The therapeutic effect of dEpoB and paclitaxel was also compared
in vivo
in a mouse model by using various tumor xenografts. dEpoB is much more effective in reducing tumor sizes in all MDR tumors tested. Analysis of dEpoF, an analog possessing greater aqueous solubility than dEpoB, showed curative effects similar to dEpoB against K562, CCRF-CEM, and MX-1 xenografts. These results indicate that dEpoB and dEpoF are efficacious antitumor agents with both a broad chemotherapeutic spectrum and wide safety margins.
Solution structure of the 2-amino-1- methyl-6-phenylimidazo[4,5-
b
]pyridine C8-deoxyguanosine adduct in duplex DNA
Authors: Brown Karen , Hingerty Brian E. , Guenther Elizabeth A. , Krishnan V. V. , Broyde Suse , Turteltaub Kenneth W. , Cosman Monique
Published: July 2002

The carcinogenic heterocyclic amine (HA) 2-amino-1-methyl-6-phenylimidazo[4,5-
b
]pyridine (PhIP) is formed during the cooking of various meats. To enable structure/activity studies aimed at understanding how DNA damaged by a member of the HA class of compounds can ultimately lead to cancer, we have determined the first solution structure of an 11-mer duplex containing the C8-dG adduct formed by reaction with
N
-acetoxy-PhIP. A slow conformational exchange is observed in which the PhIP ligand either intercalates into the DNA helix by denaturing and displacing the modified base pair (main form) or is located outside the helix in a minimally perturbed B-DNA duplex (minor form). In the main base-displaced intercalation structure, the minor groove is widened, and the major groove is compressed at the lesion site because of the location of the bulky PhIP-
N
-methyl and phenyl ring in the minor groove; this distortion causes significant bending of the helix. The PhIP phenyl ring interacts with the phosphodiester-sugar ring backbone of the complementary strand and its fast rotation with respect to the intercalated imidazopyridine ring causes substantial distortions at this site, such as unwinding and bulging-out of the strand. The glycosidic torsion angle of the [PhIP]dG residue is
syn
, and the displaced guanine base is directed toward the 3′ end of the modified strand. This study contributes, to our knowledge, the first structural information on the biologically relevant HA class to a growing body of knowledge about how conformational similarities and differences for a variety of types of lesions can influence protein interactions and ultimately biological outcome.
Identification of an autoimmune serum containing antibodies against the Barr body
Authors: Hong Bo , Reeves Peter , Panning Barbara , Swanson Maurice S. , Yang Thomas P.
Published: July 2002

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence
in situ
hybridization with a probe against
XIST
RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with
XIST
RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.
Conservation of the biochemical mechanisms of signal transduction among mammalian Notch family members
Authors: Mizutani Tomohiko , Taniguchi Yoshihito , Aoki Tomokazu , Hashimoto Nobuo , Honjo Tasuku
Published: July 2002

Mouse Notch1, which plays an important role in cell fate determination in development, is proteolytically processed within its transmembrane domain by unidentified γ-secretase-like activity that depends on presenilin. To study this proteolytic event, we established a cell-free Notch cleavage assay system using the membrane fraction of fibroblast transfectants of various Notch constructs with deletion of the extracellular portion (Notch ΔE). The cytoplasmic portion of Notch1 ΔE was released from the membrane upon incubation at 37°C, which was inhibited by the specific γ-secretase inhibitor, MW167, or by overexpression of dominant negative presenilin1. Likewise, other members of mouse Notch family were proteolytically cleaved in a presenilin-dependent, MW167-sensitive manner
in vivo
as well as in the cell-free Notch ΔE cleavage assay system. All four members of the mouse Notch family migrated to the nucleus and activated the transcription from the promoter carrying the RBP-J consensus sequences after they were released from the membrane. These results demonstrate the conserved biochemical mechanism of signal transduction among mammalian Notch family members.
Functional analysis of the
LACERATA
gene of
Arabidopsis
provides evidence for different roles of fatty acid ω-hydroxylation in development
Authors: Wellesen Kirsten , Durst Francis , Pinot Franck , Benveniste Irene , Nettesheim Klaus , Wisman Ellen , Steiner-Lange Sabine , Saedler Heinz , Yephremov Alexander
Published: July 2002

