bluuebunny/crossref_metadata_2025 huggingface dataset:
Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5
Authors: Ahlijanian Michael K. , Barrezueta Nestor X. , Williams Robert D. , Jakowski Amy , Kowsz Kim P. , McCarthy Sheryl , Coskran Timothy , Carlo Anthony , Seymour Patricia A. , Burkhardt John E. , Nelson Robert B. , McNeish John D.
Published: July 2002

Hyperphosphorylation of microtubule-associated proteins such as tau
and neurofilament may underlie the cytoskeletal abnormalities and
neuronal death seen in several neurodegenerative diseases including
Alzheimer's disease. One potential mechanism of microtubule-associated
protein hyperphosphorylation is augmented activity of protein kinases
known to associate with microtubules, such as cdk5 or GSK3β. Here we
show that tau and neurofilament are hyperphosphorylated in transgenic
mice that overexpress human p25, an activator of cdk5. The p25
transgenic mice display silver-positive neurons using the Bielschowsky
stain. Disturbances in neuronal cytoskeletal organization are apparent
at the ultrastructural level. These changes are localized predominantly
to the amygdala, thalamus/hypothalamus, and cortex. The p25
transgenic mice display increased spontaneous locomotor activity and
differences from control in the elevated plus-maze test. The
overexpression of an activator of cdk5 in transgenic mice results in
increased cdk5 activity that is sufficient to produce
hyperphosphorylation of tau and neurofilament as well as cytoskeletal
disruptions reminiscent of Alzheimer's disease and other
neurodegenerative diseases.
Patients with Alzheimer's disease have reduced activities in midlife compared with healthy control-group members
Authors: Friedland Robert P. , Fritsch Thomas , Smyth Kathleen A. , Koss Elisabeth , Lerner Alan J. , Chen Chien Hsiun , Petot Grace J. , Debanne Sara M.
Published: July 2002

The development of Alzheimer's disease (AD) later in life may be
reflective of environmental factors operating over the course of a
lifetime. Educational and occupational attainments have been found to
be protective against the development of the disease but participation
in activities has received little attention. In a case-control study,
we collected questionnaire data about 26 nonoccupational activities
from ages 20 to 60. Participants included 193 people with probable or
possible AD and 358 healthy control-group members. Activity patterns
for intellectual, passive, and physical activities were classified by
using an adaptation of a published scale in terms of “diversity”
(total number of activities), “intensity” (hours per month), and
“percentage intensity” (percentage of total activity hours
devoted to each activity category). The control group was more active
during midlife than the case group was for all three activity
categories, even after controlling for age, gender, income adequacy,
and education. The odds ratio for AD in those performing less than the
mean value of activities was 3.85 (95% confidence interval:
2.65–5.58,
P
< 0.001). The increase in time
devoted to intellectual activities from early adulthood (20–39) to
middle adulthood (40–60) was associated with a significant decrease in
the probability of membership in the case group. We conclude that
diversity of activities and intensity of intellectual activities were
reduced in patients with AD as compared with the control group. These
findings may be because inactivity is a risk factor for the disease or
because inactivity is a reflection of very early subclinical effects of
the disease, or both.
ANX7, a candidate tumor suppressor gene for prostate cancer
Authors: Srivastava Meera , Bubendorf Lukas , Srikantan Vasantha , Fossom Linda , Nolan Lisa , Glasman Mirta , Leighton Ximena , Fehrle Wilfred , Pittaluga Stefania , Raffeld Mark , Koivisto Pasi , Willi Niels , Gasser Thomas C. , Kononen Juha , Sauter Guido , Kallioniemi Olli P. , Srivastava Shiv , Pollard Harvey B.
Published: July 2002

The
ANX7
gene is located on human chromosome 10q21,
a site long hypothesized to harbor a tumor suppressor gene(s) (TSG)
associated with prostate and other cancers. To test whether
ANX7
might be a candidate TSG, we examined the
ANX7-
dependent suppression of human tumor cell growth,
stage-specific ANX7 expression in 301 prostate specimens on a prostate
tissue microarray, and loss of heterozygosity (LOH) of microsatellite
markers at or near the
ANX7
locus. Here we report that
human tumor cell proliferation and colony formation are markedly
reduced when the wild-type
ANX7
gene is transfected into
two prostate tumor cell lines, LNCaP and DU145. Consistently, analysis
of ANX7 protein expression in human prostate tumor microarrays reveals
a significantly higher rate of loss of ANX7 expression in metastatic
and local recurrences of hormone refractory prostate cancer as compared
with primary tumors (
P
= 0.0001). Using four
microsatellite markers at or near the
ANX7
locus, and
laser capture microdissected tumor cells, 35% of the 20 primary
prostate tumors show LOH. The microsatellite marker closest to the
ANX7
locus showed the highest rate of LOH, including one
homozygous deletion. We conclude that the
ANX7
gene
exhibits many biological and genetic properties expected of a TSG and
may play a role in prostate cancer progression.
Self-assembled monolayers from a designed combinatorial library of
de novo
β-sheet proteins
Authors: Xu Guofeng , Wang Weixun , Groves John T. , Hecht Michael H.
Published: July 2002

