bluuebunny/crossref_metadata_2025 huggingface dataset:
Different mutator phenotypes in
Mlh1
- versus
Pms2
-deficient mice
Authors: Yao Xiang , Buermeyer Andrew B. , Narayanan Latha , Tran Doan , Baker Sean M. , Prolla Tomas A. , Glazer Peter M. , Liskay R. Michael , Arnheim Norman
Published: July 2002

Deficiencies in DNA mismatch repair (MMR) result in increased mutation rates and cancer risk in both humans and mice. Mouse strains homozygous for knockouts of either the
Pms2
or
Mlh1
MMR gene develop cancer but exhibit very different tumor spectra; only
Mlh1
−/−
animals develop intestinal tumors. We carried out a detailed study of the microsatellite mutation spectra in each knockout strain. Five mononucleotide repeat tracts at four different chromosomal locations were studied by using single-molecule PCR or an
in vivo
forward mutation assay. Three dinucleotide repeat loci also were examined. Surprisingly, the mononucleotide repeat mutation frequency in
Mlh1
−/−
mice was 2- to 3-fold higher than in
Pms2
−/−
animals. The higher mutation frequency in
Mlh1
−/−
mice may be a consequence of some residual DNA repair capacity in the
Pms2
−/−
animals. Relevant to this idea, we observed that
Pms2
−/−
mice exhibit almost normal levels of Mlh1p, whereas
Mlh1
−/−
animals lack both Mlh1p and Pms2p. Comparison between
Mlh1
−/−
animals and
Mlh1
−/−
and
Pms2
−/−
double knockout mice revealed little difference in mutator phenotype, suggesting that
Mlh1
nullizygosity is sufficient to inactivate MMR completely. The findings may provide a basis for understanding the greater predisposition to intestinal cancer of
Mlh1
−/−
mice. Small differences (2- to 3-fold) in mononucleotide repeat mutation rates may have dramatic effects on tumor development, requiring multiple genetic alterations in coding regions. Alternatively, this strain difference in tumor spectra also may be related to the consequences of the absence of Pms2p compared with the absence of both Pms2p and Mlh1p on as yet little understood cellular processes.
An estrogen receptor-selective coregulator that potentiates the effectiveness of antiestrogens and represses the activity of estrogens
Authors: Montano Monica M. , Ekena Kirk , Delage-Mourroux Regis , Chang Weiru , Martini Paolo , Katzenellenbogen Benita S.
Published: July 2002

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its coregulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-activated transcription factor that mediates the stimulatory effects of estrogens and the inhibitory effects of antiestrogens such as tamoxifen in breast cancer and other estrogen target cells. To understand how antiestrogens and dominant negative ERs suppress ER activity, we used a dominant negative ER as bait in two-hybrid screening assays from which we isolated a clone from breast cancer cells that potentiates the inhibitory activities of dominant negative ERs and antiestrogen-liganded ER. At higher concentrations, it also represses the transcriptional activity of the estradiol-liganded ER, while having no effect on other nuclear hormone receptors. This clone, denoted REA for “repressor of estrogen receptor activity,” encodes a 37-kDa protein that is an ER-selective coregulator. Its competitive reversal of steroid receptor coactivator 1 enhancement of ER activity and its direct interaction with liganded ER suggest that it may play an important role in determining the sensitivity of estrogen target cells, including breast cancer cells, to antiestrogens and estrogens.
Lovastatin-mediated G
1
arrest is through inhibition of the proteasome, independent of hydroxymethyl glutaryl-CoA reductase
Authors: Rao Sharmila , Porter Donald C. , Chen Xiaomei , Herliczek Thaddeus , Lowe Michael , Keyomarsi Khandan
Published: July 2002

In this paper we present the finding that lovastatin arrests cells by inhibiting the proteasome, which results in the accumulation of p21 and p27, leading to G
1
arrest. Lovastatin is an inhibitor of hydroxymethyl glutaryl (HMG)-CoA reductase, the rate-limiting enzyme in cholesterol synthesis. Previously, we reported that lovastatin can be used to arrest cultured cells in the G
1
phase of the cell cycle, resulting in the stabilization of the cyclin-dependent kinase inhibitors (CKIs) p21 and p27. In this report we show that this stabilization of p21 and p27 may be the result of a previously unknown function of the pro-drug, β-lactone ring form of lovastatin to inhibit the proteasome degradation of these CKIs. The lovastatin mixture used in this study is 80% open-ring form and 20% pro-drug, β-lactone form. We show that while the lovastatin open-ring form and pravastatin (a lovastatin analogue, 100% open ring) inhibit the HMG-CoA reductase enzyme, lovastatin pro-drug inhibits the proteasome but does not inhibit HMG-CoA reductase. In addition, many of the properties of proteasome inhibition by the pro-drug are the same as the specific proteasome inhibitor lactacystin. Lastly, mevalonate (used to rescue cells from lovastatin arrest) unexpectedly abrogates the lactacystin and lovastatin pro-drug inhibition of the proteasome. Mevalonate increases the activity of the proteasome, which results in degradation of the CKIs, allowing lovastatin- and lactacystin-arrested cells to resume cell division. The lovastatin-mediated inhibition of the proteasome suggests a unique mechanism for the chemopreventative effects of this agent seen in human cancer.
θ, a novel γ-aminobutyric acid type A receptor subunit
Authors: Bonnert Timothy P. , McKernan Ruth M. , Farrar Sophie , le Bourdellès Béatrice , Heavens Robert P. , Smith David W. , Hewson Louise , Rigby Michael R. , Sirinathsinghji Dalip J. S. , Brown Nicola , Wafford Keith A. , Whiting Paul J.
Published: July 2002

