bluuebunny/crossref_metadata_2025 huggingface dataset:
Arctic “ozone hole” in a cold volcanic stratosphere
Authors: Tabazadeh A. , Drdla K. , Schoeberl M. R. , Hamill P. , Toon O. B.
Published: July 2002

Optical depth records indicate that volcanic aerosols from major eruptions often produce clouds that have greater surface area than typical Arctic polar stratospheric clouds (PSCs). A trajectory cloud–chemistry model is used to study how volcanic aerosols could affect springtime Arctic ozone loss processes, such as chlorine activation and denitrification, in a cold winter within the current range of natural variability. Several studies indicate that severe denitrification can increase Arctic ozone loss by up to 30%. We show large PSC particles that cause denitrification in a nonvolcanic stratosphere cannot efficiently form in a volcanic environment. However, volcanic aerosols, when present at low altitudes, where Arctic PSCs cannot form, can extend the vertical range of chemical ozone loss in the lower stratosphere. Chemical processing on volcanic aerosols over a 10-km altitude range could increase the current levels of springtime column ozone loss by up to 70% independent of denitrification. Climate models predict that the lower stratosphere is cooling as a result of greenhouse gas built-up in the troposphere. The magnitude of column ozone loss calculated here for the 1999–2000 Arctic winter, in an assumed volcanic state, is similar to that projected for a colder future nonvolcanic stratosphere in the 2010 decade.
A structure-based method for derivation of all-atom potentials for protein folding
Authors: Kussell Edo , Shimada Jun , Shakhnovich Eugene I.
Published: July 2002

A method for deriving all-atom protein folding potentials is presented and tested on a three-helix bundle protein, as well as on hairpin and helical sequences. The potentials obtained are composed of a contact term between pairs of atoms, and a local density term for each atom, mimicking solvent exposure preferences. Using this potential in an all-atom protein folding simulation, we repeatedly folded the three-helix bundle, with the lowest energy conformations having a C
α
distance rms from the native structure of less than 2 Å. Similar results were obtained for the hairpin and helices by using different potentials. We derived potentials for several different proteins and found a high correlation between the derived parameters, suggesting that a potential of this form eventually could be found that folds multiple, unrelated proteins at the atomic level of detail.
Positioning protein molecules on surfaces: A nanoengineering approach to supramolecular chemistry
Authors: Liu Gang-Yu , Amro Nabil A.
Published: July 2002

We discuss a nanoengineering approach for supramolecular chemistry and self assembly. The collective properties and biofunctionalities of molecular ensembles depend not only on individual molecular building blocks but also on organization at the molecular or nanoscopic level. Complementary to “bottom-up” approaches, which construct supramolecular ensembles by the design and synthesis of functionalized small molecular units or large molecular motifs, nanofabrication explores whether individual units, such as small molecular ligands, or large molecules, such as proteins, can be positioned with nanometer precision. The separation and local environment can be engineered to control subsequent intermolecular interactions. Feature sizes as small as 2 × 4 nm2(32 alkanethiol molecules) are produced. Proteins may be aligned along a 10-nm-wide line or within two-dimensional islands of desired geometry. These high-resolution engineering and imaging studies provide new and molecular-level insight into supramolecular chemistry and self-assembly processes in bioscience that are otherwise unobtainable, e.g., the influence of size, separation, orientation, and local environment of reaction sites. This nanofabrication methodology also offers a new strategy in construction of two- and three-dimensional supramolecular structures for cell, virus, and bacterial adhesion, as well as biomaterial and biodevice engineering.
Chiral protein scissors: High enantiomeric selectivity for binding and its effect on protein photocleavage efficiency and specificity
Authors: Kumar Challa V. , Buranaprapuk Apinya , Sze Ho C. , Jockusch Steffen , Turro Nicholas J.
Published: July 2002

Chiral recognition of protein-binding sites by a simple organic molecule with selectivities >100 is reported here. The
l
-isomer of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-
l
-Phe) binds to BSA with an affinity constant (
K
b
) of 3 × 10
7
M
−1
, whereas the corresponding
d
-isomer (Py-
d
-Phe) binds 100 times weaker. The enantiomers showed contrasting spectral changes when bound to BSA. Whereas hypochromism was observed with the
l
-isomer, hyperchromism was observed for the
d
-isomer, and, whereas the fluorescence of the
l
-isomer was quenched, the fluorescence of the
d
-isomer was enhanced. The induced CD spectra of the enantiomers bound to BSA bear a near mirror-image relationship. In contrast, the enantiomers show only moderate binding selectivity with lysozyme. The differences in the enantioselectivities with the two proteins indicate that the binding site of 4(1-pyrene)-3-butyroyl-phenylalanine amide (Py-Phe) in BSA is crowded, whereas that of lysozyme is more accommodative of either isomer. The enantioselective binding of Py-Phe isomers is further examined in protein photocleavage studies. Py-
d
-Phe cleaves BSA and lysozyme at a single site in a manner similar to Py-
l
-Phe, but the cleavage yields are lower for the
d
-isomer. Sequencing of the resulting fragments indicated that the photocleavage sites of Py-
d
-Phe on BSA and lysozyme are identical to those of Py-
l
-Phe. Flash photolysis studies indicated only minor differences between the two enantiomers. The large binding selectivities, therefore, do not influence cleavage specificity or cleavage site location. The strong role of the single asymmetric center of Py-Phe in recognition and its minor role in photocleavage chemistry are demonstrated.
Paracrine
in vivo
inhibitory effects of hepatitis B virus X protein (HBx) on liver cell proliferation: An alternative mechanism of HBx-related pathogenesis
Authors: Tralhao J. Guilherme , Roudier Jean , Morosan Serban , Giannini Carlo , Tu Hong , Goulenok Cyril , Carnot Françoise , Zavala Flora , Joulin Virginie , Kremsdorf Dina , Bréchot Christian
Published: July 2002