A variety of naturally occurring biomaterials owe their unusual
structural and mechanical properties to layers of β-sheet proteins
laminated between layers of inorganic mineral. To explore the
possibility of fabricating novel two-dimensional protein layers, we
studied the self-assembly properties of
de novo
proteins
from a designed combinatorial library. Each protein in the library has
a distinct 63 amino acid sequence, yet they all share an identical
binary pattern of polar and nonpolar residues, which was designed to
favor the formation of six-stranded amphiphilic β-sheets.
Characterization of proteins isolated from the library demonstrates
that (
i
) they self assemble into monolayers at an
air/water interface; (
ii
) the monolayers are dominated
by β-sheet secondary structure, as shown by both circular dichroism
and infrared spectroscopies; and (
iii
) the measured
areas (500- 600 Å
2
) of individual protein molecules in the
monolayers match those expected for proteins folded into amphiphilic
β-sheets. The finding that similar structures are formed by
distinctly different protein sequences suggests that assembly into
β-sheet monolayers can be encoded by binary patterning of polar and
nonpolar amino acids. Moreover, because the designed binary pattern is
compatible with a wide variety of different sequences, it may be
possible to fabricate β-sheet monolayers by using combinations of
side chains that are explicitly designed to favor particular
applications of novel biomaterials.
Severe iron deficiency anemia in transgenic mice expressing liver hepcidin
Authors: Nicolas Gaël , Bennoun Myriam , Porteu Arlette , Mativet Sandrine , Beaumont Carole , Grandchamp Bernard , Sirito Mario , Sawadogo Michèle , Kahn Axel , Vaulont Sophie
Published: July 2002

We recently reported the hemochromatosis-like phenotype observed in our
Usf2
knockout mice. In these mice, as in murine models of hemochromatosis and patients with hereditary hemochromatosis, iron accumulates in parenchymal cells (in particular, liver and pancreas), whereas the reticuloendothelial system is spared from this iron loading. We suggested that this phenotypic trait could be attributed to the absence, in the
Usf2
knockout mice, of a secreted liver-specific peptide, hepcidin. We conjectured that the reverse situation, namely overexpression of hepcidin, might result in phenotypic traits of iron deficiency. This question was addressed by generating transgenic mice expressing hepcidin under the control of the liver-specific transthyretin promoter. We found that the majority of the transgenic mice were born with a pale skin and died within a few hours after birth. These transgenic animals had decreased body iron levels and presented severe microcytic hypochromic anemia. So far, three mosaic transgenic animals have survived. They were unequivocally identified by physical features, including reduced body size, pallor, hairless and crumpled skin. These pleiotropic effects were found to be associated with erythrocyte abnormalities, with marked anisocytosis, poikylocytosis and hypochromia, which are features characteristic of iron-deficiency anemia. These results strongly support the proposed role of hepcidin as a putative iron-regulatory hormone. The animal models devoid of hepcidin (the
Usf2
knockout mice) or overexpressing the peptide (the transgenic mice presented in this paper) represent valuable tools for investigating iron homeostasis
in vivo
and for deciphering the molecular mechanisms of hepcidin action.
The myosin motor, Myo4p, binds Ash1 mRNA via the adapter protein, She3p
Authors: Takizawa Peter A. , Vale Ronald D.
Published: July 2002

In
Saccharomyces cerevisiae
, mRNA encoding the cell-fate determinant Ash1p is localized to the distal tip of daughter cells. Five
SHE
genes are required for proper Ash1 mRNA localization, one of which encodes the myosin Myo4p. We show that three of the five She proteins, She2p, She3p, and Myo4p, colocalize with Ash1 mRNA
in vivo
and coimmunoprecipitate with Ash1 mRNA from cell extracts. We also find that She3p binds to Myo4p in the absence of RNA and She2p is required for binding She3p-Myo4p to Ash1 mRNA. These results suggest that She3p acts as an adapter protein that docks the myosin motor onto an Ash1–She2p ribonucleoprotein complex.
Genetic dissection of polyunsaturated fatty acid synthesis in
Caenorhabditis
elegans
Authors: Watts Jennifer L. , Browse John
Published: July 2002

Polyunsaturated fatty acids (PUFAs) are important membrane components and precursors of signaling molecules. To investigate the roles of these fatty acids in growth, development, and neurological function in an animal system, we isolated
Caenorhabditis elegans
mutants deficient in PUFA synthesis by direct analysis of fatty acid composition.
C. elegans
possesses all the desaturase and elongase activities to synthesize arachidonic acid and eicosapentaenoic acid from saturated fatty acid precursors. In our screen we identified mutants with defects in each fatty acid desaturation and elongation step of the PUFA biosynthetic pathway. The fatty acid compositions of the mutants reveal the substrate preferences of the desaturase and elongase enzymes and clearly demarcate the steps of this pathway. The mutants show that
C. elegans
does not require n3 or Δ5-unsaturated PUFAs for normal development under laboratory conditions. However, mutants with more severe PUFA deficiencies display growth and neurological defects. The mutants provide tools for investigating the roles of PUFAs in membrane biology and cell function in this animal model.
Agonist-induced conformational changes in the G-protein-coupling domain of the β
2
adrenergic receptor
Authors: Ghanouni Pejman , Steenhuis Jacqueline J. , Farrens David L. , Kobilka Brian K.
Published: July 2002