γ-Aminobutyric acid type A (GABA-A) receptors are a major mediator of inhibitory neurotransmission in the mammalian central nervous system, and the site of action of a number of clinically important drugs. These receptors exist as a family of subtypes with distinct temporal and spatial patterns of expression and distinct properties that presumably underlie a precise role for each subtype. The newest member of this gene family is the θ subunit. The deduced polypeptide sequence is 627 amino acids long and has highest sequence identity (50.5%) with the β1 subunit. Within the rat striatum, this subunit coassembles with α2, β1, and γ1, suggesting that γ-aminobutyric acid type A receptors consisting of arrangements other than αβ + γ, δ, or ɛ do exist. Expression of α2β1γ1θ in transfected mammalian cells leads to the formation of receptors with a 4-fold decrease in the affinity for γ-aminobutyric acid compared with α2β1γ1. This subunit has a unique distribution, with studies so far suggesting significant expression within monoaminergic neurons of both human and monkey brain.
Homeotic genes and the arthropod head: Expression patterns of the
labial, proboscipedia,
and
Deformed
genes in crustaceans and insects
Authors: Abzhanov Arhat , Kaufman Thomas C.
Published: July 2002

cDNA fragments of the homologues of the
Drosophila
head homeotic genes
labial
(
lab
),
proboscipedia
(
pb
), and
Deformed
(
Dfd
) have been isolated from the crustacean
Porcellio scaber.
Because the accumulation domains of the head homeotic complex (Hox) genes had not been previously reported for crustaceans, we studied the expression patterns of these genes in
P. scaber
embryos by using
in situ
hybridization. The
P. scaber lab
homologue is expressed in the developing second antennal segment and its appendages. This expression domain in crustaceans and in the homologous intercalary segment of insects suggests that the
lab
gene specified this metamere in the last common ancestor of these two groups. The expression domain of the
P. scaber pb
gene is in the posterior part of the second antennal segment. This domain, in contrast to that in insects, is colinear with the domains of other head genes in
P. scaber
, and it differs from the insect
pb
gene expression domain in the posterior mouthparts, suggesting that the insect and crustacean patterns evolved independently from a broader ancestral domain similar to that found in modern chelicerates.
P. scaber Dfd
is expressed in the mandibular segment and paragnaths (a pair of ventral mouthpart structures associated with the stomodeum) and differs from insects, where expression is in the mandibular and maxillary segments. Thus, like
pb
,
Dfd
shows a divergent Hox gene deployment. We conclude that homologous structures of the mandibulate head display striking differences in their underlying developmental programs related to Hox gene expression.
Volatile anesthetics block actin-based motility in dendritic spines
Authors: Kaech Stefanie , Brinkhaus Heike , Matus Andrew
Published: July 2002

Dendritic spines form the postsynaptic contact sites for most excitatory synapses in the brain. Spines occur in a wide range of different shapes that can vary depending on an animal’s experience or behavioral status. Recently we showed that spines on living neurons can change shape within seconds in a process that depends on actin polymerization. We have now found that this morphological plasticity is blocked by inhalational anesthetics at concentrations at which they are clinically effective. These volatile compounds also block actin-based motility in fibroblasts, indicating that their action is independent of neuron-specific components and thus identifying the actin cytoskeleton as a general cellular target of anesthetic action. These observations imply that inhibition of actin dynamics at brain synapses occurs during general anesthesia and that inhalational anesthetics are capable of influencing the morphological plasticity of excitatory synapses in the brain.
PSD-95 promotes Fyn-mediated tyrosine phosphorylation of the
N
-methyl-
d
-aspartate receptor subunit NR2A
Authors: Tezuka Tohru , Umemori Hisashi , Akiyama Tetsu , Nakanishi Shigetada , Yamamoto Tadashi
Published: July 2002

Fyn, a member of the Src-family protein-tyrosine kinase (PTK), is implicated in learning and memory that involves
N
-methyl-
d
-aspartate (NMDA) receptor function. In this study, we examined how Fyn participates in synaptic plasticity by analyzing the physical and functional interaction between Fyn and NMDA receptors. Results showed that tyrosine phosphorylation of NR2A, one of the NMDA receptor subunits, was reduced in
fyn-
mutant mice. NR2A was tyrosine-phosphorylated in 293T cells when coexpressed with Fyn. Therefore, NR2A would be a substrate for Fyn
in vivo.
Results also showed that PSD-95, which directly binds to and coclusters with NMDA receptors, promotes Fyn-mediated tyrosine phosphorylation of NR2A. Different regions of PSD-95 associated with NR2A and Fyn, respectively, and so PSD-95 could mediate complex formation of Fyn with NR2A. PSD-95 also associated with other Src-family PTKs, Src, Yes, and Lyn. These results suggest that PSD-95 is critical for regulation of NMDA receptor activity by Fyn and other Src-family PTKs, serving as a molecular scaffold for anchoring these PTKs to NR2A.
Caspase activation: The induced-proximity model
Authors: Salvesen Guy S. , Dixit Vishva M.
Published: July 2002