The role of the hepatitis B virus X protein (HBx) in the pathogenesis of hepatitis B virus (HBV) infection remains unclear. HBx exhibits pleiotropic biological effects, whose
in vivo
relevance is a matter for debate. In the present report, we have used a combination of HBx-expressing transgenic mice and liver cell transplantation to investigate the
in vivo
impact of HBx expression on liver cell proliferation and viability in a regenerative context. We show that moderate HBx expression inhibits liver regeneration after partial hepatectomy in HBx-expressing transgenic mice. We also demonstrate that the transplantation of HBx-expressing liver cells, isolated from HBx transgenic mice, is sufficient to inhibit overall recipient liver regeneration after partial hepatectomy. Moreover, the injection of serum samples drawn from HBx-expressing transgenic mice mimicked the inhibitory effect of HBx on liver regeneration. Finally, the incubation of primary rat hepatocytes with the supernatant of HBx-expressing liver cells inhibits cellular DNA synthesis. Taken together, our results demonstrate a paracrine inhibitory effect of HBx on liver cell proliferation and lead us to propose HBV as one of the few viruses implicated in human cancer which act, at least in part, through paracrine biological pathways.
Altered stability of pulmonary surfactant in SP-C-deficient mice
Authors: Glasser Stephan W. , Burhans Michael S. , Korfhagen Thomas R. , Na Cheng-Lun , Sly Peter D. , Ross Gary F. , Ikegami Machiko , Whitsett Jeffrey A.
Published: July 2002

The surfactant protein C (SP-C) gene encodes an extremely
hydrophobic, 4-kDa peptide produced by alveolar epithelial cells in the
lung. To discern the role of SP-C in lung function, SP-C-deficient
(−/−) mice were produced. The SP-C (−/−) mice were viable at
birth and grew normally to adulthood without apparent pulmonary
abnormalities. SP-C mRNA was not detected in the lungs of SP-C
(−/−) mice, nor was mature SP-C protein detected by Western blot of
alveolar lavage from SP-C (−/−) mice. The levels of the other
surfactant proteins (A, B, D) in alveolar lavage were comparable to
those in wild-type mice. Surfactant pool sizes, surfactant synthesis,
and lung morphology were similar in SP-C (−/−) and SP-C (+/+)
mice. Lamellar bodies were present in SP-C (−/−) type II cells, and
tubular myelin was present in the alveolar lumen. Lung mechanics
studies demonstrated abnormalities in lung hysteresivity (a term used
to reflect the mechanical coupling between energy dissipative forces
and tissue-elastic properties) at low, positive-end, expiratory
pressures. The stability of captive bubbles with surfactant from the
SP-C (−/−) mice was decreased significantly, indicating that SP-C
plays a role in the stabilization of surfactant at low lung volumes, a
condition that may accompany respiratory distress syndrome in infants
and adults.
Large vortex-like structure of dipole field in computer models of liquid water and dipole-bridge between biomolecules
Authors: Higo Junichi , Sasai Masaki , Shirai Hiroki , Nakamura Haruki , Kugimiya Takaki
Published: July 2002

We propose a framework to describe the cooperative
orientational motions of water molecules in liquid water and around
solute molecules in water solutions. From molecular dynamics (MD)
simulation a new quantity “site-dipole field” is defined as the
averaged orientation of water molecules that pass through each spatial
position. In the site-dipole field of bulk water we found large
vortex-like structures of more than 10 Å in size. Such coherent
patterns persist more than 300 ps although the orientational memory of
individual molecules is quickly lost. A 1-ns MD simulation of systems
consisting of two amino acids shows that the fluctuations of
site-dipole field of solvent are pinned around the amino acids,
resulting in a stable dipole-bridge between side-chains of amino acids.
The dipole-bridge is significantly formed even for the side-chain
separation of 14 Å, which corresponds to five layers of water. The way
that dipole-bridge forms sensitively depends on the side-chain
orientations and thereby explains the specificity in the
solvent-mediated interactions between biomolecules.
Directed evolution of novel polymerase activities: Mutation of a DNA polymerase into an efficient RNA polymerase
Authors: Xia Gang , Chen Liangjing , Sera Takashi , Fa Ming , Schultz Peter G. , Romesberg Floyd E.
Published: July 2002