The majority of extracellular physiologic signaling molecules
act by stimulating GTP-binding protein (G-protein)-coupled receptors
(GPCRs). To monitor directly the formation of the active state of a
prototypical GPCR, we devised a method to site specifically attach
fluorescein to an endogenous cysteine (Cys-265) at the
cytoplasmic end of transmembrane 6 (TM6) of the β
2
adrenergic receptor (β
2
AR), adjacent to the
G-protein-coupling domain. We demonstrate that this tag reports
agonist-induced conformational changes in the receptor, with agonists
causing a decline in the fluorescence intensity of
fluorescein-β
2
AR that is proportional to the biological
efficacy of the agonist. We also find that agonists alter the
interaction between the fluorescein at Cys-265 and
fluorescence-quenching reagents localized to different molecular
environments of the receptor. These observations are consistent with a
rotation and/or tilting of TM6 on agonist activation. Our studies,
when compared with studies of activation in rhodopsin, indicate a
general mechanism for GPCR activation; however, a notable difference is
the relatively slow kinetics of the conformational changes in the
β
2
AR, which may reflect the different energetics of
activation by diffusible ligands.
Laurasian migration explains Gondwanan disjunctions: Evidence from Malpighiaceae
Authors: Davis Charles C. , Bell Charles D. , Mathews Sarah , Donoghue Michael J.
Published: July 2002

Explanations for biogeographic disjunctions involving South America and Africa typically invoke vicariance of western Gondwanan biotas or long distance dispersal. These hypotheses are problematical because many groups originated and diversified well after the last known connection between Africa and South America (≈105 million years ago), and it is unlikely that “sweepstakes” dispersal accounts for many of these disjunctions. Phylogenetic analyses of the angiosperm clade Malpighiaceae, combined with fossil evidence and molecular divergence-time estimates, suggest an alternative hypothesis to account for such distributions. We propose that Malpighiaceae originated in northern South America, and that members of several clades repeatedly migrated into North America and subsequently moved via North Atlantic land connections into the Old World during episodes starting in the Eocene, when climates supported tropical forests. This Laurasian migration route may explain many other extant lineages that exhibit western Gondwanan distributions.
Subunit composition of mammalian transient receptor potential channels in living cells
Authors: Hofmann Thomas , Schaefer Michael , Schultz Günter , Gudermann Thomas
Published: July 2002

Hormones, neurotransmitters, and growth factors give rise to calcium entry via receptor-activated cation channels that are activated downstream of phospholipase C activity. Members of the transient receptor potential channel (TRPC) family have been characterized as molecular substrates mediating receptor-activated cation influx. TRPC channels are assumed to be composed of multiple TRPC proteins. However, the cellular principles governing the assembly of TRPC proteins into homo- or heteromeric ion channels still remain elusive. By pursuing four independent experimental approaches—i.e., subcellular cotrafficking of TRPC subunits, differential functional suppression by dominant-negative subunits, fluorescence resonance energy transfer between labeled TRPC subunits, and coimmunoprecipitation—we investigate the combinatorial rules of TRPC assembly. Our data show that (
i
) TRPC2 does not interact with any known TRPC protein and (
ii
) TRPC1 has the ability to form channel complexes together with TRPC4 and TRPC5. (
iii
) All other TRPCs exclusively assemble into homo- or heterotetramers within the confines of TRPC subfamilies—e.g., TRPC4/5 or TRPC3/6/7. The principles of TRPC channel formation offer the conceptual framework to assess the physiological role of distinct TRPC proteins in living cells.
Defective lymphocyte chemotaxis in β-arrestin2- and GRK6-deficient mice
Authors: Fong Alan M. , Premont Richard T. , Richardson Ricardo M. , Yu Yen-Rei A. , Lefkowitz Robert J. , Patel Dhavalkumar D.
Published: July 2002

Lymphocyte chemotaxis is a complex process by which cells move within tissues and across barriers such as vascular endothelium and is usually stimulated by chemokines such as stromal cell-derived factor-1 (CXCL12) acting via G protein-coupled receptors. Because members of this receptor family are regulated (“desensitized”) by G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation and β-arrestin binding, we examined signaling and chemotactic responses in splenocytes derived from knockout mice deficient in various β-arrestins and GRKs, with the expectation that these responses might be enhanced. Knockouts of β-arrestin2, GRK5, and GRK6 were examined because all three proteins are expressed at high levels in purified mouse CD3+ T and B220+ B splenocytes. CXCL12 stimulation of membrane GTPase activity was unaffected in splenocytes derived from GRK5-deficient mice but was increased in splenocytes from the β-arrestin2- and GRK6-deficient animals. Surprisingly, however, both T and B cells from β-arrestin2-deficient animals and T cells from GRK6-deficient animals were strikingly impaired in their ability to respond to CXCL12 both in transwell migration assays and in transendothelial migration assays. Chemotactic responses of lymphocytes from GRK5-deficient mice were unaffected. Thus, these results indicate that β-arrestin2 and GRK6 actually play positive regulatory roles in mediating the chemotactic responses of T and B lymphocytes to CXCL12.
The SOS response regulates adaptive mutation
Authors: McKenzie Gregory J. , Harris Reuben S. , Lee Peter L. , Rosenberg Susan M.
Published: July 2002