Members of the caspase family of proteases transmit the events that
lead to apoptosis of animal cells. Distinct members of the
family are involved in both the initiation and execution phases of cell
death, with the initiator caspases being recruited to multicomponent
signaling complexes. Initiation of apoptotic events depends on
the ability of the signaling complexes to generate an active protease.
The mechanism of activation of the caspases that constitute the
different apoptosis-signaling complexes can be explained by an
unusual property of the caspase zymogens to autoprocess to an active
form. This autoprocessing depends on intrinsic activity that resides in
the zymogens of the initiator caspases. We review evidence for a
hypothesis—the induced-proximity model—that describes how the first
proteolytic signal is produced after adapter-mediated clustering of
initiator caspase zymogens.
Structural aspects of activation pathways of aspartic protease zymogens and viral 3C protease precursors
Authors: Khan Amir R. , Khazanovich-Bernstein Nina , Bergmann Ernst M. , James Michael N. G.
Published: July 2002

The three-dimensional structures of the inactive protein precursors
(zymogens) of the serine, cysteine, aspartic, and
metalloprotease classes of proteolytic enzymes are known.
Comparisons of these structures with those of the mature, active
proteases reveal that, in general, the preformed, active conformations
of the residues involved in catalysis are rendered sterically
inaccessible to substrates by the residues of the zymogens’ N-terminal
extensions or prosegments. The prosegments interact in
nonsubstrate-like fashions with the residues of the active sites in
most of the cases. The gastric aspartic proteases have a
well-characterized zymogen conversion pathway. Structures of human
progastricsin, the inactive intermediate 2, and active human pepsin are
known and have been used to define the conversion pathway. The
structure of the zymogen precursor of plasmepsin II, the malarial
aspartic protease, shows a new twist on the mode of inactivation used
by the gastric zymogens. The prosegment of proplasmepsin disrupts the
active conformation of the two catalytic aspartic acid residues by
inducing a major reorientation of the two domains of the mature
protease. The picornaviral 2A and 3C proteases have a
chymotrypsin-like tertiary structure but with a cysteine
nucleophile. These enzymes cleave themselves from the viral polyprotein
in cis (intramolecular cleavage) and carry out trans cleavages of other
scissile peptides important for the virus life cycle. Although the
structure of the precursor viral polyprotein is unknown, it probably
resembles the organization of the proenzymes of the bacterial serine
proteases, subtilisin, and α-lytic protease. Cleavage of the
prosegment is known to occur in cis for these precursor molecules.
Multiple, targeted deficiencies in selectins reveal a predominant role for P-selectin in leukocyte recruitment
Authors: Robinson Stephen D. , Frenette Paul S. , Rayburn Helen , Cummiskey Marge , Ullman-Culleré Mollie , Wagner Denisa D. , Hynes Richard O.
Published: July 2002

We extend our previous analyses of mice deficient in selectins by describing the generation and comparative phenotype of mice lacking one, two, or three selectins after sequential ablation of the murine genes encoding P-, E-, and L-selectins. All mice deficient in selectins are viable and fertile as homozygotes. However, mice missing both P- and E-selectins (PE−/−), and mice missing all three selectins (ELP−/−) develop mucocutaneous infections that eventually lead to death. Mice deficient in multiple selectins display varying degrees of leukocytosis, resulting in part from alterations in leukocyte rolling and recruitment.PE−/−mice,ELP−/−mice, and mice missing both P- and L-selectins (PL−/−) show drastic reductions in leukocyte rolling and in extravasation of neutrophils in thioglycollate-induced peritonitis. In a separate inflammatory model (ragweed-induced peritoneal eosinophilia), we demonstrate P-selectin to be both necessary and sufficient for the recruitment of eosinophils. The phenotype of mice missing both E- and L-selectins (EL−/−) is less severe than those seen in the other double knockouts. Comparisons among the double knockouts suggest that P-selectin normally cooperates with both E- and L-selectins. Our results indicate a preeminent role for P-selectin in regulating leukocyte behavior in mice. Data from theELP−/−mice indicate, however, that all three selectins are important to leukocyte homeostasis and efficient neutrophil recruitment.
Rapid and efficient fusion of phospholipid vesicles by the α-helical core of a SNARE complex in the absence of an N-terminal regulatory domain
Authors: Parlati Francesco , Weber Thomas , McNew James A. , Westermann Benedikt , Söllner Thomas H. , Rothman James E.
Published: July 2002

A protease-resistant core domain of the neuronal SNARE complex consists of an α-helical bundle similar to the proposed fusogenic core of viral fusion proteins [Skehel, J. J. & Wiley, D. C. (1998)
Cell
95, 871–874]. We find that the isolated core of a SNARE complex efficiently fuses artificial bilayers and does so faster than full length SNAREs. Unexpectedly, a dramatic increase in speed results from removal of the N-terminal domain of the t-SNARE syntaxin, which does not affect the rate of assembly of v-t SNARES. In the absence of this negative regulatory domain, the half-time for fusion of an entire population of lipid vesicles by isolated SNARE cores (≈10 min) is compatible with the kinetics of fusion in many cell types.
“Switch I” mutant forms of the bacterial enhancer-binding protein NtrC that perturb the response to DNA
Authors: Yan Dalai , Kustu Sydney
Published: July 2002