We describe
lacerata
(
lcr
) mutants of
Arabidopsis
, which display various developmental
abnormalities, including postgenital organ fusions, and report cloning
of the
LCR
gene by using the maize transposon
Enhancer
/
Suppressor-mutator
(
En
/
Spm
). The pleiotropic mutant
phenotype could be rescued by genetic complementation of
lcr
mutants with the wild-type
LCR
gene.
The
LCR
gene encodes a cytochrome P450 monooxygenase,
CYP86A8, which catalyzes ω-hydroxylation of fatty acids ranging from
C12 to C18:1, as demonstrated by expression of the gene in yeast.
Although palmitic and oleic acids were efficient substrates for LCR,
9,10-epoxystearate was not metabolized. Taken together with previous
studies, our findings indicate that
LCR
-dependent
ω-hydroxylation of fatty acids could be implicated in the
biosynthesis of cutin in the epidermis and in preventing postgenital
organ fusions. Strikingly, the same pathway seems to control trichome
differentiation, the establishment of apical dominance, and senescence
in plants.
Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation
Authors: Lee Yu-Chen , Martin Emil , Murad Ferid
Published: July 2002

The α1- and β1-subunits of human soluble guanylate cyclase
(sGC) were coexpressed in the Sf9 cells/baculovirus system. In
addition to the native enzyme, constructs with hexahistidine tag at the
amino and carboxyl termini of each subunit were coexpressed. This
permitted the rapid and efficient purification of active recombinant
enzyme on a nickel-affinity column. The enzyme has one heme per
heterodimer and was readily activated with the NO donor sodium
nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole
(YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other
in combination and demonstrated a remarkable 2,200-fold stimulation of
the human recombinant sGC. The effects were inhibited with
1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the
recombinant enzyme with respect to GTP was examined. The products of
the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent
of inhibition by cGMP depended on the activation state of the enzyme,
whereas inhibition by pyrophosphate was not affected by the enzyme
state. Both reaction products displayed independent binding and
cooperativity with respect to enzyme inhibition. The expression of
large quantities of active enzyme will facilitate structural
characterization of the protein.
Initial recovery of vision after early monocular deprivation in kittens is faster when both eyes are open
Authors: Mitchell Donald E. , Gingras Guy , Kind Peter C.
Published: July 2002

A comparison was made of the speed of visual recovery in the
deprived eye of kittens after a 6-day period of monocular deprivation
imposed at 5–9 weeks of age in two postdeprivation conditions. In one
condition, binocular recovery (BR), both eyes were open, whereas in the
other condition, reverse lid-suture (RLS), the formerly nondeprived eye
was closed to force the animal to use the originally deprived eye. In
littermate pairs, BR kittens began to recover form vision 12 to 30
h before those subjected to RLS. The vision of the deprived eye of the
BR animals remained superior to that of their RLS littermates for 4–8
days. Although this finding is difficult to reconcile with competitive
mechanisms of synaptic plasticity, it supports a prediction of an
alternative model of synaptic plasticity [Bienenstock, E. L.,
Cooper, L. N. & Munro, P. W. (1982)
J.
Neurosci.
2, 32–48] for slower initial recovery with RLS
because of the time required to reset the modification threshold.
Mantle dynamics and seismic tomography
Authors: Tanimoto Toshiro , Lay Thorne
Published: July 2002

Three-dimensional imaging of the Earth's interior, called seismic
tomography, has achieved breakthrough advances in the last two decades,
revealing fundamental geodynamical processes throughout the Earth's
mantle and core. Convective circulation of the entire mantle is taking
place, with subducted oceanic lithosphere sinking into the lower
mantle, overcoming the resistance to penetration provided by the phase
boundary near 650-km depth that separates the upper and lower mantle.
The boundary layer at the base of the mantle has been revealed to have
complex structure, involving local stratification, extensive structural
anisotropy, and massive regions of partial melt. The Earth's high
Rayleigh number convective regime now is recognized to be much more
interesting and complex than suggested by textbook cartoons, and
continued advances in seismic tomography, geodynamical modeling, and
high-pressure–high-temperature mineral physics will be needed to fully
quantify the complex dynamics of our planet's interior.
Negative hair-bundle stiffness betrays a mechanism for mechanical amplification by the hair cell
Authors: Martin P. , Mehta A. D. , Hudspeth A. J.
Published: July 2002