Upon starvation some
Escherichia coli
cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment. Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress. Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown. Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair. We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation. We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction. We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB. These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system.
Meiotic recombination and chromosome segregation in
Schizosaccharomyces pombe
Authors: Davis Luther , Smith Gerald R.
Published: July 2002

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast
Schizosaccharomyces pombe
and the distantly related budding yeast
Saccharomyces cerevisiae
, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.
Shotgun identification of protein modifications from protein complexes and lens tissue
Authors: MacCoss Michael J. , McDonald W. Hayes , Saraf Anita , Sadygov Rovshan , Clark Judy M. , Tasto Joseph J. , Gould Kathleen L. , Wolters Dirk , Washburn Michael , Weiss Avery , Clark John I. , Yates John R.
Published: July 2002

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.
Obligatory heterotetramerization of three previously uncharacterized Kv channel α-subunits identified in the human genome
Authors: Ottschytsch N. , Raes A. , Van Hoorick D. , Snyders D. J.
Published: July 2002

Voltage-gated K
+
channels control excitability in neuronal and various other tissues. We identified three unique α-subunits of voltage-gated K
+
-channels in the human genome. Analysis of the full-length sequences indicated that one represents a previously uncharacterized member of the Kv6 subfamily, Kv6.3, whereas the others are the first members of two unique subfamilies, Kv10.1 and Kv11.1. Although they have all of the hallmarks of voltage-gated K
+
channel subunits, they did not produce K
+
currents when expressed in mammalian cells. Confocal microscopy showed that Kv6.3, Kv10.1, and Kv11.1 alone did not reach the plasma membrane, but were retained in the endoplasmic reticulum. Yeast two-hybrid experiments failed to show homotetrameric interactions, but showed interactions with Kv2.1, Kv3.1, and Kv5.1. Co-expression of each of the previously uncharacterized subunits with Kv2.1 resulted in plasma membrane localization with currents that differed from typical Kv2.1 currents. This heteromerization was confirmed by co-immunoprecipitation. The Kv2 subfamily consists of only two members and uses interaction with “silent subunits” to diversify its function. Including the subunits described here, the “silent subunits” represent one-third of all Kv subunits, suggesting that obligatory heterotetramer formation is more widespread than previously thought.
Cysteinyl leukotriene receptor 1 is also a pyrimidinergic receptor and is expressed by human mast cells
Authors: Mellor Elizabeth A. , Maekawa Akiko , Austen K. Frank , Boyce Joshua A.
Published: July 2002

The cysteinyl leukotrienes (cys-LTs) LTC
4
, LTD
4
, and LTE
4
are a class of peptide-conjugated lipids formed from arachidonic acid and released during activation of mast cells (MCs). We now report that human cord-blood-derived MCs (hMCs) express the CysLT1 receptor, which responds not only to inflammation-derived cys-LTs, but also to a pyrimidinergic ligand, UDP. hMCs express both CysLT1 protein and transcript, and respond to LTC
4
, LTD
4
, and UDP with concentration-dependent calcium fluxes, each of which is blocked by a competitive CysLT1 receptor antagonist, MK571. Stably transfected Chinese hamster ovary cells expressing the CysLT1 receptor also exhibit MK571-sensitive calcium flux to all three agonists. Both hMCs and CysLT1 transfectants stimulated with UDP are desensitized to LTC
4
, but only partially to LTD
4
. Priming of hMCs with IL-4 for 5 days enhances their sensitivity to each agonist, but preferentially lowers their threshold for activation by LTC
4
and UDP (≈3 log
10
-fold shifts in dose-response for each agonist) over LTD
4
(1.3 log
10
-fold shift), without altering CysLT1 receptor mRNA or surface protein expression, implying the likely induction of a second receptor with CysLT1-like dual ligand specificity. hMCs thus express the CysLT1 receptor, and possibly a closely related IL-4-inducible receptor, which mediate dual activation responses to cys-LTs and UDP, providing an apparent intersection linking the inflammatory and neurogenic elements of bronchial asthma.
Raf-1 promotes cell survival by antagonizing apoptosis signal-regulating kinase 1 through a MEK–ERK independent mechanism
Authors: Chen Jing , Fujii Katsunori , Zhang Lixin , Roberts Thomas , Fu Haian
Published: July 2002