NtrC (nitrogen regulatory protein C) is a bacterial enhancer-binding protein of 469 residues that activates transcription by σ54-holoenzyme. A region of its transcriptional activation (central) domain that is highly conserved among homologous activators of σ54-holoenzyme—residues 206–220—is essential for interaction with this RNA polymerase: it is required for contact with the polymerase and/or for coupling the energy from ATP hydrolysis to a change in the conformation of the polymerase that allows it to form transcriptionally productive open complexes. Several mutant NtrC proteins with amino acid substitutions in this region, including NtrCA216Vand NtrCG219K, have normal ATPase activity but fail in transcriptional activation. We now report that other mutant forms carrying amino acid substitutions at these same positions, NtrCA216Cand NtrCG219C, are capable of activating transcription when they are not bound to a DNA template (non-DNA-binding derivatives with an altered helix–turn–helix DNA-binding motif at the C terminus of the protein) but are unable to do so when they are bound to a DNA template, whether or not it carries a specific enhancer. Enhancer DNA remains a positive allosteric effector of ATP hydrolysis, as it is for wild-type NtrC but, surprisingly, appears to have become a negative allosteric effector for some aspect of interaction with σ54-holoenzyme. The conserved region in which these amino acid substitutions occur (206–220) is equivalent to the Switch I region of a large group of purine nucleotide-binding proteins. Interesting analogies can be drawn between the Switch I region of NtrC and that of p21ras.
The kinetic mechanism of myosin V
Authors: De La Cruz Enrique M. , Wells Amber L. , Rosenfeld Steven S. , Ostap E. Michael , Sweeney H. Lee
Published: July 2002

Myosin V is an unconventional myosin proposed to be processive on actin filaments, analogous to kinesin on a microtubule [Mehta, A. D.,
et al
. (1999)
Nature
(London)
400, 590–593]. To ascertain the unique properties of myosin V that permit processivity, we undertook a detailed kinetic analysis of the myosin V motor. We expressed a truncated, single-headed myosin V construct that bound a single light chain to study its innate kinetics, free from constraints imposed by other regions of the molecule. The data demonstrate that unlike any previously characterized myosin a single-headed myosin V spends most of its kinetic cycle (>70%) strongly bound to actin in the presence of ATP. This kinetic tuning is accomplished by increasing several of the rates preceding strong binding to actin and concomitantly prolonging the duration of the strongly bound state by slowing the rate of ADP release. The net result is a myosin unlike any previously characterized, in that ADP release is the rate-limiting step for the actin-activated ATPase cycle. Thus, because of a number of kinetic adaptations, myosin V is tuned for processive movement on actin and will be capable of transporting cargo at lower motor densities than any other characterized myosin.
In silico
detection of control signals: mRNA 3′-end-processing sequences in diverse species
Authors: Graber Joel H. , Cantor Charles R. , Mohr Scott C. , Smith Temple F.
Published: July 2002

We have investigated mRNA 3′-end-processing signals in each of six eukaryotic species (yeast, rice, arabidopsis, fruitfly, mouse, and human) through the analysis of more than 20,000 3′-expressed sequence tags. The use and conservation of the canonical AAUAAA element vary widely among the six species and are especially weak in plants and yeast. Even in the animal species, the AAUAAA signal does not appear to be as universal as indicated by previous studies. The abundance of single-base variants of AAUAAA correlates with their measured processing efficiencies. As found previously, the plant polyadenylation signals are more similar to those of yeast than to those of animals, with both common content and arrangement of the signal elements. In all species examined, the complete polyadenylation signal appears to consist of an aggregate of multiple elements. In light of these and previous results, we present a broadened concept of 3′-end-processing signals in which no single exact sequence element is universally required for processing. Rather, the total efficiency is a function of all elements and, importantly, an inefficient word in one element can be compensated for by strong words in other elements. These complex patterns indicate that effective tools to identify 3′-end-processing signals will require more than consensus sequence identification.
Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene
Authors: Seeds Nicholas W. , Basham Mark E. , Haffke Susan P.
Published: July 2002

Neuronal migration is a critical phase of brain development, where defects can lead to severe ataxia, mental retardation, and seizures. In the developing cerebellum, granule neurons turn on the gene for tissue plasminogen activator (tPA) as they begin their migration into the cerebellar molecular layer. Granule neurons both secrete tPA, an extracellular serine protease that converts the proenzyme plasminogen into the active protease plasmin, and bind tPA to their cell surface. In the nervous system, tPA activity is correlated with neurite outgrowth, neuronal migration, learning, and excitotoxic death. Here we show that compared with their normal counterparts, mice lacking the tPA gene (tPA
−/−
) have greater than 2-fold more migrating granule neurons in the cerebellar molecular layer during the most active phase of granule cell migration. A real-time analysis of granule cell migration in cerebellar slices of tPA
−/−
mice shows that granule neurons are migrating 51% as fast as granule neurons in slices from wild-type mice. These findings establish a direct role for tPA in facilitating neuronal migration, and they raise the possibility that late arriving neurons may have altered synaptic interactions.
Mice cloned from embryonic stem cells
Authors: Wakayama Teruhiko , Rodriguez Ivan , Perry Anthony C. F. , Yanagimachi Ryuzo , Mombaerts Peter
Published: July 2002