The creation of novel enzymatic function is of great interest, but remains a challenge because of the large sequence space of proteins. We have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. The Stoffel fragment (SF) of
Thermus aquaticus
DNA polymerase I is displayed on a filamentous phage by fusing it to a pIII coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent pIII coat proteins. Phage particles displaying SF polymerases, which are able to extend the attached oligonucleotide primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized to streptavidin-coated magnetic beads and subsequently recovered. After four rounds of screening an SF library, three SF mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates.
Induction of apoptosis in human papillomaviruspositive cancer cells by peptide aptamers targeting the viral E6 oncoprotein
Authors: Butz Karin , Denk Claudia , Ullmann Angela , Scheffner Martin , Hoppe-Seyler Felix
Published: July 2002

Certain types of human papillomaviruses (HPVs) are closely linked to the development of human cancers. Herein, it is shown that intracellular targeting of the HPV16 E6 oncoprotein by E6-binding peptide aptamers resulted in the apoptotic elimination of HPV16-positive cancer cells, whereas HPV-negative cells were not affected. These results provide direct experimental evidence that the HPV E6 oncoprotein has antiapoptotic activity in HPV-positive tumor cells that is required for their survival. The E6-targeting molecules identified herein have implications for the development of therapeutic strategies for the treatment of HPV-associated dysplasias and cancers.
Complex formation by the human RAD51C and XRCC3 recombination repair proteins
Authors: Masson Jean-Yves , Stasiak Alicja Z. , Stasiak Andrzej , Benson Fiona E. , West Stephen C.
Published: July 2002

In vertebrates, the RAD51 protein is required for genetic recombination, DNA repair, and cellular proliferation. Five paralogs of RAD51, known as RAD51B, RAD51C, RAD51D, XRCC2, and XRCC3, have been identified and also shown to be required for recombination and genome stability. At the present time, however, very little is known about their biochemical properties or precise biological functions. As a first step toward understanding the roles of the RAD51 paralogs in recombination, the human RAD51C and XRCC3 proteins were overexpressed and purified from baculovirus-infected insect cells. The two proteins copurify as a complex, a property that reflects their endogenous association observed in HeLa cells. Purified RAD51C–XRCC3 complex binds single-stranded, but not duplex DNA, to form protein–DNA networks that have been visualized by electron microscopy.
Handoff from recombinase to replisome: Insights from transposition
Authors: Nakai Hiroshi , Doseeva Victoria , Jones Jessica M.
Published: July 2002

Bacteriophage Mu replicates as a transposable element, exploiting host enzymes to promote initiation of DNA synthesis. The phage-encoded transposase MuA, assembled into an oligomeric transpososome, promotes transfer of Mu ends to target DNA, creating a fork at each end, and then remains tightly bound to both forks. In the transition to DNA synthesis, the molecular chaperone ClpX acts first to weaken the transpososome's interaction with DNA, apparently activating its function as a molecular matchmaker. This activated transpososome promotes formation of a new nucleoprotein complex (prereplisome) by yet unidentified host factors [Mu replication factors (MRFα2)], which displace the transpososome in an ATP-dependent reaction. Primosome assembly proteins PriA, PriB, DnaT, and the DnaB–DnaC complex then promote the binding of the replicative helicase DnaB on the lagging strand template of the Mu fork. PriA helicase plays an important role in opening the DNA duplex for DnaB binding, which leads to assembly of DNA polymerase III holoenzyme to form the replisome. The MRFα2 transition factors, assembled into a prereplisome, not only protect the fork from action by nonspecific host enzymes but also appear to aid in replisome assembly by helping to activate PriA's helicase activity. They consist of at least two separable components, one heat stable and the other heat labile. Although the MRFα2 components are apparently not encoded by currently known homologous recombination genes such as
recA
,
recF
,
recO
, and
recR
, they may fulfill an important function in assembling replisomes on arrested replication forks and products of homologous strand exchange.
Adhesion induced by mobile binders: Dynamics
Authors: Brochard-Wyart F. , de Gennes P. G.
Published: July 2002

We consider a vesicle bilayer loaded with molecules that can bind (upon contact) with a solid surface, following the classical model of Bell, Dembo, and Bongrand. We are interested in situations where the contact area varies with time: we assume that binders can then migrate via diffusion. The resulting dissipation and lag create a retarded force on the contact line, which could be significant in squeezing or rolling experiments. However, there are two cases where we expect the lag force to be ineffective: (
i
)
separation
by shrinking of an adhesive patch (where the Evans “tear out” process turns out to be less costly) and (
ii
) spontaneous growth of a patch from a point contact. In this last case, the lag force is weak, and we give detailed predictions for the growth laws.
Placental overgrowth in mice lacking the imprinted gene
Ipl
Authors: Frank Dale , Fortino Weiwei , Clark Lorraine , Musalo Raymond , Wang Wenxian , Saxena Anjana , Li Chi-Ming , Reik Wolf , Ludwig Thomas , Tycko Benjamin
Published: July 2002