The Ser/Thr kinase Raf-1 is a protooncogene product that is a central component in many signaling pathways involved in normal cell growth and oncogenic transformation. Upon activation, Raf-1 phosphorylates mitogen-activated protein kinase kinase (MEK), which in turn activates mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs), leading to the propagation of signals. Depending on specific stimuli and cellular environment, the Raf-1–MEK–ERK cascade regulates diverse cellular processes such as proliferation, differentiation, and apoptosis. Here, we describe a MEK–ERK-independent prosurvival function of Raf-1. We found that Raf-1 interacts with the proapoptotic, stress-activated protein kinase ASK1 (apoptosis signal-regulating kinase 1)
in vitro
and
in vivo
. Deletion analysis localized the Raf-1 binding site to the N-terminal regulatory fragment of ASK1. This interaction allows Raf-1 to act independently of the MEK–ERK pathway to inhibit apoptosis. Furthermore, catalytically inactive forms of Raf-1 can mimic the wild-type effect, raising the possibility of a kinase-independent function of Raf-1. Thus, Raf-1 may promote cell survival through its protein–protein interactions in addition to its established MEK kinase function.
Dominant loss of responsiveness to sweet and bitter compounds caused by a single mutation in α-gustducin
Authors: Ruiz-Avila Luis , Wong Gwendolyn T. , Damak Sami , Margolskee Robert F.
Published: July 2002

Biochemical and genetic studies have implicated α-gustducin as a key component in the transduction of both bitter or sweet taste. Yet, α-gustducin-null mice are not completely unresponsive to bitter or sweet compounds. To gain insights into how gustducin mediates responses to bitter and sweet compounds, and to elicit the nature of the gustducin-independent pathways, we generated a dominant-negative form of α-gustducin and expressed it as a transgene from the α-gustducin promoter in both wild-type and α-gustducin-null mice. A single mutation, G352P, introduced into the C-terminal region of α-gustducin critical for receptor interaction rendered the mutant protein unresponsive to activation by taste receptor, but left its other functions intact. In control experiments, expression of wild-type α-gustducin as a transgene in α-gustducin-null mice fully restored responsiveness to bitter and sweet compounds, formally proving that the targeted deletion of the α
-gustducin
gene caused the taste deficits of the null mice. In contrast, transgenic expression of the G352P mutant did not restore responsiveness of the null mice to either bitter or sweet compounds. Furthermore, in the wild-type background, the mutant transgene inhibited endogenous α-gustducin's interactions with taste receptors, i.e., it acted as a dominant-negative. That the mutant transgene further diminished the residual bitter and sweet taste responsiveness of the α-gustducin-null mice suggests that other guanine nucleotide-binding regulatory proteins expressed in the α-gustducin lineage of taste cells mediate these responses.
ATP transduces signals from ASGM1, a glycolipid that functions as a bacterial receptor
Authors: McNamara Nancy , Khong Amy , McKemy David , Caterina Mike , Boyer Jose , Julius David , Basbaum Carol
Published: July 2002

The flagella of the Gram-negative bacterium
Pseudomonas aeruginosa
serve not only for motility but also to bind bacteria to the host cell glycolipid asialoGM1 (ASGM1) through the protein flagellin. This interaction triggers defensive responses in host cells. How this response occurs is unclear because ASGM1 lacks transmembrane and cytoplasmic domains and there is little information about the downstream effectors that connect ASGM1 ligation to the initiation of host defense responses. Here, we show that ASGM1 ligation promotes ATP release from the host cell, followed by autocrine activation of a nucleotide receptor. This response links ASGM1 to cytoplasmic signaling molecules and results in activation of phospholipase C, Ca
2+
mobilization, phosphorylation of a mitogen-activated protein kinase (Erk 1/2), and activation of mucin transcription. These results indicate that bacterial interaction with host cells can trigger autocrine nucleotide signaling and suggest that agents affecting nucleotide receptors may modulate host responses to bacteria.
The virulence factor AvrXa7 of
Xanthomonas oryzae
pv
. oryzae
is a type III secretion pathway-dependent nuclear-localized double-stranded DNA-binding protein
Authors: Yang Bing , Zhu Weiguang , Johnson Lowell B. , White Frank F.
Published: July 2002

AvrXa7
is a member of the
avrBs3
avirulence gene family, which encodes proteins targeted to plant cells by a type III secretion apparatus. AvrXa7, the product of
avrXa7
, is also a virulence factor in strain PXO86 of
Xanthomonas oryzae
pv.
oryzae
. Avirulence and virulence specificities are associated with the central repeat domain, which, in
avrXa7
, consists of 25.5 direct repeat units. Mutations in three C-terminal nuclear localization signal motifs eliminated avirulence and virulence activities in rice and severely reduced nuclear localization in a yeast assay system. Both pathogenicity functions and nuclear localization were restored on the addition of the sequence for the nuclear localization signal motif from SV40 T-antigen. The loss of avirulence activity because of mutations in the acidic transcriptional activation domain was restored by addition of the activation domain from the herpes simplex viral protein VP16. The activation domain was also required for virulence activity. However, the VP16 domain could not substitute for the endogenous domain in virulence assays. In gel shift assays, AvrXa7 bound double-stranded DNA with a preference for dA/dT rich sequences. The results indicate that products of the
avrBs3
-related genes are virulence factors targeted to host cell nuclei and have the potential to interact with the host DNA and transcriptional machinery as part of their mode of action. The results also suggest that the host defensive recognition mechanisms are targeted to the virulence factor site of action.
cAMP acts as a second messenger in pollen tube growth and reorientation
Authors: Moutinho Ana , Hussey Patrick J. , Trewavas Anthony J. , Malhó Rui
Published: July 2002