Cloning allows the asexual reproduction of selected
individuals such that the offspring have an essentially identical
nuclear genome. Cloning by nuclear transfer thus far has been reported
only with freshly isolated cells and cells from primary cultures. We
previously reported a method of cloning mice from adult somatic cells
after nuclear transfer by microinjection. Here, we apply this method to
clone mice from widely available, established embryonic stem (ES) cell
lines at late passage. With the ES cell line R1, 29% of reconstructed
oocytes developed
in vitro
to the morula/blastocyst
stage, and 8% of these embryos developed to live-born pups when
transferred to surrogate mothers. We thus cloned 26 mice from R1 cells.
Nuclei from the ES cell line E14 also were shown to direct development
to term. We present evidence that the nuclei of ES cells at
G
1
- or G
2
/M-phases are efficiently able to
support full development. Our findings demonstrate that late-passage ES
cells can be used to produce viable cloned mice and provide a link
between the technologies of ES cells and animal cloning. It thus may be
possible to clone from a single cell a large number of individuals over
an extended period.
Perceptual motion standstill in rapidly moving chromatic displays
Authors: Lu Zhong-Lin , Lesmes Luis A. , Sperling George
Published: July 2002

In motion standstill, a quickly moving object appears to stand
still, and its details are clearly visible. It is proposed that motion
standstill can occur when the spatiotemporal resolution of the shape
and color systems exceeds that of the motion systems. For moving
red-green gratings, the first- and second-order motion systems fail
when the grating is isoluminant. The third-order motion system fails
when the green/red saturation ratio produces isosalience (equal
distinctiveness of red and green). When a variety of high-contrast
red-green gratings, with different spatial frequencies and speeds, were
made isoluminant and isosalient, the perception of motion standstill
was so complete that motion direction judgments were at chance levels.
Speed ratings also indicated that, within a narrow range of luminance
contrasts and green/red saturation ratios, moving stimuli were
perceived as absolutely motionless. The results provide further
evidence that isoluminant color motion is perceived only by the
third-order motion system, and they have profound implications for the
nature of shape and color perception.
Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G
1
arrest after γ-irradiation
Authors: Brugarolas James , Moberg Ken , Boyd Scott D. , Taya Yoichi , Jacks Tyler , Lees Jacqueline A.
Published: July 2002

In mammalian cells, activation of certain checkpoint pathways as a result of exposure to genotoxic agents results in cell cycle arrest. The integrity of these arrest pathways is critical to the ability of the cell to repair mutations that otherwise might compromise viability or contribute to deregulation of cellular growth and proliferation. Here we examine the mechanism through which DNA damaging agents result in a G
1
arrest that depends on the tumor suppressor p53 and its transcriptional target p21. By using primary cell lines lacking specific cell cycle regulators, we demonstrate that this pathway functions through the growth suppressive properties of the retinoblastoma protein (pRB) tumor suppressor. Specifically, γ-irradiation inhibits the phosphorylation of pRB at cyclin-dependent kinase 2-specific, but not cyclin-dependent kinase 4-specific, sites in a p21-dependent manner. Most importantly, we show that pRB is a critical component of this DNA damage checkpoint. These data indicate that the p53 → p21 checkpoint pathway uses the normal cell cycle regulatory machinery to induce the accumulation of the growth suppressive form of pRB and suggest that loss of pRB during the course of tumorigenesis disrupts the function of an important DNA damage checkpoint.
Neurotrophins regulate agrin-induced postsynaptic differentiation
Authors: Wells David G. , McKechnie Beth A. , Kelkar Samir , Fallon Justin R.
Published: July 2002

The precise orchestration of synaptic differentiation is critical for efficient information exchange in the nervous system. The nerve–muscle synapse forms in response to agrin, which is secreted from the motor nerve terminal and induces the clustering of acetylcholine receptors (AChRs) and other elements of the postsynaptic apparatus on the subjacent muscle cell surface. In view of the highly restricted spatial localization and the plasticity of neuromuscular junctions, it seems likely that synapse formation and maintenance are regulated by additional, as-yet-unidentified factors. Here, we tested whether neurotrophins modulate the agrin-induced differentiation of postsynaptic specializations. We show that both brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4) inhibit agrin-induced AChR clustering on cultured myotubes. Nerve growth factor and NT-3 are without effect. Muscle cells express full-length TrkB, the cognate receptor for BDNF and NT-4. Direct activation of this receptor by anti-TrkB antibodies mimicked the BDNF/NT-4 inhibition of agrin-induced AChR clustering. This BDNF/NT-4 inhibition is likely to be an intrinsic mechanism for regulating AChR clustering, because neutralization of endogenous TrkB ligands resulted in elevated levels of AChR clustering even in the absence of added agrin. Finally, high concentrations of agrin can occlude the BDNF/NT-4 inhibition of AChR clustering. These results indicate that an interplay between agrin and neurotrophins can regulate the formation of postsynaptic specializations. They also suggest a mechanism for the suppression of postsynaptic specializations at nonjunctional regions.
An activation-specific role for transcription factor TFIIB
in vivo
Authors: Wu Wei-Hua , Hampsey Michael
Published: July 2002