The
Ipl
(
Tssc3
) gene lies in an extended imprinted region of distal mouse chromosome 7, which also contains the
Igf2
gene. Expression of
Ipl
is highest in placenta and yolk sac, where its mRNA is derived almost entirely from the maternal allele.
Ipl
encodes a small cytoplasmic protein with a pleckstrin-homology (PH) domain. We constructed two lines of mice with germ-line deletions of this gene (
Ipl
neo
and
Ipl
loxP
) and another line deleted for the similar but nonimprinted gene
Tih1
. All three lines were viable. There was consistent overgrowth of the Ipl-null placentas, with expansion of the spongiotrophoblast. These larger placentas did not confer a fetal growth advantage; fetal size was normal in Ipl nulls with the
Ipl
neo
allele and was decreased slightly in nulls with the
Ipl
loxP
allele. When bred into an
Igf2
mutant background, the
Ipl
deletion partially rescued the placental but not fetal growth deficiency. Neither fetal nor placental growth was affected by deletion of
Tih1
. These results show a nonredundant function for
Ipl
in restraining placental growth. The data further indicate that
Ipl
can act, at least in part, independently of insulin-like growth factor-2 signaling. Thus, genomic imprinting regulates multiple pathways to control placental size.
The X-linked lymphoproliferative syndrome gene product SH2D1A associates with p62dok(Dok1) and activates NF-κB
Authors: Sylla Bakary S. , Murphy Kerry , Cahir-McFarland Ellen , Lane William S. , Mosialos George , Kieff Elliott
Published: July 2002

The X-linked lymphoproliferative syndrome (XLP) is a genetic disorder in which affected males have a morbid or fatal response to Epstein–Barr virus infection. The XLP deficiency has been mapped to a gene encoding a 128-residue protein, SH2D1A, which is comprised principally of a Src homology 2 (SH2) domain. We now report that SH2D1A associates with Dok1, a protein that interacts with Ras-GAP, Csk, and Nck. An SH2D1A SH2 domain mutant that has been identified in XLP does not associate with Dok1, in accord with the hypothesis that this interaction is linked to XLP. The association of SH2D1A with Dok1 also depends on phosphorylation of Dok1 Y449in the sequence ALYSQVQK. Further, overexpression of SH2D1A is found to activate NF-κB in 293T cells. NF-κB activation by SH2D1A does not depend on the wild-type SH2 domain and is inhibited by a dominant-negative IκB kinase β. Thus, SH2D1A can affect multiple intracellular signaling pathways that are potentially important in the normal effective host response to Epstein–Barr virus infection.
Identification and characterization of a newly recognized population of high-Na
+
, low-K
+
, low-density sickle and normal red cells
Authors: Bookchin Robert M. , Etzion Zipora , Sorette Martin , Mohandas Narla , Skepper Jeremy N. , Lew Virgilio L.
Published: July 2002

We describe a population of sickle cell anemia red cells (SS RBCs) (≈4%) and a smaller fraction of normal RBCs (<0.03%) that fail to dehydrate when permeabilized to K
+
with either valinomycin or elevated internal Ca
2+
. The nonshrinking, valinomycin-resistant (
val-res
) fractions, first detected by flow cytometry of density-fractionated SS RBCs, constituted up to 60% of the lightest, reticulocyte-rich (R1) cell fraction, and progressively smaller portions of the slightly denser R2 cells and discocytes. R1
val-res
RBCs had a mean cell hemoglobin concentration of ≈21 g of Hb per dl, and many had an elongated shape like “irreversibly sickled cells,” suggesting a dense SS cell origin. Of three possible explanations for
val-res
cells, failure of valinomycin to K
+
-permeabilize the cells, low co-ion permeability, or reduced driving K
+
gradient, the latter proved responsible: Both SS and normal
val-res
RBCs were consistently high-Na
+
and low-K
+
, even when processed entirely in Na-free media. Ca
2+
+ A23187-induced K
+
-permeabilization of SS R1 fractions revealed a similar fraction of
cal-res
cells, whose
86
Rb uptake showed both high Na/K pump and leak fluxes.
val-res
/
cal-res
RBCs might represent either a distinct erythroid genealogy, or an “end-stage” of normal and SS RBCs. This paper focuses on the discovery, basic characterization, and exclusion of artifactual origin of this RBC fraction. Many future studies will be needed to clarify their mechanism of generation and full pathophysiological significance.
In vivo
activity in a catalytic antibody-prodrug system: Antibody catalyzed etoposide prodrug activation for selective chemotherapy
Authors: Shabat Doron , Lode Holger N. , Pertl Ursula , Reisfeld Ralph A. , Rader Christoph , Lerner Richard A. , Barbas Carlos F.
Published: July 2002

Effective chemotherapy remains a key issue for successful cancer treatment in general and neuroblastoma in particular. Here we report a chemotherapeutic strategy based on catalytic antibody-mediated prodrug activation. To study this approach in an animal model of neuroblastoma, we have synthesized prodrugs of etoposide, a drug widely used to treat this cancer in humans. The prodrug incorporates a trigger portion designed to be released by sequential retro-aldol/retro-Michael reactions catalyzed by aldolase antibody 38C2. This unique prodrug was greater than 10
2
-fold less toxic than etoposide itself in
in vitro
assays against the NXS2 neuroblastoma cell line. Drug activity was restored after activation by antibody 38C2. Proof of principle for local antibody-catalyzed prodrug activation
in vivo
was established in a syngeneic model of murine neuroblastoma. Mice with established 100-mm
3
s.c. tumors who received one intratumoral injection of antibody 38C2 followed by systemic i.p. injections with the etoposide prodrug showed a 75% reduction in s.c. tumor growth. In contrast, injection of either antibody or prodrug alone had no antitumor effect. Systemic injections of etoposide at the maximum tolerated dose were significantly less effective than the intratumoral antibody 38C2 and systemic etoposide prodrug combination. Significantly, mice treated with the prodrug at 30-fold the maximum tolerated dose of etoposide showed no signs of prodrug toxicity, indicating that the prodrug is not activated by endogenous enzymes. These results suggest that this strategy may provide a new and potentially nonimmunogenic approach for targeted cancer chemotherapy.
Enzyme mimicry by the antiidiotypic antibody approach
Authors: Kolesnikov Alexander V. , Kozyr Arina V. , Alexandrova Elena S. , Koralewski Frédéric , Demin Alexander V. , Titov Mikhail I. , Avalle Bérangère , Tramontano Alfonso , Paul Sudhir , Thomas Daniel , Gabibov Alexander G. , Friboulet Alain
Published: July 2002