Hearing and balance rely on the ability of hair cells in the inner
ear to sense miniscule mechanical stimuli. In each cell, sound or
acceleration deflects the mechanosensitive hair bundle, a tuft of rigid
stereocilia protruding from the cell's apical surface. By altering the
tension in gating springs linked to mechanically sensitive transduction
channels, this deflection changes the channels' open probability and
elicits an electrical response. To detect weak stimuli despite energy
losses caused by viscous dissipation, a hair cell can use active
hair-bundle movement to amplify its mechanical inputs. This
amplificatory process also yields spontaneous bundle oscillations.
Using a displacement-clamp system to measure the mechanical properties
of individual hair bundles from the bullfrog's ear, we found that an
oscillatory bundle displays negative slope stiffness at the heart of
its region of mechanosensitivity. Offsetting the hair bundle's
position activates an adaptation process that shifts the region of
negative stiffness along the displacement axis. Modeling indicates that
the interplay between negative bundle stiffness and the motor
responsible for mechanical adaptation produces bundle oscillation
similar to that observed. Just as the negative resistance of
electrically excitable cells and of tunnel diodes can be embedded in a
biasing circuit to amplify electrical signals, negative stiffness can
be harnessed to amplify mechanical stimuli in the ear.
A dual-specificity aminoacyl-tRNA synthetase in the deep-rooted eukaryote
Giardia lamblia
Authors: Bunjun Shipra , Stathopoulos Constantinos , Graham David , Min Bokkee , Kitabatake Makoto , Wang Alice L. , Wang Ching C. , Vivarès Christian P. , Weiss Louis M. , Söll Dieter
Published: July 2002

Cysteinyl-tRNA (Cys-tRNA) is essential for protein synthesis. In
most organisms the enzyme responsible for the formation of Cys-tRNA is
cysteinyl-tRNA synthetase (CysRS). The only known exceptions are the
euryarchaea
Methanococcus jannaschii
and
Methanobacterium thermoautotrophicum
, which do not
encode a CysRS. Deviating from the accepted concept of one
aminoacyl-tRNA synthetase per amino acid, these organisms employ
prolyl-tRNA synthetase as the enzyme that carries out Cys-tRNA
formation. To date this dual-specificity prolyl-cysteinyl-tRNA
synthetase (ProCysRS) is only known to exist in archaea. Analysis of
the preliminary genomic sequence of the primitive eukaryote
Giardia lamblia
indicated the presence of an archaeal
prolyl-tRNA synthetase (ProRS). Its
proS
gene was cloned
and the gene product overexpressed in
Escherichia coli
.
By using
G. lamblia
,
M. jannaschii
, or
E. coli
tRNA as substrate, this ProRS was able to form
Cys-tRNA and Pro-tRNA
in vitro
. Cys-AMP formation, but
not Pro-AMP synthesis, was tRNA-dependent. The
in vitro
data were confirmed
in vivo
, as the cloned
G.
lamblia proS
gene was able to complement a
temperature-sensitive
E. coli
cysS strain.
Inhibition studies of CysRS activity with proline analogs (thiaproline
and
5′-
O
-[
N
-(
l
-prolyl)-sulfamoyl]adenosine)
in a
Giardia
S-100 extract predicted that the organism
also contains a canonical CysRS. This prediction was confirmed by
cloning and analysis of the corresponding
cysS
gene.
Like a number of archaea,
Giardia
contains two enzymes,
ProCysRS and CysRS, for Cys-tRNA formation. In contrast, the purified
Saccharomyces cerevisiae
and
E. coli
ProRS enzymes were unable to form Cys-tRNA under these conditions.
Thus, the dual specificity is restricted to the archaeal genre of
ProRS.
G. lamblia's
archaeal-type prolyl- and
alanyl-tRNA synthetases refine our understanding of the evolution and
interaction of archaeal and eukaryal translation systems.
Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking
Authors: Fukuda Mitsunori , Moreira Jorge E. , Liu Vincent , Sugimori Mutsuyuki , Mikoshiba Katsuhiko , Llinás Rodolfo R.
Published: July 2002