A yeast mutant was isolated encoding a single amino acid substitution [serine-53 → proline (S53P)] in transcription factor TFIIB that impairs activation of the
PHO5
gene in response to phosphate starvation. This effect is activation-specific because S53P did not affect the uninduced level of
PHO5
expression, yet is not specific to
PHO5
because Adr1-mediated activation of the
ADH2
gene also was impaired by S53P. Pho4, the principal activator of
PHO5
, directly interacted with TFIIB
in vitro
, and this interaction was impaired by the S53P replacement. Furthermore, Pho4 induced a conformational change in TFIIB, detected by enhanced sensitivity to V8 protease. The S53P replacement also impaired activation of a lexA
(op)
-lacZ
reporter by a LexA fusion protein to the activation domain of Adr1, thereby indicating that the transcriptional effect on
ADH2
expression is specific to the activation function of Adr1. These results define an activation-specific role for TFIIB
in vivo
and suggest that certain activators induce a conformational change in TFIIB as part of their mechanism of transcriptional stimulation.
The meningococcal PilT protein is required for induction of intimate attachment to epithelial cells following pilus-mediated adhesion
Authors: Pujol Céline , Eugène Emmanuel , Marceau Michaël , Nassif Xavier
Published: July 2002

The ability of
Neisseria meningitidis
(MC) to interact with cellular barriers is essential to its pathogenesis. With epithelial cells, this process has been modeled in two steps. The initial stage of localized adherence is mediated by bacterial pili. After this phase, MC disperse and lose piliation, thus leading to a diffuse adherence. At this stage, microvilli have disappeared, and MC interact intimately with cells and are, in places, located on pedestals of actin, thus realizing attaching and effacing (AE) lesions. The bacterial attributes responsible for these latter phenotypes remain unidentified. Considering that bacteria are nonpiliated at this stage, pili cannot be directly responsible for this effect. However, the initial phase of pilus-mediated localized adherence is required for the occurrence of diffuse adherence, loss of microvilli, and intimate attachment, because nonpiliated bacteria are not capable of such a cellular interaction. In this work, we engineered a mutation in the cytoplasmic nucleotide-binding protein PilT and showed that this mutation increased piliation and abolished the dispersal phase of bacterial clumps as well as the loss of piliation. Furthermore, no intimate attachment nor AE lesions were observed. On the other hand, PilT
−
MC remained adherent as piliated clumps at all times. Taken together these data demonstrate that the induction of diffuse adherence, intimate attachment, and AE lesions after pilus-mediated adhesion requires the cytoplasmic PilT protein.
Calcium block of Na
+
channels and its effect on closing rate
Authors: Armstrong C. M. , Cota Gabriel
Published: July 2002

Calcium ion transiently blocks Na
+
channels, and it
shortens the time course for closing of their activation gates. We
examined the relation between block and closing kinetics by using the
Na
+
channels natively expressed in GH3 cells, a clonal line
of rat pituitary cells. To simplify analysis, inactivation of the
Na
+
channels was destroyed by including papain in the
internal medium. All divalent cations tested, and trivalent
La
3+
, blocked a progressively larger fraction of the
channels as their concentration increased, and they accelerated the
closing of the Na
+
channel activation gate. For calcium,
the most extensively studied cation, there is an approximately linear
relation between the fraction of the channels that are calcium-blocked
and the closing rate. Extrapolation of the data to very low calcium
suggests that closing rate is near zero when there is no block.
Analysis shows that, almost with certainty, the channels can close when
occupied by calcium. The analysis further suggests that the channels
close preferentially or exclusively from the calcium-blocked state.
High-efficiency gene transfer into skeletal muscle mediated by electric pulses
Authors: Mir Lluis M. , Bureau Michel F. , Gehl Julie , Rangara Ravi , Rouy Didier , Caillaud Jean-Michel , Delaere Pia , Branellec Didier , Schwartz Bertrand , Scherman Daniel
Published: July 2002

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.
Situs inversus and embryonic ciliary morphogenesis defects in mouse mutants lacking the KIF3A subunit of kinesin-II
Authors: Marszalek Joseph R. , Ruiz-Lozano Pilar , Roberts Elizabeth , Chien Kenneth R. , Goldstein Lawrence S. B.
Published: July 2002

The embryonic cellular events that set the asymmetry of the genetic control circuit controlling left-right (L-R) axis determination in mammals are poorly understood. New insight into this problem was obtained by analyzing mouse mutants lacking the KIF3A motor subunit of the kinesin-II motor complex. Embryos lacking KIF3A die at 10 days postcoitum, exhibit randomized establishment of L-R asymmetry, and display numerous structural abnormalities. The earliest detectable abnormality in KIF3A mutant embryos is found at day 7.5, where scanning electron microscopy reveals loss of cilia ordinarily present on cells of the wild-type embryonic node, which is thought to play an important role in setting the initial L-R asymmetry. This cellular phenotype is observed before the earliest reported time of asymmetric expression of markers of the L-R signaling pathway. These observations demonstrate that the kinesin-based transport pathway needed for flagellar and ciliary morphogenesis is conserved from
Chlamydomonas
to mammals and support the view that embryonic cilia play a role in the earliest cellular determinative events establishing L-R asymmetry.
Coculturing diverse clonal populations prevents the early-stage neoplastic progression that occurs in the separate clones
Authors: Chow Ming , Rubin Harry
Published: July 2002