The concept of “internal image” of antiidiotypic antibodies
has provided the basis for eliciting catalytic antibodies. A monoclonal
IgM 9A8 that was obtained as an antiidiotype to AE-2 mAb, a known
inhibitor of acetylcholinesterase, displayed esterolytic activity.
Study of recombinant Fab fragments and separate light and heavy chains
of 9A8 confirmed that the antibody variable domain encodes the
catalytic function, whereas neither part of the primary sequence of the
Fab exhibited homology with the enzyme. The specific modification of
the 9A8 variable domain by an active site-directed covalent inhibitor
revealed the presence of an active site Ser residue. A
three-dimensional modeling suggests the existence of a functional
catalytic dyad Ser-His. Comparison of active sites of 9A8 and 17E8
esterolytic abzyme raised against transition-state analog revealed
structural similarity although both antibodies were elicited by two
different approaches.
Myokymia and neonatal epilepsy caused by a mutation in the voltage sensor of the KCNQ2 K
+
channel
Authors: Dedek Karin , Kunath Bernhard , Kananura Colette , Reuner Ulrike , Jentsch Thomas J. , Steinlein Ortrud K.
Published: July 2002

KCNQ2 and KCNQ3 are two homologous K
+
channel subunits that can combine to form heterotetrameric channels with properties of neuronal M channels. Loss-of-function mutations in either subunit can lead to benign familial neonatal convulsions (BFNC), a generalized, idiopathic epilepsy of the newborn. We now describe a syndrome in which BFNC is followed later in life by myokymia, involuntary contractions of skeletal muscles. All affected members of the myokymia/BFNC family carried a mutation (R207W) that neutralized a charged amino acid in the S4 voltage-sensor segment of KCNQ2. This substitution led to a shift of voltage-dependent activation of KCNQ2 and a dramatic slowing of activation upon depolarization. Myokymia is thought to result from hyperexcitability of the lower motoneuron, and indeed both KCNQ2 and KCNQ3 mRNAs were detected in the anterior horn of the spinal cord where the cells of the lower motoneurons arise. We propose that a difference in firing patterns between motoneurons and central neurons, combined with the drastically slowed voltage activation of the R207W mutant, explains why this particular KCNQ2 mutant causes myokymia in addition to BFNC.
Chaos and stability of the solar system
Authors: Malhotra Renu , Holman Matthew , Ito Takashi
Published: July 2002

Over the last two decades, there has come about a recognition that chaotic dynamics is pervasive in the solar system. We now understand that the orbits of small members of the solar system—asteroids, comets, and interplanetary dust—are chaotic and undergo large changes on geological time scales. Are the major planets' orbits also chaotic? The answer is not straightforward, and the subtleties have prompted new questions.
A
CBF5
mutation that disrupts nucleolar localization of early tRNA biosynthesis in yeast also suppresses tRNA gene-mediated transcriptional silencing
Authors: Kendall Ann , Hull Melissa W. , Bertrand Edouard , Good Paul D. , Singer Robert H. , Engelke David R.
Published: July 2002

In the budding yeast,
Saccharomyces cerevisiae
,
actively transcribed tRNA genes can negatively regulate adjacent RNA
polymerase II (pol II)-transcribed promoters. This tRNA gene-mediated
silencing is independent of the orientation of the tRNA gene and does
not require direct, steric interference with the binding of either
upstream pol II factors or the pol II holoenzyme. A mutant was isolated
in which this form of silencing is suppressed. The responsible point
mutation affects expression of the Cbf5 protein, a small nucleolar
ribonucleoprotein protein required for correct processing of rRNA.
Because some early steps in the
S. cerevisiae
pre-tRNA
biosynthetic pathway are nucleolar, we examined whether the
CBF5
mutation might affect this localization. Nucleoli
were slightly fragmented, and the pre-tRNAs went from their normal,
mostly nucleolar location to being dispersed in the nucleoplasm. A
possible mechanism for tRNA gene-mediated silencing is suggested in
which subnuclear localization of tRNA genes antagonizes transcription
of nearby genes by pol II.
The DELLA motif is essential for gibberellin-induced degradation of RGA
Authors: Dill Alyssa , Jung Hou-Sung , Sun Tai-ping
Published: July 2002