Synaptotagmin (Syt) I , an abundant synaptic vesicle protein,
consists of one transmembrane region, two C2 domains, and a short C
terminus. This protein is essential for both synaptic vesicle
exocytosis and endocytosis via its C2 domains. Although the short C
terminus is highly conserved among the Syt family and across species,
little is known about the exact role of the conserved C terminus of Syt
I. In this paper, we report a function of the Syt I C terminus in
synaptic vesicle docking at the active zones. Presynaptic injection of
a peptide corresponding to the C-terminal 21 amino acids of Syt I
(named Syt-C) into the squid giant synapse blocked synaptic
transmission without affecting the presynaptic action potential or the
presynaptic Ca
2+
currents. The same procedure repeated with
a mutant C-terminal peptide (Syt-CM) had no effect on synaptic
transmission. Repetitive presynaptic stimulation with Syt-C produced a
rapid decrease in the amplitude of the postsynaptic potentials as the
synaptic block progressed, indicating that the peptide interferes with
the docking step rather than the fusion step of synaptic vesicles.
Electron microscopy of the synapses injected with the Syt-C peptide
showed a marked decrease in the number of docked synaptic vesicles at
the active zones, as compared with controls. These results indicate
that Syt I is a multifunctional protein that is involved in at least
three steps of synaptic vesicle cycle: docking, fusion, and reuptake of
synaptic vesicles.
Exchanges are not equally able to enhance meiotic chromosome segregation in yeast.
Authors: Ross L O , Maxfield R , Dawson D
Published: July 2002

Homologous chromosomes pair, and then migrate to opposite poles of the spindle at meiosis I. In most eukaryotic organisms, reciprocal recombinations (crossovers) between the homologs are critical to the success of this process. Individuals with defects in meiotic recombination typically produce high levels of aneuploid gametes and exhibit low fertility or are sterile. The experiments described here were designed to test whether different crossovers are equally able to contribute to the fidelity of meiotic chromosome segregation in yeast. These experiments were performed with model chromosomes with which it was possible to control and measure the distributions of meiotic crossovers in wild-type cells. Physical and genetic approaches were used to map crossover positions on model chromosomes and to correlate crossover position with meiotic segregation behavior. The results show that crossovers at different chromosomal positions have different abilities to enhance the fidelity of meiotic segregation.
Functional complementation of xeroderma pigmentosum complementation group E by replication protein A in an in vitro system.
Authors: Kazantsev A , Mu D , Nichols A F , Zhao X , Linn S , Sancar A
Published: July 2002

Xeroderma pigmentosum (XP) is caused by a defect in nucleotide excision repair. Patients in the complementation group E (XP-E) have the mildest form of the disease and the highest level of residual repair activity. About 20% of the cell strains derived from XP-E patients lack a damaged DNA-binding protein (DDB) activity that binds to ultraviolet-induced (6-4) photoproducts with high affinity. We report here that cell-free extracts prepared from XP-E cell strains that either lacked or contained DDB activity were severely defective in excising DNA damage including (6-4) photoproducts. However, this excision activity defect was not restored by addition of purified DDB that, in fact, inhibited removal of (6-4) photoproducts by the human excision nuclease reconstituted from purified proteins. Extensive purification of correcting activity from HeLa cells revealed that the correcting activity is inseparable from the human replication/repair protein A [RPA (also known as human single stranded DNA binding protein, HSSB)]. Indeed, supplementing XP-E extracts with recombinant human RPA purified from Escherichia coli restored excision activity. However, no mutation was found in the genes encoding the three subunits of RPA in an XP-E (DDB-) cell line. It is concluded that RPA functionally complements XP-E extracts in vitro, but it is not genetically altered in XP-E patients.
Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.
Authors: Bruni R , Roizman B
Published: July 2002

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.
Emergence of order in visual system development.
Authors: Shatz C J
Published: July 2002