Pollen tube growth and reorientation is a prerequisite for fertilization and seed formation. Here we report imaging of cAMP distribution in living pollen tubes microinjected with the protein kinase A-derived fluorosensor. Growing tubes revealed a uniform distribution of cAMP with a resting concentration of ≈100–150 nM. Modulators of adenylyl cyclase (AC), forskolin, and dideoxyadenosine could alter these values. Transient elevations in the apical region could be correlated with changes in the tube-growth axis, suggesting a role for cAMP in polarized growth. Changes in cAMP arise through the activity of a putative AC identified in pollen. This signaling protein shows homology to functional motifs in fungal AC. Expression of the cDNA in
Escherichia coli
resulted in cAMP increase and complemented a catabolic defect in the fermentation of carbohydrates caused by the absence of cAMP in a
cya
A mutant. Antisense assays performed with oligodeoxynucleotide probes directed against conserved motifs perturbed tip growth, suggesting that modulation of cAMP concentration is vital for tip growth.
Differential role of transcription-coupled repair in UVB-induced G
2
arrest and apoptosis in mouse epidermis
Authors: van Oosten Marijke , Rebel Heggert , Friedberg Errol C. , van Steeg Harry , van der Horst Gijsbertus T. J. , van Kranen Henk J. , Westerman Anja , van Zeeland Albert A. , Mullenders Leon H. F. , de Gruijl Frank R.
Published: July 2002

Nucleotide excision repair (NER), apoptosis, and cell-cycle
regulation are major defense mechanisms against the carcinogenic
effects of UVB light. NER eliminates UVB-induced DNA photolesions via
two subpathways: global genome repair (GGR) and transcription-coupled
repair (TCR). Defects in NER result in the human disorders xeroderma
pigmentosum (XP) and Cockayne syndrome (CS), displaying severe UV
sensitivity and in the case of XP, cancer proneness. We investigated
the impact of deficiencies in NER subpathways on apoptosis,
hyperplasia, and cell cycle progression in the epidermis of UVB-exposed
CS group B (
Csb
−/−
) mice (no TCR), XP
group C (
Xpc
−/−
) mice (no GGR), and XP
group A (
Xpa
−/−
) mice (no TCR and no
GGR). On UVB treatment (250 J/m
2
),
Xpa
−/−
and
Csb
−/−
mice revealed an extensive
apoptotic response in the skin, a blockage of cell cycle
progression of epidermal cells, and strong hyperplasia. Interestingly,
the absence of this apoptotic response in the skin of wild-type
and
Xpc
−/−
mice coincided with the
ability of epidermal cells to enter the S phase. However, only
epidermal cells of
Xpc
−/−
mice
subsequently became arrested in the G
2
phase. Our data
demonstrate that TCR (and/or restoration of UVB-inhibited
transcription) enables damaged cells to progress through S phase and
prevents the induction of apoptosis and hyperplasia.
G
2
arrest is manifest only under conditions of proficient
TCR in combination with deficient GGR, indicating that epidermal cells
become arrested in the G
2
phase as a result of persisting
damage in their genome.
P
instability factor: An active maize transposon system associated with the amplification of
Tourist
-like MITEs and a new superfamily of transposases
Authors: Zhang Xiaoyu , Feschotte Cédric , Zhang Qiang , Jiang Ning , Eggleston William B. , Wessler Susan R.
Published: July 2002

Miniature inverted-repeat transposable elements (MITEs) are widespread and abundant in both plant and animal genomes. Despite the discovery and characterization of many MITE families, their origin and transposition mechanism are still poorly understood, largely because MITEs are nonautonomous elements with no coding capacity. The starting point for this study was
P instability factor
(
PIF
), an active DNA transposable element family from maize that was first identified following multiple mutagenic insertions into exactly the same site in intron 2 of the maize anthocyanin regulatory gene
R
. In this study we report the isolation of a maize
Tourist
-like MITE family called
miniature PIF
(
mPIF
) that shares several features with
PIF
elements, including identical terminal inverted repeats, similar subterminal sequences, and an unusual but striking preference for an extended 9-bp target site. These shared features indicate that
mPIF
and
PIF
elements were amplified by the same or a closely related transposase. This transposase was identified through the isolation of several
PIF
elements and the identification of one element (called
PIFa
) that cosegregated with
PIF
activity.
PIFa
encodes a putative protein with homologs in
Arabidopsis
, rice, sorghum, nematodes, and a fungus. Our data suggest that
PIFa
and these
PIF
-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS
5
group and are responsible for the origin and spread of
Tourist
-like MITEs.
Postsegregational killing does not increase plasmid stability but acts to mediate the exclusion of competing plasmids
Authors: Cooper Tim F. , Heinemann Jack A.
Published: July 2002