RGA
and
GAI
are homologous genes that encode putative transcriptional regulators that repress gibberellin (GA) signaling in
Arabidopsis
. Previously we showed that the green fluorescent protein (GFP)-RGA fusion protein is localized to the nucleus in transgenic
Arabidopsis
, and expression of this fusion protein rescues the
rga
null mutation. The GA signal seems to derepress the GA response pathway by degrading the repressor protein RGA. The GA-insensitive, semidominant, semidwarf
gai
-
1
mutant encodes a mutant protein with a 17-amino acid deletion within the DELLA domain of GAI. It was hypothesized that this mutation turns the gai protein into a constitutive repressor of GA signaling. Because the sequences missing in gai-1 are identical between GAI and RGA, we tested whether an identical mutation (
rga
-Δ
17
) in the
RGA
gene would confer a phenotype similar to
gai
-
1
. We demonstrated that expression of
rga
-Δ
17
or
GFP
-(
rga
-Δ
17
) under the control of the
RGA
promoter caused a GA-unresponsive severe dwarf phenotype in transgenic
Arabidopsis
. Analysis of the mRNA levels of a GA biosynthetic gene,
GA4
, showed that the feedback control of GA biosynthesis in these transgenic plants was less responsive to GA than that in wild type. Immunoblot and confocal microscopy analyses indicated that rga-Δ17 and GFP-(rga-Δ17) proteins were resistant to degradation after GA application. Our results illustrate that the DELLA domain in RGA plays a regulatory role in GA-induced degradation of RGA. Deletion of this region stabilizes the rga-Δ17 mutant protein, and regardless of the endogenous GA status rga-Δ17 becomes a constitutively active repressor of GA signaling.
Prevalence of somatic alterations in the colorectal cancer cell genome
Authors: Wang Tian-Li , Rago Carlo , Silliman Natalie , Ptak Janine , Markowitz Sanford , Willson James K. V. , Parmigiani Giovanni , Kinzler Kenneth W. , Vogelstein Bert , Velculescu Victor E.
Published: July 2002

Although a small fraction of human cancers have increased rates of somatic mutation because of known deficiencies in DNA repair, little is known about the prevalence of somatic alterations in the vast majority of human cancers. To systematically assess nonsynonymous somatic alterations in colorectal neoplasia, we used DNA sequencing to analyze ≈3.2 Mb of coding tumor DNA comprising 1,811 exons from 470 genes. In total, we identified only three distinct somatic mutations, comprising two missense changes and one 14-bp deletion, each in a different gene. The accumulation of approximately one nonsynonymous somatic change per Mb of tumor DNA is consistent with a rate of mutation in tumor cells that is similar to that of normal cells. These data suggest that most sporadic colorectal cancers do not display a mutator phenotype at the nucleotide level. They also have significant implications for the interpretation of somatic mutations in candidate tumor-suppressor genes.
Cloning and expression of a gene encoding an integrin-like protein in Candida albicans.
Authors: Gale C , Finkel D , Tao N , Meinke M , McClellan M , Olson J , Kendrick K , Hostetter M
Published: July 2002

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.
Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.
Authors: Duckett D R , Drummond J T , Murchie A I , Reardon J T , Sancar A , Lilley D M , Modrich P
Published: July 2002

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.
Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide.
Authors: Janson J , Soeller W C , Roche P C , Nelson R T , Torchia A J , Kreutter D K , Butler P C
Published: July 2002

The islet in non-insulin-dependent diabetes mellitus (NIDDM) is characterized by loss of beta cells and large local deposits of amyloid derived from the 37-amino acid protein, islet amyloid polypeptide (IAPP). We have hypothesized that IAPP amyloid forms intracellularly causing beta-cell destruction under conditions of high rates of expression. To test this we developed a homozygous transgenic mouse model with high rates of expression of human IAPP. Male transgenic mice spontaneously developed diabetes mellitus by 8 weeks of age, which was associated with selective beta-cell death and impaired insulin secretion. Small intra- and extracellular amorphous IAPP aggregates were present in islets of transgenic mice during the development of diabetes mellitus. However, IAPP derived amyloid deposits were found in only a minority of islets at approximately 20 weeks of age, notably after development of diabetes mellitus in male transgenic mice. Approximately 20% of female transgenic mice spontaneously developed diabetes mellitus at 30+ weeks of age, when beta-cell degeneration and both amorphous and amyloid deposits of IAPP were present. We conclude that overexpression of human IAPP causes beta-cell death, impaired insulin secretion, and diabetes mellitus. Large deposits of IAPP derived amyloid do not appear to be important in this cytotoxicity, but early, small amorphous intra- and extracellular aggregates of human IAPP were consistently present at the time of beta-cell death and therefore may be the most cytotoxic form of IAPP.
Sin3 corepressor function in Myc-induced transcription and transformation.
Authors: Harper S E , Qiu Y , Sharp P A
Published: July 2002