Neural connections in the adult central nervous system are highly precise. In the visual system, retinal ganglion cells send their axons to target neurons in the lateral geniculate nucleus (LGN) in such a way that axons originating from the two eyes terminate in adjacent but nonoverlapping eye-specific layers. During development, however, inputs from the two eyes are intermixed, and the adult pattern emerges gradually as axons from the two eyes sort out to form the layers. Experiments indicate that the sorting-out process, even though it occurs in utero in higher mammals and always before vision, requires retinal ganglion cell signaling; blocking retinal ganglion cell action potentials with tetrodotoxin prevents the formation of the layers. These action potentials are endogenously generated by the ganglion cells, which fire spontaneously and synchronously with each other, generating "waves" of activity that travel across the retina. Calcium imaging of the retina shows that the ganglion cells undergo correlated calcium bursting to generate the waves and that amacrine cells also participate in the correlated activity patterns. Physiological recordings from LGN neurons in vitro indicate that the quasiperiodic activity generated by the retinal ganglion cells is transmitted across the synapse between ganglion cells to drive target LGN neurons. These observations suggest that (i) a neural circuit within the immature retina is responsible for generating specific spatiotemporal patterns of neural activity; (ii) spontaneous activity generated in the retina is propagated across central synapses; and (iii) even before the photoreceptors are present, nerve cell function is essential for correct wiring of the visual system during early development. Since spontaneously generated activity is known to be present elsewhere in the developing CNS, this process of activity-dependent wiring could be used throughout the nervous system to help refine early sets of neural connections into their highly precise adult patterns.
Sources of selection bias in evaluating social programs: An interpretation of conventional measures and evidence on the effectiveness of matching as a program evaluation method
Authors: Heckman James J. , Ichimura Hidehiko , Smith Jeffrey , Todd Petra
Published: July 2002

This paper decomposes the conventional measure of selection bias in
observational studies into three components. The first two components
are due to differences in the distributions of characteristics between
participant and nonparticipant (comparison) group members: the first
arises from differences in the supports, and the second from
differences in densities over the region of common support. The third
component arises from selection bias precisely defined. Using data from
a recent social experiment, we find that the component due to selection
bias, precisely defined, is smaller than the first two components.
However, selection bias still represents a substantial fraction of the
experimental impact estimate. The empirical performance of matching
methods of program evaluation is also examined. We find that matching
based on the propensity score eliminates some but not all of the
measured selection bias, with the remaining bias still a substantial
fraction of the estimated impact. We find that the support of the
distribution of propensity scores for the comparison group is typically
only a small portion of the support for the participant group. For
values outside the common support, it is impossible to reliably
estimate the effect of program participation using matching methods. If
the impact of participation depends on the propensity score, as we find
in our data, the failure of the common support condition severely
limits matching compared with random assignment as an evaluation
estimator.
Interferon-inducible protein-10 identified as a mediator of tumor necrosisin vivo
Authors: Sgadari Cecilia , Angiolillo Anne L. , Cherney Barry W. , Pike Sandra E. , Farber Joshua M. , Koniaris Leonidas G. , Vanguri Padmavathy , Burd Parris R. , Sheikh Nasreen , Gupta Ghanshyam , Teruya-Feldstein Julie , Tosato Giovanna
Published: July 2002

Human Burkitt lymphoma cell lines give rise to progressively growing subcutaneous tumors in athymic mice. These tumors are induced to regress by inoculation of Epstein–Barr virus-immortalized normal human lymphocytes. In the present study, analysis of profiles of murine cytokine/chemokine gene expression in Burkitt tumor tissues excised from the nude mice showed that expression of the murine α-chemokine interferon-inducible protein-10 (IP-10) was higher in the regressing than in the progressive Burkitt tumors. We tested the effects of IP-10 on Burkitt tumor growth in nude mice. Inoculation of established Burkitt tumors either with crude preparations of murine IP-10 or with purified human IP-10 caused visible tumor necrosis in a proportion of the animals, although no complete tumor regressions were observed. Constitutive expression of murine IP-10 in Burkitt cells reduced their ability to grow as subcutaneous tumors, and caused visible tumor necrosis in a proportion of the animals. Histologically, IP-10-treated and IP-10-expressing Burkitt tumors had widespread evidence of tumor tissue necrosis and of capillary damage, including intimal thickening and vascular thrombosis. Thus, IP-10 is an antitumor agent that promotes damage in established tumor vasculature and causes tissue necrosis in human Burkitt lymphomas established subcutaneously in athymic mice.
Role of hydrophobic interactions and desolvation in determining the structural properties of a model alpha beta peptide.
Authors: Butcher D J , Moe G R
Published: July 2002