Postsegregational killing (PSK) systems consist of a tightly linked
toxin–antitoxin pair. Antitoxin must be continually produced to
prevent the longer lived toxin from killing the cell. PSK systems on
plasmids are widely believed to benefit the plasmid by ensuring its
stable vertical inheritance. However, experimental tests of this
“stability” hypothesis were not consistent with its predictions.
We suggest an alternative hypothesis to explain the evolution of PSK:
that PSK systems have been selected through benefiting host plasmids in
environments where plasmids must compete during horizontal
reproduction. In this “competition” hypothesis, success of PSK
systems is a consequence of plasmid–plasmid competition, rather than
from an adaptive plasmid–host relationship. In support of this
hypothesis, a plasmid-encoded
parDE
PSK system mediated
the exclusion of an isogenic Δ
parDE
plasmid. An
understanding of how PSK systems influence plasmid success may provide
insight into the evolution of other determinants (e.g., antibiotic
resistance and virulence) also rendering a cell potentially dependent
on an otherwise dispensable plasmid.
Compressive nonlinearity in the hair bundle's active response to mechanical stimulation
Authors: Martin P. , Hudspeth A. J.
Published: July 2002

The auditory system's ability to interpret sounds over a
wide range of amplitudes rests on the nonlinear responsiveness of the
ear. Whether measured by basilar-membrane vibration, nerve-fiber
activity, or perceived loudness, the ear is most sensitive to small
signals and grows progressively less responsive as stimulation
becomes stronger. Seeking a correlate of this behavior at the level of
mechanoelectrical transduction, we examined the responses of hair
bundles to direct mechanical stimulation. As reported by the motion of
an attached glass fiber, an active hair bundle from the bullfrog's
sacculus oscillates spontaneously. Sinusoidal movement of the fiber's
base by as little as ±1 nm, corresponding to the application at the
bundle's top of a force of ±0.3 pN, causes detectable phase-locking
of the bundle's oscillations to the stimulus. Although entrainment
increases as the stimulus grows, the amplitude of the hair-bundle
movement does not rise until phase-locking is nearly complete. A bundle
is most sensitive to stimulation at its frequency of spontaneous
oscillation. Far from that frequency, the sensitivity of an active hair
bundle resembles that of a passive bundle. Over most of its range, an
active hair bundle's response grows as the one-third power of the
stimulus amplitude; the bundle's sensitivity declines accordingly in
proportion to the negative two-thirds power of the excitation. This
scaling behavior, also found in the response of the mammalian basilar
membrane to sound, signals the operation of an amplificatory
process at the brink of an oscillatory instability, a Hopf bifurcation.
Trafficking of Plasmodium chabaudi adami-infected erythrocytes within the mouse spleen.
Authors: Yadava A , Kumar S , Dvorak J A , Milon G , Miller L H
Published: July 2002

Plasmodium chabaudi adami causes a nonlethal infection in mice. We found that crisis, the time of rapidly dropping parasitemia, was abrogated by splenectomy, indicating the role of spleen in parasite killing. The factors that mediate spleen-dependent immunity are not known. An earlier study in Plasmodium berghei-infected rats showed an association between increased clearance of heat-treated erythrocytes and the onset of crisis [Wyler, D. J., Quinn, T. C. & Chen, L.-T. (1982) J. Clin. Invest. 67, 1400-1404]. To determine the potential effects of different vascular beds in parasite killing, we studied the distribution of parasitized erythrocytes and bacteria in the spleens of P. chabaudi adami-infected mice during precrisis (a period of rising parasitemia) and during crisis. After intravenous injection, bacteria were localized predominantly in the marginal zone. In contrast, parasitized erythrocytes were found in the red pulp. We also found that during precrisis, a time of no immunity, the uptake of radiolabeled infected erythrocytes by the spleen was increased, not decreased. These data imply that no change occurs in the flow of parasitized erythrocytes through the spleen during the transition to an immune state (crisis). Our observations suggest that immune effector mechanisms, not circulatory changes, account for spleen-dependent parasite killing during a P. chabaudi adami infection in mice.
The nu gene acts cell-autonomously and is required for differentiation of thymic epithelial progenitors.
Authors: Blackburn C C , Augustine C L , Li R , Harvey R P , Malin M A , Boyd R L , Miller J F , Morahan G
Published: July 2002

The nude mutation (nu) causes athymia and hairlessness, but the molecular mechanisms by which it acts have not been determined. To address the role of nu in thymogenesis, we investigated whether all or part of the nude thymic epithelium could be rescued by the presence of wild-type cells in nude <--> wild-type chimeric mice. Detailed immunohistochemical analyses revealed that nude-derived cells could persist in the chimeric thymus but could not contribute to cortical or medullary epithelial networks. Nude-derived cells, present in few clusters in the medulla, expressed markers of a rare subpopulation of adult medullary epithelium. The thymic epithelial rudiment of nude mice strongly expressed these same markers, which may therefore define committed immature thymic epithelial precursor cells. To our knowledge, these data provide the first evidence that the nu gene product acts cell-autonomously and is necessary for the development of all major subpopulations of mature thymic epithelium. We propose that nu acts to regulate growth and/or differentiation, but not determination, of thymic epithelial progenitors.
Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries.
Authors: Diatchenko L , Lau Y F , Campbell A P , Chenchik A , Moqadam F , Huang B , Lukyanov S , Lukyanov K , Gurskaya N , Sverdlov E D , Siebert P D
Published: July 2002