Many basic-helix-loop-helix-leucine zipper (b-HLH-LZ) proteins, including the Myc family and non-Myc family, bind a common DNA sequence CACGTG, yet have quite different biological actions. Myc binds this sequence as a heterodimer with Max in the activation of both transcription and transformation. The Myc family members Mad and Mxi1 are known to suppress Myc-induced transcription and transformation and to dimerize with Max to form ternary complexes with the mammalian Sin3 transcriptional corepressor (mSin3). The b-HLH-LZ domain of TFEB, which cannot heterodimerize within the Myc family, does not suppress Myc-induced transcription or transformation. However, transfer of a 25- to 36-aa region from Mad or Mxi1, which interacts with mSin3, to the b-HLH-LZ of TFEB, mediated profound suppression of Myc-induced transcription and transformation. These results suggest that the DNA binding specificities of the Myc family and non-Myc family b-HLH-LZ proteins, in the context of the cellular genes involved in Myc-induced transformation, are shared. The results also demonstrate that targeting mSin3 to CACGTG sites via a non-Myc family DNA binding domain is sufficient to oppose Myc activity in growth regulation.
Environmental stress sensitivity of an ascorbic acid-deficient Arabidopsis mutant.
Authors: Conklin P L , Williams E H , Last R L
Published: July 2002

L-ascorbic acid (vitamin C) is a powerful reducing agent found in millimolar concentrations in plants, and is proposed to play an important role in scavenging free radicals in plants and animals. However, surprisingly little is known about the role of this antioxidant in plant environmental stress adaptation or ascorbate biosynthesis. We report the isolation of soz1, a semi-dominant ozone-sensitive mutant that accumulates only 30% of the normal ascorbate concentration. The results of genetic approaches and feeding studies show that the ascorbate concentration affects foliar resistance to the oxidizing gas ozone. Consistent with the proposed role for ascorbate in reactive oxygen species detoxification, lipid peroxides are elevated in soz1, but not in wild type following ozone fumigation. We show that the soz1 mutant is hypersensitive to both sulfur dioxide and ultraviolet B irradiation, thus implicating ascorbate in defense against varied environmental stresses. In addition to defining the first ascorbate deficient mutant in plants, these results indicate that screening for ozone-sensitive mutants is a powerful method for identifying physiologically important antioxidant mechanisms and signal transduction pathways. Analysis of soz1 should lead to more information about the physiological roles and metabolism of ascorbate.
Immunization with DNA vaccines encoding glycoprotein D or glycoprotein B, alone or in combination, induces protective immunity in animal models of herpes simplex virus-2 disease.
Authors: McClements W L , Armstrong M E , Keys R D , Liu M A
Published: July 2002

DNA vaccines expressing herpes simplex virus type 2 (HSV-2) full-length glycoprotein D (gD), or a truncated form of HSV-2 glycoprotein B (gB) were evaluated for protective efficacy in two experimental models of HSV-2 infection. Intramuscular (i.m.) injection of mice showed that each construction induced neutralizing serum antibodies and protected the mice from lethal HSV-2 infection. Dose-titration studies showed that low doses (< or = 1 microgram) of either DNA construction induced protective immunity, and that a single immunization with the gD construction was effective. The two DNAs were then tested in a low-dosage combination in guinea pigs. Immune sera from DNA-injected animals had antibodies to both gD and gB, and virus neutralizing activity. When challenged by vaginal infection with HSV-2, the DNA-immunized animals were significantly protected from primary genital disease.
Blastic transformation of p53-deficient bone marrow cells by p210
bcr/abl
tyrosine kinase
Authors: Skorski Tomasz , Nieborowska-Skorska Malgorzata , Wlodarski Pawel , Perrotti Danilo , Martinez Robert , Wasik Mariusz A. , Calabretta Bruno
Published: July 2002

Blastic transformation of chronic myelogenous leukemia (CML) is
characterized by the presence of nonrandom, secondary genetic
abnormalities in the majority of Philadelphia
1
clones, and
loss of p53 tumor suppressor gene function is a consistent finding in
25–30% of CML blast crisis patients. To test whether the functional
loss of p53 plays a direct role in the transition of chronic phase to
blast crisis, bone marrow cells from p53
+/+
or
p53
−/−
mice were infected with a retrovirus carrying
either the wild-type BCR/ABL or the inactive kinase-deficient
mutant, and were assessed for colony-forming ability. Infection of
p53
−/−
marrow cells with wild-type BCR/ABL, but not
with the kinase-deficient mutant, enhanced formation of hematopoietic
colonies and induced growth factor independence at high frequency, as
compared with p53
+/+
marrow cells. These effects were
suppressed when p53
−/−
marrow cells were coinfected with
BCR/ABL and wild-type p53. p53-deficient BCR/ABL-infected marrow
cells had a proliferative advantage, as reflected by an increase in the
fraction of S+G
2
phase cells and a decrease in the number
of apoptotic cells. Immunophenotyping and morphological
analysis revealed that BCR/ABL-positive p53
−/−
cells
were much less differentiated than their BCR/ABL-positive
p53
+/+
counterparts. Injection of immunodeficient mice with
BCR/ABL-positive p53
−/−
cells produced a
transplantable, highly aggressive, poorly differentiated acute
myelogenous leukemia. In marked contrast, the disease process in mice
injected with BCR/ABL-positive p53
+/+
marrow cells was
characterized by cell infiltrates with a more differentiated phenotype
and was significantly retarded, as indicated by a much longer survival
of leukemic mice. Together, these findings directly demonstrate that
loss of p53 function plays an important role in blast transformation in
CML.
Synaptic vesicle endocytosis mediates the entry of tetanus neurotoxin into hippocampal neurons
Authors: Matteoli Michela , Verderio Claudia , Rossetto Ornella , Iezzi Numa , Coco Silvia , Schiavo Giampietro , Montecucco Cesare
Published: July 2002