Model AB, a 20-amino acid peptide that was designed to adopt an alpha beta tertiary structure stabilized by hydrophobic interactions between residues in adjacent helical and extended segments, exhibited large pKa shifts of several ionizable groups and slow hydrogen/deuterium exchange rates of nearly all the peptide amide groups [Butcher, D. J., Bruch, M. D. & Moe, G. T. (1995) Biopolymers 36, 109-120]. These properties, which depend on structure and hydration, are commonly observed in larger proteins but are quite unusual for small peptides. To identify which of several possible features of the peptide design are most important in determining these properties, several closely related analogs of Model AB were characterized by CD and NMR spectroscopy. The results show that hydrophobic interactions between adjacent helical and extended segments are structure-determining and have the additional effect of altering water-peptide interactions over much of the peptide surface. These results may have important implications for understanding mechanisms of protein folding and for the design of independently folding peptides.
Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins.
Authors: Blasina A , Kittell B L , Toukdarian A E , Helinski D R
Published: July 2002

The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria. The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested. The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication. A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number. Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes. To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized. Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay. It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication. These observations provide direct evidence in support of the coupling model of replication control.
Intermediate- and long-term earthquake prediction.
Authors: Sykes L R
Published: July 2002

Progress in long- and intermediate-term earthquake prediction is reviewed emphasizing results from California. Earthquake prediction as a scientific discipline is still in its infancy. Probabilistic estimates that segments of several faults in California will be the sites of large shocks in the next 30 years are now generally accepted and widely used. Several examples are presented of changes in rates of moderate-size earthquakes and seismic moment release on time scales of a few to 30 years that occurred prior to large shocks. A distinction is made between large earthquakes that rupture the entire downdip width of the outer brittle part of the earth's crust and small shocks that do not. Large events occur quasi-periodically in time along a fault segment and happen much more often than predicted from the rates of small shocks along that segment. I am moderately optimistic about improving predictions of large events for time scales of a few to 30 years although little work of that type is currently underway in the United States. Precursory effects, like the changes in stress they reflect, should be examined from a tensorial rather than a scalar perspective. A broad pattern of increased numbers of moderate-size shocks in southern California since 1986 resembles the pattern in the 25 years before the great 1906 earthquake. Since it may be a long-term precursor to a great event on the southern San Andreas fault, that area deserves detailed intensified study.
Design and synthesis of ribonucleic guanidine: a polycationic analog of RNA.
Authors: Dempcy R O , Luo J , Bruice T C
Published: July 2002

Replacement of the phosphodiester linkages of the polyanion RNA with guanidinium linkers (represented by g) provides the polycation ribonucleic guanidine (RNG). An anticipated structure for the triple-helical hybrid [r(Up)9U.r(Ag)9A.r(Up)9U] is presented. A basic strategy for the synthesis of RNG oligomers is described. Synthetic procedures are provided for tetrameric adenosyl RNG [r(Ag)3A].
Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy.
Authors: Zhang J F , Hu C , Geng Y , Selm J , Klein S B , Orazi A , Taylor M W
Published: July 2002

Treatment of a human breast cancer cell line (MDA-MB-435) in nude mice with a recombinant adenovirus containing the human interferon (IFN) consensus gene, IFN-con1 (ad5/IFN), resulted in tumor regression in 100% of the animals. Tumor regression occurred when virus was injected either within 24 hr of tumor cell implantation or with established tumors. However, regression of the tumor was also observed in controls in which either the wild-type virus or a recombinant virus containing the luciferase gene was used, although tumor growth was not completely suppressed. Tumor regression was accompanied by a decrease in p53 expression. Two other tumors, the human myelogenous leukemic cell line K562 and the hamster melanoma tumor RPMI 1846, also responded to treatment but only with ad5/IFN. In the case of K562 tumors, there was complete regression of the tumor, and tumors derived from RPMI 1846 showed partial regression. We propose that the complete regression of the breast cancer with the recombinant virus ad5/IFN was the result of two events: viral oncolysis in which tumor cells are being selectively lysed by the replication-competent virus and the enhanced effect of expression of the IFN-con1 gene. K562 and RPMI 1846 tumors regressed only as a result of IFN gene therapy. This was confirmed by in vitro analysis. Our results indicate that a combination of viral oncolysis with a virus of low pathogenicity, itself resistant to the effects of IFN and IFN gene therapy, might be a fruitful approach to the treatment of a variety of different tumors, in particular breast cancers.
Models of DNA structure achieve almost perfect discrimination between normal prostate, benign prostatic hyperplasia (BPH), and adenocarcinoma and have a high potential for predicting BPH and prostate cancer
Authors: Malins Donald C. , Polissar Nayak L. , Gunselman Sandra J.
Published: July 2002