Most human cancers are of monoclonal origin and display many genetic alterations. In an effort to determine whether clonal expansion itself could account for the large number of genetic alterations, we compared spontaneous transformation in cloned and uncloned populations of NIH 3T3 cells. We have reported that progressive transformation of these cells, which is driven by the stress of prolonged contact inhibition at confluence, occurs far more frequently in cultures of recent monoclonal origin than in their uncloned progenitors. In the present work we asked how coculturing six clones at early and late stages of progression would affect the dynamics of transformation in repeated rounds of confluence. When coculture started with clones in early stages of transformation, marked by light focus formation, there was a strong inhibition of the progression to the dense focus formation that occurred in separate cultures of the individual clones. In contrast, when coculture started after the individual clones had progressed to dense focus formation, there was selection of transformants from the clone producing the largest and densest foci. Mixing the cells of a single clone with a large excess of uncloned cells from a subline that was refractory to transformation markedly decreased the size of dense foci from clones in transit from light to dense focus formation, but had much less effect on foci from clones with an established capacity for dense focus formation. The major finding of protection against progression by coculturing clones in early stages of transformation suggests that the expansion of a rogue clone
in vivo
increasingly isolates many of its cells from genetically stabilizing interactions with surrounding clones. Such clonal isolation might account for the increase in mutation rates associated with the dysplasia in colorectal adenomas that signifies the transition between benign and malignant growth.
Use of soluble peptide–DR4 tetramers to detect synovial T cells specific for cartilage antigens in patients with rheumatoid arthritis
Authors: Kotzin Brian L. , Falta Michael T. , Crawford Frances , Rosloniec Edward F. , Bill Jerry , Marrack Philippa , Kappler John
Published: July 2002

Considerable evidence indicates that CD4+T cells are important in the pathogenesis of rheumatoid arthritis (RA), but the antigens recognized by these T cells in the joints of patients remain unclear. Previous studies have suggested that type II collagen (CII) and human cartilage gp39 (HCgp39) are among the most likely synovial antigens to be involved in T cell stimulation in RA. Furthermore, experiments have defined dominant peptide determinants of these antigens when presented by HLA-DR4, the most important RA-associated HLA type. We used fluorescent, soluble peptide–DR4 complexes (tetramers) to detect synovial CD4+T cells reactive with CII and HCgp39 in DR4+patients. The CII-DR4 complex bound in a specific manner to CII peptide-reactive T cell hybridomas, but did not stain a detectable fraction of synovial CD4+cells. A background percentage of positive cells (<0.2%) was not greater in DR4 (DRB1*0401) patients compared with those without this disease-associated allele. Similar results were obtained with the gp39-DR4 complex for nearly all RA patients. In a small subset of DR4+patients, however, the percentage of synovial CD4+cells binding this complex was above background and could not be attributed to nonspecific binding. These studies demonstrate the potential for peptide–MHC class II tetramers to be used to track antigen-specific T cells in human autoimmune diseases. Together, the results also suggest that the major oligoclonal CD4+T cell expansions present in RA joints are not specific for the dominant CII and HCgp39 determinants.
Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy
Authors: Tse Eric , Rabbitts Terence H.
Published: July 2002

Antibodies have been expressed inside cells in an attempt to ablate
the function of oncogene products. To make intracellular antibodies
more generally applicable and effective in cancer therapy, we have
devised a method in which programmed cell death or apoptosis
can be triggered by specific antibody–antigen interaction. When
intracellular antibodies are linked to caspase 3, the
“executioner” in the apoptosis pathway, and bind to the
target antigen, the caspase 3 moieties are self-activated and thereby
induce cell killing. We have used this strategy in a model system with
two pairs of intracellular antibodies and antigens.
In
vivo
coexpression of an antibody-caspase 3 fusion with its
antigenic target induced apoptosis that was specific for
antibody, antigen, and active caspase 3. Moreover, the antibody-caspase
3 fusion protein was not toxic to cells in the absence of antigen.
Therefore, intracellular antibody-mediated apoptosis should be
useful as a specific therapeutic approach for the treatment of cancers,
a situation where target cell killing is required.
Localization of a peripheral membrane protein: Gβγ targets Gα
Z
Authors: Fishburn C. Simone , Pollitt Sonia K. , Bourne Henry R.
Published: July 2002

To explore the relative roles of protein-binding partners vs. lipid modifications in controlling membrane targeting of a typical peripheral membrane protein, Gα
z
, we directed its binding partner, βγ, to mislocalize on mitochondria. Mislocalized βγ directed wild-type Gα
z
and a palmitate-lacking Gα
z
mutant to mitochondria but did not alter localization of a Gα
z
mutant lacking both myristate and palmitate. Thus, in this paradigm, a protein–protein interaction controls targeting of a peripheral membrane protein to the proper compartment, whereas lipid modifications stabilize interactions of proteins with membranes and with other proteins.
As climate changes, so do glaciers
Authors: Lowell Thomas V.
Published: July 2002