Tetanus neurotoxin causes the spastic paralysis of tetanus by
blocking neurotransmitter release at inhibitory synapses of the spinal
cord. This is due to the penetration of the toxin inside the neuronal
cytosol where it cleaves specifically VAMP/synaptobrevin, an
essential component of the neuroexocytosis apparatus. Here we show that
tetanus neurotoxin is internalized inside the lumen of small synaptic
vesicles following the process of vesicle reuptake. Vesicle
acidification is essential for the toxin translocation in the cytosol,
which results in the proteolytic cleavage of VAMP/synaptobrevin and
block of exocytosis.
The number of unidentified amacrine cells in the mammalian retina
Authors: Strettoi Enrica , Masland Richard H.
Published: July 2002

The three largest known populations of amacrine cells in the rabbit
retina were stained with fluorescent probes in whole mounts and counted
at a series of retinal eccentricities. The retinas were counterstained
using a fluorescent DNA-binding molecule and the total number of nuclei
in the inner nuclear layer were counted in confocal sections. From the
total number of inner nuclear layer cells and the known fraction of
them occupied by amacrine cells, the fraction of amacrine cells made up
by the stained populations could be calculated. Starburst cells made up
3%, indoleamine-accumulating cells made up 4%, and AII cells made up
11% of all amacrine cells. By referring four smaller populations of
amacrine cells to the number of indoleamine-accumulating cells, they
were estimated to make up 4% of all amacrine cells. Thus, 78% of all
amacrine cells in the rabbit’s retina are known only from isolated
examples, if at all. This proportion is similar in the retinas of the
mouse, cat, and monkey. It is likely that a substantial fraction of the
local circuit neurons present in other regions of the central nervous
system are also invisible as populations to current techniques.
Synergy between chronic corticosterone and sodium azide treatments in producing a spatial learning deficit and inhibiting cytochrome oxidase activity.
Authors: Bennett M C , Mlady G W , Fleshner M , Rose G M
Published: July 2002

Previously, we developed a rat model of persistent mitochondrial dysfunction based upon the chronic partial inhibition of the mitochondrial enzyme cytochrome oxidase (EC 1.9.3.1). Continuous systemic infusion of sodium azide at approximately 1 mg/kg per hr inhibited cytochrome oxidase activity and produced a spatial learning deficit. In other laboratories, glucocorticoids have been reported to exacerbate neuronal damage from various acute metabolic insults. Therefore, we tested the hypothesis that corticosterone, the primary glucocorticoid in the rat, would potentiate the sodium azide-induced learning deficit. To this end, we first identified nonimpairing doses of sodium azide (approximately 0.75 mg/kg per hr) and corticosterone (100-mg pellet, 3-week sustained-release). We now report that chronic co-administration of these individually nonimpairing treatments produced a severe learning deficit. Moreover, the low dose of corticosterone, which did not elevate serum corticosterone, acted synergistically with sodium azide to inhibit cytochrome oxidase activity. The latter result represents a previously unidentified effect of glucocorticoids that provides a candidate mechanism for glucocorticoid potentiation of neurotoxicity induced by metabolic insult. These results may have the clinical implication of expanding the definition of hypercortisolism in patient populations with compromised oxidative metabolism. Furthermore, they suggest that glucocorticoid treatment may contribute to pathology in disease or trauma conditions that involve metabolic insult.
POU domain factor Brn-3b is required for the development of a large set of retinal ganglion cells.
Authors: Gan L , Xiang M , Zhou L , Wagner D S , Klein W H , Nathans J
Published: July 2002

The three members of the Brn-3 family of POU domain transcription factors are found in highly restricted sets of central nervous system neurons. Within the retina, these factors are present only within subsets of ganglion cells. We show here that in the developing mouse retina, Brn-3b protein is first observed in presumptive ganglion cell precursors as they begin to migrate from the zone of dividing neuroblasts to the future ganglion cell layer, and that targeted disruption of the Brn-3b gene leads in the homozygous state to a selective loss of 70% of retinal ganglion cells. In Brn-3b (-/-) mice other neurons within the retina and brain are minimally or not at all affected. These experiments indicate that Brn-3b plays an essential role in the development of specific ganglion cell types.
In vitro
selection of a 7-methyl-guanosine binding RNA that inhibits translation of capped mRNA molecules
Authors: Haller Aurelia A. , Sarnow Peter
Published: July 2002

Using systematic evolution of ligands by exponential enrichment (SELEX), an RNA molecule was isolated that displays a 1,000-fold higher affinity for guanosine residues that carry an N-7 methyl group than for nonmethylated guanosine residues. The methylated guanosine residue closely resembles the 5′ terminal cap structure present on all eukaryotic mRNA molecules. The cap-binding RNA specifically inhibited the translation of capped but not uncapped mRNA molecules in cell-free lysates prepared from either human HeLa cells or from
Saccharomyces cerevisiae
. These findings indicate that the cap-binding RNA will also be useful in studies of other cap-dependent processes such as pre-mRNA splicing and nucleocytoplasmic mRNA transport.