In our previous studies of DNA, wavenumber–absorbance relationships of infrared spectra analyzed by principal components analysis (PCA) were expressed as points in space. Each point represented a highly discriminating measure of structural modifications that altered vibrational and rotational motion, thus changing the spatial orientation of the points. PCA/Fourier transform-infrared technology has now provided a virtually perfect separation of clusters of points representing DNA from normal prostate tissue, BPH, and adenocarcinoma. The findings suggest that the progression of normal prostate tissue to BPH and to prostate cancer involves structural alterations in DNA that are distinctly different. The hydroxyl radical is likely a major contributor to these structural alterations, which is consistent with previous studies of breast cancer. Models based on logistic regression of infrared spectral data were used to calculate the probability of a tissue being BPH or adenocarcinoma. The models had a sensitivity and specificity of 100% for classifying normal vs. cancer and normal vs. BPH, and close to 100% for BPH vs. cancer. Thus, the PCA/Fourier transform-infrared technology was shown to be a powerful means for discriminating between normal prostate tissue, BPH and prostate cancer and has considerable promise for risk prediction and clinical application.
Characterization and tissue-specific expression of the rat basic fibroblast growth factor antisense mRNA and protein
Authors: Knee Rai , Li Audrey W. , Murphy Paul R.
Published: July 2002

An RNA transcribed from the antisense strand of the FGF-2 gene has been implicated in the regulation of FGF-2 mRNA stability in amphibian oocytes. We have now cloned and characterized a novel 1.1-kb mRNA (
fgf-as
) from neonatal rat liver. In non-central nervous system (CNS) tissues the
fgf-as
RNA is abundantly expressed in a developmentally regulated manner. The FGF-AS cDNA contains a consensus polyadenylylation signal and a long open reading frame (ORF) whose deduced amino acid sequence predicts a 35-kDa protein with homology to the MutT family of nucleotide hydrolases. Western blot analysis with antibodies against the deduced peptide sequence demonstrates that the FGF-AS protein is expressed in a broad range of non-CNS tissue in the postnatal period. In the developing brain, the abundance of sense and antisense transcripts are inversely related, suggesting a role for the antisense RNA in posttranscriptional regulation of FGF-2 expression in this tissue.The FGF-AS is complementary to two widely separated regions in the long 3′ untranslated region of the FGF-2 mRNA, in the vicinity of the proximal and distal polyadenylylation sites. These findings demonstrate that the FGF-2 and
fgf-as
RNAs are coordinately transcribed on a tissue-specific and developmentally regulated basis and suggest that interaction of the sense and antisense RNAs may result in posttranscriptional regulation of FGF-2 in some tissues.
Defensive production of formic acid (80%) by a carabid beetle (
Galerita lecontei
)
Authors: Rossini Carmen , Attygalle Athula B. , González Andrés , Smedley Scott R. , Eisner Maria , Meinwald Jerrold , Eisner Thomas
Published: July 2002

The carabid beetle
Galerita lecontei
has a pair of abdominal defensive glands that secrete a mixture of formic acid, acetic acid, and lipophilic components (long-chain hydrocarbons and esters). Formic acid, at the concentration of 80%, is the principal constituent. The beetle ejects the secretion as a spray, which it aims accurately toward parts of the body subjected to assault. At full capacity, the glands store 4.5 mg of formic acid (3% of body mass), enough for upward of six ejections. The beetle reloads the glands at a rate of 126 μg of formic acid per day. For the approximately 500 secretory cells of the glands, this means an hourly output of 10 ng of formic acid per cell, or about 5% of cell volume. Replenishing empty glands to their full formic acid load takes the beetle an estimated 37 days. Replenishing the 0.7 mg of formic acid expended in a single discharge takes 5.5 days.
Whole genome analysis: Experimental access to all genome sequenced segments through larger-scale efficient oligonucleotide synthesis and PCR
Authors: Lashkari Deval A. , McCusker John H. , Davis Ronald W.
Published: July 2002

The recent ability to sequence whole genomes allows ready access to all genetic material. The approaches outlined here allow automated analysis of sequence for the synthesis of optimal primers in an automated multiplex oligonucleotide synthesizer (AMOS). The efficiency is such that all ORFs for an organism can be amplified by PCR. The resulting amplicons can be used directly in the construction of DNA arrays or can be cloned for a large variety of functional analyses. These tools allow a replacement of single-gene analysis with a highly efficient whole-genome analysis.