Understanding abrupt climate changes requires detailed
spatial/temporal records of such changes, and to make these records,
we need rapidly responding, geographically widespread climate trackers.
Glacial systems are such trackers, and recent additions to the
stratigraphic record show overall synchronous response of glacial
systems to climate change reflecting global atmosphere conditions.
Dopamine tone regulates D1 receptor trafficking and delivery in striatal neurons in dopamine transporter-deficient mice
Authors: Dumartin Brigitte , Jaber Mohamed , Gonon Francois , Caron Marc G. , Giros Bruno , Bloch Bertrand
Published: July 2002

In vivo
, G protein-coupled receptors (GPCR) for neurotransmitters undergo complex intracellular trafficking that contribute to regulate their abundance at the cell surface. Here, we report a previously undescribed alteration in the subcellular localization of D1 dopamine receptor (D1R) that occurs
in vivo
in striatal dopaminoceptive neurons in response to chronic and constitutive hyperdopaminergia. Indeed, in mice lacking the dopamine transporter, D1R is in abnormally low abundance at the plasma membrane of cell bodies and dendrites and is largely accumulated in rough endoplasmic reticulum and Golgi apparatus. Decrease of striatal extracellular dopamine concentration with 6-hydroxydopamine (6- OHDA) in heterozygous mice restores delivery of the receptor from the cytoplasm to the plasma membrane in cell bodies. These results demonstrate that,
in vivo
, in the central nervous system, the storage in cytoplasmic compartments involved in synthesis and the membrane delivery contribute to regulate GPCR availability and abundance at the surface of the neurons under control of the neurotransmitter tone. Such regulation may contribute to modulate receptivity of neurons to their endogenous ligands and related exogenous drugs.
The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase
Authors: Platt Adam , Ross Helen C. , Hankin Steven , Reece Richard J.
Published: July 2002

The transcriptional induction of the
GAL
genes of
Saccharomyces cerevisiae
occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the
GAL1
gene are inserted into the
GAL3
coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged
in vivo
and
in vitro
. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the
GAL
genes. Kinetic analysis of Gal3p+SA reveals that the
K
m
for galactose is similar to that of Gal1p, but the
K
m
for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.
The high copper tolerance of
Candida albicans
is mediated by a P-type ATPase
Authors: Weissman Ziva , Berdicevsky Israela , Cavari Ben-Zion , Kornitzer Daniel
Published: July 2002

The pathogenic yeast
Candida albicans
has higher resistance than the baker's yeast
Saccharomyces cerevisiae
to elevated concentrations of copper. To understand the basis of this differential resistance, we performed a functional screen for
C. albicans
genes involved in copper detoxification. Here, we report the isolation of two such genes: a metallothionein, Ca
CUP1
, and a copper-transporting P-type ATPase, Ca
CRP1
. Both genes are induced by extracellular copper. Gene disruptions indicated that the copper extrusion pump is responsible for the unusual resistance of
C. albicans
to copper, whereas the metallothionein is responsible for the residual copper resistance of the Ca
crp1Δ
mutant. We show further that under acidic and anaerobic conditions, such as prevail in the natural niche of
C. albicans
, the digestive tract of animals, Ca
CRP1
function becomes essential for survival in the presence of even very low copper concentrations. These observations suggest that copper in the gastrointestinal tract may present a toxic challenge to which enteric organisms had to adapt.
Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model
Authors: Fang Jianmin , Shing Yuen , Wiederschain Dmitri , Yan Li , Butterfield Catherine , Jackson Geraldine , Harper Jay , Tamvakopoulos George , Moses Marsha A.
Published: July 2002

Among the earliest and most important stages during tumorigenesis is the activation of the angiogenic process, an event that is termed the “switch to the angiogenic phenotype.” We have developed an
in vivo
system that can reliably recapitulate the stages in tumor development that represent this transition. Using this model, we have harvested and studied tumor nodules that can be distinguished from each other on the basis of their degree of vascularization. Angiogenic tumor nodules were characterized by the presence of capillary vessels as determined by factor VIII immunohistochemistry, and both angiogenic and proteolytic activities
in vitro
. In contrast, preangiogenic nodules were devoid of microvessels and showed little angiogenic or proteolytic activity
in vitro
. Addition of a specific metalloproteinase inhibitor resulted in the abrogation of both angiogenic and proteolytic activities of the angiogenic nodules
in vitro
. Comparative substrate gel electrophoresis detected the presence of a prominent matrix metalloproteinase (MMP-2) in the angiogenic nodules when compared with the preangiogenic ones. Suppression of MMP-2 activity by antisense oligonucleotides in the vascular nodules resulted in the loss of angiogenic potential both
in vitro
and
in vivo
in the chick chorioallantoic membrane assay. Moreover, this suppression of MMP-2 activity in angiogenic nodules inhibited tumor growth
in vivo
by approximately 70%. These results strongly implicate the activity of MMP-2 as a requirement for the switch to the angiogenic phenotype and validate this model as a reliable and reproducible tool by which to study other cellular and biochemical factors involved in the acquisition of the angiogenic phenotype.