A new and highly effective method, termed suppression subtractive hybridization (SSH), has been developed for the generation of subtracted cDNA libraries. It is based primarily on a recently described technique called suppression PCR and combines normalization and subtraction in a single procedure. The normalization step equalizes the abundance of cDNAs within the target population and the subtraction step excludes the common sequences between the target and driver populations. In a model system, the SSH technique enriched for rare sequences over 1,000-fold in one round of subtractive hybridization. We demonstrate its usefulness by generating a testis-specific cDNA library and by using the subtracted cDNA mixture as a hybridization probe to identify homologous sequences in a human Y chromosome cosmid library. The human DNA inserts in the isolated cosmids were further confirmed to be expressed in a testis-specific manner. These results suggest that the SSH technique is applicable to many molecular genetic and positional cloning studies for the identification of disease, developmental, tissue-specific, or other differentially expressed genes.
The dynamics of hepatitis B virus infection.
Authors: Payne R J , Nowak M A , Blumberg B S
Published: July 2002

We consider a cellular model of infection by the hepatitis B virus and describe how it may be used to account for two important features of the disease, namely (i) the wide variety of manifestations of infection and the age dependence thereof, and (ii) the typically long delay before the development of virus-induced liver cancer (primary hepatocellular carcinoma). The model is based on the assumption that the liver is comprised of both immature and mature hepatocytes, with these two subpopulations of cells responding contrastingly upon infection by the virus.
A distribution of tumor size at detection and its limiting form.
Authors: Yakovlev A Y , Hanin L G , Rachev S T , Tsodikov A D
Published: July 2002

A distribution of tumor size at detection is derived within the framework of a mechanistic model of carcinogenesis with the object of estimating biologically meaningful parameters of tumor latency. Its limiting form appears to be a generalization of the distribution that arises in the length-biased sampling from stationary point processes. The model renders the associated estimation problems tractable. The usefulness of the proposed approach is illustrated with an application to clinical data on premenopausal breast cancer.
Expression of Drosophila mushroom body mutations in alternative genetic backgrounds: a case study of the mushroom body miniature gene (mbm).
Authors: de Belle J S , Heisenberg M
Published: July 2002

Mutations in 12 genes regulating Drosophila melanogaster mushroom body (MB) development were each studied in two genetic backgrounds. In all cases, brain structure was qualitatively or quantitatively different after replacement of the "original" genetic background with that of the Canton Special wild-type strain. The mushroom body miniature gene (mbm) was investigated in detail. mbm supports the maintenance of MB Kenyon cell fibers in third instar larvae and their regrowth during metamorphosis. Adult mbm1 mutant females are lacking many or most Kenyon cell fibers and are impaired in MB-mediated associative odor learning. We show here that structural defects in mbm1 are apparent only in combination with an X-linked, dosage-dependent modifier (or modifiers). In the Canton Special genetic background, the mbm1 anatomical phenotype is suppressed, and MBs develop to a normal size. However, the olfactory learning phenotype is not fully restored, suggesting that submicroscopic defects persist in the MBs. Mutant mbm1 flies with full-sized MBs have normal retention but show a specific acquisition deficit that cannot be attributed to reductions in odor avoidance, shock reactivity, or locomotor behavior. We propose that polymorphic gene interactions (in addition to ontogenetic factors) determine MB size and, concomitantly, the ability to recognize and learn odors.
Ethanol causes translocation of cAMP-dependent protein kinase catalytic subunit to the nucleus.
Authors: Dohrman D P , Diamond I , Gordon A S
Published: July 2002

Short- and long-term ethanol exposures have been shown to alter cellular levels of cAMP, but little is known about the effects of ethanol on cAMP-dependent protein kinase (PKA). When cAMP levels increase, the catalytic subunit of PKA (C alpha) is released from the regulatory subunit, phosphorylates nearby proteins, and then translocates to the nucleus, where it regulates gene expression. Altered localization of C alpha would have profound effects on multiple cellular functions. Therefore, we investigated whether ethanol alters intracellular localization of C alpha. NG108-15 cells were incubated in the presence or absence of ethanol for as long as 48 h, and localization of PKA subunits was determined by immunocytochemistry. We found that ethanol exposure produced a significant translocation of C alpha from the Golgi area to the nucleus. C alpha remained in the nucleus as long as ethanol was present. There was no effect of ethanol on localization of the type I regulatory subunit of PKA. Ethanol also caused a 43% decrease in the amount of type I regulatory subunit but had no effect on the amount of C alpha as determined by Western blot. These data suggest that ethanol-induced translocation of C alpha to the nucleus may account, in part, for diverse changes in cellular function and gene expression produced by alcohol.